Silk Production in Australia

A report for the Rural Industries Research and Development Corporation

by J.G. Dingle, E. Hassan,

M. Gupta, D. George, L. Anota, and H. Begum

November 2005

RIRDC Publication No 05/145 RIRDC Project No UQ-96A

ii

© 2005 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 208 5 ISSN 1440-6845 Title of your publication: Silk Production in Australia Publication No. 05/145 Project No. UQ-96A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details J. G. Dingle School of Animal Studies The University of Queensland, Gatton Gatton, Queensland, 4343. Phone: 07 5460 1250 Fax: 07 5460 1444 Email: jgd@sas.uq.edu.au or j.dingle@uq.edu.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in November 2005 Printed on environmentally friendly paper by Canprint

iii

Foreword Silk is a highly valued natural fibre which is increasing in demand around the world. Following the positive assessment for Australia to develop a commercial silk industry (Dingle, RIRDC Publication No 00/56), this project assessed the quality of the silkworm and mulberry plant resources available in sub-tropical Australia and developed improved silkworm varieties, methods that enabled year-round availability of silkworms and mulberry leaves and improved mulberry harvesting machinery. The report reviews information already known about sericulture and moriculture and provides details of the methods that were used in the research and development work. The project was successful in identifying more productive varieties of silkworm and mulberry, in developing a Bt tolerant strain of silkworm and in developing methods of hatching silkworm eggs at any time of the year and of efficiently growing, harvesting and preserving mulberry leaves. This project was funded from RIRDC Core Funds which are provided by the Federal Government. This report, a new addition to RIRDC’s diverse range of over 1500 research publications, forms part of our New Animal Product R&D program, which aims to accelerate the development of viable new animal industries. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html Peter O’Brien Managing Director Rural Industries Research and Development Corporation

iv

Contents

Foreword ......................................................................................................................................................iii

Executive Summary ......................................................................................................................................vi Sericulture .............................................................................................................................................................. vii Moriculture............................................................................................................................................................ viii Implications and recommendations....................................................................................................................... viii

1. Introduction ...............................................................................................................................................1

Section A 2: Comparison of Silkworm Varieties in Australia and the Development of Improved Strains .......2

2. Review of literature on sericulture..............................................................................................................2 2.1 Historical background ........................................................................................................................................ 2 2.2 World silk production......................................................................................................................................... 3 2.3 Silk market features............................................................................................................................................ 4 2.4. Silkworm species and their distribution in the world ........................................................................................ 7 2.5 General morphological characteristics of silkworm........................................................................................... 9 2.6 Types of silkworm............................................................................................................................................ 10 2.7 Space, leaf, diet and sanitation for rearing ....................................................................................................... 11 2.8 Feeding behaviour of silkworm........................................................................................................................ 11 2.9 Voltinism.......................................................................................................................................................... 12 Table 2.5 The characteristics of voltinism ............................................................................................................. 12 2.10 Photoperiodic effect on hatching.................................................................................................................... 13 2.11 Moulting......................................................................................................................................................... 13 2.12 Effect of population densities on larval growth, cocoon production and adult longevity of silkworm .......... 13 2.13 Effects of temperature and humidity on silkworm development.................................................................... 13 2.14 Effect of different mounting materials on silkworm cocoon spinning ........................................................... 14 2.15 The characteristics of silkworm that affect silk production............................................................................ 14 2.16 Effect of cold storage of silkworm eggs/ or hatching..................................................................................... 15 2.17 Diapause and artificial hatching of silkworm eggs......................................................................................... 16 2.18 Biological aspects of embryonic diapause of silkworm ................................................................................. 17 2.19 Factors and stimuli for diapause induction and termination of silkworm ...................................................... 17 2.19.3.2 Metabolism of lipid .................................................................................................................................. 19 2.21 Hybridisation.................................................................................................................................................. 21 References .............................................................................................................................................................. 24

3. General methodologies .............................................................................................................................28 3.1 Silkworm, the test insect .................................................................................................................................. 28 3.2 Mulberry leaves, the silkworm food................................................................................................................. 28 3.3 The bacterium, Bacillus thuringiensis .............................................................................................................. 29 3.4 Local and exotic silkworm strains.................................................................................................................... 30 3.5 Rearing of silkworms ....................................................................................................................................... 31 3.6. Artificial hatching ........................................................................................................................................... 35 3.7 The disease bioassays....................................................................................................................................... 37 3.8 The development of disease resistance............................................................................................................. 39 3.9 Hybridization of silkworms.............................................................................................................................. 39

4. Evaluation of three silkworm varieties......................................................................................................41 4.1 Introduction ...................................................................................................................................................... 41 4.2 Materials and methods ..................................................................................................................................... 42 4.3 Results .............................................................................................................................................................. 44 4.4 Discussion ........................................................................................................................................................ 48 References .............................................................................................................................................................. 49 5.1 Introduction ...................................................................................................................................................... 50 5.2 Materials and methods ..................................................................................................................................... 51 5.3 Results .............................................................................................................................................................. 52 5.4 Discussion ........................................................................................................................................................ 56 References .............................................................................................................................................................. 57

v

6. Transfer of parental Bt-tolerant genes into hybrids of mulberry silkworm ...............................................58 6.1 Introduction ...................................................................................................................................................... 58 6.2 Materials and methods ..................................................................................................................................... 58 6.3 Results and discussion...................................................................................................................................... 59 References .............................................................................................................................................................. 63

7. Development of a method of termination of egg diapause .........................................................................64 7.1 Introduction ...................................................................................................................................................... 64 7.2 Materials and methods ..................................................................................................................................... 65 7.3 Results .............................................................................................................................................................. 67 7.4 Discussion ........................................................................................................................................................ 72 References .............................................................................................................................................................. 73

8. Development of stored and artificial feed for silkworms ...........................................................................74 8.1 Introduction ...................................................................................................................................................... 74 8.2 Mulberry leaf storage ....................................................................................................................................... 74 8.3 Artificial diets................................................................................................................................................... 74 8.4 Conclusion........................................................................................................................................................ 75 References .............................................................................................................................................................. 75

Section B: Comparison of Mulberry Varieties in Australia and Development of a Mulberry Shoot Harvester .....................................................................................................................................................76

9. Review of literature on moriculture..........................................................................................................76 9.1 Introduction ...................................................................................................................................................... 76 9.2 Overview of silk industry ................................................................................................................................. 77 9.3 Adaptability...................................................................................................................................................... 77 9.4 Breeding ........................................................................................................................................................... 78 9.5 Varieties ........................................................................................................................................................... 79 9.6 Leaf quality and yield....................................................................................................................................... 79 9.7 Mulberry cultivation......................................................................................................................................... 80 9.8 Harvesting ........................................................................................................................................................ 86 9.9. Conclusions ..................................................................................................................................................... 91 References .............................................................................................................................................................. 92

10. Evaluation of local Australian mulberry varieties ...................................................................................95 10.1 Introduction .................................................................................................................................................... 95 10.2 Materials and methods.................................................................................................................................... 95 10.3 Results ............................................................................................................................................................ 96 10.4. Conclusion..................................................................................................................................................... 98 References .............................................................................................................................................................. 98

11. Development of a mulberry shoot harvester............................................................................................99 11.1 Introduction .................................................................................................................................................... 99 11.2. The harvester components............................................................................................................................. 99 11.3 Preliminary tests............................................................................................................................................103 11.3.1 Test 1..........................................................................................................................................................103 References .............................................................................................................................................................104

12. Implications and recommendations ......................................................................................................105

vi

Executive Summary Despite the development of synthetic fibres to replace silk from the late 19th century onward, silk has maintained its appeal and value. It has many unique physical advantages and the lustre and drape of silk fabrics make silk the most attractive and highest priced natural fibre. The worldwide demand for silk is increasing but production is decreasing and an opportunity exists for Australia to use technical expertise to develop our own specialist silk industry. Australian environmental conditions are very suitable for the development of sericulture, which includes both silkworm rearing and mulberry plant cultivation for silkworm food. Silkworms (Bombyx mori) and mulberry plants (Morus spp.) are widespread in Australia but little development work has been undertaken on these resources until the present. The first step in development is to identify the most productive strains of silkworm and mulberry plants. The second step is to try to decrease the risk of disease affecting production. The third step is to try to increase production. In the case of mulberry production this latter aim is multifaceted and involves finding the best conditions under which to grow mulberry plants and the most efficient and economical way to harvest them. This project examined these aspects as a basis for establishing an Australian sericulture industry. Several attempts to establish a silk industry in Australia were made in the mid to late 1800s, but they failed due to lack of funds, poor mulberry stock, unsuitable land, disease and lack of knowledge. The Japanese economic recovery after World War II was greatly aided by developing a silk industry second only to China. However, the Japanese silk industry in rapidly declining nowadays as industrial employment offers higher incomes. In many countries sericulture is a sideline industry that offers a means for people to increase their incomes especially where prices for rural products are low. It is possible for this to occur in Australia, but it is also possible that a highly sophisticated sericultural industry would be viable. China produces about 75% of the world’s raw silk, which is valued at $1.6 billion. India produces about 15% and Korea and Turkmenistan produce just under 5% each. About 30 other countries produce commercial silk. However, it is difficult to compete with China in the international silk market and it is suggested that Australia should aim at satisfying some of its own silk needs first and then produce silk to supply niche markets overseas. In 2001 Australia imported $40 million worth of silk based products and over the last 10 years over $500 million worth of silk products have been imported. There are 400-500 species of silk-producing moths in the world, but only 8 or 9 species are cultivated commercially and the domesticated mulberry silk moth (Bombyx mori) produces 99% of the world’s silk. Several countries have state sponsored sericultural research institutes and approximately 2000 strains of mulberry silkworm have been developed. Strains differ mainly in their preferred environmental temperature and in the number of generations per year. Temperate strains usually have 1-2 generations per year (uni- and bi-voltine), produce finer stronger silk and are mainly fed on Morus alba mulberry leaves. Tropical strains usually have 4-5 generations per year (multivoltine), produce coarser and weaker silk and are usually fed on Morus nigra mulberry leaves. Within these varieties strains also differ in production in terms of eggs produced, growth rate of larvae, size of cocoon and length of thread in the cocoon. Other factors that influence silk quantity and quality include the growing conditions of the mulberry plants and silkworms. Among these factors are photoperiodicity, number of moults per generation, population density, temperature, humidity and mounting materials. Twenty-one characters of silkworm can influence silk yield. Silkworm eggs can be stored at low temperature and can be artificially hatched.

vii

Silkworms are susceptible to viral, bacterial and fungal diseases. Good hygiene and disinfection can keep growing areas clean but do not prevent the entry of infectious agents. Breeding for resistance is one way of decreasing the effects of infection. An additional hazard for any insect culture is the widespread use of a biological agent, the bacterium Bacillus thuringiensis (Bt) to control plant pests. This agent will also kill susceptible B. mori. It is possible to increase the silkworm’s natural resistance to this bacterium by exposing to the micro-organism and breeding from the survivors. In addition to direct selection of silkworm varieties for higher production and disease resistance, it is possible to obtain a boost in these features by crossing strains to produce hybrid vigour.

Sericulture All the methods used to hatch and rear silkworms and measure silk production are described. Methods of measuring disease resistance and the methods used for selecting and breeding for increased disease resistance to Bt are described. Comparison of two local Australian silkworm populations (QBill and QBite) with a population imported from Indonesia (Insab), showed that the two Australian populations had similar increases in body weight and length over the larval period, but the Insab population was superior in both characters. All three populations spent similar periods in the first four instar stages. However, Insab took two days longer to reach maturity in the fifth stage. Insab produced a heavier cocoon and a better shell ratio (cocoon shell weight: pupa weight) and greater number of eggs with a higher hatchability than QBill or QBite. The QBite population had the highest resistance to Bt. However, susceptible individuals in the Insab population took longer to die. Both characteristics should be incorporated into a breeding program for the Australian silkworm industry. The third generation Bt tolerant Insab population (30-43% larval mortality) was crossed both ways with the Bt susceptible QBill population (60-85% larval mortality). The Insab-QBill (male x female) hybrid had the higher tolerance to Bt (35% larval mortality) and the QBill-Insab hybrid population had the lowest tolerance (87.5% mortality). These mortality percentages were similar to those of the male parent and the conclusion was drawn that the Bt tolerance gene(s) was on the male sex chromosome. The variety of mechanisms involved in entering and terminating diapause (seasonal rest) enables several different procedures to be used to artificially terminate diapause. We developed three methods. (i) Cold (HCl) acid treatment was successful if eggs were refrigerated for 30 days, but the hatching period was prolonged. (ii) Hot acid treatment was successful if eggs were treated within 24 hours or refrigerated for 90 days. The hatching period of these eggs was much shorter. (iii) With no acid treatment, eggs did not hatch after 30 or 60 days refrigeration but they hatched naturally after 90 days refrigeration. All three silkworm populations produced over 97% hatch rates. Even in tropical areas mulberry plants have seasonal growth and the leaves become tough and dry in autumn and winter. Two techniques were developed to overcome this seasonal unavailability of fresh mulberry leaves: (i) Refrigeration of fresh mulberry leaves in sealed plastic bags and (ii) Artificial diets. The artificial diets contained about 30% mulberry leaves to which were added bean powder, yeast, sucrose, cellulose, agar and water. Blocks of this diet were stored in less space than mulberry leaves and could be kept refrigerated until needed. Both refrigerated leaves and the artificial diet were used to successfully maintain larval development during autumn and winter.

viii

Moriculture Mulberry (Morus sp.) is a fast growing, deciduous, deep-rooted perennial tree that grows throughout temperate, sub-tropical and tropical regions. There are at least 24 species and 100 varieties that vary in habitat, yield and nutrient content. It is estimated that one tonne of mulberry leaf is required to feed silkworms emerging from one ounce (28.3 grams) of eggs, which will yield 25-30kg of cocoons. One hectare of fertile land can yield 15-40 tonnes of mulberry leaves per year; plants in temperate regions yielding half that of plants in tropical regions. However, the higher cellulose content of tropical mulberries makes the leaf less acceptable and less nutritious. Yield and nutritional value are affected by soil type and plant density and they can be increased by fertilisation and irrigation. Twenty kilograms of raw silk per hectare can be produced from rain fed unfertilised mulberries using inferior silkworms; whereas up to 120 kg of raw silk per hectare can be produced using the best variety of mulberry with good cultivation techniques and a good silkworm breed. Harvesting is usually by picking leaves by hand or chopping branches using hand tools or machines. All these factors were examined in this project and efficient cultivation and harvesting methods were developed. Seven varieties of M. nigra (LV1-4, 6-8) and one variety of M. alba (LV5) were grown. LV2, 5 and 6 had faster growth, greater leaf mass, larger stem diameters, longer internodal lengths and fewer branches. Significant variation among varieties was apparent enabling selection for characters most suitable for various Australian conditions. A cutting machine was designed, constructed and tested. After initial testing and modification, the finished model, which is fully described and illustrated in the text, was connected to a John Deere tractor (JD 1750) with hydraulic flow rating of 47L/min at 19 MPa maximum pressure. The field test was carried out on a single row of mulberry plants 60 m long. The harvester was adjusted to an initial cutting height of 1.6m using a lifting mast and the tractor speed was 1.6km/hr. Reeling in, cutting, transferring and collection in the bin of stems cut at two heights were satisfactory.

Implications and recommendations This report described techniques and methods suited to the selection and development of suitable silkworm and mulberry varieties, and the cultivation and harvest methods that enable a sericulture industry to be commenced in Australia. It is recommended that this information be provided to potential silkworm farmers and that the silkworm and mulberry varieties and harvesting equipment developed be made available to this new rural industry. It is also recommended that the breeding and development centre established at the University of Queensland, Gatton, be funded to support the new sericultural industry.

1

1. Introduction Sericulture is practiced widely in developed countries, such as Japan and South-East Asia. In developing countries, it is essentially a village-based, and welfare oriented industry, capable of providing employment to large sections of the population. Although sericulture has been considered as a subsidiary occupation in rural areas, recent technological developments have made it possible to practice sericulture on an intensive scale, producing greater profits than most other agricultural crops and hence it can be assessed for its feasibility for establishment in Australia. FAO reports on sericulture, indicate that the 1997 world production of raw silk was valued at over 1.6 billion dollars. From the point of view of a national economy, one of the important advantages of sericulture is that it does not require much land. Australian environmental conditions are very suitable for the development of sericulture including silkworm rearing and mulberry plant cultivation for silkworm food. Silkworm (Bombyx mori L.) and mulberry plants (Morus sp.) have been found in Queensland and other states in Australia. However, establishment of a new sericulture industry might face the following problems under Australian conditions. Silkworms are highly susceptible to diseases and many of the causal agents (such as virus, fungi, bacteria), are present under Australian natural conditions. They are major constraints in silkworm disease management, as it is difficult to exclude pathogens from the silkworm rearing environment (Nataraju et al, 1999). These include infection by pathogenic micro-organisms like viruses (grasserie disease), bacteria (flacherie disease), fungi (green muscardine & white muscardine diseases) and protozoa (pebrine disease) (Shivaprakasam and Rabindra, 1996). The occurrence of disease might vary due to weather conditions, rearing season, silkworm races, feeding and rearing techniques and geographical areas. Therefore, disease prevention and the development of disease resistance in new silkworm races are extremely important for the new industry in Australia. Moreover, as present Australian silkworm races may have low silk productivity and quality, introduction or improvement for higher productivity is essential for a commercial sericulture industry. An extensive study is needed to improve existing local races for commercial purposes, and to develop new races through a breeding program, for improved silk productivity, adaptability to local environments and disease resistance/tolerance capabilities (Sen et al., 1999). Heterosis or hybrid vigour is a popularly applied method, where different silkworm races are crossed to produce stronger offspring with improve characteristics. The importation and improvement of pure race of silkworm races for higher productivity are necessary for running a profitable sericulture industry.

2

Section A: Comparison of Silkworm Varieties in Australia and the Development of Improved Strains 2. Review of literature on sericulture 2.1 Historical background Sericulture, or silk production, from the moth, Bombyx mori L., has a long and colourful history unknown to most people. Although there are several commercial species of silkworms, B. mori is the most widely used and intensively studied, and techniques for its rearing are the most improved. This insect is the sole living species in its family, Bombycidae, and has been domesticated for so long that it probably no longer survives in the wild. According to Chinese records, the discovery of silk production from B. mori occurred about 2,700 B.C. Chinese legend states that the great prince, Hoang-ti, directed his wife, Si-ling-chi, to examine the silkworm and test the practicability of using the thread. Thereafter, Si-ling-chi discovered not only the means of raising silkworms, but also the manner of reeling the silk, and of employing it to make garments. Si-ling-chi was later deified for her work and honoured with the name Seine-Than, or "The Goddess of Silk Worms". Sericulture during the following centuries spread through China and silk became a precious commodity highly sought by other countries (Krishnaswami et al., 1973; Veda et al., 1997). In 139 B.C., the world's longest highway was opened, and stretched from Eastern China to the Mediterranean Sea. In addition to tangible commodities such as gold and jade, new ideas and religions also passed along this road. This road was the historically famous "Silk Road," named after its most important commodity. Sericulture reached Japan through Korea, after the early part of the third century A.D. Shortly after 300 A.D., sericulture traveled westward and the cultivation of the silkworm was established in India. According to tradition, the egg of the insect and the seeds of the mulberry tree were carried to Tibet concealed in the headdress of a Chinese princess and from Tibet, the industry spread to India and Persia (Krishnaswami et al., 1973). The emperor Justinian gained the secrets of sericulture for the Roman Empire in 522 A.D., with the smuggling of the silkworm eggs from China by Persian monks. During the 18th and 19th centuries, Europeans also developed several major advancements in silk production. England by the 18th century led Europe in silk manufacturing because of English innovations in the textile industry. These innovations included improved silk-weaving looms, power looms and roller printing. The great French scientist, Louis Pasteur, rescued the silk industry in 1870 by showing that the epidemic Pebrine disease of silk-worms could be prevented through simple microscopic examination of adult moths. These advances set the trend for a more mechanized and scientific approach to silk production than existed previously. Sericulture has also been attempted in the United States, but these endeavours have been sporadic and largely unsuccessful. Sericulture was carried on to some extent by the early colonists of Virginia, South Carolina, and Georgia. (Cherry, 1989; Narasaiah, 1992). Many attempts have been made to establish a silk industry in Australia but none of them was viable because of:

1. Lack of funds and poor mulberry plants: In 1848, Mr Beuzeville, Sydney, attempted to establish a silk production farm but he failed because of lack of funds and poor mulberry production. It was also reported that around 1862, Charles Brady, NSW, managed to import and maintained healthy silkworm stocks (it was the time of Pebrine disease in Europe) but his project could not continue to expand because of insufficient fund.

3

2. Unsuitable land: In 1849, Mrs Bladen Neil establish the Victorian ladies “Silk Association”

a joint stock company which was granted 1000 acres of land by the Victorian government. It failed because of unsuitability of land. In 1890’s, another pioneer Louis Frederick Wurm, for the first time in Australian history, won a number of medals for his silk in Europe and America and was also awarded 100 pounds by the South Australian Government. He showed that silkworms could be reared in less time in Australia than Europe. Because of ill health, he was forced to move to a property in Adelaide which was not suitable for silkworms.

3. Diseases and lack of knowledge: In 1870, William Coote started his silk industry in Brisbane,

with funds from the Queensland Government. In 5 years, he imported improved strains of silkworms from Italy but he lost all his stocks due to Pebrine disease.

Affleck and Howard, NSW, had a similar fate. They continued a silkworm project for eight years but eventually the project failed due to the death of all stock from Pebrine disease introduced from Europe. In 1891 Sir Henry Parkes agreed to the appointment of Reginald Champ as overseer, in the first official attempt to establish commercial sericulture. Although it was profitable in its early stages, the scheme gradually failed because of Mr Champ’s insufficient knowledge and ability (Dingle, 2000).

2.2 World silk production The main silk-producing countries of the world today are, China, India, DPR Korea, Turkmenistan, Brazil, Uzbekistan, Thailand, Vietnam, Kyrgyzstan, Japan, Iran, Tajikistan, Romania and Indonesia (FAO, 2001). World silk production has approximately doubled during the last 30 years in spite of man-made fibers replacing silk for some uses. China and Japan during this period have been the two main producers, together manufacturing more than 50% of the world production each year. China during the late 1970's drastically increased its silk production and became the world's leading producer of silk. The 1970's were a period of tumultuous political and social upheaval in China, resulting in various economic reforms. Undoubtedly, these reforms are partially responsible for China's increased silk production. Thus the country that first developed sericulture approximately 4,700 years ago has again become the world's main producer of silk. Table 1. shows the world production of raw silk in 2000 was 110,316 metric tons (FAO 2001).

4

Table 2.1 FAO statistics for world production of raw silk Production (Tonnes) in

Country name 1990 1995 2000

World 86,148 115,569 110,316

Brazil 1,693 2,070 2,070

China 55,003 80,001 76,001

India 11,800 15,000 15,500

Indonesia 120 120 120

Iran 180 600 500

Japan 5,721 3,229 650

Korea (DPR) 4,200 4,000 5,000

Kyrgyzstan * 1,000 900

Romania * 140 130

Tajikistan * 200 300

Thailand 1,250 1,313 1,000

Turkey 290 271 100

Turkmenistan * 4,500 5,000

Uzbekistan * 2,000 2,000

Vietnam 500 650 900

Source: FAO Statistics 2001 * In 1990, Kyrgyzstan, Romania, Tajikistan, Turkmenistan and Uzbekistan was part of USSR, and silk production in USSR was 4,094 mt tons.

It would not be likely that Australia could compete with China in international markets. Australia should therefore aim to satisfy is own internal demand for silk and then endeavour to enter specialised niche markets which do not have large scale competition

2.3 Silk market features Ever since the invention of nylon in the late 1930s silk has faced strong competition. The nylon industry, which rapidly developed for use in the army during the war, becomes a well established industry soon after the Second World War ended. In 1946, the demand for nylon fabrics and socks covered more than fifty percent of the demand indicating a decline in demand for silk socks. Hence, raw silk fabrics exported from Japan after the World War II changed from being used to make socks to cloth. In Japan, for three years, from 1946, when the other industries had not recovered from the impact of the World War II, the export of raw silk fibres and silk fabric comprised 22% of total exports. After that the silk rearing farms were converted to food producing farms and cocoon production decreased to a mere 53,000 tons in 1947. During Korean War in 1950, the recovery of the Japanese economy steadily progressed and struggled to meet the heavy demand for silk production. The agriculture farm boom with food crops as the main item came to an end. Soon after the war ended the sericulture industry began to acquire importance. As a consequence, the export of raw silk fibres reached 16,000 tons in 1954. In the following year, cocoon production increased to 114,000 tons.

5

After 1960, the Japanese economic growth increased and there was a rapid increase in the internal demand for silk fabrics (Veda et al., 1997). Japan's silk production market was once the largest in the world, but has shrunk rapidly due to industrialization and higher labour costs. Japan is now a major importer of raw silk, cocoons etc. to meet its own domestic requirements (Narasaiah, 1992). But silk has some unique properties, which makes it able to compete against artificial silk. Bombyx mori silk is widely used for producing various silk fabrics such as dresses, kimonos, quilt covers and ties. By-products of cocoons such as inferior cocoon, un-reelable cocoon, pupal shirt, cocoon floss and waste filaments are utilized as raw materials in cosmetics, foodstuffs and in the electric industry. Silk can absorb moisture without feeling damp and its cool-in-summer, warm-in winter property has yet to be matched by cheaper synthetics. New developments in silk technology allow silk garments to be hand washed rather than dry-cleaned (Gongyin and Cui, 1996). The following table shows the properties of silk fabrics.

6

Table 2.2 Properties of silk fabrics

Property Definition Benefits and uses Abrasion resistance

Ability of fabric to withstand the rubbing inherent in everyday use.

Durability, resistance to splitting.

Absorbency

The amount of moisture a dry fibre will absorb from the air.

Comfort, warmth, water repellence, wrinkle resistance.

Draping

Ability to hang delicately. Is more appealing to the eye than other materials.

Durability

Ability to withstand wear and decay. Seemingly delicate but very strong; used for suture material.

Dyability

The fibres’ receptivity to coloration by dyes.

Able to attach to and hold colour better than any other fabric.

Elasticity

The ability of the fabric to stretch over its length without breaking.

Able to stretch up to 20% and mould itself over any shape. Used for silk stockings.

Flame resistance

Burns slowly in an open flame and is self-extinguishing once flame is removed.

Excellent fabric for wall coverings and upholstery.

Insulation Does not conduct electricity. Used to insulate electric wires. Lustre

The light reflected from the surface. Prism-shaped fibre makes silk very lustrous.

Mildew/mould resistance

Mould is white or greyish coating formed by fungi.

Resistant to mildew, moulds and rots that attack other fibres, unless left in damp conditions for long periods.

Resilience

Ability to resume an original shape after being stretched.

Tends to hang out and has good shape retention.

Size reduction

Ability to bundle or fold into a small size.

Can carry in a small space. Silk maps were hidden in clothing during the war.

Strength

Ability to resist stress. Strong, but slightly weaker when wet. Tougher than cotton or fine wools.

Weight

Silk is one of the lightest natural fabrics.

Preferred for dresses in Asia (sari, kimono, panjabi handkerchief etc.) and jockey riding jackets.

Warmth Silk feels warm on the skin. Used to line snow jackets. Source: Dingle (2000). The silk market has undergone a major change over the last decade. Silk as a raw material is traded in the form of cocoons, raw silk, waste silk, silk noils (short, tangled fibers) and spun silk or yarn. Silk products are relatively less expensive compared to other fabrics and the greater wealth of many people in the industrialized world means that silk garments are no longer bought only by the very rich. World trade in silk is divided almost equally between raw silk, fabrics and finished goods. India converts a high proportion of its raw silk production into silk fabrics of which 20 per cent are exported. China produces 90 per cent of the world's raw silk exports and 40 per cent of the world's silk fabric exports. European imports are moving away from raw silk to fabrics and finished garments. Italian and Belgian companies handle 85 per cent of European trade (FAO, 2001). Table 2.3 shows the worldwide imports and exports of raw silk and byproducts of the silk industry.

7

Table 2.3 FAO statistics for worldwide exports and imports of raw and waste silk Country name Exports in 1999

(in Mt) Imports in 1999 (in Mt)

Bangladesh - 2,200

China 12,089 173

Germany 590 967

Hong Kong 1,048 1,059

India 17 2,743

Italy 22 2,695

Japan 3 2,596

Korea (DPR) 1,000 5

Korea (South) 11 2,107

Kyrgyzstan 20 250

Paraguay 224 -

Singapore 157 120

Tajikistan 150 -

Thailand 8 75

Turkey - 154

Turkmenistan 4,100 -

Uzbekistan 240 -

Vietnam 60 90

Source: FAO Statistics 2001 There is an opportunity for Australia to enter the silk market because demand for silk is increasing but supplies are decreasing.

2.4. Silkworm species and their distribution in the world Domestication of Bombyx mori occurred in China about 2700 B.C. Once a closely guarded secret, the knowledge of sericulture eventually spread to most of the Orient and Europe. Today, most commercial silk production is in the far eastern countries where mulberry plants grow vigorously and labour costs are relatively low. Gongyin and Cui (1996) listed the species names and worldwide distribution of the main silk producing moths shown in Table 2.4. There are several families of Lepidoptera that produce silk when larvae are spinning their cocoons.

8

Table 2.4 Species and distribution of main silk-producing moths Species name Distribution 1. Bombycidae:

Bombyx mori Temperate zone, subtropical and tropical zone B. mandarina China, Japan and Korea 2. Saturniidae

Anthraea pernyi China, Korea, India, Japan and Russia A. paphia India A. yamamai Japan, China and Korea A. assamensis Assam (India) A. polyphemus Massachusetts (U.S.A) Philosamia cynthia ricini India, China, Japan, Cuba, Egypt, France and Italy P. cynthia China, Japan, India, Korea and Indonesia Attacus atlas Southern Asia Actias seleme Southern Asia Dictyopoea japonica Japan and China Eriogyna pyretorum China, Vietnam, Burma, India and Malaysia Saturnia boisduvall China and Eastern Siberia Rhodinia fugax China & Japan Hyalophora euryalus California (U.S.A) 3. Notodontidae:

Anaphe venta Zaire, Togo & neighbouring countries A. infracta Zaire, Togo & neighbouring countries A. reticulata Uganda Epanaphe carteri Cameroon E. moloneyi Zaire, Togo & neighbouring countries E. vuileti Cameroon. 4. Lasiocampidae: Borocera cajani Madagascar Gonometa postica Africa G. ruforunnea Botswana Gloreria psidii Mexico Malacosoma incarvum azteam Mexico Pachypasa otus Southeastern Europe 5. Pleridae:

Eucheira socialls Mexico Source: Gongyin and Cui (1996).

9

Silk-producing moths are members of the class Insecta, which spin cocoons the thread of which can be used by humans. In the world, there are about 400-500 species of silk-producing moths. As the surface texture of their cocoons and communal webs look like paper, leather or cloth, the idea to exploit them is very old. In fact sericulture, the process of rearing these silk-producing moths in captivity or collecting their cocoons or silk in the field for human use, has been practiced for thousands of years. There are however only 8-9 moth species which produce silk of commercial value. Nowadays the domestic silkworm or mulberry silk moth, Bombyx mori provides probably more than 99% of the silk in the world. It belongs to the Bombycoidea superfamily and Bombycidae family. There are approximately 2,000 different strains of Bombyx mori used in silk production (Reddy, 1986). Bombyx mori produces at least one generation a year. Generation time varies with the silkworm race or geographic site. Bombyx mori the domesticated silkworm is originally derived from Bombyx mandarina-moore. (Rao, 1998). The mulberry silkworm belongs to the following taxonomic classification. Phylum - Arthropoda Class - Insecta Order - Lepidoptera Superfamily - Bombycoidae Family - Bombycidae Genus - Bombyx Species - mori In contrast, other silk-producing moths are called wild silk moths or non-mulberry silk moths (Krishnaswami et al., 1973). They are not reared in captivity and cocoons are collected from field populations. In some cases, some rearing is done outdoors with little or even no protection of larvae. Most wild silk moths such as Tussar, Muga (common name) etc. belong to the families Saturniidae, Notodontidae, Lasiocampidae and Pieridae in the order Lepidoptera. It is recommended that Australia produces Bombyx mori silk because Bombyx mori silkworm is the domestic silk moth and its silk production rate is very high (more than 90%). Also it produces high quality silk. 2.5 General morphological characteristics of silkworm (i) Egg Shape Ovoid, flat with a micropyle at anterior pole Size 1.00 to 1.3 mm in length 0.9 to 1.2 mm in width Weight Single egg 0.5 mg Colour pale yellow (undeveloped), grey (embryonated) (ii) Larva Colour Black or dark brown Size 5 mm x 1 mm (early stage) and 7 cm x 1.5 cm (after hatching) Weight Early 0.4 mg and mature 2.5-3 g Head Consists of 6 - segments fused together to form cranium antennae, mandibles,

maxillae and labium are present on 2nd, 4th 5th, 6th segments respectively pairs of ocelli (simple eyes) present little above the base of antennae. Mandibles well developed for mastication.

Thorax Consists of 3 segments called pro, mesothorax and metathorax. Each segment bears a pair of jointed legs, which end in claws. Eyespot present on the 2nd thoracic segment.

Abdomen Body segments: Segments 1-9 are distinguished and the others which carry caudal legs, are fused. Pseudo legs: Present from segments 3-6 caudal horn is on dorsal side.

10

Sexual markings: In female on 8th and 9th segment silky glands are seen 8th Iswats fore gland, 9th Iswat's hind gland. 8th and 9th segment, Herold’s gland present. Tubercles: Sub-dorsal; Supra-spiracular; Infra -spiracular; and Basal tubercle Setae: Present on tubercles (3-6) Hairs: Minute, regularly distributed over the body Spiracles: 9 pairs present (1 pair thoracic and 8 pairs abdominal) Lateral line: None Mid-ventral line: None

(iii) Pupa Colour Brown Size 2.5 x 1 cm Weight 0.8-1 g Head A pair of large compound eyes and a pair of antennae present Thorax 3-pairs of legs present 2-pairs of wings Abdomen Body segments: Ten abdominal segments can be seen on the ventral side but only

nine segments are visible from dorsal side. Sexual markings: Female has fine longitudinal line on the 8th abdominal segment but it is absent in male.

Cocoon The cocoons are oval single shell slightly flossy; weight: 1-2 g, Colour: golden yellow greenish or white.

(iv) Adult: Colour Greyish Size 1.5 cm male and 2.0 cm female Head Compound eyes situated on the either side of the head, ocilli absen, antennae

conspicuous large and bi-pectinate Thorax Comprises of prothorax, mesothorax and metathorax. Each comprises of a pair of

legs and each leg consists of 5 segments, meso- and metathorax bear one pair of wing each.

Abdomen In male eight abdominal segments are visible; in female only seven. In female at the caudal end a knob like projection with sensitive hairs present; in males a pair of hooks known as “Harps” is present.

Wings Eye spot absent

Source: Rao, 1998

2.6 Types of silkworm The wide range of silkworm races and hybrids are classified into the following categories: According to place of production Native region; Chinese race, Japanese race,

European race, Tropical race etc. According to voltinism Univoltine, Bivoltine, Multivoltine (polyvoltine) According to moulting feature Trimoulter, tetramoulter, pentamoulter etc. According to cocoon colour Pure white, dirty white, yellow, buff, straw, green

and so on. According to use For high quality fabric, special texture etc. According to larval marking Plain silkworm, pattern or marked silkworm such

as strips, dark colour, zebra bands, brown spots, quail marks, multistar etc.

Source: Veda et al. (1997) and Aruga (1994).

11

The type of silkworm that would be most suited to Australia’s conditions is tetramoulter, high quality silk fabric of bivoltine/multivoltine with native region or Japanese/Chinese race.

2.7 Space, leaf, diet and sanitation for rearing Rearing space plays an important role in the success of a silkworm crop and improvement of cocoon quality. Overcrowding leads to unequal and insufficient consumption of leaf, unequal growth of worms, susceptibility to diseases and low cocoon yield. The importance of a wider rearing space has been studied both in case of multivoltine and bivoltine silkworm rearing (Sengupta and Yusuf, 1974; Roychoudhury et al., 1991). The quality of leaf is also an important factor that contributes to successful silkworm rearing. Quality of mulberry leaf depends upon the variety of mulberry and the conditions of cultivation. Moreover, the difference in the quality of mulberry leaves reflects in the difference relating to the physiological functions of larvae and the difference in the resistance against the various diseases such as dwarf disease, dogare blight, bud blight, white root rot disease and disease caused by scale insects (Aruga, 1994). It was also supported by Gabriel and Rapusas (1976) that the quality of mulberry leaf is influenced by several factors including maturity, variety, climate, season, exposure to light, irrigation and soil type in which the mulberry is grown. The proper environmental condition therefore could mean a successful cocoon crop. Das et al. (1993) observed the combined effect of rearing space, leaf quality, diet rationing and sanitation measures on the rearing performance of bivoltine silkworms at different seasons. He compared two groups; T1: involving Chinese spacing (23.34- 27.84 m 2 for 20,000 larvae.), tender leaf feeding, diet rationing (20% less of optimum) and twice bed cleaning (at 9.30 a.m. and 7.30 p. m.) per day; T2; for conventional rearing technique like Indian spacing (11.15- 22.30 m 2 for 20,000 larvae.), mature leaf feeding, no diet rationing (optimum feeding) and one time bed cleaning (at 9.30 a.m.) daily. The rearing performance of T1 was significantly better than that of T2 in all seasons, except in a hot summer period (the influence of rearing space was suppressed by other factors during hot summer). It is generally recommended to feed the silkworms with mature leaves during 5th instar, but Narayanaprakash et al. (1985) reported that bivoltine hybrid fed on tender leaves spun heavier cocoons with more fibroin content compared to those fed on mature leaves. It has also been observed that an excess of leaf causes an accumulation of unfed leaves in rearing bed leading to putrefaction and unhygienic conditions which is often more harmful than feeding less quantity of leaf. Moreover, over consumption of leaves by the larvae leads to a greater accumulation of excreta in the rearing bed and it is important to remove the excreta and unfed leaves daily. In insect rearing sanitation is very important in order to stop disease contamination in the silkworm colonies. Keep implements clean and avoid spreading contaminated material or disease organisms from infected colonies. Infected dead larvae are one of the most dangerous sources of infection and great care must be taken to cause their destruction (pers.obs.). Balavenkatasubbaiah et al. (1989) found that rearing room, tray, stand and seat paper played major roles as a sources of contamination and their effective disinfection resulted in significantly lowering the incidence of disease.

2.8 Feeding behaviour of silkworm Bombyx mori larvae feed optimally on leaves of mulberry, Morus alba L. and also on a commercial artificial diet, but reject most plants after palpating or a few bites, or even without touching (Asaoka, 2000). Stoyan et al. (1985) reported that feeding effect has also strongly influence on larval mortality and quality cocoon production as well as their larval duration as larvae feed only during their larval period. Moreover, mulberry leaf quality and feeding method play important roles in B. mori nutrition. As a nutrient, water has a major influence on the growth of immature insects (Scriber & Salansky, 1981). Silkworms utilise 85-88% of total leaf consumption during 5th instar, ranging over a period of about 8 days (Krishnaswami et al. 1973). Silkworms, being a highly domesticated insect, eat only fresh mulberry leaf and stop eating once the leaf loses its moisture and becomes unpalatable (Gangwar et al., 1993).

12

Gangwar et al. (1993) also investigated the ingestion of mulberry leaf by cross breed silkworm race MY1 x NB4D2. He observed that the leaf ingested per feed was greater during the night compared to that during the day time. Hence, the traditional practice of dividing the per day feed into four equal quantities may be recast by giving more leaf during night hours than the day hours. Maximum leaf ingestion was recorded for 22.00 hours feeding (29.8 g/100 larvae) followed by leaf ingestion at 06.00 hours feeding (28.2 g/100 larvae). They also reported that the maximum consumption of leaf occurred during first two hours of feeding, as the leaf is fresh and palatable during this period. They also noticed that Bombyx mori ingest more mulberry leaf during the night hours and early morning period when the temperature is comparatively low and water loss from the leaves is also minimum compared to the daytime.

2.9 Voltinism The mulberry silkworms may produce offspring once or many times in a single year. On the basis of the number of generations per year, they are classified in different categories such as univoltine, bivoltine and multivoltine. Univoltines produce only one generation in a year, whereas bivoltines produced two generations and multivoltines more than two generations in a year (Rao, 1998). The eggs laid by univoltine silkworms remain under diapause until next spring. The univoltine cocoons produce superior quality silk because these silkworms can rear only once in a year, the larval period is longer. Generally these varieties are used in colder regions because their life cycle suits the productive cycle of mulberry leaves in those regions. Bivoltine silkworm races have two generations in a year. The eggs hatched in spring develop to adults which lay non-diapausing eggs in the first generation and those lay dormant eggs in the second generation. The larvae of bivoltines can tolerate different environmental conditions better than univoltine (Rao, 1998). Multivoltine silkworms have a short larval period. Multivoltine females lay non-diapausing eggs and can tolerate the higher temperature and humidity. The cocoon size however is smaller. The important characteristics of voltinism are as follows in table 2.5.

Table 2.5 The characteristics of voltinism Character Univoltine and Bivoltine Multivoltine Egg Diapausing/Non-diapausing Non-diapausing Larva longer larval duration Shorter Generation One/Two generations in a year More than two generations in a year Silk filament length 1000-1500 mts and even above 600 mts and above

Source: Rao, 1998 The above data help us to understand that the silk filament length of univoltine/ bivoltine cocoon will be longer than the silk bave length of multivoltine cocoons. Further the commercial value of univoltine / bivoltine cocoons are higher than that of multivoltine. The larvae of univoltine silkworms are very sensitive to temperature and humidity and other environmental conditions. Voltinism is a heritable character and is generally controlled by hormones. Voltinism is controlled by a set of genes and the larval period is affected by environmental conditions. The longer the larval period the greater the cocoon weight, filament length and thickness of silk filament (Rao, 1998).

13

2.10 Photoperiodic effect on hatching Silkworms are photosensitive and generally have a tendency to crawl towards dim light. Rearing in either complete darkness or in bright light leads to irregularity in growth and moulting of silkworm larvae. It was recommended that larval moult is uniform when silkworms are reared in 16 hours light and remaining period in darkness (Krishnaswami et al., 1973). Bombyx mori was reported to be a short day insect (Yamashita and Hasegawa, 1985). However, Shimizu (1982) showed that an artificial diet produced long day characteristics for individuals that pass through an embryonic diapause in the next generation. It was reported that in short day photoperiodic (light:dark)combinations (L:D = 4:20), the hatching was confined to a single day. However, under all the long day (L:D = 20: 4) photoperiodic combinations, the hatching was observed on the next (second) day. Hatching was predominantly diurnal with about 95% of hatching observed at the early hours of the light part of the natural LD cycle (L:D = 12:12) (Reddy and Babu, 1990)

2.11 Moulting Silkworm larva shed their skin during development. There are trimoulters, tetra-moulters and pentamoulters, all depending upon the type of silkworm race (Rao, 1998). During moulting the silkworm stops feeding on mulberry leaves and searches for a corner and becomes dull in movements. Silkworm larvae with three moults will have a short life cycle and their silk fibre will be thin compared to the pentamoulters, which will have a longer life cycle and produce thick fibre. At the time of moulting the old skin is shed and also the tunica intima of the fore and hind intestine and the tenidium of the trachea are cast off (Rao, 1998; Aruga, 1994). The interval between two moultings is called a "stadium", and the larva at each stage is called an "instar". Casting off the old skin is called "Exuvia". In the course of moulting the content of the exuvial gland and malpighian vessel flows out between the new and old skins to help cast the exuvia (Rao, 1998). This moulting process is controlled by the secretion of hormones in brain and in turn the production of those hormones to controlled by genes. The prothoracic gland in the brain secretes ecdyson hormone. The ecdyson hormone prepare the silkworm for moulting and the juvenile hormone secreted by corpus allatum inhibits further maturation and perpetuates the silkworm larval characters to undergo moulting. The action of the prothoracic gland and corpus allatum are controlled by moulting genes and sex-linked genes respectively (Rao, 1998). Temperature and light have an influence on the moulting of silkworms. At a higher temperature, larvae enter into moulting earlier (Rao, 1998).

2.12 Effect of population densities on larval growth, cocoon production and adult longevity of silkworm It has been reported that overcrowding during the 4th and 5th larval instars decreased larval period and also might increase diseases of larvae i.e., grasserie, flacherie, pebrine and muscardine (Islam, 1981) Talukder et. al. (1990) observed that if the population density increased, the rate of cocoon production and adult longevity decreased. They recommended a population density of 100 larvae per 0.09 square metre (1 square foot).

2.13 Effects of temperature and humidity on silkworm development Among many others factors, temperature affects the growth of mulberry silkworms. Silkworm’s health was directly affected by both high and low temperature (Krishnaswami et al., 1973). Khan (1983) reported that optimum temperature (25°±2) ensured the normal growth of silkworm larvae. It was found that aged larvae were more sensitive to temperature than young ones. Pilai and Krishnaswami (1987) noticed reduced survival rate, pupation rate and cocoon quality due to high

14

temperature (above 30° ). Talukder (1993) also observed that temperature ranges above 28 ± 1OC adversely affect larval and cocoon characteristics and disease incidence as well as yield of cocoons. As well as temperature, humidity also plays an important role in silkworm rearing. The role of humidity both direct and indirect influences directly the physiological functions of the silkworm. Indirectly humidity influences the rate of withering of the leaves in the silkworm beds. It was reported that optimum humidity (75±2%) ensured the normal growth of silkworm larvae (Krishnaswami et al., 1973).

2.14 Effect of different mounting materials on silkworm cocoon spinning Data from analysis of the cocoon crop clearly indicates that among the many factors that contribute to a good yield, the mounting material used for spinning of cocoons plays a vital role. In silkworm rearing where the economic product is cocoon, the mounting material, besides affecting the yield and having a bearing on the quality of cocoon, must be economical and easily available (Rajan et al., 1996). Moreover, the aim of the mounting material is to provide many cocooning places to ripen worms. The relative impact of rearing techniques of silkworm has been worked out for B. mori under tropical conditions in India (Krishnaswami et al., 1973), but practically no information is available as to the effect of different mounting materials on the cocoon under the temperate climatic conditions. Dar et al. (1989) reported that mounting materials like glass and plastic cocoonage have a better impact on cocoon features, but these can not be afforded by poor rearers. On the other hand, mounting materials like mustard hay, pinus shootlets, and mulberry twigs are easily available. They also showed that when mustard hay and pinus shootlets are used the cocoon features were quite superior, being second only to plastic and glass cocoonages.

2.15 The characteristics of silkworm that affect silk production Commercial exploitation of the mulberry silkworm has resulted in the production of 110,316 metric tons of raw silk world-wide annually (FAO, 2001). Chatterjee et al. (1993) reported that 21 characters of Bombyx mori are recognized as contributing to silk yield quantitatively or qualitatively. These are: 1) fecundity 2) hatching percentage 3) missing percentage of young age larvae (i.e., early larval survival) 4) missing percentage of late age larvae (late larval survival) 5) total larval duration (i.e., rearing period) 6) fifth instar duration 7) cocoon number per 10,000 larvae brushed [Silkworm eggs will be either on an egg sheet or in

loose form. Brushing is the process of carefully separating the newly hatched larvae from the empty egg shells or egg sheets, and transferring them to the rearing trays with the help of a smooth brush

8) cocoon weight per 10,000 larvae brushed 9) pupation rate 10) single cocoon weight 11) single shell weight 12) shell ratio (i.e., ratio of single shell weight to single cocoon weight expressed in percentage) 13) mature larval body weight 14) floss percentage (floss is the foundation layer of the cocoon with entangled filaments from

which a continuous silk filament cannot be obtained) 15) single cocoon filament length 16) single cocoon filament weight 17) filament size 18) reelability percentage 19) raw silk percentage

15

20) neatness 21) boil-off ratio (silk thread is reeled from the cocoons by boiling them in water so that the

gummy materials are dissolved and the silk filament can be reeled without any breaks. The term is used in silk industry to classify the expected grade of raw silk with respect to reeling and weaving).

While some of these characters are heritable, others are determined by environmental factors. (Thiagarajan et al. 1993) Seasons and Silkworm strains: In India the average silk yield from indigenous strains of silkworms has been reported as around 30 kg/dfl (dfl = disease-free laying; one dfl equals approximately 500 eggs with an average 80% hatching, i.e., 400 worms), where as, in Japan the average yield is 60 kg/dfl. Thiagarajan et al. (1993) suggested that the yield in India should aim to by 50% be increased to 45 kg/dfl. This could be achieved by rearing silkworm strains most suited for particular seasons. In Japan there are 19 strains suitable for spring rearing and 22 strains suitable for summer and autumn rearing (Shimizu & Tajima, 1972). Physical factors: Physical factors such as temperature and relative humidity (RH) in different seasons also greatly influence the growth of silkworms (Gabriel & Rapusas, 1976). The first and second instars are reared at 26-28 OC temperature and 80-90% RH, they are healthier in later stages (third, fourth and fifth instars). Temperature, RH, and ventilation during the spinning of silkworms also influence the quality of silk. Silk filament length: The length of silk filament also may vary in the given strain in different seasons (Ueda et al., 1969). Recent experiments have shown that physical properties such as cocoon weight, shell weight, and filament length will be optimal when mature Bombyx mori are kept at 21-24 OC temperature and 67% RH.

2.16 Effect of cold storage of silkworm eggs/ or hatching For the successful operation of a sericulture industry, the role of the grainage (egg production and storage) segment for the continuous and proper supply of suitable seed (silkworm egg) to the rearers is very significant. The silkworm eggs should have superior and robust features suitable for the climatic conditions of the area where the operation is going on. The preservation of silkworm eggs at low temperature has been in practice for ten decades but the method of cold storage of silkworm eggs was improved in the beginning of 19th century by the use of refrigeration. The tolerance to low temperature decreased gradually with the increase in incubation period of the eggs. The hatching of silkworm eggs under incubation can be delayed safely for 2-3 days by refrigerating them at the blue egg stage. Chaturvedi and Upadhyay (1990) found that the hatchability of eggs gradually went down when they were cold stored beyond 5 days at various embryonic stages 48 h after oviposition. The early stages of embryonic development of silkworm are more resistant to low temperature than are more to advanced stages. It is recommended that silkworm production follow the following procedures: Type of silkworm; proper sanitation of rearing house and wider rearing space; good quality of leaf; optimum feeding; proper time of feeding; the length of light:dark period; optimum temperature and humidity (25°±2, 75±2%) better mounting materials.

16

2.17 Diapause and artificial hatching of silkworm eggs Bivoltine silkworm races are basically adapted to temperate regions. The number of generations in a year is genetically determined. It over-winters in the egg stage and the diapause is due to the diapause hormone secreted by the sub-esophageal ganglion (Kubota et al., 1979). The hormone plays an important role in the accumulation of 3-hydroxykynurenine and glycogen in the developing oocyte (Yamashita et al., 1972). Diapause is a special phenomenon in insects to avoid unfavourable environmental conditions. When the diapause eggs are left under natural conditions, the embryo grows to a particular stage and then stops growing to enter into diapause (Veda et al., 1997). Once the embryo enters diapause, unless it hibernates through winter and than is subjected to particular temperature, the embryo does not resume growth. However, after the eggs are laid, if they are subjected to an artificial treatment at a proper period, it is possible to stimulate further growth without involving diapause. It is this artificial method of stimulating hatching that is known as artificial hatching. As a means of artificial hatching, physical stimuli and chemical stimuli have been tested to break the diapause. Temperature and friction treatment, hot water, air pressure, electric treatment, ultraviolet treatment, supersonic treatment, are included in the physical stimuli category. On the other hand, HCl treatment, nitric acid treatment, sulphuric acid treatment, enzyme treatment, ozone treatment and perchloride treatment are included in the chemical stimuli category (Veda et al, 1997). Chilling is generally effective in termination of insect diapause (Yamashita and Yaginuma, 1991). Exposure of diapausing silkworm eggs to lower temperature of 5-7.5 OC for over 60 days completely terminates diapause (Takami, 1969). However, the appropriate chilling duration depends upon the amount of time for which the eggs were exposed to 25 OC after being laid. The rearing of bivoltine silkworms could be taken up throughout the year in tropical conditions by adopting an artificial means of termination of diapause at a specific stage of embryonic development. Manjula & Hurkadli (1995) have evolved short term and ordinary chilling procedures for bivoltine silkworm eggs for temperate and tropical conditions respectively. During favourable seasons, quality bivoltine silkworm eggs can be prepared in excess and cold stored. If all insects are considered, for insects having one generation a year (univoltine), diapause will intervene at the correct time regardless of environmental conditions and is termed obligatory diapause. In those having two or more generations a year (bi- or polyvoltine), diapause occurs in that generation which spans the unfavorable period and is called facultative diapause (Mansingh, 1971). Diapause occurs at embryonic, larval, pupal or adult reproductive stages of the life cycle and the stage is characteristically fixed in each species. However, treohole breeding mosquitos, Aedes triseriatus, overwinter primarily as diapausing embryos but have the capacity of entering diapause at the last (fourth) instar (Kappus and Venard, 1967). In the pitcherplant mosquito, Wyeomyia smithii, diapause ordinarily occurs in the third larval instar, but a second diapause can occur in the fourth instar, thereby affording an individual mosquito two diapause options in a single year (Lounibos and Bradshaw, 1975). The majority of insects are so-called long-day insects, i.e. short-day conditions induce diapause and long-day conditions terminate it. Some insects are of the short-day type and the situation is the reverse of the long-day type. Some bivoltine insects exhibit both long- and short-day responses. For example, the cabbage moth Mamestra brassicae (Masaki and Sakai, 1965) has two types of ecologically and physiologically different pupal diapause: summer diapause (aestivation) and hibernal diapause. Although the short day length induces the hibernal type of diapause in those insects, the long day length induces either pupal aestivation or non-diapause pupae. It was suggested that the pupae produced by long day length retain the dual potency of the two developmental channels; the switch is on at a low temperature during larval stages to promote metamorphosis and off at a high temperature during larval stages so that retardation occurs (Masaki and Sakai, 1965).

17

2.18 Biological aspects of embryonic diapause of silkworm In Australia the common field cricket Teleogryllus commodus is multivoltine in the far-northern latitudes but univoltine in the southern latitudes, the initiation of embryonic diapause being regulated by temperature after oviposition. It is suggested that their original habitat was the tropical region favorable for the growth and breeding of the multivoltine population and that diapause has evolved as an adaptation enabling their survival in the southern latitudes of Australia (Hogan, 1965).In the case of the western corn rootworm, Diabrotica virgifera, it is also inferred that the mechanism of egg diapause evolved in the tropics or subtropics to overcome a dry season adapting the species to a temperate climate, and subsequently it invaded the temperate regions of North America (Krysan el al., 1977). In Japan the band-legged ground cricket Pteronemobius fascipes is univoltine in the north but bivoltine in the south, the egg diapause being controlled by the photoperiod acting on the parents. The response is of the long-day type, and the critical photoperiod lies between 13 and 14 h light per day. Although this cricket appears to be a long-day insect on the basis of its egg diapause, nymphal development shows a short-day response. That is, nymphal development is retarded by long-day photoperiod and the degree of retardation increases toward the south or low latitudes, the retarding photoperiod being coincidentally shifted to a short-day range. From these results, Masaki (1973) suggested that the bivoltine population is probably the original form from which the univoltine population has evolved. Krysan, et al. (1977) also reported that diapause intensity is to be closely correlated with latitude. For example, Diabrolica virgifera (rootworm)) has shorter diapause duration in populations residing in cooler regions than those of warmer regions. Thus, diapause must have arisen so that insects could cope with environmental pressures, and the general physiological similarities among different types of diapause may result from convergent evolution. However, the adaptation mechanism is different from species to species and even within the same species. Incidentally, special attention has been paid to the egg diapause of silkworms by industrial interests. Practically, univoltine and bivoltine silkworms produce cocoons superior, in both quality and quantity, to those of polyvoltine or multivoltine larvae. In a given strain, silkworms programmed to lay diapause eggs are superior to those that lay non-diapause eggs. Thus, the programming to diapause is desirable for increased silk production but the resultant diapause eggs are not suitable for continuous rearing of larvae through a year. To overcome this contradiction, many attempts were made in Japan to elucidate diapause mechanisms in silkworm eggs over the past 70 years. Investigations concentrated on the regulation of diapause induction by environmental stimuli and the mechanism of diapause break. The successful application of these results has contributed greatly to the development of the silk industry. It would be necessary to develop methods of diapause induction and cessation to support the development of a silk industry in Australia.

2.19 Factors and stimuli for diapause induction and termination of silkworm 2.19.1 The effect of temperature It is well known in longday insects that long days and high temperatures function to avert diapause, while short days and low temperatures induce diapause; high temperature reduces the effect of short days while low temperature ensures its effect. In egg diapause occurring at young embryonic stages such as with Teleogryllus commodus (Hogan, 1965), temperature acts directly on the eggs after oviposition contrary to the case of diapause in fully developed embryos.

18

The silkworm, Bombyx mori is a short day insect and diapause determination is almost maternal, that is, temperature as well. Photoperiod has most effect at the embryonic stage of the previous generation and only supplementary in the post-embryonic stages. The sensitive embryonic stage begins just before blastokinesis (Yamashita and Hasegawa, 1985). The following table 2.6 summarises the effect of temperature and light on diapause induction in bivoltine silkworms. Table 2.6 Effects of temperature and light on egg diapause in Bombyx mori (bivoltine race) Condition of temperature and light at Embryonic stage First to second Fourth to

pupal age Rate of adults laying diapause and non diapause eggs

25° Light Dark

25° or 20° 25° or 20°

25° 25°

Diapause Diapause

20° Light Dark

25° or 20° 20° 25° 25°

25° or 20° 25° 25° 20°

Diapause Diapause < Non-diapause Diapause < Non-diapause Diapause < Non-diapause

15° Light 20° 25° 25°

25° 25° 20°

Diapause < Non-diapause Diapause < Non-diapause Diapause < Non-diapause

Dark 25° or 20° 25° or 20° Non-diapause Note: for diapause egg production in multivoltine races, high temperature (28-30° C) and light during embryonic life as well as low temperature (20O C) from late larval stage to adult emergence are required. Additional exposure of newly laid eggs to 15° C guarantees diapause; otherwise, eggs even like dark coloured diapause eggs sometimes develop and hatch. (Source: Yamashita and Hasegawa, 1985).

2.19.2 Chemical effects on diapause of silkworm eggs There are two approaches used to investigate the effects of chemicals on egg diapause: injection of test chemicals into female pupae and direct application to oviposited eggs. Diapause of Teleogryllus commodus eggs terminates on exposure to ammonia gas, but the role of these chemicals is unclear (Hogan, 1964). In the Chrysomelid beetle Atrachya menetriesi, Ando (1971) reported that egg diapause terminates upon dipping in mercuric chloride solution and that the site of action of mercuric ions is the chorion itself; presumably, the ions increase the permeability of the chorion to oxygen. HCl treatment of silkworm diapause eggs has been the method of choice for blocking diapause for about 70 years both in industry and the basic laboratory. However, it is still an open question how the acid treatment blocks diapause. HCl infiltrating into eggs was supposed to impede the embryonic protein synthesis system in yolk cells which results in the resumption of embryogenesis (Park and Yoshitake, 1970); HCl treatment was reported to stimulate the activity of specific esterase isozyme (Kai and Nishi, 1976) and RNA synthetic activity (Kurata et al, 1979) in treated diapause eggs.

2.19.3 Diapause hormone and metabolism of silkworm Organogenesis is attributable to the initiation of gene expression followed by the synthesis of new proteins, while the cessation of gene expression results in arrested development and differentiation. Along this line, nucleic acid analyses were performed to elucidate the nature of egg diapause in Bombyx mori. DNA content increases logarithmically in both non-diapause and diapause embryos at one day after oviposition. In the former, rapid DNA increases continue for several more days, but in

19

the latter no further increase is observable at 2 days after oviposition when diapause is established (Kurata et al., 1980). In another study, the RNA content of eggs of both types increased slightly during the first 2 days after oviposition and then rose steeply and linearly throughout embryogenesis in non-diapause eggs with a peak of RNA synthetic activity at day 2, but remained at a low level in diapause eggs (Kurata et al., 1979). On the other hand, the hormone was reported to reduce strikingly the rate of conversion of low-molecular RNA to high molecular RNA, in pupal silkworm ovaries (Hasegawa and Yamashita, 1967), but this result was not pursued. Therefore, the connection between nucleic acid metabolism and the diapause hormone is conjectural. 2.19.3.1 Pigment metabolism Coloration or pigmentation of insects is due mainly to melanin, pterine pigments, ommochrome and carotenoid pigments. Melanin is synthesised from phenylalanine or tyrosine, pterine pigment from guanosine5'-monophosphate and ommochrome from tryptophan, but carotenoid pigments originate from the diet. In some insects the coloration is closely correlated with diapause, and the colour pattern is sometimes used as an index to distinguish diapause from non-diapause animals! Among these pigments, ommochrome is well documented in relation to embryonic diapause (Yamashita and Hasegawa, 1985). Silkworm eggs of the diapause type are black due to ommochrome formed in the serosa cells, while non-diapause eggs are light yellow due to the lack of this pigment. Pigmentation of diapause eggs occurs during the first 2 days after oviposition the pigment precursor, 3-OHkyn (kynurenine), is accumulated in the follicle cell during pupal development (Ogawa and Hasegawa, 1975).

2.19.3.2 Metabolism of lipid Lipid and carbohydrate are the main substances utilised to supply the metabolic energy and cell constituents during dormancy and post dormancy. Kim et al., (1981) stated that the lipid content of the eggshell as well as in eggs is higher in diapause eggs than in non-diapause eggs of silkworm. The lipid concentration of silkworm diapause eggs decreases as diapause proceeds and it is thought that lipid is the major substrate consumed during diapause (Chino, 1958). 2.19.3.3. Metabolism of protein To consider protein metabolism in relation to egg diapause it is advisable to distinguish the proteins stored as nutrients from those that function as enzymes. Engelmann (1979) reported about yolk proteins, especially vitellogenin or vitellin. In adult reproductive diapause, the biosynthesis and hormonal regulation of vitellin appear to be quite important. Since vitellin is the predominant protein component in insect eggs accounting for 50-90% of the egg proteins, the metabolism of this protein seems to be closely associated with the physiological events of embryogenesis. The vitellin of Bombyx mori was purified and characterised as a glycolipoprotein with a molecular weight of 440,000 (Izumi et al., 1980). Immunotitration of vitellin in Bombyx eggs showed that vitellin degradation occurred only at the later stage of embryogenesis and about 40% of the initial amounts remained unused at larval hatching (Irie and Yamashita, 1980), indicating no utilisation of this protein throughout the diapause. Thus, vitellin metabolism appears to be independent of the diapause phenomena in Bombyx eggs, but in other eggs, which enter diapause at a later stage of embryogenesis, vitellin may play a role. Kai and Hasegawa (1971) reported that in Bombyx mori the SG (suboesophagal ganglion) or DH (diapause hormone) affects developing ovaries so as to reduce the content of specific proteins - trichloroacetic acid insoluble and acid ethanol soluble. As the content of the proteins in the ovaries and mature eggs is very small, analyses by starch-gel electrophoresis were adopted to identify the specific protein. Banding pattern and staining of mature egg proteins suggested that the specific protein is a type of albumin. In many cases, albumin fractions are reported to carry esterases, and

20

this was clearly documented in developing ovaries and mature eggs of the non-diapause type of the silkworm; the esterase activity was found in six bands and one of them which moves fastest toward the anode was reduced in activity by the presence of SG and administration of DH extracts. It was designated "esterase A" (Kai and Hasegawa, 1972). "Esterase A" activity is high in non-diapause eggs and low in diapause eggs. The low activity in the latter continues to decrease gradually during the diapause period, but it increases during chilling or by HCL treatment of diapause eggs (Kai and Hasegawa, 1972; Kai and Nishi, 1976). The function of "esterase A" of silkworms is suggested to be responsible for the lysis of yolk cells, which is essential for resumption of embryogenesis (Kai and Hasegawa, 1973). 2.19.3.4 Carbohydrate metabolism As with the case of lipid metabolism, carbohydrate metabolism is also closely correlated with diapause phenomena. In embryonic diapause much attention has been paid to carbohydrate metabolism, especially in Bombyx eggs. In Japan quite a number of studies on the relationship of glycogen to diapause were performed in the 1930s on this insect, and at that time it was reported that large amounts of glycogen in newly laid diapause eggs completely disappeared within 10 days after oviposition and reappeared during overwintering (Yamashita and Hasegawa, 1985). Chino (1957) found that glycogen originally present in Bombyx diapause eggs was nearly all converted into polyols (sorbitol and glycerol) and at the termination of diapause these polyols are utilised for the resynthesis of glycogen. Later, it was reported that diapause in mature embryos of the eastern tent caterpillar Malacosoma americanum is associated with a very high glycerol content, low carbohydrate level and the ability to withstand low temperature (Mansingh, 1974). Mansingh concluded that the induction of diapause, i.e. glycerol accumulation enhances the supercooling ability and therefore increases the cold-hardiness of the diapausing mature embryo of this insect. In Teleogryllus emma on the other hand, a mannan-like polysaccharide, the main carbohydrate component, and glycerol are accumulated in the ovaries and mature eggs during adult life. They are retained at their original level during diapause and are used for embryogenesis after diapause termination (Irie et al., 1979). This work suggests that combination of cold storage and HCL would be the most likely method of breaking diapause in univoltine or bivoltine Bombyx mori.

2.20 Diseases of silkworm Silkworm crop loss is mainly attributed to the occurrence of diseases, rather than to unfavourable weather conditions that lead to poor harvests of mulberry leaves (Watanabe, 1986). Hence, prevention of silkworm diseases is one of the most important aspects in the success of commercial sericulture. Disinfection of the rearing house and appliances before the commencement of rearing is generally recommended but it is not necessarily adequate to prevent the occurrence of disease. Therefore, along with the disinfection of the rearing house and appliances, use of disease resistance silkworm varieties would be a further step in the direction of more effective disease prevention (Eguchi, et al. 1998). Of all silkworm diseases which cause damage, viral diseases, are the most serious (Shivaprakasam and Rabindra, 1995). Hence a need has been felt to develop silkworm breeds which are resistant to the most serious viral disease caused by the Bombyx mori nuclear polyhedrosis virus (BmNPV). Very few reports are available on the development of resistance of insects to a pathogen compared to the number of reports on the development of resistance to insecticides. Uzigawa and Aruga (1966) have developed a strain of silkworm resistant to infectious flacherie virus after selecting survivors from virus fed larvae for five consecutive generations. Similarly, Watanabe (1967) had evolved a silkworm strain, resistant to cytoplasmic polyhedrosis by selecting survivors from virus fed larvae for eight generations.

21

Recently, Eguchi et al. (1998) have succeeded in evolving a silkworm hybrid "Taisei" which is completely resistant to Bombyx mori densonucleosis virus type I (BmDNV 1). Since the resistance to BmDNV I is controlled by a single major gene (Watanabe and Maeda, 1978, 1981; Eguchi et al., 1986), it is possible to introduce the resistance into a susceptible breed easily by cross breeding. In contrast, resistance of the silkworm to nuclear and cytoplasmic polyhedrosis virus (NPV and CPV) and also infectious flacherie virus (IFV) is controlled by polygenes (Watanabe, 1986, 1991). Therefore, induction of complete resistance to these viruses is practically impossible in the silkworm breeds. However, breeding of strains with comparatively more resistance to these viruses is possible by selection of survivors after virus exposure (Aratake, 1973).

2.20.1 Flacherie (Bacterial) Disease The etiology of viral and fungal diseases have been investigated thoroughly whereas the etiology of bacterial diseases is not fully understood because of the multiplicity of factors involved in bacterial infection. The mechanism of infection of flacherie is so complicated that several theories have been proposed, such as microbial and non-microbial causes interacting to cause flacherie in silkworms (Krishnaswami et al. 1973). Bacillus thuringiensis was first isolated in Japan by Ishiwata (1901) as a causative agent of the sotto disease of the silkworm, Bombyx mori. Because of economical importance of this insect in Japan, many researchers have investigated the distribution of B. thuringiensis in silkworm, in particular, rearing insectaries of the farmers. It is now well established that this organism is widely distributed in sericulture environments of Japan (Ohba et al. 1979; Ono and Watanabe 1983). Ohba found that a total of 186 B. thuringiensis colonies were isolated from 24(96.0%) out of 25 mulberry trees. Among the 186 isolates, 18 (9.7%) were active on larvae of Bombyx mori and it is included serovers Kurstaki, Kenyae, canadensis ( H5ac), aizawai and “ un typable”(no reactivity) {Ohba, 1996}.

2.20.2 Muscardine (fungal) Disease The entomopathogenic fungus, Beauveria bassiana infects many economically important insects including silkworm, Bombyx mori. The site of infection is generally the integument and the digestive tract. Kawakami (1973) reported that hyphae penetrate mainly into the epidermis and fat bodies of the silkworm larvae. Pekrul and Grula (1979) found production of certain enzymes by the hyphae of B. bassiana while penetrating through the larval integument. Sohaf et al (1993) used topical inoculation method for observing the development of the fungus B. bassiana inside the larval tissues. They reported that gut epithelium was completely filled with the hyphae, which penetrated through the intercellular spaces of the midgut tissues. According to Yanagita and Iwashita (1987), midgut tissues are destroyed by the toxins or substances secreted by the hyphae. Death of larvae occurred 108 hours after inoculation. In Australia, it would be necessary to investigate the disease resistance capability of silkworms and develop more disease resistant silkworm populations to decrease the threat of disease.

2.21 Hybridisation Gamo (1983) found silkworm races display genetic, physiological, ethological, morphological, biochemical and quantitative differences. Besides, many intensive breeding efforts aiming at improving the productivity and quality of silk have yielded improvement in a number of inbred lines (Datta, 1984). Maruyama (1984) reported that many inbred lines which carry specific improvement for a particular character have also been developed. When different varieties or strains are crossed, often the offspring have balanced features derived from the two parents or they are stronger than either of the parents and even excel in productivity. This phenomenon is known as heterosis or hybrid vigour.

22

The foregoing account shows that heterosis has been harnessed in the silkworm by crossing different breeds, inbred lines and geographical races. Maruyama (1984) reported that when the two lines selected for cocoon shell weight for 55 generations (from 1928 to 1982) were crossed, the hybrid mean for the selected trait (cocoon shell weight) was closer to the mean of the better parent. The crossing effects in two lines selected divergently for long and short filament lengths for 11 generations from the same base population. It also evaluated that the two lines selected for length of silk filament differed by 172 meters. The effect of accrued differences in filament length in the parental lines was seen in the hybrids of interline matings (Nagaraju and Pavakumar, 1995). However, the level of heterosis recorded for different traits by different workers is not consistent. Subba Rao and Sahai (1990) using 30 bivoltine hybrids showed the highest significant level of heterosis for cocoon yield, which is a function of both survival rate and cocoon weight, followed by cocoon weight and denier (fibre diameter). Studies made by various workers show that the degree of heterosis varies steeply for different characters. Such wide differences in the manifestation of heterosis suggest that the parental strains involved in the hybrids differ in their genetic make up, as reflected in their sharp differences in origin, voltinism and quantitative traits such as larval duration, single cocoon weight, cocoon shell weight and filament length (Gamo and Hirabayashi, 1983; Tayade, 1987; Sathenahalli et al., 1989). Ohi et al., (1970) showed that during the different stages of silkworm development many characters have a relationship with the qualitative and quantitative aspect of silk yield. However, the characters which reveal intense manifestation of heterosis are as follows: 1. The duration of feeding of hybrids becomes shorter than that of the parents or the midparental

values (MPV) 2. The mortality rate is lower than that of the parents 3. The double cocoon rate is higher than that of the parents 4. The cocoon weight is higher than that of MPV 5. The cocoon shell layers are heavier than that of MPV 6. The length of silk fibre is longer than that of MPV 7. The cocoon fibre weight is heavier than that of MPV. Silkworm varieties could be crossed into single cross (A x B), three way cross [(A x B) x C], and double cross [(A x B) x (C x D)]. Generally, single cross hybrids manifest the highest rate of hybrid vigour as compared to three-way and double cross hybrids (watanabe, 1961). Udupa and Gowda (1988) also recorded that three way and double cross hybrids are inferior to single cross hybrids. Such a difference between hybrids of single, three- way and double crosses could be interpreted considering the fact that one of the parents involved in three-way and both parents in the double cross hybrids are actually F1 individuals. Furthermore, the population produced by three-way or double cross hybrids is a mixture of genotypes, all of which could in principle have been produced by single crosses, but differs from single cross hybrids in the following three ways (Falconer, 1981). i) If the lines crossed have been selected, and if any of the consequent superiority of their single

crosses is due to epistatic interactions, some of this superiority is lost in three-way and double crosses.

ii) There is genetic variation within the crosses and consequent loss of phenotypic uniformity. iii) The variance between crosses is reduced and the best three-way and double cross hybrids are

consequently not as good as the best single cross hybrids. The expression of heterosis varies in the reciprocal crosses of silkworm breeds which differ in voltinism and maturity period. First, Nagatomo (1942) proposed that maturity genes linked to the Z chromosome play an important role in reciprocal hybrid differences since the larval maturity gene

23

has a close correlation with body size, cocoon weight, cocoon shell weight and body weight. Later, one writer considered that polyvoltines possess the early maturity (Im) gene and the bivoltines carry the late maturity gene (+Im) on Z chromosomes. In a cross between polyvoltine x bivoltine and its reciprocal hybrids, the reciprocal differences for maturity and related traits could be expected, although this is an oversimplified explanation. There seems to be an intricate, genetic mechanism which involves voltinism, maturity genes and temperature during silkworm rearing and hormonal interplay which seem to affect the manifestation of the traits (Oshiki, 1979). The degree of manifestation of heterosis shown by a particular cross can be influenced by the environment (Barlow, 1989). Generally, in a good environment (which includes an optimum temperature of 25°C, mulberry leaves of high nutritive value, optimum rearing space, germ-free conditions, air current and 70-75% relative humidity) silkworm parental strains register a higher silk yield of good attributes (Ueda et al. 1969). As a result, MPV is close to hybrid mean values with a little difference. In fact when one of the parental strains is a high-yielding bivoltine strain and the other one is a polyvoltine strain in a cross, heterosis over the high-yielding strain will be negative when both the parental strains and hybrids are raised in a good environment. On the other hand, when both the parental strains and the hybrids are raised in an adverse environment (for example, high temperature > 31° C), the mean of the hybrids will be much higher than the mean of the parental strains. In such cases heterosis over both MPV and better parental values will he higher. Singh et al. (1990) observed negative heterosis in the hybrid segregants for almost all the traits except denier. It is not surprising that negative heterosis was observed for all the traits, considering the fact that trimoulters have a shorter larval duration, spin smaller and lighter cocoons with a short filament length. On the other hand, tetramoulter segregant hybrids revealed a high degree of positive heterosis for all the traits except for filament length. In the reciprocal cross, i.e., trimoulter female x tetramoulter male, only tetramoulters of both sexes appeared. A high magnitude of heterosis for all the traits was observed in this hybrid. The appearance of only female trimoulters in the cross of tetramoulter x trimoulter is due to the sex-linked nature of the trimoulter gene and the non-appearance of trimoulters in the cross of trimoulter female (polyvoltine) x tetramoulter (bivoltine) is due to differences in the sex-linked maturity genes (+ m, lm) in the parental strains which act in co-operation with moulting genes. The degree of manifestation of heterosis does not seem to show marked differences between sexes in the hybrid progeny. Shimizu (1966) showed that females recorded slightly higher heterosis ratio than males both for cocoon (Female: Male = 28.36: 26.01) and shell weight (Female: Male = 26.25: 22.90). In Australia, it would be quite necessary to improve its native populations (quality and quantity) through hybridization with foreign population. The review shows that single crossing is better than double or three way crossings. Conclusions: This review indicates that sericulture is a very old practice. Although artificial silk (nylon) is becoming popular, natural silk still has a great demand in world markets. Silkworms have many species within the Lepidoptera Order. However the mulberry silkworm, Bombyx mori, provides more than 90% of the silk in the world. So, in Australia, it would be recommended to rear mulberry silkworm, Bombyx mori. Because of different environmental conditions, the voltinism of silkworms is different in different geographical regions. This review has shown that univoltine or bivoltine silkworm produce diapaused eggs, whereas the multivoltine produce non-diapaused eggs. To overcome the egg diapause in univoltine and bivoltine silkworms, it is recommended that cold treatment and HCl be used. Because diseases are a large threat to the establishment of a silkworm industry in Australia, it is recommended that disinfectants be used in the rearing house and appliances, and that disease resistant silkworm varieties be developed.

24

To improve production of local Australian populations, it is also suggested that hybridization be used. The most important research work that requires to be done to establish a silk industry in Australia is to develop highly productive silkworm populations under Australian conditions.

References Ando, Y. 1971. Distribution of mercury in the eggs of the false melon beetle, Atrachya menetriesi Faldermann (Coleoptera: Chrysomelidae), treated with mercuric chloride for breaking diapause. Appl. Ent. Zool. 6: 67-74. Aratake, Y. 1973. Strain difference of the silkworm, Bombyx mori L. in the resistance to a nuclear polyhedrosis virus. J. Sericulture Sci. Japan. 42(3): 230-238. Aruga, H. 1994. Principles of Sericulture (Translated from Japanese). A.A. Balkema/Rotterdam. Pp.376. Asaoka, K. 2000. Deficiency of gustatory sensitivity to some deterrent compounds in “polyphagous” mutant strains of the silkworm, Bombyx mori . J. Comp. Physiol. A. 186: 1011-1018. Balavenkatasubbaiah, M., Sharma, S.D., Baig, M., Singh, B.D., Reddy, S.V. and Noamani, M.K.R. 1989. Role of disinfection of rearing appliances and sunlight exposure on the inactivation of disease causing pathogen of silkworm, Bombyx mori L. Indian J. Sericulture. 28(2): 200-206. Barlow, R.1989. Experimental evidence for interaction between heterosis and environment in animals. Anim. Breed. Abst. 49:715-739. Chaturvedi, M. L. and Upadhyay, V. B. 1990. Effect of cold storage on the hatchability of silkworm (Bombyx mori) eggs. J. Advanced Zoology. 11(1) : 63-65 Chatterjee, S.N., Rao, P.R.M., Jayaswal, K P., Singh, R. and Datta, R. K. 1993. Genetic variability in mulberry silkworm, Bombyx mori L breeds with low silk yield. Indian J. Sericulture. 32. 69-86. Cherry, R. 1989. Sericulture. Bull. Entomol. Soc. Am. 35: 83-84. Chino, H. 1957. Conversion of glycogen to sorbitol and glycerol in the diapause eggs of the Bombyx silkworm. Nature. 180: 606-607. Chino, H. 1958. Carbohydrate metabolism in the diapause egg of the silkworm Bombyx mori - II Conversion of glycogeninto sorbitol and glycerol during diapause. J. Insect Physiol. 2:1-12. Dar, H. U., Singh, T.P. and Bhat, M.I. 1989. A comparative study of mounting materials for silkworm Bombyx mori L. Entomon. 14(3-4): 211-215. Das, S., Ghosh, M. K., Shamsuddin, M., Saha, P. K. and Sen, S. K. 1993. Impact of rearing space, leaf quality, diet rationing and sanitation measures on

bivoltine silkworm rearing in the plains of West Bengal India. Uttar Pradesh J. Zool. 13(1): 76-80. Datta, R.K. 1984. Improvement of silkworm races (Bombyx mori L.) in India. Sericologia. 24: 393-415. De Wilde J. 1962. Photoperiodism in insects and mites. Ann. Rev. Entomol. 7 : 1-26. Dingle, J.G. 2000. Silk Production in Australia. A report for the Rural Industries Research and Development Corporation. RIRDC publication No 00/56, Australia. Pp 48. Ehiobu, N. G. and Goddard M.E.1989. Heterosis in cross between lines of Drosophila melanogaster selected for adaptation to different environments. Theo. Appl. Genet. 77 : 253-259. Eguchi, R., Furuta, Y. and Ninaki, O. 1986. Dominant non-susceptibility to densonucleosis virus in the silkworm , Bombyx mori . J. Sericulture Sci. Japan. 55 (2) : 177-178. Eguchi, R., Hara, W., Simazaki, A., Hirota, K., Ichiba, M., Ninagi, O. and Nagayasu, K. 1998. Breeding of the silkworm race “Taisei” nonsusceptible to a densonucleosis virus type –1. J. Sericulture Sci. Japan. 67 (5) : 361-366. Engelmann, F. 1979. Insect vitellogenin : Identification, biosynthesis, and role in vitellogenesis. Adv.Insect Physiol. 14:49-109. Falconer, D.S.1981. Introduction to Quantitative Genetics. Second ed., Longman, London. FAO. 2001. Food and Agriculture Organisation Statistical Website, Rome (http://www.fao.org/). Gangwar, S.K., Somasundaram, P. and Thangavelu, K. 1993. Feeding behaviour of silkworm Bombyx mori. L. J. Adv. Zool. 14 (2):115-118. Gabriel, B.P. and Rapusas, H.R. 1976. The growth and development of Bombyx mori (L.) at different leaf maturity and variety of mulberry. Philippines Agric. 60: 130-146. Gamo, T. 1983. Biochemical genetics and its application to the breeds of the silkworm. Japan Agric. Res. Quarterly. 16: 264-272. Gamo, T. and Hirabayashi, T. 1983. Genetic analysis of growth rate, pupation rate and some quantitative characters by dialled cross in silkworm, Bombyx mori L. Japan J. Breed. 33: 178-190. Gongyin, Y. and Cui, H. 1996. Some features of silk producing moths.Tropicultura.14 (1): 30-33.

25

Hasegawa, K. and Yamashita, O. 1967. Control of metabolism in the silkworm pupal ovary by the diapause hormone. J. Sericult. Sci. Japan .36:297-300. Hogan, T.W. 1964. Further data on the effect of ammonia on the termination of diapause in eggs of Teleogryllus commodus (Walk.) (Orthoptera: Gryllidae). Aus. J. Biol. Sci.17: 752-757. Hogan, T.W. 1965. Some diapause characteristics and interfertility of three geographic populations of Teleogryllus commodus (Walk.) (Orthoptera: Gryllidae). Aus. J. Zool. 13: 455-459. Irie , K., Suzuki, K. and Miya, K. 1979. Change of carbohydrate content during ovarian and embryonic development in Emma field cricket, Teleogryllus emma. Appl. Ent. Zool. 14:278-284. Irie , K. and Yamashita, O. 1980. Changes in vitellin and other yolk protein during embryonic development in the silkworm, Bombyx mori . J. Insect Physiol. 26:811-817. Islam, B.N. 1981. Improvement of silkworm multiplication and silk production under Bangladesh condition. A monograph of sericulture. Department of Entomology, Bangladesh Agricultural University, Mymensingh. Pp. 83. Izumi, S., Tomino, S. and Chino, H. 1980. Purification and molecular properties of vitellin from the silkworm, Bombyx mori. Insect. Biochem. 10:199-208. Kai , H. and Hasegawa, K. 1971. Studies on the mode of action of the diapause hormone with special reference to the protein metabolism in the silkworm, Bombyx mori L. I - The diapause hormone and the protein soluble in ethanol containing trichloroacetic acid in mature eggs of adult ovaries. J. Sericult. Sci. Japan. 40:199-208. Kai , H. and Hasegawa, K. 1972a . Electrophoretic protein patterns and esterase zymograms in ovaries and mature eggs of Bombyx mori in relation to diapause. J. Insect Physiol. 18:133-142. Kai , H. and Hasegawa, K. 1972b. Studies on the mode of action of the diapause hormone with special reference to the protein metabolism in the silkworm, Bombyx mori L. III - Effect of acid treatment and determents on “esterase A” in diapause eggs . J. Sericult. Sci. Japan. 41:253-262. Kai , H. and Hasegawa, K. 1973. An esterase in relation to yolk cell lysis at diapause termination in the silkworm, Bombyx mori. J. Insect Physiol. 19:799-810. Kai, H. and Nishi, K.1976.Diapause development in Bombyx eggs in relation to “esterase A” activity. J. Insect Physiol. 22:1315-1320. Kappus, K.D. and Venard, C.E. 1967. The effects of photoperiod and temperature on the induction of diapause in Aedes triseriatus (Say). J. Insect Physiol. 19:1007-1019.

Kawakami, K. 1973. Studies on muscardine disease of the silkworm, Bombyx mori L. with special reference to the invasion of causative fungi and pathological changes in infected larvae. Bull. Seric. Exp. Stn. (Tokyo). 25(5): 347-370. Khan, Z.I. 1983. Resham (Special issue). Bangladesh Sericulture Board, Rajshahi. p. 8-26. Kim, S.E., Shikata, M. and Kai, H. 1981. Egg-shell lipids in relation to water evaporation and diapause in the silkworm, Bombyx mori. J. Sericult. Sci. Japan. 50:94-100 (in Japanese with English summary). Krishnaswami, S., Narsimhanna, M.N., Suryanarayana, S.K. and Kumarraj, S. 1973. Sericulture Manual 2 - Silkworm rearing. Food and Agriculture Organisation of United Nations, Rome, pp. 131. Krysan, J.L., Branson, T.F. and Castro, C.D. 1977. Diapause in Diabrotica virgifera (Coleoptera: Chrysomelidae): A comparison of eggs from temperate subtropical climates. Ent. Exp. Appl. 22:81-89. Kubota, I., Isobe, M., Imai, K., Goto, T., Yamashita, O. and Hasegawa, K. 1979. Characterization of the silkworm diapause hormone. B. Agric. Biol. Chem. 43:1075-1078. Kurata, S., Koga, K. and Sakaguchi, B. 1979. RNA content and RNA synthesis in diapause and non-diapause eggs of Bombyx mori. Insect Biochem. 9:107-109. Kurata, S., Yaginuma, T., Kobayashi, Y., Koga, K. and Sakaguchi, B. 1980. DNA content and cell number during the embryogenesis of Bombyx mori. J. Sericult. Sci. Japan. 49:107-110.( In Japanese with English summary). Kushuda, J., Tazima, Y., Onimaru, K., Ninaki, O. and Sujuki, Y. 1986. Mol. Gen. Genet. 203: 359-364. Lounibos, L.P. and Bradshaw, W.E. 1975. A second diapause in Wyeomyia smithii seasonal incidence and maintenance by photoperiod. Canada. J. Zool. 53:215-221. Manjula, A. and Hurkadli, H.K. 1995. Chilling of silkworm eggs. Indian Text. J. 11: 70-74. Mansingh, A. 1971. Physiological classification of dormancies in insects. Canada. Ent. 103: 983-1009. Mansingh, A. 1974. Studies on insect dormancy. II-Relationship of cold-hardiness to diapause and quiescence in the eastern tent caterpillar, Malacosoma americanum (Fab), (Lasiocampidae: Lepidoptera). Canada. J. Zool. 52: 629-637. Maruyama, T. 1984. Breeding of silkworm races 010 and 610 having more filament weight and filament length. Tech. Bull. Sericulture. Exp. Stn. 119:53-70. Masaki, S. 1973. Climatic adaptation and photoperiodic response in the band-legged ground cricket. Evolution 26:587-600.

26

Masaki, S. and Sakai, T. 1965. Summer diapause in the seasonal life cycle of Mamestra brassicae Linne (Lepidoptera: Noctuidae). Jap. J. Appl. Ent. Zool. 9:191-205. Nagatomo, T. 1942. On the inheritance of the voltinism in the silkworm. J. Sericult. Sci. Japan. 13: 114-115. Nagaraju, J. 2000. Recent advances in molecular genetics of the silk moth, Bombyx mori. Current Sci. 78(2):151- 161. Nagaraju, J. and Pavankumar, T. 1995. Effect of selection of cocoon filament length in divergently selected lines of the silkworm, Bombyx mori. J. Sericulture Sci., Japan. 64:103-109. Narasaiah, P. Venkata.1992.Sericulture in India. Ashish Publishing House, 8/81, Punjabi Bagh, New Delhi-110026. Pp. 214. Narayanaprakash, R., Periasamy, K. and Radhakrishnan, S. 1985. Effect of dietary water content on food utilization and silk production in Bombyx mori L. ( Lepidoptera: Bombycidae). Indian J. Sericulture. 24(1):12-17 Nataraju, B, Sivaprasad, V. and Datta, R.K. 1999. Studies on the cause of 'thatte roga' in silkworm, Bombyx mori L. Indian J. Sericulture. 38 (2):149-151 Ogawa, H. and Hasegawa, K. 1975. Kynurenine 3-hydroxylase activity and follicle development in the silkworm, Bombyx mori. Insect Biochem. 5:119-134. Onba, M., Aizawa, K. and Furusawa, T. 1979. Distribution of Bacillus thuringiensis serotypes in ehime Prefecture, Japan. Appl. Entomol. and Zool. 14:340-345. Ohba, M. 1996. Bacillus thuringiensis populations naturally occuring on mulberry leaves: A possible source of the populations associated with silkworm-rearing insectaries. J. Applied Bacteriology. 80:56-64. Ohi, H., Miyahara, J. and Yamashita, A. 1970. Analysis of various practically important characteristics in the silkworm in early breeding generations of hybrids. Variation among strains, correlation between parents and offspring as well as relation between each character. Tech. Bull. Sericult. Exp. Sta., MAFF. 93:39-49. Ono, K. and Watanabe, H. 1983. Distribution and serological identification of Bacillus thuringiensis isolated in Japan. J. Sericult. Sci. Japan. 52:47-50. Oshiki, T. 1979. Studies on the sex-linked manifestation of the quantitative characters in Bombyx mor . IV. Relationship between the sex-linked quantitative characters and the corpus allatum hormone. J. Sericult. Sci. Japan. 480:360-364. Park, K.E. and Yoshitake, N. 1970. Function of the embryo and the yolk cells in diapause of the silkworm eggs (Bombyx mori). J. Insect. Physiol. 16:2223-2239.

Pekrul, S. and Grula, E.A. 1979. Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy. J. Invertebr. Pathol. 34(3): 238-247. Pillai, S.V. and Krishnaswami, S. 1987. Indian J. Sericulture. 16: 63. Rajan, R.K., Inokuchi, T. and Datta, R.K. 1996. Manual on mounting and harvesting technology. CSRTI, Central Silk Board, Mysore. Pp. 22 Rao, M.M.M. 1998. A Text Book of Sericulture. B.S. Publications. Hyderabad, India, Pp. 197. Reddy, G.S. 1986. Genetics and breeding of silkworm, Bombyx mori L. pp. 70-80. In Boraiah, G. (ed.) Lectures on Sericulture. Suramya Publication, Bangalore, India. Reddy, S.N. and Babu, S.K. 1990. Hatching patterns in the silkworm Bombyx mori L. (PM x NB4D2) under different photoperiodic combinations. Proc. Indian Acad. Sci. (Anim. Sci). 99(4): 327-334 Roychoudhury, N., Paul, D.C. and Subbarao, G. 1991. Growth, fecundity and hatchability of eggs of Bombyx mori L. in relation to rearing space. Entomon. 16(3): 203-207 Sathenahalli, S.B., Govindan, R. and Goud, J.V. 1989. Heterosis studies for some quantitative traits in silkworm, Bombyx mori. Indian J. Sericulture. 28: 100-102. Sarker, Dilip D. 1998. The silkworm: Biology, Genetics and breeding. Vikas Publishing House PVT LTD, New Delhi. Pp.338. Scriber, J.M. and Salansky, F. 1981. The nutritional ecology of immature insects. A. Rev. Ent. 26: 183-211. Sen, R., Ahsan, M.M. and Datta, R.K. 1999. Induction of resistance to Bombyx mori nuclear polyhedrosis virus, into a susceptible bivoltine silkworm breed. Indian J. Sericulture. 38(2): 107-112. Sengupta, K and Yusuf, M.R. 1974. Studies on the effect of spacing during rearing on different larval and cocoon characters of some multivoltine breeds of silkworm, Bombyx mori L. Indian J. Sericulture. 13:11-16. Shimizu, S. 1966. Comparison of heterosis between male and female progeny in the silkworm, Bombyx mori L. J. Sericult. Sci. Japan. 35:133. Shimizu, I. 1982. Photoperiodic induction in the silkworm, Bombyx mori, reared on artificial diet: evidence for external photoreception. J. Insect Physiology. 28:841-846 Shimizu, M. and Tajima, Y. 1972. Silkworm races suitable for spring, summer and autumn rearing, In Shimizu, M. and Tajima, Y. (eds.) Handbook of silkworm rearing. Agric. Tech. Manual I., Fuji Publ. Co. Ltd. Tokyo, Japan. p. 304-307.

27

Shivaprakasam, N. and Rabindra, R.J. 1995. Incidence of grasserie in silkworm Bombyx mori L. in selected districts of Tamilnadu. Indian J. Sericulture. 34(2):100-104. Singh, R., Nagaraju, J., Premalatha, V., Rao, R.M., Vijayaraghavan, R. and Gupta, S.K. 1990. Heterosis analysis in the silkworm, Bombyx mori. Sericologia. 30 : 293-300. Sivaprakasam, N. and Rabindra, R.J. 1996. Integrated disease management methods for grasserie in silkworm Bombyx mori L. Indian J. Sericulture. 35 (2) 122-127 Sohaf, K.A., Chishti, M.Z. and Trag, A.R. 1993. Histopathology of the silkworm, Bombyx mori L. infected with Beauveria bassiana (Bals.). Vuill. Indian J. Sericulture. 32(2): 213-215. Stoyan, K., Aleksandrov, K., Zakhariva, K. and Nauki, A. Y. Z. 1985. Results of raising silkworms (Bombyx mori) at distributed environmental factors. Sericulture Test control Stn. Bulg. 22(3):76-81. Subba Rao, G. and Sahai, V. 1990. Combining ability and heterosis studies in bivoltine strains of silkworm, Bombyx mori (L.). UP J. Zoology. 9: 150-164 Takami, T. 1969. A General Text Book of Silkworm Eggs. Zenkoku Sanshu Kyokai, Tokyo. pp. 24-25. Talukder, F. A. 1993. Factors contributing to cocoon yield in Bombyx mori L. in Bangladesh. Pak. J. Sci. Ind. Res. 36(4): 148-150. Talukder, F.A., Huq, S.B. and Khan, A.B. 1990. Effect of larval population densities of mulberry silkworm, Bombyx mori on the rate of cocoon production and adult longevity. Prog. Agric. 1(1):93-6. Tanaka, Y. 1916. J. Coll. Assoc. Sapporo. 7: 129-255. Tayade, D.S. 1987. Heterosis effect on economic traits of new hybrids of silkworm, Bombyx mori under Marathwada conditions. Sericologia. 27: 301-307. Thiagarajan, V., Bhargava, S. K., Babu, M.R. and Nagaraj, B. 1993.Differences in seasonal performance of twenty six strains of silkworm, Bombyx mori, (Bombycidae). Journal Lepidopterists’ Society. 47(4):331-337. Toyama, K. 1906. Breeding Methods of Silkworm, Sango, Shimpo. 158: 282-286. Ueda, S., Kimura, R. and Sujuki, K. 1969. Studies on the growth of silkworm, Bombyx mori (L.). II. The influence of the rearing conditions upon larval growth, productivity of silk substances, eggs and boiled off loss in the cocoon shell. Bull. Sericulture Expt. Stn. Japan. 23: 290-293.

Udupa, S. and Gowda, V. 1988. Heterosis expression in silk productivity of different crosses of silkworm, Bombyx mori. Sericologia. 28: 395-399. Uzigawa, K. and Aruga, H. 1966. On the selection of resistance strains of the infectious flacherie virus in the silkworm, Bombyx mori L. J. Sericulture Sci. Japan. 35 (1): 23-26. Veda, K., Nagai, I. and Horikomi, M. 1997. Silkworm rearing (translated from Japanese). Science Publishers. Inc., U.S.A. Pp. 302 Watanabe, H. 1961. Studies on difference in the variability of larval body and cocoon weights between single cross and three way- cross or double cross hybrids in the silkworm, Bombyx mori. J. Sericult. Sci. Japan. 30: 463-467. Watanabe, H. 1967. Development of resistance in the silkworm, Bombyx mori to preoral infection of a cytoplasmic polyhedrosis virus. J Invertebr. Pathol., 9(4): 474-479. Watanabe, H. 1986. Resistance of the silkworm, Bombyx mori to viral infections. Agric. Ecosyst. Environ. 15: 131-139. Watanabe, H. 1991. The host population. In: Epizootiology of Insect diseases, Fuxa, J. and Tanada, Y. (Eds.), John Wiley and Sons, New York, pp. 7112. Watanabe, H. and Maeda, S. 1978. Genetic resistance to preoral infection with a densonucleosis virus in the silkworm, Bombyx mori J. Sericulture Sci. Japan. 47(3): 209-214. Watanabe, H. and Maeda, S. 1981. Genetically determined non-susceptibility of the silkworm, Bombyx mori, to infection with a densonucleosis virus (Densovirus). J. Invertebr. Pathol., 38(3): 370-373. Yamashita, O. and Hasegawa, K. 1985.Embryonic diapause. In: Comprehensive Insect Physiology, Vol. 1, Biochemistry and Pharmacology. G.A. Kerkut and L.I. Gilbert (Eds), Pergamon Press, Great Britain, p. 407-434. Yamashita, O. and Yaginuma, T. 1991. Silkworm eggs at low temperatures: Implication for sericulture. In: Insect at Low Temperature. R.E. Lee Jr. and D. L. Denlinger, (Eds.), Chapman and Hall, London. p.424-445. Yamashita, O., Hasegawa, K. and Seki, M. 1972. Effect of the diapause hormone on trehalose activity in pupal ovaries of the silkworm, Bombyx mori L. General Comp. Endocrinol. 18:515-523. Yanagita, T. and Iwashita, Y. 1987. Histological observations of larvae of the silkworm, Bombyx mori, orally infected with Beauveria bassiana. L J. Sericulture Sci. Japan. 56(4):285-291.

28

3. General methodologies 3.1 Silkworm, the test insect The insect used for the present research work is known as the mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). The silkworm has been domesticated over a period of more than 4000 years. The Bombyx mori was originally derived from Bombyx mandarina-moore (Rao, 1998). The mulberry silkworm belongs to the following taxonomic classification. Phylum - Arthropoda Class - Insecta Order - Lepidoptera Family - Bombycidae Genus - Bombyx Species - mori B. mori undergoes complete metamorphosis. It changes from the egg stage to larva, to pupa and then moth. The silkworm eggs are ovoid in shape and pale yellow in colour. Eggs are normally 1.0 - 1.3 mm in length and 0.9 to 1.2 mm in width. The larvae are black or dark brown in colour. They feed on mulberry leaves and go through four moultings and five larval stages (Krishnaswami et al., 1973; Shekar and Herdingham, 1995). The pupae are brown in colour and the pupal period may last for 8 - 14 days (Krishnaswami et al., 1973). The adult moth emerges by softening the fibrous cocoon shell with the aid of an alkaline salivary secretion. Adult life is short, lasting from 3-10 days. The adults do not feed and are also incapable of flight. The females are larger in size and generally sluggish while the males are somewhat smaller and more active. A female of the multivoltine variety may lay on average approximately 400 eggs while the average number for the uni-and bi-voltine varieties of silkworm moths is from 500 - 600 (Krishnaswami et al., 1973). The mulberry silkworms may produce offspring once or many times in a single year. On the basis of the number of generations per year, mulberry silkworms are classified in three different categories. The uni-voltines produce only one generation in a year, whereas bi-voltines produce two generations and multi-voltines more than two generations in a year (Rao, 1998). The eggs laid by uni-voltine silkworms remain under diapause until next spring. The uni-voltine cocoons produce superior quality silk. These silkworms can be reared once in a year only. Generally these varieties are adapted to colder regions. Bi-voltine silkworm races have two generations in a year. The eggs hatched in spring develop to lay non-diapausing eggs in the first generation and lay dormant eggs in the second generation. The larvae can tolerate different environmental conditions to a greater extent than uni-voltines. On the other hand, the multi-voltine silk worms have a short larval period. Multi-voltines lay non-diapausing eggs and can tolerate higher temperatures and humidities. Their cocoon size is smaller than other two categories.

3.2 Mulberry leaves, the silkworm food The mulberry (Latin: Morus) plant is found throughout the tropical belt. The systematic position of genus Morus is as follows Class: Dicotyledon Subclass: Archichlayindae Order: Urticlaes Family: Moracae Genus: Morus L. Morus is a deep-rooted, perennial, woody genus. The plant habit ranges from large bush to large tree. There are two main types of mulberry bush: the white mulberry, Morus alba, which produces a pinkish-red fruit, has ovate, acute or crenate serrate leaves with three basal veins; and the black mulberry, Morus nigra, which produces a purplish-black fruit used in the production of jams or

29

wine, and has wider leaves than in M. alba (Shekar and Hardingham, 1995; Sarker, 1998). The white mulberry is grown extensively for its leaf to feed silkworms. The white mulberry can be successfully grown in almost all climatic zones, in temperate as well as tropical areas, in rainfed or irrigated conditions and some varieties even thrive on arid, marginal land. Shekar and Hardingham (1995) stated that the quality of cocoon the silkworm produces depends on the quality of mulberry leaf on which it feeds.

White mulberry plant Black mulberry plant The continuous supply of fresh mulberry leaves was one of the very important factors for our research. At the beginning of our research, different categories of mulberry leaves, as needed to feed different stages of larvae, were collected from the UQ Gatton Experimental farm fields. The University has few large mulberry trees located in different parts of the farm fields, which were served as our first sources of mulberry leaves. At the beginning of experiment, we used leaves from both the white mulberry (Morus alba) and black mulberry (Morus nigra). Our preliminary research showed that the larvae reared on either species of the mulberry had similar growth pattern. After a year, we started to collect mulberry leaves from the new mulberry plantations established in the farm field, where only white mulberry cuttings were used. Though we had plenty of mulberry trees in our farm, but due the growth pattern of mulberry trees under Australian environment, fresh leaves were not available during the winter season (June – August). Therefore, we had to temporarily cease our silkworm rearing during the winter season.

3.3 The bacterium, Bacillus thuringiensis There are several types of bacterial disease found among silkworms. Among them, “Sotto disease” is one of the most important. Sotto disease occurs when the silkworm larvae ingest mulberry leaves contaminated by Bacillus thuringiensis. The bacteria are widely distributed on mulberry leaves and causes severe damage to the silkworm industry (Krishnaswami et al., 1973; Sarker, 1998). For our research, a commercial formulation of Bacillus thuringiensis var. kurstaki was used, as it is one of the widely used biopesticide in Australian crop protection. Pramanik and Somchoudhury (2001) reported that of B. thuringiensis var. kurstaki is highly lethal to Bombyx mori larvae, causing over 50% mortality of 4th instar larvae at a low concentration of 0.01% of Bt. Inagaki et al (1992) found that Bombyx mori larvae were 2,500-fold more susceptible to the B. thuringiensis var. kurstaki infection than Spodoptera litura larvae. Therefore, we have chosen it for our research. The commercial name and details of bacteria used are as follows:

Commercial name: DIPEL® Distributor: Arther Yates & Co. Limited, NSW, Australia Type: HG Bio-Insecticide Active Constituents: 4320 International Units per mg of Bacillus thuringiensis var. kurstaki

30

We used it to screen all local and foreign mulberry silkworm strains against the bacterial disease incidence. We also used the same bacteria for developing disease resistance in selected mulberry silkworm strains.

3.4 Local and exotic silkworm strains Three different silkworm strains, two Australian origins and one of Indonesian origin, coded as QuBite, QuBill and Insab, respectively, were used for our research. The QuBite and QuBill strains were provided by Dr. John Dingle, School of Animal Studies, University of Queensland, from the recently completed “Silk Production in Australia” project. The code names for all above strains were assigned on the basis of their origins, and cocoon shapes and colours. The “QuBite” code represents a silkworm strain of Queensland origin with oval shaped white coloured cocoon. The “QuBill” code represents a silkworm strain of Queensland origin with oval shaped yellow coloured cocoon. The “Insab” code represents a silkworm strain of Indonesian origin with peanut shaped white coloured cocoon. Their comparative characteristics are as follows: Table 3.1: Comparative characteristics of three Bombyx mori strains

Population code

Origin Cocoon colour

Cocoon shape

Cocoon-Shell ratio (%)

QuBite Queensland White Oval 13.51

QuBill Queensland Yellow Oval 11.70

Insab Indonesia White Peanut 18.46

After receiving the parent stocks, they were reared for two successive generations under the same laboratory conditions. The eggs from 2nd generation parents were used as parent stocks for the initiation of our research. The Indonesian strain (Insab) was received as a complement from Dr. Dingle through his one of the research student. After receiving the Insab strain, they were screened, under the direct supervision of AQIS, for one generation to screen its any disease or pest association. The 2nd generation was handed over to us and we used them as parent stocks to produce further generation to be used for all of our experiments. The cocoon appearances of the three strains are as follows:

QuBite cocoons QuBill cocoons Insab cocoons Figure 3.1: Cocoon appearances of the experimental strains of Bombyx mori

31

3.5 Rearing of silkworms Three silkworm populations, two Australian origins and one of Indonesian origin, were used for the present research study. All insect rearing and experiments were done at the School of Agronomy and Horticulture, University of Queensland and Department of Biology, Sultan Qaboos University, Sultanate of Oman. In both laboratories, a temperature of 25 ± 2 OC, relative humidity of 75 ± 2% and with a light : dark ratio of 16: 8 hours were strictly maintained. All silkworm eggs were preserved in the refrigerator at 4 - 5OC in the laboratory, until needed for the next experiment. Before starting silkworm rearing, the insect rearing facilities (insectary and growth chamber) were disinfected as follows. The walls, ceiling and roof of the silkworm rearing facilities, and the appliances used for rearing were disinfected thoroughly with 2 - 4 percent formaldehyde solutions. Before disinfecting, the rearing facilities were made airtight as far as possible. The inside temperature was maintained around 25°C for quick diffusion of the formaldehyde gas. After spraying, the facility was kept closed for 15-20 hours. Then for the next 24 hours, the door and windows of the facilities were kept open to remove all traces of formaldehyde.

3.5.1. Incubation of eggs As soon as silkworm eggs were needed for setting a new experiment, a sufficient number of eggs, laid on egg cards, were taken out of the refrigerator and placed under experimental laboratory conditions, as mentioned above. The egg cards were placed in a larger plastic container (25 L x 16 W x 5 H cm) for convenience and left for hatching. In case of natural hatching experiments, the eggs were not treated with any chemicals. On the other hand, in the case of eggs for the artificial hatching experiments, they were treated, dried and then set for incubation.

3.5.2 Larval feeding Krishnashwami et al. (1971) and Rao (1998) reported that hatching of silkworm generally starts early in the morning and the maximum number of larvae hatch around 8 a.m. According to Rao (1998), the newly hatched larvae develop a good appetite within one hour after hatching. Therefore, we thoroughly checked all egg cards every morning for signs of hatching.

Young larvae on mulberry leaf Mature larvae on mulberry leaf

Figure 3.2: Larvae feeding on fresh mulberry leaves Circular plastic containers (25 cm diameter x 8.5 cm high) were used as silkworm rearing trays. Brown paper was placed at the bottom of each rearing tray, as a rearing support for the larvae. During the rearing period, papers were changed daily to remove larval faeces and unconsumed mulberry leaves for hygiene. For early stage of larvae, especially the 1st instar, moist cotton, wrapped in plastic, was provided inside the rearing trays to prevent withering of the delicate leaves and to maintain proper humidity inside the rearing trays.

32

3.5.2.1 Transferring young larvae in leaves: As soon as we observed the first hatching of larvae on egg cards, the cards were transferred to a rearing tray and chopped young mulberry leaves were sprinkled over the egg cards. It helped the newly hatched larvae to crawl on to the mulberry leaves. In the same afternoon, the egg cards were removed from the tray and any larva remained on the cards were tapped on to the rearing tray with the help of a camelhair brush. 3.5.2.2 Leaf quality: During our experiments, we provided a sufficient amount of different categories of fresh mulberry leaves, hand-picked daily from the university farm fields at Gatton and Al-Khod. Krishnashwami et al. (1971) reported that silkworm larvae need different ages of mulberry leaves during their growth stages, to ensure their healthy and uniform growth. According to them, early-age silkworms eat leaves from the surface while late-age worms from the edges. According to Aruga (1994), the quality of mulberry leaves supplied to the young silkworm larvae greatly affects their rearing. If the first - third instar silkworm larvae were fed with poor quality mulberry leaves, they become more often afflicted with serious diseases in the fifth instar, and the physiology of silkworms gradually becomes abnormal. All collected leaves were cleaned and washed before feeding the larvae twice a day. The first instar larvae were provided with youngest tender mulberry leaves, whereas subsequent instar larvae were given more mature leaves according to the growth stages. The fifth instar larvae were provided with the largest amount of mature leaves, as leaf consumption is very high during this instar. 3.5.2.3 Bed cleaning: Bed cleaning or removal of the old mulberry leaves, faecal matter of silkworms, exuviae, any unhealthy larvae etc., from the rearing tray were done once a day to keep the larvae healthy and free from any disease. However, during the moulting stages, no bed cleaning was done, as it might interfere with the moulting process and larval growth. 3.5.2.4 Rearing spaces: As the silkworm larvae grew in weight and size very fast, their density in the rearing trays increased and overcrowding could have occurred. To overcome this situation, we allowed around 200-250 newly hatched larvae in the rearing tray, but the larval numbers were reduced to 150-200, 100-125, 50-60 and 15-20 larvae per single tray, for the 2nd, 3rd, 4th and 5th instar larvae, respectively.

3.5.3 Moulting The silkworm larvae become inactivate, shed their skins and undergo development between every two larval instars and this phenomenon is known as moulting (Aruga, 1994; Rao, 1998). We observed that after attaining the maximum growth in a specific instar, silkworm larvae stop feeding on mulberry leaves, search for a quite place or attached themselves to a part of a leaf and reduce their movements. This situation continues for a day or more and then the larva sheds its old skin and starts its new instar. To avoid any interference with this physiological activity, we did not perform bed cleaning during the moulting stages.

3.5.4 Spinning At the end of the 5th instar, the mature larvae reached their highest body weight, became translucent and started to spin their cocoons. Krishnaswami et al. (1973) reported that the mature larvae become very restless and raise their heads in search of support so as to be able to start spinning. For our observations, we found that most of the mature larvae ceased their feeding and moved to the corner of the tray. As soon as we saw this condition of the larvae, we transferred them to a paper-made mountage for spinning cocoons.

33

3.5.5 Mounting and spinning It was reported that among the many factors that contribute to a good yield, the mounting material used for spinning of cocoon plays a vital role (Rajan et al., 1996). Different materials such as grass and plastic cocoonage, hay, pine shootlets, mulberry twigs etc. can be used as mounting materials (Krishnaswami et al., 1973; Dar et al., 1989). For our research, we prepared temporary hand-made mountages with art papers, which can easily be made and fit within the rearing tray, and can be safely disposed after use. Each mountage had around 30 – 35 chambers, each of which was 4 cm x 4 cm in size.

A hand-made mountage Larval spinning in the mountage

Figure 3.3: A hand-made mountage and larval spinning activities As soon as a mature larva was found started to spin a cocoon, we transferred it immediately into the mountage. Each larva was placed in a single chamber (4 x 4 cm) to spin its cocoon for the next 2 – 3 days. We observed that at the end of spinning, silkworm larva transformed into the pupal form.

3.5.6 Cocoons During the spinning process, a silkworm larva spins a cocoon around itself for 48-72 hours. As we placed each larva in a separate chamber inside the mountage, we left them for 4 – 5 days to finish their cocoon spinning and hardening of the cocoons. After hardening of the cocoons, they were cleaned from attached flosses (upper layer of loose silk) by hand and then kept in a separate plastic container, layered with a paper towel at the bottom for completing the pupal stage. As we used three different strains of silkworms (QuBite, QuBill and Insab), we observed three different types of cocoons. The QuBite strain produced oval shaped white cocoons, the QuBill produced oval shaped yellow coloured cocoons and the Insab produced peanut shaped white cocoons.

3.5.7 Silkworm pupation After the completion of cocoon spinning, within the next day or two, the larva transforms itself into a pupa within the cocoon (Krishnaswami et al., 1973). In our experiments, we checked the pupae inside the cocoons after 5-6 days of the spinning. As the pupae were matured by that time, the cocoons were cut at their smaller end to bring out pupae for examination. The pupae were weighed and their other measurements were taken, and then they were put back into their original cocoons.

34

QuBite pupae (♀ and ♂) QuBill pupae (♀ and ♂) Insab pupae (♀ and ♂)

Figure 3.4: Male and female pupae of experimental three silkworm strains

3.5.8 Adult handling Through our experiment, we observed that after 10-12 days pupal period, silkworm adult moths emerge from the cocoon. To enhance the adult emergence and subsequent mating, we placed 8-10 pairs of cocoons (male and female) from the same stain in a rearing tray with its lid loosely attached.

Adult male moth Adult female moth

Figure 3.5: Male and female adult moths of silkworm We observed that adults normally emerged during the night time. On the following morning, the adults were found in the tray as mating / pair conditions. Our observations showed that generally male adults emerged few hours / a day earlier than their female counterparts. We allowed the pairing undisturbed for next few hours. In the afternoon, the male and female adults were separated from their pairs, and kept them separated till dusk. It was observed that in some cases, equal number of male and females did not emerged from the pupae on the same day. If the emerged male number was greater than female, no problem occurred. However, if the female numbers were greater than males, then it created a serious situation because the new female moth generally lays eggs 24 hours after their emergence irrespective of their mating. So, if they do not mate before the egg laying, all the laid eggs will be infertile and useless for producing next generation. In those specific cases, we had to use old male moths, which were emerged a day or two early. In some cases, we had to re-use a mated male in this regard, if there was no unmated male was available from the earlier emergence.

35

3.5.9 Oviposition and egg production At the evening, each female adult was separately placed on an 11 cm filter paper and covered with an inverted funnel, so that the female adult can’t escape from the filter paper, but received proper aeration through the open narrow end of the funnel. The egg laying females were kept in the darker side of the room to provide them less light during the oviposition. According to our observation, female adult start to lay eggs from 8:00 PM onward. On the following morning, we removed all female adults from the filter papers. The eggs, attached to the filter papers, were air-dried under normal room temperature. We generally observed a range of 300 - 700 eggs laid by a female overnight.

Female moth covered with funnel Finishing of egg laying

Figure 3.6: Egg oviposition experiments After the oviposition, the eggs were found golden in colour, but they started to become darker with the advance of time. Normally, all eggs were kept at laboratory temperature for a week or more and then they were transferred to 4-5OC temperature conditions in the refrigerator.

3.6. Artificial hatching During our experiments, we found that all the three strains of the Bombyx mori laid diapaused eggs, which were not hatching within normal laboratory conditions. As to establish a new sericulture industry under Australian environment, continuous supply of silkworm eggs are very necessary, we had to employ different techniques to hatch eggs by artificial stimulation of physical or chemical means. We tested following standard techniques, as cited and used by different researchers world-wide (Park and Yoshitake, 1970; Kurata et al, 1979; Yamashita and Yaginuma, 1991; Manjula and Hurkadli, 1995). After testing the referred protocols, we had to make some modifications and then establish a suitable protocol for the Australian environment.

3.6.1 Low temperature treatment To test the low temperature effects on the artificial hatching, we carried out the experiments through the modified methods of Yamashita and Yaginuma (1991). The silkworm eggs from three different strains were kept in refrigerator (around 5OC) for different time periods including 1 - 3 months. At the end of the specific times, they were taken out from the refrigerator and placed in large plastic containers and put in the insectary for hatching.

36

3.6.2 Cold acid treatment As the hydrochloric acid (HCl) treatment of diapaused eggs in sericulture industry has regularly been used, we used HCl for the treatments of silkworm eggs as suggested by different researchers including the Rao (1998). In case of cold acid treatment system, the eggs were treated at room temperature for 60, 80, 100 or 120 min with HCl acid (spec. gravity 1.075%). Then they were washed under running water and dried under shade for next 24 hours. On the next day, the eggs were set for hatching.

3.6.3 Hot acid treatment In case of hot hydrochloric acid (HCl) treatment, we followed different methods suggested by different researchers. At the end with modifications of some methods, we devised our own treatment protocols according to the hatching suitability. According to our modified method, the silkworm eggs were treated with warm HCl as follows: 3.6.3.1 Preparation HCl dilution The preparation of hydrochloric acid (HCl) dilution was done under fume cupboard condition. HCl with specific gravity of 1.16 was used for the experiment. The HCl acid was first diluted to 1.11 specific gravity, which is appropriate for diapause termination. The equation used for the dilution method was as follows:

V1 x C1 = V2 x C2 where V1 = volume of stock HCl (specific gravity 1.16) C1 = Concentration of stock HCl (32% as indicated in chart) V2 = Volume of diluted HCl solution C2 = Concentration of diluted HCl (22% as indicated in chart)

On the basis of the above equation, for preparing 100 ml of diluted HCl, the necessary amount of HCl was V1 = [(100 x 22) / 32] = 68.75 ml. Therefore, 68.75 ml of stock HCl was added into 31.25 ml of distilled water to prepare 100 ml HCl solution with specific gravity of 1.11. We used this concentration for our all experiments of artificial hatching. The exact specific gravity was checked with the help of a hygrometer. 3.6.3.2 Treatment of silkworm eggs At the beginning of experiment, the temperature of the water bath was raised to 46 OC. A thermometer was kept in water to constant monitoring of water temperature in water bath. 150 ml of prepared hydrochloric acid was poured in to a 500 ml sized glass beaker. The beaker with hydrochloric acid was then placed into the water bath. A second thermometer was put inside the beaker (containing HCl) to monitor temperature of the acid solution. When the temperature of the HCl acid (inside the beaker) reached exactly to 46 OC, then the silkworm egg cards (1 -2 egg cards in each time) were deep into the acid. The egg cards were kept in the acid in fully submerged condition, only for 5 minutes. A stopwatch was used to measure the exact time, as we observed that if the eggs are left in acid over 5 minutes, the eggs do not hatch. As soon as the 5 minute treatment time was over, the egg cards as well as the loose eggs in the beaker were quickly sieved in a second glass beaker, to drain out the HCl. The eggs hold into the sieve, were then washed in running water for next 15 - 20 minutes, until all traces of HCl was fully removed from eggs. The egg washing was done over a third container (beaker), as some loose eggs were observed to wash away from the sieve.

37

3.6.3.3. Drying of silkworm eggs After cleaning and washing, all eggs were transferred to a large and thick paper towel / filter paper, with the help of a camel hairbrush. Cautions were taken to spread eggs so that no egg was attached or over to another egg. The paper towel / filter papers with eggs were then dry under normal room temperature for next 24 hours. After 24 hours, a 9 cm petri dish was kept ready for the egg transfer. A filter paper was placed in the bottom of petri dish. Then the eggs were brushed, from the paper towel or filter papers, on to the petri dish and labeled accordingly. 3.6.3.4 Incubation of treated eggs The petri dish with silkworm eggs then was kept in the insectary under experimental temperature, humidity and light conditions for their normal hatching. For next few days, eggs were checked on regular basis to determine the period needed for hatching from HCl treated eggs.

3.7 The disease bioassays Three silkworm populations, coded as QuBite, QuBill and Insab, respectively, were used for the disease bioassay study. After hatching from the eggs, neonates were brushed and reared up to second moulting on fresh leaves of mulberry (Morus alba). One-day old 3rd instar larvae of uniform size were selected from all three races and used for the experiment. All insect rearing and experiments were done under the laboratory condition at temperature 25 ± 2 OC, relative humidity 75 ± 2% and with light : dark ratio, 16: 8 hours.

3.7.1 Preparation of Bt samples For disease bioassay, we used the Dipel®, a commercial preparation of Bacillus thuringiensis var. kurstaki, as a source of insecticide (Arthur Yates and Co. Ltd, Australia, active constituents: 4320 international units of potency per mg of B. thuringiensis var. kurstaki). The preparation of stock solution was done by dissolving 66.7 mg of Dipel® in 100 ml of distilled water (strength: 2881.44 IU / ml). All further dilutions were made from this stock. All mixtures were used at once for insect bioassays or stored for shorter period of time in refrigerator (4OC) before using. All unused solutions were disposed after 12 hours from their preparation time. The stock solution of Bt insecticide was prepared by dissolving 66.7 mg of Dipel® in 100 ml distilled water, so that 150 µl of stock solution contains 100 µg of Dipel®. Experimental concentrations (80, 70, 60, 50 and 40 µg per 150 µl solution) were made from the above stock solution, by diluting with the appropriate amount of distilled water, so that 150 µl insecticide solution contained either 80, 70, 60, 50 or 40 µg of Dipel® (equivalent to 345, 302, 259, 216 or 172 IU of Bt).

3.7.2 Preparation of leaf samples The fresh mulberry leaves were hand picked from the farm field. They were thoroughly washed under running water and then dried with paper towel. Then, with the help of a sterile cork-borer, discs of 4.50 cm diameter (15.90 sq cm areas) were cut from fresh mulberry leaves. All cut discs were immediately transferred to a plastic container with moist paper towels at the bottom, so those discs did not loose moisture. The container was kept closed with its lid and kept in refrigerator for a short period, until used. As soon as the leaf discs were ready, 150 µl solution of each concentration (80, 70, 60, 50 and 40 µg Dipel®), was separately applied to the lower surface of each leaf disc using a micro-applicator. The solution was smeared evenly with the help of a flat-tipped glass-rod and then allowed to air-dry under vacuum cupboard conditions. The control leaf discs were treated with distilled water only and smeared and air-dried in same way. Three leaf discs were treated with the same concentration, acted as 3 replications.

38

3.7.3 Bt toxicity tests Tests for Bt toxicity by leaf disc bioassay was conducted according to the method described by Sen et al. (1999) with some modifications. Immediately after the 2nd moulting, newly emerged 3rd instar healthy larvae of a similar size were collected from the rearing trays. A series of 250 ml plastic cups were properly marked and then layered with filter papers at the bottoms. After the drying of treated and control leaf discs, each disc was placed in a separate plastic cup, served as food arena for the bioassays. Ten newly moulted 3rd instar silkworm larvae were released on each leaf disc and allowed to feed for next 24 hours. All plastic cups were kept at 25 ± 2 OC temperature, 75 ± 2% relative humidity and with light : dark ratio of 16: 8 hours. At the end of 24 hours, the larval mortality was counted in each cup. After removing the dead, all surviving larvae were transferred to new plastic boxes and fed with untreated fresh mulberry leaves. Larval mortalities were also recorded at 48, 72 and 96 hours after treatment, and all dead larvae were removed from the boxes on a regular basis.

Larvae on treated leaf disc Leaf discs in boxes Larval mortality at 24 hours

Figure 3.7: Disease bioassay on silkworm larvae

3.7.4 Statistical analysis For the statistical analysis, the mortality data were corrected by Abbott's formula (1925) and then analyzed using ANOVA and Duncan's multiple range test (Duncan, 1951).

Bt Concentration – larval mortality relationships were calculated using Probit Analysis (Finney, 1971) with a log10 transformation of concentrations of Bt. Results were expressed as micrograms of Dipel®

per leaf disc (µg/disc). Two LD50s were considered to be significantly different (P < 0.05) if their 95% fiducial limits did not overlap; slopes were similarly considered to be significantly different if their standard errors did not overlap. For time-mortality relationships (LT50), the mortality data were analyzed using the probit model (Finney, 1971), which produced a straight-line fit to the probit of mortality response to the logarithm of the treatment times.

39

3.8 The development of disease resistance The gradual increase in Bt resistance in one of the experimental silkworm strain (Insab) was targeted through continuous disease pressure. As the “Insab” silkworm strain showed better performances in cocoon-shell ratio and more disease tolerance than other two strains, in preliminary screening tests. Therefore, we decided to use this strain for the development of disease resistance. Three subsequent generation (parent, F1 and F2) of the “Insab” were treated with same doses of Dipel® (80, 70, 60, 50 and 40 µg per 150 µl solution) to produce F1, F2 and F3 generations, respectively. We employed the above “3.7: Disease Bioassays” protocols for treating 3rd instar neonate “Insab” larvae in 3 subsequent generations with Dipel® and measure their resistance levels at 24, 48, 72 and 92 hours after treatment. All dead and diseased larvae were removed. Then only healthy larvae, survived from 80 µg per 150 µl solution dose treatment, were used for further rearing according to the “3.5: Rearing of Silkworms” protocols stated above. During the larval, pupal and adult stages, very careful observations had been taken to rear them, and all abnormal larvae, pupae, cocoons or adults were immediately discarded to keep the generation free from any backlash. Our long-term experiment showed an excellent and gradual resistance development in each subsequent generation of Insab strains. At the end of this experiment, we tried to transfer this resistance characteristic of Insab strains to another Bt susceptible strain namely, QuBill, through conventional hybridization system.

3.9 Hybridization of silkworms In our hybridization experiment, two different silkworm strains, of which one was a local strain coded as “QuBill” and other one was a foreign strain coded as “Insab”, were used. Both strains were chosen on the basis of their disease susceptibility and yield contributing characteristics in preliminary screening tests. Between them, the “QuBill” strains were of Queensland origin, with plain larvae, oval shaped and yellow coloured cocoons and univoltine in nature. On the other hand, the “Insab” strain had larvae marked with horse-shoe marking, peanut shaped and white coloured cocoons, and bivoltine in nature. Regarding the disease susceptibility, the “Insab” strain had shown better resistance from the very beginning. Moreover, “Insab” was also used to improve its Bt resistance characteristic through continuous Bt (Dipel®) treatment for three successive generations. The details of developing disease resistance has been given in “3.8: Developing of disease resistance” section above. The F3 generation of “Insab” strain was comparatively highly resistance to Bt, where as the “QuBill” strain was susceptible to Bt disease infection. The both strains were reared for 3 successive generations through inbreeding systems and were under control environment such as 75 ± 2 % relative humidity, 25 ± 2 ºC temperature, and 16:8 hour light and dark period, and fed with same mulberry leaves. The only difference was the “Insab” was exposed to Bt pressure for 3 generations, where as the “QuBill” strain was reared without any Bt pressure. Through this process, The “Insab” was made highly Bt resistance, but left “QuBill” as Bt susceptible.

40

3.9.1 Hybridization techniques During the experiment, we set Insab and QuBill eggs separately in the growth chamber, so that they hatched in same day. Then they were reared on mulberry leaves of same variety. All the larvae, pupae and adults rearing were done according to the protocols mentioned in section “3.5: Rearing of Silkworms” earlier. During the pupal stage, the male (♂) and female (♀) pupae were identified in both strains and then they were separately kept in different boxes, to avoid any kind of inbreeding. As soon as a male or female adult emerged from its cocoon, it used to make a pair with a female or male from either strain. The pairing system was as follows:

♂ Insab x ♀ QuBill and

♀ Insab x ♂ QuBill The pairs were separated after 3 - 5 hours and the respective female moth was transferred in a separate plastic cup, layered with paper towel at its bottom. In the evening, each female was transferred on to a marked filter paper (11 cm diameter) and covered with an inverted funnel. The female was left undisturbed overnight to lay eggs. In the following morning, the female moth was removed from the filter paper and the eggs were used for producing next generation (hybrids).

3.9.2 HCl treatments To assist egg hatching immediately without any diapause, the eggs were treated with warm hydrochloric acid (HCl) within next 24 hours after their laying. The details of acid treatment protocols are given in section “3.6.3: Hot acid treatment” above. When young hybrid larvae hatched from the treated eggs, they were reared according to protocols mentioned the section “3.5: Rearing of silkworms”. In the present experiment, we measured the performances of new hybrid (Insab x QuBill) for their cocoon productivity and disease resistance, and compared these parameters with their both parents in following ways:

3.9.3 Higher yielding characters During the rearing, the quantitative characters of hybrid eggs, larvae, pupae and cocoons, such as hatching percentages of eggs; larval weight and length in all instars; pupal length and weight; cocoon colour and weight; shell ratio etc. will be recorded to compare these characteristics with the parents (Insab and QuBill).

3.9.4 Disease resistance characteristics The disease resistance characteristics in hybrids was measured through the Dipel® treatment protocols as mentioned in section “3.7: Disease bioassays” above. The percentage of disease resistance capability, incorporated into hybrid populations from the resistant parent, was measured through the comparison between the hybrid and parents (Insab and QuBill) strains.

3.9.5 Statistical analysis For the statistical analysis, the mortality data were corrected by Abbott's formula (1925) and then analysed using ANOVA and Duncan's multiple range test (Duncan, 1951). Bt Concentration and larval mortality relationships were calculated using Probit Analysis (Finney, 1971) with a log10 transformation of concentrations of Bt. Details of the statistical analysis are given in section 3.7.4 above.

41

4. Evaluation of three silkworm varieties 4.1 Introduction In Sericulture, rearing of silkworm larva is the most important task and is the centre of the industry. The silkworm larva may need continuous care for 20-24 days in the case of the multivoltine species in tropical areas, or 24-28 days in the case of uni- and bivoltine races in temperate areas (Krishnaswami et al., 1973). The time of silkworm rearing mainly depends on the proper sprouting of mulberry leaves and plentiful supply of good quality leaves. Therefore, when tropical countries can produce 4-6 generations of silkworm in a single year, the sub-tropic and temperate countries can produce only 1 - 2 generations only in the same period (Krishnaswami et al., 1973). Therefore availability of mulberry leaves, temperature conditions and others play a major role for the establishment of sericulture industry in a specific country. The quality of leaf is an important factor that contributes to successful silkworm rearing. Elumalai et al (2001) studied the influence of medium coarse and coarse leaves on rearing parameter of silkworm and found that the medium coarse leaves enhanced most of the economic characters. Krishnaprasad et al (2003) carried out experiments to study the effect of different types of mulberry leaves on the rearing performance of Bombyx mori. They reported that silkworms fed with tender leaves showed higher cocoon yield by weight, cocoon weight and shell weight than those fed with medium leaves. These parameters were lowest in silkworms fed with soiled leaves. Rahman et al (2000) evaluated the nutritive quality of mulberry leaves through moulting test, and found that some mulberry genotypes produced high percentage of moulting ability in silkworm larvae. Chandrashekar et al (1997) found that the performance of silkworm was better when reared on mulberry shoots than on plucked leaves. Sujatha et al (2001) reported that when late age silkworm larvae were exposed to different rearing temperatures (25, 30 and 35 OC), the silk productivity was largely influenced by the rearing temperature. Kumar et al (1997) reared a bivoltine silkworm breed on tender, medium and coarse mulberry leaf separately at constant rearing temperatures of 28, 30 and 32 OC. They reported that the productivity of silkworm was largely influenced by the quality of leaf and also by rearing temperature. According to their suggestions, the tender leaf fed at low temperature (28 OC) resulted in better yield. In their other experiment, Kumar et al (2000) reported that the nutritional status (leaf quality) of mulberry leaf vary with age/leaf maturity and temperature played vital role in growth, development and productivity of the silkworm. They fed different leaf qualities (viz., tender, medium and coarse) to silkworm at different rearing temperature viz. 28°, 30° and 32°C during young age; and found that increase in leaf maturity combined with higher temperature resulted in drastic reduction in silkworm growth rate and biomass accumulation. Muniraju et al (2001) reported that combination of lower temperature during young age and higher temperature at late age of silkworm larvae was found better than constant high temperature regimes throughout the larval period. Researchers from different countries reported the effects of season on mulberry silkworm rearing. Sujatha et al (2000) carried out rearing experiment of bivoltine breed for two seasons, winter and summer in India, with six mulberry varieties. They found that larval, cocoon and post-cocoon parameters (like cocoon weight, shell weight, shell per cent, single cocoon filament weight and single cocoon filament length) varied from variety to variety. They also reported that of the two seasons, winter was more favourable for silkworm rearing. Ray et al (2000) evaluated the combined effect of seasons and mulberry varieties on rearing of silkworm and found that the productivity of silkworm was significantly influenced by varieties and rearing seasons. It is now well accepted that the rearing space plays an important role in the success of silkworm crop and improvement of cocoon quality. Roychoudhury et al. (1991) reported the importance of wider rearing space have been studied both in case of multivoltine and bivoltine silkworm rearing. Over

42

crowding leads to unequal and insufficient consumption of leaf, unequal growth of worms, susceptibility to diseases and low cocoon yield (Sengupta and Yusuf, 1974). Tribhuwan and Singh (2001) evaluated the performance of bivoltine silkworm breed under Chinese, Indian, and Japanese spacing systems on nine economic traits (weight of mature larvae, effective rate of rearing, single cocoon weight, single shell weight, shell ratio, pupation, absolute silk content, female pupal weight, and fecundity). They found that the wider spacing recommended in China was the most appropriate, as all the economic traits, except fecundity. Talukder et. al. (1990) observed that if the population density increased, the rate of cocoon production and adult longevity decreased. The pupal mortality during the rearing of silkworm is also a common phenomenon. Sugun et al (2000) reported that the pupal mortality or melting of cocoons was regularly noticed in field conditions and some times at alarming rates. They suggested that this phenomenon could be occurred due to the inherent character of the race, improper handling of silkworms from the egg stage till the cocooning, adverse environmental conditions during rearing, preservation of cocoons, low quality of mulberry leaf, etc.

4.2 Materials and methods We reviewed different published standard techniques as cited and used by different researcher’s world-wide (Krishnashwami et al., 1973; Dar et al., 1989; Aruga, 1994; Shekar and Hardingham, 1995; Rajan et al., 1996; Veda et al., 1997; Rao, 1998). After consulting the referred protocols, we made few modifications and then practiced the following modified protocols for our current experiments.

4.2.1 Materials used 1. Metal Shelves 2. Camel hair brush 3. Paper Towel 4. Filter papers 5. Rearing containers (plastic) 6. Paper Mountage 7. Plastic Funnel 8. Forceps

9. Illuminated magnifier 10. Refrigerator 11. Room Humidifier 12. Thermometer 13. Hygrometer 14. Fresh Mulberry leaves 15. Knife and chopping board

4.2.2 Insect materials used Three different silkworm strains, two Australian origins and one of Indonesian origin, coded as QuBite, QuBill and Insab, respectively, were used for the present research study. The code names for all above strains were assigned on the basis of their origins, and cocoon shapes and colours. The “QuBite” code represents a silkworm strain of Queensland origin with oval shaped white coloured cocoon. The “QuBill” code represents a silkworm strain of Queensland origin with oval shaped yellow coloured cocoon. The “Insab” code represents a silkworm strain of Indonesian origin with peanut shaped white coloured cocoon.

4.2.3 Experimental environment All insect rearing and experiments were done at the School of Agronomy and Horticulture, University of Queensland and Department of Biology, Sultan Qaboos University, Sultanate of Oman. In both laboratories, the temperature 25 ± 2 OC, relative humidity 75 ± 2% and with light : dark ratio, 16: 8 hours were strictly maintained. All silkworm eggs were preserved in the refrigerators at 4 - 5OC in the laboratory, until needed for next experiment. Before the starting of silkworm rearing, the insect rearing facilities (Insectary and growth chamber) was disinfected with 2 - 4 percent formaldehyde solutions.

43

4.2.4 Rearing techniques The detailed techniques, used for the current experiments, were discussed in Chapter 3, section 5, under title “3.5. Rearing of Silkworm”. For the convenience of understanding, we are giving short details of them below: 4.2.4.1 Incubation of eggs Silkworm eggs, laid in egg cards and preserved at 4 -5 OC temperature in a refrigerator, were placed in a plastic Insect rearing container (25 L x 16 W x 5 H cm) in the Insectary. The eggs were set for hatching under laboratory conditions, as mentioned section 4.2.3 above. All egg cards were thoroughly checked every morning for the sign of hatching. As soon as the first hatching of larvae on egg cards were observed, the cards were transferred in a rearing plastic tray (diam 14.5 cm) and chopped young mulberry leaves were sprinkled over the egg cards, so that the newly hatched larvae could crawl on to the mulberry leaves. 4.2.4.2 Larval feeding and handling During the experimental period, all larvae were provided with sufficient amount and different categories of fresh mulberry leaves (on the basis of larval instars), hand-picked daily from the university farm fields at UQ, Australia or SQU, Oman. Only the cleaned and washed leaves were provided to the larvae twice a day. The first instar larvae were provided with youngest tender mulberry leaves, where as subsequent instars’ larvae were given more mature leaves according to the growth stages. The fifth instar larvae were provided with the largest amount of matures leaves, as leaf consumption is very high during this instar. The bed cleaning (i.e. removing the old mulberry leaves, faecal matter of silkworms, exuviae, any unhealthy larvae etc. from the rearing tray) were done once a day to keep the larvae healthy and free of disease. No bead cleaning was done during the moulting stages to avoid any interfere with moulting process and larval growth. As the silkworm larvae grew in weight and size very fast, their density in the rearing trays changed within short time. In case of newly hatched insects, around 200-250 larvae were kept in a single rearing tray, and larval density was subsequently reduced to 150-200, 100-125, 50-60 and 15-20 larvae per tray, for the 2nd, 3rd, 4th and 5th instars, respectively. 4.2.4.3 Mounting and spinning As, at the end of 5th instar, the mature larvae reached highest body weight, become translucent and started to spin the cocoon. As soon as a mature larva was found started to spin cocoon, it was transferred from the rearing tray to a separate cocoon chamber (4 cm x 4 cm) of hand-made hard-paper mountages, and was allowed to spin its cocoon for next 2 – 3 days. We observed that at the end of spinning, silkworm larva transformed into pupal form. 4.2.4.4 Pupal and adult handling After placing the mature larvae on to the mountage, they were left for next 4 – 5 days for finishing of spinning and hardening of the cocoons. At day 7, the cocoons were collected from the mountage, cleaned from attached flosses manually and then kept in a separate plastic container, layered with a paper towel at the bottom for completing the pupal stage. After that, a number of cocoons were cut from their smaller end to bring out pupae for examinations. The pupae were weighed and their other measurements were taken, and then they were put back into their original cocoons. The Shell Ratio (SR) was calculated as: SR = (shell weight / cocoon weight) x 100 It was observed that after 12-14 days pupal period, silkworm adult moths emerge from the cocoon. It was also observed that adults normally emerged during the night time. On the following morning, the adults were observed in the tray as mating / pair conditions. The pairing were left undisturbed till the afternoon, when they were separated and each adult was kept in isolated condition.

44

4.2.4.5 Oviposition and egg production Each female adult, in the afternoon, separately placed on an 11-cm filter paper (labelled with population name, egg laying date etc.) and covered with an inverted funnel, to avoiding escape. The female adults were then left undisturbed for the night. On the following morning, female adults were removed from the filter papers, and eggs, attached to the filter papers, were air-dried under normal room temperature. Normally, all eggs were kept at laboratory temperature for a week or more and then they were transferred to 4-5OC temperature conditions in the refrigerator. The needed total time under normal lab temperature depends on the further use of the same egg mass. As for example, if the eggs will be needed for a longer storage period, then they will be kept for few weeks at lab temperature to ensure the normal hatchability of the eggs after the long storage period. In case of shorter storage period, the eggs were kept only a week in the lab temperature and then transferred to the refrigerator.

4.3 Results The results from current experiments are given below in table 4.3.1, 4.3.2, 4.3.3, 4.3.4 and 4.3.5. We compared the larval, pupal, adult and egg characteristics among the different silkworm populations, namely, QuBite, QuBill and Insab.

4.3.1 Larval characteristics We compared different larval characteristics during their rearing under laboratory conditions and their results are given in Tables 4.1, 4.2 and 4.3. Table 4.1 shows the difference in larval weights in different larval instars of three different populations namely, Insab, QuBite and QuBill. It was found that in most cases, statistical differences existed among the tested 3 populations. Though at the beginning, the larval weight was highest in the QuBill population, but during the later stages (3rd, 4th, 5th and mature), it performances came to second position. On the other hand, even the initial larval weight of Insab population was moderate, but from the 2nd instar and onward, the larval weight performance was statistically superior to other two populations (Table 4.1). The mature larval weight of Insab (5.009 g) was significantly higher than both the local silkworm populations. The results also showed a huge increase in larval body weight in all 3 populations, when the 1st instar and mature larvae were compared. In case of Insab, the increase in body weight was 9,632 times, followed by QuBite (6,363 times) and QuBill (4,838 times). A graphical representation of the larval body weight has been given in Figure 4.1. When larval lengths were compared among the 3 tested populations, a more or less similar trend was observed (Table 4.2). Though the initial body length of Insab was similar or lesser than the other two populations during the 1st and 2nd instars level, but they became statistically higher in following 3rd, 4th, 5th and mature larval levels (13.60, 37.32, 49.96 and 72.20 mm, respectively). When compared between two Australian populations, the QuBill showed overall better body lengths than the QuBite population. The results also showed that when the larval lengths between 1st instar and mature larvae were compared, a change in larval length was observed. The Insab achieved 24 times body length at mature larval level, followed by the QuBill (20 times) and QuBite (18.6 times). A graphical representation of the larval lengths has been given in Figure 4.2. The Table 4.3 showed the larval periods in different stages. It was found that all three silkworm populations spent nearly similar times during the 1st, 2nd, 3rd and 4th larval instars. However, at 5th instar level, the Insab population spent a significantly longer period (10.00 days) to complete this instar, followed by the QuBill and QuBite populations (8.00 days).

45

Table 4.1 Comparative larval weights in three different silkworm populations

Larval weights in different instars (in mg) Population name

1st Instar 2nd Instar 3rd Instar 4th Instar 5th Instar Mature larva

QuBite 0.44 c 9.00 a 17.47 c 154.70 c 887.60 c 2800.00 c

QuBill 0.62 a 9.00 a 20.07 b 211.80 b 1318.00 b 3000.00 b

Insab 0.52 b 9.20 a 29.41 a 584.00 a 1405.00 a 5009.02 a

Sx value 0.014 0.130 0.450 15.120 7.270 25.080

N.B: During the calculation of average weights, 100 1st instar, 20 2nd instar, 15 3rd instar, 10 4th instar, 5 5th instar and 5 mature larvae were used for each replication. In all above cases, 5 replications were used to calculate each average value. Table 4.2 Comparative larval lengths in three different silkworm populations

Larval Lengths in different instars (in mm) Population name

1st Instar 2nd Instar 3rd Instar 4th Instar 5th Instar Mature larva

QuBite 3.00 8.56 a 11.80 c 25.80 b 40.88 c 56.00 c

QuBill 3.00 8.70 a 12.40 b 25.00 b 45.00 b 60.40 b

Insab 3.00 7.80 b 13.60 a 37.32 a 49.96 a 72.20 a

Sx value - 0.071 0.163 2.521 0.648 0.650

N.B: During the calculation of average lengths, 5 larvae were used for each replication and 5 replications were used to calculate each average value.

Figure 4.3.1: Comparative Larval Weights in three different silkworm populations

0

1000

2000

3000

4000

5000

6000

1st 2nd 3rd 4th 5th MatureLarval stage

Wei

ght (

mg)

QuBite

QuBill

Insab

Figure 4.3.2: Comparative Larval Lengths in three different silkworm populations

0

10

20

30

40

50

60

70

80

1st 2nd 3rd 4th 5th Mature

Larval Instar

leng

th (m

m) QuBite

QuBill

Insab

46

It might be assumed that due to 2 extra days in larval period, the Insab larvae had opportunity to consumed more food than other two populations, and that might contribute to attain higher body weight (Table 4.1) and length (Table 4.2). The larval periods at 1st, 2nd, 3rd and 4th instars, in all populations, also included one day moulting period. The larval period was measured as the time needed from the hatching of egg to completion of 1st mounting (in case of 1st instar) or from one moulting to subsequent moulting (in case of 2nd, 3rd and 4th instars).

4.3.2 Cocoon and pupal characteristics Table 4.4 shows the cocoon and pupal characteristics of all three tested silkworm populations. For this experiment, we used 7 day-old cocoon (counting form the day of spinning). The cocoons were cleaned from associated flosses and then used for measurement. It was found that the Insab population produced heavier cocoon with significantly higher weight (2.200 g) followed by QuBill (1.671 g) and QuBite (1.560 g). When each pupa was removed from own cocoon, the cocoon’s weight & length and shell’s weight were measured. It was observed that Insab pupae were significantly higher in weight (1.850 g) and length (27.80 mm), followed by the QuBill (1.470 g and 25.833 mm) and QuBite (1.372 g and 25.00 mm, respectively). The similar trend was observed during measuring the shell weight. The Insab shells were significantly heavier (0.369 g) than the QuBill (0.195 g) and QuBite (0.181 g). When the Shell Ratio (SR) were calculated, it was found that the Insab had significantly higher shell ratio (16.773 %), followed by QuBill (11.670 %) and QuBite (11.603 %).

4.3.3 Adult properties When the adult properties were compared among the three experimental populations, it was found that adult longevity for male and female, fecundity and egg hatching rates can be varied from population to population (Table 4.5). The results showed that there was no significant difference in male or female adult longevity (in case of same sex) among the tested three populations. Though the Insab male (6.00 days) and females (11.60 days) were found as longest living adults, but the differences among 3 populations were statistically insignificant (Table 4.5). It was observed that in all populations, the female adults were longer living than their male counterparts. As for example, when he female Insab lived for 11.60 days, the male counterparts of the same population lived for 6.00 days only. In case of the fecundity (egg laying capability), the Insab female appeared to be superior among the tested three populations (Table 4.5). The Insab female laid around 750 eggs on average, which was significantly higher than the number of eggs laid by QuBite (606.20) and QuBill (558.80). However, there was no statistical difference between the numbers of eggs laid by two Australian populations. In case of hatching rates (Table 4.5), all the tested silkworm populations showed higher larval hatching rates (97.36 – 98.30%). No significant difference was observed among the local and exotic populations. The Insab eggs had 98.30% hatching rates.

47

Table 4.3: Comparative larval periods in three different silkworm populations

Larval periods in different instars (in days) Population name

1st Instar 2nd Instar 3rd Instar 4th Instar 5th Instar

QuBite 4.00 3.00 4.20 5.60 8.00 b

QuBill 4.00 3.00 4.00 5.80 8.00 b

Insab 4.00 3.00 4.00 6.00 10.00 a

Sx value - - - - 0.172

N.B: Five replications, for each population, were used to calculate each average value. The average larval period includes the total of larval active feeding period plus moulting period in each instar (except the 5th instar)

Table 4.4: Pupal and cocoon weight and length in three different silkworm populations

Pupal properties Cocoon and shell properties Population name

Weight (g) Length (mm)

Cocoon weight (g)

Shell weight (g)

Shell ratio (%)

QuBite 1.372 c 25.000 b 1.560 c 0.181 b 11.603 b

QuBill 1.470 b 25.833 b 1.671 b 0.195 b 11.670 b

Insab 1.850 a 27.800 a 2.200 a 0.369 a 16.773 a

Sx Value 0.032 0.440 0.035 0.014 0.298

N.B: Five replications, for each population, were used to calculate each average value. Table 4.5: Adult properties in three different silkworm populations

Adult longevity Population name

Male Female

Number of total eggs laid

Hatching rates (%)

QuBite 5.80 a 9.00 a 606.20 b 97.36 a

QuBill 5.40 a 9.00 a 558.80 b 97.46 a

Insab 6.00 a 11.60 a 750.00 a 98.30 a

Sx Value 0.447 0.906 27.990 0.651

N.B: Five replications, for each population, were used to calculate each average value.

48

4.4 Discussion The comparative studies on different larval, pupal, cocoon and adult characteristics among the two Australian local (QuBill and QuBite) and one exotic (Insab) populations, showed variations in measured parameters. The comparison of larval weight and length showed the superiority of exotic Insab population over the local two populations. Our study showed the huge changes in larval weight and length during different larval stages. It was interesting to observe that a mature larva attained around 4,836 to 9,632 times of the initial body weight from its 1st instar neonate larval stage. Similarly, the mature larva also gained 18 to 20 times body length than its 1st instar stage. Our report is fully in agreement with the findings of Krishnaswami et al. (1973), where they reported that a mature silkworm larva might gained about 10,000 times its own weight at the time of hatching and this phenomenal growth takes place within the short span of 20-25 days larval life. These enormous changes in larval body weight and length happened within a short period of time and highly linked with the fundamental objectives of silk productivity. Therefore, the increase in these two parameters was strictly monitored for our experiments in all three populations. It was found that both the Australian local populations showed statistically similar increase in body weight and length. The exotic Insab population showed its superiority in both parameters, which can be considered during the future selection of better population for Australian environment. The lower performances of both the local populations might be related with their long term wildness in Australia without proper care, and thereby, decrease in qualitative and quantitative characteristics. A long-term future inbreeding under proper care might help to restore their genetic purity and better performances. The overall larval period (table 4.3.3) showed that all tested populations spent a more or less similar period up to their 4th instar. However, the major differences during 5th instar period were observed in them. The Insab population spent two extra days to complete their 5th instar, which might helped them to attain highest body weight and body length due to possible extra feeding opportunity than other two populations. When both the local populations completed their larval period in 25 days, the exotic Insab population has taken 27 days to complete the same. Therefore, the longer period in 5th instar might be a key to rearing silkworm for higher silk productivity. Our results are in agreement with the finding of Krishnaswami et al. (1973). They reported that on the basis of voltanism in silkworm races (univoltine or multivoltine), the incubation period may continue for 9 - 14 days, larval period for 20 - 28 days, pupal period for 10 - 15 days and the adult stage for 3 - 10 days. As our tested silkworm populations produced 2 generations per year under natural environment, therefore, they might be categorized under bi-voltine group. The comparison of cocoon, pupal and shell properties in tested 3 populations, showed the superiority of Insab population. The Insab produced higher cocoon, pupal and shell weight than the other two local populations. In case of the Shell Ratio (SR), the Insab population showed better performances than other two, which is a one of the important and deciding factors in selecting or rearing silkworm for higher production. The adult longevity and fecundity were also important factors for the silkworm rearing. The results showed that comparatively longer life span of females over the male counterparts. Krishnaswami et al. (1973) reported that the adult life span is around 3-10 days depending upon silkworm races. They also reported that the females are larger in size and generally sluggish while the males are somewhat smaller and more active. Their findings are in agreement with our results, as we observed the life spans of different populations ranged from 5.4 – 11.60 days and in all cases, we observed the larger female than their smaller male counterparts. The fecundity performances of the Insab population over the QuBill and QuBite showed its prospect for selecting for future commercial rearing. As it can lay significantly higher number of eggs with higher hatching rates, they can be use profitably for commercial rearing. The hatching rates among

49

the tested 3 populations showed that irrespective of local or exotic population, higher hatching percentage can be achieved. Krishnaswami et al. (1973) suggested that a female multivoltine silkworm might lay on average approximately 400 eggs while the average number for the uni-and bivoltine varieties of silkworm moths is from 500 - 600.

References Aruga, H. 1994. Principles of Sericulture (Translated from Japanese). A.A. Balkema / Rotterdam. Pp. 376. Chandrashekar, S., Govindan, R. and Narayanaswamy, T.K. 1997. Influence of feeding frequency based on feeding potential in late-age of Bombyx mori L. on larval traits. Environ. Ecol. 15(3): 503-505. Dar, H. U., Singh, T. P. and Bhat, M. I. 1989. A comparative study of Mounting materials for silkworm Bombyx mori L. Entomon. 14(3-4): 211-215. Elumalai, K., Ekambaram, E., Rajalakshmi, S., Sudarsanam, D., Raja, N., Jayakumar, M. and Jeyasankar, A. 2001. Influence of age of mulberry (Morus alba) leaves on economics traits of three races of silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Uttar Pradesh J. Zool. 21(2): 159-161 Krishnaprasad, N.K., Sannappa, B. and Naik, B.C.D. 2003. Effect of feeding mulberry leaves of variable maturity on cocoon traits of Bombyx mori L. Environ. Ecol. 21(1): 112-116. Krishnaswami, S., Narsimhanna, M.N., Suryanarayana, S.K. and Kumarraj, S. 1973. Sericulture Manual 2 - Silkworm rearing. Food and Agriculture organisation of United Nations, Rome, pp. 131. Kumar, C.S., Sekharappa, B.M. and Sarangi, S.K. 1997. Influence of temperature and leaf quality on rearing performance of silkworm, Bombyx mori L. Indian J. Sericulture. 36(2): 116-120. Kumar, C.S., Sekharappa, B.M. and Sarangi, S.K. 2000. Role of temperature and leaf quality on the larval growth and development during young age of silkworm, Bombyx mori. J. Experiment. Zool., India. 3(1): 9-16. Muniraju, E., Sekharappa, B.M. and Raghuraman, R. 2001. Production potential of silkworm, Bombyx mori L. under different rearing temperatures. Indian J. Sericulture. 40(1): 15-20 Rahman, M.S., Doss, S.G. and Sau, H. 2000. Leaf quality assessment of some selected mulberry

genotypes through moulting test using silkworm larvae. Agric. Sci. Digest. 20(3): 180-182 Rajan, R.K., Inokuchi, T. and Datta, R.K. 1996. Manual on mounting and harvesting technology. CSRTI, Central Silk Board, Mysore. Pp. 22 Rao, M.M.M. 1998. A Text Book of Sericulture. B.S. Publications. Hyderabad, India pp. 197. Ray, N., Senapati, S.K. and Deb, D.C. 2000. Studies on the combined effect of mulberry varieties and seasons on performance of bivoltine silkworm Bombyx mori in Terai zone of West Bengal. Indian J. Entom. 62(1): 60-65. Shekar, P. and Hardingham, M. 1995. Sericulture and silk production: A handbook. Intermediate Technology Publications, London. Pp. 55 Sujatha, P., Prasad, P.R. and Rao, N.V. 2000. Influence of different mulberry varieties on larval, cocoon and post-cocoon parameters of Bombyx mori L. Pest Managem. Econ. Zool. 8(2): 161-165. Sujatha, K., Padmaja, P. and Rao, A.P. 2001. The cocoon and post cocoon characters of silkworm, Bombyx mori exposed to different temperature during larval development. J. Experiment. Zool., India. 4(2): 211-214. Sugun, R., Katti, S.R., Chandrakala, M.V. and Raghuraman, R. 2000. Pupal mortality in Bombyx mori - incidence and causative factors. J. Experiment. Zool., India. 2000, 3(1): 1-8. Talukder, F.A., Huq, S.B. and Khan, A.B. 1990. Effect of larval population densities of mulberry silkworm, Bombyx mori on the rate of cocoon production and adult longevity. Prog. Agric. 1(1) : 93-96. Tribhuwan, S. and Singh, T. 2001. Influence of rearing space on economic traits in the silkworm Bombyx mori L. Bull. Indian Acad. Sericulture. 5(2): 111-113. Veda, K., Nagai, I. and Horikomi, M. 1997. Silkworm rearing (translated from Japanese). Science Publishers. Inc., U.S.A. Pp. 302

50

5. Relative susceptibility of three different populations of mulberry silkworm (Bombyx mori L.) to Bacillus thuringiensis 5.1 Introduction Bacillus thuringiensis (Bt) is a gram-positive spore forming bacterium characterised by the formation of parasporal inclusions during sporulation (Aronson et al., 1986; Ohba, 1996). After being ingested by the lepidopteran larvae, the parasporal inclusions are dissolved in larval midgut juice and release protoxins. The activated toxins interact with the larval epithelial membrane and induce pore formation in the membrane, which ultimately leads to insect death (Gill et al., 1992). Bt is considered to be the causative agent for a special type of “flacherie” disease of silkworms, known as “Sotto”. Larvae affected by Bt lose their appetite, shows signs of pain, undergo convulsions and their bodies become stretched and cracked. The cadavers gradually become brown to black-brown and finally when rotting they turn black (Aruga, 1994). Because of their efficacy against insects, commercial preparations of different varieties of Bacillus thuringiensis (Bt) have been sold and used as biopesticides against crop pests worldwide for over half a century. However, growing public concern surrounding Bt use has sparked worldwide debate over current policies (Jayaraman, 1991). Ohba (1996) reported that Bt populations were commonly (96%) present in mulberry plants especially on the leaves in Japan. Van Driesche and Bellows (1996) reported that in India, fear over a potential epizootic, or microbial pathogen out-break, inspired a governmental ban on the use of Bt in silkworm/ mulberry growing areas, despite the nation's moves to reduce the use of traditional chemical pesticides. Pramanik and Somchoudhury (2001) during their study on the adverse effects of Bacillus thuringiensis var. kurstaki against different larval instars of the silkworm (Bombyx mori) reported that it is highly lethal to larvae, causing over 50% mortality of 4th instar larvae at a low concentration of 0.01% of Bt. Inagaki et al (1992) found that Bombyx mori larvae were 2,500-fold more susceptible to the Bacillus thuringiensis var. kurstaki infection than Spodoptera litura larvae. They found rapid growth of Bt in the hemolymph of B. mori. Hassan et al (1989) compared the effects of sub-lethal doses of Bt (var. kurstaki) on third instar Bombyx mori L. larvae and found that Bt significantly reduced the fecundity and fertility of surviving females.

The establishment of a new sericulture industry in Australia might face a number of disease problems including Bacillus thuringiensis, which might turn up either from natural mulberry leaf sources or from the use of Bt pesticides in nearby areas. This organism may therefore be a major constraint in silkworm disease management, because it is difficult to exclude pathogens from the silkworm rearing environment (Balavenkatasubbaiah et al., 1999). Thus, it is important to study the effects of Bt on different Australian and imported silkworm races and to develop disease resistant silkworm races. The present investigation was carried out to study the in vivo lethal effects of Bacillus thuringiensis var. kurstaki against three different races of Bombyx mori larvae under laboratory conditions.

51

5.2 Materials and methods 5.2.1. Insect rearings Three silkworm populations, two of Australian origin and one of Indonesian origin, coded as QuBite, QuBill and Insab, respectively, were used for the present research study. The experiment was set in a completely Randomized Block Design with three replications. After hatching from the eggs, neonates were brushed and reared to second moulting on fresh leaves of mulberry (Morus alba). One-day old 3rd instar larvae of uniform size were selected from all three races and used for the experiment. All insect rearing and experiments were done under the following laboratory condition at the School of Agriculture and Horticulture, University of Queensland: temperature 25 ± 2 OC, relative humidity 75 ± 2% and with light : dark ratio, 16: 8 hours.

5.2.2. Preparation of insecticide Dipel®, a commercial preparation of Bacillus thuringiensis var kurstaki, was used as a source of insecticide (Arthur Yates and Co. Ltd, Australia, active constituents: 4320 international units of potency per mg of B. thuringiensis var. kurstaki). The preparation of stock solution was done by dissolving 66.7 mg of Dipel® in 100 ml of distilled water (strength: 2881.44 IU / ml). All further dilutions were made from this stock. All mixtures were used at once for insect bioassays or stored for shorter period of time in refrigerator (4OC) before using.

5.2.3. Preparation of leaf discs With the help of a sterile cork-borer, fresh mulberry leaves were cut as discs of 4.50 cm diameter and 15.90 sq cm areas.

5.2.4. Insect bioassays Tests for Bt toxicity by leaf disc bioassay was conducted according to the method described by Sen et al. (1999) with some modifications. Immediately after the 2nd moulting, newly emerged 3rd instar larvae of a similar size were collected from the rearing trays. The stock solution of Bt insecticide were prepared by dissolving 66.7 mg of Dipel® in 100 ml distilled water, so that 150 µl of stock solution contains 100 µg of Dipel® (432 IU of Bt). Lower concentrations (80, 70, 60, 50 and 40 µg /150 µl solution) were obtained. The stock solution was diluted with the appropriate amount of distilled water, so that 150 µl insecticide solution (would apply either 80, 70, 60, 50 or 40 µg of Dipel®, equivalent to 345, 302, 259, 216 or 172 IU of Bt) was applied to the lower surface of the leaf disc using a micro-applicator. The solution was smeared evenly with the help of a flat-tipped glass-rod and then allowed to air-dry under vacuum cupboard conditions. The control leaf discs were treated with distilled water only and smeared and air-dried in same way. After drying, each leaf disc was transferred to a 250 ml plastic cup, whose bottom was already layered with a filter paper. Ten newly moulted 3rd instar silkworm larvae were released on each leaf disc and allowed to feed for next 24 hours. All plastic cups were kept at 25 ± 2 OC temperature, 75 ± 2% relative humidity and with light: dark ratio of 16: 8 hours. At the end of 24 hours, the larval mortality was counted in each cup. After removing the dead, all surviving larvae were transferred to new plastic boxes and fed with untreated fresh mulberry leaves. Larval moralities were also recorded at 48, 72 and 96 hours after treatment, and all dead larvae were removed from the boxes on a regular basis. For the statistical analysis, the mortality data were corrected by Abbott's formula (1925) and then analyzed using ANOVA and Duncan's multiple range test (Duncan, 1951). Bt Concentration – larval mortality relationships were calculated using Probit Analysis (Finney, 1971) with a log10 transformation of concentrations of Bt. Results were expressed as micrograms of Dipel®

per leaf disc (µg/disc). Two LD50s were considered to be significantly different (P < 0.05) if their 95% fiducial limits did not overlap; slopes were similarly considered to be significantly different if their standard errors did not overlap.

52

5.3 Results The preliminary investigations on the relative resistance levels of the three different populations of Bombyx mori against different doses of Bacillus thuringiensis var kurstaki were carried out. The results from the three populations are given in Tables 5.1, 5.2 and 5.3. The effects of different Bt doses against ‘QuBite’ population (Table 5.1) showed that all doses of Bt possessed lethal capabilities against this silkworm population. The mortality rate was found to be Bt dose and time dependent. The highest larval moralities (53.33%, 66.67% and 70.00% at 24, 48 and 72 HAT, respectively) were observed at the highest Bt dose (80 µg Dipel®/disc). However, no additional mortality was observed at 96 HAT. The ‘QuBill’ population (Table 5.2) had a higher mortality rate (63.33%, 86.67% and 86.67% at 24, 48 and 72 HAT, respectively) at 80 µg Dipel®/disc dose, than the ‘QuBite’ population. No further larval mortality was observed at 96 HAT. The ‘Insab’ population (Table 5.3) showed that at 24 hours after treatment (HAT), most of the Bt doses did not cause any mortality to this population. Though the highest Bt dose (80 µg/disc) showed some insect mortality at this stage, it was negligible (3.33%) in relation to the insect mortality caused by Bt in the other two populations. However, at 48 HAT and 72 HAT, a significant number (53.33% and 76.67%, respectively) of Bombyx mori larvae died due to the lethal effects of Bt. Similar to the results in the other two populations, no additional mortality was observed at 96 HAT. The ‘QuBite’ population showed most resistance to the highest Bt dose at 72 HAT (larval mortality 70.00%), followed by the ‘Insab’ (76.67%), whereas the ‘QuBill’ population showed the highest susceptibility to Bt attack at the same time (larval mortality 86.67%). Table 5.1: QuBite larval mortality due to the application of different Bt doses on leaf discs

Average larval mortality percentage (%) ± SE at Bt conc (µg/disc)

No. of treated insects 24 HAT 48 HAT 72 HAT 96 HAT

80 30 53.33 ± 9.81 a 66.67 ± 2.72 a 70.00 ± 0.0 a 70.00 ± 0.0 a

70 30 43.33 ± 7.20 ab 56.67 ± 5.44 ab 66.67 ± 7.20 a 66.67 ± 7.20 a

60 30 40.00 ± 4.71 ab 50.00 ± 4.71 ab 60.00 ± 4.71 ab 60.00 ± 4.71 ab

50 30 36.67 ± 7.20 ab 46.67 ± 2.72 b 46.67 ± 2.72 b 46.67 ± 2.72 b

40 30 26.67 ± 2.72 b 40.00 ± 4.71 b 43.33 ± 2.72 b 43.33 ± 2.72 b

Control 30 0 c 0 c 0 c 0 c

* In each replication, 10 newly moulted 3rd instar silkworm larvae were used. * SE = Standard Error * HAT = Hours after treatment * Original data corrected by Abbott's formula (1925) and then transformed into arcsine √percentage

values before ANOVA and Duncan’s Multiple Range Test (DMRT) * Each disc was treated with 150 µl of diluted insecticide mixture. * Values followed by the different letters within a column are significantly different at the 0.05

level (DMRT)

53

Table 5.2: QuBill larval mortality due to the application of different Bt doses on leaf discs

Average larval mortality percentage (%) ± SE at Bt conc (µg/disc)

No. of treated insects

24 HAT 48 HAT 72 HAT 96 HAT

80 30 63.33 ± 2.72 a 86.67 ± 2.72 a 86.67 ± 2.72 a 86.67 ± 2.72 a

70 30 60.00 ± 16.33 ab 80.00 ± 12.47 a 83.33 ± 9.81 a 83.33 ± 9.81 a

60 30 60.00 ± 4.71 ab 76.67 ± 2.72 ab 76.67 ± 2.72 ab 76.67 ± 2.72 ab

50 30 33.33 ± 5.44 b 63.33 ± 2.72 ab 66.67 ± 5.44 ab 66.67 ± 5.44 ab

40 30 33.33 ± 5.44 b 53.33 ± 9.81 b 60.00 ± 4.71 b 60.00 ± 4.71 b

Control 30 0 c 0 c 0 c 0 c

* In each replication, 10 newly moulted 3rd instar silkworm larvae were used. * SE = Standard Error * HAT = Hours after treatment * Original data corrected by Abbott's formula (1925) and then transformed into arcsine √percentage

values before ANOVA and Duncan’s Multiple Range Test (DMRT) * Each disc was treated with 150 µl of diluted insecticide mixture. * Values followed by the different letters within a column are significantly different at the 0.05

level (DMRT) Table 5.3: Insab larval mortality due to the application of different Bt doses on leaf discs

Average larval mortality percentage (%) ± SE at Bt conc (µg/disc)

No. of treated insects

24 HAT 48 HAT 72 HAT 96 HAT

80 30 3.33 ± 2.72 a 53.33 ± 9.81 a 76.67 ± 2.72 a 76.67 ± 2.72 a

70 30 0 a 46.67 ± 11.86 ab 66.67 ± 5.44 ab 66.67 ± 5.44 ab

60 30 0 a 36.67 ± 5.44 ab 63.33 ± 2.72 ab 63.33 ± 2.72 ab

50 30 0 a 26.67 ± 7.20 bc 50.00 ± 9.43 b 50.00 ± 9.43 b

40 30 0 a 16.67 ± 9.81 c 46.67 ± 7.20 b 46.67 ± 7.20 b

Control 30 0 a 0 d 0 c 0 c

* In each replication, 10 newly moulted 3rd instar silkworm larvae were used. * SE = Standard Error * HAT = Hours after treatment * Original data corrected by Abbott's formula (1925) and then transformed into arcsine √percentage

values before ANOVA and Duncan’s Multiple Range Test (DMRT) * Each disc was treated with 150 µl of diluted insecticide mixture. * Values followed by the different letters within a column are significantly different at the 0.05

level (DMRT) Probit analysis: The probit analysis, estimate of LD50 and their 95% fiducial limits for Bombyx mori larval mortality are presented in table 4. Comparison of LD50's on the basis of the probit analyses at 48 hours after treatment (48 HAT), showed that the ‘QuBill’ was the most susceptible (LD50 38.29 µg Dipel® /disc) of the three populations tested, followed by ‘QuBite’ (LD50 55.10 µg/disc). However, at 72 HAT, the susceptibility sequence changed to the ‘QuBite’ (LD50 33.93 µg/disc) followed by ‘Insab’ (LD50 45.73 µg/disc), when the ‘QuBite’ appeared as most resistant to Bt attack (LD50 48.96 µg/disc).

54

The probit regression lines for the effects of Bt on three silkworm populations showed a clear linear relationship between Y axis for larval mortality rate and X axis for Bt concentrations (Figure 5.1 and 5.2). Because the larvae were treated with more lethal agents at higher concentrations, the slopes of the probit lines were steeper as concentration increased. At the 48 HAT (Figure 1), the regression lines was Y = - 3.981 + 2.108X for the ‘QuBite’ population, Y = - 5.299 + 3.00X for the ‘QuBill’ population and Y = - 6.623 + 3.533X for the ‘Insab’ population. Comparing all three regression lines, the steeper line was found in ‘QuBill’ population. At the 72 HAT (Figure 2), the regression lines was Y = - 4.265 + 2.524X for the ‘QuBite’ population, Y = - 4.541 + 2.967X for the ‘QuBill’ population and Y = - 4.433 + 2.670X for the ‘Insab’ population were calculated. Comparing all three regression lines, the steeper line was found in ‘QuBill’ population. Table 5.4. Probit analysis for lethality of different Bt doses on three populations of mulberry

silkworm, Bombyx mori Silkworm Race

No. of larvae

LD50 (µg/disc)

X2 value 95% fiducial limit Slope +_ S.E.

QuBite:

24 HAT 150 77.43 a 0.24 55.21 - 108.64 2.11 ± 0.99

48 HAT 150 55.10 a 0.34 23.11 - 77.75 2.11 ± 0.98

72 HAT 150 48.96 a 0.32 25.34 - 59.04 2.52 ± 0.99

96 HAT 150 48.96 a 0.32 25.34 - 59.04 2.52 ± 0.99

QuBill:

24 HAT 150 58.34 a 1.97 47.44 - 71.60 3.00 ± 0.99

48 HAT 150 38.29 b 0.17 20.12 - 46.28 3.40 ± 1.06

72 HAT 150 33.93 b 0.13 9.73 - 43.52 2.97 ± 1.07

96 HAT 150 33.93 b 0.13 9.73 - 43.52 2.97 ± 1.07

Insab:

24 HAT 150 N/A N/A N/A N/A

48 HAT 150 74.94 a 0.01 64.84 - 109.03 3.53 ± 1.05

72 HAT 150 45.73 b 0.49 22.55 - 54.82 2.67 ± 1.00

96 HAT 150 45.73 b 0.49 22.55 - 54.82 2.67 ± 1.00 * No. of insects were based on five concentrations and tree replicates of 10 3rd instar larvae each. * LD50 = Median Lethal dose of Dipel® * X 2 = chi-square, goodness-of-fit test * S.E.= Standard error

55

* Values followed by the same letter within a column are not significantly different at the 0.05 level by DMRT (P < 0.05).

Fig 1: Relationship of Bt Dose to B. mori mortality in different populations at 48 HAT

-1.5

-1

-0.5

0

0.5

1

1.5

1 2Log dose

Prob

it m

orta

lity

QuBite QuBill Insab

56

5.4 Discussion The preliminary investigations on the lethality of different doses of Bt against three silkworm populations showed that the ‘QuBite’ population had highest resistance capability in relation to the other two test populations. However, among the three, the ‘Insab’ population showed a unique characteristic to be slower to kill (die) by the Bt attack for first 24 hours after treatment. Therefore, this special characteristic might be very important defence mechanisms for sericulture industry. The study confirmed that among the two Australian silkworm populations, the ‘QuBite’ population has more promising future in relation to the Bacterial (Bt) infection, which might come from natural Bt infestation on mulberry leaves or from Bt biopesticide used in neighbouring crop fields.

Fig 2: Relationship of Bt Dose to B. mori mortality in different populations at 72 HAT

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1 2Log dose

Prob

it m

orta

lity

QuBite QuBill Insab

57

References Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265-267 Aronson, A., Beckman, I. W. and Dunn, P. 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Review. 50, 1-24, 1986 Aruga, H. 1994. Principles of Sericulture (translated from Japanese). A.A. Balkema, Rotterdam. Balavenkatasubbaiah, M., Ananthalakshmi, K.V.V., Selvakumar, T., Nataraju, B. Datta, R.K. 1999. Chlorine dioxide, a new disinfectant in sericulture. Indian J. Sericulture. 38(2): 125-130. Duncan, D.B. 1951. A significance test for differences between ranked treatments in an analysis of variance. Virginia J. Sci. 2, 171-189. Finney, D.J. 1971. Statistical methods in biological assays (2nd edition). Griffin, London. Gill, S. S., Cowles, E.A. and Pietrantonio P.V. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annual Rev. Entomol. 37, 615-636. Hassan, M., Khan, A.R. and Zaman, K.U. 1989. The fecundity and fertility of the silkworm, Bombyx mori L. treated sublethally with Bacillus thuringiensis var

kurstaki. Indian J. Sericulture. 28(2): 178-181. Inagaki, S., Miyasono, M., Yamamoto, M., Ohba, K., Ishiguro, T., Takeda, R., Hayashi, Y. 1992. Induction of antibacterial activity against Bacillus thuringiensis in the common cutworm, Spodoptera litura (lepidoptera: noctuidae). Appl. Entomol. Zool, 27 (4): 565-570. Jayaraman K. and Caveat, A. 1991. Bt must be allowed, but with caution and after carefully controlled trials. Current Sci. 60, 465. Ohba, M. 1996. Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries. J. Appl. Bacteriol. 80 (1): 56-64. Pramanik, A. and Somchoudhury, A.K. 2001. Adverse effect of Bacillus thuringiensis Berliner on silkworm, Bombyx mori L. and other beneficial nontarget organisms. J. Entomol. Res. New Delhi. 25(1):53-57. Sen, R., Ahsan, M.M. and Datta, R.K. 1999. Induction of resistance to Bombyx mori nuclear polyhedrosis virus, into a susceptible bivoltine silkworm breed. Indian J. Sericulture. 38(2): 107-112. Van Driesche, R.G. and Bellows, J.T.S. 1996. Biological Control. Chapman and Hall, New York.

58

6. Transfer of parental Bt-tolerant genes into hybrids of mulberry silkworm 6.1 Introduction Different commercial preparations of Bacillus thuringiensis (Bt) have been sold and used as biopesticides against crop pests worldwide due to their efficacy against insects. It was reported that Bt is also commonly (96%) present in mulberry plants especially on the leaves (Ohba, 1996). When ingested by silkworm larvae, the parasporal inclusions of Bt are dissolved in larval midgut juice and release protoxins. The activated toxins interact with the larval epithelial membrane and induce pore formation in the membrane, which ultimately leads to insect death (Gill et al., 1992). The Bt affected larvae gradually become brown to black-brown and finally when rotting they turn black (Aruga, 1994). It was reported that silkworm larvae are highly susceptible to Bt, causing over 50% mortality of 4th instar larvae at a low concentration of 0.01% of Bt (Pramanik and Somchoudhury, 2001). The silkworm larvae were also found as 2,500-fold more susceptible to the Bt var. kurstaki infection than Spodoptera litura larvae (Inagaki et al, 1992). As the Bt is considered to be the causative agent for a special type of flacherie disease of silkworms, countries with established sericulture industries, fear over a potential Bt caused disease out-break, restricted the use of Bt in silkworm/ mulberry growing areas (Van Driesche and Bellows, 1996). The establishment of a new sericulture industry in Australia might encounter Bt related disease problems, either from mulberry leaf sources or from the use of Bt pesticides in nearby crop areas (Begum et al., 2004). As it is difficult to exclude pathogens from the silkworm rearing environment, the Bt might be a major constraint in silkworm disease management (Balavenkatasubbaiah et al., 1999). Thus, it is important to develop Bt tolerant new silkworm races for the future Australian sericulture Industry. The present investigation was carried out to develop Bt tolerant new silkworm populations through the conventional hybridization technique between a Bt tolerant and a Bt susceptible population; to compare the Bt tolerance levels in the hybrids and parents; and to note the transfer pattern of Bt tolerant character (gene) from parents to hybrids.

6.2 Materials and methods Two silkworm populations, one Australian origin and one of Indonesian origin, coded as QuBill and Insab, respectively, were used for the present research study. Dipel®, a commercial preparation of Bacillus thuringiensis var kurstaki, was used as a source of Bt insecticide (Arthur Yates and Co. Ltd, Australia, active constituents: 4320 international units of potency per mg of B. thuringiensis var. kurstaki). For two consecutive generations, the Insab populations at 3rd instar larval stages were treated with three specific doses of Bt (80, 60 and 40 µg /150 µl solution) according to the method described by Begum et al. (2004). After 72 hours of observations, only the survived larvae were separately reared and allowed them to complete the generation. As a result, at the 3rd generation, a comparatively Bt tolerant Insab population appeared (30 - 43 % larval mortality). This was used as the “Bt tolerant parent (BtTIns)”. On the other hand, the QuBill population was also treated with the same Bt doses and was found as highly susceptible to Bt infection (60 - 85% larval mortality). This QuBill population was used as the “Bt susceptible parent (BtSQuB)”. All the experiments were set in completely Randomized Block Design (CRBD) with four replications. All insect rearing and experiments were done in a growth chamber at 25 ± 2 OC temperature, 75 ± 2% relative humidity, and 16: 8 hours light : dark ratio.

59

Eggs from both the Bt- tolerant and susceptible parent populations were set for hatching during the same time and tested for the Bt tolerance at 3rd Instar level. After 72 hours, 50 surviving larvae, from each parent, were selected and were reared under above conditions. All larvae were provided with fresh mulberry leaves collected from the field grown mulberry trees. When the larvae became matured, they were transferred to specially built paper mountage (set inside a plastic box) for the spinning of cocoons. The cocoons were harvested and cleaned after a week from the start of spinning. Then the healthy and similar sized cocoons were kept in separate plastic boxes till the emergence of adults. On the morning of the day of emergence, a female (BtSQuB♀) moth of Bt susceptible QuBill parent was paired with a male (BtTIns♂) moth of Bt tolerant Insab parent. Similarly, another female (BtTIns♀) moth from Bt tolerant Insab parent was paired with a male (BtSQuB♂) moth of Bt susceptible QuBill parent. The coupled pairs were kept enclosed under an inverted funnel for next 7-8 hours. Then all couples were separated and each individual female moth was placed on a pre-labelled filter paper (Whatman, 9 cm diameter) and enclosed with an inverted funnel, to facilitate the egg laying on filter paper without escaping. The females were kept under dark condition for the overnight period and on the next morning, they were removed from the filter paper. These filter papers with silkworm eggs were used for the hatching and developing two different hybrids namely, “BtSQuB♀ x BtTIns♂ (QuB-Ins hybrid)” and “BtTIns♀ x BtSQuB♂ (Ins-QuB hybrid)”. Both the hybrid populations were separately but at the same time, were reared under growth chamber conditions, as mentioned above. Once the both hybrid populations reached at 3rd instar larval stages, they were treated with three doses of Bt (80, 60 and 40 µg /150 µl solution) according to the method described by Begum et al. (2004). Four replications were used for each Bt dose and 10 larvae were included in each replication. All treated insects were kept at 25 ± 2 OC temperature, 75 ± 2% relative humidity and using light : dark ratio of 16: 8 hours. At the end of 24 hours, the larval mortality was counted in each replication. After removing the dead, all surviving larvae were transferred to new plastic containers and fed with untreated fresh mulberry leaves. Larval moralities were also recorded at 48 and 72 hours after treatment (HAT), and all dead larvae were removed from the boxes on a regular basis. For the statistical analysis, the original mortality data were transformed into arcsin √percentage values and then analyzed using ANOVA and Duncan's multiple range test (Duncan, 1951). Bt Concentration – larval mortality relationships were calculated using probit analysis (Finney, 1971) with a log10 transformation of concentrations of Bt. Results (LD50) were expressed as micrograms of Dipel® per leaf disc (µg/disc).

6.3 Results and discussion The preliminary investigations on the relative Bt tolerance levels in two different hybrid silkworm populations (“QuB-Ins hybrid” and “Ins-QuB hybrid”) in relation to their parents (Insab and QuBill), against three different doses of Bacillus thuringiensis var kurstaki had been carried out. The results from the four populations are given in following Table 6.1 and Figures 6.1 and 6.2. The comparative Bt tolerance and susceptible responses of two new hybrids namely, “Qub-Ins hybrid” and “Ins-Qub hybrid” populations in relation to their parents (QuBill and Insab) showed that Bt possessed different levels of toxicity against these silkworm populations (Table 6.1). The mortality rate was found to be Bt dose and time dependent. The larval morality rates increased with the higher doses of Bt, and the higher mortalities were observed at 72 hours after the treatment. Jayanthi and Padmavathamma (1997) reported that larval mortality rates of B. mori was increased in higher concentrations of Bacillus thuringiensis (Bt), but decreased in lower Bt concentrations. We fully agree with the above findings, as we also observed the significant decreases in larval mortality in lower doses of Bt. Among the experimental 4 populations, the “Ins-Qub hybrid” showed the highest Bt susceptibility

60

rates (87.5% larval mortality at 72 HAT with 80 µg Dipel®/disc dose), which was closer but higher than its male QuBill parent (85% mortality). On the other hand the “Qub-Ins hybrid” showed the highest Bt tolerance (35% larval mortality at 72 HAT with 80 µg Dipel®/disc dose) level, which was closer but lower than its male Insab parent (42.50% mortality). When all the 4 silkworm populations were compared for 24, 48 and 72 hours after treatment (24, 48 & 72 HAT), it was found that the Bt-tolerance or Bt-susceptibility responses of “Qub-Ins hybrid” were similar to its male Insab parent, where as the Bt-tolerance responses of “Ins-Qub hybrid” showed very similar trends with its male QuBill parent (Fig 6.1). The probit analysis, estimate of LD50 for larval mortality of two new hybrids and their parents are presented in figure 2. Comparison of LD50's on the basis of the probit analyses at 48 and 72 hours after treatment (48 & 72 HAT), showed that the ‘Ins-QuB hybrid’ had the lowest LD50 (32.06 and 29.44 µg Dipel® /disc, respectively) among the tested 4 populations, closely followed by its male parent “QuBill” (LD50 35.48 and 31.85 µg/disc, respectively). On the other hand, the “QuB-Ins hybrid” showed the highest LD50 (137.28 and 121.40 µg/disc, respectively) during the same times, followed by its male parent “Insab” (LD50 117.38 and 105.61 µg/disc, respectively). Though at 24 HAT, both the parents showed better performances than their hybrids, with the advances of time (48 & 72 HAT), they lost their superiority to their hybrids. The sex chromosomes of the silkworm, Bombyx mori, are designated ZW for the female and ZZ for the male, i.e. female is the heterogametic and male is the homogametic (Nagaraju, 1996; Yokoyama et al., 2003). It was reported that the W-chromosome strongly determine the femininity and has been successfully applied to produce different genetic stocks with dominant alleles for egg colour, larval markings and cocoon colour (Nagaraju, 1996; Ohbayashi et al., 2002). However, references were not available regarding the association of Z or W chromosome with transfer of disease tolerance or susceptible character. Our preliminary investigations on the relative toxicity of Bt doses against new hybrids and their parent’s silkworm populations clearly showed that the “Qub-Ins hybrid” population had highest Bt tolerance capability, followed by its male Insab parent population. In case of the Bt susceptibility, we also observed the similar relationship between the “Ins-QuB hybrid” and its male parent “QuBill” population. Unfortunately, the references on the W or Z chromosome associated with the transfer of disease tolerance/ susceptible character from the parents to offspring are scanty. However, on the basis of our current experimental results, we may hypothesised that as the both hybrids were expressing their Bt- tolerance or susceptible characteristics similar to their male parents only, and opposite to their female parents, there is a possibility that this “Bt-tolerance” or Bt-Susceptible” character was acquired from their male parents. However, further detailed study is needed to establish the chromosome associated with the transfer of this character from the parents to hybrids.

61

Table 6.1: Larval mortality in hybrids and parent silkworm populations due to the application of different Bt doses on leaf discs

Larval mortality percentages at Population and Bt

concentration (µg/disc) No. of treated insects

24 HAT 48 HAT 72 HAT

A. QuBill:

80 40 60.00 ± 4.08 a 82.50 ± 4.79 a 85.00 ± 2.89 a

60 40 57.50 ± 4.79 a 75.00 ± 2.89 a 77.50 ± 2.50 a

40 40 32.50 ± 4.79 b 55.00 ± 8.66 b 60.00 ± 4.08 b

Control 40 0 0 0

B. Insab:

80 40 30.00 ± 7.07 a 40.00 ± 7.07 a 42.50 ± 7.50 a

60 40 22.50 ± 2.50 a 30.00 ± 4.08 a 30.00 ± 4.08 ab

40 40 20.00 ± 7.07 a 22.50 ± 8.54 a 22.50± 8.54 b

Control 40 0 0 0

C. QuB-Ins hybrid:

80 40 7.50 ± 4.79 a 30.00 ± 8.17 a 35.00 ± 4.08 a

60 40 5.00 ± 2.89 a 25.00 ± 5.00 a 25.00 ± 5.00 ab

40 40 0 12.50 ± 4.79 a 15.00 ± 6.46 b

Control 40 0 0 0

D. Ins-QuB hybrid :

80 40 40.00 ± 7.07 a 87.50 ± 4.79 a 87.50 ± 4.79 a

60 40 30.00 ± 7.07 ab 67.50 ± 8.54 b 67.50 ± 4.79 b

40 40 22.50 ± 4.79 b 62.50 ± 2.50 b 65.00 ± 2.89 b

Control 40 0 0 0

a,b Population means within a column followed by the same letter are not significantly different at

the 5% level (Duncan's Multiple Range Test)

62

Figure 2: Parent and Hybrid LD50 due to Bt treatment

0

50

100

150

200

250

300

24 HAT 48 HAT 72 HATHours after treatment

LD50

val

ues

(ug)

QuBill Ins-QuB QuB-Ins Insab

Figure 1: Parent and Hybrid Larval Mortality due to Bt treatment (80 ug/disc)

0

20

40

60

80

100

24 HAT 48 HAT 72 HATHours after treatment

Larv

al m

orta

lity

(%)

QuBill QuB-Ins Ins-QuB Insab

63

References Aruga, H. 1994. Principles of Sericulture (translated from Japanese). A.A. Balkema, Rotterdam. pp. 376. Balavenkatasubbaiah, M., Ananthalakshmi, K.V.V., Selvakumar, T., Nataraju B. and Datta. R.K. 1999. Chlorine dioxide, a new disinfectant in sericulture. Indian J. Sericulture. 38(2): 125-130. Begum, H.A., Hassan, E. and Dingle, J. 2004. Resistance of three different populations of mulberry silkworm (Bombyx mori L.) to Bacillus thuringiensis. J. Plant Diseases Protect. 111(3): 231-237. Duncan, D.B. 1951. A significance test for differences between ranked treatments in an analysis of variance. Virginia J. Sci. 2: 171-189. Finney, D.J. 1971. Statistical Methods in Biological Assays (2nd Edition) Griffin, London. pp. 668. Gill, S. S., Cowles, E.A. and P.V. Pietrantonio, P.V. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annual Rev. Entomol. 37: 615-636. Inagaki, S., Miyasono, M., Yamamoto, M., Ohba, K., Ishiguro, T., Takeda, R. and Hayashi, Y. 1992. Induction of antibacterial activity against Bacillus thuringiensis in the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae). Appl. Entomol. Zool. 27 (4): 565-570. Jayanthi, P.D.K. and Padmavathamma, K. 1997. Laboratory evaluation of toxicity of Bacillus

thuringiensis subsp. kurstaki to larvae of mulberry silkworm, Bombyx mori L. J. Entomol. Res. New-Delhi. 21(1): 45-50. Nagaraju, J. 1996. Sex determination and sex-limited traits in the silkworm, Bombyx mori: Their applications in sericulture. Indian J. Sericulture. 35(2): 83-89. Ohba, M. 1996. Bacillus thuringiensis populations naturally occurring on mulberry leaves: A possible source of the populations associated with silkworm-rearing insectaries. J. Appl. Bacteriol. 80 (1): 56-64. Ohbayashi, F., Suzuki, M.G. and Shimada, T. 2002. Sex determination in Bombyx mori. Current Sci. 83(4): 466-471 Pramanik, A. and. Somchoudhury, A.K. 2001. Adverse effect of Bacillus thuringiensis Berliner on silkworm, Bombyx mori L. and other beneficial nontarget organisms. J. Entomol. Res. New Delhi. 25(1): 53-57. Yokoyama, T., Abe, H., Irobe, Y., Saito, K., Tanaka, N., Kawai, S., Ohbayashi, F., Shimada, T. and Oshiki, T. 2003. Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, Female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited pB silkworm (Bombyx mori) strain. Hereditas. 138(2): 148-153.

64

7. Development of a method of termination of egg diapause 7.1 Introduction The diapause is a special adaptation in insects to avoid unfavourable environmental conditions (Manjula and Hurkadli, 1995). When the diapause eggs are left under natural conditions, the embryo grows to a particular stage and then stops growing to enter into diapause (Veda et al., 1997). In case of bivoltine silkworm race, the spring generation produces eggs, which produce offspring without delay, where as the summer generation produces eggs, which hibernated through autumn and winter and then hatch in the next spring (Kubota et al., 1979; Manjula and Hurkadli, 1995). The silkworm, Bombyx mori enters embryonic diapause induced by a hormone secreted from the subesophageal ganglion during ovarian development (Kubota et al., 1979; Miura and Shimizu, 1987). One of the most interesting problems in the embryonic diapause is the fact that the developmental stage in which diapause occurs is separated in time from that at which the humoral environment exerts its effect (Sonobe et al., 1979). On the other hand, for successful operation of sericulture industry, the continuous and proper supply of quality silkworm eggs to the rearers is very significant. Therefore, breaking of diapause in better quality silkworm races (such as univoltine and bivoltine) is one of the major tasks for the sericulture industry. It is known that, after the eggs are laid, if they are subjected to an artificial treatment at a proper period, it is possible to stimulate further growth without involving diapause (Rahman and Ahmed, 1989; Saheb et al., 1996; Hurkadli, 1997). This artificial method of stimulating the hatching is also known as artificial hatching. As a means of artificial hatching, physical stimuli and chemical stimuli have been tested to break the diapause (Veda et al., 1997). Temperature and friction treatment, hot water, air pressure, electric treatment, ultraviolet treatment, supersonic treatment, are included in the physical stimuli category. On the other hand, HCl treatment, nitric acid treatment, sulphuric acid treatment, enzyme treatment, ozone treatment and perchloride treatment, are included in the chemical stimuli category (Rahman and Ahmed, 1989; Saheb et al., 1996; Srinivasababu et al., 1996; Hurkadli, 1997; Veda et al., 1997). Chilling is generally effective in termination of insect diapause (Yamashita and Yaginuma, 1991). Exposure of diapausing silkworm eggs to lower temperature of 5-7.5 OC for over 60 days completely terminates diapause (Takami, 1969). Manjula & Hurkadli (1995) have evolved short term and ordinary chilling procedures for bivoltine silkworm eggs of temperate and tropical conditions respectively. Use of warm water treatment was also found a way of terminating egg diapause in silkworms. In China, artificial hatching of diapaused eggs of silkworm was accomplished significantly by warm water treatment (Anonymous, 1982). Rahman and Ahmed (1989) showed that when silkworm eggs were treated with warm water of 53.33 OC temperature for 3 – 7 seconds, they showed over 90% hatching rates. Srinivasababu et al. (1996) treated 24 hours old silkworm eggs at 48 OC for 10 seconds to get 88% hatching percentage. Singh et al. (1988) in experiments with 6 strains of the Bombyx mori, reported that eggs dipped for 4-8 seconds in hot water at 56 OC terminated egg diapause. During their experiment with the effect of hot water treatment on diapausing eggs of 3 multivoltine and 3 bivoltine races of Bombyx mori, Mathur et al (1992) showed that diapause could be broken by immersing of the eggs in water at a temperature of 53.1OC for 5 seconds without any adverse effect on hatchability. Among the various physical and chemical stimuli tried to induce artificial hatching, the most successful and practical method is the acid treatment (Krishnaswami et al., 1973). After oviposition, the eggs were preserved at 25OC and the acid treatment was done about 20-48 hours after the egg

65

laying (Anonymous, 1997). Saheb et al (1991) suggested that on diapausing eggs of Bombyx mori, both the cold and hot hydrochloric acid treatments could be used effectively in the age range 9-32 and 17-32 hours, respectively, to obtain a minimum of 90% hatching ratio. The specific gravity of the acid was found suitable in range of 1.100-1.120 specific gravity. In case of cold HCl acid treatment, Saheb et al (1991) reported that immersed diapausing eggs in HCl of specific gravity 1.0964 at 25 OC for 80 minutes showed 90% hatching rates. Hurkadli (1997) treated the eggs of bivoltine Bombyx mori with hydrochloric acid of 1.11 specific gravity (at 15 OC) for a period of 37 ± 2 minutes at 35 OC. Similarly, Aijaz et al (1999) reported that when uni- and bivoltine silkworm eggs, aged 3-27 hours, were treated with cold hydrochloric acid with a specific gravity of 1.110 at 25 ± 1 °C for 70 minutes showed a 90 – 97% hatchability. On the other hand, Manjula & Hurkadli (1995) reported successful hot acid treatment of bivoltine silkworm eggs with hydrochloric acid (HCl) of specific gravity of 1.110 at 48 OC for 5 min and 15 sec. Saheb et al (1996) treated diapaused silkworm eggs with HCl of 1.075 specific gravity at 46.1 OC temperature for 5 minutes. All researchers suggested that the treated eggs should be rapidly washed with water after acid treatment. All traces of acid should be removed and then the eggs should be dried. Eggs treated in this manner should be immediately transferred for incubation.

7.2 Materials and methods During our experiments, we found that all the three strains of the Bombyx mori laid diapaused eggs, which were not hatching within normal laboratory conditions. As to establish a new sericulture industry under Australian environment, continuous supply of silkworm eggs are very necessary, we had to employ different techniques to hatch eggs by artificial stimulation of physical or chemical means. We tested following standard techniques, as cited and used by different researcher’s world-wide (Park and Yoshitake, 1970; Kurata et al, 1979; Yamashita and Yaginuma, 1991; Manjula and Hurkadli, 1995). After testing the referred protocols, we had to make some modifications and then establish the following suitable protocol for the Australian environment.

7.2.1 Materials used 1. Water bath 2. Thermometer - 2 3. Sieve - finest mesh 4. Stop watch 5. Camel hair Brush 6. Paper Towel (filter paper) 7. Glass beaker (500 ml size) -) 8. Wire net (for drying eggs) 9. Hydrometer 10. Insect Rearing room 11. Insect trays 12. Hydrochloric acid (specific gravity of

1.16)

66

7.2.2 Insect materials used For the current experiment, eggs from the two silkworm populations (namely, QuBite and QuBill) were used. The eggs were less than 24 hours old after the oviposition.

7.2.3 Low temperature treatment To test the low temperature effects on the artificial hatching, we carried out the experiments through the modified methods of Yamashita and Yaginuma (1991). The silkworm eggs from three different strains were kept in refrigerator (around 5OC) for different time periods including 1 - 3 months. At the end of the specific times, they were taken out from the refrigerator and placed in large plastic containers and put in the insectary for hatching.

7.2.4 Cold acid treatment As the hydrochloric acid (HCl) treatment of diapaused eggs in sericulture industry has regularly been used, we used HCl for the treatments of silkworm eggs as suggested by different researchers including the Rao (1998). In case of cold acid treatment system, the eggs were treated at room temperature for 60, 80, 100 or 120 min with HCl acid (spec. gravity 1.11). Then they were washed under running water and dried under shade for next 24 hours. On the next day, the eggs were set for hatching.

7.2.5 Hot acid treatment In case of hot hydrochloric acid (HCl) treatment, we followed different methods suggested by different researchers. At the end with modifications of some methods, we devised our own treatment protocols according to the hatching suitability. According to our modified method, the silkworm eggs were treated with warm HCl as follows: 7.2.5.1 Preparation of HCl dilution: The preparation of HCl dilution was done under fume cupboard condition. The HCl used for the current experiment had specific gravity of 1.16. The acid was diluted to 1.11 specific gravity, as it was reported as appropriate for diapause termination. The dilution was done on the basis of the following equation:

V1 x C1 = V2 x C2 where, V1 = volume of stock HCl (specific gravity 1.16) C1 = Concentration of stock HCl (32% as indicated in bottle) V2 = Volume of diluted HCl solution C2 = Concentration of diluted HCl (22% as indicated in chart)

For preparing 100 ml of diluted HCl, the necessary volume of stock HCl was:

V1 = (100 x 22) / 32 = 68.75 ml

Therefore, 68.75 ml of stock HCl was added into 31.25 ml of distilled water to prepare diluted 100 ml amount of HCl with specific gravity 1.11. The final specific gravity was checked and confirmed with the help of a hydrometer. 7.2.5.2 HCl treatment of the eggs: At the beginning of experiment, the temperature of the water, in the water bath, was set at 46 OC. A thermometer was kept in water for constant monitoring of water temperature. Then 150 ml of diluted hydrochloric acid (with specific gravity 1.11 as prepared above) was poured in to a beaker (500 ml size). The beaker with hydrochloric acid was then placed into the water bath. A second thermometer was put inside the beaker (containing HCl) to monitor the temperature of the acid solution.

67

When the temperature of the HCl acid (inside the beaker) was exactly be 46 OC, then 1 - 2 silkworm egg cards was deep into the acid. The egg cards were fully submerged into the acid only for 5 minutes (actually 4 min 50 sec). It was observed that some eggs were lying loosen at the beaker bottom. A stopwatch was used to measure the exact time, as from our previous experience we knew that if the eggs were kept in acid over 5 minutes, the eggs would not be hatched. In the mean time, a second glass beaker (500 ml size) and a sieve was kept ready. As soon as the time crossed 4 min 50 sec, the egg cards, as well as the loose eggs in the beaker, were quickly poured off from the beaker and sieved to drain out the HCl (as we observed that this process takes approximately 10 sec). The eggs in the sieve were then washed in running water for 15 - 20 minutes, until all trace of HCl was removed from eggs. The egg washing was done over a third beaker (500 ml size), to avoid the loss of loose eggs to be washed away from the sieve. After that, the cleaned and washed eggs were transferred on a large and thick paper towel with the help of a camel hairbrush. 7.2.5.3 Drying of silkworm egg: The washed eggs were evenly spread over the paper towel, so that no egg was attached to or sat over another egg. The eggs on paper towel were dried under normal room temperature for next 24 hours. After drying, a filter paper was placed on the bottom of a 9 cm petri dish. Then, the dried eggs, from the paper towel, were brushed on to the filter paper of the petri dish. 7.2.5.4 Incubation of treated silkworm egg: The petri dish with treated silkworm eggs was kept in the Insectary for hatching at temperature 25 ± 2 OC, relative humidity 75 ± 2% and with light : dark ratio, 16: 8 hours. The eggs were checked regularly. It was observed that within next 10 -12 days, newly hatched larvae started to hatch from the HCl treated eggs.

7.3 Results As a part of the quest for developing a suitable artificial hatching system, different types of experiments were set up. As most of the researchers described hydrochloric acid (HCl) treatments as one of the best artificial hatching technique for the sericulture industry, we focused on different acid treatment methods for our present experiments. The results from our experiments are given below in table 7.3.1, 7.3.2, 7.3.3, 7.3.4, 7.3.5 and 7.3.6. Table 7.3.1 shows the effects of cold acid treatment on hatching silkworm larvae from diapaused eggs of two different populations namely, QuBite and QuBill. We compared the efficacy of HCl in respect to different refrigeration periods, intermediate periods and acid treatment times. The results showed that when the silkworm eggs were refrigerated at 5 OC for 7 days and then placed 19 days for Intermediate care, the cold HCl treatment did not initiate the larval hatching. The similar results were observed in both the QuBite and QuBill populations. When the refrigeration period was increased from 7 days to 10 days, no changes in results were observed (Table 7.3.1). When the refrigeration period was increased up to 14 days and at the same time intermediate care period was decreased to 10 days, again no hatching was observed in different cold acid treatment times i.e. 60, 80, 100 and 120 minutes. Similar results were also observed when the refrigeration period increased to 21 days (Table 7.3.1). However, when refrigeration period was finally increased to 30 days, by keeping other parameters unchanged, larval hatching from the diapaused eggs was observed in both populations. However, it was interested to note that the hatching pattern for this parameter was different from normal hatching

68

pattern. In this case, larval hatching were continued over a long period of time, in respect to normal hatching. The table 7.3.2 showed the larval hatching pattern due to the use of cold acid treatment after 30 days of refrigeration and 10 days of intermediate care period. The results showed that due to this treatment, larval hatching started 19 days after the treatment and continued for next 22 days, i.e. up to 41 days after the acid treatment. In relation to the hatching period spans (Day 11 - 22), the highest hatching rates were observed during the ‘day 14 – 16’ period (18.69, 29.34 and 29.6% for 80, 100 and 120 min treatment times, respectively). Among the different acid treatment times (60, 80, 100 & 120 min), the highest larval hatching percentage (84.28%) was observed in 120 min treatment times, followed by 100 min treatment times (73.65%). On the other hand, the lowest hatching percentage (33.70%) was observed in 60 min treatment time. A graphical representation of the results has been given in figure 7.3.1.

Table 7.3.1: Termination of diapause through cold acid treatment systems Population name

Refrigeration period

Intermediate care (Insectary conditions)

Cold acid treatment (min)

Hatching rates (%)

QuBite 7 days 19 days 100 min No hatching

QuBill 7 days 19 days 100 min “

QuBite 10 days 19 days 100 min “

QuBill 10 days 19 days 100 min “

QuBite 14 days 10 days 60, 80, 100, 120 min “

QuBill 14 days 10 days 60, 80, 100, 120 min “

QuBite 21 days 10 days 60, 80, 100, 120 min “

QuBill 21 days 10 days 60, 80, 100, 120 min “

QuBite 30 days 10 days 60, 80, 100, 120 min Gradual hatching

QuBill 30 days 10 days 60, 80, 100, 120 min Gradual hatching

Table 7.3.2: Larval hatching rates in cold acid treatment systems

larval hatching rates (days after first larval hatching) Time of treatment (minutes) Day 11-13 Day 14-16 Day 17-19 Day 20-22

Total Hatching rates (%)

60 min 3.3 8.29 5.52 9.39 33.7

80 min 5.6 18.69 17.75 13.55 63.55

100 min 12.57 29.34 17.96 9.58 73.65

120 min 10.36 29.6 22.24 13.55 84.28

N.B. The first hatching was observed on day 19 after incubation. i.e. Day 11–13 means larvae hatched between day 11 and day 13 after the first larval hatching or 30 to 32 days after incubation

In case of hot acid treatment, different types of results were observed. The results were given in tables 7.3.3, 7.3.4 and 7.3.5.

69

When the combination of refrigeration and intermediate care periods were used along with different temperature and treatment times, variation in larval hatching were observed. When the diapaused eggs, after a period of 19 days refrigeration and 6 days intermediate care, were treated with HCl at 35 OC for 37 minutes, no larval hatching were observed. But, when the refrigeration period increased to 90 days, over 90% larval hatching rates were observed in both silkworm populations due to the HCl treatment at 46 OC for 5 minutes (Table 7.3.3). The same results were observed for both the populations of silkworm (i.e. QuBite and QuBill). It was also interested to note that the first larval hatching was observed only 4 days after acid treatment. In a different set of experiments, diapaused silkworm eggs were treated within 24 hours of their oviposition, without waiting for any refrigeration or intermediate care period. The results were surprising and mentioned in table 5.3.4. When we treated less than 24 hours old silkworm eggs with hot HCl at 46 OC for 5 minutes, over 97% larval hatching rates were observed in both populations. It was interested to note that the hatching patterns were also different from the cold HCl treatment patterns. The first larval hatching for hot acid treatment was observed within 11 days after the treatment (Table 7.3.5). Moreover, most of the hatching (68.89% in case of QuBite and 72.35% in QuBill) occurred within the 2-3 days from the first sign of hatching, which was completely different from the patterns observed in cold acid treatment. A graphical representation of the hatching pattern has been given in Figure 7.3.2. Besides the cold or hot acid treatment, larval hatching in relation to different refrigeration periods, were observed through another set of experiments. The results were given in the table 7.3.6. The results showed that the termination of egg diapause also influenced by the time of refrigeration. When diapaused eggs were refrigerated for 30, 60 or 90 days, no hatching were observed in first two treatments (table 7.3.6). However, the diapaused eggs, after refrigerating for 90 days, produced healthy larvae within 11 days after bringing out from the refrigeration system. In all three silkworm populations, over 97% hatching rates were observed.

70

Table 7.3.3: Termination of diapause through hot acid treatment systems Population name

Refrigeration period

Intermediate care

Hot acid treatment Hatching rates (%)

QuBite 19 days 6 days 35OC for 37 min No hatching

QuBill 19 days 6 days 35OC for 37 min No hatching

QuBite 90 days 6 days 46OC for 5 min > 90%

QuBill 90 days 6 days 46OC for 5 min > 90%

N.B. The hatching of larvae (for 90 days refrigeration only) were started from the 4th day

after acid treatment. Table 7.3.4: Termination of diapause through hot acid treatment systems without refrigeration Population name

Refrigeration period

Intermediate care

Hot acid treatment Hatching rates (%)

QuBite 0 0 46OC for 5 min 99.18

QuBill 0 0 46OC for 5 min 97.59

Table 7.3.5: Larval hatching rates in hot acid treatment (at 46OC for 5 min)

Larval hatching rates after acid treatment Population name

Day 11 Day 12 Day 13 Day 14-15

Total hatching rates (%)

QuBite 13.40 72.35 10.02 3.44 99.18

QuBill 15.23 68.89 8.54 1.80 97.59

71

Table 7.3.6: Termination of diapause through different refrigeration periods Population name Refrigeration period Treatment Hatching rates (%)

QuBite 30 days No treatment No hatching

QuBill 30 days “ “

Insab 30 days “ “

QuBite 60 days No treatment No hatching

QuBill 60 days “ “

Insab 60 days “ “

QuBite 90 days No treatment 98.23%

QuBill 90 days “ 97.21%

Insab 90 days “ 98.54%

N.B: The hatching of larvae were started from the 11th day after incubation.

0102030405060708090

Hat

chin

g ra

tes

D11-13 D14-16 D17-19 D20-22 Hatch %

Days after first hatching

Figure 5.3.1: Artificial hatching rates in different silkworm populations through cold acid treatment

60 Min 80 Min 100 Min 120 Min

72

7.4 Discussion To develop a suitable artificial hatching technique for uni- and bivoltine silkworms, a series of experiments were carried out. The results showed a variety of outcomes. During the cold hydrochloric acid (HCl) treatment experiments, we found that a long time refrigeration (30 days) along with shorter intermediate care period (10 days), cold HCl treatment helps to terminate egg diapause in silkworms. The results are in agreement with the findings of Hurkadli (1997), where after 150 days refrigeration period, 5 days intermediate care and then HCl treatments were done at 35 OC for 35-40 minutes to achieve over 90% hatching rates. Aijaz et al (1999) reported that hydrochloric acid treatment in uni- and bivoltine silkworm eggs at 25 °C for 70 minutes showed a 90-97% hatchability. We also found that larval hatching rates are gradual in case of cold HCl treatment, where the hatching occurred for a long span of times. In case of hot HCl treatment experiments, we found that a longer refrigeration period (i.e. 90 days) helps the hot acid treatment effectively to terminate diapause in silkworm eggs. However, if fresh diapausing eggs, which are less that 24 hours old after oviposition, immediate hot acid treatment can yield a better results. Our experiments showed that if less than 24 hours old silkworm eggs were treated with hot HCl at 46 OC for 5 minutes, a successful and over 97% larval hatching rate can be achieved. We also recorded that in this case, the first hatching occurred within 11 days of treatment. Our results are in agreement with the finding of Saheb et al (1996), where they treated 0-2 day old diapaused eggs with HCl at 46.1 OC for 5 minutes to achieve over 94% hatchability. Manjula & Hurkadli (1995) reported successful hot acid treatment of bivoltine silkworm eggs with HCl at 48 OC for 5 min and 15 sec. It was also found that if the less than 24 hours old eggs were treated with hot acid, most of the hatching occurred between day 11 and 12 after treatment. Our last experiment showed that if diapaused eggs were refrigerated for around 90 days, they started to hatch within 11 days of their removal from the refrigerator. Therefore, if the acid treatments are not available or not usable, these longer refrigeration techniques can be used for producing healthy larvae from diapaused eggs.

0102030405060708090

100

Hat

chin

g ra

tes

Day 11 Day 12 Day 13 Day 14-15 Hatching %

Days of hatching

Figure 5.3.2: Larval hatching rate s due to hot acid tre atme nt

QuBite QuBill

73

References Aijaz, M., Rather, A.Q. and Aijaz, M. 1999. Selection of egg sensitivity in diapausing silk worm (Bombyx mori) to cold hydrochlorization for efficient hatchability. J. Ecobiology. 11(1): 3-12. Anonymous. 1982. Silkworm egg production. Regional Sericulture Training Center, Guanghou, China. Pp. 87 Anonymous. 1997. Silkworm Rearing. Science Publishers Inc., USA. Pp. 302. Hurkadli, H.K. 1997. Artificial hatching of silkworm eggs (Bombyx mori L.) after different chilling periods. Indian J. Sericulture. 36(1): 47-50. Krishnaswami, S., Narashinna, M.N., Suranarayan, S.K. and. Kumararaj, S. 1973. Silkworm eggs. FAO Agric Service Bull. 15: 42-53. Mathur, S.K., Kumar, K., Rao, G.S., Singh, B.D. and Lall, S.B. 1992. Hot water treatment to break diapause in silkworm eggs. Indian Textile Journal. 103(2): 46-48.

Rahman, S.M. and Ahmed, S.U. 1989. Artificial hatching of hibernated eggs of silkworm, Bombyx mori L. by warm water treatment. Bangladesh J. Zool. 17(2): 117-121. Saheb, N.M.B., Kumar, V., Negi, B.B.S., Sengupta, K. and Noamani, M.K.R. 1991. Comparative study on age-specific response of hot and cold hydro-chlorization methods on the diapausing eggs of silkworm, Bombyx mori L. Indian J. Sericulture. 30(2): 151-153. Singh, R., Nagaraju, J., Noamani, M.K.R. and Vijayaraghavan, K. 1988. Termination of egg diapause in the silkworm, Bombyx mori with hot water treatment. Current Science. 57: 1139 - 1140.

74

8. Development of stored and artificial feed for silkworms 8.1 Introduction In all countries mulberry plants have seasonal growth. Some means of making available mulberry leaf contents or substitutes is necessary to grow silkworms in autumn and winter. It is possible to attempt to refrigerate mulberry leaves or to manufacture artificial diets. With regard to the latter, the formulation and use of artificial diets for insect rearing has greatly expanded since the 1950s. They are used for laboratory rearing of insects for research into insect physiology, ecology, genetics, pathogen production, hormone and pheromone manipulation and physiological and biological control techniques (Singh, 1977). The only equivalent to the use of artificial diets to increase an insect product worn or consumed by humans is in apiculture. Artificial diets for silkworms also date for the 1950s and many of the diets developed have been reviewed by Singh (1977) and Hamamura (2001). Ito and colleagues developed artificial diets for silkworms based both on mulberry leaves and without mulberry leaves (e.g. Ito et al. 1974, 1975). Most artificial diets for silkworms have a similar basis. For example, they often contain around 25% dried mulberry leaf powder together with soybean powder, potato starch, cellulose, agar, plus vitamin and mineral mixtures and an antifungal or antiseptic agent. The mixtures are heated from 90°C to autoclaving temperature and pressure for from 10 to 30 minutes. They are then stored under refrigeration.

8.2 Mulberry leaf storage Storage of fresh mulberry leaves in a large plastic bag, which was inserted into another plastic bag to seal it, was successful, with silkworms spinning cocoons and pupating after eating mulberry leaves stored for six months in a cold room at 0ºC.

8.3 Artificial diets Several variations of artificial media were prepared, using mulberry leaves as a base. Trial 1. The leaves were prepared by drying fresh leaves in a drying oven for three days and then crushing them to a powder. The ingredients used in all diets were: A. Water 200ml Mulberry leaves 60g Canolini bean powder 36g Brewer’s yeast 28g Sucrose 24g Cellulose powder 24g Ascorbic acid 2.8g Sorbic acid 0.66g Nipagen (inhibitor) 0.66g Pentavite 0.262ml B. Water 400ml Agar 18g Mix A + B and refrigerate.

75

When the artificial diet above was presented to silkworms of different developmental stages, it was observed that some larvae ate the diet and some did not. Larvae were observed twice a day until all worms died. Trial 2. The quantities of some of the ingredients were modified. For example the quantity of agar was halved because it was thought that the medium may have been too hard for the silkworms to eat. The second modification was to use fresh mulberry leaves instead of dry powdered leaves. More of this diet was eaten by silkworms but not all, and those that ate it did not reach the pupal stage. Trial 3. Other modifications tried were to (a) freeze dry the mulberry leaves before powdering them, and (b) to use the extract of boiled mulberry leaves. The diet using the latter extract was well accepted by silkworms. However, their growth was slow and some died before reaching the pupal stage. Trial 4. A methanol extract of fresh mulberry leaves was prepared. The methanol was evaporated overnight, the residue filtered and the filtrate was used in preparing the diet. Silkworms ate this diet well, grew well and developed to the pupal stage.

8.4 Conclusion Artificial diets could be formulated for silkworms in Australia that were suitable for young worms through to the pupal stage.

References Ganga, G. (2003). Comprehensive Sericulture. Vol.1. Moriculture. Science Publishers, Enfield, NH. Hamamura, Y. (2001). Silkworm Rearing on Artificial Diet, Science Publishers, Enfield, NH. Ito, et al. (1974) Rearing of larvae of silkworm, Bombyx mori, entirely on semi-synthetic diets. J. Agric. Chem. Soc. Japan. 48:403-407.

Ito, et al. (1975). Growth, development, and cocoon production in artificial diet-rearing of the silkworm, Bombyx mori, with or without application of a synthetic juvenile hormone analog. J. Agric. Chem. Soc. Japan. 49:39-48.

76

Section B: Comparison of Mulberry Varieties in Australia and Development of a Mulberry Shoot Harvester

9. Review of literature on moriculture 9.1 Introduction Mulberry is a fast growing deciduous, deep rooting perennial tree that grows throughout the temperate, subtropical and tropical regions. The trees when allowed to grow reach heights of 20 - 25 m with girths of 8 m. Depending on cultivation conditions, the tree can be grown as a bush, middling or tree (Rangaswami et al. 1976). The mulberry plant belongs to the genus Morus of the family Moraceae. There are at least 100 known varieties. Generally, mulberry is a diploid plant with 28 chromosomes (2n = 28). However, it is rich in ploidy and many triploid varieties have been found especially among Morus bombysis. The mulberry (Morus sp.), a perennial plant, is a native of the Indo-China region and is distributed widely in both hemispheres. Although mulberry plant may be as old as man's existence, the earliest record of cultivation was in 2800 B.C., in China where the technique was taught by Ching Nong (Rangaswami et al. 1976, Ganga and Chetty 1999). The earliest significance to men could be found in the chronicles of Chou-king of China in 2200 B.C. when silk was found by accident. Later the silk industry spread to Korea in 1200 B.C., Japan in 300 B.C, India in 140 B.C., and to the rest of the world in 618 - 907 A.D (Boraiah 1994). Mulberry forms the basic food source for silkworms and most of the silk goods in the world market are produced from the mulberry silkworms (Rangaswami et al. 1976). It is estimated that one tonne of mulberry leaf is required to feed silkworms emerging from one ounce (28.3 g) of eggs which will yield about 25 - 30 kg of cocoons (Rangaswami et al. 1976). One hectare of fertile land yields about 15 - 40 tons (15.24 - 40.64 tonnes) of mulberry leaves per annum depending on the climatic conditions. In temperate and subtropical regions like in Europe and Japan, about 15 - 20 tons (15.24 - 20.32 tonnes) of leaves per hectare are produced whereas in tropical areas under intensive cultivation, about 30 tons (30.48 tonnes) per hectare annually could be harvested. In tropical environments where the climate is favourable it has been shown that yield can be twice that of temperate regions. The limitation of moisture in tropical areas gives poor yield and quality. In temperate and subtropical regions, mulberry is available 2 - 3 times a year whereas in tropical regions the leaves are available throughout the year for silkworm rearing (Rangaswami et al. 1976). Proteins from the mulberry leaf provide nearly 70 % of the silk proteins produced by the silkworm. With the objective of increasing the production of mulberry and reducing the cost of production, work into intensive cultural operations such as application of fertilisers, irrigation systems, breeding of mulberry and variety selection, training, pruning and regulation method, harvesting methods have been carried out. Mechanisation of cultural practises and harvesting methods has helped to reduce the production costs. Irrigation (Rangaswami et al. 1976) has a big impact on the production of mulberry. Observations show that the yield can be increased 7 - 8 times through irrigation over dry land cultivation in the tropics, also improving the quality of the leaves. Japanese farmers through the adoption of technology are able to produce average yield of 15 tonnes of leaves per hectare, which produces about 712 kg of cocoons yielding 117 kg of silk. In India, in tropical regions, it has been shown that a yield of 30 tonnes of leave per hectare per annum can be achieved. For optimum growth, the mean atmospheric temperature should be 23 - 27oC and rainfall of 635 - 2500 mm/year.

77

Mulberry silk is the most common of the four silk types in the world accounting for nearly 95 % of the world supply. The other three types of silk of commercial value are tasar, eri and muga. The mulberry leaves are rich in protein, carbohydrate and amino acids and are a source of food for the silkworm. Besides feeding silkworm, mulberry leaves, shoots and branches can be utilised as feed for other animals, the fruits as human food, the wood for firewood or for arts and craft purposes and for their medicinal properties. In India about 60 % (Narasaih 1992, Rangaswami et al. 1976) of cocoon cost is absorbed in production of mulberry leaves. In Japan 63 % (Gopal 1998) of cocoon production is incurred through labour expenses. Of the various mulberry production operations, cultivation of mulberry takes up 21 % (Gopal 1998) and mulberry collection takes up 22.8 % of labour time. One of the advantages of sericulture is that it does not require a large area of land. The area required to produce one tonne of raw silk depends on the intensity of cultivation and the quality of silkworm reared. Twenty kilogram of raw silk is produced from rain fed unfertilised mulberry production, and using inferior silkworms. Whereas up to 120 kg of raw silk per hectare can be produce using the best variety of mulberry together with good cultivation techniques and a good silkworm breed. On average, a yield of 40 - 60 kg of raw silk can easily be produced using a good mulberry variety, cultivation technique and silkworm race (Rangaswami et al. 1976).

9.2 Overview of silk industry Over 30 nations produce silk, China being the largest producer followed by India (Table 9.1). The annual world production in 1992 was reported to be about 84,000 t of raw silk (Boraiah 1994). This is 0.16 % of the world textile fibre production. The future of silk is promising with developing nations benefiting through labour employment. Table 9.1 World silk production (t) (Boraiah 1994). Silk Production (t) Country 1938 1978 1987 1992 China 4855 12,000 35,000 54,500 India 690 3,475 8,455 13,000 Japan 43,150 15,960 7,864 5,085 USSR 1,900 3,200 4,000 4,000 South Korea 1,875 4,235 1,650 1,200 Brazil 35 1,250 1,780 2,373 Thailand - - - 1,700 Other Countries 4,045 2,200 2,874 2,210 Total 56,500 49,360 62,503 84,068 Australian scene Dingle (2000) reported that opportunities exist for Australia to enter the world silk trade with the demand for silk increasing and the supply decreasing. There is also scope to diversify into alternate agricultural production. In 2001, Australia imported $40.195 million (ABS 2002) worth of silk based products. Over the past ten year period from 1992 - 2001, $529.746 million worth of silk products were imported (ABS 2002). Australia through the establishment of a silk industry could replace these imports and be self reliant as well as exporting.

9.3 Adaptability Mulberry is found to grow in various climatic conditions from temperate regions to the tropics. Mulberry growth is dictated by the agro-climatic conditions, soil type and condition in which the plant is cultivated (Reddy et al. 2000, Chakraborty et al. 2000, Baksh et al. 2000, Phukan et al. 2000, and Bindroo et al. 2000). The mulberry tree is widely spread throughout all regions from the tropics (above 30oC) through to the sub-arctic (-0oC), from sea level to high altitudes as high as 4000 m above sea level adapting to various soil and climatic conditions.

78

The optimum conditions (Ganga and Chetty 1999, Rangaswami et al. 1976) for mulberry cultivation are at temperatures of 24 - 29oC, water supply of 280 - 400 ml or a rainfall range of 600 - 2500mm per year, elevation of about 700m above MSL, humidity of 65 - 80 % and sunlight of 5 - 10 hours in temperate regions and 9 - 13 hours in tropical regions. Soils of loamy, clayey loam or sandy loam are best with pH of 6.2 - 6.8. In field situation, the optimum conditions may not be available; hence mulberry growth will be restricted. In conditions which are limiting, managerial practices are applied such as pH correction, fertiliser application, and irrigation. On the other hand, various plant breeding techniques are used to develop breeds, which are adaptable to given conditions.

9.3.1 Soil The soil in which mulberry grows varies from location to location. Physical and chemical characteristics of soil, its water holding capacity, pH, lime, humus content and nutrient status have a direct influence on mulberry leaf productivity and life. A hectare of good land can produce about 15 - 40 t of leaves per year (Rangaswami et al. 1976). This is dependent on the climatic factors of the region. The plant grows well in drained flat fertile land of clayey loam to loamy soil with pH of 6.5 - 7.0 (Rangaswami et al. 1976). Mulberry can be established on gentle to steep slopes and can tolerate slightly alkaline or acidic conditions. Chakraborty et al. (2000) reported better growth and yield by certain varieties in saline coastal soils of West Bengal. The soil quality (Rangaswami et al. 1976) not only influences the leaf yield but also the growth of the silkworm which then is reflected by the quality of the cocoon.

9.3.2 Climate The seasonal variations have direct effect on the growth of mulberry plants. Some varieties perform much better while others will perform poorly (Baksh et al. 2000, Bindroo et al. 2000 and Phukan et al. 2000). Boraiah (1994) and Rangaswami et al. (1976) states that mulberry will grow best in regions with rainfall ranging from 600 - 2500 mm and temperatures of 23 - 28oC. Any rainfall or temperature above or below will limit growth and leaf production. The plant also performs best in areas receiving sun light duration of 5 - 12.5 hours in temperate regions and 9 - 13 hours in the tropics. A relative humidity of 65 - 80 % and altitude of 700 m above sea level is ideal for mulberry cultivation. In temperate regions the plants remain dormant during winter whereas in tropical regions the mulberry tree grows continuously throughout the year.

9.3.3 Australian environment Australia has climatic conditions suitable for mulberry cultivation. Mulberry trees can be seen growing successfully in various locations. The trees are grown either as ornamentals or for its fruit or to feed silkworms for recreational purposes. There are three main species of mulberry tree in Australia, the white (Morus alba), and the black (Morus nigra) which are both imported and a native species (Morus australis).

9.4 Breeding Most species of mulberry are diploids having 28 chromosomes but a few are polyploid. Many polyploids, especially triploids cultivated in Japan, are noted to be resistant to cold weather and diseases. They are rich in vegetative growth and their quality of leaves is better than those of diploids. Triploids are produced from crossing diploids and the artificially induced tetraploids. Tetraploids are good breeding material as well as good feed for silkworms. Attempts made in India (Rangaswami et al. 1976) to directly plant in the field imported varieties in order to reduce the cost of production were not successful due to differing cultural practises as well as the climatic environment. Crossing of imported varieties with promising local varieties resulted in developing breeds, which were high yielding, high quality and were able to adapt to the local environment.

79

The purpose of mulberry breeding is to develop a variety that is an efficient user of nutrients and water, has a high yield, high quality, adaptability to environment, is pest and disease resistant, has a high return and lower production costs. The desirable traits are spread among many species and varieties and repeated hybridisation is done to evolve a variety featuring most of the desired traits. A breeding program starts with the collection of as many local and exotic varieties as possible and evaluation of their characters.

9.5 Varieties The mulberry (Morus sp.), a perennial plant is a native of the Indo-China region and is distributed widely in both hemispheres. The mulberry plant belongs to the genus Morus of the family Moraceae. Koidzumi (1917) classified the genus Morus into 24 species and one subspecies (Hiroaki et al. 2000) with at least 100 known varieties. More than 1000 varieties/genotypes (Rajan et al. 2000) are preserved in germplasm banks all over the world but only 10 % are cultivated with popular varieties constituting only 1 %. Generally, mulberry is a diploid plant with 28 chromosomes (2n = 28). However, it is rich in ploidy and many triploid varieties have been found especially among Morus bombysis. The suitability of mulberry cultivars has to be considered from two aspects, 1. Agronomic and silkworm rearing, 2. Leaf quality and suitability of feeding to different age silkworms. The aim of plant selection is to achieve a better yield, a more nutritious product, disease resistance, and frost and drought tolerant. Agronomic characters such as good rooting, fast growth, high yields, adaptability, pest/disease resistance and drought tolerance are factors in the selection of cultivars. A good variety may not alone be good enough as farming practices, agro-climatic zones as well as inputs affect the performance. The selection of different varieties depends on various factors such as fertiliser (Krishnaswami et al. 1971, Yamamoto 1985), season (Yamanouchi et al. 2001), density (Krishnaswami et al. 1971, Yamanouchi et al. 2001), culture techniques (Zaman et al. 1997), water use (Mira and Sharmain 1999), pest and disease, drought tolerance and soil conditions. Morphological and biological characterising are important for further breeding and cultivation purposes. Tests and evaluation work indicating differences and similarities in characters have been profiled for reference (Zito et al. 1983, Ming-FangFa et al. 1994, Hiroaki et al. 2001). The purpose of varietal selection is to select a variety with traits of environmental suitability, large leaves, short internodal lengths, quick sprouting, high leaf yield, high crude protein, high free amino acids, high total minerals and high total soluble sugar content. Selections of high yield varieties consider the leaf size as an important character. The sizes of the leaves differ from species to species.

9.6 Leaf quality and yield The quality of the mulberry leaf fed to the silkworm is reflected by the quality of the silk. It is vitally important to produce quality leaves to feed the silkworm. The quality of the leaf is a direct result of prudent agronomic management aspects in the cultivation of mulberry. A complete agronomic package of variety selection, planting techniques, nutrient management, water application, pest and disease control measures, and harvesting techniques ensures high yield and good quality mulberry leaf (Krishnaswami et al. 1971, Yamamoto et al. 1980, Ming-FangFa et al. 1994). One hectare of good land can produce 15 - 40 t of leaves per year (Rangaswami et al. 1976). This is dependent on the climate of the region. In temperate and subtropical regions about 15 - 20 t/ha of leaves can be produced. Irrigation in combination with use of fertiliser has increased the production by 7 - 8 times in dry farming conditions as well as increasing the quality of the leaves. The quantity and quality of water directly influence leaf yield. The yield in an irrigated field can be double that of

80

a rain fed field. This can be increased to 3 - 4 times more when used with fertiliser (Rangaswami et al. 1976). The chemical composition and the rearing evaluation of the silkworm determine nutritive value of leaves. Leaves from irrigated fields have more moisture, protein, and other nutrients than rain fed leaves. Silkworm reared on leaves from irrigated fields showed better health, and an increase in weight, cocoon weight, and shell weight (Rangaswami et al. 1976). Nitrogen is the most important element that contributes to the increase in leaf yield. Nitrogen increases vegetative growth and leaf number, size and weight (Rangaswami et al. 1976).

9.7 Mulberry cultivation Mulberry can be either grown vegetatively or through seeds. Vegetative propagation is the most practised method because of the speed in raising saplings, maintenance of plant character, and adaptability. Different vegetative propagation methods such as bark grafting, veneer grafting, root grafting, layering, cuttings, are applied depending on location and country. In India (Rangaswami et al. 1976), most propagation uses the cutting method. A good harvest is a result of proper cultivation techniques of land selection, clearing, preparation, planting, watering, nutrient application, pest and disease control, weeding and harvesting. Increases in production of mulberry leaf (Hisao 1994) are due to advances in breeding, cultivation and pruning techniques as well as management techniques. Shoots or branches of 8 - 15 mm diameter are selected from the mulberry plant. The shoots are cut into 100 - 250 mm lengths with 2 - 4 active buds and planted directly in the field or raised in a nursery first and transplanted after 2 - 3 months into the field. Raising in a nursery is preferred because when transplanted they have a higher survival rate and uniform growth than those directly field planted. To induce rooting, root hormones are used. Mulberry plants can grow on a variety of soils. The preferred soil should be a deep fertile, well drained, clayey loam to loam in texture friable, porous and with good moisture holding capacity. Slightly acid (6.2 - 6.8 pH) soils are ideal for good growth. Although mulberry grows best in flat and fertile land it can be grown in marginal and slopey land where other crops cannot be grown. The land should be cleared of all vegetation and roots uprooted and removed. The land should be deep ploughed and levelled out with good drainage. Mulberry is a deep rooting plant; hence with deep ploughing the roots grow deep enabling faster growth.

9.7.1 Mulching Of the many objectives of mulching, conservation of soil moisture by preventing or hindering evaporation, retention of nitrogen in the soil and keeping out weeds are important aspects of dry land farming. Mulching using vegetative or plastic means have been practiced in mulberry fields to minimise moisture evaporation and the need for weed control. Kasiviswanathan et al. (1971) and Matsusi (1997) using polyethylene mulch reported increases (43 % and 20 - 67 % respectively) in leaf production and efficiency in retaining moisture. Also the quality of the leaf is increased by producing more succulent leaf in drier periods. Fresh and dry weight with moisture content of leaves under mulch was reported to be higher than those under no mulch. They also reported that on average the soil temperature under mulch increased during winter months and decreased during summer months compared to no mulch. Moisture level under mulch was higher ranging from 0 - 6 % than under no mulch. Weed control under plastic mulch showed less than 1 weeds/ft2 compared to 24 weeds/ft2 under no mulch.

9.7.2 Fertiliser The growth and production of leaf in mulberry are known to be responsive to intensive management. The naturally occurring fertility of the soil can not be entirely depended upon for good growth. Organic and inorganic fertiliser is relied on to supplement the nutrients for good growth and high leaf yield. Adding organic and farmyard manure as well as growing mulch crop especially leguminous plants and turning into the ground also assist in providing nutrients. Mulberry being a

81

photosynthetic plant requires about 13 nutrient elements absorbed from the soil in addition to C, O, and H which are absorbed through water and the air. Nitrogen is the most important which contributes to the increase in leaf yield, vegetative growth, leaf number, size and weight. It has been shown that with the application of fertiliser, the leaf yield can be doubled (Rangaswami et al. 1976). In many cases, the correct amounts of nutrient elements are not applied which affects the overall growth and production of quantity and quality of the leaf. Also various bad soil conditions cause immobilisation or fixation of nutrients in the soil. This results in various nutritional disorders, which are expressed in various symptoms. Excess nutrient on the other hand may cause toxicity effects. In both cases, the growth and production of good quality mulberry leaves is affected (Bongale 1994). A general formula for replenishment of fertiliser of mulberry fields based on depletion through the leaf harvest has been worked out in Japan (Yokoyama 1962) and in India (Ray et al. 1973). However these formulas are not directly applicable to any other country due to differences in the method and frequency of harvest as well as the soil and climatic conditions (Islam et al, 1982). Yield and quality Islam et al. (1982) reported significant differences in leaf yields. As the dosage of NPK nutrients increased from 0:0:0 - 400:200:100 kg/ha, the leaf yield also increased. Similarly Shankar and Rangaswami (1999) reported best quality foliage and highest yield of silkworm, cocoon weight, and single filament length at 400 kg N and 200 kg K2O/ha/yr. Similar responses were recorded for potassium (K2O) addition at 240 kg/ha/yr (Shankar and Sriharsha 1999). In Japan, a standard application of chemical fertiliser to mulberry fields is 30 kg of N, 14 kg of phosphate, 12 kg of K per 0.1 hectare for alluvial soils and 30 kg of N, 16 kg of phosphate, 20 kg of K per 0.1 hectare for volcanic ash soils. In either case an application of at least 1,500 kg of compost per 0.1 hectare is recommended. (Hiroaki et al. 2000) In India, Krishnaswami et al. (1971) reported significant improvement in economic characters with a heavy application (900 kg/ha) of nitrogen fertiliser together with closer spacing compared to a combination of local varieties, wider spacing and 100 kg/ha of nitrogen. There was no harmful effect in cocoon production as is commonly believed. Significantly higher leaf yields were achieved when farm yard manure (FYM) of 20 t/ha/yr combines with 280:120:120 NPK t/ha/yr were used (Shankar et al. 2000) compared with using only FYM. Also N, K and crude protein content were higher compared with treatments of 10 - 40 t/ha/yr of FYM. Total soluble sugars were high at FYM of 20, 30, 35, 40 t/ha/yr than at 10 t/ha/yr. On the other hand, Shankar et al. (2000) reported silkworm weight, cocoon weight, shell weight and filament length were much superior when fed with leaves incorporating only FYM to FYM plus inorganic fertiliser. This highlights the importance of organic manure and its significance in the eco-system. Crude protein content was reported to be highest in leaves receiving ammonium sulphate, least in unfertilised plots and intermediate in plots receiving calcium ammonium nitrate, ammonium nitrate and urea as the N component of the fertiliser (Subbarayappa et al. 1994). Similar trends of high protein content, high yield and high cocoon yield were reported for leaves treated with ammonium sulphate, compared with plots fertilised with calcium ammonium nitrate and urea (Subbaswamy et al. 1999). Leaves receiving 400 kg/ha/yr N in combination with sulphate of potash of 180 kg/ha/yr gave highest leaf yield and cocoon yield compared with lesser doses. Also silkworm performed better on leaves treated with higher levels of N and sulphate of potash (Shankar et al. 2001)

82

9.7.3 Irrigation Mulberry can grow in all types of soil and climatic conditions. The trees can be either grown as rain fed or water supplemented with irrigation system. Where mulberry is grown as a rain fed crop, the soil is poor and the water holding capacity is also poor. Rain water percolates fast through the profile with little moisture left in the root zone. This problem can be minimised by adding organic and farmyard manure. Growing a mulch crop especially leguminous plants and turning into the ground can also assist. Two other benefits are achieved through this practice, namely nitrogen fixing and mulching. Under different water regimes, mulberry performance differs. The amount and frequency of getting water to the plant is dependent on the soil moisture content level, the weather, soil type, stages of growth and population of the plants. Many types of irrigation practice have been used to bring water to the mulberry trees. The type applicable is based on the economic threshold of the developer, soil type, topography, technical know how, water availability, climate, associated costs and the ease of application and other factors. Many systems are already in place, varying from location to location and still more research is being conducted to identify systems that suit particular needs, requirements and environments. Where uniform rainfall at the rate of 100 - 250 mm per month is received, there is no need for supplementary irrigation. In most regions of the world such rainfall conditions do not exist. In China, much emphasis has been placed on irrigation. Even areas with sufficient rainfall have irrigation system in place. Irrigation is combined with drainage to remove excess water. The most common spacing practiced under irrigation conditions are as follows, using ridge and furrow planting practice in India (Dandin 1994). Space (m x m) Plant population/acre (ha) 0.6 x 0.25-0.3 22,000 (55,000) 0.6 x 0.6 11,000 (27,000) 0.9 x 0.9 5,000 (12,000) 9.7.3.1 Soil moisture For efficient use of water enough water is to be applied to bring the soil moisture level to field capacity (FC). The amount of moisture held in the soil is limited by its FC and wilting coefficient. The water between these two limits is the available water for the plant. The amount of available water that can be stored in the root zone of the soil depends upon its depth and the soil texture and structure. At field capacity the mulberry extracts more water and grows rapidly whereas the growth slows down as it approaches wilting point. 9.7.3.2 Water requirements The amount of water required by the mulberry plant is dependent on various factors such as soil moisture content level, weather, soil type, stage of growth, plant population and available water. It has been estimated that 6 - 7 litre of water is lost every day in the form of water vapour through evapo-transpiration. Also it is estimated that 0.4 - 0.6 (Dandin 1994) or 0.28 - 0.4 (Rangaswami et al.1976) litre is required to produce 1 gram of dry matter. In order to compensate for the above two activities, 6 - 8 litres of water/day are supplied through irrigation depending on the soil type and the season (Dandin 1994). It is observed that mulberry grows best producing high quality leaf at 70 - 80 % field capacity. To maintain this, additional water is applied through irrigation. A sufficient amount of water has to be applied to bring the moisture level to the FC and to cover the entire root zone. For mulberry it is necessary to bring the moisture level to FC down to one meter in depth (Rangaswami et al.1976). About 50 - 75 mm per acre (123 - 185 mm/ha) has to be applied to

83

bring the soil moisture level up to the root zone. Water is frequently required during the active growing period. The recommended weekly irrigation is 22,000 - 33,000 gallons/acre (247,000 - 370,000 L/ha) amounting to 11 115 m3/yr for black loam soil, 12 968 m3/yr for red loam soil and 16 673 m3/yr for sandy soil (Dandin 1994). Rangaswami et al. (1976) suggested rates of 30 900 - 37 064 m3/yr whilst Soo-Ho-Lim et al. (1990) suggested 14 600 - 18 250 m3/yr. The suggested different water requirements highlight the importance of locality or region. Different locations have different irrigation needs based on weather, soil, plant variety, plant growth and plant population. 9.7.3.3 Irrigation systems Irrigation can be applied in many different ways such as furrows, flat beds, basins, sprinklers, sprayers, or drips. In the furrow method, a series of ridges and furrows is prepared where water is channelled into the base and the water seeps into the ridge by capillary action. In the flat bed method the field is split into small areas with built up bunds trapping the water in the enclosed area. The basin method involves forming a basin around the plant with water channelled to each basin using interconnecting channels. More recently water has been piped to the mulberry plants and applied in the form of sprayers, sprinklers or a drip system. Drip, spray and sprinkler systems are more efficient water applicators but the purchase price is high, pushing beyond the reach of small farmers from developing countries. In arid regions, water is scarce and an efficient way of using water had to be developed. It was found that a drip system is an efficient way of conserving water. A conventional drip system costs $2470/ha, which is costly for small farmers in developing countries. A study involved in developing a simpler drip system, which reduced the cost by 90 % (costing about $247/ha), found that uniformity of flow from the emitters was 73 - 80 % efficient. 9.7.3.4 Drip irrigation In water-scarce regions, drip irrigation provides an efficient way to conserve irrigation water but cost is the limiting factor (Polak et al. 1997) for small farmers in developing countries. Cheaper alternative drip techniques (Polak et al. 1997) have been developed and found to reduce the labour cost by half and also twice the area irrigated with the same amount of water. Muralidhara et al. (1994) suggested that a drip irrigation system would be an economically viable method of irrigation in water-scarce regions. With its high net present value, cost-benefit ratio above one, and internal rate of return higher than the discount rate, drip irrigation forms a viable alternative technology 9.7.3.5 Leaf yield and quality The quantity and quality of water directly influence the leaf yield and quality. The yield in an irrigated field can be double that of a rain fed field. This can be increased by 3 - 4 times when used with fertiliser (Rangaswami et al. 1976). Leaves from irrigated fields have more moisture and protein, and are more nutritious than rain fed leaves. Silkworm reared on leaves from irrigated fields showed better health and an increase in weight, cocoon weight and shell weight (Rangaswami et al. 1976). Das et al (1996) reported a high leaf yield of 7.83 t/ha/yr in fertiliser/irrigation combination and low yield of 5.48 t/ha/yr in rain-fed fields. Benchamin and Begum (1990) found that silkworm reproductive rate was greater when fed with irrigated mulberry leaves. Also Yamamoto et al. (1980) showed that mulberry leaves from irrigated plots achieved higher nutrient content, good growth of the mulberry tree and better growth of silkworm. Mira et al. (1999) reported a mulberry variety (Sujanpuri) yielding maximum biomass under irrigation while a local variety was superior to the other two varieties (Kanva-2 and Sujanpuri) under rain fed conditions. 9.7.3.6 Alternate irrigation water Where there is a water resource limitation, alternate water could be utilised. A comparative study using bore well water and polluted (domestic and industrial) river water showed leaves contained

84

significantly higher concentrations of protein, sugar, phenol and chlorophyll, and mineral elements from the polluted water irrigated plots than from the bore well irrigated plots (Shree et al. 2000, Bogale and Krishna 2000). No adverse effects on silkworm were noticed when fed with leaves from gardens irrigated with sewage effluent (Shree et al. 2000, Bongale and Krishna 2000). In fact gardens irrigated with sewage water recorded significant and positive correlations of cocoon yields with increased leaf N and non-protein N contents (Bongale and Krishna 2000). 9.7.3.8 Water stress Water stressing (Reddy and Sudhakar 1992, Reddy and Sreenivasulu 1993, Kumari 1995, Kumari and Veeranjaneyulu 1996, Singhal and Roopa 1998, Ramanjulu et al. 1998, Ramanjulu et al. 1998, Barathi et al. 2001) has adverse effects on physiological and biochemical characters (photosynthesis, carbohydrate and starch, protein, ascorbic acid and proline accumulation). Reductions are noticed in the photosynthesis rate, carbohydrate and starch production, protein and ascorbic acid production. Drought sensitive varieties are more responsive (Ramanjulu et al. 1998**) than drought tolerant varieties. Short term water stressed plants can recover (Ramanjulu et al. 1998*). Silkworms reared on leaves with different moisture content showed increased consumption and growth rates (Paul et al. 1992) by worms feeding on higher MC leaves than leaves with low MC. 9.7.3.9 Water logging Water logged fields minimise oxygen uptake by the roots. Drainage of the field aids in microbial activities that improve the soil structure and fertility. Drainage also aids in leaching of excess salt. Water logging (Das et al. 2000) showed relative water content of leaves increased gradually with the duration of flooding. An increase in loss of electrolytes and amino acids into the incubation medium indicated impaired membrane integrity with excess water stress. Among the photosynthetic pigments, chlorophylls, but not the carotenoids, appeared most vulnerable to flooding. A negative correlation was found between the duration of flooding and titre of some primary metabolites (total soluble sugars, total soluble proteins and RNA 9.7.3.10 Soil moisture extraction pattern (SMEP) Effective root zone for water absorption was up to 70 cm although the maximum extraction was from the upper layers. Soil hardness is lowest at 10 cm and hardest at 30 cm depth. Soil moisture tension (pF) is highest at 10 cm and decreases downward becoming stable at about 50 - 70 cm depth. pF of spring pruned gardens was higher than in summer pruned gardens. pF was also found to be higher for close spacing before irrigation than wider spacing after irrigation (Rama et al. 1998). 9.7.3.11 Poor/slopey/arid land On land of poor soil, slopey landscape and arid regions where high value crops perform poorly, mulberry plants are an alternative. Various trials indicate mulberry performs much better with better returns (Jia and Qian 1992) than other crops (soybean, millet and potatoes).

9.7.4 Plant density Mulberry plant growth, yield and quality differ with different planting densities. Methods of pruning, harvesting, mulberry variety, climate as well as the soil condition influence the spacing between trees. To decide upon the optimum plant population, the variety of mulberry, training and harvesting methods to be adopted are decided first. In general, varieties with vigourous branching are spaced further apart. In rain fed dry farming conditions, where moisture is limiting, wider spacing is practised. In semi-tropical regions, densities of up to 150,000 trunkless trees/ha have been grown. In other regions, it is common to see very low height plants (0.15 m), spacing at 1m x 0.3m or densities of 30,000 trees/ha. Mulberry trees of low height (0.4 m) are spaced at 1.7m x 0.8m or densities of 7,500

85

trees/ha and medium cut trees (1 m) at 2.2m x 1m or densities of 1,500 trees/ha. The current tendency is to have wider spacing to allow for mechanisation with a density rate of 7,500 trees/ha. Boraiah (1994) recommends a spacing of not less than 0.9m x 0.9m. At this spacing, the plants get better lighting and develop well. He further states that Japanese farmers have learnt through experience that proper spacing helps improve the quality of leaf. The Japanese have proved through experimentation that qualitative and quantitative yields of mulberry depend upon the plant population in a given area and the number and length of branches on a plant. The leaf yield increases but the quality decreases where it is densely populated. The leaves from densely populated plants are usually small and thin hence easy to wither. The qualities of such leaves are low and thus affect the yield and quality of the cocoon. Depending on the height of pruning, different spacings are practised. For instance in Japan (Rangaswami et al. 1976, Hisao 1994), for low (root) pruning (<0.5 m height), a distance of 1.2 - 1.8 m row spacing and 0.6 - 1.2 m between plants are practised (450 - 1350 trees per 0.1 ha). In medium pruning (0.5 - 1.5 m high), 280 - 540 trees per 0.1 ha and for high pruning (>1.5 m high) cases, tree population of 240 - 480 are maintained. By careful management the Japanese have reduced the leaf cocoon ratio to 20:1 (Boraiah 1994). In India the spacing is not strictly maintained and hence farmers go for the quantity, sacrificing the quality. The average cocoon yield/hectare is 300 kg in India compared to that in Japan of 650 - 700 kg (Boraiah 1994). The maintenance of quality of leaves is one of the main reasons for the quality of cocoon production. Different countries practise different densities of cultivation due largely to varying traditional practices and the agro-climatic elements. Recently, with more emphasis on mechanisation, wide spacing is practised to accommodate machinery. 9.7.4.1 Leaf yield Work by Ghosh et al. (1997) Choudhury et al. (1991) and Bongale et al. (2000) reported higher yield at a spacing of 0.6m x 0.6m whilst also noting a sharp decline with spacing beyond 0.6m x 0.6m whereas Madan and Satyawati (1999) reported greater leaf yield at 0.5m x 0.5m spacing and subsequently increasing with wider spacing. Katsuji (1983) reported, for summer pruning, 1/3 leaf number, dry weight, and leaf area in dense plots (treatments of 833, 3996 and 6666 plants per 0.1 ha) than sparse plots and sum of length and dry weight of branches were less than 1/2 of those in sparse plots. For spring pruning, in dense plots, decrease of branch numbers, elongation and less leaf spread were observed. The growing point of the apical bud tended to become smaller in dense plots. On a per plant basis, there was an increase in new shoot number, new shoot yield, leaf yield (Lin et al. 1994 and Rao et al. 2000) and total length (Rao et al. 2000) as the plant spacing increased whereas leaf yield per hectare was higher for denser plantings. However, leaf water content (Bongale et al. 2000 and Rao et al. 2000), leaf water retention at 6 and 12 hours after harvest (84.04 and 76.15 %, respectively) and leaf yield (7650.50 kg/ha) were highest for denser (0.6m x 0.6m) spacing (Rao et al. 2000). Generally, the performance of silkworms was best during the winter season at 0.6m x 0.6m spacing. Mira et al. (1999) reported initial high yields with dense trees (0.5m x 0.5m spacing) but subsequently the yield increased with wider spaced planting (1 x 1, 1.5 x 1.5, 2 x 2 m2). This suggests the scope of initial dense planting and thinning after the 1st, 2nd or 3rd harvest. 9.7.4.2 Chemical composition Typical composition of mulberry leaves is moisture (75 - 82 %), and of the dry matter, crude protein (24 - 36 %), crude fat (3 - 4 %), crude fibre (9 - 11 %), ash (mineral) (7 - 8 %), and carbohydrate (12 - 20 %) (Ganga and Chetty 1999). This composition varies with age of leaf, climate, soil type, ground water, sunlight, pruning method and the variety. Ghosh et al. (1997), Katsuji (1983) and Lin et al. (1994) reported trends of mineral, dry matter and crude protein contents in leaves and branches

86

decreasing as plant spacing decreased. On the other hand, Lin et al. (1994) reported that plant spacing did not reveal any significant effect on the content of crude fat, crude fibre and total ash. With regard to season, in India nitrogen contents in the upper leaves were raised in June but fell in August in the densest plots. In June the dry weight and the nitrogen contents of the tree tops per unit area in dense plots were 3 times as much as the sparse plots. These results indicate that large amounts of leaf production in dense conditions rapidly changed the light condition in the plant community, which was unfavourable for the transportation of nitrogen and other nutrients. Katsuji (1983) further reported that in dense plots the morphology of leaves varied with the leaf position. The content of chlorophyll in the dense plot in the upper leaves was raised in June and July but fell in August. Leaf weight per area became less especially in the middle position. In dense plots, the cell layers of branch tissues such as cortex, xylem, and phloem fibre were less in number and vascular bundles were poorly developed. The general trend is to use sound agronomy practices to maintain the balance between yield and quality. It is commonly found that dense plots yield higher leaf mass with lower nutritional qualities and vice versa with wider spaced mulberry plots. It would be worth considering the idea of establishing initially a dense plot and thinning toa sparser plot after the 1st, 2nd or third harvest based on the findings of Mira et al. (1999). This technique would cater both for quantity and quality without sacrificing one or the other.

9.8 Harvesting To feed the silkworm, mulberry leaves are brought to the feed house after being harvested in the field. Leaves are harvested either through manual or mechanical means. In most mulberry producing nations, the harvesting operation is manual. Because of an increase in labour costs, research and trials are being conducted to mechanise the harvesting operation. The technique of harvesting mulberry leaves depends on the rearing practice for the silkworm. Three harvesting techniques used are, leaf picking, branch cutting and whole shoot harvesting. The very young silkworms feed on softer tender leaves whereas older worms feed on matured leaves as well as stems/branches. In manual harvesting, selective picking of the leaves is done using hands or with the aid of hand tools. Hand tools are required when harvesting shoots or branches. A mechanised harvester can be either of a leaf stripping or a shoot and branch pruning/cutting type. The stripper type strips only the leaf, leaving the branch or shoot in the field to continue production of leaves, whereas the cutter/pruner type cuts or prunes the whole shoot or branch. The harvester design and configuration should be such as to suit the mulberry tree/bush physical nature, either for harvesting of shoots, branches or leaves from a tree or a shrub. Configuration and training of the plant, plant vigour, vegetative nature, pruning and shoot population would need to be studied and catered for in the harvester design stage. The size of harvester is also influenced by the number of silkworms and the stages of growth besides plant properties, plant density and field size. Minimum bruising and disturbance to the plant shoot, branch and root during harvest must be maintained to continue leaf production. Bruising would encourage infestation of diseases and dehydration consequently leading to death of the plant. Disturbance of the root anchorage could also reduce continuity of vigorous growth thus reducing leaf production. The physical properties of the leaves, branches and shoots need to be known such as strength, moisture content, leaf area, shoot diameter and hardiness. In the leaf strip harvesting system, the physical characteristics of the petiole and its association to the leaf and stem must be studied. Force or torque required in stripping the leaf with minimum damage to the shoot and branch as well as root disturbance must be evaluated. Different aged leaves from different mulberry varieties exhibit different physical characters and properties.

87

Time of harvest The time of harvesting is important to maintain quality. The leaf quality is affected by the rate of withering. The time of harvesting influences the length of time the leaf maintains its quality. The fresher the leaf, the better is the feed quality. Due to photosynthesis and respiration activity, metabolism takes place, and the nutritional foods (proteins) are changed to amino acids, and carbohydrates to simple sugar in the leaves. The leaves therefore become poorer in nutritional content over a period of time. It is recommended to harvest early in the morning and the harvest transported quickly to the feeding or storage site. Storage of harvest must be kept in a cool environment at 20oC and relative humidity of over 90 % (Rangaswami et al. 1976, Boraiah 1994) to slow down respiration and transpiration. These should also be covered with a wet cloth and heaping avoided to minimise decomposition. Mulberry plants are pruned in different ways according to the climate, geographic conditions and forms of silkworm rearing. In dense planting, mechanical harvesting is so essential that low pruning at a point near to the ground to prevent stump formation is desirable (Hiroaki et al. 2000). The relationship between pruning method and harvesting amount shows that low pruning gives the highest harvest followed by medium and high pruning (Aruga 1994). Influence of harvesting on physiology Harvesting causes losses of plant organs for photosynthesis, leading to loss of rootlets which leads to reduction of nutrients needed for recovery (Lim et al. 1990). The leaves act as food producers for tree growth. Average growth rate of mulberry shoots is 1 and 3 cm during spring and summer respectively (Lim et al. 1990). During the growing season the roots absorb nutrients and water while during the dormant season they store the nutrients. When the leaves and branches are removed, there would be a decrease in leaf yield the following season due to the nutrient loss. It is advisable to leave some leaves and branches on the mulberry tree to minimise the stress on plants and for continuity of production (Lim et al. 1990).

9.8.1. Harvest methods There are two distinct methods of harvesting mulberry leaves, being either manual or mechanical. The manual method involves picking individual leaves, branches or shoot-lets by hand or assisted with hand tools. The mechanical method involves use of machines to harvest the leaves, branches and shoots. The mechanical harvester is either attached to a tractor powered by the power take off (PTO), or a self propelled walk behind type or driven by an operator on a platform. In most sericulture industry countries, harvesting operation is manually practised using hands and hand tools. With an increase in labour costs, there is a trend to mechanise the harvesting phase. Sixty percent of the cost (Boraiah 1994) of silk production is taken up by mulberry production (harvesting inclusive). Therefore any labour saving exercise applied would reduce costs thereby increasing net earnings. There is an increasing amount of research and trials conducted in the area of mechanising the harvesting operation. The preference for the method of harvesting depends on the availability of labour and the associated costs. 9.8.1.1. Manual harvesting Manual harvesting is commonly practised in most mulberry producing countries. With the aid of hand tools, mulberry leaves, shoots or branches are harvested and taken to the silkworm rearing house. It is a costly method but on the other hand, it is most reliable as well as providing jobs. In developing countries where there is plenty of manual labour available at low wages, it is cheap as well as a solution for some social issues. In traditional silk producing countries (China, India, Japan) the traditional methods of mulberry leaf harvesting are picking leaves, plucking shootlets or cutting branches by hand. Use of small hand

88

tools such as pruning shears, sickles, knives and leaf pluckers or strippers are used to assist in harvesting. The manual harvesting method takes up 30 % (Tooru, 1981) of the total working time for silkworm rearing. In Japan, traditional harvesting was by means of plucking shootlets or cutting shoots for spring rearing and picking leaves for summer and autumn rearing. Manual harvesting is reliable for selective harvesting to cater for different silkworm stages. Selective harvest of either the leaf only, shootlet only, tips only or combination of either is practised. It is also flexible in catering for different rearing season. This allows mulberry plants to be continuously productive in leaf supply throughout the breeding period. A plant would have its leaves harvested at different times depending on the age of the silkworm. Also shootlets are harvested at different position of the plant. This selective harvesting enables the one plant to be continuously productive during the season. Hand tools are used either as leaf pluckers or shoot and branch cutters. Finger and spike frame blades (Lim et al. 1990) are used for leaf picking. Secateurs, pruning sickles, shears, and saws (Tooru 1981, Lim et al. 1990) are used to prune the shoots to a low level for new shoots to emerge when productivity falls or when the shoots reach an unmanageable size and height. i Leaf picking Leaf picking is a labour intensive harvesting method where leaves are selectively picked or the shoots are plucked (Lim et al. 1990, Aruga 1994). The terminal buds are removed after leaf picking, enabling auxiliary buds to develop to lateral shoots. The main advantage of this method is that leaves are selected to suit the growth stage of silkworm, the tender young leaves being fed to young worms and older mature leaves to older worms (Ganga and Chetty 1999). The disadvantage is that it requires more labour and also the leaves wither quickly. In India 6-7 leaf pickings a year is the practice but in Japan and Russia only three pickings are possible. In India the leaf picking starts about 10 weeks after the bottom pruning with other pickings at 7 - 8 week intervals, achieving 6 - 7 harvests in the year. The tree is then pruned to near ground level. Picking the leaf can be either with or without the petiole. Finger blades for cutting the leaf and a spike frame for holding the leaves (Lim et al. 1990) are tools used in picking leaves. ii Branch harvesting Branch cutting involves less labour than leaf picking. The amount of branch harvesting undertaken depends on the rearing season, the physiological condition of plant, labour efficiency and the development of new shoots (Lim et al. 1990, Aruga 1994). The whole branches with leaves are cut and fed to worms after the third moult (Ganga and Chetty, 1999). This is practised in parts of India, Russia and Japan. Branch harvesting interrupts normal growth of the plant. However, by combining branch harvesting and leaf picking (Lim et al. 1990), it is possible to minimise the decrease in yield. In recent times, the shoot harvesting and rearing method is becoming more popular (Aruga 1994). Manual harvested fields have a denser plant population than mechanically harvested fields. The advantages of this type of harvesting are that it saves labour in harvesting, in distribution of feed and bed and space changing, gives maximum use of plant matter, minimum rearing equipment used, better hygienic conditions and better feed quality due to slower respiration (Lim et al. 1990, Aruga 1994). iii Whole shoot harvesting In this method the whole mulberry shoots are cut close to the ground level by bottom pruning and fed to silkworms near the 4th moult stage (Ganga and Chetty 1999). On the other hand, harvesting by topping the shoot enable the remaining leaves to mature uniformly. Shoots are harvested every 10 - 12 weeks, achieving 4 - 6 harvests per year. This is suitable in regions where sprouting happens throughout the year, as in some parts of India and tropical and sub-topical regions. In Japan and Russia, shoot harvests are done mechanically using machines. In recent times, the practice of rearing silkworms on shoots is becoming more popular (Aruga 1994). Sharp knives or sickles (Boraiah

89

1994) are used to cut the shoots or branches to minimise splitting or breaking the bark. Manual labour is reduced to 50 % of leaf picking. 9.8.1.2. Mechanical harvesting In response to recent labour shortages and the rising cost of labour, mechanisation of shoot harvesting has been keenly pursued (Tooru 1981). As in manual harvesting, either the leaf alone or the shoot or branches can be harvested mechanically. A leaf picking machine was developed and tested in Japan (Tawara et al. 1972). In about 1958 a technique of shoot harvesting was developed as a labour saving system. At present in Japan 95 % of all sericulture farmers have adopted this method (Tooru 1981). Another type of harvester that was looked at in 1997 as reported by Hong and others was a carry type mulberry harvester developed in Korea. Tractors of either 2 or 4 wheel drive are used achieving harvesting rates of 30 - 50 minutes per 0.1 hectare (Gopal 1998). A small shoot reaper mounted on a 4 wheel drive tractor was developed in 1963 and this was used as a prototype to do further trials and tests. Different types of shoot reapers have been developed since then. The main types developed are of 4 wheel drive tractor mountable, small hand tractor mountable, and shoot reapers mounted on hand carriers (Tooru 1981). A study conducted by Naio and Kobabayashi (1995) looked at developing a harvesting machine to produce 10 tonnes of cocoon by rearing silkworm 10 times in a year with the farmer only having to harvest once a week in each silkworm rearing period. In this study the mulberry leaves are harvested 2 - 4 times during 2 years in 3 different ways - once a year, twice a year, and 3 times in 2 years. A binder type harvester was developed to harvest shoots in a mulberry plantation of medium density (1.5m x 0.5m) and high density (1m x 0.5m). The study found that 8.5 tonnes of cocoon could be produced by rearing 270 boxes of eggs in a year with leaf harvesting and worm rearing performed 10 times. On average 1.209 tonnes of cocoon were produced per hectare. These results indicated that binder-type harvester can be utilised in multiple worm rearing. Also a model system for leaf harvesting suitable for multiple silkworm rearing by riding a stride type harvester was suggested based on the experiments. i Four wheel drive mountable In 1963 - 1967, several models of shoot reapers mounted on 4 wheel drive tractors of 20 p.s were developed (Okabe 1968). The main components of this reaper type consisted of a cutter lift mechanism, drawing conveyor mechanism, cutting mechanism and binding part. The cutter lift mechanism consists of a lift frame and wire winder with other accessories which is adjustable to cutting heights of 0.4 - 0.5 m above the ground. The drawing conveyor consists of a pair (doubled layered) of cylindrical steel conveyor which carriers the mulberry shoots to the cutting mechanism located at the rear of the conveyor which cuts the shoots with reciprocating knife. Only the upper blade with strokes of 0.1 m cut the shoots. The binding part follows the cutting mechanism where the shoots are bound by hand into bundles. This reaper is attached to the right rear side of the four wheel tractor and powered through the tractor PTO. The reaper runs through a furrow and harvests the shoot on one side of the ridge and cuts the other side of ridge upon returning in the other traffic lane thereby completing the harvest of a ridge. In a field of 2.5 m furrow space (inter ridge space) it worked at a rate of 1 hr per 0.1 ha, harvesting more than 90 % of shoots. Although this harvester was introduced into the farmer's field, it was not readily accepted due to the need for narrow lanes, its cost, and two workers required for the operation, as well as the difficulty in fitting and operation (Tooru 1981). ii Hand tractor mountable To meet the farmers’ needs to operate on narrow spacing and smaller farms, a small reaper (Tooru 1981) mounted on a hand tractor was developed in 1969. Although the main components were similar to the four wheel tractor mountable, the size was smaller and the fitting mechanism much improved. The components consisted of a cutter lift mechanism, drawing conveyor mechanism, cutting mechanism and binding table that was fitted to the right rear of the tractor.

90

The lift mechanism consisted of a basic lift frame, auxiliary wheel, stand mast and wire winder which was used for attaching the reaper to the hand tractor as well as for lowering and lifting the cutter. The drawing conveyor consisted of a divider, a pair of cylindrical steel conveyor star wheels and others which carry mulberry shoots to the cutter and lies the cut shoots on the binding table. The cutting mechanism consisted of a reciprocating knife of 0.4 m with its upper blade moving through an 80 mm stroke. The L-shape binding table was located at the rear of the cutter where the shoots are bound by hand into bundles of 15 kg. Specification of the shoot harvester is given in Table 9.2. Table 9.2 Specification of mulberry shoot reaper mounted on hand tractor (Tooru 1981) Description Data Whole length 2.6 m Whole width 1.5 m Whole weight 180 kg Cutting height range 0.35 - 1.3 m Cutter speed 0.80 m/s Inside conveyor speed 0.47 m/s Outside conveyor speed 0.22 m/s Stalk speed 0.30 m/s Tractor power 4 - 5 ps The harvesting of mulberry was done in the following sequence, 1. the cutting height is adjusted as required between 0.3 - 1.2 m, 2. the reaper runs in the furrow and harvests one side of a ridge, 3. reaper is stopped when the harvest shoots reached 15 kg and the shoots bundled, bound and placed on the furrow, 4. the tractor runs on the other side of ridge harvesting the remainder of the mulberry shoots completing one full cycle, and 5. the bundled shoots are transported to the rearing house at the end of the harvest. The harvesting using this machine was done by one person with a working rate of 2 hour per 0.1 ha which was 4 times faster than manual harvesting with more than 95 % of the shoots harvested. This harvester was popular. However, problems were reported such as damage to the steel conveyor and difficulty of cutting large shoots but these have all been improved (Tooru 1981). Table 9.3. Work efficiency of mulberry shoot reaper mounted on hand tractor (Tooru 1981) Test Season

Length of harvested shoots

Cutting height

Reaping Time

Binding Time per 0.1 ha

Total time

(m) (m) (min) (min) (min) Spring rearing 1.2 - 1.3 0.6 37 77 114 Summer rearing 1.9 - 2 0.8 37 53 90 Late autumn rearing

1.7 - 1.8 1.2 39 56 95

Planting density: inter-ridge space = 2.5 m, inter-plant space = 0.5 m, ridge length = 50 m iii Shoot reaper mounted on small carrier Tooru (1981) reported that in 1974, a new type of reaper was developed which had a rubber finger belt and rubber fingered chain as the drawing conveyor and a rotary knife as the cutter. This was mounted on a small vehicle specially designed for the reaper and was cheaper to operate. The operation and the efficiency was almost similar to the hand tractor mountable, but with less problems in practice.

91

iv Mechanical leaf picker A leaf picking machine (Tawara et al. 1972) was designed and developed and tested for its effectiveness. It was composed of a threshing drum and feeder roller, motors and transmission, and a frame. Tests indicate that as the rotational speed of the drum increased, the unpicked leaf ratio and the damaged leaf ratio decreased and increased respectively. Comparing the V-shaped and plate shaped teeth in picking ability, the former was inferior to the latter. Damaged leaf ratio was high in the case of the plate shaped teeth machine. Under high rotational speed, the unpicked leaf ratio and the damaged leaf ratio were about 10 % and 50 % respectively in the V-shaped tooth and the plate-shaped teeth. Vibration had no effect on the leaf picking except by poorer cutting action.

9.8.2 Other crop harvesters Other crop harvesters for commercial products such as tobacco, tea and some vegetables have been developed. Some of these harvesters are widely used in the commercial production of the respective crops whereas others are going through the final process of being recommended for commercial production. Ideas can be adopted from these harvesters to develop a better mulberry harvester. The plants harvested have similar characteristics to mulberry, so the same principles and features can be used to develop a mulberry harvester.

9.9. Conclusions Mulberry (Moraceae morus) is a deciduous perennial plant, which grows throughout the temperate, subtropical, and tropical regions, growing to a height of 25 m and covering an 8m diameter. The plant is able to grow in poor soils, in arid regions, from sea level to as high as 4000 m above sea level, adapting to varying soil and climatic conditions. Depending on the cultivation, the tree can be grown as a tall tree, middling tree or a bush. The plant provides the nutrients for the silk producing organism, the silkworm (Bombyx mori). The leaf yield harvest is dependent very much on the soil, weather condition, and management practices. From a hectare of fertile land, yields of 15 - 40 tonnes of leaves can be harvested. In tropical regions the leaves are available all year round whereas in temperate regions only 2 - 3 harvests are obtained. With the objective of increasing the production and reducing the cost, work into intensive production operations such as fertiliser application, pesticide and weedicide application, irrigation system, breeding, training, pruning, and harvesting techniques have been carried out. Mechanisation of the cultural operations has been carried out to reduce the cost of production. Over 30 nations produce silk. China is the largest producer followed by India and Japan respectively. The annual world raw silk production was about 84,000 t in 1994 which is 0.16 % of world fibre production. Australia imports about $40.2 million worth of silk products annually. With its diversity in climate and soil, there is an opportunity for Australia to enter into silk production. Superior mulberry varieties have been developed through breeding processes, which are efficient users of nutrients and water with high yields of quality leaves and with traits of environment adaptability and pest and disease resistance. The desirable traits are spread among many species, varieties and repeated hybridisation is undertaken to evolve a variety featuring most of the desired traits. The quality of the leaf fed to the silkworm is dependent upon the complete agronomic package of variety selection, planting techniques, nutrient management, water application, pest and disease control measures and harvesting techniques. Irrigation, in combination with fertiliser, has increased the production by 8 times in dry farming systems as well as increasing the quality. Ideal conditions for optimum growth are at mean temperatures of 23 - 27oC, rainfall of 600 - 2500 mm/year, humidity of 65 - 80 % and at 700 m above sea level. Well drained soil of loamy, clayey loam or

92

sandy loam at pH of 6.2 - 6.8 is best. On average, 40 - 60 kg of raw silk can be produced by use of a quality variety of mulberry with good cultural practices and good quality silkworm races. Nitrogen is the most important nutrient for increasing the yield. Organic manure and mulch are added to supplement the soil nutritional requirements as well as improving the soil conditions. Water is applied using different irrigation methods. Recently water has been piped to the plants and applied in the form of sprayers, sprinklers and drippers. Where water is scarce, efficient use of water is important. Water use efficiency and cost savings are two major benefits in fields where drip irrigation systems have been utilised recently. The mulberry plant is adaptable to poor soil where other plants perform poorly. The leaf yield is high in dense populated plots but this increase is at the sacrifice of quality. The spacing is dependent upon harvesting, pruning methods, plant variety and other agro-climatic elements. Although the leaf yield is found to be highest between 0.5 - 1.0 x 0.5 - 1.5 m spacing, the plant spacing is also dependent on cultivation techniques, the level of mechanisation as well as the individual farmer’s preference. The harvesting of mulberry leaf to feed silkworms is commonly performed by hand either stripping the leaves alone, branch cutting or cutting part of or all the shoot. With the rise in labour cost and shortage in labour, alternative leaf harvesting methods are been considered such as mechanical harvesting. Mechanical harvesters have been developed as tractor mounted or self propelled walk behind types. Sixty percent of the cocoon cost is associated with mulberry leaf production. Any effort involved in reducing this cost such as mechanisation of mulberry production operations (including harvesting) would result in a rise in the net earnings of the farmer.

References Australian Bureau of Statistics (ABS). (2002). International accounts and trade. Merchandise exports and imports by commodity. Baksh, S., Mir, M.R., Darzi, G.M. and Khan, M.A., 2000. Performance of hardwood stem cuttings of mulberry genotypes under temperate climatic conditions of Kashmir, India. Ind. J. of Seri. 39(1), 30-32. Barathi, P., Sundar, D. and Reddy, A.R., 2001. Changes in mulberry leaf metabolism in response to water stress. Biologia Plantarum. 44(1), 83-87 Benchamin, K.V. and Begum, A.N., 1990. Reproductive rate of silkworm under irrigated and non-irrigated conditions of mulberry cultivation. Ind. J. of Eco. 17(2) 140-143 Bindroo, B.B., Anil, D., Koul,S., Trag, A.R. and Dhar, A. 2000. Studies on dormancy and sprouting behaviour of mulberry (Morus sp) under sub-tropical agroclimate. Ind. J. of For. 23(4), 411-414. Bongale, U.D. and Krishna, M. 2000. Leaf quality of mulberry (Morus indica L.) and cocoon crops of the silkworm (Bombyx mori L.) as influenced by sewage and borewell water irrigation. Ind.J. of Seri. 39(2), 165-168 Bongale, U.D., Gowda, S.N. and Narayana, V. M., 2000. Effect of different plant densities and nitrogen levels on Viswa (DD), S-36 and M-5 mulberry (Morus indica L.)

varieties under irrigated condition. Ind. J. of Seri., 39(2), 103-107. Boraiah, G., 1994. Lectures on Sericulture. SBS Publishers Distributors. Bangalore, India. Chakravorty, S.P., Biswas, B.R., Vijayan, K., Roy, B.N., and Saratchandra, B. 2000. Evaluation of mulberry varieties for coastal saline soils of West Bengal. Bul. of Ind. Aca. of Seri. 4(2), 41-45. Choudhury, P.C., Shukla, P., Ghosh, A., Mallikarjuna, B. and Sengupta, K., 1991. Effect of spacing, crown height and method of pruning on mulberry leaf yield, quality and cocoon yield. Ind. J. of Seri. 30(1), 46-53 Das, C., Das, N.K., Chattopadhyay, S, Sengupta, T. and Sen, S.K., 2000. Effect of water logging on physiobiochemical attributes of mulberry (Morus alba L). Ind. J. of Pl. Physio. 5(1), 79-81 Dingle, .J.G.., 2000. Potential for Silkworm Production in Australia. RIRDC, Canberra. Ganga, G., and Chetty, J.S., 1999. An Introduction to Sericulture. Oxford & IBH Publishing., New Delhi. Ghosh, A., Ambika, P.K. and Mishra, R.K., 1997. Effect of varieties, spacings and fertilizer doses on growth, yield and quality of mulberry. Ind. J. of Seri., 36(2, 138-141.

93

Gopal, A. 1998. Illustrated Textbook on Sericulture. Science Publishers Inc. USA Hisao, A., 1994. Principles of Sericulture. A.A. Balkema, Rotterdam. Jia, H.L. and Qian, G.S., 1992. An investigation of soil and water conservation in a mulberry orchard on sloping land in north Shaanxi. Shaanxi J. of Ag. Sc. 1(35), 42 Krishnaswami, S., Kumararaj, K., Vijayaraghavan, and Kasiviswanathan, K., 1971. Silkworm feeding trials for evaluating the quality of mulberry leaves as influenced by variety, spacing and nitrogen fertilisation. Ind. J. of Seri. 10(1), 79-89. Kumari, B.D.R., 1995a. Physiological responses of Morus alba L. to water stress. J. of Phyto. Res. 8(1), 43-47 Kumari, B.D.R., 1995b. Changes in nucleic acid levels in mulberry under moisture stress. Crop Improvement. 22(2), 142-145

Kumari, B.D.R., and Veeranjaneyulu, K., 1996. Changes in leaf water potential, osmotic adjustment, and proline metabolism in mulberry during water stress. Israel J. of Pl. Sc. 44(2-3), 135-141 Lim, Soo-Ho., Kim, Young-Taek., Lee, Sang-Poong., Rhee, In-Jun., Lim, Jung-Sung., and Lim, Bhung-Ho., 1990. Sericulture Training Manual. FAO Agricultural Services Bulletin 80, Rome. Lin, Jinn-Tsair., Yu, Shin-Jin. and Hsieh, F-K.., 1994. Effects of plant spacing on the yield and chemical composition of mulberry leaves. 10(167), 43-49 Machii, H., Koyama, A. and Yamanouchi, H. 2000. Mulberry breeding, cultivation and utilisation in Japan. FAO Elect. Conf. Mulberry for Animal Production. Ming, F., Zhang, F.Y., Wang, Z.Y., and Cao, H.F. 1994. A study on the selection of introduced good varieties of morus alba L. Ningxia J. of Ag. and For. Sc. and Tech. 5, 19-23. Mira, M., Satyawati, S., Madan, M. and Sharma., 1999. Biomass yield of hybrid varieties of mulberry in a non-moriculture area. Biomass and Bioenergy. 17(5), 427-433. Muralidhara, H.R., Gundurao, D.S., Sarpeshkar, A.M. and Ramaiah, R.. 1994. Is drip irrigation viable for mulberry cultivation - an economic analysis. Mysore J. of Ag. Sc. 28(3), 256-260. Naoi, T. and Kobayashi, T., 1995. Development of mulberry harvesting system suitable for frequent silkworm rearing. Bul. of Nat. Inst. of Seri. and Ento. Sc., 15:7-21. Narasaih, P.V., 1992. Sericulture in India. Ashish Publishing House. New Delhi, India. Nath, B.S., Raju, C.S., Ramanjulu, S., Chowdhury, C.C. and Prakash, N.B.V., 1997. Influence of sewage water

irrigation on mulberry leaf quality and its impact on the silkworm, Bombyx mori L. Ind. J. of Seri. 36(1), 57-59 Paul, D.C., Rao, G.S. and Deb, D.C., 1992. Impact of dietary moisture on nutritional indices and growth of Bombyx mori and concomitant larval duration. J. of Ins. Physio. 38(3), 229-235 Phukan, J.D., Handique, P.K., Hazarika, U., Chakravorty, R., Sikdar, A.K. and Mahanta, J.C. 2000. Growth and leaf yield of a few improved mulberry strains as influenced by soil and agroclimatic conditions in N. E. India. Ind. J. of Seri. 39(1), 84-85. Polak, P., Nanes, B. and Adhikari, D., 1997. A low cost drip irrigation system for small farmers in developing countries. J. of the American Water Resources Ass. 33(1), 119-124 Rajan, M.V., Dandin, S.V., Magadum, S.V. and Datta, R.K., 2000. Nutritional evaluation of mulberry (Morus spp) genotypes through silkworm growth studies. Uttar Pradesh J. of Zoo. 20(1), 47-53 Rama, K., Naoi, T. and Kant, R., 1998. Studies on the soil moisture extraction pattern (SMEP) of spring and summer pruned mulberry (Morus spp.) gardens. Ind. J. of Seri. 37(2), 167-170 Ramanjulu, S., Sreenivasalu, N., Kumar, S.G. and Sudhakar, C., 1998a. Photosynthetic characteristics in mulberry during water stress and rewatering. Photosynthetica. 35(2), 259-263 Ramanjulu, S., Sreenivasulu, N. and Sudhakar, C., 1998b. Effect of water stress on photosynthesis in two mulberry genotypes with different drought tolerance. Photosynthetica. 35(2), 279-283 Rangaswami, G., Narasimhanna, S.M.N., Kaviviswanathan, S.K., Sastry, S.C.R. and Manjeet, S.J. 1976. Mulberry Cultivation. FAO. Ag. Ser. Bul. Rao, A.P., Mallikarjunappa, R.S. and Dandin, S.B., 2000. Studies on effect of different plant spacings on leaf yield and quality of M5 mulberry genotype under semi-arid conditions of Karnataka. Karnataka J. of Ag. Sc. 13(4), 882-886 Reddy, H.T., Prabhuraj, D.K., Bangale, U.D., Mahadevappa, L., Lingaiah.and Sanaulla, H. 2000. Fertility status of mulberry growing soils of Siddlaghatta Taluk of Kolar District, Karnataka. Bul. of Ind. Aca. of Seri. 4(2), 46-49. Reddy, P.S. and Sreenivasulu, R.P., 1993. Differential response of two mulberry (Morus alba) cultivars to water stress. Nat. Aca. Sc. Letters. 16(3), 97-100 Reddy, P.S. and Sudhakar, C., 1992. Modulation in levels of inorganic ions and dry matters in local and K2 mulberry varieties under low moisture regimes. Ind. J. of Pl. Physio. 35(4), 341-344 Shankar, M.A., Devaiah, M.C., Peter, A. and Rangaswami, B.T., 2000. Effects of

94

graded levels of organic manure on growth, yield and quality of mulberry in relation to silkworm growth and cocoon production. Crop Research Hisar. India. 19(1), 128-132 Shankar, M.A. and Rangaswami, B.T., 1999. Effects of applied nitrogen and potassium on mulberry leaf yield and quality in relation to silkworm cocoon. Better Crops International. 13(2), 20-21 Shankar, M.A., Rajegowda, Jayaramaiah, M., Rangaswami, B.T., and Maibaum,. 2001. Response of mulberry to nitrogen and sulphate of potash on yield and quality of leaf, in relation to sustainable cocoon production and grainage parameters. Plant Nutrition Food Security and Sustainability of Agro-eco-systems Through Basic and Applied Research. Netherlands. 326-327 Shankar, M.A. and Sriharsha, S.A., 1999. Potassium for yield and quality of mulberry leaf in relation to silkworm cocoon production. Better Crops International. 13(2), 18-19 Shree, M.P., Venkatesh, C.M. and Subbarayappa, C.T., 2000. Nutritional status of mulberry leaves as influenced by sewage water irrigation. Bul. of Ind. Aca. of Seri. 4(1), 61-66 Singhal, B.K. and Roopa., 1998. Changes in of promising mulberry (Morus indica L.) under water stress condition. Philip. J. of Sc. 127(4), 277-286 Subbarayappa, C.T., Kenchannagowda, S.K. and Reddy, M.M., 1994. Effect of nitrogen sources on the nutrient concentration of mulberry leaf. Madras Ag. J. 81(10), 566-568 Subbaswamy, M.R, Singhvi, N.R., Naidu, B.V., Suryanarayana, N., and Datta, R.K., 1999. Relative efficiency of different nitrogenous fertilisers on mulberry leaf yield and quality. Ind. J. of Seri. 38(1), 62-63

Sudo, M. and Kamaruddin, A.M., 1971. Fundamental studies on the leaf picking machine for mulberry. 9 Differences of detachment forces by means of pulling, shear, cutting and impact. J. of Seri. Sc. of Jap. 40(2), 111-119 Sudo, M., Ai, F. and Tawara, T., 1969. Fundamental studies on the leaf picking machine for mulberry. 5 Impact energy of a leaf detachment. J. of Seri. Sc. of Jap. 38(3), 199-204 Sudo, M., Tawara, T., Kamaruddin, A.M., 1970. Fundamental studies on the leaf picking machine for mulberry. 7 Impact energy of a shoot detachment. J. of Seri. Sc. of Jap. 39(2), 109-114 Tawara, T., Ai, F., Sudo, M., Watanabe, K. and Ito, K., 1972. Development studies on the leaf picking machine of cylinder type for Moru trees. 1 Design and trial manufacture. J. of Seri. Sc. of Jap. 41(6), 452-460 Tooru, Okabe., 1981. Development of Mulberry Shoot Reapers. J. of Jap. Ag. Res. Quarter. 14(4), 235-238 Yamamoto, A., Yamamoto, T., Mabuchi, K., Nishida, J. and Adachi, S. 1980. On the effects of irrigation on the leaf quality of the mulberry tree. Bul, Fac. Textile Sc. Kyoto. 9(2), 174-182. Yamanouchi, H., Koyama, A., Naganuma, K., Tsunoda, F., Oyama, M., Shimada, T., Tsukada, K., Nakamura, K., Umezawa, M., Hashimoto, K., Hijikata, K., Nozaki, M. and Machii, H., 2001. Agronomic Characteristics of new Mulberry Cultivars: Comparison of new Cultivars Norin No 10 to 16 at 15 Locations. Bul. of the Nat. Inst. of Seri. and Entom. Sc. 23:1-104. Zito, S., Koitsu, K., Hisashi, H., Nagao, M., Kenji, M. and Kiyoshi, K., 1983. Morphological and biological differences among four mulberry species from Java. J. of Seri. Sc. 52(3), 198-202.

95

10. Evaluation of local Australian mulberry varieties 10.1 Introduction Sericulture is practised widely in Asian countries, with China and India the two largest producers of raw silk. The annual world production of raw silk in 2000 was reported to be about 110,316 tonnes (Anon. 1999). The demand for high quality silk is increasing while the production is decreasing. Recently, in many developed countries there has been a renewed interest in silk production due to its unique natural properties, cultural significance and its use in the fashion business (Aruga 1994; Ganga and Chetty 1999) and thus opportunities exist for Australia to enter the world silk trade (Dingle 2000). Australian environmental conditions are very suitable for sericulture development including silkworm rearing and mulberry cultivation for silkworm food. The most important benefit of sericulture is that it can be practiced on small to medium sized land holding in rural areas, either as a subsidiary or main occupation. The mulberry belongs to the genus Morus of the family Moraceae. There are 24 species of Morus and one subspecies with at least 100 known varieties (Machii et al. 2000). Mulberry trees can grow in a wide range of climatic, topographical and soil conditions. These are widely spread throughout all regions from the tropics to the sub-arctic and from sea level to altitudes as high as 4000 m (FAO 1980). Mulberry performance is dictated by the agroclimatic conditions, the soil type and conditions under which the plant is cultivated (Phukan et al. 2000: Chakravorty et al. 2000). Characterisation and evaluation of mulberry is important for silkworm production (Thangavelu et al. 1997). No data is available in Australia on the value of domestic mulberry cultivars for silk production even though mulberry trees have been grown for over a hundred years. The main objective of this study was to evaluate a range of local mulberry varieties for their potential for silkworm production.

10.2 Materials and methods The study commenced in August 2001 at the University of Queensland, Gatton Campus in the Lockyer Valley, Queensland. The field soil is a heavy, dark cracking clay with slope of less than 1%. It receives an average rainfall of 763 mm which is summer dominant whilst evaporation rate is high, about double the average rainfall. Summer months are warm to hot with maximum temperatures of 20-33oC although heat waves can reach 41oC. Minimum winter temperatures of 6-10oC are reached and frosts are possible from May to September. Mulberry planting material was located and sourced from various locations in South East Queensland. Ten varieties were identified and selection was based on physical characters such as leaf, stem, vigour and health. Planting material was taken from branches ranging from 5 to 15 mm in diameter. The cuttings from these 10 varieties were placed in five litre poly bags filled with potting media of pine bark, saw dust and sand in the ratio of 2.5:0.75:3 together with osmocote fertiliser and propagated in a glasshouse for 18 weeks. All varieties survived except the shahtoot and the weeping mulberry. The remaining 8 varieties (LV1 to LV8) were transplanted into the field on raised plastic mulch beds at a row by tree spacing of 4 m × 2 m. Varieties LV1, 2, 3, 4, 6, 7 and 8 belong to Morus nigra and LV5 to Morus alba. Water was applied through a drip system. A completely randomised design with eight varieties and three replications was used. Plots consisted of ten plants. Agronomic characters, plant height, growth rate, dry leaf mass, stem diameter, number of branches and internodal length were determined. Diameter was measured 400 mm from the ground. Leaf samples were taken for the 3rd and 4th fully developed leaves from the top of the plant. Internodal length was the overall height divided by the number of internodes. Two samples per plot were taken for all parameters except leaf mass where two leaves were sampled from each of the ten plants in a plot. The fresh leaves were oven dried at 60oC for 72 hours to determine the average mass of a dry leaf (Phukan et al. 2000). Data were subjected to analysis of variance. Means were compared by Tukey’s Simultaneous Test at the 5% level of probability.

96

10.3 Results Means of agronomic characters of mulberry varieties at twenty weeks after transplanting are shown in Table 10.1. LV5 and LV6 were significantly taller (3.02 and 3.10 m respectively) than other varieties. LV4 and LV8 were the shortest (2.05 and 2.14 m respectively). There were significant differences in height of about 1 m between the two extreme varieties. Stem diameter for LV5, LV6 and LV7 varieties was largest (28 mm) and LV4 was smallest (16 mm). Other varieties ranged between 22 and 25 mm in diameter. Leaf mass was highest for LV2 (2.82 g) and LV5 (2.75 g) and LV8 was lowest (2.22 g). Generally, varieties with the highest growth tended to have highest leaf mass. Branch number was highest for LV7 (9.4) and LV8 (9.3). LV6 had 1.8 branches which was the lowest of all varieties. The rest of the varieties had between 2 to 5 branches. LV3 and LV5 had longer inter-nodal lengths of 46.2 and 47.1 mm respectively. LV1 and LV9 recorded lower lengths of 37.2 and 38.3 mm. Internodal length for other varieties was intermediate. Plant height up to 8 weeks after transplanting showed little variation among varieties (Figure 10.1). Thereafter, differences in plant height became significant and increased with time. Among all the varieties, the growth rates of LV5 and LV6 were highest at 15.3 and 14.5 mm/day, respectively (Figure 10.2). LV4 and LV8 had the lowest growth rates of 8.5 and 8.1 mm/day, respectively. Other varieties were intermediate in growth rate. Table 10.1 Agronomic characters of mulberry varieties at twenty weeks after transplanting Variety Plant Height

(m) Stem Diameter (mm)

Inter-nodal Length (mm)

Avg No. of Branches

Leaf Mass (g)

LV1 2.60 abc* 25 ab 37.2 c 4.7 abc 2.45 bc LV2 2.72 abc 25 ab 42.1 b 2.3 bc 2.82 a LV3 2.80 abc 22 ab 46.2 a 2.8 bc 2.74 ab LV4 2.05 c 16 b 41.2 bc 4.3 abc 2.55 abc LV5 3.02 a 28 a 47.1 a 4.8 abc 2.75 ab LV6 3.10 a 28 a 40.7 bc 1.8 c 2.75 ab LV7 2.76 abc 28 a 44.7 ab 9.4 a 2.62 abc LV8 2.14 bc 23 ab 38.3 bc 9.3 a 2.22 c *Means followed by the same letter are not significantly different according to Tukey’s Simultaneous Tests at 5% probability.

97

Figure 10.1 Mulberry growth after transplanting for the two tallest and two shortest varieties

Figure 10.2 Average growth rate of mulberry varieties during the first twenty weeks after transplanting

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 5 10 15 20 25

Time after transplanting (weeks)

Plan

t hei

ght (

m)

LV4LV5LV6LV8

02468

1012141618

LV1 LV2 LV3 LV4 LV5 LV6 LV7 LV8

Variety

Gro

wth

rate

(mm

/day

)

98

10.4. Conclusion These investigations showed LV5 and LV6 to have faster growth, higher leaf mass, larger stem diameters, longer internodal lengths and fewer branches. LV2 also showed similar trends to LV5 and LV6. LV4 and LV8 showed slower growth rates with low levels of leaf mass, smaller stem diameters, more branches and shorter internodal lengths followed closely by LV1. Significant variation among varieties is apparent even at this early stage of growth. It appears that these characters would be useful for selection of mulberry cultivars for sericulture. The characterisation and evaluation process would identify mulberry cultivars suitable for Australian agro-climatic conditions and as feed for silkworms.

References Anon. 1999. FAO Production Yearbook, Vol. 53:241. Aruga, H. 1994. Principles of Sericulture. A.A. Balkema, Rotterdam. Chakravorty, S.P., Biswas, B.R., Vijayan, K., Roy, B.N., and Saratchandra, B. 2000.Evaluation of mulberry varieties for coastal saline soils of West Bengal. Bulletin of Indian Academy of Sericulture. 4(2):41-45. Dingle, J.G. 2000. RIRDC Publication 00/56:6.Australia. FAO 1980. China Sericulture. FAO Agriculture Service Bulletin 42. Ganga,G., Chetty,J,S. 1999. An Introduction to Sericulture, Oxford and IBH, New Delhi.

Machii, H., Koyama, A., Yamanouchi, H. 2000. FAO Electronic Conference: Mulberry for Animal Production. Available on http://www.fao.org/livestock/agap/frg/mulberry accessed on 28/06/02. Phukan, J.D., Handique, P.K., Hazarika, U., Chakravorty, R., Sikdar, A.K. and Mahanta, J.C. 2000. Growth and leaf yield of a few improved mulberry strains as influenced by soil and agroclimatic conditions in N. E. India. Indian Journal of Sericulture. 39(1):84-85. Thangavelu, K., Mukherjee, P., Tikader, A., Ravindran, S., Goel, A.K., Rao, A.A., Naik, G.V. and Sekar, S. 1997. Catalogue on Mulberry (Morus spp) Germplasm. Vol. 1, Central Silk Board, Hosur, India.

99

11. Development of a mulberry shoot harvester 11.1 Introduction In traditional silk producing countries (China and India), the common method of mulberry leaf harvesting is picking leaves, plucking shootlets or cutting branches by hand with the use of small hand tools such as pruning shears, sickles, knives and leaf pluckers or strippers. The manual harvesting method takes up to 30% of the total working time for silkworm rearing (Tooru 1981). Either leaves are selectively picked or the whole shoot is harvested. Leaf picking is labour intensive because leaves are selectively picked or the shoots are plucked (Lim et al. 1990; Aruga 1994). Branch cutting involves less labour than leaf picking and depends on the rearing season, physiological condition of plant, labour efficiency and development of new shoot (Lim et al. 1990; Aruga 1994). In recent times, shoot harvesting for rearing is becoming more popular (Aruga 1994). New harvesting techniques have been developed in order to mechanise the field operation. Mechanisation of the mulberry harvesting operation reduces the harvesting time to 25% of manual harvesting (Naoi and Kobayashi 1995). The mechanical method involves use of a machine to harvest the leaves, the branches or the whole shoots. The machine is either attached to a tractor powered by the PTO, a self propelled machine on wheels or tracks walk behind type or driven by an operator on a platform. The mechanical leaf harvester can be categorised in two types, a leaf stripper or a shoot pruner/cutter. The stripper type strips only the leaf leaving the naked branch in the field to continue production of leaves. The pruner type would prune or cut the whole branch/shoot above the ground. In response to recent labour shortage, mechanisation of shoot harvesting has been keenly desired (Tooru 1981). At present 95% of all sericulture farmers in Japan adopt this method (Tooru 1981). A small shoot reaper mounted on a 4 wheel drive tractor was developed in 1963 and this was used as a prototype to do further trials and tests. Different types of shoot reapers have been developed since then. The main types developed are 4 wheel drive tractor mountables, small hand tractor mountables, and shoot reapers mounted on hand carriers (Tooru 1981).

11.2. The harvester components A mechanical mulberry leaf harvester system was developed with the aim of reducing labour costs, being robust to be attached on any average standard tractor and being able to harvest large mulberry fields in a short time. It was thought that it would be operated off the tractor hydraulics taking advantage of the hydraulic power system. The harvester developed was a branch/shoot harvester rather than a leaf stripper. A leaf stripper would be more expensive to develop due to the staggered nature of leaf on shoots as well as the deflective nature of the shoots and the growth character (bunching). The harvester developed had the feature of a forklift where the harvester could be adjusted to any desired cutting height. The aim was to produce a maximum plant length of cut of 500 mm which would be fed to a batch of silkworm of same age. This was to minimise double handling (cutting to length) to feed different age groups if it were to be harvested in long lengths. The harvester is composed of 7 components, 1. cutter knife, 2. conveyor, 3. reel, 4. hydraulic system, 5. lifting mast, 6. collection bin, and 7. the structures to support the system. The whole harvester is attached to the 3 point linkage of a standard tractor of 30-40 HP both for transportation and field operational mode. The knife, conveyor, reel and hydraulics are inter-connected on one support that is pivoted from one point at the mast. When in transport mode, the cutting system would swing in line with the tractor ensuring no part protrudes beyond the tractor. In the field, the cutter system would be swung out at 90o to the direction of travel straddling the row of mulberry plants. Height adjustments are made to the desired cutting height using the lifting mast. It is designed to cut the mulberry shoots to a maximum length of 500 mm starting at a height of 1500 mm above the ground

100

and decreasing to as low as 600 mm above the ground. A collection bin of 0.5 tonne is located on the fork lift bars with a clearance of about 200 mm from the ground. Figure 11.1. Schematic layout of mulberry leaf harvester.

11.2.1 Cutter knife The reciprocating knife has a stroke of 70 mm and an effective length of 1500 mm. The knife blades are 70 mm x 70 mm triangular in shape and pin fixed to a holding plate. These blades slide between fingers that are connected to a plate. Brackets hold the finger arrangement firmly onto a backing plate. One end of the knife is connected to a hydraulic motor through an arm/flywheel configuration with an offset of 30 mm radius to the axis of the motor shaft. The rotational motion of the motor is converted into linear motion using the flywheel/arm arrangement with an eventual reciprocating cutting action of the cutter knives.

11.2.2 Conveyor The conveyor is a chain type, 500 mm wide and 1500 mm long with a 45o elevated length of 600 mm. Horizontal bars each with 2 x 100 mm fingers spaced at 400 mm apart are connected to chain links every 5 chain link equally spaced apart. The conveyor is opened toward the knife end with side plates at the opposite end as well as to the opposite of the discharge end. The conveyor is powered by a hydraulic motor coupled directly onto the drive shaft via a 200 mm diameter sprocket. The top of the conveyor is 50 mm above the knife.

11.2.3 Reel The reel is 1200 mm in diameter and 1600 mm in length. Six radial arms each with a 100 mm galvanised rake plate are bolted at 400 mm radius. On the plate are bolted double finger tines which are 200 mm in length. The tines are staggered located on alternate rake plates. In total there are 33 tines which are equally spaced. The reel is located above and forward of the knife with a clearance

Cutter knife

Tractor forward travel

Collection Bin

Reel Motor

Mat

Conveyor

Conveyor motor

Reel

Control valve

101

of 20 mm from the top of the knife. Also a clearance of 20 mm is allowed from the top of conveyor finger. The whole reel is located on bearings on both ends with one end directly connected to the drive motor.

11.2.4 Hydraulic system The hydraulic system consists of 3 hydraulic motors, a load-independent proportional valve, pilot valve, hydraulic ram, hoses, and fittings. The power required to operate the knife, conveyor, and reel is derived from hydraulic motors connected to the tractor hydraulics via the control valve. The mast (forklift) is also connected to the tractor hydraulics via the control valve without motor. The speed at which the knife, conveyor and reel operate is regulated using levers on the proportional valve in controlling the hydraulic fluid flow to the respective motors.

11.2.5 Lifting mast A fork lift mast of 1 tonne capacity is incorporated into the harvesting system. A one meter hydraulic ram, pivot connected at both ends actuates the top connection to enable it to slide up and down in channel slides using a chain and sprocket drive. The ram is actuated by a lever on the proportional valve which is connected to the tractor hydraulics. The height adjustment is adjusted using this lifting device. The whole harvester system is supported by the 3 point linkage both in transport and harvest mode.

11.2.6 Collection bin Two forklift legs 1000 mm apart and 1200 mm long support a collection bin of 500 kg. The legs are stationary fixed to the lower end of the fork lift frame.

11.2.7 Structural frame/support There are two frame supports, one supporting the knife, reel and conveyor, the other supporting the whole harvester system together with the forklift mast. The knife and the conveyor part are attached to the forklift mast through a pin joint which is loosely pivoted to allow manoeuvrability into either the transport or operation mode. A support for the proportional valve is attached to the forklift mast frame such that the operator would easily access the controls.

11.2.8 Transportation mode Transportation of the harvester on the road is through the use of tractor with the harvester attached to its 3 point linkage. The overall length of the harvester is 2.5 m and its gross mass is about one tonne. A support with a locating pin located on the forklift leg is placed under the harvester. This minimises the bounce effect. A bar lever with locking pins at both ends stops the cutter/conveyor combination from moving laterally whilst in transport

11.2.9 Harvesting mode In harvesting mode, the knife and conveyor are swung at 90 to the tractor’s line of travel and locked into place using a pin. These straddle the mulberry plant row. As the tractor travels forward with the knife and conveyor operating, the mulberry plant shoots lodge into the fingers of the knife and feed positively onto the reciprocating knife which cuts the shoots. These would in turn be pushed/thrown onto the conveyor by the reel with the aid of the forward motion of tractor which conveys and discharges into the collection bin. Upon filling of the bin, the cut mulberry plants are then bagged and transported to the feeding house where the silkworms are kept.

102

Figure 11.2 Front (angled) view of harvester

103

11.3 Preliminary tests

11.3.1 Test 1 The original design of the harvester did not include a reel. The first field test was conducted on a 30 m row with mulberry plants at 1 m apart. The test was performed on 14 1/2 week plants after pruning which had reached a height of 2.5 m from the ground. The trial test was conducted using a Case C60 tractor (47 kW) with the harvester attached to the 3 point linkage and powered off the tractors hydraulic system (52 L/min) operating at a high engine idle speed of 2200 rpm with the tractor at its slowest forward speed. The handling of the harvester on the road during transportation experienced no major difficulty or problem. However, during the test it was observed that:

1. very little plant material was feeding onto the knife 2. mulberry shoots were been pushed forward and under the cutter knife 3. there was very little cutting of material 4. of the cut plant, practically no material was transferred onto the conveyor 5. cut material were building up at the knife and conveyor area.

Upon these observations it was decided to include a reel.

11.3.2 Test 2 A second field test was conducted after the addition of the reel. This test was carried out on a single row 60 m long with mulberry plants at 1 m spacing. The mulberry plants were 22 weeks after pruning with the tallest at 3.2 m from the ground. The harvester was adjusted to the cutting height of 1.6 m using the lifting mast, positioning it to cut ½ - ¾ row width from the nearest side of tractor. The tractor speed at which the harvesting test was performed was at 1.6 km/h. The aim was to determine whether harvester was able to perform the required task of:

1. reel in mulberry shoots onto the cutter 2. cleanly cut plants 3. transfer cut material on to the conveyor 4. conveyor to collect and transfer into the collection bin 5. collection of transferred material into bin with minimum wastage.

It was observed that the above five aims were satisfactorily achieved until jamming of a branch into the conveyor and the cover plate rendered it un-operational preventing further test and data collection. The preliminary data collected showed cut material of 4.03 kg over the 12 m with the tractor travelling at 1.6 km/h. Of this 3.3 kg were collected and 0.73 kg wasted which is 82% efficient. Over the two cutting heights, a total of 308 shoots were cut with 252 being collected into the bin and 56 missing the bin which also is an collection efficiency of 82%. It was observed that the harvester did perform satisfactorily until the damage to the conveyor and cover plate. Modification and repair work were done on the conveyor, the conveyor motor, knife finger and the knife drive cover after the damage during the second test above. The damage to the conveyor involved the chain link, conveyor arm and the base plate. A broken knife finger was replaced with a new finger. The motor on the conveyor was changed from a 60 L/min to 40 L/min flow model.

104

11.3.3 Test 3 The machine was tested using a John Deere tractor (JD 1750) with hydraulic flow rating of 47 L/min at 19 MPa maximum pressure. The third field test was carried out again on the single row 60 m long. The harvester was adjusted to the cutting height of 1.6 m using the lifting mast. A tractor speed of 1.6 km/h was used to conduct the test. It was observed that reeling in, cutting, transferring and collection of material into the bin during cutting at 2 different heights were satisfactory. Data was not collected due to 50% of leaves senesced due to cold weather. Further testing will be done during the next season (November 2004).

References Aruga, H., 1994. Principles of Sericulture. A.A.

Balkema, Rotterdam Lim, S., Kim, Y., Lee, S., Rhee, I., Lim, J. and Lim, B., 1990. Sericulture Training Manual. FAO Agricultural Services Bulletin 80, Rome

Naoi, T. and Kobayashi, T., 1995. Development of mulberry harvesting system suitable for frequent silkworm rearing. Bulletin of National Institute of Sericulture and Entomological Science. 15:7-21. Tooru, O., 1981. Development of Mulberry Shoot Reapers, Journal of Japan. Agricultural Research Quarter. 14(4): 235-238

105

12. Implications and recommendations This report described techniques and methods suited to the selection and development of suitable silkworm and mulberry varieties, and the cultivation and harvest methods that enable a sericulture industry to be commenced in Australia. It is recommended that this information be provided to potential silkworm farmers and that the silkworm and mulberry varieties and harvesting equipment developed be made available to this new rural industry. It is also recommended that the breeding and development centre established at the University of Queensland, Gatton, be funded to support the new sericultural industry.