THE HELICOVERPA ARMIGERA NPV

PRODUCTION MANUAL

D Grzywacz, R J Rabindra, M Brown, K A Jones

& M Parnell

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TABLE OF CONTENTS

Page

1 Executive Outline 1

1.1 Using the manual 1

2 Insect Viruses - an Introduction 3

2.1 Baculoviruses 4

2.2 Nucleopolyhedroviruses (NPV) 5

2.3 Granuloviruses (GV) 7

2.4 Non-occluded Baculoviruses 9

2.5 Cytoplasmic Polyhedrosis Viruses 9

2.6 The Life Cycle of Baculoviruses 10

2.7 Introduction to H. armigera NPV 13

2.8 Commercial Products 13

2.9 References 14

3 Insect Culture for Virus Production and Testing 15

3.1 Importance of Clean Culture, Sanitation and Hygiene 15

3.2 Insectary Design 15

3.2.1 Quarantine room 17

3.2.2 Entry preparation room 17

3.2.3 Adult moth room 17

3.2.4 Larval holding room 18

3.2.5 Egg handling room 18

3.2.6 Diet preparation room 18

3.2.7 Washing area 18

3.2.8 Sterilisation room 18

3.3 Equipment 19

3.4 Insect culture in the laboratory 19

3.4.1 Establishment of a culture from the wild 20

3.4.2 Quarantine and colony clean up 20

3.5 Diet 23

3.5.1 Procedure for larval diet 24

3.5.2 Adult diet 24

3.6 Insect Rearing Protocol 25

3.6.1 Egg production 25

3.6.2 Larval rearing 25

3.6.3 Rearing of late stage larvae 26

3.6.4 Selection for vigour 27

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3.6.5 Pupation 27

3.7 Supply of Insects for Virus Production 28

3.8 Problems in Rearing Larvae 28

3.8.1 Mould on diet caused by Aspergillus niger 29

3.8.2 Phorid flies 29

3.9 References 29

4 The Production of Insect Viruses 30

4.1 Introduction 30

4.2 Tissue Culture Production 30

4.3 Production in Whole Insects 30

4.4 Insects 31

4.5 Inoculation 32

4.6 Inoculum Purity 32

4.6.1 Protocol for purifying inocula 33

4.6.1.1 Primary processing 33

4.6.1.2 Gradient centrifugation 34

4.7 Incubation and rearing 38

4.8 Sanitation of production facilities 38

4.9 Harvesting 39

4.10 Model protocol for the production of Helicoverpa armigera NPV

In multicell trays 40

4.10.1 Preparation of production trays 40

4.10.2 Inoculation of production trays and loading larvae 42

4.10.3 Harvesting infected larvae 43

4.10.4 Processing of infected larvae 44

4.11 Processing insect viruses for use as biopesticides 44

4.11.1 Homogenising and filtering 45

4.11.2 Centrifugation 46

4.12 Storage of insect viruses 48

4.12.1 Freeze-drying 48

4.12.2 Spray drying 52

4.13 Summary of Main Points 56

4.14 References 56

5 Quality Control in NPV Production 57

5.1 The Importance of Quality Control 57

5.1.1 The importance of routine protocols 57

5.2 Counting NPV 57

5.3 Larval Equivalents 58

5.4 Quality Control Techniques 58

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5.4.1 Observation 58

5.4.2 Microscopy 59

5.4.3 Bioassay 59

5.4.4 DNA identification 60

6 Microscopic examination of NPV & GV 61

6.1 Microscopes and choice of illumination 61

6.2 Counting baculoviruses and the use of standards 61

6.3 Microscopic staining techniques 62

6.3.1 Sikorowski quick stain for CPV polyhedra (& NPV) 62

6.3.2 Method for differential Giemsa staining of occluded insect

Viruses and other pathogenic micro-organisms in smears of

Insect tissues 63

6.3.3 Giemsa stain with acid hydrolysis for nucleopolyhedrovirus

(granulovirus) and cytoplasmic polyhedrosis virus inclusions 65

6.4 Counting NPV using the improved Neubauer haemocytometer or

Counting chamber 66

6.5 Identifying contaminants under the microscope 70

6.6 References 70

7 Bioassay Techniques and Microbial Pesticides 72

7.1 Introduction 72

7.1.1 Bioassay 72

7.1.2 Lethal dose50 and lethal concentration50 72

7.1.3 Types of assay 74

7.1.4 Summary of main points 74

7.2 Surface Dose Bioassay to Determine LC50 in 1st Instar Larvae 74

7.2.1 Introduction 74

7.2.2 Procedure 76

7.3 Droplet Bioassay 76

7.3.1 Introduction 76

7.3.2 Sample preparation 77

7.3.3 Administration of dose 78

7.3.4 Artificial diet for polypots 81

7.4 Diet Plug Bioassay to Determine LD50 82

7.4.1 Introduction 82

7.4.2 Procedure 83

7.5 Leaf Dip Bioassay 83

7.5.1 Introduction 83

7.5.2 Procedure 84

7.6 Analysis of bioassay data 85

7.6.1 Graphical method for estimation of the median lethal

Concentration (LC50) 85

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7.6.2 Transformation of percentages to probits 86

7.7 References 86

8 Identification of Insect Viruses with Restriction Endonucleases 88

8.1 Introduction 88

8.2 DNA Profiling 88

8.3 DNA Extraction and Identification Procedure 89

8.3.1 Extraction and dissolution of virus 89

8.3.2 Phenol extraction of DNA 90

8.3.3 Dialysis 91

8.3.4 Ethanol extraction as an alternative to dialysis 92

8.3.5 Digestion 92

8.3.6 Electrophoresis 93

8.3.7 Preparation of solutions 95

8.3.8 Additional Notes 96

9 Microbiological examination of viral pesticides 98

9.1 The Problem 98

9.2 Counting Bacteria and Other Contaminants 98

9.3 Equipment 99

9.4 Sampling and Dilution 99

9.5 Total Viable Count 100

9.6 Pathogen Screening 102

9.7 Coliforms, Shigella and Salmonella 102

9.7.1 Motility determination 104

9.7.2 Oxidase test 104

9.7.3 Catalase test 104

9.8 Staphylococcus aureus 104

9.9 Bacillus Species 105

9.10 Yeasts 106

9.11 References 106

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1 EXECUTIVE OUTLINE This manual details the methods and techniques required to produce Helicoverpa armigera nucleopolyhedrovirus (NPV) for controlling H. armigera on crops. This virus is a highly virulent natural pathogen of the insect. When applied to crops in the field in sufficient quantity the virus acts as a natural pesticide infecting and killing H. armigera larvae and protecting the crop. The virus is a pathogen specific to H. armigera and a few closely related lepidoptera and will therefore not harm other insects, animals or man. It is therefore extremely safe and is compatible with all other biological control techniques using insect parasites, predators or pathogens. The virus can be produced both in live insects or tissue culture but the latter is currently not economic for mass production so that only production methods based upon live insects are detailed here. Production is achieved by feeding larvae with a small quantity of NPV, and then rearing them for a period. During this time the larvae grow, the infection develops and the virus multiplies. When the larvae are harvested the virus has increased 10,000-fold over the initial dose. These infected larvae are then processed to release the virus and as such are used as the basis for the biopesticide. The high efficiency of this technique of virus replication means that the equivalent of 250-500 infected larvae can be used successfully to protect one hectare of crop. This production process for viral biopesticides can be carried out using relatively simple techniques and low capital cost equipment. It is, therefore, appropriate for small scale production or for production where technological resources are limited. Using these systems the cost of such biopesticides can be comparable or cheaper than conventional chemical alternatives. In addition, unlike chemical or bacterial insecticides, no significant history of resistance development to viral biopesticides has occurred. However while production is, in principle, simple it does in practice require considerable care and strict adherence to quality control standards to achieve a consistent product. Failure to adhere strictly to production procedures can result in loss of production efficiency and contamination by micro-organisms. Contamination of the insect production facilities by other viruses, bacteria and insect parasites is the most common cause of production failure. Heavy contamination in batches of NPV biopesticide results invariably in reduced pesticidal action and failure to control the pests when used in the field. This manual details the range of procedures needed to maintain and monitor the quality of the virus. 1.1 USING THE MANUAL This manual describes a number of procedures used to produce H. armigera NPV. However this manual does not detail all the possible modifications and adaptations that may be required for specific local production situations and should not be taken as a replacement for good product development. The availability of local ingredients, equipment and resources may determine that some modifications, however minor, will almost always be required in practice. Each modification will need to be validated, specifically, before being implemented. This information in this manual should be used as a guide and to assist local product development not as a replacement for good applied research.

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While we have endeavoured to describe as clearly and comprehensively as possible all the procedures detailed in this manual, many of them do require a level of expertise and skill that is best acquired only through hands-on training and experience with someone already competent in the techniques. Actual training alongside experienced staff in such procedures is always preferable to attempting to replicate procedures from this manual alone. The authors are always willing to offer advice on suitable training opportunities if users have difficulty in locating appropriate training. Many of these procedures can be used in the production of other insect viruses either unchanged or with some modification and it is hoped that the information contained here will stimulate and assist workers developing other viruses as pest control agents. The authors welcome enquiries and feedback on modifications or new procedures for producing other viruses.

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2 INSECT VIRUSES - AN INTRODUCTION Over 600 viruses have been reported as infecting insects, these are drawn from 15 families of viruses. Important characteristics used to differentiate these viruses are:

(a) The nature of the genetic material (RNA or DNA) and its form (single or double stranded).

(b) The size and shape of the virus particles. (c) Whether or not the virus particle is enveloped by a membrane. (d) Whether or not the virus particle is occluded within a proteinaceous inclusion

body. Table 1. Families of insect viruses and their characteristics.

Family Nucleic acid

Particle morphology Shape Size (nm)

Envelope Occl'n. body

Parvoviridae SsDNA Isometric 18-26 - - Iridoviridae DsDNA Icosahedral 125-300 - -

Baculoviridae DsDNA Bacilliform 40-60 x 200-400 + + Poxviridae DsDNA Ovoid 165-300 x150-470 + + Polydnaviridae DsDNA Ovoid 150x350 + - Nucleocapsid 85x330 + - Ascoviridae DsDNA Allantoid/ 130x400 + - Bacilliform Nodaviridae SsRNA Icosahedral 29 diam - - Picornaviridae SsRNA Spherical 22-30 diam - - Tetraviridae SsRNA Icosahedral 35-39 diam - - Reoviridae DsRNA Icosahedral 55-69 diam - + Birnaviridae DsRNA Icosahedral 60 diam - - Rhabdoviridae SsRNA Bullet shaped/ 50-95 x 130-380 + - Bacilliform Togaviridae SsRNA Spherical 60-65 diam + - Flaviviridae SsRNA Spherical 35-45 diam + - Bunyaviridae SsRNA Spherical/Oval 90-100 diam + -

For the purposes of pest control, interest has concentrated on those families of viruses that do not infect mammals, for obvious safety reasons. Of the 15 families infecting insects only three, the baculoviruses, the polydnaviruses and the ascoviruses are not suspected of having mammalian hosts. The baculoviruses have been the most studied as these are both highly infectious by ingestion and show good horizontal transmission (from adult to egg). They also have the advantage of infecting many important species of lepidopteran and other insect pests. The ascoviruses are considered

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poor candidates as they have a low infectivity. The polydnaviruses are also poor candidates as they are known to replicate only in the ovaries of parasitic wasps. 2.1 BACULOVIRUSES Biopesticide development is concerned almost exclusively with members of one family, the Baculoviridae (the Baculoviruses). The two groups of viruses found in this family are the nucleopolyhedroviruses (NPV) and the granuloviruses (GV). The baculoviruses infect >600 species of insect, mainly Lepidoptera, including many important pest species. Insect species in other orders infected by baculoviruses include Hymenoptera (31 species), Diptera (27) and Coleoptera (5). Baculoviruses also infect a few other species of arthropods such as prawns and other crustacea, but do not infect any non-arthropod species. This specificity is one attraction of baculoviruses as biopesticides; they are known to be completely safe to man, animals and important beneficial insects such as bees, predatory insects and parasitoids. The baculoviruses are rod-shaped (baculo = rod) particles containing double stranded DNA enclosed by a lipoprotein envelope. Both the NPV and GV are found occluded within proteinaceous inclusion bodies which provide some environmental protection. Over 20 species of baculovirus have been developed or registered as commercially available insecticides and over 30 different products, based upon NPV or GV, have been registered as commercial insecticides. This text will concentrate mainly on the Helicoverpa (Heliothis) armigera NPV (HaNPV). Another important reason for the interest in NPV as potential insect control agents is that, unusually for viruses, they are relatively easy to see and count using a light microscope. Almost all other viruses are too small to be seen except with an Electron Microscope, and so are much more difficult to identify and isolate. NPV attracted the attention of pest control scientists, interested in looking for an alternative to chemical pesticides, because they can cause highly infectious disease that kills in 5-7 days. These viruses attack some of the most important Lepidopteran crop pests, including species of Heliothis, Helicoverpa and Spodoptera. Some of the related GV species are also highly infectious, including Cydia pomonella (apple codling moth) GV (CpGV) and Plutella xylostella (diamond back moth) GV (PxGV). However not all GV are as fast acting as NPV and some such as Spodoptera littoralis (cotton leafworm) GV (SlGV) produce chronic infections that may only kill after an extended period 20-30 days and thus are much less suitable for biopesticide use. The other important characteristic of baculoviruses is that most kill only target pest species. This can be seen as an advantage or a drawback. This specificity and safety to most other insects means it is completely compatible with other biological control options. The virus has no effect on beneficial insects, predators or parasites, so that its use does not produce any of the secondary pest problems encountered when broad spectrum chemicals are used. Its specificity also means it is safe to humans and can be sprayed onto crops right up to time of harvesting with no residue problems.

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2.2 NUCLEOPOLYHEDROVIRUSES (NPV) These are the viruses most studied and used commercially in pest control. They infect over 400 species of insect and are well known to cause major lethal epizootics in important lepidopteran pest species. They are commonly isolated from insects in the field and being visible with light microscopy can be easily detected if present. They also have, for viruses, a relatively fast mode of action, killing infected insects within 4-7 days. NPV are rod shaped double stranded DNA viruses of the family Baculoviridae which infect a wide range of insect species, chiefly Lepidoptera but also some members of the Hymenoptera and Diptera. These viruses are the cause of a highly infectious lethal disease in the larvae of susceptible species. The NPV are highly specific and most infect only a few species of closely related insects. The viruses are named after the insect in which they were first isolated and identified. The NPV are seen in dead or dying larvae as bright irregular crystals called occlusion bodies (OB) or polyhedral inclusion bodies (PIB) (Plate 1). These are protein crystals normally 1-7µm across that show up at x400 as bright refractive crystals, especially under phase contrast. These crystals are many sided (polyhedra in Latin) and are composed of a protein with a molecular weight of 25-30 kilodaltons called polyhedrin. The whole OB crystal is surrounded by a calyx or membrane of protein.

Within this PIB are found embedded up to 200 virus particles or virions. These virions are the actual virus infective particles and are composed of a rod-shaped DNA/protein structure called a nucleocapsid (approximately 50 x 250-400nm), inside a membrane envelope (Plate 2). The circular double stranded DNA is 88-200 kilobase pairs (kbp) in length and 50-100 megadaltons in weight. The nucleocapsids may be found either enveloped singly (SNPV) by the lipoprotein membrane envelope or, in some species, multiple (MNPV) nucleocapsids are enveloped within each envelope. Between 20 to 200 virions are embedded in the polyhedrin matrix depending upon species, e.g. in HaNPV there are commonly up to 30 virions in each OB.

Plate 1 Helicoverpa armigera NPV. Phase contrast illumination at X1000 magnification. NPV arrowed.

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These OB are the infective stage of the virus, designed to transmit the infection from insect to insect. The polyhedrin crystal helps to protect the vulnerable virions from inactivation by environmental factors. Baculoviruses in this form are extremely stable and can retain infectivity for many years, if not exposed to UV light or high temperatures(>50°C). For more detail on the biology of the Baculoviruses the reader is recommended to refer to one of the standard works such as Tanada & Kaya (1993), Adams & McClintock (1991), Hunter-Fujita et al (1998) or the two volume work by Granados and Federici (1986). The virus infects the insect in the form of a polyhedral inclusion body (PIB). When the polyhedra enters the mid gut of an insect it dissolves under the alkaline conditions (pH 9-11) releasing the virions. These virions enter the cells of the mid gut and proceed to multiply in the nucleus. From this initial infection new virions are produced which proceed to spread the infection to other body tissues such as haematocytes, tracheal cells, fat body cells and hypodermis. It is in these tissues during the later stages of the infection that polyhedra are produced in which virions become embedded. When the insect dies it ruptures releasing these polyhedra to infect other insects. Insects killed by NPV will commonly contain up to 100 million PIB. In the wild, infection occurs through the ingestion of PIB. Insects infected with NPV show few symptoms for the first 2-4 days after ingestion of the virus. The larvae then progressively cease to feed and become less active. During advanced stages of the infection, as the epidermis is infected, the skin becomes very fragile and ruptures easily. The larvae become wilted and the body contents become a fluidised mass of decomposed tissues and polyhedra. Just prior to death infected larvae often climb to the highest parts of the substrate they are located, e.g. tops of plants and attach themselves by their prolegs. On death they hang in a characteristic V-shape (Plate 3). Plate 2 Electron micrograph of NPV. Virions arrowed.

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Plate 3 Classic symptoms of an H.armigera larva killed by NPV infection

2.3 GRANULOVIRUSES (GV) The GV are slightly different to NPV in that there is normally one virion (rarely two) in each protective inclusion crystal. The crystals, called granules or capsules, are much smaller than the polyhedra and are rod-shaped with rounded ends (Plate 4). Each granule measures 0.2-0.5µm x 0.05µm and are just visible under the light microscope at x1000 (Plate 5). The single virion within each granule is surrounded by a crystalline protein matrix very similar to that of the polyhedra of NPV. In GV this protein is called granulin. It is chemically very similar to polyhedrin being a 25-30 kilodalton (Kda) polypeptide. The genome of GV is double stranded DNA of 90-160 kbp.

Plate 4 Plate 5 Electron micrograph of Granulovirus (R. Bateman) Granulovirus. Dark field at X1000

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Over 150 species of insect, mainly lepidoptera, are known to be susceptible to GV. These species are principally the Noctuidae (approx. 50 species) and Tortricidae (20 species). GV usually infect the insects through the midgut epithelia cells, similar to the action of NPV. The capsules dissolve under the alkaline conditions of the midgut releasing the virions which then invade the midgut epithelium cells. The virus replicates in these cells and the virions produced then go on to infect the major target organ, the fat body. Some species of GV may also attack the hypodermal and tracheal tissues. Where the fat body is the major organ invaded, the main symptom of infection is reduced growth rate. In some species growth may continue beyond the normal larval life time, even producing larvae which are eventually larger than uninfected larvae. These larvae do eventually die after becoming progressively weaker and sluggish, and showing mottling or other colour changes (Plate 6). These infections are much slower than with NPV, often taking 20-30 days to kill. In those GV species where tissues other than the fat body are attacked, the symptoms resemble those of NPV with death resulting in 4-7 days. The larvae, once infected, become wilted with the skin being very fragile and tending to rupture in the later stages.

Plate 6 Spodoptera littorals larva infected with GV

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There is one GV, affecting the grape leaf skeletoniser (Harrisinia brillians), in which the infection and virus replication is confined to the midgut tissue alone. These larvae become wilted, and often are shrunken and darken in colour. A diahorreal discharge from the gut, containing many infective GV capsules, is common. Thus, it is only those GV which attack tissues other than the fat body which produce rapid death and are, therefore, of interest as microbial insecticides. Of these, the Cydia pomonella GV has already been commercialised for control of the apple codling moth Cydia pomonella. Other GV of commercial interest include Pieris rapis GV and Plutella xylostella GV. 2.4 NON-OCCLUDED BACULOVIRUSES There was until recently a third group of baculoviruses, the non-occluded baculoviruses (NOB), in which no occlusion bodies had been observed. In these viruses only virions are found. Three species have been studied in any detail, these include NOB affecting the rhinoceros beetle (Oryctes rhinoceros) and Heliothis zea. The O. rhinoceros NOB has been used as an agent for control of O. rhinoceros in Melanesia. However now these viruses have been removed from the baculovirus group and re-designated as a separate family of non-affiliated viruses. 2.5 CYTOPLASMIC POLYHEDROSIS VIRUSES There is one group of viruses that under the light microscope can be confused with NPV, and these are the cytoplasmic polyhedrosis viruses (CPV). These are from a completely different family of viruses, the Reoviridae. These differ from baculoviruses in having RNA as their genetic material and the infectious particles are icosahedral (many sides, all equal in shape) of 55-70nm in diameter rather than rod-shaped, as in the baculoviruses. The genetic material is composed of double stranded RNA found in 9-10 distinct segments of total molecular weight 13-18 Kda and 20-30 kbp in length. However these icosahedral CPV virus particles are also embedded in a large crystal protein protective crystal similar to OB, though they can be larger than the OB of NPV at 3-15µm. The biology and life cycle of CPV are different to that of NPV as these viruses infect only certain tissues, producing chronic infections of the gut and are much slower to kill and therefore less useful as insecticides than baculoviruses (although they may be effective as classical biological control agents). CPV are important as they can infect the same species as NPV, can become contaminants in the production of NPV and may be a problem in insect rearing. Distinguishing the OB of CPV from NPV is not easy under a light microscope (Plate 7) and correct identification of CPV requires special staining techniques, nucleic acid analysis or an electron microscope. As the name suggests these viruses multiply in the cytoplasm of infected cells. While there are some superficial similarities with NPV in that the polyhedra dissolve in the midgut to release the virions and these then infect the midgut epithelial cells, in CPV the infection is confined to the midgut alone and does not spread to other tissues. CPV do not kill the insect quickly but give rise to chronic infections in which insects continually shed infective polyhedra in the faeces.

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As a result of the slow speed of kill there has been little interest in developing them as insect control agents. They are, however, commonly found infecting lepidoptera including the Heliothis species. 2.6 THE LIFE CYCLE OF BACULOVIRUSES An appreciation of the life cycle of baculoviruses like NPV and its mode of replication is essential for an understanding of virus production dynamics. Baculoviruses infect insects when the OB are ingested by a susceptible species along with the food. It must be emphasised that these viruses have no contact effect and cannot infect an insect unless eaten. In the alkaline conditions of the insect midgut the protective polyhedral crystal dissolves releasing the infectious virions into the gut. The infective dose for NPV varies with the age of the larvae, in some species we know that a single polyhedra contains sufficient virions to infect a newly hatched neonate larva. In older, larger larvae the dose needed to produce an infection increases and for later instars up to 10,000 OB may be required. The released virions pass through the peritrophic membrane of the gut and fuse with the microvilli on the columnar epithelial cells. This releases the nucleocapsids, the DNA/protein rods contained in the virions, which enter the cell and migrate to the nucleus, entering through the nuclear pores. This initiates the first replication cycle of the virus that is restricted to the cells in the midgut. Here the nucleocapsid unwinds, the DNA is exposed and virus replication begins. Like all viruses, NPV and GV need to use a living insect cell to replicate. Within as little as one hour post infection (PI), virus replication can begin. During this time the virus must halt the host cells’ normal pattern of activity, enabling it produce virally encoded proteins and replicating the viral DNA. The nucleus of the infected cell becomes swollen and enlarged at 8 hours PI as progeny virus start to be produced. These progeny virus particles are called extra cellular virus (ECV) or budded virus (BV) and consist of naked nucleocapsids. These migrate to the outside of the cell, where they acquire an envelope and protein structures, called peplomers, of a particular 64K protein at one end

Plate 7 CPV at X1000 magnification

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of the virion before leaving the cell. Some production of polyhedra may also occur in the first 48 hours PI but these OB are usually small and defective containing no virions. At this point the first cycle of viral replication is complete and the second phase of infection in the other body tissues follows. Just as the OB is the form of the virus designed to carry the infection from insect to insect, the ECV or BV is the form in which the virus spreads from the initial site of infection in the midgut to the other tissues of the body of the insect. It is in the midgut tissue that the host insects are sometimes able to contain and halt the infection by destroying or shedding infected cells before the first cycle is completed. The immune system of the host insects can detect infected cells and then try to destroy these cells before viral replication is complete thus halting the infection. However, some NPV are known to have the genes for proteins that block this infected cell recognition system thus helping the virus defeat the host’s immune system. Even in successfully infected insects the destroyed midgut tissues are replaced in a day or two after initial infection so the gut recovers and the insect resumes feeding. After the midgut cycle the BV particles spread throughout the body in the haemolymph and infects, in turn, the haematocytes, and cells of the fat body, trachea and hypodermis. It is in these tissues that a second cycle of infection occurs and that the OB are produced. If the infection gets established and this second cycle of virus replication occurs, it is often found that 90% or more of susceptible cells may become infected. In these the nuclei become swollen and nucleocapsids are produced, but unlike in the first cycle large amounts of polyhedrin are also synthesised and condense to form crystals in which the nucleocapsids become embedded, to form new OB and so complete the cycle. Cells containing large numbers of OB burst and disintegrate. This OB production in a species such as H. armigera starts around 5 days after infection, peaking at 7-8 days PI. The massive destruction of body tissue that accompanies the production of OB eventually kills the insect. The virus also produces in the last stages various protease enzymes that aid the breakdown of insect cells, thus releasing the OB to help them spread and infect other insects. In a typical nucleus 10-50 OB are produced giving 109-1010 OB per insect. On death these OB may comprise in total 10% of the insect’s weight. This astonishing high productivity of NPV in larvae is another reason for their attraction as biopesticides and is unmatched by any other type of virus. Successfully infected insects secrete NPV during the later stages of infection and move about the host plants spreading virus extensively before dying. OB also remain active if eaten and passed through predators and the activities of birds, mammals and other larval predators may be important in spreading NPV epidemics. In some GV, such as CpGV or PxGV, the cycle is similar but in others, such as SlGV, a smaller range of tissues is infected and instead of rapidly destroying the insect they produce chemicals to prolong its larval life. In these viruses an infected insect produces virus at a much slower rate than in NPV, but as the larvae may live twice as long as normal before dying this can also be a successful strategy for spreading the GV widely. The OB are the persistent form of the virus with the crystal protecting the vulnerable nucleocapsids. Under the right conditions, for example in soil, these OB can remain active for years. However while OB are relatively resistant to heat, desiccation and some other environmental factors, they are

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quickly denatured and inactivated by UV light which rapidly inactivates the DNA, without destroying the OB. NPV and GV have strictly limited host ranges and most species successfully infect only a few related species of host insect. HaNPV for example can replicate in H. armigera, H. zea and H. virescens as can HzNPV. NPV and GV are named after the insect species they were first described from, and also on the basis of whether one (singly enveloped) or more than one (multiple enveloped) nucleocapsids are contained in a single envelope, e.g. H. armigera Singly enveloped Nucleopolyhedrovirus, HaSNPV. Apart from the multiple or singly enveloped feature, NPV or GV cannot be identified visually from either light or EM microscopy. To identify viruses beyond the grouping into GV or NPV you need to look at the DNA sequence using restriction enzymes or molecular probes. Examination of the DNA using these techniques has shown that many variants of a species may exist. The existence of these genetic variants with different biological activities may have important implications for the development of biopesticides, both in the possibility to select better naturally occurring strains and as a source of material for genetic manipulation. 2.7 INTRODUCTION TO H. ARMIGERA NPV The Helicoverpa armigera nucleopolyhedrovirus (HaNPV) is a baculovirus that infects and kills H. armigera (podborer, gram podborer, American boll worm, tomato fruit worm). The pest is endemic to Africa, Southern Europe, Asia and Australia, and the HaNPV has also been isolated from all of these areas. H. armigera is susceptible to both the H. armigera NPV (HaNPV), originally isolated from it, and also the Heliothis zea NPV. The HaNPV virus itself also infects both H. virescens and H. zea. This characteristic specificity confers both advantages and limitations. It means the NPV are harmless to man, domestic animals and other wildlife making them extremely safe and environmentally acceptable. They will not harm domestic insects such as bees or silkworms or beneficial insects such as parasitoids and predacious insects, and they are ideal candidates for use within an Integrated Pest Management (IPM) strategy. The limitation is that a NPV capable of killing H. armigera will not infect other pest insects present on the same crop, even other Lepidoptera such as Spodoptera spp., Erias spp., or Agrotis spp. 2.8 COMMERCIAL PRODUCTS A number of commercial products based upon NPV have been developed, mainly for controlling lepidopteran pests (Plate 8). These have included the Heliothis virus product Elcar, the first one registered as a true commercial NPV product in 1975, but since discontinued. More recently a new generation of baculovirus-based products have appeared, including another Heliothis NPV product Gemstar and Spod-X, based upon Spodoptera exigua NPV. Other similar products are expected to appear in due course.

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Plate 8 Commercial NPV products produced in Thailand and India 2.9 REFERENCES Adams, J R & McClintock, J T (1991) Nucleopolyhedroviruses of insects. In: Atlas of

invertebrate viruses, J R Adams & J R. Bonami (eds.), CRC Press, Boca Raton Copping, L G (1993) Baculoviruses in crop protection. Agrow Business report, DS 85, PJB

Publications Ltd. Granados, R R & Federici, B A (eds.) (1986) Biology of Baculoviruses, Volume 1: Biological

properties and molecular biology, and Volume 2: Practical Applications for Insect Control, CRC Press, Boca Raton, Florida.

Hunter-Fujita, F R, Entwistle, PF, Evans, H F and Crook, N E (eds.) (1998) Insect Viruses and

Pest Management. Wiley & Sons, Chichester. King, L A & Possee, R D (1992) The Baculovirus expression system. Chapman Hall, London. O’Reilly, D R; Miller, L K & Luckow, V A (1994) Baculovirus expression vectors: a laboratory

manual. Oxford University Press, New York. Shuler, M L; Wood, H A; Granados, R R & Hammer, D A (eds.) (1995) Baculovirus expression

systems and biopesticides, Wiley Liss, New York.

14

Tanada, Y & Kaya, H K (1993) Insect Pathology, Academic Press, San Diego.

15

3 INSECT CULTURE FOR VIRUS PRODUCTION AND TESTING A supply of healthy, disease-free insects is the first requirement of any programme to either produce or test insect viruses. In practice the establishment of such a culture, even with ideal facilities, can be a surprisingly long process and often at least three months is required to achieve this, even if no serious problems are encountered. Where conditions are not ideal, i.e. where strict quarantine facilities for acclimating field collected insects are not available, it can take much longer or become impossible. Even with an apparently vigorous and successfully widespread insect pest such as H. armigera, it can prove difficult to establish a culture in the laboratory. While the major enemies of culture establishment are disease, fungus and parasites, phenomena such as diapause and infertility can also be serious problems.

3.1 IMPORTANCE OF CLEAN CULTURE, SANITATION AND HYGIENE The establishment and production of a healthy colony of insects is only possible if basic facilities are available and rigorous sanitation and hygiene protocols are established and followed. The production facility must be maintained in very clean conditions. All working surfaces, flooring and plastic ware must be surface sterilised with 0.5 % sodium hypochlorite daily. Glassware and metal ware can be sterilised in a hot air oven at 180°C for 2 hours. Plastics, if autoclavable, can be sterilised by autoclaving for 30 minutes at 121ºC and 12 bar. Various types of plastic disposable rearing vessels are available. These range from individual cups (approximately 25ml) to multi-cavity trays with suitable lids. Disposables may be expensive in developing countries, but have an advantage of making disease control in the colony very much easier. If reusable containers are used, they should be sterilised, washed thoroughly in a separate room and then sterilised again prior to re-use. All waste from the insectary or virus production unit should be autoclaved or burned before disposal. 3.2 INSECTARY DESIGN The insectary for the production of host insects should be designed in such a way as to avoid contamination of the colony with pathogenic diseases. The rearing area should be well isolated with restricted access. A plan of a model insectary with dimensions is shown in Figure 1.

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Figure 1. Basic plan of a model Insectary for Helicoverpa armigera

STERILIZATION WASHING

STORE DIET PREPARATION EGG HANDLING

LARVAL HOLDING MOTH ROOM

QUARANTINE OFFICE

ENTRYPREPARATION

10’

10’

16’

14’ 14’

16’

10’

10’

While a smaller less dedicated facility may have to serve, especially when a research or production programme is in its early days, as far as possible insectaries should try to incorporate the isolation characteristics and protocols of the dedicated facility as described below.

17

The requirements of the different sections are as follows. 3.2.1 Quarantine room Laboratories used for establishing new colonies must have a separate special quarantine facility. The greatest potential source of disease is from insects newly introduced from the wild. No insects should be introduced into the main culture until they have undergone quarantine to determine that they are disease-free, or a selection process has been carried out to eradicate any disease. Wild colonies are held here for observation and examination of insects dying in the process of rearing, and later on for the examination of reproducing female moths to check for microsporidia infection1. Staff working in the quarantine room should not enter any other part of the facility. The office as well as the quarantine area should be isolated from the main rearing facility.

3.2.2 Entry preparation room Access to the main insect culture facilities should be through a room where the workers can wash their hands and change to sterile overall clothing before entering the rearing area. If full changes of clothing are impractical then the wearing of clean laboratory coats and shoe covers or special indoor footwear should be adopted as routine. An air shower may also be built into this part of the facility for the staff to pass through after changing and before entering the rearing facilities. Shower facilities may also be included, particularly where insects that are known to produce allergenic reaction in humans are reared.

3.2.3 Adult moth room This room should have temperature and humidity control that maintains optimum conditions for the host insect; for H. armigera and most important noctuid species these are 24 + 2°C and a relative humidity of 90 + 5%). Air coolers with humidistats may be used to regulate the humidity. The filters in the air coolers trap the moth scales and can be periodically removed and cleaned. A suitable air circulation and filtration system can also be installed to trap moth scales which may otherwise pose health hazards to workers. Allergy to insect scales and hairs do occur, and are most pronounced with those insect species with urticating hairs. However, even such species as H. armigera that are not associated with urticating hairs may initiate dermal or pulmonary allergic responses in staff. This can be overcome by the use of particle masks and regular cleaning of air filters to reduce scale and hair problems. In some cases though, it may be necessary for sensitised staff to wear full protective suits and wear air filter apparatus.

1 Insects are infected with microsporidia through ingestion of contaminated food, however, microsporidia can be transmitted transovarially and is therefore very difficult to eradicate from a colony once infection is established. Infection is normally chronic and results in reduced longevity and fecundity. This is one of the most common problems in insect rearing systems; eradication normally requires re-establishment of the colony from single, uninfected breeding pairs (see section 3.4.2).

18

3.2.4 Larval holding room This room should be maintained at a temperature of 24 + 2°C and a low relative humidity of 45 + 5%. It should be large enough to hold larval holding trays arranged in racks (see Section 3.6). 3.2.5 Egg handling room This facility should be isolated from the moth room. The temperature should be 24 + 2°C. Humidity is not critical. 3.2.6 Diet preparation room This is where the diet is prepared, cooked blended and stored. This room should have a balance for weighing out the diet components, a stove or microwave to cook the diet and a blender of suitable size to mix the diet. Annexed to this room should be the store for the diet materials, and should contain a refrigerator for storing items such as vitamins and antibiotics. A large refrigerator for holding prepared diet prior to use is also advisable.

3.2.7 Washing area This should be an isolated area to wash the larval trays, moth cages, oviposition cages, and all other rearing appliances. 3.2.8 Sterilisation room This facility for sterilisation of equipment should be located next to the wash area.

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3.3 EQUIPMENT The following items should be considered as essential, or at least highly desirable, equipment in any insectary. More specialised, industrial scale equipment may also be used for large-scale commercial production. Equipment

Purpose

1. Research microscope with phase contrast and

dark field facility x400 - x1000

Examination of tissues for disease diagnosis.

2. Horizontal laminar hood(s) Clean, contaminant-free air during cooling of diet and infesting the diet with eggs/larvae.

3. Electronic balance(s)

0.1g to 300g for weighing diet ingredients; 0.001g to 30g for weighing larvae/pupae.

4. Gas stove/microwave oven Cooking diet. 5. Refrigerator For storing larval diet and adult feed;

Vanderzant vitamin mix and antibiotics. 6. Water distillation \ osmosis unit Distilled water for diet. 7. Hot air oven Sterilisation of glass and metal ware. 8. Autoclave Sterilisation of autoclavable rearing materials. 9. Room air conditioners Temperature control in rearing rooms. 10. Room air coolers/humidifiers Humidity control in rearing room. 11. Thermohygrographs or electronic temperature/humidity monitors with data logger

Monitoring conditions in moth and rearing rooms.

12. Blender Mixing diet components.

3.4 INSECT CULTURE IN THE LABORATORY Great care should be taken while establishing a colony of H. armigera. There are two ways of initiating a colony.

• Acquiring insects from a well established insectary.

• Starting a new colony from wild populations.

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3.4.1 Establishment of a culture from the wild In most developing countries, starting a culture by obtaining insects from a disease-free established colonies may not be possible. The only other option is to initiate the colony from wild populations. H. armigera is polyphagous, attacking several crops and can be collected in sufficiently large numbers on chickpea, pigeon pea and sunflower. Populations collected from the early broods can be expected to be largely free from diseases, particularly microsporidiosis. In India, late broods collected in the months of January and February can often be infected with Vairimorpha spp. Ideally isolated breeding pairs should be kept separately, so that the origin of all eggmasses are known and source of disease isolated and destroyed (see next section).

3.4.2 Quarantine and colony clean up Field-collected insects should be reared in isolation in the quarantine room following the routine protocols (see Section 5: Quality Control), and closely watched for any disease symptoms. Larvae which show symptoms of disease should be removed from the colony and destroyed by autoclaving. Strict sanitation should be maintained to avoid infection and the spread of the disease. Slow-growing and stunted larvae, as well as malformed pupae and adults, should be destroyed by autoclaving prior to disposal (caution: Never discard directly into the waste bin without autocalving). Forceps used for collection of diseased insects should also be sterilised. All the spent moths should be examined for microsporidian infection and only the progeny from disease-free moths should be selected for further rearing, and the rest should be destroyed by autoclaving. The microsporidia (Plate9), particularly Vairimorpha spp., commonly occur in laboratory colonies of H. armigera and weaken the insects, leading to reduced fecundity. The production of NPV in microsporidian infected larvae can be reduced substantially. In order to maintain the vigour and productivity of the colony, it is essential that this pathogen is kept out of the colony. Since the pathogen can be transmitted vertically to the next generation through the eggs, the utmost care should be taken to select insects free from microsporidiosis. This is achieved through maintaining separate breeding groups (or, alternatively, single breeding pairs) and careful microscopic examination of smears of moths after egg laying. Pupae are segregated in groups of 20 and kept in separate emergence cages with identification numbers. The progeny groups are maintained separately and the abdomen of spent moths are pooled, macerated in distilled water (l0ml), filtered through muslin, and a droplet smeared on a slide and examined under a phase contrast research microscope (Figure 2). The spores of the microsporidian are elliptical measuring about 2 x 4.5µm. All progeny from infected moths should be discarded. Progeny eggs and larvae obtained from moths free from microsporidians are reared very carefully and the second generation moths screened again for microsporidians. The selection process is repeated with the third generation moths, and pupae from the clean colony are moved to the regular rearing facility after surface sterilisation with 0.5% sodium hypochlorite.

21

Plate 9 Microsporidia (arrowed), a common contaminant.

22

Figure 2. Selection of microsporidian-free H. armigera population Moth groups

1 2 3 4 5 6 7 8 9 n

Egg 1a 2a 3a 4a 5a 6a 7a 8a 9a na Batches 1b 2b 3b 4b 5b 6b 7b 8b 9b nb 1c 2c 3c 4c 5c 6c 7c 8c 9c nc 1d 2d 3d 4d 5d 6d 7d 8d 9d nd 1e 2e 3e 4e 5e 6e 7e 8e 9e ne Spent Moth groups

1 2 3 4 5 6 7 8 9 n

â â â â â â â â â â

Pool abdomens from each group separately and prepare smears in distilled water

Examine under Phase Contrast microscope (x45 objective) Vairimorpha spores*

1 2 3 4 5 6 7 8 9 n

*If present (+), the sub-colony must be

+ - â

- â

- â

+ + + - â

- â

+

destroyed. Select microsporidian-free groups for

further rearing

â

REPEAT SCREENING - II GENERATION

â

SELECT

â

REPEAT SCREENING - III GENERATION

â

SELECT FOR COLONY

23

3.5 DIET Larvae can be reared on either natural host plants or artificial diet. Development times are often reported as shorter for insects reared on host plants but in most insectaries artificial diets are preferred for convenience, standardisation and hygiene reasons. The larvae can be grown on a wide variety of semi-synthetic diets, most of which are variants on the original semi-synthetic diets of Vanderzant et al (1962). Diets based upon wheat germ have been used very successfully but as this item is often not available in developing countries a diet based upon a more generally available alternative such as chickpea is preferable. The composition of one such diet used for many generations in India is given in Table 1, below. Table 1. Composition of semi-synthetic diet for H. armigera COMPONENT QUANTITY Chickpea flour 100 g* Yeast 30 g Wesson's salt mix 7 g Methyl Paraben2 2 g Sorbic acid 1 g Ascorbic acid 3 g Agar 13 g Vanderzant vitamin solution 8 ml** Streptomycin sulphate 40 mg Carbendazim 675 mg Formalin 2 ml*** Water 720 ml * Whole chickpea seeds can also be used (soak in distilled water overnight). ** 28% solution in distilled water. *** Not included in diets used for inoculation of larvae with virus and post-inoculation rearing. Other bases for diet have been tested including haricot bean flour, soybean flour and sorghum. Chickpea has often been found to be the best while sorghum has been reported as tending to produce deformed pupae.

2 4-methyl parahydroxy benzoate

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3.5.1 Procedure for larval diet 1. Boil agar in 360ml of water, until completely dissolved. 2. Cook chickpeas in 360ml of water for 10 minutes and then transfer to a mixer. 3. Add the agar to the chickpea flour and mix. 4. The Wesson’s salt mix, methyl paraben, sorbic acid and yeast are added together and

homogenised for 5 minutes. 5. After the diet has cooled to about 70°C, add the vitamin mix, ascorbic acid, carbendazim,

formalin and streptomycin sulphate, and mix the diet well for 5 minutes. 6. While still warm, dispense the diet into the rearing vessels, place (preferably) in a laminar flow

hood and leave to cool and solidify. 7. Cover the diet with clingfilm and store in a refrigerator until use.

3.5.2 Adult diet The natural food of adult moths is nectar. In the laboratory they are commonly fed on a 10% sugar or honey solution, however a syrup such as that detailed below in Table 2, containing vitamins and a preservative is better. Adult diet pots should be changed every day, because if pots are left the diet ferments. Table 2. Composition of adult diet Sucrose 50g Methyl paraben 1g* Absolute alcohol 1ml* Vanderzant vitamin solution (28% in distilled water) 10ml Distilled water 500ml *Dissolve methyl paraben in absolute alcohol before mixing with water. This syrup should be stored in a refrigerator.

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3.6 INSECT REARING PROTOCOL 3.6.1 Egg production 1. Place the pupae in a dish of about 200ml capacity, filled with damped, autoclaved vermiculite (or

sand) inside netlon emergence cages (40 x 40 x 40cm). 2. Dispense the adult diet in to suitable sterile glass vials containing sterile absorbent cotton; then

place these inside the emergence cages. 3. Select 10 adult males and 10 females from the emerged moths. The females have a brownish

tinge and from batches of pupae of the same age will emerge first, followed by the males which have a greenish tinge. To keep a continuous culture, egg batches from at least 20-30 pairs are required. In the absence of humidity controlled rooms the cages can be held in large plastic tents with open pots containing water pots with wicks. With small cages, similar water pots can be placed inside the cages.

4. Release the 10 pairs of moths into each of the egg laying cages. These are plastic containers (20

x 30cm) and covered with a fine muslin cloth. Adult feed is again provided inside in two glass vials.

5. Check the cages daily and remove the cloth on which the eggs are laid, replace it with a fresh

one. Also replace the diet pots with fresh ones. 6. After five days of egg laying the moths are generally spent and should be discarded. 7. After collection, store the egg cloths in humid plastic containers (>95% RH). 8. After 24 hours, surface sterilise the eggs by immersing them in 10% formalin for 10 minutes.

Then wash the cloth in running tap water for 15 minutes before hanging it up to dry. Do not wash too vigorously or the eggs will be washed off. As an alternative to the use of formalin immersion of eggs in 0.1% freshly prepared sodium hypochlorite is also effective in sterilising eggs.

9. Replace the cloth in the humidified storage box. Normally the eggs hatch on the third day at 24 +

2°C. In-breeding has been reported as a problem in cultures and efforts should be made to avoid sibling matings. Bartlett (1985) gives guidelines for achieving this. To improve breeding success some authorities recommend only maintaining adults as pairs, singly to a cage, i.e. one pair to a cage. However, the multiple pair cage system described above has been used very successfully in NRI, India and Thailand.

3.6.2 Larval rearing

26

Larvae can be grown in groups (Plate 10) until the early third instar stage at about 7 days post hatching. After this they turn cannibalistic and must be reared separately.

Plate 10 Group rearing of H. armigera larvae

1. After hatching starts, transfer the egg cloth with the neonate larvae to boxes (30 x 20cm)

containing a 4 blocks of diet. 2. Close with a tight fitting lid and keep on the bench. 3. Alternatively cut up the cloth into parts with 25-50 neonate larvae on, put one-to-a-tub in a

200ml tubs with a block of diet (20x20mm) at the bottom and store as above. The larvae will climb on to the diet and settle to feed.

4. Allow the larvae to grow until the third instar stage.

3.6.3 Rearing of late stage larvae The containers carrying the third instar larvae should be carefully examined for any disease incidence and even if a single larva is found infected, the entire lot of larvae in the container should be destroyed and the container either sterilised carefully or disposed of. 1. Transfer Larvae from disease free batches individually to the preferred individual rearing

containers.

27

2. Hold the larvae in their individual containers at 24 + 2°C and 45 + 5% RH to complete their life cycle and pupate.

There are a number of options for individual rearing containers. Glass penicillin or freeze drying vials of 10ml or 25ml capacity, containing about 5g of diet and sealed tightly with sterile absorbent cotton are very effective and have the advantage of being autoclavable. Or, 25ml plastic cups with clip-on lids are also widely used (Plates 11 & 12). These are often disposed of after use but can be sterilised chemically and re-used. Another alternative is to use plastic multicell rearing trays specially made for bioassay and insect rearing.

Plates 11 & 12 Individual rearing of late stage larvae 3.6.4 Selection for vigour Apart from regular examination of the colony for infectious diseases, the larvae should be subjected to selection for vigour. All larvae being reared on diet for breeding purposes should be examined at the fourth and fifth instar stages, and only the healthy, vigorously growing larvae should be selected to continue. Any slow growing or stunted larvae should be discarded.

3.6.5 Pupation The larvae usually pupate inside the diet and if there is excess humidity, pupation will be affected. Hence the humidity should be maintained at around 45%. In plastic pots the addition of a piece of filter paper fixed between the lid and the pot can help control excess moisture.

28

1. Discard larvae pupating late. (The slow growing insects usually harbour infectious diseases) 2. Four days after pupation, collect the pupae but discard all cracked, malformed or undersized

pupae. 3. Wash pupae selected for further culturing in running tap water and surface sterilise them by

immersing in 0.5% sodium hypochlorite solution, containing 0.1% Teepol. 4. Wash pupae thoroughly in water to remove the hypochlorite and keep them on filter paper to

dry. 5. Place pupae inside the emergence cages and continue the culturing as described above (Plates

13 & 14).

Plates 13 & 14 Pupae prepared for emergence and rearing cages for adult H. armigera

3.7 SUPPLY OF INSECTS FOR VIRUS PRODUCTION Contamination of the colony with NPV can destroy the cultures and lead to serious set back in the production programme. One of the main sources of contamination is the movement of materials and staff between the Helicoverpa insectary and the virus production facility. Hence it is essential that two separate set of personnel are employed for the two programmes. Movement of staff and re-usable materials between the two facilities should be avoided. It is best to supply eggs to the virus production unit where an isolated clean space may be created for culturing the insects until it they are inoculated with the virus for large scale production at third instar stage.

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3.8 PROBLEMS IN REARING LARVAE Under tropical conditions, apart from infectious diseases such as NPV and granulovirus, two other major problems often encountered when mass rearing insects are mould on the diet and phorid flies.

3.8.1 Mould on diet caused by Aspergillus species If carbendazim is not added to the diet Aspergillus species can start growing on the faecal pellets and subsequently spread to the whole diet surface interfering with the larval growth and pupation. It is presumed that the mould inhibitors (Methyl paraben and sorbic acid) are digested in the insect gut making the faecal pellets amenable for the growth of the fungus. The incidence can sometimes be as high as 50-60%. Addition of carbendazim has be shown to give significant control of the mould (Kennedy and Rabindra, unpublished data). The fungicide did not affect the larval development, pupation, adult emergence, fecundity and fertility.

3.8.2 Phorid flies Phorid flies are attracted to the diet and the flies drop their eggs on the diet if it is exposed. Multi-cavity trays with holes in the lids are particularly vulnerable for fouling by the phorid fly. The flies can drop their eggs through even minute holes and the larvae can spoil the diet and make it unfit for H. armigera. This problem can be solved by using glass vials plugged with cotton. In commercial scale production this may not be cost-effective and hence the entire insectary should be made fly-proof. However, one should remember that the flies are very small and can sneak through the smallest of holes. UV fly traps installed inside the insectary are helpful in reducing the intensity of the problem. These phorid flies also attack the pupae and hence, pupal cages should be phorid fly-proof.

3.9 REFERENCES Armes, A J; Bond, G S & Cooter, R J (1992) The laboratory culture and development of

Helicoverpa armigera. NRI Bulletin No.57. Natural Resources Institute, Chatham Maritime, Kent. ME4 4TB.

Nagarkatti, S & Prakash, S (1974) Rearing Heliothis on artificial diet. Tech. Bull. of the

Commonwealth Institute of Biological Control, Bangalore. 17, 169-173. Singh, P & Moore, R F (1985) Handbook of Insect Rearing. Elsevier, Amsterdam. Vanderzant, E S; Richardson, C D & Fort, S W (1962) Rearing of bollworm on artificial diet. J.

Econ. Entomol. 55, pp. 140.

30

31

4 THE PRODUCTION OF INSECT VIRUSES

4.1 INTRODUCTION Insect viruses, like all other viruses, can only replicate by infecting a living cell (see Section 2.6: The Life Cycle of Baculoviruses). In this they differ from other biopesticides like Bacillus thuringiensis (Bt) or fungi that can be mass-produced by fermentation on simple media. Both the nucleopolyhedroviruses (NPV) and the granuloviruses (GV) that have been developed as pest control agents can, therefore, only be produced in living insect tissues. These can either be in whole insects (in vivo) or in isolated insect cell culture lines (in vitro).

4.2 TISSUE CULTURE PRODUCTION Tissue culture of insect cells is still only at the research and development stage and while an important research tool it is not yet either reliable or economic enough for large-scale production of viruses for use as pesticides. However, tissue culture may provide a useful tool for production of inocula that is subsequently used for in vivo production. The cell culture of NPV is the most developed with a number of well-established cell lines capable of supporting the replication of Spodoptera and Heliothis NPV. The cell culture of GV is less advanced and only a few cell lines capable of replicating these viruses are as yet established. Currently tissue culture has the drawbacks that it needs expensive media that mimic insect blood and the problems of maintaining oxygen levels in large vessels (>250 litres) without killing the delicate insect cells have not yet been overcome. Also there is a problem sustaining occlusion body (OB) production in tissue culture. With prolonged tissue culture the virus has a tendency to revert to budded virus (BV) only mutants and loose the capacity to produce occluded virus. Considerable efforts are underway in USA & Europe to develop large industrial scale systems of >20,000 litres for pesticide production but achievement of this goal seems some way off yet. For information on techniques for tissue culturing NPV two excellent sources of detailed information are the books by King and Possee (1992) and O’Reilly et al. (1994). 4.3 PRODUCTION IN WHOLE INSECTS All industrial production of NPV for use as biopesticides is therefore currently done in vivo by infecting, rearing and harvesting whole insects. Systems range from simple home made pesticides, involving farmers using field collected larvae, to computer controlled robot operated mass production facilities with capacity of 0.25 - 1 million ha per annum. Examples of such modern mass production facilities have been built in France, Canada and the USA.

32

The procedure for in vivo production is very simple in principle:

1. Infect larvae of a susceptible species. 2. Grow infected larvae for a time to allow the infection to develop and the virus to

replicate. 3. Harvest the larvae and extract the virus.

However, in practice maintaining a sustained production of virus has not been found to be easy. It is only with well-trained staff, adequately developed production procedures, the appropriate equipment and a high standard of process quality control that effective production can be both attained and maintained. Typical problems found with production include:

• Reduced production rate per insect over time. • Contamination of the system by other competing pathogens reducing the NPV yield as

well as lowering the quality of the product. • Failure to maintain a supply of healthy insects.

The causes of problems are various but important factors are:

• Poor quality insects. • Impure inocula. • Inappropriate dosing. • Poor rearing. • Unsuitable harvesting. • Poor sanitation.

4.4 INSECTS Good quality NPV can only be produced in healthy, disease-free larvae. For this reason most commercial production in USA, S. E. Asia and Europe uses insects cultured specifically for biopesticides production. The costs are higher than in using field collected larvae but there are advantages especially where sustained production is needed. A major advantage is that insects can be bred disease-free and so, when infected, give maximum production of NPV without any other contaminant. They can also be cultured all year round in contrast to many species of field collected larvae which may be available only seasonally. Seasonal production can be a serious drawback where capital investment is high or there is a need to retain trained staff. A common problem in using poorly cultured or field collected insects is that they are often infected with other pathogens such as GV, cytoplasmic polyhedrosis virus (CPV), microsporidia, fungi or bacteria. When these insects are inoculated with NPV, under the stress of infection the other

33

pathogens can also multiply at the expense of NPV production and may also kill the larvae before maximum NPV production is achieved.

34

A major advantage with cultured insects is that their growth is under close control so that one can select just the right age and size of larvae for maximum production. This is important as there is often a crucial window of larval size and age that produces the maximum amount of virus. If larvae are too small, they die too quickly of infection and the NPV yield can be only 5-10% of the optimum. If larvae are too large they need too great an inocula to produce an infection and may pupate before full infection, so again production efficiency suffers. With field collected larvae it is found that collectors tend to select larvae too large for efficient production as these are more obvious and preferentially collected. With Helicoverpa armigera NPV produced in H. armigera, it was found that optimum size is very important in maximising yield. At optimum infection size, using III/IVth instar larvae of 40-70 mg in weight, one can get maximum yield and high infection rate with a low dose of 105 OB per insect (surface dosing). 3 x 109 OB per insect can be harvested. With early instars (<IIIrd), 20mg larvae are easily infected with a small dose but tend to die small having produced few OB. In these instances production per larvae struggles to reach 1 x 109 OB per insect. In contrast late instars (IV/Vth) are difficult to infect and need very large inocula. Even then the rapid growth means that many do not succumb to infection and again average yields rarely exceed 1 x 108 OB per larva.

4.5 INOCULATION Most insects are surface dosed by placing uninfected larvae on trays of artificial diet surface-sprayed with the correct inoculating dose of virus. It is possible to use diet in which virus has been incorporated to a specific concentration but in most cases this is more wasteful of inocula, however, it may be appropriate for larvae that burrow into the diet. In most systems larvae placed on sprayed diet are then reared through on the same tray until harvesting. However some producers use virus sprayed onto leaves on which larvae are allowed to feed for 24 hours and then transferred to clean diet for rearing on until harvesting, the main objection to this is that it adds another stage and is therefore more labour intensive. The dose used for inoculation is important and should be optimised. Too much virus is wasteful and inefficient. Also, overdosing will kill small larvae before they can grow big enough to produce maximum virus. Too little will leave some larvae uninfected. Each virus/pest system will have its own optimum dose and an important part of setting up production is carrying out trials to determine the best dose and size of insect. In the HaNPV production system developed at the NRI, 105 OB per 60mg IIIrd instar larva sprayed onto diet trays was found to be the best dose.

4.6 INOCULUM PURITY It is extremely important that good quality inocula is used. Probably one of the major reasons for failure in production is the use of poor, impure or low-activity inocula. Inocula should be highly purified by sucrose gradient centrifugation to ensure it is free from contamination by other pathogens, (such as GV, Microsporidia, Fungi, etc.). The practice of recycling virus from production, without purifying, is particularly dangerous as contaminants can build up every time it is recycled until eventually it may contain 90% other contaminants and 10% NPV. Using such an inocula is a

35

guarantee of poor production. Therefore virus should never be recycled without being checked and purified. If such purification equipment as centrifuges for gradient purification are lacking, this problem can still be reduced if all the infected larvae to be used for the inocula are individually examined microscopically and any showing signs of microsporidia, CPV, GV or other pathogens are discarded.

4.6.1 Protocol for purifying inocula Most purification procedures are based on centrifugation and the possession of a centrifuge should be considered as essential to any properly equipped insect virus facility be it laboratory or production plant. The procedure employed will depend on the nature of the sample, on the purity required and on the equipment available. If high and low speed centrifugation are available the following procedure for purification of NPV or GV from individual infected insects should be used. 4.6.1.1 Primary processing.

1. Place insect cadaver in 1-1.5ml 0.1% sodium dodecyl sulphate (SDS) in distilled water in a

microtube. 2. Homogenise thoroughly using a micropestle to disrupt the cuticle then vortex for

1 - 2 minutes. 3. In a microfuge, spin at 100g for just 5 - 10 seconds to pellet large debris such as pieces of

cuticle. 4. Decant supernatant into a clean tube, re-suspend the loose pellet in 0.1% SDS and repeat

the above step. 5. Combine supernatants and either re-suspend again or discard the pellet. 6. Centrifuge the supernatant at 2500g for 5 minutes (or 5000g for 10 minutes for GV) to

pellet the virus. 7. Discard the supernatant with the layer of fat above and re-suspend the pellet in

1-1.5ml of distilled water. The pellet may need to be vortexed to achieve complete re-suspension.

8. Pellet virus again at 2500 - 5000g for 5 minutes (10 minutes for GV), discard the

supernatant, and re-suspend pellet in a small volume of distilled water, as above.

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4.6.1.2 Gradient centrifugation If NPV is destined for inoculation it is recommended that NPV be further purified to remove any GV, bacteria, microsporidia or other parasites. To do this a long spin, using gradient centrifugation is needed. The purification of NPV by this technique is wasteful and usually entails loss of 50-75% of the NPV OB, so the procedure is only used where high purity is essential. To carry out gradient centrifugation much higher g forces in the order of >50,000g for at least an hour are needed. Such centrifuges are very expensive costing UK£60-90,000 with, in addition, special rotors that also cost at least UK£20,000 each. It can help also to sediment the semi-pure NPV through 10% sucrose prior to gradient purification with a single long spin. The NPV is pelleted but lower density material left above the sucrose. The principle of gradient centrifugation is to place the sample in a tube that contains liquid of different densities and then subject it to a high g force over a prolonged period. The particles of different density then move through the liquid column until they reach a layer of equal density at which point they halt (Figure 3). The different density liquid layers can be produced by dissolving different quantities of a cheap inert solute such as sucrose or glycerol in water. The gradient can be made up of discrete bands of liquid on top of one another, e.g. 40%, 45%, 50%, 55% and 60% sucrose, or it can be in the form of a continuous gradient down the tube with the lightest at the top, e.g. 40-60 % sucrose. Figure 3. Gradient separation of impure NPV Pre- Centrifugation Post centrifugation Centrifugal force Density Gradient Gradient Centrifugation B.D Hames pp 45-93. In “ Centrifugation: A Practical Approach” Ed. D Rickwood (Second Edition, 1989) Reprinted by permission of Oxford University Press. Continuous gradients can be made using a gradient former that itself can be home made from two plastic bottles and a connecting tube. The other items needed are a magnetic stirrer and peristaltic pump (Figure 4). Glycerol can be used in the gradient but food grade sucrose is generally adequate.

Impurevirus

Bands of virus, insect debris & contaminants

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Gradient

If a gradient former is not used, a series of suspensions needs to be made up covering the range of densities, e.g. 40, 45, 50, 55, 60 and 65% sucrose. Start with the densest and add a measure to each tube, e.g. 10ml in 100ml tube. Then carefully add 15ml of each successively lighter fraction by decanting the suspension carefully down the inside of the tube wall taking care not to disturb the lower layers. The tube should then be left for an hour to allow the layers to diffuse. Figure 4. A simple apparatus for the preparation of linear gradients (a gradient former)

B.D Hames pp 45-93. In “ Centrifugation: A Practical Approach” Ed. D Rickwood (Second Edition, 1989) Reprinted by permission of Oxford University Press. Loading the sample is crucially important to successful centrifugation. Overloading produces poor separation of fractions and poor results. The best separation is obtained if the sample volume is small compared to the gradient volume, so the sample volume should be 2-3% of the gradient volume. This makes the cost of the process high, as a run with 6 x 150ml tubes will only process 20-30ml of semi-pure NPV with a concentration of no more than 1 x 1010 OB ml-1. The sample itself should be loaded carefully to avoid disturbing the gradient or layers, again by slowly running it down the side of the tube.3

3 To get the NPV to move into and through the gradient, forces of 50,000g are required and runs are 1-3 hours long. When NPV is put through a gradient it settles or bands out at 54-56% sucrose, at a density of 1.25 g ml -1. To process large batches of NPV, a special rotor called a zonal rotor is used and this can purify 50-100ml of sample per run. Gradients can also be used to purify GV which has a similar density to NPV. However, as the particles are smaller, greater g forces (90,000g) are needed to obtain good bands.

Stirrer

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PREPARATION OF GRADIENTS A sucrose gradient is best generated using a gradient former. A simple gradient maker can be prepared without having to purchase costly equipment. For a 40 - 65% linear, continuous gradient two sucrose suspensions are prepared at 40% and 65% w/w in 0.1% SDS in distilled water. The 65% sucrose is placed in the reservoir and the 40% sucrose into the mixing chamber. A multichannel peristaltic pump is used to fill several tubes at the same time. Light sucrose is fed first to the bottom of tubes via long needles and is then displaced upwards by progressively heavier suspensions. If a gradient maker is unavailable then a step gradient can be substituted with results that can be as good. For a 40 - 65% gradient suspensions at 40, 45, 50, 55, 60 and 65% sucrose (w/w) are prepared. Starting with the 65%, 5ml are added to each tube. Then using a digital pipette, each layer is overlaid with 5ml of each successively lighter suspension by pouring the suspension down inside of tube wall, carefully and slowly. The gradient is left to stand for an hour to allow the layers to diffuse. Step gradient stage

1. Prepare solutions of 50% and 60% (w/w) sucrose in 0.1% SDS. 2. Add 0.5ml of 60% sucrose to a microtube. Carefully overlay a similar volume of 50%

sucrose and ensure there is no mixing. 3. Take a pelleted sample (as described in 4.6.2) and re-suspend it in an equal volume of 20%

sucrose in 0.1% SDS. 4. Take 100 microlitres of the resulting sample and carefully place it on to the 50% sucrose in

the microtube. Carefully place the tubes into a microfuge and centrifuge at maximum speed for at least 15 minutes (for GV, at least 30 minutes).

5. NPV and GV will band at the interface. Heavier material will pellet while light material will

remain in the sample layer. 6. Extract the band and dilute with distilled water. Centrifuge at high speed to pellet virus.

Discard the supernatant, re-suspend in distilled water and repeat the centrifugation. Discard the supernatant and re-suspend in distilled water.

This process will provide greater purity than primary processing but for even greater purity a continuous sucrose gradient can be used . For this you will need access to an ultracentrifuge with capacity to reach a relative centrifugal force (RCF) in excess of 50,000g.

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Continuous sucrose gradient purification

1. Pellet the virus sample and re-suspend in a small volume (0.5 - 1.0ml) of 10 - 20% sucrose

(w/w) in 0.1% SDS. 2. Prepare a linear 40 - 65% sucrose gradient in 30ml polyallomer centrifuge tubes. 3. Centrifuge for one hour at 50,000g (two hours at 90,000g for GV) in a swing-out rotor.

This technique of isopycnic density gradient centrifugation will provide NPV/GV of the highest purity particularly if attention is paid to preliminary preparation and purification of the sample before this stage.

4. Occluded virus will form a thick white band at their isopycnic density point at about 54 -

56% sucrose, equivalent to approximately 1.25g/ml. 5. Collect the NPV band using a long needle and syringe or pipette, and dilute with distilled

water. 6. Pellet the virus by centrifugation at 2500 - 5000g for 5 - 10 minutes. Re-suspend pellets in

distilled water and store at 4°C or in the freezer. 7. After sucrose gradient centrifugation, purified PIB tend to aggregate easily. Sonication

should be used to disperse occlusion bodies. It may be useful to centrifuge samples through a single concentration of 40 or 45% sucrose prior to isopycnic centrifugation. Alternatively, rate zonal centrifugation can be used to give greater sample purity prior to an isopycnic run. For large quantities, zonal rotors are preferred but are VERY expensive. They have a high capacity and eliminate much of the time consuming preparation required when using swing out rotors. LOADING SAMPLES IN TUBES FOR SWING-OUT ROTOR Sample loading is a critical step in centrifugation. Narrow sample zones relative to gradient length produce optimum resolution in fractionation. Sample volume is a function of the cross-sectional area of the tubes, but is usually about 2-3% of the gradient volume. When loading the sample, the pipette containing the sample is touched against the meniscus at the tube wall and the sample allowed to run slowly out. Tubes should be filled to within 3mm of the rim to prevent collapse and should be precisely balanced. UNLOADING BANDS Unloading the NPV band requires both care and skill. Where a number of bands form, it is advisable to unload each in turn, lightest first and then to examine them to determine which one

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contains the NPV. Nothing is more frustrating than after all the effort taken to produce the virus, to find out later you have harvested a band full of bacteria and discarded the NPV!

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Commercial unloaders are available. Satisfactory unloading can be achieved with either a syringe and large bore needle, or with a pipette. Layers above the required band can be drawn off first to avoid unnecessary contamination. After collecting the band of NPV, the sucrose/NPV suspension is mixed in 10 times its volume of water, centrifuge and pelleted to get rid of the sucrose. This pure NPV should then be stored in a freezer at -20°C. It should be noted that pure NPV clumps easily so sonication is needed to separate the sample when defrosting and taking sub-samples.

4.7 INCUBATION AND REARING Conditions should be kept to the optimum so that infected insects are not stressed but left to feed, grow naturally and develop the infection. Insects subjected to heat, high humidity and other stresses tend to die early before the NPV can complete its cycle, and so contain few polyhedra. Cannibalism is a common problem in the later stages of infection and can severely reduce production. For this reason most production of Heliothines is carried out in multicellular, divided containers. Trays are subdivided into individual compartments. Each may be 1 - 2cm x 1 - 2cm and a tray may contain 150-500 such compartments. These trays are best fitted with removable dividers so that the diet can be poured onto them and then sprayed with virus; after which the dividers are pressed into the diet producing the compartments and the insects introduced. These trays can be made out of commercially available stacking trays with insertable plastic dividers adapted from such sources as light defuser grids in fluorescent lighting units. Alternatively, specially made rearing trays can be designed. The placing of larvae in such trays is very labour intensive and in advanced plants, robots and other machines are used to carryout this task.

4.8 SANITATION OF PRODUCTION FACILITIES It is very important that good sanitation practice is carried out and all equipment sterilised before re-use and facilities cleaned regularly. Dirty plants do not produce clean products. Good sanitation is the key in preventing and controlling the outbreaks of contaminants such as microsporidia, bacteria, fungi and insect pests that can impede production. There should be set procedures with specified routines of regular cleaning of all surfaces and rooms involved in insect rearing, diet production and virus production. Autoclaving or low pressure steaming of equipment is the most effective sterilising technique, but simple domestic industrial hypochlorite disinfectants are highly effective. They must however, be made up fresh to prescribed strength (5%) every day. If left, and especially if exposed to diet or dead insects, this becomes inactive and then if used in washing becomes itself a major source of contamination. Production equipment should be washed then sterilised before re-use. Screening of windows and the use of insecticuters can be important in controlling flies, which can be a real problem in some climates. These lay their eggs in the diet on production trays and their larvae then compete with the virus host larvae for food, thus reducing virus production. UV tubes to disinfect rooms are also useful but should not be relied upon alone.

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Where insects are reared for virus production it is very important to keep insectary staff from going into the production or cleaning areas and vice versa. The biggest source of potential contamination to the insectary facility of a virus production plant is the virus production areas themselves. If virus is allowed to contaminate the insectaries, production of larvae for virus infection will rapidly collapse thus halting production.

4.9 HARVESTING It would seem logical to leave the larvae to die naturally so that you are sure that the virus has completed its cycle and maximum production has been achieved. This in principle is correct and is done in some production systems. However it also creates problems. Dead larvae are very fragile due to the effect of virally expressed proteases produced during the last stages of infection and on handling easily burst and leak the body contents so that NPV is lost. Another problem is that after death larval corpses are rapidly invaded and colonised by bacteria which multiply and begin to decompose the larvae. The decomposition action produces the bad smell often associated with impure NPV products and the multiplication of bacteria produces bacterial spores that resemble NPV. In live larvae bacterial levels are relatively low with around 106 bacteria per insect and few spores (<102). In dead larvae, bacterial totals can be very much higher at around 1x109 total bacteria per larvae and in excess of 106 spores. To remove these contaminants after harvesting and get rid of the unacceptable smell is difficult and expensive. It can be done using sucrose or glycerol gradient centrifugation but this may involve losses of up to 50% of the NPV content and increase the cost of the final product by 200%. Interestingly much of the bad smell often found in poor quality aqueous NPV products probably comes from decomposing fats in the insect fat body being broken down by bacteria. If the infection is correctly established most of this fat is metabolised during the infection so the smell is often worse in samples poorly infected with virus containing low titres of virus and few mature OBs. For these reasons it can be better to use larvae harvested alive or before they die of virus and wash them to remove the surface bacterial contamination. By this simple technique it is possible to get a low contamination product which does not develop a bad smell. However, in live harvesting, timing is crucial if you are to get good yields. Significant numbers of mature OBs do not appear until five days post infection (PI). For optimum production there is a need to harvest before significant mortality occurs but must be done when production of mature OBs has reached its maximum. The determination of the best harvesting time has to be done individually in each host/virus system. In any system the precise combination of conditions varies with inocula, temperature, larval age and virus species. In temperate species with long life expectancies it can take a prolonged time, e.g. for producing the NPV of Euproctis chrysorrhoea it takes up to 70 days. However in most tropical species the time is much shorter, for example in HaNPV one can get 2.5 x 109 OB per larvae by harvesting after seven days, when 80% larvae are still intact for harvesting. In some cases where it is difficult to get good live harvesting a procedure known as post harvesting incubation can be useful to ensure OB are mature. It is known that polyhedra harvested early from live larvae are often much less infectious than those from larvae that have died and therefore are probably mature. If live larvae

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are washed and held for a second incubation away from diet, faeces, etc., this can often improve the infectivity of the OB after harvesting, without unduly increasing the bacterial burden.

4.10 MODEL PROTOCOL FOR THE PRODUCTION OF HELICOVERPA

ARMIGERA NPV IN MULTICELL TRAYS Helicoverpa armigera NPV (HaNPV) is produced at NRI by inoculating third instar Helicoverpa armigera larvae of 40-80mg weight and rearing them on artificial diet in production trays. Infected larvae are harvested after a period of incubation. Using the virus production method, it is possible to gain a 1x105 fold return on inoculum used. It is important to note that the method described below applies only to the insect colony and NPV strain mentioned. A different colony dosed with a different strain of HaNPV may give different results due to variation in potency of strain and susceptibility of insect colony. This protocol uses plastic trays into which a metal dividing grid is inserted to produce the required individual rearing cells. There are four stages to production of HaNPV:

1. Preparation of production trays.

2. Inoculation of production trays and loading larvae.

3. Harvesting infected larvae from production trays.

4. Treatment of infected larvae.

4.10.1 Preparation of production trays The preparation of production trays involves pouring molten artificial diet into plastic trays of size 420mm x 300mm; the trays should be filled with diet to a depth of at least 1cm. The surface of the diet should be completely flat and smooth. When the trays are filled, the diet must be left to set and they are then ready to be inoculated with NPV. The volume of diet prepared below is sufficient to make three production trays for 1500 larvae.

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PREPARATION OF ARTIFICIAL DIET Ingredients

2400ml distilled water

50g agar fine powder

290g stabilised wheat germ

270g dried active yeast

50g white table sugar

50g casein powder

1.2g cholesterol powder

10g sorbic acid

12g methyl-4-hydroxybenzoate

24µl linoleic acid (24 drops) Method of cooking Cooking diet is best done using a microwave oven as this provides even cooking of the diet without the need of constant stirring, however if one is not available, electric or gas rings can be used but care must to taken to stir the diet frequently to prevent burning.

1. Measure out the water and mix in the agar.

2. Boil the mixture to fully dissolve the agar.

3. While boiling, measure out the rest of the ingredients. When all have been weighed mix them together while dry to avoid casein forming lumps when added to agar.

4. When the agar has dissolved, mix the dry ingredients into it using an electric hand mixer and boil gently for one hour.

5. Cover the bowl with cling film (not if using gas/electric heating) to avoid the mixture drying out, but make a small hole in the film to allow the pressure to escape.

6. Once the diet has cooked it needs to cool to 60°C, below this the diet may begin to solidify causing problems when pouring into trays.

7. On reaching 60°C add the following vitamins and minerals and mix in thoroughly:

24g of Wesson's salt mix

24ml of Vanderzant vitamin mix

30g of ascorbic acid

2.4g of choline chloride

8. Once mixed, pour the diet into the trays immediately to avoid lumps occurring due to cooling and solidification of the diet.

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4.10.2 Inoculation of production trays and loading the larvae The production trays are inoculated with pure HaNPV sprayed onto their surface using a chromatography sprayer operated with rubber bellows (Plate 15) (If a chromatography sprayer is not available a small hand-held plant or perfume sprayer can be used). The dose is applied at a rate of 2x105 PIB per larva. Approximately 500 larvae are placed per tray, therefore a total of 1x108 PIB of inoculum is used. The inoculum is delivered in 5ml of water. INOCULATION OF THE PRODUCTION TRAYS Procedure

1. Autoclave the chromatography sprayer.

2. Prepare the inoculum (1x108 PIB in 5ml of distilled water for a tray of 500 larvae).

3. Place a production tray into a clean air cabinet (if available) at an angle of 60°.

4. Fill the sterile sprayer with the inoculum and spray half of the diet surface very evenly. Then turn the tray the opposite way up and spray the other half in the same manner. Repeat this procedure until the contents of the sprayer has been expelled.

5. Lay the production tray flat and allow the inoculum to dry in the clean air cabinet.

Plate 15 Applying NPV inoculum to a virus production tray using a chromatography sprayer

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LOADING THE LARVAE To prevent cannibalism the larvae must be reared in isolation. Segregation is achieved by pressing an aluminium honeycomb, called an aeroweb, into the diet. This creates individual cells exposing an area of diet inoculated with approximately 2x105 PIB. Procedure

1. Press a sterile sheet of aeroweb into the artificial diet.

2. Using featherlight forceps, place one third instar larvae into each cell (Plate 16).

3. When all the cells are full, place a sterile sheet of woven polythene over the top of the aeroweb. Then clamp a perforated aluminium lid over the whole assembly to prevent escapees.

4. Incubate the production trays at 26°C.

Plate 16 A production tray loaded with H. armigera larvae

4.10.3 Harvesting infected larvae On the fifth day, production trays should be examined. If the larvae are at maximum weight, showing heavy infection all live larvae should be harvested. Only live larvae should be collected in order to keep any bacterial contaminants to a minimum. The number of larvae recovered should be

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noted to enable yield values to be calculated. If the larvae have not reached maximum weight and no prominent signs of infection are apparent, then the lid should be replaced and the tray checked the next day. Procedure

1. Remove the lid and polythene mesh.

2. Remove the aluminium honeycomb from the diet and tip the larvae onto a tray

3. Remove all live (infected) larvae from the diet surface, tray and honeycomb and place them into a suitable container.

4. Incubate the collected larvae for a further 24 hours at 26°C to allow full expression of NPV in infected larvae.

5. Place infected larvae in to the freezer until processing can be done.

4.10.4 Processing of infected larvae Procedure

1. Thoroughly homogenise harvested infected larvae in an equal volume of sterile distilled water using a commercial blender or food mixer.

2. Slowly filter the resulting slurry through a double layer of muslin supported in a large funnel and wash the residue twice with sterilised water.

3. Finally, gather the muslin and gently squeeze the residue to extract the remaining liquid. Discard the muslin. Avoid excessive squeezing as this will cause the residue to pass through the muslin and into the filtrate.

4. Deep freeze the slurry to await formulation or purification as required.

4.11 PROCESSING INSECT VIRUSES FOR USE AS BIOPESTICIDES The processing of NPV or GV covers the turning of virus-infected larvae into a useable form ready for formulation as a biopesticide. It involves various processes, typically filtration and centrifugation, to remove unwanted contaminants and insect wastes while retaining the active ingredients, the NPV or GV. Typically, the processing of NPV-infected insects involves reduction in water content, removal of insect debris, reduction in bacterial numbers and the release of NPV from insect tissues and cells. The product of processing is a paste or suspension rich in the NPV occlusion bodies (OB). This is then stored in a suitable form for later formulation either as a paste in water, which needs refrigeration or as a dried powder. All processing stages involve some loss of polyhedra. Therefore there is always a need to balance what you gain from each stage against the loss of activity. To obtain pure virus you typically loose 50-70% of your starting material during processing. It is important therefore not to carry out more processing than is necessary.

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When working with newly harvested insects or crude unpurified homogenates it is important that all the processing steps are carried out at 4-10°C. Keeping the process cool prevents the multiplication of bacteria that cause decomposition. Also, it prevents the enzymes present in the dead insects from aiding further the decomposition of the insect body contents. When warm, (above 10°C), the bacteria and the insects’ enzymes will breakdown the cell contents of the insect, including fats, to produce a range of degradation products including those responsible for the foul smell found in poor quality NPV. These decomposition products do not appear to directly affect the NPV, which is protected in its OB crystals. However, until either the NPV is finally purified or dried, it is essential that it is kept in a refrigerator or cool on ice at all times, if the production of bad odours is to be avoided.

4.11.1 Homogenisation and filtering The first step in processing involves homogenising or blending the larvae to disrupt all the cells releasing the NPV, and help break up any clumps of NPV. The NPV are produced in the nuclei of infected cells, so the tissues of the dead insects and the nuclei of the cells in them need to be disrupted to release the NPV. This process is aided by protease enzymes that the virus produces late in the infection cycle, that help to breakdown the cells thus allowing the OB to escape. However, if larvae are harvested alive prior to the completion of the cycle, this process may take longer. This step can be carried out in scientific blenders such as a Waring blender, and the 5 litre capacity models are excellent for processing large batches. However, normal domestic or industrial catering blenders are adequate. All equipment should be cleaned between batches and disinfected to reduce bacteria contamination and smell. The material should be blended when still cool from the refrigerator. Blending of collected insects is normally done in two or three times their own volume of water, preferably sterile distilled water that is cooled to 3 0r 4 ºC but this is not essential. Disruption in water with a small amount of detergent added, (e.g. 0.1% sodium dodecyl sulphate or Teepol or Tween), often helps to promote good homogenisation and does not affect the NPV, but again is not essential. However, highly chlorinated tap water should not be used as the chlorine can inactivate the NPV. The next stage is to filter the homogenate through muslin to remove pieces of the skin, legs and other hard parts of the insect that can block the nozzles of sprayers. Simple cotton muslin folded over to twice thickness is adequate to filter out most of these larger fragments. The muslin is placed in a large filter funnel and the homogenate allowed to drip through. At the end of this filtration process, the residue is washed with distilled water to carry through any NPV present. Finally, the muslin is gathered together from the top and gently squeezed to extract the remaining liquid. Caution here is important as excessive squeezing will force the solid waste to pass through the muslin. After filtering, the used cotton muslin is best discarded as it will begin to smell very quickly and if used again could contaminate other batches. Again the liquid should be kept cold throughout to avoid any decomposition during this process.

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Keeping the mixture cold also helps to solidify any insect body fats so that they get filtered out. This is useful because the decay or rancidity of insect body fats is believed to be a major source of some of the strongly smelling compounds that give poorly prepared NPV such a bad smell. Also, when these fats chemically decay they can produce superoxide radicals that can inactivate viral DNA. Thus removing the insect fats can both improve the smell of NPV and its storage qualities. 4.11.2 Centrifugation Centrifugation is the process of concentrating the solid particles in a suspension by spinning them in a centrifuge, thus separating them from the liquid fraction. As NPV are particles this is used to concentrate and separate them from the water, added to assist homogenisation, and the insect waste. The virus also needs to be separated from the homogenisation liquid, which contains insect proteins, enzymes, etc. Only good centrifugation carried out in cool conditions can help to remove the bacteria, insect body fats and contaminants that are a major cause of bad smell. These fats break down over time and produce a smell, and high concentrations of fat breakdown products, such as organic acids may interfere with the processing or formulation later on. However, where a centrifuge is not available prolonged settling followed by the pouring off of the liquid may be effective but it must be done under cooled conditions, (<10°C), in a refrigerator if decay and subsequent bad smells are to be avoided in the final product. DIFFERENTIAL CENTRIFUGATION The basic procedure is to use a short, low speed spin to separate off any remaining hard parts; followed by a longer, high speed spin to sediment the NPV and separate it from the water, fats and liquid insect wastes. The initial centrifugation is best done using a large volume, low speed centrifuge. An appropriate machine would be one capable of 3000-6000 RPM (capable of producing forces of 5000g)4 with 4 x 750ml buckets capable of processing 3 litres of homogenate at a time (equivalent to 1000-2000 infected insects). Such a machine costs £10,000 for a refrigerated model, which is best as it guarantees that the samples can be kept cool for reasons discussed above. A non-refrigerated centrifuge with similar specifications costs half as much, but more care and effort is required, (e.g. pre cooling-samples, buckets etc.), to ensure the samples do not become too warm for too long.

4 The speed setting needed to get this gravitational force depends upon the design of machine used and its instruction manual should be consulted. There is also a standard equation that can be used to determine the necessary speed, based upon the dimensions of the rotor. RCF (g) = 11.18 x r x (N/1000)2

where r = radius in cm from centre of rotor to point at which RCF value is required N = speed of rotation in revolutions per minute. For a required g force, the speed in RPM can be calculated by re-arranging thus: N = √(RCF/11.18 x r ) x 1000

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For pelleting virus, angle rotor are fastest but swing-out buckets are adequate, with most large buckets of >100ml being of the swing out type. Therefore using large swing-out buckets of 500-750ml capacities, may be much quicker than having to make a number of runs with small angled rotors. A short, low speed spin is used initially to pellet any hard parts or skin that have got through the filtering, but this should last no more than 1-2 minutes at 100g. After this, the liquid which is still turbid and full of NPV is poured off and retained, while the pellet of hard parts left in the bottom of the bucket is washed out and discarded. The NPV OB themselves are then pelleted using 2500g for 20-30 minutes. At the end of this run, the liquid supernatant is poured off and discarded. This discarded supernatant contains soluble material, some bacteria, lipids (especially if done cold), and other insect wastes. The pellet of NPV is re-suspended in a small quantity of water (preferably sterile, distilled water). This pellet should contain concentrated semi-pure NPV at a concentration of 109-1010 OB/ml The following procedure can be used. 1. Weigh and homogenise infected insect cadavers in an equal volume (weight) of 0.1% sodium

dodecyl sulphate (SDS) in sterile, iced, distilled water.

2. Filter through cheese cloth or muslin to remove gross debris.

3. Wash debris through with additional 0.1% SDS and squeeze. 4. Spin the crude virus extract at 100g for 30 seconds to pellet gross contaminants. Decant and

retain the supernatant. Re-suspend the loose pellet in 0.1% SDS and sediment again at 100g for 30 seconds. Combine the supernatants and discard the final loose pellet. NB: g force is determined by the angular velocity of the rotor and the distance of the sample from the axis. See Footnote 4 for the formula to calculate g.

5. Spin the supernatants at 2500g for 10 minutes to pellet viruses and other similar sized particles.

Discard the supernatant which contains very small particles and a floating lipid layer.

6. Re-suspend the pellet in a small volume of distilled water. This procedure will often give virus of adequate purity.

Greater purity can subsequently be obtained by sedimenting the product through a 40% sucrose solution in a long, high speed spin. Low density material will remain above the sucrose solution while heavier particles, including the virus, will sediment. One centrifuge run is usually sufficient to give NPV of adequate purity for pesticide formulation. Clean-up is easiest with larvae harvested and frozen before death. In these samples bacterial levels (< 106 bacteria ml-1) and bacterial spore levels (<102 m1-1) are low. In some samples which are very dirty, e.g. where a high proportion of dead larvae have been included, it may be necessary to repeat this stage twice to remove the gross contamination. In samples containing many dead

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harvested larvae, bacterial levels are high (> 109 ml-1), and spore forming bacteria will have invaded the cadavers and multiplied (> 106 ml-1). As a result fermentation of insect body contents has often started and the clean-up becomes more difficult. If bacterial spores are present it is impossible to separate these from the NPV without expensive gradient centrifugation, as their size and pelleting characteristics are similar to OB. It is also difficult to inactivate the spores because they are highly resistant and you cannot destroy them without inactivating NPV. In well-infected samples of insects, NPV infection degrades fat body cells and the lipid content of the homogenate is very low. If much lipid is seen to be present at the centrifugation stage it usually means the insects in the sample are either poorly infected with NPV or the infection has not been allowed to develop properly. Thus, getting the infection and harvesting stages right reduces processing problems enormously. This simple primary centrifuged material is ideal for use as pesticide and there is no need to process the sample further. In fact, further processing can reduce the effectiveness of NPV as a pesticide. It has been found in studies of UV protectants that this primary purified NPV has a better UV stability than pure NPV with the addition of UV protectants.

4.12 STORAGE OF INSECT VIRUSES For ease of storage and subsequent formulation, harvested infected larvae are homogenised and then dried. Drying can be achieved by either freeze-drying or spray-drying.

4.12.1 Freeze-drying Freeze drying is one of the standard methods of drying biological material while retaining full biological activity. Freeze drying is used to dry samples of bacteria and viruses where it is required to reduce their bulk or to produce a dry, stable active powder for long term storage. It is also commonly used to help purify or concentrate unstable biological materials such as enzymes or antibiotics. Thus in freeze drying, the sample dries while remaining at sub-zero temperatures so that no heat degradation of biologically active molecules occurs. At the very low vacuums used, even with samples frozen to -40°C, samples of several litres can be dried out completely within 24 hours without ever raising their temperature above freezing. Typically the water vapour coming from the sample is drawn from the sample chamber by the vacuum pump into a frozen condenser chamber at -60°C, where the water crystallises out on the walls as ice and can be removed separately when the run is over. When freeze drying NPV from insects, the infected insects are first macerated and filtered to remove gross insect debris (Refer to Section 4.11.1). It has been found that trying to freeze insects that are intact and whole is slower than with macerated insects, presumably because the intact skin acts as a barrier and slows down the escape of the water vapour from the insect bodies.

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Freeze drying can be applied to samples of any size, however the process gets increasingly slow as the thickness of the sample increases. Thus samples of depth no greater than 1-1.5cm are easily dried out in 24 hours but samples deeper than that take very much longer, (the corpse of a baby elephant has successfully been freeze dried at London Zoo but that took 18 months to complete the drying!) Insect material, when freeze dried, is often very light and flaky and can be difficult to handle. The process is made easier by using a starting paste containing as little liquid as possible (no more than 85-90% total water). Another way to make samples easier to handle is to freeze dry them with an inert carrier such as china clay or lactose to help give the final product bulk. For freeze drying to be successful the sample must be completely frozen. If any part remains unfrozen it will not dry out completely and the whole batch will warm up and melt during the drying run. Usually if a homogenate of infected insects is cooled to below -20°C it will completely freeze. However if the sample contains many insects harvested after death in which there has been microbial degradation of insect tissues, some of the bacterial fermentation products may act as an antifreeze and a lower temperature is required. If freezing thoroughly becomes a problem it can often be overcome by pelleting the NPV and discarding the liquid phase, which will contain the freezing point depressant fermentation products, and re-suspending the pelleted NPV in clean water. Once a freeze drying run starts it must be continued until the process is complete. If stopped halfway through, e.g. by a power cut, the sample can rapidly warm up and melt, making it impossible to continue until the sample is re-frozen. It is important to note that leakage of virus into the vacuum pump occurs if the ice melts and the vacuum is reinstated. This will damage the pump and will require expensive repair or replacement. It is recommended that a trip switch is incorporated into the system that will cut power in the event of a power cut during out of office hours.

Plate 17 Trays of NPV homogenate ( ) in a freeze-dryer, Edwards Super-Modulyo.

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FREEZE DRYING PROTOCOL FOR VIRUS-INFECTED INSECTS This protocol is based upon a freeze dryer with integral freezing water trap and separate freezer trays, e.g. Modulyo 12K with tray freezer attachment (Plate 17).

1. Macerate the infected insect sample in water, (approximately two parts water to one part insects).

2. Filter through muslin to remove gross debris, skin and hard parts such as jaws.

3. Pellet the NPV by centrifugation at 3000g for 20 minutes.

4. Discard the supernatant and re-suspend the pellet in one part water. At this point add the inert carrier if desired.

5. Mix thoroughly and pour into the freeze dryer trays. These should be filled no deeper than 10-15mm. If the freeze dryer has temperature probes, place them so they are frozen into the sample but not in direct contact with the metal of the freezer trays (taping them in place before you add the sample to the tray is usually necessary).

6. Freeze at -40°C until fully frozen, usually 8-15 hours, e.g. overnight. (If the freeze dryer has a drying chamber with a built in high capacity freezer unit, this can take only a couple of hours).

7. Check that the oil in the freeze-drier's vacuum pump is at the right level and that it is clean.

8. Start the freezing unit freeze dryer water trap and allow it to run for 30 minutes to cool the water condenser chamber to working temperature (-50°C). Be aware that the temperature gauge on most freeze dryers shows the temperature of the coolant in the system not that of the chamber. If you run the freeze dryer before the condenser chamber is cold enough to trap all the water vapour the water will get into the oil of the vacuum pump and this will ultimately cause the pump to seize.

9. When ready load the frozen trays into the dryer racks, close the chamber and start the vacuum pump immediately. Watch the pressure gauge reading and this should start to fall rapidly within a minute or two. If not check for leaks or a valve left open.

10. The sample temperature should start to fall as sublimation starts and the sample cools even further. If there is any part of the sample not frozen it may cause a meltdown and liquefaction of the sample at this point. However it may alternatively completely freeze during this initial fall in temperature and the run will be OK. Keep the sample under observation for the first 30 minutes as this is when meltdowns occur.

11. If the freezer trays have thermostatically controlled heaters set these at -20° initially. This will feed heat into the sample to drive the sublimation of the water without heating it up enough to melt the sample.

12. Once the drying process is successfully started after 2-3 hours the heater setting can be raised to 10°C to speed things up. Some freeze dryers have programmable heater trays and a progressive rise in the temperature profile back to room temperature can be set to ensure rapid drying.

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13. On successful completion the sample will return to room temperature or the heater tray set temperature, and this indicates all the frozen water has sublimed and the sample has completely dried.

14. To stop the run switch off the vacuum pump and open the valve to allow air to enter the drying chamber. Do this slowly or some of the dry sample may blow out of the tray all over the drying chamber and into the condenser chamber.

15. Open the drying chamber and remove the dried sample. You should quickly transfer it to a sealed container as crude insect virus samples are very hygroscopic and will quickly re-absorb water from the air which will de-stabilise them and causes the powder to clump and clog.

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4.12.2 Spray drying NPV is commonly produced in vivo through a process of larval inoculation and subsequent harvest. For ease of storage, harvested infected larvae are homogenised and then dried. Drying can be achieved by either freeze drying (see Section 4.12.1) or spray drying. Spray drying involves atomisation of feed into a spray, and contact between the spray and a drying medium results in moisture evaporation. With baculoviruses, a suspension of homogenised infected larvae (the feed) is combined with a high pressure air flow and atomised through a hydraulic nozzle. The spray generated combines with a hot air stream and moisture evaporation takes place from droplet surfaces. Evaporation is rapid due to the vast surface area of droplets in a spray. Dried particles suspended in hot air are then removed in a cyclone. To achieve rapid evaporation the hot air stream can exceed input temperatures of 200°C. Like almost all biological material, baculoviruses such as NPV are heat sensitive. However, because these viruses are encapsulated in a protective protein crystal they are much less sensitive than most viruses or vegetative bacteria. However, there is a risk of denaturing virus if exposure to heat is excessive. Rapid evaporation, on the other hand, produces cooling, which mitigates the heating effect. Once dry however, particles will be heated to the output temperature, and rapid separation of virus from the hot air stream is essential. Careful control of input temperature and feed flow rates is essential. Output temperature is critical to the spray drying of heat-sensitive material. Despite the above comments about virus heat sensitivity we have found that NPV is surprisingly heat tolerant. Output temperatures in the range 70 - 100°C do not appear to destroy activity although exposure to these temperatures is extremely short. Spray drying is an efficient alternative to freeze drying and grinding. Careful control of temperatures is required and bench top models such as the Buchi, (see below), should not be left entirely unattended and constant attention should be paid to the spraying conditions such as inlet and outlet temperature to prevent overheating or clogging. Failure to watch this could lead to the material being lost or inactivated. Spray drying eliminates the need for the separate grinding stage required for freeze drying, provided the homogenates are first filtered to remove large particles. The powder produced is sufficiently fine to be passed through knapsack sprayer filters without causing blockage. Moisture content of dried powders is acceptable at between 1 - 4% and does not seem to be related to output temperature. SPRAY DRYING WITH BUCHI 190 MINISPRAY DRYER The following protocols are for a Buchi 190 mini spray dryer, the commonest research scale machine in development laboratories (Plate 18). This is used for preparing small batches of material for testing purposes. For mass production much larger machines, often used in the food industry, with capacities of at least 15 -100 litres per hour can be used for preparing bulk batches. When using these machines exact procedures may differ and the instruction manuals should be consulted

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but the principles and general technique are the same. A high-pressure air supply is required for this process. Spray drying carried out in a Buchi 190 mini spray drier is in an open cycle. Air drawn into the spray drier under control of an aspirator, is heated to the required temperature and then passed into a drying chamber. The suspension to be dried is delivered by a peristaltic pump through silicone tubing to a hydraulic nozzle (0.5mm diameter orifice), where it is atomised by high pressure air (6-7 bar). Evaporation takes place in the drying chamber where the atomised sample and heated air combine. Suspended particles in the drying air are drawn out of the drying chamber and into a cyclone under the action of the aspirator. Here, product particles are spun out of the air stream and passed on to a product collector. Humid air passes upwards and out through a filter before being exhausted. Suspension flow rates of between 0.3 and 1.5 litres per hour can be achieved in the Buchi 190. This should be seen as a guide only. Differences in suspension viscosities and pump roller adjustments may alter the flow rates seen with distilled water alone. At high feed flow rates, even at the highest input temperatures, the process is inefficient as wet feed accumulates on the drying chamber walls. Output temperature is principally controlled by feed flow rate and feed water content. Higher feed flow rates lead to lower output temperatures because of increased evaporative cooling. For any one feed flow rate, higher input temperatures give rise to higher output temperatures. Minimum product residence time in the drying chamber can be assumed to be the average residence time of the air (Masters, 1985). Throughput of air, assuming linear response of the aspirator control setting, is approximately 33.75m3 hr-1. Residence time is calculated by dividing the chamber volume (3.29x10-

3m3) by throughput, giving 0.35 seconds. Most of the product however has a much higher residence time because of particle re-circulation and retention in areas of lower air velocities. PROTOCOL TO SPRAY DRY UNPURIFIED SUSPENSIONS OF INSECT VIRUSES:

Plate 18 Spray drying NPV using a mini-spray dryer, Buchi-190.

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1. Prepare a homogenous suspension of infected larvae in distilled water.

2. Using a double layer of muslin, filter out all particles greater than 0.5mm diameter which could otherwise accumulate and block the hydraulic nozzle.

3. Centrifuge the suspension and discard the supernatant with any fat layer.

4. Re-suspend the pellet with distilled water, the suspension should not be more than 25% suspended solids. Above this the viscosity will be too great for efficient flow and atomisation.

5. Stabilise the input and output temperatures using distilled water before starting to dry the sample using the procedure below.

6. Turn on the power supply and switch on the spray dryer; turn on the high pressure air supply; attach the cooling water supply to hydraulic nozzle assembly; open the air supply to the nozzle and set at approximately 700 NI h-1.

7. Switch on the aspirator and heater and allow the input temperature to stabilise at approximately 150°C by adjusting the heater temperature control little by little as necessary. Ensure that any residual moisture from cleaning has evaporated and that there is no contamination from previous use.

8. Place a flask containing approximately 500ml distilled water on the shelf below the peristaltic pump and place the feed tube in the water.

9. Switch on the pump and, with water flowing, adjust the gap between the rollers and the tube seating such that there is no reverse flow. The gap should be approximately 0.2mm less than twice the tube wall thickness. A gap greater than this will lead to inefficient delivery and reverse flow; a lesser gap than this will risk straining the pump and rupturing the delivery tube.

10. With distilled water being pumped through the nozzle adjust the pump speed a little at a time such that the output temperature is between 70 and 75°C.

11. The input temperature should remain at approximately 150°C. If not, adjust the heater temperature accordingly. Note that this will effect the output temperature also, so additional adjustments to the flow rate will be needed.

12. When the input and output temperatures have stabilised at 150 and 70°C, replace the distilled water with the suspension of crude virus.

13. Activate the automatic nozzle cleaning. Because the suspension contains a lower water content, there is likely to be a rise in output temperature. This should be compensated for by slightly increasing the pump speed.

14. Note that some suspensions are prone to sedimentation, in this case the suspension should be mounted on a magnetic stirrer.

15. When the suspension has finished drying, replace the empty flask with distilled water but do not allow the spray dryer to run dry at this stage as this will raise the output temperature, putting the dried product at risk. The change from virus suspension to water must be immediate.

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16. Allow the water to flow until the feed supply tube and nozzle are clear of suspension.

17. Turn off the pump. At this point, output temperatures will rise quickly and it is important that all dried product has collected in the product collector below the cyclone where it is isolated from increases in output temperature.

18. Once all traces of atomised water have disappeared, (within a few seconds), turn off the heater but do not turn off the aspirator. The aspirator must be left running until the input temperature has fallen to below 70°C.

19. Once this point is achieved the aspirator is turned off.

20. When air flow has stopped, the product can be removed in its collector and should be stored in a sealed sachet or container immediately, preferably with silica gel, as spray dried crude virus will rapidly absorb ambient humidity which leads to loss of activity during storage.

21. A small sample should be retained separately for moisture analysis.

22. Disassemble the spray dryer and clean all glassware and metal components with a proprietary disinfectant, rinse thoroughly with distilled water, dry and re-assemble.

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4.13 SUMMARY OF MAIN POINTS

• Production of NPV can only be carried out in live insect cells, either in whole insects or in cell culture.

• In whole insects virus production can be highly productive and all commercial production is done this way, as cell culture is still under development as a practical technique.

• Key to production in insects is good inocula, healthy insects, correct choice of NPV dose, harvesting time and stable rearing conditions.

• While harvesting live insects from production takes more care it results in a cleaner more consistent product.

• All processing involves loss of NPV do what you need, no more. Gradient purified NPV is needed for production inocula but not for field use.

• All processing must be carried out at 4-10°C to avoid the development of bad smells and the build up of bacterial contamination.

• Properly processed centrifuged NPV is better for formulation as it has high activity and low smell.

• It is easier to clean up well-infected, live harvested insects into a low smelling product. • Drying of virus suspension is the most convenient way of storage.

4.14 REFERENCES Dobrata, M & Hinton, R (1992) Conditions for density gradient separations. In: Preparative

centrifugation a practical approach. Rickwood D. (ed.) IRL Press, pp. 77-142. Hunter, F R; Crook, N E and Entwistle, P F (1984) Viruses as pathogens for the control of

insects. In Microbiological methods for environmental biotechnology. (ISBN 0-12-295040-2).

Masters, K (1991) Spray drying handbook. (5th Edition), Longman Scientific, Harlow England. Rickwood, D (ed.) (1987) Centrifugation - a practical approach. (2nd edition) IRL Press, 354 pp. Roth, H & Rickwood, D (1992) Centrifuges and rotors. In: Preparative centrifugation - a

practical approach. Rickwood, D (ed.) IRL Press, pp 42-76. Rowe, T W G & Snowman, J W (1978) Edwards freeze drying handbook. Edwards High

Vacuum, Crawley, England.