journal of research in biology volume 4 issue 4

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List of Editors of Editors in the Journal of Research in Biology

Managing and Executive Editor:

Abiya Chelliah [Molecular Biology] Publisher, Journal of Research in Biology.

Editorial Board Members:

Ciccarese [Molecular Biology] Universita di Bari, Italy.

Sathishkumar [Plant Biotechnologist] Bharathiar University.

SUGANTHY [Entomologist] TNAU, Coimbatore.

Elanchezhyan [Agriculture, Entomology] TNAU, Tirunelveli.

Syed Mohsen Hosseini [Forestry & Ecology] Tarbiat Modares University (TMU), Iran.

Dr. Ramesh. C. K [Plant Tissue Culture] Sahyadri Science College, Karnataka.

Kamal Prasad Acharya [Conservation Biology] Norwegian University of Science and Technology (NTNU), Norway.

Dr. Ajay Singh [Zoology] Gorakhpur University, Gorakhpur

Dr. T. P. Mall [Ethnobotany and Plant pathoilogy] Kisan PG College, BAHRAICH

Ramesh Chandra [Hydrobiology, Zoology] S.S.(P.G.)College, Shahjahanpur, India.

Adarsh Pandey [Mycology and Plant Pathology] SS P.G.College, Shahjahanpur, India

Hanan El-Sayed Mohamed Abd El-All Osman [Plant Ecology] Al-Azhar university, Egypt

Ganga suresh [Microbiology] Sri Ram Nallamani Yadava College of Arts & Sciences, Tenkasi, India.

T.P. Mall [Ethnobotany, Plant pathology] Kisan PG College,BAHRAICH, India.

Mirza Hasanuzzaman [Agronomy, Weeds, Plant] Sher-e-Bangla Agricultural University, Bangladesh

Mukesh Kumar Chaubey [Immunology, Zoology] Mahatma Gandhi Post Graduate College, Gorakhpur, India.

N.K. Patel [Plant physiology & Ethno Botany] Sheth M.N.Science College, Patan, India.

Kumudben Babulal Patel [Bird, Ecology] Gujarat, India.

CHANDRAMOHAN [Biochemist] College of Applied Medical Sciences, King Saud University.

B.C. Behera [Natural product and their Bioprospecting] Agharkar Research Institute, Pune, INDIA.

Kuvalekar Aniket Arun [Biotechnology] Lecturer, Pune.

Mohd. Kamil Usmani [Entomology, Insect taxonomy] Aligarh Muslim university, Aligarh, india.

Dr. Lachhman Das Singla [Veterinary Parasitology] Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.

Vaclav Vetvicka [Immunomodulators and Breast Cancer] University of Louisville, Kentucky.

José F. González-Maya [Conservation Biology] Laboratorio de ecología y conservación de fauna Silvestre, Instituto de Ecología, UNAM, México.

Dr. Afreenish Hassan [Microbiology] Department of Pathology, Army Medical College, Rawalpindi, Pakistan.

Gurjit Singh [Soil Science] Krishi Vigyan Kendra, Amritsar, Punjab, India.

Dr. Marcela Pagano [Mycology] Universidade Federal de São João del-Rei, Brazil.

Dr.Amit Baran Sharangi [Horticulture] BCKV (Agri University), West Bengal, INDIA.

Dr. Bhargava [Melittopalynology] School of Chemical & Biotechnology, Sastra University, Tamilnadu, INDIA.

Dr. Sri Lakshmi Sunitha Merla [Plant Biotechnology] Jawaharlal Technological University, Hyderabad.

Dr. Mrs. Kaiser Jamil [Biotechnology] Bhagwan Mahavir Medical Research Centre, Hyderabad, India.

Ahmed Mohammed El Naim [Agronomy] University of Kordofan, Elobeid-SUDAN.

Dr. Zohair Rahemo [Parasitology] University of Mosul, Mosul,Iraq.

Dr. Birendra Kumar [Breeding and Genetic improvement] Central Institute of Medicinal and Aromatic Plants, Lucknow, India.

Dr. Sanjay M. Dave [Ornithology and Ecology] Hem. North Gujarat University, Patan.

Dr. Nand Lal [Micropropagation Technology Development] C.S.J.M. University, India.

Fábio M. da Costa [Biotechnology: Integrated pest control, genetics] Federal University of Rondônia, Brazil.

Marcel Avramiuc [Biologist] Stefan cel Mare University of Suceava, Romania.

Dr. Meera Srivastava [Hematology , Entomology] Govt. Dungar College, Bikaner.

P. Gurusaravanan [Plant Biology ,Plant Biotechnology and Plant Science] School of Life Sciences, Bharathidasan University, India.

Dr. Mrs Kavita Sharma [Botany] Arts and commerce girl’s college Raipur (C.G.), India.

Suwattana Pruksasri [Enzyme technology, Biochemical Engineering] Silpakorn University, Thailand.

Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries] Yogeshwari Mahavidyalaya, Ambajogai, India.

Dr. Pankaj Sah [Environmental Science, Plant Ecology] Higher College of Technology (HCT), Al-Khuwair.

Dr. Erkan Kalipci [Environmental Engineering] Selcuk University, Turkey.

Dr Gajendra Pandurang Jagtap [Plant Pathology] College of Agriculture, India.

Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry] Shree Shivaji Arts, Commerce & Science College, India.

Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy] Bio-Science Research Foundation, India.

Nasroallah Moradi Kor [Animal Science] Razi University of Agricultural Sciences and Natural Resources, Iran

T. Badal Singh [plant tissue culture] Panjab University, India

Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture] AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya,

Kalyani, Nadia, West Bengal, India.

Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous

practices, Ethno botany] V. C., Vidyasagar University, Midnapore.

M.Sugumaran [Phytochemistry] Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District.

Prashanth N S [Public health, Medicine] Institute of Public Health, Bangalore.

Tariq Aftab Department of Botany, Aligarh Muslim University, Aligarh, India.

Manzoor Ahmad Shah Department of Botany, University of Kashmir, Srinagar, India.

Syampungani Stephen School of Natural Resources, Copperbelt University, Kitwe, Zambia.

Iheanyi Omezuruike OKONKO Department of Biochemistry & Microbiology, Lead City University,

Ibadan, Nigeria.

Sharangouda Patil Toxicology Laboratory, Bioenergetics & Environmental Sciences Division,

National Institue of Animal Nutrition

and Physiology (NIANP, ICAR), Adugodi, Bangalore.

Jayapal Nandyal, Kurnool, Andrapradesh, India.

T.S. Pathan [Aquatic toxicology and Fish biology] Department of Zoology, Kalikadevi Senior College, Shirur, India.

Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA.

Dr. Amit Bandyopadhyay [Sports & Exercise Physiology] Department of Physiology, University of Calcutta, Kolkata, INDIA .

Maruthi [Plant Biotechnology] Dept of Biotechnology, SDM College (Autonomous),

Ujire Dakshina Kannada, India.

Veeranna [Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India.

RAVI [Biotechnology & Bioinformatics] Department of Botany, Government Arts College, Coimbatore, India.

Sadanand Mallappa Yamakanamardi [Zoology] Department of Zoology, University of Mysore, Mysore, India.

Anoop Das [Ornithologist] Research Department of Zoology, MES Mampad College, Kerala, India.

Dr. Satish Ambadas Bhalerao [Environmental Botany] Wilson College, Mumbai

Rafael Gomez Kosky [Plant Biotechnology] Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas

Eudriano Costa [Aquatic Bioecology] IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil

M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India

Rajib Roychowdhury [Plant science] Centre for biotechnology visva-bharati, India.

Dr. S.M.Gopinath [Environmental Biotechnology] Acharya Institute of Technology, Bangalore.

Dr. U.S. Mahadeva Rao [Bio Chemistry] Universiti Sultan Zainal Abidin, Malaysia.

Hérida Regina Nunes Salgado [Pharmacist] Unesp - Universidade Estadual Paulista, Brazil

Mandava Venkata Basaveswara Rao [Chemistry] Krishna University, India.

Dr. Mostafa Mohamed Rady [Agricultural Sciences] Fayoum University, Egypt.

Dr. Hazim Jabbar Shah Ali [Poultry Science] College of Agriculture, University of Baghdad , Iraq.

Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics]

Agronomy and Plant Breeding Dept., Razi University, Iran

Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution,

Forest protection] College of Life Sciences, Nankai University, China.

María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico

Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology] New University of Lisbon, Caparica, Portugal

Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech] Visva-Bharati (a Central University), India

Table of Contents (Volume 4 - Issue 4)

Serial No Accession No Title of the article Page No

1 RA0446 Laboratory evaluation and comparative study of herbal mosquito coils

against the filarial vector, Culex quinquefasciatus (Diptera: Culicidae).

Susheela P and Radha R.

1332-1337

2 RA0447 Daily Activity Budget of Nicobar Long-tailed Macaque (Macaca

fascicularis umbrosa) in Great Nicobar Island, India..

Rajeshkumar S, Raghunathan C, Kailash Chandra and Venkataraman K.

1338-1347

3

RA0454

Analysis on protein fingerprint, RAPD and fruit quality of tomato

mutants by ion beam implantation.

Duan HY, Wang CF, Yu YA, Li XW and Zhou YQ.

1348-1356

4

RA0452

The leaping behavior of the sally lightfoot crab Grapsus grapsus

(Crustacea: Decapoda: Brachyura) at an oceanic archipelago.

Marina de Sá Leitão Câmara de Araújo.

1357-1364

http://jresearchbiology.com/documents/RA0446.pdf




Article Citation: Susheela P and Radha R. Laboratory evaluation and comparative study of herbal mosquito coils against the filarial vector, Culex quinquefasciatus (Diptera: Culicidae) Journal of Research in Biology (2014) 4(4): 1332-1337

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Biology

Laboratory evaluation and comparative study of herbal mosquito coils

against the filarial vector, Culex quinquefasciatus (Diptera: Culicidae)

Keywords: Mosquito, Culex quinquefasciatus, repellency, Plant essential oil.

ABSTRACT: Synthetic insecticides employed for the control of insect pests are toxic to man and livestock acting as pollutants to the environment, killing all beneficial insects thereby causing a disturbance to the ecosystem. The use of natural products such as plant essential oils has assumed significance as an important component of insect pest management because of their financial viability and eco-friendly nature. They hold promise as alternatives to chemical insecticides to reduce pesticide load in the environment. A laboratory experiment was conducted to investigate the efficacy of three essential oils -eucalyptus oil, lemon grass oil and thyme oil for the repellent activity against the filarial vector, Culex quinquefasciatus. Among the essential oils, Lemon grass oil showed good repellency property when compared to the other two plant oils. Hence, the results of the investigation would indicate a significant potential for lemon grass oil as a possible source of natural products that could be used as an alternative to synthetic insecticides.

1332-1337 | JRB | 2014 | Vol 4 | No 4

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

www.jresearchbiology.com Journal of Research in Biology

An International

Scientific Research Journal

Authors:

Susheela P* and Radha R.

Institution:

Department of Zoology,

PSGR Krishnammal College

for Women Coimbatore,

Tamilnadu, India.

Corresponding author:

Susheela P.

Web Address: http://jresearchbiology.com/

documents/RA0446.pdf.

Dates: Received: 01 April 2014 Accepted: 31 May 2014 Published: 20 Jun 2014

Journal of Research in Biology

An International Scientific Research Journal

Original Research

ISSN No: Print: 2231 –6280; Online: 2231- 6299

INTRODUCTION

Mosquitoes are considered as an important insect

pests that affect the health and well being of human

beings and other animals worldwide. Mosquitoes are

cosmopolitan in distribution and have occupied many

niches including higher altitudes. Mosquitoes are always

considered as a nuisance because they consume blood

from living vertebrates, including human beings

(Bernhard et al., 2003). In India, annually around 40

million people suffer from mosquito borne diseases. The

extensive use of mosquito repellents and insecticides in

public health programmes has caused extensive level of

environmental pollution and serious health hazards.

Many of them are alarmingly toxic to human beings and

also other non-target organisms.

Controlling the mosquitoes in an effective manner is

often complex and expensive task which requires support

from communities and also from different groups such as

industry, agriculture, state and local governments

(Joseph et al., 2004). The harmful effect of the pesticides

on the environment, animals, plants and human beings is

an issue of great concern. As far as India is concerned,

many of the insecticides and larvicides are

commercialized in the form of dust, powder or sprays

that contain chemicals such as organochlorine,

organophosphates and synthetic pyrethroid. Yet

mosquitoes, due to a prolonged use of these insecticides

become resistant and thus it becomes a difficult task to

eradicate them totally (Prajapati et al., 2005). They also

pose a threat to the human population by carrying vector

borne diseases and sometimes out break as epidemics.

Hence to control the vector mosquitoes, efforts are

being taken to look for an alternate solution which

will ultimately minimize the use of synthetic

insecticides.

The development of eco-friendly insecticides will

serve its purpose as a new alternate to substitute the

synthetic insecticides essentially cutting down the

chemical pollution. The pyrethrum flower extracts

contain active materials that are potential enough to

control the mosquito population. (Sutthanont et al.,

2010). In recent times, plant products are used as novel

chemo therapeutants in pest management in different

parts of the world, because of their biodegradable nature.

(Hardin and Jackson, 2009).Therefore, the present study

was aimed to investigate the mosquito repellent nature of

three essential oils: Eucalyptus tereticornis (Eucalyptus),

Cymbopogon citratus (lemon grass) and Thymus vulgaris

(thyme) against C. quinquefasciatus.

MATERIAL AND METHODS

Plant Oils:

The plant oils were purchased from the Aromatic

Oil Stores, Coimbatore, Tamil Nadu and formulated for

the experiment. A stock solution at 1000 ppm is prepared

by dissolving the essential oils in distilled water using

2 ml of 100% acetone respectively. The serial dilutions

of essential oils at the concentration of 5%, 15% and

25% and three replicate of each concentration were

made.

Preparation of herbal mosquito coils:

Mosquito coils were prepared using cow dung,

sawdust, neem leaves, flower waste and tulsi leaves.

Then the essential oils, Thymus vulgaris, Lemon grass,

and Eucalyptus oils were sprayed (w/w) on top of the

coil by using a hand spray pump in different

concentration of 5%, 15% and 25 % separately and they

were used for its efficacy against C. quinquefasciatus

mosquito. The coil was dried in the oven at 70°C for

6 hours was dried for half an hour at room temperature.

These coils were then packed in suitable air tight plastic

folders and kept for 2 – 3 days for even spread of the

essential herbals on the coil.

Test Organisms

The test organism, C. quinquefasciatus, was reared in

the laboratory in the Department of Zoology, PSGR

Krishnammal College for Women, Coimbatore, Tamil

Nadu. Dog biscuits and yeast powder in a ratio of 3:1

Susheela and Radha, 2014

1333 Journal of Research in Biology (2014) 4(4): 1332-1337

http://en.wikipedia.org/wiki/Vertebrates

were given as feed for the mosquito larvae. On the other

hand, adult mosquitoes were fed with a 10% sucrose

solution and a 1 week-old chick. Mosquitoes were kept

at relative humidity of 28-30°C, 75 ± 5%, with 14-h light

and 10-h dark, photo period ( Kitzmiller et al., 1954).

Bioassays

Repellency Test

The experiment was conducted in a closed room,

with a volume of 92.8 m3 in the Department of Zoology,

PSGR Krishnammal College for Women, Coimbatore,

Tamil Nadu. The human volunteers sat at 1 m, 2 m, 4 m,

and 8 m from the herbal mosquito coil. The mosquito

coil was put in the middle of one side of the room. For

control, 50 female unfed, 5 days old mosquitoes were

released in the centre of the room. Then the number of

landing mosquitoes on the bare legs of the human

volunteers was counted for a period of 2 min. For testing,

the mosquito coil was ignited, then counting of the

number of landing mosquitoes on the bare legs of the

human volunteers began and was recorded at periodic

intervals. Three replications were done by changing the

positions of the human volunteers, and then repeating the

procedure the next day.

Journal of Research in Biology (2014) 4(4): 1332-1337 1334


Figure-1 Repellency of lemon grass oil against C. quinquefasciatus

Concentration of lemon grass oil

Concentration of eucalyptus oil

Figure-2 Repellency of eucalypus oil against C. quinquefasciatus

RESULTS AND DISCUSSION

The results of repellency test of thyme oil against

C. quinquefasciatus (Say) after one hour of treatment are

presented in Figure-3. The results clearly indicated that

the highest repellency was reported at 25% concentration

of thyme oil when compared to 5% concentration and

10% concentration. As the concentration of the plant oil

formulation increases the total mortality of

C. quinquefasciatus also gets increased. Figure-2

revealed the efficacy of eucalyptus oil against

C. quinquefasciatus. The lowest repellency was observed

at 5% concentration of eucalypus oil and the highest

repellency was observed at 25% concentration. But the

essential oil, eucalypus oil is more effective than thyme

oil. Increase in the concentration of the plant oil

formulation was found to increase the total repellency of

Culex quinquefasciatus. The different concentrations of

the lemon grass oil was recorded against

Culex quinquefasciatus in Figure-1. The percentage of

repellency was found to be high in 25 % concentration

than 5 % concentration of the plant oil. The results of

this study clearly indicated that lemon grass oil had high

repellency potential to control the mosquitoes than the

other two essential oils.

A number of studies have been focused on lemon

grass oil for controlling mosquitoes as a larvicide and a

repellent with varied results. Hanifah et al., (2011)

demonstrated C. citratus extract has more acaricidal

activity against Dermatophagoides farina and

D. pteronyssinus than Azadirachta indica at 50%

concentration. This proves the efficiency of

Cymbopogon citratus in controlling the insect pests.

Oyedele et al., (2002) evaluated the ointment and cream

formulations of lemon grass oil in different classes of

base and the oil in liquid paraffin solution for mosquito

repellency in a topical application. Cilek et al., (2011)

studied the efficacy of several commercially formulated

essential oils against caged female Aedes albopictus and

Culex quinquefasciatus. Mgbemena (2010) found that

the essential oil O. gratissimium had a greater larvicidal

activity than C. citratus. Purwal et al., (2010) tested the

activity of C. citratus and Mentha piperita essential oils

in a combination against Pediculus humanus and found

a mean time to death of 60 minutes. Therefore the

essential oils can be used as an alternative to synthetic

insecticides for vector control programmes.

The essential oils (EO) eucalyptus oil, lemon

grass oil, thyme oil were evaluated for repellent activity

against the Culex quinquefasciatus. Essential oils of

Concentration of thyme oil

Figure-3 Repellency of thyme oil against C. quinquefasciatus



http://www.ncbi.nlm.nih.gov/pubmed?term=Oyedele%20AO%5BAuthor%5D&cauthor=true&cauthor_uid=12046869

http://www.ncbi.nlm.nih.gov/pubmed?term=Cilek%20JE%5BAuthor%5D&cauthor=true&cauthor_uid=22017089

many plants were observed to have mosquito larvicidal

property and have received attention as potentially

controlling vectors of mosquito borne disease (Zhu et al.,

2006). Therefore, the use of lemon grass oils in insect/

mosquito control is an alternative pest control method for

minimizing the harmful effects of pesticidal compounds

on the environment. The present study has identified

more plant oils showing larvicidal activity against

Culex mosquito. The results obtained suggest that the

plant oils are promising as larvicides against

Culex mosquito. The present study also suggests the use

of Lemon grass oil as the most effective alternative in

controlling mosquitoes.

CONCLUSION

The results of the present investigation proved

that the all essential oils at higher concentration are

effective but lemongrass oil exhibit a significant knock

down activity at higher concentration when compared to

the other oils. For the commercialization of these herbal

mosquito coils, further simulated and actual field trials

are required. Hence, Lemongrass essential oil, alone or in

combinations with those obtained from other mosquito

repellent plant species, could be potentially used for the

preparation of mosquito repellent products.

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filariasis in north-eastern Tanzania:alternative

approaches. Physiotherapy. 89(12): 743–749.

Cilek JE, Hallmon CF and Johnson R. 2011. Semi-

field comparison of the BG lure, nonanal, and 1-octen-3-

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Am Mosq Control Assoc., 27(4):393–397.

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Omar MH. 2011. Acaricidal activity of Cymbopogon

citratus and Azadirachta indica against house dust mites.

Asian Pac J Trop Biomed., 1(5):365-369.

Hardin JA and Jackson FLC. 2009. Applications of

natural products in the control of mosquito-transmitted

diseases. Afr J Biotechnol., 8(25): 7373-8.

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MH. 2004. Larvicidal and mosquitocidal extracts, a

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Oyedele AO, Gbolade AA, Sosan MB, Adewoyin FB,

Soyelu OL and Orafidiya O. 2002, Formulation of an

Effective Mosquito-repellent Topical Product from

Lemongrass Oil, Phytomedicine. 9(3):259-262.

Prajapati V, Tripathi AK, Aggarwal KK and

Khanuja SPS. 2005 Insecticidal, repellent and

oviposition-deterrent activity of selected essential oils

against Anopheles stephensi, Aedes aegypti and

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Jitpakdi A, Chaithong U, Riyong D and Pitasawat B.

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edible plant-derived essential oils against the pyrethroid-

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Article Citation: Rajeshkumar S, Raghunathan C, Kailash Chandra and Venkataraman K. Daily Activity Budget of Nicobar Long-tailed Macaque (Macaca fascicularis umbrosa) in Great Nicobar Island, India. Journal of Research in Biology (2014) 4(4): 1338-1347

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Biology

Daily Activity Budget of Nicobar Long-tailed Macaque

(Macaca fascicularis umbrosa) in Great Nicobar Island, India

Keywords: Macaca fascicularis umbrosa, Daily activity budget, Great Nicobar Island

ABSTRACT: Nicobar long-tailed macaques (Macaca fascicularis umbrosa Miller, 1902) are distributed in three Islands of Nicobar namely Great Nicobar, Little Nicobar and Katchal. Their insular population requires special attention from research and management perspectives. Daily activity budget of M.f. umbrosa in the Great Nicobar Island was studied from October 2011 to September 2013 by intensive direct observation method. Study revealed that Nicobar long-tailed macaque, undergoes most of the time for Locomotion (36.07%), followed by feeding (22.35%), resting or being inactive (15.74%), grooming (11.14%), vocalization (7.03%), playing (5.64%), sexual arousal (1.46%) and agonistic (0.56%). All daily activities have significant difference (χ2 = 1156.22; df = 7, P = 0.05). Chi-square test demonstrated that the daily activity budget differed significantly among the behaviours. Qualitative results found that the interaction within the group was fighting and grabbing food. The significant observation of disability in their legs was noticed in Nicobar Long-tailed Macaque. The relation between their behaviour and disability is also discussed.

1338-1347 | JRB | 2014 | Vol 4 | No 4



An International


Authors:

Rajeshkumar S1*,

Raghunathan C1,

Kailash Chandra2 and

Venkataraman K2.

Institution:

1. Zoological Survey of

India, Andaman and Nicobar

Regional Centre, Port Blair-

744 102, Andaman and

Nicobar Islands, India.

2. Zoological Survey of

India, M-Block, New

Alipore, Kolkatta-700 053,

India.


Rajeshkumar S.

Email Id:



Dates: Received: 01 Apr 2014 Accepted: 30 May 2014 Published: 24 Jun 2014



Original Research


INTRODUCTION

Primates are maintaining the sustainable

ecosystem and play as indicator for ecosystem health;

hence, they help in making of conservation and

management plans. Non-human primates of undisturbed

areas are having great behavioural variation (Thomas,

1991) which are closely related to human beings such as

eating, playing, fighting, keeping young ones etc. (Rod

and Preston-Mafham, 1992). The daily activities and

behaviour of primates differ between residential, non-

residential and undisturbed areas (Krebs and Davies,

1993). Large group size, poor habitat quality, seasonal

variation in food availability may affect their daily

activity budget (Peres, 1993; Passamani, 1998). The

Long-tailed macaques (Macaca fascicularis umbrosa

Miller, 1902) are the only non-human primates found on

Nicobar Islands (Umapathy et al., 2003). Other

subspecies occur in Myanmar, Cambodia, Laos,

Vietnam, Thailand, Malaysia, Indonesia and the

Philippines (Rodman, 1991; Tikader and Das, 1985).

This species varies in their behaviour, social

organisations, habitat consumption, morphology and

genetic variation due to wide distribution (Brent and

Veira, 2002; Hamada et al., 2008). Previous researches

in Nicobar subspecies are available for population status

and distribution profiling (Umapathy et al., 2003;

Sivakumar, 2010; Narasimmarajan and Raghunathan,

2012) Study on ecology and behaviour are also focused

in the other subspecies of Long-tailed Macaque in South

East Asian countries. Reports are available on the

aggressive and social behaviour of M. fascicularis

(Nordin and Jasmi, 1981; Zamzarina, 2003; Brent and

Veira, 2002; Khor, 2003; Md-Zain et al., 2003; Siti,

2003). The present study is focused on the daily activity

budgets of M f. umbrosa in Great Nicobar Island.

MATERIALS AND METHODS

Study Area

The Great Nicobar Island is about 1045.1 sq km

comprises of Campbell bay National Park and Galathea

National Park (Fig. 1). These two National Parks

embrace Great Nicobar Biosphere Reserve (GNBR). The

study site covers about 3 km2 and is composed of low

hills near dense semi evergreen forest, Maggar Nallah

river and human Settlements at Govind Nagar (06°

59.985' N 093° 54.459' E) and it is 6 km away from

Campbell Bay (Fig. 1). GNBR has richest faunal and

Rajeshkumar et al., 2014


Fig 1. Study area and Study site.

floral communities. Great Nicobar is the home for plants

like Albizia chinensis, Albizia lebbeck, Artocarpus

chaplasha, Calophyllum soulattri, Dipterocarpus sp.,

Pterocarpus sp., and Sterculia campanulatum. In fauna,

other than the long-tailed macaques, the endemic

mammals recorded are Nicobar wild boar (Sus scrofa

nicobaricus), Nicobar Tree shrew (Tupaia nicobarica),

Nicobar shrew (Crocidura nicobarica) and Nicobar

Flying fox (Pteropus faunulus).

Behaviour Sampling Method

Following the methods of Hambali et al., (2012);

Md-Zain et al., (2008b) and Brent and Veira (2002) daily

activity observations of macaque were made during 2 to

3 days in a week at 0500 hours until 1630 hours for 78

days from October 2011 to September 2013 to determine

the behaviour categories. A study group categories and

its composition of the three consecutive years are given

in Table. 1. The total number of individuals in study

group increased year by year i:e from 37 to 51

individuals. Every year the numbers of females were

more than that of males. This group was marked by their

dominant male who had a distinctive large and elongated

white area between the eyes and white eyelids compared

to the other groups. Focal animal sampling method was

adopted to collect the quantitative data at ten minutes

interval (Altmann, 1974; Lehner, 1979). During

torrential rain and adverse weather condition, the

observation was discontinued until the weather resumes

normally, because the animals were partially obscured or

moved completely from the observation sites. The data

on the observations of locomotion, feeding, resting,

grooming, vocalization, playing, sexual arousal and

agonistic were collected during the study. Chi-square test

was applied to analyse the behaviour data set obtained.

The nonparametric χ2 test was used to analyze the

significance of activity budgets.


Result on the percentage of eight daily activities

of Nicobar long-tailed macaques monitored is given in

Table 2. Chi-square analysis upon the present study

indicated that all the eight behavioural observation shows

significant differences (Table 2). Jaman and Huffman

(2008) observed that, activities of Japanese macaque

(M. fuscata) in captivity varied between age-sex classes.

Similarly the behavioural variation occurred in

individual with different age-sex observed in the present

study. The most observed daily activity for all the age

group was locomotion. The locomotion is the highest

portion of daily activity in long-tailed macaques

compared to other activities (Hambali et al., 2012; Md-

Zain et al., 2010; Sia, 2004; Suhailan, 2004). This is

because of diurnal in nature as they are very active

during the day as they use their maximum time in

searching for food.

Locomotion

According to Menard (2004) and Wheatley

(1980) Long-tailed Macaques are the primates spending

most of their time for moving as they are mainly

frugivorous and occupy more space. It was also observed

that the study group’s moving choice is varied day by

day to different location and range. When they move out



Group categories Adult (Mature) Immature

Total No. of Individual

Year Male Female Total Sub Adult Juvenile Infant

2011 (October) 10 13 23 10 3 1 37

2012 (March) 12 15 27 12 4 2 45

2013 (August) 13 16 29 12 6 4 51

Table 1. Year wise group composition and total number of Individuals in the study group.

of their home range, there was a shortage of food sources

and availability of fruits. According to O’Brien and

Kinnaird (1997), availability of food source significantly

affects their locomotion in daily activity pattern.

Sometimes these animals visit human settlement areas

and raids crop land, coconuts farms and banana farms

which lead to their destruction. The result indicates that

the macaque spent most of the time in moving due to the

insufficient food sources in their habitat. Likewise this

study group also spend most of their time to visit

different localities because of their diminishing natural

food sources.

Feeding

Besides locomotion, feeding was observed as one

of the major activities of macaque during the study (Fig.

2 A). It resembles with the other subspecies studied by

Hambali et al., (2012), Md-Zain et al., (2010), Suhailan

(2004) and Tuan-Zaubidah (2003) who all found that

feeding is the second most occurrence activity compared

to other. However this finding was contradict with other

macaque species. For example Southern India wild lion-

tailed macaque (Kurup and Kumar, 1993) and captive

Japanese macaque (Jaman and Huffman, 2008) spend the

highest proportion of time in resting rather than feeding

depending on the food and weather factor. An increase in

one activity may pose some influence on other activities

(Jaman and Huffman, 2008). The main food sources are

fruits, flowers, tender leaves, insects, crabs, beetles,

butterflies, some spiders, grasshopper etc. Usually

macaque feed insects in afternoon period between resting

and grooming. When the food sources are less long-

tailed macaque usually rest.

Resting

Resting is the third most activity observed in our

study (Fig. 2 B). The result of the study revealed that

prolonged feeding activity considerably reduced the

resting behaviour during the observation from macaque

in Great Nicobar as noticed by Hambali et al., (2012) in

Malayan long-tailed macaque and Kurup and Kumar

(1993) in lion-tailed macaque. Resting includes activities

like sleeping, lying down and to sit idle. Macaques were

observed resting on tree branches, dead woods, bushes,

rocks and sometimes resting on the roads. Also they use

to take a few minutes rest after walking continuously.

Rainy season and unusual climate directly affect their

feeding and moving activities and increase their resting



Activity Observation Percentage (%) Expected frequency χ2 = (O-E)2/E

Locomotion 518 36.07 179.5 638.34*

Feeding 321 22.35 179.5 111.54*

Resting 226 15.74 179.5 12.04*

Grooming 160 11.14 179.5 2.12*

Vocalization 101 7.03 179.5 34.33*

Playing 81 5.64 179.5 54.05*

Sexual 21 1.46 179.5 139.95*

Agonistic 08 0.56 179.5 163.85*

Total 1436 100 1436 1156.25

Table 2. Percentage and Chi-square value of Nicobar long-tailed macaque’s daily activity.

* Showing significant differences (p<0.05), by using the chi-square test (χ2).

Degrees of freedom (df) = 7, O-Observation, E-Expected frequency.

activity. During night time, macaques sleep on the top of

tree branches. This behaviour indicates that the macaque

protect themselves from the predators. The only known

predator is reticulated python (Broghammerus

reticulatus) as no other higher predators are found in

Great Nicobar Island, but the anthropogenic activity and

domestic predators like dogs also affects their normal

activity.

Grooming

Grooming is the fourth highest activity observed

after resting (Fig. 2 C). This result is similar with M.

fascicularis found in Kuala Selangor Nature Park,

Malaysia (Hambali et al., 2012). Most of their grooming

activity occurs at the time of resting period. It was

predominantly observed at late afternoon when the

macaques return to the home range. At the time of

grooming one monkey picks up lice from other’s body.

Most of the individuals often prefer to self-groom rather

than social grooming. Social grooming highly noticed

between the adult female and adult male. Observations

on grooming between the adult female with infants were

least due to the presence of only few infant in the group.

There was a least observation on grooming between

adult female and juveniles as well as sub adults. Self-

grooming was also often observed in sub adults and

secluded male at the time of resting. In addition, after

mating, the dominant male is groomed by female.

According to Lazaro-perea et al., (2004) this behaviour

can be a way to get protection from others while fighting

and also for sharing of food.

Vocalization

Vocalization is the fifth behaviour that has been

observed. When the agonistic interaction occurs between

the group individuals, dominant adult male produce loud

calls and all the other individuals sound continuously. In

general, macaque produces loud calls especially for



Fig 2. Various activities of Long-tailed Macaque in Great Nicobar Island

A. Feeding, B. Resting, C. Grooming, D. Playing, E. Mating, F. Agonistic.

grabbing and snatching food item and fighting with their

group member. In addition during agonistic interaction

within the group or entrance of predatory animals such as

dogs in their territory, macaque used to make

vocalization. Normally vocalization can be treated as a

warning signal to protect themselves from predators as

observed by Md-Zain et al., (2010). Due to the

observer’s or the human’s activity in their range,

macaque produce different sounds and mainly the sub

adults seem to be most active as they used to climb very

quickly and keep other individuals alert. Members of the

group after hearing the vocal call warning used to climb

to higher ground to escape or hide in bushes. We

observed a least number of calls produced by macaques

while playing activity. Kipper and Todt (2002) and Md-

Zain et al., (2010) also found that the vocal call was

produced by macaques while playing. In the present

study the male long-tailed macaques were found to

produce vocal calls while grooming after mating. No

females were observed producing vocals during mating.

On the other hand observation made by Md-Zain et al.,

(2010) showed that females were found to produce vocal

during and after mating. The possible reason for this

behaviour can be a hormonal effect (Engelhardt et al.,

2005).

Playing

Playing activity is the sixth behaviour that has

been observed during the study period (Fig. 2 D). We

found predictable differences in playing activity in the

juveniles and sub adults. Juveniles were found to play

more than sub adults. Adult macaques were not involved

in playing activity. The playing behaviour may form a

social competition and juveniles in their active age

period will learn on social relations (Kipper and Todt,

2002). Usually, playing behaviour was observed in the

late afternoon, when adult long-tailed macaques are

inactive. Wrestling, chasing, tickling, swinging on the

tree branches, pulling their tails to play with one another

and invert hanging and jumping were the playing

categories observed during the study. It was also

observed that these animals prefer playing on the

selected trees like Casuarinas, Pandanus, Guava and

Coconut. In the evening, all the group member moves

near sleeping site and while moving many were found

collecting and eating some insects in the bushy area.

Sexual Arousal

Sexual behaviour like mating, mount, inspect

copulation are the categories were observed as the

seventh activity (Fig. 2 E). In our study period dominant

males were actively involved in mating with adult

females as this may help females in giving birth to

healthy generation. Females use to live with multimale

group, focused in copulating with dominant males as

observed by Hambali et al., (2012), Lawler et al., (1995),

Md-zain et al., (2010) and Van Noordwijk and Van

Schaik (1999). Sexual behaviour observed is only a small

portion of daily activity in long-tailed macaque.

Normally the adult male was found to smell or observe

the adult female genitalia first to make sure that the

females are ready to mate or not which is in corroborated

with the report of Brent and Veira (2002), Md Zain et al.,

(2010) and Hambali et al., (2012). The long-tailed

macaque takes a few seconds for mating activity.

Agonistic Activity

The least observed activity is the agonistic

behaviour (Fig. 2 F). During our study chase, grab, hit,

bite and fight are the categories of agonistic behaviour

observed as the eighth activity. Though these behaviours

are supported by Hambali et al., (2012), Md-Zain et al.,

(2010), Suhailan (2004) and Tuan-Zaubidah (2003) they

found that mating is the least observed activity. Fighting

behaviour occurred while gaining foods and mates.

Hambali et al., (2012) found that Malay wild long-tailed

macaque has a hierarchy in the group, so that they have

their own way to avoid fight when looking for food

together. Chasing and biting occur sometime between the

males and sub adults. Adult male were more aggressive

when their food was grabbed by other males, this shows



that the aggression appeared in males higher than

females which is agreed with the Brent and Veira (2002)

from macaque observed at Indo-China population.

Significantly we observed few aggressive activities in the

Nicobar long-tailed macaque against human beings

especially women and children during the study period.

Disability and Behaviour

During our study period several disabled

macaques were spotted (Fig 3). They were not able to

move properly due to their disability. These disabilities

may cause some changes in their daily activities which in

turn will cause changes in their behaviour like

locomotion, disability in finding mates, foraging

activities, etc. The relation between disability and

behaviour is also reported in Japanese macaques

(Macaca fuscata) by Turner et al., (2012). The possible

causes of disabilities are congenital defects, dog chasing

and anthropogenic activities. However, exact cause of

disability was not known. But this significant

observation may throw some light on the threats and

their status of these monkeys.

CONCLUSIONS

The present study enlightened behavioural and

activity patterns of the long-tailed macaque population

living in the Great Nicobar Island. It is revealed that



Fig 3. Disability in Nicobar Long-tailed macaque

A. Forearm partially disabled, B. Foreleg disabled, C. Hindleg partially disabled.

locomotion, feeding and resting were the most common

daily activities of these monkeys. Disabled macaques

spotted during our study period may give some

information on the changes in their behaviour that occur

due to disability as well as on the threats they use to

encounter. This study also found that the aggressive

behaviour against humans may raise the issue of human-

macaque conflict. Further studies on the specific impact

of crop raiding and feeding behaviour will derive the

implication of its conservation and management

strategies.

ACKNOWLEDGMENTS

The authors are grateful to the Ministry of

Environment and Forests, Government of India. The

logistic support provided by Divisional Forest officer,

Nicobar Division, Campbell Bay is duly acknowledged.

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Article Citation: Duan HY, Wang CF, Yu YA, Li XW and Zhou YQ. Analysis on protein fingerprint, RAPD and fruit quality of tomato mutants by ion beam implantation. Journal of Research in Biology (2014) 4(4): 1348-1356

Jou

rn

al of R

esearch

in

Biology

Analysis on protein fingerprint, RAPD and fruit quality of

tomato mutants by ion beam implantation

Keywords: Ion beam, tomato, SDS-PAGE, RAPD, fruit quality.

ABSTRACT: In this research, seeds of tomato were irradiated by ion beam or treated with ion beam and soybean DNA, and some tomato mutants with morphological variations were analyzed. Protein analysis in the leaves of mutants showed, changes of protein pattern in mutants were different as compared with the control, the main variation of protein pattern were darkening of bands, increase of protein bands were detected in mutant 12, mutant 14 and mutant 15 and lose of a band in mutant 15. Genomic DNA of mutants were analyzed by RAPD, and total number of amplification bands, number of differential bands and rate of differential bands were studied among mutants. Compared with the control, rate of differential bands was 100.0 % in mutant 9 and 15, also high in mutant 14 and 12, but was 20.0-50.0 % in other mutants except for mutant 3 and 11 without differential bands. In addition, content of vitamin C, soluble saccharide and protein were different, and fruit quality was multifarious in the fruit of mutants compared with the control; mutant 7 has better comprehensive nutritional quality of fruit, whereas mutant 12 and 14 stand second. The above results showed that effects of ion beam or soybean DNA on tomato genomic DNA would lead to the changes in gene expression, protein synthesis and fruit quality, moreover some tomato plants with better fruit quality or special characters were achieved, which would provide basis for the application of ion beam technology in tomato breeding.

1348-1356 | JRB | 2014 | Vol 4 | No 4


www.jresearchbiology.com


An International


Authors:

Duan HY*, Wang CF,

Yu YA, Li XW and

Zhou YQ.

Institution:

College of Life Science,

Henan Normal University,

Xinxiang 453007, China.


Duan HY.

Email Id:


documents/RA0454.pdf. Dates: Received: 03 Jun 2014 Accepted: 06 Jun 2014 Published: 26 June 2014



Original Research


INTRODUCTION

In recent years, mutation breeding has been a

novel way in plant genetic improvement, especially low

energy ion beam implantation which exhibits many

advantages, such as low damage, high mutation rate,

wide mutational spectrum, and so on (Yu, 2000). At

present, ion beam mutation breeding technology has

been successfully applied to a lot of crop breeding, such

as rice, wheat, tobacco, cotton, soybean, rape and others

(Zhou, 2009). In addition, the etching and sputtering

effects of ion beam on cells would be very beneficial to

foreign DNA entering into the cells (Wang et al., 2009,

Li and Sun, 2011) and some transgenic plants have been

achieved by ion beam implantation (Duan et al., 2012),

thus the transgenic technology mediated by ion beam is a

simple and feasible transgenic method.

Tomato is one of the most important vegetables

and fruits that contain abundant nutrients, such as

lycopene, vitamin C, trace elements and other nutrients

(Xue et al., 2004, Wang et al., 2010). In order to meet

the need of people, germplasm resources or genetic

improvement breeding of tomato is required to be

studied and new cultivar of tomato should be cultivated.

In our laboratory, it was found that nitrogen ion (N+) or

argon ion (Ar+) had obvious influences on cell mitosis

and chromosome structure, and lead to various types of

chromosome aberrations (Duan et al., 2013). Thus, dry

seeds of tomato (tomato Zhongza No. 9) were irradiated

by N+ or Ar+ ion beam and soak into soybean DNA after

ion beam implantation to obtain a series of new

germplasm and cultivars with important application

value, and some tomato mutants with the variations of

morphologic characters were found in M1 present

generation. In this research, tomato mutants with

morphologic variations were analyzed by SDS-PAGE

and RAPD, and several indexes of fruit quality were also

detected, which would provide foundation for new

cultivars of tomato and theoretical basis for ion beam

mutation breeding of tomato.

MATERIALS AND METHODS

Plant materials

In this study, seeds of tomato (tomato Zhongza

No. 9) were provided by Vegetable Flower Institute of

Agricultural Sciences, Beijing, P. R. China, and were

respectively irradiated by N+ or Ar+ ion beam in the 30

kev energy conditions. Seeds of soybean (soybean

Zaoshu No. 2) were preserved in our laboratory, soybean

seedlings with single-leaf were used to extract genomic

DNA with CTAB method, and DNA fragments of

soybean genomic DNA were obtained by ultrasonication.

Culture of tomato plants

Tomato seeds implanted with N+ or Ar+ ion beam

were treated as described in research (Ji et al., 2001), at

first were respectively immersed into 0.1×SSC buffer

solution or 300 µg ml-1 DNA working solution which

was composed of soybean DNA and 0.1×SSC buffer

solution, and then were separately washed several times

with sterile water, but the control was only immersed

into sterile water. The above seeds were sowed in the test

field and cultured under the greenhouse conditions with

20°C light and 10°C dark temperature cycle. Seven days

later, seeds germinated, seedlings with two leaves were

transplanted in nutritive bowl and continued to be

cultured. When cultured for two months, seedlings with

five or six leaves were transplanted in the test field and

cultured at the above culture condition.

In addition, the variations of morphologic

characters in tomato plant were found, such as tall plant,

fat leaves, thick stalk, and so on, moreover protein and

DNA fingerprint of some tomato mutants were

respectively analyzed by SDS-PAGE or RAPD, and

several indexes of fruit quality were also detected.

SDS-PAGE of protein in leaves

Proteins were extracted from the fresh leaves of

tomato plants with morphologic variations as described

previously (Ji et al., 2001) with modifications. 1.0 g

leaves were mixed together with 1ml sterile water and

grinded in the mortar on ice-bath, and then the

Duan et al., 2014


homogenate of leaves were centrifuged for 20 min by

12000 rpm at 4°C. The supernatant in the centrifuge tube

was transferred to 5 ml volumetric flask, furthermore, the

precipitate in the centrifuge tube was extracted again

with sterile water and then the supernatant was also

transferred to the above 5 ml volumetric flask, in which

the supernatant was diluted with sterile water to a

constant volume, then the solution was mixed and

preserved at -20°C. The content of soluble protein in the

above solution was determined by Bradford colorimetric

method (Bradford, 1976) at 595 nm, and the standard

curve of soluble protein was drawn with Bovine Serum

Albumin (BSA). In this research, SDS-PAGE of protein

was performed under experiment conditions of 3 %

stacking gel (pH6.8), 12 % separating gel (pH8.8) and

Tris-Glycine buffer solution (pH8.3), and Coomassie

Brilliant Blue method was used in this research.

RAPD amplification

In this study, leaves of tomato mutants were used

to extract DNA by CTAB extraction procedure (Ausubel

et al., 1987). RAPD amplification was performed as the

method (Kangfu et al., 1994). Reaction system of RAPD

amplification was 25 μl and composed of 20 ng DNA,

0.2 μmol L-1 primer, 0.2 μmol/L dNTPs, 2.0 mmol L-1

Mg2+, 1U Taq DNA polymerase and double distilled

water. RAPD amplification was performed as follows:

initial denaturalization at 94°C for 5 min, followed by 35

cycles of 94°C for 1 min, 36°C for 1 min and 72°C for

1.5 min, with a final extension cycle of 72°C for 8 min.

In addition, 100 primers were screened to obtain primers

by which amplification bands are most distinctive,

numbers of amplification bands are more and the

repeatability is preferable.

Determination of soluble saccharide in fruit

Assay of soluble saccharide was performed by

enthrone colorimetric method (Liu et al., 2013) with

improvement. Mature fruit of tomato mutants was

crushed with juicer, 0.5 g tomato juices were mixed

together with 5 ml sterile water in test tube, subsequently

the test tube was sealed with plastic film and put in

boiling water for 30 min to extract soluble saccharide.

The crude extract was filtered into 10 ml volumetric

flask, simultaneously the text tube and residues were

rinsed repeatedly with sterile water, and then the extract

was diluted with sterile water to constant volume. The

content of soluble saccharide was determined with

spectrophotometry at 485 nm, and the standard curve of

soluble saccharide was drawn with sucrose. In addition,

determination of soluble saccharide content was repeated

three times.

Determination of vitamin C and protein in fruit

Mature fruits of tomato mutants were crushed

with juicer, 0.5 g tomato juices were diluted with sterile

water to 100 ml volumetric flask, then extracted by

vacuum extrusion machine and preserved for the

determination of fruit protein and vitamin C.

Determination of fruit protein was performed as

determination of leaf protein in tomato, content of

vitamin C was assayed by spectrophotometry (Chen

et al., 2012) with modification and the standard curve of

vitamin C was drawn with standard vitamin C.

Moreover, determination of vitamin C and protein was

repeated three times.


Protein fingerprint in the leaves of tomato mutants

It is well known that, effects of ion beam on

plant are very obvious and could cause versatility, such

as stem diameter, flowering phase, plant height, quality

characteristic, and so on (Phanchaisri et al., 2012). In this

research, protein in the leaves of tomato mutants were

analyzed by SDS-PAGE (Figure 1), and the electro

photograph was drawn in Figure 2 to more clearly

observe changes of the protein pattern. As compared

with the control, the main variation of protein pattern in

the mutants were some bands darkening, especially the

band with 0.350 Rf value obviously darkened, however

lose and increase of protein band was less found, only


Duan et al., 2014

app:ds:besides

two bands increased in mutant 12, mutant 14 and mutant

15, and the Rf values were 0.05 and 0.083 respectively,

furthermore mutant 15 lost one band (Rf=0.133) in

comparison with the control and other mutants. The

above results suggest the effects of ion beam or soybean

DNA on leaf protein of tomato mutants were various,

which was same to other researchers (Ji et al., 2001).

Owing to the effects of ion beam on chromosome

structure (Huang et al., 1994), we infer that variation of

protein pattern in the leaves of tomato mutants might be

caused by the changes of genomic DNA due to the

damage of ion beam or integration of soybean DNA.

RAPD analysis of genomic DNA in tomato mutants

RAPD technology is actually PCR amplification,

and any organism could be identified by RAPD markers

(Williams et al., 1990, Welsh et al., 1991). Hither to,

some plant mutants induced by ion beam implantation

have been already analyzed by RAPD markers, such as

Nicotiana tabacum (Zhang et al., 1998), Cucumis melo

(Chen et al., 2002), Arabidopsis thaliana (Chang et al.,

2003), Dahlia pinnata Cav. (Yu et al., 2008), Jatropha

curcas (Pamidimarri et al., 2010), Balsamine (Gao et al.,

2012), and so on. In this research, genomic DNA of

tomato mutants was also analyzed with RAPD markers

in order to explore changes in the genomic DNA.

100 random primers were used in the RAPD

amplification, but only bands amplified by S11 primer

(GTAGACCCGT) and S45 primer (TGAGCGGACA)

could be variant between the control and tomato mutants,

and numbers of amplification bands and length of

amplification fragment were different in the mutants by

different primer (Figure 3). As shown in the Figure 3 (a),

only one DNA fragment with 550 bp was amplified by

primer S11 in the control, mutant 3 and mutant 11.

Compared with the control, DNA fragment with 850 bp

increased in mutant 2, mutant 4-8, mutant 10 and mutant

13, DNA fragment with 550 bp lost in mutant 9, mutant

12, mutant 14 and mutant 15, and numbers of

amplification bands and length of amplification fragment

were same in mutant 12 and mutant 14. Furthermore,

four DNA fragments were amplified in mutant 9, in

which DNA fragment with 700 bp was also amplified in

Duan et al., 2014


M: marker, 1: the control, 2-11: tomato mutant induced by ion beam and soybean

DNA, 12: tomato mutant induced with 2×1017N+/cm2 ion beam, 13: tomato mutant

induced with 4×1017N+/cm2 ion beam, 14: tomato mutant induced with 2×1017Ar+/cm2

ion beam, 15: tomato mutant induced with 4×1017Ar+/cm2 ion beam.

Figure 1: Protein pattern in the leaves of tomato mutants by SDS-PAGE

mutant 12 and mutant 14. On the other side, bands

amplified by S45 primer were shown in Figure 3 (b); two

bands were amplified from the control, mutant 2-6,

mutant 11 and mutant 13, one special band was

amplified in mutant 8, mutant 10, mutant 12 and mutant

15 compared with the control. Moreover, three bands

were amplified in mutant 9, but their lengths were

different from the control and other mutants. Meanwhile,

there were two bands in mutant 14 in which one DNA

fragment with 700 bp was also found in mutant 12 and

mutant 15, yet other DNA fragment with 500 bp was

only amplified in mutant 14.

In addition, RAPD amplification bands of tomato

mutants by S11 and S45 primer were given in Table 1,

total number of amplification bands, number of

differential bands and rate of differential bands in tomato

mutants were found to be different. Compared with the

control, rate of differential bands were 100.0 % in mutant

9 and mutant 15, and number of differential bands were 7

and 3, respectively. Secondly, rate of differential bands

in mutant 14 and mutant 12 were also high, the number

of differential bands were 5 and 4, respectively.

However, rate of differential bands in the mutant 3 and

mutant 11 was 0.0 %, moreover rate of differential bands

in other mutants was in the scope of 20.0-50.0 %. Further

more, although rate of differential amplification bands

was 100.0 % in mutant 9, some protein bands only

darken and number of protein bands did not change in

mutant 9. In addition, the variation of protein pattern in

mutant 12, mutant 14 and mutant 15 were relatively

large, and rate of differential amplification bands was

respectively 66.7 %, 83.3 % or 100.0 %. Therefore, the

differential DNA fragments amplified by RAPD might

be related to the expression of some genes by encoding

some proteins or regulating protein synthesis, but it is not

clear whether differential DNA fragments could

influence fruit quality.

Fruit quality of tomato mutants

As everyone knows, tomato is rich in nutrition,

such as saccharide, vitamin C, protein, etc. (Xue et al.,

2004, Wang et al., 2010). In this research, fruit quality of

tomato mutants were assayed, content of vitamin C,

soluble saccharide and protein in the fruit of tomato

mutants were respectively listed in Table 2. As compared

Duan et al., 2014


Figure 2: Protein ideograph in the leaves of tomato mutants

1: the control, 2-11: tomato mutant induced by ion beam and soybean DNA, 12: tomato mutant induced with

2×1017N+/cm2 ion beam, 13: tomato mutant induced with 4×1017N+/cm2 ion beam, 14: tomato mutant induced

with 2×1017Ar+/cm2 ion beam, 15: tomato mutant induced with 4×1017Ar+/cm2 ion beam.

with the control, content of vitamin C in 50 % mutants

was low, such as mutant 2-4, mutant 6, mutant 9, mutant

13 and mutant 15, especially lower in mutant 2, mutant 9

and mutant 4, and was 66.60 μg g-1, 69.65 μg g-1 and

74.43 μg g-1, respectively. However, content of vitamin

C was high in mutant 5, mutant 7, mutant 8, mutant 10-

12 and mutant 14, especially was higher in mutant 8

(152.03 μg g-1), mutant 10 (167.09 μg g-1) and mutant 12

(174.49 μg g-1), moreover content of vitamin C was the

highest in mutant 11 (242.24 μg g-1). In addition, content

of soluble saccharide in 64 % mutants was lower than the

control, but was high in mutant 2, mutant 5, mutant 7,

mutant 9 and mutant 10, particularly higher in mutant 7

(58.84 mg g-1) and mutant 2 (46.96 mg g-1). Furthermore,

content of protein was high in 64 % mutants in

comparison with the control, especially was the highest

Duan et al., 2014


Figure 3: Results of RAPD amplification by S11 primer and S45 primer

(a) Results of RAPD amplification by S11 primer, (b) Results of RAPD amplification by S45 primer. M: DM2000,

M: marker, 1: the control, 2-11: tomato mutant induced by ion beam and soybean DNA, 12: tomato mutant induced

with 2×1017N+/cm2 ion beam, 13: tomato mutant induced with 4×1017N+/cm2 ion beam, 14: tomato mutant induced

with 2×1017Ar+/cm2 ion beam, 15: tomato mutant induced with 4×1017Ar+/cm2 ion beam.

a b

Table 1: RAPD amplification bands of tomato mutants by

S11 and S45 primer

Plants Total number

of bands

Number of

differential bands

Rate of differential

bands (%)

1 3 0 0.0

2 4 1 25.0

3 3 0 0.0

4 4 1 25.0

5 5 2 40.0

6 5 2 40.0

7 6 3 50.0

8 4 2 50.0

9 7 7 100.0

10 5 2 40.0

11 3 0 0.0

12 6 4 66.7

13 5 1 20.0

14 6 5 83.3

15 3 3 100.0

1: the control, 2-11: tomato mutant induced by ion beam and soybean

DNA, 12: tomato mutant induced with 2×1017N+/cm2 ion beam, 13: tomato

mutant induced with 4×1017N+/cm2 ion beam, 14: tomato mutant induced

with 2×1017Ar+/cm2 ion beam, 15: tomato mutant induced with 4×1017Ar+/

cm2 ion beam.

in mutant 9 (46.57 mg g-1), yet content of protein in

mutant 2, mutant 5, mutant 8, mutant 10 and mutant 11

was lower than the control, and only 6.17 mg g-1 protein

in mutant 10.

On the other side, content of vitamin C, soluble

saccharide and protein were different in mutants, and

fruit quality of mutants was multifarious. As shown in

Table 2, compared with the control, content of vitamin

C, soluble saccharide and protein in mutant 7 was all

higher, so mutant 7 has better comprehensive quality of

fruit, secondly were mutant 12 and mutant 14 because

content of vitamin C and protein was both higher.

Moreover, content of soluble saccharide and protein in

mutant 9 was both higher, especially content of protein

was the highest (46.57 mg g-1). However content of

vitamin C in mutant 11 was the highest (242.24 μg g-1),

and content of soluble saccharide and protein was only

11.06 mg g-1 and 18.58 mg g-1. In addition, content of

vitamin C and soluble saccharide was low in mutant 15

and mutant 3, one other thing to note is that nutritional

quality of mutant 3 and mutant 11 are obviously different

with the control, but rate of differential amplification

bands was 0.0 % in mutant 3 and 11 which were treated

with ion beam and soybean DNA, inferring some big

insert segment of soybean DNA might be not amplified,

perhaps there might be a more complicated relationship

between nutritional quality of fruit and genomic DNA of

tomato irradiated with ion beam or treated with ion beam

and soybean DNA, moreover the effect mechanism of

ion beam or foreign DNA was very complex and need to

be further studied and explored.

CONCLUSION

This study shows that ion beam or soybean DNA

could influence leaf protein, genomic DNA and fruit

quality of tomato mutants, inferring the variation of leaf

protein and fruit quality in tomato mutants might be

caused by the changes of genomic DNA which would

happen due to damage of ion beam or integration of

soybean DNA. However the effects of ion beam or


Duan et al., 2014

Table 2: Content of vitamin C, soluble saccharide and protein in the fruit of tomato

Plant Content of vitamin C

(μg/g)

Content of soluble

saccharide (mg/g)

Content of protein

(mg/g)

1 111.95 19.18 18.88

2 66.60 46.96 13.98

3 95.07 13.17 20.51

4 74.43 17.09 26.44

5 114.28 21.37 18.48

6 95.66 14.19 20.12

7 116.91 58.84 29.19

8 152.03 16.35 17.45

9 69.65 37.48 46.57

10 167.09 40.51 6.17

11 242.24 11.06 18.58

12 174.49 16.46 25.48

13 92.36 13.65 21.19

14 122.95 19.12 24.86

15 99.96 12.62 20.35

The average content

in mutants 119.71 23.87 21.88

1: the control, 2-11: tomato mutant induced by ion beam and soybean DNA, 12: tomato

mutant induced with 2×1017N+/cm2 ion beam, 13: tomato mutant induced with 4×1017N+/cm2

ion beam, 14: tomato mutant induced with 2×1017Ar+/cm2 ion beam, 15: tomato mutant

induced with 4×1017Ar+/cm2 ion beam.

app:ds:perhaps

app:ds:mechanism

soybean DNA were different, and the changes among

protein, DNA and fruit quality was not consistent with

each other, thus it is necessary to further study effect

mechanism of ion beam or foreign DNA, which would

contribute to provide foundation for ion beam mutation

breeding of tomato.

ACKNOWLEDGMENT

This research was kindly supported by Science

Fund from Henan province (122300410025), and the

grant of young teachers in Henan province institution of

higher learning (2011GGJS-063), in P. R. China.

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Advantages


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Article Citation: Marina de Sá Leitão Câmara de Araújo. The leaping behavior of the sally lightfoot crab Grapsus grapsus (Crustacea: Decapoda: Brachyura) at an oceanic archipelago. Journal of Research in Biology (2014) 4(4): 1357-1364

Jou

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esearch

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Biology

The leaping behavior of the sally lightfoot crab Grapsus grapsus

(Crustacea: Decapoda: Brachyura) at an oceanic archipelago

Keywords: Crab behavior, Fernando de Noronha Archipelago, Red rock crab, Semi-terrestrial crab.

ABSTRACT: The genus Grapsus includes a total of nine recognized species of semi-terrestrial crabs. Among them, Grapsus grapsus (Linnaeus, 1758) stands popularly known as sally lightfoot crab. It is very abundant in Oceanic Islands, such as the Fernando de Noronha Archipelago, Brazil. The present study registered the behavior of jumping between the rocks by G. grapsus in the supralittoral of Fernando de Noronha Archipelago. Field observations were performed in May 2012, including video footage. The crabs, juveniles and adults, males and females, jump from a rock to another. This can be related to a defense habit, but it seems that the crabs also jump to avoid entering into the sea, or to escape from wave wash. Other registers on crabs jumping from literature are also discussed. However, more studies on this behavior are still necessary for understanding them completely.

1357-1364 | JRB | 2014 | Vol 4 | No 4



An International


Authors:

Marina de Sá Leitão

Câmara de Araújo.

Institution:

Departamento de Ciências

Exatas e Naturais, Faculdade

de Ciência, Educação e

Tecnologia de Garanhuns

(FACETEG), Campus

Garanhuns, Universidade de

Pernambuco (UPE), Brazil.


Marina de Sá Leitão

Câmara de Araújo.

Email Id:



Dates: Received: 20 May 2014 Accepted: 30 May 2014 Published: 26 Jun 2014



Original Research

ISSN No Print: 2231 –6280; Online: 2231- 6299

INTRODUCTION

The genus Grapsus Lamarck, 1801 (Grapsidae)

includes a total of nine recognized species of semi-

terrestrial crabs: G. adscensionis (Osbeck, 1765),

G. albolineatus Latreille, in Milbert, 1812,

G. fourmanoiri Crosnier, 1965, G. granulosus H. Milne

Edwards, 1853, G. grapsus (Linnaeus, 1758),

G. huzardi Desmarest, 1825, G. intermedius de Man,

1888 , G. longi tars is Dana , 1851 and

G. tenuicrustatus (Herbst, 1783) (WORMS, 2013; Ng

et al., 2008). Among these species, G. grapsus, stands

out popularly and are known as red rock crab, sally

lightfoot crab, "aratu" (in Portuguese) and "abuete negro"

or "sayapa" (in Spanish). This species is found in the

Pacific Ocean, from Baja California to Northern Chile,

and Galapagos Islands, and in the Atlantic Ocean, from

Bermudas, Florida, Gulf of Mexico, Antilles, Colombia,

and from Venezuela to Brazil. In the Brazilian coast, this

crab is found from the States of Ceará to Espírito Santo,

but it is more abundant in the Oceanic islands (Fernando

de Noronha Archipelago, Rocas Atoll and Saint Peter

and Saint Paul Rocks) (Melo, 1996; Freire et al., 2011).

At Saint Peter and Saint Paul Rocks, (Ross 1847, apud

Holthuis et al., 1980) cited that this species is a predator

of the eggs of birds that nest at the area, and Viana et al.,

(2004) cited that this is one of the most abundant animal

species on the rocks. Melo (1996) also signals the

occurrence of this species at Trindade, a Brazilian

volcanic island distant 1,167 km from the continent, but

probably the species inhabiting this island is, in fact,

G. adscensionis (Hartnoll, 2009). Ratti (2004) believed

that the differences between G. adscensionis and

G. grapsus were not enough to support two different

species, but more recently, several authors such as Ng

et al., (2008) and Freire et al., (2011), recognized the

taxonomic validity of both species.

Among the oceanic island this species can be

found, stands out the Fernando de Noronha Archipelago

(FNA) (3°51′S, 32°25′ W), a complex of volcanic islands

and rocks, which is found under jurisdiction of the State

of Pernambuco, Northeast of Brazil. The benthic fauna

of FNA was studied by Lopes and Alvarenga (1955) and

Matthews and Kempf (1970) (Mollusca), Pires et al.,

(1992) (Cnidaria), Mothes and Bastian (1993) and

Muricy and Moraes (1998) (Porifera), among others.

Several oceanographic expeditions explored the

archipelago, such as H.M.S. Beagle Challenger

Expedition, Hartt Expedition, Branner-Agassiz

Expedition, Calypso, Canopus and Almirante Saldanha.

The results of the Crustacea sampled on these

expeditions can be found at several publications, such as

Smith (1869), Miers (1886), Henderson (1888), Bate

(1888), Rathbun (1900, 1918, 1925, among others),

Forest and de Saint-Laurent (1967) and Coelho et al.,

(2006, 2007, 2008). Fausto-Filho (1974) presented a list

of the Decapoda and Stomatopoda collected by himself

and based on some of the cited publications, which

resulted in a total of 66 species (3 Stomatopoda and 63

Decapoda) for FNA. Included, there is G. grapsus. The

species was considered very abundant, being found in all

beaches. There is no doubt that the species inhabiting

FNA is G. grapsus. They are commonly observed in the

rocky shores of the islands that compose the archipelago,

sharing the habitat with Plagusia depressa (Fabricius,

1775) (Plagusiidae). The present study aims to describe

the jumping behavior of Grapsus grapsus at FNA during

field observations.

MATERIAL AND METHODS

The archipelago is distant 545 km from the

capital of Pernambuco, the Municipality of Recife,

occupies an area of 26 km² and the main island,

Fernando de Noronha, has an area of 17 km², being 6

miles long and 2 miles wide (Matthews and Kempf,

1970; Fausto-Filho, 1974). In May 2012, during three

days, field observations and footages of this species were

performed at Sueste Bay, FNA (Figure-1) (3º52'01" S;

32º25'19" W). At the bay, the Sueste Beach and the

Araújo, 2014


Sueste Mangrove are included, the last one being

considered the only oceanic mangrove of South Atlantic.

In the seawater of the bay, there are several islets, such

as Cabeluda, Chapéu, Ovos and Trinta-Réis.

The individuals of Grapsus grapsus were

observed in the rocky shore of the bay. These rocks are

mainly distributed in the extremities of the bay, and also

serve as habitat for Plagusia depressa. The water was

transparent and shallow, with a depth of 1m. The footage

was performed with a Panasonic camera, DMC-FT10

model. After that, a bibliographic research was

performed to seek possible registers of the jumping

behavior of crabs in the literature.

The air temperature and tidal heights for the

dates of study were obtained through the Integrated

System of Environmental Data (SINDA).


The air temperature for the study period varied

from 25.5 to 30ºC (Figure-2), characterizing a tropical

climate. The observations were performed during the dry

period, equatorial summer. According to Ribeiro et al.,

(2003, 2005), the FNA climate is of the type Aw of

Köppen's classifications, i.e. tropical with semi-arid


Araújo, 2014

A B

C D

Figure 1. Brazilian coast with the location of the Fernando de Noronha Archipelago, FNA

(A); FNA with the location of the Sueste Bay (B); Aerial view of the Sueste Bay (C);

Rocky shore at Sueste Bay, where the field observations of Grapsus grapsus (Linnaeus,

1758) were perfomed (D).

characteristics, having well defined dry and rainy

periods.

The tidal level for the study period varied from

1.25 to 2.75 m (Figure-3). The tidal regime can be

characterized as semi-diurnal tide, since there are two

high tides in each lunar day (Thurman, 1997). According

to Souza (2011), the maximum height of the tide in FNA

is 2.80 m, and the minimum, 0.0m. Thus, regarding its

amplitude, the tide of FNA can be classified as

mesotides.

The observed population consisted of Grapsus

grapsus juveniles and adults of both sexes. They were

found sharing the habitat with Plagusia depressa.

Besides the size, adults and juveniles are also

distinguished by the color of the carapace. Juveniles of

G. grapsus are dark green, dark gray or almost black,

which is important for they camouflage on the

black volcanic rocks of oceanic islands, and with light

yellow spots. On the other hand, adults are quite variable

in color; some are dark red or bright red (especially

Araújo, 2014


Figure 2. Air temperature by dates and hour during the study period,

at Fernando de Noronha Archipelago.

Figure 3. Tidal level by dates and hour during the study period,

at Fernando de Noronha Archipelago.

males), others are dark green. Some lines and spots can

be observed (Fausto-Filho, 1974; Freire et al., 2011)

(Figure-4).

During the field observations, an unusual

behavior in Brachyura could be noticed: the sally

lightfoot jumps from a rock to another. Two scenes of

G. grapsus jumping were recorded (Videos 1, 2 and 3).

This behavior was observed for both males and

females, and juveniles and adults. A total of 12

observations were performed. In a first moment, it can be

an useful strategy to prevent predation, as described to

the species which will be discussed below. Besides, this

type of movement could be important to escape from the

wave wash (Video 1) or to avoid entering into the water

(Video 2), instead of walking through the water to reach

another point of the rocks. They also seem to jump from

a lower to a higher rock (Video 3). Kramer (1967) also

observed this behavior in a population of G. grapsus

from Galapagos. He noticed that the jumpy crabs had an

average carapace width of 30 cm. The crabs from FNA

were not measured, but it was clear that they did not

reach 10 cm CW. Before jumping, the crab aligns the

body by stretching the front running pairs of legs on

(Kramer, 1967), which was also noticed in the present

study.

Some other interesting information was found in

the literature, regarding the locomotion of crabs. The

species Armases roberti (H. Milne Edwards, 1853)

(Sesarmidae) is found along river banks between rocks

and stones, as well as on the vegetation (Chace and

Hobbs, 1969). According to Schubart and Diesel (1998),

when these crabs are disturbed, they jump from the trees

into the water, and due to this behavior, they are know in

the Caribbean as “jumpy crabs”. Thus, this behavior

could be related to a defensive attitude. A similar

behavior was also registered for Percnon gibbesi (H.

Milne-Edwards, 1853) (Percnidae) by Deudero et al.,

(2005); the specimens, observed in shallow waters, run

and jump when threatened, seeking for shelter from

predators.

The crabs Sesarma trapezoideum H. Milne

Edwards, 1837 (Sesarmidae) occur preferentially in

riverine cliffs near water streams (Jeng et al., 2003).

According to these authors, these crabs retreat into

crevices or jump into the water below them when

disturbed; few minutes after that, they climb back to the

cliff. The species Leptograpsus variegatus (Fabricius,

1793) (Grapsidae), a supralittoral crab of rocky shores as

G. grapsus, jump into tidal pools or into the sea to escape

from predation (Greenaway et al., 1992).

CONCLUSIONS

All these mechanisms described in literature are

related to a fast escape from danger, such as predation,

including jumping into the water. But during the field

observations of G. grapsus, it could be noticed that the

specimens also jump from a rock to another, which could

be useful to escape from the wave wash or to avoid

entering into the water. They also seem to jump to a

higher rock. However, further studies on this feature are

still necessary. For example, to test if there is difference

in the jumping frequency between sexes and age classes,

as well as or to correlate the distance or amplitude of the

jumps with the body size of the crab.

Araújo, 2014


Figure 4. Crabs of the species Grapsus grapsus

(Linnaeus, 1758) from the rocky shore at Sueste

Bay, Fernando de Noronha Archipelago.

ACKNOWLEDGEMENT

The author is thankful to Maurício de Sá Leitão

Dévé, Silvia de Sá Leitão Dévé e Jean Luc Dévé for

aiding in the field work and footage of the species. I also

thank Dr. Christoph Schubart for bringing me

informations regarding crabs' behavior, which helped me

describing the 'jumpy' grapsoids of Fernando de Noronha

Archipelago.

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Araújo, 2014


http://www.marinespecies.org/aphia.php?p=taxdetails&id=106963

http://www.marinespecies.org/aphia.php?p=taxdetails&id=106963

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