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As. Pac. J. Mol. Biol. & Biotech., June 2010 Vol. 18, 241-246

Letter to the Editor: Discussion on the proposed hypothetical risks in relation to open field release of a self-limiting transgenic Aedes aegypti mosquito strains to combat dengue

Prabhakargouda B. Patil1, Mohammad Shafiul Alam2, Prakash Ghimire3, Renaud Lacroix4, Pahalagedera H. Dona Kusumawathie5, Rajib Chowdhury6, I. Pulikkottil Sunish7, Xu Libo8, Phong Tran Vu9, Moe Myint Aung10, Buyankhishig Burneebaatar11, Kanutcharee Thanispong12, D.V. Dharamsingh13 and Siraj Ahmed Khan14*

1Entomology Research Unit, Maharashtra Hybrid Seed Company Ltd, Jalna, 431203, Maharashtra, India;
2Parasitology Laboratory, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka-1212, Bangladesh;
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal;
4Oxitec Sdn Bhd, Plaza See Hoy Chan, Suite 1502, Jalan Raja Chulan, Kuala Lumpur 50200, Malaysia;
5Regional Office, Anti Malaria Compaign, Dutugemunu Mawatha, Watapuluwa, Kandy, Sri Lanka;
6WHO Regional Office for South East Asia, World Health House, Indraprastha Estate, Mahatma Gandhi Marg, New Delhi, 110002, India;
7Centre for Research in Medical Entomology, Field Station, 222, Kamaraj Road, Arakandanallur, 605752, Villupuram, Tamil Nadu, India;
8Chinese Academy of Inspection and Quarantine (CAIQ), No.a3, Gaobeidian Beilu, Chaoyang, Beijing 100025, China;
9National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Vietnam;
10Department of Preventive and Social Medicine, Defense Services Medical Academy, Mingaladon, Yangon, Myanmar;
11National Centre for Communicable Disease, NCCD, Campus Nam-Yan-Ju street, 210648, Ulaanbaatar city, Magnolia;
12Bureau of Vector-Borne Disease, Department of Disease Control, Ministry of Public Health, Tiwanon Road, Nonthaburi, Thailand;
13Madurai Institute of Social Sciences, 9, Alagar Koil Road, Madurai, 625002, India;
14Regional Medical Research Centre (NE Region), Dibrugarh, 786001, Assam, India

*Author for Correspondence.
Siraj Ahmed Khan,
Regional Medical Research Centre (NE Region) ICMR,
PO Box no. 105, Dibrugarh-786001,
Assam, India.
Email: sirajkhanicmr@gmail.com.

Abstract.
Aedes aegypti is the major mosquito vector of dengue fever (DF), dengue haemoraghic fever (DHF), dengue shock syndrome (DSS) and chikungunya around the globe (Chaturvedi and Nagar, 2008). In spite of considerable efforts to control dengue and chikungunya, these diseases remain of considerable importance around the world and the absence of specific treatment or preventive vaccine for DF/CHIK has led to a search for new technologies for vector control. Successful and effective disease-vector control strategies have been very difficult to achieve using conventional methods, due to various factors including insecticide resistance, poor knowledge of the biology of the vectors, and also limited human capacity as all the breeding sites of mosquitoes are not easily accessible particularly Ae. aegypti which prefers to breed in clean water like flower pots, tree holes etc. To over¬come these difficulties, it becomes necessary to apply selec¬tive, target-specific and site-specific control strategies based on increased knowledge of the biology of vector-pathogen-human interactions, on the improvement of existing control tools and on the development of new and innovative vector control tools and strategies. The use of innovative technolo¬gies like Genetically Modified Mosquitoes (GMM) within integrated control strategies is feasible, however for proper implementation studies on efficacy, safety for humans and the environment, ethical legal and social implications (ELSI) concerns and public acceptance should be properly addressed (Toure et al., 2004; Vasan, 2009; Wilke et al., 2009).

Earlier a UNDP–sponsored workshop on the risk assess¬ment of transgenic insects (series – 1) was co-hosted in No¬vember 2008 by Malaysia’s Ministry of Natural Resources and Environment, the Institute for Medical Research (IMR) under the ministry of Health Malaysia, and the Centre for Research in Biotechnology for Agriculture at the University of Malaya. The workshop extensively discussed the risks and benefits of three case studies: hypothetical field release of genetically modified fruit flies (Tephritidae sp.), pink bollworm (Pectinophora gossypiella) and mosquitoes (Ae. aegypti). The participants determined the potential hazards associated with these hypothetical trials, and then applied the tools of risk assessment and risk mitigation / management to determine the likelihood and consequences of the identified potential hazards, and to prepare an overall risk assessment (Beech et al., 2009). The overall risk was ranked accordingly (Viz. High – Rank 1, Medium – Rank 2, Low – Rank 3, and Negligible – Rank 4). The risk assessment carried out is concerned to the use of a transgenic Ae. aegypti mosquito expressing a fluorescent marker gene (DsRed) and a repressible lethal trait (known as RIDL) in order to suppress the target field population of Ae. aegypti in Peninsular Malaysia (Thomas et al., 2000; Phuc et al., 2007 and Lee et al., 2008). To create a pool of regional scientists well-trained in the assessment and management of biosafety issues and imple¬mentation of genetically modified disease vectors for the control of vector-borne diseases, courses on biosafety train¬ing related to potential release of genetically modified dis¬ease vectors are being conducted in Africa, Latin America and Asia and are sponsored by TDR. Recently a UNICEF / UNDP / World Bank / WHO – sponsored second Asian training course on “Biosafety for Human Health and the Environment in Relation to Potential Release of Genetically Modified Vectors” was hosted and organized on 22nd February – 5th March 2010 by the Centre for Research in Medical Entomology (CRME), Madurai, India, an Institute of In¬dian Council of Medical Research (ICMR). These 12 days of training were attended by 14 scientists working in the field of entomology, medical entomology, infectious diseases, law, social sciences, medicine, vector control and microbiology. The objectives of this Biosafety Training Course were:
1. To increase the awareness of Asian researchers and decision-makers of issues and challenges such as ethical, le¬gal and social implications related to the development and implementation of this technology.
2. To ensure the feasibility and safety of genetically mod¬ified disease vectors in Asian countries.
3. To build capacity in Asia for the safe development and implementation of this technology.

During the training program an approach was made to add to the discussion and conclusions of the previous workshop on risk assessment mentioned by Beech et al. (2009) associated with the implementation of GM technology. In this brief note, we discussed the report of risk assessment in Beech et al. (2009) for few additional potential consequences, risk mitigation / management, overall risk and ranking in response against potential hazards, and in addition we proposed and discussed other possible potential hazards as¬sociated with the release of genetically modified mosquitoes (Appendix I).

Other than the risk assessment associated with the implementation of RIDL technology we also discussed potential hazards associated with the implementation of GM mosquitoes with a female-specific flightless phenotype for population control (Fu et al., 2010) within an integrated program of mosquito management (Appendix II).

The trainees were divided into two groups of 7 in a way that ensured balance in terms of gender. Each group independently prepared and discussed risk assessments for the potential hazards identified by Beech et al. (2009) and additional potential hazards proposed by the groups. The out¬puts from each group were then discussed, combined and harmonized into a single document and summarized (Ap¬pendix I and II).

Additional hazards identified to supplement the UNDP workshop output
1. Multiple mating behaviour of female Ae. aegypti with sterile males or wild males or both: The female Ae. aegypti mosquito mates once in her lifetime and stores the sperm in the spermathecae. Release of sterile males in the environment could lead to competition between sterile and wild males. As sterile males are released at a much greater ratio than that of wild males (10:1 or greater) there is much less chance for wild male to come across a female partner for mating leading to sex-starved wild males. However it is in¬trinsic property of the male insects to search for female part¬ner for mating which may lead and/or increase the chances of forced mating by wild males with the wild females which have been already mated with the sterile males. This behav-ioural change may affect the efficacy of the SIT program and may lead to multiple mating behavioural activity in female mosquitoes. We suggest that forced mating experiments be examined under laboratory conditions. This can be done by allowing one wild female to mate with a sterile male and then introduce it into cage containing a known number of wild males and look for any possible forced mating. If forced mating occurs then one should look for the successful intru¬sion of sperm into the female genital tract / spermatheca. Further successful intrusion of sperm, if any, by the wild male mosquito into the spermatheca of the mated (by sterile male) female will lead to mixing of sperms in the spermath¬eca which will lead to competition between the fertile and ‘infertile’ sperms. Studies into this possibility are suggested.
2. Presence of tetracycline in the environment: This was thought by participants in the training to be of low risk. However, many commercially available animal feeds contain tetracycline which may lead to contamination of Ae. aegypti breeding sites leading to successful emergence of adult mos¬quitoes and affect the efficacy of the RIDL technology.

CONCLUSION
The ethical principle dictates that we should be reasonably cautious about premature use of a technology before poten¬tial risks are understood (Macer, 2007). Some have advo¬cated a total precautionary principle for genetic engineer¬ing, which means that no technology with any known risk should be attempted (Ho, 1998). The Cartagena Protocol on Biosafety, an international, legally binding agreement that regulates international movement of living modified organisms (LMOs), advises this extreme caution (CBD, 2000). Since no human action can be guaranteed to have zero risk, in practice, these principles are used to assess the relative safety of technology and are central to any public health program (Callahan, 2002). In view of this the training participants have discussed the potential hazards and risks associated with the implementation of the GM technol ogy with reference to RIDL technology and female-specific flightless phenotype for mosquito control. We reviewed the list of risks in Beech et al. (2009) and our group identified two additional risks for RIDL technology to add to the overall risk register. Both of these were related to the potential of the overall efficacy of the product to be compromised, although were thought overall to have low risk potential. In addition an attempt was made to find out the potential hazards associated with the implementation of GM mosquitoes with a female-specific flightless phenotype for population control within an integrated program of mosquito management. The groups identified eight potential hazards and discussed which were thought overall to have negligible risk.

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