Mosquito in the ointment

A U.K.-based biotechnology company is planning a large-scale trial release of modified A. aegypti mosquitoes in Maharashtra though the method has not been conclusively tested for its safety or efficacy to control wild populations of the insect vector.

Published : Jan 31, 2018 12:30 IST

A technician counting mosquito larvae in the laboratory at the Oxitec Ltd facility in Campinas, Brazil, on March 1, 2016.

A technician counting mosquito larvae in the laboratory at the Oxitec Ltd facility in Campinas, Brazil, on March 1, 2016.

YEAR after year in different parts of India, there are recurring epidemics of dengue and chikungunya. While State health departments and research organisations are trying their best to control the vector mosquito, Aedes aegypti (and also A. albopictus ), not much success has been achieved because the mosquito has a remarkable ability to adapt to changing environments: in forests, it breeds in water collected in tree holes/axils of plant leaves and in urban areas it breeds wherever rainwater collects or water is stored. The eggs can survive for very long periods in a dry state, often for more than a year. A. aegypti females take multiple little sips during multiple bites, thus increasing the number of people a single mosquito carrying a virus can infect. Conventional insecticidal measures cannot control the vector effectively, and environmental control by destroying all its breeding places is virtually impossible. Genetic engineering is a method that is being advocated vociferously.

Among numerous organisations trying to make inroads into the field of mosquito control is Oxitec, a United Kingdom-based biotechnology company that carries out genetically manipulated mosquito releases (GM release).

What is GM release? It is based on the successful control of the screwworm fly ( Cochliomyia hominivorax ), a destructive obligate parasite of animals, mainly livestock, in the Midwestern United States in the last century. These are not biting flies, but they cause extensive damage to the skin of livestock, thus affecting the hide and skin industry. The female fly mates only once in its lifetime, retaining the sperm for fertilisation of all subsequent egg batches, and after mating it seeks an open wound on the cattle host. The eggs are laid on the edge of the wound in a shingle-like manner and these develop into larvae, or maggots, that eat the flesh of the host. Since the cattle industry is important, large numbers of sterilised flies were released to control this fly.

Sterile insect technique This was the start of the sterile insect technique (SIT) programme. Edward Knipling (1960) of the U.S. Department of Agriculture (USDA) was the first to successfully use this technique against screwworm flies, which involved releasing into the environment both males and females that had been irradiated. However, SIT and other genetic strategies designed to eliminate large populations of mosquitoes rely on efficient inundative releases of competitive, sterile males into the natural habitat of the target species. It is essential that no females are released since sterile females do not contribute to sterility in natural field populations and they bite humans. Therefore, it is absolutely necessary that there is stringent separation of males from females before release.

R.S. Patterson (1970) of the USDA was the first to try this when he made a small-scale release of Culex quinquefasciatus (males sterilised by irradiation) on Seahorse Key, an island off the Florida coast. But no control was achieved.

The first large-scale release of sterilised C. quinquefasciatus mosquitoes was planned in Delhi following an agreement between the World Health Organisation (WHO) and the Indian Council of Medical Research (ICMR) under which the Genetic Control of Mosquitoes Unit (GCMU) was started in June 1970. The main reason the project was started was that PL-480 rupee funds had accumulated over a period of years and were lying unspent with the U.S. embassy in India. While the WHO had an agreement with the ICMR, it also had a separate agreement with the U.S. Public Health Service (which the ICMR later claimed it was not aware of at the time). For all meetings, a representative from the USPHS was also present in addition to WHO and ICMR officials. The objective of this project according to the agreement was to control the vectors of malaria ( Anopheles ) and filariasis ( Culex ).

But the major release programme was only with C. quinquefasciatus . Hundreds of thousands of these mosquitoes, supposedly only males, were released every day over a period of time. Even though sexual dimorphism in the size of pupae is more marked in Culex than in Aedes or Anopheles , there was about 5 to 10 per cent contamination, that is about 5,000 to 10,000 females were released per day for every 100,000 mosquitoes. The females released are likely to bite humans, and if the mosquitoes are infected (even deliberately with a dangerous pathogen), they can spread disease to humans. Thus, even an innocuous mosquito control programme can be a biohazard. It was also found during the GCMU study that the survival rate, sexual competitiveness, flight range and longevity of the released males were very much reduced compared with the wild males, and therefore they were not competitive. Thus, the whole programme to control the Culex mosquitoes was a failure.

The release of Anopheles stephensi , the vector of malaria in urban areas, was not attempted. Since there is no sexual dimorphism in the size of the pupae in Anopheline and much less so in Aedes mosquitoes, mechanical separation of males from females is just not possible. However, a lot of work was done by the project on factory-scale mass production techniques for A. aegypti , the natural vector of several arboviruses found in India such as dengue, chikungunya, yellow fever (human cases not reported in India) and Zika. A. aegypti is a well-known biological warfare weapon, according to numerous publications. Therefore, there was real apprehension in the minds of many about why the GCMU was working on A. aegypti .

It may be mentioned that the whole programme was at the initiative of the USPHS, which operated from Fort Detrick, the centre for U.S. biological warfare research. The WHO’s role was purely that of a broker. A controversy started when Hans Laven, who did research work on C.quinquefasciatus , wrote an article for the German media condemning the use of thiotepa for chemosterilisation by the U.S. group in the project, led by Patterson, claiming it was carcinogenic. (Irradiation was also used to sterilise the males.) There was also a lead story in the Indian newspaper National Herald about the carcinogenic effects of thiotepa, which was used for sterilising mosquitoes in the GCMU.

These stories led K.S. Jayaraman, the science reporter of the Press Trust of India, to investigate. His detailed probe exposed many chinks in the armour of the WHO’s project, and this finally led to a detailed parliamentary inquiry led by the Public Accounts Committee (PAC). After all aspects of the work of the project were analysed, the 167th and 200th reports of this committee recommended the closure of the GCMU in June 1975. The then Prime Minister of India, Indira Gandhi, then set up a committee that included M.S. Swaminathan and M.G.K. Menon, both renowned scientists of international repute, to prepare guidelines for future collaborative research. There were stringent conditions to be enforced by a committee in the Health Ministry for all collaboration with foreign agencies.

The GCMU project initially made valuable studies on the ecological and behavioural aspects of C. quinquefasciatus . It did not do anything to control the malaria vector Anopheles stephensi . But it concentrated on Aedes aegypti , which is not a vector of human malaria or filariasis. Techniques for the production of A. aegypti mosquitoes in the millions were perfected, and gadgets that could be mounted on cycles were developed for their mechanical dispersal. A large-scale release of this dangerous mosquito was planned in the crowded city of Sonepat, Haryana (but was stopped at the last minute by the government). The full details of the GCMU from its opening until its closure are available in “The Story Behind WHO Exposure”, an article written by C. Raghavan, former Chief Editor of the Press Trust of India, in Mainstream Weekly (May 17, 1975).

Oxitec has been in the picture for a long time with its GM releases. It was active in Brazil during the 2014 Zika epidemic, but there are conflicting reports about whether it succeeded or not in controlling mosquito populations. A June 2012 report in Lankaweb by Shenali Waduge said that in 2009 Oxitec released large numbers of genetically modified mosquitoes to combat dengue fever in Grand Cayman, an island in the Caribbean. In 2010, three million genetically modified mosquitoes were released. The catch is that the release was done in secret.

Secret releases EnviroNews World News stated that Oxitec had released GM mosquitoes into the wild in several places in the past, and the release was conducted in secret, but “when locals became aware of it, they were left feeling upset and disenfranchised. Following the Grand Cayman release, Oxitec went forward and ‘released their frankenskeeters in Malaysia, Brazil, Panama, India, Singapore, Thailand, and Vietnam’, wrote Dr [Joseph] Mercola…. It should be stated that those tests abroad, although secretive and out of the public eye in most cases, were not carried out by Oxitec alone, rather, in tandem with enthusiastic and compliant participating mosquito control agencies in those countries. For example, The Mosquito Research Control Unit (MRCU) was behind the release in Cayman. The Institute of Medical Research (IMR) in Malaysia—Moscamed and University of Sao Paulo in Brazil and the Gorgas Memorial Institute in Panama, all were behind the tests in their respective countries. In the case of the U.S., the Florida Keys Mosquito Control District decided to propose the experiment, despite widespread opposition in the targeted Key Haven area. Since millions of these mosquitoes have already been released into wild populations, using mostly poverty-stricken countries as the guinea pigs, some people want more comprehensive research to be done on the long-lasting ramifications in those areas first, places where the horse has already been let out of the barn, before any more of these ‘frankenskeeters’ are put on the loose. Oxitec and their enabling counterparts were able to go forward releasing the GM mosquitoes into the wild, in part, because there are currently little to no regulations in place prohibiting it. In the U.S., until legislators get involved and bring EPA [U.S. Environmental Protection Agency] and/or FDA [U.S. Food and Drug Administration] regulations up to speed on the issue of releasing GM insects and animals into the open environment, companies like Oxitec will continue their experiments in the wilderness areas surrounding local communities, utilising Americans, the ecosystem and the whole planet, for that matter, as their test subjects.”

Recently, there have been a series of articles on the use of mosquitoes infected with the bacterium Wolbachia for mosquito vector control and to interrupt the transmission of the dengue virus. There are many parasites and pathogens naturally occurring in the environment that infect mosquito larvae. Wolbachia is only one of them, and it is reported to stop the virus from replicating in mosquitoes that transmit dengue. Therefore, to reduce infections in humans, one can release Wolbachia -infected mosquitoes to mate and pass the bacterium to future generations. If the method works, vast numbers of wild mosquitoes will eventually carry Wolbachia and will be unable to transmit dengue. But where is the technology to do this?

In a detailed study on Wolbachia , S. Sabesan and others of the ICMR (2017) said that the idea of releasing mosquitoes infected with Wolbachia was not practical in the field, and added: “At the moment, we do not have much claim to success in our war against mosquito vectors, except in situations where there has been vector habitat destruction. The mosquito vector host(s) and parasites and/or endosymbionts will try to adapt themselves to maintain a balance in nature. However, it is possible to keep the vector mosquitoes at bay by adopting simple environmental management and sanitation.”

Some more experiments are planned in India, and the health authorities seem to have signed a memorandum of understanding with Monash University, Melbourne, Australia, to use Wolbachia -infected mosquitoes. They say that the dengue, Zika or chikungunya viruses cannot replicate when mosquitoes have Wolbachia . It is claimed that unlike the Release of Insects carrying Dominant Lethal (RIDL) genes technology, Wolbachia is self-sustaining, making it a low-cost intervention. The downside is that the release of even a single female mosquito infected with Wolbachia could “potentially lead to the alien bacteria spreading in the target population”, said a report. Megan Carter ( Science , August 11, 2015) said that Wolbachia releases also had dangerous potential. Gabriel Hamer of Texas A&M University said “it’s so hard to go from conceptual, theoretical consideration that this should work to a field trial” that actually demonstrates less human disease. “So far, with all these Wolbachia efforts around the world, I haven’t seen any overwhelming success stories.”

But history has shown that it is not necessary to release genetically manipulated mosquitoes to control wild populations. Why not emphasise environmental control, supplemented with the judicious use of insecticides? India has been doing this for years. Only more efforts are needed. But the problem is enormous in a large country like India with many different ecological diversities. It is extremely difficult, if not impossible, for the authorities to effectively control mosquitoes.

Alarming news A most alarming piece of news, which prompted this story, is a report that appeared in the Media Scanning and Verification Cell of the Integrated Disease Surveillance Programme, National Centre for Disease Control, Directorate General of Health Services, (ID no. 2317 of 7.1.2018) on how Oxitec is working again in India.

It states: “In the normal course, one would expect that the imperialism that sees Third World countries as suitable guinea pigs for risky biological research would arise from the United States, but in the case of GM mosquitoes, the threat has arisen from the Oxford University in the U.K., an institution that after 71 years of ‘freedom’ India’s political, administrative and intellectual leadership still venerates. A subordinate department of Oxford University, Oxitec began experimenting with genetically modified Aedes aegypti mosquitoes, which are carriers for the virus that causes dengue and Zika. Soon they began creating problems, which seem not to have been brought to the attention of the authorities in India, who for long have been falling over each other to pander to the demands of foreign ‘health’ researchers, especially in the hugely risky field of vaccines. The modified Aedes aegypti mosquitoes were released into the atmosphere in Brazil in 2014 and created severe problems there. However, they have now been brought for trials to India. The trials began in Maharashtra on 23 January 2017. These insects were created in Oxford labs using the RIDL technology (Release of Insects carrying Dominant Lethal genes).”

It goes on: “In the case of the RIDL technology, the genetically altered male mosquitoes have to be released in large numbers at regular intervals. Only then can they compete with the wild normal male insects for mating. Since the larvae die before reaching adulthood, the technology is a ‘self-limiting approach (the genetic modifications are not perpetuated in wild populations)’ notes a piece published in The Lancet (February 1, 2016).” Recently appointed Deputy Director General of Programmes of the WHO, Dr Soumya Swaminathan, has been on record saying that in any genetic releases more than 100 such GM mosquitoes would be needed per person in the targeted region, that too for months together, to sustain any hope of exterminating A. aegypti mosquitoes. This gives one an idea about the magnitude of the problem.

When it was reported that Oxitec conducted small-scale field trials in Maharashtra on January 23, 2017, and planned to carry out large-scale field trials in three districts in the State, Jayaraman wrote to the company for details. It replied: “Following the positive laboratory assessment previously described, we are now ready to conduct the next stage of evaluation under natural environmental conditions.... Progress towards evaluations outside laboratory conditions will only be possible following approvals by the Indian regulatory bodies: the Review Committee on Genetic Manipulation (RCGM) and the Genetic Engineering Approval Committee (GEAC).” Oxitec called the January 23 cage trials “positive laboratory assessment”.

The results of the cage experiments are not explicit, particularly about the “separation of sexes”. In a small laboratory cage experiment, you can release males only after separating the sexes of a small number of mosquitoes mechanically by hand in a Petri dish. But in all Oxitec trials in other countries, it has always been significantly silent on the percentage of contamination by females in any release. Very large numbers of males have to be released for them to be competitive. Before any approval is given for field trials, the authorities should insist that not a single female mosquito is released as they bite and can transmit infection (natural or induced).

On the documents released by Oxitec, GeneWatch said the information showed that the project release was ineffective and posed risks to the community. These data were not available to the National Conservation Council when it cleared the way for the proposed island-wide roll-out of the GM mosquito project and revealed how in some cases notable numbers of biting female mosquitoes had found their way into the batches for release of male mosquitoes. The activists said that Oxitec had struggled to suppress the wild population of mosquitoes, and the technique had only had an effect in the dry season, when the numbers of mosquitoes were low, combined with insecticidal spraying. “Plans to roll out Oxitec’s GM mosquito releases anywhere must be halted whilst this new information is properly considered,” said Helen Wallace, director of GeneWatch UK. “Oxitec’s GM technology is failing in the field and poses unnecessary risks.”

Testing ground As far as India is concerned, Jayaraman warned: “If approved by its regulators, India will become the testing ground for a new but controversial technology.… Using gene-editing technology, researchers at the University of California (UC)—at its campuses in Irvine (UCI) and San Diego (UCSD)—modified the Anopheles stephensi mosquitoes to create a strain that resists infection by Plasmodium falciparum , the parasite that causes malaria…. And employing what is called ‘Gene Drive’, they showed that the gene which prevents malaria transmission in these genetically modified mosquitoes can be passed on to their offspring. This implies that, in several generations, the entire population of Anopheles stephensi in the wild can potentially be replaced by one incapable of transmitting malaria. But, will this silver bullet that works in laboratory perform in the real world? That is what the U.S. team is planning to find out using malaria endemic India as the test bed for their experiment under a generous $70 million (Rs.460 crore) grant from India’s Tata Trusts.”

Therefore, it is obvious that the release of genetically manipulated vector mosquitoes not only is ineffective but also poses a great danger to society. As already stated, A. aegypti mosquitoes and the viruses they transmit are potential biological warfare agents, and countries like the U.S. have been working on this. Bharat Dogra wrote (in Mainstream Weekly , March 27, 2010): “Senior American journalist William Blum reported: ‘In 1956 and 1958, declassified documents have revealed that the U.S. Army let loose swarms of specially bred mosquitoes in Georgia and Florida to see whether disease carrying insects could be weapons in a biological war. The mosquitoes bred for the tests were of Aedes aegypti , the precise carrier of dengue fever as well as other diseases. In 1967, it was reported by Science magazine that at the U.S. government centre in Fort Detrick, Maryland, dengue fever was amongst those diseases that are at least the object of considerable research and that appear to be among those regarded as potential biological warfare agents.’”

How could India allow such things to happen, especially after its unpleasant experience in 1970 with the GCMU project? There are many other projects being undertaken at present in India in the health sector with foreign collaboration. In one long-term project by a research institute in India in collaboration with a U.S. university, the main role of the Indians is to ship P. falciparum -infected blood samples to their counterparts in the U.S. Of course, the Indian scientist collaborators do make frequent visits to the U.S., for “discussions”. As far as I know, the outcome of this long-term collaboration is inconclusive.

Are any of these projects scrutinised by the special committee in the Health Ministry that was supposed to be set up in accordance with the PAC recommendations? No wonder Oxitec gets a free ride in India. The ICMR does not seem to have much of a say in the matter, though its representative attends all the meetings. The Department of Biotechnology’s RCGM prepares guidelines to regulate programmes on GM organisms and applications, including in industry, with a view to ensuring environmental safety. The ICMR participates in the meetings, and one would have thought that it would have blocked Oxitec from carrying out these trials.

India publishes a large number of scientific papers in the health field every year, many of which pertain to genetics, molecular biology, biochemistry, biotechnology, etc., and, in recent years particularly, to genetic control of mosquitoes, gene transfers, etc. Projects on ecological and environmental studies for which one has to carry out fieldwork have been denied funding.

How can one dream of eradicating the mosquito, which adapts itself to any environmental condition and which has evolved over millenia, through modern genetic tools? Gone are the days when scientists such as Fred Soper, Paul Russell, Lewis Hackett and Karl Meyer and, in recent years, L.J. Bruce-Chwatt, Ramachandra Rao and the many scientists of the erstwhile Malaria Institute of India who worked in the field achieved a lot in controlling malaria and mosquitoes. Great work was done before Independence, for example, by Ronald Senior-White, who carried out extensive studies on mosquitoes while constructing the Jeypore (Odisha)-Visakhapatnam (Andhra Pradesh) railway line; P.J. Barraud and S.R. Christophers, who wrote books; and Stuart Baker, Ronald Ross and others. Fred Soper controlled malaria in Brazil and yellow fever in the Panama Canal Zone through environmental sanitation. New research schemes with utopian goals are formulated and get funding.

The WHO and its tropical diseases research programmes are now product-development oriented with commercial motives. There are many examples of this in the health field. The Oxitec story is one such. Many people in India make a lot of money in the name of mosquito control (like the pesticides lobby), and why would Oxitec also not want a finger in the pie?

P.K. Rajagopalan is a former Director of the Vector Control Research Centre in Puducherry, an institute of the Indian Council of Medical Research.

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