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A letter from Pesticide Action Network Asia/Pacific on the application by the Malasian Institute for Medical Research to release GM mosquitoes into the wild. We refer to the public announcement by the National Biosafety Board of Malaysia about the application by the Institute for Medical Research (IMR) for the release of genetically modified male Aedes aegypti mosquitoes in Pahang and Melaka (referred to as Living Modified Organisms or LMOs of the OX513A strain) (Reference No. NRE(S) 609-2/1). We have serious concerns and objections. First of all, there is a lack of transparency and information about the genes involved in the genetic engineering of the mosquito. For example, how is this male LMO ‘created’? Is there not the risk of a margin of error that might allow female LMOs to be selected in the process? What are the sources of the molecular marker and the ‘lethal’ gene that will make the offspring of the LMO and a female Aedes aegypti die? This is very critical. The technique apparently employed in this IMR project seems to be the one called “Released Insects with a Dominant Lethal” (RIDL) which is a tetracycline-repressible lethal system, utilizing the piggyBac transposon. If the key gene that confers the dominant lethal trait is tTAV, a protein, — and we do not know this for sure since the IMR refuses to release the information — then in the absence of tetracycline, the mosquito offspring of the LMO will likely die from the toxic effects of the over-production of tTAV. If such a gene is the one causing fatality to the offspring of the LMO, then what is the precise mode of action of the tTAV protein? Its mode of action and how it leads to the death of the mosquito offspring/organism exactly appear unclear and little understood. This should be clarified and investigated before any open releases are considered, as it may have environmental or health consequences as well as carry risks arising from horizontal gene transfer. The public announcement and fact sheet do not look at the possibility of new health risks to humans and animals arising from the genetically modified mosquitoes, in particular if female LMOs are released accidentally or female progenies from the released male LMOs somehow survive. In relation to the latter, Phuc et al. [1] state that 3-4% of the first larval instar of OX513A do survive to adulthood. Thus the IMR fact sheet is not quite accurate in stating that the presence of the “conditional lethality trait” in OX513A progenies is fatal; “resulting in the death of the progenies in the absence of tetracycline”. The figure for 3-4% is given for laboratory experiments. What is the figure for field cage trials? Different conditions (biotic and abiotic stresses) need to be tested for changes in (a) the survival rate of OX513A mosquitoes and (b) phenotypic and behavioral characteristics. Please let us briefly explain our concern regarding the use of a seemingly untested protein. As an example, Bt crops like cotton and corn are genetically engineered with the Bt-toxin gene from the soil-bacterium Bacillus thuringiensis (Bt). There are many different forms of and genes for Bt toxins—the most commonly used are Cry1Ab and Cry1Ac. Cry1Ac has been found to be a potent immunogen. It binds to gut cells and is capable of causing changes in the permeability of the gut (e.g. [2-5]). Other examples of unpredicted immunogenicity or toxicity are two food products. In the 1990s, in feeding trials with rats (and mice), genetically engineered (GE) tomatoes in the US (Clagene) as well as GE potatoes in the UK [6,7] were found to cause damage to the gut and its mucosal cell lining. In both cases, the transgenes used were coding for proteins regarded as harmless when ingested by mammals. Another major risk in the IMR project is horizontal gene transfer of the piggyBac insert, which contains the two transgenes. According to a paper by Ho and Cummins [9], the risk of the transgenes being transferred horizontally to other species is highly increased due to their combination with the piggyBac transposon. The risks of such transposons transferring to the genomes of the mammalian hosts should be investigated, including the possible transfer to laboratory animals used as blood meal donors for female LMO mosquitoes. This is relevant at this present stage as there will potentially be females amongst the released LMO mosquitoes. The male LMOs have to be sorted from the females, and this takes place at the pupae stage, when males are generally smaller than females. This, however, is unlikely to be 100 per cent accurate. It is obvious that transgene escape can readily occur, whether horizontally or vertically (via sexual reproduction). The enhanced possibility of horizontal gene transfer is only one possible effect of genetic engineering. Transgenes as well as the insertion of transgenes via genetic engineering are known to give rise to other unexpected, unintended, positional, synergistical, or pleiotropic effects [10]. As an example, one study in 2005 looked at GE peas that had been genetically engineered with a bean gene. Unexpectedly, the protein product from the bean gene changed its characteristics when produced in peas and caused immune reactions and inflammation in mice, not seen with the bean [11]. This provides evidence that a gene may behave differently when transferred from one organism to another, even if the two organisms are very close from an evolutionary standpoint. The relevance of this for the given situation is that there are likely to be changes in the GE mosquito other than the intended or expected ones. These would include changes in genoptypic, phenotypic or metabolic levels as well as behavioural levels. Genetically engineering a mosquito, which is a vector of disease, may give rise to unexpected effects that may include negative impacts on human and animal health, for example, the insect may become more virulent, aggressive or its bite might have different effects on the host. The proposal by the IMR to do fogging after the release is also fraught with contention. Fogging with resigen (active ingredients: S-bioallethrin and permethrin) means spraying communities and the environment with poisonous pesticides. Both are pyrethroids which have been linked to toxicity in humans including carcinogenicity, reproductive and developmental toxicity, and neurotoxicity as well as acute toxic effects such as coughing, redness, burning sensation/pain in the eyes and skin, dizziness, headache, fatigue, nausea, listlessness, vomiting, epigastric pain, muscular fasciculation [12,13]. These pyrethroids can be inhaled or ingested (directly or through water). Permethrin has also been found to have potential to be an endocrine disrupter [14]. Besides this, fogging is ineffective in controlling mosquitoes because it is not targeted but simply sprayed all over the area, allowing a large proportion of mosquitoes to escape. Last but not least, involving the communities that will be affected by the release as well as the public at large is a matter of public trust. The effects of the genetically engineered mosquito including its molecular marker and the ‘lethal’ gene (assumed to be tTAV) on fish, frogs or other organisms present in the environment that might feed on it, and its possible effects on humans or other mammals have not been tested. Before any open release, this information must be determined, especially since there is risk of survival of the GE mosquito offspring. continued added by: JanforGore