Science in Society Archive

Important Books & Reports

Banishing Glyphosate

Banishing Glyphosate - Dr. Eva Sirinathsinghji, Dr. Mae-Wan Ho and others

Glyphosate/Roundup, falsely claimed by Monsanto to be safe and harmless, has become the world’s most widely and pervasively used herbicide; it has brought rising tides of birth defects, cancers, fatal kidney disease, sterility, and dozens of other illnesses - more

Ban GMOs Now

Ban GMOs Now - Dr. Mae-Wan Ho and Dr. Eva Sirinathsinghji

Health & environmental hazards especially in the light of the new genetics - more

Living Rainbow H2O

Living Rainbow H2O - Dr. Mae-Wan Ho

A unique synthesis of the latest findings in the quantum physics and chemistry of water that tells you why water is the “means, medium, and message of life” - more

The Rainbow and the Worm - the Physics of Organisms

The Rainbow and the Worm - the Physics of Organisms - Dr. Mae-Wan Ho

“Probably the Most Important Book for the Coming Scientific Revolution” - more

Transgenic Mosquitoes Not a Solution

All attempts at controlling the spread of diseases by transgenic mosquitoes are ineffective, inefficient, costly, and hazardous to varying degrees Dr. Mae-Wan Ho

Considerable controversy has been aroused by Oxitec, a UK company based in Oxford, over its releases of transgenic mosquitoes designed to control the spread of dengue fever, which at the same time, highlighted the gross inadequacy of the regulation of transgenic insects releases worldwide  [1] (Regulation of Transgenic Insects Highly Inadequate and Unsafe, SiS 54).

More important than the adequacy of regulation is whether transgenic insects are a solution to the problem of disease control.

Population reduction by introducing dominant lethal gene

Transgenic mosquitoes such as those created by Oxitec are designed to reduce or eradicate natural populations of disease vectors, whereas newer approaches are designed instead to replace natural populations with the minimum of ecological disturbance.

For far too long, the extermination of the insect vector has been the preferred option to controlling the spread of infectious disease, starting with DDT and other toxic insecticides.  Not surprisingly, the first efforts in creating transgenic mosquitoes followed the same path, and Oxitec was no exception. It consisted in the ‘release of insects with a dominant lethal’ (RIDL) intended to reduce natural populations [2] (see Terminator Mosquitoes to Control Dengue? SiS 39). Oxitec’s transgenic mosquitoes were created with the transposon (jumping gene) piggyBac, the dominant lethal gene incorporated is tTA, coding for the tetracycline-repressible transcription activator protein, which when expressed at high levels kills the developing embryo, for reasons still unknown ([2, 3] Can GM Mosquitoes Eradicate Dengue Fever, SiS 50). When expressed, the tTA protein binds to the tetO operator upstream of the tTA transgene and drives more synthesis of tTA in a positive feedback loop. In the presence of tetracycline, however, tetracycline binds to the tTA protein, and prevents it from binding to tetO, thereby turning off the synthesis of more tTA, allowing the insect embryo to survive.

Thus, the transgenic mosquitoes can be grown in the presence of tetracycline. The adult male mosquitoes (which do not bite humans) are sorted from the females that do bite and transmit disease.  The sorted males are then released into the field to mate with wild-type female mosquitoes; whereupon the progeny will be killed in the absence of tetracycline, and the wild population is decimated. That is the theory, as far as it goes.

Oxitec transgenic mosquitoes inefficient and unsafe

Oxitec claims that results from Cayman trials showed a reduction in Aedes aegypti populations by 80 % [4], while the journal Nature reported on its News Blog [5] that “the controlled release of male mosquitoes genetically engineered to be sterile has successfully wiped out dengue fever in a town of around 3, 000 people, in Grand Cayman”. Both reports are inaccurate [6], if not outright false.

The mosquitoes are not sterile and Oxitec never successfully eradicated dengue fever from any population; furthermore, dengue is not endemic in the Cayman Islands.

The mosquitoes, engineered to be dependent on tetracycline, are not completely killed in its absence; as some 3 to 4 % of the mosquitoes embryos survive to adulthood in the laboratory [7]. Tetracycline is a very common antibiotic in the environment, particularly in sewers, septic tanks and water treatment plants, common breeding grounds for A. aegypti. Oxitec admitted that survival rates could be as high as 15 % in the presence of contaminating levels of tetracycline [8]. Moreover, Oxitec’s system of sorting males from females is not perfect and up to 0.5 % of the released mosquitoes could be female [7].

Much of that was confirmed in publications that appeared in the biotech-friendly 

journal Nature Biotechnology since. The journal’s news report of the experiment presented a positive spin, if you only read the headline [7]: “Results from the first open-field trial of transgenic mosquitoes bode well for large-scale release to fight infectious disease.” Only in the final paragraphs are the problems revealed. The transgenic males were only half as successful as wild-type males in mating with the wild-type females, but still, they were better than the sterile male Mediterranean fruit flies used worldwide to protect fruit and vegetable crops.

“The results are promising, but whether they portend successful sterile insect release against A. aegypti is questionable” [7], the commentators stated, for three reasons. First, they questioned the reliability of the field data; too few traps were set, and only a small number of larvae were scored for paternity, to determine whether they came from the transgenic males or wild-type males. Second, implementation of RIDL-based dengue control will require large-scale systems for producing the insects, transporting and releasing them. Not only is basic research for optimal procedures missing, there are not even established standards for assessing insect quality along the entire rearing to release pathway; for example, the sorting of males from females based on size of the pupae nevertheless left 0.5 % females in the released population. And most importantly, the progeny of crosses between transgenic males and wild type females in the laboratory survived at the “disturbingly high rate of 3.5%.” This certainly does not sound like a ringing endorsement.

There was nothing in Oxitec’s own report in the journal [9] that contradicted the assessment of the commentators.

One immediate concern [6] is that decline in the natural populations of A. aegypti could leave an ecological niche to be filled by other, possibly more harmful pests. For example, the Asian Tiger mosquito, A.  albopictus is considered one of the most invasive species and carries many diseases including dengue fever and the West Nile virus. Another possibility is that the dengue virus could also evolve and become more virulent.

Serious potential health hazards are associated with high levels of the transgene product, the tTA protein, which could easily be injected into the bloodstream of humans by biting females, not to mention the propensity of the piggyBac vector integrated transgene -sequences to remobilize and transfer horizontally to human cells (see [1]).

The problems associated with Oxitec’s transgenic mosquitoes are to varying extent generic to transgenic mosquitoes and other insects.

Problems generic to transgene insect technologies

As mentioned in relation to Oxitec’s transgenic mosquitoes, the technology requires continual releases of lab-reared transgenic males in order to keep the natural population down [8].  We shall see that even with strategies involving population replacement, continued releases are also required.

A review published in 2012 states [10]: “Of particular concern for practical transgenesis applications are the difficulties in defining and standardizing the long-term effectiveness of transgenic manipulations.”

Apart from its relative inefficiency, transgene expression is variable and can even be lost from established transgenic lines. For example, the activity of a gene that suppresses dengue virus in a transgenic strain of A. aegypti was lost after 17 generations [11].

Loss of transgene activity could be due to gene silencing or actual loss of transgene through remobilization of integrated transgenes. Transgenic lines created with transposon- vectors – applying to most of them including the Oxitec transgenic mosquitoes - are subject to instability due to remobilization of the transposon-derived sequences. Remobilization can result in horizontal gene transfer to unrelated species. The first generation of transposon vectors are probably the worst offenders as I have commented [12] (Terminator insects give wings to genome invaders, I-SIS report): “These artificial transposons are already aggressive genome invaders, and putting them into insects is to give them wings, as well as sharp mouthparts for efficient delivery to all plants and animals and their viruses.”

Safer transgenic options still inefficient

There are now more up-to-date, safer vectors for creating transgenic mosquitoes and other insects  that can target the transgenes more precisely into the genome and increase their stability of expression, as well as decrease their tendency to remobilize [10].

One of the best researched examples is the ‘selfish gene’ Medea model [13], which aims to replace the natural population with one that confers resistance to disease or parasite, rather than decimate it. The goal is to create the minimum ecological disturbance, and if the genetic modification is precise and well designed, it also causes minimum physiological and biochemical changes to the mosquito vector.

Medea elements were first identified in the flour beetle Tribolium castaneum through crosses between geographically isolated strains. They are located at a fixed position in the genome, and when present in females, only progeny that inherit the element-bearing chromosome from either the maternal and/or paternal genome survive. In contrast, Medea-bearing males give rise to wild-type and Medea-bearing progeny with equal frequency when mated to wild-type females. One Tribolium  Medea gene, MedeaM1 has been mapped, and is associated with a composite Tc1 transposon that includes a number of genes. Genetic analysis suggests a model in which Medea consists of two tightly linked loci: one that encodes a toxin passed on to all progeny via the egg, and a second that encodes an antidote active in the zygote.        

A synthetic Medea element Medeamyd88 was created, in which a modified version of maternal-specific bicoid promoter was used to drive the expression of a transcript encoding two synthetic microRNAs designed to silence the expression of myd88, a maternally expressed protein required for dorso-ventral pattern formation (by binding to complementary base sequences on the transcript, thereby preventing its translation into protein). This kills the embryo if left un-neutralized. The linked antidote gene encodes a microRNA-insensitive version of the myd88 transcript lacking the target sites present in the maternal transcript, and is placed under the control of a transient early zygote-specific promoter. Expression of the antidote occurs early enough that the rescued embryos develop normally. In a lab experiment, introducing Medeamyd88 into a wild-type population with a 1:1 homozygous Medea male/wild-type male ratio resulted in the entire population carrying at least one copy of Medea after 10-12 generations.

Elements designed so that the maternally expressed microRNAs were located in an intron of the antidote also showed Medea-like behaviour. This configuration prevents recombination from creating Medea elements that lack the Medea effector or the antidote-only elements, each of which can lead to the appearance of wild-type individuals. Other implementations are possible.

Thus, no foreign proteins are introduced into the natural population; there is only a shift of a gene from mother to the zygote. The use of miRNAs to generate a pre-toxic state provides an important degree of redundancy because multiple microRNAs each processed and functioning as an independent unit can be linked into a polycistronic transcript.

However, as the researchers are aware, transgene silencing can still occur. The best that can be done is to try and wall off the transgenes from the effects of repressive chromatin that would silence the expression of genes. This can be achieved, at least to some extent by flanking the Medea construct with sequences that confer boundary/insulator function.

Still, as the researchers point out, populations subject to replacement will always need maintenance and modification over time. In particular, it is likely that first generation transgenes will mutate to inactivity, become silenced and/or lose effectiveness as the pathogens adapt and become resistant. Further rounds of population replacement with novel toxin-antidote combinations are necessary.

To conclude

Transgenic mosquitoes as a strategy to controlling natural infectious disease vectors are ineffective, inefficient, costly to implement, and hazardous to varying degrees. More than a decade of dedicated efforts have not resulted in major advances. It is against such a background that the latest non-transgenic approach appears to be such a perfect solution (see [14] Non-transgenic Mosquitoes to Combat Dengue, SiS 54).

Article first published 07/03/12



References

  1. Ho MW. Regulation of transgenic insects highly inadequate. Science in Society 54 2012.
  2. Cummins J and Ho MW. Terminator mosquitoes to control dengue? Science in Society 39, 33-35, 2008.
  3. Cummins J. Can GM mosquitoes eradicate dengue fever? Science in Society 50, 48-49, 2011.
  4. March 2011 Newsletter.” Oxitec, Mar. 2011. Web. <http://www.oxitec.com/our-news/newsletters/march-2011-newsletter
  5. “GM Mosquitoes Wipe out Dengue Fever in Trial.” Nature News Blog. Nature, 11 Nov. 2011. Web. http://blogs.nature.com/news/2010/11/gm_mosquitoes_wipe_out_dengue.html
  6. Genetically engineered mosquitoes in the U.S. Issue Brief, Friends of the Earth, Washington DC, http://www.biosafety-info.net/file_dir/21277023124f348b11ef3c4.pdf
  7. “Road test for genetically modified mosquitoes”, Todd Shelly & Don McInnis, (News and Views), Nature Biotechnology 2011, 29, 984-5.
  8. Oxitec statement in response to NGO allegations. Press release, 12 January, http://www.oxitec.com/2012/01/press-release-oxitec-statement-in-response-to-ngo-allegations/#more-3170
  9. Harris, AF, Nimmo D, McKenny AR, Kelly N, Scaife S Donnelly CA, Beech C, Petrie WD and Alphey L. Field performance of engineered male mosquitoes. Nature biotechnology 2011, 29, 1034-9.
  10. Fraser Jr MJ. Insect transgenesis: current applications and future prospects. Ann Rev Entomol 2012, 57, 267-89.
  11. Franz AW, Sanchez-Vargas I, Piper J, et al. Stability and loss of a virus resistance phenotype over time in transgenic mosquitoes harbouring an antiviral effector gene. Insect Mol. Biol. 2009, 18:661–72.
  12. Ho MW Terminator insects give wings to genome invaders. I-SIS Report, 19 March 2001,   http://www.i-sis.org.uk/terminsects-pr.php
  13. Ho MW. Non-transgenic mosquitoes to combat dengue. Science in Society 54 2012.

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There are 3 comments on this article so far. Add your comment above.

Todd Millions Comment left 11th March 2012 09:09:09
I can't restrain from asking-As well as the transpond leaks,has oxitec/epa quango completed their survey showing no background levels of tetracycline ozze from the swamp water bacterial culture that the drug was origianaly developed from?I await such biblical works of fiction with-quivering anticipation.

henrymark101 Comment left 19th April 2013 08:08:05
Nice title of the post as well as content on Transgenic Mosquitoes. I like your post and the content that i want to get. Thanks for the sharing. You just describe the topics are wonderful and more useful topic is the insect technologies. We hope that we will get more from you...!!!

Alice Br Comment left 31st January 2016 07:07:33
You do realize that this article explains why all the babies with missing frontal lobes are being born is area where these mosquitoes have been released. Its not the Zika virus its the tTA protein gene expression kill switch in the mosquito. The author of this article was right.

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