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Here Come the Terminator Mosquitoes, Genetically Engineered Insects 5-16-08

Why take a dangerous, costly, and untested high tech option when safe, effective, and affordable alternatives are available?

Millions of transgenic mosquitoes are to be released into the fishing village of Pulau Ketam off Selangor, Malaysia, as part of an international series of field trials to fight dengue fever [1]. The Malaysian field trials will be undertaken by the Health Ministry's Institute of Medical Research (IMR) in collaboration with Oxitec Ltd., a spin-off biotech company from the University of Oxford in the UK. This follows the reported success of confined laboratory trials conducted under the supervision of the IMR over the past year.

The technique, which has won Oxitec the Technology Pioneers 2008 award at the World Economic Forum, involves releasing transgenic male Aedes mosquitoes carrying a ‘killer' gene to mate with wild female mosquitoes, which causes (nearly) all their progeny to die. This is a variant of the Sterile Insect Technique (SIT) that has been successfully used in wiping out other insect vectors in the past [2], though the sterile males were created by X-irradiation, and not by transgenesis.

The release of sterile males is considered “environmentally benign” [2], as only female mosquitoes bite and suck blood and transmit the disease-causing virus; not the male mosquitoes.

If the Pulau Ketam trials are successful, the transgenic killer mosquitoes will be released in bigger towns which have a high incidence of dengue [1]. Dengue is reported to be the fastest growing vector-borne disease in the world, affecting 55 percent of the global population with an estimated 100 million cases in over 100 countries. Chikungunya, a disease similar to dengue fever and also spread by the Aedes mosquito, has become a major problem, at least in India, where there were 140 000 cases in 2007.

Oxitec has received regulatory and import permits for confined evaluation in the US, France and Malaysia, while still holding discussions with regulators of other endemic countries such as India.

Environmental groups fear that releasing the transgenic mosquitoes may affect the ecosystem and cause further damage. But there has been remarkably little informed reporting on the nature of the potential hazards involved.
What is dengue?

Dengue fever is an illness caused by an RNA flavivirus spread by the bites of mosquitoes. The symptoms include fever, headache, rash, severe pains in the muscles and joints, and pain behind the eyes. Dengue fever is rarely fatal, while the related dengue hemorrhagic fever is a severe disease that leads to death in approximately 5 percent of cases. Dengue hemorrhagic fever is seen most often in children younger than 15 years old. It is also seen most often in individuals who were previously infected with simple dengue fever [3].

The dengue flavivirus occurs in four different serotypes, DEN-1, DEN-2, DEN-3, and DEN-4. Contracting one form of dengue fever provides lifelong immunity from that serotype, but not from the other serotypes. Cases of dengue fever occur primarily in urban areas in the tropics. Humans contract dengue fever from bites of infected female mosquitoes of the genus Aedes . Aedes aegypti is the primary vector in most regions. When a female Aedes mosquito bites a person infected with dengue, the virus incubates in the insect body for 8-11 days, after which the mosquito can spread the disease to other humans for the remainder of its life span (15-65 days). Once the virus enters a human, it circulates in the bloodstream for two to seven days, during which time the virus can spread. Aedes albopictusis was originally the primary vector of dengue fever, and remains a major vector in Asia. The species has recently spread to Central America and the US, where it is a secondary vector of the disease . Aedes aegypti is primarily urban, and Aedes albopictusis rural, thereby increasing the ecological range of habitats in which people can become infected. Humans are the primary reservoir for the virus [3].

In recent years, dengue has spread extensively in North and South America. In Mexico the number of dengue cases increased 600 percent between 2001 and 2007. In 2007 alone, there was a 40 percent increase in dengue cases. The disease has also spread to Hawaii and along the border in Texas. Even though the impact of climate change on the increased incidence and spread of dengue is less obvious than is the increase of malaria, it is reasonable to assume that global warming will greatly extend the range of the virus disease [4 ], though this assumption has been contested [5].
The blueprint for exterminating mosquitoes

Exterminating the mosquito vector is the preferred approach to controlling dengue according to those promoting genetic modification of mosquitoes [2]. The Stanford Business School proposed that releasing genetically modified (transgenic) male mosquitoes could eliminate dengue fever and other mosquito-borne diseases within a year in communities of up to a million people. Stanford Business School is promoting the work of researchers at Stanford's Institute for Computational and Mathematical Engineering, and Oxford University and London School of Hygiene and Tropical Medicine in the UK. The technique employed is called “Released Insects with a Dominant Lethal” (RIDL), a variant of SIT. The dominant lethal male mosquitoes developed for RIDL are more sexually attractive to female mosquitoes than the dominant lethal males produced by X-irradiation [2], and they cause death of the progeny during the late larval stages, thereby allowing the transgenic larvae to compete with the normal insect larvae for food. The mathematical model analysing the control of mosquito-borne diseases by a RIDL predicts eradication of dengue disease in one year [2, 6].

The mathematical model cannot be trusted to make reliable predictions, however, simply because the genetics and even more so, the ecology and host-parasite relationship of dengue disease are complex and poorly understood; in particular, there are silent as well as overt infections [5]. More seriously, the optimistic mathematical model says nothing about the genetic modification involved in RIDL, and there lies the devil in the detail.
Genetic modification to produce RIDL

The RIDL trait was created using a transposon. Transposons are mobile genetic elements (‘jumping genes'). They are similar to viruses, but lack the ability to form viral coats. RIDL was created by the piggyBac transposon originally isolated from a culture of cells of the cabbage looper, and has been used extensively in insect genetic-engineering. The piggyBac vector is prevented from replicating independently of the chromosome bearing it (non-autonomous) by removing its transposase enzyme that enables it to multiply and move among the chromosomes of the cells that it infects (though this is by no means a safeguard, see below) .

The transgenic male mosquito to be released has incorporated a gene for a red fluorescent marker protein for easy identification, but the key gene that confers dominant lethal trait is tTAV , encoding a tetracycline repressible transcription activator protein, driven by the promoter tetO of a Drosophila heat shock protein gene. In the presence of tetracycline, tTAV binds tetracycline and the complex does not bind to tetO , so no further expression of tTAV takes place. In the absence of tetracycline, there is a positive feedback loop in which tTAV binds to tetO , driving more expression of tTAV. The over production of tTAV is toxic, and kills the insect. But it is uncertain why excessive tTAV is lethal. In summary, RIDL is a tetracycline-repressible lethal system [7]. It has been suggested that the lethality of excessive transcription activator is due to transcriptional ‘squelching' or interference with ubiquitin-dependent breakdown of proteins. Mice modified with a gene for the tetracycline repressible transcription activator were not killed when the gene was activated by removing tetracycline [8].
Is this terminator insect safe?

The most glaring aspect of the proposed release is that the lethally acting transcription activator tTAV has a rather ill-defined action. The information presently available does not tell us what is killing the target animals. Even though a homologous tetracycline-repressed gene was not toxic to mice upon its activation, the killing toxin in the mosquito should certainly be identified before released to the environment is contemplated.

Another major hazard is horizontal gene transfer of the piggyBac insert. This issue has been thoroughly addressed in ISIS' submissions to the USDA with regard to the release of the pink bollworm in 2001 [9, 10]. We provided evidence that the disabled vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, basically because the transposase function enabling the piggyBac inserts to move can be supplied by ‘helper' transposons. Such helper transposons are potentially present in all genomes [10], including that of the mosquito. The main reason for using transposons as vectors in insect control is precisely because they can spread the transgenes rapidly by ‘non-Mendelian' mean within a population [11], i.e., by replicating copies and jumping into genomes, including those of the mammalian hosts. Although each transposon has its own specific transposase enzyme that recognizes its terminal repeats, the enzyme can also interact with the terminal repeats of other transposons, and evidence suggest “extensive cross-talk among related but distinct transposon families” within a single eukaryotic genome [12].

It is disingenuous to claim that because only male mosquitoes are released that don't bite people or other mammals, the technique is “environmentally benign” [2]. First of all, the transgenic mosquitoes, both males and females, have to be mass-produced in the laboratory. In order for transgenic females, also carrying the dominant lethal in double dose, to propagate the line, they have to take blood meals from laboratory animals such as mice or rabbits, not to mention the odd lab worker, which gives plenty of opportunity for horizontal gene transfer. Second, the transgenic males have to be sorted from the females, and this takes place at the pupae stage, when males are generally smaller than females, but this may not be 100 percent accurate. Third, the tetracycline-dependence of the transgenic lines is not absolute. In the absence of tetracycline, 3 to 4 percent of transgenic progeny actually survive to adulthood [2].

It is obvious that transgene escape can readily occur. As Ho commented [10]: “ 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.”

One cannot stress enough that horizontal gene transfer and recombination is the main highway to exotic disease agents.

The piggyBac inserts may also be mobilized by the transposase of piggyBac transposons already carried by Baculovirus (a common soil-borne insect virus) that infect insect cells, and this possibility has not been evaluated in the laboratory. Baculovirus not only carries piggyBac transposons, it has also been used in human gene therapy as it is capable of infecting human cells. It is indeed strange that the mobility and horizontal gene transfer of the piggyBac vector has not been thoroughly studied even though the activity of the vector is widely recognized.

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia , the cabbage looper, where it causes high mutation rates in the Baculovirus infecting the cells by jumping into its genes [9]. The piggyBac itself is 2.5 kb long with 13 bp inverted terminal repeats. It has specificity for the base sequence TTAA (at which it inserts); the probability of this sequence occurring is (0.25) 4 or 0.4 percent in any stretch of DNA, where it can cause insertion mutations: disrupting and inactivating genes, or inappropriately activating genes. This transposon was later found to be active in a wide range of species, including the fruit fly Drosophila , the mosquito transmitting yellow fever A aegypti , the medfly Ceratitis capitata , and the original host, the cabbage looper. The piggyBac vector gave high frequencies of transpositions, much higher than other transposon vectors in use, such as the mariner and Hirmar [13]. The piggyBac transposon is also active in human and mouse cells, and in the mouse germline; and a version with minimal terminal repeats exhibited greater transposition activity in human cells than another, well-characterised hyperactive Sleeping Beauty transposon system widely used for preclinical gene therapy studies [14].
Recent alternatives to RIDL

There are recent effective and affordable alternatives to RIDL in controlling mosquitoes that spread dengue fever and other diseases. Extracts from the paradise tree Melia azedarach showed promising larvicide and oviposition deterrent effects on the mosquito [15]. Essential oil from mullila and zedoary plants also proved effective in treating mosquito larvae [16]. Euphoriaceae extracts, particularly Euphorbia tirucalli can be applied as an ideal larvicide against Aedes aegypti [17].

A study in Thailand surveyed water-filled containers where Aedes mosquitoe pupae were found. Large water containers held 90 percent of pupae in rural areas and 60 percent in urban areas. Covering and treating such large containers should greatly reduce the mosquito population [18]. Bacillus thuringiensis israelensis VectoBac proved effective in treating water jars to combat dengue mosquitoes in Cambodia [19]. In Cuba, studies on the social and environmental determinants of Aedes aegypti confirmed that the greatest risks were associated with failure to treat stored water, and water in flower vases for religious practices. Efforts to reduce infestation should therefore focus on preventive practices [20].

These low tech practices may prove much more effective than the expensive high technology solutions, which are also far from safe.
 

References

1. “‘Warrier' mosquitoes to fight dengue scourge”, P. Selvaranti, New Sunday Times NST Online, 27 April 2008.
2. Atkinson MP, Su Z, Alphey N, Alphey LS, Coleman PG, Wein LM. Analyzing the control of mosquito-borne diseases by a dominant lethal genetic system. PNAS 2007, 104, 9540-5.
3. Kyle JL, Harris E. Global spread and persistence of dengue. Annu Rev Microbiol . 2008 Apr 22. [Epub ahead of print] doi:10.1146/annurev.micro.62.081307.163005
4. Barclay E. Is climate change affecting dengue in the Americas ? Lancet . 2008 371.973-4.
5. Halstead SB. Dengue virus-mosquito interactions. Ann Rev Entomol 2008, 53, 273-91.
6. STANFORD GRADUATE SCHOOL OF BUSINESS Blueprint Proposed for Wiping Out Disease-bearing Mosquitoes 2007, http://www.gsb.stanford.edu/news/research/wein_mosquitos....
7. Phuc HK, Andreasen MH, Burton RS, Vass C, Epton MJ, Pape G, Fu G, Condon KC, Scaife S, Donnelly CA, Coleman PG, White-Cooper H, Alphey L. Late-acting dominant lethal genetic systems and mosquito control. BMC Biol. 2007, 5, 11.
8. Gong P, Epton MJ, Fu G, Scaife S, Hiscox A, Condon KC, Condon GC, Morrison NI, Kelly DW, Dafa'alla T, Coleman PG, Alphey L.A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly. Nat Biotechnol . 2005, 453-6.
9. Cummins J. Terminator insects a primer, piggyBac a name to remember. ISIS Report 15 March 2001, http://www.i-sis.org.uk/piggybac-pr.php
10. Ho MW Terminator insects give wings to genome invaders. ISIS Report, 18 March 2001, http://www.i-sis.org.uk/terminsects-pr.php
11. Aparecida M, and Capurro ML. Perspectives in the control of infectious diseases by transfenic mosquitoes in the post-genomic era – A review. Mem Inst Oswaldo Curs, Rio de Janeiro 2007, 102, 425-33.
12. Feschotte C, Osterlund MT, Ryan P and Wessler SR. DNA-binding specificity of rice mariner-like transposases and interactions with Stowaway MITEs. Nucleic Acids Research 2005, 33, 2153-65.
13. Lobo N, Li X and Fraser Jr. MJ. Transposition of the piggyBac element in embryos of Drosophila melanogaster , Aedes aegypti and Trichoplusia ni . Mol Gen Genet 1999: 261: 803-10.
14. Wilson MH, Coates CJ and George AL. PiggyBac transposon-mediated gene transfer in human cells. Molecular Therapy 2007, 15, 139-45.
15. Coria C, Almiron W, Valladares G, Carpinella C, Ludueña F, Defago M, Palacios S.Larvicide and oviposition deterrent effects of fruit and leaf extracts from Melia azedarach L. on Aedes aegypti (L.) (Diptera: Culicidae). Bioresour Technol . 2008, 99(8), 3066-70.
16. Pitasawat B, Champakaew D, Choochote W, Jitpakdi A, Chaithong U, Kanjanapothi D, Rattanachanpichai E, Tippawangkosol P, Riyong D, Tuetun B, Chaiyasit D. Aromatic plant-derived essential oil: an alternative larvicide for mosquito control. Fitoterapia. 2007, 78(3), 205-10.
17. Rahuman AA, Gopalakrishnan G, Venkatesan P, Geetha K Larvicidal activity of some Euphorbiaceae plant extracts against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res . 2008,102(5), 867-73.
18. Barbazan P, Tuntaprasart W, Souris M, Demoraes F, Nitatpattana N, Boonyuan W, Gonzalez JP. Assessment of a new strategy, based on Aedes aegypti (L.) pupal productivity, for the surveillance and control of dengue transmission in Thailand. Ann Trop Med Parasitol. 2008, 102(2),161-71.
19. Setha T, Chantha N, Socheat D. Efficacy of Bacillus thuringiensis israelensis , VectoBac WG and DT, formulations against dengue mosquito vectors in cement potable water jars in Cambodia. Southeast Asian J Trop Med Public Health . 2007, 38(2), 261-8.
20. Spiegel JM, Bonet M, Ibarra AM, Pagliccia N, Ouellette V, Yassi A.Social and environmental determinants of Aedes aegypti infestation in Central Havana: results of a case-control study nested in an integrated dengue surveillance programme in Cuba. Trop Med Int Health 2007, 12(4), 503-1