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Here Come the Terminator Mosquitoes, Genetically Engineered
- Terminator Mosquitoes to Control Dengue?
By Prof. Joe Cummins and Dr. Mae-Wan Ho
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 . 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 , though the sterile males were created by X-irradiation,
and not by transgenesis.
The release of sterile males is considered “environmentally benign” , 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 . 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 .
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 .
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 .
The blueprint for exterminating mosquitoes
Exterminating the mosquito vector is the preferred approach to controlling
dengue according to those promoting genetic modification of mosquitoes . 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 , 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 . 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 . 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 .
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 , 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 ,
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 .
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”
. 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 .
It is obvious that transgene escape can readily occur. As Ho commented : “
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
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 . 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 . 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 .
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 . Essential oil from mullila and zedoary
plants also proved effective in treating mosquito larvae . Euphoriaceae
extracts, particularly Euphorbia tirucalli can be applied as an ideal larvicide
against Aedes aegypti .
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 . Bacillus thuringiensis israelensis
VectoBac proved effective in treating water jars to combat dengue mosquitoes in
Cambodia . 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 .
These low tech practices may prove much more effective than the expensive high
technology solutions, which are also far from safe.
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