ISIS Report 12/03/12
Non-transgenic Mosquitoes to Combat Dengue
New research shows how a common symbiotic bacterium can
stop the dengue virus multiplying in the mosquito host, making hazardous and
inefficient transgenic mosquitoes obsolete Dr. Mae-Wan
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A team of researchers at several universities in Australia
have made a breakthrough in controlling the spread of dengue fever. They
infected the mosquito vector with a symbiotic bacterium that stops the dengue
virus from multiplying in the mosquito host thereby effectively blocking the
transmission of the virus from mosquitoes to humans.
This makes obsolete all attempts
at controlling natural disease vectors through transgenic mosquitoes, which are
by comparison ineffective, inefficient, costly, and hazardous to varying
degrees (see  Transgenic
Mosquitoes Not a Solution, SiS 54).
mosquito protects against infection
strategy adopted by the Australian research team led by Scott O’Neill at Monash
University, Victoria, is simple. The symbiotic bacteria living inside insect
cells naturally induce resistance to pathogens, so infecting natural
populations of insect vectors with such bacteria could be a way of controlling
the spread of the disease. The bacteria belong to the genus Wolbachia, a
widespread endosymbiont of many insects, and known to induce resistance to
infections by viruses and other pathogens in the host (see Box).
is a genus of bacteria that infect a wide range of arthropods and some
nematodes. They are found in 17 to 76 % of surveyed arthropods and estimated to
be present in 66 % of all arthropod species. This makes Wolbachia one of
the most widespread intracellular bacteria, where it has evolved a symbiotic
relationship with the host. The bacterium was first identified by Marshall
Hertig and Burt Wolbach in the mosquito Culex pipiens in 1924 . In
1971, Janice Yen and Ralph Barr at University of California Los Angeles
discovered that Culex mosquito eggs were killed by cytoplasmic
incompatibility when the sperm of Wolbachia-infected males fertilized
infection-free egg. This cytoplasmic incompatibility is maternally inherited,
and provides a driving mechanism for infection to spread rapidly through the
has been linked to viral resistance in Drosophila melanogaster and
mosquitoes. Flies infected with the bacteria are more resistant to RNA vruses
such as the Drosophila C virus, Nora virus, Flock House virus, cricket
paralysis virus Chkungunya virus, and West Nile virus, as well as a Plasmodium
that cause bird malaria [3-5].
In a previous
study , the team found that a laboratory strain of Wolbachia pipientis,
wMelPop-CLA, isolated from Drosophila
melanogaster and introduced into the dengue mosquito vector Aedes
aegypti shortened the mosquito’s lifespan by 50 %, but also inhibited
infection with dengue and Chikungunya viruses as well as the avian malaria
parasite, Plasmodium gallinaceum.
Population replacement as
opposed to reduction
Shortening the lifespan of the
insect vector host was initially deemed desirable as a means of reducing the
potential for transmitting dengue by making the wild populations less
long-lived, thereby effectively reducing the natural population.
discovery that Wolbachia infection can make the mosquito vector
resistant to the pathogen is more important than any effect on the host
lifespan. In terms of population replacement - the option now favoured by most
researchers - an infected host with normal lifespan would be more effective in
controlling disease, as they will invade and infect natural populations more readily.
In the new
study [6, 7], a different strain (wMel) isolated from Drosophila
melanogaster that did not shorten host lifespan was introduced into A.
aegypti. The infected mosquitoes were first tested in caged populations under
semi-field conditions before they were released and successfully invaded two
natural populations, reaching near-fixation in a few months.
control measures focus on reducing populations of the disease transmission
vector, but these have largely failed to slow the current dengue pandemic, with
more than 50 million estimated to be affected each year. Wolbachia pipientis
can spread rapidly into uninfected host populations by inducing cytoplasmic
incompatibility. This causes embryos from Wolbachia-uninfected females to die
when they are mated with infected males, whereas the embryos of eggs from
infected females are unaffected. Because Wolbachia is maternally inherited,
this effect provides a transmission advantage for the symbiont, resulting in
rapid invasion of insect host populations.
Creating new mosquito strains
infected with endosymbiont Wolbachia
To facilitate the infection of A.
aegypti, the wMel strain was first transferred from D. melanogaster
embryos into a mosquito cell line and grown for approximately two years to
allow the bacteria to adapt to the mosquito intracellular environment. Wolbachia
bacteria purified from the cell line were then injected into A aegypti
embryos, and stably infected mosquito lines were established in 2 to 8
generations. One of them, MGYP2, was selected for further experiments.
mosquitoes were grown in the presence of tetracycline, and after 8 generations,
a ‘tetracycine cured’ line MGYP2tet was established that no longer has
Wolbachia in it. An outbred line MGYP2OUT was created by backcrossing the MGYP2
line for three generations to the F1 progeny of wild-caught A. aegypti
eggs from Cairns, Australia. The F1 progeny from wild-caught eggs were used as
control, non-infected ‘wild-type’, to be compared with wMel-infected MGY2OUT
of the wMel Wolbachia in all infected lines was about 10 bacteria to
each mosquito cell, about 3-fold lower than mosquitoes infected with the
life-shortening wMelPop-CLA. The
tissue distribution of the bacteria in the adult female mosquitoes was
visualized with fluorescence in situ hybridization, and shown to be
widespread, with infection levels in ovaries and salivary glands similar to
those infected with wMelPop-CLA.
The heavy infection in ovaries would support high levels of maternal
transmission, while the absence in saliva ensures that it will not be passed
onto humans . However, in contrast to the wMelPop-CLA infection, wMel is not present at high levels in Malphigian
tubules and fat bodies, thoracic ganglia or brain tissue, which may account for
the non-pathogenic nature of wMel.
Mass reciprocal crossing experiments between MGYP2 and MGYP2tet
showed that the wMel bacteria induce strong cytoplasmic incompatibility in that
no hatched eggs result from the crosses between infected males and uninfected
females. In contrast, wMel-infected females mated to uninfected and
wMel-infected males resulted in hatch rates of approximately 90 %.
Under semi-field conditions - consisting of cages that provide
environments simulating the natural habitat of A. aegypti in north
Queensland Australia - the fecundity and viability of wMel-infected MGYP2OUT
were not different from wild-type mosquitoes. But wMelPop-CLA infected female
mosquitoes showed drastically reduced fecundity, and its eggs were also much
potential of Walbachia in mosquito lines infected with wMel and wMelPop-CLA respectively were then tested
in the same semi-field facility. Starting with a frequency of infected
mosquitoes at 0.65, the wMel infection increased rapidly and reached fixation
within 30 days in cage A and 80 days in cage B, while the wMelPop-CLA reached fixation after 40 days in cage A, but reached
only 80 % replacement in cage B, possibly due to predation by two geckos found
in cage B.
To test whether the infected mosquitoes were resistant to the dengue
virus, a dengue virus infected blood meal was fed to females infected with
wMel, and the levels of virus in whole bodies, legs and saliva were assessed
after 14 days. The wMel mosquitoes had approximately 1 500-fold fewer virus
compared with uninfected controls. Levels of virus in wMelPop-CLA infected mosquitoes were even lower, by 10 000-fold.
Infectious virus was present in 29 out of 36 saliva samples (80.2 %) from uninfected
control mosquitoes, whereas only 2 out of 48 saliva samples from wMEL-infected
mosquitoes contained low levels of virus. It turns out that the positive
samples were from an individual that escaped infection through imperfect
maternal transmission. Saliva from wMelPop-CLA infected mosquitoes were
completely free of virus. Thus, there was practically complete blockage of
dengue virus transmission by Wolbachia-infected mosquitoes.
Deliberate release after
extensive consultation at multiple sites and regulatory approval
A deliberate release of wMel-infected
A. aepgypti was carried out in early January 2011 during the wet season.
Adult male and female mosquitoes were released near Cairns in north-eastern
Australia, a total of 141 600 at 184 sites in Yorkeys Knob and 157 300 at 190
sites in Gordonvale over a period of 10 to 11 weeks (one release per week). The
open releases were approved by the Australian Pesticides and Veterinary
Medicines Authority and were preceded by an extensive period of community
engagement and subsequent strong community support. Following the initial
release, Wobachia frequencies were monitored every 2 weeks and monitoring
continued after releases were terminated.
The frequency of Wolbachia-infected
A. aegypti increased rapidly to more than 80 to 95 % by the end of
colleagues point out that the Wolbachia-infected mosquito populations
can act as nursery areas for future collection and dispersal of mosquitoes
without having to rear additional Wolbachia-infected mosquitoes in
special insectariums. Because the resistance to pathogens depends on a complex
interaction between the mosquito host and endosymbiont bacteria , it is not
easy for the pathogens to overcome the resistance by mutation or recombination.
The next step
is to test whether the infected mosquitoes are effective for controlling dengue,
and further ahead, whether other mosquito-borne diseases such as malaria could
be similarly controlled.
question is whether Wolbachia itself can be passed onto human hosts, or become
a pathogen for human beings. A Fact Sheet published by O’Neill’s group states
research Wolbachia infected insects are feeding on our researchers all
the time and there is no sign of any human illness associated with insect Wolbachia.
is an insect bacterium that has not been detected living inside humans or any
other vertebrates. It can be made to infect human tissue culture cells in the
laboratory but these laboratory systems are very artificial and do not predict
the actual ability of Wolbachia to infect an actual human being.”
The safest and most effective
way to control the transmission of mosquito-borne infectious diseases such as
dengue may be by infecting mosquito vectors with a common natural endosymbiont
bacterium Wolbachia. The bacterium spreads readily by maternally
inherited cytoplasmic incompatibility that kills the offspring of uninfected females
fertilized by sperms from infected males. It also induces high levels of viral
resistance in the mosquito host, thus blocking disease transmission. This approach
leads to rapid replacement of natural populations of disease vectors with
disease-resistant insects that remain stable in the wild, with minimum
disturbance to the ecological niche, and without the need for continual
replenishment with disease-resistant strains.
It makes all
attempts at engineering transgenic mosquitoes for reducing or replacing natural
There are 4 comments on this article so far. Add your comment
|Rory Short Comment left 12th March 2012 16:04:43|
The contents of this article are music to my soul. As creatures imbued with consciousness we should be working in cooperation with Nature, not against it, and this article gives an excellent example of doing so.
|John Farion Comment left 13th March 2012 21:09:06|
Cheers for this. Now let's work on bedbugs!
|Todd Millions Comment left 21st March 2012 07:07:01|
Message for John Farion-Bed bugs furthertherunto
Mr Farion,The matter is well in hand-Ants.Find a local mound of predetory ants,and leave the clothes ,bedding ect you suspect harbouring bed bugs over it for a couple days.Old russian trick from the steppes.It also works well with politicians,clergy and other advertizers and bankers-if you stake em down firmly and leave for a week.Cheers
|chewaik Comment left 15th December 2013 09:09:29|
everyone is desperate to get rid of dengue burden soonest possible, how come this non-gm to combat dengue is so slow to implement in Asia????