genetically modified mosquitoes
TRANSCRIPT
Genetically modified mosquitoes:
Demystified
Topical discussion for August
Overview
Introduction
Mosquito Life Cycle
Transmission cycle for Vector-borne diseases
Overview of Vector Control
Impair Pathogen Development
Wolbachia infected mosquitoes
Wolbachia and its ability to suppress DENV2 in mosquitoes
Can Wolbachia control malaria
Key safety concerns on the spread of Wolbachia to humans
Release of Insects carrying a Dominant Lethal (RIDL) and
Sterile Insect Technique (SIT)
Introduction
Mosquitoes are vectors of serious human infections
Dengue
Malaria
Yellow Fever
Vector control is crucial and important in the fight against vector-borne diseases
From 1950s to 1970s, there were optimistic views that such diseases could be controlled using insecticides and drugs
But there were increasing problems of:
Increasing mosquito resistance to pesticides
Parasite resistance to drugs
Slow progress in vaccine development
Genetic modification of mosquitoes was thus looked at since 1955
Mosquito life cycle Culex and Culiseta species,
the eggs are stuck together in rafts of up to 200
Anopheles, Ochlerotatusand Aedes , as well as many other genera, do not make egg rafts, but lay their eggs singly
Culex, Culiseta, and Anopheles lay their eggs on the water surface while many Aedes and Ochlerotatus lay their eggs on damp soil that will be flooded by water.
Overview of transmission cycle for vector-
borne diseases
Mosquito lifecycle
(Egg to Adult)
Adult
Emerges
Find a mate
within 24-48
hours
Mating behaviour source: http://library.wur.nl/frontis/disease_vectors/17_takken.pdf
First blood meal
from infective host
Extrinsic Incubation
PeriodIntrinsic Incubation
Period
Oviposition
within 48
hours
Onset of Disease
Bites naive host
Mosquito infective period
(remaining lifespan)
Next mating
cycle
Extrinsic incubation period in mosquitoes
Vector-borne pathogens typically enter midgut, nerve tissue, body fat and ovaries before invading the salivary glands.
The pathogens will continue replicating in the salivary glands until the end of the mosquito’s lifespan.
Overview of Vector Control
Vector Control
Physical intervention Chemical intervention Biological intervention
PesticidesSource Reduction
Mosquito nets
Insecticides Treated Nets (ITN)
Release of Insects
carrying a Dominant
Lethal (RIDL) and Sterile
Insect Technique (SIT)
Impair pathogen
development
Indoor Residual Spraying
Education
Enforcement
Process flow
Laboratory experiments to establish stable Wolbachia
infected Aedes aegypti mosquitoes
Find out the effectiveness and spread of Wolbachia
within native mosquito population
Find out the extent of
dengue virus suppression
in mosquitoes
Phenotypical features of
Wolbachia infected
mosquitoes
Transmission of
Wolbachia to humans
(safety concerns)
Ability of transgenic
mosquitoes to infect
humans with DENV
Impair pathogen development
Impairing pathogen development (vector-borne pathogens)
was proposed by Laven H. et al as early as 1967
The use of Wolbachia pipentis, a intracellular insect bacterium
which was isolated in 1924 in the ovaries of Culex pipens
It confers 4 different phenotypes:
Male killing: males are killed during larval development
Feminization: infected males develop as either females or infertile
pseudo-females
Parthenogenesis: reproduction of infected females without the
need for male
Cytoplasmic incompatibility: inability of infected males to mate
with uninfected females or females who are infected with another
Wolbachia strain
Wolbachia-induced cytoplasmic
incompatibility in mosquitoes
Wolbachia-infected male mosquitoes mates with an uninfected female mosquito
Wolbachia-infected females produce infected progeny in all matings allowing the infection to rapidly spread through mosquito population.Walker, T. and L.A. Moreira, Mem Inst Oswaldo
Cruz, 2011. 106 Suppl 1: p. 212-7
Dengue virus suppression in Wolbachia
infected mosquitoes midgut
Wolbachia (WB1) infected mosquitoes midgut show no significant increase in the DENV titers even after 18 days post infection.
Bian, G., et al, PLoS Pathog, 2010. 6(4)
Dengue virus suppression in Wolbachia
infected mosquitoes thorax (salivary glands)
Wolbachia (WB1) infected mosquitoes thorax show no significant increase in the DENV titers even after 18 days post infection.
Thorax is where the salivary glands are present.
Bian, G., et al, PLoS Pathog, 2010. 6(4)
Why was the previous 2 slides important?
Midgut
Salivary glands
If the dengue virus is unable to
transverse to the salivary glands,
passing on the virus to human host
would not be possible.
What are the factors leading to DENV
suppression?
17-fold increase in Defensin and 4.49-fold increase in Cecropin
Other Toll pathway genes in mosquito fat body are upregulated which may represent a potential mechanism underlying the suppression of dengue infection by Wolbachia
Bian, G., et al, PLoS Pathog, 2010. 6(4)
Can Wolbachia be used to control malaria?
In laboratory conditions, malaria infection is reduced in
Wolbachia infected Anopheles mosquitoes.
As Anopheles mosquitoes are not natural hosts of
Wolbachia, it is hard to attain stable Wolbachia infected
mosquitoes to be released into the wild
Due to the above limitation present, field trials are not
able to be performed.
Key safety concerns on the spread of
Wolbachia to humans PCR amplification of the
Wolbachia wsp gene or mosquito apyrase has shown only the presence of Wolbachia in salivary glands, but not in saliva.
These results are supported by the size of the intracellular Wolbachia (around 1mm in diameter) and the diameter of mosquito salivary ducts (also about 1 mm)
Wolbachia infected mosquitoes are not able to infect humans with the Wolbachiabacterium
Moreira, L.A., et al., PLoS Negl Trop Dis, 2009. 3(12): p. e568.
wsp
apyrase
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Infe
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Unin
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Infe
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Field trial to test the effectiveness and spread of
Wolbachia within native mosquito population
Wolbachia
infected
mosquitoes
spread the
disease
relatively quickly
over a period of
18 weeks in 2
separate sites
(Ten releases
were made over
the monitoring
period)
York
ey’
sK
nob
Gord
onva
le
Hoffmann, A.A., et al. Nature, 2011. 476(7361): p. 454-7
Field trial to test the effectiveness and spread of
Wolbachia within native mosquito population
Proof of concept that stable Wolbachia infected mosquitoes can introduce the infections to native mosquito population quickly.
York
ey’
sK
nob
Gord
onva
le
Hoffmann, A.A., et al. Nature, 2011. 476(7361): p. 454-7
Conclusions on pathogen development
impairment
Wolbachia infected mosquitoes are an interesting natural biological concept to control the spread of vector borne diseases
Laboratory reared stable Wolbachia infected mosquitoes are able to effectively introduce and infect the native mosquito population
DENV-2 is observed to be inhibited in Wolbachia-infected mosquitoes midgut and thorax. This proves to be promising as DENV-2 does not seem to be able to spread by Wolbachia-infected mosquitoes.
Stable Wolbachia infected Anopheles have to be developed before the suppression effectiveness of Wolbachia on Plasmodium could be tested out.
Overview of Vector Control
Vector Control
Physical intervention Chemical intervention Biological intervention
PesticidesSource Reduction
Mosquito nets
Insecticides Treated Nets (ITN)
Release of Insects
carrying a Dominant
Lethal (RIDL) and Sterile
Insect Technique (SIT)
Impair pathogen
development
Indoor Residual Spraying
Education
Enforcement
Sterile Insect Technique (SIT)
Invented by Edward F Kipling
By releasing sterile males to mate with wild females,
reducing their reproductive potential and ultimately, if
enough sterile males are released, it would bring about
eradication of the pest population.
Progeny of GM insects with wild-type insects are targeted
to possess the following traits:
Reduced competition in mating
Sterile progeny
Progeny with development defects
Reduced lifespan
Flightless phenotypes etc
Sterile Insect Technique (SIT) continued…
Traditional SIT involves mass rearing of mosquitoes to produce
equal numbers of the 2 sexes
Females are generally separated and discarded before release
they are not thought to help control efforts and may be detrimental.
Various mechanical and genetic sexing methods were
employed but fairly yield single sex population
Radiation induced translocations to the Y chromosome as dominant
selectable markers
Pupal mass sorting – females generally have larger mass
Time of eclosion – females generally emerge later than males
A better approach would be incorporating a transgene system
which lead to development of RIDL
Release of Insects carrying a Dominant
Lethal (RIDL®)
Using a transgene system to induce repressible female
specific lethality without requiring sterilization by
irradiation
Requires that a strain of the target organism carries a
conditional, dominant, sex-specific lethal trait,
where the permissive conditions can be created in the
laboratory or factory but will not be encountered in the
wild population.
Science behind RIDL
Tetracycline-repressible lethal system coupled with a
marker to identify those which are genetically modified
tTAV is a tetracycline-repressible transcriptional activator
which drives the over-expression of tTAV in absence of
tetracycline
High levels of tTAV is toxic due to interaction with key
transcription factors
Gong P et al Nat Biotechnol. 2005 Apr
Science behind RIDL
Oxitec uses a piggyBac transposon construct in their GM mosquitoes which is as shown in the picture below
piggyBac is a stable transposase system which is widely adopted in many cancer and insect studies
tTAV component is conjoined with a female specific sterility gene [fs(1)K10] – to achieve single sex population
fs(1)K10 is required in the dorsal-ventral patterning of the embryo and over-expression will result in progeny having double dorsal regions, and not surviving past the fourth –instar larval stage
LA513 constructPhuc Hk et al, BMC Biol. 2007 Mar 20; 5:11
Science behind RIDL
2nd component is for marking of all GM mosquitoes
which will be released into the wild
It is a constitutively expressed gene which can be
detected under fluorescence in the mosquitoes’
eyes
Progeny of the GM males and wild type females will also
inherit the gene and can be detected upon capture
LA513 constructPhuc Hk et al, BMC Biol. 2007 Mar 20; 5:11
500G – GM mosquitoes made
Wild type female
GM males released
into wild
If there are
sufficient male GM
mozzies released
in the wild….
Various examples of GM mosquitoes
Aedes aegypti OX513A
Male sterile GM mosquitoes
Aedes aegypti OX3604
Female flightless phenotype
Aedes albopictus OX3688
Anopheles spp – arabiensis, albimanus, quadrimaculatus
Malaria vector
Culex spp – quinquefasciatus, pipens
West Nile, Ross River, Murine Fever, Japanese Encephalitis, Rift
Valley, Bana
Dengue, Chikungunya,
Yellow Fever
Chikungunya
Tetracycline repressibility lethality in LA513
Progeny of LA513/+ males with WT female survives better in Tetracycline supplemented media
Survivability of progeny of heterozygous crosses reduces in tetracycline free media
Tetracyclin
e
w/o
Tetracyclin
e
Phuc Hk et al, BMC Biol. 2007 Mar 20; 5:11
Field trial of Aedes aegypti OX513A at
Cayman Islands
OX513A males are released in a 10-ha area at an avg rate
of 465 males/ha/wk starting in Nov 16
Before release, mosquitoes are screened again to prevent
accidental release of OX513A female mosquitoes
Fluorescent larvae detected from ovitraps recovered
would suggest that they are progeny of the GM males
with a wild type female
Mating outcomes was determined by ovitrapping
Adult trapping was also done to find out the proportion
of GM males in the sample population
Field trial of Aedes aegypti OX513A at
Cayman Islands - Results
OX513A males represented ~16% of the total adult males in the 7 week trial
9.6% of 1316 larvae captured had the heterozygous OX513A insertion
Roughly 2-fold difference in progeny fraction and OX513A male fraction in field
Field trial of Aedes aegypti OX513A at
Cayman Islands - Conclusions
Limitations
Can only sample eggs or larvae and it is difficult to estimate the
relationship between the eggs analyzed and the number of females
which they derive
Moving forward
The data allows researcher to estimate how many OX513A males
might need to be released in the area to suppress the population
Based on models described in Phuc HK et al, mating fractions of 13-
57% is required for suppression
Based on their data, a sustained release of ~1.4-12 times the release
rate for this experiment is required
However, the release has to be combined with integrated vector
management to achieve maximal results.
Conclusions
GM mosquitoes still has a long way to go before it could
be used as an effective means of vector control
Wolbachia infected mosquitoes looks most promising and
there are a few studies that are going on in Australia
Model studies on vector population dynamics should be
looked at closely before mass numbers of GM
mosquitoes are released into the wild
On a final note, we need to bear in mind that this
technology will create a shift in the equilibrium of nature
and vector-borne diseases
References - Wolbachia1) Bian, G., et al., The endosymbiotic bacterium Wolbachia induces resistance to
dengue virus in Aedes aegypti. PLoS Pathog, 2010. 6(4): p. e1000833.
2) Hoffmann, A.A., et al., Successful establishment of Wolbachia in Aedespopulations to suppress dengue transmission. Nature, 2011. 476(7361): p. 454-7.
3) Iturbe-Ormaetxe, I., T. Walker, and O.N. SL, Wolbachia and the biological control of mosquito-borne disease. EMBO Rep, 2011. 12(6): p. 508-18.
4) Popovici, J., et al., Assessing key safety concerns of a Wolbachia-based strategy to control dengue transmission by Aedes mosquitoes. Mem Inst OswaldoCruz, 2010. 105(8): p. 957-64.
5) Walker, T. and L.A. Moreira, Can Wolbachia be used to control malaria? MemInst Oswaldo Cruz, 2011. 106 Suppl 1: p. 212-7.
6) Laven, H., Eradication of Culex pipiens fatigans through cytoplasmicincompatibility. Nature, 1967. 216(5113): p. 383-4
7) Moreira, L.A., et al., Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia. PLoS Negl Trop Dis, 2009. 3(12): p. e568.
References – Sterile Insect Technique
8. Alphey, L., Re-engineering the sterile insect technique. Insect Biochem Mol Biol, 2002. 32(10): p. 1243-7.
9. Benedict, M.Q. and A.S. Robinson, The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol, 2003. 19(8): p. 349-55.
10. Fu, G., et al., Female-specific flightless phenotype for mosquito control. Proc Natl AcadSci U S A, 2010. 107(10): p. 4550-4.
11. Gong, P., et al., A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly. Nat Biotechnol, 2005. 23(4): p. 453-6.
12. Harris, A.F., et al., Field performance of engineered male mosquitoes. Nat Biotechnol, 2011. 29(11): p. 1034-7.
13. Heinrich, J.C. and M.J. Scott, A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc Natl AcadSci U S A, 2000. 97(15): p. 8229-32.
14. Horn, C., et al., piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics. Genetics, 2003. 163(2): p. 647-61.
15. Labbe, G.M., D.D. Nimmo, and L. Alphey, piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse). PLoS Negl TropDis, 2010. 4(8): p. e788.
References – Sterile Insect Technique15. Marrelli, M.T., et al., Mosquito transgenesis: what is the fitness cost? Trends Parasitol, 2006. 22(5): p.
197-202.
16. Marshall, J.M., The Cartagena Protocol and genetically modified mosquitoes. Nat Biotechnol, 2010. 28(9): p. 896-7.
17. Nolan, T., et al., Developing transgenic Anopheles mosquitoes for the sterile insect technique. Genetica, 2011. 139(1): p. 33-9.
18. Phuc, H.K., et al., Late-acting dominant lethal genetic systems and mosquito control. BMC Biol, 2007. 5: p. 11.
19. Rad, R., et al., PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science, 2010. 330(6007): p. 1104-7.
20. Subbaraman, N., Science snipes at Oxitec transgenic-mosquito trial. Nat Biotechnol, 2011. 29(1): p. 9-11.
21. Thomas, M.C., et al., The biology and evolution of transposable elements in parasites. Trends Parasitol, 2010. 26(7): p. 350-62.
22. Tu, Z., Insect Transposable Elements. Insect Molecular Biology and Biochemistry, 2012. 2012: p. 57-89.
23. Tu, Z. and C. Coates, Mosquito transposable elements. Insect Biochem Mol Biol, 2004. 34(7): p. 631-44.
24. Venner, S., C. Feschotte, and C. Biemont, Dynamics of transposable elements: towards a community ecology of the genome. Trends Genet, 2009. 25(7): p. 317-23.
25. Zayed, H., et al., Development of hyperactive sleeping beauty transposon vectors by mutational analysis.Mol Ther, 2004. 9(2): p. 292-304.