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- 1. Genetically modified mosquitoes: Demystified Topical discussion for August
- 2. Overview Introduction Mosquito Life Cycle Transmission cycle forVector-borne diseases Overview ofVector 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)
- 3. 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
- 4. Mosquito life cycle Culex and Culiseta species, the eggs are stuck together in rafts of up to 200 Anopheles, Ochlerotatus and 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.
- 5. 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 Period Intrinsic Incubation Period Oviposition within 48 hours Onset of Disease Bites naive host Mosquito infective period (remaining lifespan) Next mating cycle
- 6. 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 mosquitos lifespan.
- 7. Overview of Vector Control Vector Control Physical intervention Chemical intervention Biological intervention PesticidesSource Reduction Mosquito nets InsecticidesTreated Nets (ITN) Release of Insects carrying a Dominant Lethal (RIDL) and Sterile InsectTechnique (SIT) Impair pathogen development Indoor Residual Spraying Education Enforcement
- 8. 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
- 9. 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
- 10. 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
- 11. 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)
- 12. 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)
- 13. 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.
- 14. 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)
- 15. 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.
- 16. 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 theWolbachia bacterium Moreira, L.A., et al., PLoS Negl Trop Dis, 2009. 3(12): p. e568. wsp apyrase Uninfectedmosquito Infectedmosquito UninfectedSaliva InfectedSaliva Uninfectedsalivaryglands Infectedsalivaryglands
- 17. 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) YorkeysKnobGordonvale Hoffmann,A.A., et al. Nature, 2011. 476(7361): p. 454-7
- 18. 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. YorkeysKnobGordonvale Hoffmann,A.A., et al. Nature, 2011. 476(7361): p. 454-7
- 19. Conclusions on pathogen development impairment Wolbachia infected mosquitoes are an interesting natural biological concept to control the spread of vector borne diseases Laboratory reared stableWolbachia 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. StableWolbachia infected Anopheles have to be developed before the suppression effectiveness of Wolbachia on Plasmodium could be tested out.
- 20. Overview of Vector Control Vector Control Physical intervention Chemical intervention Biological intervention PesticidesSource Reduction Mosquito nets InsecticidesTreated Nets (ITN) Release of Insects carrying a Dominant Lethal (RIDL) and Sterile InsectTechnique (SIT) Impair pathogen development Indoor Residual Spraying Education Enforcement
- 21. 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
- 22. 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 theY 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
- 23. 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.
- 24. 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
- 25. Science behind RIDL Oxitec uses a piggyBac transposon construct in their GM mosquitoes which is as shown
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