Theoretical Approaches to Using Genetic Manipulation to Control Mosquito Disease Transmission
Michael Riehle University of Arizona Why do we need novel control strategies for mosquitoes? Current Malaria Control Strategies Vector Control Parasite Control
Anopheles gambiae Anopheles stephensi
Insecticide treated Indoor residual Antimalarial drugs Vaccine bednets spraying Artemisinin combination RTS,S/AS01 therapy (ACT) (39% efficacy) Challenges with current control strategies
Artemisinin Resistance
WHO World Malaria Report 2015 Pyrethroid Resistance
Ashley et al., N Engl J Med 2014; 371:411-423 Current Arboviral Control Strategies
Vector Control Pathogen Control
Aedes aegypti Aedes albopictus
No drug treatment Vaccine ULV spraying Larviciding Dengue-Dengvaxia® Yellow fever Challenges with current control strategies
RANSON, Hilary; BURHANI, Joseph; LUMJUAN, Nongkran and BLACK IV, William C. Insecticide resistance in dengue vectors. TropIKA.net [online]. 2010, vol.1, n.1 What are our other options???
1. Sterile insect technique (SIT) • Release of Insects carrying a Dominant Lethal (RIDL)
2. Wolbochia infected mosquitoes
3. Population replacement strategies Sterile Insect Technique (SIT)
Dr. Edward Knipling's theorized that insect pests, such as the screwworm, could be controlled by inundating wild populations with the introduction of numerically overwhelming sterile males.
The egg masses laid by fertile females mated with sterile males would never hatch into the destructive larvae.
Reproduction would drop with each generation, eventually to reach zero if sterile flies continued to be introduced.
Known as the Sterile Insect Technique (SIT), Knipling focused on ways to use his method with the screwworm fly. Sterile Insect Technique (SIT) By the late 1930's, most elements for screwworm eradication were in place:
1. Knowledge of screwworm as a separate species with a dependence on live hosts
2. Bushland's technique for raising large numbers of the flies
3. Knipling's understanding of the mating patterns of the species
4. Knipling's theory for controlling the pest by the mass release of sterile males.
5. A fifth crucial element, a way to sterilize large numbers of screwworms, arrived with the assistance of the Oak Ridge National Laboratory and the loan of cobalt-60 gamma ray equipment Sterile Insect Technique (SIT) Sterile Insect Technique (SIT)
While this concept should apply to mosquitoes there are two main problems
Survival Release of and fitness potential disease vectors Release of Insects carrying a Dominant Lethal (RIDL) How does it work?
Wilke, André Barretto Bruno, et al. (2009) Release of Insects carrying a Dominant Lethal (RIDL)
Wilke, André Barretto Bruno, et al. "Mini-review: genetic enhancements to the sterile insect technique to control mosquito populations." AsPac J Mol Biol Biotechnol 17.3 (2009): 65-74. Release of Insects carrying a Dominant Lethal (RIDL) Pros and Cons
Pros Cons Minimal non-target effects Can’t release females Based on a proven technology Must manually sort males Males will seek out females Infrastructure for large scale breeding Allows for immature competition Concerns with releasing transgenic insect Reduces nuisance biting Leaves ecological void Self-limiting Self-limiting RIDL - Field examples
2009 Grand Cayman
Harris et al. Nat. Biotech. 30(9) 828-30
2012 Iteberaba Brazil
Nimmo - Oxitec Sex specific RIDL – AKA RIDL 2.0 Sex specific RIDL – AKA RIDL 2.0
Non-transgenic Transgenic Sex specific RIDL – AKA RIDL 2.0 Pros and Cons
Pros Cons Minimal non-target effects Can’t release females Based on a proven technology Infrastructure for large scale breeding Males will seek out females Concerns with releasing transgenic insect Allows for immature competition Leaves ecological void Automatic sorting – female lethal Self-sustaining Reduces nuisance biting Self-sustaining
Has not been tested in the field Using bacteria to control dengue and Zika Wolbachia
Wolbachia is an intracellular bacterial symbiont found in a wide range of arthropods.
wMel infected ovaries
Credit: Simon Moule/Eliminate Dengue Using bacteria to control dengue and Zika How it works
Eliminate Dengue: Our Challenge Using bacteria to control dengue and Zika Lab Results
Walker, Thomas, et al. "The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti Dengue virus populations." Nature 476.7361 (2011): 450-453. Using bacteria to control dengue and Zika Lab Results
Aliota, Matthew T., et al. "The w Mel Strain of Wolbachia Reduces Transmission of Chikungunya Virus in Aedes aegypti." PLoS Negl Trop Dis 10.4 Chikungunya virus (2016): e0004677. Using bacteria to control dengue and Zika Lab Results
Aliota, Matthew T., et al. "The wMel strain of Wolbachia Reduces Zika virus Transmission of Zika virus by Aedes aegypti." Scientific reports 6 (2016). Using bacteria to control dengue and Zika Field Results Using bacteria to control dengue and Zika Field Results
In 2011 two field releases were performed in Australia
Sustained releases of wMel-infected mosquitoes over a 10-week period.
Five weeks following the last release, Wolbachia frequencies had reached almost 100 percent in wild populations at both locations.
Persisted over time.
Low level of uninfected Immigration? Using bacteria to control dengue and Zika Pros and Cons
Pros Cons Minimal non-target effects Mode of anti-viral action unknown Unique killing mechanism Resistance? Males will seek out females Studies on effectiveness against No ecological void arboviruses needed Self-sustaining with drive system Not recallable Does not affect nuisance biting Population replacement with genetically engineered mosquitoes Population replacement – how does it work?
Genetically engineer a mosquito that:
• Kills the pathogen in the mosquito
• Blocks the pathogen from developing within the mosquito
• Shortens the mosquito’s lifespan so that pathogens do not have time to complete their development How to engineer a malaria resistant mosquito
1. A method for genetically engineering mosquitoes
2. Anti-pathogen molecules
3. An effective genetic drive mechanism Transposable elements MEDEA CRISPR/Cas9 Components of a transformation system DNA delivery system: electroporation microinjection lipofection biolistics Components of a transformation system Active Class II elements:
P element: First identified in Drosophila Rubin and Spradling (1982) used the P element to restore color to Drosophila white eye mutants Researches spent ~10 years trying to get the P element to work in other insects Only works in Drosophila Components of a transformation system Active Class II elements:
~Size Length of Target-site Element Family Origin (kb) ITRs specificity Hermes hAT Musca 2.7 17 GTNCAGAC domestica MosI Tc1- Drosophila 1.3 ~30 TA mariner mariner mauritiana Minos Tc1- Drosophila 1.4 254 TA mariner hydei piggyBac novel Lepidopteran 2.5 13 TTAA baculovirus Components of a transformation system Marker genes:
Physiological (select) antibiotic resistance genes (neomycin, hygromicin) insecticide resistance (OPD, dieldrin) Components of a transformation system Marker genes: Visible (screen) fluorescent proteins eye color Components of a transformation system Marker genes: Fluorescent markers predominate Greatest reason multiple markers can be used Components of a transformation system Promoters:
Actin 3XP3 Testes specific
Carboxypeptidase = Midgut specific Vitellogenin = Fat body specific Transformation procedures involve donor and helper constructs
Mos 1 mariner donor plasmid
MLH SV40 EGFP ACT 5C VG MAL-I N2 SV40 MRH
Marker gene Effector gene
Plasmid backbone
Mos 1 mariner helper plasmid
HSP82 promoter Mos 1 transposase
Plasmid backbone Transformation Procedures
• Donor and helper plasmids are injected into insect embryos (G0 generation) before formation of nuclear membranes (~2 h).
• Embryos are heat-shocked to induce transposase (often not necessary)
• Embryos are reared to adult stages (transient expression)
• Adults are mated to non- transgenic animals
• Progeny (G1) are scored for presence of marker genes and families established Microinjection of DNA into newly laid An. Stephensi eggs Other transformation systems Gene targeting systems Advantages: Known insertion site Reduced mortality Increased fitness Improved transformation efficiency
Disadvantages: More complex Additional marker is required Additional lines maintained Cre/loxP phiC31 Other transformation systems CRSPR/Cas9
Advantages: Site directed insertion No docking strains required Efficient knock-out Inexpensive
Disadvantages: Low efficiency for knock-ins
New England Biolabs How to kill the pathogen Effector Molecules
Malaria Parasites Arboviruses Antimicrobial peptides Antimicrobial peptides Single chain antibodies RNAi constructs Reactive Oxygen Species RNAi Pathway RNAi constructs Promoters/lethal genes How to kill the pathogen Drive Systems
Mendelian inheritance is not going to be sufficient Too many mosquitoes need to be released Fitness costs
Thus we need a genetic drive system that can induce inheritance of the anti-pathogen gene at an accelerated rate.
These can include: Transposable elements Wolbachia MEDEA CRISPR/Cas9 Maternal-Effect Dominant Embryonic Arrest MEDEA Crispr/Cas9 Drive
Gantz et al. PNAS 2015 112 (49) E6736-E6743 Other concerns
Logistics:
Can you rear enough mosquitoes?
Can you transport the mosquitoes to areas of need? Poor/no roads Political unrest Lack of public cooperation Other concerns
Ethics:
You are releasing a flying transgenic vector
Does not respect international boundaries
Will require the combined support of governments, health organizations, philanthropy and the general public Other concerns
Multiple mosquito subspecies:
For example - The most important malaria vector is the Anopheles gambiae complex
Consists of at least seven species
Many reproductively isolated subspecies Where are we at?
RIDL – Multiple field releases with a proven reduction in Ae. aegypti populations wMel – Multiple field releases with persistant wolbachia establishment. Antiviral effectiveness must still be proven
Population replacement – Ability to engineer pathogen resistant mosquitoes. However, no drive mechanism and numerous ethical and logistical issues. Acknowledgements
Jenya Koch Vanessa Corby-Harris
UC-Davis Group U. Georgia Group Support Dr. Shirley Luckhart Dr. Mark Brown Dr. Nazzy Pakpour Dr. Andrew Nuss Dr. Cecilia Giulivi Dr. Bo Wang Kong Wai Cheung Eric Hauck Jose Pietri Anna Drexler