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Genetically modified mosquitoes: Demystified Topical discussion for August

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Page 1: Genetically modified mosquitoes

Genetically modified mosquitoes:

Demystified

Topical discussion for August

Page 2: Genetically modified mosquitoes

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)

Page 3: Genetically modified mosquitoes

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

Page 4: Genetically modified mosquitoes

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.

Page 5: Genetically modified mosquitoes

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

Page 6: Genetically modified mosquitoes

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.

Page 7: Genetically modified mosquitoes

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

Page 8: Genetically modified mosquitoes

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

Page 9: Genetically modified mosquitoes

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

Page 10: Genetically modified mosquitoes

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

Page 11: Genetically modified mosquitoes

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)

Page 12: Genetically modified mosquitoes

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)

Page 13: Genetically modified mosquitoes

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.

Page 14: Genetically modified mosquitoes

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)

Page 15: Genetically modified mosquitoes

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.

Page 16: Genetically modified mosquitoes

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.

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apyrase

Unin

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Infe

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osq

uito

Unin

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ed S

aliv

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Infe

cted S

aliv

a

Unin

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Page 17: Genetically modified mosquitoes

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

Page 18: Genetically modified mosquitoes

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

Page 19: Genetically modified mosquitoes

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.

Page 20: Genetically modified mosquitoes

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

Page 21: Genetically modified mosquitoes

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

Page 22: Genetically modified mosquitoes

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

Page 23: Genetically modified mosquitoes

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.

Page 24: Genetically modified mosquitoes

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

Page 25: Genetically modified mosquitoes

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

Page 26: Genetically modified mosquitoes

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

Page 27: Genetically modified mosquitoes

500G – GM mosquitoes made

Page 28: Genetically modified mosquitoes

Wild type female

GM males released

into wild

If there are

sufficient male GM

mozzies released

in the wild….

Page 29: Genetically modified mosquitoes

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

Page 30: Genetically modified mosquitoes

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

Page 31: Genetically modified mosquitoes

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

Page 32: Genetically modified mosquitoes

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

Page 33: Genetically modified mosquitoes

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.

Page 34: Genetically modified mosquitoes

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

Page 35: Genetically modified mosquitoes

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.

Page 36: Genetically modified mosquitoes

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.

Page 37: Genetically modified mosquitoes

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.