this presentation deals with transgenic mosquitoes,strategies of vector control by transgenic mosquitoes, methods of transgenesis in mosquitoes, its drawbacks and its limitations

1. Genetic Modified
Organisms :
Transgenic Mosquitoes
Presented by:Presented by:
ANIL KUMARANIL KUMAR
M.Sc. PHE iiM.Sc. PHE iindnd SemSem
2015-172015-17
VCRC(ICMR)VCRC(ICMR)

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Terminologies
TransgeneTransgene ::
Gene orgenetic material from one source added to otherGene orgenetic material from one source added to other
organism.organism.
Transgenesis :Transgenesis :
the process of introducing an exogenous gene (transgene) into athe process of introducing an exogenous gene (transgene) into a
living organism so that the organism will exhibit a new propertyliving organism so that the organism will exhibit a new property
and transmit that property to its offspringand transmit that property to its offspring..
Genetically modified Organisms :Genetically modified Organisms :
Organisms (i.e. plants , animals ormicroorganisms) in which theOrganisms (i.e. plants , animals ormicroorganisms) in which the
genetic material (DNA) has been altered in a way that does notgenetic material (DNA) has been altered in a way that does not
occurnaturally by mating and/ornatural recombination.occurnaturally by mating and/ornatural recombination.

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Continued…
Transgenic Mosquitoes :Transgenic Mosquitoes :
Mosquitoes in which genetic material (DNA) has been altered in aMosquitoes in which genetic material (DNA) has been altered in a
way that does not occurnaturally by mating and/ornaturalway that does not occurnaturally by mating and/ornatural
recombination.recombination.
It is used in interruption of VectorBorne Disease (VBD) cycle.It is used in interruption of VectorBorne Disease (VBD) cycle.

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Need???
 Inspite of more than 70 years of vectorcontrolInspite of more than 70 years of vectorcontrol
operations disease burden is still persisting.operations disease burden is still persisting.
 Development of resistance against Chemicals usedDevelopment of resistance against Chemicals used
in vectorcontrol operations.in vectorcontrol operations.
 Environment and health hazards related to ChemicalEnvironment and health hazards related to Chemical
control of mosquito vectors.control of mosquito vectors.
 Biological methods are not much effective.Biological methods are not much effective.

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Table of Contents
2. Methods2. Methods
3. Role in vector control3. Role in vector control
4. Drawbacks4. Drawbacks
1. Introduction1. Introduction

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Introduction
 Formore than two decades, scientists have beenFormore than two decades, scientists have been
working to develop genetically modified mosquitoesworking to develop genetically modified mosquitoes
foruse as public health tools to prevent theforuse as public health tools to prevent the
transmission of mosquito borne diseases.transmission of mosquito borne diseases.
 Recent advances in the development of geneticallyRecent advances in the development of genetically
modified mosquitoes have raised hopes forthemodified mosquitoes have raised hopes forthe
availability of new, potent and cost-effective tools toavailability of new, potent and cost-effective tools to
aid in the fight against Dengue, Zika and malaria.aid in the fight against Dengue, Zika and malaria.
 Initial lab trails and small scale field trials haveInitial lab trails and small scale field trials have
shown a good result inshown a good result in AedesAedes aegyptiaegypti andand Ano phelesAno pheles
ablimanuablimanuss control.control.

7. FIRST RELEASE OF TRANSGENIC
MOSQUITOES

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Strategies by transgenic mosquitoes
PopulationPopulation
REPLACEMENTREPLACEMENT
PopulationPopulation
suppressionsuppression
TransgenicTransgenic
mosquitoesmosquitoes

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SELF –SELF –
PERSISTINGPERSISTING
Self - limitingSelf - limiting
Transgenic
mosquitoes
Population
Population
suppression
suppression
Po
pulatio
n
Po
pulatio
n
REPLAC
EM
EN
T
REPLAC
EM
EN
T

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NATURAL ALLELE
SELF-LIMITING
PERSISTENSE
SELF-PERSISTAINING
INVASIVENESS
Types of transgenesis in mosquitoesTypes of transgenesis in mosquitoes

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Sterile insect technique
 The most strongly self-sustaining strategies involeThe most strongly self-sustaining strategies invole
the use of sterile insects.the use of sterile insects.
 Mostly sterile-male strategies involve the use ofMostly sterile-male strategies involve the use of
heritable lethal genes ormutations.heritable lethal genes ormutations.
 Fully penetrant sterile/lethal traits the modificationFully penetrant sterile/lethal traits the modification
is strongly self-limiting ,as it disappears within ais strongly self-limiting ,as it disappears within a
generation unless additional modified insects aregeneration unless additional modified insects are
released.released.
 Irradiation, chemosterilization, transgenicIrradiation, chemosterilization, transgenic
sterilization were the ancient methods of malesterilization were the ancient methods of male
sterilization.sterilization.
 Mosquitoes are modified by adding a gene thatMosquitoes are modified by adding a gene that
produces a protein called tetracycline repressibleproduces a protein called tetracycline repressible
activatorvariant tTAV.activatorvariant tTAV.

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Continued….
 tTAV can able to act as a SWITCHthat controls thetTAV can able to act as a SWITCHthat controls the
activity of othergenesactivity of othergenes
 GMmosquito produces higheramount of this tTAVGMmosquito produces higheramount of this tTAV
proteinprotein
 As it activates its own gene, this will ties up some ofAs it activates its own gene, this will ties up some of
the cell machinery.the cell machinery.
 The modified gene is actually a lethal gene.The modified gene is actually a lethal gene.
 Tetracycline acts as an antidot to this gene.Tetracycline acts as an antidot to this gene.
 Hence the mosquitoes are supplimented withHence the mosquitoes are supplimented with
tetracycline and mate with the wild mosquitoes.tetracycline and mate with the wild mosquitoes.
 The offsprings inherit the lethal gene and due to theThe offsprings inherit the lethal gene and due to the
insufficiency of tetracycline in the field they will die.insufficiency of tetracycline in the field they will die.

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RIDL
 Release of insects carrying a dominant lethal geneticRelease of insects carrying a dominant lethal genetic
system (RIDL)system (RIDL)
 Two types :-Two types :-
 fsRIDL (femalespecific RIDL)
 bi-sex RIDL
 It uses a lethal gene expressed in the zygot, ratherIt uses a lethal gene expressed in the zygot, rather
than in the paternal parent.than in the paternal parent.
 It releases transgenic mosquito homozygous fortheIt releases transgenic mosquito homozygous forthe
repressible dominant lethal gene.repressible dominant lethal gene.
 In fsRIDL the lethal gene is female specific;In fsRIDL the lethal gene is female specific;
 Femaleoffspringswill die
 Heterozygousmaleoffsprigswill bealive
 If theseheterozygousmalesmatehalf of their offspring inherit the
femalelethal transgene

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Continued
In bi-sex RIDLthe heterozygous offspringsIn bi-sex RIDLthe heterozygous offsprings
are killed by the inherited lethal gene.are killed by the inherited lethal gene.

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Incompatible Insect Technique (IIT)
 WolbachiaWolbachiainfected males were successfully used forinfected males were successfully used for
vectorcontrol.vectorcontrol.
 Sterility can also be created by artificial infectionSterility can also be created by artificial infection
with various strains ofwith various strains of Wolbachia.Wolbachia.
 WolbachiaWolbachiain effects conducts a sterile-male attack inin effects conducts a sterile-male attack in
uninfected females.uninfected females.
 ForFor Wolbachia,Wolbachia, all embryos produced by an infectedall embryos produced by an infected
motherare infected.motherare infected.
 In addition, embryos are nonviable if fertilized by aIn addition, embryos are nonviable if fertilized by a
male carrying amale carrying a WolbachiaWolbachiastrain not carried by thestrain not carried by the
femalefemale

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Homing Endonuclease Gene
 HEGs are selfish DNA elements encoding proteinsHEGs are selfish DNA elements encoding proteins
(endonucleases) that recognise and cleave specific(endonucleases) that recognise and cleave specific
DNA sequences of ˜20-30 nucleotides.DNA sequences of ˜20-30 nucleotides.
 HEGs may drive recessive lethal orsterile alleles toHEGs may drive recessive lethal orsterile alleles to
the population, potentially forcing it to extinction.the population, potentially forcing it to extinction.
 Homing :- when the HEG is inserted into theHoming :- when the HEG is inserted into the
potential site, repairof the HEG induced cut can copypotential site, repairof the HEG induced cut can copy
the HEG-bearing allele.the HEG-bearing allele.
 Occurrence of homing in the germ line causes super-Occurrence of homing in the germ line causes super-
Mendelian inheritence, whereby more than 50% ofMendelian inheritence, whereby more than 50% of
the offspring of a HEG heterozygote carry the HEG.the offspring of a HEG heterozygote carry the HEG.
 It is of two types :-It is of two types :-
 X-shredder
 Sterileallele

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Continued…
 If the HEGs cut X-linked sequence and the HEG isIf the HEGs cut X-linked sequence and the HEG is
expressed during spermatogenesis, this may resultsexpressed during spermatogenesis, this may results
inin
 Nonviabledaughters
 Theelimination of X-bearing system
 If the X-shredderis Y-linked, all male offspringsIf the X-shredderis Y-linked, all male offsprings
carry it, which can lead to invasion through meioticcarry it, which can lead to invasion through meiotic
drivedrive

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Transmission blocking vaccines
 It consist of antibodies that are ingested by theIt consist of antibodies that are ingested by the
mosquito with the blood meal and interfere withmosquito with the blood meal and interfere with
parasite development.parasite development.
 Proteins expressed on the surface of gametes ofProteins expressed on the surface of gametes of
parasites (eg., Pfs47/Pfs230) and ookinetes (eg.,parasites (eg., Pfs47/Pfs230) and ookinetes (eg.,
Pfs25 &Pfs28) have been tested forsuch vaccines.Pfs25 &Pfs28) have been tested forsuch vaccines.
 Antibodies against these protein bind to the parasiteAntibodies against these protein bind to the parasite
and block ookinete invasion of the midgutand block ookinete invasion of the midgut
epithelium.epithelium.
 Mosquito midgut antigens could be targeted.Mosquito midgut antigens could be targeted.

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Paratransgenesis
 It is the genetic manipulation of commensal orsymbioticIt is the genetic manipulation of commensal orsymbiotic
bacteria to alterthe host’s ability to transmit a pathogenbacteria to alterthe host’s ability to transmit a pathogen
 It is an alternative means of preventing malariaIt is an alternative means of preventing malaria
transmissiontransmission
 Bacteria can be engineered to express and secreteBacteria can be engineered to express and secrete
peptides orproteins that block parasite invasion orkillpeptides orproteins that block parasite invasion orkill
the parasite in the midgutthe parasite in the midgut
 Gram-positive and gram negative bacteria includingGram-positive and gram negative bacteria including
Escherichia, Alcaligenes, Pseudomonas, SerratiaandEscherichia, Alcaligenes, Pseudomonas, Serratiaand
BacillusBacillus have been identified in the midgut of wildhave been identified in the midgut of wild
mosquitomosquito
 Eg.,Eg., An. stephensiAn. stephensi fed withfed with E. ColiE. Coli that express a fusionthat express a fusion
protein of ricin and single chain antibody againstprotein of ricin and single chain antibody against P.P.
BergheiBerghei ookinete surface protein and inhibited the oocystookinete surface protein and inhibited the oocyst

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Promoters and Effectorgenes
 To produce GMculicine mosquitoes and AnophelineTo produce GMculicine mosquitoes and Anopheline
mosquitoes which interfere withmosquitoes which interfere with PlasmodiumPlasmodium
development promoters and effectorgenes can be useddevelopment promoters and effectorgenes can be used
 Effectergenes products are those which hindertheEffectergenes products are those which hinderthe
parasite development.parasite development.
 Promoters are those which drive the transgenesPromoters are those which drive the transgenes
 Single chain antibodies that recognize surface proteinsSingle chain antibodies that recognize surface proteins
show promise in interfering with parasite developmentshow promise in interfering with parasite development
 An anti-Pbs21 single chain antibody inhibited oocystAn anti-Pbs21 single chain antibody inhibited oocyst
formation by upto 95%formation by upto 95%
 A single chain antibody againstA single chain antibody against P. gallinaceumP. gallinaceum
Circunsporozoite protein(CSP), expressed from a SinbdisCircunsporozoite protein(CSP), expressed from a Sinbdis
virus vector, reduced numberof parasite in the salivaryvirus vector, reduced numberof parasite in the salivary
glands by 99%glands by 99%

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Continued…
 Transgenic mosquitoes expressing bee venom PLA2Transgenic mosquitoes expressing bee venom PLA2
from the carboxypeptidase promoterreducedfrom the carboxypeptidase promoterreduced P.P.
bergheiberghei oocyst formation by 87%.oocyst formation by 87%.
 TransgenicTransgenic An. stephensiAn. stephensi expressing PLA2 fromexpressing PLA2 from
AgAper1 promoterinhibited oocyst formation.AgAper1 promoterinhibited oocyst formation.
 An alternative strategy is to use synthetic moleculesAn alternative strategy is to use synthetic molecules
to interfere with parasite development.to interfere with parasite development.
 An. stephensiAn. stephensi engineered with a synthetic geneengineered with a synthetic gene
expressing a SM1 tetramerunderthe control of theexpressing a SM1 tetramerunderthe control of the
carboxypeptidase promoterreduced thecarboxypeptidase promoterreduced the P.P. bbegheieghei
development.development.

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Refractory genes
 These are genes that conferring the ability toThese are genes that conferring the ability to
transmit the pathogen orparasitetransmit the pathogen orparasite
 Refractory genes may be used where modestRefractory genes may be used where modest
prevalence and duration are adequateprevalence and duration are adequate
 It can be used with a self-limiting driversystem eg.,It can be used with a self-limiting driversystem eg.,
Killer-rescueKiller-rescue
 Orwithout a driversystem eg., Multi locusOrwithout a driversystem eg., Multi locus
assortment(MLA)assortment(MLA)
 Dissociation of refractory gene:- loss of refractoryDissociation of refractory gene:- loss of refractory
gene DNA from a gene drive construct such that thegene DNA from a gene drive construct such that the
refractory gene no longerfunctionsrefractory gene no longerfunctions

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Gene Drive Systems forSpreading
Refractory Genes
Using Transposible elements (Tes)Using Transposible elements (Tes)
One of the first gene drive systems to gainOne of the first gene drive systems to gain
widespread attentionwidespread attention
TEs replicate within a host genome and hence areTEs replicate within a host genome and hence are
inherited more frequently in offspring’s genomeinherited more frequently in offspring’s genome
The increase in inheritance enables TEs to spreadThe increase in inheritance enables TEs to spread
even in the presence of fitness cost to the hosteven in the presence of fitness cost to the host
This has lead to widespread prevalence among manyThis has lead to widespread prevalence among many
taxataxa
Various families of TEs represent 47% of theVarious families of TEs represent 47% of the AedesAedes
aegyptiaegypti mosquito genomemosquito genome

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Medea elements
 Maternal effect dominant embryonic arrest is aMaternal effect dominant embryonic arrest is a
selfish gene composed of a toxin and an antidoteselfish gene composed of a toxin and an antidote
 A mothercarrying Medea will express the toxin inA mothercarrying Medea will express the toxin in
hergermline and kill herprogenyhergermline and kill herprogeny
 The offspring with medea allele produce copies ofThe offspring with medea allele produce copies of
the antidote that saving theirlivesthe antidote that saving theirlives
 In a heterozygous motherwith one medea alleleIn a heterozygous motherwith one medea allele
and one non-medea allele,and one non-medea allele,
 half of theoffspringswill inherit themedeaalleleand survive
 Other half will inherit thenon-medeaand die

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drawbacksdrawbacks
 Self –limiting factor of transgenic mosquitoes will lead to periodic
releaseof transgenic mosquitoes.
 Periodic releaseof transgenic mosquitoeswill beacostlier affair .
 The SMT requires a very large number of males i.e, 5 sterile males to 1
wild femaleto mate.
 Invasive genetic system requires more (wolbachia or UD) modified
mosquitoesfor their intial establishment.
 About 3-4% of Sterile Modified Mosquito (SMM) carrying a
dominant lethal gene escapes its activation. (Source: Department of
Biosafety, Ministry of Natural Resources& Environment of Malaysia)
 Useof antibioticsin GMM can lead to drug resistance.

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 Selection of transgenic mosquitoes is a manual work.Selection of transgenic mosquitoes is a manual work.
 Selection of transgenic male and female larvae orSelection of transgenic male and female larvae or
pupae is manual work.pupae is manual work.
 Mosquito fitness is a majorconcern.Mosquito fitness is a majorconcern.
 Immigration into otherareas.Immigration into otherareas.
 Community involvement, social and ethical issues.Community involvement, social and ethical issues.

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DARK SIDE
OF
COIN

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OFF THE RECORDS….
(Coincidene Or .........!!)
 OX513A was the name given to the modified male
mosquitoes that were released in Brazil. They were
bred in a lab in the UK and modified to require
tetracycline, an antibiotic, to stay alive and destined
to dieif they didn’t get aregular supply of it.
 Oxitech did two trialsin Brazil i.e, 2012 and 2015.
 Zika is not new to world, it was discovered in
Uganda in 1947, microcephaly is a complication of
zika which was first reported from the same area of
Brazil whereOxitech did it trials.
 Colombia, a neighbour of Brazil has recorded more
than 20,000 cases of zika but has only reported a
small increase of microcephaly related to zika. (up
to April 16th 2016)

31. Epicentreof Zikain Brazil

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QUESTIONS YETTO BEANSWERED...!!
 Are we prepared for any epidemics because of gmm?
 Are we prepared against gmm, if it is used as
bioterrorism weapon?
 Ecological niche created by wiping a mosquito species will
be filled by other species so can gmm be used as only
option?
 If we are wiping a mosquito species from an area it may lead
to infiltration on the mosquitoes from the neighboring
areas, so release of gmm will it be a continuous process?
 if you are releasing gmm (Aedes aegypti) in an area and in the
same place any epidemic occurs because of other vector
what control measures one will take ??
 15 % of transgenic mosquitoes escapes the activation of
lethal genes as tetracyclin (antidote) is also widely used in
the field of agriculture , so if these mosquitoes will bite
human then?? It may lead to resistance in mosquitoes too!!