GENETIC MANIPULATION OF MOSQUITO A VECTOR CONTROL OPTION

8/3/2019 GENETIC MANIPULATION OF MOSQUITO A VECTOR CONTROL OPTION

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DEDICATION

This work is dedicated to Almighty GOD for his infinite mercy

towards me. I also dedicate this work to my parents; HRH EZE

P.C. UKONU AND UGOEZE HELEN UKONU.

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ABSTRACTIn the ongoing fight against vectors of human diseases, diseaseendemic countries may soon benefit from innovative controlstrategies involving modified insect vectors. For instance, three

promising methods (viz. RIDL [Release of Insects with a DominantLethal], Wolbachia infection, and refractory mosquito technology)are being developed by researchers around the world to combat

Aedes aegypti, the primary mosquito vector of viral fevers such asdengue (serotypes 14), Japanese encephalitis and yellow fever.Some of these techniques are already being extended to othervectors such as Aedes albopictus (the secondary vector of thesediseases) and Anopheles mosquito species that transmit malaria.

To enable Disease Endemic Countries (DECs) to take advantageof these promising methods, initiatives are underway that relate

to biosafety, risk assessment and management, and ethicalsocialcultural (ESC) aspects to consider prior to and during thepossible deployment of these technologies as part of anintegrated vector control programme. There has beenconsiderable progress over the last decade towards developingthe tools for creating a refractory mosquito. Accomplishmentsinclude germline transformation of several important mosquitovectors, the completed genomes of the mosquito Anophelesgambiae and the malaria parasite Plasmodium falciparum, and

the identification of promoters and effector genes that conferresistance in the mosquito. Better control measures such asfunding the development of Mathematical models, provide fundfor the sensitive of mosquito surveillance and funding aerialrelease equipment.

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TABLE OF CONTENTS

DEDICATION--------------------------------------------------------------------- I

ABSTRACT -------------------------------------------------------------------- II

TABLE OF CONTENT---------------------------------------------------------- III

CHAPTER ONE

INTRODUCTION--------------------------------------------------------------- 5

CHAPTER TWO

METHODS OF GENETIC MANIPULATORS---------------------------- 12

CHAPTER THREE

USE OF GENETIC MANIPULATION IN THE CONTROL OF MOSQUITO AND

MOSQUITO-BORNE DISEASES

-MALARIA----------------------------------------------------------------------- 20

-FILARIASIS--------------------------------------------------------------------- 21

-DENGUE FEVER-------------------------------------------------------------- 22

-JAPANESE ENCEPHALITIS------------------------------------------------ 25

-YELLOW FEVER-------------------------------------------------------------- 26

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CHAPTER FOUR

SUCCESSES RELATED TO GENETIC MANIPULATION OF MOSQUITO

VECTOR--------------------------------------------------------------------------- 28

CHALLENGES RELATED TO GENETIC MANIPULATION OF MOSQUITO

VECTORS------------------------------------------------------ 31

CHAPTER FIVE

CONCLUSION------------------------------------------------------------------ 37

REFERENCES --------------------------------------------------------------- 39

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CHAPTER ONE

INTRODUCTION

Mosquito is a devastating vector that attacks between one andthree million people annually and causes massive economiclosses (Curtis 1996). Moreover, the number of cases is increasing,due to the emergence insecticide-resistant mosquitoes, althoughintensive research to develop new drugs and insecticides isongoing (Federici 1995). Furthermore, despite promisingdevelopments, no effective vaccines have yet been developedand existing control measures are inadequate. Mosquitoes areobligatory vectors for malaria paarasite and this part of the

parasite cycle represents a potential weak link in transmission.Therefore, control of parasite development in the mosquito hasconsiderable promise as a new approach in the fight againstmalaria. Development of the malaria parasite in the mosquito iscomplex (Fig.1; Ghosh et al., 2003) and for the most part occursin the midgut (gamete to oocyst stages). Although thousands ofgametocytes are acquired with the blood meal, only a fewsuccessfully mature into oocysts, but each of them producesthousands of sporozoites (Ghosh et al., 2001). Because oocyst

formation is a bottleneck in sporogonic development, targetingpre-sporozoite stages could be a more effective strategy to blockparasite transmission.In recent years, methods for the genetic modification ofmosquitoes have been developed, and effector genes whoseproducts interfere with Plasmodium development in the mosquitoare beginning to be identified. While many of theinitial hurdles have been overcome, major questions remain to beanswered, foremost among which is how to introduce refractorygenes into wild mosquito populations. Here strategies to altermosquito vector competence and consider issues related totranslating this knowledge to field applications.

The overarching goal of malaria vector control is to reduce thevectorial capacity of local vector populations below the critical

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threshold needed to achieve a malaria reproduction rate (R0, theexpected number of human cases that arise from each human.Because of the long extrinsic incubation time ofPlasmodium in its

Anopheles vectors, the most effective vector control strategies in

use today rely on insecticide interventions like indoor residualinsecticide sprays (IRSs) and long-lasting insecticide-treated nets(LLINs) that reduce vector daily survival rates (Enayati andHemingway, 2010). For many malaria-endemic regions, thesetools can make substantial contributions to malaria control andmay be sufficient for local malaria elimination. These were theonly regions considered by the recent Malaria Elimination Group(MEG). Regions where existing interventions will not besufficiently effective include those where high rates oftransmission occur. For example, in much of sub-Saharan Africa,

where the entomological inoculation rates (EIRs) can reach levelsapproaching 1,000 infective bites per person per year (Hay et al,2000), the best use of existing interventions can only help toreduce annual inoculation rates by approximately an order ofmagnitude. Additional interventions will clearly be required,however, both for regions with extremely high rates oftransmission and for regions where the major vectors are notsusceptible to current control tools [Shaukat et al, 2010].

To develop vector-targeted interventions in support of malariaeradication in all disease endemic settings that are unfettered bythese limitations, three challenges need to be recognized andaddressed with great urgency today. The first challenge, for whichnear-term product development is essential, is the preservationand improvement of the utility of existing insecticide-basedinterventions. This challenge will require a vibrant researchagenda that develops a broader range of insecticides with novelmodes of action that can circumvent emerging resistance toexisting insecticides, particularly the pyrethroids. This agenda

must include the creation of strategies for the use of newinsecticides that minimize the emergence of resistance. A relatedand critical focus of the agenda will be the development of rapidand affordable methods for detecting the emergence ofepidemiologically important levels of insecticide resistance.Because of the fundamental dependence of many current malaria

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control and elimination programs on pyrethroid insecticidebasedLLINs and the emerging problem of pyrethroid insecticideresistance in many vector species, especially in sub-SaharanAfrica, development of new insecticides that can be used in LLINs

is the most immediate need [Ranson et al, 2009].The second challenge is development of interventions that affectvector species not effectively targeted by current tools. At leastthree dozen different species of Anopheles mosquitoes areimportant in malaria transmission worldwide. Many of thesespecies are not susceptible to tools like IRS and LLINs, whichtarget indoor feeding and/or resting vectors [Terenius et al,2008]. Control of malaria transmitted by these vectors will requirenew interventions that target other aspects of their biology,

including outdoor feeding and resting, oviposition site preference,mating behavior, or sugar meal selection. Major features of theagenda to tackle this challenge will be defining the vector speciesfor which such new tools are most important and devising toolsthat will be effective for multiple important vector species.

The most difficult research challenge for vector control during allphases of malaria elimination/eradication but particularly duringthe final stages of eradication is development of novel

approaches that will permanently reduce the very high vectorialcapacities of the dominant malaria vectors in sub-Saharan Africa.Without such approaches, local elimination in Africa will beextremely challenging. Even when elimination is achieved, theresidual vectorial capacities of local mosquitoes will pose alingering threat of massive epidemics should malaria bereintroduced to a population that has lost partial immunity.Measures to reduce vectorial capacities will need to be eitherextremely cost-effective, if they are to be sustained untileradication is achieved, or able to effectively yield a long-term,

sustained reduction of transmission following a one-timeapplication. Genetic control programs (which could be achievedby a variety of genetic manipulation approaches) designed topermanently reduce the vectorial capacities of natural vectorpopulations have received the most attention to date, andcurrently represent some of the most promising ideas in this area

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[sinkins and Gould, 2006], but the development of other novelapproaches must be strongly encouraged.

It is these three challenges that the Malaria Consultative Group onVector Control concentrated on during its deliberations.

Disease endemic countries (DECs) are showing interest in thepossible benefits of innovative control methods involving modified(either as a genetic drive mechanism or through infection)mosquito vectors of human diseases. For instance, threepromising methods Release of Insects (mosquitoes) carrying aDominant Lethal gene (viz. RIDL, Wolbachia, and refractorymosquito technology) are being developed by researchers aroundthe world to combatAedes aegypti, the

primary mosquito vector of viral fevers such as dengue (serotypes1-4), chikungunya and yellow fever. Some of these techniques arealready being extended to other vectors such asAedes albopictus(the secondary vector of these diseases) and Anopheles speciesthat spread malaria. Therefore, these innovative strategies, andtheir agents, are coming to the attention of the regulators, vectorcontrol agencies, and policy- makers in DECs. To enable DECs totake advantage of these promising methods, initiatives areunderway that relate to biosafety, risk assessment and

management, and aspects to consider prior to and during thepossible deployment of these technologies as part of anintegrated vector control programme.

Alternatively, it may be possible to alter the mosquito populationto a less harmful form, for example by making the mosquitoesunable to transmit specific pathogens. Such approaches, knownas population replacement strategies, have two essential steps.

The first of these is to identify a heritable modification that willmake the mosquitoes less harmful; the second is to introgress

this modification into a wild mosquito population (Alphey et al.,2002; Riehle et al., 2003).Persistence and spread of the modification: modifications may beself-limiting or self-sustaining in the target wild population. Self-limiting systems will by design be eliminated from the targetpopulation over time, e.g. by natural selection. The modification isthen maintained in the wild population only by periodic release of

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additional modified mosquitoes. The speed of this eliminationmay vary from one strategy to another; for example a dominantlethal orsterilizing transgene will be completely eliminated in one

generation, whereas a construct with a milder fitness penaltymay persist for several generations. Nonetheless, self-limitingsystems will neither persist indefinitely nor spread significantlybeyond the target area. In contrast, self-sustaining systems areintended to persist indefinitely, and indeed to increase inprevalence, e.g. allele frequency, in the target area and beyond.

These properties may make deployment of such systemsrelatively inexpensive, as they may be able to spread from arelatively small release. However, their indefinite presence in theenvironment, and potential to spread into new populations, may

raise additional regulatory and social concerns (Angulo and Gilna,2008a; Angulo and Gilna, 2008b).

The African Malaria mosquito, An. Gambiae is probably the mostdangerous of all animals (Curtis 1996).This mosquito is aparticularly dangerous vector as it is anthropophilic and has along life-span. Mosquitoes are also the vectors for such diseasesas yellow fever (Ae. aegypti), LaCrosse encephalitis (Ae.triseriatus), and dengue (Ae. albopictus). Control of these vectors

by employing our knowledge of host-seeking behavior, is animportant means of improving public health.

Effective vector control is dependent upon knowledge of aspecies' specific ecology. Five elements of a program for vectorcontrol include (1.) determining the vector species, (2.)knowledge of the mosquitos behavior and ecology, (3)surveillance (4.) education for the people affected, and (5.)control measures (Mitchell 1996).

Effective vector control is fundamental to the suppression ofmany epidemic and endemic diseases of man such as Malaria,

Yellow fever, and Filariasis. Before the second world war,transmission of some diseases has been interrupted by the use ofnon-persistent insecticides or by environmental manipulation butthis is usually on a limited scale and at a high cost. The discovery

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of long lasting organic synthetic insecticides, revolutionalized thewhole concept of vector control and for the first time it waspossible to contemplate the control or even eradication ofmajority of arthropod-borne diseases throughout the world.

However, the development of insecticide-resistance and thediscovery of behavioural characters which impede control haveseriously modified this optimistic outlook and vector control hasagain become one of the important problems confronting healthauthorities.

Some of the worlds most important infectious diseases aretransmitted by insects (Sinkins and Gould 2006). The burden ofthese diseases is especially heavy in the developing countries ofthe tropics. Control programs have commonly relied on

insecticides to control the vector, vaccines if available, andtherapeutic drugs to treat infected patients. Insecticides, whichare still arguably the best control weapon, are becomingincreasingly ineffective as vector speciesdevelop resistance. New insecticides are becoming prohibitivelyexpensive to develop and the public is concerned about thedetrimental effects they may have on the environment. To furthercompound these problems many human parasites are becomingdrug resistant (e.g. Plasmodium and chloroquine) and the

promise of safe, inexpensive and efficacious vaccines has notmaterialised.Furthermore, human populations are becoming increasinglymobile; living in ever expanding cities with little public healthinfrastructure. The end result is the current worsening globalvector-borne disease situation making the need for innovative,sustainable control strategies urgent.

The techniques of molecular biology and genetic engineeringhave the potential to play a role in the development of new,integrated control programs. The idea of controlling vector-bornediseasesthrough the genetic manipulation of insects dates back to the1940s (Vanderplank 1944). The potential of genetic techniquesfor use in vector control programs is evident from the successful

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eradication of the screwworm from the USA, Mexico and Libya(Vargas-Teran et al., 1994; Baumhover 1966). However, sterilemale release is unlikely to be a cost effective strategy formosquito populations due to their high reproductive rates and

ability to rapidly colonise new areas. Rapid advances in DNAbasedtechnology are now allowing scientists to envision novel geneticcontrol strategies involving the creation and release of transgenicinsects (Crampton et al, 1994; Gwadz 1994). Instead of focusingon the eradication of the vector, these strategies aim to replacenatural vector populations with transgenic insects unable totransmit disease, thereby effectively immunising the vectorrather than the human population.

Three interconnected research objectives must be achievedbefore a disease control strategy involving the release oftransgenic vectors could be attempted. First, genes whichencode traits that render the vector refractory to a particularpathogen must be identified. Second, methods to introduce andexpress these genes in insects in a stable, heritable fashion mustbe developed. Third, a means for spreading these genes to highfrequency in natural vector populations must be accomplished.

At the present time much of the research being conducted on thegenetic modification of insect disease vectors is focusing on theAfrican malaria vectorAnopheles gambiae. This is not surprisingconsidering that malaria is by far the most important vector-bornedisease in the world today. As this paper will consider, thetechnology needed to manipulate mosquito vector populations islikely to be developed in the near future. In planning a vectorcontrol strategy involving transgenic insects, care must be takenin selecting the disease system with which to first apply thisstrategy. The complex epidemiology of malaria together with theextremely high transmission rates common in much of Africasuggest that this technology might have a better hope of successif utilized initiallyin a less ambitious disease context. Lessons learned from initialinterventions could then be used to develop a sustainable

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integrated strategy for malaria control in Africa and other subsaharans.

CHAPTER TWO

METHODS OF GENETIC MANIPULATORS

2.1. TRANSMISSION BLOCKING VACCINES

Transmission blocking vaccines consist of antibodies that areingested by the mosquito with the blood meal and interfere withparasite development. Proteins expressed on the surface ofgametes (e.g. Pfs47/48, Pfs230) and ookinetes (e.g. Pfs25 andPfs28) have been tested for such vaccines ( Healer et al., 1999).Antibodies

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Fig.1. Life cycle of Plasmodium in the mosquito. The approximate developmental time atwhich each stage occurs in Plasmodium berghei (maintained at 20C) is indicated.

Transmission starts when the mosquito ingests an infected bloodmeal (0h). Within minutes,gametocytesdevelop into gametes (the star-shaped figure illustrates exflagellation, which isthe formation of male gametes) that fuse to form the zygote. At24h, the motile ookineteinvades the midgut epithelium and differentiates into an oocyst. About 2 weeks later, the

oocyst ruptures, releasingthousands of sporozoites into the mosquito body cavity. Of all thetissues that sporozoites come in contact with, they can invade only thesalivary gland. Whenthe mosquito bites another vertebrate host, transmission is completed by release ofsporozoites from the salivary glands(not shown). Reprinted from Ghosh et al. (2003), withpermission from Elsevier Science.

against these proteins bind to the parasite and presumably blockookinete invasion of the midgut epithelium.Alternatively, mosquito midgut antigens could be targeted.Various reports that polyclonal antibodies against mosquitomidgut proteins interfere with Plasmodium oocyst formation have

been published ( Lal et al., 1994, 2001), but in no case have therelevant antigens been identified (Jacobs-Lorena and Lemos,1995). It should be noted that transmission blocking vaccines donot protect the immunized individual but act by preventinginfection of people in the surrounding community. Thus, forethical reasons and for increased effectiveness, transmissionblocking antigens will have to be incorporated into conventionalvaccines that target the vertebrate stages of the parasite.

2.2. PARATRANSGENESIS

Paratransgenesis, the genetic manipulation of commensal orsymbiotic bacteria to alter the hosts ability to transmit apathogen, is an alternative means of preventing malariatransmission. Bacteria can be engineered to express and secretepeptides or proteins that block parasite invasion or kill theparasite in the midgut. This strategy has shown promise incontrolling transmission of Trypanosoma cruzi by Rhodnius

prolixus under laboratory conditions (Beard et al., 2002).

Furthermore, symbiotic bacteria in the tsetse fly have beenisolated, transformed with a reporter gene, and reinserted intothe fly (Beard et al., 1998). For this strategy to be used in malariacontrol, bacteria that can survive in the mosquitos midgut mustbe identified. Gram-positive and -negative bacteria, includingEscherichia, Alcaligenes, Pseudomonas, Serratia and Bacillus,have been identified in the midgut of wild anopheline adults

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(Demaio et al., 1996; Straifet al., 1998). These bacteria are easilycultured in the laboratory and may be suitable targets for geneticmanipulation. Whether these bacteria are stable or transientresidents of the midgut of adult mosquitoes remains to be

determined. To successfully control malaria the refractoryproteins or peptides expressed by the bacteria must act on themidgut stages of the malaria parasites, maintain their bioactivityin the midgut environment, and be expressed in sufficientquantities. When An. stephensi mosquitoes were fed E. coli thatexpress a fusion protein of ricin and a single-chain antibodyagainst Pbs21 (a P. berghei ookinete surface protein), oocystformation was inhibited by up to 95% (Yoshida et al., 2001). Othereffector molecules, such as SM1 and PLA2, are considered below(see Genetically modified mosquitoes). The use of

paratransgenesis for the control of malaria will require thedevelopment of methods to introduce genetically modifiedbacteria into field mosquitoes.

2.3. GENETICALLY MODIFIED MOSQUITOESAnother promising approach is to genetically modify mosquitoesto express proteins or peptides that interfere with Plasmodiumdevelopment. Methods to produce transgenic culicine (Jasinskieneet al., 1998) and anopheline (Catteruccia et al., 2000). Promoters

to drive the transgenes and effector molecules whose productshinder parasite development are considered below :

2.3.1. PROMOTERSAn essential step in engineering mosquitoes with reduced vectorcompetence is the identification of suitable promoters to drive theexpression of anti-parasitic genes. During its development in themosquito, the parasite occupies three compartments: midgutlumen, hemocoel and salivary gland lumen. Thus, promoters thatdrive synthesis and secretion of proteins into these compartmentsneed to be identified. In addition to spatial considerations, thetime of protein synthesis relative to arrival of the parasite in eachof these compartments needs to be considered.Control of transmission has the best chance of success if pre-sporozoite stages in the midgut lumen are targeted. Studies inthe laboratory demonstrated that carboxypeptidase, a digestive

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enzyme, and a peritrophic matrix protein, are activated inresponse to a blood meal and the proteins secreted in the midgutlumen (Edwards et al., 1997).Later stages of parasite development can be targeted in the

hemocoel and salivary glands. The vitellogenin promoter andsignal sequences were shown to drive strong gene expression inthe fat body and protein secretion into the hemocoel (Kokoza etal., 2000). However, this gene has a restricted temporal profile ofexpression that peaks around 24h after a blood meal and returnsto basal level by 48h. Soon after traversing the midgutepithelium, the ookinete transforms into an oocyst that is coveredby a thick capsule. Sporozoites are liberated from the oocyst asearly as 10 days later. These characteristics of parasitedevelopment limit the choice of effector genes that can be used

in conjunction with the vitellogenin promoter to those encodingproteins with exceptionally long half-lives. Re activation of thevitellogenin promoter by additional blood meal(s) may lessen thisshortcoming. The availability of a strong promoter with peakexpression in the hemolymph at about the time of sporozoiterelease from oocysts would be ideal. Two salivary glandpromoters have been characterized in transgenic mosquitoes:Maltase-I and Apyrase (Coates et al., 1999).

2.3.2. STERILE INSECT TECHNIQUE (SIT)Insect populations can be controlled by the release of largenumbers of sterile males. Thus, if a female mates with a male thathas no sperm or whose sperm was rendered unviable, this femalewill have fewer or no progeny. When many sterile males arereleased, the local population tends to decline or become extinct.

There are a number of cases of the successful local application ofthis technique, for example, in the control of the Mediterraneanfruit fly in Latin America, the New World screwworm in theAmericas and Libya, and for tsetse in Zanzibar, Africa. SIT alsohas been applied, on a limited scale, to Culex in India and

Anopheles albimanus in El Salvador.For population control, the crucial parameter is the ratio of thenumber of released sterile males to the number of males in thelocal population, which ideally should be around 10:1. Therefore,sterile-insect control is only effective when the resident

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population to be controlled is small relative to the number ofsterile males that can be mass-produced for release or when itcan be reduced to very low levels with conventional control toolsbefore the start of releases. It is highly desirable that only males

be released for two reasons: (1.) In most cases only females biteand transmit disease while, moreover, sterile females can alsotransmit (2.) Males would court and mate with the released sterilefemales (instead of local females), thus reducing the efficacy ofthe programme (Alphey and Andreasen 2002). Large-scaleproduction in the laboratory of a pure male population by non-genetic means may be problematic. It may rely on sex-specificdifferences of pupal size (culicine mosquitoes) or adult eclosiontimes (tsetse), but these protocols rarely yield a 100% malepopulation. Clearly, genetic sexing methods (see below) are far

superior. The most commonly used technique for malesterilization is exposure to high doses of radiation, a procedurethat damages chromosomes and results in unviable sperm.Sterilization by chemical means also has been employed. Becauseof the large numbers of insects that need to be released, it iscrucial that the effectiveness of the sterilization procedureapproaches 100%. However, the large doses of radiation andchemicals needed to achieve this effectiveness may reduce insectfitness, survival and mating competitiveness. These strategies

can fail if the laboratory-reared males do not mate as effectivelyas their field counterparts. The advent of germ-linetransformation for a number of different insects has led to thedevelopment of genetic alternatives for production of sterileinsects (Thomas et al., 2000). In one version of this approach(Release of Insects carrying a Dominant Lethal or RIDL, Thomaset al., 2000), a conditional dominant lethal gene is introduced intothe target insect genome. This gene has two importantproperties: 1) it is expressed only in females (or it kills onlyfemales); and 2) the gene is effectively repressed by a compoundthat does not occur normally in nature (e.g. tetracycline). Largeinsect populations are maintained by rearing them in thepresence of tetracycline, which represses the dominant lethalgene and allows the survival of equal numbers of males andfemales. Prior to release, the insects are reared in the absence oftetracycline, a condition that allows the expression of the

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dominant lethal gene and the death of all females. The resultingmales can be released without further manipulation or treatment.Males carry two copies (homozygous) of the dominant lethalgene. When these males mate in nature, all female progeny will

be killed and only males will be produced. Since these survivingmales are heterozygous for the dominant lethal gene, thepopulation-reducing effect is still manifested in the secondgeneration.It should be emphasized that the effectiveness of the SterileInsect Technique (SIT) is dependent on population structure anddynamics. Furthermore, this technique leaves intact the biologicalniche in which the target insect is found. SIT is most likely tosucceed in cases where target populations are small, the numberof target insects is low, and the target area is sufficiently isolated,

thereby reducing the likelihood of re-invasion. It is unlikely to beeffective for controlling mosquito populations in highly endemicareas of Africa where the mosquito population consists of severalvector species in high densities, where access to breeding sites isdifficult and where poorly interbreeding mosquito populations co-exist.

2.3.3. GERM-LINE TRANSFORMATIONDrosophila melanogaster was the first multicellular organism to

be stably transformed (Spradling and Rubin 1982). The samegeneral principles that were used in this pioneering work are stillemployed today for all germ-line transformation work in insects(Atkinson and James 2002). Embryos are injected with two DNAconstructs. One construct contains a gene encoding a dominantselectable marker (e.g., eye color, a fluorescent protein) and thegene of interest, each driven by a separate promoter, and bothsequences are together flanked by the inverted repeats of atransposable element. The second construct encodes atransposase, which is an enzyme that recognizes the invertedrepeats and catalyses the insertion of the intervening sequencesinto the genome of the host insect. It took from 1982 until the mid1990s to develop two crucial technologies: an appropriatetransposable-element system (at first scientists did not realizethat the P transposable element is not active in non-Drosophilaorganisms) and a suitable transformation marker (e.g., GFP).

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Since then, germ-line transformation of many insects has beenaccomplished but mosquitoes (Aedes, Anopheles, Culex) whichare the only insects of medical importance in this list. Importantly,bothAn. stephensi and An. gambiae can be transformed, though

the success rate in the latter case is still low. Improvement of thetransformation efficiency ofAn. gambiae is a high-priority topicfor future research. It would also be desirable to develop germ-line transformation procedures for other medically importantinsects such as sand flies and black flies. Current technologycannot be applied to germ-line transformation of tsetse becausethese do not lay eggs (that would need to be injected), only fullyformed larvae. However, genetic modification of tsetse vectorialcapacity could be achieved via genetic modification of one of itssymbionts.

The net result of germ-line transformation is the integration intothe genome of the host organism of a relatively large DNAsequence, flanked by inverted repeats of the transposableelement. The inserted DNA contains at least two genes, the geneto be investigated and a transformation marker gene (e.g., eyecolor, GFP) that allows transformed individuals to be identified.

2.3.4. EFFECTOR GENES

The term effector gene is used here for genes whose productsinterfere with the development of a pathogen. At least fourclasses of effector genes can be identified:( 1) Genes whoseproducts interact with insect host tissues crucial for parasitedevelopment: Examples of this class are SM1, a peptide thatoccupies putative salivary-gland and midgut receptors for themalaria parasite (Ghosh and Ribolla;Jacobs-Lorena 2001) andphospholipase A2 (PLA2), which is a protein that interferes withthe malaria ookinete invasion of the midgut (Zieler et al., 2001).( 2.) Genes whose products interact with the pathogen: Examplesof this class are genes encoding single chain monoclonalantibodies that bind to the parasites outer surface thus blockingtheir development ( De Lara Capurro et al., 2000).( 3.) Geneswhose products kill the pathogen: Examples are peptides from theinsects innate immune system such as defensins and cecropins,and peptides from other sources that act as selective toxins to

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parasites but do not affect the host insect, such as magainins,Shiva-1, Shiva-3 and gomesin (Kim et al., 2004). Most publishedwork on effector genes deals with effects on the malaria parasiteand little is known about such genes for other pathogens. In

particular, it is not clear what class of effector genes would beuseful for nematodes (filaria). Since these may be encapsulated incertain mosquito strains, genes that activate encapsulation couldbe considered as possible effector genes. For viruses, genes ofthe first class (interference of host-tissue invasion) or genes thatinterfere with virus replication (Olson et al., 1996) are possiblecandidates.4) Another possible strategy to reduce vector competence is bymanipulation of its immune genes, for instance by using RNAinterference or smart sprays (Christophides,Vlachou and Kafatos

2004).Another important strategic consideration is the stage of malariaparasite development to target. When a mosquito ingests aninfected blood meal, it acquires thousands of gametocytes ofwhich only few (usually less than ten) manage to cross the midgutand form oocysts. Later, each oocyst produces thousands ofsporozoites, a significant proportion of which invade the salivarygland. Because the strong bottleneck at the level of midgutinvasion, this stage of parasite development constitutes a prime

target for intervention.Midgut invasion is also a strong bottleneck in the process ofarboviral transmission.

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CHAPTER THREE

USE OF (GENETIC MANIPULATION) IN THE CONTROL OFMOSQUITO AND MOSQUITO-BORNE DISEASES

3.1. MALARIA:Successful development of the technology described above(transgenesis, promoter characterization and effector-geneidentification), permitted the creation of genetically modifiedmosquitoes impaired in their ability to transmit the malariaparasite. An early example was the creation of an Ae. aegyptiexpressing defensin in the haemolymph (Kokoza et al., 2000).However, the effect of defensin on malaria parasite developmenthas not been reported. At about the same time, the Jameslaboratory reported that a single-chain monoclonal antibody thatrecognizes a

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sporozoite surface protein inhibits invasion of the salivary gland(De Lara Capurro et al., 2000). In this instance, the effector genewas transiently expressed from a viral vector that is not inheritedby the mosquito progeny. The Jacobs-Lorena laboratory showed

that a stably integrated gene encoding SM1 strongly inhibitsparasitedevelopment in transgenic mosquitoes (Ito et al., 2002). Inanother example, transgenic mosquitoes expressing PLA2 alsohad much reduced vectorial competence (Moreira et al., 2002).Recently, it was demonstrated that the capacity to transmit themalaria parasite is reduced by about 60% in transgenic An.gambiae expressingcecropin from a carboxypeptidase promoter (Kim et al., 2004).

Thus, it is clear that mosquitoes can be genetically modified to

reduce their vectorial competence. To date, most reportedexperiments have been done with non-human malaria parasites.An important next step is the transfer of this technology to humanpathogens.

Wolbachia. Wolbachia are intracellular bacteria that inhabit thegerm line of a number of insects and distort reproduction bykilling progeny that do not contain it, by a phenomenon known ascytoplasmic incompatibility (CI). Compelling evidence in favour of

Wolbachia as a drive mechanism comes from Drosophila. Turelliand Hoffmann (1991) observed that Wolbachia swept through theD. simulans population in California at the rate of 100 km peryear. In principle, Wolbachia could provide a powerful drivingmechanism. However, no Wolbachia have yet been identified inanopheline mosquitoes (these are the exclusive vectors forhuman malaria), although they have been observed in culicinemosquitoes. A major limitation ofWolbachia is that it inhabits thegerm line while the pathogen develops in the soma. Thus, it isdifficult to target parasites with genes introduced into Wolbachia.A possible solution to this problem is the identification of genesthat cause CI. Currently little is known at the molecular levelabout how CI functions or how many genes are involved. Whenidentified, such gene(s) could conceivably be used to create adriving mechanism viatheir insertion into the mosquito genome.

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Meiotic drive. Population replacement can be driven by certaingenes, such as the Drosophila segregation distorter gene, thatfavour its inheritance over individuals not containing the gene.

Unfortunately, very little is known about such genes in insects ofmedical importance. One complication is that at least in modelsystems (Drosophila, mouse), the drive mechanism depends onmultiple genes (e.g., distorter andresponder) and this could complicate the implementation of thissystem in mosquitoes. Moreover, if such genes were to beemployed to drive effector genes into populations, all meioticdrive and effector genes would have to be tightly linked to avoidloss of effectiveness due to recombination.

3.2. FILARIASISMalaria and Bancroftian filariasis rank amongst the world's mostprevalent tropical infectious diseases. An estimated 300500million people are infected with malaria annually, resulting in 1.53 million deaths (WHO, 2000). Lymphatic filariasis is probably thefastest spreading insect-borne disease of man in the tropics,affecting about 146 million people (WHO, 1992). Many biologicalcontrol agents have been evaluated against larval stages ofmosquitoes, of which the most successful ones comprise bacteria

such as Bacillus thuringiensis israelensis and B. sphaericus(Becker and Margalit, 2003), mermithid nematodes such asRomanomermis culicivorax(Zaim et al, 1988), microsporidia suchas Nosema algerae (Undeen and Dame, 1987), and severalentomopathogenic fungi (Federici, 1995). Among these fungi, theoomycete Lagenidium giganteum has proven successful forvector control in rice fields (Hallmon et al,2000) and is currentlyproduced commercially (Khetan,2001). Other mosquito-pathogenic fungi that target larval instars include thechytidriomycetes Coelomomyces (Shoulkamy and Lucarotti,1998), and the deuteromycetes Culicinomyces (Sweeney, 1981),Beauveria (Clark et al, 1968) and Metarhizium (Robert, 1970). Ofthe few fungi known to infect adult Diptera, the majority belong tothe group of Zygomycetes (Entomophthoraleans) (Low andKennel, 1972).

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Only a handful of studies have evaluated biological controlagents/methodologies to control adult stages of tropicaldisease vectors. Soars (1982) infected adult Ochlerotatussierrensis with the deuteromycete Tolypocladium cylindrosporum,

resulting in 100% mortality after 10 days,whereas (Clark et al ,1968). showed in a laboratory study that adult mosquitoes ofCulex tarsalis, Cx. pipiens,Aedes aegypti, Ochlerotatus sierrensis,Ochlerotatus nigromaculis, and Anopheles albimanus weresusceptible to Beauveria bassiana.Recently, (Scholte et al, 2003)reported that adult An. gambiae is susceptible to B. bassiana, aFusarium spp., and Metarhizium anisopliae.

Several current techniques appear capable of reducingtransmission of filarial parasites, but most still require both

validation of their impact in largescale control programmes andassessment of their cost-effectiveness. Among the mostpromising are the following: biocides, especially Bacillussphaericus (a self-reproducing, toxin-producing bacterium) for thecontrol of Culex quinquefasciatus mosquitos (Hougard, 1993);polystyrene beads to limit the breeding of vectors, especially inenclosed urban breeding sites, such as latrines and cesspits.

3.3. DENGUE FEVER:Dengue fever (DF) and its more serious form, denguehemorrhagic fever (DHF) and dengue shock syndrome (DHF/DSS)are caused by four closely related but antigenically distinct,single-strand RNA viruses transmitted by mosquitoes to humans.Dengue Virus(DV) cause more human morbidity and mortalitythan any other vector-borne viral disease with 2.5-3.0 billionpeople at risk of infection and 50-100 million DF and 250,000-500,000 DHF/DSS annual cases (Gubler 1996; 1998). All four DVserotypes cause disease and case-fatality rates for untreatedDHF/DSS can be 30-40%. The risk of DHF/DSS is highest in areaswhere two or more DV serotypes are transmitted ( Rigau-Perez etal., 1998). At this time, there is no licensed vaccine and no clinicalcure for the disease. Ae. aegypti is by far the most important and

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efficient vector of DV because of its affinity for humans (Gubler,1998). Dengue control currently depends on reduction orelimination of Ae. aegypti. In the 1940-1960s most tropicalAmerican countries used integrated programmes of

environmental management and insecticides to eliminatemosquitoes (Gubler, 1998), but many of these were abandoned inthe early 1970s (Reiter and Gubler 1997).Several Government Viral Control(GVC) strategies for reducing DVtransmission have been identified as potential dengue diseasecontrol methods and are designed either to reduce the overallpopulation of DV-transmitting vectors or to replace existing vectorpopulations with populations that cannot transmit the virus. Twovector population reduction approaches are currently beinginvestigated and are in early laboratory cage trials. The first

population reduction strategy is the development and use ofnatural or genetically engineered densoviruses that arepathogenic to Ae. aegypti (Carlson, Afanasiev and Suchman2000).

The second population reduction strategy is the development anduse of insects carrying dominant lethal mutations ( Thomas et al. ,2000). This approach would require mating of GeneticallyModified Vectors (GMV)-RIDL males with local vector populationsproducing offspring that die prior to becoming adults. Both

approaches are designed to reduce transmission of DVs byreducing the vector population. Approaches designed to replacepopulations of vectors are more long-term, but could havesignificant consequences for dengue disease control in the future(James, 2000). In these approaches, an effector gene, such as ananti-DV gene, is appropriately expressed to block transmission bythe vector. GVC approachesrequire identification of tissue-specific promoters, anti-pathogeneffector genes, and genetic drive mechanisms such as synthetictransposable elements (TE) to introgress the effector gene intothe population, eliminating vector competence. Successful GVCstrategies will require knowledge of vector ecology in DECs andlarge cage trials in DECs prior to release of biocontrol agents.

Current state of the artGenetic approaches leading to vector population reduction

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Mosquito densoviruses as tools for population reduction andtransduction.

The Aedes densonucleosis virus (AeDNV; family Parvoviridae) ismosquito specific and does not infect vertebrates or non-target

invertebrates. Larvae are infected in oviposition sites and die in adose-dependent manner depending on viral titre and stage ofinfection. AeDNV is maintained through metamorphosis and istransmitted vertically to offspring (Barreau, Jousset and Bergoin1997). Infected female mosquitoes deliver viruses to multiplebreeding sites and viral concentrations increase as larvae becomeinfected and shed, thus increasing horizontal transmission toother larvae. Survival of infected adult females also decreasessignificantly in a dose-dependent manner (Kuznetsova andButchasky 1988, Suchman and Carlson, unpublished). Shortening

the female adult lifespan would reduce vectorial capacitysince a significant proportion of females would not survive theextrinsic DV incubation period. Recently, a number of otherdensoviruses have been discovered that also may be adapted asbiocontrol and transducing agents (Kittayapong, Baisleyand O'Neill 1999).AeDNV research has the most immediate potential to deliverproducts for an effective field trial once a field site is selected andmore extensive cage experiments completed. Prototype

population cage experiments testing the ability of AeDNV topersist, spread and reduce mosquito populations have alreadybeen performed and areencouraging: a relatively low inoculum of virus in a larval rearingsite replicates to levels that reduce the mosquito population, andfemale mosquitoes originally from the site inoculate virus intonew sites.

3.4. JAPANESE ENCEPHALITIS

To the best of our knowledge, this is the first attempt to study themidgut microbiota of a medically important insectsuch as the Cx. quinquefasciatus using culture-independentmethods. Earlier results and the isolation ofA. culicicola fromthe midgut of both Cx. quinquefasciatus andAe. aegypti indicatethat at least a fraction of mosquito midgut inhabitants could becommon for different mosquito species inhabiting the same

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environment (Demaio et al., 1996) Mosquitoes are known to illicita specific immune response against parasites, Gram-positivebacteria, and Gram-negative bacteria(Dimopolos et al., 1997)Some of these immune responsive genes are expressed in

response to both protozoa and bacteria, and this raises thepossibility that the presence of specific bacteria in the gut mayhave an effect on the efficacy at which a pathogen is transmittedby a vector mosquito(Pumpuni et al.,1996)The present studyassumes importance in the light of earlier studies39 and our ownobservations that the composition of midgut microbiota has asignificant effect on the survival of pathogens in the gutlumen(Mourya et al., 2002).Furthermore, studies involving the effect of the isolates of themidgut bacterial flora from this study on the susceptibility

of Cx. quinquefasciatus to Japanese encephalitis virus havealready been undertaken. Our results indicate that theincorporationof the Pseudomonas and Acinetobacter isolates in the mosquitoblood meal resulted in an increased susceptibility of Cx.quinquefasciatus to this virus(Mourya et al.,2002) Isolatesaffiliated with the genus Acinetobacter form a major part of themidgut microbiota of various mosquito species, as have beenreported in this study and many of the earlier reports.

Moreover, the availability of reliable expression systems andtransposable elements for the genus Acinetobacter provides aneasy tool for the manipulation of the bacteria to produce anti-parasitic proteins. Once established in the mosquito midgut usingartificial means, these modified bacteria can thus be used for thegeneration of transgenic mosquitoes refractory to transmission ofdiseases. Thus, proteobacteria, especially those related to

Acinetobactercould be considered as the candidate bacteria forthe genetic manipulation of mosquitoes.

3.5. YELLOW FEVERWolbachia spp. are maternally inherited, obligately intracellularbacteria that commonly infect invertebrates, including 20% ofinsect species (Bourtzis and Miller, 2003). A hypothesizedexplanation for the evolutionary success ofWolbachia is its abilityto affect host reproduction; cytoplasmic incompatibility (CI) is one

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of the most widely reported effects (Sinkins, 2004). UnidirectionalCI can occur when the Wolbachia infection type present in a malediffers from that in his mate. Although the precise mechanism isunknown, a lock/key model has been proposed in which the

Wolbachia infection modifies the sperm during spermatogenesis(Werren,1997). If the male inseminates a female lacking acompatible Wolbachia type, the modified sperm fail to achievekaryogamy.In contrast, rescue of the modified sperm occurs in embryosfrom females infected with compatible Wolbachia types. Thus,in populations that include both infected and uninfectedindividuals, Wolbachia-infected females can mate successfullywithall males in the population. In contrast, uninfected females can

reproduce successfully only with uninfected males. This patternof unidirectional CI allows Wolbachia to spread rapidly throughhost populations. Previous studies of insects with multipleWolbachia types have demonstrated that unidirectional CI can beadditive (Dobson,2003). Multiple Wolbachia infection types withinan individual male may independently modify sperm, requiring asimilar combination of infection types in female mates forcompatibility. Additive unidirectional CI can result in repeatedpopulation replacement events, in which single- or double-

infection cytotypes are replaced by a Wolbachia superinfection(i.e.,individuals harboring two or more infections). The concept ofpopulation replacement has attracted attention for its potentialapplications. A frequently referenced strategy is based on thereplacement of natural populations with modified populations thatare refractory to disease transmission (Enserlink, 2001). AWolbachia-based population replacement strategy requires thegeneration of artificial infection types that differ from those of thetargeted populations.

Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tigermosquito, is native to Asia and is a globally invasive insect.Examples of introduction and establishment include North andSouth America (Gratz, 2004), and recent invasions have extendedto Africa, Australia, and Europe. In addition to being an invasive

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pest, this mosquito is an aggressive daytime human biter and hasbeen implicated as a vector of animal (Scoles and Kambhampati,1995) and human (Gratz, 2004) disease. Recent reports havehighlighted its role as a primary vector during recent chikungunya

virus epidemics (Simon et al, 2008).Aedes albopictus populationsare naturally infected with two Wolbachia types: wAlbA and wAlbB(Sinkins et al ,1995). Therefore, to employ Wolbachia as a vehiclefor population replacement, additional, incompatible infectionmust be introduced into the natural infection types. Previously,Wolbachia strain wRi was successfully established inA. albopictusby microinjecting the cytoplasm of Drosophila simulans(Riverside) into the embryos of aposymbiotic (i.e., withoutWolbachia) A. albopictus mosquitoes (Xi et al, 2006). Ashypothesized, the resulting artificial infection displayed a pattern

of bidirectional CI when these mosquitoes were crossed with thenaturally double infected strain. Thus, the modification/ rescuemechanism(s) of the wRi infection is known to differ from those ofthe naturally occurring infection types. Therefore, wehypothesized that individuals harboring the combined wRi, wAlbA,and wAlbB infections would be unidirectionally incompatible withthe naturally infected mosquitoes.

CHAPTER FOUR

SUCCESSES AND CHALLENGES OF GENETIC MANIPULATIONOF MOSQUITOES

4.1. SUCCESSES RELATED TO GENETIC MANIPULATIONStill, the mortality and morbidity rates associated with thesepathogens remain high in low-income countries in tropical and

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subtropical regions (Guinovart et al., 2006). In total, more thanhalf of the global population is affected by mosquito-bornediseases resulting in millions of deaths and hundreds of millions ofcases every year. These statistics provide the impetus to study

mosquitoes with the expectation that new knowledge couldcontribute to the alleviation of this disease burden.

The application of genetic analyses and molecular biologicaltechniques for research on mosquitoes provides opportunities forthe development of new disease control strategies (Hill et al.,2005). Among these opportunities are novel vector controlmethods for population reduction or replacement (Curtis andGraves, 1988). Population reduction seeks to decrease theabsolute number of mosquitoes and, therefore, lower the

probability of contact between mosquitoes and their human hosts.

Population replacement strategies are designed to replacesusceptible mosquitoes (can transmit a pathogen) with refractorymosquitoes (cannot transmit a pathogen), and such strategies donot require changes in mosquito population densities. For any ofthese strategies to be effective, it is important to reduce thenumber of infectious mosquitoes below a threshold level so thatthe probability of transmission falls to a point where the parasite

population declines steeply and irreversibly.

One population replacement strategy has the goal to geneticallymodulate vector competence and is based on the hypothesis thatan increased frequency in a vector population of a gene thatinterferes with a pathogen will result in the reduction orelimination of transmission of that pathogen (Collins and James,1996). A key objective was to establish routine methods forgenerating transgenic mosquitoes, and this was achieved with anumber of species using class II transposable elements(Grossman et al.,2001). These successes stimulated debate andresearch to measure the impact of introduced genes on mosquitofitness. For example, integration of a transgene may disrupt oralter expression characteristics of endogenous genes, transgeneproducts may be toxic, or transgene transcription and translationmay usurp resources needed for normal survival or reproductive

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functions. Accordingly, the effects of transgenes integration andexpression on mosquito fitness vary (Irvin et al., 2004). Mostgenetic approaches require the transformed insects to exhibit aslow a fitness cost as is possible (Lambrechts et al., 2008).

Therefore, the expression of a transgene should be limited to aspecific tissue and time in the mosquito to achieve the maximumeffect on the pathogen, while minimizing the potential load on thevector. Recent advances in the areas of genomics and geneticengineering are expected to enable the design and production ofmosquitoes expressing antipathogen effector molecules under thecontrol of synthetic or hybrid (chimeric or mosaic) promoter-regulatory DNA to achieve optimum performance. Functionalsynthetic plant promoters that combine a collection of DNAsequence elements from pathogen-activated genes (Rushton et

al., 2002) serve as a conceptual model for similar developmentsin mosquitoes.

While mass release (inundation) is required for those approaches(SIT and RIDL) that do not propagate genes through a targetpopulation, implementation of population replacement requiresan effective system for gene drive to spread genes into wildpopulations. Gene drive involves the introduction andestablishment of a population replacement effector gene using

genetic mechanisms that circumvent Mendelian inheritance(Braig and Yan, 2002). Standard genetic approaches based ongene segregation and selection require fitness advantages linkedtightly to the antipathogen gene and are likely to be tooprotracted in time to be useful. The bases for gene drive systemscome from known genetic phenomena, two of which arediscussed here. Mobile genetic elements, such as the previouslymentioned class II transposons, may move rapidly intopopulations, for example the P elements of D. melanogasterspread worldwide in a period of _50 years (Kidwell, 1983). Thesemobile genetic elements spread through populations byreplicative transposition in the germline, which means that amating between an insect with an active transposon in itsgenome and one without can result in progeny that all have theelement. However, mobilization of naturally occurring or insertedtransposons has yet to be observed in wild populations or

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transgenic mosquitoes (Sethuraman et al., 2007). This could be anatural consequence of strong selective pressures limitingtransposon movement (ODonnell and Boeke, 2007). Movinggenes into a population without relying on transposon

mobilization is possible with MEDEA (maternal-effect dominantembryonic arrest).

Another genetic control approach that combines populationreplacement with lethality, death-on-infection, demands thata population of mosquitoes carry a conditional lethal gene that isactivated by the presence of parasites or viruses. This results inthe selective death of the infected vectors. Death-on-infectiondiminishes possible ecological effects associated with populationreduction while reducing transmission rates. Specific pathogen

responsive promoters to drive the expression of toxic proteins,apoptosis effectors, or double-stranded RNA (dsRNA) targetingvital gene transcripts are essential for this approach. Analyseshave been conducted of global changes in gene expression inmosquitoes exposed to parasites or viruses (Xi et al., 2008) with agoal to identify genes with enhanced levels of expression uponinfection. Continued studies of mosquito immune responses willcontribute the necessaryregulatory elements for the development of this approach.

SIT is a species-specific and environmentally nonpolluting methodof insect control that relies on the release of large numbers ofsterile insects (Dyck et al., 2005). Mating of released sterile maleswith native females leads to a decrease in the femalesreproductive potential and ultimately, if males are released insufficient numbers over a sufficient period of time, to the localelimination or suppression of the pest population. Highlysuccessful, area-wide SIT programmes have eliminated orsuppressed a range of major veterinary and agricultural pestsaround the world. These programmes can succeed on very largescales the largest rearing facility alone produces around 2 billionsterile male Mediterranean fruit flies per week (~20 tons/week)primarily for use in California and Guatemala. For these pests, SITis a proven cost-effective strategy for eradication or suppression

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of target populations, or to protect areas against invasion or re-invasion.

4.2. CHALLENGES OF GENETIC MANIPULATION

Although major advances have been accomplished in recentyears, it is important that the search for new effector moleculesand promoters continue for two reasons. First, considering howeasily parasites acquire drug resistance, it is likely that parasiteswill be selected that can overcome the barrier imposed by theeffector molecules. Secondly, maximum efficiency of blockingparasite development (ideally 100%) is important for thetransgenic mosquito strategy to have a significant impact ondisease transmission. Furthermore, while many of the tools forgenetic modification of mosquitoes have been developed, an

extensive gap exists in our ability to transfer this technology tothe field for the control of malaria.Others includes:

1. The fitness cost of refractorinessTo maximize the likelihood of successfully introducing refractorygenes into a wild mosquito population, transgenesshould impose minimal fitness load. We assessed fitness oftransgenic An. stephensi expressing the SM1 and the PLA2transgenes by a variety of criteria, including measurements of

longevity and fertility, and use of population cages (L. A. Moreira,J. Wang, F. H. Collins and M. Jacobs-Lorena, manuscript submittedfor publication). The SM1 transgene did not impose a detectablefitness load, but transgenic PLA2 mosquitoes had much reducedfertility and competed poorly with non-transgenics in cageexperiments. The reasons for this reduced fitness remain to beinvestigated. Catteruccia et al., (2003) reported that four differenttransgenic mosquito lines expressing fluorescent reporterproteins from an actin promoter are less fit than the wild type.However, reduced fitness was most likely due to inbreeding. Theyisolated homozygous lines soon after transgenic mosquitoes wereobtained, which may have caused recessive deleterious genesresiding near the point of transgene insertion to becomehomozygous (hitchhiking effect). Conversely, in the experimentsof Moreira et al., the transgenic mosquitoes were kept asheterozygotes, being continuously crossed to mosquitoes from

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laboratory population cages. This demonstrates the importance ofmosquito outcrossing. In addition, the experiments of Catterucciaet al., (2003) used a transgene driven by the strong andubiquitous actin promoter. The abundant synthesis of a foreign

protein throughout the organism may conceivably havedeleterious effects on fitness (Liu et al., 1999). For this reason,SM1 expression was restricted to posterior midgut cells for only afew hours after a blood meal and the protein was secreted fromthe cells, thus minimizing fitness load. Absolute absence of fitnessload may not be essential for introducing genes into wildpopulations. Theoretical modeling suggests that given anappropriate drive mechanism, a gene could have a significantfitness cost and still be driven through the population (Ribeiro andKidwell, 1994; Boete and Koella, 2003). This is fortunate, since

this same model suggests that any released mosquitoes wouldneed to be nearly 100% refractory to have any impact on malariatransmission, necessitating multiple refractory genes that mayincur greater fitness costs.

2. Developing an effective drive mechanism

Two general strategies can be considered for introducingtransgenic mosquitoes in the field: population replacement or a

genetic drive mechanism. Population replacement, or inundatoryrelease, requires a significant reduction of the resident mosquitopopulation (for instance ,with insecticides), followed by therelease of large numbers of refractory mosquitoes to fill thevacated biological niche. This strategy is promising as a researchtool and as a field test to assess the effectiveness of thetransgenic mosquito approach for interrupting malariatransmission. However, this strategy cannot be considered forlarge-scale control purposes, because it is not possible to producesufficient numbers of mosquitoes to achieve populationreplacement on a country- or continentwide level. Transposableelements may incur a substantial fitness cost. Transpositioncauses random integration across the genome, some of whichmay disrupt genes and lead to mutations that could be lethal,reduce fecundity or decrease fitness. Predictive

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models suggest that transposable elements would be able todrive refractory genes from a small number of transgenicmosquitoes into the wild population even if a fitness cost waspresent (Ribeiro and Kidwell, 1994). However, there is

considerable lack of experimental data to corroborate or disprovethe models. Another consideration is that mobility of thetransposable element may be negatively regulated by arepressor. For instance, mobility of the P element in D.melanogaster decreases after several generations because aninhibitor of transposition gradually accumulates and the fly is saidto acquire the P (refractory) cytotype. This is of practicalimportance because in such cases the gene(s) can be driventhrough a population only once. If the effector gene(s) acquiresmutations or the parasite becomes resistant to the effector gene

product another gene cannot be driven into the same populationwith the same transposable element.

(3.)Mass production of transgenic mosquitoes andgenetic sexing mechanisms

Transgenic-based methods to reduce or eradicate vectorpopulations, such as the release of insects carrying a dominant

lethal (RIDL; Thomas et al., 2000), show promise for somespecies. However, their use as a malaria control program in Africawould be difficult to implement due to reproductivelyincompatible subspecies and migration of mosquitoes amongvillages. Even if successful, this approach would leave anecological vacuum that another malaria vector could quickly fill.

Therefore, replacement of wild populations with transgenicmosquitoes carrying refractory genes instead of populationsuppression or eradication methods would be more appropriate.Unfortunately, this approach still requires the release of vastnumbers of biting insects, which is ethically questionable due totheir nuisance factor and potential role as vectors for secondarydiseases. Thus, widespread release of genetically modifiedmosquitoes is best done using only non-biting males,necessitating an efficient system for male selection. Moreover,the ability to release only males would provide a more realistic

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prospect of making the use of transgenic mosquitoes acceptableto the local communities and to the public in general.

(4.) Avoiding resistance to the refractory genesParasites facing a refractory mosquito population would be understrong selective pressure, similar to the one posed by anti-malarials, and thus resistance may develop. Engineering amosquito with multiple refractory genes that target differentaspects of parasite development could minimize resistance to therefractory genes. For example, a transgenic mosquito might beengineered to express a peptide to disrupt midgut and salivarygland invasion, have an enhanced encapsulation response totarget the oocyst, and express defense peptides to target the

sporozoites. Furthermore, chances of success will be greatlyincreased if each refractory element is as close to 100% effectiveas possible and if introduction of the refractory genes is coupledwith traditional control methods, such as reduction of wildpopulations with insecticides prior to a transgenic release, drugtreatment of infected individuals, and use of bed nets. Theeffectiveness of transposable elements may decrease with timeafter field release. Immediately after the introduction of a noveltransposable element into a population the element enjoys a

period of unrestrained activity and spreading. Eventually,individuals with mutations in the transposase or those that haveenacted regulatory inactivation of the element will be selected.

Transposase silencing has been well studied in the mariner familyand has been hypothesized to occur byseveral mechanisms, including overproduction inhibition wherebyan increase in transposase activity correlates with decreasedtransposition or random transposase mutations. Randomtransposase mutations may lead to open reading framedisruptions and inactive transposases that compete with activetransposase for substrate (competitive inhibition) or reduce theactivity of wild-type transposase (dominant negativecomplementation; (Hartl et al., 1997). The mechanism oftransposable element silencing will need to be well understoodbefore transposable elements are used in the field.

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SterilizationRecent advances allow several potential improvements over themethods available in early trials. All current SIT programmes use

radiation to sterilize the insects. However, it has proven difficultto irradiate mosquitoes to near-complete sterility withoutsignificantly weakening them (Andreasen and Curtis, 2005).

Economic cost-benefit analysis, which is needed to support use ofnovel interventions, is difficult because of lack of reliable data onthe economic burden of disease for dengue and other neglectedtropical diseases, and because of uncertainty arounddevelopment and implementation costs. Ideally it would bepossible to analyse not only the cost-effectiveness of the stand-

alone novel strategy, but also to compare it with existingalternate strategies and to model its incorporation in integratedvector management (IVM) programmes, and indeed integrateddisease management programmes including drugs and vaccines,where available.

As genetics-based population suppression moves from laboratoryto field, the lack of a clear regulatory framework for field use ofmodified mosquitoes is a significant challenge. This issue is not

restricted to developing countries, or to strategies dependent onthe use of recombinant DNA technology. Once regulatoryframeworks are in place, risk assessments and public consultationalso will be lengthy processes due the novelty of technologies andlack of experience by regulating agencies. The route toimplementation of control programmes based on thesetechnologies is not obvious. Agricultural SIT programmes havegenerally been established and operated by governments, thoughthere is limited private-sector involvement. Existing vector controlprogrammes are generally government-funded and -operated,though they purchase vector control products and services fromthe private sector. The development of new vector controlapproaches is generally in the private sector. The current genetic-based technologies are perceived as too high-risk for largecompanies

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to bring them into their portfolios. This risk is a combination of thetechnical and regulatory risks of bringing the technologies tomarket and the market risk or uncertainty regarding customersand prices.

A myriad of logistic, financial and technical challenges face theseprogrammes though not particularly related to human health orregulation. To test any technology on a large scale with littleexperience in a developed country is difficult ; to do so in adeveloping country is more so. The availability of trainedpersonnel, materials and infrastructure present country-specificdifficulties. Some of this cannot be anticipated and may not beeasy to remedy.

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CHAPTER FIVE

CONCLUSION

Major advances in recent years, including successful germlinetransformation and characterization of promoters, are allowingresearchers to test putative refractory genes. One important taskfor the near future is the identification of additional effectorgenes, and this will be greatly facilitated by the availability of the

An. gambiae and P. falciparum genome sequences. Thisknowledge can be used to engineer a mosquito that inhibits orkills the malaria parasite during multiple developmental stages.With this ideal mosquito on the horizon, the most important taskis to begin laying the groundwork for its introduction into the wild.A high priority should be devoted to the topic of how to introducethe relevant genes into wild mosquito populations. Also neededare ecological studies to evaluate population structure and geneflow. In addition, we must grapple with the ethical and political

concerns involved with a large-scale release of a geneticallymodified organism. Considerable challenges lay ahead but thereare reasons to be optimistic that we will be able to add geneticmodification of mosquitoes to our arsenal in the fight againstmalaria.While most are in the realm of guidance or guidelines, some haveregulatory status and are legally binding. Each can provide usefulbackground for upcoming national decisions regarding applicationof genetic strategies for vector control,as the most promisingtechnologies move from laboratory to confined or open field trialsand, if successful, eventual widespread field programmes forvectored disease control.The effectiveness of this method can bebest achieved by:(1.) Provide funding to develop the general mathematical modeldescribed above. While there are several efforts to developmodels for specific technologies, these are neither sufficiently

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flexible nor accessible by project planners to make them of valuefor routine programme implementation.(2.) Provide funding for sensitive methods for Anophelessurveillance. These must be robust and capable of being applied

over large areas.(3.) Provide funding for development of aerial release equipment.The same equipment can likely be used for all genetic releaseprogrammes. Such equipment allows the spatial extent of vectorreleases to be realistically considered.

Planning vector release programmes will be facilitated bydevelopment of general mathematical models of SIT that areupdated and modified to include characteristics such as larvaldensity dependence and survival, reduced mating

competitiveness, species bionomics and semi-sterility. Theseprogrammes should be accessible via a simple interface to end-users who are planning release programmes, not only softwaredevelopers or modellers. Surveillance methods for Anopheles atvery low population densities will be challenging. During theeradication ofAn. arabiensis from Egypt, the absence of reboundof populations to cessation of control efforts confirmedelimination, but this will not be suitable for routine assessment ofprogramme effectiveness. Sensitive methods and materials such

as attractants are needed.

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