GENETIC MANIPULATION OF MOSQUITO AS A VECTOR CONTROL OPTION

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.

ABSTRACT

In the ongoing fight against vectors of human diseases, disease endemic countries may soon benefit from innovative control strategies involving modified insect vectors. For instance, three promising methods (viz. RIDL [Release of Insects with a Dominant Lethal], 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 as dengue (serotypes 1–4), Japanese encephalitis and yellow fever. Some of these techniques are already being extended to other vectors such as Aedes albopictus (the secondary vector of these diseases) and Anopheles mosquito species that transmit malaria. To enable Disease Endemic Countries (DECs) to take advantage of these promising methods, initiatives are underway that relate to biosafety, risk assessment and management, and ethical–social–cultural (ESC) aspects to consider prior to and during the possible deployment of these technologies as part of an integrated vector control programme. There has been considerable progress over the last decade towards developing the tools for creating a refractory mosquito. Accomplishments include germline transformation of several important mosquito vectors, the completed genomes of the mosquito Anopheles gambiae and the malaria parasite Plasmodium falciparum, and the identification of promoters and effector genes that confer resistance in the mosquito. Better control measures such as funding the development of Mathematical models, provide fund for the sensitive of mosquito surveillance and funding aerial release equipment.

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

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

                                     CHAPTER ONE

INTRODUCTION

Mosquito is a devastating vector that attacks between one and three million people annually and causes massive economic losses (Curtis 1996). Moreover, the number of cases is increasing, due to the emergence insecticide-resistant mosquitoes, although intensive research to develop new drugs and insecticides is ongoing (Federici 1995). Furthermore, despite promising developments, no effective vaccines have yet been developed and existing control measures are inadequate. Mosquitoes are obligatory 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 has considerable promise as a new approach in the fight against malaria. Development of the malaria parasite in the mosquito is complex (Fig.·1; Ghosh et al., 2003) and for the most part occurs in the midgut (gamete to oocyst stages). Although thousands of gametocytes are acquired with the blood meal, only a few successfully mature into oocysts, but each of them produces thousands of sporozoites (Ghosh et al., 2001). Because oocyst formation is a bottleneck in sporogonic development, targeting pre-sporozoite stages could be a more effective strategy to block parasite transmission.

In recent years, methods for the genetic modification of mosquitoes have been developed, and effector genes whose

products interfere with Plasmodium development in the mosquito are beginning to be identified. While many of the

initial hurdles have been overcome, major questions remain to be answered, foremost among which is how to introduce refractory genes into wild mosquito populations. Here strategies to alter mosquito vector competence and consider issues related to translating this knowledge to field applications.

The overarching goal of malaria vector control is to reduce the vectorial capacity of local vector populations below the critical threshold needed to achieve a malaria reproduction rate (R₀, the expected number of human cases that arise from each human. Because of the long extrinsic incubation time of Plasmodium in its Anopheles vectors, the most effective vector control strategies in use today rely on insecticide interventions like indoor residual insecticide sprays (IRSs) and long-lasting insecticide-treated nets (LLINs) that reduce vector daily survival rates (Enayati and Hemingway, 2010). For many malaria-endemic regions, these tools can make substantial contributions to malaria control and may be sufficient for local malaria elimination. These were the only regions considered by the recent Malaria Elimination Group (MEG). Regions where existing interventions will not be sufficiently effective include those where high rates of transmission occur. For example, in much of sub-Saharan Africa, where the entomological inoculation rates (EIRs) can reach levels approaching 1,000 infective bites per person per year (Hay et al, 2000), the best use of existing interventions can only help to reduce annual inoculation rates by approximately an order of magnitude. Additional interventions will clearly be required, however, both for regions with extremely high rates of transmission and for regions where the major vectors are not susceptible to current control tools [Shaukat et al, 2010].

To develop vector-targeted interventions in support of malaria eradication in all disease endemic settings that are unfettered by these limitations, three challenges need to be recognized and addressed with great urgency today. The first challenge, for which near-term product development is essential, is the preservation and improvement of the utility of existing insecticide-based interventions. This challenge will require a vibrant research agenda that develops a broader range of insecticides with novel modes of action that can circumvent emerging resistance to existing insecticides, particularly the pyrethroids. This agenda must include the creation of strategies for the use of new insecticides that minimize the emergence of resistance. A related and critical focus of the agenda will be the development of rapid and affordable methods for detecting the emergence of epidemiologically important levels of insecticide resistance. Because of the fundamental dependence of many current malaria control and elimination programs on pyrethroid insecticide–based LLINs and the emerging problem of pyrethroid insecticide resistance in many vector species, especially in sub-Saharan Africa, 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 affect vector species not effectively targeted by current tools. At least three dozen different species of Anopheles mosquitoes are important in malaria transmission worldwide. Many of these species are not susceptible to tools like IRS and LLINs, which target indoor feeding and/or resting vectors [Terenius et al, 2008]. Control of malaria transmitted by these vectors will require new 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 the agenda to tackle this challenge will be defining the vector species for which such new tools are most important and devising tools that will be effective for multiple important vector species.

The most difficult research challenge for vector control during all phases of malaria elimination/eradication but particularly during the final stages of eradication is development of novel approaches that will permanently reduce the very high vectorial capacities of the dominant malaria vectors in sub-Saharan Africa. Without such approaches, local elimination in Africa will be extremely challenging. Even when elimination is achieved, the residual vectorial capacities of local mosquitoes will pose a lingering threat of massive epidemics should malaria be reintroduced to a population that has lost partial immunity. Measures to reduce vectorial capacities will need to be either extremely cost-effective, if they are to be sustained until eradication is achieved, or able to effectively yield a long-term, sustained reduction of transmission following a one-time application. Genetic control programs (which could be achieved by a variety of genetic manipulation approaches) designed to permanently reduce the vectorial capacities of natural vector populations have received the most attention to date, and currently represent some of the most promising ideas in this area [sinkins and Gould, 2006], but the development of other novel approaches must be strongly encouraged.

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

Disease endemic countries (DECs) are showing interest in the possible benefits of innovative control methods involving modified (either as a genetic drive mechanism or through infection) mosquito vectors of human diseases. For instance, three promising methods Release of Insects (mosquitoes) carrying a Dominant Lethal gene (viz. RIDL, Wolbachia, and refractory mosquito technology) are being developed by researchers around the world to combat Aedes aegypti, the

primary mosquito vector of viral fevers such as dengue (serotypes 1-4), chikungunya and yellow fever. Some of these techniques are already being extended to other vectors such as Aedes albopictus (the secondary vector of these diseases) and Anopheles species that spread malaria. Therefore, these innovative strategies, and their agents, are coming to the attention of the regulators, vector control agencies, and policy- makers in DECs. To enable DECs to take advantage of these promising methods, initiatives are underway that relate to biosafety, risk assessment and management, and aspects to consider prior to and during the possible deployment of these technologies as part of an integrated vector control programme.

Alternatively, it may be possible to alter the mosquito population to a less harmful form, for example by making the mosquitoes unable to transmit specific pathogens. Such approaches, known as ‘population replacement’ strategies, have two essential steps. The first of these is to identify a heritable modification that will make 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 be self-limiting or self-sustaining in the target wild population. Self-limiting systems will by design be eliminated from the target population over time, e.g. by natural selection. The modification is then maintained in the wild population only by periodic release of additional modified mosquitoes. The speed of this elimination may vary from one strategy to another; for example a dominant lethal or

sterilizing transgene will be completely eliminated in one generation, whereas a construct with a milder fitness penalty

may persist for several generations. Nonetheless, self-limiting systems will neither persist indefinitely nor spread significantly beyond the target area. In contrast, self-sustaining systems are intended to persist indefinitely, and indeed to increase in prevalence, e.g. allele frequency, in the target area and beyond. These properties may make deployment of such systems relatively inexpensive, as they may be able to spread from a relatively small release. However, their indefinite presence in the environment, 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 most dangerous of all animals (Curtis 1996).This mosquito is a particularly dangerous vector as it is anthropophilic and has a long life-span. Mosquitoes are also the vectors for such diseases as 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 an important means of improving public health.

                                                                                                                       

Effective vector control is dependent upon knowledge of a species' specific ecology.  Five elements of a program for vector control include (1.) determining the vector species, (2.) knowledge of the mosquito’s 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 of many 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 of non-persistent insecticides or by environmental manipulation but this is usually on a limited scale and at a high cost. The discovery of long lasting organic synthetic insecticides, revolutionalized the whole concept of vector control and for the first time it was possible to contemplate the control or even eradication of majority of arthropod-borne diseases throughout the world. However, the development of insecticide-resistance and the discovery of behavioural characters which impede control have seriously modified this optimistic outlook and vector control has again become one of the important problems confronting health authorities.

Some of the world’s most important infectious diseases are transmitted by insects (Sinkins and Gould 2006). The burden of these diseases is especially heavy in the developing countries of the tropics. Control programs have commonly relied on insecticides to control the vector, vaccines if available, and therapeutic drugs to treat infected patients. Insecticides, which are still arguably the best control weapon, are becoming increasingly ineffective as vector species

develop resistance. New insecticides are becoming prohibitively expensive to develop and the public is concerned about the detrimental effects they may have on the environment. To further

compound these problems many human parasites  are becoming drug resistant (e.g. Plasmodium and  chloroquine) and the promise of safe, inexpensive  and efficacious vaccines has not materialised.

Furthermore, human populations are becoming increasingly mobile; living in ever expanding cities with little public health infrastructure. The end result is the current worsening global vector-borne disease situation making the need for innovative,

sustainable control strategies urgent.

The techniques of molecular biology and genetic engineering have the potential to play a role in the  development of new, integrated control programs.  The idea of controlling vector-borne diseases

through the genetic manipulation of insects dates  back to the 1940s (Vanderplank 1944). The  potential of genetic techniques for use in vector  control programs is evident from the successful

eradication of the screwworm from the USA,  Mexico and Libya (Vargas-Teran et al., 1994; Baumhover 1966). However, sterile male release  is unlikely to be a cost effective strategy for mosquito populations due to their high  reproductive rates and ability to rapidly colonise  new areas. Rapid advances in DNA based

technology are now allowing scientists to envision  novel genetic control strategies involving the creation and release of transgenic insects (Crampton et al, 1994; Gwadz 1994). Instead of focusing on the  eradication of the vector, these strategies aim to replace natural vector populations with transgenic insects unable to transmit disease, thereby effectively “immunising” the vector rather than the human population.

Three interconnected research objectives must  be achieved before a disease control strategy  involving the release of transgenic vectors could  be attempted. First, genes which encode traits that  render the vector refractory to a particular  pathogen must be identified. Second, methods to  introduce and express these genes in insects in a  stable, heritable fashion must be developed. Third,  a means for spreading these genes to high frequency in natural vector populations must be accomplished.

At the present time much of the research being  conducted on the genetic modification of insect disease vectors is focusing on the African malaria vector Anopheles gambiae. This is not surprising

considering that malaria is by far the most important vector-borne disease in the world today. As this paper will consider, the technology needed to manipulate mosquito vector populations is likely to be developed in the near future. In planning a vector control strategy involving transgenic insects, care must be taken in selecting the disease system with which to first apply this strategy. The complex epidemiology of malaria together with the extremely high transmission rates common in much of Africa suggest that this technology might have a better hope of success if utilized initially

in a less ambitious disease context. Lessons learned from initial interventions could then be used to develop a sustainable integrated strategy for malaria control in Africa and other sub saharans.

CHAPTER TWO

                 

                   METHODS OF GENETIC MANIPULATIONS

2.1.           TRANSMISSION BLOCKING VACCINES

Transmission blocking vaccines consist of antibodies that are ingested by the mosquito with the blood meal and interfere with parasite development. Proteins expressed on the surface of gametes (e.g. Pfs47/48, Pfs230) and ookinetes (e.g. Pfs25 and Pfs28) have been tested for such vaccines ( Healer et al., 1999). Antibodies

2.2. PARATRANSGENESIS

Paratransgenesis, the genetic manipulation of commensal or symbiotic bacteria to alter the host’s ability to transmit a pathogen, is an alternative means of preventing malaria transmission. Bacteria can be engineered to express and secrete peptides or proteins that block parasite invasion or kill the parasite in the midgut. This strategy has shown promise in controlling transmission of Trypanosoma cruzi by Rhodnius prolixus under laboratory conditions (Beard et al., 2002). Furthermore, symbiotic bacteria in the tsetse fly have been isolated, transformed with a reporter gene, and reinserted into the fly (Beard et al., 1998). For this strategy to be used in malaria control, bacteria that can survive in the mosquito’s midgut must be identified. Gram-positive and -negative bacteria, including Escherichia, Alcaligenes, Pseudomonas, Serratia and Bacillus, have been identified in the midgut of wild anopheline adults (Demaio et al., 1996; Straif et al., 1998). These bacteria are easily cultured in the laboratory and may be suitable targets for genetic manipulation. Whether these bacteria are stable or transient residents of the midgut of adult mosquitoes remains to be determined. To successfully control malaria the refractory proteins or peptides expressed by the bacteria must act on the midgut stages of the malaria parasites, maintain their bioactivity in the midgut environment, and be expressed in sufficient quantities. When An. stephensi mosquitoes were fed E. coli that express a fusion protein of ricin and a single-chain antibody against Pbs21 (a P. berghei ookinete surface protein), oocyst formation was inhibited by up to 95% (Yoshida et al., 2001). Other effector molecules, such as SM1 and PLA2, are considered below (see ‘Genetically modified mosquitoes’). The use of paratransgenesis for the control of malaria will require the development of methods to introduce genetically modified bacteria into field mosquitoes.

2.3. GENETICALLY MODIFIED MOSQUITOES

Another promising approach is to genetically modify mosquitoes to express proteins or peptides that interfere with Plasmodium development. Methods to produce transgenic culicine (Jasinskiene et al., 1998) and anopheline (Catteruccia et al., 2000). Promoters to drive the transgenes and effector molecules whose products hinder parasite development are considered below :

2.3.1. PROMOTERS

An essential step in engineering mosquitoes with reduced vector competence is the identification of suitable promoters to drive the expression of anti-parasitic genes. During its development in the mosquito, the parasite occupies three compartments: midgut lumen, hemocoel and salivary gland lumen. Thus, promoters that drive synthesis and secretion of proteins into these compartments need to be identified. In addition to spatial considerations, the time of protein synthesis relative to arrival of the parasite in each of 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 in the laboratory demonstrated that carboxypeptidase, a digestive enzyme, and  a peritrophic matrix protein, are activated in response to a blood meal and the proteins secreted in the midgut lumen (Edwards et al., 1997).

Later stages of parasite development can be targeted in the hemocoel and salivary glands. The vitellogenin promoter and signal sequences were shown to drive strong gene expression in the fat body and protein secretion into the hemocoel (Kokoza et al., 2000). However, this gene has a restricted temporal profile of expression that peaks around 24·h after a blood meal and returns to basal level by 48·h. Soon after traversing the midgut epithelium, the ookinete transforms into an oocyst that is covered by a thick capsule. Sporozoites are liberated from the oocyst as early as 10 days later. These characteristics of parasite development limit the choice of effector genes that can be used in conjunction with the vitellogenin promoter to those encoding proteins with exceptionally long half-lives. Re activation of the vitellogenin promoter by additional blood meal(s) may lessen this shortcoming. The availability of a strong promoter with peak expression in the hemolymph at about the time of sporozoite release from oocysts would be ideal. Two salivary gland promoters 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 large numbers of sterile males. Thus, if a female mates with a male that has no sperm or whose sperm was rendered unviable, this female will have fewer or no progeny. When many sterile males are released, the local population tends to decline or become extinct. There are a number of cases of the successful local application of this technique, for example, in the control of the Mediterranean fruit fly in Latin America, the New World screwworm in the Americas and Libya, and for tsetse in Zanzibar, Africa. SIT also

has 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 the number of released sterile males to the number of males in the local population, which ideally should be around 10:1. Therefore, sterile-insect control is only effective when the resident population to be controlled is small relative to the number of sterile males that can be mass-produced for release or when it can be reduced to very low levels with conventional control tools before the start of releases. It is highly desirable that only males be released for two reasons: (1.) In most cases only females bite and transmit disease while, moreover, sterile females can also transmit (2.) Males would court and mate with the released sterile females (instead of local females), thus reducing the efficacy of the programme (Alphey and Andreasen 2002). Large-scale production in the laboratory of a pure male population by non-genetic means may be problematic. It may rely on sex-specific differences of pupal size (culicine mosquitoes) or adult eclosion times (tsetse), but these protocols rarely yield a 100% male population. Clearly, genetic sexing methods (see below) are far superior. The most commonly used technique for male sterilization is exposure to high doses of radiation, a procedure that damages chromosomes and results in unviable sperm. Sterilization by chemical means also has been employed. Because of the large numbers of insects that need to be released, it is crucial that the effectiveness of the sterilization procedure approaches 100%. However, the large doses of radiation and chemicals needed to achieve this effectiveness may reduce insect fitness, survival and mating competitiveness. These strategies can fail if the laboratory-reared males do not mate as effectively as their field counterparts. The advent of germ-line transformation for a number of different insects has led to the development of genetic alternatives for production of sterile insects (Thomas et al., 2000). In one version of this approach (Release of Insects carrying a Dominant Lethal or RIDL, Thomas et al., 2000), a conditional dominant lethal gene is introduced into the target insect genome. This gene has two important properties: 1) it is expressed only in females (or it kills only females); and 2) the gene is effectively repressed by a compound that does not occur normally in nature (e.g. tetracycline). Large insect populations are maintained by rearing them in the presence of tetracycline, which represses the dominant lethal gene and allows the survival of equal numbers of males and females. Prior to release, the insects are reared in the absence of tetracycline, a condition that allows the expression of the dominant lethal gene and the death of all females. The resulting males can be released without further manipulation or treatment. Males carry two copies (homozygous) of the dominant lethal gene. When these males mate in nature, all female progeny will be killed and only males will be produced. Since these surviving males are heterozygous for the dominant lethal gene, the population-reducing effect is still manifested in the second generation.

It should be emphasized that the effectiveness of the Sterile Insect Technique (SIT) is dependent on population structure and dynamics. Furthermore, this technique leaves intact the biological niche in which the target insect is found. SIT is most likely to succeed in cases where target populations are small, the number of target insects is low, and the target area is sufficiently isolated, thereby reducing the likelihood of re-invasion. It is unlikely to be effective for controlling mosquito populations in highly endemic areas of Africa where the mosquito population consists of several vector species in high densities, where access to breeding sites is difficult and where poorly interbreeding mosquito populations co-exist.

2.3.3. GERM-LINE TRANSFORMATION

Drosophila melanogaster was the first multicellular organism to be stably transformed (Spradling and Rubin 1982). The same general principles that were used in this pioneering work are still employed today for all germ-line transformation work in insects (Atkinson and James 2002). Embryos are injected with two DNA constructs. One construct contains a gene encoding a dominant selectable marker (e.g., eye color, a fluorescent protein) and the gene of interest, each driven by a separate promoter, and both sequences are together flanked by the inverted repeats of a transposable element. The second construct encodes a transposase, which is an enzyme that recognizes the inverted repeats and catalyses the insertion of the intervening sequences into the genome of the host insect. It took from 1982 until the mid 1990s to develop two crucial technologies: an appropriate transposable-element system (at first scientists did not realize that the P transposable element is not active in non-Drosophila organisms) and a suitable transformation marker (e.g., GFP). Since then, germ-line transformation of many insects has been accomplished but mosquitoes (Aedes, Anopheles, Culex) which are the only insects of medical importance in this list. Importantly, both An. stephensi and An. gambiae can be transformed, though the success rate in the latter case is still low. Improvement of the transformation efficiency of An. gambiae is a high-priority topic for future research. It would also be desirable to develop germ-line transformation procedures for other medically important insects such as sand flies and black flies. Current technology cannot be applied to germ-line transformation of tsetse because these do not lay eggs (that would need to be injected), only fully formed larvae. However, genetic modification of tsetse vectorial capacity could be achieved via genetic modification of one of its symbionts.

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

2.3.4. EFFECTOR GENES

The term effector gene is used here for genes whose products interfere with the development of a pathogen. At least four classes of effector genes can be identified:( 1) Genes whose products interact with insect host tissues crucial for parasite development: Examples of this class are SM1, a peptide that occupies putative salivary-gland and midgut receptors for the malaria parasite (Ghosh and Ribolla;Jacobs-Lorena 2001) and phospholipase A2 (PLA2), which is a protein that interferes with the malaria ookinete invasion of the midgut (Zieler et al., 2001).( 2.) Genes whose products interact with the pathogen: Examples of this class are genes encoding single chain monoclonal antibodies that bind to the parasite’s outer surface thus blocking their development ( De Lara Capurro et al., 2000).( 3.) Genes whose products kill the pathogen: Examples are peptides from the insect’s innate immune system such as defensins and cecropins, and peptides from other sources that act as selective toxins to parasites but do not affect the host insect, such as magainins, Shiva-1, Shiva-3 and gomesin (Kim et al., 2004). Most published work on effector genes deals with effects on the malaria parasite and little is known about such genes for other pathogens. In particular, it is not clear what class of effector genes would be useful for nematodes (filaria). Since these may be encapsulated in certain mosquito strains, genes that activate encapsulation could be considered as possible effector genes. For viruses, genes of the first class (interference of host-tissue invasion) or genes that interfere with virus replication (Olson et al., 1996) are possible candidates.

4) Another possible strategy to reduce vector competence is by manipulation of its immune genes, for instance by using RNA interference or ‘smart sprays’ (Christophides,Vlachou and Kafatos 2004).

Another important strategic consideration is the stage of malaria parasite development to target. When a mosquito ingests an infected blood meal, it acquires thousands of gametocytes of which only few (usually less than ten) manage to cross the midgut and form oocysts. Later, each oocyst produces thousands of sporozoites, a significant proportion of which invade the salivary gland. Because the strong bottleneck at the level of midgut invasion, this stage of parasite development constitutes a prime target for intervention.

Midgut invasion is also a strong bottleneck in the process of arboviral transmission.

CHAPTER THREE

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

3.1. MALARIA:

Successful development of the technology described above (transgenesis, promoter characterization and effector-gene identification), permitted the creation of genetically modified mosquitoes impaired in their ability to transmit the malaria parasite. An early example was the creation of an Ae. aegypti expressing defensin in the haemolymph (Kokoza et al., 2000). However, the effect of defensin on malaria parasite development has not been reported. At about the same time, the James laboratory reported that a single-chain monoclonal antibody that recognizes a

sporozoite surface protein inhibits invasion of the salivary gland (De Lara Capurro et al., 2000). In this instance, the effector gene was transiently expressed from a viral vector that is not inherited by the mosquito progeny. The Jacobs-Lorena laboratory showed that a stably integrated gene encoding SM1 strongly inhibits parasite

development in transgenic mosquitoes (Ito et al., 2002). In another example, transgenic mosquitoes expressing PLA2 also had much reduced vectorial competence (Moreira et al., 2002). Recently, it was demonstrated that the capacity to transmit the malaria parasite is reduced by about 60% in transgenic An. gambiae expressing

cecropin 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 reported experiments have been done with non-human malaria parasites. An important next step is the transfer of this technology to human pathogens.

Wolbachia. Wolbachia are intracellular bacteria that inhabit the germ line of a number of insects and distort reproduction by killing progeny that do not contain it, by a phenomenon known as cytoplasmic incompatibility (CI). Compelling evidence in favour of Wolbachia as a drive mechanism comes from Drosophila. Turelli and Hoffmann (1991) observed that Wolbachia swept through the D. simulans population in California at the rate of 100 km per year. In principle, Wolbachia could provide a powerful driving mechanism. However, no Wolbachia have yet been identified in anopheline mosquitoes (these are the exclusive vectors for human malaria), although they have been observed in culicine mosquitoes. A major limitation of Wolbachia is that it inhabits the germ line while the pathogen develops in the soma. Thus, it is difficult to target parasites with genes introduced into Wolbachia. A possible solution to this problem is the identification of genes that cause CI. Currently little is known at the molecular level about how CI functions or how many genes are involved. When identified, such gene(s) could conceivably be used to create a driving mechanism via

their insertion into the mosquito genome.

Meiotic drive. Population replacement can be driven by certain genes, such as the Drosophila segregation distorter gene, that favour its inheritance over individuals not containing the gene. Unfortunately, very little is known about such genes in insects of

medical importance. One complication is that at least in model systems (Drosophila, mouse), the drive mechanism depends on multiple genes (e.g., distorter and

responder) and this could complicate the implementation of this system in mosquitoes. Moreover, if such genes were to be employed to drive effector genes into populations, all meiotic drive and effector genes would have to be tightly linked to avoid loss of effectiveness due to recombination.

3.2. FILARIASIS

Malaria and Bancroftian filariasis rank amongst the world's most prevalent tropical infectious diseases. An estimated 300–500 million people are infected with malaria annually, resulting in 1.5–3 million deaths (WHO, 2000). Lymphatic filariasis is probably the fastest spreading insect-borne disease of man in the tropics, affecting about 146 million people (WHO, 1992). Many biological control agents have been evaluated against larval stages of mosquitoes, of which the most successful ones comprise bacteria such as Bacillus thuringiensis israelensis and B. sphaericus (Becker and Margalit, 2003), mermithid nematodes such as Romanomermis culicivorax (Zaim et al, 1988), microsporidia such as Nosema algerae (Undeen and Dame, 1987), and several entomopathogenic fungi (Federici, 1995). Among these fungi, the oomycete Lagenidium giganteum has proven successful for vector control in rice fields (Hallmon et al,2000) and is currently produced commercially (Khetan,2001). Other mosquito-pathogenic fungi that target larval instars include the chytidriomycetes Coelomomyces (Shoulkamy and Lucarotti, 1998), and the deuteromycetes Culicinomyces (Sweeney, 1981), Beauveria (Clark et al, 1968) and Metarhizium (Robert, 1970). Of the few fungi known to infect adult Diptera, the majority belong to the group of Zygomycetes (Entomophthoraleans) (Low and Kennel, 1972).

Only a handful of studies have evaluated biological control agents/methodologies to control adult stages of tropical

disease vectors. Soarés (1982) infected adult Ochlerotatus sierrensis 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 of Culex tarsalis, Cx. pipiens, Aedes aegypti, Ochlerotatus sierrensis, Ochlerotatus nigromaculis, and Anopheles albimanus were susceptible to Beauveria bassiana. Recently, (Scholte et al, 2003) reported that adult An. gambiae is susceptible to B. bassiana, a Fusarium spp., and Metarhizium anisopliae.

Several current techniques appear capable of reducing transmission of filarial parasites, but most still require both validation of their impact in largescale control programmes and assessment of their cost-effectiveness. Among the most promising are the following: biocides, especially Bacillus sphaericus (a self-reproducing, toxin-producing bacterium) for the control of Culex quinquefasciatus mosquitos (Hougard, 1993); polystyrene beads to limit the breeding of vectors, especially in enclosed urban breeding sites, such as latrines and cesspits.

3.3. DENGUE FEVER:

Dengue fever (DF) and its more serious form, dengue hemorrhagic 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 mortality than any other vector-borne viral disease with 2.5-3.0 billion people at risk of infection and 50-100 million DF and 250,000-500,000 DHF/DSS annual cases (Gubler 1996; 1998). All four DV serotypes cause disease and case-fatality rates for untreated DHF/DSS can be 30-40%. The risk of DHF/DSS is highest in areas where two or more DV serotypes are transmitted ( Rigau-Perez et al., 1998). At this time, there is no licensed vaccine and no clinical cure for the disease. Ae. aegypti is by far the most important and efficient vector of DV because of its affinity for humans (Gubler, 1998). Dengue control currently depends on reduction or elimination of Ae. aegypti. In the 1940-1960s most tropical American countries used integrated programmes of environmental management and insecticides to eliminate mosquitoes (Gubler, 1998), but many of these were abandoned in the early 1970s (Reiter and Gubler 1997).

Several Government Viral Control(GVC) strategies for reducing DV transmission have been identified as potential dengue disease control methods and are designed either to reduce the overall population of DV-transmitting vectors or to replace existing vector populations with populations that cannot transmit the virus. Two vector population reduction approaches are currently being investigated and are in early laboratory cage trials. The first population reduction strategy is the development and use of natural or genetically engineered densoviruses that are pathogenic to Ae. aegypti (Carlson, Afanasiev and Suchman 2000).

The second population reduction strategy is the development and use of insects carrying dominant lethal mutations ( Thomas et al. ,2000). This approach would require mating of Genetically Modified Vectors (GMV)-RIDL males with local vector populations producing offspring that die prior to becoming adults. Both approaches are designed to reduce transmission of DVs by reducing the vector population. Approaches designed to replace populations of vectors are more long-term, but could have significant consequences for dengue disease control in the future (James, 2000). In these approaches, an effector gene, such as an anti-DV gene, is appropriately expressed to block transmission by the vector. GVC approaches

require identification of tissue-specific promoters, anti-pathogen effector genes, and genetic drive mechanisms such as synthetic transposable elements (TE) to introgress the effector gene into the population, eliminating vector competence. Successful GVC strategies will require knowledge of vector ecology in DECs and large cage trials in DECs prior to release of biocontrol agents.

Current state of the art

Genetic approaches leading to vector population reduction

Mosquito densoviruses as tools for population reduction and transduction.

The Aedes densonucleosis virus (AeDNV; family Parvoviridae) is mosquito specific and does not infect vertebrates or non-target invertebrates. Larvae are infected in oviposition sites and die in a dose-dependent manner depending on viral titre and stage of infection. AeDNV is maintained through metamorphosis and is transmitted vertically to offspring (Barreau, Jousset and Bergoin 1997). Infected female mosquitoes deliver viruses to multiple breeding sites and viral concentrations increase as larvae become infected and shed, thus increasing horizontal transmission to other larvae. Survival of infected adult females also decreases significantly in a dose-dependent manner (Kuznetsova and Butchasky 1988, Suchman and Carlson, unpublished). Shortening the female adult lifespan would reduce vectorial capacity

since a significant proportion of females would not survive the extrinsic DV incubation period. Recently, a number of other densoviruses have been discovered that also may be adapted as biocontrol and transducing agents (Kittayapong, Baisley

and O'Neill 1999).

AeDNV research has the most immediate potential to deliver products for an effective field trial once a field site is selected and more extensive cage experiments completed. Prototype population cage experiments testing the ability of AeDNV to persist, spread and reduce mosquito populations have already been performed and are

encouraging: a relatively low inoculum of virus in a larval rearing site replicates to levels that reduce the mosquito population, and female mosquitoes originally from the site inoculate virus into new sites.

3.4. JAPANESE ENCEPHALITIS

To the best of our knowledge, this is the first attempt to study the midgut microbiota of a medically important insect

such as the Cx. quinquefasciatus using culture-independent methods. Earlier results and the isolation of A. culicicola from

the midgut of both Cx. quinquefasciatus and Ae. aegypti indicate that at least a fraction of mosquito midgut inhabitants could be common for different mosquito species inhabiting the same environment (Demaio et al., 1996) Mosquitoes are known to illicit a specific immune response against parasites, Gram-positive bacteria, 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 the possibility that the presence of specific bacteria in the gut may have an effect on the efficacy at which a pathogen is transmitted by a vector mosquito(Pumpuni et al.,1996)The present study assumes importance in the light of earlier studies39 and our own observations that the composition of midgut microbiota has a significant effect on the survival of pathogens in the gut lumen(Mourya et al., 2002).

Furthermore, studies involving the effect of the isolates of the midgut bacterial flora from this study on the susceptibility

of Cx. quinquefasciatus to Japanese encephalitis virus have already been undertaken. Our results indicate that the incorporation

of the Pseudomonas and Acinetobacter isolates in the mosquito blood meal resulted in an increased susceptibility of Cx. quinquefasciatus to this virus(Mourya et al.,2002) Isolates affiliated with the genus Acinetobacter form a major part of the midgut microbiota of various mosquito species, as have been reported in this study and many of the earlier reports.

Moreover, the availability of reliable expression systems and transposable elements for the genus Acinetobacter provides an easy tool for the manipulation of the bacteria to produce anti-parasitic proteins. Once established in the mosquito midgut using artificial means, these modified bacteria can thus be used for the generation of transgenic mosquitoes refractory to transmission of diseases. Thus, proteobacteria, especially those related to Acinetobacter could be considered as the candidate bacteria for the genetic manipulation of mosquitoes.

3.5. YELLOW FEVER

Wolbachia spp. are maternally inherited, obligately intracellular bacteria that commonly infect invertebrates, including 20% of insect species (Bourtzis and Miller, 2003). A hypothesized explanation for the evolutionary success of Wolbachia is its ability to affect host reproduction; cytoplasmic incompatibility (CI) is one of the most widely reported effects (Sinkins, 2004). Unidirectional CI can occur when the Wolbachia infection type present in a male differs from that in his mate. Although the precise mechanism is

unknown, 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 a compatible Wolbachia type, the modified sperm fail to achieve karyogamy.

In contrast, “rescue” of the modified sperm occurs in embryos from females infected with compatible Wolbachia types. Thus,

in populations that include both infected and uninfected individuals, Wolbachia-infected females can mate successfully with

all males in the population. In contrast, uninfected females can reproduce successfully only with uninfected males. This pattern

of unidirectional CI allows Wolbachia to spread rapidly through host populations. Previous studies of insects with multiple Wolbachia types have demonstrated that unidirectional CI can be additive (Dobson,2003). Multiple Wolbachia infection types within an individual male may independently modify sperm, requiring a similar combination of infection types in female mates for compatibility. Additive unidirectional CI can result in repeated population 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 of population replacement has attracted attention for its potential applications. A frequently referenced strategy is based on the replacement of natural populations with modified populations that are refractory to disease transmission (Enserlink, 2001). A Wolbachia-based population replacement strategy requires the generation of artificial infection types that differ from those of the targeted populations.

Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito, is native to Asia and is a globally invasive insect. Examples of introduction and establishment include North and South America (Gratz, 2004), and recent invasions have extended to Africa, Australia, and Europe. In addition to being an invasive pest, this mosquito is an aggressive daytime human biter and has been implicated as a vector of animal (Scoles and Kambhampati, 1995) and human (Gratz, 2004) disease. Recent reports have highlighted its role as a primary vector during recent chikungunya virus epidemics (Simon et al, 2008). Aedes albopictus populations are naturally infected with two Wolbachia types: wAlbA and wAlbB (Sinkins et al ,1995). Therefore, to employ Wolbachia as a vehicle for population replacement, additional, incompatible infection must be introduced into the natural infection types. Previously, Wolbachia strain wRi was successfully established in A. albopictus by microinjecting the cytoplasm of Drosophila simulans (Riverside) into the embryos of aposymbiotic (i.e., without Wolbachia) A. albopictus mosquitoes (Xi et al, 2006). As hypothesized, the resulting artificial infection displayed a pattern of bidirectional CI when these mosquitoes were crossed with the naturally double infected strain. Thus, the modification/ rescue mechanism(s) of the wRi infection is known to differ from those of the naturally occurring infection types. Therefore, we hypothesized that individuals harboring the combined wRi, wAlbA, and wAlbB infections would be unidirectionally incompatible with the naturally infected mosquitoes.

CHAPTER FOUR

SUCCESSES AND CHALLENGES OF GENETIC MANIPULATION OF  MOSQUITOES

4.1. SUCCESSES RELATED TO GENETIC MANIPULATION

Still, the mortality and morbidity rates associated with these pathogens remain high in low-income countries in tropical and subtropical regions (Guinovart et al., 2006). In total, more than half of the global population is affected by mosquito-borne diseases resulting in millions of deaths and hundreds of millions of cases every year. These statistics provide the impetus to study mosquitoes with the expectation that new knowledge could contribute to the alleviation of this disease burden.

The application of genetic analyses and molecular biological techniques for research on mosquitoes provides opportunities for the development of new disease control strategies (Hill et al., 2005). Among these opportunities are novel vector control methods for population reduction or replacement (Curtis and Graves, 1988). Population reduction seeks to decrease the absolute number of mosquitoes and, therefore, lower the probability of contact between mosquitoes and their human hosts.

Population replacement strategies are designed to replace susceptible mosquitoes (can transmit a pathogen) with refractory

mosquitoes (cannot transmit a pathogen), and such strategies do not require changes in mosquito population densities. For any of these strategies to be effective, it is important to reduce the number of infectious mosquitoes below a threshold level so that the probability of transmission falls to a point where the parasite population declines steeply and irreversibly.

One population replacement strategy has the goal to genetically modulate vector competence and is based on the hypothesis that an increased frequency in a vector population of a gene that interferes with a pathogen will result in the reduction or elimination of transmission of that pathogen (Collins and James, 1996). A key objective was to establish routine methods for generating transgenic mosquitoes, and this was achieved with a number of species using class II transposable elements (Grossman et al., 2001). These successes stimulated debate and research to measure the impact of introduced genes on mosquito fitness. For example, integration of a transgene may disrupt or alter expression characteristics of endogenous genes, transgene products may be toxic, or transgene transcription and translation may usurp resources needed for normal survival or reproductive functions. Accordingly, the effects of transgenes integration and expression on mosquito fitness vary (Irvin et al., 2004). Most genetic approaches require the transformed insects to exhibit as low a fitness cost as is possible (Lambrechts et al., 2008). Therefore, the expression of a transgene should be limited to a specific tissue and time in the mosquito to achieve the maximum effect on the pathogen, while minimizing the potential load on the vector. Recent advances in the areas of genomics and genetic engineering are expected to enable the design and production of mosquitoes expressing antipathogen effector molecules under the control of synthetic or hybrid (chimeric or mosaic) promoter- regulatory DNA to achieve optimum performance. Functional synthetic plant promoters that combine a collection of DNA sequence elements from pathogen-activated genes (Rushton et al., 2002) serve as a conceptual model for similar developments in mosquitoes.

While mass release (inundation) is required for those approaches (SIT and RIDL) that do not propagate genes through a target population, implementation of population replacement requires an effective system for gene drive to spread genes into wild populations. Gene drive involves the introduction and establishment of a population replacement effector gene using genetic mechanisms that circumvent Mendelian inheritance (Braig and Yan, 2002). Standard genetic approaches based on gene segregation and selection require fitness advantages linked tightly to the antipathogen gene and are likely to be too protracted in time to be useful. The bases for gene drive systems come from known genetic phenomena, two of which are discussed here. Mobile genetic elements, such as the previously mentioned class II transposons, may move rapidly into populations, for example the P elements of D. melanogaster spread worldwide in a period of _50 years (Kidwell, 1983). These mobile genetic elements spread through populations by replicative transposition in the germline, which means that a mating between an insect with an active transposon in its genome and one without can result in progeny that all have the element. However, mobilization of naturally occurring or inserted transposons has yet to be observed in wild populations or transgenic mosquitoes (Sethuraman et al., 2007). This could be a natural consequence of strong selective pressures limiting transposon movement (O’Donnell and Boeke, 2007). Moving genes into a population without relying on transposon mobilization is possible with MEDEA (maternal-effect dominant embryonic arrest).

Another genetic control approach that combines population replacement with lethality, ‘‘death-on-infection,’’ demands that

a population of mosquitoes carry a conditional lethal gene that is activated by the presence of parasites or viruses. This results in the selective death of the infected vectors. Death-on-infection diminishes possible ecological effects associated with population reduction while reducing transmission rates. Specific pathogen responsive promoters to drive the expression of toxic proteins, apoptosis effectors, or double-stranded RNA (dsRNA) targeting vital gene transcripts are essential for this approach. Analyses have been conducted of global changes in gene expression in mosquitoes exposed to parasites or viruses (Xi et al., 2008) with a goal to identify genes with enhanced levels of expression upon infection. Continued studies of mosquito immune responses will contribute the necessary

regulatory elements for the development of this approach.

SIT is a species-specific and environmentally nonpolluting method of insect control that relies on the release of large numbers of sterile insects (Dyck et al., 2005). Mating of released sterile males with native females leads to a decrease in the females’ reproductive potential and ultimately, if males are released in sufficient numbers over a sufficient period of time, to the local elimination or suppression of the pest population. Highly successful, area-wide SIT programmes have eliminated or suppressed a range of major veterinary and agricultural pests around the world. These programmes can succeed on very large scales – the largest rearing facility alone produces around 2 billion sterile male Mediterranean fruit flies per week (~20 tons/week) primarily for use in California and Guatemala. For these pests, SIT is a proven cost-effective strategy for eradication or suppression 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 recent years, it is important that the search for new effector molecules and promoters continue for two reasons. First, considering how easily parasites acquire drug resistance, it is likely that parasites will be selected that can overcome the barrier imposed by the effector molecules. Secondly, maximum efficiency of blocking parasite development (ideally 100%) is important for the transgenic mosquito strategy to have a significant impact on disease transmission. Furthermore, while many of the tools for genetic modification of mosquitoes have been developed, an extensive gap exists in our ability to transfer this technology to the field for the control of malaria.Others includes:

1. The fitness cost of refractoriness

To maximize the likelihood of successfully introducing refractory genes into a wild mosquito population, transgenes

should impose minimal fitness load. We assessed fitness of transgenic An. stephensi expressing the SM1 and the PLA2 transgenes 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 submitted for publication). The SM1 transgene did not impose a detectable fitness load, but transgenic PLA2 mosquitoes had much reduced fertility and competed poorly with non-transgenics in cage experiments. The reasons for this reduced fitness remain to be investigated. Catteruccia et al., (2003) reported that four different transgenic mosquito lines expressing fluorescent reporter proteins from an actin promoter are less fit than the wild type. However, reduced fitness was most likely due to inbreeding. They isolated homozygous lines soon after transgenic mosquitoes were obtained, which may have caused recessive deleterious genes residing near the point of transgene insertion to become homozygous (‘hitchhiking effect’). Conversely, in the experiments of Moreira et al., the transgenic mosquitoes were kept as heterozygotes, being continuously crossed to mosquitoes from laboratory population cages. This demonstrates the importance of mosquito outcrossing. In addition, the experiments of Catteruccia et al., (2003) used a transgene driven by the strong and ubiquitous actin promoter. The abundant synthesis of a foreign protein throughout the organism may conceivably have deleterious effects on fitness (Liu et al., 1999). For this reason, SM1 expression was restricted to posterior midgut cells for only a few hours after a blood meal and the protein was secreted from the cells, thus minimizing fitness load. Absolute absence of fitness load may not be essential for introducing genes into wild populations. Theoretical modeling suggests that given an appropriate drive mechanism, a gene could have a significant fitness cost and still be driven through the population (Ribeiro and Kidwell, 1994; Boete and Koella, 2003). This is fortunate, since this same model suggests that any released mosquitoes would need to be nearly 100% refractory to have any impact on malaria transmission, necessitating multiple refractory genes that may incur greater fitness costs.

2. Developing an effective drive mechanism

Two general strategies can be considered for introducing transgenic mosquitoes in the field: population replacement or a genetic drive mechanism. Population replacement, or inundatory release, requires a significant reduction of the resident mosquito population (for instance ,with insecticides), followed by the release of large numbers of refractory mosquitoes to fill the vacated biological niche. This strategy is promising as a research tool and as a field test to assess the effectiveness of the transgenic mosquito approach for interrupting malaria transmission. However, this strategy cannot be considered for large-scale control purposes, because it is not possible to produce sufficient numbers of mosquitoes to achieve population replacement on a country- or continentwide level. Transposable elements may incur a substantial fitness cost. Transposition causes random integration across the genome, some of which may disrupt genes and lead to mutations that could be lethal, reduce fecundity or decrease fitness. Predictive

models suggest that transposable elements would be able to drive refractory genes from a small number of transgenic mosquitoes into the wild population even if a fitness cost was present (Ribeiro and Kidwell, 1994). However, there is considerable lack of experimental data to corroborate or disprove the models. Another consideration is that mobility of the transposable element may be negatively regulated by a repressor. For instance, mobility of the P element in D. melanogaster decreases after several generations because an inhibitor of transposition gradually accumulates and the fly is said to acquire the P (refractory) cytotype. This is of practical importance because in such cases the gene(s) can be driven through a population only once. If the effector gene(s) acquires mutations or the parasite becomes resistant to the effector gene product another gene cannot be driven into the same population with the same transposable element.

(3.)         Mass production of transgenic mosquitoes and genetic sexing mechanisms

Transgenic-based methods to reduce or eradicate vector populations, such as the release of insects carrying a dominant lethal (RIDL; Thomas et al., 2000), show promise for some species. However, their use as a malaria control program in Africa would be difficult to implement due to reproductively incompatible subspecies and migration of mosquitoes among villages. Even if successful, this approach would leave an ecological vacuum that another malaria vector could quickly fill. Therefore, replacement of wild populations with transgenic mosquitoes carrying refractory genes instead of population suppression or eradication methods would be more appropriate.

Unfortunately, this approach still requires the release of vast numbers of biting insects, which is ethically questionable due to their nuisance factor and potential role as vectors for secondary diseases. Thus, widespread release of genetically modified mosquitoes 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 prospect of making the use of transgenic mosquitoes acceptable to the local communities and to the public in general.

(4.)         Avoiding resistance to the refractory genes

Parasites facing a refractory mosquito population would be under strong selective pressure, similar to the one posed by anti-malarials, and thus resistance may develop. Engineering a mosquito with multiple refractory genes that target different aspects of parasite development could minimize resistance to the refractory genes. For example, a transgenic mosquito might be engineered to express a peptide to disrupt midgut and salivary gland invasion, have an enhanced encapsulation response to target the oocyst, and express defense peptides to target the sporozoites. Furthermore, chances of success will be greatly increased if each refractory element is as close to 100% effective as possible and if introduction of the refractory genes is coupled with traditional control methods, such as reduction of wild populations with insecticides prior to a transgenic release, drug treatment of infected individuals, and use of bed nets. The effectiveness of transposable elements may decrease with time after field release. Immediately after the introduction of a novel transposable element into a population the element enjoys a period of unrestrained activity and spreading. Eventually, individuals with mutations in the transposase or those that have enacted regulatory inactivation of the element will be selected. Transposase silencing has been well studied in the mariner family and has been hypothesized to occur by

several mechanisms, including overproduction inhibition whereby an increase in transposase activity correlates with decreased transposition or random transposase mutations. Random transposase mutations may lead to open reading frame disruptions and inactive transposases that compete with active transposase for substrate (competitive inhibition) or reduce the activity of wild-type transposase (dominant negative complementation; (Hartl et al., 1997). The mechanism of transposable element silencing will need to be well understood before transposable elements are used in the field.

Sterilization

Recent advances allow several potential improvements over the methods available in early trials. All current SIT programmes use radiation to sterilize the insects. However, it has proven difficult to irradiate mosquitoes to near-complete sterility without significantly weakening them (Andreasen and Curtis, 2005).

Economic cost-benefit analysis, which is needed to support use of novel interventions, is difficult because of lack of reliable data on the economic burden of disease for dengue and other neglected tropical diseases, and because of uncertainty around development and implementation costs. Ideally it would be possible to analyse not only the cost-effectiveness of the stand-alone novel strategy, but also to compare it with existing alternate strategies and to model its incorporation in integrated vector management (IVM) programmes, and indeed integrated disease management programmes including drugs and vaccines, where available.

As genetics-based population suppression moves from laboratory to field, the lack of a clear regulatory framework for field use of modified mosquitoes is a significant challenge. This issue is not restricted to developing countries, or to strategies dependent on the use of recombinant DNA technology. Once regulatory frameworks are in place, risk assessments and public consultation also will be lengthy processes due the novelty of technologies and lack of experience by regulating agencies. The route to implementation of control programmes based on these technologies is not obvious. Agricultural SIT programmes have generally been established and operated by governments, though there is limited private-sector involvement. Existing vector control programmes are generally government-funded and -operated, though they purchase vector control products and services from the private sector. The development of new vector control approaches is generally in the private sector. The current genetic-based technologies are perceived as too high-risk for large companies

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

A myriad of logistic, financial and technical challenges face these programmes though not particularly related to human health or regulation. To test any technology on a large scale with little experience in a developed country is difficult ; to do so in a developing country is more so. The availability of trained personnel, materials and infrastructure present country-specific difficulties. Some of this cannot be anticipated and may not be easy to remedy

CHAPTER FIVE

CONCLUSION

Major advances in recent years, including successful germline transformation and characterization of promoters, are allowing researchers to test putative refractory genes. One important task for the near future is the identification of additional effector genes, and this will be greatly facilitated by the availability of the An. gambiae and P. falciparum genome sequences. This knowledge can be used to engineer a mosquito that inhibits or kills the malaria parasite during multiple developmental stages. With this ideal mosquito on the horizon, the most important task is to begin laying the groundwork for its introduction into the wild. A high priority should be devoted to the topic of how to introduce the relevant genes into wild mosquito populations. Also needed are ecological studies to evaluate population structure and gene flow. In addition, we must grapple with the ethical and political concerns involved with a large-scale release of a genetically modified organism. Considerable challenges lay ahead but there are reasons to be optimistic that we will be able to add genetic modification of mosquitoes to our arsenal in the fight against malaria.

While most are in the realm of guidance or guidelines, some have regulatory status and are legally binding. Each can provide useful background for upcoming national decisions regarding application of genetic strategies for vector control,as the most promising technologies move from laboratory to confined or open field trials and, if successful, eventual widespread field programmes for vectored disease control.The effectiveness of this method can be best achieved by:

(1.) Provide funding to develop the general mathematical model described above. While there are several efforts to develop models for specific technologies, these are neither sufficiently flexible nor accessible by project planners to make them of value for routine programme implementation.

(2.) Provide funding for sensitive methods for Anopheles surveillance. 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 release programmes. Such equipment allows the spatial extent of vector releases to be realistically considered.

Planning vector release programmes will be facilitated by development of general mathematical models of SIT that are updated and modified to include characteristics such as larval density dependence and survival, reduced mating competitiveness, species bionomics and semi-sterility. These programmes should be accessible via a simple interface to end-users who are planning release programmes, not only software developers or modellers. Surveillance methods for Anopheles at very low population densities will be challenging. During the eradication of An. arabiensis from Egypt, the absence of rebound of populations to cessation of control efforts confirmed elimination, but this will not be suitable for routine assessment of programme effectiveness. Sensitive methods and materials such as attractants are needed.

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