International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 14
International Journal of Advance Research, IJOAR .org Volume 1, Issue 3, March 2013, Online: ISSN 2320-916x
THE VIABILITY OF TRANSGENESIS FOR CONTROL OF MALARIA Himansha Singh*1, Mustafa Ahmad2
*1School of Translational Medicine, University of Manchester, Manchester, M13 9PT, Lancashire, UK [email protected] 2School of Biological Sciences, University of Salford, Greater Manchester, M4 4WT, UK
Abstract
Malaria is a serious problem and a reason for severe mortality especially in developing countries. Several techniques such as insecticides, drugs, sterile insect technique, etc. have been employed by researchers to eradicate the lethal disease. However, the parasites and vectors gained significant immunity against the treatment. Incorporation of genetically modified vectors (GMVs) into disease control could be promising. Genetic modification (GM) of mosquitoes offers opportunities for controlling malaria. Transgenic strains of mosquitoes have been developed and evaluation of these to 1) replace or suppress wild vector populations and 2) reduce transmission and deliver public health gains are an imminent prospect. Identification of promoters, effectors, and reporter genes affecting malaria in mice further developed hopes for this process. This review focuses on malaria and the use of transgenesis for its prevention and cure, along with highlighting the associated challenges. Unlike other current therapies, there are no precedents (yet) of successful transgenesis application, leading to the classification of this strate gy as linear technology-push, but researchers are optimistic that these strategies could be further improved.
Keywords
Malaria, Genetically modified vectors, Technological challenges, and genetic modification
IJOAR© 2013 http://www.ijoar.org
International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 15
on exploitation of symbiotic microorganisms, viruses, INTRODUCTION and transposons (8). Insect transmitted diseases have been posing serious health problems and are one of the leading causes of
mortality in many developing countries (1). These Malaria – a serious problem emerging and resurgent vector-borne diseases can give
rise to infectious diseases affecting crop plants, animals Malaria is one of the major causes of death in children and humans (2). Plants are shown to produce toxic or in the first 28 days of life (9). Nchinda (1998) reported inhibitory substances as an aid against insects and from an estimate of 300-500 million cases per year with 1.5- the past decades, even humans have developed various 2.7 million deaths per year and also documented that insecticides such as dichlorodiphenyltrichloroethane African children below 5 years constitute 90 % of the (DDT) (3). Genetic process such as sterile insect deaths (10). Until the late 1960s, malaria was found in technique (SIT), in which radiation-sterilized males parts of Europe, USA and north Australia along with its confer sterility to the pest population on release, is base in tropical areas of Africa, Latin America and Asia. expanding for pest control (4). However, in a wider After that, it has spread across sub-Saharan Africa, sense, it is not very desirable to look for preferences to Central and South America, Middle East, the Indian purge insect population as they may occupy an subcontinent, South and South-east Asia, Papua New imperative niche in the food chain, but it is required to Guinea and some islands in the West Pacific. In fact, develop strategies to overcome the diseases spread by malaria has affected about 100 countries and about one- insect pests (2). Incorporation of genetically modified third of the world‟s population is at stake (11). In a vectors (GMVs) into disease control programs could be report by World Heath Organization, it‟s recorded that 5 promising. Germ-line transformation in insects of % of African children are likely to die by malaria before medical importance is already feasible in Aedes, age 5. This constitutes 25 % of child mortality rate in Anopheles, Culex, Rhodnius, and Glossina spp. Africa and it‟s been noticed that most of the deaths Paratransgenesis, in which host symbiotic bacteria are occurs by anaemia and cerebral malaria (12, 13). There genetically modified to kill the parasite, is already used were estimated 225 million cases of malaria worldwide to control African and American forms of in 2009 and in 2011; W.H.O reported 2.23 % of deaths tyrpanosomiasis (sleeping sickness and Chagas disease) worldwide caused by malaria (14). (5). Genetic modification (GM) of mosquitoes offers Since 1992 four principles are implemented and kept in opportunities for controlling malaria. Transgenic strains mind to control malaria: early diagnosis and treatment, of mosquitoes have been developed and evaluation of selective and sustainable preventive measures, vector these to 1) replace or suppress wild vector populations control techniques, containment and prevention of and 2) reduce transmission and deliver public health epidemics, and building up of local capacity (11). Apart gains are an imminent prospect (6). The initial steps for from healthcare damage, malaria is an economic burden transformation of malarial mosquitoes have already led and it has been documented that households in Africa to the identification of promoters, effectors, and reporter spend €2–25 on treatment annually and €0.20–15 on genes affecting malaria in mice (7). In mosquitoes, the prevention per month, and in small farm households in drive system is based on genetic phenomena (e.g., Kenya and Nigeria 5 and 13% of the household competitive displacement, reduced heterozygous fitness, expenditure, respectively, is spent on malaria (12, 13). under-dominance, and meiotic drive) and systems based
IJOAR© 2013 http://www.ijoar.org
International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 16
Endemic countries bear a huge loss of percentage points malariae generally causes milder disease (18). However, of gross domestic product every year due to malaria (5, P. vivax is responsible for largest number of infections 12, 13). worldwide (19). P. knowlesi is a zoonosis and cause malaria in macaques but can also infect humans (20).
Life Cycle of malaria parasite
Malaria parasite has two hosts- primary and secondary. Female mosquitoes Anopheles sp. are primary hosts and Malaria- causative agent they carry the Plasmodium sporozoites in their salivary glands (See Fig. 1) (21). After feeding on an infected Malaria is an infection caused by a single-celled human, the parasite gametocytes taken up in the blood parasite and is spread by female mosquito Anopheles sp. differentiate into male and female gametes and fuse in There are five species of the parasite: Plasmodium vivax, the mid-gut of mosquito, where it produces okinetes Plasmodium falciparum, Plasmodium ovale, that penetrates the gut lining and produces an oocyte in Plasmodium malariae and Plasmodium knowlesi the gut wall. Upon the rupture of the oocyte, sporozoites (15,16). Plasmodium falciparum causes severe damage are produced that migrates to salivary gland and ready and is responsible for vast majority of deaths associated to infect a human (22, 23). with malaria (17), while P. vivax, P. ovale and P.
Figure 1 Life Cycle of Malarial Parasite (Source: http://www.malariasite.com/malaria/lifeCycle.htm) (21)
sporozoites migrate to hepatocytes and divide and grow When mosquito bites a healthy human (secondary host), within parasitophorous vacuoles to form merozoites. This is several invasive sporozoites introduce in the skin. They called a pre-erythrocytic phase with little or no pathological fight against the immune system and those who survive actions (See Fig. 1) (21, 26). These merozoites eventually then find a blood vessel leading to liver. It has recently released into blood stream and initiate blood stage of been shown that the sporozoites travel by a continuous infection thereon (27). Within red sequence of stick-and-slip motility, using the thrombospondin-related anonymous protein (TRAP) family and an actin–myosin motor (24, 25). These
IJOAR© 2013 http://www.ijoar.org
International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 17
blood cells, the parasite expands rapidly with a sustained cycling of the parasite population modifying the cell membrane permeability and cytosolic composition. During each cycle, each merozoite grows and divides within a vacuole into 8-32 fresh merozoites, through the stages of ring, trophozoite, and schizont. At the end of each cycle RBC ruptures and release new merozoites that, in turn, infect more RBCs (24, 25, 26).
Current therapies and genetic approach
Three basic strategies have been attempted to contain insect borne diseases: 1) treat infected people with drugs that kill the pathogen (e.g. chloroquine for
IJOAR© 2013 http://www.ijoar.org
International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 18
malaria); 2) control insect vector populations (e.g. refractoriness (40). Genetic manipulation would then be Insecticides); and 3) develop vaccines that prevent achieved through identification of genes responsible for infection (28). The only successful vaccine against encapsulation, melanization and death of early malarial arthropod-transmitted pathogen is yellow-fever vaccine infection in mosquito (41, 42) and identification of and even in this case, the disease has not been possible mechanisms of introducing these genes into eradicated. Also, the research for vaccines against mosquito genome is critical (43, 44) malaria and dengue fever has failed considerably. Two approaches are considered: 1) population suppression and 2) population replacement (29). Population suppression is achieved through insecticides like DDT, elimination of breeding sites or interference with reproduction (28) However, insects are now becoming resistant against these line of attacks (30, 31) and even other biological methods, such as the sterile-insect techniques (SIT), have been rendered less effective by changes in insect behavior (32). . This integrated approach with a combination of traditional control methods, preventive measures and pharmaceutical regimes has been utilized by vector control programs in past (33), but not that they have shown to be limited in their success against the diseases, it necessitates the inclusion of an alternative control method. As Knols and Bossin (2006) put it “The continued threat of vector-borne diseases calls for both reactive and proactive efforts to mitigate the significant morbidity and mortality they cause” (34, 35).
High genetic diversity or genetic virtuosity, variability of potential target molecules, and intracellular sequestration are strategies adapted by pathogens to evade immune attack (28). Second action is to attack malaria pathogen directly rather than killing mosquito. Since the advent of genetically modified organism (GMO), research has directed to use the opportunity to control insect borne disease by creating lines of genetically modified disease vectors (6, 29, 36). Genes that can alter the behavior and biology of insects are identified and when these genes are inserted into the insect‟s genome, they are called transgenes and the insect is described as transgenic insect. Introduction of transgenes into the population of wild mosquitoes will render the mosquitoes resistant to infection by the malaria parasite (28). Gene drive is a process by which the transgenic mosquitoes must be assimilated into the wild malaria-transmitting mosquito population. Gene drive requires introgression of anti-parasite genes into the insect vector populations. Such genes, if present at high enough frequencies, will impede transmission of the target parasites and result in reduced human sickness or death (37). Hopes for this possibility is raised due to reporting of genetic variability for mosquitoes against the malaria parasite (38) i.e. there is an observation that most species of mosquitoes do not transmit malaria, and even among the species that do, many individual seem incapable of transmitting the disease and are „refractory‟. Alteration in genes responsible to permit malaria transmission in mosquitoes may, then, help in rendering mosquito population refractory to the parasite, eventually halting malaria transmission (39). For instance, it is noted that melanotic encapsulation is one of the reasons for this IJOAR© 2013 http://www.ijoar.org
International Journal of Advance Research, IJOAR .org IJOARBP- Volume 1, Issue 4, May 2013 ISSN 2320-916x 19
inhibiting parasite development and showing reduction in vectorial competence is also reported in transgenic Transgenic insects mosquitoes (55, 56). Ito and colleagues showed that
Drosophila melanogaster was rhe first multicellular transgenic mosquitoes were less susceptible to infection organism to be stably transformed (45). The same germ- (oocyte load) and had fewer sporozoites in their salivary line transformation principle is used currently to work glands. They suggested that where mosquitoes carry in insects (46). In brief, the embryo is injected with two DNA-constructs: 1) Containing gene of interest and gene encoding selective marker, both driven by separate promoter and both sequences are flanked by inverted repeats of a transposable element, and 2) Containing transposase enzyme that recognizes inverted repeats and catalyses the insertion of the intervening sequences into insect‟s genome (28). The final result is the integrated host genome with the gene of interest and marker gene (…allows identification of transformed individuals e.g. eye colour, GFP). The integrated DNA is stable and transmitted in a mendelian manner from one generation to another. However, population replacement or reduction should occur in short time (8) and therefore, it should be linked to mechanisms for selection (e.g. meiotic drive) or the traits itself providing a fitness advantage (29).
Promoter involved in expression drive should be strong for abundant transcription. Out of the two types: 1) Ubiquitous, and 2) Tissue-specific promoters, the latter is observed to be advantageous as it is restricted to specific tissue (28). Strong tissue-specific promoters that have been characterized in mosquitoes include gut carboxypeptidase (47), fat body vitellogenin (48) and gut peritrophic matrix protein 1 (PM1) (28). Similarly, effector genes are equally important. They could be: 1) Gene products interfere with the development of pathogen. For e.g., SM1 (a peptide occupying salivary- gland and midgut receptors for the malaria parasite) (49), Phospholipase A2 (PLA2; protein that intereferes with malaria ookinete invasion of the midgut (50), 2) Gene products interact with pathogen. For e.g., gene encoding monoclonal antibodies that block parasite‟s development by binding to its outer surface (51), 3) Gene products kill the pathogen. Examples are peptides from the host immune system such as defensins and cecropsins and peptides from other sources such as magainins, Shiva-1, Shiva-3 and gomesin (52). Another possible strategy is to reduce vector competence by manipulation of its immune genes, for instance by using RNA interference or „smart sprays‟ (28).
Development of effectiveness in the aforementioned technology (transgenesis, promoter characterization and effector-gene identification) permits creation of genetically modified mosquitoes impaired to transmit parasites i.e. refractory mosquitoes. A variety of engineering refractory mosquitoes are currently studied for malarial (53). An early example is Anopheles Aegypti that was developed to express defensin in the heamolymph (48). Another creation was a single chain monoclonal antibody reported to recognize sporozoite surface protein inhibiting invasion of the salivary gland (54). Transformation by gene encoding SM1 and PLA2
IJOAR© 2013 http://www.ijoar.org less than five oocytes, inhibition of transmission is very (59). So, if the trait is linked to one of these aggressive effective (55). Kim et al, 2004 reported 60 % reduction genes, it can rapidly and effectively be spread among in transmission of malaria parasite in transgenic the mosquito population. Meiotic drive distorting sexual Anopheles gambiae expressing cecropin from a behaviour is detected in mosquitoes raising hopes for its carboxypeptidase promoter (52). Another method for use in GMM strategies (60). However, the molecular generating refractoriness involves discovering genes mechanism of meiotic drive is complex involving that govern refractoriness in natural population (7). multiple genes and is still elusive. Intracellular From the above discussion, it is evident that mosquitoes symbiont such as Wolbachia is also considered can be genetically modified to reduce vector promising for gene drive using cytoplasmic competence, however, it requires overcoming certain incompatibility (CI) (61). Although, it has not been hurdles to provide desirable efficacy. identified in mosquitoes yet, research shows experimental transfer of bacterium over genus barriers including complete CI in the new host (62).
CHALLENGES Based on these, Jacobs-Lorena (2006) formulated
priorities for research in transgenesis of insects (28)-
Parasite resistance Fitness and Gene drive “…a method to drive effector genes into field mosquito populations needs to be devised. Parasites have heterogenous genome and favors selection of individuals to overcome barrier such as -The efficiency of An. gambiae transformation needs to drugs and effector gene products. It is therefore, be improved. important to insert more than one effector gene to block -Anopheline mosquitoes cannot be stored frozen or parasite development by different mechanism (28). desiccated. Establishment of repository centres for For effective transformation in mosquitoes, it is transgenic lines is desirable. important that there is least possible detrimental effect -Whenever possible, work should be conducted with the on their survival and reproduction (fitness load) (28). organisms that are most relevant to human disease (An. However, till now, its not yet achieved. Mosquitoes gambiae, P. falciparum)”. with PLA2 gene-transformation show reduced egg production (47, 55, 56). Mosquitoes with GFP from actin promoter may also have fitness disadvantage (28). Translational challenges Introduction of novel phenotypic traits in transgenic mosquitoes may induce these fitness impairments There are several translational challenges surrounding impeding gene drive and spreading of transgenes (57). transgenesis for malaia control (See Fig. 2) (6). Ethical, Expression of effector genes, especially using legal and social issues involved in transgenesis are ubiquitous promoters, may be detrimental for the noteworthy (63). mosquito. Also, transgenes may interfere with gene Even though transgenesis is promising in laboratory and expression of mosquito strain because of random research bench to eradicate malaria, it is essential that genomic insertion. This intertional mutagenesis may be countries evaluate and adopt this approach as a potential naturally selected and can be overcome by selection on tool for malaria control. This will increase the lines with higher fitness. Whatever approach is adopted investment in knowledge and skills acquisition with in the coming years, compensation for fitness loss, respect to transgenic mosquitoes. Ultimately, it aims to preferably by conferring fitness advantages to transfer the problem-ownership to the disease endemic transgenics that outweigh cost-benefit equilibria in country (DEC) (64). Another thing to consider is susceptible wild-type mosquitoes, will remain a critical containment and risk management. Although, challenge (6). guidelines are available for handling, transport, and Effective transformation also requires efficient gene laboratory confinement for transgenic mosquitoes, no drive system, which is undoubtedly, a major technical such guidelines are drafted for DEC (6). issue. Many features are required to make a gene drive In the above sections, several challenges that transgenic ideal for competitive displacement of population (28, mosquitoes will face in future are discussed. Although, 29). For instance, gene drive should compensate any forecasting is often inaccurate, some features that may fitness load incurred by transgenesis (6, 9, 28, 55); it affect GM mosquitoes are emphasized by Knol et al, should carry large fragments of DNA to accommodate 2007 (6): 1) By 2025, 90 % of world‟s population will all the elements controlling driver characteristics; it live in developing countries, 2) 52 % of African should have minimal side-effects and incur elimination population will live in urban environment, 3) of any unforeseen phenotype and ecological effects (29). Environmental modification may aid in malaria control, Transposable elements are widely appreciated for gene 4) High population density may increase the interest drive as they not only rapidly move and propagate towards case management, personal protection within the genome, but also colonize other genome measures, or larval control, 4) Genetic strategies may through horizontal trasnsfer (29). To date three malaria become attractive. vectors have been transformed using transposable elements: the Asian vector Anopheles stephensi (44), the Latin American vector A. albimanus and African vector A. gambiae (55, 57, 58). Another mechanism in insects is meiotic drive, where genes (Drivers) can overrule random segregation of chromatides and forcefully drive their inheritance through populations IJOAR© 2013 http://www.ijoar.org
for population replacement in future and may eliminate malaria. Holistic, tedious, resource-intensive, time- consuming approach is needed to drive genetic control trials to maximize the efficiency and minimise the obstrucles (6), but they will ultimately decide whether transgenesis is viable for controlling insect-borne diseases.
Figure 2 Views from the malaria research and References control community (N = 196) in response to statements presented during a plenary debate at the 1. (WHO) World Health Organization. 2004. Global 4th MIM conference held in Yaoundé, Cameroon, in burden of disease. November 2005. The debate was titled: “Is the http://www.who.int/malaria/wmr2008/MAL2008- transgenic mosquito as a weapon against malaria Chap1-EN.pdf. ever going to fly?” Black, yes; dark gray, no; light 2. Durvasula et al. (1997) Prevention of insect-borne gray, no opinion; white, no answer (Adapted from 6) disease: An approach using transgenic symbioticbacteria. Proc Natl Acad Sci U S A. 94:3274– 3278. 3. Georghiou, G P (1997) In Overview of Insecticide Resistance to Agrochemicals, American Chemical Society Symposium Series, eds. Green, M.B., LeBaron, H.M. & Moberg, W.K. (Am. Chem. Soc., Washington, DC) 1990.Godhwani S, Godhwani JL, Vyas DS. J. Ethnopharmacol. 21: 153-163. 4. Dyck VA, Hendrichs J, Robinson AS (2000) Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. Dordrecht: Springer.2005 5. Anonymous: Malaria-economic implications. Weekly Epidemiological Record . 75:318 – 319. 6. Knols BGJ, Bossin HC, Mukabana WR and Robinson AS (2007) Transgenic Mosquitoes and the Fight Against Malaria: Managing Technology Push in a Turbulent GMO World. Am J Trop Med Hyg. 77:232-242. 7. Riehle MM, Markianos K, Niare O, Xu J, Li J, et al (2006) Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region. Science. 312: 577-579 8. James AA, Beerntsen BT, Capurro ML, Coates CJ, Coleman J, Jasinskiene N and Krettli AU (1999) Con- trolling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system. Parassitologia. 41: 461–471. 9. Bryce J, Boschi-pinto C, Shibuya K, Black R (2005) WHO estimates of the causes of death in children. The Lancet. 365:1147-1152. 10. Nchinda TC (1995) Malaria: a reemerging disease in Africa. Emerging Infectious Diseases. 4:398 – 403. 11. Kager PA (2002) Malaria control: constraints and opportunities. Tropical medicine & International health. 7:1042-1046. 12. World Health Organization (1999) The World Health Report 1999. Making a Difference. WHO, Geneva 13. World Health Organization (2000) WHO Expert Committee on Malaria, Twentieth report. WHO, Geneva (at http://mosquito.who.int/docs)
14. WHO (World Health Organization). 2011. World Malaria Report summary. http://www.who.int/malaria/world_malaria_report_2010/ malaria2010_summary_keypoints_en.pdf.
15. Ullah I: Malaria and its Causative Agent. 3 December Conclusion 2010. Health Education [online]. [Accessed 26 February
The concept of transgenesis for malaria control is 2012 ]. Available from:
(2005) "The global distribution of clinical episodes of potential approach for control of vector-borne diseases. Plasmodium falciparum malaria". Nature. 434 (7030): Proc. Nat. Acad. Sci. USA. 97:10295-10297. 214–7. 37. ISIS Reports. (2001). Two takes on malaria-transgenic 18. Sutherland CJ, Tanomsing N, Nolder D, et al (2010) mosquitoes coming. Available: http://www.i- "Two nonrecombining sympatric forms of the human sis.org.uk/malaria.php#roll. Last accessed 26th Feb 2012. malaria parasite Plasmodium ovale occur globally". J. 38. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley Infect. Dis. 20:1544–1550. DC, Miller LH, Collins WE, Campbell CC, Gwadz RW 19. Mendis K, Sina B, Marchesini P, Carter R (2001) The (1986) Genetic selection of a Plasmodium-refractory neglected burden of Plasmodium vivax malaria. Am J strain of the malaria vector Anopheles gambiae. Science. Trop Med Hyg. 64:97–106. 234:607–610. 20. Singh B, Kim Sung L, Matusop A et al (2004) "A large 39. Marshall JM, Taylor CE (2009) Malaria control with focus of naturally acquired Plasmodium knowlesi transgenic mosquitoes. PLoS Med. 6(2):0164-0168. infections in human beings". Lancet. 363:1017–1024. 40. Paskewitz SM, Brown MR, Lea AO, Collins FH (1988) 21. http://www.malariasite.com/malaria/LifeCycle.htm Ultrastructure of the encapsulation of Plasmodium (unknown). Life cycle of malaria parasite [online]. cynomolgi (B strain) on the midgut of a refractory strain [Accessed 26th Feb 2012]. Available from: of Anopheles gambiae. J Parasitol. 74:432–439.
54. De Lara Capurro M, Coleman J, Beerntsen BT, et al Mboera LEG, Knols BGJ (2004) Development of (2000) Virus-expressed, recombinant single-chain genetically modified mosquitoes in Africa.Lancet Infect antibody blocks sporozoite infection of salivary glands in Dis. 4: 264–265. Plasmodium gallinaceum-infected Aedes aegypti. American Journal of Tropical Medicine and Hygiene, 62, 427-433. 55. Ito J, Ghosh A, Moreira LA, et al (2002) Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature. 417 (6887): 452-455. 56. Moreira LA, Ito J, Ghosh A, et al (2002) Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes. Journal of Biological Chemistry 277 (43):40839-40843. 57. Marrelli MT, Moreira CK, Kelly D, Alphey L, Jacobs- Lorena M (2006) Mosquito transgenesis: what is the fitness cost? Trends Parasitol. 22:197–202. 58. Fosdick RB (1946) Foreword. In: Practical Malariology (eds PFRussell, LSWest & RD Manwell) W.B. Saunders Company, Philadelphia, PA. 7-8. 59. Hurst GD, and Werren JH (2001) The role of selfish genetic elements in eukaryotic evolution. Nat Rev Genet. 2: 597–606. 60. Hickey and Craig (1996) Distortion of sex ratio in populations of Aedes aegypti. Can J Genet Cytol. 8: 260– 278. 61. Curtis CF, and Sinkins SP (1998) Wolbachia as a possi- ble means of driving genes into populations. Parasitology 116 (Suppl. ): S111–S115. 62. Zabalou S, Riegler M, Theodorakopoulou M, Stauffer C, Savakis C and Bourtzis K (2004) Wolbachia-induced cytoplasmic incompatibility as a means for insect pest pop- ulation control. Proc Natl Acad Sci USA. 101: 15042– 15045. 63. Takken W, Scott TW eds. (2003) Ecological Aspects for Application of Genetically Modified Mosquitoes. Dordrecht: Springer.. 64. Mshinda H, Killeen GF, Mukabana WR, Mathenge EM,
IJOAR© 2013 http://www.ijoar.org