Vet. World, 2012, Vol.5(9):571-576 REVIEW Genetic manipulation of endosymbionts to control vector and vector borne diseases

Vector borne diseases (VBD) are on the rise because of failure of the existing methods of control of vector and vector borne diseases and the climate change. A steep rise of VBDs are due to several factors like selection of insecticide resistant vector population, drug resistant parasite population and lack of effective vaccines against the VBDs. Environmental pollution, public health hazard and insecticide resistant vector population indicate that the insecticides are no longer a sustainable control method of vector and vector-borne diseases. Amongst the various alternative control strategies, symbiont based approach utilizing endosymbionts of arthropod vectors could be explored to control the vector and vector borne diseases. The endosymbiont population of arthropod vectors could be exploited in different ways viz., as a chemotherapeutic target, vaccine target for the control of vectors. Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting arthropods may serve as a powerful approach to control certain arthropod-borne diseases. Genetic transformation of symbiotic bacteria of the arthropod vector to alter the vector's ability to transmit pathogen is an alternative means of blocking the transmission of VBDs. In Indian scenario, where dengue, chikungunya, malaria and filariosis are prevalent, paratransgenic based approach can be used effectively.


Introduction
the control cost in India has been estimated in the tune of 2000 crore rupees per annum [5].Ticks are also Vector borne diseases (VBD) are of global vectors of a variety of pathogens that are implicated in importance because of their economic and health severe pathologies in many mammalian species [6].implications in livestock, human and companion Amongst the VBDs of animals, the impact of tick animals [1].Tick and Tick Borne Diseases (TTBDs) borne diseases viz.theileriosis, babesiosis, anaplasmosis, affect 80% of the world's cattle population and are ehrlichiosis and cowdriosis is ranked high.The widely distributed throughout the world, particularly traditional methods to control vector and vector borne in the tropics and subtropics [2]; they represent an diseases (VVBDs) are not sufficient enough to curb important proportion of all animal diseases affecting the menace caused by the VVBD's [7].The antigenic the livelihood of poor farmers in tropical countries.variation present in most of the vector borne parasites/ The impact of TTBDs on the livelihood of resource pathogens impedes the development of effective poor farming communities has been ranked high [3].
vaccine against these VBDs [8,9].Besides other Cattle ticks are responsible for severe economic losses factors, the global warming has been facilitating the in both dairy and beef cattle enterprises in tropics [4].
breeding of arthropod vectors and their entry into new Ticks are responsible for a variety of losses, caused by geographical areas is likely to disturb the delicate the direct effect of attachment ('tick worry'), by the equilibrium and contribute to new epidemics of VBDs injection of toxins, or through the morbidity and [10].Selection of insecticide resistant vector population mortality associated with the diseases that they and drug resistant vector borne parasite/pathogen transmit.The global cost of Tick and Tick-Born population has tremendously contributed to the Diseases (TTBDs) has been roughly estimated in the reoccurrence of VBDs.Due to the reluctance of tune of 14000-18000 million US$ per annum of which multinational national companies to fund insecticide arthropod host may not be major or essential.Various research which is becoming uneconomical, the authors reported different endosymboints in diverse number of insecticides available for use in field has arthropod vectors.Wolbachia spp, a rickettsia which is declined significantly [11] and there is a strong present in 20-70% of arthropods stated to be a reproductive possibility of not having any new insecticide in near parasite of arthropods rather than symbiont [15].future.The continuous and indiscriminate use of the These organisms cause a number of interactions with available limited number of insecticides may lead to a the host and alter the host response that includes stage at which the genotype of entire mosquito and tick cytoplasmic incompatibility (CI), parthenogenesis populations would turn into homozygous resistant to induction [16] and male killing.the available insecticides, a situation that can

Transmission of symbionts in various arthropod
jeopardize the lives of man and animals globally.The vectors: The different symbionts in the various public health preparedness to meet out any such arthropods are transmitted to their young ones by eventualities in future should explore the possibilities various ways and means.The predominantly present of alternative control strategies or the integration of Wolbachia organisms of Aedes and Culex spp.different strategies for the control of VVBDs.mosquitoes are transmitted by vertical manner via Amongst the various alternative control strategies gametes to their next generations [17].Though the [12], genetically modified symbiont based control tsetse flies are larviparous, their young ones get some strategy is under explored and appears to have great of the symbiotic organisms through the secretion of potential to curb the VVBDs in an integrated format.
milk glands during their intrauterine life [18].Endosymbionts of arthropod vectors: Endosym-Rhodococcus rhodhini, gut symbionts of the biosis is a specific type of symbiosis in which the triatomine bug (Rhodinus prolixus) vector of Chagas' microbial partner lives within its host and represents disease, transmitted from the adults to the progenies the most intimate contact between interacting through coprophagy [19].Understanding of the organisms [13].Endosymbionts are commonly found biology of the transmission of symbionts from the in arthropods that always depend on single source of adult vectors to progenies is imperative for the diet that is deficient of some nutrients and the symbiont based control approach to be effectively beneficial endosymbionts provide the essential used in the field conditions.nutrients to arthropods.An endosymbiont can be

Endosymbiont-arthropods-pathogen interaction:
either intracellular (Wigglesworthia spp. in side Perturbation of the interaction between endosymbiont, bacteriome) or extracellular (Sodalis spp. in midgut vector and parasite is the ultimate idea of the symbiont lumen of tsetse fly) organism.Endosymbionts are based approach to control VVBDs.reported to be present in midgut, haemolymph, fat body and ovaries of the different arthropod vectors.
Endosymbiont-Vector interaction: Arthropod vectors The arthropod host domesticates the endosymbenefit from the symbiosis and augment their bionts for its own welfare by utilizing the functions functional capabilities to facilitate their expansion in that are present in the symbionts but lacking in the to novel niches.The best examble is Wigglesworthia host.Scientists classify insect endosymbionts in two glossinidia an intracellular endosymbiont of Glossina broad categories, primary and secondary.Primary fly which synthesises plethora of vitamin biosynthetic endosymbionts have been associated with their insect products that may promote host reproduction.hosts for many millions of years, they form obligate Wolbachia organism's biosynthesis of riboflavin and association and display co-speciation with insect FAD, are essential for the Brugia malayi coenzymes hosts.Primary endosymbionts are essential for the and cytochromes.survival of the arthropod host and elimination of the Endosymbiont-pathogen interaction: The presence organism has a deleterious effect on the host [14].Because of symbionts and the pathogenic organisms together in the host is dependent on symbiosis, endosymbionts are the vector and the possible role of the interaction transmitted vertically or maternally to the young ones between the two in favour of disease transmission had with no horizontal transmission between hosts.
been reported Amongst the bacterial gut flora the S. Secondary endosymbionts exhibit a more recently marcescens chitinase has the ability to digest the developed association and are not obligate.Secondary peritrophic membrane of mosquito [20].Plasmodium endosymbionts are organisms appear to be the resultant ookinetes produce chitinase to invade the midgut of multiple independent infections and horizontal epithelium of mosquito.transmission and their contribution to the welfare of Vector-Parasite interaction: The competence of extensively studied symbiont of mosquitoes and the the vector to transmit a parasite/pathogen requires a same is the widely explored Wolbachia of arthropod molecule(s) within the vector that interacts with the vectors.cytoplasmic incompatibility phenomenon of parasite/pathogen.Proteolytic lectin present in the the Wolbachia of mosquito species is presently being midgut of tsetse fly stated to have trypanosome explored as a means to control the mosquito population.transforming activity [21].Creation of paratransgenic Wolbachia cytoplasmic incompatibility (CI): Its tsetse fly with the phenotype to check the activity of is a phenomenon in which mating between Wolbachia proteolytic lectin of the fly would limit the transmission infected male insect and female insect of the same of African trypanosmiosis.
species without Wolbachia infection (Unidirectional CI) and mating between insects of the same species

Control strategies
with different Wolbachia strain infection (Bidirectional Vector control: Endosymbionts of arthropod vectors CI), result in embryonic mortality [23].The reciprocal can be targeted and/or manipulated in different ways mating (infected female x uninfected male) and mating to control the VVBDs of man and animals.The vector between infected individuals are fully compatible.control can be attempted in three different ways by Mechanism of CI -Although the mechanism of utilizing Wolbachia by Chemotherapeutic, Immuno-CI has not been elucidated in the molecular level, presently logical and Wolbachia cytoplasmic incompatibility it is explained with two terminologies, modification (CI) based approach.and rescue.Modification is the process in which the Wolbachia modifies the sperm of the infected male Chemotherapeutic approach: This approach exploits during spermatogenesis by an unknown process.The the endosymbionts of arthropods vectors as a modified mature sperm is devoid of Wolbachia.If a chemotherapeutic target with the aim to disturb the modified sperm enters an incompatible egg (uninfected symbiosis Sterility was observed in healthy tsetse flies or infected with different strain) a delay in break down fed with tetracycline (2500µg/ml) due to damage to of nuclear membrane of pronuclei of sperm resulting the mycetome bacterial endosymbionts [14].Antibiotic in mitotic asynchrony [24].Amongst the incompatible (Tetracycline group) against the endosymbionts of crosses, bidirectional CI could be exploited to control nematode parasites when administered to the the mosquito population which in turn limits the mammalian host disrupt the symbiosis and lead to transmission of mosquito borne diseases.growth retardation and/or infertility of the parasite.

Vector borne diseases control
Immunological approach: Immunization of animals with the whole killed endosymbionts or purified antigens Paratransgenesis: Genetic transformation, of or recombinant antigens of the endosymbionts would commensal or symbiotic bacteria of the arthropod vector render them immune to tick vectors.The concealed is to alter the vector's ability to transmit pathogen.It is antigens or the mid gut antigens of the blood feeding an alternative means of blocking the transmission of arthropods like ticks, glossina, mosquitoes are the VBDs.The mid gut bacteria of arthropod vectors can potential vaccine targets being exploited by the be engineered to express and secrete effector proteins researchers for the control of vectors [22].Instead of which block the parasite invasion or kill the parasite in targeting the host (vector) antigens, the endosymbionts the mid gut or hemolymph or reproductive tract.The could be targeted to disturb the symbiotic relationship arthropod vectors that harbor the genetically transformed between the vector and the symbiont.Following endosymbionts are called as paratransgenic vector.ingestion of the blood from immunized animals, these For this strategy to be used in vector borne disease antibodies together with other components of the control, bacteria that survive inside the vector's body immune system such as complement, will destroy the have to be identified.The endosymbionts of arthropod symbionts inside the vector, leading either to death or vectors can be cultured and genetically transformed to to disruption of normal gut physiology of the tick and express the effector gene inside the vector in such a reduce growth and egg-laying ability.
way the gene product kills the parasite/pathogen that Wolbachia cytoplasmic incompatibility (CI) based the vector transmits.Isolation and characterization of approach: Wolbachia infections in arthropods can the endosymbionts present in midgut, hemolymph and manipulate reproduction of their hosts in a variety of reproductive tracts is the very first step towards the ways e.g., induced parthenogenesis, male killing, paratransgenic apparoach.In this strategy, the normal parthenogenesis, and cytoplasmic incompatibility symbiont population of the arthropod vector can be (Cl) [20].Wolbachia of mosquito species is the replaced with genetically modified symbionts, resulting in population of arthropod vectors refractory biotechnology will find safer solutions to the to the particular vector borne parasite/pathogen.This aforementioned problems in near future.strategy has shown promise in controlling the Whether transformed symbionts, can replace rhodnii was delivered into the asymbiotic first instar non-transformed in natural insect populations, can't be nymph orally in such a manner to express an said with certainty, despite of the early success with antimicrobial peptide L-cecropin A, inside the gut the transformation of insect vector symbionts.lumen which conferred resistance status to the Symbionts perhaps have no fitness load on insect hosts paratransgenics [25].Wolbachia might be used to and are capable of being transmitted via transovarian prevent the transovarian transmission of arboviruses transmission or lateral transmission.Thus, a strong viz., dengue virus, La crosse virus, rift valley fever gene drive system can potentiate the effectiveness of virus.With the paratransgenic approach, African paratransgenesis.An example of such gene drive trypanosomosis can be controlled using the tsetse system can be Wolbachia Endosymbionts [31].Although symbiont.Here the genes are not inserted to tsetse there has been a great advances in the development of chromosome, but instead to the tsetse symbiont, stable lines of genetically modified disease vectors Sodalis.Sodalis is well suited for paratransgenesis [32][33][34], there exists many challenges in to the because a) it resides in the gut in close proximity to application of transgenesis to control vector-borne pathogenic trypanosomes, b) a system for culturing diseases outside the laboratory.Future studies Sodalis in vitro has been developed [26] and can be mimicking field conditions likely will uncover the used in conjunction with standard molecular biology importance of fitness to the establishment of techniques to insert and express foreign genes of transgenic mosquitoes in natural habitats.India which interest in this bacterium [27] c) Sodalis is highly suffers lot due to dengue, chikungunya, malaria and resistant to many trypanocidal peptides, [28,29] d) filariosis need a programme on Wolbachia CI and recombinant Sodalis (recSodalis) can be reintroduced paratransgenic based approach to encounter aforeinto tsetse by thoracic microinjection and passed on to mentioned VBDs.The main constraints to the future progeny where they successfully express the establishment of an efficient transgenic vector approach marker gene product [30] and e) its genome is completely are the scarcity of a transgenes that effectively reduce sequenced and annotated, and this information will pathogen load.Again further studies utilizing QTL serve as a valuable resource that can be exploited to mapping, reverse genetics, gene knockdown, or other improve the efficiency of this expression system.techniques to identify traits associated with vector Because these commensal bacteria live naturally in competence can reveal candidate genes that, when close proximity to where trypanosomes develop and targeted, may effectively block pathogen development replicate in the tsetse midgut, expression of trypanocidal and transmission.products in Sodalis has the potential to block parasite Conclusion development in the fly.
Though this technology is useful in many A variety of very effective methods have been aspects, it has some demerits viz., socio-ethical issues, employed for suppressing arthropod vector populations, fitness of the genetically modified (GM) symbiont, including the application of biological control agents alteration of ecosystem, horizontal gene transfer and the elimination of breeding sites, with a among compatible bacteria, transfer of GM symbiont continuing and heavy reliance on the use of chemical to non-target arthropods and subsequently getting into insecticides.However, the development of insecticide the food chain, it can not be kept aside of our list of resistance by vector insects, the cost of developing and arsenals to fight against VBDs.The endosymbionts of registering new insecticidal compounds, and the arthropod are having small genome and/or more increase in legislation to combat the detrimental psuedogenes which indicates the long term coeffects of insecticidal residues on the environment, evolution with loss of considerable size of their have emphasized the need to assess alternative genome and functional genes by conserving the genes strategies for vector control which are cost effective essential to adapt to the arthropod microenvironment.and safer.Endosymbionts of the arthropod vector are This genome erosion and adaptive degeneration identified as a potential source for the control of phenomenon questions the stability of the transgene/ VVBDs and symbiont based approaches are considered effector gene in long run.The advancements in the as cost effective.Bioprospecting of molecules involved Trypanosoma cruzi by Rhodnius prolixus.The genetically transformed Rhodococcus 17. Townson H.(2002). Wolbachia as a potential tool in the interaction between vector, symbiont and for suppressing filarial transmission, Annals Trop parasite/pathogen will offer more targets to check the Med Parasitol., 96: 117-127.transmission of VBDs through paratransgenic approach