The incidence of malaria has been increasing at an alarming rate. Nearly 3000 million people are reported to be affected annually and 1/3 of them die. Confronting malaria has achieved good progress in the elimination of mosquito species that would carry Plasmodium parasites by eradiation of breeding grounds and using DDT (Crampton et al., 1992). But, the problem is not yet solved due to the emerging new incident cases. The control of malaria has been a daunting task for health care professionals. This could be due to the resistance potential acquired by the wide range of mosquitoes that may act as vectors. With the advancements in science and technology, much emphasis was given to molecular biology approaches. Earlier, a synthetic, non-radioactive-based DNA probe has been a choice for the efficient management of vector, Anopheles gambiae complex. Similarly, gene manipulation was preferred to disrupt the disease transmittance (Crampton et al., 1992).
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Carlson et al (1995) have developed tools and methods for genetic alteration of mosquitoes, and RNA and DNA virus gene-targeting vehicles, and potential antiviral gene constructs, as they appear promising for the transformation of mosquitoes that show resistance to pathogens. Hence, arthropod genomes have become favorite targets for genetic manipulation of malaria control. However, literature is still limited on the strategies intended for exploring the genetic aspects/ control of mosquitoes.
- Aims and Objectives: The aims and objectives of the proposal are 1. To apply the modern biotechnology tools to understand the key molecular machinery /mechanisms that may be contributing to the development of disease 2. To develop and establish a standardized inexpensive protocol that could address the problem.
- Approaches: Boete and Koella (2002) described a theory based on genetic modification where encapsulation was the strategy to control malaria. Here, genes connected to the encapsulation process were studied as their distribution remained unknown in the natural populations. So, a model needs to be developed based on population genetical and epidemiological processes as this would facilitate to assess gene transmission intensity, resistance obtained through evolution, and tools driving the genes (Boete & Koella, 2002). Genetic markers, sexing and genetic sterilization need to be employed to better understand the epidemiology of malaria vectors and pathogens. Initially, the genome sequence of arthropods needs to be understood to gain insights into the key gene pathways responsible for disease transmission. Here, the transposon sequence will be deciphered to design suitable probes for gene silencing or elimination (Sparagano & De Luna, 2009). This is to ensure the method of post-integration elimination of transposon sequences that would stabilize any insertion in genetically-modified insects (Sparagano & De Luna, 2009). This is nothing but the Sterile Insect Technique where certain metabolic pathways would be altered to better sidestep or block the development of offspring released from the parent insect (Sparagano & De Luna, 2009). The next strategy is the use of bee venom phospholipase A2 (PLA2) which would inhibit ookinete invasion of the mosquito midgut.
Here, the protein-coding sequence would be mutated to deactivate the PLA2. The DNA sequence specific to the mutated PLA2 (mPLA2) would be finally placed in the downstream region of a mosquito midgut-specific promoter (Anopheles gambiae peritrophic protein 1 promoter, AgPer1) (Sparagano & De Luna, 2009). This manipulated gene construct would be used to transform mosquitoes. Transgenic cell lines would be developed and checked whether they would circumvent Plasmodium gallinaceum oocyst development (Rodrigues et al., 2008).
The other methods to be followed are that we would be altering the non-structural genes 2A and 4B and the 3’non-coding region of the yellow fever virus to determine the role of genetic markers of viral dissemination from the mosquitoes, Aedes aegypti midgut (McElroy et al., 2006). For this purpose, we would be obtaining the clones of disseminating (Asibi) and non-disseminating (17D) yellow fever viruses (YFV), to develop chimeric viruses for testing the attenuation process of YFV and its dissemination (McElroy et al., 2006). Sexing lines harboring novel genes would be produced for the mosquito under investigation, for example, Anopheles stephensi, the most important human vector (Catteruccia, Benton & Crisanti, 2005). Male mosquitoes, at their 3rd instar larval stage, with green fluorescent protein (EGFP) expression under the control region of beta2-tubulin promoter would be recognized by their fluorescent gonads (Catteruccia, Benton & Crisanti, 2005). These would be sorted out from females both manually and through machines. To better understand the microbial diversity of midgut, a conventional culture method would be applied followed by an analysis of a 16S ribosomal RNA (rRNA) gene sequence library (Pidiyar et al., 2004). This strategy would be to identify the microbiota that may serve as important targets for the genetic control of malaria about disease transmission (Pidiyar et al., 2004). Gene expression would be controlled by removing certain DNA sequences from the integrated transgenes in insects (Jasinskiene et al., 2003). This is to allow the analysis of vital structural elements that could regulate gene expression (Jasinskiene et al., 2003). Similarly, control elements would be manipulated along with the single integration such that the overall genome would be benefitted (Jasinskiene et al., 2003). Here, a recombination system matching with a location of the ‘core-lox site would be employed to excise a gene from transgenic mosquitoes (Jasinskiene et al., 2003). Next, to check the influence of the incorporated foreign genes on the development of new alleles, the spread rate of introduced genes would be determined (Zhong et al.,2006).
For this purpose, the kinetics of gene incorporation between two different geographical populations of mosquitoes would be determined with techniques like microsatellite markers and amplified fragment length polymorphisms (AFLPs). This could better help us to gain insights into the role of genetic markers in the development of a large genetic differentiation between the two populations (Zhong et al., 2006). A phage display library would be constructed to detect peptide –SM1-sequences that would bind to the midgut surfaces and salivary glands of mosquitoes (Jacobs-Lorena, 2003). The transgenic mosquitoes thus obtained would be checked to determine the expression of an SM1 tetramer that is specific to the region of the gut-specific promoter (Jacobs-Lorena, 2003). This result would be correlated with the determination of phospholipase A2 that acts as another effector region (Jacobs-Lorena, 2003). This would be followed by another experiment described earlier. According to this method, certain secreted factors and putative receptors would be isolated from the salivary glands of mosquitoes (Arcà et al., 1999). By employing the Signal Sequence Trap technique, the expression of cDNAs in the salivary gland region would be identified. Similarly, the expression of certain homologs of genes namely the apyrase and D7 would be determined in parallel with cDNAs (Arcà et al., 1999). Finally, to better dissect the connection between the digestive system pathways and the storage of feed in the midgut of mosquitoes, factors that control the digestion physiology would be focused on. This will be done by the introduction of Soy trypsin inhibitor(STI) to a protein meal.To assess whether this method inhibited digestion or not, SDS –PAGE analysis of feces would be carried out to check for the expression of indigested proteins.
The period required for carrying out this project would three years. The first year for data and sample collection.The subsequent years for laboratory analysis. The estimated cost would be nearly 10,000 USD.
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