Supervisor : Prof. Dr. Iftikhar Hussain Isolation and molecular characterization of Wolbachia Pipientis from local insects population Umair Hassan MPhil Microbiology 2006-ag-1143 Supervisor : Prof. Dr. Iftikhar Hussain

Vector borne diseases; A major threat Arthropod-borne viruses (arboviruses) have become significant public health problems, with the emergence and re-emergence of arboviral diseases nearly worldwide. DENV, JE, WNV, CHIKV, CCHF Half of global population at risk of getting DENV infection. Currently very limited control options for dengue which focus on reducing population abundance of the major mosquito vector, Aedes aegypti using insecticides and larvicides. Drawback Repeated exposure of mosquitoes to insecticides has allowed insecticide resistance to develop, increasing the need to use more expensive alternative compounds. Biological control; RIDL (Release of Insects carrying Dominant Lethal genes) and Wolbachia based system.

Wolbachia 1924: Hertig and Wolbach Host range: Arthropoda and Nematoda; reproductive tissues Maternally transmitted w/in species; Horizontally transmitted between species Obligate intracellular lifestyle Phylum Arthropoda Insects: up to 67% of species Isopods: 35% of terrestrial isopods Mites: 43% of mites Lice: ~100% of species

Phenotypic effects due to wolbachia trans-infection Cytoplasmic incompatibility Life shortening Blood feeding alteration Bendy proboscis Plasmodium, DENV, CHIKV interference Embryo mortality Lower hatch rate Reduced fecundity

Objectives Isolatation of Wolbachia endobacteria from mosquitoes as well as from fruit flies. Detection of Wolbachia strains with the help of PCR by targeting Wsp gene. Clone based Sequencing of partial Wsp gene Phylogenetic analysis

Materials and Methods Sample collection Sample preparation Isolation of Wolbachia PCR based detection Cloning Sequencing Phylogenetic analysis

PCR amplification and agarose gel electrophoresis of Wsp gene fragment 592 bp 100bp MW ladder PCR amplification and agarose gel electrophoresis of Wsp gene fragment

Neighbor-Joining Phylogenetic tree of locally isolated wolbachia strain

Future perspective To control Mosquito population Biological control Less cost (Insecticides etc) Mosquito-Borne diseases Malaria Dengue Chikungunia Bed Bug Tsetse fly Yellow fever

Eliminate Dengue Project, Australia The EDP was originally established in Australia with the aim of using Wolbachia-based strategies to curb the spread of dengue, a mosquito-borne disease. The success of the EDP has inspired a broad push to identify applications for Wolbachia in other disease vectors.