2. 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

3. 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.

4. 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

6. 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

7. 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

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

9. 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

10. Neighbor-Joining Phylogenetic tree of locally isolated wolbachia strain

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

12. 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.