Browsing by Author "Rodrigo, W.W.P."

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Bacterial Diversity in the Midgut of Field Caught Culex tritaeniorhynchus
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranasinghe, H.A.K.; Gunathilaka, P.A.D.H.N.; Amarasinghe, L.D.; Rodrigo, W.W.P.
Ingestion of blood meal by female mosquitoes triggers a series of physiological processes in midgut where symbiotic microbes also exist. These symbiotic microbes can be engineered to produce molecules that inhibit pathogens; through paratransgenic approach. Little is known about the midgut microbes of Culex mosquitoes and no attempt has been made so far in Sri Lanka. Cx. tritaeniorhynchus mosquitoes were separated from the entomological surveys conducted at Kelaniya Medical officer of Health (MOH) area from June – August 2019. Unfed adult female mosquitoes were sacrificed using a cold shock and were surface sterilized using 70% ethanol followed by rinsing with phosphate buffer saline (PBS). Midgut of mosquitoes were dissected and midgut of ten mosquitoes were pooled in sterile PBS (250 μL) to make a homogenized lysate. A dilution series (100- 10-7) was made from lysate. 100 μL from each dilution was plated on Plate Count Agar (PCA) and were incubated for 48 hours at 37 0C. Pure cultures for each microbe were obtained from the primary plates using streak plate method, sub culturing in Nutrient Agar. The experiment set up was repeated 25 times with ten mosquito pools at each effort. Colony separation was done based on phenotypical differences and basic biochemical tests. Stab cultures of isolates were sequenced for 16S ribosomal RNA partial gene. To identify the closest related sequence, obtained sequences were analyzed by Bioedit software package and completely aligned sequences were compared with the BLAST database. The evolutionary history was inferred using the neighbor-Joining method and the evolutionary analyses were conducted in MEGA X. A total of eight bacterial strains namely; Staphylococcus pasteuri, Bacillus megaterium, Staphylococcus cohnii, Pantoea dispersa, Staphylococcus chromogenes, Bacillus aquimaris, Staphylococcus arlettae, Staphylococcus scuiri was isolated from Cx. tritaeniorhynchus (n=250). All of these species were belonged to two phyla; Firmicutes and Proteobacteria. Phylum Firmicutes was the dominant phyla which include seven species. The evolutionary distances which were computed using Tajima-Nei method were used to infer the phylogenetic tree. It represented a close relationship between the species of two genera; Staphylococcus and Bacillus while the relationship was distant for genus Pantoea. The present data strongly encourage further investigations to explore the potential usage of these microbes through the paratransgenic approach which is a novel eco-friendly vector control strategy
Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata
(Asian Pacific Tropical Medicine Press , China, 2014) Rodrigo, W.W.P.; Dassanayake, R.S.; Karunanayake, E.H.; Gunawardene, Y.I.N.S.; Weerasena, O.V.D.S.J.
OBJECTIVE: To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses. METHODS: To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently. RESULTS: Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa. CONCLUSIONS: SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.
Molecular Characterization of Midgut Bacteria in Larval and Adult Stages of Aedes albopictus in Gampaha District, Sri Lanka
(Institute of Biology Sri Lanka, 2021-09) Ranasinghe, H.A.K.; Gunathilaka, P.A.D.H.N.; Amarasinghe, L.D.; Rodrigo, W.W.P.
Bacterial species that are acquired from the aquatic larval and adult stages are established in the midgut of mosquitoes, exhibiting different functional tasks retaining in the gut as symbiotic species. The present study aimed on screening of midgut bacteria of larval and adult Ae. albopictus, as a fundamental pre-requirement to support the Sterile Insect Technique (SIT) and Incompatible Insect Technique (IIT) approaches which are in progress, in Sri Lanka. In novel techniques such as SIT, IIT or the use of genetically modified mosquitoes need artificial rearing of the life cycle stages of disease vectors followed by open releases into the environment and thereby reduce vector densities through population replacement or suppression. Sampling sites included Brandiyamulla, Gampaha, and Miriswaththa in Gampaha Medical Office of Health (MOH) area of Sri Lanka. Unfed adults and 3rd instar larvae, 250 in number were sacrificed using a cold shock and 70% Ethanol respectively. 70% ethanol followed by phosphate buffer saline (PBS) were used for surface sterilization. A homogenized lysate was prepared in sterile PBS (250μL), by pooling dissected midguts of ten individuals of larvae/adult mosquitoes. A dilution series (100- 10-7) was made from lysate and 100 μL from each dilution was plated on Plate Count Agar and pure cultures for each microbe were obtained. Isolated bacteria were subjected to 16S rRNA amplification. A total number of 6 bacterial strains (Microbacterium trichothecenolyticum, Kocuria kristinae, Elizabethkingia miricola, Staphylococcus sciuri, Pantoea dispersa, Neisseria flavescens) were identified from 5 bacterial families; Flavoacteriaceae (22.05%), Neisseriaceae (11.76%), Micrococcaceae (10.29%), Staphylococcaceae (14.70%), and Erwiniaceae 35.29%) from field-collected adults, while 6 strains (Agromyces sp., Microbacterium paraoxydans, Microbacterium sp., Bacillus megaterium, Bacillus nanhaiensis, Bacillus sp.) were identified from the field-captured larvae. Species composition of gut microbes isolated from larvae was dominated by family Bacilliaceae (76.76%). Pantoea dispersa and Bacillus megaterium were the most prominent bacterial species isolated from midgut of adults and larvae respectively. Microbacterium genera was found as common for both adults and larvae, although no common bacteria were found up to species level. Midgut bacteria belonged to Bacteroidetes (Elizabethkingia miricola) and Proteobacteria (Pantoea dispersa, Neisseria flavescens) were only recorded from the midgut of adults. Larvae and adults in Ae. albopictus denoted different midgut bacterial species.