International Postgraduate Research Conference (IPRC)
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Item Mosquito Breeding Habitat Diversity and Distribution of Species in Selected Areas in the Districts of Kurunegala, Gampaha, Kegalle and Kandy in Sri Lanka(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.; Udayanga, N.W.B.L.Sri Lanka has been suffering from mosquito-borne diseases since ancient times with the high prevalence of malaria, filariasis and Japanese Encephalitis (JE). As a result of successive efforts in control programs, Sri Lanka has received remarkable achievements. However, with all these successful efforts some arbovirus infections majorly the dengue has increased rapidly over last few decades. Some control programs only target the specified vectors for that disease and report of other vector species is ignored. The prevalence of different mosquito species from surveillance has been limitedly document is Sri Lanka which is grossly inadequate in providing evidence for potential health risks. The present investigation attempts to cater for this knowledge gap by documenting the prevalence of mosquito species in four selected districts of Sri Lanka. Entomological surveys were conducted from a total of 160 temporary and permanent mosquito breeding habitats identified in selected areas, in the districts of Kurunegala, Kegalle, Kandy and Gampaha from June 2017 to October 2018. Mosquito immature stages were sampled using standard dipping, siphoning or pipetting methods according to the nature of breeding habitat and collected specimen were identified up to the species level. Chi-square test of independence was used to evaluate the significance in the distribution of different mosquito species among different breeding sites in the studied districts. The diversity indices for breeding habitats were subjected to a Kruskal Wallis test followed by Dunn’s Multiple Comparison to identify the significance in the variations of diversity indices across breeding sites and studied districts. A total of 4663 mosquito larvae belonging to seven genera and fifteen species of mosquitoes were collected from natural and artificial water holding macro and micro habitats located in study sites. The relative distribution of different mosquito species differed significantly among the four studied districts (X2=143.248; df= 33; P< 0.001). Mosquito assemblages in the districts of Gampaha and Kandy indicated a similarity of 83.16%, while the district of Kegalle shared a similarity of 75.45% with the above cluster. A total of 21 permanent/temporary key breeding sites were found from the study areas. As suggested by the Kruskal Wallis statistics (P < 0.05 at 95% of significance), all the diversity indices for immature stages of mosquitoes varied significantly across different breeding sites recorded during the study. Meanwhile, none of the diversity indices indicate significant differences in terms of locality. The highest values of Pielou's Index (2.01 ± 0.6), Menhinik's Index (3.34 ± 0.83) and Margelef Index (0.59 ± 0.19) and highest species richness (4.0 ± 2.82) in terms of mosquito larvae were shown by paddy fields within the studied four districts. Findings of the current study would be useful to identify the entomological potential for disease transmission and facilitate implementation of appropriate vector control interventionsItem 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 strategyItem Susceptibility among Dengue Vectors to Malathion and Deltamethrin Insecticides in Three Selected Medical Officer of Health (MOH) Areas in the District of Gampaha, Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Dalpadado, C.P.R.D.; Amarasinghe, L.D.; Gunathilaka, P.A.D.H.N.Dengue has become the most important public health problem in Sri Lanka at present. As there is no effective vaccine or drug up to now, vector control is the main arm of disease control. Of these approaches, insecticide based control is used widely. However, excess use of chemicals and inappropriate application without determining the discriminative doses has given rise to the insecticide resistance among vector mosquitoes. Entomological surveys (Ovitap) were conducted in Negombo, Attanagalla and Dompe Medical Officer of Health (MOH) areas representing Urban, Sub urban and rural conditions, respectively from December 2016 December to June 2019. A total of 100 ovitraps each was placed outdoor and indoor in randomly followed by the collection after 5 - 7days. Eggs collected from ovitraps were reared under laboratory condition the laboratory (Temperature; 27 ± 10C: Relative Humidity 70 - 80%). The reared larvae were identified using standard taxonomic keys. Identification was further confirmed by identification of adults emerged from larvae . Adult mosquitoes were reared under optimized insectary conditions provided with sugar feeding. Batch of 140 mosquitoes from Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse) was taken separately from the colony 24 hour after a sugar feeding. Each batch of mosquito was exposed to malathion (0.8%) and Deltamethrin (0.03%) insecticide susceptibility following the World Health Organization (WHO) defined procedures. The experiment setup was repeated for 2 times. Mortality and corrected morality was calculated using Abbortt’s formula after 24 hours’ exposure period. The results indicate that both dengue vectors have evolved high level of insecticide resistance to malathion (0.8%) and deltamethrin (0.03%) in the District of Gampaha except Ae. albopictus mosquitoes in rural areas. Ae. albopictus mosquitoes were found fully susceptible for 0.03% deltamethrin in Dompe (rural) MOH area and emergence of resistance (95.9%) was observed in Attanagalla (suburban) area. It was found resistant in Negombo area (83.8%) which is highly populated. Ae. albopictus mosquitoes were found fully resistant for 0.8% malathion in both urban (71.5%) and Sub urban (85.3%) areas while emergence of resistance was observed in Dompe MOH area (90.8%). Ae. aegypti was found resistance to both 0.03% deltamethrin and 0.8% malathion in Negombo area (88.1%, 53.5% respectively) and resistance was suggested for 0.03% deltamethrin in Attanagalla MOH area (91.8%) while it was found resistant for 0.8% malathion (89.8%) in the same area. Ae. aegypti was not recorded in Dompe MOH area during the study period. Since both the dengue vectors have developed resistance to WHO diagnostic discriminative doses for Malathion and deltamethrin intensity bioassays (five times and ten times of discriminative dose) are needed to be done to confirm the level of resistance in these vectors. Therefore, results highlight the requirement of continuous insecticide resistance monitoring incorporated with the surveillance and control efforts. Hence, use of evidence based discriminative doses of insecticide for each area should be vital to delay the development of resistance among natural populationItem Identification of Volatile Compounds in Different Parts of the Brinjal Plant (Solanum melongena)(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Nusra, M.S.F.; Paranagama, P.A.; Amarasinghe, L.D.; Udukala, D.N.Plants synthesize and emit a large variety of volatile compounds, which produce a wide range of behavioral responses in insects. In some plants, these volatile organic compounds are the key compounds that are involved in attraction of insect pests. In addition, the synergism between insect pheromones and plant volatiles can increase the attraction of insect pest, offering new strategies for the development of more effective and reliable pest control programs. Volatile mediated foraging behavior is important in insect pests when they target host plants. The volatiles of brinjal may be functionally more important in host-finding behavior of their insect pest. Samples of brinjal plant leaves, fruits, shoots and flowers were collected from the unsprayed brinjal field. Steam distillation method was used to extract the volatiles from shade dried brinjal leaves, shoots, flowers and fresh fruits. The small pieces of the plant material were subjected to steam distillation for 4 hours at a distillation rate of 50 ml/hour. In this study, gas chromatography coupled with mass spectrometry technique was used for the identification of volatiles extracted from host plant brinjal. The GC oven was programmed at an initial oven temperature of 40 0C increased at a rate of 1 0C/min to 50 0C, then increased at a rate of 4 0C/min to 210 0C, and then raised at a rate of 8 0C/min to a final temperature of 230 0C. The MS data of eluted compounds were acquired and identified by the comparison of their retention times to those of authentic standards and with mass spectra from NIST library. Results indicated that brinjal plant produces 1-Methyl-5,8-dimethoxy-1,2,3,4-tetrahydro-1,4-iminonaphthalene, Benzyl alcohol, Phenylethyl Alcohol, Benzeneacetaldehyde, Butylated hydroxytoluene and Methyl salicylate from leaves; Butylated hydroxytoluene, n-Octyl formate, Butyl phthalate and 1,3-Dioxolane, 2-(methoxymethyl)-2-phenyl from fruits; Benzyl alcohol, 1,6-Octadien-3-ol, 3,7-dimethyl, Butylated hydroxytoluene, Hydroquinone and Methyl salicylate from shoots; and 1,6-Octadien-3-ol, 3,7-dimethyl, Methyl salicylate, Butylated hydroxytoluene, Hydroquinone and Benzyl alcohol from flowers as major compoundsItem Oviposition Preference of Aedes albopictus (Diptera: Culicidae) at Domestic and Peri-Domestic Settings in Gampaha Medical Officer of Health (MOH) of Sri Lanka(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Dalpadadoa, C.P.R.D.; Amarasinghe, L.D.; Bandaranayake, W.A.C.L.There are varying ranges of water filled man-made and natural container habitats with varying physico chemical parameters available for the breeding of Aedes mosquitoes. House to house cross-sectional entomological survey was carried out monthly in Gampaha MOH area from April 2017 to June 2018 in order to investigate oviposition preference of dengue vector mosquitoes in domestic and peri-domestic areas. Larval collections were done indoors and outdoors using random sampling technique for minimum 100 houses within a radius of 300 m and potential breeding places in each site were identified and categorized accordingly. Out of 1500 premises examined, 17.4% (n= 261) premises were found positive for Ae. albopictus and Ae. aegypti was not recorded during the study period. The House Index (HI), Container Index (CI), and Breteau Index (BI), varied from 3 - 39, from 5.4 - 44.4, and from 4 - 67, respectively during the study period and there was a strong correlation between the HI and the number of dengue patients reported in Gampaha MOH area according to pearson correlation analysis. (r = 0.64). Overall 2828 potential breeding places were examined in which 18.2% (n=405) containers were found positive for Ae. albopictus larvae. The result indicates that most of these positive breeding places were located outdoors 91.2% (n=369) and only 8.8 %( n=36) positive breeding places were located indoor which includes non- used cisterns/ commodes and refrigerator trays. There were 26.5% (n=750) discarded receptacles, 22.6% (n=640) temporary removal items, 13.2% (n=373) natural breeding places, 8.6% (n=242) water storage items, 8.3% (n=235) covering polythenes, 6.2% (n=176) pet feeding cups, 4.8% (n=136) ornamentals, 2.8% (n=78) tyres, 2.4%( n=68) non used cisterns and commodes and 1.9%( n=53) refrigerator trays. Results Indicates that tyres (51.9%), non- used cisterns/commodes (47.7%), ornamentals (26.2%) were the most preferred breeding localities forAe. albopictus followed by discarded receptacles (24.3%) irrespective to the number of potential breeding places found. According to the results CI for discarded items, temporary removals and covering items increased drastically prior to dengue epidemics and four to five weeks lag period was identified between increased container index and increased number of dengue patients. Non- used cisterns/commodes and natural breeding places showed year around productivity and positivity for Ae. albopictus which indicated that they contributed to maintain and survival of mosquito vector population between intermediate dry periods. Therefore, this study emphasizes the control of mosquito breeding in key breeding places such as non-used cisterns, commodes and, natural breeding places may be beneficial to reduce vector densities during dry period hence, leading to reduce the spreading mosquito vectors in to other made artificial containers during rainy season leading to major out breaksItem Antagonist Potential of Vorticella microstoma on the larval development of Culex gelidus and Aedes aegypti(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Ranasinghe, H.A.K.; Amarasinghe, L.D.Selection of effective biological agents which can suppress the growth of immature stages of mosquito vectors has drawn a wide attention in disease control programmes. Vorticella microstoma is a parasitic ciliate which has an ability to parsitize on larval stages of mosquitoes and inhibit their growth. However, the potential antagonist activity has not been evaluated in detail in Sri Lanka. Fifteen each of first, second, and third instar larvae of Cx. gelidus and Ae. aegypti were placed in separate V. microstoma culture bottles at room temperature (27± 2 ºC) and mortality rate of mosquito larvae was observed after 48 h. Bioassay was done in completely randomized design with controls. The first, second, and third instar larvae of Cx. gelidus were allowed to be infested with the trophont stage of V. microstoma. Heavily infested larvae with V. microstoma were identified by the presence of parasites attached all over the body surface. V. microstoma trophonts did not usually attach to siphon region of mosquito larvae when they are alive. None of the instar levels of Ae. aegypti showed susceptible to infection with V. microstoma. In the first instar larvae of Cx. gelidus, thorax and the abdominal segments had the attachment of 30–35 trophonts of V. microstoma, whereas 2–4 trophonts had attached to the anal papillae. Higher densities of V. microstoma were found in abdominal segments of second instar larvae of Cx. gelidus, whereas total of 45–50 trophonts had attached to thoracic region and abdominal segments. Fifty to fifty-five trophonts of V. microstoma had attached to third instar larvae of Cx. gelidus, with higher densities in the anal papillae followed by abdominal segments; in the anal papillae, 20–25 trophonts were found to be attached. V. microstoma infection was positively correlated with the body size of mosquito larvae. The percentage mortality of the first, second, and third instar larvae of Cx. gelidus did not significantly differ from each other (One-Way ANOVA: IBM SPSS Statistic Software, P<0.05). Therefore, the study results would be a new avenue to work on environmentally agreeable manner in reducing the Cx. gelidus vector mosquito populations.