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Item Pheromone baited biopesticide for control of Leucinodes orbonalis Guenee in brinjal plant(Frontiers in Bioscience, Elite, 2020) Nusra, M.S.F.; Paranagama, P.A.; Amarasinghe, L.D.; Udukala, D.N.Leucinodes orbonalis Guenee, Brinjal fruit and shoot borer (BFSB), is the major pest on brinjal world wide. Larvae of this pest cause the damage; which at initial stages adversely affect the shoot growth, and in later stages diminish fruit quality. Spraying of insecticides is the main pest control method. This has been absolutely ineffective due to concealed habit of the larvae. Such a phenomenon apprehends to the development of insecticides resistance which demands continuously increasing doses. Manipulating the insect behavior using semiochemicals could be an opportunity for better management of insect pest. Application of BFSB sex pheromone components (E)-11-hexadecenyl acetate (E11-16: Ac) alone or in combination with (E)-11-hexadecen-1-ol (E11-16: OH) in traps can be used to suppress the populating growth. Goal-oriented interdisciplinary research on semiochemicals for sound administration of BFSB will depend on a better understanding of the key chemical ecology stimuli of relevance to the pest.Item Comparative Effect of Parasitism of Ciliated Protists Associated with Selected Vector Mosquito Larvae in Selected Rice Field Habitats in Ganewatte, Kurunegala District, Sri Lanka(Institute of Biology Sri Lanka, 2021-09) Wijesinghe, S.; Amarasinghe, L.D.Mosquito breeding sites provide habitats for diversifying naturally occurring microbiota and favour different types of interactions including parasitism. This study was carried out to determine the natural occurrence of ciliated protist parasites associated with selected vector mosquito species inhabiting rice fields in Ganewattha area in Kurunegala district in Sri Lanka and to compare their parasitic effects on the selected vector mosquito larvae. Mosquito samples were collected from five rice field sites during the period from June to December 2020. The samples were observed for epibionts/parasitic infections under the microscope. Moribund or dead mosquito larvae in each sample collection were observed for internal parasitic infections. A comparative study was carried out to determine the lethal effect of ciliated organisms on Culex tritaeniorhynchus (n=50) and Culex gelidus larvae (n=50) in replicated trials (R=3). The minimum number of V. microstoma that cause a lethal effect on Cx. tritaeniorhynchus third instar larvae (n=100) was determined by a bioassay with ten replicates and two controls per each replicate. A total of 1650 third instar mosquito larvae of Culex tritaeniorhynchus (62.8%) and Culex gelidus (37.2%) were collected in this study. Ciliated protists namely Vorticella microstoma, Zoothamnium spp. and Chilodonella spp. associated with both species of mosquito larvae were identified. Results revealed that V. microstoma has the potential of infection to cause 71.33 (±5.23) mean percentage mortality of Cx. tritaeniorhynchus larvae. A minimum of 1000 V. microstoma is required to kill a third instar larva of Cx. tritaeniorhynchus at 69.60 (±2.40) hours of exposure. Culex gelidus larvae showed only 41.33 (±3.43) mean percentage mortality. This study concludes that V. microstoma is the most successful ciliated parasite as a killing agent of Culex tritaeniorhynchus vector mosquito larvae. Their abundance and effectiveness may contribute to develop them as an effective biocontrolling agent in the future.Item Diversity of midgut bacteria in larvae and females of Aedes aegypti and Aedes albopictus from Gampaha District, Sri Lanka(Parasites & Vectors volume 14, 2021) Ranasinghe, K.; Gunathilaka, N.; Amarasinghe, L.D.; Rodrigo, W.; Udayanga, L.Abstract Background: The midgut microbiota of mosquitoes maintain basal immune activity and immune priming. In recent years, scientists have focused on the use of microbial communities for vector control interventions. In the present study, the midgut bacteria of larvae and adults of Aedes aegypti and Ae. albopictus were assessed using both fieldcollected and laboratory-reared mosquitoes from Sri Lanka. Methods: Adults and larvae of Ae. aegypti and Ae. albopictus were collected from three selected areas in Gampaha Medical Officer of Health area, Gampaha District, Western Province, Sri Lanka. Bacterial colonies isolated from mosquito midgut dissections were identified by PCR amplification and sequencing of partial 16S rRNA gene fragments. Results: Adults and larvae of Ae. aegypti and Ae. albopictus harbored 25 bacterial species. Bacillus endophyticus and Pantoea dispersa were found more frequently in field-collected Ae. aegypti and Ae. albopictus adults, respectively. The midgut bacteria of Ae. aegypti and Ae. albopictus adults (X2 = 556.167, df = 72, P < 0.001) and larvae (X2 = 633.11, df = 66, P < 0.001) were significantly different. There was a significant difference among the bacterial communities between field-collected adults (X2 = 48.974, df = 10, P < 0.001) and larvae (X2 = 84.981, df = 10, P < 0.001). Lysinibacillus sphaericus was a common species in adults and larvae of laboratory-reared Ae. aegypti. Only P. dispersa occurred in the field-collected adults of Ae. aegypti and Ae. albopictus. Species belonging to genera Terribacillus, Lysinibacillus, Agromyces and Kocuria were recorded from Aedes mosquitoes, in accordance with previously reported results. Conclusions: This study generated a comprehensive database on the culturable bacterial community found in the midgut of field-collected (Ae. aegypti and Ae. albopictus) and laboratory-reared (Ae. aegypti) mosquito larvae and adults from Sri Lanka. Data confirm that the midgut bacterialItem 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.Item Microbiota Diversity associated with Mosquito Breeding Habitats in Kegalle District, Sri Lanka(2021-07) Ranasinghe, H.A.K.; Amarasinghe, L.D.The distribution, abundance, and individual fitness of mosquitoes are known to be dependent on associated microbiota composition with developing larvae. Identification of such naturally occurring microbiota and their interactions on mosquito larvae, in terms of parasitic, pathogenic, competitive or predatory organisms against larvae is beneficial. Such agents can be used for potential larval controlling approaches, in an environmental- friendly manner. Thus, the present study aimed to investigate the diversity of microbiota associated with mosquito larval habitats. Sampling was performed from a variety of breeding habitats in Kegalle district, Sri Lanka. Microbiota in water samples were preserved using Rose Bengal solution and Lugol‟s iodine, and were identified observing under the microscope, using standard identification keys. A variety of mosquito breeding habitats indicated the presence of 37 microbiota species under 9 phyla, belong to; Amoebozoa, Arthropoda, Ciliophora, Charophyta, Chlorophyta, Cyanobacteria/ Cyanophyta, Euglenozoa, Ochrophyta/ Heterocanthophyta and Rotifera. Except in coconut shells, every breeding habitat type investigated had the prevalence of rotifers, at least with one species. Thus the phylum Rotifera displayed the highest percentage of abundance (30.83%) of total microbiota. Species under phylum Charophyta had the lowest abundance among recorded (2.19% of total microbiota). Philodina citrina and Euglena pisciformis were found as species showing all three categories of abundance types; constant, common and accidental/or rare. Although the highest number of microbiota species were recorded from paddy field breeding habitats, all the species were existed as accidental or rare species in the habitat type according to their abundance. Only ponds and tree holes were recorded with beta diversities over 50% (high heterogeneity) in microbiota composition among the systems. Paddy fields, marshy lands, blocked drainages, metal containers, and leaf axils had beta diversities between 20 and 50%, indicating intermediate heterogeneity. The rest of the other habitats had beta diversities below 20%, indicating low heterogeneity. Paddy fields exhibited the highest gamma diversity (16) and Shannon-Weiner diversity (52.17) values. From the microbiota species recorded, Zoothamnium sp. was identified as an epibiont in Culex gelidus and Culex tritaeniorhynchus mosquito larvae. Updated information from the present investigation would be facilitated for implementing appropriate vector control interventions.Item Spatial and temporal variations of ground water quality in Kalpitiya peninsula(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Dilshara, R.M.P.; Handapangoda, H.M.A.K.; Swarnathilake, D.S.G.G.C.; Amarasinghe, L.D.; Herath, H.M.I.K.; Udayanga, N.W.B.A.L.Intensive agricultural practices in Kalpitiya have caused excessive usage of fertilizers. The sandy regosol soil and the over irrigation due to the presence of semi-arid climatic conditions in the area have led to nutrient leaching conditions in Kalpitiya. Since 1990s, the groundwater aquifers in Kalpitiya remain contaminated with higher levels of nitrate due to intensive agricultural practices and the current status of groundwater pollution remains poorly studied. Therefore, this study was conducted to evaluate the current status of groundwater pollution in Kalpitiya, with special emphasis on spatial and seasonal variations. Groundwater samples of 50 wells (potable and agricultural) located in five sentinel sites, namely Nawakkadu, Narakkalli, Thalavila, Kandakuli and Kalpitiya town were collected at monthly intervals from November 2018 to March 2020. In addition, the existing land use practices surrounding the respective wells were also recorded. Selected water quality parameters, namely pH, Electrical Conductivity (EC), Total Dissolved Solids (TDS), salinity, nitrate and phosphate concentrations were analyzed using standard methods. The General Linear Model (GLM) followed by Tukey’s pairwise comparison was used for statistical analysis. Significant spatial variations (p< 0.05) at 95% level of confidence. EC, nitrate, phosphate and TDS levels in groundwater samples of Nawakkadu, Narakkalli, and Kalpitiya town areas remained above the permissible levels for potable water quality given by the Central Environmental Authority (CEA). Therefore, adequate treatment of groundwater is recommended prior to use for drinking purposes.Item Toxic potential of green synthesized silver nanoparticles using Annona glabra leaf extract against Daphnia magna(Proceedings of the 1st International Conference on Frontiers in Chemical Technology, Institute of Chemistry Ceylon, 2020) Paragodaarachchi, Y.L.; Wickramarachchi, P.A.S.R.; De Silva, C.R.; Amarasinghe, L.D.This study aims to evaluate the acute toxicity of biosynthetic AgNPs produced using Annona glabra leaf extract, against a selected aquatic-indicator, Daphnia magna. The toxicity of the biosynthetic AgNPs was compared against the Ag+ ion solution. Aqueous leaf extracts of A. glabra were prepared by heating chopped leaves (20.0 g) with water (100.0 mL) at 100 oc for 1 hour. AgNPs were synthesized by incubating the leaf extract of A. glabra (2 mL) with AgN03 solution (l mM, 20 mL) for 3 hours. The formed AgNPs were separated using centrifugation (6000 rpm, 20 minutes) and the separated AgNPs were freeze dried. AgNPs were Characterized by UV — visible Spectrometer, particle size analyzer and scanning electron microscopy. acute toxicity tests against D. magna were conducted according to the Organization for Economic Co-operation and Development (OECD) standard procedure using neonates aged less than 24 hours reproduced using parthenogenesis. D. magna neonates were exposed to solutions within the concentration range Of 0.01 — 10 mg/L Of AgNP solutions and concentration range Of 0.5 — 2 gg/L Of ion solutions for 48 hours. Tests were conducted in quadruplicate and 95% confidence interval was calculated. Biosynthesized AgNPs showed a plasmon resonance peak at 419 nm, were spherical in shape and the size ranged between 10 — 190 nm. The EC50 value against D. magna for Agt ions were 1.41 ± 0.2 gg/L and for AgNPs it was 3.96 ± 1.1 mg/L after 48 hours. Results obtained from this study suggests that the toxicity ofbiosynthetic A. glabra - AgNPs exerts lesser toxic effect to D. magna than the Agt ions.Item Potential of bio fabricated Annona glabra - silver nanoparticles to control Aedes aegypti and Aedes albopictus(, Proceedings of the 1st International Conference on Frontiers in Chemical Technology, Institute of Chemistry Ceylon, 2020) Aberathna, A.A.A.U.; Amarasinghe, L.D.; Sithara, W.S.; Wickramarachchi, S.; De Silva, C.R.Bio synthesized silver nanoparticles using Annona glabra leaf extract (An-AgNPs) as the reducing and stabrlizing agent were assessed as a novel larvicide against dengue vector mosquito larvae. The fresh leaves of A. glabra were crushed well and heated with distilled water under 70 0C for I hour. The supernatant was mixed (10% and 20% v/v of total volume) with 5 mM AgNO, to synthesize AnAgNPs. Formation ofAn-AgNPs were confirmed bythe color change and by appearance ofSPR peak in the Uv-vis spectrum and characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FTIR). lhe larvicidal activity of An-AgNPs was assessed against the third instar larvae of Ae. aegypti ^nd, Ae. albopictus, under the laboratory conditions (2 -10 mg/L). The change ofsolution color from yellow to dark brown/black and the SPR band at 435 nm confirmed the formation of An-AgNPs. According to SEM; An-AgNPs were spherical in morphology. FTIR analysis detected that An-AgNPs are capped and stabilized with biomolecules of A. glabraleaf ltZl9 (C=O stretching), 1366 (CH2 bending) and 3500-3200 (N-H and O-H stretching) and l2l7 cm-' (C-O stretching)i. The LCro of An-AgNPs (10%) and An-AgNPs (20%)againstthethirdinstarslarvaeofAe.aeglptiafter24hourswere5.2gand2.43mglland of Ae. albopictus were 3.02 and 2.51 mg/L respectively. The LC* of An-AgNPs (I0%) and An-AgNPs (20%)againstthethirdinstarslarvaeof Ae.aegyptiafter48hourswerel.5l andl.lT mg/Landof Ae. albopictus were 1.14 and 2.i0 mg/L respectively. The LC,n ofleafcrude plant extract against the third instars larvae of Ae. aegTpti after 24 and 48 hours were 5.9455 and 3.5485 mg/L and for the third instars iaruae of Ae. albopictus were 5.0040 arrd2.7346 mg/L respectivdy. This study shows that AnAgNPs is efficient in controlling mosquito larvae.Item Comparative study on larvicidal activity of green synthesized silver nanoparticles and Annona glabra (Annonaceae) aqueous extract to control Aedes aegypti and Aedes albopictus (Diptera: Culicidae)(Heliyon 6, 2020) Amarasinghe, L.D.; Wickramarachchi, P.A.S.R.; Aberathna, A.A.A.U.; Sithara, W.S.; De Silva, C.R.The present study reports mosquito larvicidal potential of green synthesized silver nanoparticles by using Annona glabra leaves (An-AgNPs). Synthesized An-AgNPs were characterized by Ultraviolet-Visible spectroscopy (UVVIS), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS) technique and Fourier transform infrared spectroscopy (FTIR). Colur change from pale yellow to brick red of the plant extract and AgNO3 solution indicated the formation of An-AgNPs initially. Surface Plasmon Resonance (SPR) band at 435 nm in the UV-Vis confirmed the formation of An-AgNPs. SEM images showed that An-AgNPs were spherical in shape. FTIR proved that An-AgNPs were functionalized with biomolecules in A. glabra leaves. Based on DLS analysis the average size range of synthesized An-AgNPs was determine to be 10–100 nm and 100–1000 nm. Third instar larvae of dengue vector mosquitoes, Aedes aegypti and Aedes albopictus were subjected to larvicidal bioassays in a range of concentrations of An-AgNPs and A. glabra crude aqueous leaf extract (2–10 mg/L). AnAgNPs exhibited very high larvicidal activity against dengue vector mosquito larvae; LC50 value for Ae. aegypti at 24 h exposure to An-AgNPs (Plant extract: AgNO3 1 : 10) 5.29 mg/L; An-AgNPs (Plant extract: AgNO3 2 : 10) 2.43 mg/L while LC50 value for Ae. albopictus at 24 h exposure to An-AgNPs (Plant extract: AgNO31:10) 3.02 mg/ L; An-AgNPs (Plant extract: AgNO3 2:10) 2.51 mg/L. LC50 values obtained for A. glabra leaf extract tested against Ae. aegypti and Ae. albopictus are 5.94 mg/L and 5.00 mg/L respectively at 24-hour exposure. This study further revealed that Ae. albopictus is more susceptible than to Ae. aegypti to a given concentration of An-AgNPs and to crude aqueous leaf extract of A. glabra. Larvicidal effect of An-AgNPs is superior to the crude aqueous leaf extract of A. glabra. An-AgNPs is a potent larvicide for dengue vector control.Item Contarinia maculipennis as an emerging threat to Dendrobium in Sri Lanka - A case study.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Dias, M.A.; Amarasinghe, L.D.; Jayalath, W.G.H.; Attanayake, R.N.Blossom midge, Contarinia maculipennis which belongs to Order Diptera: Family Cecidomyiidae is considered as one of the major threat to ornamental and several crop plant species due to its wide host range. For the first time C. maculipennis was recorded from Dendrobium sp. in 1992 from Florida, but the origin of this species is considered as Southeast Asian region. In Korea, it is officially nominated as a quarantine pest since 2007 due to it’s sever economic impact on vegetable crops and ornamental plants. For the first time in Sri Lanka, completely damaged Dendrobium cultivation was found in an ornamental plant nursery at Gampaha district in 2017. It was noted that the symptoms were similar to that of blossom midge damage. Maggots were found to be feeding inside unopened flower buds, causing deformed, discolored buds and blossoms causing premature bud or blossom drop. Floral buds were often found to be rotted. Samples from immature bud stage to fully opened flowers were randomly collected from infected fields into polythene bags. To identify causative agent, floral buds with larval stages were kept in glass containers to allow them to complete their life cycle and thereby morphological characters were studied to confirm the pest species. In addition, yellow color grease sheets were kept inside the greenhouses to trap any adult stages of the pest species. Samples were collected and preserved using 70% ethanol for identification. Since all the damaged flower buds displayed symptoms of bacterial rots, to determine if any bacterial infection is also associated with the symptoms, bacterial isolation procedure was carried out. Different stages of floral samples were separately surface sterilized for two minutes using 70% ethanol and three serial washings with sterilized distilled water. Tissue macerate was prepared and kept for 3 hours before culturing on Nutrient Agar (NA) plates, Potato Dextrose Agar (PDA) and Luria-Bertani (LB) plates. Each sample had three replicates and ten samples were cultured. Growth from the tissues were observed and pure cultures were obtained. Relative length of the first and second flagellomeres, wing length and pattern, larval sternal spatula and its associated papillae and larval eighth abdominal segments were compared with identification keys which were used to identify the genera, Contarinia. The adult stages of trapped insects and adult stages of insect immerged from the larval stages were useful in confirming the species as C. maculipennis. Basic biochemical tests and Gram’s staining assisted in identifying the bacterium as belonging to the genera, Erwinia sp. and it appears that the bacterial infection occurs as a secondary infection after larval stages of C. maculipennis damage the floral tissues. This is the first record of C. maculipennis infecting orchid nurseries in Sri Lanka and if proper care is not taken it will invade other crop species as the pest has a broad host range. It is not clear whether the pest was a recent introduction through the imports of plant material or whether it is a result of host jump and therefore, it warrants further research.