Browsing by Author "Ranasinghe, H. A. K."

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Antibacterial efficacy and phytochemical properties of Citrus crenatifolia (Heen Narang) and Citrus reticulata (Jama Narang)
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Salwathura, I. A.; Deraniyagala, N. G. I. A.; Fernando, N. H.; Perera, M. D.; Perera, E. H. L.; Ranasinghe, H. A. K.
The Citrus family encompasses various plant species known for their antibacterial properties. Various parts of Citrus plants have been used in traditional Sri Lankan medicine for an extended period. Citrus reticulata was selected for its established antibacterial properties, providing a foundation to further investigate its potential alongside the less-researched Citrus crenatifolia. The present study was conducted to investigate the antibacterial properties of C. crenatifolia and C. reticulata found in Sri Lanka. Active phytochemicals were extracted from the peels, using the reflux method employed in an aqueous medium. The antibacterial properties of plant extracts were assessed using the well diffusion method against six common bacterial pathogens; Staphylococcus aureus (ATCC 23235), Pseudomonas aeruginosa (ATCC 15442), Proteus mirabilis (ATCC 35659), Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 25922) and Enterococcus faecalis (ATCC 29212). Double distilled water was used as the negative control, while commonly used antibiotics (ampicillin, penicillin, gentamicin, vancomycin, augmentin, and ciprofloxacin) served as the positive controls. A McFarland standard was employed for each test microbe. The diameter of inhibition zones was measured after incubation, and statistical analysis was performed using the SPSS software package. The independent sample t-test revealed that there was no significant difference between the mean values of the control and both peel sample extractions for all tested bacteria. (p = 0.115). There was no statistically significant difference in antibacterial activity among the six microbes according to the One-Way ANOVA test for C. reticulata (p = 0.271). But there was a statistically significant difference in the antibacterial activity of C. crenatifolia (One-Way ANOVA: p = 0.021). Tukey’s Post-Hoc analysis of C. crenatifolia extract showed a significant difference in the mean inhibition zone diameters between S. aureus and P. aeruginosa (p = 0.044) as well as S. aureus and K. pneumoniae (p = 0.049). The antibacterial activities of both extracts were supported by the presence of phytochemicals such as alkaloids, steroids, flavonoids, phenol, and tannins which showed combination effects. Anyhow, saponins were not detected in both extracts. The present findings highlight the potential of C. crenatifolia and C. reticulata extracts as valuable sources of antibacterial agents, warranting potential applications in the field of microbial control. Further investigation to elucidate its specific mechanisms of action is recommended.
Diversity of microbiota associated with dengue vector mosquito breeding habitats in Udapalatha MOH Division, Sri Lanka
(Kumari J. Y.; Amarasinghe L. D.; Ranasinghe H. A. K.; (2021), Diversity of microbiota associated with dengue vector mosquito breeding habitats in Udapalatha MOH Division, Sri Lanka, Proceedings of the International Conference on Applied and Pure Sciences (ICAPS 2021-Kelaniya) Volume 2, Faculty of Science, University of Kelaniya Sri Lanka. Page 3., 2022) Kumari, J. Y.; Amarasinghe, L. D.; Ranasinghe, H. A. K.
Microbiota associated with mosquito breeding habitats are among biotic factors and serve as parasites, pathogens, predators, competitors, non-competitors, and food items for developing larvae. Therefore, there are naturally occurring microbiota species that serve as potential controlling agents against mosquito larvae, causing lethal effects on them. This information can be used in developing novel mosquito vector control strategies. Besides, the degree of parasitic, pathogenic, or predatory effects may also vary with the geographical location, influencing environmental conditions, including abiotic factors and other biotic factors within the habitat. Therefore, the current study was carried out to determine the diversity of microbiota associated with different breeding habitats of dengue vector mosquitoes; Aedes aegypti and Ae. albopictus. Sampling was performed from a variety of breeding habitats at Udapalatha MOH division in Gampola urban area from November 2021 to January 2022. Microbiota in water samples were preserved using Rose Bengal solution and Lugol’s iodine and were identified under microscope, using standard identification keys. During the survey, twelve different breeding habitats, namely plastic containers, metal containers, concrete slabs, glassware, tires, leaf axils, tree holes, coconut shells, bamboo trees, ornamental ponds, discarded roof tiles and clay pots, were identified as key breeding sites of Aedes mosquitoes. From the collected samples 54.54% of habitats were positive for Ae. albopictus and 45.46% were positive for Ae. aegypti. Eleven microbiota species; Canthocamptus staphylinus, Canthocamptus sp., Parastenocaris sp., Lepadella ovalis, L. patella, Rotatoria rotatoria, Rotatoria sp., Asplanchna brightwelli, Trichocerca rattus, Euglena sp., and Flagilaria sp. were encountered from breeding habitats associated with Ae. aegypti. Meanwhile, Canthocamptus staphylinus, Parastenocaris sp., L. ovalis, L. patella, R. rotatoria, Rotatoria sp., Euglena sp. and Flagilaria sp. recorded from breeding habitats associated with Ae. albopictus. The highest Shannon Weiner diversity index and gamma diversity relating to the Ae. aegypti were recorded from leaf axils, while for Ae. albopictus, it was from metal containers. For Ae. aegypti, the highest heterogeneity in microbiota diversity was recorded from tire habitats and it was from plastic containers for Ae. albopictus. Out of the microbiota species recorded, no species was identified as parasitic or pathogenic to Aedes mosquito larvae. Updated information from the present investigation would facilitate implementing appropriate vector control interventions.
Diversity of midgut symbiotic bacteria in Aedes mosquito genera: A meta-analysis
(Faculty of Science, University of Kelaniya Sri Lanka, 2022) Kumari, J. Y.; Amarasinghe, L. D.; Udayanga, N. W. B. A. L.; Ranasinghe, H. A. K.
The gut bacterial composition of mosquitos has the capability of altering mosquito pathogen transmission through numerous mechanisms, which is useful in novel transmission control strategies. Therefore, understanding the diversity of gut bacteria in different mosquito genera is highly important. The present analysis was carried out to evaluate the variations in gut-bacteria in field-caught Aedes adult mosquitos from different geographical locations. A literature survey was conducted to identify quantitative data related to the midgut bacterial abundance in Aedes, using standard search terms and keywords. The collected data were arranged at the bacterial class level and were subjected to a square-root transformation. A resemblance matrix was constructed based on the Bray-Curtis resemblance. Cluster analysis followed by the Analysis of Similarities (ANOSIM) and Distance based Redundancy Analysis (dbRDA) were used for data analysis. Gut bacterial abundance from six different countries; Netherlands, USA, Sri Lanka, India, Brazil, and Panama, were used in the study. A total of 12 bacterial classes, were recorded and bacterial classes of Alphaproteobacteria and Gammaproteobacteria accounted for the highest abundance in Aedes mosquitoes from most of the countries. Interestingly, Aedes mosquitoes from Brazil, India and Sri Lanka, did not report the abundance of Alphaproteobacteria in their gut contents. Based on the Bray-Curtis similarity-based cluster analysis, four major clusters were identified at a similarity level of 48.9%, based on the overall abundance of bacterial classes. Aedes mosquitoes of Sri Lanka and India formed one cluster, sharing a similarity of 81.6%, while Brazil and Panama formed another cluster. Meanwhile, Aedes mosquitoes from Netherland and USA formed two separate clusters. The statistical significance of aforementioned clustering status was further verified by the Analysis of Similarities (ANOSIM), which reported a Global R value of 0.96. The clustering status suggested by the cluster analysis was further verified by the findings of the dbRDA. As indicated by the loadings of the dbRDA axes, the abundance of Gammaproteobacteria and Bacilli bacterial classes were found to be responsible for the similarity between gut bacterial diversity in Aedes mosquitoes from India and Sri Lanka. Further studies on the gut-microbial diversity of Aedes mosquitoes are recommended to support the implementation of novel vector control strategies in Sri Lanka.
Effect of aqueous extracts of Annona glabra plant and extracellular metabolites of two Trichoderma species on the survival of larval Culex quinquefasciatus and eggs of Aedes aegypti
(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Aberathna, A. A. A. U.; Ranasinghe, H. A. K.; Rajapaksha, R. D. T.; Amarasinghe, L. D.
Phytochemicals constitute a rich source of bioactive chemicals and some microbial products carry selective toxicity to many insect species, hence those are considered as alternatives to chemical insecticides. This study aimed to determine the potential of concentration gradient of Annona glabra aqueous leaf and fruit extracts for the mortality of third instar larvae of Culex quinquefasciatus; and to determine the ovicidal activity of extracellular metabolites of Trichoderma harzianum and Trichoderma viride at concentration gradient of 100, 200, 300, 400, 500 g/L on field collected eggs of Aedes aegypti with four replicates including controls for all bioassays. For the bioassays, aqueous crude extraction of plant materials was done by overnight shaking and extraction of extracellular metabolites of T . harzianum and T . viride was done using Richard ’ s broth . Study revealed that LC50 for A . glabra aqueous leaf extract for third instar larvae of Cu . quinquefasciatus was 16.26 g/L and 8.29 g/L in 24 hr and 48 hr exposure respectively (Minitab 14, Probit analysis). LC50 for A . glabra aqueous fruit extract, for third instar larvae of Cu . quinquefasciatus were higher than the LC50 for A . glabra leaf extract; 422.54 g/L and 114.01 g/L in 24 hr and 48 hr exposure respectively. 100% mortality was observed in mosquito larvae under 69.86 g/L of leaf and 1000g/L of fruit extract treatments after 24 hours of exposure. Mosquito larvae mortality compared to those in the control group reveals that the mortality of mosquito larvae may be due to the toxic compounds found in the plant. Although literature has shown that most Trichoderma strains produce volatiles and non-volatile toxic metabolites, significant ovicidal activity of extracellular metabolites of T . harzianum and T . viride on field collected eggs of Ae . aegypti was not observed (One-Way ANOVA; P>0.05) after 24 hr of exposure. The results of the study revealed that, both A . glabra fruit and leaf extracts are potential mosquito larvicides for Cu . quinquefasciatus. This may contribute to the formulation of an environmentally friendly measure to control Cu . quinquefasciatus vector mosquito species, which is a vital need to overcome the use of synthetic insecticides.
Mosquito larvicidal effect of Annona glabra fruit extract on dengue vector, Aedes aegypti
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Wijebandara, N. R. Y.; Ranasinghe, H. A. K.; Amarasinghe, L. D.; Wickramarachchi, S. R.
Annona glabra is a plant of Family Annonaceae and many plant species belong to this family have been intensively studied since they were discovered to contain compounds with important biological properties. These properties include cytotoxic, antitumor, antiparasitic, antifungal, antispasmodic, repellent, and insecticidal activities. Insecticidal effects of plant extracts vary depending on the plant parts (leaf, seed, fruit, bark) used for the extraction. Larvicidal effect of A. glabra aqueous extract from leaves and seeds have been evaluated against Aedes aegypti mosquitoes in Sri Lanka, and larvicidal effect of fruit is not determined yet. Therefore, the present study was conducted to evaluate the effect of A. glabra fruit extract for its larvicidal efficacy on third instar larvae of Ae. aegypti. Fresh ripen fruits of A. glabra plant were collected from Hunupitiya, (N 06º 58.904/, E 079° 54.281/) in Gampaha district, Sri Lanka. The aqueous crude extract was prepared from powdered A. glabra fruits. Seven concentrations were prepared between the range of 3 g/L – 300 g/L. Batches of 25 third instar larvae of Ae. aegypti were used for larval bioassays. Ae. aegypti mosquito larvae were evaluated in accordance with guidelines of World Health Organization. The experimental setup was repeated four times per each concentration. Probit analysis was used to evaluate the relationship of mortality with the concentration of aqueous crude extract. The A. glabra fruit extract showed a dose-dependent effect against Ae. aegypti larvae after the 48-hour exposure period. The percentage mortality rates have shown a significant variance among different concentrations (P = 0.000). The recorded LC50 and LC90 values for aqueous crude extract were 87.71 ± 5.41 gL-1 and 510.79 ± 12.56 gL-1 respectively, after 48 hours of exposure period. Larvicidal activity of A. glabra plant is supported by previous literature, where the presence of phytochemicals such as saponins, flavonoids, steroids and tannins have shown combined effects in terms of larvicidal action against mosquitoes. Hence, there is a potential of A. glabra aqueous fruit extract as a potential source for the development of an environment-friendly plant-based larvicide against Aedes aegypti.
Naturally Occurring Microbiota in Dengue Vector Mosquito Breeding Habitats and Their Use as Diet Organisms by Developing Larvae in the Kandy District, Sri Lanka
(BioMed Research International, 2020) Ranasinghe, H. A. K.; Amarasinghe, L. D.
Naturally occurring microbiota in mosquito larval habitats are among biotic factors which affect the population dynamics of developing larvae. Many microbiota species serve as food items for vector mosquito larvae, and food limitations within habitats adversely affect larval survival, developmental rate, adult fitness, and thereby vector competence. Therefore, identification of microbiota as associates with larvae reveals their relationship between each other as parasites, pathogens, epibionts, or diet organisms. Analysis of associated microbiota species in the dengue vector larval breeding habitats (n = 40) and the mosquito larval gut content were conducted in Kandy District in Sri Lanka. Study revealed that a total of 22 microbiota species belong to nine phyla (Amoebozoa, Bacillariophyta, Ciliophora, Chlorophyta, Sarcodina, Cyanobacteria/Cyanophyta, Euglenozoa, Ochrophyta/Heterokontophyta, and Rotifera) were encountered from different Ae. aegypti mosquito breeding habitats while 26 microbiota species that belonged to ten phyla were recorded from Ae. albopictus mosquito breeding habitats with one additional phylum Arthropoda. Considering Ae. aegypti breeding habitats, only Philodina citrina in low roof gutters existed as constant species. Considering Aedes albopictus breeding habitats, Volvox aureus in plastic containers, Lecane luna in coconut shells, Phacus pleuronectes in concrete slabs, and Pinnularia sp. in tree holes existed as constant species. The rest of the microbiota existed as common or accidental/rare species in a variety of habitat types. The Shannon-Weiner diversity (21.01 and 19.36) and gamma diversity (eight and eight) of the microbiota associated with Ae. aegypti and Ae. albopictus larvae, respectively, in ponds were found to be higher than other types of breeding habitats recorded during the study. Twelve microbiota species were recorded from larval gut analysis as food organisms of both species of mosquito larvae. However, the distribution of gut microbiota species differed between Ae. aegypti and Ae. albopictus (Chi − square = 21:294, P = 0:002). Identification of microbiota as food items of vector mosquito larvae led to a focus on larval food limitation by introducing food competitors, which could be a potential additional tool for integrated vector control approaches within the country.
Prevalence of micro-fauna associated in rice field habitats in Kurunegala district in Sri Lanka and potential mosquito larvicidal effect of Vorticella microstoma
(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Ranasinghe, H. A. K.; Amarasinghe, L. D.
Micro-fauna are associated with most of the mosquito larval breeding habitats in tropical climate. There may be parasitic, pathogenic or even predatory species among these biota to mosquito larvae and their potential use against mosquitoes is a new avenue of study in vector control. Rice fields are common breeding sites for certain vector mosquito species in Sri Lanka. The objective of the present study was to find the prevalence of micro-fauna in order to search for potential parasitic or pathogenic taxa/ species associated with mosquito larvae inhabiting rice field breeding habitats and to carry out bioassays for potential taxa. Sampling was done from paddy fields (n=12) in Kurunegala district using a metal larval scooper and the samples were preserved using Rose Bengal solution. Twelve micro-fauna species were recorded; the highest abundance was recorded from Vorticella microstoma (62.68%). Other micro-fauna species recorded were Zoothamnium sp., Diurella stylata, Keratella valga, Lecane luna, Monostyla bulla, Philodina citrina, Acanthocystis aculeata, Canthocamptus staphylinus, Daphnia longis, Notholca acuminata, Arcella arenaria . Also, 98 . 60 % of mosquito larvae which were collected from rice fields belonged to the genus Culex . Culex gelidus and Culex tritaeniorynchus represented the majority of samples (76.26%) and the highest abundance was recorded from Cu . gelidus . Study revealed that Vorticella microstoma trophont stage was lethal to Cu . tritaeniorynchus and Cu . quinquefasciatus larvae. Bioassay resulted that, 32.14% of Cu . quinquefasciatus died 05 days after infection with lab maintained V . microstoma cultures . Pupation was also delayed compared to the duration of normal life-cycle and only 10.71% of population emerged as adults. 100% mortality was recorded from Cu . quinquefasciatus in 2 days of infection with V . microstoma. The mean mortality percentage of two mosquito species studied were significantly different from each other (IBM SPSS Software, One-Way ANOVA: P<0.05). Trophonts of V . microstoma were infected to thorax, abdominal segments and anal papillae regions of larvae. The results of study revealed that Vorticella microstoma could be used as killing agent of mosquito larvae in future studies and would be a new avenue to work on environmentally agreeable manner in reducing the Culex spp . mosquito populations .