RESCON 2023

Permanent URI for this collectionhttps://ir.lib.pdn.ac.lk/handle/20.500.14444/5953

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Morphological changes in eggs and larvae of aedes aegypti adapting to salinity
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Kokila, S.; Surendran, S.N.; Karunaratne, S.H.P.P.
Aedes aegypti, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development in freshwater (FW) and has recently been observed to develop in coastal brackish water (BW) habitats, evolving various physiological mechanisms that enable these mosquitoes to survive and breed in BW habitats. The present study investigates the morphological changes of eggs and larvae of Ae. aegypti reared in FW and BW under laboratory conditions. Five different laboratory colonies were used for the subsequent experiments: two freshwater colonies named JFW (reared in 0 g/L tap water) and NFW (reared in tap water purified by reverse osmosis) and one brackishwater colony (reared in 10 g/L water) named JBW and two reversal colonies where FW larvae transferred and continued to reared in BW designated as JBWR colony, and BW larvae transferred and continued to reared in FW designated as JFWR colony. Cuticle thickness of the 6th to 8th abdominal segments of ten mid-L4 larvae from each experimental setup was measured. The results demonstrated that the JBW colony had significantly thicker (p < 0.0001) cuticle (JBW; [70th Generation; G70] = 1530 ± 111 nm) than the two freshwater colonies (JFW [G70] = 996 ± 132 and NFW [G20] = 945 ± 75 nm). Where salinity conditions had been reversed, the cuticle thickness differed significantly from the parent JBW and JFW colonies (JBWR [G3] = 1512 ± 132, [G11] = 1167 ± 159, JFWR [G3] = 1196 ± 92, [G11] =1402 ± 122 nm). The maximum length and width of four anal papillae of 40 L4 larvae from each of the five colonies were measured. The mean anal papilla length of JBW larvae [G69] was 0.62 ± 0.04 mm, and this was significantly greater (p < 0.05) than the mean anal papilla length of two FW colony larvae (JFW [G69] = 0.49 ± 0.06 mm, NFW [G22] = 0.58 ± 0.05 mm). JFWR colony ([G4] = 0.59 ± 0.05, [G11] = 0.6 ± 0.05 mm) tended to have longer lobes than FW larvae maintained in FW and vice versa in JBWR compared to the JBW colony (JBWR [G4] = 0.54 ± 0.03, [G11] = 0.55± 0.05 mm) and this difference was statistically significant (p < 0.05). A total of 100 eggs from ten iso females were analyzed, and the mean egg lengths of all five colonies significantly differed (p < 0.05) from each other. It is anticipated that changes to the cuticles of salinity-tolerant Ae. aegypti larvae may reduce the effectiveness of insecticides used to control arboviral infections. The morphological changes in anal papillae and eggs may alter ion, water, and gas transport mechanisms to allow Ae. aegypti to adapt to increased salinity in natural habitats. The results highlight the necessity for further research on the ultrastructure and physiological mechanism of the cuticle and anal papillae in relation to insecticide resistance and the genomic biology of salt tolerance in Ae. aegypti.
Morphometric analysis of genus piper (piperaceae), Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Gopallawa, B.; Yakandawala, D.M.D.; Wickramasuriya, A.; Samuel, R.
The genus Piper is represented in Sri Lanka by five wild species, three of which are endemic. Five Piper species have been introduced for various medicinal and economic purposes. According to the National Red List 2020, three of these species are threatened: P. hymenophyllum Miq., P. walkeri Miq., and P. trineuron Miq. However, due to recent taxonomic revisions, some species have been delimited to specific taxa, causing confusion. As a result, a field-based taxonomic study was initiated to determine the species limits of the genus Piper in Sri Lanka. The fieldwork was conducted throughout the country during 2022-2023, encompassing wildlife reserves, forest reserves, and cultivations. The collected specimens were tentatively identified using available published literature and herbarium specimens. The plant specimens were extensively examined, and morphological characters, both vegetative and reproductive, were coded into a data set. A population was coded with at least three mature individuals. Using PAST software (version 2.15), a multivariate analysis was performed on the data set. Hierarchical cluster analysis was performed on data collected from 58 populations using 60 qualitative and quantitative characteristics. The cluster solution was chosen from the most suitable algorithm, which employed Gower Distance to calculate similarity measures with the 'paired group' (UPGMA) option and the Single Linkage Algorithm based on the highest Cophenetic Correlation Value. The hierarchical cluster analysis resulted in 11 phenotypic groups, of which 10 corresponded to already-described species. The most contributing characters for the grouping, based on the SIMPER analysis, were leaf width, leaf length, and inflorescence length. The study suggests that the species limits of the known species remain stable while some possible new species resulting from intraspecific groups are identified, especially in P. betle, P. zeylanicum, and P. nigrum that need confirmation through molecular studies.
Evaluation of primers designed for nested PCR to detect p2 clade of leptospires causing leptospirosis in humans and animals
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Nadeeshani, W.M.M.; Gamage, C.D.; Rajapakse, R.G.S.C.
The pathogenic Leptospira (P) bacterium can transmit to humans through direct or indirect pathways and cause leptospirosis. This zoonosis is considered endemic in developing countries like Sri Lanka. Each year, more than one million leptospirosis cases, including 60,000 deaths, are recorded globally. Leptospirosis diagnosis is predominantly based on pathogen detection in clinical samples by flaB nested PCR. Intermediate Leptospira species (P2) can also cause leptospirosis but are undetectable through current molecular tests. Therefore, this study aimed to evaluate the primers designed for nested PCR to detect the P2 clade using extracted DNA samples collected from the Department of Microbiology, Faculty of Medicine, University of Peradeniya, Sri Lanka. The primers used for leptospirosis-related research in the past 10 years were analyzed in the literature survey. Intermediate primers (I-flaB) were initially evaluated using the in silico-based method through NCBI (National Center for Biotechnology Information) Primer-BLAST. Both known and unknown samples were used for the wet lab primer evaluation. DNA from 12 Leptospira species used in the Microscopic Agglutination Test (MAT), P2 human and animal clinical samples, and intermediate pathogen (P011) were used as the known samples. Seventy-two stored clinical blood samples that were flaB-PCR negative were used as the unknown samples. 16s rRNA-PCR was conducted for the unknown samples to detect the existence of bacterial DNA. The five P2 species, L. inadai, L. broomii, L. wolffii, L. licerasiae, and L. fainei were explicitly detected by I-flaB primers in the ‘in silico’ evaluation, whereas the detection was non-specific in the wet lab evaluation using known samples where two P2 samples gave negative results. Of the unknown samples, six samples were positive for 16s rRNA-PCR, implying the presence of bacterial DNA. However, I-flaB nested PCR was negative for all these six samples. Therefore, the detection of available I-flaB primers is non-specific. The ‘in silico’ method should be used cautiously. Primer evaluation before the application of newly designed primers is vital.
Isolation and identification of an efficient cellulose-degrading fungal species, aspergillus nomius from soil
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Herath, H.M.A.S.; Samaraweera, P.
Cellulase is a group of enzymes consisting of exoglucanase, endoglucanase, and β-glucosidase that act on cellulose sequentially to degrade to glucose monomers. Due to the high activity, availability, and stability of fungal cellulases, they have many potential applications in industries such as bioethanol, textiles, paper, food and beverage. Since there is limited work on glucose production from leaf litter and plant biomass using fungal cellulases, this study aimed at the molecular-based identification of efficient cellulolytic fungal species. Different fungal species were isolated from soil samples collected from four different locations in Sri Lanka: 1) the “MiniSinharaja” premises, 2) Botany Department in University of Peradeniya, 3) a compost soil sample and 4) a leaf litter contaminated soil sample from Nikaweratiya and screened for their cellulolytic activity using Gram’s iodine and Congo red tests and identified up to the genus level by observing the colony morphology and microscopic features of fungal cultures. The filter paper assay quantitatively determined the most efficient cellulose-degrading fungal species. During the molecular identification, fungal genomic DNA was extracted, and Internal Transcribed Spacer (ITS) region-based PCR amplification was carried out using ITS1 and ITS2 primer pair followed by sequence analysis. Eleven different fungal species were isolated, and ten species of these gave positive results upon the formation of a yellow colour halo region around the colonies in both Congo red and Gram’s iodine tests, thus confirming their cellulolytic activity. The fungal isolate with the highest cellulolytic activity was identified as Aspergillus nomius by a phylogenetic analysis. Since fungi have gained attention as prominent cellulase producers, this study contributed to the molecular identification of an efficient cellulose-degrading fungal species in plant biomass-contaminated soil samples. Further research can characterize industrially valuable properties of cellulases and media optimization for Aspergillus nomius.
Calmodulin-binding transcription activator (camta) genes in soybean and their expression analysis
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Jayarathne, D.P.; Ratnayake, S.; Dhanapala, M.P.C.S.; Wijeratne, A.J.
Calmodulin Binding Transcription Activator (CAMTA) genes encode a special class of transcription factors (TFs) in eukaryotes, mainly in plants and animals. CAMTA TFs have been reported to mediate transcriptional regulation of plant stress response genes. However, the functions of CAMTA genes are yet to be well characterized in crop plants such as soybeans. Soybean is the world's fourth-largest commercial crop, which provides primary protein and oil sources. However, yield loss due to various stresses is a rising burden to farmers. It is crucial to employ various strategies to address this problem, including incorporating CAMTA genes into breeding programs. Therefore, it is necessary to characterize these genes in soybeans. However, gene redundancies in soybeans due to recent genome duplications pose a challenge in identifying gene functions. Therefore, finding the recently duplicated CAMTA is imperative to expedite the functional characterization. This study employed phylogenetic and expression analysis to investigate the evolutionary relationship and function of identified soybean CAMTA genes. The evolution of these genes compared to CAMTA genes of closely related species were analyzed. Furthermore, the study explored the expression patterns of these genes in different plant parts. The soybean CAMTA TFs were grouped into six major groups (with bootstrap support 100%), each containing two TFs. Genes in these clusters share a common evolutionary ancestor with Glycine soja but form a separate clade with other legumes (with bootstrap values above 60%), indicating they originated from a gene duplication event. Furthermore, two members of the four duplicated gene groups showed no statistically significant difference in expression levels in different parts. This suggests that the duplicated CAMTA gene retained the same expression patterns thus likely retained the same function. The expression data can be used in future studies to identify candidate genes to enhance soybean resistance to different diseases.
Google street view reveals new insight into the distribution of panicum maximum in Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Dhanasekara, K.E.H.A.; Athukorala, A.D.S.N.P.; Gama-Arachchige, N.S.
The alien invasive species (AIS) Panicum maximum causes negative impacts on the ecosystem by out-competing native vegetation, creating fire hazards during dry periods, and blocking access to animals. Panicum maximum has a wide distribution in Sri Lanka, while an accurate estimation of its distribution has not yet been determined. Estimating the current distribution of P. maximum in the island is vital to develop management strategies to control its spread. Thus, the objectives of the current study were to map and model the distribution of P. maximum in Sri Lanka. Google Street View (GSV) images have been used previously as a low-cost method to map the distribution of several animal and plant species. Thus, the mapping of P. maximum was conducted using GSV data, Google Earth Pro and QGIS software. The presence and absence data of P. maximum covering the entire island were recorded from 50,231 and 30,950 data points from GSV images taken during 2015-2016 and 2020-2022, respectively. The distribution maps were generated separately for each district, depicting the presence and absence of P. maximum. The density distribution maps of P. maximum were prepared for each district and the entire country using the same data sets. Heat maps can be used to predict the density of the species distribution. P. maximum is mainly distributed in the wet and intermediate zones of the country, with a limited distribution in the dry zone. Compared to 2015-2016, a higher distribution of P. maximum was observed in 2020-2022. During 2015-2016, the lowest distribution (<10%) was reported in the Northern Province, particularly Trincomalee and Batticaloa districts. The highest distribution of P. maximum (>80% of the total area) was reported from Polonnaruwa, Kurunegala. Kandy and Galle districts. A comparison of the distribution of P. maximum during 2015-2016 and 2020- 2022 revealed a 2-11% increase in the spread of P. maximum in 11 districts thus a further spreading of the species can be expected, especially in wet and intermediate zones where its distribution is high.
Microplastics contamination in Indian backwater oysters(Magallana bilineata) in the Panadura Estuary, Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Nawarathna, M.K.R.A.W.S.N.; Rathnasuriya, M.I.G.; Kapukotuwa, G. K.; Rajakaruna, R.S.
Microplastics (MPs) are fragments of plastic less than 5 mm in length, and MP pollution in marine environments is widespread. As oysters are sessile filter feeders, they could serve as an indicator species of MP pollution and a proxy for marine environment contamination. The objectives of this study were to determine the effect of different depth strata and attached substrates on the level of MP contamination of the Indian backwater oyster Magallana bilineata in the Panadura estuary, Sri Lanka. Oysters were sampled from three depth strata (Surface to 4 m, 4–8 m, 8–12 m depth), according to their soft tissue wet weight class (0 to 4 g, 4 –8 g, 8 –12 g) and substrate type (natural vs plastic) in the estuary. The soft tissues of oysters were digested individually in 10% KOH, and MPs were separated using the density separation method (NaCl solution), extracted onto a filter paper (11 µm), and enumerated under the dissecting microscope. The MPs were classified according to colour and shape, and the polymer types were identified using FTIR spectroscopy. The average pieces of MPs were 5.133±4.37 items/individual, and the mean concentration was 1.212±1.188 items/g. Fibres (95%) were the dominant MPs category, and black (52%) was the dominant colour. Polypropylene, Polyethylene terephthalate, and polyvinylchloride were the predominant polymers observed in the oyster tissues. Substrate type did not affect the abundance of MPs (Two sample t-test; t=-0.33 p=0.743); however, the depth and size of the oysters had a significant effect on the content of MPs (One-way ANOVA, p<0.05). According to the post hoc test, the 8 – 12 m depth range had a higher mean content of MPs (9.78±7.26), and the weight class 8 – 12 g had a higher mean content of MPs (10.00 ±7.11). There was an interaction between two factors: depth range and weight class of oysters. The results showed that the larger the oyster and the deeper it lives, the higher the content of MPs. The results suggest that the wild-caught oysters in the Panadura estuary contained a higher number of MPs than those reported globally, with an average of 1.4 pieces per individual.
Genotyping and phenotyping of selected rice germplasm for blast disease resistance under local conditions
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Ruwanthika, A.M.C.; Jayawardana, N.U.; Bulathsinhalage, V.N.D.; Dissanayaka, D.M.H.R.; Ariyarathne, H.A.C.K.
Rice blast caused by fungal pathogen Magnaporthe oryzae is the most destructive disease in rice cultivation worldwide. Even though the pathogen affects the entire plant, neck and panicle blasts are the most severe forms, causing 100% yield losses in server infections. Zenith and Usen are blast-resistant varieties that are widely used in blast-resistant international rice breeding programs worldwide. This study screened panicle blast resistance genes Pikh and Pita/Pita-2 in local rice varieties Bg352, At362, and Usen and Zenith. Pachchaperumal was used as the susceptible control. Dominant marker YL155/YL87 and co-dominant marker Pikh were used to amplify the Pita/Pita-2, and Pikh genes, respectively, from the selected rice germplasm. The same varieties were phenotyped for blast resistance in local environmental conditions. Three replicates of each variety were inoculated at two weeks after the heading stages. The plants were inoculated by spraying the mycelium suspension of two isolates of M. oryzae that were previously Koch’s postulated. After inoculation, plants were incubated for 72 hours in humid chambers to facilitate disease development. A neck blast was observed on Bg352 and At362; however, no symptoms were observed on Usen, Zenith, and Pachchaperumal. Panicle blast symptom development was inconsistent in the replicates in any of the varieties. Hence, these observations need to be verified. The resistant Pita/pita-2 gene was present in Bg352 and At362. However, Pikh resistant allele was not amplified from Bg352 and At362. Verification of resistance phenotypes of Usen and Zenith and identification of allelic variations will assist in identifying potential donor parents for blast resistance in rice crop improvement.
Interrogation of saltol loci from FL 478 to elite variety bg 352: early screening data
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Dissanayaka, D.M.H.R.; Dasanayaka, B.I.; Abeysiriwardena, D.S. De Z.; Ariyarathna, H.A.C.K.
Soil salinization significantly affects local rice production, posing a dual threat to the country's economy and food security due to the high sensitivity of rice to salinity. The cultivation of saltsensitive rice varieties intensifies the above situation. Thus, genetic improvement of elite rice varieties for salinity tolerance is significant. The SalTol Quantitative Trait Loci (QTL) is collocated with the OsHKT1;5, a gene encoding a high-affinity Potassium transporter that is associated with salinity tolerance at the seedling stage. The objective of the present study was to introgress SalTol QTL from rice variety FL 478 into popular high-yielding rice variety Bg352. A backcrossing program was initiated using FL 478 and Bg 352, respectively, as the donor and the recurrent parent. The plants were manually crossed, and the F1 generation was advanced to BC1F1. Leaf samples were collected from the individual seedlings of the BC1F1 population, DNA was extracted, and Polymerase Chain Reaction was performed using RM 493 (SalTol linked SSR marker). The success rate of the manual crossing program was 90.5%. BC1F1 population was similar to the recurrent parent in plant height, architecture, and days to flowering, with a high uniformity within the population. From 38 BC1F1 plants studied, nine were heterozygous for SalTol. The nine plants where SalTol was successfully introgressed were advanced to BC2F1. The use of QTL linked RM 493 marker was effective, improved breeding efficiency and accuracy, and accelerated the breeding process. Further, backcrossing and markerassisted selection are needed for the recipient genome recovery
Pest and disease management strategies of vegetable growers in the Central Province of Sri Lanka: a survey
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Dahanayake, U.D.; Daundasekera, W.A.M.; Damunupola, J.W.
Sri Lanka faces significant obstacles from pests and pathogens in vegetable cultivation, which leads to substantial crop losses. Synthetic agrochemicals used to manage pest and disease outbreaks have many adverse effects on human health and the environment. The objective of the study was to assess the pre- and postharvest pest and disease management strategies used for vegetables through a questionnaire survey. Twenty growers per district in the Central province (Kandy, Matale and Nuwara-Eliya districts) were selected and their responses were recorded. Almost all farmers practice different kinds of land preparation techniques to prepare their lands before vegetable cultivation. Of the 60 farmers surveyed, about 53% responded that different land preparation methods impact pests and disease control. About 98% use synthetic pesticides in disease management. Although all farmers know the health risks associated with synthetic pesticides, about 10% of them still do not take proper precautions when handling them. Even though majority of those surveyed depend on synthetic pesticides for pest and disease management, most of them had a fair knowledge of how to handle them. Farmers used fewer non-chemical pest and disease control methods due to limited knowledge and confidence in these techniques. Government regulations banning agrochemicals have forced farmers towards more organic cultivation, while most of them face problems in crop production and pest and disease management. Integrated pest management (IPM), which has been introduced to minimize pesticide usage, is common among farmers. According to the responses of eight exporters, commodities from IPM-certified farmers are always selected for vegetable exports, as they use a minimal concentrations of synthetic pesticides.
Characteristic wing vein patterns of common mosquito genera in Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Bandaranayake, M.B.K.K.; Dissanayake, H.M.J.N.; Amarasekara, H.M.S.S.; De Silva, W.A.P.P.; Chathuranga, W.G.D.
Mosquitoes play a significant role in transmitting many devastating diseases to humans and wildlife. Proper identification is crucial in understanding mosquito epidemiology and disease transmission. Morphological taxonomic keys which are commonly employed in identification, may not be accurate in differentiating sibling species. The study investigated unique wing vein lengths and fork angles to develop a mosquito identification key. Larvae were collected from breeding sites and reared under laboratory conditions. Adult mosquitoes were collected using animal-baited traps and human landing catches. Right and left wings of 40 individual mosquitoes (females: 65%; males: 35%) representing seven common genera [Culex (n = 18), Aedes (n = 12), Armigeres (n = 12), Orthopodomyia (n = 12), Toxorhynchites (n = 4), Coquilletidia (n = 12), and Anopheles (n = 10)] were used for this study. Slide- mounted wings were photographed, and 21 wing vein length measurements and the folk angle were taken in pixels using a microscopic camera (CuA, MCu, MCu cross vein, M3+4, M1+2, M2, M1, R2+3 - M1+2 Cross vein half 1 & 2, R4+5, R2+3, R3, R2, 1A-CuA, CuA- M3+4, M3+4-M2, M2-M1, M1-R4+5, R4+5-R3, R3-R2, R2-R1, R2+3-M1+2). Results of the Multivariate Analysis of Variance (MANOVA) showed a significant difference in the length of selected wing veins between genera (P < 0.001). The Canonical Variate Analysis (CVA) suggested that the lengths of the Mcu cross vein, M1+2 vein, CuA vein, McU vein, and R2+3 are specific to each genus. The folk angle of R2+3 – M1+2 was excluded from MANOVA and CVA due to unit discrepancies. Taxonomic keys were generated based on the non-overlapping minimum and maximum values of the length of wing veins. Toxorhynchites were characterized by Mcu Cross Vein (range: 0.04 – 0.11 mm), while Orthopodomyia was distinguished with the M 1+2 vein (range: 0.86 – 0.95 mm). The identity of Aedes was confirmed using the CuA vein (range: 0.39 - 0.56 mm). Armigeres was characterized with the McU vein (range: 0.48 – 0.59 mm), while Coquillettidia was distinguished with the R 2+3 vein, which ranged from 0.51 – 0.67 mm. The folk angle of R 2+3 – M 1+2 can be used to separate Culex (118.74° – 144.63°) from Anopheles species (172.52° – 178.72°). These findings provide valuable and cost-effective insights into the taxonomy of common mosquito genera, while further studies are warrented to determine the applicability in identifying mosquito species/sibling-level.
Diversity of ichthyo fauna at the upper northern basin of Thondamanaru lagoon, Jaffna, Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Aniththa, S.; Piratheepa, S.; Gobika, S.; Subanky, S.; Niranjan, K.
An assessment of fish diversity is essential to maintain ecological stability and the lagoon fisheries. This study examined the fish fauna at the upper northern basin of Thondamanaru Lagoon. Samples were collected fortnightly from November 2021 to October 2022 from three selected sampling sites (L1- beyond the barrage, in front of the fieldwork centre, L2- near the barrage and in front of Selva Sannathikovil and L3-near the sand bar) at the upper northern basin of Thondamanaru lagoon, with the help of fishermen. Collected fishes were identified to the species level at the laboratory by using their morphological characters. This survey showed the occurrence of 45 species belonging to 27 families and eight orders. Perciformes is numerically the largest with 32 species, followed by Clupeiformes with five species, Beloniforms and Cichiliformes with two species each. Beryciformes, Pleuronectiformes, Gonorynchiformes and Siluriformes were represented by single species only. Sardinella albella (14%) was the most abundant fish species in L3. Oreochromis mossabicus (29%) and Gerres abbreviates (55%) were abundant in L1 and L2, respectively. The highest Shannon Diversity index (H’) was in L3 (H’= 5.9257), whereas the minimum was found at L2 (H’= 1.0207). At the same time, the highest Simpson’s index was in L1 (D = 0.1966), while the minimum was at L3 (D = 0.0561). The highest abundance of ichthyo fauna was recorded in L3, followed by L1, and L2. This study revealed the impact of barrage construction across the lagoon on the diversity, distribution and abundance of ichthyo fauna, supporting the conservation and management of fishery resources in this lagoon.
Bioactivity of solvent extracts of piper nigrum seeds
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Gamaarachchi, S.; Samarakoon, K.; Adikaram, N.K.B.; Marikkar, N.; Jayasinghe, L.
The spices’ history is the history of humankind itself. Spices have been valued for their flavouring and medicinal properties for thousands of years. Among the many spices available, Piper nigrum, commonly known as Black Pepper, is used as a food ingredient mainly due to its specific pungent taste of Piperine, which is the main compound of Black Pepper. This study was carried out to determine the bioactivities of different extracts of seeds of P. nigrum. Black Pepper seeds were purchased from the local market, air dried and ground to obtain a homogenous powder using a home-use grinder. The powdered sample was sequentially extracted into Dichloromethane (CH₂Cl₂ ) and Methanol (MeOH) by sonicating for 30 minutes. This procedure was repeated twice, and the filtrate was combined and evaporated to dryness using a rotary evaporator. Subsequently, the crude extracts were subjected to DPPH radical scavenging activity, Brine Shrimp lethality bioassay using Artemia salina, phytotoxicity against germination of lettuce seeds (Lactuca sativa), and enzyme inhibitory assays against α-amylase and lipase enzymes for 1000 mg L⁻¹ to 31.25 mg L⁻¹ concentrations for each extract. The results showed that the CH₂Cl₂ extract exhibited the highest DPPH radical scavenging activity with an IC₅₀ value of 178.33 mg L1 followed by the MeOH extract with IC₅₀ 221.25 mg L⁻¹. The CH₂Cl₂ extract exhibited IC₅₀ values of root and shoot inhibition in phytotoxicity assay with 1313.7 mg L⁻¹and 796.51 mg L⁻¹, respectively. The MeOH extract exhibited IC₅₀ values for root and shoot inhibition at 914.79 mg L⁻¹ and 1524.26 mg L⁻¹, respectively. Both extracts demonstrated 100% cytotoxicity up to 31.25 mg L⁻¹. Therefore, an assay was conducted for lower concentrations up to 3.125 mg L-1. CH₂Cl₂ extract showed 100% inhibition at 3.125 mg L⁻¹, while MeOH extract showed cytotoxicity with IC₅₀=133.01 mg L⁻¹. None of the extracts showed enzyme inhibitory activity against α -amylase and lipase enzymes. This study suggests the potential bioactivity of P. nigrum seeds for medicinal and agricultural purposes.
Antioxidant activity of endophytic fungi isolated from leaves of citrus aurantiifolia
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Samarakoon, K.; Atapattu, N.; Adikaram, N.K.B.; Yakandawala, D.; Jayasinghe, L.
Endophytic fungi are sources of natural products which can be utilized in medicine, agriculture and industry. The main objective of this study was to isolate endophytic fungi from the leaves of Citrus aurantiifolia and determine their bioactivities and molecular identification. Fresh and healthy leaves of C. aurantiifolia were collected from the Central Province of Sri Lanka. Segments (5 mm × 5 mm) of leaves were tripled sterilized and placed on the Potato Dextrose Agar and incubated in the dark for seven days at room temperature (27 °C) until the appearance of fungal mycelium. Three endophytic fungi emerged and were labelled as Fungus 10, Fungus V’ and Fungus B, and sub-cultured to obtain pure cultures. Pure fungi were cultured on a large scale in Potato Dextrose Broth and kept for 21 days in shakers at room temperature. Subsequently, the medium was filtered, and the broth was extracted using EtOAc (1:1). Mycelium was crushed and extracted with EtOAc and MeOH sequentially. EtOAc extracts of the broth and mycelium and MeOH extract of the mycelium were subjected to antioxidant activity assay using the DPPH radical scavenging method. EtOAc extracts of broth and mycelium of Fungus 10 gave IC₅₀ of 52.1± 23.18 mg L⁻¹ and 52.61 ± 18.73 mg L⁻¹, respectively. EtOAc extracts of both broth and mycelium of Fungus V’ were combined based on similar TLC patterns and IC₅₀value of 23.8 ± 8.98 mg L⁻¹ was obtained. MeOH extract of the mycelium of Fungus V’ gave IC₅₀of 104.54 ± 63.26 mg L⁻¹. EtOAc extracts of broth and mycelium of the Fungus B gave 27.11 ± 0.44 mg L⁻¹ and 112.4 ± 14.65 mg L⁻¹ IC₅₀ values respectively. Fungus V’ and Fungus B were identified as Biscogniauxia capnodes and Curvularia dactyloctenicola by amplifying ITS regions of the rDNA gene, suggesting the potential use of these endophytes as sources of antioxidants.
α- Amylase, antioxidant, cytotoxicity, lipase, and phytotoxicity studies of alpinia calcarata and gloriosa superba leaves extracts
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Ekanayaka, E.M.T.A.; Siriwardhane, U.; Adikaram, N.K.B.; Marikkar, J.M.N.; Jayasinghe, L.
Many natural sources, such as plants, fungi, bacteria, and algae, have been proven to possess pharmaceutical values with fewer adverse effects. Both medicinal and non-medicinal plants have been proven to contain pharmaceutical properties with low toxicity and high efficiency. This study was conducted to determine the bioactivities of the leaves of Alpinia calcarata (Zingiberaceae) and Gloriosa superba (Colchicaceae) (GS). The plants were collected from home gardens of Kandy district, central province, Sri Lanka. Plant samples were washed, air-dried, and ground into a fine powder. Extracts were obtained using dichloromethane (CH₂Cl₂) and methanol (MeOH) by sonication. α- amylase inhibitory activity, cytotoxicity against brine shrimp, and 2-2–Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging antioxidant activity, lipase inhibitory activity, phytotoxicity against germination of lettuce seeds were assessed for dilution series of each crude extract ranging from 1000 mg L ⁻¹ to 31.25 mg L⁻¹. CH₂Cl₂ extract of G. superba (GSC) showed considerable lipase inhibition (IC₅₀= 782± 6.31 mg L ⁻¹). MeOH extract of A. calcarata (ACM) showed the highest antioxidant activity (IC₅₀= 0.14 ± 0.24 mgL ⁻¹). CH₂Cl₂ extract of A. calcarata (ACC), MeOH extract of G. superba (GSM), and GSC also showed strong antioxidant activities (IC₅₀= 158.73 ± 12.41 mg l⁻¹, 25.03 ± 2.68 mg L ⁻¹, 192.24 mg L⁻¹ respectively). GSC and GSM both showed the highest lethality against brine shrimp (IC₅₀= 41.45 mg L ⁻¹ and 122 mg L⁻¹ respectively). ACC and ACM also showed a moderate lethality against brine shrimp (IC₅₀= 300.54 mg L⁻¹and 428.95 mg L⁻¹, respectively). GSC extract showed the highest phytotoxicity ability (Root – IC₅₀= 203.84 mg L⁻¹, Shoot- IC₅₀ =97.58 mg L⁻¹) and GSM extract also showed a considerable phytotoxicity ability (Root– IC₅₀=338.12 mg L⁻¹, Shoot-IC₅₀= 217.93 mg L⁻¹). The results suggest that leaves of A. calcarata and G. superba can be used as promising sources to isolate therapeutic compounds.
In silico drug discovery using the knime analytics platform to identify phytochemicals with VEGFR-2 inhibitory properties
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Perera, M.R.V.; Kumari, L.R.LS.; Wijesinghe, W.R.P.
Conventional drug discovery is a challenging process. Computer-aided drug design (CADD), especially Quantitative Structure-Activity Relationship (QSAR), offers an efficient alternative by correlating chemical structure to biological activity using statistical and computational models. Lately, QSAR has been integrated with KNIME, making CADD straightforward. The Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) binds to the Vascular Endothelial Growth Factor (VEGF) and promotes angiogenesis, which is crucial for tumour progression. Thus, VEGFR-2 inhibitors suppress angiogenesis, interrupting cancer progression. This study incorporated a modified-TeachOpenCADD workflow of KNIME to identify phytochemicals with VEGFR-2 inhibitory properties. The data of 343 phytochemicals, 12 VEGFR-2 inhibitors, and 10 non-cancer drugs were acquired from the ChEMBL database, and MACCS fingerprints were generated using the RDKit toolkit. The compounds were classified based on activity using Random Forest (RF), Artificial Neural Network (ANN) and Support Vector Machine (SVM), each with 10-fold cross-validations. The predicted phytochemicals’ drug likeliness was assessed using Lipinski’s rule of five. The ANN, RF and SVM performed with accuracies of 96.36%, 96.64% and 97.20%, respectively, which aligns with existing CADD studies. The root mean square error (RMSE) of RF, ANN and SVM were, respectively, 0.183, 0.191 and 0.167. The multilayer feedforward ANN with one hidden layer (each with 10 neurons) identified three active phytochemicals. With the split criterion as information gain ratio, RF only returned known VEGFR-2 inhibitors. Similarly, SVM with Radial Basis Function (RBF) in the kernel only predicted VEGFR-2 inhibitors. This is because ANN, compared to RF and SVM, excels in decision-making based on training data. All three predicted phytochemicals comply with Lipinski’s rule and could be potential VEGFR-2 inhibitors. Notably, two predicted phytochemicals are found in Butea monosperma and Saraca indica which are native to Sri Lanka. However, subsequent in vitro and in vivo studies are required before these can be established as anticancer agents.
Efficacy of the insect growth regulator novaluron in the control of dengue vector mosquitoes
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Herath, J.M.M.K.; De Silva, W.A.P.P.; Weeraratne, T.C.; Karunaratne, S.H.P.P.
Sri Lanka is experiencing an alarming rate of dengue incidence with several outbreaks. Vector resistance to synthetic neuro-inhibitory insecticides has become a major problem in vector control programmes. Novaluron is an Insect Growth Regulator (IGR) which inhibits chitin synthesis in immature stages of insects. The present study aimed to evaluate the efficacy of novaluron and determine the dosages to be used in dengue vector control programs. Batches of 25 laboratory-reared third instar larvae of Ae. aegypti and Ae. albopictus were exposed to formulated emulsifiable novaluron (Rimon ® EC10) (0.00001 ppm - 4ppm) for two fixed exposure periods of 7 days and 14 days to determine LC50 and LC99 values. Percentage inhibition of adult emergence values (IE₅₀ and IE₉₉) were determined by exposing a 14-day period to 0.0005ppb-0.00005ppb concentrations. Mortality data and emergence inhibition data were subjected to probit-regression analysis using SPSS. Water storage buckets (10 L) and plastic barrels (200 L) with 25 third instar larvae per 1L were treated with IE₉₉, 2x IE₉₉ and 10x IE99 under semi-field conditions to assess the residual effect of novaluron on larval emergence. For both larval species, LC₅₀ values were 0.044 – 0.049, and LC99 values were 0.144 - 0.151ppm for the 7-day exposure. For 14-day exposure, LC₅₀ values were 0.002 to 0.005 ppm, and LC₉₉ values were 0.006-0.01 ppm. For both species, IE₉₉ was 0.001 ppb. Under semi-field conditions, the discriminating dosage, 0.002 ppb (2 x IE₉₉) gave 100% emergence inhibition for up to 7 days, >50% inhibition for up to 86 days. After 118 days, it gave 97% adult emergence, declining the IE to 3%. Currently, novaluron Rimon ® EC10 recommended dosage is 200 ppm in Sri Lanka. The results highlight the need for authorities to reduce recommended dosages of novaluron Rimon ® EC10 in minimizing environmental pollution and to preserve non-target insect larvae.
Ligand-based virtual screening of potential drug combinations against aminoglycoside resistance in bacteria
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Rasangan, J.P.P.S.; Wijesinghe,W.R.P.
Antibiotic resistance in bacteria is an international crisis estimated to have 10 million annual deaths by 2050. Aminoglycosides (AGs) are becoming inefficient with the widespread of multidrug-resistant bacteria. The resistance mainly occurs due to the enzyme modification or inactivation of AGs, increased expression of efflux pumps that remove AG from the cell, reduced drug permeability or alteration in the drug target site. Many studies have determined that one of the fastest routes to evade AG resistance is restoring the effectiveness of existing AGs by combining them with some chemical compounds. The main purpose of this study was to predict the bioactivity of phytochemicals and a few other drugs against AG resistance using Machine Learning (ML) methods and predict their synergy with existing AGs (Amikacin, Tobramycin), using an efficient and less expensive pipeline. Open-source nodes in KNIME software were used to create a pipeline including Random Forest, Support Vector Machine and Artificial Neural Network ML models and RDKit tools. The ChEMBL site was used to retrieve structural data of 500 phytochemicals, 18 AGs, and 18 Non-antibiotic drugs. The drug likeliness of potential drug candidates was determined using Lipinski’s rule of five by the CDK toolkit. The synergies were predicted using the bioactivity data obtained from the PubChem site. Eleven compounds resulted as potential drug candidates by the pipeline: Allicin, Aspartic acid, Fosbretabulin, Myoinositol, Lysine, Alpha-pinene, Acetoside, Linoleic acid, Histidine, Choline and Polymyxin B. Out of them only Fosbretabulin and Lysine respectively having 0.033 and 0.36 individual bioactivities showed a higher synergistic effect only when combined with Tobramycin. The synergy scores are 0.24 and 0.78, respectively. Fosbretabulin is a cancer drug, and not known for having antimicrobial properties. This pipeline can screen many molecules to predict potential drug combinations against AG resistance before laboratory experiments, reducing cost, time and resources
A computational approach to identify beta-lactam-like compounds from phytochemicals
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Gunathilaka, K.A.M.A.G.D.M.; Abeysundara, S.P.; Wijesinghe, W.R.P.
Antimicrobial resistance (AMR) is one of this century's most severe global health problems. It has been estimated that AMR is directly responsible for 700,000 deaths annually, which is predicted to be 10 million by 2050. Present antibiotics are failing due to various reasons while phytochemicals are gaining attention due to their favorable attributes as antimicrobials. Many studies have shown the importance of applying Computational Biology to Microbiology. In this context, the KNIME (Konstanz Information Miner) analytics platform was used for our analysis. The primary goals of this research were to develop a KNIME workflow using a Machine Learning approach to discover potential compounds with betalactam properties from a set of phytochemicals and to use a 96-well plate assay to screen such candidate compounds biochemically. Compound data were acquired for beta-lactams, non-antibiotic drugs and phytochemicals from ChEMBL. Cells of molecule string column were converted into SMILES (Simplified Molecular Input Line Entry System). RDKit molecule column was generated from SMILES. Fingerprints were generated, and data was prepared for Machine Learning. Machine Learning algorithms were used to train the filtered ChEMBL dataset to discriminate between active and inactive compounds. After comparing the structural similarities, a set of phytochemicals (decanoic acid, alpha-pinene) was identified as hits for beta-lactams with more than 85% overall accuracy. Decanoic acid was screened biochemically. A slight growth inhibition was observed with increasing decanoic acid concentration from 100-700 µgmL-1. The developed KNIME workflow can identify possible beta-lactam-like compounds from a set of phytochemicals. Ninety-six well plate assay is a significant method to screen a large number of hits simultaneously. In conclusion, the intended KNIME workflow has been successfully developed and seven phytochemicals as hits for beta-lactams were found. As future directions other candidate compounds will be screened and further analyzed.
Identification of potential HER2 inhibitors from phytochemicals using computer-aided drug design
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2023-11-03) Kumari, L.R.L.S.; Amarasinghe, A.A.H.E.; Wijesinghe, W.R.P.
Computer-aided drug Design (CADD) combined with Machine Learning (ML) is a drug discovery technique that is more efficient than nonautomated traditional drug recognition methods. Small molecule Tyrosine Kinase Inhibitors (TKIs) act on Human Epidermal growth factor Receptor 2 (HER2) and suppress cell proliferation in breast cancer. Even though many synthetic drugs are available, few natural chemicals have been identified as TKIs for HER2. This analysis was carried out to recognize phytochemicals structurally analogous to available TKIs. Modified TechOpenCADD KNIME workflow combined Machine Learning (ML) with Quantitative Structure-Activity Relationship (QSAR). Version 4.7.0 of KNIME with Server Space, Cheminformatics, and RDKit extensions was utilized to examine the input data. A total of 422 phytochemicals were used together with 19 already existing TKIs and 20 drugs of some non-communicable diseases to feed in as Simplified Molecular Input Line Entry System (SMILES). “RDkit fingerprint node” was used to convert the dataset into FeatMorgan molecular fingerprints to make data readable for ML. Fundamental ML techniques Random Forest (RF), Support Vector Machine (SVM), and Artificial Neural Network (ANN) were employed to train the model with 98.26%, 97.18%, and 98.48% accuracy, respectively. Predicted data were tested for drug-likeness using Lipinski’s Rule of Five to determine the eligibility as oral drugs. From RF and SVM, one phytochemical each and three phytochemicals from ANN were predicted to be structurally similar to TKIs. The results obtained overlapped in three ML algorithms and followed at least three or more of Lipinski's Rules, indicating the possibility of developing them as TKIs. In several previous studies, two of the three predicted phytochemicals have already been shown to be HER2 inhibitors. The plant sources of predicted phytochemicals are commonly found in Sri Lanka. However, further studies should be done to ensure the effectiveness and validity of the results.

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