Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

BACKGROUND: Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. METHODOLOGY: A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. PRINCIPAL FINDINGS: Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. CONCLUSIONS: Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Morphological and Molecular Analysis of Fungal Species Associated with Blast and Brown Spot Diseases of Oryza sativa.

Fungal diseases blast and brown spot in rice cause severe yield losses worldwide. Blast is caused by Magnaporthe oryzae, and Bipolaris oryzae is reported as the main causal organism of brown spot. Both diseases cause leaf lesions that are difficult differentiate until the later stages. Early detection and differentiation of the lesions would help the adoption of disease management strategies specific to the pathogens and prevent reductions in the quality and quantity of rice yields. This study was conducted in the Northern Province of Sri Lanka over five consecutive rice cultivating seasons to characterize the causal fungi of rice blast and brown spot diseases by morphological and molecular means and to develop a visual guide to differentiate the two diseases. Disease incidence was recorded in 114 fields from 2017 to 2019, and fungal isolates associated with the lesions of both diseases were cultured and subjected to morphological and molecular characterization. Competitive growth interactions between M. oryzae and the more common individual fungal isolates of the brown spot lesions were evaluated. Fungal metagenomic analysis was conducted for the fungal species isolated from brown spot lesions. A suppression of blast accompanied by an increased incidence of brown spot disease was observed during the study period. M. oryzae was confirmed to be the causal organism of the blast, and >20 species of fungi were identified to be associated with brown spot lesions through morphological and molecular studies and metagenomic analyses. Fungal internal transcribed spacer region sequencing revealed genetic variation in the highly conserved region of DNA sequences of blast and brown spot fungal isolates. B. oryzae, Curvularia, and Microdochium species were commonly isolated from brown spot lesions. In vitro competitive growth interactions between the fungal isolates revealed growth suppression of M. oryzae by the fungal isolates associated with brown spot lesions. Similarly, it can be speculated that the abundance and severity of blast in the field may have an influence on brown spot-associated fungi. A simple visual guide was developed to differentiate blast and brown spot lesions. The findings would be highly useful in the timely management of these major fungal diseases affecting rice.

In silico molecular and morphological analysis of rice blast resistant gene Pi-ta in Sri Lankan rice germplasm.

BACKGROUND: Pi-ta is a major blast resistant gene, introgressed from indica rice varieties. In this study, diversity of the Pi-ta gene of 47 Sri Lankan rice accessions was studied by bioinformatics, and the results were validated with molecular and disease reaction assays. Sequences of rice accessions at the locus Os12g0281300 were retrieved from Rice SNP-Seek Database, and the coding sequence of reference Pi-ta gene of cultivar Tetep (accession no. GQ918486.1) was obtained from GenBank. Comparisons were made at nucleotide, amino acid, and protein structure level, and the 3D models predicted using Phyre2 software were superimposed using TM-align software. RESULTS: In silico analysis revealed that 10 accessions possessed resistant allele of the Pi-ta gene. The remaining accessions recorded high polymorphism in the leucine-rich domain resulting in 9 allele types, leading to single-amino acid substitutions at 27 different positions including a functional mutation of alanine to serine at the 918th amino acid position. None of the genotypes led to truncations in the amino acid sequence. The in silico analysis results were validated on 23 accessions comprising resistant and susceptible genotypes and another 25 cultivars from Northern Sri Lanka, by molecular assay using YL183/YL87 and YL155/YL87 resistant and susceptible allele-specific markers. Resistance of Pi-ta gene for the causal fungus, Magnaporthe oryzae, was further validated through pathogenicity assay. CONCLUSION: The Pi-ta gene, especially the LRD region, revealed significant variations within Sri Lankan rice cultivars leading to high levels of resistance against blast. This information would be highly useful in breeding programmes for resistance against rice blast.

A functional molecular marker for detecting blister blight disease resistance in tea (Camellia sinensis L.).

KEY MESSAGE: Identification of an EST-SSR molecular marker associated with Blister blight, a common fungal disease of tea, facilitating marker-assisted selection, marking a milestone in tea molecular breeding. lister blight (BB) leaf disease of tea, caused by the fungus Exobasidium vexans, results in 25-30% crop loss annually. BB is presently controlled by Cu based fungicides, but genetic resistance is the most viable option in disease management. Tea is a naturally out-crossing, woody perennial necessitating a long time for completion of a breeding programme. Marker-assisted selection (MAS) is vital to expedite breeding programmes and also for better accuracy in gene identification. The aim of the current research was to derive marker-trait associations using an F1 population segregating for BB. The population was genotyped at 11 expressed sequence tag simple sequence repeat loci followed by detecting the alleles by fragment analysis. The genotypic and phenotypic data were subjected to single-marker analysis resulting in the identification of EST-SSR073 as a diagnostic marker amplifying three alleles of the sizes, 168, 170 and 190 bp in F1. Of them, alleles 190 and 168 bp were confirmed to concur BB resistance and susceptibility, respectively. The alleles were validated in a panel of 64 tea cultivars, resulting in the amplification of 12 alleles at EST-SSR073. The EST-SSR073 allele sequences matched with Camellia sinensis photosystem-I reaction center subunit-II. The marker EST-SSR073 can be effectively used in breeding tea against BB, recording a milestone in MAS in tea.

In silico structural homology modelling of EST073 motif coding protein of tea Camellia sinensis (L).

BACKGROUND: Tea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server. RESULTS: The primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure. CONCLUSIONS: Camellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

Genetic characterization of Aedes aegypti (Diptera: Culicidae) in Sri Lanka based on COI gene.

BACKGROUND & OBJECTIVES: Aedes aegypti is the most prominent vector for dengue virus worldwide. Accurate identification of the species and understanding its colonization pattern are essential prerequisites in vector control. Thus, the present study was aimed to genetically characterize Ae. aegypti mosquitoes collected from different regions of Sri Lanka based on mitochondrial COI gene. METHODS: Thirty-three Ae. aegypti larval samples were collected from 19 districts. A 735bp region of the mitochondrial COI gene was amplified and analyzed for genetic diversity indices. Phylogenetic trees were constructed using Sri Lankan samples and also including mosquito samples reported from other parts of the world. RESULTS: High genetic diversity was observed within the samples analysed (gene diversity: 0.949; average number of nucleotide differences: 6.371). There were 20 haplotypes presented within the 19 localities investigated. The phylogenetic tree derived two main clades. However, no distinguishable clustering pattern was observed in the phylogenetic tree except for the districts in the northern corner indicating extensive admixing among different populations. When samples from other countries were included in the phylogenetic tree, Anuradhapura, and Mannar samples were clustered together with samples from India, Venezuela, USA, Portugal and Cambodia while Rathnapura was clustered with Bolivia and France. INTERPRETATION & CONCLUSION: Our results suggest that Sri Lanka has undergone multiple invasions of Ae. aegypti from various parts of the world over an extensive period. Further, the mosquito control campaigns had not caused a significant effect on the Ae. aegypti populations which is existing in mutation-drift equilibrium.

Utility of pf/pan RDT for diagnosis in the prevention of re-establishment of malaria in Sri Lanka.

The utility of CareStartTM Malaria Pf/PAN (HRP2/pLDH) Ag Combo Test, in detecting non-endemic clinical malaria cases was evaluated in Sri Lanka, a country in prevention of re-establishment of malaria following elimination. RDT, microscopy and nested PCR were performed for 350 suspected malaria patients recruited prospectively. There were 173 PCR confirmed malaria patients and 177 PCR negative subjects. Plasmodium falciparum amounted to 48% of infections with 44% P. vivax, 6% P. ovale and 2% P. malariae. Performance characteristics of RDTs and microscopy were compared with nested PCR. Sensitivity and specificity of RDT with 95% confidence intervals (CI) were as follows: any malaria infection 95.95% (CI = 91.84-98.36) and 94.92% (CI = 90.57-97.65); P. falciparum 100% (CI = 95.65-100) and 97.00% (CI = 94.18-98.70) and other species 92.22% (CI = 84.63-96.82) and 99.62% (97.88-99.99) respectively. A significant difference between sensitivities of HRP2 (100%, CI = 95.65-100) and pan pLDH line (68.67%, CI = 57.56-78.41) was seen for P. falciparum, parasite densities less than 1000 parasites/microliter being detected only by HRP2. Sensitivity and specificity of microscopy with 95% CI were as follows: any malaria infection, 94.22% (CI = 89.63-97.19) and 99.44% (CI = 96.89-99.99); P. falciparum 89.16% (CI = 80.40-94.90) and 99.63% (CI = 97.94-99.99); other species 98.89% (CI = 93.96-99.97) and 100% (CI = 98.59-100) respectively. The low sensitivity of pan specific pLDH for P. falciparum, P. ovale and P. malariae should be taken in to consideration when using this RDT as a point of care test when and wherever microscopy facilities are not readily available. Considering the low sensitivity of microscopy for P. falciparum, it is preferable to perform both tests, when malaria is highly suspected.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC50 8.4 ± 0.2 µM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 µM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 µM. Vateriferol (1) exhibited weak cytotoxic potency (<50% growth inhibition) against the tested cell lines at 147.2 µM.

Development of a DNA probe and a PCR based diagnostic assay for Rhizoctonia solani using a repetitive DNA sequence cloned from a Sri Lankan isolate.

Rhizoctonia solani is a destructive fungal pathogen of many economically important plants all over the world and the causative organism of sheath blight of rice in many tropical countries including Sri Lanka. A repetitive sequence from the genome of R. solani was cloned and characterized with a view to develop a DNA probe and a PCR diagnostic assay for detection of the fungus. The cloned sequence was 1550 bp long and appeared to be interspersed throughout the genome. The cloned sequence hybridized only to R. solani DNA and was sensitive enough to detect 100 pg of R. solani genomic DNA. PCR primers were designed from the cloned sequence and it was possible to develop a PCR assay for the specific detection of the fungal DNA with 10 pg sensitivity.

Plasma pharmacokinetics of a generic amoxycillin and the innovator brand: a comparison.

OBJECTIVE: To compare the bioavailability and plasma pharmacokinetics of a generic brand of amoxycillin (State Pharmaceutical Manufacturing Coporation) selected from the lower price range, with that of the innovation brand (Amoxil, Beecham). DESIGN: Sixteen healthy adult volunteers were allotted to two groups and each group was given a test dose of amoxycillin from each brand. Blood samples were collected at 0, 2, 3 and 4 hours thereafter plasma levels were assayed using high performance liquid chromatography. RESULTS: Analysis of results show that the generic product had similar bioavailability and pharmacokinetics when compared with the innovator product. CONCLUSION: The quality assured generic amoxycillin tested had similar bioavailability as a more costly branded version.