Phylogeographic genetic variation of Indoplanorbis exustus (Deshayes, 1834) (Gastropoda: Planorbidae) in South and Southeast Asia.

The freshwater snail Indoplanorbis exustus play an important role as the sole intermediate host of several medically- and economically-important trematodes, especially zoonotic schistosomes and echinostomes, which can infect and cause diseases in livestock and people. This study aims to explore the mitochondrial cytochrome c oxidase subunit 1 sequence variation of I. exustus collected from new geographical areas; 459 specimens of I. exustus were collected from 43 localities in South and Southeast Asia. The 42 haplotypes (Ie1 - Ie42) we detected were classified into haplogroups I - V. Phylogenetic analyses revealed five major clades, A - E, in concordance with all previous studies. Clade E contained two subclades, E1 (haplogroup I) and E2 (haplogroup II). The most widespread genetic group was subclade E1. Clade A, clade B (haplogroup V), and clade C (haplogroup IV) were found only in South Asia, whereas clade D (haplogroup III) was specifically found in Southeast Asia. In Thailand, I. exustus showed high genetic divergence with 21 haplotypes. Several isolates showed significant genetic differences from others with unique haplotype(s). Hence, we confidently conclude our findings support all previous studies that I. exustus is a species complex with at least four major lineages and five haplogroups. Our additional analyses of 35 samples from Sri Lanka showed these were indeed an independent genetic group as previously found, but they can now be classified as a unique group forming subclade E2 (haplogroup II) of I. exustus sensu lato.

Preliminary screening of the aqueous extracts of twenty-three different seaweed species in Sri Lanka with in-vitro and in-vivo assays.

BACKGROUND: Seaweeds are an important source of bioactive compounds which are applied in various aspects of medicinal investigations. The present study was conducted to investigate cytoxicity (in-vitro and in-vivo) and wound healing activity of different seaweed species in Sri Lanka. METHODS: Twenty-three seaweed samples, belonging to Phaeophyta (Brown), Chlorophyta (Green) and Rhodophyta (Red) were used for the experiments. Samples were collected from the inter-tidal and the sub-tidal habitats around Sri Lankan coast (Southern, Northern and North-western). Aqueous seaweed extracts were tested for cytotoxic and wound healing activity; in-vitro and in-vivo. To determine toxicity of aqueous seaweed extracts, brine shrimp lethality assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on mouse fibroblasts (L929) cell line were performed. Cell migration induction of seaweed extracts was assessed by scratch wound healing assay using L929 cell line. Based on the our previous experiments S.ilicifolium (SW23) was selected for the in vivo study to confirm our hypothesis. Albino mice (BALB/c) were divided into three groups (12 in each) and a circular area (44.07 ± 02.51 mm2) of full skin was excised to create a wound in mice group II and III. Group III received aqueous extract of Sargasum illicifolium (400 mg/kg BW/day for 12 days, orally), Group II received distilled water for 12 days whereas Group I was used as the control group and it was tested without forming wounds and without providing any treatment. Further, the expression level of Tumor Necrosis Factor (TNF-α) and Transforming Growth Factor-ß (TGF-ß) via RT-PCR were measured every three days until the end of the experiment. RESULTS: Phytochemical tests showed positive results to flavonoids in all the selected green seaweeds and alkaloids were observed in red seaweeds. In the toxicity assay, red seaweed, Acanthophora spicifera (SW17) was found to be highly effective on nauplii of brine shrimp (LC50 = 0.072 µg/µl). LC50 value of green seaweed species, Caulerpa racemosa (SW02 and SW08) and Caulerpa sertularioides (SW10) was not found within the tested concentration series. The highest cytotoxic effect on L929 cell line was exhibited by aqueous extracts of red seaweed; Jania adhaereus with 50.70 ± 7.304% cell viability compared with control group. The highest cell migration activity was observed in L929 cell line group treated with extracts of green seaweed namely; Halimeda opuntin (SW07) and extracts of brown seaweed namely; Stoechospermum polypodioides (SW11). Extracts of S. illicifolium (SW23) exhibited a significantly enhanced wound healing activity in mice group III within three days (P < 0.05) with an open wound area of 17.35 ± 1.94 mm2 compared with control group (26.29 ± 2.42 mm2). TGF-ß gene expression peaked on 6th day of post-wound and subsequently decreased on 9th day of post-wound in mice group III. TNF-α expression was suppressed in mice group III whereas it was elevated in group II. TGF-ß expression is enhanced in the treatment group compared to the control group. CONCLUSIONS: Aqueous extracts of selected seaweeds are a significant source of potential compounds with wound healing properties, which might be helpful in the healing of various wounds. This also infers that many species of brown and red seaweeds have the potential of wound healing, specifically, Sargasum illicifolium and Jania adhaereus could be a potential candidate for in-vivo studies related to wound healing and cancer therapy in the near future.

Molecular characterization of Fasciola jacksoni from wild elephants (Elephas maximus maximus) of Sri Lanka: a taxonomic evaluation.

Fasciola jacksoni is a significant contributor to the health and mortality of Asian elephants, particularly those in Sri Lanka. Despite the impact of fascioliasis on elephant populations, it is a neglected veterinary disease with limited taxonomic understanding. Molecular characterization and phylogenetic analysis of F. jacksoni were carried out to evaluate its suggested basal position in the Fasciolidae. Adult worms were collected during post-mortem of elephants, and eggs were collected from living elephants in National parks across Sri Lanka. Using the mitochondrial genes nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1), and a partial 28S ribosomal DNA (28S rDNA), DNA sequences were generated from the F. jacksoni adult and egg material. Maximum likelihood (ML) phylogenetic analyses did not resolve F. jacksoni to be basal to the Fasciolidae. Furthermore, the ML analyses showed that the genus Fasciola was not monophyletic and that F. jacksoni was a sister species to the deer liver fluke Fascioloides magna. A clear framework is required to determine the taxonomic status of F. jacksoni and this current study provides the first detailed application of molecular techniques from multiple hosts across Sri Lanka with the production of reference DNA sequences for this important parasite.