DNA bar coding of Aplousobranchiata and Phlebobranchiata Ascidians (Phylum:Chordata) inferred from mitochondrial cytochrome oxidase subunit I (COI) gene sequence approach in Andaman and Nicobar Islands, India: a first report.

Ascidians (Phylum: Chordata) are sessile and filter-feeding marine animal, species identification of ascidians is possible by observing various morphological and anatomical features in various stages of life span. However, this method is labor intensive, time-consuming and very difficult for non-specialists particularly when dealing with field collections. Suborder Aplousobranchiata and Phlebobranchiata is the largest group of tunicates within, morphological and molecular data suggest that Didemnidae and Ascidiidae are monophyletic, but the monophyly of each genus and their phylogenetic relationships are still poorly understood. Therefore, this study was aimed to develop DNA barcodes of ascidians belonging to the orders of Aplousobranchiata and Phlebobranchiata species namely Diplosoma listerianum, Lissoclinum fragile, Didemnum psammatode, Phallusia fumigata and Phallusia ingeria collected from Andaman and Nicobar Islands were sequenced and submitted in Gen Bank. Colony structure, Scanning Electron Microscope (SEM) for spicules of colonial ascidians, larval type and zooids formation were found to be the most useful morphological characters for discriminating the species. Our BLAST results proved D. Listerianum KP842724 (98%) L. fragile KP842726 (100%) D. psammatode KP779902 (99%), P. fumigata KP779904 (99%) and P. ingeria KP842727 (100%) similarity and this is the first report of mitochondrial COI gene of these ascidians from Andaman and Nicobar Islands. We explored the usefulness of CO1 gene sequences for molecular level identification and mtDNA data in assessing a phylogenetic relationship of ascidian species.

Molecular phylogeny of four ascidian species inferred from mitochondrial Cytochrome Oxidase subunit I (COI) sequence.

Ascidians is a crucial group for the studies of deuterostome evolution and the origin of chordates, yet little molecular work has been done to determine the evolutionary relationships and largely unexplored beyond a few species. The phylogenetic analysis are presented for four different species of solitary ascidians, Ascidia virginea, Ascidiella aspersa, Clavelina oblonga and Aplidium fuscum, obtained from Great Nicobar Biosphere reserve (GNBR). Mitochondrial Cytochrome Oxidase subunit 1(COI) gene was amplified and the genetic diversity at the phylogenic level was measured. Bar-coded sequences were extracted with BLAST format from NCBI and the genetic diversity of the submitted sequences were compared with the related ascidian species. Maximum divergences measured among the four species were as follows: Ascidia virginea (96%), Ascidiella aspersa (96%), Clavelina oblonga (94%) and Aplidium fuscum (97%). This is the first report of molecular phylogeny of ascidians from Great Nicobar Biosphere Reserve, Andaman and Nicobar Islands. From this study, we found some stable clades on the evolutionary relationships among these ascidian species that may prompt a reevaluation of some morphological characters.

Aplousobranchia ascidians in Andaman and Nicobar Islands: a combined morphological and molecular discrimination.

Aplousobranchia ascidians from two different families were integrated with morphological characteristics and molecular phylogenetic analysis for the first time. The present study employed morphological descriptions (colony structures, tunic, zooids, spicules stigmata and test) and a molecular approach, using a fragment of the mitochondrial cytochrome oxidase I (COI) gene of four Aplousobranchia colonial ascidians Aplidium conicum (98%), Aplidium elegans (98%), Didemnum fulgens (92%) and Trididemnum cyanophorum (94%) from Andaman and Nicobar Islands. Bar-coded sequences were extracted with BLAST format from NCBI and the heritable diversity of the submitted sequences were compared with associated ascidian species. Study revealed that the evolutionary relationship among the ascidian species exhibited the constant clades, which may help for rapid reassessment of morphological characters of the species distributed worldwide.

In vitro antioxidant activities of an exopolysaccharide from a salt pan bacterium Halolactibacillus miurensis.

Exopolysaccharide (EPS) designated as HMEPS was isolated from Halolactibacillus miurensis and purified through gel filtration chromatography. EPS extracted from the supernatant yielded a 56.1% total carbohydrate content. The ash and moisture content were 12.1% and 1.5% respectively. Galactose and glucose were found as main monosaccharides of the HMEPS through HPLC analysis. FT-IR spectra of the HMEPS revealed its composition with hydroxyl, alkenes, amide and carboxyl as functional groups and aliphatic amine and alkynes at the fingerprint region. In vitro antioxidant activity was investigated against hydroxyl, DPPH, superoxide free radicals and the scavenging activity against all were found to be dose dependent proportionately. HMEPS showed higher reducing ability against superoxide radical and potency in chelating the ferrous ions. 10mg/ml of HMEPS was found equivalent to 2.7units of ascorbic acid through the total antioxidant assay. Phylogenetic relation of H. miurensis SEEN MKU3 (GenBank number KT803852) was plotted with MEGA 5.0.

Molecular identification of ascidians from the Palk Bay Region, Southeast coast of India.

BACKGROUND: More than 50 ascidian species distributed in the Palk Bay, Southeast coat of India. Up to a very few molecular work has been performed to determine the evolutionary relationships of ascidians in the world. OBJECTIVES: Present study explored the value of mtDNA data in assessing phylogenetic relationships within the family Ascidiacea, Didemnidae, Styelidae, and molecular identification of ascidians from the Palk Bay, Southeast coast of India. MATERIALS AND METHODS: The phylogeny analysis was executed based on mitochondrial cytochrome oxidase subunit I (COI) sequences of ascidian species. A BLASTN search can be run to determine the similarity of an unknown DNA sequence (query) with the collection of all known DNA sequences in GenBank. RESULT: The BLASTN results showed that Didemnum candidum, Ascidia ahodori and Styela clava match the other listings for these species in GenBank. Mitochondrial COI gene sequences of collected ascidians were submitted to GenBank and obtained the accession numbers. CONCLUSION: This study is the first report of molecular identification of ascidian species of the Palk Bay, Southeast coast of India. This information about Palk Bay ascidian communities provides a baseline of general biodiversity of that ecosystem.

A statistical approach for optimization of alkaline lipase production by ascidian associated-Halobacillus trueperi RSK CAS9.

A marine ascidian-associated bacterium, Halobacillus trueperi RSK CAS9, was optimized for lipase production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum lipase production (1355.81 U/ml) were determined to be tuna powder (14.58 g/l), olive oil (5.05 ml/l); NaCl (72.42 g/l), temperature (45 °C) and pH 9.0. An alkaline lipase was purified to 8.46 fold with 1193.59 U mg-1 specific activities with the molecular weight of 44 kDa. The activity was substantially inhibited by EDTA and PMSF, indicating that it was a metalloenzyme serine residue which was essential for catalytic activity. Thus, lipase production by microbial conversion of marine fish wastes in this study suggested its potential utilization for the production of high value products.