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Morphological and Molecular Characterization of Several Neogastropod Species from Coastal Waters of Bangladesh with One New Record

K. S. Sultana
Aquatic Bioresource Research Lab., Department of Fisheries Biology and Genetics, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh.

P. S. Brishti
Aquatic Bioresource Research Lab., Department of Fisheries Biology and Genetics, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh.

S. Ahmed
Department of Zoology, Jagannath University, Dhaka-1100, Bangladesh

M. B. Billah
Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

K. A. Habib
Aquatic Bioresource Research Lab., Department of Fisheries Biology and Genetics, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh

Abstract/ Executive Summary:

The present study reports the morphological and molecular characterization of marine neogastropods collected from the South-East to the South-West coasts of Bangladesh. A total of 21 species under 13 families were identified morphologically, of which 7 species were barcoded successfully using a partial sequence of mitochondrial cytochrome oxidase subunit-I (COI) gene. The average nucleotide frequencies of these species were G (guanine) = 20.57%, C (cytosine) = 18.44%, A (adenine) = 23.65%, T (thymine) = 37.35% and the GC content was 39.01%. The average Kimura two-parameter (K2P) distances of the COI barcode sequences within species, genera, and families were 20.7%, 22.0%, and 22.0%, respectively. The average inter-species genetic divergence was calculated as 21.0%. In addition, the COI barcode sequence of Nassarius stolatus was identified and submitted to GenBank for the first time. The study also reports the new record of a species, Indothais rufotincta Tan & Sigurdsson, 1996 from Bangladesh. This finding greatly extends the distributional range of this species from the West coast to the East coast of the Bay of Bengal.

Keywords: Bangladesh, DNA barcoding, Genetic distance, Indothais rufotincta, Neogastropoda, Phylogeny

Location: Aquatic Bioresource Research Laboratory, Department of Fisheries Biology and Genetics, Sher-e-Bangla Agricultural University, Bangladesh.

Start Date: 00-06-2017

End Date: 00-12-2019

Program Area: Resource Development and Management

Commodity: Aquatic animal

Objectives:

In the present study, morphological identification and molecular characterization of the collected neogastropods from SE and SW coastal waters of Bangladesh were carried out to differentiate one species from the other using genetic distance and phylogenetic assessment tools.

Methodology:

Samples were collected by direct hand-picking from the intertidal or subtidal zones of Sonadia Island (21°28'38.8"N; 91°55'23.1"E), Dublar Char (21°45'03.8"N; 89°33'15.8"E), Cox’s Bazar sea beach (21°27'48.6"N; 91°56'47.1"E) and Saint Martin’s Island (20°36'34.7"N; 92°19'40.0"E) located in the southeast coast of Bangladesh. The diversity of molluscan fauna in these areas is higher than in the other coastal sites of the country. These areas possess sandy, sandy-muddy to muddy beaches, mangroves, seagrass beds, and dunes, which make these places a unique habitat for marine molluscan fauna. Samples were also collected from Dublar Char of the Sundarbans mangrove forest area on the South-West coast of the country. The present study was conducted from June 2017 to December 2019. After collecting, the samples were initially preserved in an ice bucket with crushed ice until they were transferred to -18°C in Aquatic Bioresource Research Laboratory, Department of Fisheries Biology and Genetics, Sher-e-Bangla Agricultural University, Bangladesh. All the collected samples were tagged and photographed by a digital camera. Tissue samples were collected from the specimens that had muscles inside the peel and were preserved in 99% ethanol for molecular study. Specimens were identified mostly on the basis of peel characteristics following Rao et al., Tan and Sigurdsson (1996), Poutiers (1998), Ramakrishna, et al., and Siddiqui et al.. All observations were recorded in datasheets for final analyses. Genomic DNA was extracted from the collected tissues using a TIANamp Marine Animals DNA Kit (TIANGEN, China) following the protocol provided inside the kit box. The concentration of genomic DNA was then measured by Qubit 3.0 fluorometer. The polymerases chain reaction (PCR) was performed in a 25 µl reaction mixture in 0.2 ml reaction tubes in the thermal cycler. The barcode region of mtDNA COI gene was amplified by using universal primers commonly known as ‘Folmer primers’ (LCO1490 5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3' and HCO2198 5'-TAA ACT TCA GGG TGA CCA AAA ATC A-3') designed by Folmer et al. (1994). The thermal cycling conditions included an initial denaturation temperature of 95°C for 5 minutes, followed by 35 cycles of 95°C for 1 min for denaturation, 49°C for 1 min for annealing, 72°C for 1:30 min for extension, and 72°C for 5 min for a final extension. For the species that were not amplified by the universal COI primers, another primer set (COXAF 5'-CWA ATC AYA AAG ATA TTG GGA-3' and COXAR 5'-AAT ATA WAC TTC WGG GTG ACC-3') designed by Colgan et al. (2001) were used. In this case, the thermal cycling condition included an initial denaturation temperature of 94°C for 3 min followed by 35 cycles of 94°C for the 30s, 48°C for 1 min and 72°C for 1 min with the final extension of 72°C for 10 min. PCR products were visualized on 1% agarose gel stained with ethidium bromide in the gel documentation chamber. The flow UV-ray was kept on to watch the band in the connected computer by using GeneSys software. PCR samples with a single and clear visible band were purified with the PCR purification kit (TIANGEN- Universal DNA Purification Kit, China) for sequencing. The extracted DNAs were sequenced from a local commercial sequencer. The software Geneious (version 9.0.5) was used for editing nucleic acid sequences. The obtained consensus sequences were then edited based on the chromatogram peak clarities with the help of Chromas Lite. BLAST (Basic Local Alignment Search Tool) was used to check sequence identity between the obtained sequences and database of sequences from the GenBank at the National Center for Biotechnology Information (NCBI) and BOLD database. This helped to identify sequence similarity across gene sequence data of the NCBI. Sequence data were assembled and edited using the MEGA-7 software and subsequently submitted to BOLD and GenBank. Specimens and collected data, along with sequences and trace files, were provided to the project ‘MSK DNA Barcoding of Marine Molluscs, Bangladesh in BOLD. Sequences were aligned using ClustalW in MEGA 7 software, and different parameters such as the sequence length, polymorphic loci, and parsimony-informative sites were calculated. Sequence composition analysis was performed in BOLD. Sequence divergence was calculated using the Kimura two-parameter (K2P) model in MEGA 7 software, which was also used for constructing the phylogeny by Neighbor-Joining (NJ) method with 10000 bootstrap replications. The COI sequences of conspecifics reported to GenBank from other countries were used for evaluating genetic distance and phylogenetic assessment. The barcode sequences of the present study made a single clade in the phylogeny with low (usually <2%) or no genetic distance with the conspecifics of other localities which will validate the accuracy of the species identification.

Source of Information: J. Bio-Sci. 29(1): 79-91, 2021 (June)

Article Link (Online Journal): DOI: https://doi.org/10.3329/jbs.v29i0.54824

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The outcome of the present study reveals a significant contribution to DNA barcode reference sequences of marine neogastropods of Bangladesh. In addition, the species Indothais rufotincta Tan & Sigurdsson, 1996 has been recorded for the first time in Bangladesh which greatly extends its distributional range from the West coast to the East coast of the Bay of Bengal. Studies on the biology and taxonomy of gastropod species of Bangladesh are needed with the assistance of DNA barcoding in order to provide vital information for assisting conservation efforts.

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