Lepidocephalus Thermalis (V.) — DNA Barcode Approach
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Indian Journal of Experimental Biology Vol. 57, August 2019, pp. 573-579 Deciphering the morphological and molecular characteristics of fresh water fish Lepidocephalus thermalis (V.) — DNA barcode approach Shobana Manoharan, Raghavan Kuppu & Ramesh Uthandakalaipandian* Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai–625 021, Tamil Nadu, India Received 16 August 2017; revised 12 July 2018 Lepidocephalus thermalis (Valenciennes), commonly called the spiny loach or the spotted loach, is a small, edible fresh water fish, and phylogeography based differences in their morphology are not so apparent. Hence, in the present study, along with the morphometric and meristic characterization of L. thermalis collected from 11 sites in the southern Tamil Nadu region, their genomic adaptation using DNA barcode were analyzed. Initial molecular characterization of these fishes was carried out by RAPD analysis which showed polymorphism in their banding pattern. Further, PCR amplification of mitochondrial gene cytochrome oxidase subunit I (CO I) for DNA barcoding was carried out, which revealed distinct sequences within the species and were submitted to BOLD (Barcode of Life Database). Five Barcode Index Numbers (BIN’s) ACY8615, ACZ6233, ACZ7491, ADB3001 and ADC6754 were obtained in positive correlation to their species distribution and biogeography of southern Tamil Nadu region, India based on Mantel’s test with significant P value 0.01. Thus, this study helps in better understanding of the phylogeography based genetic diversity in L. thermalis. Keywords: Aquarium, Ayira meen, BOLD, cytochrome oxidase I gene, Indian spiny loach, Ornamental fishery Lepidocephalus thermalis (Valenciennes 1846), changes in the relative growth of their various commonly called the Indian spiny loach (locally, body parts during development9,10. Morphometric Ayira meen), is an IUCN Least Concern status1 measurement of fishes and the study of statistical Cobitid fish valued for its indigenous flavour, relationship between them are essential for the taste and few species for ornamental fishery taxonomic identification of a species11. In recent available in fresh water ecosystems of South India decades, integrated taxonomic approach is and Sri Lanka2. They are of socioeconomic widely relied by taxonomists as it employs both importance with considerable market value in morphological and molecular studies. Hence, Tamil Nadu, India. Nutraceutically, they serve as a employing conventional molecular biology techniques valuable source of proteins, lipids, calcium, in taxonomy studies have become inevitable. Among vitamin A, iron in their edible forms more abundant them, cytochrome oxidase - I (CO I) gene of than other large sized fresh water fish do3-8. However, mitochondrial DNA, cytochrome b12 are applied to only very few authenticated reports about different study the systematics, phylogeny13, DNA barcoding aspects of these small indigenous fish species of fish products and ornamental fish trade14-16. Based are available. on this overall importance of taxonomy, in the present Fish taxonomy is typically accomplished using study, we analyzed the morphometric, meristic and morphometric measurements and meristic counts. molecular characteristics of the Indian spiny loach Morphometric and meristic study is the classical Lepidocephalus thermalis. taxonomy method depending upon the shape variation and its covariation with other variables. It is a Materials and Methods fundamental part of most biological research as Sample collection historical diversity of living organisms were based on L. thermalis (n=30 from each site) were collected descriptions of morphological forms. Moreover, from 11 different locations of southern sexual maturity in many fishes is found to cause Tamil Nadu rivers, such as Vaigai (7 sites), ————— Cauvery (1 site), Tamirabarani (2 sites) and *Correspondence: Kousiga (1 site) using dip nets during the year E-mail: [email protected] September-December, 2015. 574 INDIAN J EXP BIOL, AUGUST 2019 Morphometric and meristic character analysis at 7000 rpm for 5 min and air dried And suspended Morphometric analysis was carried out for the DNA in sterile Milli Q water or TE buffer. parameters, such as total length, standard length, head length, dorsal fin length, caudal fin length, head width DNA quantification and body width were calculated using a standard The DNA extracted was quantified using UV measuring scale and microscope. Meristic parameters Spectrophotometer (Hitachi) at 260/280 nm. The such as dorsal, pelvic, pectoral, anal, caudal fin ray quality and intactness of DNA was checked and barbel counts were recorded17(Fig. 1). using 0.8% TAE (Tris-Acetate-EDTA) Agarose Gel Electrophoresis. Molecular analysis DNA extraction PCR amplification Four DNA extraction protocols based on PCR amplification and sequencing were carried out Asahida et al.18, Ruzzante et al.19, Kumar et al.20 and using DNA extracted in one representative fish method IV [a slight modification to the methodology sample from each site for molecular analysis. 18 of Asahida et al. ] were adapted. In the modified Random amplification of genomic DNA using RAPD primers protocol, 10 mg caudal fin tissue using was Polymerase chain reaction was performed to homogenised using 500 µL TNES-Urea buffer randomly amplify the extracted genomic DNA (10 mm Tris, pH 7.5, 125 mm NaCl, 10 mm EDTA, using 50 different arbitrary RAPD primers 1% SDS and 4M Urea), 60 µg/mL Proteinase K from Operon Technologies Ltd. USA. PCR was followed by incubation at 37ºC for 3 h. RNAse to a carried out in a 20 µL reaction volume containing final concentration of 60 µg/mL was added and 10 µL of 2X PCR mix (Ampliqon), 4 µL of incubated at room temperature 37°C for 1 h. Added 5 picomole primer, 2 µL of 20 ng template DNA and equal volume of phenol: chloroform: isoamyl alcohol 4 µL of sterile Milli Q in an Agilent Thermocycler21. (25:24:1) to the digest and centrifuged at 13200 rpm The thermal cycle profile was as follows: 94ºC for for 10 min at 4ºC. Transferred the supernatant to a 2 min followed by 35 cycles of denaturation at 94ºC fresh tube and treated with equal volume of for 30 s, annealing at 37ºC for 45 s and extension at chloroform: isoamyl alcohol (24:1) mixture. 72ºC for 1 min with a final extension for 10 min at Centrifuged and transferred the supernatant. 72ºC. PCR products were electrophoresed on a 3% The DNA was precipitated using 95% ethanol th TAE agarose gel stained with ethidium bromide (Molecular Grade) and 1/15 volume of 3M sodium (0.5 µg/mL), visualized using UV Trans-illuminator acetate, pH 5.2 and incubated at 4ºC for 2 h. and documented using the Bio-Rad Gel Centrifuged the tubes at 13200 rpm for 10 min at Documentation System. 4ºC, removed the supernatant carefully and the pellet was washed twice with 70% ice cold ethanol Cytochrome oxidase I (CO I) gene amplification PCR was performed to amplify the conserved Cytochrome Oxidase subunit I gene 650 base pairs (bp) from the extracted DNA. The nucleotide sequence of primers for CO I22,23, used in this study are as follows: forward primer (5'-TCAACCAACCAC AAAGACATTGCCAC-3') and reverse primer (5'-CGG TCTGAACTCAGATCACGT-3'). PCR was carried out in a 20 µL reaction volume containing 10 µL of 2X PCR mix (Ampliqon), 2 µL of 5 picomole forward primer and 2 µL of 5 picomole reverse primer, 2 µL of 20 ng template DNA and 4 µL of sterile Milli Q in Fig. 1 — Schematic description of morphometric and meristic an Agilent Thermal Cycler. The thermal cycle profile characters studied in Lepidocephalus thermalis. [Standard length was as follows: 94ºC for 2 min followed by 35 cycles (SL), Total length (TL), Snout length (SnL), Head width (HW), of denaturation at 94ºC for 30 s, annealing at 55ºC for Body width (BW), Head depth (HD), Body depth (BD), Eye 45 s and extension at 72ºC for 1 min with a final diameter (ED), Pectoral fin rays (PFR), Pelvic fin rays (PFR), Dorsal fin rays (DFR), Anal fin rays (AFR), Caudal fin rays extension for 10 min at 72ºC. PCR products were (CFR), Barbels (BrB)] electrophoresed on a 1.2% TAE agarose gel stained MANOHARAN et al.: MORPHOMETRIC AND MERISTIC CHARACTERIZATION OF INDIAN SPINY LOACH 575 with ethidium bromide (0.5 µg/mL) visualized using closely related species17,26. The fish samples analyzed UV Trans-illuminator and documented using the Bio- in the 11 sites had almost similar morphological Rad Gel Documentation System. The PCR band was characters in their length and width. Morphometric gel eluted using Favorgen Gel Extraction Kit and analysis revealed the significant variation in male sequencing was performed using an Applied and female fishes, where the female had a slightly Biosystems Automated DNA sequencing system. The greater body width than the male. The meristic sequences were submitted to BOLD and NCBI characters slightly varied in their fin counts, whereas database24,25. all the fish had a uniform barbel count of 6 in number. There was no difference in their fin ray formula Statistical and bioinformatics analysis based on male or female. Table 1 describes the All the experiments were carried out in morphological and meristic data for 30 fish samples triplicates and statistical analysis of one sample from 11 different sites. ‘t’ test was performed using SPSS 22.0 version. Online tools from BOLD workbench Molecular analysis (http://www.barcodinglife.org/) were utilized for Extraction of DNA is a fundamental process for Barcode data analysis and phylogeny. any molecular study to be progressed ahead like DNA barcoding, hybridization, restriction endonuclease Results and Discussion mapping27-29 cloning, characterization and direct 30,31 Morphometric and meristic characteristics sequencing of target genes or DNA fragments Morphologically, these fishes were identified based requires a good quality, genomic DNA for analysis. on their pointed ‘V’ shaped head, presence of spots DNA extraction from fish tissues was carried out throughout their lateral line, the presence of a dark using different protocols17,32-33 to obtain high quality round spot surrounded with a yellow-white ring near and quantity of DNA.