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. The use of caudal fin tissue for the caudal region and the pointed square edged caudal DNA extraction made possible to maintain the fish fin region whereas in Lepidocephalus guntea (H.), alive for further experiments. Among the DNA their head is ‘U’ shaped with few scales in their head extraction methods compared, method IV (modified region which helps to differentiate them from their protocol of Asahida et al.18) appeared to be most

Table 1 — Morphometric and Meristic characteristics of Lepidocephalus thermalis from 11 sites Morphometric (cm) Meristic (Number counts) Site Standard Total Snout Head Body Head Body Eye Pectoral Pelvic Dorsal Anal Caudal No. Barbel length length length width width depth depth diameter fin fin fin fin fin 5.50± 6.60± 1.70± 2.00± 2.67± 2.03± 2.60± 0.40± 8.20± 7.60± 7.90± 7.60± 13.70± 1. 6.00±0 0.79 0.72 0.51 0.40 0.82 0.68 0.83 0.08 0.40 0.72 0.84 0.61 1.17 5.09± 5.92± 1.51± 2.23± 2.41± 1.84± 2.28± 0.42± 7.94± 7.97± 7.94± 8.10± 14.20± 2. 6.00±0 1.05 1.06 0.53 0.84 1.15 0.78 1.47 0.09 0.69 0.55 0.63 0.61 0.80 5.14± 6.01± 1.47± 1.61± 1.99± 2.15± 1.80± 0.42± 8.00± 7.97± 7.17± 8.13± 14.56± 3. 6.00±0 0.72 0.68 0.52 0.66 0.80 0.45 0.55 0.13 0.59 0.56 0.46 0.35 0.72 5.53± 6.31± 0.91± 1.41± 1.76± 1.55± 2.10± 0.44± 8.20± 7.10± 8.03± 8.00± 14.03± 4. 6.00±0 0.60 0.62 0.16 0.20 0.41 0.43 0.24 0.12 0.48 0.35 0.32 0.95 0.41 5.53± 6.36± 1.01± 1.55± 2.38± 1.30± 2.15± 0.42± 7.96± 8.03± 7.90± 7.33± 13.33± 5. 6.00±0 0.91 0.81 0.39 0.30 0.28 0.50 0.56 0.12 0.67 0.32 0.88 0.48 0.70 5.71± 6.57± 1.43± 2.26± 2.21± 1.64± 1.68± 0.41± 8.50± 7.66± 8.10± 8.27± 13.93± 6. 6.00±0 0.47 0.49 0.40 0.11 0.30 0.24 0.19 0.08 0.50 0.55 0.48 0.74 0.74 5.94± 6.79± 1.67± 2.02± 1.96± 1.61± 1.80± 0.44± 8.20± 8.26± 7.86± 7.50± 14.70± 7. 6.00±0 0.48 0.39 0.18 0.29 0.22 0.24 0.50 0.16 0.55 0.52 0.63 0.57 0.60 5.47± 6.30± 1.86± 1.54± 2.08± 1.61± 1.96± 0.40± 8.70± 8.03± 7.57± 7.47± 14.06± 8. 6.00±0 0.79 0.80 0.50 0.33 0.13 0.20 0.48 0.09 0.47 0.32 0.68 0.57 0.98 5.54± 6.37± 1.67± 2.59± 1.80± 1.63± 2.41± 0.42± 8.00± 7.36± 8.03± 8.50± 14.63± 9. 6.00±0 0.77 0.70 0.50 0.48 0.11 0.44 0.65 0.08 0.26 0.56 0.32 0.57 0.67 5.76± 6.77± 2.21± 2.53± 2.08± 1.60± 2.33± 0.44± 8.00± 8.13± 8.06± 7.73± 14.20± 10. 6.00±0 1.09 1.12 0.23 0.23 0.30 0.16 0.46 0.12 0.00 0.43 0.37 0.58 0.80 5.45± 6.35± 1.38± 2.04± 2.02± 1.48± 2.21± 0.49± 8.00± 8.16± 8.80± 8.33± 14.96± 11. 6.00±0 0.97 0.99 0.50 0.32 0.92 0.80 0.58 0.11 0.26 0.95 0.57 0.67 1.20 n= 30 from each site, values are denoted as mean ± standard deviation, one sample t test p values were significant at 0.05 level.

576 INDIAN J EXP BIOL, AUGUST 2019

effective, 10 mg of caudal fin tissue yielded around Table 2. RAPD based banding pattern was analysed 1657 ng/µL of DNA in a total extraction time period initially (data not shown), since they were dominant of around 6 h 15 min. Moreover, the method of markers and not reproducible21,34, further proceeded to Kumar et al.20 helped to extract DNA within 2 h but DNA barcode analysis which helps to identify the the yield was little low 469 ng/µL compared to the uniqueness of genus even at species level35. modified protocol. DNA barcode analysis RAPD analysis DNA barcode - mitochondrial CO I gene There are very few research works available in the approximately 650 base pairs were amplified (Fig. 3). literature on L. thermalis based on genetic diversity and BOLD assigned 5 unique Barcode Index Number’s their genome sequence is not well studied, hence, RAPD (BIN’s) for the samples from 11 sites in southern analysis was performed. Among the 50 RAPD primers Tamil Nadu region. Although, the sequences with less used for the study only 10, namely OPA01, OPA02, than 500 bp were not assigned with BIN’s (BOLD), OPA05, OPA14, OPA15, OPA19, OPAC09, OPAC14, they have their GenBank Accession Numbers, with OPAH05 and OPAH07 (Fig. 2) showed significant sequence length ≥400 bp. The sequence details are polymorphic bands. This evidently demonstrated the summarised in Table 3. Consequently, all the genomic variations among the 11 samples based on dominant molecular marker analysis. The nucleotide sequence of primers used in this study are shown in

Fig. 3 — Cytochrome oxidase I gene PCR amplified product (around 650bp) in 1.2% agarose gel. [Lanes: M, 1 kb DNA ladder (New England Biolabs); and 1-11, DNA samples from their respective 1-11 sites]

Table 2 — Primer sequences - RAPD analysis Primer Sequence 5' – 3' OPA01 CAGGCCCTTC OPA02 TGCCGAGCTG OPA05 AGGGGTCTTG OPA14 TCTGTGCTGG OPA15 TTCCGAACCC OPA19 CAAACGTCGG

Fig. 2 — Polymorphic bands amplified by OPAC09 (Top lane) OPAC09 AGAGCGTACC and OPAH07 (Bottom lane) in 3% agarose gel. [Lanes: M, 1 kb OPAC14 GTCGGTTGTC DNA ladder (New England Biolabs); and 1-11, DNA samples OPAH05 TTGCAGGCAG from their respective 1-11 sites] OPAH07 CCCTACGGAG Table 3 — Accession number and Sequence details of CO I gene Site No. Sample ID Location River (District) Barcode index Sequence Genbank number (BIN) length (bp) accession no. 1. DBMKU001-15 N 9.9932; E 77.9861 Vaigai (Madurai) BOLD:ACY8615 603 KU693182 2. DBMKU002-15 N 10.0443; E 77.6022 Vaigai (Madurai) - 484 KX070757 3. DBMKU003-15 N 10.0309; E 77.9456 Vaigai (Madurai) BOLD:ACY8615 594 KU693181 4. DBMKU004-15 N 9.93032; E 78.165 Vaigai (Madurai) BOLD:ADB3001 645 KX283344 5. DBMKU005-15 N 9.93474; E 78.1856 Vaigai (Madurai) BOLD:ACZ6233 587 KX070756 6. DBMKU006-16 N 9.967; E 77.4355 Vaigai (Theni) BOLD:ACZ7491 657 KX070755 7. DBMKU007-16 N 9.5539; E 77.961 Kousiga (Virudhunagar) BOLD:ACZ7491 537 KX070754 8. DBMKU008-16 N 10.191; E 77.634 Vaigai (Theni) BOLD:ACZ7491 648 KX070753 9. DBMKU009-16 N 11.149; E 79.452 Cauvery (Tanjavur) - 462 KX586318 10. DBMKU010-16 N 8.7450; E 78.069 Tamirabarani (Tuticorin) - 420 KX586317 11. DBMKU011-16 N 8.6940; E 77.469 Tamirabarani (Tirunelveli) BOLD:ADC6754 639 KX586316 MANOHARAN et al.: MORPHOMETRIC AND MERISTIC CHARACTERIZATION OF INDIAN SPINY LOACH 577

sequences (11 numbers) were used for further shared concordant BIN. Sites 6, 7 and 8 had the evaluation. concordant BIN as they were sharing same river flow The sampling efficiency, analysed by systems geographically36,37. Accumulation curve of BOLD based on Kimura 2 The Geographic Distance Correlation acquired by parameter, BOLD alignment and pairwise deletion Mantel Test (Mantel38) between the geographic method stated significant accumulation of barcode distance matrix (in kilometres) and the genetic index numbers within the species (Fig. 4). The divergence matrix (Blagoev et al.39) using Multiple sequence divergence analysis showed a maximum Sequence Comparison by Log Expectation Alignment divergence of 8.53% and mean of 3.84% (Table 4). (MUSCLE)40,41 revealed positive correlation with Phylogenetic analysis was done using BOLD Mantel P value 0.01 (Fig. 6). Interestingly, it was also alignment and Kimura 2 distance parameter (Fig. 5). found that within the flow of Vaigai river three unique The phylogenetic tree revealed two distinct major BIN’s were obtained from L. thermalis with clades with Vaigai River-based population clustered significant correlation value based on geographic as one, the other showed the clusters based on Tamirabarani, Kousiga and Cauvery River flow along the regions of southern Tamil Nadu. The major clade included sample DBMKU004, 005, 006, 007 and 008 of Vaigai River in Theni and Madurai District, Kousiga river in Virudhunagar District, another clade DBMKU001, 002, 003 from Vaigai river, Madurai District and finally DBMKU009, 010 and 011 of Tamirabarani in Tuticorin, Tirunelveli District, Cauvery of Tanjavur District. Sampling site 1 and 3 of Vaigai River shared concordant BIN. Sites 6, 7 and 8 of Vaigai and Kousiga River had the concordant BIN as they were sharing River flow systems that are not geographically so far apart. In this study, in order to substantiate if there is any relation with respect to species uniqueness with biogeography, most of the samples were collected from Vaigai river and to distinguish their authenticity only a few sites were chosen in other riverine systems. Sampling site 1and 3

Fig. 5 — BOLD generated phylogenetic tree for Lepidocephalus Fig. 4 — BOLD based accumulation curve for sampling efficiency. thermalis based on BOLD alignment and kimura 2 distance parameter. Table 4 — BOLD generated sequence divergence analysis within the species No. of samples Taxa Comparisons Minimum Distance Mean Distance Maximum Distance Standard Error Distance (%) (%) (%) (%) 11 1 55 0 3.84 8.53 0.04 578 INDIAN J EXP BIOL, AUGUST 2019

Fig. 6 — Geographic distance and genetic divergence correlation using Mantel test. distance correlation. The positive correlation obtained 4 Sarojnalini CH, Nutritive values of two indigenous cobitid by Mantel test between the geographic distance fishes Botia berdmorei and Lepidocephalus guntea of Manipur. The Bioscan, 2 (2010) 391. matrix (in kilometres) and the genetic divergence 5 Singh MB, Sarojnalini C & Vishwanath W, Nutritive values matrix along with comparison of spread by Minimum of sun-dried Esomus danricus and smoked Lepidocephalus Spanning Tree of collection sites and maximum guntea. Food Chem, 36 (1990) 89. intra-specific divergence revealed the existence of 6 Shobana M, Raghavan K & Ramesh U, Bioprospecting the association between the species and biogeographic anti-microbial properties of Lepidocephalus thermalis (V.). specificity. This evidently demonstrated the unique J Biol Active Products Nature, 7 (2017) 270. molecular identity among the native fish species 7 Shobana M, Raghavan K & Ramesh U, Comparative assessment of nutritional composition in raw and based on their geographical locations. cooked Indian freshwater fish Lepidocephalus thermalis (V.). Proc Zool Soc, Springer India (In Press) https://doi.org/ Conclusion 10.1007/s12595-017-0249-4 Above detailed analyzes of morphometric, meristic 8 Muzaddadi AU, Taye RK & Bhattacharjya BK, Traditional and molecular characteristics of the Indian spiny knowledge associated with numsing, an ethnic fish product loach Lepidocephalus thermalis revealed the prepared by Mising tribes of Upper Assam, India. Indian J Tradit Knowle, 12(1) (2013) 91. molecular variation exhibited by these species based 9 Bhuiyan AS & Islam MN, Fecundity of Xenentodon cancila on phylogeography. Thus, the present study evidently (Hamilton) (Belonidae: Beloniformes). Environ Ecol (India), demonstrated the unique molecular identity among 8 (1990) 1004. the native fish species for the geographical locations. 10 RitaKumari, SD & Nair BN, Maturation and spawning in a As there are only few molecular reports available on tropical loach, Lepidocephalus thermalis (Cuv. and Val). L. thermalis, the present work adds significant Proc Indian Natl Sci Acad, 44 (Part B) (1978) 111. knowledge on the basic aspects of morphometry and 11 Tandon KK, Johal MS & Bala S, Morphometry of Cirrhinus reba (Hamilton) from Kanjli wetland, Punjab, molecular taxonomy to elicit reliable taxonomic India. Res Bull Punjab Univ Sci, 43 (1993) 73. studies in the indigenous, native fish species 12 Kartavtsev Y & Lee JS, Analysis of nucleotide diversity L. thermalis with unique BIN’s. Further, molecular at the cytochrome b and cytochrome oxidase 1 genes studies can be progressed to identify wild, native at the population, species, and genus levels. Russ J Genet, 42 species from different other sites. (2006) 341. 13 Hebert PD, Ratnasingham S & de Waard JR, Barcoding Acknowledgement animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Roy Soc Lond B, 270 The Educational Fellowship by UGC - NON-NET (2003) S96. Scheme is gratefully acknowledged. 14 Smith PJ, McVeagh SM & Steinke D, DNA barcoding for the identification of smoked fish products. J Fish Biol, Conflict of Interest 72 (2008) 464. The authors declare no conflict of interest. 15 Steinke D, ZemLak TS & Hebert PD, Barcoding Nemo: DNA-based identification for the ornamental fish trade. PLoS References One, 4 (2009) e6300. 1 Shaji CP, Lepidocephalus thermalis. The IUCN Red 16 Paabo S, Ancient DNA: extraction, characterization, List of Threatened Species 2011e. T172360A6876283, molecular cloning, and enzymatic amplification. Proc Natl http://dx.doi.org/10.2305/IUCN.UK.20111.RLTS.T172360A Acad Sci USA, 86 (1989) 1939. 6876283.en. Accessed on 29 December 2016. 17 Babu RM & Yazdani OM, Specific identity of 2 Kumari SDR & Nair NB, Length-weight relationship of the Lepidocephalus guntea (Hamilton) (Cypriniformes: loaches Noemacheilus triangularis (Day) and Lepidocephalus Cobitidae) with considerations of L. thermalis (Val.) as its thermalis (Cuv. & Val). Matsya, 4 (1978) 52. synonym. J Zool Soc India, 30 (1980)13. 3 Rahman MH, Hossain MA, Kawai K & Hossain M, A 18 Asahida T, Kobayashi T, Saitoh K & Nakayama I, Tissue Morphometric characteristic and reproductive periodicity of preservation and total DNA extraction from fish stored at freshwater fish, Lepidocephalus guntea (Hamilton). Bull Mar ambient temperature using buffers containing high Sci Fish Kochi Univ, 17 (1997) 141. concentration of urea. J Fish Sci, 62 (1996) 727. MANOHARAN et al.: MORPHOMETRIC AND MERISTIC CHARACTERIZATION OF INDIAN SPINY LOACH 579

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