Analysis of DNA Content of Wild and Cultured Labeo Calbasu Hamilton, 1822 Using Nanophotometer

1. International Journal of Trend in International Open Access Journal International Open Access Journal | www.ijtsrd.com International Journal of Trend in Scientific Research and Development (IJTSRD) Research and Development (IJTSRD) www.ijtsrd.com ISSN No: 2456 - 6470 6470 | Volume - 3 | Issue – 1 | Nov – Dec 2018 Dec 2018 Analysis of DNA Calbasu (Hamilton Content of Wild and Cultured Labeo Hamilton, 1822) Using Nanophotometer Nisha Rana1, Seema Jain2 nd Cultured Labeo Using Nanophotometer 1Research Scholar Research Scholar, 2Associate Professor Department of Zoology RGPG College, Meerut, Uttar Pradesh, India Department of Zoology RGPG College , India ABSTRACT Labeo calbasu is an important food fish and commonly known as the “calbasu” or “Black Rohu”. The present study was conducted on DNA extraction and determination of DNA quantity of both male and female of wild and cultured Labeo calbasu Nanophotometer. DNA isolation was done and gel electrophoresis was carried out. Extracted DNA was analyzed using nanophotometer (Nanophotometer P330; Implen, Germany) concentration of DNA and its purity level. Several different methods have been employed in the quantification of nuclear DNA over the past 50 years. The ease of use of this technique not only makes it a feasible option for small volume analysis of DNA but also a practical alternative for spectrophotometric measurement. Significant progress was made to measure micro volume liquid samples (<1µl) in biotechnology & pharmaceutical applications. For the present study both male and female of wild calbasu(from middle Ganga Region) and cultured Labeocalbasu(from culture ponds and/ hatchery) were taken. The value of DNA concentration in female of wild Labeocalbasuwas in between 58 ng/µl and of male was in between range of 64 ng/µl. The value of DNA concentration in female of cultured Labeocalbasuwas 60- 66 ng/µ 68- 74 ng/µl. KEY WORDS: DNA Quantity, wild, cultured, Nanophotometer, Labeo calbasu. INTRODUCTION Labeo is a genus of carps in the family Labeo calbasu locally called Kalibasu or Calbasu or Black rohu, is a freshwater fish species and is the most important carp species next to three Indian Major Carps; Labeo rohita, Catla catla mrigala. The synonyms used for this species are Cyprinus calbas (Hamilton, calbasu (Day, 1878), Labeo calbasu Shebbeare, 1937). It is a popular food fish and also is admired as a sport fish. Recently this fish species has also made its entry in ornamental fish markets of India and abroad. In last few years, the natural populations of this fish species has seriously declined due to fishing and other anthropological reasons. The dorsal profile of Calbasu is more convex than of abdomen.Other identifying features includes; thick and fringed. Two pairs of barbells pair longer than maxillary pair (Rahman, 19 pores on snout and eye situated a bit anterior from the half of the head (Bhuiyan, 1964). Caudal peduncle is short an lateral line well marked, scales in size. There are 20 rows of scales before dorsal fin and 22 rows round the caudal 1964). Mouth is moderately wide and Gill openings of Kalibasu is wide a villiform, short and feeble (Bhuiyan, 1964). Colour body is dark-black but the ventral part light dark. ventral surface of the opercular region is white iris coppery. This fish attains a length of (Talwar and Jhingran, 1991). feeder. It feeds on vegetable matter, crustaceans, insect larvae etc. Labeo calbasu higher plants 48%, protozoa 12%, crustacean 10%, mollusca 5%, mud and sand 15% (Bhuiyan, 1964). It does inhabit rivers and tributaries. They are also seen in deep pools clear sluggish streams, creeks. It can be reared in ponds and also in tanks (Bhuiyan, 1964 and Rahman, 1989). Of wide distribution in India, it is one of the major Indian carps. It is an important food fish and at several Indian carps. It is an important food fish and at several is an important food fish and commonly known as the “calbasu” or “Black Rohu”. The present study was conducted on DNA extraction and determination of DNA quantity of both male and The synonyms used for this species are Hamilton, Labeo calbasu (Shaw and It is a popular food fish and also is admired as a sport fish. Recently this fish species has also made its entry in ornamental fish markets of India and abroad. In last few years, the natural populations of this fish species has seriously declined due to over fishing and other anthropological reasons. 1822), 1822), Labeo Labeo calbasuusing Nanophotometer. DNA isolation was done and gel electrophoresis was carried out. Extracted DNA was analyzed using nanophotometer (Nanophotometer P330; Implen, Germany) concentration of DNA and its purity level. Several e been employed in the quantification of nuclear DNA over the past 50 years. The ease of use of this technique not only makes it a feasible option for small volume analysis of DNA but also a practical alternative for spectrophotometric cant progress was made to measure micro volume liquid samples (<1µl) in biotechnology & pharmaceutical applications. For the present study both male and female of wild Labeo (from middle Ganga Region) and cultured to to determine determine the the The dorsal profile of Calbasu is more convex than of Other identifying features includes; Lips are pairs of barbells present, rostal pair longer than maxillary pair (Rahman, 1989). No ye situated a bit anterior from the 964). Caudal peduncle is ateral line well marked, scales are moderate in size. There are 20 rows of scales before dorsal fin and 22 rows round the caudal peduncle (Bhuiyan, 1964). Mouth is moderately wide and also inferior. Gill openings of Kalibasu is wide and gill rakers are villiform, short and feeble (Bhuiyan, 1964). Colour of black but the ventral part light dark. The the opercular region is white iris coppery. This fish attains a length of nearly 90 cm and/ hatchery) were taken. The value of DNA concentration in was in between 58 -62 l and of male was in between range of 64- 68 l. The value of DNA concentration in female of 66 ng/µl and of male Generally it is a bottom feeder. It feeds on vegetable matter, crustaceans, Labeo calbasu feeds on algae 10%, higher plants 48%, protozoa 12%, crustacean 10%, mollusca 5%, mud and sand 15% (Bhuiyan, 1964). It does inhabit rivers and tributaries. They are also seen in deep pools clear sluggish streams, creeks. It can be DNA Quantity, wild, cultured, in the family Cyprinidae. locally called Kalibasu or Calbasu or is a freshwater fish species and is the most important carp species next to three Indian tanks (Bhuiyan, 1964 and Of wide distribution in India, it is one of the major and Cirrhinus @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 3 | Issue – 1 | Nov-Dec 2018 Dec 2018 Page: 906

2. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 places is referred to as the "Black Rohu". It is an important game fish in the tanks where it is s and is cultivated along with other species. It thrives better in tanks and lakes than in running waters; can tolerate slightly brackish water also. It does not normally breed in ponds; induced bred by hypophysation. It is essentially a bottom feeder attains a length of 90 cm. It can be taken on small fly spoon (Talwar and Jhingran 1991). OBJECTIVE To extract DNA of both male and female of wild and cultured Labeo calbasu. To determine concentration of DNA of both male and female of wild and cultured Labeo calbasu Nanophotometer. MATERIALS AND METHODS In the present study, Labeo calbasu collected from different water bodies of Western Uttar Pradesh (Bijnor district, district Hapur and from district Meerut), morphologically identified (with the help of standard literature of Day’s fauna 1875 78,1889; Jayaram, 1981) and preserved studies (for DNA isolation and quantification). Approx. 100mg of muscle tissue and fin clip from 2 individuals of each species were preserved in 95% ethanol until used and will be kept in molecular analysis. Voucher preserved in 10% formalin solution.DNA isolation was done by following the method of Ruzzante (1996) with minor modifications. The DNA was diluted to a final concentration of 100ng/µL. electrophoresis was carried out by 1.5-2% agarose gel. The extracted DNA was further analyzed using Nanodrop spectrophotometer (Nanophotometer P330; Implen, Germany) to determine the concentration of DNA and its purity level.Total DNA quantification was carried by nanophotometetrically absorbance of 260 and 280nm. A total of of Labeo calbasu of wild and cultured analyzed. RESULTS The present study was conducted on the isolation of DNA and determination of DNA concentration of both male and female of wild and cultured calbasu using Nanophotometer.DNA quantification was done by using nanophotometer and all data was noted (Table 1). The value of DNA concentration in female of wild Labeocalbasu was between 58 places is referred to as the "Black Rohu". It is an important game fish in the tanks where it is stocked and is cultivated along with other species. It thrives better in tanks and lakes than in running waters; can tolerate slightly brackish water also. It does not normally breed in ponds; induced bred by hypophysation. It is essentially a bottom feeder. It attains a length of 90 cm. It can be taken on small fly- ng/µl and of male was between (Fig.3). The value of DNA concentration in female of cultured Labeocalbasu was 60 68- 74 ng/µl (Fig. 4). Table1.Quantity of DNA in all 20 samples of Labeo calbasu in Nanogram/ Sample No. Female Male 1 60 66 2 58 64 3 61 66 4 62 68 5 59 64 Nucleic acids play a major role in growth an development. The amount of de (DNA), the carrier of genetic information, is stable under changing environmental situations and has been used as an indicator of biomass and cell number (Regnault and Luquet 1974). study revealed that the highest content of DNA was seen in female fishes of wild and also of cultured However slight change was seen in male fish. The comparative result revealed that the highest content was in cultured fishes, where all optimal conditions were maintained and also in female fishes of cultur fishes. l and of male was between ranges of 64- 68 ng/µl The value of DNA concentration in female of was 60 - 66 ng/µl and of male Quantity of DNA in all 20 samples of in Nanogram/ µl/individual Cultured Wild Wild Cultured Male 68 70 74 72 74 Female 66 65 60 66 62 To extract DNA of both male and female of wild and To determine concentration of DNA of both male and Nucleic acids play a major role in growth and development. The amount of deoxyribonucleic acid ), the carrier of genetic information, is stable under changing environmental situations and has been used as an indicator of biomass (Dortch et al. 1983) and cell number (Regnault and Luquet 1974). The study revealed that the highest content of DNA was en in female fishes of wild and also of cultured. However slight change was seen in male fish. The comparative result revealed that the highest content was in cultured fishes, where all optimal conditions were maintained and also in female fishes of cultured Labeo calbasu using Labeo calbasu fishes were collected from different water bodies of Western Uttar district, district Hapur and from , morphologically identified (with the help of standard literature of Day’s fauna 1875- 78,1889; Jayaram, 1981) and preserved for molecular quantification). tissue and fin clip from 2-5 individuals of each species were preserved in 95% ethanol until used and will be kept in -20 °C for molecular analysis. Voucher specimens specimens DNA isolation method of Ruzzante et.al., modifications. The DNA was were were diluted to a final concentration of 100ng/µL.Gel 2% agarose gel. The extracted DNA was further analyzed using hotometer P330; to determine the concentration of Total DNA quantification by nanophotometetrically A total of 20 samples of wild and cultured fishes were Fig. 1 Quantity of DNA content in Wild L. calbasu Fig. 1 Quantity of DNA content in Wild in Nanogram/ µl/individual in Nanogram/ µl/individual was carried taking taking The present study was conducted on the isolation of DNA and determination of DNA concentration of both male and female of wild and cultured Labeo Nanophotometer.DNA quantification nanophotometer and all data was The value of DNA concentration in Quantity of DNA content in Cultured L. Fig. 2 Quantity of DNA content in Cultured calbasu in Nanogram/ in Nanogram/ µl/individual was between 58-62 @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 3 | Issue – 1 | Nov-Dec 2018 Dec 2018 Page: 907

3. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 and in case of males value was between 88-91 ng/µl. and in case of males value was between 88 Similar study on diminution level of RNA/DNA ratio in tissue of Labeo rohita endocrine disrupting compounds was also done by Verma et al., 2016. REFERENCES 1.A. Adedunnijanet, “Isolation and determination of DNA concentration of African catfish ( gariepinus) using nanodrop and agarose gel”. A project report submitted to the department of aquaculture management, University of Agriculture, Nigeria 2014. 2.F. Day, The Fishes of India: being a natural history of the fishes known to habitat the sea fresh water of India, Burma and Ceylon. Text and Atlas in 4 parts, Today and Tomorrow’s Book Agency, London, 1875-78, 3.J. S. Nelson,. Fishes of the world, Fourth edition. Wiley, New York, 2006. 4.N. Pratima, A. Chaturvedi, Sreedhar, A. Ravicharan, and “Identification of fresh water fish species commonly consumed in Andhra pradesh using PCR-RFLP profile of cytochrome b gene food Nut. Sci. 2014. 5.M. Prado, A. Boix, and approach for the simultaneous detection of DNA from different fish species based on a nuclear target: quantification potential Chem., 403: 2012, pp 3041 6.B. Shi, X. Liu, Y. Xu, and characterization of three gonadotropin and their expression patterns during ovarian maturation in Cynoglossus semilaevis Mol. Sci., 16: 2015, pp 2767 7.R. Verma, A. K. Singh, and “Preliminary study on diminution RNA/DNA ratio in tissue of exposure to some compounds(EDCs)”, Aceh Science 1(1): 2016,pp 16-20. 8.D. E. Ruzzante, C. T. Taggart, C. Cook, and Goddard. (1996): Genetic differentiation between inshore and offshore Atlantic cod off new found land: microsatellite DNA variation and antifreeze level. Canadian J 53: 634-645. level of RNA/DNA ratio by exposure to some endocrine disrupting compounds was also done by Isolation and determination of African catfish (Clarias ) using nanodrop spectrophotometer . A project report submitted to the department of aquaculture management, University of Agriculture, Nigeria, Fig. 3 Comparative DNA Quantification in nanogram/µl in all 20 samples of both male and female L. calbasu Fig. 3 Comparative DNA Quantification in in all 20 samples of both male and and and fisheries fisheries DNA measurement can provide valuable tool for monitoring the health and condition of fish. knowledge from such investigations can be used in optimizing and sustaining yield, stock management and conservation of genetic diversity. DISCUSSION Nanodrop spectrophotometry powerful technology that allows Quantification of DNA, RNA (A260) concentrations and sample purity (260/280 ratio) over a large concentration range of 2 - 15,000 ng/L double standards DNA (Pratima et. al,. 2014). DNA concentration in female of wild Labeo was between 58-62 ng/µl and of male was between ranges of 64- 68 ng/µl. The value of DNA concentration in female of cultured Labeo was 60 - 66 ng/µl and of male 68- 74 ng/ al., (2012) used nanodrop method quantification from different fishes bas target. Nanodrop technique was also used by Shi al., (2015) for DNA quantification in the process of molecular characterization of Cynoglossus semilaevis. In the project report submitted by Aderibigbe Adedunni (2014) to the department of aquaculture and fisheries management, University of agriculture, Nigeria determination of DNA concentration of Clarias gariepinus was done by nanodrop. DNA quantification of Male and Female Clarias batrachus, Clarias gariepinus andClarias hybrids was also by Shobhna (2017). She observed the value of DNA concentration in female of C. batrachus between 59 and 61 ng/µl and of male was in between range of 70- 76 ng/µl. The value of DNA concentration in female of C. gariepinus 63 ng/µl and of male 75- 78 ng/µl. Highest DNA concentration was seen in hybrid individuals, in females the concentration was in between 74 females the concentration was in between 74-76 ng/µl DNA measurement can provide valuable tool for monitoring the health and condition of fish. The knowledge from such investigations can be used in optimizing and sustaining yield, stock management The Fishes of India: being a natural history of the fishes known to habitat the seas and fresh water of India, Burma and Ceylon. Text and Atlas in 4 parts, Today and Tomorrow’s Book , XX + pp.778. . Fishes of the world, Fourth edition. Nanodrop powerful technology that allows Quantification of A (A260) concentrations and sample purity (260/280 ratio) over spectrophotometry is is an an extremely extremely Chaturvedi, K. Manorama, M. Ravicharan, and , K. Bhagavatula,. Identification of fresh water fish species commonly consumed in Andhra pradesh using RFLP profile of cytochrome b gene”. Int. J. and and protein protein (A280) (A280) 15,000 ng/L double 2014). The value of Labeocalbasu l and of male was between l. The value of DNA Labeocalbasu 74 ng/µl. Prado et. method for DNA Boix, and C.V. Holst,. “Novel for the simultaneous detection of DNA from different fish species based on a nuclear target: quantification potential”. Anal Bioanal 3041–3050. quantification from different fishes based on nuclear target. Nanodrop technique was also used by Shi et. (2015) for DNA quantification in the process of Xu, and S. Wang, “Molecular characterization of three gonadotropin subunits and their expression patterns during ovarian Cynoglossus semilaevis”. Int. J. 2767-2793. Cynoglossus semilaevis. In the project report submitted by Aderibigbe aquaculture and fisheries management, University of agriculture, Nigeria determination of DNA concentration of was done by nanodrop. DNA Singh, and K. Jaiswal, Preliminary study on diminution level; of RNA/DNA ratio in tissue of Labeo rohita by exposure to some Clarias batrachus, hybrids was also done he value of DNA C. batrachus was in l and of male was in between The value of DNA C. gariepinus was 60 and µl. Highest DNA endocrine endocrine AcehJournal of Animal 20. disrupting disrupting Taggart, C. Cook, and Goddard. (1996): Genetic differentiation between inshore and offshore Atlantic cod Gadus morhua off new found land: microsatellite DNA variation Canadian J. Fish. Aq. Sci., concentration was seen in hybrid individuals, in @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 3 | Issue – 1 | Nov-Dec 2018 Dec 2018 Page: 908

4. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 9.Shobhna, Molecular Characterization and DNA quantification of Clarias batrachus, C. gariepinus and their hybrids. PhD thesis CCS Meerut, 2017. 10.A. L. Bhuiyan,. Fishes of Dacca. Asiatic Society of Pakistan, Dacca. 1964, 148 pp. 11.F. Day, 1878. Fishes of India, being a natural history of fishes known to inhabit the seas and freshwaters of India, Burma and Ceylon. William Dawson & Sons Ltd., London, Vol. I 12.F. Hamilton, An account of the fishes found in the river Ganges and its branches, Edinburgh & London, Fishes Ganges, 1822, p 279. 13.A. K. A. Rahman, of Bangladesh. The Bangladesh, Department of Zoology, University of Dhaka, Dhaka-1000. 1989 p 115. 14.A. K. A. Rahman, of Bangladesh (Second edition). The Zoological Molecular Characterization and DNA Clarias batrachus, C. gariepinus and their hybrids. PhD thesis CCS University, Society of Bangladesh, Department of Zoology, University of Dhaka, Dhaka 15.Shaw and Shebbeare, The Fishes of Northern Bangal. Journal of Royal Asiatic Society of Bengal Science. 1937, p 22. 16.P. K. Talwar, and A. G. Jhingran, India and Adjacent Countries. Volume 1. Oxford & IBH Publishing Co. Pvt. Ltd. New Delhi, Calcutta. 1991, p 203. 17.Q. Dortch, T. L Roberts, J. R. J Ahmed,: RNA /DNA concentrations as indicators o f grow planktonic marine organisms. 13, 1983, pp 61-71. 18.M. Regnault, P, Luquet, nucleic acid content of prepuberal shrimp, Crangon vulgaris 291—298. Society of Bangladesh, Department of Zoology, University of Dhaka, Dhaka-1000. 2005, 394 pp. The Fishes of Northern Journal of Royal Asiatic Society p 22. . Asiatic Society Jhingran, Inland Fishes of Day, 1878. Fishes of India, being a natural history of fishes known to inhabit the seas and India and Adjacent Countries. Volume 1. Oxford & IBH Publishing Co. Pvt. Ltd. New Delhi, Ceylon. William London, Vol. I: p 536. s, J. R. Jr Clayton, S. I. A ratios ratios and and DNA DNA An account of the fishes found in the , Edinburgh & p 279. ntrations as indicators o f growth rate in planktonic marine organisms. Mar. Ecol Pros Ser Freshwater Zoological Zoological Freshwater Fishes Fishes . The Society Society of of Study by evolution of of prepuberal growth in the Crangon vulgaris. Mar. Biol. 25, 1974, h, Department of Zoology, University Freshwater Freshwater Fishes Fishes (Second edition). The Zoological @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 3 | Issue – 1 | Nov-Dec 2018 Dec 2018 Page: 909