Annual Research & Review in Biology 35(4): 19-31, 2020; Article no.ARRB.57207 ISSN: 2347-565X, NLM ID: 101632869 Morphology and Molecular Biology of Benthic Java Sea Shark Ray Rhina ancylostoma Bloch and Scheider 1801 (Elasmobranchia: Rhinidae) Agus Hartoko1*, Delianis Pringgenies2, Amelia Cahya Anggelina2 and Takashi Matsuishi3 1Department of Fisheries, Faculty of Fisheries and Marine Science, Diponegoro University, Semarang, Indonesia. 2Department of Marine Science, Faculty of Fisheries and Marine Science, Diponegoro University, Jalan Prof. Sudarto, SH Kampus Undip Tembalang, Semarang, Indonesia. 3Department of Fisheries Science, Hokkaido University, Japan. Authors’ contributions The work had been carried out in collaboration among all authors responsibility. Author AH as the first author is research coordinator, main concept and manuscript finalization. Author DP as second author contribute for DNA analysis and discussion. Author ACA contribute for laboratory analysis. All authors read and approve the final manuscript. Article Information DOI: 10.9734/ARRB/2020/v35i430208 Editor(s): (1) Paola Angelini, University of Perugia, Italy. (2) Dr. Manikant Tripathi, Dr. Ram Manohar Lohia Avadh University, India. Reviewers: (1) B. Gunalan, Thiru Kolanjiappar Government Arts College, India. (2) Adeyeye, Samuel Ayofemi Olalekan, Ton Duc Thang University, Vietnam. Complete Peer review History: http://www.sdiarticle4.com/review-history/57207 Received 10 March 2020 Accepted 17 May 2020 Original Research Article Published 23 May 2020 ABSTRACT Aims: Morphology and molecular biology of a rare shark-rays Rhina ancylostoma caught from Java sea.Indonesia. Study Design: Morphology, morphometry and DNA analysis of the fish specimen. Place and Duration of Study: Sample wascollected from fish auction hall at north Java, specimen now stored in Lab of Ichtiology. Department of Fisheries. Faculty of Fisheries and Marine Science. Diponegoro University, between March to December 2019. Methodology: DNA extraction, amplification and molecular identification of fish sample.Yield of DNA supernatant transferred in an eppendeorf tube and stored in 4°C for further process. PCR _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; Hartoko et al.; ARRB, 35(4): 19-31, 2020; Article no.ARRB.57207 amplification. Part of Mithocondrial DNA COI (Cytochrome Oxidase subunit-I) gen was amplified using Polymerase Chain Reaction (PCR) method. Then matched up with GenBank database at NCBI using Basic Local Alignment Search Tool (BLAST) analysis. Philogenetic tree was set using Neighbor-Joining method, Kimura-2 parameter model and 1,000 bootstrap replication. Some sequence from NCBI GenBank were inputted into philogenetic tree as comparison. Results: Rhina ancylostoma as one of demersal shark rays catch from tropical shallow Java sea. Range of Total Length 73 – 200 cm and dominant Total Length catch 125 cm. Morphologyis characterised with three lines of spine thorn on the head, morphometry characteriswide of head to TL ratio 0.77. DNA analysis had confirmed the specimen of AH2 as Rhina ancylostoma based on homological match up of sequence of Gen Bank database with reference accesion number KU721837.1 with length sequence of 665 bpand identical similarity of 99.84% for specimen Accession number LC 505461. Conclusion: Rhina ancylostoma as one of demersal shark rays catch from Java sea. Range Total Length (TL) of catch 73 – 200 cm and dominant TL catch range 101 – 125 cm. Morphologically character of wide head ratio with three lines of spine thorn on the head. Nearest genetic distance of 0.02 to Rhincobatushorkelii and 0.017 to R. australiae. Longest genetic distance of 0.243 to Potamotrygon motoro. Keywords: Morphology; molecular biology; shark-ray; Java-sea; Rhinidae. 1. INTRODUCTION regardedwill damage the net unit by its thorny skin, but stillcollect the catch forhigh price of its High biodiversity of tropical seawater teleost and fins only. Habitat degradation and the fishing elasmobranch of Indonesia is very well known. operations had more challenge to these sharks High biodiversity and high consumption of the survival and populationsespescialy at Java Sea. fish isneed to be carefully managed for Sharks known as demersal fish and mostly sustainable fish resource use [1]. Consumption inhabit on the bottom of the sea and thus need in nowadays demand for the elasmobrach assumed to have correlation with the depth of the fish was even higher thanlast decades demands. sea as the habitat [10]. Aim of study is to explore Specifically for elasmobranch sharks market morphology and molecular biology ofdemersal demand as high price commodity is mainly for R. ancylostoma catch from Java Sea landed at the sharkfins for Asean demand market [2]. Cirebon, Tegal and Rembang fishing port. These highdemand had driven vastly for fisherman to catch more sharks and mainly for its 2. METHODOLOGY fin inconsiderably. In other sector had increasing capability of sharks fish processing for food and 2.1 Samples and Studysites vitaminsextraction [3]. Indonesia had been known as one of the bigest sharks catch country The data ofdemersalshark-rays R. ancylostoma in the world with 103,245 tons in 2011 and were collected from fisherman catch from Java 105,230 tons in 2018. Sharks can be as the main sea landed at Cirebon, Tegal and Rembang target as well as by catch infishingoperations [4]. fishing port (Fig. 1). Quantitative yearly data of As mention by [5] high demand for shark fins and sharks catch was collected from three fishing rays for soup, celebratory dish in Asian cuisines port at north Java for period of 2011-2018. Shark and more specifically of the pre-branchial catch operation by fishermen at Java sea were appendages or gill rakers of mobulid rays (Family mostly using bottom gillnet, locally called as Mobulidae) is reality. Also the demand for ‘liong bun’ net. Morphology and morphometric medicinal purposes had drives to uncontrolled measurement involving of the main taxonomical exploitation of the vulnerable elasmobranch measurements such as Total Length, Standard fishes [6-9]. One of rare demersal shark catch Length, fin length, distance between dorsal fin-I from Java sea is Rhina ancylostoma. and II, caudal fin and ventral finand ratios [11]. The International Union for Conservation of Nature (IUCN) had classified R. ancylostoma DNA extraction, amplification and molecular as in category of vulnerable since it was widely identification of fish specimen. Tissue sampling caught by artisanal and commercial fishery and collection of fish specimen using scalpel and especiallyfor its valuable fins. As by-catch of pinset and sterilized using ethanol 96% bottom trawlersand tuna long liners it was washing with sterilized seawater. Genomic DNA 20 Hartoko et al.; ARRB, 35(4): 19-31, 2020; Article no.ARRB.57207 Fig. 1. Bathymetry of Java Sea and fish landing site extraction using modified Chelex 100 method electrophoresis method using 1% agarose and [12,13]. One gram of sample of muscle tissue put gel-rad colouring. PCR specimen with DNA in the Eppendorf tubes filled with 500 µl of visualization then purified and sequenced at Chelex 10% solution combined with 7 µl Genetica Science Indonesia company. proteinase K (10 mg/ml). Specimen sample then Examination on result of forward and reverse incubated in heating block at 55℃ for 90 minute sequence, sorted and edited using Mega 7.0 for DNA release, followed with 100℃ for 20 software [13,15]. Then matched up with minute for proteinase-K deactivation and GenBank database at NCBI using Basic Local centrifuge at 9,000 RPM for 15 minute. Yield of Alignment Search Tool (BLAST) analysis. DNA supernatant transferred intoeppendeorf Phylogenetic tree was set using Neighbor- tube and stored in 4℃ for further process. PCR Joining method, Kimura-2 parameter model and amplification. Part of Mithocondrial DNA COI 1,000 bootstrap replication. Some sequence from (Cytochrome Oxidase subunit –I) gen was NCBI Gen Bank were inputted into phylogenetic amplified using Polymerase Chain Reaction tree as comparison. (PCR) method. 3. RESULTS AND DISCUSSION An amount of 26 µl mix solution of 1µl DNA template, 12.5 µl Bioline My Taq™ HS Red Mix, 3.1 Morphology 1.25 µl forward primer 10 mM, 1.25 µl reverse primer 10 mM and 10 µl aquabidest. Primer As widely known that teleost group of Gobiidae used was Fish BCL: 5’-TCA ACY AAT CAY AAA as the most rich species group with about 2,228 GAT ATY GGC AC-3’ forward and Fish BCH: 5’- species found in the tropical seas of Iran, ACT TCY GGG TGR CCR AAR AAT CA-3’ Malaysia, Indonesia and Fiji [16]. The species of reverse [13,14]. The mix then put into Thermal Rhina ancylostoma group of elasmobranchia Cycle for PCR process, with pre-denaturation at Rhinidae inhabit the bottom of up to 200 m 95 ℃ for 5 minute, followed with 38 cycles of depth, was regarded as one rare and not well denaturation at 94℃ for 30 second. Annealing documented species and references. Unlike the process at 48 ℃ for 30 second then extention at case of Mediteransea receiving many migratory 72℃ for 45 second and final extention at 72℃ for and climate cycle changes species [17]. The 5 minutes. Sample of PCR then visualizedto demersal elasmobranch of R. ancylostoma in the determine the presence DNA band by study was regarded as a semi enclosed 21 Hartoko et al.; ARRB, 35(4): 19-31, 2020; Article no.ARRB.57207 ecosystem distributed in a shallow Java sea (Fig. and