Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021 DOI : 10.20473/jafh.v10i1.20905 Molecular Identification of the Genus Molicola Larvae from Swordfish (Xiphias gladius) Captured in Sri Lanka by Ribosomal Subunit Gene Sequencing D.P.N. De Silva1, 2*, J.L.C.S. Perera3, H.S.D. Fernando1, R.R.M.K.P. Ranatunga1 and B.G.D.N.K. De Silva1 1Center for Biotechnology, Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka 2Department of Animal Science, Faculty of Animal Science and Export Agriculture, Uva Wellassa University, Badulla, Sri Lanka 3Laboratory of Veterinary Public Health, Graduate School of Agricultural & Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan *Correspondence : Abstract [email protected] Swordfish (Xiphias gladius) is a migratory fish Received : 2020-07-22 commercially exploited due to its high export value. The Accepted : 2020-09-05 presence of parasites in fish leads to economic losses in the export market and public health issues. This study was Keywords : conducted to identify the parasite larvae inhabiting swordfish Molicola, Swordfish, and to determine its phylogenetic origin using ribosomal Trypanorhyncha, rRNA subunit gene sequence. Parasite samples were extracted from swordfish muscles and five larvae belong to Molicola genus, confirmed by scolex morphology, were used for genomic DNA extraction. Polymerase chain reaction (PCR) was performed to amplify 18S and 28S ribosomal RNA (rRNA) subunit genes followed by Sanger sequencing. DNA sequences were edited by BioEdit software and assembled by CLC genomics version 8.0. Consensus sequences were aligned with NCBI blast to determine the species status. Isolated larval sequences were best aligned with genus Molicola followed by genus Gymnorhyncha. Out of the two published Molicola rRNA gene sequences, 99% identity was observed with Molicola sp. HP5 isolate from Indonesia. Due to the lack of sequence data on other Molicola species (except M. thyristes) for comparison, our sequences were published as Molicola sp. Sri Lankan isolates. This is the first record of Molicola sp. in swordfish from Sri Lanka and the results will enhance the knowledge on the distribution of Molicola species while contributing to expanding the genetic information on rRNA coding sequences. INTRODUCTION Capture fisheries provide 65% of the Among food fish Tuna species is the animal protein requirement of the people highest catch while swordfish (Xiphias in Sri Lanka. According to the fisheries gladius) is one of the seasonal fish which statistics in 2016, its contribution to gross has higher demand from importing domestic production (GDP) was around countries (Ministry of Fisheries & Aquatic 1.8 % (marine 1.6 %, inland 0.2 %). Resources Development, 2019). Swordfish De Silva et al. (2021) 66 Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021 DOI : 10.20473/jafh.v10i1.20905 is also called Broadbill, belongs to the discarded due to parasitic infestation in family Xiphiidae of order Perciformes. muscles. A whitish, thread-like parasite They can be found in the Indian, Atlantic found in muscles often mistook as the and Pacific oceans as well as in the zoonotic Anisakis species until a Mediterranean, Marmara, Black, and Azov preliminary study using morphological seas (FAO, 2019). Due to over-fishing, this evaluation of this parasite revealed as species reduced by 28% over the past two plerocercoid larvae of the genus Molicola decades (IUCN, 2019). Swordfish migrate belong to order Trypanorhyncha (De Silva to cold waters for feeding and returns to et al., 2017). But due to higher warm waters during summer for morphological similarity within the genus spawning. They eat small to relatively Molicola, this study was aimed at the large fishes and occasionally consume molecular identification methods to crustaceans and squids (Williams and identify up to the species level and to Bunkley-Williams, 1996). These feeding construct its phylogenetic tree. and breeding patterns increase their risk of The main morphological getting exposed to parasites. Most of the characteristics of order Trypanorhyncha marine helminths, especially are, scolex (with two or four bothria) and Trypanorhyncha species have multiple tentacular apparatus (consists of four hosts during their life cycle including tentacles covered with hooks and attached swordfish and crustaceans as intermediate to four bulbs by tentacle sheaths) (Palm et hosts (Palm and Caira, 2008). al., 2009). There are about 277 valid The parasitism in fish is an Trypanorhyncha species identified so far ecological coexistence between two and new species are being added organisms in close contact. Some parasites continuously. Based on a more detailed establish an equilibrium of host-parasitic cladistic analysis, five Trypanorhyncha relationship being encysted or dormant superfamilies and 15 families were found. until it gets a favorable condition to The five super families are, multiply (Schaperclaus, 1992). It is found Tentacularioidea, Eutetrarhynchoidea, that there are 49 parasitic species infecting Gymnorhynchoidea, Lacistorhynchoidea, swordfish. About 13 of them are host- and Otobothrioidea (Palm, 1997). specific while six are super family-specific. Taxonomic reports of the genus Swordfish harbors about 20 larval Molicola indicated the presence of tapeworm species particularly due to the parasitic larvae in teleost muscles and consumption of intermediate hosts liver. Sunfish (Mola mola) were reportedly infested with parasitic larvae. There are 14 infected by Molicola in France, the cestodes (tapeworms) recorded in Mediterranean region, Japan, New swordfish including Trypanorhynch Zealand, India, and Canada. Molicola was (Williams and Bunkley-Williams, 1996). also reported on muscles of Thyrsites sp. Parasites infesting swordfish from Holland (Knoff et al., 2004). Molicola muscles include Pennella sp. (Copepod) horridus possess elongated scolex, four (Hogans et al., 1985; Castro-Pampillόn et auriculate and elongated bothridia which al., 2002), cestode Trypanorhyncha is curved and apically inclined with round- plerocercoid larvae of Molicola edged, thick rims that are highly similar to (Gymnorhyncus) horridus, and the morphological examination of the Sri Gymnorhynchus gigas, and flukes, Lankan isolate of Molicola sp. reported Maccalumtrema xiphiados. A zoonotic previously (Knoff et al., 2004; De Silva et parasite Anisakis nematode larvae were al., 2017). But there are other Molicola also reported in swordfish muscle (Hogans species (Molicola uncinatus (also called et al., 1983; Castro-Pampillόn et al., 2002; Molicola thyrsitae) (Johns et al., 2009) and Garcia et al., 2011). Molicola walteri) which share similar In Sri Lanka, swordfish catch is morphological characteristics (Palm, seasonal, and a large amount of flesh was 2004), indicating the necessity of https://e-journal.unair.ac.id/JAFH 67 De Silva et al. (2021) Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021 DOI : 10.20473/jafh.v10i1.20905 molecular identification for the Biotechnology, Department of Zoology, determination of species. University of Sri Jayewardenapura, Sri In Sri Lanka, very few studies have Lanka, where all morphological analysis, been conducted on marine capture fish DNA extraction and polymerase chain diseases. The presence of cestode and reaction (PCR) were performed. PCR nematode parasites from Sri Lanka was products were sent to Microgen, Korea for reported as early as 1906 (Shipley and the Sanger sequencing. Hornell, 1906). The earliest report of Trypanorhyncha species from Sri Lanka, Research Material named Halysiorhynchus microcephalus was The whitish, thread-like parasitic isolated from Himantura imbricate (Scaly samples obtained from swordfish muscle Whipray) from the Sri Lankan coast were confirmed as Molicola plerocercoid (Southwell, 1929). A specimen at the larvae by morphological analysis. To British Museum of Natural History, which examine the morphology of the parasite was received from Sri Lanka had been using wet mounts, compound light identified as another Trypanorhynch microscope (Olympus, CX22LED, U.S.A.) larva, Pseudo-gilquinia pillersi (Beveridge and microscope slides were utilized and et al., 2007). Since there are a number of the parasitic samples were fixed in 10% Trypanorhynch parasites inhabiting formalin 1.5 ml. Five larvae were various host species, it was a timely confirmed as genus Molicola based on necessity to identify the parasitic species their scolex characteristics and used for present in swordfish muscles. genomic DNA extraction. For DNA This study focused on the extraction, reagents mentioned in the identification of Molicola plerocercoid methods of Ballinger-Crabtree et al. larvae up to the species level and to (1992) were used. develop a phylogenetic tree to determine In order to amplify ribosomal its origins. Ribosomal RNA genes of 18S subunit genes, primers were designed for and 28S subunits were sequenced for 18S rRNA (ssrRNA) and 28S rRNA comparative analysis due to their (lsrRNA) gene sequences as shown in conserved nature during evolution. Based Table 1. Primer sequences and PCR on this study Molicola sp. Sri Lankan protocols for 18S rRNA and 28S rRNA isolates found in swordfish enhances the gene amplification were based on Palm et rRNA gene database of Trypanorhyncha al. (2009) with slight modifications. parasites. PCR reagents such as magnesium chloride (MgCl2), deoxyribose nucleotide METHODOLOGY triphosphate