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 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 , 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 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

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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 (dNTP), buffer (5x), Taq Place and Time DNA polymerase (University of Colombo, Parasitic samples from the muscles Sri Lanka) were used to prepare samples of swordfish (captured from the FAO for amplification. Thermal cycler (Master statistical zone 57 of the Indian Ocean), cycler personal, Eppendorf, USA) was were obtained from a fish processing used to run the PCR. Agarose gel factory in Sri Lanka. Samples were (Promega, USA) was used in gel collected twice during 2015 and 2016, electrophoresis. ABI sequencer (U.S.A.) directly from the fish processing factory was utilized to perform Sanger sequencing and transferred on ice to the Center for at Microgen, Korea.

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Table 1. Primers used to amplify and sequencing of 18S rRNA and 28S rRNA gene segments. Primer type Primer ID Primer sequence ssrDNA (18S rRNA gene) Forward primer ssrDNA F1 5`-GCGAATGGCTCATTAAATCAG-3` Reverse primer ssrDNA R1 5`-CTTGTTACGACTTTTACTTCC-3` Internal primers 300F2 5`-AGGGTTCGATTCCGGAG-3` 600R 5`-ACCGCGGCKGCTGGCACC-3` 1270F 5`-ACTTAAAGGAATTGACGG-3` 930F 5`-GCATGGAATAATGGAATAGG-3` 1200F 5`-CAGGTCTGTGATGCCC-3` lsrDNA (28S rRNA gene) Forward primer lsrDNA F1 5`-ACCCGCTGAATTTAAGCATAT-3` Reverse primer LsrDNA R1 5`-GCTATCCTGAGGGAAACTTCG-3` Internal primers 300F 5`-CAAGTACCGTGAGGGAAAGTTG-3` ECD2 5`-CTTGGTCCGTGTTTCAAGACGGG-3` 400R 5`-GCAGCTTGACTACACCCG-3` 1090F 5`-TGAAACACGGACCAAGG-3`

Research Design distilled water to make it 25µl of total Parasitic samples from swordfish volume. muscles were obtained by adhering to the The thermocycler was run according complete randomization method. A total to the following conditions: To amplify of 20 parasitic samples was obtained for 18S rRNA gene, initial denaturation at morphological diagnosis and from them, 94°C for 2 minutes followed by 40 cycles five confirmed Molicola samples (by scolex of 94°C, 30 seconds, 54°C, 30 seconds and morphology) were selected for DNA 72°C, 2 minutes. For 28S rRNA gene extraction followed by a molecular amplification, 95°C, 5 minutes initial examination. Each parasitic sample was denaturation followed by 40 cycles of tested for both 18S rRNA and 28S rRNA 95°C, 30 seconds, 55°C, 30 seconds and genes and sequences were compared for 72°C, 2 minutes. Both PCRs were finally determination of their individual extended at 72°C for 7 minutes before it variations and phylogenetic distance. was kept at 4°C until electrophoresis. Final PCR products were observed by one Work Procedures percent (1%) Agarose gel (Promega, USA) electrophoresis. Genomic DNA from five parasites All amplicons of 18S rRNA and 28S was separated into two sets for the rRNA genes were sequenced by the Sanger detection of two regions of the ribosomal method (ABI sequencer at Microgen, RNA gene, 18S rRNA and 28S rRNA Korea) and sequences were analyzed to respectively. PCR reaction was performed confirm the species and constructed the using the reagents and amounts as below: phylogenetic tree. Total volume of the PCR product was 25 µl. Genomic DNA 2 ng were used from Data Analysis each sample as the template. Double Poor quality bases on both ends of distilled water was added as the negative control. PCR master mix was prepared by the sequences were trimmed by using Bio adding 1 µl each from forward and reverse Edit software version 7.2.5 (Hall, 1999). primers, 6 µl of magnesium chloride Multiple alignments was performed using Clustal W and assembled by de novo (MgCl2), 1.25 µl deoxyribose nucleotide triphosphate (dNTP), buffer (5x) 5 µl, Taq assembly of contigs by CLC Bio genomics work bench version 8.0 (www.clcbio. DNA polymerase (University of Colombo, Sri Lanka) 0.2 µl into 8.55 µl of double com). Consensus sequences were compared with the reference sequences https://e-journal.unair.ac.id/JAFH 69 De Silva et al. (2021) Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021 DOI : 10.20473/jafh.v10i1.20905 deposited in National Center for The amplicon for 18S rRNA and 28S Biotechnology Information (NCBI) by rRNA gene, obtained by PCR using region- basic local alignment tool (BLAST). specific universal primers for trematodes Phylogenetic analysis and the taxonomic showed electrophoresis band size over tree were performed by using Molecular 1500 bp which formed the consensus Evolutionary Genetics Analysis (MEGA) sequences of 18S rRNA and 28S rRNA version 6.0 (Tamura et al., 2013) genes of about 2030 bp and 1500 bp neighbour-joining method (Saitou and respectively. Based on the sequence Nei, 1987). analysis of 18S rRNA and 28S rRNA gene, all five isolates from Sri Lanka were RESULTS AND DISCUSSION aligned to Molicola sp. HP5 (Indonesia) Morphological identification of the with 99% and 100% identity, followed by parasitic larvae found on swordfish muscle Molicola thyristes isolate Moli (Australia) was reported previously by our research and Gymnorhynchus isuri. All three species group as Molicola plerocercoid larvae belonged to the family Gymnorhynchidae belong to the Order Trypanorhyncha (De (Table 2). Silva et al., 2017).

Table 2. Species comparison using NCBI blast of 18S and 28S rRNA gene sequences of Sri Lankan isolates with species belong to superfamily Gymnorhynchoidea. 18S rRNA 28S rRNA Species name Family NCBI NCBI Identity Identity accession accession Molicola sp. HP5 Gymnorhynchidae 99% FJ572913 100% FJ572949 Molicola thyristes Gymnorhynchidae 99% DQ642908 98% DQ642746 Gymnorhynchus isuri Gymnorhynchidae 98% DQ642909 97% DQ642747 Pintneriella Rhopalothylacidae 98% FJ572912 96% FJ572948 musculicola Chimaerarhynchus Gymnorhynchidae 95% DQ642906 93% DQ642744 rougetae Vittirhynchus squali Gilquiniidae 95% DQ642905 93% DQ642743 Sagittirhynchus Gilquiniidae 95% DQ642907 93% DQ642745 aculeatus Gilquinea robertsoni Gilquiniidae 95% FJ572910 93% FJ572944 Aporhynchus Aporhynchidae 95% FJ572911 93% FJ572947 norvegicus

This clearly confirmed that the samples, there were less than ten plerocercoid larvae isolated from nucleotide variations found (more than swordfish from Sri Lanka (Indian ocean) 95% identity) indicating that they all belonged to the genus Molicola, family belong to the same species. The significant Gymnorhynchidae. There were point identity of Sri Lankan isolates with mutations and insertions in 18S and 28S Molicola sp. Hp5 from Indonesia obtained rRNA gene sequences possibly due to the from Taractes rubescens, suggests that individual variations. The evolutionary swordfish captured near the Sri Lankan distance between the sequences of five coast might have migrated via Indonesian isolates was not significantly different (p coasts. Furthermore, both T. rubescens and > 0.05). Therefore, all the parasitic larvae swordfish are pelagic fish that shows isolated from swordfish in this study similar feeding patterns (Scott and Tibbo, belong to the same species. 1968; Gòmez-Morales et al., 2008), Though the analysis of 18S rRNA justifying the possible entry of Molicola and 28S rRNA gene sequences were larvae through their food chain. The other performed in five individual parasitic species of close identity with Sri Lankan https://e-journal.unair.ac.id/JAFH 70 De Silva et al. (2021) Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021 DOI : 10.20473/jafh.v10i1.20905 isolates was Molicola thyristes isolated Currently, in the NCBI database only two from Australia in Thyrsites atun. It is also Molicola species 18S and 28S rRNA gene an intermediate host of Molicola sp. which sequences were available for comparison. feeds on pelagic crustaceans (Nakamura Therefore, our results were submitted as and Parin, 1993). Molicola sp. SL 01 to 05. The country of Phylogenetic analysis of 18S rRNA origin, Sri Lanka was stand by ‘SL’ and one and 28S rRNA sequences of Molicola Sri to five indicates the individual Lankan isolates were compared with 20 plerocercoid larval isolates. The Sri Trypanorhyncha species belonged to Lankan isolates identified from this study different families. Based on the were deposited at NCBI GenBank with the phylogenetic analysis, the highest accession numbers for 18S rRNA partial significance with Molicola sp. HP5 isolate gene sequences starting from KX712332 to from Indonesia was observed and Sri KX 712336 and for 28S rRNA partial gene Lankan isolates shared the common sequences from KX712337 to KX712341. ancestor with members from the This is the first study on rRNA genes of Gymnorhynchidae family (Figure 1). The Molicola sp. infested in the muscles of comparison of both phylogenetic trees swordfish captured from the Sri Lankan showed similarity in 18S rRNA and 28S coast to the best of our knowledge. Further rRNA genes. Accordingly, it confirms the studies on other Molicola species are morphologically recognized genus of the needed to develop a better sequence parasite. Nucleotide sequences of 18S and comparison and speciation of the genus 28S ribosomal subunit gene of Molicola sp. Molicola.

A

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B

Figure 1. Evolutionary relationships of Molicola Sri Lankan isolates with other species belong to different taxonomic families of order Trypanorhyncha, obtained by Neighbor-joining method using (A) 18S rRNA and (B) 28S rRNA gene sequences. Scientific names of the species and their NCBI accession numbers were written at respective nodes and the branch lengths indicate the evolutionary distance drawn to a scale bar of 0.01 for 18S and 0.02 for 28S rRNA gene. E. harfordi belong to the order Diphyllidea was used as an outgroup.

It is mandatory to perform fish similarity to Molocola sp. HP5 from quality checks before exporting to other Indonesia. This is the first record of countries and the difficulty arose when molecular analysis of ribosomal subunit both Trypanorhyncha and Anisakis species genes in Molicola species isolated from look alike grossly in color and consistency swordfish captured near the Sri Lankan (Muscolino et al., 2012). Although coast. Molicola is not a zoonotic parasite as Anisakis studies on mice have shown ACKNOWLEDGMENT anaphylaxis due to Molicola horridus The authors would like to allergens (Gomez-Morales et al., 2008). acknowledge the staff members of the However, the chance of allergic reactions Center for Biotechnology, Department of to occur in consumers by using frozen Zoology, University of Sri swordfish slices is very low, but the Jayewardenapura, Sri Lanka. parasitized flesh reduces the consumer preference (Muscolino et al. 2012). Hence REFERENCES this method can be used for confirmatory Ballinger-Crabtree, M.E., Black, W.C. and and differential diagnosis of Miller B.R., 1992. Use of genetic Trypanorhyncha parasites from other fish polymorphisms detected by the parasites. random-amplified polymorphic DNA

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