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First Molecular Characterization of Hysterothylacium Fabri Larvae (Nematoda: Raphidascarididae) in the Mediterranean Sea Based on the Small Subunit Ribosomal RNA Gene Sequence

Hysterothylacium fabri is commonly reported in various Mediterranean fish. However, there is no data on the mitochondrial ribosomal RNA ( rrnS ) gene sequence of H. fabri larvae in the Mediterranean Sea waters. Therefore, we aimed to reveal molecular characterization of H. fabri based on the rrnS gene sequence in the current study. Firstly, the fourth stage of H. fabri larvae were identified at parasitological examination from Mullus barbatus in the Mediterranean Sea. Morphologically identified H. fabri larvae herein were also supported by the sequencing of the nuclear ribosomal ITS gene regions. In the next step, the rrnS gene of these larvae was molecularly analysed. Our H. fabri isolate (MK886659) showed 96.42% identity with H. fabri reported from China (MF140349) based on the rrnS gene. The nucleotide difference based on the rrnS gene between Chinese isolate (MF140349) and our H. fabri isolate (MK886659) was determined as 3.2%. Consequently, for the first time, the mitochondrial rrnS gene sequencing of H. fabri from the Mediterranean waters were performed in the current study.


INTRODUCTION
Hysterothylacium species are marine ascaridoids and over 70 recognizable species have been described in the worldwide (Moravec and Justine 2015). Hysterothylacium species were reported as larvae and adults in various marine teleost from the Mediterranean Sea (Barcala et al. 2018;Costa et al. 2018;Gazzonis et al. 2017;Keskin et al. 2015;Pekmezci et al. 2014;Petter and Maillard 1988;Roca-Gerones et al. 2018;Simsek et al. 2018;Szostakowska et al. 2001;Tedesco et al. 2018). Until now, five Hysterothylacium larval morphotypes (III, IV, V, VIII, IX) and H. reliquens, H. fabri, H. aduncum, and H. girgenti have been genetically identified using ribosomal and mitochondrial DNA markers in the Mediterranean waters (Barcala et al. 2018;Costa et al. 2018;Keskin et al. 2015;Pekmezci et al. 2014;Roca-Gerones et al. 2018;Simsek et al. 2018;Tedesco et al. 2018;Khammassi et al. 2020). There has been limited knowledge on the H. reliquens, H. fabri, and H. aduncum in marine teleost from Turkish Mediterranean waters (Keskin et al. 2015;Pekmezci et al. 2014;Simsek et al. 2018). Although there are molecular studies for the certain identification of marine ascaridoid species, the taxonomy of the genus Hysterothylacium in Mediterranean waters remains incomplete and unresolved (Costa et al. 2018). Moreover, there is no study on the rrnS gene sequence of molecular characterisation of H. fabri larvae in Mediterranean waters. Therefore, for the first time, we performed the molecular characterization of rrnS mitochondrial gene of H. fabri from the Mediterranean waters.

Fish Sampling, Parasite Collection, and Morphological Examination
For this study, ethics committee approval was not needed because no handling of live marine teleost specimens was involved. A total of forty-five fresh and dead Mullus barbatus (L.) caught from the Turkish Mediterranean coast were purchased from local fishermen between March 2018 to May 2018 and examined under a stereomicroscope for the presence of Hysterothylacium spp. larvae in the digestive tract and visceral cavity. All nematodes were mechanically removed, washed in saline, and stored in ethanol solution. A middle portion of nematodes was used for genetic identification and other portions were individually cleared with lactophenol for using morphological identification. The nematodes were identified using the location of the excretory pore, the digestive systems, and the posterior ends (Petter and Maillard 1988;Tedesco et al. 2018). A subsample of five specimens of the fourth stage of H. fabri larvae was randomly selected and subjected to molecular analysis using a small middle portion of the nematodes.

DNA Extractions, PCR Amplifications, DNA Sequencing, and Phylogenetic Analysis
Genomic DNA was extracted from individual nematodes using the DNA extraction kit (GeneJET Genomic DNA Purification Kit, Thermo Scientific, Waltham, MA, USA) following to the manufacturer's instructions. The PCR amplification targeting the nuclear ribosomal ITS gene and the mitochondrial ribosomal RNA (rrnS) gene were performed with the primers NC5/NC2 and MH3/MH4.5 primer pairs, respectively (D'Amelio et al. 2007;Zhu et al. 1998). For ITS gene, PCR mixture was prepared according to previously described by Pekmezci et al. (2014). PCR was carried out in a final volume of 50 μl, containing 10-50 ng of extracted DNA, 1× Taq Buffer with KCI (Thermo Scientific), 3 mM of MgCl2 (Thermo Scientific), 0.3 mM dNTPs (Thermo Scientific), 2 pmol of each primer, 2.5 U of Taq DNA Polymerase (Thermo Scientific), and DEPC-treated water. The PCR conditions were as follows: 15 min at 95 °C, then 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C followed by a final elongation of 5 min at 72 °C. For rrnS gene, PCR mixture was prepared according to previously described by D' Amelio et al. (2007) and PCR reaction (50 μl) was contained 20-40 ng of extracted DNA, 10 mM Tris-HCl (pH 8. 3), 50 mM KCl (Thermo Scientific), 3 mM MgCl2 (Thermo Scientific), 1 mM of dNTPs (Thermo Scientific), 50 pM of each primer, 0. 5 ml of Taq DNA Polymerase (Thermo Scientific). The PCR conditions were as follows: 10 min at 95 °C, then 35 cycles of 30 sec at 95 °C, 30 sec at 55 °C and 30 sec at 72 °C followed by a final elongation of 7 min at 72 C. Positive PCR products were purified and sent to Macrogen company for DNA sequencing. The raw sequence data were analysed using Geneious R11 (Kearse et al. 2012). The contigs were compared with all Hysterothylacium species sequences using the BLASTn algorithm via GenBank (Altschul et al. 1997). Pairwise distances using a Kimura 2-parameter and best of the nucleotide substitution model were calculated in MEGA 7.0 (Kumar et al. 2016). The TN93+G+I model for rrnS gene sequences was selected using Akaine Information Criterion. The rrnS datasets were used for maximum likelihood phylogenetic tree by bootstrap of 1000 replicates (Felsenstein 1985). The newly generated rrnS sequence was deposited in the GenBank under the accession number: MK886659.

RESULTS
The prevalence of the Hysterothylacium larvae in the Mediterranean Sea was detected as 10% (4/40) in M. barbatus. The morphology of selected specimens agrees well with the description of the fourth stage of H. fabri larvae (Figure 1).

DISCUSSION
The paper on morphological identification of Hysterothylacium larvae published by Petter and Maillard (Petter and Maillard 1988) described the third (L3) and fourth (L4) stage of H. aduncum larvae, L3 and L4 of H. fabri larvae, L4 of H. arnoglossi larvae and L3 and L4 of Hysterothylacium sp. I and II larvae from the Mediterranean Sea. Recently, L3 and L4 stage and adult of H. fabri were also morphologically identified from the Mediterranean Sea (Tedesco et al. 2018). In the present study, our specimens were morphologically identified as L4 of H. fabri according to the identification key (Petter and Maillard 1988;Tedesco et al. 2018). Moreover, the morphological identification of H. fabri reported herein was also supported by molecular analyses. Our ITS regions sequence of L4 of H. fabri isolate displayed 98.90-100% identity (97% query cover) with the ITS sequences of H. fabri (JX974558, MH211474-94, MF539787-89, JQ520158, KU948632-37, KC852206) from the Mediterranean Sea, South Korean waters, and Chinese waters Pekmezci et al. 2014;Tedesco et al. 2018;Zhang et al. 2018). Therefore, previously morphologically classified L4 of H. fabri larvae were also genetically confirmed as H. fabri based on the ITS regions. Herein, for the first time, we also determined the rrnS mitochondrial sequences of H. fabri from the Mediterranean waters. However, there was one sequence for the rrnS gene of H. fabri (MF140349) recorded previously from China deposited in GenBank for comparison . Interestingly, the %3.2 genetic distance was observed among rrnS sequences of H. fabri comparable to those reported ) and the present study. Nucleotide identity of the ribosomal and mitochondrial DNA higher than 96% for Anisakidae and Raphidascarididae are considered to be the same species (da Fonseca et al. 2016). Moreover, other known H. aduncum species exhibited low level of intraspecific nucleotide differences (p distance=0.004 to 0.009) between the Black Sea (MK886768) and the Mediterranean Sea isolates (MF000685-MF000691) for rrnS gene, whereas; high intraspecific genetic diversity (p distance=0.021) in the same species was also detected between Black Sea (MK886768) and China (MF140344) isolates (Pekmezci 2019). Therefore, the rrnS mitochondrial nucleotide differences between the China and our Mediterranean isolates of H. fabri should be considered as intraspecific nucleotide variation. Additionally, there may be intraspecific nucleotide differences between the remote communities of the same Hysterothylacium species from different geographic area. Moreover, several authors accept H. fabri collected from marine fish in the Mediterranean and Chinese waters to be a complex comprising at least two or three sibling species (Guo et al. 2014;Martin-Sanchez et al. 2003).
In the present study, high nucleotide variability (3.2 % nucleotide difference) was detected in the rrnS sequences of Mediterranean (MK886659) and the China (MF140349) isolates of H. fabri. Thus, we also considered H. fabri as sibling species according to rrnS gene. In conclusion, for the first time, we determined the mitochondrial rrnS data of H. fabri from the Mediterranean Sea in the present study. These molecular results provided species-specific genetic identification of H. fabri from the Mediterranean waters based on rrnS gene. More studies are needed to investigate the nucleotide variability by using various gene markers both larvae and adults of Hysterothylacium species from the Mediterranean waters.
Financial Support: This research received no grant from any funding agency/sector.

Ethics Committee Information:
This study is not subject to the permission of HADYEK in accordance with Article 8 (k) of the "Regulation on Working Procedures and Principles of Animal Experiments Ethics Committees".