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Molecular Characterization of Anisakis typica (Nematoda: Anisakidae) from the Mediterranean Sea Coasts of Turkey: First Mitochondrial rrnS Sequence Data

: There is no study about the molecular characterization of the mitochondrial rrnS gene of Anisakis typica from the Turkish Mediterranean coasts. Thus, molecular characterization of the rrnS gene of A. typica from Turkish waters is aimed for the first time in the present study. Ascaridoid marine nematodes were collected and morphologically identified by light microscope. Identified Anisakis type I larvae were randomly selected and molecularly analysed by Restriction Fragment Length Polymorphism (RFLP) for the internal transcribed spacer (ITS) region. Anisakis typica larvae identified by PCR-RFLP analyses were amplified and sequenced with both directions for the rrnS gene. The rrnS sequence of A. typica from the Mediterranean coasts of Turkey (MT395672) showed 99.80% identity (100% query coverage) with rrnS gene of A. typica (JX500052) from the Caribbean Sea, Atlantic Ocean. Between A. typica from the Mediterranean Sea (MT395672) isolate and the Caribbean Sea isolate differed by only one nucleotide (C‒T, at alignment position 437). Pairwise genetic distance between the rrnS sequence of A. typica herein and other A. typica from the Caribbean Sea was 0.002. Consequently, we determined the mitochondrial rrnS data of A. typica from the Mediterranean Sea in the present study for the first time. The valid genetic data (MT395672) reported here can be used to molecular identification of Anisakis species from the Mediterranean Sea and worldwide.


INTRODUCTION
Adult nematodes of Anisakis Dujardin, 1845 are mainly found in the gastrointestinal canal of marine mammals. Different species of marine mammals and fish and squids serve as definite and intermediate or paratenic hosts, respectively. Until now based on molecular genetic markers such as nuclear and mitochondrial DNA, nine distinct Anisakis species have been reported in the world. Among the mitochondrial markers, the cytochrome oxidase I (cox1), II (cox2) and the small subunit of rRNA (rrnS) genes are often prepared for molecular characterizations of Anisakis species. The specimen of A. typica has been widely distributed in definitive and intermediate or paratenic hosts at marine waters between 35-40°N to 36°S as geographic coordinates (Mattiucci & Nassetti 2008, Mattiucci et al., 2018. Both mitochondrial cox2 and nuclear ITS data of Anisakis species from marine fish were previously reported from coasts of Turkey (Pekmezci et al., 2014). Moreover, molecular characterization of rrnS loci of only Hysterothylacium aduncum and Contracaecum overstreeti among ascaridoids nematodes of marine fish was made in the Turkish waters (Pekmezci & Yardimci, 2019;Pekmezci, 2019). Up to date, there is no study about the molecular characterization of the mitochondrial rrnS gene of A. typica from the Turkish Mediterranean coasts. Therefore, the molecular characterization of the rrnS gene of A. typica from Turkish waters is aimed for the first time in the current study.

Morphological examinations, PCR and RFLP analysis:
Nematodes were collected from Merluccius merluccius in the Mediterranean coasts of Turkey. These were individually cut into three parts. The anterior and posterior parts were used for morphological identifications. The middle parts were used for DNA extractions using commercial kits. Nematodes were morphologically identified by light microscope according to Berland, (1961) and Petter and Maillard, (1988). Morphologically identified representative specimens were randomly selected and genetically analysed. The ITS regions of nuclear DNA were amplified using NC5/NC2 primers (Zhu et al., 1998). The ITS regions were then digested with HhaI and HinfI enzymes using RFLP analysis to identify the species (D'Amelio et al., 2000). All PCR amplicons and RFLP patterns were electrophoresed on 2% gel and visualized under UV illumination. The rrnS gene of the mitochondrial DNA of the selected Anisakis species was amplified with MH3/MH4.5 primer pairs (D'Amelio et al.,

DNA Sequencing and Genetic analysis:
Selected three individuals were sequenced both directions with MH3/MH4.5 primer pairs using Sanger methods for the rrnS gene. We checked the sequence quality, assembled and then trimmed to remove primers in Geneious R11 (Kearse et al., 2012). The assembled sequence was blasted in GenBank database to examine the nucleotide similarity (Altschul et al., 1990). Obtained rrnS data from Genbank were aligned by ClustalW in MEGA X multiple sequence alignments (Kumar et al., 2018) and adjusted manually. Pairwise distances were estimated using the K2P model in MEGA X (Kumar et al., 2018).

RESULTS
Ascaridoid nematodes were morphologically identified as third stage of Anisakis type I larvae. Some specimens of Anisakis larvae were classified as A. typica by RFLP analyses with HhaI and HinfI enzymes as previously described by D'Amelio et al., (2000). The amplified rrnS gene of A. typica was produced ~500 bp in the PCR analyses. After DNA sequencing of rrnS gene and trimmed to primers, the 494 bp length products were obtained in the present study. There were no intraspecific nucleotide differences detected within rrnS gene of three representatives. Therefore, the rrnS sequence of one representative was submitted to GenBank was given the accession number: MT395672. Nucleotide difference in the rrnS sequences between A. typica from the Mediterranean Sea (MT395672) and the Caribbean Sea differed by one nucleotide (C-T, at alignment position 437) (Figure 1). Pairwise genetic distance between the rrnS sequence of A. typica herein and other A. typica from the Caribbean Sea was 0.002.

DISCUSSION
This study represents first data on the molecular characterization of the rrnS gene region of A. typica isolated from the Mediterranean Sea. The sequence analysis of the rrnS loci of A. typica was firstly characterized from the Caribbean Sea, Atlantic Ocean by Mattiucci et al., (2014) and previously submitted as accession number JX500052 in GenBank database. A new valid rrnS sequence of A. typica (accession number MT395672) in the present study was the second record in the GenBank database. Our rrnS sequence of A. typica from the Mediterranean coasts of Turkey (MT395672) showed 99.80% identity (100% query coverage) with rrnS gene of A. typica (JX500052) from the Caribbean Sea, Atlantic Ocean (Mattiucci et al., 2014). Moreover, a search in GenBank database showed that rrnS sequence of A. typica in the present study were ranged from 88.91% to 91.11% identical to rrnS sequences of A. physeteris pegreffii (LC222461) and A. simplex (AY994157). These results showed that mitochondrial rrnS gene could be used effectively in the genetic distinction of Anisakis species. Pairwise genetic distance for mitochondrial rrnS loci showed a very low-level variation among the Mediterranean Sea isolate of A. typica (MT395672) herein and the Caribbean Sea isolate of A. typica previously found (JX500052) (p distance=0.002). We considered the very low level of nucleotide differences (0.2%) between those isolates of A. typica as intraspecific nucleotide differences because of the different geographical locations.

CONCLUSION
We determined the mitochondrial rrnS data of A. typica from the Mediterranean Sea in the present study for the first time. Moreover, the new valid rrnS sequence (accession number MT395672) was the second record in the GenBank and this unique data can be also used to molecular identification of Anisakis species from the Mediterranean Sea and worldwide.