First morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from marine fishes in Vietnamese water

Abstract Anisakid nematodes are parasites of cetaceans, their larval stages live in marine fishes. The third-stage larvae of some Anisakis species are also the etiological agents of human anisakiasis caused by consumption of raw or undercooked infected fish. Thus, identiﬁcation of Anisakis larvae at the species level is crucial for their ecology and epidemiology. In Vietnam, although Anisakis larvae have been reported, they have not been identified to the species level. The aim of this study was, therefore, to identify third-stage larvae of Anisakis collected from marine fishes in Vietnamese water, based on morphological characteristics and molecular analysis. All Anisakis larvae found in this study were morphologically similar to each other and identical to Anisakis typica. In addition, molecular analysis based on ITS1-5.8S-ITS2 sequences confirmed them as A. typica. Vietnamese A. typica population was genetically close to those from Asian countries and Australia. The third-stage larvae of A. typica were collected from eight fish species from three localities in the South of Vietnam. Among them, seven were recorded as new intermediate hosts of A. typica. This is the first identification of A. typica larvae in Vietnamese water with records of new fish hosts.

Nematodes of the genus Anisakis (Nematoda: Anisakidae) are parasites of marine organisms. The life cycle of these nematodes requires marine mammals, mainly cetaceans, as the definitive hosts, and crustaceans, fish, and cephalopods as intermediate/ paratenic hosts (Klimpel and Palm, 2011). Humans are accidental hosts due to ingestion of raw or undercooked fish containing the third infective-stage larvae (L3). Human anisakiasis patients suffer from abdominal pain, nausea, vomiting, and/or diarrhea (Dorny et al., 2009). In addition, allergic reactions may occur due to exposure to the nematode antigens (Aibinu et al., 2019;Audicana et al., 2002). Given the influence on human health, Anisakis nematodes are of interest. Anisakid larvae can be morphologically identified at the genus level by typical characteristics of anterior and posterior regions, and are classified into two types, type I and II, based on the length of the ventriculus and presence/absence of the tail spine (mucron): Anisakis type I larva has a longer ventriculus and a mucron, while type II larva has a shorter ventriculus and no mucron (Berland, 1961). Type I consists of A. simplex, A. pegreffii, A. typica, A. ziphidarum, and A. nascettii, while type II consists of A. paggiae, A. physeteris, and A. brevispiculata (Mattiucci and Nascetti, 2008). Previously, it was not easy to identify anisakid larvae at the species level, because there is a lack of distinct morphological characteristics First identification of Anisakis typica larvae in Vietnam: Hien et al. required for species identification (Farjallah et al., 2008). Recent studies indicated morphological differences between Anisakis species (Chen and Shih, 2015;Sonko et al., 2019;Tunya et al., 2020). In addition, molecular tools allow the accurate identification of anisakid larvae by using sequences of the internal transcribed spacer (ITS) region of ribosomal DNA Mattiucci and Nascetti, 2008).
In Vietnam, data on Anisakis nematodes are scarce. There have been a few reports on Anisakis larvae without morphological description and identification to species level (Arthur and Te, 2006;Ngo et al., 2009). During our recent comprehensive survey for parasites of marine fishes in Vietnamese water, we collected Anisakis larvae from eight fish species. The aim of the present study was to identify these Anisakis specimens from Vietnamese water by morphological and molecular approaches.

Fish examination and larval collection
Marine fish were bought in 10 fish ports located in 10 provinces along the seashore of Vietnam where fishing vessels docked (Fig. 1). All fish specimens were placed on ice and transferred to the laboratory under good aeration. Fish were dissected, their body cavities and internal organs were examined under a stereomicroscope. Third-stage larvae were isolated from the body cavity and visceral organs. The larvae were washed in phosphate-buffered saline. For morphological identification, larvae were preserved in 4% formalin. Representative specimens were preserved in 70% ethanol for DNA isolation.

Molecular and phylogenetic analysis
DNA of three representative larvae from three localities was extracted using QIAamp DNA stool Minikit (Qiagen, Hilden, Germany). Two primers NC5-GTAGGTGAACCTGCGGAAGGATCATT (forward) and NC2-TTAGTTTCTTTTCCTCCGCT (reverse) were used in a polymerase chain reaction (PCR) to amplify the rDNA region of the first to the second internal transcribed spacer (ITS1-5.8S-ITS2) (Zhu et al., 2000). PCR products were electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide staining. Positive PCR products were sent to Macrogen Company (Korea) for sequencing. The nucleotide sequences obtained in this study were deposited in GenBank under accession numbers LC592876-LC592878.
BLAST searches were performed at NCBI (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) to find sequence similarities. Sequences of Anisakis species available in GenBank were downloaded for analysis. The analysis involved 42 nucleotide sequences, including a sequence (KM491173) of Contracaecum osculatum as an out-group. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model in MEGA software v.7.0. (Kumar et al., 2016). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter = 0.8044)). All positions containing gaps and missing data were eliminated. There were a total of 656 positions in the final dataset.

Results and discussion
A total of 3,775 fish of 138 species from 10 study sites were examined. Anisakis larvae were found from eight fish species from three localities, Khanh Hoa, Vung Tau, and Bac Lieu, in the South of Vietnam. Prevalences of infection ranged from 10 to 50% with intensity varied from 1 to 19 larvae/fish (Table 1), non-infected fish species were presented in the supplemental material.
All Anisakis larvae were morphologically similar to each other. The body of the larvae was cylindrical in shape, attenuated at both ends, and measured 17.2 to 20.3 (18.6 ± 1.1) mm long and 0.26 to 0.34 (0.29 ± 0.03) mm width (n = 30 larvae). The lips were inconspicuous, with a prominent boring tooth at the anterior extremity. The esophagus had an anterior muscular part and measured 1.52 to 1.58 (1.54 ± 0.02) mm long and a glandular ventriculus measured 0.58 to 0.82 (0.64 ± 0.04) mm long. Long intestinal caeca with clear demarcation were present. The body of larvae ended at a short cylindrical mucron measuring 0.021 to 0.030 (0.025±0.005) mm long (Figs. 2, 3). These characteristics of the third-stage larvae were identical to Anisakis larvae type I (Berland, 1961). It has previously been noted that it is difficult to distinguish between Anisakis species belonging to type I because they look quite similar to each other (Farjallah et al., 2008). However, recent studies based on morphological and molecular approaches provided descriptions and microphotographs showing differences between L3 larvae of A. pegreffii and A. typica (Chen and Shih, 2015;Sonko et al., 2019). In addition, Tunya et al. (2020) suggested that the protruded mucron of L3 larvae can be used to identify anisakid larvae at the species level: the protruded mucron of A. simplex was cone-shape, while that of A. typica was cylindrical-shape, which is narrower and longer than that of A. simplex. According to these, L3 larvae of Anisakis specimens found in this study were identified as A. typica. Because the Anisakis larvae collected in the present study were all morphologically similar to each other, three larvae representative for three locations were used for molecular analyses. Three ITS1-5.8S-ITS2 sequences obtained from three L3 larvae were 771 bp and completely identical (100%) with each other. In agreement with morphological identification, the BLAST searches revealed that the ITS1-5.8S-ITS2 sequences of Anisakis larvae from Vietnam showed the highest similarity (100%) with that of A. typica available in GenBank. The analysis of genetic distances demonstrated that inter-specific genetic distances between A. typica and other Anisakis species were: A. paggiae 17.2%; A. ziphidarum 17.3%; A. pegreffii 18.0%; A. simplex 18.0%; A. physeteris 18.4%; and A. brevispiculata 18.7%. In the phylogenetic tree (Fig. 4), A. typica made a distinct clade that was far distant from other Anisakis species. Vietnamese A. typica were genetically close to those from China, Thailand, Indonesia, Papua New Guinea, and Australia, to make a common group that was separated from another group of America, Brazil, Turkey, and Portugal. Our analysis is in agreement with a previous report that the separation of A. typica populations related to geographical origins (Tunya et al., 2020).
Molecular studies of Anisakis from various parts of the world Oceans confirmed the validity of nine Anisakis species (Klimpel and Palm, 2011). Most of them distribute in the Atlantic and the Mediterranean Sea with several records of some species in the South American, African, and Australian sea, and SW Pacific Ocean. A unique distribution pattern has been known for A. typica which has been reported in warmer temperate and tropical waters (Mattiucci and Nascetti, 2006). In Asian countries, A. typica larvae have been reported in Japan, Korea, China, Taiwan, Indonesia, and Thailand Palm et al., 2008Palm et al., , 2017Sonko et al., 2019;Tunya et al., 2020;Umehara et al., 2010;    Zhu et al., 2007). In the present study, we firstly identified A. typica larvae in Vietnamese water. Although a limited number of larval samples were molecularly analyzed, we speculated, based on their morphological similarity, that the Anisakis larvae found in this study are all A. typica. It is highly possible that A. typica is the most dominant species or the only Anisakis species in the South of Vietnamese water

Conclusion
The present study firstly identified A. typica larvae, based on morphological characteristics and molecular analysis, from eight marine fish species in the South of Vietnamese water and recorded seven fish species as new intermediate hosts of this Anisakis nematode. Genetically, the ITS1-5.8S-ITS2 sequences of Vietnamese A. typica were close to those from Asian countries and Australia, to make a common group separated from another group from America and Europe.