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

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.

Figure 2:

Figure 3:

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).

Figure 4:

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 (Lee et al., 2016; Palm et al., 2008, 2017; Sonko 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 similar to reports in Thailand water where A. typica was the only species found in the Gulf of Thailand (Eamsobhana et al., 2018; Tunya et al., 2020).

Regarding intermediate fish hosts, the third-stage larvae of A. typica are found in various fish species. They differ from place to place depending on geographical locations. Outside of Vietnamese water, 32 fish species have been reported as intermediate hosts of A. typica (Table 2). In this study in Vietnam, A. typica larvae were found from eight fish species, Carangoides malabaricus, Dcapterus macarellus, Sargocentron rubrum, Lutjanus johnii, Megalaspis cordyla, Priacanthus hamrur, Pristipomoides filamentosus, and Trichiurus lepturus. Among these, only T. lepturus has been previously reported as an intermediate host of A. typica in Korean and Taiwanese waters, the other seven species are reported as new hosts.

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.