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Original Article

Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand

Rattanachai Tunya1, Chalobol Wongsawad1,2,*, Pheravut Wongsawad1,3, Jong-Yil Chai4,5
The Korean Journal of Parasitology 2020;58(1):15-25.
Published online: February 29, 2020

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*Corresponding author (wchalobol@gmail.com)
• Received: September 24, 2019   • Revised: November 22, 2019   • Accepted: November 22, 2019

Copyright © 2020 by The Korean Society for Parasitology and Tropical Medicine

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Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand
Korean J Parasitol. 2020;58(1):15-25.   Published online February 29, 2020
DOI: https://doi.org/10.3347/kjp.2020.58.1.15
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Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand
Korean J Parasitol. 2020;58(1):15-25.   Published online February 29, 2020
DOI: https://doi.org/10.3347/kjp.2020.58.1.15
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Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand
Image Image Image Image Image Image Image
Fig. 1 Ornate threadfin bream (N. hexodon) and Japanese threadfin bream (N. japonicus) (A and B, respectively) from the Gulf of Thailand purchased from a department store in Chiang Mai province. Fish sold in the department store in Chiang Mai province (C) and fresh specimens of L3 anisakid larvae viewed under a stereo microscope (D).
Fig. 2 A map showing the location of Chachoengsao and Chiang Mai province, Thailand. Distance between the 2 locations is about 750 km.
Fig. 3 Fresh specimens of L3 A. typica from N. japonicus viewed under a light microscope are shown (A–D). Drawings on specimen from permanent slides (E–H) indicate the nerve ring, oblique esophago-intestinal junction (A, E) and tail (B, F), boring tooth and inconspicuous lips (C, G), rectal gland, anus and rounded tail with terminal mucron (D, H). ao, anus opening; bt, boring tooth; ed, excretory duct; ep, excretory pore; es, esophagus; int, intestine; mu, mucron; nr, nerve ring; pv, proventriculus; rg, rectal gland; vt, ventriculus.
Fig. 4 Scanning electron micrographs of L3 A. typica from N. hexodon (A–D) and N. japonicus (E–H) from the Gulf of Thailand. Mouth parts have been shown in A and E, cuticle structures in the midsection with longitudinal linear cuticular striations, small papillae (white arrow) and grooves (black arrow) (B, F). Cuticle structures of the posterior end with longitudinal linear cuticular striations (C), the posterior end including the anal opening and grooves (black arrow) (G) and posterior extremity with mucron (D, H) ao, anus opening; ap, amphid; bt, boring tooth; cp, cephalic papillae; dl, dorsal lip; ep, excretory pore; lvl, left ventrolateral lip; m, mouth; mu, mucron; rvl, right ventrolateral lip.
Fig. 5 A comparison of the protruded mucron of L3 anisakid larvae using a scanning electron micrograph. Anisakis simplex from Ishii et al., 1989 has been shown in (A) and A. typica from the present study has been shown in (B).
Fig. 6 Maximum likelihood (A) and neighbour-joining (B) phylogenetic trees for Anisakis spp. based on the ITS1-5.8S-ITS2 region of rDNA. Accession numbers are reported in the text and Hysterothylacium aduncum was used as an outgroup. Number on node is 1,000 bootstrap replications.
Fig. 7 Comparisons of L3 A. typica ITS1-5.8S-ITS2 rDNA sequences in present study with others (A). Maximum likelihood and neighbour-joining phylogenetic tree (B) of A. typica based on ITS1-5.8S-ITS2 of rDNA with accession numbers, hosts and locations (C). Number of bootstrap replications was 1,000.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand

Average length and weight of sampled fish. Data regarding prevalence, intensity and sites infected with the L3 anisakid larvae have been given.

Fish species Mean fish length (cm) Mean fish weight (g) Prevalence of anisakid larvae Intensity of anisakid larvae Abundance of anisakid larvae Infected sites (%)
Body cavity Liver
Ornate threadfin bream (Nemipterus hexodon) 19.18 200.16 68% 3.47 2.36 100.00 -
Japanese threadfin bream (N. japonicus) 18.42 162.63 60% 4.17 2.50 99.20 0.80

Comparison of morphometric data obtained from L3 anisakid larvae (mm)

Anisakis simplex A. simplex A. simplex A. simplex A. typica A. typica A. typica A. typica
Hosts Oncorhynchus keta Tribolodon hakanensis Merluccius merluccius lessepsianus O. keta Pseudopercis numida - Trichiurus lepturus Nemipterus hexodon & N. japonicus
Body length 19.73–28.41 32.96–35.66 14.1–25.6 12.75–29.94 - - 15.34–22.43 12.31–18.00
Body width 0.42–0.68 0.6 0.48–0.62 0.45–0.75 - - 0.35–0.60 0.34–0.45
Proventriculus length 1.03–2.59 2.58 1.18–2.68 1.18–2.58 1.52 1.32–1.57 1.10–1.81 1.36–1.88
Proventriculus width - - - - - - - 0.08–0.12
Ventriculus length 0.83–1.56 1.16–1.52 0.71–0.92 0.90–1.50 0.62 0.48–0.88 0.50–0.76 0.55–0.94
Ventriculus width 0.14–0.35 0.20–0.25 - 0.13–0.31 - - - 0.10–0.20
Mucron length 0.01–0.03 0.015 0.019–0.032 0.02–0.03 - - - 0.01–0.02
References [18] [23] [24] [25] [14] [26] [27] Present study

Genetic distance of Anisakis spp. based on ITS1-5.8S-ITS2 of rDNA

Taxa 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Anisakis typicaa - 0.000 0.000 0.000 0.000 0.000 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. typicab 0.000 - 0.000 0.000 0.000 0.000 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. typica (AB432909) 0.000 0.000 - 0.000 0.000 0.000 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. typica (HF911524) 0.000 0.000 0.000 - 0.000 0.000 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. typica (JX523715) 0.000 0.000 0.000 0.000 - 0.000 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. typica (KC928262) 0.000 0.000 0.000 0.000 0.000 - 0.021 0.021 0.021 0.022 0.022 0.023 0.023 0.202
A. ziphidarum 0.159 0.159 0.159 0.159 0.159 0.159 - 0.016 0.018 0.019 0.009 0.009 0.009 0.205
A. paggiae 0.159 0.159 0.159 0.159 0.159 0.159 0.124 - 0.009 0.008 0.017 0.017 0.018 0.202
A. physeteris 0.164 0.164 0.164 0.164 0.164 0.164 0.140 0.054 - 0.007 0.019 0.019 0.021 0.208
A. brevispiculata 0.172 0.172 0.172 0.172 0.172 0.172 0.143 0.047 0.035 - 0.019 0.019 0.021 0.225
A. simplex 0.175 0.175 0.175 0.175 0.175 0.175 0.053 0.127 0.145 0.144 - 0.001 0.011 0.205
A. pegreffii 0.177 0.177 0.177 0.177 0.177 0.177 0.052 0.125 0.143 0.146 0.001 - 0.011 0.204
A. nascettii 0.182 0.182 0.182 0.182 0.182 0.182 0.053 0.141 0.162 0.160 0.070 0.068 - 0.243
H. aduncumc 0.910 0.910 0.910 0.910 0.910 0.910 0.916 0.925 0.928 0.994 0.916 0.912 1.033 -

aAnisakis typica from Nemipterus hexodon,

bA. typica from N. japonicus,

cHysterothylacium aduncum was used as an outgroup, distance value (below diagonal), and standard error (above diagonal).

Table 1 Average length and weight of sampled fish. Data regarding prevalence, intensity and sites infected with the L3 anisakid larvae have been given.
Table 2 Comparison of morphometric data obtained from L3 anisakid larvae (mm)
Table 3 Genetic distance of Anisakis spp. based on ITS1-5.8S-ITS2 of rDNA

Anisakis typica from Nemipterus hexodon,

A. typica from N. japonicus,

Hysterothylacium aduncum was used as an outgroup, distance value (below diagonal), and standard error (above diagonal).

Table 1

Table 2

Table 3

PHD : Parasites, Hosts and Diseases

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