(Nematoda, Anisakidae) in the Common Ponyfish Leiognathus Equulus Forsskã

Home , Anisakidae, Lethrinus lentjan, Terranova (nematode)

Microbial Pathogenesis 149 (2020) 104597

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Microbial Pathogenesis

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First record of third-stage Terranova larval type II (Nematoda, Anisakidae) in the common ponyfish Leiognathus equulus Forsskål

Nawal Al-Hoshani a, Saleh Al-Quraishy a, Mohamed A. Dkhil a,b, Ahmed A. Baiomy c, Rewaida Abdel-Gaber a,c,* a Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia b Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt c Zoology Department, Faculty of Science, Cairo University, Cairo, Egypt

ARTICLE INFO ABSTRACT

Keywords: The current study was carried out to investigate the natural occurrence of nematode parasites that infect the Leiognathus equulus common ponyfishLeiognathus equulus from Jeddah, Saudi Arabia. Third-stage nematode larvae were found to be Terranova species encysted in the peritoneum of the fish studied, with the prevalence of infection being 25%. Light microscopy Morphological studies revealed that this parasite belongs to the Anisakidae family within the genus Terranova by having all the generic Molecular analysis characteristic features. Based on the intestinal caecum ratio to the length of the ventriculus being 2:1, the excretory pore with ventral location below the boring tooth, the body ended with a conical tail; the larvae found in the present study were identified as Terranova larval type. To validate its taxonomic position within Anisa kidae, this Terranova species’ morphological features were combined with the ITS-1 gene’s molecular analysis. It demonstrated sequence similarities 94.38–76.57% with taxa of Anisakidae. A preliminary genetic comparison between the present parasite and other ascaridoids placed it as a putative sister taxon to the previously described Terranova species. The first record of the current anisakid larvae in the common ponyfish with a unique genetic sequence for the partial sequence of the ITS-1 gene was observed in this study. Its taxonomic position was confirmed in Anisakidae.

1. Introduction fish (second intermediate host) consume infected copepods, and the larvae become the third larval stage (L3) in different tissues. Fish are The superfamily Ascaridoidea Baird [1] is a group of parasitic then eaten by humans (accidental host) via the ingestion of raw or nematodes that infect a wide range of vertebrates, containing more than half-cooked fish and the anisakiasis results [19–21]. Awareness of 800 species among families within the order Ascaridida [2]. In both fish-borne parasitic diseases has been obtained after previous studies temperate and cold waters, nematodes of the Family Anisakidae Railliet documented the pathogenic effect of anisakid species on humans and Henry [3] are widely distributed ascaridoid parasites [4]. In Egypt, [22–28]. Libya, and Yemen, few papers have previously been published on Red The taxonomy of the anisakid larvae has recently been significantly Sea fish that study anisakid larvae of medical significance to humans redefined using nuclear rDNA sequence data and mitochondrial genes [5–13]. Only one study in Saudi Arabia by Al-Quraishy et al. [14] on the due to the lack of established morphological characteristics [20–36]. occurrence of anisakid nematodes larvae in the Red spot emperor Previous studies by Shamsi et al. [37,38] showed that for specific ani Lethrinus lentjan with full morphological description and molecular sakid nematodes identification and life cycle analysis, ITS-1 and ITS-2 phylogenetic analysis is available. are considered useful genetic markers. Anisakid nematodes have complex (indirect) life cycle [15–18]. They The present study aimed to record, for the first time, the anisakid become the first-stage larvae (L1) after the eggs have been ejected into nematode larvae as parasites of the common ponyfish Leiognathus the water with the feces of the definitivehost (with the adult worms) and equulus marketed in Jeddah, Saudi Arabia. Furthermore, the third-stage are then consumed by tiny crustaceans, primarily copepods (first in Terranova larval type II is described as morphologically and genetically termediate host), and differentiated into second-stage larvae (L2). Then, characterized.

* Corresponding author. Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia. E-mail address: [email protected] (R. Abdel-Gaber). https://doi.org/10.1016/j.micpath.2020.104597 Received 21 September 2020; Received in revised form 23 October 2020; Accepted 25 October 2020 Available online 27 October 2020 0882-4010/© 2020 Elsevier Ltd. All rights reserved. N. Al-Hoshani et al. Microbial Pathogenesis 149 (2020) 104597

Fig. 1. Photomicrographs for different body parts of Terranova sp. (L3). (A) Whole-mount preparations. (B–I) High magnificationsshowing: (B–F) Anterior part of the body. (G) Transverse annulations of the cuticle. (H) Oesphago-intestinal junction (arrows). (I) Posterior part of the body. Note: AN, anus; BT, boring tooth; ED, excretory duct; EP, excretory pore; IC, intestinal caecum; IN, intestine; L, lips; MO, mouth opening; OS, oesophagus; PA, papillae; RG, rectal glands; RT, rectum; T, tail; TA, transverse annulations; VT, ventriculus.

2. Materials and methods 2.2. Fish and parasite collection and morphological studies

2.1. Description of the study area Eighty specimens of the common ponyfish Leiognathus equulus (Family: Leiognathidae) were fished alive during January–December Jeddah Islamic Port is the main gateway to the holy cities of Makkah 2019 from the commercial fishermen from the studied area, the city of and Madinah in Saudi Arabia. Its unique location on the Red Sea coast is Jeddah, Saudi Arabia. The weight and length of the collected samples distinguished in the middle of the international shipping route between were recorded appropriately. All fish were kept alive in glass aquaria, east and west. It stretched for about 12.5 square kilometers along the supplied with chlorine-free tap water with continuous aeration and ◦ ′ ◦ ′ seashore at 21 28 N, 39 10 E for latitude and longitude, respectively. filtration, and then transported directly to the Research Parasitology Laboratory in the Zoology Department, College of Science, King Saud

2 N. Al-Hoshani et al. Microbial Pathogenesis 149 (2020) 104597 (0.1) 0.01) ± 0.130 0.150 0.122 – – – 0.17 0.18 0.25 0.15 0.14 length – – – – – Tail 0.060 0.07 0.12 (0.15) 0.22 (0.25) 0.10 (0.12) 0.12 (0.13) 0.109 – 0.089 (0.112 (2:1) (0.51) (2:1) caecum of 0.843 – 0.74 0.68 – – ratio The intestinal length/ventriculus length – 0.43 – – – – 0.34 0.411 smoothly. 0.01) length ± tapering 0.983 0.825 0.748 – – – 0.28 0.39 0.62 0.90 0.85 – – – – – and Intestinal caecum 0.350 0.12 (0.19) – 0.37 (0.38) 0.39 (0.49) 0.50 (0.71) 0.612 0.61 (0.68) 0.671 (0.855 0.01) annulations ± 0.521 0.400 0.435 – – – 0.49 0.18 0.35 0.32 0.54 0.38 – – – – – – strong Ventriculus length 0.200 0.26 (0.37) 0.12 (0.15) 0.27 (0.30) 0.22 (0.27) 0.24 (0.38) 0.340 0.29 (0.34) 0.339 (0.466 with tail length (14.3) of length 14.56% 15.54% – – 19% 10.14% conical – – ratio 26.5% – body The oesophagus to 11.5 6.71 (7.89) – – 9.5 – 11.01 (12.82) 10.98 (13.55) pore, 0.02) (0.88) ± 0.891 1.00 0.952 excretory – – – 1.37 0.87 1.15 0.85 1.03 – – – – – 1.14 – the Oesophagus length 0.440 0.86 (1.12) 0.60 (0.72) 0.82 (0.95) 0.60 (0.71) 0.4 0.802 0.73 (0.85) 0.524 (0.756 of previously. of (0.20 location (0.21) (0.38) (0.23) (0.24) (0.37) (0.25) ring anterior 0.286 0.25 – 0.29 0.45 0.25 0.29 0.72 0.32 the – – – – – – – distance nerve described 0.01) anterior The the from end – 0.17 0.35 0.20 0.20 0.22 0.231 0.22 0.19 ± parts those lips, body 0.01) with ± 0.250 0.286 – width – 0.39 1.10 0.19 0.21 0.28 0.28 – – – – – – different Body – 0.22 (0.30) 0.78 (0.94) 0.14 (0.18) 0.12 (0.16) 0.18 (0.24) 0.204 0.18 (0.23) 0.197 (0.221 inconspicuous for understudy II 0.2) (6.6) present, ± 7.98 length 8.15 20.03 0.36 7.00 9.02 8.3 type – – – – – – – 6.75 9.0 – – Measurements Body 3.80 9.93 (14.20) 0.26 (0.28) 4.50 (6.0) 4.5 (4.92) 3.0 6.61 5.42 (6.63) 5.43 (6.01 tooth larval by , , , , and (New , , Terranova Echeneis furcosus (Saudi (Brazil) , Caesio Scomber (New (Kuwait) , Scomberoides bicarinatus (Yemen) Caranx , characterized commerson , and mate equulus (Locality) Epinephelus (Australia) fulvoguttatus fulvoguttatus orthogrammus (Australia) blochi carponotatus, areolatus , squamosissimus , qunie whitleyi Nemipterus guatucupa L. third-stage , stellaris argentimaculatus larvae , Atule fish ignobilis of , species and bohar fulviflamma Trachinotus Host Plagioscion (Brazil) Teleost Abalistes Cynoscion Abudefduf cuning Carangoides Caranx melampygus cyanopodus Grammatorcynus Lutjanus L. L. australasicus Carangoides Caledonia) Carangoides Scomberomorus Epinephelus naucrates Sphyraena sp. Caledonia) Leiognathus Arabia) Terranova ] ] ] a 46 51 al. [ 47 al. [ [ from ] measurements third- al. et et Sey al. II al. 49 al. [ et sources et study and et et and type comparable Terranova ] ] ] 1 ] 45 8 48 50 [ [ [ Suthar [ All Petter Tavares Comparable stage larval different Jabbar Al-Zubaidy Fontenelle Shamsi Moravec Shamsi Present a Table Comparative

3 N. Al-Hoshani et al. Microbial Pathogenesis 149 (2020) 104597

Table 2 Nematode species used in the phylogenetic analysis of the third-stage Terranova larval type II for their corresponding ITS-1 gene region.

Parasite species Host species Accession no. Identity (%) GC content (%) Reference

Terranova pectinolabiata Sphyrna mokarran MK542878.1 93.87% 47.7% Shamsi et al. [52] Terranova sp. Rhomboplites aurorubens MF668767.1 93.39% 48.2% Quiazon et al. [53] Terranova sp. Sciaenops ocellatus MF680005.1 94.38% 48.6% Quiazon et al. [53] Terranova sp. Micropogonias undulatus MF680024.1 93.17% 48.4% Quiazon et al. [53] Terranova sp. Balistes capriscus MF680008.1 94.08% 48.1% Quiazon et al. [53] Terranova sp. Lagodon rhomoides MF668762.1 94.23% 47.7% Quiazon et al. [53] Terranova sp. Stenotomus caprinus MF668763.1 90.77% 47.7% Quiazon et al. [53] Terranova sp. Thunnus atlanticus MF668775.1 90.52% 48.1% Quiazon et al. [53] Terranova sp. Trichiurus lepturus MF668784.1 90.50% 48.1% Quiazon et al. [53] Terranova sp. Scomberomorus cavalla MF668837.1 90.47% 47.7% Quiazon et al. [53] Terranova sp. Scomberomorus maculatus MF668780.1 93.23% 47.6% Quiazon et al. [53] Terranova sp. Pagrus pagrus MF668772.1 90.46% 48% Quiazon et al. [53] Terranova sp. Stenotomus chrysops MF680027.1 92.60% 48.2% Quiazon et al. [53] Pulchrascaris sp. Sphyrna lewini MK890764.1 87.72% 48.8% Shamsi et al. [54] Pulchrascaris chiloscyllii Sphyrna lewini MF061687.1 87.34% 48.5% Li et al. [55] Anisakis physeteris Coryphaena hippurus MF668926.1 77.22% 50.2% Quiazon et al. [53] Anisakis brevispiculata Kogia breviceps MK325199.1 77.40% 50.1% Shamsi et al. (52) Anisakis physeteris – AB201789.1 77.61% 49.2% Kijewska et al. [56] Anisakis paggiae Kogia breviceps MK325218.1 77.83% 49.2% Shamsi et al. [52] Pseudoterranova cattani – KF781285.1 79.80% 46.2% Weitzel et al. [57] Pseudoterranova azarasi Oncorhynchus keta LC511600.1 78.35% 46.9% Jin [58] Pseudoterranova decipiens Gadus morhua MG272335.1 78.59% 46.7% Klapper et al. [59] Contracaecum osculatum Halichoerus grypus MT258529.1 76.57% 45.8% Mohamed et al. [60] Contracaecum rudolphii Pelecanus occidentalis JF424597.1 77.28% 49.6% D’Amelio et al. [61]

University, Riyadh, Saudi Arabia. All applicable international and 3. Results institutional guidelines for the care and use of animals were followed according to the ethical committee at King Saud University. Fish were Twenty out of eighty (25%) specimens of the examined fishspecies, dissected and examined macroscopically for larval nematodes on the Leiognathus equulus, were commonly found to be naturally infected with surface of the internal organs. The gastrointestinal tract was examined peritoneum-encysted third-stage Terranova larval type . Based on the under a stereomicroscope (Nikon SMZ18, NIS ELEMENTS software) for appearance of the intestinal caecum and ventriculus, absence of devel the presence of nematodes. The nematode parasites were collected, oped labia and ventricular appendix, and location of the excretory pore washed in saline, and then fixedfor subsequent analysis in 70% ethanol. being at the anterior end, these larvae were classified as type II (Fig. 1 The parasitological indices of prevalence and intensity were calculated (A-I)). The per-fish number of these larvae ranged from 3 to 15. The according to the equations of Bush et al. [39]. The length of the larval weight and length of the collected samples were ranged between 210 body was measured directly. Larval nematodes were cleared in lacto and 260 g and 14–17 cm, respectively. The highest parasitic intensity phenol and analyzed under the microscope of LEICA-DM 2500 (NIS was found in fish hosts with a body length of >12 cm, referring to the ELEMENTS software, version 3.8), according to Murata et al. [40]. positive relationship between the host length and the intensity of nem Terranova larvae were identified according to the identification key to atode infection. The morphological description of these larvae was Cannon [41]. The measurements were expressed as a range in milli summarized (see Table 1). meters (mm) with a mean ± SD in parentheses. A total of 859 bp was analyzed and deposited in GenBank with 49.4% GC content of the ITS-1 gene region (accession number MT533277.1). 2.3. Molecular analysis Using the partial ITS-1 sequence data, phylogenetic analysis was per formed to establish the relationships of the anisakid larvae described Genomic DNA (gDNA) was extracted from larvae preserved in herein with other related nematode species (Table 2). The current ethanol using a Qiagen kit (QIAamp® DNA Mini-kit) (Hilden, Germany) phylogeny was based on the maximum likelihood method, representing following the recommended protocol. Polymerase chain reaction (PCR) one chromadorean order of Ascaridida with two superfamilies of was employed to amplify the internal transcribed spacer-1 (ITS-1) gene Ascaridoidea and Cosmocercoidea (Figs. 2 and 3). Our phylogenetic ′ region using the primer set NC5F (5 -GTA GGT GAA CCT GCG GAA GGA analysis using the partial ITS-1 gene region is split into two clades ′ ′ ′ TCA TT-3 ) and NC2R (5 -TTA GTT TCT TTT CCT CCG CT-3 ), and the (Fig. 3). The major one showed that the Ascaridoidea members with the cycling conditions following Zhu et al. [42]. The amplicons’ analysis Anisakidae family, including fivegenera, were: Terranova, Pulchrascaris, was then conducted on 1.5% w/v agarose gel labeled with SYBR green. Anisakis, Pseudoterranova, and Contracaecum. All members of these PCR amplicons were subjected to bi-directional and automatic groups are of monophyletic origin. The minor clade was employed sequencing (BigDye Terminator v3.1, Applied Biosystems, USA) using within Cosmocercoidea by an outgroup of Falcaustra sinensis comprising the same primer set employed in PCR reaction. The sequencing data the Kathlaniidae family. The ML topology showed that the Anisakidae resulted from the analysis was deposited with accession number family was divided into two subfamilies: Anisakinae (represented by MT533277.1 in GenBank. The partial sequence of the ITS-1 gene was Anisakis, Pseudoterranova, Pulchrascaris, and Terranova) and Contra aligned with additional sequences selected from GenBank using BioEdit caecinae (represented by Contracaecum), each strongly supported with a 7.0.1 [43] and then used for the phylogenetic analysis. The Maximum value of 99, respectively. Pair distances for the ITS-1 sequence data Likelihood (ML) method was used to construct a phylogenetic tree that alignment revealed 94.38–76.57% for Anisakidae taxa (Table 2). focused on the best-fitsubstitution analysis model (Tamura 3-parameter Sequence variation in ITS-1 among all specimen sequences was model). Falcaustra sinensis was used as an outgroup. With 1000 repli 0–0.12%, and the GC content was 45.8–50.2%. The sequence similarities cates, the reliability of the ML tree was tested using the bootstrap for genera within the Anisakidae family were: 90.46–94.38% with Ter method. The software for Molecular Evolutionary Genetics Analysis ranova taxa, 87.72–87.34% with Pulchrascaris, 77.83–77.22% with (MEGA 7.0) was used to perform this phylogenetic analysis [44]. Anisakis, 79.80–78.35% with Pseudoterranova, and 77.28–76.57% with Contracaecum. Our analysis showed that the present Terranova species

4 N. Al-Hoshani et al. Microbial Pathogenesis 149 (2020) 104597

Fig. 2. Sequence alignment of the partial ITS-1 gene of Terranova sp. type II with the most closely related larval species (Only variable sites are shown. Dots represent bases identical to those of the first sequences, and dashes indicate gaps).

5 N. Al-Hoshani et al. Microbial Pathogenesis 149 (2020) 104597

Fig. 3. Molecular Phylogenetic analysis by Maximum Likelihood method using the Tamura 3- parameter model based on the partial ITS-1 sequence demonstrating the position of Terranova sp. type II with other related species within Ascaridoidea. The tree with the highest log likelihood ( 2290.46) is shown. The percentage of trees in which the associ ated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise dis tances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary ana lyses were conducted in MEGA7.

were located in the same Terranova larval type clade as Anisakidae and phylogenetic study of the ITS-1 query data was conducted for the considered closely related but distinct species. recovered anisakid species compared with other ITS-1 data for the previously deposited sequences for anisakid larval stages on the Gen 4. Discussion Bank database, which agreed with Shamsi et al. [37,38] reported that both ITS-1 and ITS-2 larval stages sequences for different anisakid Nematode identification is often not completed down to the species nematodes allowed unequivocal identificationof those larvae to species level, particularly for non-zoonotic marine ascaridoids [49], and mo level. Monophyly of Anisakidae was recovered by the current ML phy lecular analyses are often not precisely classified larvae [62]. The Ter logeny, which agreed with Nadler and Hudspeth [70], and Nadler et al. ranova larvae specimens recovered in the current study were following [31] stated that the combined analysis of SSU and LSU sequences using the previously reported specimens by Cannon [41], Petter and Sey [45], ML inference provided a monophyletic origin of Anisakidae. In agree Tavares et al. [46] in which the third-stage Terranova larvae had all the ment with Feldman and Bowman [71], our phylogeny supports the characteristic features for these larval type, except for the occurrence of distinction between subfamilies Anisakinae and Contracaecinae within caudal papillae for the adult Terranova species which is not observed in Anisakidae. Furthermore, Fagerholm [65], Nadler et al. [72], Mohandas this study. There are several reports of morphotypes of Terranova larval et al. [34], Liu et al. [35,73], Zhao et al. [74]; Li et al. [36,55] reported types (I and II) in various fish species [41] with no existing precise that the Anisakidae family is split into two subfamilies with a substantial identificationsince larval anisakids cannot be identifiedat species level support bootstrap value dependent on the morphological characters in due to the absence of taxonomically relevant features and comparable conjunction with the ITS-1, ITS-2, 5.8S, 28S, 18S, COX1, COX2, and 12S sequences data from a well-identified adult [49]. Our specimens are query sequences, which were: the Anisakinae subfamily that parasitizes morphologically distinct based on the ratio of intestinal caecum length semiaquatic tetrapod (clustered Anisakis, Pulchrascaris, Pseudoterranova, to ventriculus length (2:1) and the absence of the mucron, which is and Terranova) and the Contracaecinae subfamily that parasitize consistent with the data obtained by Cannon [41] and Shamsi et al. [51], fish-eatingbirds (clustered Phocascaris and Contracaecum). All members to be recognized as the third-stage Terranova larval type II. The preva of the Terranova genus clustered herein in the distinct clade comprising lence rate of infection (25%) of this larval type with the host length/ all type II larval stages and the recovered Terranova species is deeply weight can be found in a positive correlation; this agreed with embedded in this clade supported by substantial bootstrap value, which Purivirojkul [63] and Pardo-Gandarillas et al. [64], which reflectshigh agreed with data obtained by Shamsi and Suthar [49] that the Terranova values for the larval nematode abundance. larval type was resolved as a distinct clade with substantial bootstrap Due to the lack of characteristic morphological features for identi value and none of the other anisakid species (Anisakis spp., Contra fying larval stages of anisakid nematodes, this led to considerable con caecum spp., Pseudoterranova spp.) with similar morphological features troversy over the classificationof the family Anisakidae [65]. Molecular to Terranova spp. (excretory pore opened at the basal level of the labia) approaches have recently been employed to explore the taxonomic were grouped in the same clade as Terranova larval type reported herein. status and genetic variation of anisakids [47], consistent with the cur rent study concept. Furthermore, Zhu et al. [66], Anshary et al. [67], 5. Conclusion Mattiucci et al. [68], Palm et al. [69] suggested that genomic DNA encoding ribosomal RNA (18S, ITS-1, 5.8S, ITS-2, 28S) sequencing was It could be concluded that the parasite species found in L. equulus was used to create a phylogenetic hypothesis for Ascaridoidea superfamily Terranova type II larvae and termed as third-stage Terranova larvae with members. Using the Maximum Likelihood (ML) method, the current a unique genetic sequence and new locality data in Saudi Arabia.

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