First record of Pseudoterranova decipiens (Nematoda, Anisakidae) infecting the Red spot emperor Lethrinus lentjan in the Red Sea

The current parasitological study was carried out to investigate helminth parasites infecting the Red spot emperor Lethrinus lentjan inhabiting Hurghada City at the Gulf of Suez, Red Sea, Egypt. Third-stage larvae of nematode parasite was isolated from the intestine as well as body cavity of the examined fish. Light and scanning electron microscopy revealed that this parasite belonged to Anisakidae family within the genus Pseudoterranova. The present species is named Pseudoterranova decipiens based on the presence of triangular mouth aperture with prominent boring teeth and soft swellings of the cuticle, long muscular esophagus, ventrally excretory pore, and narrow transverse slit of anal opening followed by a short mucron. The morphological characteristics of this species were confirmed by molecular analysis of 18S rDNA gene region of the present parasite. It demonstrated a close identity ≥89% with taxa under family Anisakidae, 85% with Raphidascarididae, and 79-84% with Toxocaridae. A preliminary genetic comparison between gene sequence of the present parasite and other oxyurid species placeed it as a putative sister taxon to other Pseudoterranova decipiens described previously. This study demonstrated that the 18S rDNA gene region of Pseudoterranova decipiens yielded a unique sequence that confirmed its taxonomic position in Anisakidae.


Introduction
Anisakid nematodes have a global distribution among a wide variety of more than 200 marine fish species as intermediate host (MCCLELLAND et al., 1990) or as paratenic hosts (KØIE et al., 1995;KUHN et al., 2011). Cephalopod, marine mammals, and humans can become accidental hosts for anisakids by ingesting fish infected with third-stage larvae and their prevalence's can be very high (WHARTON et al., 1999;ABOLLO et al., 2001;MCCLELLAND, 2002;SZOSTAKOWSKA et al., 2002). The life cycles of marine ascaridoid nematodes involve a number of stages
Palavras-chave: Peixe marinho, Lethrinus lentjan, Pseudoterranova spp., estudos morfológicos, análise molecular. and hosts. Adult and other life stages of these anisakid nematodes can be found in almost any part of the fish including the body cavity, internal organs, swim bladder, deeper layers of the skin or fins and external muscle layers (SMITH & WOOTTEN, 1978;. Rocka (2004) followed by Nada & Abd El-Ghany (2011) stated that anisakid nematodes commonly found in bony fish are represented by the following genera: Anisakis (Dujardin, 1845), Contracaecum (Railliet & Henry, 1912), Hysterothylacium (Ward & Magath, 1917), Paranisakiopsis (Yamaguti, 1941) and Pseudoterranova (Mozgovoy, 1951). Anisakid larvae are usually very difficult to identify species using morphology due to the lack of differential characters, but when adults are described and genetically characterized, then such larva assigned to a species based on molecular studies (MATTIUCCI et al., 1997). Anisakid nematodes belonging to the Pseudoterranova decipiens species complex (also known as sealworms or codworms) mature and reproduce in the digestive tract of pinnipeds (LAUCKNER, 1985;DI AZEVEDO et al., 2017;IRIGOITIA et al., 2018). As far as it has been known, the life cycle of Pseudoterranova species also includes crustaceans as the first hosts, and fish as second hosts (SUKHDEO, 2012). Third-stage larvae (L3) of sealworms have commonly been reported in marine teleosts worldwide (GEORGE-NASCIMENTO, 1987;MATTIUCCI & NASCETTI, 2008). The complex of Pseudoterranova decipiens is composed of six sibling species with four species occurring in the Northern Hemisphere are namely Pseudoterranova azarasi (Yamaguti & Arima, 1942), Pseudoterranova bulbosa (Cobb, 1888), Pseudoterranova decipiens sensu stricto (s.s.) (Krabbe, 1868), and Pseudoterranova krabbei Paggi et al. (2000). While, Mattiucci & Nascetti (2008) reported that the remaining two species present in the Southern Hemisphere are namely Pseudoterranova cattani (George-Nascimento & Urrutia, 2000), and Pseudoterranova decipiens Bullini et al. (1997). Additional species in the genus Pseudoterranova include Pseudoterranova cattani, identified using allozyme markers in the Pacific (SUKHDEO, 2012). Therefore, the present study aimed to report the natural occurrence of anisakid nematodes in the Red spot emperor Lethrinus lentjan. Additionally, identify the recovered worms by using light and scanning electron microscopy in order to determine its characteristic morphology, which may contribute valuable information to knowledge of the anisakids. Also, clarify the taxonomic position of the present anisakid nematodes using molecular phylogenetic analysis.

Fish samples collection and parasitological examinations
Forty two specimens of the Red spot emperor Lethrinus lentjan (Family: Lethrinidae) were collected during the period of August 2017-May 2018 from boat landing sites and fishermen of Hurghada City at the Gulf of Suez, Red Sea, Egypt. The collected fish specimens were transported immediately to the Laboratory of Parasitology Research at Zoology Department, Faculty of Science, Cairo University, Egypt, using special boxes for parasitological examination. All procedures contributing to this work comply with the ethical standards authorized by the Institutional Animal Care and Use Committee (IACUC) at Faculty of Science, Cairo University in Egypt with no. CUFS/S/Para/38/2014.
Gross microscopic examinations of all abdominal fish organs were done. The contents of the digestive tract were examined under a binocular microscope for the detection of any parasitic worms, which then removed with a fine forceps or a pipette. After isolation of parasitic worms from the infected fish, washing several times with an isotonic saline solution was done to get rid of any mucous and debris wastes. Isolated worms were fixed in 70% ethanol and subsequently clarified with lactophenol for morphological identification, in accordance with standard reference keys (PETTER & QUENTIN, 2009). Parasite prevalence was calculated according to Bush et al. (1997). Photomicrographs of adult specimens were made with the aid of microscope Leica DM 2500 (NIS ELEMENTS software, ver. 3.8) in the Laboratory of Parasitology Research in Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia.
For scanning electron microscope, some of nematode worms were fixed with 3% buffered glutaraldehyde, dehydrated in ascending series of ethyl alcohol (70%, 80%, 95%, 100%), infiltrated with amyl acetate, processed in a critical point dryer "LEICA, EM CPD300", sputter-coated with gold-palladium in a gold sputtering system (JEOL, JEC-3000FC) for 30 s at ~70 mTorr pressure, and finally examined under an Etec Autoscan at 10-kV JEOL scanning electron microscope (JSM-6060LV). Measurements were carried out in millimeters, presented as a range followed by the arithmetic mean±SD in parentheses, and unless otherwise were stated.

Results
Thirty one (73.80%) out of forty two specimens of the Red spot emperor Lethrinus lentjan (Family: Lethrinidae) were found to be infected with anisakid nematoda parasite. The infection was recorded in the intestine as well as the body cavity of the examined fish. The infection was increased during summer to be 95.23% (20 out of 21) followed by winter season to be 52.38% (11 out of 21). The number of parasites per fish was ranged from 8 to 16.
Description (Figures 1-10) The body of the recovered third-stage larvae was yellowish to reddish in color, medium-sized, and elongated. Body measured 6.8-8.6 (7.2±1.1) mm in length and 0.16-0.21 (0.18±0.01) mm in width. The anterior end is rounded with triangular mouth aperture. Lips were absent with prominent boring teeth at the  anterior extremity provided with soft swellings of the cuticle at the level of papillae surrounding the tri-radiate mouth opening. The worm's esophagus had a long anterior muscular part, 0.79-0.96 (0.91±0.01) mm long. The excretory pore opened ventrally, below the boring teeth at the anterior end. The covering cuticle was rigid with transverse annular striations. The anal opening was in the form of a fairly long and narrow transverse slit. Tail was short, conical, pointed, 0.10-0.20 (0.15±0.01) mm long. The worm body ended by a short mucron, 0.01-0.03 (0.02±0.002) mm long.

Molecular analysis
A total of 350 bp of 18S rDNA gene sequence with 54.6% GC content was recovered from the present anisakid species and deposited in GenBank (gb| KR864891.1). Pairwise comparison of the nucleotide sequences and divergence showed that the present anisakid species revealed sequence identities ≥89% with taxa under family Anisakidae, 85% with Raphidascarididae, and 79-84% with Toxocaridae. Among Anisakidae members, the maximum identity with low divergent values was recorded as 96% with the previously studied Pseudoterranova decipiens (gb| KF806033.1, U94766.1, JQ673263.1), followed by 95% with Pseudoterranova sp. (gb| KC013597.1), 93% with Anisakis simplex (gb| LL324431.1), 91% with Anisakis typica (gb| HF911524.1), and 89% with Terranova caballeroi (gb| U94767.1), which had the highest blast scores with small number of nucleotide differences (Figure 11). Phylogenetic analysis produced a neighbor-joining tree constructed with partial sequences consistently formed two major lineages ( Figure 12). The first major clade represent monophyletic origin for Ascaridoidea species and consisted of two larger subclades, showed that Raphidascarididae (sampled genera included Raphidascaris and Hysterothylacium) being sister to Anisakidae (sampled genera included Anisakis, Pseudoterranova, Contracaecum, and Phocascaris). The other clade represents the monophyletic origin of Toxocaridae represented by genus Toxocara. Phylogenetic relationship showed that the present anisakid species is deeply embedded in the genus Pseudoterranova with close relationship to all previously studied species of Pseudoterranova species especially Pseudoterranova decipiens as a more related sister taxon.

Discussion
The anisakid nematode Pseudoterranova decipiens was recovered during the current study to infect the intestine as well as the body cavity of the Red spot emperor Lethrinus lentjan fish with 73.80% as a percentage of infection. This observation agreed with data obtained by Klimpel & Palm (2011) followed by Najda et al. (2018) whom stated that the digestive tract considered as the preferred site of anisakid infection in marine mammals. The rate of parasitic infection increased during summer and fall during winter; these results coincided with data obtained by Ólafsdóttir & Hauksson (1998) who stated that the common seal infected by Pseudoterranova decipiens with a percentage of 44.7% and development of ascaridoid larvae are highly influenced by temperature and the generation time likely to be longer in the warm water.
The morphology of the present parasite has the same diagnostic generic features of genus Pseudoterranova by having an elongated body provided with a rounded anterior end with triangular mouth aperture provided with lateral boring teeth and ended by a short pointed tail with mucron. It was compared morphologically and morphometrically to other Pseudoterranova species recorded previously. Description of the present parasite species agreed much more with Pseudoterranova decipiens described previously by Timi et al. (2001) from Engraulis anchoita in morphological and morphometric data of the different body parts. In addition, it resembled to records of Felizardo et al. (2009)  Molecular approaches to delimiting and identifying anisakid nematodes have markedly influenced our understanding of their systematics and biodiversity (NADLER & HUDSPETH, 1998KELLERMANNS et al., 2007;BRUNET et al., 2017). In the present study, a nuclear rDNA region of 350 bp was amplified by using NC5 and NC2 primers and revealed sequence similar to homologous regions within the nuclear ribosomal sequence of other Pseudoterranova decipiens described previously. Apparently, the tree estimated in this study strongly supported several of the higher taxonomic groups. Phylogenetic tree constructed with partial sequences consistently formed two major lineages one represent monophyletic origin for Ascaridoidea species and consisted of two larger subclades, showed that Raphidascarididae is a sister group to Anisakidae with low genetic variations between them; while, the other clade represent the monophyletic origin of Toxocaridae, these results are in accordance with that obtained by Paggi et al. (2000) followed by Abollo et al. (2003), Nadler et al. (2005), Kellermanns et al. (2007) and Madanire-Moyo & Avenant-Oldewage (2013). The MP tree supported the taxonomic position of the present Pseudoterranova species which is deeply embedded in the genus Pseudoterranova with a close relationship with other Pseudoterranova decipiens described previously as a more related sister taxon. Therefore, this anisakid species has a unique genetic sequence for one of Pseudoterranova species. Therefore, it could be concluded that the parasite species found in Lethrinus lentjan was identified as Pseudoterranova decipiens with a unique genetic sequence and having new locality records in Egyptian water.