2.2. Studied species and dissection

A total of 139 specimens were examined (n = 35 specimens of each Lagocephalus sceleratus, L. suezensis, and Torquigener flavimaculosus, n = 34 specimens of L. guentheri). Dissections were performed following standard procedures (Cribb and Bray, 2010; Palm and Bray, 2014; Klimpel et al., 2019). First, basic morphometric data, total length (L_T), standard length (L_S), and total weight (W_T) were taken. Hereafter, the body surface and openings (eyes, skin, fins, gills, nostrils, anus, and mouth cavity) were investigated macroscopically. For dissection, gill arches and eyes were removed first, afterwards, the body cavity was opened and the internal organs (liver, gall bladder, digestive tract, gonads, swim bladder, and heart) were removed. The organs were opened and inspected macroscopically and with a stereomicroscope (Zeiss Stemi 508 0.63–5.0, Carl Zeiss Microscopy GmbH, Jena, Germany). All found parasites were mechanicaly cleaned of stomach content and transferred into buffered saline solution (0.9% NaCl) and morphologically gross sorted. Parasites were transferred and stored in 96% EtOH for molecular identification or 80% EtOH for morphology. The stomach content was also identified to the lowest possible taxonomic level.

2.3. Mounting

Parasites were either cleared and mounted in glycerine (after Klimpel et al., 2019) and sealed with paraffin or Aceto-Carmine stained and mounted in Canada balsam (after Palm, 2004). Parasites were identified using a light microscope (OLYMPUS BX53), camera (OLYMPUS DP74) and photo software (OLYMPUS cellSens Dimension 1.6 (Olympus Corporation, Shinjuku, Tokyo, Japan), and assorted literature (Pearse, 1952; Deardorff and Overstreet, 1981; Dojiri and Cressey, 1987; Moravec, 1998; Euzet, 1994; Palm, 2004; Boxshall and El-Rashidy, 2009; Ben Ahmed et al., 2015; Martin et al., 2018; Guardone et al., 2020; Noël and Sittler, 2021).

2.4. Molecular identification of cestodes and nematodes

Total genomic DNA was extracted using a Qiagen® DNeasy Blood & Tissue Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer's protocol. In the case of very high parasite counts, a representative subsample, as described in Santos at al. (2017), was analysed. Extracted DNA was used to amplify the ITS1-5.8s-ITS2 sequence region (for nematodes) and the 28S rRNA gene (for cestodes) For PCR protocols see Table 2. The samples were purified with Qiagen QIAqick® PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and sent to SEQLAB (Microsynth SEQLAB GmbH, Göttingen, Germany) for sequencing. The obtained sequences were edited with MEGA X ((Kumar et al., 2018) and DNA Baser V4 Software (Heracle BioSoft SRL, Arges, Romania). The results were compared to the GenBank™ gene database and selected sequences were additionally uploaded to the GenBank™ for reference. Voucher specimens were deposited in the ‘Zoologische Lehrsammlung’ in Berlin, Germany, for further research.

3.2. Types and prevalence of parasites

The parasitological analyses of the 139 pufferfish retrieved ten species of parasites and an additional protozoan parasite. The parasites were identified to the lowest possible taxonomic level (Table 3). The metazoan parasite groups/taxa were Digenea (1), Cestoda (3), Nematoda (3), Hirudinea (1), and Copepoda (2).

The adult Digenea were found in the digestive tract lumen of L. guentheri and T. flavimaculosus, with a prevalence and intensity of 11.8% and 1–9 for L. guentheri and 2.9% and 1 for T. flavimaculosus. Total length (L_T) of Digenean (n = 2) was 1772–2225 μm, body width 895–915 μm, but when alive, distinctly more elongated (L_T > 2x body width). The oral sucker was subterminal, a diameter of 110–251 μm. The following prepharynx was surrounded by a prominent, muscular post-oral ring. The pharynx length x width was 123 μm × 110 μm. The ventral sucker was closely located to mid-body and larger than the oral sucker, with a diameter of 290–425 μm. Vitelline follicles measured 22 μm × 21 μm in diameter, reaching from the pharynx to the end posterior of the body cavity. Pigment granules were present, and sparsely distributed throughout the body. Testes were located opposite each other, measuring 160 μm × 114 μm and 146 μm × 116 μm. The ovary overlapped the right testis dorsally and was located posterodorsally to the ventral sucker. Eggs were oval and operculated, length x width was 26–43 μm x 14–30 μm (Fig. 1). The presence of the muscular post-oral ring around the prepharynx, body length over 2x body width, and sparse pigment vesicles throughout the body identified the Digenea as genus Maculifer Nicoll, 1915 according to Martin et al. (2018). The body size, size, and position of the measured internal organs either pointed towards M. indicus or M. dayawanensis since the exact position of the genital pore in relation to the intestinal bifurcation was not recognizable. The extent of the vitelline follicles from the pharynx, not just from the intestinal furcation to the posterior end of the body and its hosts identified as Maculifer dayawanensis Sheng and Tong, 1990 according to Sheng and Tong (1990) and Madhavi and Bray (2018). Specimens are deposited in the collection of the Museum für Naturkunde Berlin (catalogue nos. ZMB E.7672; ZMB E.7673).

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Fig. 1

Habitus of alive (A) and mounted line-drawn (B) specimens of Maculifer dayawensis from the intestinal tract of Lagocephalus guentheri, including all visible characteristics. The black spots in the alive specimen – the pigment granules. Diagnostic characteristics of the parasite - the muscular post-oral ring (1), genital pore (2), cirrus sack (3), ovary dorsally behind left testes (4) and opposite testes (5), as well as vitelline follicles reaching up to the pharynx. Follicles extend over the whole body and are partially omitted in the drawing for the sake of clarity.

All three found cestode species were plerocercoid larval stages from two families, Trypanorhyncha and Tetraphyllidea. Two species of Trypanorhyncha were encysted on various organs, the Tetraphyllidea were free inside the digestive system. The first species was found encysted on the intestines (mesenteries, swim bladder) surrounded by connective tissue capsules in the body cavity in L. sceleratus. Prevalence was 22.9% and intensity 1–3. Freed from the blastocysts (n = 3), L_T was 3.4–10.2 mm with a transparent long and slender body (width 618.7–1000.8 μm at pars bothrialis). Scolex is long, slender, and well-developed. Two opposing posteriorly slightly notched bothridia are with length of 529.1–839.0 μm. Four long tentacles (length 1081.4–1934.5 μm; basal width 25.2–63.7 μm) are bearing hollow hooks. Tentacle sheaths are as spirals inside the scolex, ending in four elongated (length 484.6–1934.5 μm; width 131.9–224.4 μm) bulbs. The appendix behind the pars bulbosa is long and slender. All measurements and general morphological traits were consistent with the description by Palm (2004). The morphology, morphometric measures, and shape of the tentacle sheaths identified the specimens as plerocerci of Callitetrarhynchus gracilis. Blasting on the GenBank against 28S rRNA sequence (n = 1, accession no. OM867573) also gave 100% Query Cover and 98.06–100% Identity for C. gracilis (accession nos. FJ572957.1; DQ642758.1-DQ642759.1; MG694210.1; MN488532.1).

The second species was encysted on the stomach wall of L. guentheri. P was 2.9% and intensity was one individual. L_T was 766.0 μm, width 457.6 μm at pars bothrialis. A compact, well-developed scolex with four opposing triangular bothridia (length 379.5 μm). Four straight and short tentacles with solid hooks (length 272.8 μm; basal width 29.7 μm), retracted in straight sheathes, ending in four short bulbs (length 231.8 μm). Appendix behind the pars bulbosa short. The measurements fitted the ones by Palm (2004) for plerocercoids of Nybelinia africana Dollfus, 1960. The small size, morphological features, and morphometric measurements identified it as a plerocercoid of N. africana.

The third cestode were plerocercoids in the digestive tracts of Lagocephalus spp. Prevalence and intensity ranged from 11.4% and from one to nine individuals in L. suezensis to 50.0% and from one to 200 individuals in L. guentheri. L_T was 0.2–2 mm, with the bodies being transparent and brittle, showing a diverse body shape, with several distinct morphotypes. Four bothria were present on the scolex, sometimes sitting on peduncles, sometimes retracted into the scolex or completely absent and a central apical sucker was present on the very top of the scolex. The morphological features were identical to the characteristics described in Euzet (1994) and used for his diagnosis of Tetraphyllidea Carus, 1863. The four bothria in combination with the apical sucker and the location of the plerocercoids attributed the cestodes to the order Tetraphyllidea.

Among the nematodes, three species were present. The first species was present in all four pufferfish species, living freely inside the intestine. Only two specimens were found enrolled on the liver and gills in L. suezensis. Prevalence and intensity ranged from 31.4% and from one to eleven individuals in L. suezensis to 88.6% and from one to 80 parasites in L. sceleratus. Larval stages (L3/L4) were present in all pufferfish, adults only in larger specimens of L. sceleratus and only a single adult male in L. guentheri. L_T was 2–40 mm, with a long slender body reaching its greatest width near the middle. The cuticle was smooth, with no ornaments on the conical tail. Both posteriorly directed ventricular appendix and anteriorly directed internal caecum were present. Adults with three pronounced lips and spicules (males), or visible ovaries (females). Measurements and morphological features fell within the description of Deardorff and Overstreet (1981) provided for Hysterothylacium reliquens (Norris & Overstreet, 1975) Deardorff and Overstreet, 1981. Blasting on the GenBank™ against ITS1-5.8s-ITS2 sequence regions (n = 5, accession nos. OM888581- OM888585) resulted in a 94–100% Query Cover and 99.8–100% Identity for deposited vouchers of H. reliquens from the Mediterranean Sea (accession nos. MF062507.1-MF062509.1), Arabian Gulf (accession nos. KX786286.1-KX786293.1) and the Atlantic US-coast (accession nos. MF668856.1; MF668873.1; MF668876.1; MF668880.1) and confirmed the identity of H. reliquens.

The second nematode was indistinguishable from H. reliquens larvae and could only be identified through molecular identification after blasting. The host was L. guentheri, with a prevalence of 5.8% and an intensity of two individuals. Only two specimens could be identified (accession nos. OM888586- OM888587). Query Cover was 87–88% and identity 100% for an unidentified larval form of the genus Hysterothylacium Ward & Magath, 1917 from the Mediterranean Sea (accession nos. MT365529.1-MT365537.1) described by Guardone et al. (2020).

The third nematode was only present in L. sceleratus, with a prevalence of 2.9% and intensity of two parasites, with two specimens found. All measured specimens were incomplete, so no L_T was measurable. The body of the nematode was very elongated and slender, reaching the greatest width near the middle. A posteriorly directed ventricular appendix was present, and intestinal caecum was absent. Length of ventricle was 136–162 μm, width of ventricle was 97–100.6 μm, and oesophagus length was 1488–2009 μm. Three prominent hooked lips were located on the head. One measured specimen was an adult female, having fully developed ovaries and a gonopore. The shape of the lips, the presence of a posteriorly directed ventricular appendix, the absence of an intestinal caecum, and the measurements defined the parasite as belonging to the genus Raphidascaris Railliet & Henry, 1915 as described by Moravec (1998). Specimen are stored in the institute's collection.

The single Hirudinea specimen was found on the skin of L. sceleratus under the pectoral fin, with a prevalence of 2.9% and an intensity of one individual. L_T was 3.5 cm and the body was of brown-green color. The anterior part of the body was distinctly slenderer and of a contrasting yellow in color. On the oral sucker was one pair of eyes. The body greatly widened towards the rear third, at the location of a pair of contractive vesicles, and then tapered down into a small posterior sucker without ocelli. The skin was smooth, except for 14 pairs of contracting lateral vacuoles on the side of the body. The size, presence of vesicles and vacuoles, coloration and present morphological features distinguished the leech as Trachelobdella lubrica (Grube, 1840) according to the description given by Ben Ahmed et al. (2015) and Noël and Sittler (2021).

Two copepod species were free-living on the gill filaments of the pufferfish. The first copepod was present on Lagocephalus spp. Prevalence and intensity ranged from 20.0% and from one to two individuals in L. suezensis to 38.2% and from one to eight individuals in L. guentheri. Mostly adults and rarely chalimus stages were present. The genus could be identified as Caligus Müller, 1785 by the presence of two suction organs (lunnules) on the base of the antennas in combination with a visible single nauplius eye on top of the cephalosome. Measurements for adult ♀ (n = 4) were: 3.1–3.5 mm for L_T, cephalothorax length x width 1.0–1.3 mm x 1–1.2 mm. The length of the genital complex was 1.3–1.6 mm, 1.36–1.90 times as long as the two-segmented abdomen (0.7–1.0 mm), while the first segment of the abdomen was 2.11–2.59 times as long as the second. The maxillipeds showed a well-developed, tapering process proximally on the medial margin. The measurements, traits, and hosts are consistent with the description for Caligus fugu Yamaguti and Yamasu, 1959 by Boxshall and El-Rashidy (2009).

The second copepod exclusively inhabited L. guentheri. Prevalence was 88.2% and intensity was from one to 42 individuals. Egg-bearing females were predominantly present, whereas males were significantly fewer. Measurements for ♀ (n = 2) were: L_T 2.3–2.7 mm, cephalothorax length x width 0.5–0.7 mm x 0.7–0.8 mm, 22–26% of L_T. Body three-segmented, with segments almost as wide as cephalothorax, abdomen with four segments. Caudal ramus length x width 66–74 μm × 40 μm, 1.7–1.9 times longer than wide. Four rows of spinulae on the dorsoventral surface of the genital segment, with a fifth close to the base of the caudal ramus. The diagnosis for Taeniacanthus lagocephali Pearse, 1952 by Dojiri and Cressey (1987) fitted the measured specimens. The measurements, distinct three free thoracic segments equal in width to the cephalothorax, armature at the anal segment, and its presence solely on L. guentheri, a close relative of the originally described hosts, confirmed the species as T. lagocephali.

In Table 4 the quantitative parasite data of the four pufferfish species from the Israeli Mediterranean Coast is provided. Regarding the communities, four parasite species could be classified as common (core species) with an abundance of >2.0 in at least one of the pufferfish species, one species was secondary, with an abundance of 0.6–2, two species were satellite species with an abundance of 0.2–0.6 and four were rare species with an abundance of <0.2 (Tab 4). The parasite assemblages were dominated by H. reliquens (P: 3.43–88.57%, mI: 3.00–17.68), parasitic Protozoa (P = 5.71–51.43%), Tetraphyllidea (P = 11.43–50.00%, mI = 2.75–45.12), Caligus fugu (P = 20.00–38.24%, mI = 1.14–2.34) and T. lagocephali (P = 88.24%, mI = 7.73).

All four pufferfish species were infected with less ecto-than endoparasites. The E/E ratio for Torquigener flavimaculosus, which had no ectoparasites at all, was 0 (0/2). For Lagocephalus guentheri, it reached 0.5 (2/4), for L. sceleratus 0.67 (2/3), and for L. suezensis 1.0 (1/1). For all species combined, the E/E ratio was 0.50 (3/6).

3.3. Differences in parasite assemblages

The differences in parasite assemblage between the pufferfish species were mostly determined by the most common and abundant parasites (H. reliquens, Tetraphyllidea, protists, and T. lagocephali). ANOSIM analysis showed that the parasite assemblies of each of the four species differed from the others, but with some overlaps for each species (R = 0.48; p = 0.001–0.015). SIMPER analysis showed that assemblies were the most similar between T. flavimaculosus and L. sceleratus (Average dissimilarity = 61.4%), followed by T. flavimaculosus and L. suezensis (70.1%) being equally different from L. suezensis and L. sceleratus (70.3%). The differences between L. scleratus and L. guenteri were larger (79.5%) and the biggest dissimilarity was found between T. flavimaculosus and L. guentheri (83.7%) as well as L. suezensis and L. guentheri (84.5%). Other factors such as size, size classes, or catch date failed to yield significant differences. An MDS plot (Fig. 2) also supported the results by showing a clustering in dependence of species with a sufficiently low-stress level (0.15). All infected L. guentheri formed a distinct cluster, as well as L. sceleratus. L. suenzensis clustered partially with L. sceleratus, whereas T. flavimaculosus showed the biggest overlap with L. suezensis.

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Fig. 2

Multidimensional scaling (MDS) plot displaying the similarity of the parasite fauna of the four pufferfish species. Only infected pufferfish shown and one extreme outlier of Torquigener flavimaculosus excluded for a more compact display.

4.1. Digenea

The opecoelid Digenea Maculifer dayawanensis was either a rare or a satellite parasite found in the pufferfish, with a prevalence (2.9–11.8%) similar to previous reports of Indo-pacific pufferfish (11.0%) (Martin et al., 2018). Only mature specimens were present in L. guentheri and T. flavimaculosus. This record represents the fifth report of a macroscopic Lessepsian endoparasite from the Mediterranean Sea. All previous reports were also about Digenea in fish, have been found exclusively as adults or could not be properly identified (invalid larval stages) (Fischthal, 1980; Merella et al., 2010, 2016). In the case of the adult Digenea, a possible co-invasion with the hosts from the Red Sea could not be excluded (Pais et al., 2007; Merella et al., 2010, 2016). For the genus Maculifer, only adults have been reported from the Indo-Pacific (Nicoll, 1915; Cribb, 2005; Liu et al., 2010; Martin et al., 2018). Since M. dayawanensis was both present in the non-migratory T. flavimaculosus and the more migratory and habitat-changing L. guentheri, it is so far the strongest example of a Lessepsian endoparasite successfully establishing itself. The parasite was not found by El-Lamie and Abdel-Mawla (2012), who dissected 294 specimens of L. guentheri. Accordingly, M. dayawanensis either may have entered the Mediterranean Sea after 2012 or at an unknown point in the past, most likely coinciding with the immigration of L. guentheri in 1930 (Sanzo, 1930). Another possible introduction pathway is ballast water of ships instead of direct immigration, given the extremely long distance between the first reported location, Daya Bay in the South-Chinese Sea (Sheng and Tong, 1990), and the Israeli Mediterranean Sea. The Suez Canal is a major global shipping corridor, and rules for ballast water treatment only were implemented in 2017 (Galil et al., 2018). Ballast sediments in particular have not been studied as thoroughly as ballast water, are not always filtered, and may provide a suitable habitat for the first hosts, infected snails, releasing them during ballast water change (Gollasch et al., 2019). At least one opecoelid Digenea is reported to have a shortened life cycle, omitting intermediate and even final hosts in its ontogenesis and leaving the primary intermediate host already as adults (Barger and Esch, 2000). Both infected pufferfish species consumed gastropods, which are possible first intermediate hosts for Maculifer spp. A possible shortened life cycle would also explain the absence of encysted larval stages in the pufferfish stomachs from ingestion, as described for a closely related genus of Digenea (Cable, 1956). This study provides L. guentheri and T. flavimaculosus as new host records for M. dayawanensis.

4.2. Copepoda

The copepod Caligus fugu was among either the secondary or satellite parasites, and the presence of egg-carrying females and chalimus stages proved a successful reproduction in Israeli waters. This parasitic copepod was previously reported from L. guentheri and L. suezensis in the Mediterranean Sea and the Suez Canal, and other pufferfish species in the Indo-Pacific (mostly the genus Takifugu (Boxshall and El-Rashidy, 2009)). The prevalence was slightly lower than previously described for the Mediterranean Sea (34.7–80.0%) and similar to the Suez Canal (34.6%) (El-Lamie and Abdel-Mawla, 2012; Özak et al., 2012). Only Lagocephalus spp. were infected, while the sand-bound T. flavimaculosus (Farrag et al., 2016) either did not come into contact with the parasite or was no suitable host. The presence of C. fugu on L. sceleratus either presented a possible successful host change after its original immigration, or it was previously just unreported from this species. This study described L. sceleratus as a new host for C. fugu.

The copepod Taeniacanthus lagocephali was one of the common parasites of L. guentheri. The prevalence was similar to previous reports from the Mediterranean Sea (94%) and significantly higher than in the Suez Canal (34.6%). The presence of egg-carrying females confirmed the successful reproduction of this species in Israeli waters, as previously reported in Turkey and the Suez Canal (El-Lamie and Abdel-Mawla, 2012; Özak et al., 2012). The infection pathway was identical, based on the habitat preference, to C. fugu (Farrag et al., 2016).

4.3. Cestoda

All cestodes found were from specialized parasite families (Trypanorhyncha and Tetraphyllidea) with multiple-host life cycles. Pufferfish are particularly susceptible to trypanorhynch cestodes, showing a high diversity of these parasites in their native habitats, but also for other cestodes (Palm and Bray, 2014). The trypanorhynch cestode Callitetrarhynchus gracilis was among the satellite parasites and only present in larger specimens of L. sceleratus (L_T ≥ 45 cm). The scolex was well developed; distinguishing L. sceleratus as a third intermediate host according to Palm (2004). The cestode has been reported from over 140 intermediate hosts, including pufferfish (Palm, 2004; Beveridge et al., 2014). Small clupeids (sardines), engraulids (anchovies), or carangids (jacks) are second intermediate hosts, larger predatory fish are third intermediate hosts, and carcharinid sharks are the final hosts (Palm, 2004). Since large L. sceleratus fed on clupeids, carangids, and engraulids in the Mediterranean Sea, the infection pathway was accomplished by consumption of second intermediate hosts. C. gracilis has been globally reported, including in the Mediterranean and Red Sea (Palm, 2004; El-Sayed Abdou and Palm, 2008; Abdelsalam et al., 2016). This study identified L. sceleratus as a new intermediate host.

The trypanorhynch cestode Nybelinia africana was a rare parasite and only a single specimen was found in L. guentheri. The specimen had a fully developed scolex typical for plerocercoids in a third intermediate host (Palm, 2004). The second intermediate hosts are either small fish or euphausid shrimp (“krill”), a larger fish is the third intermediate host, and a large predatory fish or cephalopod is the fourth intermediate or paratenic host. The final hosts are a diverse group of elasmobranchs (Palm, 2004). Fish, including goatfish, and cephalopods were important prey items for L. guentheri in this study and are documented as intermediate hosts for this parasite (Palm, 2004). The genus Nybelinia Poche, 1926 is widely distributed and N. africana has been reported from the Mediterranean Sea, Atlantic, and the Indian Ocean surrounding Africa (Palm, 2004; Merella et al., 2010, 2016). The genus has previously been reported in L. sceleratus (Beveridge et al., 2014) and N. africana also infects the Lessepsian predatory fish Fistularia comersonii in the Mediterranean Sea, with a much higher prevalence (82.4–100.0%) (Merella et al., 2016). This study provided L. guentheri as a new host for N. africana and therefore was the first report for this species in a tetraodontid fish.

Larval Tetraphyllidea were either common or satellite species infecting Lagocephalus spp. Tetraphyllidea were reported to infect L. guentheri in the Suez Canal (El-Lamie and Abdel-Mawla, 2012), having a much lower prevalence than in the present study (prevalence of 6.1%). Several distinct morphotypes were present, possibly representing several species infecting the pufferfish. Tetraphyllidea have life cycles involving multiple hosts, including copepods, small fish, squid, and larger fish as paratenic hosts, with elasmobranchs as final hosts (Dick et al., 2006; Pardo-Gandarillas et al., 2009). They are reported globally, including the Mediterranean Sea (Keser et al., 2007; Pardo-Gandarillas et al., 2009; Felizardo et al., 2010). The high numbers of up to 200 plerocercoids in the pufferfish assigned them as paratenic hosts. The sand-bound T. flavimaculosus was not infected, so Lagocephalus spp. may have got infected in the Posidonia oceanica meadows they inhabit (Kalogirou et al., 2010, 2012; Kalogirou, 2013; Farrag et al., 2016). The comparatively higher prevalence and intensity of the infection in L. guentheri could be attributed to its earlier switch and a stronger emphasis on fish (several confirmed small intermediate Mediterranean hosts (Radujković and Šundić, 2014)) and cephalopods (suggested important intermediate hosts of Mediterranean shark tapeworms (Tedesco et al., 2020)) in its diet, thereby accumulating more plerocercoids over time. L. sceleratus and L. suezensis were new records for the Tetraphyllidea as paratenic hosts.

4.4. Nematoda

The nematodes belonged to the Raphisascarididae, maturing in teleost fish. Hysterothylacium reliquens was present in all four pufferfish species. It was one of the common or a secondary parasite. While adult nematodes were only present in L. sceleratus and one single specimen was found in L. guentheri, marking them as final hosts. The two smaller pufferfish species only acted as intermediate hosts. The entire life cycle and first hosts of H. reliquens are unknown, but larval stages are described in various fish, cephalopods, and decapod crustaceans (Deardorff and Overstreet, 1981). For the related H. aduncum, small crustaceans and polychaetes are known as the first and intermediate host (Køie, 1993; González, 1998). All pufferfish species in this study fed on crustaceans. The high prevalence and intensity in L. sceleratus could be attributed to the high percentage of crustaceans in its diet. H. reliquens is reported globally (Deardorff and Overstreet, 1981; Zhao et al., 2016), including the Mediterranean Sea (El-Sayed et al., 2004; Simsek et al., 2018). Reported hosts include pufferfish in the Atlantic Ocean (Rodríguez et al., 2019). Sequence analyses revealed high similarity (Query Cover 94–100% and identity 99.8–100%) with various vouchers from the Atlantic Ocean, Arabian Gulf and the Mediterranean Sea, confirming this nematode as globally distributed and not as a Lessepsian migrant species. This study identified L. guentheri, L. sceleratus, L. suezensis, and T. flavimaculosus as new hosts of H. reliquens.

The second nematode also belonged to the genus Hysterothylacium, being different from H. reliquens. The sequence analysis showed a high similarity to a Hysterothylacium sp. larval form reported from octopus (Eledone spp.) in the Mediterranean Sea with a similarly low prevalence (6.7%) by Guardone et al. (2020). The sole presence and low prevalence of larvae in L. guentheri could be attributed to the high percentage of cephalopods in its diet. L. guentheri thereby is a paratenic host for Hysterothylacium sp. Taxonomic status of this nematode as a native parasite to the Mediterranean Sea could also be confirmed by molecular markers. This study presented L. guentheri as a new host for this parasite and shed light on the life cycle of this not yet fully identified taxon.

The third nematode belonged to the genus Raphidascaris and was a rare parasite of L. sceleratus. The presence of an adult female specimen identified L. sceleratus as a final host of this parasite. Raphidascaris sp., R. mediterraneus and R. macrouri, were reported from predatory fish in the Mediterranean Sea (Dallarés et al., 2014; Pérez-i-García et al., 2015). Since the isolated parasite specimens lacked significant morphological features, identification to species level was impossible. The full life cycle of this genus is only known for one species, with invertebrates as the first hosts, with small fish as the secondary or paratenic hosts, and with large predatory fish as final hosts (Anderson, 2000). Pufferfish are known as final hosts to the genus Raphidacaris in the Pacific Ocean (Moravec and Justine, 2020). Therefore, the found Raphidascaris may theoretically be a Lessepsian migrant. This study demonstrated the second report of the genus Raphidascaris in Tetraodontidae and also described L. sceleratus as a new host.

4.5. Hirudinea

The marine leech Trachellobdella lubrica was a rare parasite of L. sceleratus. The species has a circumglobal distribution in warm and temperate waters, including the Arabian and the Mediterranean Sea (Saglam et al., 2003; Öktener and Utevsky, 2010), and prefers shallow waters (Noël and Sittler, 2021). Trachelobdella lubrica parasitizes on a wide range of hosts of all sizes (Epshtein, 1973) and has been reported to parasitize another Lessepsian migrant, the cornetfish Fistularia commersonii (Merella et al., 2016). This study identified L. sceleratus as a new host for this species and the first report of a T. lubrica parasitizing a member of the Tetraodontidae.

We are very grateful to the sport/professional fishermen and the staff of the IOLR who collaborated and made available the specimens of the pufferfish We are also grateful to Dr. Rodney A. Bray for his help and advice to identify the Digenea. We thank the associate editor and anonymous reviewers for providing valuable and constructive comments on our manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.