Introduction

Among many parasites found in fish, nematodes are those of the highest importance (Dural et al. 2011; Abdel-Ghaffar et al. 2013). Ascaridoid nematodes (family Anisakidae) have been recorded worldwide naturally parasitizing approximately 200 fish species (Køie et al. 1995) and 25 cephalopod species (Hochberg 1990). Marine mammals as well as humans can also become accidental hosts by ingesting fish infected with third-stage larvae (Szostakowska et al. 2002). Anisakid nematodes which are common in bony fish are represented by the following genera: Anisakis Dujardin, 1845; Contracaecum Railliet and Henry, 1912; Hysterothylacium Ward and Magath, 1917; Paranisakiopsis Yamaguti, 1941; and Pseudoterranova Mozgovoy, 1951. The genus Hysterothylacium comprises more than 59 species and is considered as one of the most ubiquitous parasitic nematode species in fishes of the North Atlantic (Navone et al. 1998; Balbuena et al. 2000; Klimpel et al. 2006). It can be assumed that life cycles within the genus Hysterothylacium are principally similar in all species. Sexually mature adults and the fourth larval stages (L4) are mainly found in the lumen of the stomach and intestine of the fishes, which act as definitive hosts, while the third larval stages (L3) are found in the fish mesenteries. More than 100 different benthic and planktonic invertebrate species act as intermediate and/or paratenic hosts (Navone et al. 1998). In the North Atlantic, the North Sea, the Baltic Sea, the Mediterranean Sea and in adjacent temperate and cold waters, the species Hysterothylacium aduncum Rudolphi, 1802, is a very common fish parasite, but its taxonomic status is highly ambiguous (Margolis and Arthur 1979; Palm et al. 1999; Klimpel et al. 2006). Several authors assumed that H. aduncum is a single species parasitizing opportunistically in various marine fish species (Szostakowska et al. 2002; Morsy et al. 2013). In contrast, Hartwich (1975) recognized three distinctive species: H. aduncum mainly found in clupeid hosts like sprat Sprattus sprattus and herring Clupea harengus, Hysterothylacium gadi from gadoid fish, and Hysterothylacium auctum, which was frequently identified in the eelpout Zoarces viviparous. Petter and Cabaret (1995) reported a great variability between parasitic specimens collected from different fish species in North Atlantic waters, but they proposed only two subspecies on the basis of biometrical data. Thus, it would not be surprising if future molecular studies revealed that H. aduncum represents a complex of an unknown number of sibling species (Balbuena et al. 2000). Recently, various studies have demonstrated that the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) and polymerase chain reaction-based results provide genetic markers of the appropriate DNA target sequence for the exact identification and delineation of parasitic nematodes of the family Anisakidae and Ascarididae (Mašová et al. 2010; Testini et al. 2011).

A variety of organisms have been investigated to evaluate their potential as biological indicators for different types of pollution in the aquatic environment. The relationship between pollution and parasitism in aquatic organisms and the potential role of parasites as water quality indicators have received increasing attention during the past two decades (Dural et al. 2011). However, until now, little is known on the accumulation of toxins within parasites. A variety of wild freshwater and marine fish are subjected to infection by different species of parasites. Particularly, intestinal helminths can accumulate heavy metals at concentrations that are magnitudes higher than those in the host tissues or the environment (Schludermann et al. 2003; Morsy et al. 2012). This aspect suggests that parasites may serve as useful indicators for biologically available metals (Galli et al. 2001).

In the present study, morphological and molecular analyses of H. aduncum parasitizing the common sole S. solea captured at the coasts of Alexandria City in the Mediterranean Sea, Egypt were investigated. Also, the bioaccumulation effect of these parasites for some heavy metals was studied to determine whether this anisakid species is a useful bio-indicator species for water pollution or not.

Materials and methods

In the period between May to September 2013, anisakid worms of the genus Hysterothylacium were collected from 80 specimens of the common sole Solea solea (Soleidae) captured at the coasts of Alexandria City along the Mediterranean Sea, Egypt. The fishes were transported alive with good aeration and cooling to the Parasitology Laboratory of the Zoology Department, Faculty of Science, Cairo University. The fishes were identified according to Desoutter (1992) and examined externally for external lesions or parasitic cysts. They were dissected carefully and examined thoroughly for endoparasitic infections. The contents of the digestive tract were poured into physiological saline solution and examined by dissecting microscopy. The nematode larvae were collected, fixed, and stored in 70 % ethanol. For light microscopy, worms were cleared in lactophenol and photographed by help of a Zeiss microscope supplied by a Canon digital camera. The taxonomic identification followed the guidelines of Petter and Maillard (1988), Incorvaia and Díaz de Astarloa (1998), Timi et al. (2001), Bicudo et al. (2005), and Felizardo et al. (2009). For scanning electron microscopy, samples were fixed in 2.5 % glutaraldehyde solution buffered in 0.1 M sodium cacodylate. Samples were dehydrated in an ethanol series, CO2 critical-point dried, coated by gold, and finally examined and photographed using an Etec Autoscan Jeol SEM under an accelerating voltage of 20 Kv. Measurement ranges were taken in millimeters—mean values were given in parentheses unless otherwise indicated. For molecular analysis, worms were washed with phosphate buffer saline and homogenized in liquid nitrogen. DNA was extracted using a QIAamp® DNA Mini Kit (Quiagen, GmbH, Germany) following the manufacturer’s protocol. Polymerase chain reaction (PCR) was carried out to amplify the target 18S rDNA using the primers NC5 (forward: 5’-TAGGTGAACCTGCG GAAGGATCATT-3’) and NC2 (reverse: 5’-TTAGTTTCTTTTCCTCC GCT-3’). The PCR product were directly sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) with 310 Automated DNA Sequencer (Applied Biosystems, USA) using the same primers for annealing. The net sequence data was aligned using CLUSTAL-X multiple sequence alignment (Thompson et al. 1997) and compared with some of the previously recorded sequence data for species of the same genus obtained from the Genebank to analyze intra-specific differences. The dendrogram was built up using the multiple-alignment algorithm in Megalign (DNASTAR, Window version 3.12e). Phylogenetic relationships were inferred using maximum-likelihood (ML) (Felsenstein 1981). The obtained sequences have been deposited in the Genebank under accession number KC004227. Heavy metal analyses in different organs (liver, gills, kidney, and muscles) of infected and non-infected fish and also in nematode tissues were carried out according to the procedure described by UNEP/FAO/IOC/IAEA (1984). Tissues were digested with specific volumes of concentrated nitric acid and per-chloric acid (2:1 v/v) at 60 °C for 3 days. All samples were diluted with bi-distilled water and assayed using inductively coupled plasma-atomic emission spectrometry (Varian model-Liberty Series II). The metals concentrations of Pb, Zn, Fe, Cd, Cu, and Ni were recorded as microgram metal per gram wet weight and carried out at band width of 0.2 nm and wavelength range of 190–900 nm with 250 nm Elbert mount diffraction grating monochromator.

Results

Thirty five out of eighteen specimens of the examined fish were found to be naturally infected by nematode worms with a percentage of 43.75 %. The infection was recorded in the intestine of infected fish and increased during winter to be 47.5 % (19 out of 40) and fall to 40.0 % (16 out of 40) during summer.

Morphological examination of the recovered worms revealed that the adult worms are characterized by an elongated cylindrical body with its anterior end characterized by the presence of a mouth surrounded by three lips, one dorsal and two sub-ventral ones, which can be interlocked leaving interlabia in between (Figs. 1, 2, 3, 9, 10, 11). Lips (approximately equal in size) were provided with papillae-like structures (Figs. 1, 2, 3, 10). The worm possess a long anterior muscular esophagus, which measured 1.5–3.1 mm (2.5 ± 0.02) in length, was longer in females than in males, and was followed by the intestinal caecum. An excretory pore occurred slightly posterior to the level of the nerve ring. The worm body was covered by a cuticle which was transversely striated (Fig. 4). The tail of the worms was tipped by a single minute thorn called mucron. The body of the male worm measured 13.9–18 mm (16.2 ± 0.2) in length and 0.26–0.34 mm (0.30 ± 0.01) in width at the level of the esophagus. The posterior end was provided with two unequal spicules (Figs. 5, 12, 13). The left one measured 0.69–0.85 mm (0.76 ± 0.2) in length while the right one reached 0.73–0.88 mm (0.81 ± 0.2) in length and possessed caudal papillae (Figs. 6, 12, 14). The body of the female worm was 20.5–24.5 mm (22.7 ± 0.2) long and 0.41–0.52 mm (0.45 ± 0.01) wide and terminated in a conical tail bearing the anus and a short mucron (Figs. 7, 15). The larvae ended in a long cactus-like tail (Fig. 8).

Figs. 1–8
figure 1

Photomicrographs of Hysterothylacium aduncum showing high magnifications of 1–3 the anterior end being equipped by three interlocked lips (L) with interlabium (IL) in between and provided with papillae (P). Note the presence of a surrounding cuticle (C), esophagus (OE). 4 Mid-part of the body showing transverse striations (TS) of cuticle. 5–8 Posterior parts of 5 male with two spicules (SP), short mucron (M), and caudal papillae (P). 6 Caudal papillae (P). 7 Female terminated with short mucron (M) with rectum (R) and anal region (A). 8 Larva with a cactus tail or mucron (M)

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Figs. 9–15
figure 2

Scanning electron micrographs of H. aduncum at high magnifications. 9–11 The anterior end with the three interlocked lips (L), interlabium (IL), and papillae (P). 10–15 Posterior parts of 12–14 male with long spicules (SP), caudal papillae (P), and mucron (M). 15 Female with short mucron (M) and anus (A) region

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Taxonomic summary

H. aduncum Rudolphi, 1802.

Family: Anisakidae Railliet and Henry, 1912.

Host: Common sole S. solea fish Linnaeus, 1758 (Family: Soleidae).

Locality: Coasts of Alexandria City, Mediterranean Sea, Egypt.

Site of infection: The adult worms as well as their larvae were found in the intestine of the infected fish.

Prevalence rate of infection: 35 (47.5 %) out of 80 specimens of the examined fish were infected.

Molecular analyses of the recovered worms demonstrated that the 18S rDNA sequences (771–789 bp) showed 95.5 % similarity to HQ270430, JX413597, and HM437225 for different deposited sequences of previously described H. aduncum, 95.0 % similarity to HQ270433 of Hysterothylacium sp., 94.3 % similarity to AF115571 of H. auctum, and 92.3 % similarity to AB277824 of Pseudoterranova decipiens. These nematodes revealed low genetic divergent values. The highest BLAST scores were aligned and compared with our present sample shown in Fig. 16. Sequence alignment resulted in the fact that the major clade of the constructed dendrogram clustered all Anisakidae species with sequence similarities between 95.5–94.3 %. This showed that H. aduncum is deeply embedded in the genus Hysterothylacium with close relationships to other H. aduncum worms and to Hysterothylacium sp. Thus, they are sister taxons with high bootstrap values (Fig. 17). The minor clade contains 18S rDNA sequences of Porrocaecum depressum and Ascaris suum as outgroups and in addition other taxons with high divergence values.

Fig. 16
figure 3figure 3

Sequence alignment of H. aduncum (present study) infecting S. solea with the most related species. Note: Only variable sites are shown. Dots represented bases identical to those of the first sequences and dashes indicate gaps. Continued

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Fig. 17
figure 4

Dendrogram based on SSU rDNA showing the phylogenetic relationship between the present Hysterothylacium aduncum and other species belonging to the family Anisakidae. Bootstrap percentages of clades as inferred by ML shown above internal nodes

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Heavy metal analyses of the infected fish organs (liver, gills, kidney, and muscles) showed that toxic metals like Pb, Zn, Fe, Cd, Cu, and Ni were detected in all of the tested samples. The concentration rates (mg/g) of these metals in the organs of the examined fish tissues and its parasitizing nematodes were given in Table 1. It was shown that trace metal accumulations in fish tissues occurred in the following descending order: Fe > Cu > Cd > Zn > Ni > Pb. The concentrations of Zn, Fe, Cd, and Cu in fish tissues were found in the following grading order: liver > gills > muscle > kidney. Gills can accumulate large amounts of both Pb and Ni when compared to other fish tissue. The concentrations of Fe, Cu, Cd, and Ni were higher in the nematode parasites than in the tissues of infected fish and were found to be exceeding the US Environmental Protective Agency (0.1 μg−1 (epa)).

Table 1 Concentration (mean ± SD, mg/g) of heavy metals in different organs of the infected fish as well as in their parasitic nematode worms
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Discussion

The common sole S. solea Linnaeus (1758) is a representative of flatfishes which are normally distributed in the continental shelf seas of the Eastern Atlantic Ocean (Froese and Pauly 2007). In the present study, it was found that S. solea, which inhabits also the coasts of the Mediterranean Sea, was infected by H. aduncum which is a common nematode parasite that is not very host-specific neither as adult nor as larval stages (Klimpel et al. 2006). The fish host can heavily accumulate different stages of H. aduncum by ingesting crustaceans, chaetognaths, and small fish that are infested and serve as carriers (Rossin et al. 2011). The percentage of infection with this anisakid parasite was found here to reach 47.5 % being similar to the results of Genc et al. (2005), who stated that Hysterothylacium sp. infected different species of sparid and elasmobranch fishes with percentage of infection ranging between 7.69–78.57 %. Vidal-Martínez et al. (1994) reported that morphological differentiation of nematode parasites belonging to the genus Hysterothylacium is extremely difficult, since all species show the same characteristics. The present parasite is similar to H. aduncum Rudolphi, 1802; Hysterothylacium seriolae Yamaguti, 1941; Hysterothylacium fortalezae Klein, 1973; Hysterothylacium arnoglossi Petter and Maillard, 1987; Hysterothylacium scomberoidei Bruce and Cannon, 1989; Hysterothylacium physiculi Moravec and Nagasawa, 2000; and Hysterothylacium winteri Torres and Soto, 2004, since it shows also three cephalic lips as long as wide, an identical position and anatomy of the foregut, an excretory pore just behind the nerve ring, a very short intestinal caecum (slightly longer than the ventriculus), a very long ventricular appendix, distinct lateral alae, relatively short spicules (not over 1.0 mm), and tail tips of both sexes being covered with numbers of nodular protuberances. Furthermore, this worm is morphometrically very similar to other comparable species of H. aduncum reported by Rudolphi (1802) and Morsy et al. (2012) and is located in the same range of measurements for different body parts.

Molecular approaches such as PCR and direct sequencing of ITS for rDNA have proven to be particularly useful for the accurate and effective identification of all life stages of ascaridoid nematodes (Mašová et al. 2010; Testini et al. 2011; Knoff et al. 2012). In the present study, the lengths of the PCR products of H. aduncum were found to be 771–789 bp being identical to that reported previously in other studies by Martin-Sánchez et al. (2005) and Setyobudi et al. (2011). Valentini et al. (2006) showed that nucleotide differences in the ITS sequences among anisakid species were significantly greater than that within a species. Interspecific differences in 5.8S rDNA sequence were considerably lower than in the ITS, which is not unexpected because the rDNA gene is relatively conserved in nematodes. Similarly, the present study demonstrated that nucleotide differences were also highly conserved. Interspecific differences were 1.4–14.9 % with minor differences between H. aduncum and other Hysterothylacium species. The phylogenetic tree topologies observed within two clades of Hysterothylacium species grouped specimens of H. aduncum, H. auctum, Pseudoterranova decipiens, and Raphidascaris trichiuri in the same group. This is similar to the 18S rDNA MP tree topology obtained by Smythe et al. (2006). This demonstrates that H. aduncum represents a taxonomic unit genetically distant from other anisakids, even from other Hysterothylacium species. In agreement with the observation of Nadler and Hudspeth (2000), Hysterothylacium does not represent a monophyletic group, since the presence of P. decipiens and R. trichiuri suggest the existence of a polyphyletic group for H. aduncum on the 18S rDNA tree.

Some organisms are able to provide information on the chemical state of their environment through their presence or absence (Florence et al. 1992; Muir et al. 1992). Recent studies have been focused on the use of parasites as biological indicators for heavy metal accumulation (Trucekova et al. 2002). In the present study, the heavy metal analysis demonstrated that distribution of Zn, Fe, Cd, and Cu in different organs of infected fishes followed the order: liver > gills > muscles>. These results are in agreement with those obtained by Taweel et al. (2011) and Morsy et al. (2012) who studied the accumulation of different heavy metal in Tilapia and European seabass fish. The investigation shows that the liver and gills are the most important organs for assessing metal accumulation, since the levels of heavy metals in the gills reflect the concentrations of metals in the surrounding water, where the fish live, while the concentrations of metals in the liver represent the storage of metals in the fish body. These obtained results are consistent with those reported by other authors (Florence et al. 1992; Muir et al. 1992; Roméo et al. 1999; Yilmaz et al. 2007). In the present study, comparisons were done between the metal concentrations in the recovered nematode parasites and those in the different organs of the infected fish. These results showed that these nematodes accumulate more Fe, Cu, Ni, and Cd than the tissues of the infected fish. These results agreed with Azmat et al. (2008), who reported that the high level of toxic metals in Echinocephalus sp. and Ascaris sp. within its host suggests that these nematode parasites may be used as sensitive indicators for heavy metals in the aquatic ecosystem.

Recent field study have demonstrated that the 18S rDNA gene of H. aduncum yielded a unique sequence that confirms the taxonomic position in Raphidascarididae with new host and locality record in Egypt. In addition, particular fish nematode parasites can accumulate toxic metals from the aquatic environment. Thus, the application of certain parasites as sentinel organisms could provide a promising new domain for future research in environmental research.