Ultramorphology and molecular studies of Contracaecum larvae (Nematoda: Anisakidae) collected in five Cyprinid fish species from Sulaimani Province, Kurdistan Region-Iraq

Sulaimani Province is located in the northeast of Iraq; it is situated between latitudes 35° 05’ and 36° 30’ and between longitudes ‘44° 25’ and 46° 20’. It is located close to the Iraqi-Iranian border. There are many water bodies in this province in addition to the two large rivers (the Lesser Zab River and the Sirwan River) that pass through this province (Fig. 1).

Fig. 1

During the period from January to the end of December 2018, 1134 fish were collected and examined for infection with anisakid larvae (Contracaecum). The fishes comprised 13 species of Cyprinidae as follows: Acanthobrama marmid (n=20), Alburnus caeruleus (n=7), A. mossulensis (n=62), Arabibarbus grypus (n=123), Barbus lacerta (n=7), Capoeta trutta (n=222), C. umbla (n=161), Carasobarbus kosswigi (n=5), C. luteus (n=89), Carassius auratus (n=54), Chondrostoma regium (n=52), Cyprinion kais (n=10) and C. macrostomum (n=322). They were caught by a pulsed DC electro-shock device (SAMUS 1000). The fish were identified based on their morphometric and meristic characteristics (Coad, 2010) and the scientific names for fishes were identified according to Froese and Pauly (2019). The fish were immediately transported to the laboratory for parasitological examinations, where they were dissected from the ventral side. The body cavity, stomach, spleen, liver, kidneys, heart, muscles, swim bladder and gonads were all examined for anisakid cysts. The gastrointestinal tract was removed, from the rectum to the esophagus, opened longitudinally, and carefully examined under stereoscope (Amlacher, 1970). The cysts were collected and washed with physiological solution (saline solution 0.9 %) in a glass Petri dish. The cysts were opened under a stereoscope with the aid of a fine needle to release the Contracaecum larva, and then washed with saline solution and preserved. The prevalence and intensity of infection were calculated for each fish species based on Margolis et al. (1982). The infection level was calculated as prevalence (percentage of investigated fish infected) and mean intensity (mean number of Contracaecum larva per infected fish).

A total of 1134 fish were examined for parasitic larval Contracaecum nematodes. Only 17 fishes (belonged to five species) were infected with this parasite. The parasitic larvae were found in the intestinal wall, liver, ovaries, swim bladder, gallbladder and mesenteries of 1.49 % (17\1134) of fishes. The prevalence of Contracaecum larvae and the mean of intensity per infected fish varied among the different fish species (Table 1).

A total of 7 fish (prevalence 35 %, mean intensity 5.57) out of the 20 Acanthobrama marmid specimens examined were infected with Contracaecum larvae, which represented the highest prevalence among the collected fishes. While, only one fish (prevalence 0.81 %, mean intensity 7) out of 123 Arabibarbus grypus specimens examined was infected with this larva, which represented the smallest prevalence among the collected fishes in the present study. These results agree with Abdullah and Mhasein (2011), who recorded Contracaecum larvae in Acanthobrama marmid and Chondrostoma regium with prevalence of 16.6 % and 36.4 %, respectively, among ten fish species in the Lesser Zab River. This variation in the prevalence may be related to the temperature, water level, intensity of both the intermediate host and migratory bird (final host), and the types of food and feeding habits of the fish (Younis et al., 2017).

The present investigation shows that prevalence and intensity of Contracaecum larvae in the examined fishes are relatively low. This may be because only visual examination was used to isolate Contracaecum larvae in this study. The prevalence and number of larvae may have been higher than Contracaecum larvae found in the collected fishes in the present study if an incubation method had been used (Shamsi & Suthar 2016; Buchmann, 2007). Third larval stage of the anisakid nematode Contracaecum rudolphii Hartwich, 1964, are commonly infect a range of fish species and mainly cyprinids particularly in area where the final host (piscivorous birds such as cormorants) are found (Moravec, 1994).

Morphological examination and measurements were conducted by optical microscopy and identified that the Anisakid larvae in the present study were Contracaecum larvae (L3), as described by Moravec (2009). Additionally, there were no significant morphological differences among the larvae that were recorded in different fishes. The Contracaecum larvae were light brownish-yellow in colour. They had an elongated cylindrical body and short tail with conical and pointed tip. The bodies had fine, dense transverse striation of the cuticle. The larvae were encapsulated within a slender body, they have distinct boring tooth. Excretory pore situated anteriorly, cuticular striations were observed through the whole length of the body. The esophagus consisted of a long muscular part and a short glandular ventriculus. The esophageal caecum was extended posteriorly and the intestinal caecum was extended anteriorly. The intestine was filled with numerous small brownish granules, and the gonads and other parts of the reproductive system were not developed (Fig. 2).

Fig. 2

The total length of the larvae was 3.5 – 11 mm, with a width of 0.10 – 0.25 mm. The esophagus length was 0.7 – 1.3 mm, the intestinal caecum length was 0.20 – 0.35 mm and the esophageal caecum length was 0.25 – 0.40 mm (Table 2). Since this parasite was in the larval stage and lacked the reproduction system, it was difficult to morphologically determine the exact classification at the species level. The specimens showed a close resemblance in both measurements and characteristics to those recorded by Moravec (2009) in Cyprinus carpio from the Czech Republic. In the microscopy studies, there were no significant morphological differences among the Contracaecum larvae in the five different fish species; the photomicrograph of the third larval stage of Contracaecum in Carasobarbus luteus is only an example (Fig. 2).

The SEM study revealed a regular striation pattern in the cuticular striations of larvae from all fishes. The striations were narrow in the anterior of the larvae and in the pre-tail region, and they became gradually wider when extended posteriorly. There were no significant different ornamentations among the larvae. There were transverse striations between the striated cuticular rings (Fig. 3). The mouth was transverse in shape the lips are not developed and provided by four papillae, a well-defined boring tooth present. Excretory pore is anteriorly located just below the mouth. Anal opening is located near the posterior end. Tail is short. The body was free of any projections, such as a spine or papillae. The cloacal regions had no papillae because they were in the larval stage and not yet mature.

Fig. 3

The ultrastructural characteristics of the present specimens showed great similarity with the specimens (Contracaecum larva) of Younis et al. (2017), who collected them from different freshwater fishes in Lake Nasser, Egypt. Furthermore, the ultrastructural features of the present Contracaecum larvae were similar to those of Rahemo and Nawab Al-Din (2009), who used SEM to study larvae from Acanthobrama marmid collected in the Tigris River. The SEM study revealed no significant ultra-morphological differences among the Contracaecum larvae recorded in the present investigation in these five different fish species. The scanning electron photomicrograph of the third larval stage of Contracaecum in Carasobarbus luteus is used as an example (Fig. 3).

Agarose gels analyses demonstrated the same size for each ITS-1, ITS-2 and COX-2 region. The amplicons were ~530 bp, ~430 bp and ~630 bp for ITS-1, ITS-2, and COX-2 respectively, confirming that all sequences were of the same genus.

Alignment of the resulting sequences revealed that there was no significant variation in the ITS-1, ITS-2 and COX-2 regions, which indicated the presence of only one type of larva. Based on percentage identities of nucleotides from GenBank, the on-line BLAST tool showed the ITS-1 sequences obtained from larvae-infected A. marmid, A. grypus, C. trutta, C. luteus and C. regium matched 99.78 %, 100 %, 99.76 %, 100 % and 100 %, respectively to the previously reported reference gene sequences for the ITS-1 in Contracaecum rudolphii B (Zhang et al., 2009) from the stomachs of great cormorant Phalacrocorax carbo sinensis from the Guangzhou Zoo in Guangdong in China, which was examined previously and deposed in GenBank (FJ467618) (Zhang et al., 2009). The ITS-2 sequences obtained from larvae-infected A. marmid, A. grypus, C. trutta, C. luteus and C. regium matched 100 % to the previously reported reference gene sequences for the ITS-2 in Contracaecum rudolphii B (Li et al., 2005) from the stomachs of great cormorant Phalacrocorax carbo sinensis from the Venice Lagoon in northeastern Italy and from Monaci Lake in central Italy, which was examined previously and deposed in GenBank (AJ634786) (Li et al., 2005), and also matched 100 % to the previously reported reference gene sequences for the ITS-2 in Contracaecum rudolphii B from the same host from from the Guangzhou Zoo in Guangdong in China, which was examined previously and deposed in GenBank (FJ467618) (Zhang et al., 2009). The COX-2 sequences obtained from larvae-infected A. marmid matched 98.52 % to the previously reported reference gene sequences for the COX-2 in Contracaecum rudolphii B (Mattiucci et al., 2008) from the great cormorant Phalacrocorax carbo sinensis from Italy, which was examined previously and deposed in Gen-Bank (EF122203) (Mattiucci et al., 2008). The COX-2 sequences obtained from larvae-infected A. grypus, C. trutta and C. regium matched 100 %, 99.19 % and 100 % respectively to the Contracaecum rudolphii B from Phalacrocorax carbo sinensis which was examined previously and deposed in GenBank (EF513509). While, the COX-2 sequences obtained from larvae-infected C. luteus matched 99.79 % to the Contracaecum rudolphii B from the same host which was examined previously and deposed in Gen-Bank (EF558894) (Mattiucci et al., 2008).

The genetic characterisation of the parasite in the present study is available in the GenBank database; the ITS-1, ITS-2 and COX-2 sequences obtained were deposited in GenBank, and their accession numbers are stated in Table 3. The ITS-1, ITS-2 and COX-2 sequence analysis confirmed that third larval stage of Contracaecum (L3) parasitizing the Cyprinid fish (A. marmid, A. grypus, C. trutta, C. luteus and C. regium) from the present study belong to species C. rudolphii type-B, a parasite at the adult stage of the great cormorant Phalacrocorax carbo sinensis mainly from Italy water (Li et al., 2005; Mattiucci et al., 2008). ITS-1, ITS-2 and COX-2 markers may provide reliable evidence for specific species identification of Contracaecum larvae occurring in fish (Mattiucci et al., 2010). Therefore, the occurrence of C. rudolphii B larvae from Iraqi waters was also proved by molecular evidence inferred from the ITS-1, ITS-2 and COX-2 markers used in the present study. Furthermore, the sequences of ITS-1 and ITS-2 obtained from the collected larvae in different fish species were aligned with each other (the same gene). Pairwise comparisons of all nucleotides sequence among the Contracaecum larvae collected in the five different fish hosts revealed that only one nucleotide variation (0.2 %) in alignment position 161 for ITS-1 (Fig. 4) and there was no nucleotide variations for ITS-2 (Fig. 5).

Fig. 4

Fig. 5

Fig. 6

Contracaecum rudolphii Hartwich, 1964 is a species complex it consist of several sibling species. C. rudolphii sensu lato (s.l.) named C. rudolphii A and C. rudolphii B (D’Amelio et al. 1990), they could also be differentiated from each other based on the ITS-1 and ITS-2 sequence data (Li et al. 2005). D’Amelio et al. (2007) indicated the existence of a third cryptic species of C. rudolphii complex (C. rudolphii C) in double-crested cormorants from west-central Florida based on PCR-RFLP and sequencing of the rrnS mitochondrial gene and nuclear ribosomal spacers. Shamsi et al. (2009) described two new sibling species of the C. rudolphii complex, C. rudolphii D from Phalacrocorax carbo and Contracaecum rudolphii E from Phalacrocorax varius in Australia, based on the ITS-1 and ITS-2 sequence data. Recently, D’Amelio et al. (2012) recorded new isolate of C. rudolphi complex (C. rudolphii F) from brown pelican Pelecanus occidentalis in the northern Gulf of Mexico.

Various studies demonstrated that internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA (rDNA) provide genetic markers for the accurate identification of a range of species of Ascaridoids. In addition, more studies indicated that sibling species can be differentiated based on the ITS sequences (Jacobs et al., 1997; Zhu et al., 2000; 2001; 2002). The ITS-1 sequences of the obtained larvae (C. rudolphii B) in the present study show 11 (2.46 %) nucleotide differences with the previously reported reference gene sequence for the ITS-1 in C. rudolphii A which was examined and deposed in GenBank (Accession number: AJ634782). While, ITS-2 shows 14 (5.22 %) nucleotide differences with the C. rudolphii A which previously reported reference gene sequence for the ITS-2 in C. rudolphii A which was examined and deposed in GenBank (Accession number: AJ634785) (Li et al., 2005) (Fig. 6). This clear genetic differentiation support previous sequence analyses (Li et al., 2005) that there are sequence differences (1.8 %) in the ITS-1 and (5.1 %) in ITS-2 between the sibling species of C. rudolphii A and B (Li et al., 2005). Extending these studies, we investigated that there is no significant sequence variation in the ITS-1 and ITS-2 within and among the larvae collected from different fish host in the present investigation. The molecular finding of the present investigation support that the present finding larvae belong to C. rudolphii type-B.

In the phylogenetic analysis, the sequence data aligned with the data sequences of ITS-1, ITS-2 and COX-2 form other different Contracaecum species (different genotypes) and Ascaris suum used as out group detected in GenBank (Table 4, 5). Phylogenetic analyses were conducted in MEGA X (Kumar et al., 2018). The evolutionary histories were inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura & Nei, 1993). The tree with the highest log likelihood (-6713.31, -3848.19 and -385797 for ITS-1, ITS-2 and COX-2 respectively) are shown (Fig. 7, 8, 9). Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances 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 (next to the branches). The Contracaecum larvae from all different fish hosts were clustered in the same clade of Contracaecum rudolphii B. Moreover, the concatenated phylogenetic tree of the ITS-1, ITS-2 and COX-2 sequences using ML analyses indicated that Contracaecum larvae clades were distinct species by high bootstrap values (Fig. 7, 8, 9).

Fig. 7

Fig. 8

Fig. 9

The first information on Contracaecum larvae from Iraqi freshwater fishes was provided by Herzog (1969) from ten fish species collected from different inland water bodies in Iraq. In the Kurdistan Region, larvae were recorded from Dukan Lake in Sulaimani Province by Abdullah and Rasheed (2004) in Arabibarbus grypus, Carasobarbus luteus, Chondrostoma regium, Cyprinion macrostomum, Cyprinus carpio, Luciobarbus barbulus, L. esocinus, L. kersin, L. subquincunciatus, L. xanthopterus, and Squalius lepidus. Thus far, a total of 21 host fish species are known for Contracaecum larvae in the Kurdistan Region of Iraq (Mhaisen & Abdullah, 2017). In addition, molecular identification of Contracaecum larvae in fish species have also not been studied, and there is still no specific identification of the Contracaecum species in fresh and marine water fish species in Iraq. Till now, morphological identification based on only optical microscopy has been used to identify larval stage of Contracaecum only at genus level in Iraq. Recently, 42 different fish species were known as hosts for Contracaecum sp. larvae in Iraq from north to south including marine water fish (Mhaisen, 2019). Therefore, the authors provide the first molecular evidence of Contracaecum infection in fish from Iraqi waters in the present study. Notably, the Contracaecum larvae can infect many types of fish in Iraq. This nonspecific infection ability towards a variety of different organs and fish species may lead to the infection of a variety of piscivorous birds and mammals in the region. Anisakidosis is a disease caused by the accidental ingestion of larval anisakid nematodes in raw fish. All fishes which infected with Contracaecum larvae in the present study are edible, particularly A. marmid (prevalence 35 %) in Sulaimani Province. This may affect human health in this region, because this fish is used by local people and other consumers as a food source.

It is noteworthy that the adult Contracaecum rudolphii sensu lato was recorded previously for the first time in Iraq in the digestive tract of the great black cormorant Phalacrocorax carbo from Baghdad province in Iraq (Al-Moussawi & Mohammad, 2011). Furthermore, three other species of this genus (Contracaecum) were reported in birds (final host) in Iraq, namely, C. microcephalum, C. multipapillatum and C. ovale (Habish, 1977; Al-Hadithi & Abdullah, 1991).