Ultramorphology and Molecular Studies of Contracaecum Larvae (Nematoda: Anisakidae) Collected in Five Cyprinid Fish Species from Sulaimani Province, Kurdistan Region-Iraq

Abstract

A total of 1134 freshwater fishes belonging to Cyprinidae (Acanthobrama marmid (n=20), Alburnus caeruleus (n=7), Alburnus 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)) were collected in different water bodies in Sulaimani Province, Kurdistan Region-Iraq for the presence of larval nematode of the genus Conteacaecum. This investigation revealed that 17 fishes belonged to five species (A. marmid, A. grypus, C. trutta, C. luteus and C. regium) were infected with Contracaecum larvae with prevalence of 35 %, 0.81 %, 0.90 %, 4.49 % and 5.76 %, respectively. The third- larval stage was morphologically studied by optical microscopy, and the ultrastructure was investigated using scanning electron microscopy (SEM). In addition, molecular analysis was carried out by amplifying, sequencing and comparing different gene loci, including internal transcribed spacers (ITS-1 and ITS-2) and cytochrome oxidase c subunit-II (COX-2), of the different isolated Contracaecum larvae. These sequences were also compared with closely related nematode sequences from the GenBank. Fifteen sequences were obtained for this study from the collected Contracaecum larvae. ITS-1, ITS-2 and COX-2 were amplified by polymerase chain reaction (PCR) and sequenced. The sequences of ITS-1, ITS-2 and COX-2 revealed that the collected Contracaecum larval specimens from all infected fish species represented one species (Contracaecum rudolphii B) based on the identity percentage in the GenBank database. The genetic characterisation of the parasite in the present study is available in the GenBank database, and the obtained ITS-1, ITS-2 and COX-2 sequences were deposited in GenBank. The present study provides information on the accurate identification and molecular analysis of Contracaecum larvae in the infected fish species in Sulaimani Province, Kurdistan Region-Iraq.

Keywords: Contracaecum rudolphii B; Cyprinidae; Genetics; Nematode; Systematics.

Fig. 1

Map of north Iraq showing the study area.

Fig. 2

Photo micrograph of Contracaecum larva in Carasobarbus luteus. A - Anterior region of the larva (I00×); B - Posterior region of the larva (I00×); C - Mouth region of the larva (400×); D - Tail region of the larva (400×) an = anus; bt = boring tooth; er = cuticle ridges; eso = esophagus; esoc = esophageal caecum; int = intestine; intc = intestinal caecum; mo = mouth opening; p = papillae; rec = rectum; ta = tail.

Fig. 3

Scanning electron micrograph of Contracaecum larva in Carasobarbus luteus. A - Anterior region of the larva; B - Head region of the larva; C - Tail region of the larva; D - Cuticle of the larva an = anus; bt = boring tooth; cu = cuticle; exp = excretory pore; mo = mouth opening; p = papillae; ta = tail.

Fig. 4

Multiple sequence alignment for ITS-1 in C. rudolphii B in the five different fish species.

Fig. 5

Multiple sequence alignment for ITS-2 in C. rudolphii B in the five different fish species.

Fig. 6

Alignment of the ITS-1 and ITS-2 sequences representing genotype 1 (C. rudolphii B) from the present study and genotype 2 (C. rudolphii A) sequences have been deposited in GenBank under the accession numbers AJ634 782 and AJ634 785. Nucleotide differences between the aligned equences are indecated by having no asterisks.

Fig. 7

Phylogenetic relationships between Contracaecum larvae from the present study and other Contracaecum species as inferred by maximum likelihood obtained from ITSl. Ascaris suum was used as outgroup.

Fig. 8

Phylogenetic relationships between Contracaecum larvae from the present study and other Contracaecum species as inferred by maximum likelihood obtained from ITS2. Ascaris suum was used as outgroup.

Fig. 9

Phylogenetic relationships between Contracaecum larvae from the present study and other Contracaecum species as inferred by maximum likelihood obtained from COX2. Ascaris suum was used as outgroup.