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

Y. S. Abdullah
Y. S. Abdullah
, 
S. M. A. Abdullah
S. M. A. Abdullah
 and 
R. H. Hussein
R. H. Hussein
   | Feb 10, 2021
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Article Category: Research Article
Published Online: Feb 10, 2021
Page range: 41 - 58
Received: Feb 01, 2020
Accepted: Aug 11, 2020
DOI: https://doi.org/10.2478/helm-2021-0001
Keywords
© 2021 Y. S. Abdullah, S. M. A. Abdullah, R. H. Hussein, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Map of north Iraq showing the study area.
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.
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.
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.
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.
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.
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.
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.
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.
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.

Contracaecum nematodes and specimens/accession number (ITS) of taxa used to build phylogenetic trees and the nucleotide (bp) differences with the Contracaecum larvae collected from the present study.

Parasite GeneBank accession no. (ITS) No. of nucleotide differences (bp) Host Source
C. rudolphii A ITS1 (AJ634782) ITS2 (AJ634785) 9 14 Phalacrocorax carbo sinensis Phalacrocorax carbo sinensis Li et al. (2005) Li et al. (2005)
C. rudolphii B ITS1 (AJ634783) ITS2 (AJ634786) 1 0 Phalacrocorax carbo sinensis Phalacrocorax carbosinensis Zhang et al., 2009 Li et al. (2005)
C. rudolphii D ITS1 (FM210251) ITS2 (FM210268) 6 17 Phalacrocorax varius Phalacrocorax carbo sinensis Shamsi et al. (2009b) Shamsi et al. (2009b)
C. rudolphii E ITS1 (FM210257) ITS2 (FM210271) 6 15 Phalacrocorax varius Phalacrocorax varius Shamsi et al. (2009b) Shamsi et al. (2009b)
C. rudolphii F ITS (JF424597) 21 16 Pelecanus occidentalis Pelecanus occidentalis D'Amelio et al. (2012) D'Amelio et al. (2012)
C. ogmorhini ITS1 (AJ291468) ITS2 (AJ291471) 10 15 Arctocephalus pusillus doriferus Zalophus californianus Zhu et al. (2001) Zhu et al. (2001)
C. eudyptulae ITS1 (AJ007461) ITS2 (FM177565) 8 17 - Eudyptula minor Zhu et al.(unpublished) Shamsi et al. (2009a)
C. chubutensis ITS1 (HQ389546) ITS2 (HQ389548) 13 23 Phalacrocorax brasilianus Phalacrocorax atriceps Garbin et al. (2011) Garbin et al. (2011)
C. variegatum ITS1 (MK424804) 22 Bird Hbaiel & Mohammad (unpublished)
C. microcephalum ITS2 (FM177537) ITS1 (FM177523) ITS2 (FM177527) 14 50 72 Anhinga melanogaster Phalacrocorax melanoleucos Phalacrocorax melanoleucos Shamsi et al. (2009a) Shamsi et al. (2009a) Shamsi et al. (2009a)
C. septentrionale ITS1 (AJ634784) ITS2 (AJ634787) 24 35 Phalacrocorax carbo sinensis Alca torda Li et al. (2005) Li et al. (2005)
C. bioccai ITS (JF424598) 30 40 Pelecanus occidentalis Pelecanus occidentalis D'Amelio et al. (2012) D'Amelio et al. (2012)
C. radiatum ITS (AY603529) 35 65 Leptonechotes weddlli Leptonechotes weddlli Kijewska et al. (2008) Kijewska et al. (2008)
C. osculatum ITS (AB277825) 77 105 Arabesque greenling Arabesque greenling Umehara et al. (2008) Umehara et al. (2008)
C. multipapillatum ITS1 (AM940056) ITS2 (AM940060) 146 132 Pelecanus conspicillatus Pelecanus conspicillatus Shamsi et al. (2008) Shamsi et al. (2008)
C. pyripapillatum ITS1 (AM940062) ITS2 (AM940066) 141 136 Pelecanus conspicillatus Pelecanus conspicillatus Shamsi et al. (2008) Shamsi et al. (2008)
Contracaecum larva ITS1 (MN557376) ITS2 (MN526259) - - Acanthobrama marmid Acanthobrama marmid Present study Present study
Contracaecum larva ITS1 (MN557377) ITS2 (MN563727) - - Arabibarbus grypus Arabibarbus grypus Present study Present study
Contracaecum larva ITS1 (MN557378) ITS2 (MN563728) - - Capoeta trutta Capoeta trutta Present study Present study
Contracaecum larva ITS1 (MN557379) ITS2 (MN563729) - - Carasobarbus luteus Carasobarbus luteus Present study Present study
Contracaecum larva ITS1 (MN557380) ITS2 (MN563730) - - Chondrostoma regium Chondrostoma regium Present study Present study
Ascaris suum ITS1 (AB110023) ITS2 (FJ418786) - - Pig Pig Ishiwata et al. (2004) Wickramasinghe et al. (2009)

Prevalence of Contracaecum larva and mean of intensity among infected fish species.

Host Fish
 Prevalence Mean intensity
Examined Infected %
Acanthobrama marmid 20 7 35.00 5.57
Arabibarbus grypus 123 1 0.81 7.00
Capoeta trutta 222 2 0.90 3.00
Carasobarbus luteus 89 4 4.49 4.25
Chondrostoma regium 52 3 5.76 9.00

Contracaecum nematodes and specimens/accession number (COX-2) of taxa used to build phylogenetic trees and the nucleotide (bp) differences with the Contracaecum larvae collected from the present study.

Parasite GeneBank accession no. (COX-2) No. of nucleotide differences (bp) Host Source
C. rudolphii A EF122201 34 Phalacrocorax carbo sinensis Mattiucci et al. (2008)
C. rudolphii B EF558894 1 Phalacrocorax carbo sinensis Mattiucci et al. (2008)
C. rudolphii C EF014283 272 Phalacrocorax auritus D'Amelio et al. (2007)
C. rudolphii F JF727879 40 Pelecanus occidentalis D'Amelio et al. (2012)
C. ogmorhini MN624184 32 Zalophus californianus Mladineo et al. (under press)
C. chubutensis HQ328504 46 Phalacrocorax atriceps Garbin et al. (2011)
C. microcephalum EF122208 71 Phalacrocorax pygmaeus Mattiucci et al. (2008)
C. septentrionale EF558898 60 Phalacrocorax carbo carbo Mattiucci et al. (2008)
C. bioccai EF558899 50 Pelecanus occidentalis Mattiucci et al. (2008)
C. osculatum KC412224 58 Chionodraco hamatus Santoro et al. (2013)
C. multipapillatum AF179910 72 - Nadler & Hudspeth (2000)
C. micropapillatum EU852350 70 Pelecanus onocrotalus Mattiucci et al. (2010)
C. austral GQ847539 55 Phalacrocorax brasilianus Garbin et al. (2011)
C. pelagicum EF122210 60 Spheniscus magellanicus Mattiucci et al. (2008)
Contracaecum larva MN589997 - Acanthobrama marmid Present study
Contracaecum larva MN589998 - Arabibarbus grypus Present study
Contracaecum larva MN589999 - Capoeta trutta Present study
Contracaecum larva MN590000 - Carasobarbus luteus Present study
Contracaecum larva MN590001 - Chondrostoma regium Present study
Ascaris suum HQ704901 - swine Liu et al. (2012)

Comparison of measurements in Contracaecum larvae in different infected fish species in the present study (in millimeter).

Host Total length Maximum width Tail length Rectum length Boring teeth length Esophagus length Esophageal caeca length Intestinal caeca length
A. marmid 4.10 0.20 0.07 0.07 0.005 0.480 0.50 0.38
A. grypus 5.07 0.25 0.07 0.07 0.010 0.650 0.30 0.20
C. trutta 4.20 0.23 0.08 0.07 0.005 0.450 0.48 0.33
C. luteus 4.62 0.25 0.08 0.07 0.005 0.600 0.55 0.40
C. regium 7.75 0.35 0.08 0.09 0.010 0.600 0.60 0.45

Accession numbers provided by NCBI for the collected Contracaecum larvae in different fish hosts in the present study.

Host of Contracaecum rudolphii Accession numbers for ITS-1 sequences Accession numbers for ITS-2 sequences Accession numbers for COX-2
Acanthobrama marmid MN557376 MN526259 MN589997
Arabibarbus grypus MN557377 MN563727 MN589998
Capoeta trutta MN557378 MN563728 MN589999
Carasobarbus luteus MN557379 MN563729 MN590000
Chondrostoma regium MN557380 MN563730 MN590001
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