Parasitology International 63 (2014) 127–131

Contents lists available at ScienceDirect

Parasitology International

j ourna l homepage: www.e lsev ie r .com/ locate /par in t

Molecular characterization of Hysterothylacium fabri(Nematoda: Anisakidae) from Zeus faber (Pisces: Zeidae)caught off the Mediterranean coasts of Turkey based onnuclear ribosomal and mitochondrial DNA sequences

Gokmen Zafer Pekmezci a,⁎, Banu Yardimci a, Ertan Emek Onuk a, Sinasi Umur b

a Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun 55139, Turkeyb Department of Parasitology, Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun 55139, Turkey

⁎ Corresponding author. Tel.: +90 362 3121919; fax: +E-mail address: zpekmezci@omu.edu.tr (G.Z. Pekmezc

1383-5769/$ – see front matter © 2013 Elsevier Ireland Lhttp://dx.doi.org/10.1016/j.parint.2013.10.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 April 2013Received in revised form 8 October 2013Accepted 11 October 2013Available online 19 October 2013

Keywords:Hysterothylacium fabriRibosomal DNA ITS regionMitochondrial DNA cox2 geneSequencingMediterranean Sea

In the present study, Hysterothylacium fabri was found in the coasts of the Mediterranean Sea, Turkey andcharacterized by sequencing of nuclear (internal transcribed spacer, ITS) and mitochondrial (cytochrome coxidase subunit 2, cox2) markers. Pairwise comparison between the entire ITS fragment including ITS-1, 5.8S,ITS-2 sequences of theH. fabri isolates from theMediterranean Sea (Turkey, KC852206) and otherH. fabri isolatesfrom the South China Sea (JQ520158), the South Korea waters (JX974558) showed differences ranged from 0.1and 1.1%. With the present study, H. fabri from the Mediterranean Sea was characterized for the first time bysequencing of the cox2 gene.

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Anisakid nematodes of the genus Hysterothylacium Ward & Magath,1917 are common parasites in different fish species in marine, brackish,and fresh water environments [1,2]. Infection with Hysterothylaciumspecies can affect the health of the fish hosts and even cause theirdeath, with consequent economic repercussions [3]. In addition, acci-dental ingestion of Hysterothylacium spp. can rarely cause anisakidosisand hypersensitivity in humans [4,5]. The genus Hysterothylacium,currently consisting of approximately 70 recognizable species, is con-sidered one of the largest of the ascaridoid genera parasitizing fish[6–17]. However, in Turkish waters, reports of Hysterothylacium spp.from freshwater and marine fish are still limited. Hysterothylacium spp.is morphologically represented by two species and one subspecies.Among them, Hysterothylacium aduncum from Gadus sp., Merlangiusmerlangus euxinus and Trachurus trachurus in the Black Sea [18–20],and T. trachurus, Solea solea, Sparus aurata, Pomatomus saltatrix, Lizasaliens, Engraulis encrasicolus in the Dardanelles strait [21], Diplodusvulgaris, S. aurata in the Northeast Mediterranean Sea [22] andHysterothylacium fabri (reported as Contracaecum fabri) from Boopsboops, D. annularis, Oblado melanur, Pagellus erythrinus, Spoondyliosomacantharus from the Northern Aegean Sea, Turkey [23] and H. gadiaduncum from cultured rainbow trout in the Derbent Dam Lake,

90 362 4576922.i).

td. All rights reserved.

Samsun, Turkey [24]. However, to date there has been only one studyof molecular characterization of H. aduncum from different marine fishcaught off the Turkish coast of the Black Sea [20]. Recently, moleculartechniques, using DNA sequencing of ribosomal internal transcribedspacer (ITS) regions and the mitochondrial cytochrome c oxidasesubunit 2 (cox2) gene, have been proven to be particularly usefulfor the accurate identification of ascaridoid nematodes at the specieslevel for eggs, larvae, and adults [20,25–40]. Nevertheless, beforethe present study, there had been no reports of characterizing theH. fabri from the Mediterranean Sea using well-defined ITS region andcox2 gene sequences. Therefore, in the present study, anisakid speciesfrom the Mediterranean Sea were genetically characterized for the firsttime by sequencing of mitochondrial (cox2) markers.

2. Materials and methods

2.1. Sample collection and parasitological examination

Parasitological examination of Hysterothylacium spp. was performedon different groups of fish samples. Twenty-one Trachurusmediterraneus,8Mullus barbatus, 4 Sphyraena sphyraena, 3 P. erythrinus, 3Mugil cephalus,3 Liza ramada, 3Umbrina cirrosa and 1 Zeus faberwere caught from coastsof theMediterranean Sea, Turkey fromOctober 2012 toMarch 2013. Fishwere dissected carefully and examined for nematodes in the stomach,intestine, abdominal cavity, and muscles. Parasites were only recoveredfrom the intestine of Z. faber. The nematodes were repeatedly washed

128 G.Z. Pekmezci et al. / Parasitology International 63 (2014) 127–131

in 0.9% saline solution, fixed in 70% ethanol, and cleared withlactophenol. Parasites were identified using the morphology of thelabia, the position of the excretory pore, the intestinal cecum,ventricular appendix and the tail [41,42]. Measurements (the range,followed by the mean in parentheses) are given. Specimens have beendeposited in the Department of Aquatic Animal Diseases, Faculty ofVeterinary Medicine, Ondokuz Mayis University, Samsun, Turkey(Voucher OMU.DAA.2013.05).

2.2. DNA extraction, PCR amplification, and sequencing

Five of fourth-stage larvae were randomly selected among thetotal larvae samples and were subjected to the molecular analysis.Genomic DNA was extracted from individual larvae using the DNApurification kit (Genomic DNA Purification Kit, Thermo Scientific)according to the manufacturer's instructions. Polymerase chainreaction (PCR) targeting the ITS region and cox2 gene were performed.DNA content was determined using a NanoDrop 2000 spectro-photometer (Thermo Scientific) at 260 nm. PCR was carried out in afinal volume of 50 μl, containing 10–50 ng of extracted DNA, 1× TaqBuffer with KCI (Thermo Scientific), 3mMofMgCl2 (Thermo Scientific),0.3 mM dNTPs (Thermo Scientific), 2 pmol of each primer, 2.5 U ofTaq DNA Polymerase (Thermo Scientific), and DEPC-treated water.Fragments of ~1000 bp of the ITS region were amplified using theprimers NC5 (forward: 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′)and NC2 (reverse: 5′-TTAGTTTCTTCCTCCGCT-3′) [25]. The PCR wasperformed in a Thermo PxE 0.2 thermal cycler (Thermo Scientific) andthe conditions were as follows: 15 min at 95 °C, then 30 cycles of1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C followed by a finalelongation of 5 min at 72 °C. The cox2 gene fragment (~630 bp) wasamplified using 210 (reverse: 5′-CACCAACTCTTAAAATTATC-3′) and211 (forward: 5′-TTTTCTAGTTATATAGATTGRTTTYAT-3′) [43] and 1×Taq Buffer with KCI (Thermo Scientific), 2.5 mM MgCl2 (ThermoScientific), 0.4 mM dNTPs (Thermo Scientific), 2 pmol of each primer,

Fig. 1.H. fabri larvae from Z. faber. These images are exemplary for all larvae found in thepresentpore and nerve ring; (C) esophagus–ventriculus–intestine junction; (D) tail tip of fourth-stage lv, ventriculus; va, ventricular appendix (scale 50 μm).

5 U of Taq DNA Polymerase (Thermo Scientific) and 10–50 ng ofgenomic DNA, in a volume of 50μL. Reaction conditionswere as follows:3min at 94 °C, then 34 cycles of 30 s at 94 °C, 1min at 51 °C and 90s at72 °C followed by a final elongation of 10 min at 72 °C. PCR productswere electrophoresed in 1.5% agarose gel (Prona) in a TBE buffer(89mMTris, 89mMboric acid, 2mMEDTA, pH8.3) (Thermo Scientific),stained with ethidium bromide (Sigma) and visualized by UVillumination. The size of the amplified fragments was estimated bycomparisons with the 1000 bp DNA Ladder (Thermo Scientific). TheITS region and cox2 gene amplification products were sent to asequencing company (Iontek Istanbul, Turkey) for purification andsequencing in both directions using NC5-NC2 and 211–210 primers,respectively.

2.3. Data analysis and phylogenetic tree construction

The obtained sequences were verified by forward and reversecomparisons, assembled and editedwith using Contig Express in VectorNTI Advance 11.5 (Invitrogen). Sequences were compared withpreviously published data for identification by using the Basic LocalAlignment Search Tool (BLAST) via GenBank and aligned withpreviously characterized sequences of Hysterothylacium species, usingClustalW in Mega 5.0 multiple sequence alignments [44]. Nucleotidecomposition was calculated using Mega 5.0 [45]. Genetic distanceswere calculated using the Kimura two-parameter model with pairwisedeletion in Mega 5.0 [45]. Phylogenetic analysis with other knownHysterothylacium species was conducted using maximum-likelihood(ML) analysis in Mega 5.0 [45]. The aligned sequences were testedwith Mega 5.0 model test to find the best DNA model to infer thephylogenetic trees [45]. The general-time reversible model (GTR+G)was selected using Akaine Information Criterion (AIC). The evolutionaryhistory was inferred using the ML method based on the GTR+Gmodelfor ITS sequences with Contracaecum osculatum as an out group. Adiscrete Gamma distribution was used to model evolutionary rate

study. Lightmicroscopy images show: (A) cephalic end of fourth-stage larva; (B) excretoryarva. ep, excretory pore; i, intestine; ic, intestinal cecum; nr, nerve ring; oe, esophagus;

Table 1Morphometric comparison between the fourth stage larvae of H. fabri in the present study and previous record.

Characters Present study Petter and Maillard, 1987

Group 1 Group 2

Body length, mm 12.5–18.4 (14.7)a 5.7–22 (11.0) 12.5–28 (21.4)Body width, μm 284–352 (310) – –

Nerve ring, μmb 220–282 (246) – –

Excretory pore, μmb 268–335 (296) – –

Esophagus length, μm 780–840 (820) 400–1150 (742) 660–1230 (1024)Ventricular appendage length, mm 1.0–1.38 (1.06) 500–1450 (884) 550–1275 (971)Intestinal cecum length, μm 80–125 (100) 75–240 (129) 0–170 (109)Tail length, μm 92–116 (103) 85–170 (122) 70–250 (129)Length esophagus/ventricular appendage 0.6–0.78 (0.77) 0.6–1.3 (0.8±0.1) 0.9–1.3 (1.0±0.1)Length esophagus/intestinal cecum 6.7–9.7 (8.2) 2.7–17 (6.4±1.1) 5.7–20.8 (10.1±1.9)Total body length/esophagus length 16–21.9 (17.9) 10.8–20.0 (14.6±0.9) 15.7–27.0 (20.8±1.1)Host Zeus faber c Uranoscopus scaberLocalities Mediterranean Mediterranean Mediterranean

a Range and mean in parentheses.b From anterior extremity.c Various fish species (i.e. Atherina boyeri, Trachurus trachurus, Mullus surmuletus).

129G.Z. Pekmezci et al. / Parasitology International 63 (2014) 127–131

differences among sites (5 categories [+G, parameter = 0.4476]).Confidence in the ML trees was determined by analyzing 1000bootstrap replicates [46] using the Mega 5.0 program. The sequencesof ITS and cox2 of H. fabri have been deposited in GenBank databasesunder accession numbers KC852206 and KC862609, respectively.

3. Results

3.1. Parasitological result

This species analyzed in the present paper clearly belongs to thegenus Hysterothylacium. The morphology of our specimens agrees wellwith the description of H. fabri [41]. In the present study, fourth-stageof larvae was small to medium, whitish nematodes with finelytransversely striated cuticle. The larvae (n = 10) measured about12.5–18.4 (14.7) mm in long and 284–352 (310) μm in width. Lateralalae starting, as very narrow, a short distance below level of base ofsubventral lips, and extending posteriorly to caudal region. Anteriorendwith three lips, approximately equal in size, with postlabial groovesand narrow lateral flanges. Dorsal lip with two lateral double papillae,subventral lips each with one lateral amphid, one single papilla andone double papilla. Interlabia very small, about 1/3 length of lips.Esophagus short, slightly broader posteriorly than anteriorly. Theesophagus was measured 780–840 (820) μm in length, representing4.5–6.2 (5.5) % of body length. Excretory pore just posterior to nervering. Nerve ring and excretory pore located 220–282 (246) and268–335 (296) μm, respectively, from anterior extremity. Ventriculusalmost spherical, approximately as wide as esophagus. Ventriculus82–94 (90) μm long and 71–80 (75) μm wide. Intestinal cecum veryshort, slightly longer than ventriculus. Intestinal cecum 80–125 (100)μm long, representing 10.2–14.8% (12.1) % of esophageal length.Ventricular appendix narrow and much longer than intestinal cecum.Ventricular appendix 1.0–1.38 (1.06) mm long. Ratio of intestinalcecum to ventricular appendix 1:11.0–12.5 (1:10.6). Ratio of length

Table 2Pairwise comparison of nucleotide sequence differences (%) in the ITS among H. fabriisolates and various geographical isolates.

1 2 3 4

1—KC852206 Mediterranean Sea, Turkey2—JQ520158 South China Sea 0.0023—JX974558 South Korean waters 0.001 0.0034—JN005769 Northeast Atlantic 0.011 0.013 0.012

esophagus to ventricular appendage 1: 1.2–1.6 (1:1.2). Tail 92–116(103) μm long, conical, relatively short, tip of tail covered by numerousspines. Therefore, we have not hesitated to make the morphologicalidentification of H. fabri (Fig. 1, Table 1).

3.2. Molecular results

The amplifications of the cox2 gene and ITS region (spanning the ITS-1, ITS-2, and the 5.8S subunit) produced a fragment of approximately630 and 1000 bp from different individuals, respectively, on agarosegels. While the cox2 products were subjected to direct sequencinggiving products 585 bp long, ITS products were 940 bp long. Nointraspecific nucleotide variability within different individuals wasobserved in the cox2 gene and ITS region. The length of the ITS-2 andITS-1 sequences of H. fabri were 345 and 438 bp. The 5.8S sequenceswere all 157 bp long. The G + C contents were 51.3% (ITS-1), 50.9%(5.8S), and 54.2% (ITS-2). H. fabri isolates from the Mediterranean Sea,Turkey (KC852206) showed 98.9 to 99.9% identity with variousgeographical isolates of H. fabri from the South China Sea (JQ520158)and South Korean waters (JX974558) and with Hysterothylacium sp.from the Northeast Atlantic (JN005769) from GenBank. Pairwisecomparison between the entire ITS fragment including ITS-1, 58S, ITS-2 sequences of the H. fabri isolates of Z. faber from the MediterraneanSea (Turkey, KC852206) and other H. fabri isolates from the SouthChina Sea (JQ520158) and South Korean waters (JX974558) andHysterothylacium sp. from the Northeast Atlantic (JN005769) showeddifferences ranging from 0.1 to 1.1% (Table 2). ITS region sequencing(KC852206, Mediterranean Sea, Turkey) results showed that there wasa difference in one nucleotide, alignment positions 140 (JX974558,South Koreanwaters) (p=0.001), two nucleotides, alignment positions344 and 705 (JQ520158, South China Sea) (p = 0.002) and tennucleotides, alignment positions 136, 311, 321, 334, 411, 668, 687,806, 807, 813, and two indel, alignment positions 93 and 635(JN005769, Northeast Atlantic) (p = 0.011) examined in the presentstudy (Table 3). Phylogenetic relationships among H. fabri isolatesfrom Z. faber of the Mediterranean Sea, Turkey and the other H. fabriisolates and Hysterothylacium sp. as inferred by ML analysis of the ITSsequence, and based on the entire ITS fragment including ITS-1, 5.8S,and ITS-2 sequences are presented in Fig. 2. There were no previoussequences for the cox2 of H. fabri deposited in GenBank for comparison.Moreover, H. fabri from Z. faber showed 83 to 84% identity of partialcox2 sequence with the GenBank sequences of Hysterothylacium sp.Cox2 sequence of H. fabri in fish from the Mediterranean Sea is thefirst cox2 sequence for this species in GenBank.

Table 3Alignment of the variable sites in the ITS-1/5.8S/ITS-2 sequences of H. fabri specimens. One representative of each unique sequence was included for comparison.

93

136

140

311

321

334

344

411

635

668

687

705

806

807

813

1—JX974558 South Korea waters – T C G G C G T A T T G T G A2—JQ520158 South China Sea – . T . . . A . . . . A . . .3—JN005769 Northeast Atlantic C G . . T T . A – C A . A A T4—KC852206 Mediterranean Sea – . T . . . . . . . . . . . .

Dots indicate identity with the first sequence, and dashes are inferred insertion–deletion events. The consensus ITS-1/5.8S/ITS-2 sequences for theH. fabriwere included in the alignmentfor comparative purposes.

130 G.Z. Pekmezci et al. / Parasitology International 63 (2014) 127–131

4. Discussion

Accurate identification of a parasite at any stage of its developmenthas important implications for studying parasite epidemiology andresolving taxonomic problems [47]. Different studies, have dem-onstrated that the ITS region and the cox2 gene provide usefulgenetic markers for the accurate identification of sibling speciesand morphospecies within ascaridoid species [20,25–40]. The genusHysterothylacium Ward and Margath (1917) belongs to the Anisakidaefamily, which is frequently mistaken for the Contracaecum genus [48].Hysterothylacium species were considered members of Contracaecumin the past [1]. Hysterothylacium are the parasites of several freshwaterand marine fish families. The definitive hosts of Hysterothylaciumare piscivorous fishes, not birds or mammals as for Contracaecum.Hysterothylacium and Contracaecum have now been differentiatedmorphologically by the location of the excretory pore; the excretorypore of Hysterothylacium spp. is located at or near the level of thenerve ring, whereas in Contracaecum spp., it is located at the anteriorend near the base of the lips [1]. In the Turkish waters, Akmirza [23]investigated the parasites of somemarine teleostfish from theNorthernAegean Sea, Turkey and identified the nematodes as belonging to thegenus Contracaecum, which was reported as C. fabri. In the presentstudy, our reported species herein is classified as H. fabri. The specieshas been found in Z. faber, Uranoscopus scaber, Trisopterus minutus inthe Adriatic Sea and Mediterranean Sea [42,49,50], Phycis phycis andPhycis blennoides from the Mediterranean coast of Eastern Andalucía(Spain) [51] and Trachurus japonicus, Pennahia argentata, Congermyriaster and Chelidonichthys kumu in the Chinese waters [52]. A largenumber of fish species seem to act as intermediate and/or paratenic

Fig. 2. Phylogenetic relationships between H. fabri from theMediterranean Sea, Turkey and othThe evolutionary history was inferred using the ML method based on the GTR + G model forindividual sequences determined in the present study are shown in each tree. A scale bar indclustered together in the bootstrap test (1000 replicates) are shown at the internal nodes (N50

hosts, including Atherina boyeri, Mullus surmuletus, Mullus barbatus, T.trachurus, U. scaber, P. erythrinus, Spicara maena flexuosa, etc. [41,53].H. fabri is a little studied anisakid species for which important aspectsof the biological cycle remain unknown. It is a complex comprising atleast three sibling species [54]. Although this species was previouslydescribed in the Mediterranean Sea by morphological identification[41,42], our study provides sequencing of the ITS region and cox2 geneof this species in the Mediterranean Sea. In this study, bootstrappingof the sequences with ML revealed significant support for one clade(100% bootstrap support) containing H. fabri isolates from theMediterranean Sea, Turkey (KC852206), South Korean waters(JX974558) and the South China Sea (JQ520158), revealing a closerelationship between these isolates (Fig. 2). As conclusions, thecox2 gene sequences of H. fabri have been obtained for the firsttime in thepresent study, and further researches usingmorepolymorphicgenetic markers are required to examine the genetic variability andpopulation genetic structure within H. fabri from different marinefish species and geographical locations in the Black Sea, Aegean andMediterranean Turkish coasts.

Conflict of Interest

The authors do not have any potential conflicts of interest to declare.

References

[1] Deardorff TL, Overstreet RM. Review of Hysterothylacium and Iheringascaris (bothpreviously = Thynnascaris) (Nematoda: Anisakidae) from the Northern Gulf ofMexico. Proc Biol Soc Wash 1980;93:1035–79.

er Hysterothylacium species as inferred bymaximum-likelihood (ML) analysis of ITS rDNA.ITS sequences with Contracaecum osculatum as an out group. The accession numbers oficates estimated distance. The percentage of replicate trees in which the associated taxa% only).

131G.Z. Pekmezci et al. / Parasitology International 63 (2014) 127–131

[2] Moravec F. Parasitic nematodes of freshwater fishes of Europe. Dordrecht: Academiaand Kluwer; 1994.

[3] Balbuena JA, Karlsbakk E, Kvenseth AM, Saksvik M, Nylund A. Growth andemigration of third-stage larvae of Hysterothylacium aduncum (Nematoda:Anisakidae) in larval herring Clupea harengus. J Parasitol 2000;86:1271–5.

[4] Yagi K, Nagasawa K, Ishikura H, Nagasawa A, Sato N, Kikuchi K, et al. Female wormHysterothylacium aduncum excreted from human: a case report. Jpn J Parasitol1996;45:12–23.

[5] Valero A, Terrados S, Díaz V, Reguera V, Lozano J. Determination of IgE in the serumof patients with allergic reactions to four species of fish-parasite anisakids. J InvestigAllergol Clin Immunol 2003;13:94–8.

[6] Bruce NL, Adlard RD, Cannon LRG. Synoptic checklist of ascaridoid parasites(Nematoda) from fish hosts. Invertebr Taxon 1994;8:583–674.

[7] Torres P, Andrade P, Silva R. On a new species of Hysterothylacium (Nematoda:Anisakidae) from Cauque mauleanum (Pisces: Atherinidae) by brightfield andscanning electron microscopy. Mem Inst Oswaldo Cruz 1998;93:745–52.

[8] Moravec F, Nagasawa K. Some anisakid nematodes from marine fishes of Japan andthe North Pacific Ocean. J Nat Hist 2000;34:1555–74.

[9] Torres P, Soto MS.Hysterothylaciumwinteri sp. n. (Nematoda: Anisakidae), a parasiteof Chilean rock cod, Eleginops maclovinus (Perciformes: Eleginopidae), from SouthChile. Folia Parasitol 2004;51:55–60.

[10] Gopar-Merino L, Osorio-Sarabia D, García-Perieto L. A new species of Hysterothylacium(Nematoda: Anisakidae) parasite of Ariopsis guatemalensis (Osteichthyes: Ariidae)from Tres Palos lagoon, Mexico. J Parasitol 2005;91:909–14.

[11] Li L, An RY, Zhang LP. A new species of Hysterothylacium (Nematoda: Anisakidae)from marine fishes from Yellow Sea, China, with a key to the species of the genusHysterothylacium. Zootaxa 2007;1614:43–52.

[12] Li L, Xu Z, Zhang LP. A new species of genus HysterothylaciumWard et Magath, 1917(Nematoda, Anisakidae) from Liparis tanakae (Scorpaeniformes, Liparidae) from theYellow Sea, China. Acta Parasitol 2007;52:371–5.

[13] Li L, Liu Y, Zhang L. Morphological and genetic characterization of H. zhoushanensissp. nov. (Ascaridida: Anisakidae) from the flatfish Pseudorhombus oligodon(Bleeker) (Pleuronectiformes: Paralichthyidae) in the East China Sea. ParasitolRes 2012;111:2393–401.

[14] Li L, Zhang L, Liu Y. Hysterothylacium simile n. sp. and H. aduncum (Rudolphi, 1802)(Nematoda: Raphidascarididae) from marine fishes in the Bohai and Yellow Sea,China, with comments on the record of H. paralichthydis (Yamaguti, 1941) fromChinese waters. Syst Parasitol 2013;84:57–69.

[15] Raffel TR, Anderson TK. A new species of Hysterothylacium (Nematoda: Anisakidae)from the stomach of the red-spotted newt, Notophthalmus viridescens, fromPennsylvania fishless ponds. J Parasitol 2009;95:1503–6.

[16] Rossin MA, Datri LL, Incorvaia IS, Timi JT. A new species of Hysterothylacium(Ascaridoidea, Anisakidae) parasitic in Zenopsis conchifer (Zeiformes, Zeidae) fromArgentinean waters. Acta Parasitol 2011;56:310–4.

[17] Knoff M, Felizardo NN, Iniguez Jr AM, Torres EJL, Pinto RM, Gomes DC. Genetic andmorphological characterisation of a new species of the genus Hysterothylacium(Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of theNeotropical Region, State of Rio de Janeiro, Brazil. Mem I Oswaldo Cruz2012;107:186–93.

[18] Doganay A. A record of Hysterothylacium aduncum (Rudolphi, 1802) in cod-fish(Gadus sp.) from Black Sea. Vet J Ankara Univ 1994;41:208–17.

[19] Ismen A, Bingel F. Nematode infection in the whiting Merlangius merlangus euxinusoff Turkish coast of the Black Sea. Fish Res 1999;42:183–9.

[20] Pekmezci GZ, Bolukbas CS, Gurler AT, Onuk EE. Occurrence and molecularcharacterization of Hysterothylacium aduncum (Nematoda: Anisakidae) fromMerlangius merlangus euxinus and Trachurus trachurus off the Turkish coast ofBlack Sea. Parasitol Res 2013;112:1031–7.

[21] Keser R, Bray RA, Oguz MC, Celen S, Erdogan S, Doguturk S, et al. Helminth parasitesof digestive tract of some teleost fish caught in the Dardanelles at Çanakkale, Turkey.Helminthologia 2007;44:217–21.

[22] Kalay M, Donmez AE, Koyuncu CE, Genc E, Sahin G. Seasonal variationof Hysterothylacium aduncum (Nematoda: Raphidascarididae) infestation insparid fishes in the Northeast Mediterranean Sea. Turk J Vet Anim Sci2009;33:517–23.

[23] Akmirza A. Seasonal distribution of parasites detected in fish belonging to thesparidae family found near Gokçeada. Turk Parazitol Derg 2000;24:435–41.

[24] Pekmezci GZ. Metazoan parasite fauna of rainbow trout (Oncorhynchus mykiss)reared in Samsun Province, Turkey. (PhD dissertation) Samsun, Turkey: Universityof Ondokuz Mayis; 2010.

[25] Zhu XQ, Gasser RB, Podolska M, Chilton N. Characterization of anisakid nematodeswith zoonotic potential by nuclear ribosomal DNA sequences. Int J Parasitol1998;28:1911–21.

[26] Zhu XQ, Gasser RB, Jacobs DE, Hung GC, Chilton NB. Relationships among someascaridoid nematodes based on ribosomal DNA sequence data. Parasitol Res2000;86:738–44.

[27] Zhu XQ, Podolska M, Liu JS, Yu HQ, Chen HH, Lin ZX, et al. Identification of anisakidnematodes with zoonotic potential from Europe and China by singlestrandconformation polymorphism analysis of nuclear ribosomal DNA. Parasitol Res2007;101:1703–7.

[28] D'Amelio S, Mathiopoulos KD, Santos CP, Pugachev ON, Webb SC, Picanco M, et al.Genetic markers in ribosomal DNA for the identification of members of the genus

Anisakis (Nematoda: Ascaridoidae) defined by polymerase chain reaction-basedrestriction fragment length polymorphism. Int J Parasitol 2000;30:223–6.

[29] D'Amelio S, Cavallero S, Dronen NO, Barros NB, Paggi L. Two new species ofContracaecum Railliet & Henry, 1912 (Nematoda: Anisakidae), C. fagerholmi n. sp.and C. rudolphii F from the brown pelican Pelecanus occidentalis in the northernGulf of Mexico. Syst Parasitol 2012;81:1–16.

[30] Farjallah S, Ben Slimane B, Blel H, Amor N, Said K. Anisakid parasites of twoforkbeards (Phycis blennoides and Phycis phycis) from the eastern Mediterraneancoasts in Tunisia. Parasitol Res 2006;100:11–7.

[31] Farjallah S, Ben Slimane B, Busi M, Paggi L, Amor N, Blel H, et al. Occurrence andmolecular identification of Anisakis spp. from the North African coasts ofMediterranean Sea. Parasitol Res 2008;102(3):371–9.

[32] Farjallah S, Merella P, Ingrosso S, Rotta A, Ben Slimane B, Garippa G, et al. Molecularevidence for the occurrence of Contracaecum rudolphii A (Nematoda: Anisakidae) inshag Phalacrocorax aristotelis (Linnaeus) (Aves: Phalacrocoracidae) from Sardinia(western Mediterranean Sea). Parasitol Int 2008;57:437–40.

[33] Valentini A, Mattiucci S, Bondanelli P, Webb SC, Mignucci-Giannone AA,Colom-Llavina MM, et al. Genetic relationship among Anisakis species (Nematoda:Anisakidae) inferred from mitochondrial cox2 sequences, and comparison withallozym data. J Parasitol 2006;92:156–66.

[34] Kellermanns E, Klimpel S, Palm HW. Molecular identification of ascaridoidnematodes from the deep-sea onion-eye grenadier (Macrourus berglax) from theEast Greenland Sea. Deep-Sea Res I 2007;54:2194–202.

[35] Klimpel S, Kleinertz S, Hanel R, Rückert S. Genetic variability in Hysterothylaciumaduncum, a raphidascarid nematode isolated from sprat (Sprattus sprattus)of different geographical areas of the northeastern Atlantic. Parasitol Res2007;101:1425–30.

[36] Zhang L, Hu M, Shamsi S, Beveridge I, Li H, Xu Z, et al. The specific identificationof anisakid larvae from fishes from the Yellow Sea, China, using mutationscanning-coupled sequence analysis of nuclear ribosomal DNA. Mol Cell Probes2007;21:386–90.

[37] Mattiucci S, Nascetti G. Advances and trends in the molecular systematics ofAnisakid nematodes, with implications for their evolutionary ecology and host–parasite co-evolutionary processes. Adv Parasitol 2008;66:47–148.

[38] Amor N, Farjallah S, Merella P, Said K, Ben Slimane B. Molecular characterization ofHysterothylacium aduncum (Nematoda: Raphidascaridae) from different fish caughtoff the Tunisian coast based on nuclear ribosomal DNA sequences. Parasitol Res2011;109:1429–37.

[39] Setyobudi E, Jeon CH, Lee CH, Seong KB, Kim JH. Occurrence and identification ofAnisakis spp. (Nematoda: Anisakidae) isolated from chum salmon (Oncorhynchusketa) in Korea. Parasitol Res 2011;108:585–92.

[40] Smrzlić IV, Valić D, Kapetanović D, Kurtović B, Teskeredžić E. Molecular char-acterisation of Anisakidae larvae from fish in Adriatic Sea. Parasitol Res2012;11:2385–91.

[41] Petter AJ, Maillard C. Ascarides de poissons de Mediterranee occidentale. Bull MusHist Nat 1987;9:773–98.

[42] Petter AJ, Maillard C. Larves d'ascarides parasites de poissons en Méditerranéeoccidentale. Bull Mus Hist Nat 1988;4:347–69.

[43] Nadler SA, Hudspeth DSS. Phylogeny of the Ascaridoidea (Nematoda: Ascaridida)based on three genes and morphology: hypotheses of structural and sequenceevolution. J Parasitol 2000;86:380–93.

[44] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80.

[45] Tamura K, Peterson D, Peterson N, Steker G, Nei M, Kumar S. MEGA5: molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance,and maximum parsimony methods. Mol Biol Evol 2011;28:2731–9.

[46] Felsenstein J. Confidence intervals on phylogenies: an approach using the bootstrap.Evolution 1985;39:783–91.

[47] Kijewska A, Rokicki J, Sitko J, Wegrzyn G. Ascaridoidea: a simple DNA assay foridentification of 11 species infecting marine and freshwater fish, mammals, andfish-eating birds. Exp Parasitol 2002;101:35–9.

[48] Lopes LP, Pimpao DM, Takemoto RM, Malta JC, Varella AM. Hysterothylacium larvae(Nematoda, Anisakidae) in the freshwater mussel Diplodon suavidicus (Lea, 1856)(Mollusca, Unioniformes, Hyriidae) in Aripuana River, Amazon, Brazil. J InvertebrPathol 2011;106:357–9.

[49] Nikolaeva VM, Naidenova N. Nématodes de poissons pélagiques et bathypélagiquesdes mers du bassin Méditerranéen. Tr Sevastop Biol Stn 1964;17:125–58.

[50] Petter AJ, Radujkovic BM. Nematodes parasites de poissons de la mer Adriatique.Bull Mus Hist Nat 1986;3:487–99.

[51] Valero A, Paniagua MI, Hierro I, Díaz V, Valderrama MJ, Benítez R, et al. Anisakidparasites of two forkbeards (Phycis blennoides and Phycis phycis) from theMediterranean coasts of Andalucía (Southern Spain). Parasitol Int 2006;55:1–5.

[52] Li L, Xu Z, Zhang LP. Redescription of three species of Hysterothylacium (Nematoda:Anisakidae) from marine fishes from the Yellow Sea, China, with the synonymy ofHysterothylacium muraenesoxin (Luo, 1999). Zootaxa 1878;2008:55–67.

[53] Petter AJ, Lébre C, Radujkovic BM. Nématodes parasites de poissons osteichthyensde l'Adriatique méridionale. Acta Adriat 1984;25:205–21.

[54] Martín-Sánchez J, Díaz M, Artacho ME, Valero A. Molecular arguments forconsidering Hysterothylacium fabri (Nematoda: Anisakidae) a complex of siblingspecies. Parasitol Res 2003;89:214–20.