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190Russian Journal of Nematology, 2020, 28 (1), 45 - 52
Third-stage juveniles of Contracaecum sp. (Anisakidae, Ascaridomorpha) from the round goby Neogobius melanostomus of the Black Sea
1 2 Natalia V. Pronkina and Sergei E. Spiridonov
1A.O. Kovalevsky Institute of Marine Biological Research, Russian Academy of Sciences, Nakhimov Avenue 2, 299011, Sevastopol, Russia 2A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 119071, Moscow, Russia e-mail: [email protected]
Accepted for publication 25 May 2020
Summary. A long-term parasitological survey carried on the round goby, Neogobius melanostomus, from the Black Sea has revealed its high level of infection with anisakid juveniles. In previous studies, some of the juvenile stages of the genus Contracaecum were identified as C. microcephalum according to available identification keys based on morphology, although the latter species is considered more common in fresh-water habitats. Analysis of nucleotide sequences of LSU rDNA and ITS rDNA of Contracaecum juveniles from the round goby Neogobius melanostomus demonstrated their identity with the cryptic species Contracaecum rudolphii A of a species complex Contracaecum rudolphii Hartwich, 1964 sensu lato.
Key words: ascaridids, ITS rDNA, LSU rDNA, morphotype, phylogeny.
The nematodes of the genus Contracaecum Railliet & Henry, 1912 are common parasites of marine fish-eating birds and mammals and can also be found in fresh-water habitats. This taxon is the largest in the family Anisakidae Railliet & Henry, 1912, comprising more than 50 valid species. Adult nematodes and fourth-stage juveniles can be found in the alimentary tract of birds and mammals, while invertebrates and fishes serve as paratenic/intermediate hosts for third-stage juveniles. The identification of species with the use of the third-stage juveniles is hindered by their monotonous morphology. Often, Contracaecum juveniles can be identified only to the generic level as descriptions of the juvenile stages are still not available for all known Contracaecum species. Three species from definitive hosts in the coastal waters of the Crimean peninsula: C. microcephalum, C. micropapillatum and C. rudolphii (considered as C. spiculgerum) were reported earlier (Gaevskaya et al., 1975).
The examination of the Contracaecum juvenile diversity in the Black Sea fishes revealed the presence of two morphotypes. One of these morphotypes was regularly found in the golden grey mullets (Chelon auratus) of the Black Sea, and was
identified on the basis of nucleotide sequence analysis as Contracaecum multipapillatum (Pronkina & Spiridonov, 2018). The identification of another morphotype, which was especially common in the round goby, as Contracaecum microcephalum was questioned, as usually this species was reported from fresh-water habitats (Mozgovoy et al., 1968; Kornyushin et al., 2004).
The results of morphological and molecular study of these juveniles are presented below.
MATERIAL AND METHODS
The round gobies (439 specimens in total) were caught in 2007-2017 in shallow (0.3-1.5 m depth) waters of Karkinit Bay (offshore Lebjazhii islands -45°52'30" N; 33°32'30" E) in the water territory of the Crimean Nature Reserve, under a permit issued by the Reserve administration. The dissection of round gobies revealed the presence of anisakid juveniles in capsules on the serosa covers and in the abdominal cavity. Parasite prevalence of 3% with parameters of the intensity of 1-2 worms per host and abundance of 0.04 worms per individual host were observed.
© Russian Society of Nematologists, 2020; doi: 10.24411/0869-6918-2020-10004
The nematodes recovered were fixed in 70% ethyl alcohol and mounted in glycerin-lactic acid 1:1 mixture. The middle body parts of three specimens were fixed in 95% ethanol for DNA extraction and remaining body parts were mounted to serve as a voucher specimen, 'hologenophore' (Pleijel et al., 2008). The voucher specimen was deposited in the Collection of marine parasites (CMP IMBR № 1267-1273 .N.2p.V 1-7) of the
A.O. Kovalevsky Institute of Biology of the Southern Seas, Sevastopol, IBSS collection (Dmitrieva et al., 2015; http://marineparasites.org/). All the measurements are in micrometers. Line-art images were made on the basis of images obtained from an Olympus CX-41 microscope with a CAM-SC50 camera. The images were processed in Inkscape 0.48.2.-1 (2011. Scalable Vector Graphics (SVG): http://www.inkscape.org/).
Nematode species Host Geographic origin NCBI accession number Reference
LSU rDNA
C. eudyplulae Eudyptula minor AF226586 Nadler et al., 2000
C. ogmorhini Arctocephalus pusillus pusillus South Africa AF226582 Nadler et al., 2000
C. rudolphii B Phalacrocorax carbo Policoro, Italy AF226579 Nadler et al., 2000
C. rudolphii Gasterosteus aculeatus North Sea, Germany KT767121 Schade et al., 2015
C. rudolphii Gasterosteus aculeatus North Sea, Germany KT767120 Schade et al., 2015
C. rudolphii A Phalacrocorax carbo Policoro, Italy AF226585 Nadler et al., 2000
C. septentrionalis Phalacrocorax carbo Husavik, Iceland AF226588 Nadler et al., 2000
C. septentrionalis Sprattus sprattus North Sea, Germany KT767122 Schade et al., 2015
C. micropapillatum Pelecanus onocrotalus Assuan, Egypt AF226587 Nadler et al., 2000
C. microcephalum Phalacrocorax pygmaeus Scutari lake, Yugoslavia AF226573 Nadler et al., 2000
C. multipapillatum Pelecanus crispus Psatatopi, Greece AF226574 Nadler et al., 2000
C. miroungae Mirounga leonina King George Island, Antarctica AF226581 Nadler et al., 2000
C. radiatum Leptonychotes weddelli Weddell Sea, Antarctica AF226577 Nadler et al., 2000
C. osculatum Sprattus sprattus North Sea, Germany KT671118 Schade et al., 2015
C. osculatum Phoca sibirica Lake Baikal, Russia AF226589 Nadler et al., 2000
C. osculatum B Phoca groenlandica Newfoundland, Canada AF226580 Nadler et al., 2000
ITS rDNA
C. multipapillatum Chelon auratus Black Sea, Crymea MH400190 Pronkina & Spiridonov, 2018
C. radiatum Laptonychotes weddeli South Shetlands, Antartica AY603529 Kijewska et al., 2002
C. bioccai Pelecanus occidentalis Florida, USA JF424598 D'Amelio et al., 2007
C. rudolphii C Phalacrocorax carbo sinensis Florida, USA FJ589792 Zhang et al. (NCBI GanBank data)
C. rudolphii C Phalacrocorax carbosinensis Florida, USA FJ467620 Zhang et al. (unpublished)
C. rudolphii Phalacrocorax carbosinensis Florida, USA FJ589790 Zhang et al. (unpublished)
C. rudolphii Phalacrocorax aristotelis Sardinia, Italy EU678869 Farjallah et al., 2008
C. rudolphii Phalacrocorax carbo Poland AY603535 Kijewska et al., 2002
C. rudolphii F Pelecanus occidentalis Florida, USA JF424597 D'Amelio et al., 2007
Contracaecum sp. AY821750 Kijewska et al., 2002
C. osculatum Sprattus sprattus Baltic Sea, Denmark KU306717 Zuo et al., 2016
C. osculatum Sprattus sprattus Baltic Sea, Denmark KU306680 Zuo et al., 2016
Table 1. Accession numbers and related information for the Contracaecum sequences used in the phylogenetic analysis.
For DNA extraction, the middle parts of three nematode specimens were rinsed in distilled water and crushed with a pestle in 1.5 ml Eppendorf tube and then processed with Wizard® SV Genomic DNA Purification Kit (Promega, Madison, USA) according to the producer's protocol. The nucleotide sequences of two loci of ribosomal repeats were amplified: LSU and ITS rDNA. Two pairs of primers were used to amplify LSU rDNA: the forward primer LSU391 (5'-AGC GGA GGA AAA GAA ACT AA-3') with the reverse one LSU390 (5'-ATC CGT GTT TCA AGA CGG G-3'), and the standard pair for D2-D3 segment of LSU rDNA: forward primer D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and reverse one D3B (5'-TCG GAA GGA ACC AGC TAC TA-3'). ITS rDNA region was amplified with primers TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3'). Annealing parameters for LSU primers were 50°C for 60 s and for ITS primers 52°C for 35 s. The
obtained chromatograms were processed with Chromas 2.4.4 to obtain sequences. ITS and LSU rDNA sequences were deposited in NCBI GenBank as MT331855 and MT329686, respectively. Similar sequences were searched for in NCBI GenBank with BLAST (Altshul et al., 1990). The obtained sequences were aligned with Clustal X and analysed with MEGA 7.0.14 (Kumar et al., 2016).
RESULTS Contracaecum sp. juveniles (Fig. 1)
Dissected from the intermediate host, a round goby Neogobius melanostomus (Pallas, 1814) (Gobiidae).
Locality. Karkinit Bay (45°52'30" N; 33°32'30" E).
Localisation in host. Inside capsules of serosa including intestinal mesentery and in the abdominal cavity.
Material. 1 hologenophore, 7 voucher specimens (CMP IMBR №1267-1273.N.2p.V 1-7).
Fig. 1. Morphology of Contracaecum juveniles from the Black Sea Neogobius melanostomus. A - anterior end, laterally; B - tail end, laterally.
Molecular characterisation. Two nucleotide sequences of approximately 660 and 760 bp length were obtained for LSU rDNA of the studied Contracaecum juveniles with primers LSU391-LSU390 and D2A-D3B. These two sequences were completely identical in overlapping 415-420 bp. The sequence obtained with primers LSU391-LSU390 was completely identical to the sequence of Contracaecum rudolphii A (AF226585). This sequence was obtained from adult nematodes obtained after dissection of the cormorant Phalacrocorax carbo in Policoro, Italy (Table 1). Several sequences of C. rudolphii and unidentified Contracaecum sp. and also the sequence of C. eudyptulae from Australia (AF226586) differed from the studied one in a single nucleotide while the nucleotide differences with all remaining species of the genus were 36-67 bp.
Partial LSU rDNA sequence of the studied juveniles obtained with primers D2A-D3B was found to be identical with that of C. rudolphii 'A' (AF226585) and C. rudolphii '371_N' (KT767121). The last sequence was obtained from juveniles found in marine fishes in the German sector of the North Sea (Table 1). Contracaecum sequence for C. rudolphii 178N, KT767120 from the North Sea differed in 1 bp, and a set of other Contracaecum sequences (C. rudolphii 'B', C. ogmorhini, C. eudyptulae) differed in 2 bp. All remaining
Contracaecum spp. differed in 44-71 bp. The sequences of four species, C. rudolphii, C. eudyptula, C. ogmorhini and C. septentrionalis, constituted a separate clade with strong support (Fig. 2).
Partial 798 bp long ITS rDNA sequence of the Contracaecum juveniles from the round goby was obtained and found to be completely identical to the sequences of C. rudolphii deposited under accession numbers EU678869 and AY603535 from Sardinia, Italy and Poland, respectively (Table 1). These three sequences formed the strongly supported subclade in the clade consisting of several C. rudolphii sequences (Fig. 3). The latter ones have only weak or modest support.
Morphological characterisation. Body length 2755-3522, maximum diameter 166-247; width at nerve ring level 81-109, width at anal level 53-65. Body surface annulated. Excretory pore located at anterior end. Cephalic end with three protruding cuticular projections and dorsal tooth. Nerve ring near pharynx middle, 18-28 long and 43-50 wide, situated in 133-164 from apex. Ventriculus globular, 32-53 wide and 19-44 long. Ventricular appendix 299-440 long and 33-45 wide. Intestinal appendix 155-238 long and 39-50 wide (length ratio of ventricular to intestinal appendix 1.5:1). Genital primordium not discernible. Prominent rectal glandular cells. Conical tail, 81-101 long.
69/92/82
100/100/77
/ / v'.vter-o/// > /c/c - f i/m /¿V/c// /cs/.s PC I J 527061 7^cff?/ifc/c/.sc cv/ v\v .«L^/iLfc/,s AB55848O
- c// c/\ y ? ii / / crc AF226586
C. ftor ljin/ /K I;226582 C. / /1c/(>//?ft// AF226579 C. /-//¿/o//?//// KT767 12 1 C. / //?//if KTV&V 1 2O
C \ f t/¿/<>//?/iir AF226S8S
C. ///cJcj/f?///"/" /7/ /oim cl goby
C \ sc /s/c/ ///-it /.s 8
d \
KT767 122
93/62/67
100/93/89 |
94/95/94
10
99/100/95
rf
' C. 77 7 / C'7Y>/J>£ f/?///CI i I f 77 7 AF226587
- C. 777/VvY^t'c/?/7cr/1//tt AF226573
- C \ mi / /i i/?ci/?i//¿/it t in AF226574
C. 777if~<>7/7/c' AF2265 8 1 t'cic/ici/1 //r/ AI;226577
C. o.vc7//<r //7/777 KT67 1118 I C. -7//cv/7/777 'baicalensis'AF226589
L C. c>vS'<i*7//<://77777 AF226580
Fig. 2. Phylogenetic relationships of Contracaecum juveniles from the Black Sea Neogobius melanostomus, as inferred from the analysis of LSU rDNA. Bootstrap support near nodes in the format MP/NJ/ML.
Fig. 3. Phylogenetic relationships of Contracaecum juveniles from the Black Sea Neogobius melanostomus, as inferred from the analysis of ITS rDNA. Bootstrap support near nodes in the format MP/NJ/ML.
DISCUSSION
The obtained sequences of the Contracaecum sp. juveniles from the round goby were found to be identical with the LSU and ITS rDNA sequences of C. rudolphii deposited in NCBI GenBank. Morphological features of these juveniles are also similar to those of C. rudolphii (Moravec, 1994). Therefore, we can identify these juveniles as C. rudolphii.
Anisakid nematodes are quite widespread as parasites of marine and fresh-water animals and even considered as 'emerging zoonoses' (McCarthy & Moore, 2000). Knowledge of the local anisakid diversity and the identification of these parasites are crucial for the proper understanding of parasite burden in ecosystems. It is especially important for the anisakid genus Contracaecum, as up to 50 nominal species of this genus are known and it has been recently demonstrated that some species in fact represent complexes of several cryptic species (D'Amelio et al., 2007). It is also important to understand the level of specificity of Contracaecum parasitism.
The life cycle of Contracaecum rudolphii was only studied in experimental conditions and the list of possible intermediate hosts was also compiled after experimental inoculations (Mozgovoy et al., 1968; Moravec, 2009). Juvenile stages of anisakids identified as C. rudolphii were reported from fish
hosts, but such findings were not confirmed with molecular data. The identifications of C. rudolphii based on both morphological and molecular data were obtained for brackish-water fishes Neogobius melanostomus (Szostakowska & Fagerholm, 2007) in Poland and Dicentrarchus labrax, Anguilla anguilla, Atherina boyeri, Aphanius fasciatus in Italy (Mattiucci et al., 2002; 2020).
Several forms comprising the species complex Contracaecum rudolphii sensu lato were reported exclusively from shags and cormorants (Phalacrocorax spp.). Five potential definitive hosts of C. rudolphii s.l. are common in the Black and Azov seas: great cormorant (P. carbo), European shag or common shag (P. aristotelis), pygmy cormorant (P. pygmaeus), great white pelican (Pelecanus onocrotalus) and Dalmatian pelican (P. crispus) (Beskorovajnyj, 2012). The latter species is quite rare in the area where great cormorant is considered as an invasive species, first reported at the Crimean peninsula only in the 1970s. For C. rudolphii, the prevalence of 35% and the parasite intensity range 1-295 nematodes per host in the common shag P. aristotelis were reported (Kornyushin et al., 2004). Comparative data for different Crimean Phalacrocorax species are absent and at this stage we can presume that all three species can serve as hosts for C. rudolphii A.
The ITS rDNA sequence of C. rudolphii from the Crimean round goby was 100% identical to that
of specimens identified as C. rudolphii A from the same fish host, collected in the Gdansk area in Poland (Kijewska et al., 2002). As indicated above, the LSU rDNA sequence amplified with primers LSU391-LSU390 was identical to the sequence of C. rudolphii A (AF226585) from Italy. Definitive hosts for these nematodes were not defined in Poland, but the host for the adult nematode specimens from Italy was Phalacrocorax carbo. It was proven that this bird species was a host for all other C. rudolphii A specimens (Shamsi et al., 2009). Initially, it was presumed that C. rudolphii A can be found only in cormorants inhabiting marine and brackish habitats and C. rudolphii B distribution was limited to fresh-water habitats (Mattiucci et al., 2002). Later, Szostakowska & Fagerholm (2007; 2012) demonstrated the presence of both these anisakid species in marine, brackish and fresh-water habitats of northern Poland. Some level of habitat specificity was only found in the distribution of juvenile stages: C. rudolphii B was only found in fresh-water fishes, whereas C. rudolphii A was common in both fresh-water and saltwater fishes.
Currently, Contracaecum rudolphii sensu lato represents a complex of genetically close species and not all these cryptic species have full scientific descriptions. These separate species are still treated as operational taxonomic units (OTU) designated with Latin binomial for species complex and letter code, as: C. rudolphii A and B (D'Amelio et al., 1990; Cianchi et al., 1992; Mattiucci et al., 2002; Li at al., 2005, Zhu et al., 2007; Farjallah et al., 2008) distributed mainly in Europe; C. rudolphii С and F (D'Amelio et al., 2007; 2012) found in Atlantic coastal waters of North America; and C. rudolphii D and E (Shamsi et al., 2009) from Australia. Morphometric differences between specimens of some these OTU are quite pronounced (Garbin et al., 2013), but it is the common opinion that for e.g. C. rudolphii A and C. rudolphii B "there are no definitive diagnostic morphological characters for their specific identification" (Li et al., 2005). With a deficit of diagnostic features for adult nematodes, there is no wonder that juvenile stages of Contracaecum from the Black Sea were not identified correctly. An analysis of nucleotide data proved its decisive value in anisakid identification worldwide. Further studies in Contracaecum diversity in the Black Sea and the Sea of Azov area are needed.
ACKNOWLEDGEMENTS
This work is a part of the state-supported studies of the Black Sea biota in the A.O. Kovalevsky Institute of Biology of the Southern Seas, Russian Academy of Sciences (project no. АААА-А18-
118020890074-2). The molecular work was supported by the RSF grant to S.E.S. (project no. 19-74-20147). We thank Dr Paolo Merella (Dipartimento di Medicina Veterinaria Universita' di Sassari, Italy) for his support in the editorial preparation of this paper.
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Н.В. Пронькина и С.Э. Спиридонов. Личинки третьей стадии Contracaecum sp. от черноморского бычка Neogobius melanostomus.
Резюме. В процессе многолетних исследований зараженности рыб Черного моря личинками нематод сем. Anisakidae, часть обнаруживаемых личинок третьей стадии нематод рода Contracaecum определяли в соответствии с их морфологическими особенностями как Contracaecum microcephalum. Такое видовое определение вызывало сомнения, поскольку имеются указания на циркуляцию представителей этого вида в пресноводных биотопах (Kornyushin et al., 2004). Анализ нуклеотидных последовательностей двух участков рибосомальных повторов (LSU rDNA и ITS rDNA) неполовозрелых нематод рода Contracaecum, паразитирующих в Черном море у бычка кругляка Neogobius melanostomus, показал их принадлежность к криптическому виду Contracaecum rudolphii 'A' из комплекса видов Contracaecum rudolphii Hartwich, 1964 sensu lato.
