Спросить
Войти
Russian Journal of Nematology, 2018, 26 (1), 87 - 92
Morphological and molecular characterisation of anisakid juveniles from the golden grey mullet of
the Black Sea
Natalia V. Pronkina1 and Sergei E. Spiridonov2
&A-O. Kovalevsky Institute of Marine Biological Research, Russian Academy of Sciences, Nakhimov Avenue 2, 299011, Sevastopol, Russia 2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 119071, Moscow, Russia e-mail: natalya-pronkina@yandex.ru
Accepted for publication 29 June 2018
Summary. The anisakid juveniles of the genus Contracaecum were regularly reported from liver ducts of the golden grey mullet Chelon auratus (Risso, 1810) collected in the Black Sea coastal waters of Crimean peninsula. Primarily, these juveniles were identified as C. rudolphii (spiculigerum) Hartwich, 1964. After additional morphological study and analysis of nucleotide sequences the juveniles were identified as Contracaecum multipapillatum (Drasche, 1882) Lucker, 1941. Phylogenetic analysis revealed, that partial sequence of LSU rDNA of Contracaecum juveniles from golden grey mullet is identical to the deposited LSU rDNA sequence (AF226574) of C. multipapillatum from pelicans in Greece. According to the analysis of ITS rDNA this sequence of C. multipapillatum from the Black Sea is similar to those of C. multipapillatum from Australian pelicans.
The genus Contracaecum Railliet Henry, 1912 is the largest taxon of a generic level within Anisakidae Railliet Henry, 1912, with more than 50 valid species. The definitive hosts for the majority of the Contracaecum species are fish-eating birds with crustaceans as first-, and fish as second-intermediate hosts. The Contracaecum juveniles (the third-stage) were found in liver ducts of golden grey mullet Chelon auratus (Risso, 1810) during our examination of these fish in 2001. Primary identification as C. rudolphii Hartwich, 1964 by Pronkina and Belofastova (2005) was based on the diagnoses and keys from the monograph describing local fauna of parasites (Gaevskaya et al., 1975). A recent revision of Contracaecum by Moravec (2009) demonstrated that this previous identification was probably erroneous for these juveniles. An analysis of nucleotide sequences was applied to identify these Contracaecum juveniles from Black sea mullets, which therefore were identified as Contracaecum multipapillatum. Morphological and molecular characteristics supporting the identification are presented below.
MATERIAL AND METHODS
More than five hundred of 30-50 mm long golden grey mullet specimens were obtained from Crimea coastal waters in 2001-2017. The nematodes obtained after dissections were fixed in 70% ethanol and mounted in glycerin-lactic acid 1:1 mixture. The middle part of the body of one specimen was fixed in 95% ethanol for DNA extraction, and the remaining parts were mounted to serve as the voucher specimen, or &hologenophore& (Pleijel et al., 2008). The voucher specimen was deposited in the Collection of marine parasites (CMP IMBR № 1059-1080.N.2q.V 1-22) of the Kovalevskii Institute of Marine Biological Research of the Russian Academy of Sciences (Dmitrieva et al., 2015). All the measurements are in micrometers as (range) mean ± SE.
For molecular studies, a portion of gonadal tube was excised from ethanol fixed nematode. An extraction of DNA was made with &Promega& columns (Wizard® SV Genomic DNA Purification System). For amplification of LSU rDNA the
© Russian Society of Nematologists, 2018; doi:10.24411/0869-6918-2018-10008
following primers proposed by Nadler et al. (2006) were used: LSU 391 (5 &-AGCGGAGGAAAAGAA ACTAA-3&) with LSU 501 (5&-TCGGAAGGAACC AGCTACTA-3&), or LSU 391 with LSU 390 (5&-ATCCGTGTTTCAAGACGGG-3&). PCR cycling parameters for the amplification of partial LSU rDNA included primary denaturation at 94°C for 4 min followed by 35 cycles 94°C for 30 s, 56°C for 30 s and 72°C for 60 s, followed by postamplification extension at 72°C for 5 min. To amplify ITS rDNA the primer 18S (Vrain et al., 1992) together with primer AB28 (5&-ATATGCTT AAGTTCAGCGGGT-3&) (Joyce et al., 1994) were used. PCR cycling parameters for the amplification of ITS rDNA included primary denaturation at 94°C for 5 min followed by 35 cycles 94°C for 30 s, 54°C for 35 s and 72°C for 70 s, followed by postamplification extension at 72°C for 5 min. The PCR products were purified in 0.8% agarose gel, the bands were excised and DNA was extracted with columns Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). After the precipitation (ethanol + ammonium acetate) the samples were directly sequenced by &Genotech& company (Moscow). Similar sequences were searched for in NCBI GenBank with BLAST algorithm (Altschul et al., 1990). Obtained sequences were analysed in MEGA7.0.14 (Kumar et al., 2016) with three methods: maximum parsimony (MP), neighbour joining (NJ) and maximum likelihood (ML). Obtained sequences were deposited in NCBI GenBank (partial ITS rDNA as MH400190 and partial 28S rDNA as MH398587).
Contracaecum multipapillatum (Drasche, 1882) Lucker, 1941 juveniles
Dissected from the second intermediate host, golden grey mullet Chelon auratus (Risso, 1810) (Mugilidae).
Locality. Black Sea coastal water, i) near Sevastopol (44°36&24&& N, 33°28&11&& E; 44°30&18&& N, 33°35&54&& E; 44°36&39&& N, 33°29&51&& E; 44°36&31&& N, 33°31&51&& E; 44°36&60&& N, 33°30&44&& E) and ii) Karkinitski Bay (45°52&30&& N, 33°32&30&& E).
Localisation in host. Biliary ducts in liver. Up to eight juveniles per fish. The youngest infected fish were two years old.
Material. One juvenile, deposited as the hologenophore and 22 voucher specimens of juveniles (CMP IMBR № 1059-1080.N.2q.V 1-22).
Description (n = 19; intact third-stage juveniles and also the head end and tail of the hologenophore specimen). Body length 14274-29750 (23266 ± 816.3), maximal dia. 575-1125 (849 ± 29); nerve ring dia. 256-425 (343 ± 7.9), anal opening dia. 88200 (148 ± 8.9). Under light microscope the body surface is transversally annulated. Coarse annulation on head end (Fig. 1A). Cuticle 13-38 (26.8 ± 1.3) thick. Oral opening slit-like, with four flattened protrusions of body surface around (Fig. 1A). Excretory pore opening near head end. Prominent juvenile tooth, 12-25 (17.4 ± 0.7) high (Fig. 1A). Nerve ring 52-96 (76 ± 3) x 129-221 (175 ± 6), in 179-375 (253.3 ± 10.5) from anterior end. Intestinal
Fig. 1. Surface structures of Contracaecum multipapillatum juveniles from golden grey mullet. A -anterior end; B - tail.
caecum well developed, wider than ventricular appendix, 1527-3500 (2227 ± 106) long, 125-550 (327 ± 21) wide. Subglobular ventriculus on pharynx end 88-188 (123 ± 6.0) long and 88-188 (142 ± 6.1) wide with the protruding part 500-760 (658 ± 17.7) long and 100-159.6 (132 ± 3.3) wide. Length ratio of intestinal caecum to pharyngeal ventriculus appendix 3:1. Reproductive system not developed. Rectal gland discernible. Tail end cupola-shaped 125-280 (208 ± 13) long, with conical terminal process (Fig. 1B). Juvenile enclosed into capsule with coarse fibrillar structure of walls.
Primer pairs LSU391-LSU501, and LSU391-LSU390 amplified nearly identical sequences. BLAST search against this sequence revealed a set of close sequences, all of which originated from nematodes of the genus Contracaecum and related genera. The length of obtained LSU rDNA sequence was about 700 bp, but because of different lengths of sequences from GenBank the final length of obtained alignment was 669 bp, with 106 informative characters. The topology of the trees
obtained with three methods of analysis (MP, NJ and ML) was identical. The sequence obtained for juveniles from golden grey mullet in all three topologies was in the group composed of Contracaecum multipapillatum LSU rDNA sequences (Fig. 2). The bootstrap support for the entire group of four sequences and the subgroup of three sequences including that from Black Sea is strong (Fig. 2).
The length of ITS rDNA amplicon obtained with primers 18S and AB28 was about 1000 bp. Alignment of obtained sequence with those found in GenBank was only 451 bp long as some deposited sequences were quite short. The number of informative characters is 198. The topologies of trees constructed with all three methods (MP, NJ and ML) were identical. The ITS rDNA sequence from golden grey mullet clustered with two C. multipapillatum sequences and was a part clade with strong support consisting mainly of the sequences belonging to this species. Some sequences of C. ovserstreeti, C. pyripapillatum and an unidentified Contracaecum sp. were also inside this clade (Fig. 3).
Baylisascarispotosis AB893608
Contracaecum microcephalum AF226573 Contracaecum radiatum AF226577 Contracaecum miroungae AF226581 Contracaecum sp. KC013599 Contracaecum miroungae KC013596 Contracaecum osculatum AF226576 Contracaecum osculatum AF226580 Phocascaris sp. AF226575 Contracaecum sp. KC013604 Contracaecum osculatum AF226583 Contracaecum sp. KC013600 Contracaecum osculatum KC013595 Contracaecum osculatum AF226589 Contracaecum sp. MG696301
Contracaecum micropapill atum AF226587 Contracaecum septentrionale AF226588 Contracaecum eudyptulae AF226586 Contracaecum rudolphii AF226585 Contracaecum rudolphii AF226579 Contracaecum sp. AY821771 Contracaecum ogmorhini AF226582 Contracaecum sp. AY821769 Contracaecum sp. AY821768 Contracaecum sp. AY821770
Contracaecum multipapillatum U94755 Contracaecum multipapillatum U94756 Contracaecum sp. ex Golden grey mullet Contracaecum multipapillatum AF226574
Fig. 2. Phylogenetic relationships of Contracaecum multipapillatum from Black Sea golden grey mullet inferred from an analysis of partial LSU rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), K2+G model. For MP and NJ - 1000 bootstrap replications.
Hysterothylacium tetrapteri KF601901 Pseudanisakis rajae JN3 92470 Contracaecum sp. FM210434 Contracaecum sp. FM210433 Contracaecum sp. FM210435 Contracaecum sp. KY703768 Contracaecum sp. KX580607 Contracaecum overstreeti MG515224 Contracaecum multipapillatum AM940059 Contracaecum sp. ex Golden grey mullet Contracaecum multipapillatum AM940056 Contracaecum pyripapillatum AM940062 Contracaecum multipapillatum AM940063 Contracaecum pyripapillatum AM940064 Contracaecum sp. KX580603 Contracaecum sp KX580602 Contracaecum multipapillatum MHO 18546 Contracaecum multipapillatum MH018557 Contracaecum multipapillatum MHO 18551 Contracaecum multipapillatum MHO 18547 Contracaecum multipapillatum MH018550 Contracaecum multipapillatum MH018555 Contracaecum multipapillatum MH018556 Contracaecum multipapillatum MH018548 Contracaecum multipapillatum MH018558 Contracaecum multipapillatum MH018559 Contracaecum multipapillatum MH018554 Contracaecum multipapillatum MH018553 Contracaecum multipapillatum MHO 18549 Contracaecum multipapillatum MH018552 rudolphii FJ467618 rudolphii FJ467620 rudolphii EU678869 rudolphii FJ589790 rudolphii FJ589792 Contracaecum sp. AY821751 Contracaecum rudolphii JF424597 Contracaecum sp. AY821752 Contracaecum sp. AY821750 Contracaecum sp. AY821753 Contracaecum osculatum KU3 06701 Contracaecum osculatum KU306715 Contracaecum osculatum MF980981 Contracaecum osculatum KU306718 Contracaecum osculatum KU3 06706 Contracaecum osculatum KU3 06696 Contracaecum osculatum KU306717 Contracaecum sp. KJ561666 Contracaecum sp. KJ561671 Contracaecum sp. KJ561670 Contracaecum sp. KJ561662 Contracaecum sp. KJ561663 Contracaecum sp. KJ561661 Contracaecum sp. KJ561667 Contracaecum sp. KJ561665 Contracaecum sp. KJ561668 Contracaecum sp. KJ561669
Contracaecum Contracaecum Contracaecum Contracaecum Contracaecum
Fig. 3. Phylogenetic relationships of Contracaecum multipapillatum from Black Sea golden grey mullet inferred from an analysis of ITS rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), K2+G model. For MP and NJ - 1000 bootstrap replications.
DISCUSSION
Previous erroneous identification of juveniles of Contracaecum found in golden grey mullet in the Black Sea off Crimea as C. rudolphii (earlier was known under the synonym C. spiculigerum) (Pronkina Belofastova, 2005) was based on the incorrect compilation of the data on the morphology of juveniles representative of this genus (Gaevskaya et al., 1975). Measurements of anisakid juveniles obtained in the course of experimental inoculations with these nematodes of dragon-fly larvae and freshwater fishes (Mozgovoy et al., 1968) were given in this work. These data were illustrated with the figures of anisakid specimens found in the body cavity of catfish from Tumak district in Volga estuary (Dogiel Bykhovskii, 1939). The same measurements of Contracaecum juveniles identified as C. rudolphii accompanied with original drawings were given in a monograph by Moravec (1994). The measurements of Contracaecum juveniles found in golden grey mullet near Crimea coast (Pronkina Belofastova, 2005) correspond with the data presented in the above mentioned work, and their morphology to those illustrated by Dogiel and Bykhovskii (1939). Finally, the comparison of Contracaecum juveniles from Black Sea golden grey mullet with C. rudolphii juveniles described in detail by Moravec (2009) has demonstrated that the former did not fit to the corrected diagnosis of C. rudolphii.
Phylogenetic analysis of the sequences of the two nuclear loci obtained from the newly collected specimens of Contracaecum from golden grey mullet demonstrated their close similarity with the deposited sequences of C. multipapillatum.
Phylogenetic analysis of the obtained sequences demonstrated the affinity of the juveniles studied to the species C. multipapillatum. This affinity was unequivocal in the analysis of the partial LSU rDNA (Fig. 2), although only three sequences of this species are deposited in NCBI GenBank. No nucleotide differences in this LSU rDNA sequence was found between Black Sea Contracaecum in this study and the deposited sequence AF226574 of adult C. multipapillatum from Pelecanus crispus, originating from Psatatopi, Greece (Nadler et al., 2000). The difference in 10-11 bp in LSU rDNA was found between juveniles in our material and juveniles of C. multipapillatum from Mugil curema mullets of the Grand Lagoon, Horn Island, Mississippi, USA.
The ITS rDNA analysis of Contracaecum multipapillatum was based on the larger number of
sequences deposited and showed that the relationships of the studied Contracaecum juveniles from Black Sea mullets were complicated (Fig. 3). Our samples formed the strongly supported clade with two sequences of adult C. multipapillatum from pelicans collected and dissected in Australia (Victoria). The difference in 7 bp of ITS rDNA was observed between our samples and the juveniles from USA, whilst all other deposited sequences of C. multipapillatum and related species differed at least by 22 bp. The clade of these three sequences of C. multipapillatum from mullets was a part of the wider group of deposited sequences with representatives of several identified and unidentified up to species level Contracaecum samples (Fig. 3). The phylogenetic relationships of our C. multipapillatum sample with other species of the genus revealed the obscure taxonomic status of this species, which was sometimes treated as a &species complex& (Nadler et al., 2000; Mattiucci et al., 2006; 2010; D&Amelio et al., 2007).
An accumulation of data about the nucleotide diversity of anisakid nematodes in the Black Sea and adjoining waters is needed.
ACKNOWLEDGEMENTS
This work is a part of the state supported studies of Black Sea biota in the Kovalevsky Institute of Marine Biological Research, Russian Academy of Sciences (project no. AAAA-A18-118020890074-2). The work was partially supported by the RFBR grant to S.E.S. (project no. 17-04-00095a). ). SEM studies were carried out at the Joint Usage Center "Instrumental Methods in Ecology" at A.N. Severtsov Institute of Ecology and Evolution.
REFERENCES
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. Lipman, D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410. D&Amelio, S., Barros, N.B., Ingrosso, S., Fauquier, D.A., Russo, R. Paggi, L. 2007. Genetic characterization of members of the genus Contracaecum (Nematoda: Anisakidae) from fish-eating birds from west-central Florida, USA, with evidence of new species. Parasitology 134: 1041-1051. Dmitrieva, E.V., Lyakh, A.M., Kornyychuk, Yu.M., Polyakova, T.A., Popyuk, M.P., Yurakhno, V.M. Pronkina, N.V. 2015. IMBR Collection of Marine Parasites. Collection of Marine Parasites Maintained by the Institute of Marine Biological Research. URL: www.marineparasites.org (accessed: January 29, 2018.)
Dogiel, V.A. Bykhovskii, B.E. 1939. [Parasites of Fishes of the Caspian Sea]. USSR, the USSR Academy of Sciences Publishing House. 151 pp. (in Russian).
Gaevskaya, A.V, Gusev, A.V., Delyamure, S.L., Donets, Z.S., Iskova, N.I., Kornyushin, V.V., Kovaleva, A.A., Margaritov, N.M., Markevich, A.P., Mordvinova, T.N., Naidenova, N.N., Nikolaeva, V.M., Parukhin, A.M., Pogoreltseva, T.P, Smogorzhevskaya, L.A., Solonchenko, A.I., Shtein, G.A. Shulman, S.S. 1975. [Key to the Parasites of Vertebrata of the Black and Azov Seas]. USSR, Naukova Dumka. 552 pp. (in Russian).
Joyce, S.A., Reid, A., Driver, F. Curran, J. 1994. Application of polymerase chain reaction (PCR) methods to the identification of entomopathogenic nematodes. In: COST 812: Biotechnology. Genetics of Entomopathogenic Nematode - Bacterium Complexes: Proceedings of a Symposium and Workshop Held at St. Patrick&s College, Maynooth, Co. Kildare, Ireland and National Reports 1990-1993 (A.M. Burnell, R.-U. Ehlers J.-P. Masson Eds). pp. 178-187. Brussels, Belgium, Office for Official Publications of the European Communities.
Kumar, S., Stecher, G. Tamura, K. 2016. MEGA7: molecular evolutionary genetics analysis. version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
Mattiucci, S., Olivero, J., Paoletti, M., Arrollo, B. Nascetti, G. 2006. Genetic evidence for new species of Contracaecum (Nematoda, Anisakidae) parasites of the brown pelican, Pelecanus occidentalis, from Columbia: genetic relationships between congeners and larval identification. in: Abstracts of the 11th International Congress of Parasitological Associations. p. 56. Glasgow, Scotland, ICOPA XI.
Mattiucci, S., Paoletti, M., Solorzano, A.C. Nascetti, G. 2010. Contracaecum gibsoni n. sp. and C. overstreeti n. sp. (Nematoda: Anisakidae) from the Dalmatian pelican Pelecanus crispus (L.) in Greek
waters: genetic and morphological evidence. Systematic Parasitology 75: 207-224.
Moravec, F. 1994. Parasitic Nematodes of Freshwater Fishes of Europe. The Netherlands, Academia and Kluwer Academic Publishers. 473 pp.
Moravec, F. 2009. Experimental studies on the development of Contracaecum rudolphii (Nematoda: Anisakidae) in copepod and fish paratenic hosts. Folia Parasitologica 56: 185-193.
Mozgovoy, A.A., Shakhmatova, V.I. Semenova, M.K. 1968. [Life cycle of Contracaecum spiculigerum (Ascaridata: Anisakidae), a parasite of domestic and game birds]. Trudy Gel&mintologicheskoi Laboratorii AN SSSR 19: 129136 (in Russian).
Nadler, S.A., D&Amelio, S., Fagerholm, H.P., Berland, B. Paggi, L. 2000. Phylogenetic relationships among species of Contracaecum and Phocascaris (Nematoda, Ascaridoida) based on nuclear rDNA. Parasitology 121: 455-463.
Nadler, S.A., Bolotin, E. Stock, S.P. 2006. Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data. Systematic Parasitology 63: 161-181.
Pleijel, R., Jondelius, U., Norlinder, E., Nygeren, A., Oxelman, B., Schander, C., Sundberg, P. Thollesson, M. 2008. Phylogenies without roots? A plea for the use of vouchers in molecular phylogenetic studies. Molecular Phylogenetics and Evolution 48: 369-371.
Pronkina, N.V. Belofastova, I.P. 2005. [New data about nematodes of the Black Sea golden grey mullet Liza aurata (Pisces: Mugilidae)]. Ekologiya Morya 46: 77-82 (in Russian).
Vrain, T.C., Wakarchuk, D.A., Levesque, A.C. Hamilton, R.J. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15: 563-573.
Н.В. Пронькина и С.Э. Спиридонов. Морфологическая и молекулярная характеристика анизакидных личинок от черноморского сингиля.
Резюме. В протоках печени сингиля Chelon auratus (Risso, 1810) у побережья Крыма постоянно обнаруживаются личинки паразитических анизакидных нематод рода Contracaecum. Первоначально эти личинки были определены как C. rudolphii (spiculigerum) Hartwich, 1964. После дополнительного морфологического изучения и анализа полученных нуклеотидных последовательностей личинки от сингиля были определены как Contracaecum multipapillatum (Drasche, 1882) Lucker, 1941. По нуклеотидным последовательностям LSU rDNA личинки Contracaecum от сингиля оказались идентичными депонированной (AF226574) последовательности C. multipapillatum от пеликанов из Греции. По ITS-участку rDNA C. multipapillatum оказались близки к последовательностям взрослых особей C. multipapillatum от пеликанов Австралии.
