Parasitol Res (2013) 112:731–735 DOI 10.1007/s00436-012-3193-2
ORIGINAL PAPER
Morphological and molecular characterization of Ceratomyxa gurnardi sp. n. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the grey gurnard Eutrigla gurnardus (L.) (Scorpaeniformes, Triglidae) Ewa Sobecka & Beata Szostakowska & Marek S. Ziętara & Beata Więcaszek
Received: 20 March 2012 / Accepted: 8 November 2012 / Published online: 25 November 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract The myxosporean specimens were noted in grey gurnard Eutrigla gurnardus (L.) from the area near the Shetland Islands. The structure and dimensions of its vegetative stage differ from earlier descriptions. A sequence of small subunit ribosomal RNA gene obtained during the current study differs from other Ceratomyxa spp. available in GenBank. A phylogenetic position of parasite based on the 18S rDNA fragment was estimated. The proposed name for this myxosporean is Ceratomyxa gurnardi sp. n.
Introduction The grey gurnard, Eutrigla gurnardus (L.), is a widely distributed demersal species in the eastern Atlantic from Iceland, Norway, the southern Baltic Sea, the North Sea to southern Morocco, and the Mediterranean and Black Seas (Blanc and Hureau 1973). Heessen and Daan (1994) suggest that three sub-populations of grey gurnard exist in the North Sea and the Skagerrak/Kattegat: one to the northwest of Dogger Bank, one around the Shetlands, and one in the E. Sobecka (*) : B. Więcaszek Department of Hydrobiology, Ichthyology and Biotechnology of Breeding, West Pomeranian University of Technology, Szczecin, Poland e-mail:
[email protected] B. Szostakowska Department of Tropical Parasitology, Inter Faculty Institute of Maritime and Tropical Medicine, Medical University of Gdańsk, Gdańsk, Poland M. S. Ziętara Department of Molecular Evolution, University of Gdańsk, Gdańsk, Poland
Danish Straits. In the North Sea, E. gurnardus has been ranked frequently among the ten dominant species. Since the late 1980s, grey gurnard is considered to be responsible for the most predation mortality on young Atlantic cod Gadus morhua and whiting Merlangius merlangus (Floeter et al. 2005). Myxosporean represents a significant group of fish parasites (Kent et al. 2001). Only one myxosporean parasite has been reported from the gallbladder of E. gurnardus from the North Sea. It was Alataspora lepidum (Gaevskaâ and Kovaleva 1979) from the Celtic Sea, which is the area off the south coast of Ireland (Gaevskaâ and Kovaleva 1979). The original description of A. lepidum is limited only to line drawings based on light microscopy. This study aims to determine if E. gurnardus hosts only one species of myxosporean parasite.
Materials and methods Forty individuals of grey gurnard E. gurnardus were caught as by-catch in commercial catches of Atlantic cod G. morhua, near the Shetland Islands (61°10′N and 002°E) at a depth of 200 m. The fish were necropsied, and the gallbladder epithelium and bile were examined microscopically (×400, ×600, ×1,000) to detect the presence of myxosporean developmental stages. Fresh spores were measured and photographed using a Nikon Eclipse TE 2000-S microscope and processed with NIS-Elements software. Measurements (in micrometers) were taken of 30 fresh spores following the guidelines of Lom and Arthur (1989). The drawings were made using a drawing tube and Corel Draw 9.0. The gallbladder mucosa and bile material reserved for molecular studies were frozen at −20 °C. After thawing, the
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DNA was isolated with a Genomic Mini kit (A&A Biotechnology, Gdynia, Poland). A fragment of a small subunit of ribosomal RNA gene (18S rDNA) was amplified using primers MyxF2 5′ CGC GCA AAT TAC CCA ATC CAG AC 3′ (Køie et al. 2007) and Myxgen4r 5′ ACC TGT TAT TGC CAC GCT 3′ (Kent et al. 2000). The DNA amplification reaction mixture consisted of 5 μl of 10× PCR buffer (A&A Biotechnology), 0.25 mM of each dNTP (Fermentas, Vilnius, Lithuania), 0.4 μM of each primer, 1 U of RUN polymerase (A&A Biotechnology), and 2 μl of DNA template, all in 50 μl of reaction volume. PCR reaction conditions were as follows: 5 min at 94 °C (initial denaturation), 40 cycles of 30 s at 94 °C (denaturation), 30 s at 58 °C (annealing), 90 s at 72 °C (extension), and a final elongation step of 10 min at 72 °C. Before sequencing, PCR products were cleaned with the Clean Up Kit protocol (A&A Biotechnology). The products of the sequencing reaction were cleaned using an ExTerminator Kit (A&A Biotechnology) and subjected to analysis on an automatic ABI PRISM 310 DNA sequencer (Applied Biosystems) using standard procedures as described by the manufacturer and the amplification primers. The sequence obtained were then analyzed, aligned, and compared with data from GenBank using GeneStudio Pro Software (GeneStudio, Inc., Suwanee, Georgia) and MEGA5 (Tamura et al. 2011). The consensus sequence was blasted, and all related sequences of Ceratomyxa spp. were downloaded and aligned by Clustal W (Thompson et al. 1994) as implemented in MEGA5. An initial phylogenetic tree was constructed from maximum composite likelihood (MCL) distances (Tamura et al. 2004) by neighborjoining (NJ) algorithm (Saitou and Nei 1987) (not shown). The closest related species to the new species were taken into consideration. Twelve most related Ceratomyxa species with the overall mean MCL distance of 0.109 (p distance=0.1) with Ceratomyxa labracis (AF411472) as the most basal in the clade were selected. The alignment was manually improved resulting in the final length of 1,056 bp. The shortest sequence included was 915 bp long (Ceratomyxa diamanti FJ204246) and the longest, 1,038 bp long (Ceratomyxa gleesoni EU729693). The final phylogenetic tree was estimated from MCL distances by NJ algorithm (Tamura et al. 2004), utilizing the pairwise deletion option. The validity of the
Fig. 1 Line diagram of the spore of C. gurnardi sp. n
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Fig. 2 Line diagram of the spore of C. gurnardi sp. n
branches was evaluated by bootstrapping 500 replicates (Felsenstein 1985).
Results Ceratomyxa gurnardi sp. n. Type host: Eutrigla gurnardus (L.) Type location: 61°10′N and 002°E to the northeast of the Shetland Islands, at a depth of 200 m Site of infection: gallbladder, bile Prevalence: only four fish were infected (10 %) Intensity: low in bile (1–11 spores per a few microscopic fields at a magnification of ×400) Type material: the slide with the infected bile is deposited in the collection at the Museum of Natural History, Wrocław University, Poland, under accession number MB 1009. Vegetative stages: vegetative stages were not observed; the majority of immature spores were observed free floating in the bile. Spore description: spores arcuate, transversely elongate with indistinct sutural lines in immature spores and more distinctly notable in mature ones (Figs. 1 and 2); anterior and posterior margins of shell valves taper gradually; spores measure 5.8 ± 0.8 (4.9–7.4) μm in length and 26.4 ± 4.0 (20.3–31.1)μm in thickness; polar capsules almost spherical
Fig. 3 Fresh spores of C. gurnardi sp. n
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Fig. 4 Abnormal spores with three polar capsules
(subspherical), symmetrical, equal in size at 3.4±0.4 (2.7– 3.8) μm in length, and 2.9 ± 0.2 (2.9–3.3) μm in width (Fig. 3); polar filament with three coils visible inside polar capsules; aberrant spores with three polar capsules occur occasionally (Fig. 4). Phylogenetic characterization: the amplified fragment of 18S rDNA was ∼1,100 bp long (not shown). The sequence obtained (949 bp) was deposited in GenBank under accession number JQ071439. The phylogenetic relationship of the studied Ceratomyxa species is presented in Fig. 5. The most similar sequences (MCL evolutionary distance: 0.1, 0.104, and 0.109) belong to Ceratomyxa jonesi (FJ204250), Ceratomyxa ernsti (FJ204247), and C. diamanti (FJ204246) or Ceratomyxa dennisi (EU440358), respectively, but C. gurnadi sp. n. (JQ071439) is associated (bootstrap 91 %) with the Ceratomyxa species with longer MCL evolutionary distances.
Discussion The genus Ceratomyxa, representing the coelozoic Myxozoa, is widely distributed in marine hosts (Eiras 2006). To date, there are approximately 200 described species of Ceratomyxa. The comparison of C. gurnardi sp. n. with other
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Ceratomyxidae was based on morphological features and geographical distribution. Phylogenetic relationship among host species were also compared (Gunter 2009). Some species fall within the morphological and dimensional ranges of the present species; these include: Ceratomyxa auerbachi Kabata, 1962; Ceratomyxa choleospora Landsberg, 1993; Ceratomyxa chromis Lubat, Radujkovic, Marques, Bouix, 1989; Ceratomyxa dissostichi Brickle, Kalavati, MacKenzie, 2001; Ceratomyxa entzerothi Abdel-Graffar, Ali, Al Quraishy, Rasheid, Al Farraj, Abdel-Baki, Bashar, 2008; Ceratomyxa sparusaurati Sitjà-Bobadilla, Palenzuela, Alvarez-Pellitero, 1995. These species differ from C. gurnardi sp. n. described in the current paper. The differences include the lengths and thicknesses of C. auerbachi which are more than the maximum ranges of C. gurnardi sp. n.. C. choleospora is another morphologically similar species that generally differs in it smaller dimensions and widely rounded valve ends. The most similar is C. chromis particularly in size and shape, even though its valves are more elongate and the polar capsules are rounded and smaller than those in the species described in this paper. The spore thickness of C. dissostichi is smaller, its polar capsules are smaller, more elongate, and its posterior diverges significantly. The spores of C. entzerothi are thicker and are distinguishable from C. gurnardi sp. n. by their longer, wider, pyriform polar capsules. C. sparusaurati has thinner spores and polar capsules with a higher number of filament coils (six as compared to three). The shape of A. lepidum Gaevskaâ and Kovaleva, 1979, a myxosporean from E. gurnardus from the Celtic Sea is definitely different from the specimens described in this paper. The thickness of A. lepidum was 12.0–13.3 μm, but the total thickness of spores with wings ranged from 42.6 to 58.5 μm. In addition, the A. lepidum spores are slightly twisted, and wings have not been recorded in C. gurnardi sp. n.. Aberrant spores with three polar capsules are also present in other species (Ceratomyxa bassoni, Abdel-Graffar et al. 2008; Ceratomyxa protopsettae Fujita,1923; Cho et al. 2004).
Fig. 5 Hypothetical phylogenetic position of C. gurnadi sp. n. based on the 18S rDNA fragment. Bootstrap support for 500 replicates
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Numerous sequences of 18S rDNA from many species of Ceratomyxa are deposited in GenBank; however, none is identical to that obtained during the present study. Sequences from C. choleospora, C. chromis, C. dissostichi, and C. entzerothi that are the most morphologically similar to the species described in this work, as well as A. lepidum that was found in E. gurnardus from the Celtic Sea and C. bassoni and C. protopsettae which have aberrant spores with three polar capsules similar to those described in the specimens in the present work are not deposited in GenBank. The most comprehensive Ceratomyxa phylogeny inferred from 18S rDNA sequences is presented recently by Gunter et al. (2009). Their parasitological studies of the parasite fauna of fish from the Great Barrier Reef indicate Ceratomyxa as one of the most cohesive lineages within the Myxozoa although the tree remained largely unresolved. They also revealed tight host specificity for the Ceratomyxa species and stated that no parasite radiation had occurred that could be associated with co-evolution with host families. The species analyzed in this paper belong to the main subclade of the marine lineage of the Ceratomyxa genus. They all except Ceratomyxa robertsthomsoni and Ceratomyxa thalassomae take a rather basal unresolved position in the clade. In our analysis restricted only to related species of C. gurnadi sp. n., they form two evolutionary lineages with the bootstrap values over 90 %. Although C. gurnadi sp. n. seems to be associated to the lineage encompassing C. gleesoni, Ceratomyxa hallettae, and the sister species—C. thalassomae and C. robertsthomsoni, we may not exclude the possibility that it will form an independent lineage when the sequences of more related species will be once available. The evolutionary distances among this group are higher than among Ceratomyxa species associated in its sister group. Further studies are needed to explain such an association. The shape of spores of C. gleesoni, C. hallettae, C. thalassomae (Heiniger et al. 2008), C. robertsthomsoni as well as of C. jonesi, C. ernsti, C. diamanti, and C. dennisi differ markedly from C. gurnadi sp. n.. Similar in size are only the spores of C. hallettae. They are the parasites of fish species from other orders, inhabiting the Great Barrier Reef off Australia, whereas the C. gurnardi sp. n. described in the present study was found in grey gurnard from the North Sea near the Shetland Islands. Grey gurnard from the North Sea might be well separated from those in the Channel, and almost absent from south of the Southern Bight. The new species described in this paper was not noted to have inflicted pathological changes in the host fish in this study. The members of the genus Ceratomyxa that were used for comparison in this study were isolated from hosts that were phylogenetically unrelated to E. gurnardus (three host species belonged to Perciformes and single host species belonged to Clupeiformes, Gadiformes, and Mugiliformes) (Froese and Pauly 2011). There has been no record of
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Ceratomyxa infecting fishes of the family Triglidae. Even if the diet of some of the host fishes is similar to the diet of the grey gurnard, it is unlikely that the transmission of myxosporean stages could result in grey gurnard infection, because the majority of Ceratomyxa spp. are host specific (Lom and Dykova 2006). Based on the differences reported presently between the newly found species and its congeners, the authors propose it is a new species named C. gurnardi. Acknowledgments The authors would like to thank Mr. W. Szaszkiewicz for providing the specimens of E. gurnardus. Thanks are also due to Dr. J. Szulc and Mr. M. Więcaszek for technical support.
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