Parasitol Res (2013) 112:289–296 DOI 10.1007/s00436-012-3136-y
ORIGINAL PAPER
Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China Y. Liu & C. M. Whipps & Z. M. Gu & M. J. Huang & C. He & H. L. Yang & K. Molnár
Received: 22 June 2012 / Accepted: 19 September 2012 / Published online: 30 September 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract During a survey of myxozoan parasites of common carp Cyprinus carpio in Honghu Lake, Hubei Province, China, a parasite was collected that was identified as Myxobolus dispar based on an earlier description from China. However, the small subunit ribosomal DNA of this species shared only 90 % similarity with M. dispar, instead matching M. musseliusae with 100 % identity. To resolve this apparent taxonomic conflict, the validity of M. dispar reported from China was investigated. The species encountered here and in the earlier report from China both bear spores that are notably smaller than those of M. dispar in Europe. In the present study, a mucous envelope was adhered to the posterior of many fresh spores and was observed to expand and
surround the spore. This structure has never been reported from fresh spores of M. dispar. Histology showed extravascular plasmodia in the gill filaments in close contact with the cartilaginous ray of the filament, which contrasts with the plasmodia of M. dispar which develop in the arteries of the gill filaments. Phylogenetically, the current species is distinct from M. dispar, instead forming a sister group with M. musseliusae. The data presented here allow us to conclude that the species isolated is M. musseliusae and that prior reports of M. dispar in China are unsubstantiated.
Y. Liu : Z. M. Gu (*) : M. J. Huang Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei Province 430070, People’s Republic of China e-mail:
[email protected]
Myxozoan parasites infect both marine and freshwater fishes the world over. In the most recent comprehensive review of the taxonomy of these diverse parasites, Lom and Dyková (2006) reported 2,180 nominal species of Myxosporea, the most speciose genus being Myxobolus Bütschli, 1882 at 792 species, around a third of the total. Literature searches conducted for the current study identified at least 39 more Myxobolus species that have been described since 2006, indicative of the yet undiscovered diversity within this genus. This incredible diversity coupled with the simplicity of the diagnostic stage of these species, the myxospore, creates challenges for identification. The problem is that, using spore morphology alone, it is often difficult to determine the validity of morphologically similar Myxosporea even from systematically distant fishes (Molnár et al. 2011). In order to avoid the above conundrum, recent studies have suggested that host and organ specificity and also tissue tropism should be taken into consideration in species identification (Molnár 1994, 2002;
C. M. Whipps SUNY-ESF, State University of New York College of Environmental Science and Forestry, Environmental and Forest Biology, 1 Forestry Drive, Syracuse, NY 13210, USA C. He : H. L. Yang Beijing Aquatic Product Technology Promotion Department, Room 303# no 48A Building West of Huawei Chaoyang District, Beijing 100021, People’s Republic of China K. Molnár Institute for Veterinary Medical Research, Centre for Agricultural Research, HAS, POB 18, 1581, Budapest, Hungary
Introduction
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Lom and Dyková 2006; Dyková and Lom 2007; Ferguson et al. 2008; Molnár et al. 2011). However, when the spores develop in plasmodia within identical organs and tissues of the same or closely related fishes, the identification of a given species may be especially difficult (Molnár et al. 2011). As such, molecular biological methods are becoming mainstream in Myxobolus species identification (Eszterbauer and Székely 2004; Székely et al. 2009; Liu et al. 2010b, 2011b). In addition to the problems above, the myxozoan taxonomist is also faced with the burden of many species that have been described inadequately, making accurate comparisons challenging (Liu et al. 2011a). Moreover, owing to poor communication between scientists in different countries and regions, many synonyms and misidentification possibly exist (Zhang et al. 2010; Molnár 2011). In the current study, a Myxobolus species was found during a survey of myxozoan parasites of common carp Cyprinus carpio L. in Honghu Lake, Hubei Province, China. Its spore morphology is consistent with that of Myxobolus dispar described by Chen and Ma (1998); however, its small subunit DNA sequence is identical with that of M. musseliusae Yakovchuk 1979. In order to resolve this conflict, we studied the morphology of a new sample of M. dispar collected in Europe, as well as to the Chen and Ma description of M. dispar from China, and compared this to our species collected from China.
Materials and methods Ninety specimens of common carp ranging from 31 to 34 cm in length and 453–559 g in weight were harvested by a fine-meshed seine from Honghu Lake, Hubei Province, China in March to April 2011. Fish were transported to the Laboratory of Fish Diseases in College of Fisheries, Huazhong Agricultural University, Wuhan City, Hubei Province and held in aquaria, where they were euthanized with 0.2 mg/ml tricaine methanesulfonate (MS-222, Sigma) prior to dissection. The work involving animals was conducted at the Laboratory of Fish Diseases at the Huazhong Agricultural University in China. It is the personal goal of the researchers at this university to handle animals with care and use humane methods of euthanasia. The method used in this study is listed as an approved method by the American Fisheries Society (2004). Standard procedures (Lom and Dyková 1992) were used for myxosporean examination within 24 h after transportation. Morphological methods Cysts containing myxospores consistent with those of the genus Myxobolus were collected from the gills of a common carp. Fresh spores from one cyst were measured according to Lom and Arthur (1989). Spore maturity was checked by
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placing them into a 0.4 % urea solution. Spores of a given plasmodium were regarded as mature when at least 90 % of them extruded polar filaments in this solution. Measurements of spores were performed using an Olympus BH2 microscope equipped with an ocular micrometer and image analysis software (Motic images 3.2). Mean and standard deviations of each spore dimension were obtained from fresh mature spores (n030). Line drawings were made based on the fresh wet mounts with the help of a Canon IXUS210 camera. All measurements are given in micrometers unless otherwise indicated. For histological examination, tissue samples from infected organs containing developing and mature plasmodia were fixed in Bouin’s solution, embedded in paraffin wax, sectioned at 5–6 μm, and stained with hematoxylin and eosin. DNA isolation and sequencing For DNA extraction, cyst contents from tissue of one fish containing several cysts fixed in 100 % ethanol were suspended in 500 μl lysis buffer (100 mM NaCl, 10 mM Tris, 10 mM EDTA, 0.2 % SDS, and 0.4 mgml−1 proteinase K) and incubated at 55 °C overnight. Genomic DNA was extracted using a phenol-chloroform protocol (Eszterbauer et al. 2001). The ssrRNA gene was amplified with primers MX5/MX3 of Andree et al. (1999) in a polymerase chain reaction (PCR) of 50 μl, which contained approximate 200 ng of extracted genomic DNA, 1× Taq Buffer (MBI Fermentas, Vilnius, Lithuania), 2.5 mM MgCl 2 , 0.2 mM dNTPs (MBI Fermentas), 2 μM each primer, and 5 U of Taq DNA polymerase (MBI Fermentas) in MilliQ purified water. A PTC-100 DNA Engine (MJ Research Inc., Watertown, MA, USA) was used to control the cycling conditions: 95 °C for 50 s, 56 °C for 50 s, and 72 °C for 60 s for 35 cycles, with an initial denaturation at 95 °C for 5 min and a terminal extension at 72 °C for 10 min. The PCR products were purified using the High Pure PCR Product Purification Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) and sequenced in both directions with primers MX5 and MX3 using the ABI PRISM® 3730 DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA). Forward and reverse sequence segments were aligned, and a contiguous sequence was deposited in GenBank. A standard nucleotidenucleotide BLAST (blastn) search was conducted to query posted sequences. DNA sequence similarities were calculated with the MegAlign of DNASTAR Version 7.1. Phylogenetic analysis Sequences were assembled in BioEdit (Hall 1999) and verified as myxozoan by GenBank BLAST search. To evaluate the relationship of the current species to existing myxobolids, 35 sequences were aligned with Clustal X version 1.8 (Thompson et al. 1997). The alignment consisted of the top BLAST search matches and representatives of neighboring
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clades based on earlier analyses of the myxobolids (Zhao et al. 2008; Ferguson et al. 2008; Liu et al. 2010b). Myxobolus musculi and Myxobolus ampullicapsulatus were designated as outgroup taxa. Phylogenetic analyses were carried out on this 1,548 character alignment as follows: Optimal evolutionary models for maximum likelihood (ML) and Bayesian analysis were determined using jModeltest (Posada 2008) which identified the optimal evolutionary model using the Akaike information criteria, as the general time reversible model (GTR + I + G). Nucleotide frequencies were estimated from the data (A00.2315, C00.1855, G00.2915, T00.2915); six rates of nucleotide substitution were [AC]01.1269, [AG]0 4.3801, [AT]01.8502, [CG]00.3734, [CT]06.2731, [GT]0 1.0000; proportion of invariable sites00.3550; and gamma distribution00.3580 estimated with four rate categories. ML analysis was performed using PhyML (Guindon and Gascuel 2003). Bootstrap confidence values were calculated with 100 replicates. Bayesian analyses were conducted in Mr. Bayes (Ronquist and Huelsenbeck 2003) using the evolutionary model as above, with 106 generations, tree sampling every 100 generations, with a burn-in of 100 trees. Trees were initially examined in TreeView X (Page 1996) and edited and annotated in Adobe Illustrator (Adobe Systems Inc., San Jose, CA, USA).
Results Supplemental description of M. musseliusae Yakovchuk, 1979 in China Plasmodia (Fig. 1) bearing spores morphologically consistent with those of the myxozoan genus Myxobolus were found in the gill filaments of 19 of 90 carp. These plasmodia are whitish, round, or elongate ellipsoidal, measuring 150– 1,600 μm in diameter.
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Fig. 2 Photomicrograph of fresh spores of M. musseliusae. Scale bar, 5 μm
(5.9–6.7) thick (all measurements reported in microns as mean, standard deviation, and range in parentheses). Mucous envelope attached to the posterior end of most fresh spores (Fig. 4). Under observation, mucous envelope enlarged and surrounded the spore (Fig. 5), then disappeared after several minutes. Deformed spores with caudal appendages infrequently observed (Fig. 6). Pyriform unequal polar capsules. Larger polar capsule 4.9±0.4 (4.0–5.9) long by 3.4±0.3 (3.0–3.9) wide. Smaller capsule 2.8±0.4 (2.3–3.7) long by 1.9±0.3 (1.3–2.5) wide. Polar filaments coiled five to six turns in large polar capsule and four to five turns in the small polar capsule. Fresh spores with 4 to 5 distinct sutural edge markings. Single binucleate sporoplasm with round iodinophilous vacuole. Intercapsular appendix distinct, triangular, 0.8±1.2 μm long, and positioned at anterior of spore.
Description of spores Myxospores (Figs. 2 and 3) round or ellipsoidal in frontal view and lemon-shaped in lateral view, measuring 10.2±0.5 (10.0–12.8) long, 9.4±0.4 (8.8–10.0) wide, and 6.3±0.3
Fig. 1 Gill filament cut from the hemibranchia infected by a large M. musseliusae plasmodium (arrow). Fresh mount. Scale bar, 2 mm
Fig. 3 Line drawing of fresh spores of M. musseliusae. Scale bar, 5 μm
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Fig. 4 Photomicrograph of mucous envelope (arrow) adhering to the posterior end of fresh spores. Scale bar, 10 μm
Fig. 6 Photomicrograph of a M. musseliusae spore with caudal appendages (arrow). Scale bar, 10 μm
Taxonomic summary
neighboring plasmodia fused together to form a larger plasmodium (Fig. 11).
Host: Common carp C. carpio L. Locality: Honghu Lake, Honghu City, Hubei Province, China Site of infection: Gill filaments. Date of sampling: March to April 2011. Host size: 31 to 34 cm in length and 453 to 559 g in weight Prevalence: 19 of 90 specimens examined were infected (21 %). Specimens deposited: Spores on a microscope slide stained with Giemsa deposited in Laboratory of Fish Diseases, College of Fisheries, Huazhong Agricultural University, accession no. MTR20110520. Histology Heart or cylinder-shaped plasmodia developed extravascularly in the gill filaments in close contact with the cartilaginous gill ray of the filament and contained late plasmodial developmental stages and some spores (Fig. 7). In some infected filaments, several (two to six) plasmodia (Figs. 8, 10, and 11) developed together, which were segregated by their ectoplasm (Fig. 9). Frequently observed were small
Fig. 5 Photomicrograph of mucous membrane (arrow) surrounding the spore. Scale bar, 10 μm
Sequence analysis A total of 1,389 bases of ssrRNA gene were generated, not inclusive of primers MX5 and MX3, and the contiguous sequence was deposited in GenBank (JQ040301). A BLAST search indicated that this sequence was identical (100 % similarity) to an existing GenBank entry for M. musseliusae from the gill of common carp (FJ710801) and shared 90 % similarity with that of M. dispar (AF507972). Phylogenetic analysis corroborates the sister relationship of this sequence to M. musseliusae and a weakly supported sister relationship to Myxobolus algonquinensis (Fig. 12). There was no support for a sister relationship of these three sequences grouping to other gill infecting species or any other myxobolids. M. dispar is more distantly related to M. musseliusae, forming a poorly supported group with Myxobolus cyprinicola.
Discussion M. dispar was first described by Thélohan (1895), from the gill of common carp in France. This parasite is one of the
Fig. 7 Heart-shaped plasmodium (p) of M. musseliusae developing close to the cartilaginous gill ray (arrows) of the gill filament. Scale bar, 150 μm
All measurements are in micrometers, with mean ± standard deviation (if available) and range in parentheses
7 – 5 –
Cyprinus carpio Cyprinus carpio
(9.0–14) (7.5–10) (6–7) (5–8) (3–4.5) (2.5–4.5) (2–2.7) Small (10–12) 8
Cyprinus carpio Cyprinus carpio
13.3 (13.0–13.6) 9.0 (9.7–10.5) 6.5 6.8 (6.5–8.0) 3.9 (3.8–4.0) 5.4 (5.2–6.1) 2.6 (2.5–2.6) 1.2 (1.0–1.3) 10.2±0.5 (10.0–12.8) 9.4±0.4 (8.8–10.0) 6.3±0.3 (5.9–6.7) 4.9±0.4 (4.0–5.9) 3.4±0.3 (3.0–3.9) 2.8±0.4 (2.3–3.7) 1.9±0.3 (1.3–2.5) Small Spore length Spore width Spore thickness Large polar capsule length Large polar capsule width Small polar capsule length Small polar capsule width Intercapsular process
Cyprinus carpio Cyprinus carpio Host
(10.5–11.1) (8.8–10) 7.2 (3.9–4) – (1.7–2.2) – Small, but visible
Myxobolus dispar Donec and Shulman (1984) Myxobolus dispar Present study (Hungary) Myxobolus musseliusae Yakovchuk (1979) Myxobolus musseliusae Present study (China) Parasite Source
Fig. 9 Higher magnification of the box in Fig. 8 showing two plasmodia segregated by their ectoplasm. Scale bar, 30 μm
Table 1 Comparison of M. musseliusae with M. dispar in morphology
most commonly occurring myxosporeans of common carp, causing economic losses in fish farms (Ivasik et al. 1967; Molnár and Szakolczai 1980). In China, parasitologists isolated a similar species from the gill of common carp and identified it as M. dispar according to the spore morphology, host, and organ specificity (Chen and Ma 1998). Here we collected specimens of what could be attributable to M. dispar according to Chen and Ma (1998). Yet, the morphological data of M. dispar in the present study and that from Chen and Ma (1998) are different from a fresh sample of M. dispar collected from the common carp in Hungary (Table 1). In addition, a mucous envelope adhering to the posterior end of most fresh spores was observed in present study (Fig. 4), and it could become larger and surround the spore on slide (Fig. 5). Although the taxonomic utility of the mucous envelope is ambiguous (Salim and Desser 2000), this structure has never been reported from fresh spores of M. dispar. The size differences between our contemporary sample and that of Thélohan (1895) may be due to fixation which is known to decrease spore size (Parker and Warner 1970) although it is not known with certainty whether Thelohan’s samples were fixed. Regardless, it was reasonable for Chen and Ma (1998) to identify their species as M. dispar, but with the novel data presented here, this is likely
Myxobolus dispar Thélohan (1895)
Fig. 8 Two plasmodia (p) of M. musseliusae developing close contact with each other in the gill filament. Scale bar, 200 μm
10.0 (8.4–10.8) 8.9 (8.2–10.2) 6.0 (5.8–6.2) 5.1 (4.6–5.4) 3.5 (3.0–3.6) 2.9 (2.4–3.6) 1.8 (1.4–2.2)
293 Myxobolus dispar Chen and Ma (1998)
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plasmodia in roach, however, plasmodia of M. sommervillae developed in the filamental arteries while plasmodia of M. rutili located in the multilayered epithelium with close contact to the cartilage. Similarly, Ferguson et al. (2008) reported both Myxobolus insidiosus and Myxobolus fryeri from the muscle of coho salmon Oncorhynchus kisutch, but the former was intramuscular and the latter in the peripheral nerves of the muscle. Thus, exact site of infection is important, and as such, Fig. 10 Four plasmodia (p) of M. musseliusae developing close to the cartilaginous gill ray (arrows) of the gill filament. Scale bar, 200 μm
Myxobolus musculi AF380141 Myxobolus ampullicapsulatus DQ339482
an incorrect placement. Instead, our morphological data bear striking consistencies with that of M. musseliusae (Table 1). Myxobolus musseliusae was described by Yakovchuk (1979) from C. carpio farmed in the Krasnodar territory adjacent to the Black Sea. Both the host species and spore morphology that are consistent with our species were consistent with M. musseliusae, and although we do not have type material to compare DNA sequences from, it is reasonable to conclude conspecificity based on these data. Tissue tropism was also taken into consideration. Histology showed that between 1 and 6 plasmodia were found outside the blood vessels in the multilayered epithelium but in close contact with the cartilaginous ray of the gill filament (Figs. 7, 8, 10, and 11) and smaller plasmodia appear to fuse (Fig. 11). This does not correspond to any of the exact sites of infection described by Molnár (2002), but instead M. musseliusae develops in the connective tissue deep in the filaments and as plasmodia grow, they expand and are found among the epithelial cells. This developmental pattern is more similar to Myxobolus rutili (Molnár et al. 2010). Large plasmodia are typically observed in M. dispar infections of common carp, except this species develops in the arteries and are located at the tip of the infected gill filaments (Molnár 2002; Dayoub et al. 2007). Myxobolus spp. infecting the same organ and having similar sized and shaped spores are found also in other fishes. Molnár et al. (2010) described that M. rutili and Myxobolus sommervillae formed large intrafilamental
Thelohanellus kitauei GQ396677
**
Thelohanellus hovorkai DQ231155
**
**
Thelohanellus nikolskii DQ231156 Thelohanellus nikolskii GU165832
Myxobolus dispar AF507972
-/-
**
Myxobolus cyprinicola DQ439805 Myxobolus pavlovskii AF507973 Myxobolus algonquinensis AF378335
73/-
Myxobolus musseliusae FJ710801
**
79/-
Myxobolus musseliusae JQ040301 -/-
Myxobolus muelleri AY325284
**
Myxobolus muelleri DQ439806 -/-
88/96
Myxobolus bramae AF507968
**
Henneguya cutanea AY676460 Myxobolus macrocapsularis FJ716095 **
90/75 90/*
-/-
Myxobolus macrocapsularis AF507969 Myxobolus susanlimae EU598805 Myxobolus feisti EU598804
64/* -/90
Myxobolus margitae EU598803 Myxobolus muellericus DQ439808
**
Myxobolus impressus AF507970 Myxobolus rotundus FJ851448
** **
**
Myxobolus parviformis AY836151 Myxobolus wootteni DQ231157
61/-
Myxobolus cycloides DQ439810
**
Myxobolus gayerae DQ439809 -/-
Myxobolus dogieli EU003977 **
Myxobolus leuciscini DQ439811 Myxobolus alburni EU567313 **
-/61
Myxobolus shaharomae EU567312
89/*
Myxobolus erythrophthalmi EU567311
0.1
Myxobolus ellipsoides DQ439813
** **
Fig. 11 Six plasmodia (p) developing together in the gill filament. A rupture between two plasmodia is the first sign of the future fusion of solitary plasmodia to form a single large plasmodium (black arrow). White arrow shows the sixth plasmodium. Scale bar, 200 μm
Myxobolus ellipsoides DQ439812
Fig. 12 Phylogenetic tree generated from Bayesian analysis of small subunit ribosomal RNA gene sequences of M. musseliusae and related Myxobolids. GenBank accession numbers are listed adjacent to species names. Support values in percent units at branching points are listed as: Bayesian posterior probabilities/bootstrap values from ML analysis. Asterisks are shown where values exceeded 95 %. Dashes are shown for values under 60 %
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the current species is unlikely to be M. dispar because of the distinct site of infection. Perhaps the most convincing evidence comes from analysis of the ribosomal DNA gene sequence. Phylogenetically, the current species is clearly distinct from M. dispar, instead forming a sister group with M. musseliusae (Fig. 12). Furthermore, a BLAST search revealed that the sequence of our myxosporean is identical with the sequences of M. musseliusae (FJ710801, 100 %), but just shared 90 % similarity with that of M. dispar (AF507972). As pointed out by a number of researchers (Eszterbauer et al. 2001; Molnár et al. 2002, 2009; Whipps et al. 2004; Adriano et al. 2009; Liu et al. 2010a), molecular biological methods have become important taxonomic tools for differentiating between morphologically similar myxosporean species, and the work here illustrates this point. Considering the relationship of M. musseliusae to other myxobolids, the work here is consistent with that of Zhang et al. (2010), placing this species at the base of a lineage of mostly gill infecting myxobolids, but showing no strong affinity to any other particular species or lineage. In conclusion, the data presented here allow us to confidently conclude the species isolated is indeed M. musseliusae and that the presence of M. dispar in China is unsubstantiated.
Acknowledgments The authors thank Gao Yu (College of Fisheries, Huazhong Agricultural University) for his help with the drawing of spores and Xiao Peng (Institute of Hydrobiology, Chinese Academy of Sciences) for providing the applied microscope. The authors are also thankful to Edit Eszterbauer (Veterinary Medical Research Institute, Hungarian Academy of Sciences) for providing helpful suggestions on this article. This study was supported by the Nature Science Foundation of China (project numbers 31172052 and 30800096), the Science Fund for Distinguished Young Scholars of Hubei Province (project number 2011CDA091), the New Teacher Foundation of Chinese Education Department (project number 200805041025), the Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (project number 2009SC017), the Fundamental research funds for the central universities (project numbers 2011PY011 and 2012PY004), and the Technology Project for strengthening innovation of Yunnan Province (project number 2009AD009). The Hungarian partner was financed by the grant OTKA, project no. K71837.
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