Vol. 141: 39–46, 2020 DISEASES OF AQUATIC ORGANISMS Published online September 17 https://doi.org/10.3354/dao03514 Dis Aquat Org

Morphological, histological and molecular characterization of Myxidium cf. rhodei infecting the kidney of Rutilus rutilus

Marina Dashi-Dorjievna Batueva1,#, Xiaoyi Pan2,#, Jinyong Zhang3,4, Xinhua Liu3, Wu Wei3, Yang Liu3,4,*

1Institute of General and Experimental Biology of the Siberian Branch of the Russian Academy of Sciences, 670047 Ulan-Ude, Russia 2Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs; Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, 313001 Huzhou, PR China 3Key Laboratory of Aquaculture Diseases Control, Ministry of Agriculture and Rural Affairs; State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 430072 Wuhan, PR China 4School of Marine Science and Engineering, Qingdao Agricultural University, 266109 Qingdao, PR China

ABSTRACT: In the present study, we provide supplementary data for Myxidium cf. rhodei Léger, 1905 based on morphological, histological and molecular characterization. M. cf. rhodei was ob - served in the kidneys of 918 out of 942 (97%) roach Rutilus rutilus (Linnaeus, 1758). Myxospores of M. cf. rhodei were fusiform with pointed ends, measuring 12.7 ± 0.1 SD (11.8−13.4) μm in length and 4.6 ± 0.1 (3.8−5.4) μm in width. Two similar pear-shaped polar capsules were positioned at either ends of the longitudinal axis of the myxospore: each of these capsules measured 4.0 ± 0.1 (3.1−4.7) μm in length and 2.8 ± 0.1 (2.0−4.0) μm in width. Polar filaments were coiled into 4 to 5 turns. Approximately 18−20 longitudinal straight ridges were observed on the myxospore surface. The suture line was straight and distinctive, running near the middle of the valves. Histologically, the plasmodia of the present species were found in the Bowman’s capsules, and rarely in the inter- stitium of the host. Phylogenetic analysis revealed that M. cf. rhodei was sister to M. anatidum in the Myxidium clade including most Myxidium species from freshwater hosts.

KEY WORDS: Myxidium cf. rhodei · Rutilus rutilus · · Kidney · SSU rDNA

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1. INTRODUCTION torical and technical reasons, most of the reported myxosporean species have been described solely Myxosporeans are a group of morphologically and by myxospore morphology (Eiras et al. 2011), which biologically diverse cnidarian endoparasites that pri- makes accurate identification and discrimination of marily infect both freshwater and marine fishes, with species challenging. Recently, biological traits (host over 2600 species described throughout the world specificity, organ specificity, and tissue tropism) and (Okamura et al. 2018). Of these, Myxidium Bütschli, molecular characterization have been suggested to 1882 represents a species-rich genus including over provide more accurate myxosporean species discrim- 230 nominal species recorded (Eiras et al. 2011, ination and identification (Atkinson et al. 2015). Un - Espinoza et al. 2017, Fariya et al. 2020). Most Myxid- fortunately, molecular data for most myxosporean ium species are typically coelozoic and generally species are lacking. Small subunit ribosomal DNA develop in the gallbladder, urinary bladder, or uri- (SSU rDNA) sequences are available for only ~30 of nary tubules of fish hosts (Eiras et al. 2011). For his- the Myxidium spp. reported to date.

*Corresponding author: [email protected] © Inter-Research 2020 · www.int-res.com # These authors contributed equally to this work 40 Dis Aquat Org 141: 39–46, 2020

Myxidium rhodei Léger, 1905 is an economically Donets & Shul’man (1973). Morphological and mor- important myxosporean parasite primarily infecting phometric analyses of myxospores were carried out the kidney of fish hosts. It was described for the first using Carl Zeiss Axio Lab.A1 (Carl Zeiss Microscopy) time from the kidney and ureter of Rhodeus amarus with Nikon-Elements BR software according to Lom (Bloch, 1782) in France (Léger 1930). Subsequently, & Arthur (1989). All measurements are given in micro - infections of M. rhodei were reported from more than meters (μm) as mean ± SD, followed by range in 40 freshwater fish species with a wide geographical parentheses. distribution (Jaysari & Hoffman 1982, Shul’man 1984, For scanning electron microscopy, myxospores Dyková et al. 1987, Alvarez-Pellitero 1989, Kepr 1991, were transferred to a poly-L-lysine-coated coverslip Athanassopoulou & Sommerville 1993a,b, Chen & Ma and allowed to stand for 15 min, then fixed in 2.5% 1998, Longshaw et al. 2005, Pazooki & Masoumian glutaraldehyde buffered in 0.1 M sodium cacodylate 2012). Although most of these infections were reported buffer (pH 7.4) at 4°C for 24 h, and then dehydrated from the kidneys of hosts, other organs including gills, in a series of increasing concentrations of ethyl al- muscle, liver, spleen, heart, gallbladder, intestine, cohol. Finally, coverslips were critical point dried swim bladder, gonads, urinary bladder, and ureter and broken on a stub before coating with gold. The have also been recorded as infection sites of M. rhodei fixed myxospores were observed and photographed (Kepr 1991, Athanassopoulou & Sommerville 1993b, using a Hitachi S-800 SEM with TM-1000 Ver.02-01 Chen & Ma 1998). Given the many detections of M. software. rhodei from various organs of more than 40 fish species that were identified by myxospore morphology alone, the reliability of M. rhodei species identification is 2.3. Histopathology questionable (Longshaw et al. 2005). Therefore, it is not clear whether M. rhodei is a widely distributed Infected kidney tissue of 50 fish collected during parasite infecting various organs of diverse fishes or 2016− 2018 was fixed in 4% formalin over 24 h, whether some infections represent other morphologi- gradient- dehydrated, embedded in paraffin, sec- cally similar but different myxosporean species. tioned at 5−6 μm, stained with hematoxylin and To facilitate the accurate species identification of eosin, and then examined and photographed with M. rhodei, here we provide supplementary morpho- a Carl Zeiss Axio Lab.A1 with Nikon-Elements BR logical, histological, and molecular characterization software. All measurements are given in μm. of M. cf. rhodei.

2.4. DNA extraction, amplification, and sequencing 2. MATERIALS AND METHODS Genomic DNA was extracted using the DNeasy 2.1. Fish collection Blood & Tissue Kit (Qiagen), following the manu- facturer’s recommended protocol for tissue. A total of 942 roach Rutilus rutilus individuals were PCR amplification of SSU rDNA sequence was per- caught by gill nets from Chivyrkui Bay (53° 46’ N, formed using the primer pair MyxospecF (Fiala 2006) 109° 02’ E) in Lake Baikal, Russia, during all 4 seasons and 18R (Whipps et al. 2003). PCR was carried out in from 2008 to 2018. All specimens were transported a 25 μl reaction mixture, containing 30 ng of ex- alive to the laboratory and euthanized by an overdose tracted genomic DNA, 1× PCR mixture (CWBiotech), of tricaine methanesulfonate (MS222, Sigma) buffered and 10 pmol of each primer. The PCR reaction was with bicarbonate. Comprehensive examination of the performed with initial denaturation for 4 min at 95°C, skin, fins, gills, muscle, intestine, liver, spleen, swim followed by 35 cycles of denaturation at 95°C for bladder, gallbladder, kidney, heart, gonads, urinary 1 min, annealing at 48°C for 1 min, extension at 72°C bladder, and ureter for myxosporean infections were for 2 min, and a final elongation step at 72°C for performed by gross examination and light microscopy. 10 min. The amplified PCR products were excised from an agarose gel, purified using a PCR purifica- tion kit (CWBiotech), and cloned into the PMD18-T 2.2. Morphological examination vector system (Takara). Positive clones were then selected and sequenced in both directions using the Fresh myxospores of Myxidium cf. rhodei were fixed amplification primers from the ABI BigDye Termi- in glycerol-gelatin as a slide preparation according to nator v 3.1 Cycle Sequencing Kit with an ABI 3100 Batueva et al.: Supplementary data for Myxidium cf. rhodei 41

Genetic Analyzer. The contiguous sequences were with 6 rate categories. BI analysis was conducted in assembled according to the corresponding chroma - ‘MrBayes’ v.3.2.6 (Ronquist et al. 2012), with 106 gen- tograms with the SeqMan™ utility of the ‘Lasergene’ erations, tree sampling every 100 generations, with a software package (DNAStar) and submitted to the burn-in of 2500 trees. ML analysis was performed National Center for Biotechnology Information nucleo - using ‘PhyML’ 3.0 (Guindon et al. 2010). Bootstrap tide database. confidence values were calculated with 1000 repli- cates. Trees were initially examined in ‘FigTree’ v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) and then 2.5. Phylogenetic analysis edited and annotated in Adobe Illustrator (Adobe Systems). To explore the phylogenetic relationship of the present species with the existing myxosporean spe- cies, 49 SSU rDNA sequences of myxosporeans were 3. RESULTS obtained from GenBank and aligned with ClustalX 1.8 (Thompson et al. 1997) using the default setting. 3.1. Morphological description Tetracapsuloides bryosalmonae (U70623) was chosen as the outgroup taxon. Phylogenetic trees were con- Plasmodia of Myxidium cf. rhodei were observed structed using maximum likelihood (ML) and Bayesian in the kidneys of 918 out of 942 (97%) Rutilus rutilus. (BI) analyses. The optimal evolutionary model for ML Myxospores of M. cf. rhodei were fusiform with and BI analyses was determined using ‘jModeltest’ pointed ends (Figs. 1 & 2), measuring 12.7 ± 0.1 3.7 (Posada 2008), which identified the optimal evo- (11.8−13.4) in length and 4.6 ± 0.1 (3.8−5.4) in width. lutionary model as the general time-reversible model Two equal pear-shaped polar capsules were posi- using Akaike’s information criterion. Nucleotide fre- tioned at either end of the longitudinal axis of the quencies were estimated from the data (A = 0.2606, myxospore, measuring 4.0 ± 0.1 (3.1−4.7) in length C = 0.1535, G = 0.2617, T = 0.3242), and the 6 rates and 2.8 ± 0.1 (2.0−4.0) in width. Polar filaments were of nucleotide substitution were AC = 1.3879, AG = coiled 4 to 5 turns. Sporoplasm was visible in the cen- 4.4878, AT = 1.9191, CG = 0.7712, CT = 5.6449, and ter of the myxospore. Approximately 18−20 longitu- GT = 1.0000; gamma distribution = 0.3581 estimated dinal straight ridges were observed on the myxo- spore surface (Fig. 2). The suture line was straight and distinctive, running near the middle of the valves. The myxospore measurements of M. cf. rhodei are summarized in Table 1, and comparisons of M. cf. rhodei with morphologically similar Myxidium spe- cies infecting the kidney of freshwater fish are shown in Table 2.

Fig. 2. Myxidium cf. rhodei myxospores in (A) lateral and (B) Fig. 1. Myxospores of Myxidium cf. rhodei in (A,B,D) frontal frontal view, observed by scanning electron microscope. and (C) lateral view. Scale bars = 10 μm Scale bars = 10 μm 42 Dis Aquat Org 141: 39–46, 2020

Table 1. Morphological comparison of Myxidium cf. rhodei in different records. Dimensions are mean (± SD, where available) and/or ranges (where available). IS: infection site; SL: spore length; SW: spore width; PCL: polar capsule length; PCW: polar capsule width; NR: number of ridges on the spore surface; NPF: number of polar filament turns; (−) data not available

Host(s) IS SL (μm) SW (μm) PCL (μm) PCW (μm) NR NPF Locality Reference

Rutilus rutilus Kidney 12.7 ± 0.1 4.6 ± 0.1 4.0 ± 0.1 2.8 ± 0.1 18−20 4−5 Russia Present study (11.8−13.4) (3.8−5.4) (3.1−4.7) (2.0−4.0) Rhodeus amarus Kidney, ureters 14−15 3.8−4 4.5 − − − France Léger (1930) R. rutilus Kidney 10−15 4.6−5.4 3.6−4.4 2.8−3.6 21−23 5 Czech Dyková et al. Republic (1987) R. rutilus Kidney 12.47 5.15 3.96 3.22 − − Czech Kepr (1987) (11.38−13.36) (4.46−5.94) (3.46−4.95) (2.48−3.96) Republic R. rutilus Muscle 12.78 4.21 4.25 3.05 − − Czech Kepr (1987) (11.88−13.86) (3.96−4.95) (3.46−4.95) (2.77−3.46) Republic Leuciscus cephalus, Kidney 12.1 ± 0.83 5.1 ± 0.55 3.44 ± 0.45 2.5 ± 0.31 − 4−6 Spain Alvarez-Pellitero Chondrostoma polylepis (10.5−15) (4−6) (2.8−4.5) (2−3.5) (1989) R. rutilus Kidney, 12.1 4.6 3.9 3.0 − − Czech Kepr (1991) muscle, liver Republic R. rutilus Kidney 10.03 ± 0.91 4 ± 0.83 3.66 ± 0.5 3.45 ± 0.52 21−33 4−5 Greece Athanassopoulou (9−12) (3−5) (3−4) (3−4) & Sommerville (1993a) R. rutilus Kidney 9.65 ± 1.05 3.6 ± 0.66 3.37 ± 0.52 3.77 ± 0.45 21−33 4−5 England Athanassopoulou (9−13) (3−5) (3−4) (3−4) & Sommerville (1993a) Carassius auratus, Gallbladder, 15.61 4.73 4.41 3.43 12 6 China Ma et al. (1998) Leptobotia elongata kidney, gonads (13.94−17.43) (3.49−5.81) (3.49−5.24) (2.91−3.49) Abramis brama, Kidney 11.82 ± 0.48 4.25 ± 0.30 3.36 ± 0.29 2.37 ± 0.27 20−30 4−5 England Longshaw et al. L. cephalus, L. leuciscus, (10.94−12.82) (3.69−4.68) (2.80−3.83) (1.79−3.07) (2005) Phoxinus phoxinus, R. rutilus

Table 2. Comparison of Myxidium cf. rhodei with morphologically similar Myxidium species infecting the kidney of freshwater fish. Dimensions are mean (±SD for M. rhodei), with ranges in parentheses. SS: spore shape; SL: spore length; SW: spore width; PCL: polar capsule length; PCW: polar capsule width

Species Host(s) SS SL (μm) SW (μm) PCL (μm) PCW (μm) Locality Reference

M. cf. rhodei Léger, Rutilus rutilus Fusiform 12.7 ± 0.1 4.6 ± 0.1 4.0 ± 0.1 2.8 ± 0.1 Russia This study 1930 (11.8−13.4) (3.8−5.4) (3.1−4.7) (2.0−4.0) M. cirrhinae Chen Cirrhinus Ellipsoidal 13.9 5.8 3.7 3.6 China Chen & Ma & Hsieh, 1984 molitorella (12.2−15.3) (5.3−6.8) (3.4−3.9) (3.1−4.1) (1998) M. mendehi Fomena Barbus guirali, Fusiform 9.9 4.1 3.4 2.3 Cameroon Eiras et al. & Bouix, 1994 B. martorelli (7.8−13.2) (3.1−4.9) (2.7−4.5) (1.8−3.1) (2011) M. pseudobagrusi Pelteobagrus Ellipsoidal 12.9 5.7 3.7 3.2 China Chen & Ma Chen & Hsieh, 1984 fulvidraco (11.4−14.4) (5.4−6.4) (3.6−3.8) (3.0−3.4) (1998) M. tictoi Fariya, Puntius ticto Fusiform 13.10 4.67 3.25 2.43 India Fariya et al. Kaur & Abidi, 2020 (11.53−14.21) (4.13−5.37) (2.48−3.91) (1.94−2.87) (2020)

3.2. Histopathology plasmodia of M. cf. rhodei surrounded by the con- nective tissue further sporulated and completely Histological analysis showed that the plasmodia replaced the glomeruli (Fig. 3B). A granulomatous of M. cf. rhodei were found in the Bowman’s cap- inflammatory reaction characterized by the pres- sules (Fig. 3A,B,D) and rarely in the interstitium ence of a wide zone of epithelioid cells was evident of the host (Fig. 3C). At the beginning of summer, around the plasmodia of M. cf. rhodei in the inter- the plasmodia of M. cf. rhodei compressed the stitium (Fig. 3C). In the late winter and early glomeruli in the distended Bowman’s capsules spring, the plasmodia of M. cf. rhodei were filled (Fig. 3A). In late summer and early autumn, the with mature myxospores (Fig. 3D). Batueva et al.: Supplementary data for Myxidium cf. rhodei 43

Fig. 3. Histology of the kidney of Rutilus rutilus infected by Myxidium cf. rhodei. (A) At the beginning of summer, the plas- modia (arrow) of M. cf. rhodei compress the glomeruli (asterisks) in the Bowman’s capsules. Scale bar = 50 μm. (B) In late sum- mer and early autumn, the plasmodium of M. cf. rhodei is surrounded by connective tissue (black arrow) in the Bowman’s cap- sule (white arrow). Scale bar = 50 μm. (C) Plasmodium of M. cf. rhodei (black arrow) surrounded by epithelioid cells (white arrow) in the interstitium. Scale bar = 20 μm. (D) In late winter and early spring, the plasmodium of M. cf. rhodei (arrow) is filled with mature myxospores. Scale bar = 20 μm

3.3. Molecular analysis (EF602629, 89.11%). ML and BI analyses produced similar tree topologies in several cases with different Two partial SSU rDNA sequences of M. cf. rhodei bootstrap values. Phylogenetic analysis revealed M. of 1656 bp (MK102097) and 1718 bp (MK102096) in cf. rhodei was sister to M. anatidum in the Myxidium length were generated and shared 99.3% sequence clade including most Myxidium species from fresh- identity between them. A megaBLAST search indi- water hosts (Fig. 4). cated that the sequences of M. cf. rhodei did not match any sequence in the GenBank database. The most similar SSU rDNA sequences were M. hardella 4. DISCUSSION Garner, Bartholomew, Whipps, Nordhausen & Raiti, 2005 (AY688957, 89.81%), M. peruviensis Espi- A Myxidium species was collected from the kidney noza, Mertins, Gama, Fernandes & Mathews, 2017 of Rutilus rutilus and was described by morphologi- (KY996746, 89.25%), M. amazonense Mathews, Silva, cal, histological, and molecular characterization. Maia & Adriano, 2015 (KT625442, 89.18%), and M. After morphological comparison with other recorded anatidum Bartholomew, Atkinson, Hallett, Lowens- Myxidium spp., the present species most resembled tine, Garner, Gardiner, Rideout, Keel & Brown, 2008 M. rhodei. Although some differences were found in 44 Dis Aquat Org 141: 39–46, 2020

Fig. 4. Phylogenetic tree generated by Bayesian analysis of aligned partial SSU rDNA sequences of Myxidium cf. rhodei (in bold) and related species. GenBank accession numbers are listed adjacent to species names. Support values at branching points are listed as Bayesian posterior probabilities/bootstrap values from maximum likelihood analysis. Dashes indicate values <50%. FW: freshwater hosts; M: marine hosts; T: terrestrial mammal. The scale bar indicates 5 substitutions per 10 nucleotide positions Batueva et al.: Supplementary data for Myxidium cf. rhodei 45

some dimensions, the ranges of the dimensions over- water hosts. As several Myxidium species clustered lapped (Table 1), which was more suggestive of intra- with Zschokkella and Sphaeromyxa species, the than interspecific variation. Intraspecific variation in polyphyly of the genus Myxidium and other genera myxospore dimensions of M. rhodei from different of the family Myxidiidae was evident, which has been geographic locations and different organs of the observed in previous reports (Gunter & Adlard 2008, same fish was also observed (Table 1). Intraspecific Hartigan et al. 2011, Espinoza et al. 2017, Woodyard morphometric variation has been reported in several et al. 2020). This suggests that the taxonomy of the myxosporeans (Zhai et al. 2016, Stilwell et al. 2019). family Myxidiidae based on myxospore morphology Scanning electron microscopy revealed 18−20 longi- is problematic, and that the genera in this family tudinal straight ridges on the myxospore surface warrant taxonomic revision. However, molecular of the present species, i.e. fewer than in M. rhodei data of most species in the family Myxidiidae are not (21−23) reported by Dyková et al. (1987) and in available, which makes resolving the taxonomic M. rhodei (21−33) reported by Athanassopoulou & ambiguities challenging. Accordingly, it is necessary Sommerville (1993a), but consistent with M. rhodei and urgent to supplement the molecular data for (18−20) reported by Alvarez-Pellitero (1989). The most species in the family Myxidiidae. number of ridges may vary in different samples. Although various organs of fish hosts had been Acknowledgements. The present work was financially described as infection sites of M. rhodei (Kepr 1991, supported by a project of the Russian Ministry of Science and Higher Education (No. AAAA-A17-117011810039-4) Athanassopoulou & Sommerville 1993b, Chen & Ma awarded to M.D.D.B.; the Open Foundation of Key Labora- 1998), M. rhodei was mainly isolated from the kidney tory of Healthy Freshwater Aquaculture, Ministry of Agri- (Dyková et al. 1987, Alvarez-Pellitero 1989, Long- culture and Rural Affairs (2016ZJK02, ZJK201504); and the shaw et al. 2005, Pazooki & Masoumian 2012). Histo- Open Foundation of Key Laboratory of Aquaculture Dis- eases Control, Ministry of Agriculture and Rural Affairs logically, the plasmodia of the present Myxidium awarded to Y.L.; the young experts of Taishan Scholars in species were mainly found in kidney in this study, Shandong province and Initiative grant for high-level per- consistent with the original description of Rhodeus sonnel recruitment in Qingdao Agricultural University amarus (Dyková et al. 1987, Alvarez-Pellitero 1989, awarded to J.Z.; the project of ‘First class fishery discipline’ program in Shandong Province, China; and a special top Dzika et al. 2006, Batueva et al. 2015). In addition talent plan ‘One Thing One Decision (Yishi Yiyi)’ program in to M. rhodei, the present species is morphologically Shandong Province. similar to several other Myxidium species that infect the kidney of freshwater fish (Table 2). However, LITERATURE CITED given the similar myxospore morphology, identical in fection site, and repeated descriptions from R. Alvarez-Pellitero P (1989) Myxidium rhodei (Protozoa: Myxo- rutilus, the present species is attributed to M. rhodei. zoa: Myxosporea) in cyprinid fish from NW Spain. Dis Aquat Org 7: 13−16 Owing to the absence of molecular data in previous Athanassopoulou F, Sommerville C (1993a) A comparative reports of M. rhodei and other morphologically simi- study of the myxosporeans Myxidium rhodei Léger, 1905 lar Myxidium spp., it is difficult to verify their relat- and Myxidium pfeifferi Auerbach, 1908 in roach, Rutilus edness solely based on classical, morphology-based rutilus L. J Fish Dis 16: 27−38 Athanassopoulou F, Sommerville C (1993b) The significance methods. In the present study, we supplemented the of myxosporean infections in roach, Rutilus rutilus L., in first SSU rDNA sequence of M. rhodei infecting the different habitats. 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Editorial responsibility: Dieter Steinhagen, Submitted: March 14, 2020; Accepted: July 15, 2020 Hannover, Germany Proofs received from author(s): September 14, 2020