Vol. 142: 75–82, 2020 DISEASES OF AQUATIC ORGANISMS Published online November 19 https://doi.org/10.3354/dao03534 Dis Aquat Org

Morphological, histological, and molecular aspects of Myxobolus zaikae n. sp., a parasite of the roach rutilus, in Lake Baikal

Marina Dashi-Dorjievna Batueva*

Institute of General and Experimental Biology of the Siberian Branch, Russian Academy of Sciences, 670047 Ulan-Ude, Russia

ABSTRACT: A new myxobolid , Myxobolus zaikae n. sp., was found in the connective tis- sue near the kidney and liver blood vessels of the common roach Rutilus rutilus, while fish myxo - sporean fauna were being investigated in Lake Baikal, Russia. The parasites were studied on the basis of spore morphology as well as with histological and molecular methods. Mature spores of M. zaikae n. sp. are round or ellipsoidal in the frontal view and lemon-shaped in the lateral view, measuring 11.37 ± 0.11 µm (10.2−14.0 µm) in length, 10.29 ± 0.10 µm (9.6−11.0 µm) in width, and 6.3 ± 0.08 µm (5.8−7.1 µm) in thickness (mean ± SD; n = 50). Polar capsules are equal and pyriform, measuring 4.5 ± 0.07 µm (3.4−5.2 µm) in length and 2.9 ± 0.03 µm (2.6−3.3 µm) in width. Polar cap- sules contained polar filaments coiled with 5 to 6 turns. Phylogenetic analysis showed that this newly described species clusters with other myxobolid species infecting the connective tissue of different organs from Palearctic cyprinid fish.

KEY WORDS: Myxobolus zaikae n. sp. · Roach · Kidney · Liver · Lake Baikal

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1. INTRODUCTION A long-term survey of fish parasites in Lake Baikal found a parasite in the kidneys and liver of Myxosporeans (Cnidaria) are metazoan parasites common roaches and identified it as M. muelleri that have complex life cycles in vertebrate (mainly (Dugarov et al. 2011) according to the methodology fish, less often in amphibians, reptiles, birds, and in Donec & Schulman (1984). However, Donec & mammals) and invertebrate (aquatic annelid worms Schulman (1984) found rather a wide range of hosts, and bryozoans) hosts (Lom & Dyková 2006, Hallett et and the description of the site of infection by al. 2015). A total of 2425 myxosporean species have myxosporean parasites was inaccurate. The pres- been described, and at least 900 species belong to the ence of a cryptic species of Myxobolus spp. has myxobolids; a significant amount of them have been since been revealed, which has a similar spore described from fish hosts of the order shape, but differs in SSU rDNA se quences and tis- (Eiras et al. 2005, 2014, Liu et al. 2019). sue tropism (Molnár 1994, Eszterbauer 2002, 2004, The common roach Rutilus rutilus (L.) is one of the Molnár et al. 2002, 2010, Forró & Eszterbauer 2016). most common fish in the Palearctic region. Donec & The present study de scribes a new myxobolid spe- Schulman (1984) recorded more than 20 Myxobolus cies infecting the kidney and liver of roaches col- spp. found in common roach. These species require lected in Lake Baikal, Russia, and presents data on revision, as they were determined based on spore its spore morphology, tissue tropism, and molecular morphology only. characteristics.

*Corresponding author: [email protected] © Inter-Research 2020 · www.int-res.com 76 Dis Aquat Org 142: 75–82, 2020

2. MATERIALS AND METHODS coated with gold. The fixed spores were observed and photographed by a scanning electron micro- 2.1. Fish collection scope (S-800, Hitachi).

A total 236 specimens of Rutilus rutilus were col- lected in summer period of 1998−2018 in Chiv yr kuy 2.4. Molecular characterization Bay, Lake Baikal (53° 46’ N, 109° 02’ E). The fish were caught using gill nets and were measured, weighed, To determine the SSU rDNA sequences of the myxo - sexed, and aged. Plasmodia of Myx o bolus zaikae n. zoan samples, DNA was extracted from the spores sp. from the kidney and liver were studied by com- preserved in ethanol for DNA extraction with DNA- pressing pieces between 2 slides and magnified Extran-2 (Syntol) according the manufacturer’s rec- 10−25× under an MBS-15 dissecting microscope ommended protocol for tissue. The SSU rDNA (LOMO). was targeted for amplification using nested PCR assay with general myxozoan primer combination sets for partial SSU rDNA: 18e+18g (Hillis & Dixon 1991) in the 2.2. Histologic and morphometric study first run; Myx1f + Act1R, ACT1f + Myx4R, and Myx1F + Myx4R (Hallett & Diamant 2001) in the second run. Plasmodia were mechanically isolated from fish PCR products were combined with gel dye (0.25% tissue with preparation needles under a dissecting bromophenol blue, 30% glycerol) and electropho- microscope. Spores from the isolated and crushed resed on a 0.9% agarose gel (agarose, deionized pseudocysts were first studied in a wet mount, and water, 1× Tris-acetate buffer, and ethidium bromide) then some spores were fixed in glycerol−gelatin as a alongside a molecular weight standard (100 bp DNA slide preparation ac cording to Donec & Schulman ladder, Bioron) and UV visualized. PCR products (1973). Spores were identified and measured from 1 were purified using a PCR Product Purification Kit to 2 mounts for each of 10 hauls (20–25 specimens). (Biosilica) and directly sequenced with the primers Histological studies were made only on plasmodia which were used in the second run. Sequencing was developing in the kidney; in the liver we were not able made using a BigDye Terminator Cycle Sequencing to find vegetative forms of M. zaikae n. sp. in sections. Kit from Applied Biosystems (Thermo-Fisher Scien- Spore measurements were determined from 50 tific) and run on an ABI3700 DNA analyzer from spores as recommended by Lom & Arthur (1989); Applied Biosystems (Thermo-Fisher Scientific). The measurement was carried out from a single mount sequences were deposited in GenBank. using Nikon Elements BR software. All measure- ments are given in mean ± SD (µm), followed by a parenthesized range. 2.5. Phylogenetic analyses For histology, pieces of infected kidney and liver were fixed in 10% formalin and Bouin solution, The various forward and reverse sequence seg- embedded in paraffin, sectioned, stained with hema- ments were aligned in BioEdit (Hall 1999), and am - toxylin and eosin, stained with periodic acid-Schiff biguous bases were clarified using corresponding ABI reagent, and then examined and photographed. His- chromatograms. The consensus sequences were de- tological analysis of kidney tissues and spore mor- posited in GenBank with accession numbers MT - phology was performed with a light microscope 141124 and MT141128. The SSU rDNA se quences of (Axio Imager M.2, Carl Zeiss). myxosporeans were identified using BLAST. This dataset contained 37 ingroup taxa and outgroup spe- cies (Chlo ro myxum thymalli JX131381). Multiple 2.3. Scanning electron microscopy alignments were performed with Clustal X (Thomp- son et al. 1997) and manually edited applying BioEdit Spores from plasmodia were transferred to a poly- (Hall 1999). The alignment of the SSU rDNA sequence L-lysine-coated coverslip, incubated for 15 min at dataset included 565 characters after the removal of room temperature, fixed in 2.5% glutaraldehyde ambiguously aligned regions using trimAL (Capella- buffered in 0.1 M sodium cacodylate (pH 7.4) at 4°C Gutiérrez et al. 2009). Maximum likelihood (ML) for 24 h, and dehydrated in a series of ascending con- analysis was performed with MEGA 6 software centrations of ethanol. Finally, coverslips were criti- (Tamura et al. 2013) with the Tamura 3−parameter cal-point dried and broken on a stub before being (T92) +G+I model, and ML bootstrap support was cal- Batueva: Morphological, histological, molecular aspects of Myxobolus zaikae n. sp. 77

culated in 1000 replicates. Bayesian inference (BI) Prevalence: The prevalence of infection in the kid- analysis was carried out using Mr Bayes (Ronquist et neys was 20% (47/236) and in the liver 11% (26/236). al. 2012) under the best-fit model GTR+I+G, which Plasmodia of Myxobolus zaikae sp.n. were found in was selected by jModeltest 3.0 software (Posada 2008) both the liver and kidneys in 5% (12/236) of fish. using the Akaike information criterion. Posterior prob- Type material: Syntype spores in glycerine− gelatine, abilities were calculated over 5 000 000 generations via histological slides and a photo series were deposited 2 in dependent runs of 4 simultaneous Markov chain in the Parasitology Laboratory, Institute of General Monte Carlo chains with every 100th tree saved. and Experimental Biology Siberian Branch of the Trees were visualized in FigTree v.1.3.1 (https:// tree. Russian Academy of Science (accession numbers bio. ed. ac.uk/ software/ figtree/). A phylogenetic tree CHIV/MYX 2019.1-7 and CHIV/MYX 2001). SSU was generated from sequences of species of the rDNA sequences were de posited in GenBank (acces- Myxo bolus belonging to the ‘muelleri-like’ spores. sion numbers MT141128 and MT141124). Etymology: The species is named after Victor E. Zaika (1936−2014), a well-known Russian fish para- 3. RESULTS sitologist. Spores: Spores were ellipsoidal in the frontal view Myxobolus zaikae n. sp. and lemon-shaped in the sutural one (Figs. 1 & 2). Phylum Cnidaria Hatschek, 1888 Spore measurements are given in Table 1. Polar cap- Unranked Sub-phylum Myxozoa (Grassé,1970) sules were pyriform, equal in size, and slightly con- Class Myxosporea Bütschli, 1881 verging anteriorly. The polar filaments form 5 coils. Order Bivalvulida Shulman, 1959 The front ends of the polar capsules are located Family Myxobolidae Thélohan, 1892 at a distance of about 0.6−1.2 µm from each other. Genus Myxobolus Bütschli, 1882 The intercapsular appendix is clearly visible. A sutural protrusion formed a circular rim around the spore. This rim protruded above the surface in 3.1. Taxonomic summary the suture at the same distance over the entire cir- cumference. Its thickness measured ca. 0.8 µm in Type host: Rutilus rutilus (L.) () the sutural view. Seven sutural ridge markings of Locality: Chivirkuy Bay, Lake Baikal, Russia (53° 38’ N, the rim are clearly visible on the spores (Figs. 1B 109° 01’ E) & 2). A mucous envelope was not found (Fig. 1A). Site of infection: The plasmodia were located in the The iodophilic vacuole was poorly visible, located on interstitium near the blood vessels, sometimes the side (Fig. 2). attached to the walls of the blood vessels. Remarks: M. zaikae n. sp. partially resembles the Category of plasmodia: Round or oval plasmodia Myxobolus species listed in Table 1. Spores of M. from 50 to 300 µm in size. zaikae n. sp. are similar to M. erythrophthalmi in

Fig. 1. Myxobolus zaikae n. sp. (A) Fresh spores. (B) Scanning electron micrographs of a spore. Arrow identifies the sutural ridge of the rim. Scale bars = 10 µm 78 Dis Aquat Org 142: 75–82, 2020 Present 7−0.9 Molnár et 7−0.8 Molnár et − Molnár et 0.7 Molnár et − Borzák et − Molnár et − Molnár et − Molnár et al. (2009) − − Molnár spore wall; CESW: caudal extension spore wall; CESW:

Fig. 2. Schematic drawing of a spore of Myxobolus zaikae n. sp. in (A) frontal view and (B) sutural view. Scale bar = 10 µm terms of spore length and width, but there are differ- ences in the length of the polar capsules and in the number of coil turns (Table 1). Both species have a sutural protrusion forming a circular rim around the spore. However, M. zaikae n. sp. has a sutural rim with the same length along the entire circumference

(Fig. 3), while M. erythrophthalmi has a sutural rim in (5.6−6.0) (0.8−1.0) al. (2016) (7.0−7.6) al. (2009) (5.8−7.1) (0.6−1.2) (0.7−1.5) (0.7−1.5) study ST TSR NCF AESW CESW Ref. (2000) (5.5−6) al. (2006) (5.0−6.0) al. (2007) (5.7−6.7) (1.1−1.3) al. (2008) (6.0−9.0) al. (2010) the anterior extension similar in length to that of M. ) (5.0−5.5) al. (2006) zaikae n. sp., and in the caudal extension it is slightly reduced (Molnár et al. 2009). The remaining species of Myxobolus, which belong to the same phyloge- netic cluster (Fig. 4), have a larger spore size, with the exceptions of M. leuciscini and M. muelleri. sporen. sp. SL: spore width; LPC: length of polar capsule; WPC: width length; SW:

3.2. Histology M. zaikae

Examination of squashed kidney tissue showed that plasmodia with mature spores were localized in the connective tissue near large blood vessels, form- ing groups at some distance from each other (Fig. 3A). We observed elongated plasmodia up to 300 µm

(Fig. 3B). The plasmodia in histological sections were 13.7 11.0 5.3 3.3 8.0 − 4 13 ± 0.89 10.4 ± 0.80 6.1 ± 0.31 3.8 ± 0.18 7.3 ± 0.25 0.7 5 0.9 9.83 ± 0.24 7.5 ± 0.24 4.6 ± 0.51 3.6 ± 0.5 5.2 ± 0.24 − 5 to 6 − 11.0 ± 0.52 9.5 ± 0.44 5.7 ± 0.46 3.4 ± 0.26 7.0 ± 0.48 0.7 5 1.0−1.2 0. 12.1 ± 0.55 11.4 ± 1 5.5 ± 0.28 3.2 ± 0.21 5.8 ± 0.20 0.9 ± 0.1 5 − 10.1 ± 0.75 9.0 ± 0.24 4.2 ± 0.26 2.9 ± 0.29 5.3 ± 0.49 0.5 6 0.7−0.8 0. 11.72 ± 0.9 9.7−0.51 4.77−0.26 2.6 ± 0.1 5.8 ± 0.31 − 6 − 14.0 ± 0.41 12.7 ± 0.57 4.13 ± 0.22 2.2 ± 0.26 6.2 ± 0.72 1.2 ± 0.11 5 − 13.1 ± 1.26 9.9 ± 0.77 5.9 ± 0.9 3.4 ± 0.62 6.8 ± 1.17 − 6 − apparent as conglomerates, consisting of grouped 11.37 ± 0.11 10.29 ± 0.1 4.5 ± 0.07 2.9 ± 0.03 6.3 ± 0.08 0.86 ± 0.06 6 1.05 ± 0.02 1.05 ± 0.02

mature spores, and plasmodia boundaries were not (10.4−12.0) (8.3−10.2) (5.0−6.3) (3.1−3.7) (6.3−7.5) distinct, without ectoplasm. There was no connective spp. morphologically similar to tissue capsule around the plasmodium. The host im- mune response was expressed as melanomacro - phages and mast cell infiltrations (Fig. 3C). of spore wall. All measurements are in µm with mean ± SD (if available) and range parentheses. –: data not available Myxobolus Rutilus rutilus erythrophthalmus 3.3. Molecular characterizations and

phylogenetic analysis n. sp.

The DNA sequences of M. zaikae n. sp. samples

MT141128 (1292 nt) from kidney and MT141124 thalmi (11.6−13.2) (10.8−12.5) (5.2−6.0) (2.8−3.4) (13.0−14.0) (9.5−12.0) (5.0−5.6) (3.0−3.5) (9.0−11.0) (8.5−9.5) (4.0−4.5) (2.5−3.5) (10.5−13.5) (9.0−11.0) (4.5−5.0) (2.5−2.7) (9.5−10.0) (7.5−8) (4.0−5.0) (3.0−4.0 (12.1−14.5) (9.0−11.3) (5.6−7.0) (3.5−4.1) (13.2−14.4) (12−13.5) (4−4.45) (2−2.5) (10.2−14.0) (9.6−11.0) (3.4−5.2) (2.6−3.3) (11−15.7) (8−10.8) (4.0−7.2) (3.0−3.7) M. alburni Alburnus alburnus M. muelleri Leuciscus cephalus M. erythroph- Scardinius M. peleci Pelecus cultratus M. leuciscini Leuciscus cephalus M. ellipsoides Leuciscus cephalus M. dogieli Abramis brama M. shaharomae Alburnus alburnus M. rutili Rutilus rutilus Species Host SL SW LPC WPC M. zaikae capsule; ST: spore thickness; TSR: thickness of sutural rim; NCF: no. of coils of polar filaments; AESW: anterior extension of spore thickness; TSR: thickness of sutural rim; NCF: no. coils polar filaments; AESW: capsule; ST: (824 nt) from liver demonstrated 99.8% similarity. 1.Table Comparison of Batueva: Morphological, histological, molecular aspects of Myxobolus zaikae n. sp. 79

Fig. 3. Sections of the kidney of Rutilus rutilus with Myxobo- lus zaikae n. sp. (A) Several plasmodia (black arrows) adja- cent to one blood vessel (blue arrow). Fresh preparation; scale bar = 1 mm. (B) Large plasmodium located in renal in- terstitium, near the blood vessel (P: plasmodium; BV: blood vessels). H&M; scale bar = 100 µm. (C) Plasmodium infil- trated by mast cells (arrows). Periodic acid-Schiff reagent stain; scale bar = 10 µm

BLAST search revealed that these SSU rDNA se- with a posterior probability of 0.77 and bootstrap quences were unique among all myxozoans, and the value of 51%. highest similarities of sequences MT141124 and MT141128 were 93.7 and 95.4% with M. peleci KU170934, 93.5 and 95.4% with M. shaharomae 4. DISCUSSION KF515726, and 93.3 and 95.4% with Myxobolus ery- throphthalmi KF 515727, respectively. The sample Myxobolus Bütschli, 1882 is the largest genus in MT141124 showed 93.9% similarity with M. ellip- the phylum Myxozoa (Eiras et al. 2005, 2014). The soides ex Leuciscus cephalus DQ439813. The topolo- number of species in this genus is continuing to in- gies of phylogenetic trees inferred from BI and ML crease globally (Carriero et al. 2013, Cech et al. 2015, were the same, and thus both were incorporated into Székely et al. 2015, Liu et al. 2016a, 2019, Rosser et one tree based on the ML tree. al. 2017, Mathews et al. 2020, Milanin et al. 2020). We recovered 2 main clades: Clade A, with poste- The present study is the first record for a new species rior probabilities in the BI (1.00) and bootstrap values of Myxobolus infecting the common roach. M. zaikae in the ML analysis (51%), and Clade B, with 0.99 and n. sp. differs in spore morphology from the known 87%, respectively. M. intimus and muscle-infecting Myxobolus species, and the SSU rDNA sequences M. pseudodispar and M. musculi were distant from available in the nucleotide sequence database are other species (Fig. 4). Clade A consists of 23 species, also different. which is divided into 2 subclades. M. zaikae n. sp. is Donec & Schulman (1984) recorded more than 20 located in a subclade of myxobolid species, localized Myxo bolus spp. from common roach, and Eiras et al. in the connective tissue and in the blood vessels of (2005, 2014) listed 15 species described from the the gills, liver, kidneys, and other internal organs roach as a type host in their synopsis on the genus 80 Dis Aquat Org 142: 75–82, 2020

Fig. 4. Phylogenetic tree generated by maximum likelihood (ML) analysis inferred from SSU rDNA sequences of Myxobolus zaikae n. sp. and related species. Numbers near branches indicate posterior probability (Bayesian inference; BI) and bootstrap values (ML). Chloromyxum thymalli JX131381 was used as the outgroup

Myxobolus. These species were mainly determined tion and molecular data are presented by Molnár et on the basis of spore morphology. al. (2006). The spores of M. muelleri ex L. cephalus are Previously, researchers defined M. zaikae n. sp. less than those of M. zaikae n. sp. in length and width as M. muelleri (Dugarov et al. 2011). M. muelleri (Table 1). in the kidneys and liver of roaches was also men- Currently, 8 gill-infecting species, one species from tioned by Kepr (1991); however, the measurements fins and several lineages of a M. pseudodispar-intra- of the spores were not reported. According to Donec muscular parasite, infecting roach are present in the & Schulman (1984), spores having a regular ellip- SSU rDNA sequence data (Molnár et al. 2002, 2010, soid or spherical shape with narrow sutural rims Liu et al. 2016b, Fórro & Eszterbauer 2016, Lisnerová with radial folds and pyriform polar capsules with et al. 2020). There is no molecular or detailed mor- converging distal ends that are equal to or less phological data on Myxobolus spp. found in the kid- than half the length of the spores are assigned to ney and liver of the roach. For cyprinids, only 2 species, M. muelleri. The spores had large ranges (length M. shaharomae from the Alburnus 8−14.5 µm; width 7−11 µm). M. muelleri was found alburnus and M. erythrophthalmi from the common by Donec & Schulman (1984) in 56 species of fish in rudd Scardinius erythrophthalmus (Molnár et al. the gills, subcutaneous connective tissue, fins, intes- 2009), are marked in the kidney and the liver intersti- tinal wall, gall and bladder, liver, kidneys, gonads, tial tissue. spleen, and eyes. However, using molecular biolog- Young plasmodia of M. shaharomae and M. ery- ical techniques, several ‘muelleri-like’ species hav- throphthalmi develop in blood vessels, and mature ing tissue and host specificity were found later plasmodia are localized in the interstitium. M. zaikae (Molnár 2000, Molnár et al. 2006, 2007, 2008, 2009, n. sp. as well as M. rutili, M. peleci, and M. elegans 2010, Borzák et al. 2016). The species having the are localized in the connective tissue near the blood closest morphological and genetic similarity are vessel walls (Molnár et al. 2012, 2018, Borzák et al. listed in Table 1. 2016). The structure of M. zaikae n. sp. plasmodium A type host of M. muelleri Bütschli, 1882 is Leucis- is similar to that described for M. peleci (Borzák et al. cus cephalus, and a detailed morphological descrip- 2016). The spore conglomerates, called pseudocysts Batueva: Morphological, histological, molecular aspects of Myxobolus zaikae n. sp. 81

in M. peleci according to Borzák et al. (2016), are ton, Hungary. Syst Parasitol 93: 667−677 characteristic of ageing plasmodia, and some plas- Borzák R, Molnár K, Cech G, Székely C (2018) Myxobolus infection in the cornea of the roach (Rutilus rutilus) in modia are destroyed and spores are visible in the Lake Balaton. Acta Vet Hung 66:250−257 surrounding tissue and blood. We did not find dam- Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009). aged plasmodia caused by M. zaikae n. sp., unlike trimAl: a tool for automated alignment trimming in large- Borzák et al. (2016), who reported this for M. peleci scale phylogenetic analyses. Bioinformatics 25:1972−1973 Carriero MM, Adriano EA, Silva MRM, Ceccarelli PA, Maia in connective tissue. The absence of a connective tis- AAM (2013) Molecular phylogeny of the Myxobolus and sue capsule causes infiltration of plasmodia by Henneguya genera with several new South American melanomacro phages and mast cells, but their num- species. 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Editorial responsibility: Dieter Steinhagen, Submitted: May 7, 2020; Accepted: September 10, 2020 Hannover, Germany Proofs received from author(s): November 17, 2020