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Article

Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan

by
Mariko Sekiya
1,
Haruya Sakai
1,
Ying-Chun Li
2,†,
Imron Rosyadi
2,‡,
Muchammad Yunus
3 and
Hiroshi Sato
1,2,3,*
1
Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan
2
Joint Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan
3
Faculty of Veterinary Medicine, Airlangga University, Mulyorejo, Surabaya 60115, Indonesia
*
Author to whom correspondence should be addressed.
†
Present address: Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang 524091, China.
‡
Present address: Faculty of Veterinary Medicine, Gadjah Mada University, Sleman, Yogyakarta 55281, Indonesia.
Life 2024, 14(8), 974; https://doi.org/10.3390/life14080974
Submission received: 3 June 2024 / Revised: 27 July 2024 / Accepted: 29 July 2024 / Published: 2 August 2024
(This article belongs to the Special Issue Diagnosis and Management of Microbial Infections)
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Versions Notes

Abstract

:
The majority of myxosporean species (Cnidaria: Myxozoa) of the genera Myxobolus (35 species), Henneguya (8 species), and Myxidium (9 species) from freshwater or brackish fish in Japan were recorded more than 30 years ago (accumulatively 81.1% [43/53]). The re-discovery and molecular–genetic characterization of these species is a current research priority. During our myxosporean survey in Japanese freshwater fish, we detected three species that had never been recorded in Japan, but in the Russian Far East (Sakhalin Island, and Maritime Province): Myxobolus tribolodonus sp. n., forming cysts in the gills of Tribolodon sachalinensis (syn. M. marinus sensu Aseeva, 2000; M. marinus sensu Sokolov et Frolova, 2015, recorded from the gills of Pseudaspius (syn. Tribolodon) spp.); Henneguya pungitii Achmerov, 1953, forming cysts in the subcutis of external skin and buccal submucosa of Pungitius sinensis; and Myxidium salvelini Konovalov et Shulman, 1966, in the urinary bladder of Oncorhynchus masou ishikawae. These new isolates were characterized by integrated taxonomic approaches, i.e., myxospore morphology and molecular–genetic characterization of the small subunit ribosomal RNA gene (SSU rDNA). These new isolates were phylogenetically differentiated from any species whose SSU rDNA sequences were deposited in the DNA databases, and concurrently compared with recorded species based on classical morphological criteria. All three species were differentiated from myxosporeans previously recorded in Japan, indicating new distribution records out of the Russian Far East. For reliable species identification, accumulation of at least SSU rDNA sequences of known species worldwide is critically important.

1. Introduction

Endoparasitic cnidarians of the subclass Myxosporea Bütschli, 1881 (phylum Cnidaria Hatschek, 1888: class Myxozoa Grassé, 1970), take invertebrate (annelids) and vertebrate hosts (mainly fish) alternatively in their life cycles in aquatic environment [1]. They are often incriminated as causatives of a variety of symptomatic or moribund diseases of farmed fish, causing significant economic damage to the aquaculture and fishery industries [2,3,4]. Except for such symptomatic cases, myxosporean infection is generally latent, with patchy spatial distribution, and geographical knowledge of myxozoans can be biased, reflecting predilections of investigators. There are few incentives to investigations on parasites of fishes, as they have little commercial value [5]. Nevertheless, more than 2600 myxozoan species have been described, and extensive uncharted biodiversity of myxozoans remains to be investigated [5,6,7].
Two orders—Bivalvulida Shulman, 1959, and Multivalvulida Shulman, 1959—are divided in the subclass Myxosporea based on the numbers of shell valves (SVs) and polar capsules (PCs) in a myxospore [6,8]. Members of Bivalvulida are characterized by two SVs and two PCs in a myxospore (exceptionally, four PCs in a myxospores with two SVs in the genus Chloromyxum Mingazzini, 1890), and currently, 57 genera are classified in Bivalvulida [6,8]. The most speciose genus, Myxobolus Bütschli, 1882, contains more than 970 nominal species [8,9,10,11]. The genera Ceratomyxa Thélohan, 1892; Myxidium Bütschli, 1882; Henneguya Thélohan, 1892; Chloromyxym; and Thelohanellus Kudo, 1933 are also speciose, counting more than 270, 230, 210, 140, and 100 species, respectively [6,12,13,14,15,16,17,18,19,20].
Approximately sixty myxosporean species of the Bivalvulida have been recorded from freshwater and brackish fish in Japan, but most of them were recorded more than 30 years ago when specific descriptions were made solely based on myxospore morphology. For example, among nominal species of the genera Myxobolus (35 species), Henneguya (9 species), and Myxidium (9 species), 49.1% (26/53) were recorded more than 90 years ago and 81.1% (43/53) more than 30 years ago, as shown in Supplementary Table S1. Furthermore, the majority of these species have never been re-isolated or re-characterized with modern taxonomic viewpoints, i.e., specific characterization based on myxospore morphology and nucleotide sequence of the ribosomal RNA gene (rDNA), as has been recommended recently [21]. To date, only 37.7% (20/53) of species of the aforementioned three genera in Japan have been characterized using molecular–genetic techniques (Supplementary Table S1).
To address the taxonomic issues mentioned above, we are conducting a survey of myxosporean infection in freshwater fish in Japan. In the present study, we characterize three myxosporean species of the genera Myxobolus, Henneguya, and Myxidium and compare them with previously recorded species from Japan and other regions, particularly in the Russian Far East and China, where multiple myxosporean species recorded in freshwater and brackish fishes are shared with Japan [22,23,24].

2. Materials and Methods

2.1. Fish Samples

The World Fresh Water Aquarium Gifu (Aquatotto Gifu) in Kakamigahara City, Gifu Prefecture, Japan, which exhibits approximately 28,500 freshwater fish of 260 species worldwide, regularly collects native Japanese fish species for exhibition. After careful acclimatization in aquarium water, these introduced fish are transferred to exhibition tanks. Surveillance checks for dead fish are carried out daily and the dead specimens are subsequently frozen and provided to us, as described previously [25]. For this study, the bodies of 92 dead fish of 34 species (13 families) were examined between 10 October 2017 and 22 September 2018 (Supplementary Table S2).

2.2. Parasitological Examination

The frozen fish bodies were thawed, and the body weights and total and standard body lengths were recorded. The external and internal organs were then individually examined with the naked eye and under a dissection microscope. To detect myxosporeans microscopically, the contents of the luminal organs, such as the gallbladder, urinary bladder, and gastrointestinal tract, were smeared on glass slides and treated with Diff-Quik™ stain (Sysmex Co., Kobe, Japan).
Myxosporean cysts found in the gills and subcutis/submucosa were opened using fine forceps in physiological saline. Similarly, the contents of the urinary bladder containing myxospores were diluted with physiological saline. These were then observed using a light microscope (OLYMPUS BX60; OLYMPUS Co., Hachioji, Tokyo, Japan) equipped with differential interference contrast optics, photographed at a magnification of ×800, then transformed into digital images using Adobe® Photoshop® ver. 11.0 (Adobe Systems, San Jose, CA, USA). The photographs were then printed at a high magnification. Measurements were conducted on multiple printed photographs following the guidelines of Lom and Arthur [26]. All measurements are expressed in micrometers (µm) unless otherwise stated. Ranges with the means in parentheses are presented. Following the removal of a portion of the myxospores for DNA extraction, the parasite was fixed in either 10% neutral-buffered formalin solution or 70% ethanol solution. The specimens collected in the present work were deposited in the Meguro Parasitological Museum, Tokyo, Japan, under collection nos. 21656–21658.

2.3. DNA Extraction, Polymerase Chain Reaction (PCR), and Sequencing

Parasite DNA was extracted from collected myxospores using an Illustra™ tissue and cells genomicPrep Mini Spin Kit (GE Healthcare UK, Buckinghamshire, UK) according to the manufacturer’s instructions. PCR amplification of the small subunit (SSU) rDNA was performed in a 20 µL volume containing a DNA polymerase, Blend Taq-Plus- (TOYOBO, Dojima Hama, Osaka, Japan), and a combination of universal eukaryotic primers, Eurib1 (5′-ACCTGGTTGATCCTGCCAG-3′) and reverse Eurib2 (5′-CTTCCGCTGGTTCACCTACGG-3′), was used to amplify almost the complete length of the SSU rDNA at once [27,28]. The PCR products were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Tokyo, Japan), and the purified PCR products were cloned into the plasmid vector pTA2 (TArget Clone™; TOYOBO) and transformed into Escherichia coli JM109 cells (TOYOBO) according to the manufacturer’s instructions. Following propagation, the plasmid DNA was extracted using a FastGene Plasmid Mini Kit (NIPPON Genetics Co., Tokyo, Japan), and inserts from multiple independent clones, at least three, were sequenced using universal M13 forward and reverse primers (5′-GTAAAACGACGGCCAGT-3′; and 5′-GGAAACAGCTATGACCATG-3′, respectively). For sequencing purposes, some additional primers were chosen from our stocks used in the previous works [25,28,29]; NSR581/18 (5′-TCTCAGGCTCCCTCTCCGG-3′), Myxo18S_794R (5′-CGCCTGCTTTGAGCACTCTGT-3′), Myxo18S_1009R (5′-CGCATCTGTTAGTCCTTGG C-3′), Myxo 18S_575F (5′-CGCGGTAATTCCAGCTCCAG-3′), Myxo18S_887F (5′-AATGG TCGAGGGCAACTTTG-3′), Myxo18S_1028F (5′-GCCAAGGACTAACAGATGCG-3′), or Myxo18S_1217F (5′-GGGAGAGTATGGTCGCAAGT-3′).
The nucleotide sequence obtained in the present study is available from the DDBJ/EMBL/GenBank databases under accession nos. LC544125–LC544127.

2.4. Phylogenetic Analysis

Fragments of the newly obtained rDNA sequences were analyzed to identify highly similar nucleotide sequences using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information website (NCBI; https://www.ncbi.nlm.nih.gov/ (accessed on 7 July 2020). For phylogenetic analysis, the newly obtained SSU rDNA nucleotide sequences in the present study and related myxosporean sequences retrieved from the DDBJ/EMBL/GenBank databases were aligned using the MEGA7 software [30], with subsequent manual adjustments. The accession numbers of the sequences analyzed in the present study are provided in the figure showing a phylogenetic tree. Regions judged to be poorly aligned and characters with a gap in any sequence were excluded; 920 characters, of which 416 were variable, were retained for subsequent analysis. Maximum likelihood (ML) analysis was performed using the PhyML program ver. 3.0 [31,32] provided on the ‘phylogeny.fr’ website (http://www.phylogeny.fr/). The probability of inferred branch was assessed by the approximate likelihood-ratio test (aLRT), an alternative to the non-parametric bootstrap estimation of branch support [33].

3. Results

Three myxosporean species of the genera Myxobolus, Henneguya, and Myxidium were found separately in three fish species (Figure 1). Four Myxobolus plasmodia were found in the gills of a rosyface dace, Pseudaspius sachalinensis (Nikolskii, 1889) (syn. Tribolodon sachalinensis (Nikolskii, 1889)), measuring 11.0 cm in standard body length (BL) and 20.6 g body weight (BW), collected originally in May 2018 from a river running through Akkeshi-cho Town, Hokkaido Prefecture, Japan. Four cysts at the subcutis of the external surface in the anterior body and more than 20 cysts in the oral submucosa were found in an Amur stickleback, Pungitius sinensis (Guichenot, 1869), measuring 4.9 cm BL and 1.2 g BW, collected originally in May 2018 from the same river running through Akkeshi-cho Town. These cysts contained Henneguya plasmodia. Numerous Myxidium myxospores were found in the urinary bladder of a masu salmon, Oncorhynchus masou (Brevoort, 1856) (syn. Oncorhynchus masou ishikawae (Jordan et McGregor, 1925)), measuring 30.5 cm BL and 599 g BW, collected originally in May 2018 from the Nagaragawa River running through Gifu Prefecture, Japan. Since the myxospore morphology and SSU rDNA nucleotide sequences of the aforementioned species were differentiated from previously recorded species in Japan, we carefully conducted their specific identification with species recorded from other locations.
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Figure 1. Stylized illustrations of Myxobolus tribolodonus n. sp. from Pseudaspius sachalinensis (a), Henneguya pungitii from Pungitius sinensis (b,c), and Myxidium salvelini from Oncorhynchus masou (d). Frontal view (a,b,d) and sutural view (c) at the same magnification.
Figure 1. Stylized illustrations of Myxobolus tribolodonus n. sp. from Pseudaspius sachalinensis (a), Henneguya pungitii from Pungitius sinensis (b,c), and Myxidium salvelini from Oncorhynchus masou (d). Frontal view (a,b,d) and sutural view (c) at the same magnification.
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3.1. Myxobolus tribolodonus sp. n. (Myxosporea: Bivalvulida: Myxobolidae)

(syn. Myxobolus marinus sensu Aseeva, 2000; M. marinus sensu Sokolov et Frolova, 2015)
(Figure 1a, Figure 2 and Figure 3)
Four large-sized oval plasmodia were detected between the gill filaments at their upper half portion of a rosyface dace (Figure 2). The largest plasmodium measured 2.35 mm by 0.87 mm, and the other three were smaller, measuring 1.13–1.29 (1.24) mm by 0.44–0.77 (0.64) mm.
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Figure 2. Plasmodia (P) of Myxobolus tribolodonus n. sp. attached to the gill filaments of Pseudaspius sachalinensis under a dissection microscope.
Figure 2. Plasmodia (P) of Myxobolus tribolodonus n. sp. attached to the gill filaments of Pseudaspius sachalinensis under a dissection microscope.
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3.1.1. Description

Bivalvular myxospores pyriform from the frontal view, measuring 8.7–9.6 (9.2) in length, 6.5–7.5 (7.0) in width (n = 11). Valvular surface smooth. Two almost equal PCs, bullet-shaped, 4.6–6.0 (5.1) in length and 1.7–2.1 (1.9) in width, pointing towards the apical end, and the posterior end of PCs beyond the mid-line of myxospore length. Binucleated sporoplasm in the remaining space. A small-sized intercapsular appendix, and 4–5 turns of polar tubules.

3.1.2. Molecular–Genetic Characterization

A newly obtained, almost complete SSU rDNA nucleotide sequence was 2007 bp in length (DDBJ/EMBL/GenBank accession no. LC544125), and showed the highest identities (up to 95.11% [1538/1617]) with sequences from certain Myxobolus spp. such as M. alvarezae (accession no. FJ716097), M. intimus (accession no. FJ716098), M. sitjae (accession no. JF311898), M. obesus (accession no. AY325286), and M. szentendrensis (accession no. KP025684), which had 5–11 nucleotide insertion/deletion sites (indels) over an rDNA sequence comparable with that of the new species. The nucleotide identity of the SSU rDNA sequence of Myxobolus macrocapsularis Reuss, 1906, from Abramis brama (L., 1758) or Blicca bjoerkna (L., 1758) in Hungary (accession nos. AF507969 and FJ716095, respectively) with that of the present new species was 83.58% (1308/1565) with 33 indels, or 81.01% (1058/1306) with 27 indels. Shulman [22] considered M. macrocapsularis as a senior synonym of M. marinus Dogiel, 1948, from the gills of Alburnus alburnus (L., 1758) in the Russian Far East (Amur River basin and other rivers flowing to the Sea of Japan).

3.1.3. Remarks

Myxospores of this myxobolid species were pyriform, with a length of 8–10 µm, apparently classified in the genus Myxobolus according to Lom and Dyková [6]. More than 40% of ca. 970 nominal Myxobolus spp. showed an organ/tissue preference for the gills [9,10,11]. The morphologically identical myxospores were recorded from P. sachalinensis (type host of the present new species), Pseudaspius hakonensis (Günther, 1877) (syn. Tribolodon hakonensis (Günther, 1877)), and Pseudaspius brandtii (Dybowski, 1872) (syn. Tribolodon brandtii (Dybowski, 1872)) from the rivers on the Sakhalin Island or in the Maritime Province (Primorsky Kray) of the Russian Far East [34,35], as shown in Table 1. These isolates (M. tribolodonus sp. n., M. marinus sensu Aseeva, 2000, and M. marinus sensu Sokolov et Frolova, 2015) had identical myxospore dimensions and proportions (myxospore length/width), with an index of approximately 1.2 (1.1–1.3). Myxobolus marinus Dogiel, 1948, was originally described from the gill lamellae of Alburnus alburnus (Cypriniformes: Leuciscidae: Leuciscinae) in the basin of the Amur River and the Sea of Japan [22,36,37], and is currently considered to be a junior synonym of Myxobolus macrocapsularis [22,37]. The dimensions of M. macrocapsularis myxospores, including the original description of M. marinus, are apparently larger than the myxospores found in Pseudaspius spp. in the Russian Far East region (Maritime Province (Primorsky Kray) and Sakhalin Island) and Hokkaido Island of Japan (Table 1). Although Shulman [22] characterized myxospores of the former species as pyriform with narrow and pointed anterior ends, none of the isolates from the gills of Pseudaspius spp. (M. tribolodonus sp. n., M. marinus sensu Aseeva, 2000, and M. marinus sensu Sokolov et Frolova, 2015) showed such traits, but they showed comparatively bluntly pointed anterior ends of pyriform myxospores (Figure 1a and Figure 3 in the present study; also see [34,35]). Ellipsoidal plasmodia of M. macrocapsularis and M. tribolodonus sp. n. exhibited almost similar dimensions and localization near the tips of the gill filaments (Figures 3–5 in [37]; Figure 2 of the present study).
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Figure 3. Photomicrographs of myxospores of Myxobolus tribolodonus n. sp. from Pseudaspius sachalinensis. Frontal view at the same magnification.
Figure 3. Photomicrographs of myxospores of Myxobolus tribolodonus n. sp. from Pseudaspius sachalinensis. Frontal view at the same magnification.
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Further morphological comparisons with nominal Myxobolus spp. were conducted with 17 Myxobolus spp. (Supplementary Table S3), selected based on the myxospore shape (non-circular in the frontal view) and the myxospore length of between 8–10 µm using synoptic references [9,10,11]. Only four species (M. bengalensis, M. branchialis, M. flavus, and M. tambroides) have deposited SSU rDNA nucleotide sequences (DDBJ/EMBL/GenBank accession nos. KJ476883, MK412934, JQ388887, KF296346, KF296347, and JX028236), and these sequences show less than 90% identity with that of M. tribolodonus sp. n. (accesssion no. LC544125), in addition to a few to several indels within the 1520 bp to 1935 bp long sequences. Morphologically, the PCs of M. bengalensis, M. branchialis, M. flavus, and M. tambroides are wider than those of the present species (Supplementary Table S3) and situated closer to each other within a myxospore, in contrast to the distantly placed PCs in M. tribolodonus n. sp. For the remaining 13 species, no nucleotide sequences are available in the DNA databases. All of these species, except for M. funsienensis (Ma, 1998) Erias et al., 2005 (syn. Myxosoma fusienenesis Ma in Chen et Ma, 1998), differed apparently from M. tribolodonus sp. n. in their elliptical myxospore shape, wider PCs, smaller angle of two PCs, number of turns of polar tubules, and/or small dimensions of cysts (Supplementary Table S3). Although histological localization of cysts in the gills is known to have high importance in species identification [38], such data are not always available. The present new species shows similar myxospore morphology to that of M. fusienensis recorded in the gills of Spinibarbichthys yunnanensis (Tsü, 1977) (syn. Spinbarbus denticulatus yunnanensis (Tsü, 1977)) from the southernmost province in China (Yunnan), but these two species differ from each other in the shape of PCs (pyriform vs. bullet-shaped), the angular arrangement of PCs (wide vs. narrow), and the absence and presence of small intercapsular processes [9,23].
Table 1. Comparison of Myxobolus tribolodonus sp. n. with its related species a.
Table 1. Comparison of Myxobolus tribolodonus sp. n. with its related species a.
SpeciesHost FishLocation in HostLocalitySLSWSTPCLPCWSFIPNTCyst SizeReference
M. tribolodonus sp. n.Pseudaspius sachalinensis (syn. Tribolo-don sachalinensis)GillsJapan (Hokkaido)8.7–9.6 (9.2)6.5–7.5 (7.0)—4.6–6.0 (5.1)1.7–2.1 (1.9)PyriformSmall4–5max. 2.35 by 0.87 mmPresent Study
M. marinus sensu Aseeva, 2000Pseudaspius brandtii (syn. Tribolodon brandtii); Pseudaspius hakonensis (syn. Tribolodon hakonensis)GillsMaritime Province (Primorsky Kray) of Russian Far East9.7–12.08.3–10.3—5.5–6.3—pyriform———[34]
M. marinus sensu Sokolov et Frolova, 2015Pseudaspius sachalinensis (syn. Tribolodon sachalinensis); Pseudaspius hakonensis (syn. Tribolodon hakonensis)GillsSakhalin Island (Russian Far East)9.9–11.3 (10.7)8.1–9.2 (8.6)—5.0–6.1 (5.7)2.5–3.4 (3.0)pyriformsmall——[35]
M. marinus Dogiel, 1948Alburnus alburnusGillsThe basin of the Amur River and the Sea of Japan12.5–13.07.0–8.0—6.0–7.02.5–3.0pyriformsmall——[22,36]
M. macrocapsularis
(syn. Myxobolus physophilus Reuss, 1906; M. multiplex Achmerov, 1960; M. vescus Achmerov, 1960; M. oviformis Thélohan in Rostovschikov, 1952; M. marinus Dogiel, 1948) b		Rutilus rutilus (L., 1758) ; Leu-ciscus leuciscus (L., 1758); Leu-ciscus idus (L., 1758); Squalius cephalus (L., 1758) (syn. Leuci-scus cephalus (L., 1758); Scardi-nius erythrophthalmus (L., 1758); Leuciscus aspius (L., 1758) (syn. Aspius aspius (L., 1758); Barbus barbus (L., 1758); Gobio gobio (L., 1758) ; Abbottina rivularis (Basilewsky, 1855) (syn. Pseudo-gobio rivularis (Basilewsky, 1855)); Alburnus alburnus; Alburnoides bipunctatus (Bloch, 1782) (syn. Alburnus bipunctatus (Bloch, 1782)); Chondrostoma nasus (L., 1758); Pelecus cultratus (L., 1758): Opsariichthys uncirostris (Temminck et Schlegel, 1846); Carassius carassius (L., 1758); Cyprinus carpio L., 1758; Hypophthalmichthys molitrix (Valenciennes, 1844) ; Gasterosteus aculeatus L., 1758Gills, mesentery, intestinal wall, swimbladderBasins of rivers in southern Karelia, Rivers flowing to the Baltic Sea, Rivers flowing to the Azov Sea, or Caspian Sea; and Amur River,9.0–14.56.0–9.54.5–6.05.0–8.62.4–3.6pyriformsmall—Reaching 1.5 mm in diameter[22]
a Abbreviation: SL, myxospore length; SW, myxospore width; ST, myxospore thickness; PCL, polar capsule length; PCW, polar capsule width; SF, myxospore form; IP, intercapsular projection; NT, number of coil turns (polar tubles). All measurements are expressed in micrometers (µm) unless otherwise stated. Ranges are presented, with the means in parentheses. For M. flavus, means and standard variations are shown in parentheses. b All synonyms follow Shulman (1966).

3.1.4. Taxonomic Summary

  • Host: Pseudaspius sachalinensis (syn. Tribolodon sachalinensis), rosyface dace (Actinopterygii: Cypriniformes: Leuciscidae: Pseudaspininae).
  • Additional hosts: Pseudaspius hakonensis (syn. Tribolodon hakonensis) and Pseudaspius brandtii (syn. Tribolodon brandtii) [34,35].
  • Type locality: Collected in Aquatotto Gifu, Gifu Prefecture, Japan, following its relocation from a river running through Akkeshi-cho Town, Hokkaido Prefecture, Japan. Circumstantially, the infection might originate from the water in the natural habitat and not in the aquarium.
  • Additional locality: Rivers on the Sakhalin Island or in the Maritime Province (Primorsky Kray) of Siberia (Russia) [34,35].
  • Site of infection: Gill filaments (histozoic).
  • Materials deposited: Hapantotype no. 21656 (specimens in fixatives), Meguro Parasitological Museum, Tokyo, Japan.
  • Deposited rDNA sequence: DDBJ/EMBL/GenBank accession no. LC544125.
  • Etymology: The species name refers to the former genus name of the host fish.

3.2. Henneguya pungitii Achmerov, 1953 (Myxosporea: Bivalvulida: Myxobolidae)

(Figure 1b,c, Figure 4 and Figure 5)
Oval whitish cysts, measuring 1.1–1.5 mm in diameter, were found in the subcutis of the anterior body and oral submucosa of an Amur stickleback, Pungitius sinensis (Figure 4). From the subcutis of the ninespine stickleback, Pungitius pungitius (L., 1758), and Sakhalin stickleback, Pungitius tymensis (Nikolskii, 1889), in the Russian Far East (Rivers flowing to the Sea of Okhotsk on Kamchatka Peninsula and Sakhalin Island), similar cysts containing myxospores of Henneguya pungitii Achmerov, 1953, were recorded [22,35]. Recently, Dorovskikh [39] recorded myxospores of the same species from plasmodial cysts localized in the gills and liver of the ninespine sticklebacks from rivers on the Kolguyey Island, near the Kanin Peninsula, in northwestern Russia. Myxospores of H. pungitii recovered from the subcutis and submucosa, or from the gills and liver of Pungitius spp., distributed in northeastern and northwestern Russia, respectively, are relatively closer in morphology with each other, but apparently different in size (Table 2). For further research in the future, H. pungitii myxospores recovered from the cutaneous subcutis and oral submucosa of Amur Stickleback are described in detail here, along with its molecular–genetic characterization.
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Figure 4. Photomicrographs of plasmodia (arrowheads) of Henneguya pungitii at the subcutis of the external surface (a,b) and oral submucosa (c) of Pungitius sinensis under a dissection microscope. One grid = 5 mm.
Figure 4. Photomicrographs of plasmodia (arrowheads) of Henneguya pungitii at the subcutis of the external surface (a,b) and oral submucosa (c) of Pungitius sinensis under a dissection microscope. One grid = 5 mm.
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Table 2. Henneguya spp. recorded from freshwater fish, resembling H. pungitii in spore shape and dimension a.
Table 2. Henneguya spp. recorded from freshwater fish, resembling H. pungitii in spore shape and dimension a.
SpeciesHost FishLocation in HostLocalityTSLSBLSBWSBTPCLPCWCPNTCyst SizeReference
H. pungitii Achmerov, 1953Pungitius sinensisSubcutis of External Surface and Oral SubmucosaJapan (Hokkaido)47.5–56.1 (49.6)19.3–22.9 (21.0)6.3–7.6 (6.8)4.8–6.0 (5.6)7.8–9.5 (8.4)1.8–2.5 (2.1)28.2–33.2 (28.6)7–81.1–1.5 mm in Diameter, OvalPresent Study
H. pungitii Achmerov, 1953Pungitius pungitiusSubcutisBasins of Kamchatka and Paratunka Rivers (Russia)—13.0–17.04.5–6.0—6.5–8.01.8–2.018.0–20.0—1–2 mm in diameter, round[22]
H. pungitii Achmerov, 1953Pungitius tymensis (Nikolskii, 1889)SubcutisSakhalin Island (Russia)52.9–62.2 (58.2)16.7–20.6 (18.4)4.7–5.7 (5.2)—6.7–9.0 (8.2)2.1–2.6 (2.3)36.2–45.5 (39.8)——[35]
H. pungitii sensu Dorovskikh 2022Pungitius pungitiusGillsKolguyev Island, near Kanin Peninsula (Russia)35.5–40.2 (37.5)14.7–16.7 (16.1)5.4–6.0 (5.8)4.06.7–7.4 (7.1)1.3–2.0 (1.6)20.8–23.5 (21.4)——[39]
H. pungitii sensu Dorovskikh 2022Pungitius pungitiusLiverKolguyev Island, near Kanin Peninsula (Russia)27.5–32.8 (30.0)11.4–13.4 (12.7)6.1–7.7 (6.8)5.4–6.7 (5.7)5.4–7.4 (6.6)2.0–2.7 (2.5)14.7–19.4 (17.1)——[39]
H. alexeevi Shulman, 1962Hypophthalmichthys molitrix (Valenciennes, 1844); Perccottus glenii Dybowski, 1877 (syn. Percottus glehni Dybowski, 1877)Gills, ovaryAmur basin40.8–52.8 (48.0)16.8–19.2 (18.2)5.0–7.2 (6.0)4.0–5.2 (4.8)7.8–10.8 (8.9)1.8–2.2 (1.9)24.0–33.6 (29.8)6–7Cyst: round to oval[23]
H. sinensis Chen et Hsieh, 1960Channa argus (Cantor, 1842); Clarias batrachus (L., 1758); Misgurnus anguillicaudatus (Cantor, 1842)Gills, external surface, fin, oral cavity, intestine, swimming bladderChina31.8–52.2 (42.7)13.8–16.2 (15.1)4.8–6.0 (5.4)3.6–4.2 (3.7)7.2–8.4 (7.9)1.4–2.4 (1.9)18.0–36.0 (27.6)7–80.04–0.28 mm in diameter, round[23]
H. rhinogobii Li et Nie in Li et al., 1973Rhinogobius giurinus (Rutter, 1897); Rhodeus ocellatus (Kner, 1866)Gills, intestineChina, Japan (Gifu)34.7–59.3 (47.2)15.4–19.3 (16.9)4.6–6.2 (5.5)4.2–5.4 (4.3)6.9–9.2 (7.9)1.5–2.0 (1.9)19.3–40.0 (30.3)9–100.42–1.20 mm by 0.29–0.52 mm, oval[23]
H. pseudorhinogobii Kageyama et al., 2009Rhinogobius kurodai (Tanaka, 1908) (syn. Rhinogobius sp. OR)GillsJapan (Gifu)39.5–60.7 (50.7)14.2–17.8 (15.8)4.7–5.8 (3.5)4.5–5.4 (4.8)5.9–7.6 (6.5)1.1–1.7 (1.4)25.3–42.9 (34.9)80.05–0.25 mm in diameter[40]
H. pilosa Azevedo et Matos, 2003Serrasalmus altuvei Ramírez, 1965GillsBrazil52.3–56.0 (54.2)20.0–23.1 (21.1)5.5–6.3 (5.9)1.9–2.6 (2.2)7.1–7.6 (7.4)1.0–1.3 (1.2)30.5–34.9 (31.1)11–12max. 0.2 mm in diameter, round to ellipsoidal[41]
a Abbreviation: TSL, total spore length; SBL, spore body length; SBW, spore body width; SBT spore body thickness; PCL, polar capsule length; PCW, polar capsule width; CP, length of caudal processes; NT, number of coil turns (polar tubules). All measurements are expressed in micrometers (µm) unless otherwise stated. Ranges are presented, with the means in parentheses.
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Figure 5. Photomicrographs of myxospores of Henneguya pungitii. Frontal view nearly at a sagittal plane (a,d), and sutural view nearly at surface (b,c). Photographs (a–c) at the same magnification with scale bar in (b). Photograph (d) is a twice-magnified view of the spore body shown in a photograph (a). Suture lines (arrowhead) and polar tubules (arrow) are shown.
Figure 5. Photomicrographs of myxospores of Henneguya pungitii. Frontal view nearly at a sagittal plane (a,d), and sutural view nearly at surface (b,c). Photographs (a–c) at the same magnification with scale bar in (b). Photograph (d) is a twice-magnified view of the spore body shown in a photograph (a). Suture lines (arrowhead) and polar tubules (arrow) are shown.
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3.2.1. Description

Bivalvular myxospores with elongated spindle-shaped myxospore bodies and long caudal processes of individual valves. Myxospore bodies measuring 19.3–22.9 (21.0) in length, 6.3–7.6 (6.8) in width, and 4.8–6.0 (6.8) in thickness (n = 20). Two almost equal PCs, elongated pyriform, 7.8–9.5 (8.4) in length and 1.8–2.5 (2.1) in width. Binucleated sporoplasm in the remaining space. Turns of polar tubules 7–8. Caudal processes long and fine, measuring 28.2–33.2 (28.6) in length, and total myxospore length 47.5–56.1 (49.6). Valvular surface smooth.

3.2.2. Molecular-Genetic Characterization

A newly obtained, almost complete SSU rDNA nucleotide sequence was 2060 bp in length (accession no. LC544126) and showed the highest identities (91.7% [1741/1899] with 26 indels, or 91.6% [1878/2050] with 16 indels) with sequences from an unidentified Henneguya sp. and the aurantiactinomyxon KAB-2001 isolate (accession nos. U13826 and AF378356, respectively).

3.2.3. Remarks

Bivalvular myxospores of this species had two caudal processes which continued from each valve, and two elongated PCs were placed on the sutural plane, consistent with the definition of the genus Henneguya according to Lom and Dyková [6]. It is important to note that H. pungitii Achmerov, 1953, is characterized by fusiform myxospore bodies tapering to a point at both ends, although Henneguya spp. with myxospores having ellipsoid and rounded myxospore bodies in the frontal view are apparently predominant [12,18,20]. Henneguya spp. with myxospores exhibiting similar morphological characteristics to those of H. pungitii are selected in Table 2. Henneguya pungitii sensu Dorovskikh, 2022 [39], and the other five species have different organ preferences, e.g., the gills or visceral organs such as the liver, ovary, intestine, or swimming bladder and their myxospores are characterized by shorter (H. pungitii sensu Dorovskikh, 2022, H. sinensis, H. rhinogobii, and H. pseudorhinogobii) and/or thinner myxospore bodies (H. alexeevi, H. sinensis, H. rhinogobii, H. pseudorhinogobii, and H. pilosa), smaller PCs (H. pseudorhinogobii, and H. pilosa), and/or more turns of polar tubules (H. rhinogobii, and H. pilosa), as shown in Table 2.
Availability of the SSU rDNA nucleotide data of aforementioned Henneguya spp. with fusiform myxospore bodies (Table 2) is currently limited to three species (the current isolate of H. pungitii, H. rhinogobii, and H. pseudorhinogobii), and the latter two species show relatively high identity of the SSU rDNA (95.89% [1843/1922] with 10 indels over a 1930 bp length between accession nos. AB447993 and AB447994) with each other, whereas the SSU rDNA of the current H. pungitii isolate (accession no. LC544126) shows relatively low identities with those of H. rhinogobii and H. pseudorhinogobii (82.84% [1569/1894] with 61 indels over a 1958 bp length, and 87.58% [1664/1900] with 53 indels over a 1958 bp length, respectively). The phylogenetic relationships between H. pungitii from the subcutis of Pungitius spp., distributed in the Russian Far East, and H. pungitii sensu Dorovskikh 2022 from the gills and/or liver of Prungitiu pungitius, distributed in the northwestern Russia (Kolguyev Island, near Kanin Peninsula), should be elucidated in future research, since these two species show distinct tissue preferences and somewhat different myxospore morphology regardless of their identical host preference [22,35,39].

3.2.4. Taxonomic Summary of the Present Isolate

  • Host: Pungitius sinensis, Amur stickleback (Actinopterygii: Gasterosteiformes: Gasterosteidae).
  • Locality: Collected in Aqua Totto Gifu, Gifu Prefecture, Japan, following its relocation from a river running through Akkeshi-cho Town, Hokkaido Prefecture, Japan. Circumstantially, the infection might originate from the water in the natural habitat, and not in the aquarium.
  • Site of infection: Subcutis of external surface and oral submucosa (histozoic).
  • Materials deposited: Hapantotype no. 21657 (specimens in fixatives), Meguro Parasitological Museum, Tokyo, Japan.
  • Deposited rDNA sequence: DDBJ/EMBL/GenBank accession no. LC544126.

3.3. Myxidium salvelini Konovalov et Shulman, 1966 (Myxosporea: Bivalvulida: Myxidiidae)

(Figure 1d and Figure 6)

3.3.1. Morphological Characterization

Numerous free myxospores of M. salvelini Konovalov et Shulman, 1966, were found in the contents of the urinary bladder from the red spotted masu trout, Oncorhynchus masou, while coelozoic plasmodia were never found in the contents or wall of the organ. Bivalvular myxospores were fusiform (Figure 1d and Figure 6), or slightly more arcuate on one side, in the frontal view, measuring 13.6–16.7 (15.0) by 4.9–6.6 (5.7) (n = 20). Fusiform myxo-spores tapered to pointed ends at an angle 60°, and had fine striations (5–7 in number) on the surface of each valve. Two pyriform PCs, measuring 4.0–5.2 (4.4) in length and 2.1–2.8 (2.5) in width, were oriented towards the pointed ends of spores. Binucleated sporo-plasms were located in the middle parts of spores between PCs. Polar filaments formed 4–5 turns in PCs.
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Figure 6. Photomicrographs of myxospores of Myxidium salvelini. Frontal view nearly at midline of sagittal plane (a,b), and spore surface (c) at the same magnification. Scale bar is shown in (c).
Figure 6. Photomicrographs of myxospores of Myxidium salvelini. Frontal view nearly at midline of sagittal plane (a,b), and spore surface (c) at the same magnification. Scale bar is shown in (c).
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3.3.2. Molecular-Genetic Characterization

A newly obtained, almost complete SSU rDNA nucleotide sequence was 2059 bp in length (accession no. LC544127), and showed the highest identity (95.02% [1812/1907] with 9 indels) with Sphaerospora onchorhynchus (accession no. AF201373).

3.3.3. Remarks

Myxospores of this myxosporean species were fusiform with pointed ends, and were classified in the genus Myxidium according to Lom and Dyková [6]. A synopsis of the Myxidium spp. by Eiras et al. [14] included 232 nominal species, of which a majority parasitize the gall bladder as coelozoic myxosporeans, whereas only a small proportion (12.5%) parasitize the urinary system, including the kidney, ureter, and urinary bladder. Similarly, among 19 Myxidium spp. recorded in Japan (nine species from freshwater fish, and 12 species from marine fish; see Supplementary Table S1), only M. lentiforme (Fujita, 1927) Fujita, 1929 (syn. M. fusiforme Fujita, 1927), and M. uchiyamae Fujita, 1927, parasitize the urinary system, i.e., kidney, of the Japanese eel (Anguilla japonica Temminck et Schlegel, 1846) [42]. These species have morphologically distinct myxospores from those of the present new species (Table 3). Among Myxidium spp. recorded in the urinary bladder of freshwater fish worldwide, M. rimskykorsakowi Shulman, 1962, and M. salvelini have myxospores similar in morphology to those of the current isolate, but the myxospores of the former species are shorter and wider than those of the current isolate, with pointed ends at an angle of 70°–80° and rounded PCs [22]. In contrast, M. salvelini recorded from the urinary bladder and ureters of salmonids from the river basin of Kamchatka [22] and the current isolate from the masu salmon in the central Japan (Gifu, Honshu Island of Japan) are coincident in morphology with each other. In Hokkaido, Japan, M. oncorhynchi Fujita, 1923, was isolated from the gall bladder of the cherry salmon, Oncorhynchus masou masou (Brevoort, 1856) [43]. Mixidium oncorhynchi has myxospores with oval to globular PCs, differentiated from M. salvelini reported in the present study. No myxosporean species showed highly identical SSU rDNA sequences to that of the current M. salvelini isolate (accession no. LC544127). Exclusively, Sphaerospora onchorhynchus (accession no. AF201373) showed a high nucleotide identity with a limited number of indels (95.02% [1812/1907] with 9 indels).

3.3.4. Taxonomic Summary of the Present Isolate

  • Host: Oncorhynchus masou, masu salmon (syn. Oncorhynchus masou ishikawae, satsukimasu salmon, or red spotted masu trout) (Actinopterygii: Salmoniformes: Salmonidae).
  • Locality: Collected in Aqua Totto Gifu, Gifu Prefecture, Japan, following its relocation from the Nagaragawa River running through Gifu Prefecture, Japan. Circumstantially, the infection might originate from the water in the natural habitat, and not in the aquarium.
  • Site of infection: Urinary bladder (coelozoic).
  • Materials deposited: Hapantotype no. 21658 (specimens in fixatives), Meguro Parasitological Museum, Tokyo, Japan.
  • Deposited rDNA sequence: DDBJ/EMBL/GenBank accession no. LC544127.

3.4. Phylogenetic Analysis

The phylogenetic relationships of the three new isolates described above with other myxospoean species are shown in Figure 7. Myxobolus tribolodonus sp. n., parasitizing the gills of the rosyface dace, formed a highly supported clade with M. intimus Zaika, 1965, from the gill lamellae of the common roach Rutilus rutilus (L., 1758); M. hungaricus Jaczó, 1940, from the gill filaments of Abramis brama; M. obesus from the gills of Alburmus alburmus; M. sitjae Cech et al., 2012, from the gill filaments of Blicca bjoerkna; and M. szentendrensis from the gills of the common nase Chondrostoma nasus (L., 1758). All these myxobolid species with pyriform myxospores parasitize the gill filaments of cyprinid fishes of the subfamily Leuciscinae, distributed in the northern Far East (M. tribolodonus sp. n.) and Europe (the other five species). This clade is the sister group to the clades of myxobolid species parasitizing the gills of cyprinid fishes of the subfamilies Cyprininae (China and Japan) and Labeoninae (India).
Henneguya pungitii, parasitizing the external subcutis and oral submucosa of the Amur stickleback, forms a clade with Henneguya creplini (Gurley, 1894) Labbé, 1899, from the gills of the zingel Zingel zingel (L., 1758) in Europe, and with Myxobolus lepomis Rosser et al., 2017, from the gills of the redspotted sunfish Lepomis miniatus (Jordan, 1877) and the dollar sunfish Lepomis marginatus (Holbrook, 1855) in Texas, USA. The SSU rDNA sequences of these three species showed 86.14% [1392/1616] to 90.30% [1461/1618] identities and 9–18 indels with each other. This clade is the sister group to the clade of Henneguya spp. from marine fish.
Myxidium salvelini, parasitizing the urinary bladder of the satsukimasu salmon, formed a highly supported clade with Sphaerospora onchorhynchus Kent et al., 1993, from the kidney of the sockeye salmon Oncorhynchus nerka (Walbaum, 1792) in the North Pacific Ocean, and Myxidium lieberkuehni Bütschli, 1882, from the kidney of the northern pike Esox lucius L., 1758, in the north parts of the Northern Hemisphere. This clade is rather isolated from other myxosporeans (Figure 7; see also Figure 4 of Sekiya et al. [25]).

4. Discussion

Classical taxonomy of Myxozoa based on morphological criteria uses phenotypical characteristics of myxospores such as the number of SVs and PCs, arrangement of the PCs, and SV ornamentation, together with host specificity, site preference of the plasmodium, and geographical distribution [6,8]. A high degree of phenotypical plasticity of myxospores and multiple examples of convergent evolution often render the specific identification difficult [37,49,50,51,52,53,54,55,56,57]. Currently, phylogenetic analyses based on nucleotide sequences of SSU rDNA and/or other genes, e.g., the internal transcribed spacer region 1, the elongation factor 2 gene, mitochondrial cytochrome c oxidase (cox-1), and mitochondrial small and large subunit ribosomal RNA genes, have become ineluctable techniques for reliable species differentiation [5,21,58,59]. An apparent obstruction for the current taxonomic approach is the unavailability of sufficient molecular–genetic data of myxosporeans, particularly those of the species recorded from wild fish.
In the present study, three new myxosporean isolates from Japan, i.e., Myxobolus tribolodonus sp. n. (syn. M. marinus sensu Aseeva, 2000; and M. marinus sensu Sokolov et Frolova, 2015 from the gills of Pseudaspius spp. in the rivers on the Sakhalin Island and Maritime Province (Primorsky Kray) of Russian Far East), Henneguya pungitii, and Mixidium salvelini, could be differentiated based on the classical taxonomic criteria. All of these species from freshwater fishes in Japan are new distribution records, but their host species have been recorded for each species from the Russian Far East [24,35]. Concurrent molecular–genetic characterization of these new isolates of known species could make it possible to perform reliable identification of the species isolated from the field and clarify their phylogenetic relationships with closely-related species in morphology and/or other biological traits (host specificity, site preference, geographical distribution, etc.). As discussed above, Russian researchers [34,35] identified Myxobolus sp. from the gills of Pseudaspius spp. (Cyprinidae) in the Russian Far East as M. marinus Dogiel, 1948, which had been synonymized by Shulman [22] to Myxobolus macrocapsularis, parasitizing the gills and other visceral organs of a variety of cyprids. Morphologically, these two isolates, i.e., M. marinus sensu Aseeva, 2000 and M. marinus sensu Sokolov et Frolova, 2015, are identical to the new isolate from the gills of Pseudaspius sachalinensis in Hokkaido, Japan, although the myxospores of the former isolates are marginally larger than those of the Japanese isolate. It is interesting to explore phylogenetic relationships of species collected from geographically separated host populations (Russian Far East and Hokkaido Island of Japan).
Henneguya pungitii isolates from the subcutis of Prungitius spp., distributed in the Russian Far East and Hokkaido Island of Japan, show highly identical morphology of myxospores (Table 3). On the other hand, H. pungitii from Pungitius pungitius in the rivers on Kolguyev Island, near Kanin Peninsula (Russia), which was reported recently by Dorovskikh [39], showed site preferences to the gills and liver, and its myxospores were apparently smaller than the isolates from the subcutis of the same host distributed in the Russian Far East and Hokkaido. Prungitius spp. are known to have multiple evolutionarily, geographically, and ecologically separated populations [60]. It is interesting to reveal coevolutional relationships of myxosporeans with their hosts using phenotypically and geographically separated populations of H. pungitii. In addition to the possible coevolutionary speciation, sympatric speciation of myxosporeans through occupation of distinct microhabitats within a single host species has also been suggested [53,61].
Currently, it is generally recognized that there is no phylogenetically consistent pattern congruent with host family, tissue tropism, or myxospore morphology [53,61]. At individual clade levels, however, apparent clustering of myxosporeans originating from the same order/family of the host fish has been demonstrated by many studies [3,62,63,64]. Moreover, polyphyly or paraphyly of multiple myxosporean genera such as Myxobolus, Henneguya, Sphaerospora, Myxidium, Zschokkella, and Chloromyxum have been demonstrated [8,25,58,59,61,65,66]. These phylogenetic relationships of taxa classified in different genera are reflected in Figure 7 in the present study.

5. Conclusions

The majority of myxosporean species of the genera Myxobolus, Henneguya, and Myxidium from natural freshwater fish in Japan (Supplementary Table S1) remain to be molecular-genetically characterized. Synchronized efforts to accumulate at least SSU rDNA sequences of known or unknown species worldwide will be invaluable, not only for reliable species identification, but also for further understanding of evolutional history and global dispersion of different myxosporean species.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/life14080974/s1, Supplementary Table S1: Myxosporean species of the genera Myxobolus, Henneguya, and Myxidium, recorded in Japan; Supplementary Table S2: Fish specimens examined in our survey of myxosporeans in freshwater fish provided by Aquatotto Gifu, Japan; and Supplementary Table S3: Myxobolus spp. parasitizing the gills of freshwater fish, with myxospore length (SL) ranging between 7 µm and 10 µm and almost equal-sized polar capsules, like M. tribolodonus sp. n. All references cited in the supplementary materials [9,22,23,28,29,40,42,43,44,47,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118] are included in the reference list below.

Author Contributions

Conceptualization, M.S., H.S. (Haruya Sakai), and H.S. (Hiroshi Sato); methodology, M.S., H.S. (Haruya Sakai), Y.-C.L., and I.R.; software, I.R. and M.Y.; validation, I.R., M.Y., and H.S. (Hiroshi Sato); data curation, M.S., H.S. (Haruya Sakai), and Y.-C.L.; writing—original draft preparation, M.S., H.S. (Haruya Sakai), and I.R.; writing—review and editing, Y.-C.L., M.Y., and H.S. (Hiroshi Sato); visualization, H.S. (Hiroshi Sato); supervision, H.S. (Hiroshi Sato); project administration, H.S. (Hiroshi Sato); funding acquisition, H.S. (Hiroshi Sato). All authors have read and agreed to the published version of the manuscript.

Funding

This study was partially supported by a Grant-in-Aid for Food Science and Research 2019 from The Toyo Suisan Foundation (HS), JSPS Kakenhi (grant number 18K05995; HS), The Yanmar Environmental Sustainability Support Association (grant number KI0222007; HS), and a Grant-in-Aid for International Collaboration Research in Asia 2023 from the Heiwa Nakajima Foundation (M.Y., Y.C.L., and H.S.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Myxosporean specimens collected in this study are deposited in the Meguro Parasitological Museum, Tokyo, Japan, under collection nos. 21656–21658. The nucleotide sequence obtained in this study is available from the DDBJ/EMBL/GenBank databases under accession no. LC544125–LC544127. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We are indebted to the staff of Gifu World Fresh Water Aquarium (Aqua Totto Gifu) for kindly providing the preserved fish materials for this study. Their daily health checks and dedicated care of the fish under their supervision enabled freshly dead samples to be promptly available for parasitological examination. We thank the anonymous reviewers for their careful reading of our manuscript and their many insightful comments and suggestions, which have helped improve the quality of our manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Life 14 00974 g007
Figure 7. Maximum likelihood phylogenetic tree based on the SSU rDNA sequence of representative myxosporeans of the Bivalvulida. Species names are followed by host fish species, DDBJ/EMBL/GenBank accession numbers in parentheses, and country names as the collection localities. Abbreviations of country names: BR, Brazil; CA, Canada; CN, China; CZ, Czech Republic; DE, Germany; HU, Hungary; IN, India; IS, Iceland; JP, Japan; MR, Mauritania; KR, Korea; MY, Malaysia; PL, Poland; PT, Portugal; TN, Tunisia; and US, United States. Sequences newly obtained in this study are marked with gray background.
Figure 7. Maximum likelihood phylogenetic tree based on the SSU rDNA sequence of representative myxosporeans of the Bivalvulida. Species names are followed by host fish species, DDBJ/EMBL/GenBank accession numbers in parentheses, and country names as the collection localities. Abbreviations of country names: BR, Brazil; CA, Canada; CN, China; CZ, Czech Republic; DE, Germany; HU, Hungary; IN, India; IS, Iceland; JP, Japan; MR, Mauritania; KR, Korea; MY, Malaysia; PL, Poland; PT, Portugal; TN, Tunisia; and US, United States. Sequences newly obtained in this study are marked with gray background.
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Table 3. Myxidium spp. resembling M. salvelini in spore shape and dimension a.
Table 3. Myxidium spp. resembling M. salvelini in spore shape and dimension a.
SpeciesHost FishLocation in HostLocalitySLSWSTPCLPCWNTNVSReference
M. salveliniOncorhynchus masou (syn. Oncorhynchus masou ishikawae)Urinary BladderJapan (Gifu)13.6–16.7 (15.0)4.9–6.6 (5.7)—4.0–5.2 (4.4)2.1–2.8 (2.5)4–55–7Present Study
M. salveliniSalvelinus alpinus (L., 1758); Salvelinus leucomaenis (Pallas, 1814); Oncorhynchus mykiss (Walbaum, 1792) (syn. Salmo mykiss Walbaum, 1792)Urinary bladder and uretersBasin of Kamchatka River12.0–16.05.2–6.44.6–6.04.6–5.32.7–3.5—ca. 6[22]
M. americanum Kudo, 1920Apalone spinifera
(Lesueur, 1827) (syn. Trionyx spinifera Lesueur, 1827)		Urinary tubules (Kidney)USA (Illinois)15–165.5–6—43.538–10[44]
M. carinae Alvarez-Pellitero et al., 1983Luciobarbus bocagei (Steindachner, 1864) (syn. Barbus bocagei Steindachner, 1864)Gall bladderSpain12.0–14.8 (13.4)5.0–7.0 (6.0)5.5–8.0 (6.7)3.0–5.3 (4.5)2.8–4.6 (3.7)4–510–11/8–9[45]
M. clariae Landsberg, 1987Clarias gariepinus Burchell, 1822 (syn. Clarias lazera Valenciennes, 1840)Gall bladderIsrael13.4–15.1 (14.3)4.5–6.0 (5.3)—3.6–4.8 (4.2)3.0–3.8 (3.3)5–67–9[46]
M. oncorhynchiOncorhynchus masou (syn. Oncorhynchus masou masou)Gall bladderJapan (Hokkaido)11–125–83–53–4 in diameter (oval to globular)—ca. 5[43]
M. rimskykorsakowiPerccottus glenii Dybowski, 1877Urinary bladderAmur basin12.0–13.06.5–7.05.04.53.0–3.5—ca. 5[22]
M. lentiforme (syn. Myxidium fusiformis Fujita, 1927)Anguilla japonicaKidneyJapan (Shiga)19544———[42,47]
M. sinilabi Feng et Xiao, 1998Decorus rendahli (Kimura, 1934) (syn. Sinilabeo rendahli rendhali (Kimura, 1934); Pseudogyrinocheilus prochilus (Sauvage et Dabry de Thiersant, 1874) (syn. Semilabeo prochilus (Sauvage et Dabry de Thiersant, 1874)Gall bladderChina15.5–18.0 (16.0)5.0–7.0 (6.0)—3.5–5.5 (5.0)3.0–4.1 (4.0)47–10[23]
M. striatusi Sarkar, 1982Channa striata (Bloch, 1793) (syn. Ophicephalus striatus Bloch, 1793)Gall bladderIndia11.1–18.7 (14.5)4.7–7.0 (5.6)—3.7–5.6 (4.5)2.8–3.7 (3.0)——[48]
M. truttae Lèger, 1930Salmo trutta L., 1758 (syn. Salmo trutta fario L., 1758)Gall bladderFrance11–127–7.3—3.7 in diamer in diameter (oval)——[14]
M. uchiyamaeAnguilla japonicaKidneyJapan (Shiga)13.5866.5———[42]
a Abbreviation: SL, spore length; SW, spore width; ST spore thickness; PCL, polar capsule length; PCW, polar capsule width; NT, number of coil turns (polar tubles); and NVS, number of vulvar striations. All measurements are expressed in micrometers (µm) unless otherwise stated. Ranges with the means in parentheses are presented. For M. flavus, means and standard variation are shown in parentheses.
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Sekiya, M.; Sakai, H.; Li, Y.-C.; Rosyadi, I.; Yunus, M.; Sato, H. Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan. Life 2024, 14, 974. https://doi.org/10.3390/life14080974

AMA Style

Sekiya M, Sakai H, Li Y-C, Rosyadi I, Yunus M, Sato H. Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan. Life. 2024; 14(8):974. https://doi.org/10.3390/life14080974

Chicago/Turabian Style

Sekiya, Mariko, Haruya Sakai, Ying-Chun Li, Imron Rosyadi, Muchammad Yunus, and Hiroshi Sato. 2024. "Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan" Life 14, no. 8: 974. https://doi.org/10.3390/life14080974

APA Style

Sekiya, M., Sakai, H., Li, Y.-C., Rosyadi, I., Yunus, M., & Sato, H. (2024). Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan. Life, 14(8), 974. https://doi.org/10.3390/life14080974

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