短報
Microsporidium sp. in the Trunk Muscle of Coho Salmon Oncorhynchus kisutch Raised in Sea Cages in Japan
2025 年 60 巻 1 号 p. 20-23
詳細
2025 年 60 巻 1 号 p. 20-23
Sea-caged coho salmon Oncorhynchus kisutch in Tottori Prefecture, Japan, afflicted by microsporidiosis, exhibited clinical symptoms that include cyst formation and partial myoliquefaction of the trunk muscle. Analysis of small subunit ribosomal RNA gene sequences revealed that the isolated microsporidium showed high similarity to Microsporidium spp. of marine fishes, including M. seriolae, the causative agent of beko disease in Seriola fishes. Phylogenetic analysis placed the isolated microsporidium in the same clade as M. seriolae, indicating that the microsporidium infecting O. kisutch is an undescribed species closely related to M. seriolae. This is the first report of beko disease in salmonids.
Coho salmon (Oncorhynchus kisutch) is the most farmed salmonid species in Japan, with an annual production of 22,100 metric tons reported in 2023 (data from the statistics of Japan: https://www.e-stat.go.jp). Whereas other salmonid species such as rainbow trout (O. mykiss) and yamame trout (landlocked masu salmon O. masou masou) are mostly farmed in freshwater until harvest in Japan, O. kisutch is transferred from freshwater to seawater after smoltification from autumn to winter and farmed until harvest. As the water temperature > 22°C is lethal to O. kisutch (Richter and Kolmes, 2005), the farming period in sea cages is generally limited to a few to six months in Japan.
Recently, clinical symptoms, including whitish cyst formation and partial myoliquefaction in the trunk muscle, have been observed in O. kisutch after its transfer to seawater in Japan (Figs. 1A and 1B). Moreover, numerous microsporidian spores were detected in affected areas of the trunk muscles (Fig. 2). Although these symptoms resemble beko disease in Japanese amberjack Seriola quinqueradiata and greater amberjack S. dumerili caused by Microsporidium seriolae, no similar causal agents have been found in O. kisutch or other salmonid species to date.
In the present study, a phylogenetic analysis based on the small subunit ribosomal RNA (SSU rRNA) gene sequence was conducted to estimate the taxonomic position of an unidentified microsporidium (Microsporidium sp. COSTT1) in the trunk muscles of O. kisutch.
In May 2024, three spore samples were collected from cysts formed in the trunk muscles of three O. kisutch individuals that were maintained in the same sea cage in Tottori Prefecture. The fish were raised from artificial seed stock and had an average body weight of 1,119 ± 423 g (mean ± standard deviation). The water temperature at the time of sampling was 16.8°C. Prior to their transfer to sea cages in December 2023, no clinical symptoms such as cyst formation or myoliquefaction were observed during the freshwater phase.
The collected spores were imaged at ×1,000 magnification and their sizes were measured using NIS-Elements (Nikon Solutions Co., Ltd.). Thereafter, the spores were stored at -30°C for DNA extraction.
Molecular analysisDNA was extracted from spores derived from the three fish using a QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was amplified through PCR using the primers m350f (5′-CCAAGGACAGCAGCAGGCGCGAAA-3′) and m1492r (5′-AGCTGCCTTGTTACGACTT-3′) specific to a partial sequence of SSU rRNA gene. These primers were modified from 350f (5′-CCAAGGA(T/C)GGCAGCAGGCGCGAAA-3′) reported by Weiss and Vossbrinck (1998) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) reported by Weiss et al. (1994), based on the microsporidian DNA sequences available in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/).
The PCR cycles included a denaturation at 95°C for 10 s and extension at 59°C for 10 s, repeated 40 times, using KOD One PCR Master Mix -Blue (TOYOBO CO., LTD.). PCR was followed by the purification of the target amplicon performed using a Fast Gene Gel/PCR Extraction Kit (NIPPON Genetics Co, Ltd.). DNA was sequenced by Eurofins Genomics K. K., and the similarity of the acquired sequence to those available in the NCBI database was analyzed using a Basic Local Alignment Search Tool (BLAST).
Phylogenetic analysisPhylogenetic trees were constructed using the maximum likelihood (ML) and maximum parsimony (MP) methods. The 987 bp sequence of SSU rRNA gene was aligned with related 19 microsporidian species (accession numbers are shown in Fig. 3) using MUSCLE (Edgar, 2004). The ambiguous and gap regions were deleted, and the resulting SSU rRNA gene data set corresponding to each 778 bp sequence was used for phylogenetic analysis. Myosporidium merluccius was considered an outgroup. Bootstrapping analysis was conducted with 1,000 replicates. In the MP method, a max-mini branch-and-bound search was adopted to generate MP trees. In the ML method, a general time-reversible model with gamma-distributed rates and invariant sites (GTR + G + I) was adopted considering the Akaike information criterion (AIC). The number of discrete gamma categories was set to five. Default settings were used for the other parameters in each method. MEGA 11 was used for a series of analyses (https://www.megasoftware.net/).
The spores of Microsporidium sp. COSTT1 were oval to pyriform in shape (Fig. 2). The mean length and width of the spores (n=30) were 3.35 (2.93–3.76) and 2.15 (1.91–2.29) μm, respectively. These values were similar to those observed for M. seriolae spores, which were 3.3 (2.9–3.7; length) and 2.2 (1.9–2.4; width) μm, respectively (Egusa, 1982).
The DNA sequences were classified into three types characterized by gap formation and nucleotide substitutions (Fig. 3). Form 1 was obtained from all three samples, while forms 2 and 3 were obtained from one and two of the three samples, respectively. Form 1 was accordingly designated the target SSU rRNA gene sequence of Microsporidium sp. COSTT1 (accession number LC836060).
The SSU rRNA gene sequences of Microsporidium sp. COSTT1 obtained from O. kisutch showed high sequence similarity to Microsporidium spp. found in marine fishes, which include M. seriolae (Table 1). The obtained sequence showed the highest homology to Microsporidium sp. isolated from the striped jack Pseudocaranx dentex (100.0%), followed by that found in the Pacific bluefin tuna Thunnus orientalis (99.9%), and M. seriolae in S. dumerili (99.8%). Based on the phylogenetic analysis, Microsporidium sp. COSTT1 was placed in the same clade as M. seriolae in S. dumerili (LC704899) and S. quinqueradiata (AJ295322), with bootstrap values of 100 in both the maximum likelihood and maximum parsimony methods (Fig. 4). Molecular analyses indicate that Microsporidium sp. COSTT1 is an undescribed species belonging to the genus Microsporidium, which is closely related to M. seriolae. This is the first report of beko disease-like microsporidiosis in salmonids.
| Microsporidium species (accession no.) | Host | Percentage identity (bp matched/compared) | ||
|---|---|---|---|---|
| Microsporidium sp. | (LC556109) | Pseudocaranx dentex | 100.0 | (711/711) |
| Microsporidium sp. | (LC556102) | Thunnus orientalis | 99.9 | (744/745) |
| Microsporidium seriolae | (LC704899) | Seriola dumerili | 99.8 | (985/987) |
| Microsporidium sp. | (LC556103) | Thunnus orientalis | 99.7 | (720/722) |
| Microsporidium sp. | (LC556105) | Thunnus orientalis | 99.6 | (742/745) |
| Microsporidium sp. | (LC556104) | Seriola quinqueradiata | 99.6 | (742/745) |
| Microsporidium sp. | (LC556101) | Seriola dumerili | 99.6 | (742/745) |
| Microsporidium sp. | (LC556100) | Coryphaena hippurus | 99.6 | (742/745) |
| Microsporidium sp. | (LC556107) | Seriola quinqueradiata | 99.5 | (741/745) |
| Microsporidium sp. | (EU871680) | Verasper variegatus | 99.4 | (894/899) |
| Microsporidium sp. | (LC556108) | Pseudocaranx dentex | 99.2 | (702/708) |
| Microsporidium sp. | (GU124636) | Thunnus orientalis | 99.1 | (891/899) |
| Microsporidium sp. | (LC556106) | Seriola dumerili | 98.9 | (741/745) |
| Microsporidium seriolae | (AJ295322) | Seriola quinqueradiata | 98.7 | (888/900) |
| Microsporidium sp. | (AJ295323) | Pagrus major | 98.4 | (886/900) |
Given the southern limit of the geographical distribution of O. kisutch (Sandercock, 1991), this species does not appear to be the original host of Microsporidium sp. COSTT1. In Japan, O. kisutch is not naturally distributed in Tottori Prefecture and is captured only occasionally in Hokkaido. Furthermore, the likelihood of the introduction of Microsporidium sp. COSTT1 via O. kisutch is considered extremely low, as the O. kisutch was of artificial seed origin and had been raised in freshwater prior to transfer to sea cages in the affected area. The prevalence of Microsporidium sp. COSTT1 in wild fish in the area in which O. kisutch is farmed has not been sufficiently determined and, therefore, warrants further study.
In addition to Microsporidium sp. COSTT1, undescribed microsporidia, similar to M. seriolae, have been observed in various marine fish species, such as P. dentex, T. orientalis, red sea bream Pagrus major, and spotted halibut Verasper variegatus (Mekata et al., 2021; Yokoyama et al., 2008; Zhang et al., 2010). In this study, limited to SSU rRNA gene-based phylogenetic analysis, the homology of Microsporidium sp. COSTT1 to M. seriolae or the undescribed Microsporidium spp. remains unknown. Further phylogenetic analysis of other DNA sequences, such as those of the large subunit rRNA gene and the internal transcribed spacer (ITS), can clarify the correlation among Microsporidium sp. COSTT1, M. seriolae, and undescribed Microsporidium spp.
Salmonid microsporidiosis explored in the present study may become a serious concern with the increasing sea-cage salmon farming involving O. kisutch and O. mykiss in Japan. Although some cysts in S. quinqueradiata mildly infected by M. seriolae disappeared in the following year (Yokoyama et al., 2011), similar recovery is not expected in salmonid species owing to their shorter farming period in seawater, which is limited by the water temperature. Recently, the efficacy of the oral administration of benzimidazoles, such as albendazole and febantel, used to treat M. seriolae infection in Seriola fishes was demonstrated (Yanagi et al., 2021); albendazole has been approved for treating M. seriolae infection in Perciformes fishes in Japan. Although these antiprotozoal drugs have not yet been approved for salmonids, they can be considered a potentially effective measure for treating salmonid microsporidiosis.
The authors are deeply grateful to the staff of the Tottori Prefectural Fisheries Fish Farming Center and the Oita Marine Biological Technology Center, Nissui Corporation, for their assistance throughout the experiment.
