Abstract
Inodosporus fujiokai is a pathogenic microsporidian causing cyst formation within the muscles of salmonids. Confirmed infections exist in Oncorhynchus mykiss, O. masou ishikawae, and O. masou subsp. (Biwa trout), yet its full host range and differences in susceptibility among salmonid species remain undefined. This study assessed the susceptibility of various freshwater fish species to I. fujiokai, providing a risk assessment for local freshwater aquaculture in Shiga Prefecture, Japan. Salmonids (O. mykiss, O. masou ishikawae, O. masou subsp., and Salvelinus leucomaenis) and the cyprinid honmoroko Gnathopogon caerulescens were collectively exposed to infective spores of I. fujiokai from common prawn Palaemon paucidens, and the mortality and the infection levels were compared. Additionally, the susceptibility of ayu Plecoglossus altivelis (Plecoglossidae) was investigated using spore immersion and intubation methods. All tested salmonids exhibited high susceptibility, with S. leucomaenis being particularly vulnerable. Some fish died from the infection without evident cyst formation, suggesting the involvement of multiple pathogenic factors. Infection levels in G. caerulescens and P. altivelis were low, suggesting that I. fujiokai does not pose a significant risk to their aquaculture. These findings expand the understanding of the host range of I. fujiokai and offer valuable insights for risk assessment in freshwater aquaculture.
The muscle-infecting microsporidian, Inodosporus fujiokai, induces a fatal disease in farmed salmonids, characterized by the formation of spore-containing cysts in muscle tissues (Yamamoto et al., 2021, 2023). This microsporidian disease exhibited considerable lethality in certain fish species, with mortality rates reaching up to 100% in our experimental infection trials involving rainbow trout Oncorhynchus. mykiss, Biwa trout O. masou subsp., and amago trout O. masou ishikawae (Yamamoto et al., 2023; Kinami et al., 2025).
Previous studies have identified the common prawn Palaemon paucidens from Lake Biwa as the invertebrate host responsible for transmitting I. fujiokai to fish host (Yamamoto et al., 2021; Yanagida et al., 2023). While direct fish-to-fish transmission has not been achieved by feeding infected fish muscle tissue or by cohabitation with infected fish, fish acquire the infection either through ingestion of infected prawns, oral intubation or immersion using spores from P. paucidens (Kinami et al., 2025; Yamamoto et al., 2021, 2023).
Although salmonids appear particularly vulnerable to I. fujiokai, the precise host range of this microsporidian and the variability in susceptibility across fish species remain uncertain. Furthermore, the natural fish host for I. fujiokai in Lake Biwa remains unidentified. In this study, we aim to compare the susceptibility of various freshwater fish species farmed or bred in Shiga Prefecture to I. fujiokai, intending to provide a comprehensive risk assessment of this microsporidian disease for freshwater aquaculture.
Materials and Methods
Study animals
A total of six freshwater fish species were used across three experimental infection trials (Table 1): four salmonids (O. mykiss, O. masou ishikawae, O. masou subsp., and whitespotted char Salvelinus leucomaenis), honmoroko Gnathopogon caerulescens (Cyprinidae), and ayu Plecoglossus altivelis (Plecoglossidae). These fish species represent important targets for aquaculture and capture fisheries in Shiga Prefecture. All the fish utilized in the experiments were artificially bred at hatchery facilities, including Samegai Trout Farm of the Shiga Prefectural Fisheries Experiment Station (Shiga FES) and the Shingu Station of the Aquaculture Research Institute at Kindai University (Shingu ARIKU). The only exception was the P. altivelis juveniles used in Trial 3, which originated from larvae captured in Lake Biwa. Fish were raised from eggs in either underground water (Shiga FES) or stream water sourced about 150 km from Lake Biwa (Shingu ARIKU) and fed formulated pellet feeds.
Table 1. Summary of three trials experimentally infecting various freshwater fishes to
Inodosporus fujiokai | Fish species | Body weight
(g, mean ± SD) | Infection method & duration | Spore dose
(/mL or /fish) | Mortality | Cysts detection* | Infection rate
(Cysts + PCR) |
|---|
| Trial 1 | Oncorhynchus mykiss | 4.5 ± 1.1d | Immersion 4 h | 1.3 × 106 | 80.0% (12/15)c | 80.0% (12/15)b | 100% (15/15)a |
| 17.4–18.1°C | O. masou ishikawae | 6.4 ± 0.8a | Immersion 4 h | 1.3 × 106 | 93.3% (14/15)b | 66.7% (10/15)b | 100% (15/15)a |
| Shiga FES | O. masou subsp. | 3.9 ± 1.3d | Immersion 4 h | 1.3 × 106 | 85.7% (12/14)abc | 50.0% (7/14)b | 92.8% (13/14)a |
| Salvelinus leucomaenis | 5.9 ± 0.9ab | Immersion 4 h | 1.3 × 106 | 100% (15/15)a | 100% (15/15)a | 100% (15/15)a |
| Gnathopogon caerulescens | 5.4 ± 1.3c | Immersion 4 h | 1.3 × 106 | 5.9% (1/17)d | 5.9% (1/17)c | 47.1% (8/17)b |
| Trial 2 | O. masou ishikawae | 7.5 ± 1.6 | Immersion 4 h | 1.8 × 106 | 100% (19/19)a | 36.9% (7/19)a | 89.5% (17/19)a |
| 15.0–17.3°C | | | Intubation | 3.3 × 108 | 94.7% (18/19)a | 63.2% (12/19)a | 100% (19//19)a |
| Shingu, ARIKU | Plecoglossus altivelis1 | 7.7 ± 1.1 | Immersion 4 h | 1.8 × 106 | 0.0% (0/5)b | 0.0% (0/5)b | 0.0% (0/5)b |
| | | Intubation | 3.3 × 108 | 0.0% (0/8)b | 0.0% (0/8)b | 0.0% (0/8)b |
| Trial 3 | O. mykiss tank 1† | 4.6 ± 1.3a | Immersion 1 h | 1.9 × 106 | 100% (26/26)a | 57.7% (15/26)a | 96.2% (25/26)a |
| 17.6–18.8°C | tank 2† | 4.4 ± 1.1a | Immersion 1 h | 1.9 × 106 | 100% (27/27)a | 85.2% (23/27)b | 96.3% (26/27)a |
| Shiga FES | P. altivelis2 tank 1 | 2.9 ± 0.6b | Immersion 1 h | 1.9 × 106 | 9.4% (3/32)b | 9.4% (3/32)c | 15.6% (5/32)b |
| tank 2 | 3.1 ± 0.9b | Immersion 1 h | 1.9 × 106 | 3.7% (1/27)b | 11.1% (3/27)c | 25.9% (7/27)b |
* Found in the trunk muscle.
† Tank 1 and 2 represent duplicate of the same treatment.
1. Amphidromous type, 2. Landlocked type from Lake Biwa.
The information below each trial name indicates the water temperature during the experiment and the trial locations: FES = Fisheries Experiment Station; ARIKU = Aquaculture Research Institute, Kindai University.
Numbers in parentheses indicate actual counts.
Different superscript letters indicate statistical difference within each trial (p < 0.05).
Trial 1 assessed the susceptibility of the salmonids and Lake Biwa’s indigenous small cyprinid G. caerulescens to I. fujiokai. Trial 2 and Trial 3 focused on the susceptibility of P. altivelis, a species of significant importance in the local aquaculture. In the latter two trials, different types of P. altivelis were used: hatchery-reared juveniles from amphidromous brood fish in Trial 2 (referred to hereafter as amphidromous type) and a landlocked type from Lake Biwa in Trial 3. The amphidromous type juveniles were raised from eggs in land-based tanks at the Shingu ARIKU. The landlocked type juveniles, which originated from larvae (approximately 0.5 g) captured in Lake Biwa, were raised in ponds at the Shiga FES for about a half year using underground water. In all trials, either O. mykiss or O. masou ishikawae served as a positive control to ensure the experimental infection was successfully implemented.
Palaemon paucidens individuals infected with I. fujiokai were collected from Lake Biwa to serve as a source of the fish-infective microsporidian spores. Prawns exhibited an opaque appearance were selectively captured with a dipnet and kept alive until the experiment commenced (Yanagida et al., 2023). The spore suspension for each infection trial was prepared as previously described (Kinami et al., 2025). Briefly, a portion of the tail muscle tissue from each prawn was inspected under a light microscope and the tissues of individuals infected with I. fujiokai were collectively homogenized in phosphate-buffered saline to create spore suspension.
Experimental infection of I. fujiokai to freshwater fishes
Experimental infection trials of I. fujiokai in freshwater fishes were conducted at the Shiga FES and the Shingu ARIKU using previously established methods of spore immersion and gavage intubation methods (Kinami et al., 2025) (Table 1). Spore immersion facilitates the simultaneous infection of multiple fish species, allowing for direct comparison among them, whereas gavage intubation ensures the ingestion of relatively large amount of spores. The detailed procedures for these two infection methods were in accordance with the methodologies outlined by Kinami et al. (2025). Specifically, fish species were either collectively immersed in a 50 L cylindrical plastic container (42 cm diameter × 40 cm depth) filled with 25 L of water containing I. fujiokai spores derived from infected P. paucidens for up to 4 h or subjected to individual oral intubation with the spore suspension. Spore dosages were determined according to Kinami et al. (2025) and detailed in Table 1. Post-infection, each fish species was maintained in separate tanks (65 L or 100 L depending on trial), with flow-through underground water. To reduce fish density, fish in Trial 3 were split into two tanks (Table 1). Fish were fed pellet feeds, with mortality monitored daily. Dead fish were stored in individually labeled plastic bags in a freezer. The experiment concluded approximately a month after the infection, when microsporidian cysts were verified in the positive control group, and significant mortality due to the disease was noted. At the end of the experiment, all remaining fish were collected and stored frozen for further examination.
Examination of I. fujiokai in fishes
Each fish, whether deceased or surviving, underwent microscopy to check for microsporidian infection. Defrosted fish samples were filleted into three pieces, and the entire skinned left-side trunk muscle was flattened between two glass plates for detailed examination of cysts under a dissecting microscope. The heart was also examined. Upon detecting cysts, a tissue subset was inspected under a light microscope to confirm the presence of microsporidian spores. Fish with microsporidian cysts in muscle tissues were identified as infected. The severity of infection was semi-quantified by categorizing cyst density into five levels: 0 (no visible cysts found), I (a few cysts per ×40 microscopy field), II (approximately 5–10 cysts per field), III (several dozens of cysts per field), and IV (numerous cysts covering the entire tissue). In cases without detectable cysts, approximately 30 mg of homogenized trunk muscle tissue underwent I. fujiokai-specific PCR (Kinami et al., 2025). Molecular confirmation of I. fujiokai was also performed on haphazardly selected three cyst-harboring individuals from each fish species using the same PCR. To verify that the experimental fish were not infected prior to the experiment, 10 individuals from the same production batch were also tested for I. fujiokai via the specific PCR.
Statistical analysis
To assess interspecies susceptibility to the microsporidian disease, Kaplan-Meier survival analysis was conducted using mortality data (Dudley et al., 2016). This analysis involved a log-rank test, followed by post hoc pairwise comparisons. Differences in body weight among the tested fish and the correlation between body size and infection level within each species were analyzed using ANOVA with Tukey’s-HSD and Spearman’s correlation analysis, respectively. Cyst intensity levels, ranging from 0 to IV, between fish species were compared using a Z-test with Bonferroni correction. The proportions of individuals testing positive for cysts and through PCR were compared using the Chi-square test. Statistical analyses were performed using EZR, an R-based statistic software, and JMP ver. 11 (SAS Institute), with a significance level set at 0.05.
Results
The results of the three trials are summarized in Table 1. In Trial 1, all four tested salmonids exhibited high levels of infection with I. fujiokai, with the pathogen being detected in every individual either by microscopy or PCR, except for one O. masou subsp. individual. Mortality among these salmonids began to increase approximately 2 wk after infection, reaching 80–100% in the subsequent 2 wk (Fig. 1). Some dead O. mykiss displayed symptoms of hypoxia, such as opened mouth and opercula. However, these symptoms were less pronounced in other species, with many becoming lethargic, lying at the tank bottom, and eventually dying. No other pathogens beside I. fujiokai were noticed in the dead fish through brief microscopy. Kaplan-Meier survival analysis indicated significantly poor survivals in O. masou ishikawae (p = 0.0004) and S. leucomaenis (p = 0.0165) compared to O. mykiss. The mortality between O. mykiss and O. masou subsp., as well as between the two O. masou variants, were statistically similar (p > 0.05) (Table 1, Fig. 1).
Fig. 1. Kaplan-Meier survival curves in Trial 1 after exposure of Inodosporus fujiokai spores from Palaemon paucidens to different fish species: Oncorhynchus mykiss (Rainbow, gray broken line), O. masou ishikawae (Amago, dotted line), O. masou subsp. (Biwa, gray solid line), Salvelinus leucomaenis, (Char, black broken line), and Gnathopogon caerulescens (Honmoroko, black solid line).
Despite the high infection prevalence exceeding 92% among the four salmonid species in Trial 1, the intensity of infection, as indicated by cyst density, varied between species (Fig. 2). Microsporidian cysts were not microscopically detected in some individuals of O. mykiss (20%) and the two O. masou variants (33% and 50%), yet all individuals of S. leucomaenis exhibited high numbers of cysts (level III or IV). Statistical analysis revealed no significant differences in cyst levels among the three Oncorhynchus species (Z test, p > 0.05). No clear association was found between cyst density and fish size for any of the species. Cysts were observed in the hearts of some individuals, although this finding was not consistent across all cases, possibly due to factors such as small heart size and tissue degradation from defrosting, which made precise determination of infection prevalence or intensity in the heart difficult.
Fig. 2. Proportions of individual fish possessing microsporidian cysts in their trunk muscle in Trial 1; white bars represent individuals with no visible cysts (level 0) and light gray, gray, dark gray, and black bars represent cyst intensity with level I to IV, respectively. Biwa, Oncorhynchus masou subsp.; Amago, O. masou ishikawae; Rainbow, O. mykiss; Char, Salvelinus leucomaenis; Honmoroko, Gnathopogon caerulescens. Different letters indicate significant differences between fish species for each cyst intensity (p < 0.05).
In Trial 1, I. fujiokai infection was also confirmed in G. caerulescens, marking the first instance of infection in a non-salmonid fish. However, the infection in G. caerulescens appeared milder compared to that in salmonids, with only 1 out of 17 individuals (5.9%) displaying cysts and a total of 8 individuals (47.1%) testing positive by PCR (Table 1). The cyst-possessing individual, which presumably died from the infection, also exhibited cysts in the heart. In Trial 2, no I. fujiokai infection was detected in the amphidromous type of P. altivelis, regardless of the spore immersion or intubation methods. The high infection and mortality rates observed in the positive control, O. masou ishikawae confirmed successful implementation of both infection methods (Table 1, Fig. 3). Conversely, Trial 3 demonstrated some infections in the landlocked type P. altivelis, though at significantly lower rates (15.6% and 25.9%) compared to O. mykiss (96.2% and 96.3%, Table 1) (Chi-square test, p < 0.05). Mortality rates for O. mykiss in Trial 3 reached 100%, with most deaths occurring around 2 wk post-infection (Fig. 4). Out of 59 P. altivelis from two tanks, only 4 died (Table 1), but the precise cause of death remains uncertain because no cysts were found in these dead fish, even which were tested positive for PCR in the trunk muscle. Microsporidian cysts were confirmed in the trunk muscle of a total of 6 surviving P. altivelis individuals, but none were detected in the heart. Furthermore, 5 surviving P. altivelis individuals from both tanks tested positive for PCR in the heart (9.1%). This rate is considerably lower compared to the PCR positive rate in the trunk muscle, which stood at 43.6% (Chi-square test, p < 0.05).
Fig. 3. Kaplan-Meier survival curves in Trial 2 after exposure of Inodosporus fujiokai spores from Palaemon paucidens through immersion (black lines) or intubation (gray lines) method to different fish species: Oncorhynchus masou ishikawae (Amago, solid lines) and Plecoglossus altivelis (Ayu, broken lines, on top of each other).
Fig. 4. Kaplan-Meier survival curves in Trial 3 after exposure of Inodosporus fujiokai spores from Palaemon paucidens to Oncorhynchus mykiss (Rainbow, solid lines) and Plecoglossus altivelis (Ayu, broken lines). Each line represents duplicate tank for each fish species.
Discussion
The present study supports our earlier findings that salmonids are highly susceptible to I. fujiokai infection (Kinami et al., 2025; Yamamoto et al., 2023). To reduce disease risk in trout farms, it is advisable to avoid using raw prawns as feed or freezing the prawn before feeding them to fish (Yamamoto et al., 2021). Nonetheless, the unintentional introduction of infected P. paucidens, whether alive or dead, could trigger a local outbreak due to potential waterborne transmission from spores excreted from infected prawns or fish that have consumed infected prawns (Kinami et al., 2025; Yamamoto et al., 2021). This indicates the importance of maintaining vigilance in salmonid rearing facilities. The notably high prevalence, intensity, and mortality of the infection observed in S. leucomaenis highlight the need for close monitoring of this species for signs of the disease.
The present study further clarifies interspecific variations in susceptibility to I. fujiokai among salmonids, building on our preliminary findings which suggested a potential difference in susceptibility between O. mykiss and O. masou subsp., based on observed variations in mortality and cysts occurrence in trunk muscle (Yamamoto et al., 2023). The previous study, however, made direct comparison challenging due to separate tank infections via prawn feeding. In contrast, the present study adopted unified spore immersion infection method, enabling direct interspecific comparisons. Despite minor size differences among tested fish species, no size-related correlation of infection levels was found, suggesting that observed variations in susceptibility are likely inherent to each species. Similar interspecific variations have also been documented for Kabatana takedai, a microsporidian closely related to I. fujiokai, with O. mykiss showing greater vulnerability than O. masou (Miyajima et al., 2007). Despite these observations, the specific mechanisms underlying these interspecific variations in resistance to microsporidia, even among closely related fish species, remain undefined. Further physiological and immunological research could reveal these mechanisms, potentially leading to innovative control strategies against fish microsporidian diseases.
Previously, we hypothesized that the primary pathology of I. fujiokai involves cyst development in the heart, causing impaired cardiac function and leading to mortality from hypoxia (Yamamoto et al., 2023). This hypothesis seems relevant to O. mykiss, which exhibited hypoxia symptoms in this study. However, individuals from the two O. masou variants showed high mortality without obvious cysts in the trunk muscle or heart. Furthermore, these species, along with S. leucomaenis, did not display clear hypoxic symptoms. The precise cause of mortality in these cases remains uncertain, suggesting the possibility of multiple pathogenic factors associated with I. fujiokai infections that might explain the observed interspecific susceptibility variation. Further research is necessary to understand the exact pathology of I. fujiokai infections across different fish species.
This study has also demonstrated that I. fujiokai is capable of infecting non-salmonid fishes, namely the cyprinid G. caerulescens and the plecoglossid P. altivelis, marking the first documented cases of such infections. The observed low infection levels in these species suggest they are more resistant to the pathogen compared to salmonids. Also, given the use of unnaturally high spore doses for the infection challenges in this study, the actual risk of I. fujiokai-related disease in farmed G. caerulescens and P. altivelis is likely minimal.
There seems to be a difference in susceptibility between the amphidromous and landlock types of P. altivelis. The amphidromous type showed no infections even when direct oral spore intubation was conducted, suggesting significant resistance to the pathogen. Conversely, the landlocked type did exhibit infectious though at a low level. This variation may partially be due to the limited numbers of amphidromous P. altivelis individuals used in the experiment, with n = 5 and 8 for immersion and intubation experiments, respectively. Further research is necessary to conclusively ascertain if physiological and genetic differences between these types influence their susceptibility to I. fujiokai.
Microsporidia are generally known to exhibit host specificity with narrow ranges (Shaw and Kent, 1999; Willis and Reinke, 2022). However, the ability of I. fujiokai to infect multiple fish families suggests a broader host range for this species, unlike its close relative K. takedai, which has been reported only from salmonids (Miyajima et al., 2007). This broader host range aligns with the reports for some Pleistophora spp., which infect a variety of hosts across different fish families. For example, P. hyphessobryconis has been documented in more than twenty fish species such as tetras (Characiformes), zebrafish Danio rerio (Cypriniformes), and medaka Oryzias latipes (Beloniformes), while P. finisterrensis has been reported to infect blue whiting Micromesistius poutassou (Gadiformes) and turbot Scophthalmus maximus (Pleuronectiformes) (Kent et al., 2014; Fujiwara et al., 2024). The ability of I. fujiokai to infect species from at least three fish families, Salmonidae, Cyprinidae, and Plecoglossidae, illustrates that this species may also be considered a generalist, exhibiting a high degree of variability in susceptibility across fish hosts.
The natural fish host for I. fujiokai in Lake Biwa remains unidentified. Considering the low abundance of salmonids in the lake and the lack of reported microsporidian infections in wild O. masou subsp., it is plausible that I. fujiokai may depend on hosts other than salmonids to sustain its life cycle in the lake. However, G. caerulescens and P. altivelis are unlikely natural hosts in the lake, due to their specific feeding habits and habitat preferences. Their limited interaction with P. paucidens further diminishes the likelihood of natural infection. Consequently, the risk of I. fujiokai infections spreading through the translocation of wild G. caerulescens and P. altivelis appears to be minimal; however, wild fish surveillance is necessary to ensure the absence of infection risk. It is possible that other fish species, especially those that routinely consume prawns or share a benthic habitat, could act as natural hosts for this microsporidian. To further investigate this, we intend to perform experimental infections on a broader range of fish species residing in Lake Biwa. These studies, along with continuous surveillance of wild fish populations aided with the development of a sensitive I. fujiokai specific real-time PCR assay, will deepen our insight into the ecological dynamics and host preference of I. fujiokai, shedding light on its natural reservoirs and transmission pathways in the lake.
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