<i>Ichthyobodo</i> Infection Status of Juvenile Chum Salmon in the Chitose River, Japan, During Stocking

Ichthyobodo is a complex of species known to infect the skin and gills of various fish hosts (Todal et al. 2004; Isaksen et al. 2012). Severe Ichthyobodo infections can lead to skin or gill damage, resulting in a disease known as ichthyobodosis (Urawa et al. 1998). This disease has been linked to fish mortalities, particularly among hatchery-reared fish (Urawa 1993, 1995; Mizuno et al. 2017). In northern Japan, chum salmon Oncorhynchus keta (Walbaum, 1792) is reared for stocking at a large scale, and ichthyobodosis caused by Ichthyobodo salmonis has been a major problem for years (Mizuno et al. 2017). The sources of infection in hatchery fish are assumed to be both wild juvenile and adult chum salmon returning to the river to spawn (Mizuno et al. 2017). However, it is still not clear how the parasites are transmitted from wild to hatchery fish, and the chronological progression of infection in wild juvenile fish in rivers is unknown. Moreover, this lack of information currently applies to the relationships between other hatchery-reared salmonids and Ichthyobodo species on a global scale. Therefore, the objective of this study was to ascertain the temporal parasitic conditions of juvenile chum salmon in streams located upstream of hatchery sources and downstream of stocked streams. The release of hatchery fish for the purpose of stocking occurred from March 1 to April 23, 2024 (Figure 1). Concurrently, juvenile chum salmon were collected from February 20 to May 14, 2024 using a backpack electrofisher (Model 12 B; Smith-Root Inc., Vancouver, WA, USA). The sampling sites were located either upstream (“upper” site) or downstream (“lower” site) of the hatchery (Chitose Salmon Field Station) along the Chitose River in southwestern Hokkaido, northern Japan (Figure 2). The collected fish were immediately euthanized by spiking. Mucus from the dorsal fin and the area around its base was scraped directly onto a glass slide, after which the fork length was measured to the nearest 1 mm. These smear samples were air-dried and then stained with Diff-Quik (Sysmex, Kobe, Japan) at the laboratory of the Salmon Research Department of the Fisheries Resources Institute, Japan Fisheries Research and Education Agency (Hokkaido, Japan). Ichthyobodo cells were examined under a light microscope at 1000× magnification. The fish from the lower site, where there were both hatchery and wild fish, were distinguished by checking for artificial thermal marking of the otolith (Volk et al. 1999). As the number of fish released from the hatchery increases, hatchery fish become dominant over wild fish. Therefore, the number of samples was intentionally increased as the hatchery release progressed at the lower site to ensure that both wild and hatchery fish were collected (Table 1b). At the upper site, where only wild fish were expected to exist, five fish were collected on each sampling date (Table 1a). Water temperature was recorded at each site on each sampling date except for 20 February (Table 2). Beginning in late February, some fish at the hatchery began to scrape the bottom of the holding pond, which indicates a suspected infection by Ichthyobodo. Since then, hatchery staff have been monitoring the infection status of the fish in the hatchery and reported that the first infection of the year occurred on March 11. On March 13, 20 fish (Table 1c) from the pond were examined using the microscopy method described above, revealing that 90% of the fish were infected with Ichthyobodo. The morphometrics of the parasites detected on these fish from the hatchery were used for species identification because only a few specimens were collected from the river, and these were unsuitable for morphological observations. At the hatchery, the rearing water was supplied from either the river or a spring without filtration or UV treatment, and the effluent was directly discharged into the river. The infection status of each individual (infected or uninfected) was analysed by using a generalised linear mixed-effects model with a binomial probability distribution and logit link function where fork length, sampling point (upper or lower), origin (wild or hatchery) and temperature were fixed effects, and individual identification number was a random effect. The data from February 20 were excluded from this analysis because there were no temperature data. The models were estimated by using the “lme4” package in R (Bates et al. 2015). Furthermore, the correlation between the prevalence (the proportion of infected fish) of wild and hatchery fish on each sampling day was analysed using Pearson's correlation test. All statistical analyses were conducted using R 4.3.0 (R Core Team 2023), and the alpha level was set to p = 0.05. Morphological parameters of parasites were assessed according to Isaksen et al. (2011) and measurements were made from photographs by using a line drawn manually in ImageJ software (ImageJ 1.53 k; National Institutes of Health, Bethesda, MD, USA). Samples for morphometric measurements were selected from two heavily infected fish in the hatchery when principal characters (cytostomeal protrusion, nucleus, flagellar pocket) were recognisable. Morphometric parameters of the parasites were compared with the values for Ichthyobodo salmonis (Isaksen et al. 2011) by using the Student's t-test after a normality test. We used measurements from free-swimming forms from freshwater for analysis, and all statistical analyses were conducted by using R 4.3.0 (R Core Team, Vienna, Austria). Animals were handled in accordance with the guidelines for animal experiments at the Fisheries Resources Institute, Japan Fisheries Research and Education Agency (FRI-R6-19). The morphometrics of the parasites made it highly likely that they were Ichthyobodo salmonis (Table 3). While the prevalence of both wild and hatchery fish generally increased after first detection in April, the prevalence of hatchery fish was higher compared to that of wild fish (Figure 3). Meanwhile, no infections were detected at the upper site throughout the survey period. The generalised linear mixed-effects model showed that fork length and site did not have significant effects on the infection (F = 0.51, p = 0.09 and F = 0.00, p = 0.99, respectively). In contrast, origin and temperature both significantly affected the infection (F = 1.11, p = 0.00 and F = 7.23, p = 0.01, respectively). A significant correlation was identified between the prevalence of wild fish and that of hatchery fish (r = 0.98, p < 0.05). Overall, because the infection was not detected in juvenile chum salmon at the upper site, the main source of infection for chum salmon in the hatchery is presumed to be something else, such as the adult chum salmon in the river. The infections in the river were primarily found in hatchery fish, so the effects of stocking on the infection of wild fish seem to be limited. This implies that I. salmonis is basically sustained through hatchery fish and thus, reducing the infection in the hatchery is the priority for mitigating the impact of ichthyobodosis. The primary morphological characteristics of our specimens were equivalent to those of I. salmonis, although the nucleus area was comparatively small (Table 3). In addition, a previous study reported that Ichthyobodo infections in chum salmon are mostly caused by I. salmonis in Hokkaido, where we obtained the samples (Mizuno et al. 2022). Therefore, it is highly likely that the parasites investigated in this study were I. salmonis. The hatchery uses river water for rearing fish, so the water from the river upstream of the hatchery could be the source of infection. Although both juveniles and adult chum salmon were present upstream, the infection rarely occurred among upstream juveniles, judging from our results. Thus, the main source of the infection could be adult chum salmon, which migrate from the ocean and remain until around March (Ito and Nakajima 2009). The protocol employed in this study cannot detect infections if the number of parasites is too low; a PCR-based approach is needed for precise determination of infection status (Mizuno et al. 2017). Still, this study demonstrated that there were few fish infected with parasites from the upper site. Furthermore, given that adult fish are introduced into the hatchery from the river to produce hatchery fish, it is possible that these fish serve as a source of infection. Considering that the infection was not significantly affected by the sampling site, the effects of stocking on the infection of wild juvenile chum salmon in the river are limited. Nevertheless, there were also infected wild fish at the lower site in the river, and thus the infection might originate from the water in the upper river. Specifically, the observed correlation between wild and hatchery fish prevalence suggests that the presence of infected hatchery fish may be a potential contributing factor to infected wild fish in the river. Even though infection did not depend on fork length, the fish caught at the upper site tended to be smaller than those from the lower site (Table 1a,b). In addition, the significant effect of water temperature might reflect elapsed time, indicating that time spent in the river also has an impact on infection status. Yuhei Ogura: conceptualization, investigation, writing – original draft, methodology, visualization, writing – review and editing, formal analysis, data curation. I gratefully acknowledge Dr. Koh Hasegawa and Dr. Junpei Okado for help with sampling. The author declares no conflicts of interest. Data are available from the author upon reasonable request.

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