2025 Volume 60 Issue 2 Pages 50-60
In October 2022, a disease associated with a sudden increase in morbidity and mortality occurred among one-year-old red-spotted grouper Epinephelus akaara cultured in land-based tanks in Tottori Prefecture, Japan. Daily monitoring of the disease showed that fish losing equilibrium at the bottom of the tanks appeared one after another, and chronic mortality continued over a period of three months. All moribund fish examined had a bloated swimbladder and were heavily infected with monogeneans of the genus Pseudorhabdosynochus on the gills (P. epinepheli and P. lantauensis), with infection intensities up to over one thousand worms per fish. In vitro and in vivo experiments showed that hydrogen peroxide was effective, removing over 98% of the worms through bathing in 350 ppm for 60 min or 700 ppm for 30 min at around 20°C. Histopathological examination revealed that the monogenean infection did not induce significant host reactions in the gills. In contrast, lesions were found in the brain and retina, and morbid fish tested positive for nervous necrosis virus (NNV) by RT-PCR. These results suggested that the morbidity and mortality associated with loss of equilibrium were caused by infection with NNV rather than that with the monogeneans.
The coastal area of Tottori Prefecture experiences strong winds and waves, which are the characteristics of the Japan Sea in winter, with 60% of the coastline consisting of nearly straight sandy beaches with few coves. This unique geography renders the region unsuitable for offshore cage culture, negatively affecting the development of the aquaculture industry there. Since the 2010s, the prefecture has been promoting land-based aquaculture such as chub mackerel Scomber japonicus, olive flounder Paralichthys olivaceus, and red-spotted grouper Epinephelus akaara using underground seawater in coastal areas. In 2013, a private company in the prefecture launched a land-based aquaculture for red-spotted grouper, using a recirculating system with waste heat generated from the incineration of industrial wastes to regulate water temperature.
In October 2022, one-year-old red-spotted grouper started to show morbidity and mortality in the fish farm. The morbid fish lost their equilibrium, swimming upside down or lying on their side at the bottom of the tanks. Since the fish farm has never experienced such morbidity in the cultured grouper, we investigated the case and found a large numbers of monogeneans on the gills. Infection of the monogenean Pseudorhabdosynochus epinepheli on the gills was previously reported in broodstock of red-spotted grouper raised in land-based tanks (Isshiki et al., 2007). Similar to the study, we initially thought that the morbidity was caused by the severe monogenean infection. Therefore, we identified the monogeneans and performed in vitro and in vivo deworming tests. However, our further investigation suggested that another pathogen was likely the cause of the morbidity rather than the monogenean infection. This is a case report that the severe monogenean infection was not the direct cause of the morbidity observed in the cultured red-spotted grouper.
The red-spotted grouper in the fish farm originated from juveniles produced by the Tottori Prefectural Fish Stock Enhancement Association. In the hatchery, fertilized eggs were disinfected using electrolyzed seawater, and the hatched larvae were raised in ultraviolet (UV)-treated seawater. Ninety-eight days old fingerlings (total length: TL = 8.5 ± 5.6 cm, body weight: BW = 11.4 ± 2.2 g) were introduced into two land-based, 4 m3 circular tanks (Tank A and Tank B) in the fish farm on September 30, 2021. The fish farm features a recirculating aquaculture system (RAS), which was supplied with coral sand-filtered underground seawater (salinity; 33‰) without UV treatment. In the RAS, underground seawater pumped up from the coastal underground was supplied, and an equal amount of circulating water was drained out from the facility. The entire volume of rearing water in tanks was replaced 8.64 times per day. The water temperature was maintained at 24–26°C. Tank A and Tank B share the rearing water supplied by a single circulating system. Before the emergence of the morbid fish, Tanks A and B held 891 and 643 fish, respectively. In the fish farm, additional batches of red-spotted grouper were reared separately from the RAS of the two tanks and those fish showed no signs of morbidity.
Occurrence of morbidity and mortalityIn the fish farm, fish started to exhibit loss of equilibrium (morbid fish) in Tank A on October 3, 2022, approximately one year after the juveniles were introduced. A similar event occurred in Tank B on November 1, 2022. Morbid and dead fish were removed daily from each tank, and their numbers were recorded. The Kaplan-Meier method was used to calculate the cumulative probability rate of mortality in fish. In this study, this method was also applied to estimate cumulative morbidity, as morbid fish were removed daily together with dead fish.
Transportation of morbid fishLive fish that lost their equilibrium were transported multiple times to the Tottori Prefectural Fish Farming Center (TPFFC) between October 10 and December 9, 2022, without being sacrificed. The morbid fish were housed in two 300 L tanks (sampling tanks S1 and S2) with a continuous flow of seawater maintained at 20–22.5°C. The morbid fish in the sampling tanks S1 and S2 were from Tanks A and B in the fish farm, respectively. For the following examinations, fish were taken from the tanks S1 and/or S2.
Observation and identification of monogeneans on gillsSince a large number of monogeneans were observed on the gills in the morbid fish, eleven fish collected from Tanks A and B on October 5, October 26, and November 14 were examined (Table 1). The fish were anesthetized by 2-phenoxyethanol (Kanto Chemical Co., Inc.) for sampling the gills. Gills were separated, and each gill was placed in a petri dish filled with seawater, and the worms on the gill were counted under a stereo microscope. The numbers of monogenean in the whole or half set of gills were counted, and the infection intensity (i.e. the number of monogeneans in a single infected host) were estimated. For identification of the monogenean, specimens collected from a single fish from Tank A (n=107) were fixed in ammonium picrate-glycerin (AP-G). After being dehydrated and mounted in Canada balsam, selected AP-G fixed specimens (n=17) were morphologically examined and measured. Methods of measurement and terms of body parts of the parasites followed Justine (2009).
| Sampling date | Days after morbidity* | Tank | Fish condition | Number of fish | Total length (cm) | B ody weight (g) | Examination |
|---|---|---|---|---|---|---|---|
| 2022/10/3 | 0 | A | Morbid | 5 | 18.3–28.0 | 81.0–181.2 | Bacterial isolation |
| 2022/10/5 | 2 | A | Morbid | 5 | 22.8–26.3 | 133.8–283.3 | Monogenean observation |
| 2022/10/11 | 8 | A | Morbid | 2 | 18.0–23.5 | 110.8–188.7 | Bacterial isolation |
| 2022/10/26 | 23 | A | Morbid | 3 | 21.6–24.0 | 141.1–214.7 | Monogenean observation |
| 2022/11/4 | 32 | A | Morbid | 1 | 24.3 | 190.2 | NNV RT-PCR in kidney |
| 2022/11/14 | 13 | B | Morbid | 1 | 22.9 | 217.4 | Monogenean observation, Bacterial isolation, NNV RT-PCR in kidney |
| 2022/11/14 | 13 | B | Morbid | 2 | 18.9–25.7 | 105.3–220.0 | Monogenean observation, Bacterial isolation |
| 2022/12/9 | 38 | B | Morbid | 2 | 21.3–24.5 | 134.7–198.2 | Histopathology |
| 2022/12/9 | 38 | B | Morbid | 2 | 19.8–28.3 | 130.4–353.4 | NNV RT-PCR in brain |
| 2023/4/10 | 189 | A | Apparently healthy | 2 | 23.6–23.7 | 174.2–178.3 | Monogenean observation, NNV RT-PCR in brain |
Two morbid fish exhibiting loss of equilibrium, collected from tank B on December 9, 2022 were used for histopathological examination (Table 1). They were kept in the sampling tank S2 in TPFFC for 32 days without recovering from the morbidity. The fish were anesthetized by 2-phenoxyethanol for dissection, and the tissues and organs described below were excised, fixed in Davidson’s solution (3:2:1:3 mixture of 95% ethanol, commercial formaldehyde solution containing 37% formaldehyde and 8% methanol, glacial acetic acid and distilled water) for one day, dehydrated in an alcohol series, and embedded in paraffin as usual. Two sets of 3 μm-thick sections were prepared from each tissue block. The duplicated sections were stained with hematoxylin and eosin (HE) or May-Grunwald-Giemsa (MG), and examined under a microscope. The examined tissues included the brain, eye, gills, heart, liver, stomach, pyloric caeca, pancreas, intestine, spleen, kidney, swimbladder, and skin. For the brain, the mesencephalon, cerebellum, and medulla oblongata were observed.
Bacterial isolationBacterial isolation was performed in the morbid fish to assess whether a bacterial infection was associated with the morbidity. A total of ten morbid fish derived from Tanks A and B on October 3, October 11, and November 14 were examined as described in Table 1. Bacterial isolation was performed by streaking the kidney tissue onto TCBS agar supplemented with 2% NaCl, BHI agar, and nutrient agar, followed by incubation at 20°C for 48 h.
RT-PCR for nervous necrosis virus (NNV)Since viral nervous necrosis (VNN) was suspected based on the loss of equilibrium and tissue lesions in the morbid fish, an RT-PCR test for the viral agent was performed. A total of four morbid fish collected on November 4, November 14, and December 9 were examined as shown in Table 1. Total RNA was extracted from the kidney or brain tissue using Quick-DNA/RNA Pathogen Kit (Zymo Research). RT-PCR targeting T4 region of NNV RNA2 was performed with F2 (5′- CGT GTC AGT CAT GTG TCG CT -3′) and R3 (5′- CGA GTC AAC ACG GGT GAA GA-3′) primers (Nishizawa et al., 1995). Twenty-five microliters of the reaction mixture consisting of SuperScriptTM III One-Step RT-PCR system (Invitrogen) contained 2 μL RNA template and 500 nM of each primer. The amplification thermal cycling condition was as follows: reverse transcription at 55°C for 30 min, one cycle of 94°C for 2 min, and 40 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 1 min, followed by the incubation at 68°C for 5 min. The PCR amplicon was electrophoresed by 2% agarose gel supplemented with ethidium bromide and observed under an UV transilluminator. The amplicon (approximately 400 bp) was purified by illustra ExoProStar (GE Healthcare) and directly sequenced using the RT-PCR primers by Sanger sequencing services (Azenta Life Sciences).
Diagnosis of apparently healthy fish after ceasing the morbidityTo confirm the monogeneans parasitism and NNV infection after ceasing the morbidity, two apparently healthy fish were haphazardly sampled from Tank A on April 10, 2023 (Table 1), which was 189 days after the onset and 95 days after the cessation of morbidity. The monogeneans on the gills were examined and the NNV RT-PCR test was performed as described above.
Treatment tests for gill monogeneanTo treat the severe monogeneans infection on the gills, in vitro and in vivo deworming tests were performed according to the following methods. For the in vitro tests, eight worms attaching to a small piece of gill were used for each treatment group without species identification. The worms were exposed to 4 mL of six different reagents in 6-well plates: freshwater, 32‰ or 52‰ NaCl-added seawater (salinities of 62‰ or 82‰, respectively), 350 ppm or 700 ppm hydrogen peroxide (H2O2) in sea water, or 32‰ seawater (control). The experiment was performed at room temperature (20–22°C). The motility of the worms was observed under a dissecting microscope every 10 min for 70 min. Subsequently, all the worms, both attached to the gill piece, and those that had detached, were transferred to normal seawater (32‰) and observed for additional 30 min to assess whether they regained motility.
The morbid fish showing lost equilibrium kept in the sampling tanks S1 (18.5–25.3 cm TL, 81.6–292.0 g BW) and S2 (20.1–24.5 cm TL, 109.2–228.3 g BW) in TPFFC as described above were used for the in vivo deworming tests by hydrogen peroxide (H2O2) bath treatment. The in vivo tests consisted of two trials (Trial 1 and 2). Trial 1 consisted of five groups with different H2O2 concentrations and immersion durations, such as 158 ppm-60 min, 158 ppm-120 min, 350 ppm-60 min, 350 ppm-120 min, and no treatment (control). Trial 2 consisted of four groups with different H2O2 concentrations and immersion durations, such as 700 ppm-15 min, 700 ppm-30 min, 700 ppm-45 min, and no treatment (control). Three morbid fish were haphazardly selected for each treatment group. They were immersed in 30 L of each tested solution at 20.4–21.0°C with continuous aeration. After the treatment, fish from each treatment were immediately transferred to separate tanks with flowing seawater. After 24 hours, all fish were anesthetized by 2-phenoxyethanol and sacrificed to examine half set of gills to determine the monogenean infection as described above.
In Tank A of the fish farm, fish exhibiting lost equilibrium (morbid fish) started to be observed on October 3, 2022. The disease persisted until January 5, 2023. A total of 281 fish lost their sense of equilibrium over 95 days, with a cumulative morbidity of 38% (Fig. 1A). The morbid fish were removed daily, and the disease ceased three months after initiation. In Tank B, morbid fish appeared approximately one month after the onset in Tank A and increased in number at a faster rate than in Tank A. As a result, a total of 325 fish showed the same symptom over 66 days, which represented a 64% cumulative morbidity rate (Fig. 1A). In Tank B, the disease ceased after two months. The total numbers of dead fish in Tank A and B were 51 and 47, respectively, representing cumulative mortality rate of 10% and 12%, respectively (Fig. 1B).
All the morbid fish that were used for parasitological observation (n = 11) were severely infected with monogeneans on their gills. The intensity of monogenean infection in the examined fish from Tank A (n = 8) and Tank B (n = 3) ranged from 329 to 1,168 (mean ± SD = 719 ± 255.5) and from 248 to 330 (295 ± 42.4), respectively with a significantly higher intensity in Tank A (Mann-Whitney rank-sum test, p < 0.05).
The monogeneans were identified as the genus of Pseudorhabdosynochus by having a pair of squamodiscs in the haptor and a quadriloculate male copulatory organ. Furthermore, we found that there were two species (P. epinepheli and P. lantauensis) on the gills, distinguished by the shape of the vagina and squamodiscs (Beverley-Burton and Suriano, 1981; Justine, 2009). Measurements of the two monogenean species are given in Table 2. Pseudorhabdosynochus epinepheli had larger ventral and dorsal squamodiscs and larger row numbers of both squamodiscs than those of P. lantauensis (Figs. 2, 3). No fundamental differences were found between the present measurements and those of their original descriptions (Yamaguti, 1938; Beverley-Burton and Suriano, 1981). Among the 107 monogeneans morphologically examined in this study, 104 (97.2%) and 3 (2.8%) specimens were identified as P. epinepheli and P. lantauensis, respectively.
| P. epinepheli | P. lantauensis | |
|---|---|---|
| Body (Length) | 618–842 (15) | 655–667 (2) |
| Haptor (Width) | 170–236 (15) | 223–248 (2) |
| Male quadriloculate organ (Inner Length) | 58–69 (14) | 51–54 (2) |
| Vagina (Max Length) | 36–46 (15) | 46–52 (2) |
| Ventral hamulus (Outer Length) | 38–42 (15) | 47 (2) |
| Ventral hamulus (Inner Length) | 31–35 (15) | 40–41 (2) |
| Dorsal hamulus (Inner Length) | 34–38 (15) | 42–44 (2) |
| Dorsal hamulus (Outer Length) | 22–26 (15) | 28 (2) |
| Lateral bar (Length) | 46–64 (15) | 63–65 (2) |
| Ventral bar (Length) | 63–84 (15) | 95–100 (2) |
| Ventral squamodisc (Length) | 76–108 (12) | 52–60 (2) |
| Ventral squamodisc (Width) | 65–83 (12) | 44–45 (2) |
| Ventral squamodisc (Row number) | 14–18 (12) | 10 (1) |
| Dorsal squamodisc (Length) | 68–98 (8) | 46–71 (2) |
| Dorsal squamodisc (Width) | 56–81 (9) | 38 (2) |
| Dorsal squamodisc (Row number) | 13–17 (7) | 9 (1) |
Infection of the monogeneans was observed in clusters which consisted of two to four individuals attaching side by side on the same gill filaments (Fig. 4). Although the gills were infected with numerous monogeneans, no significant host reactions such as proliferation of epithelial cells or hypertrophy of filaments were observed. We found histopathological lesions in the central nervous system; clusters of microglia exhibiting foci of glial nodules were found scattered in the brain, with many in the tegmentum and medulla oblongata, fewer in the corpus cerebelli, and almost none in the optic tectum, valvula cerebelli and inferior lobe (Fig. 5). In addition, the arrangement of cell layers in the retina of one individual was disordered, and vacuoles were frequently observed (Fig. 6). The lesions in the brain and retina were moderate and no bacteria and parasites were observed in these organs.
In all the examined morbid fish, no apparent gross disease sign was observed in the visceral organs except for bloated swimbladder. No bacterium was isolated from the kidney. Because the histopathological observation suggested that fish morbidity was associated with NNV infection, we performed the NNV-specific RT-PCR. All the examined fish (n = 4) sampled at 32, 42, and 67 days after the onset of morbidity were tested positive by the RT-PCR (data not shown). The nucleotide sequence of the PCR amplicon excluding the primer regions (381 bp) was highly homologous to NNV from fourfinger threadfin Eleutheronema tetradactylum (OR553889.1) with 99.21% identity and to that from red-spotted grouper (AY510457.1) and sevenband grouper E. septemfasciatus (AB373029.1) with 98.95% identity. They are all classified into RGNNV genotype (Nishizawa et al., 1997).
Diagnosis of seemingly healthy fishApparently healthy fish (n = 2) sampled at 95 days after ceasing of the disease had no symptoms on the external appearance or in internal organs, except for severe monogenean infections on the gills (1,280 and 730 worms). The NNV genome was detected in both fish by the RT-PCR.
Treatment tests for gill monogeneanThe result of the in vitro test is shown in Fig. 7. All the worms treated with H2O2 at 700 ppm or 350 ppm stopped moving within 30 min or 70 min, respectively. Those worms never regained motility after being transferred to seawater, therefore they were considered dead (100% mortality rate). Five out of eight worms immersed in freshwater became immotile within 70 min and were never recovered after being transferred to seawater (62.5% mortality rate). All eight worms treated with 62‰ or 82‰ saltwater stopped moving within 30 min or 10 min, respectively, but five worms in each group (62.5%) regained motility after being transferred to seawater (37.5% mortality rate).
In the in vivo test of trail 1, all three individuals treated with H2O2 at 158 ppm for 60 min or 120 min still harbored the monogeneans, but the mean worm numbers were about half of that in the untreated fish (Fig. 8A). After being treated with 350 ppm H2O2 for 60 min, monogeneans remained in 2 out of 3 fish, but the number of monogeneans was less than 4% of that in the control group. No monogeneans were detected in any of the fish treated with 350 ppm H2O2 for 120 min (Fig. 8A). In trail 2, fish treated with 700 ppm H2O2 for 15 min still had monogeneans, but the mean worm number was considerably lower, approximately one-sixth, compared to that in the untreated fish (Fig. 8B). After being treated with 700 ppm H2O2 for 30 min, the monogeneans remained in only 1 out of 3 fish, and the mean worm number was less than 4% of that of the control group. Worms found at 24 h after these treatments were still alive. In contrast, treatment with 700 ppm H2O2 for 45 min achieved complete monogenean eradication, with no monogeneans found on the gills in any fish (Fig. 8B). No fish showed abnormalities within 24 h after the H2O2 bath treatment.
Despite the high load of parasites on the gills, the infection of Pseudorhabdosynochus spp. was not directly associated with the morbidity and mortality of cultured red-spotted grouper in the present case. In cases of ectoparasites such as skin flukes Benedenia seriolae and Neobenedenia girellae (Ogawa and Shirakashi, 2017) and the salmon lice Lepeophtheirus salmonis (Torrissen et al., 2013), the severity of fish morbidity is often associated with parasite load. Therefore, we initially suspected the Pseudorhabdosynochus monogeneans as the causative agent of fish morbidity. Three species of Pseudorhabdosynochus (P. epinepheli, P. lantauensis and P. satyui) have been recorded from red-spotted grouper in Japanese waters (Justine, 2009). Our observation confirmed that the monogeneans found in this study consisted of P. epinepheli and P. lantauensis. Infection of P. epinepheli has previously been reported to cause morbidity in red-spotted grouper (Isshiki et al., 2007). Hence, it was reasonable to suspect that the monogenean infection was the primary cause of morbidity in the present case. However, our investigations revealed that another disease, VNN, was the actual cause of morbidity. Although this highlights an initial misdiagnosis, we believe that documenting the entire diagnostic process is valuable for future reference, as diagnosing co-infected fish is highly challenging.
This is the first report assessing the effects of Pseudorhabdosynochus spp. on gills via histopathology. Our examinations detected no apparent abnormalities or pathological changes, such as the proliferation of epithelial cells or clubbing of gill filaments, despite Pseudorhabdosynochus spp. being known to feed on epithelial tissue of the host (Whittington and Chisholm, 2008). Furthermore, severe parasitism of Pseudorhabdosynochus spp. was confirmed in apparently healthy fish even after morbidity had ceased. These results suggest that parasitism by Pseudorhabdosynochus spp. alone is not highly pathogenic, at least by the two species detected in this study, and is unlikely to cause loss of equilibrium in red-spotted grouper, as observed in this case. After close examination on the effects of the monogenean infection using histopathology, we noticed that the abnormal swimming behavior (lost equilibrium) and the gross sign (bloated swimbladder) observed in this case differed from those in a previous study (Isshiki et al., 2007). Given the absence of histopathological changes associated with Pseudorhabdosynochus spp. infection in this study, the previously reported fish morbidity might also have been attributed to other factors, such as Vibrio spp. infection, which was mentioned as an occasional secondary infection in the previous report (Isshiki et al., 2007). On the other hand, both cases involved severe monogenean infection on the gills. Therefore, we cannot rule out the possibility that monogenean infections affected fish health and triggered different secondary infections in each case.
The primary cause of morbidity and mortality in the present case was presumed to be VNN. The lesions characteristic of the NNV infection, including tissue degeneration in the brain and retina, were confirmed by histopathological analysis. Furthermore, morbid fish were tested positive for the NNV-specific RT-PCR. Although the number of fish examined in this study was small, all the morbid fish showed the same disease sign, loss of equilibrium at the bottom of the tanks, suggesting a common etiological agent. Since VNN causes damage in the central nervous system (Nakai and Mori, 2016), it is plausible that the VNN was the direct cause of the morbidity in this case. VNN is also known to cause mass mortality in juvenile Epinephelus spp. including red-spotted grouper (Fukuda et al., 1996; Chi et al., 2003; Khumaidi et al., 2019). It has been reported that one-year-old or even older sevenband grouper E. septemfasciatus lost equilibrium and died from VNN (Fukuda et al., 1996; Tanaka et al., 1998). In this study, one-year-old red-spotted grouper showed the lost equilibrium symptom and died chronically over the three-month period. Although the accurate cumulative mortality rate in Tank A and Tank B was uncertain, as morbid fish were removed daily, the observed mortality was not as severe as that reported in juvenile groupers, where mortalities of up to nearly 100% have been documented (Chi et al., 2003; Khumaidi et al., 2019).
Deworming tests for Pseudorhabdosynochus spp. were conducted as the morbidity was initially considered to be caused by monogeneans. As mentioned above, the morbidity was found to be unrelated to the severe monogenean infections. However, considering the possibility that the monogenean infection may have unknown effects on morbidity, the authors believe it is important to document the results of this experiment. In vitro experiments suggested that freshwater and NaCl-added seawater had no distinct lethal effects on the two Pseudorhabdosynochus spp. Yang et al. (2003) reported efficacy of formalin bath treatment against P. epinepheli infecting E. coioides, but the use of formalin is prohibited in Japan. Therefore, we examined H2O2 which has been widely applied to treat Pseudorhabdosynochus spp. infections in groupers (Isshiki et al., 2007; Cruz-Lacierda et al., 2012). In our in vivo test, a few monogeneans remained following the shorter treatments of 350 ppm for 60 min and 700 ppm for 30 min. On the other hand, complete deworming was achieved with 350 ppm H2O2 for 120 min and 700 ppm for 45 min, without negative effects on the fish. Therefore, it is considered that safe and complete deworming of the two Pseudorhabdosynochus spp. is possible by these H2O2 bath, at least under conditions of 20°C. In contrast, previous study (Isshiki et al., 2007) reported that H2O2 bath at 700 ppm for a shorter duration of 15 min at 20°C or 25°C killed all P. epinepheli on red-spotted grouper. The reason for this discrepancy between the two studies remains unclear. It is uncertain why the worms were not completely eradicated in our study, despite the longer exposure time at the same H2O2 concentration. In the experiment by Isshiki et al. (2007), the efficacy of the treatment was assessed by examining the proportion of swollen, no-motile worms at 3 h after treatment. In contrast, we evaluated efficacy based on the number of worms present on the gills at 24 h post-treatment, and confirmed the presence of motile worms even in the group treated with 700 ppm H2O2 for 30 min. These observations suggest that the worms considered dead in the experiment by Isshiki et al. (2007) may have still been alive and could have regained motility over time.
Although speculative, it is possible that juveniles had already been subclinically infected with NNV at the hatchery before being transferred to the farm, where viral replication was triggered by the severe monogenean infection. In 2015, an outbreak of VNN occurred in the hatchery from which the red-spotted grouper juveniles were introduced into the fish farm. However, after the implementation of biosecurity measures including disinfecting fertilized eggs by electrolyzed seawater and using UV-treated seawater for rearing the juveniles, VNN has not recurred in the hatchery. Implementing biosecurity measures is quite effective in controlling viral agents such as NNV, but it does not appear to be flawless. There was a case where subclinically infected red-spotted grouper tested positive for NNV, despite the disinfection of fertilized eggs by electrolyzed seawater (Nishioka, 2019). A possible scenario for the present case is that the fish were harboring NNV for about a year, and the viral replication was triggered when they experienced severe infection with Pseudorhabdosynochus spp. It has also been reported that VNN can develop in NNV-infected white trevally, Pseudocaranx dentex, due to stressors such as high rearing density (Mushiake, 2000). Given the information of a previous study on P. epinepheli infection in red-spotted grouper (Isshiki et al., 2007), the monogenean infection may act as a trigger for the onset of VNN in subclinically infected fish. Thoney and Hargis Jr. (1991) also noted that fish mortality may not be caused directly by monogeneans, but by secondary infections of bacteria or viruses.
In the present study, although histopathological examination showed no direct adverse effects on the fish, the monogenean parasites may facilitate secondary infections by feeding on gill epithelial cells. Miyoshi et al. (2019) reported that skin damage in Japanese amberjack Seriola quinqueradiata caused by B. seriolae parasitism facilitates infection by the causative agent of nocardiosis. As gills are reported to be one of the portals of entry for NNV (Lampou et al., 2020), gill damage caused by monogenean infection may have facilitated horizontal transmission of NNV in the tanks in this case. The case reported by Isshiki et al. (2007) could also be a secondary bacterial infection caused by gill damages from P. epinepheli infection. Although the present study has shown that monogenean infection did not cause disease in red-spotted grouper, further study is required to elucidate the actual effects of Pseudorhabdosynochus spp. on host fish and association with other viral and bacterial diseases. We believe that in cases of Pseudorhabdosynochus spp. infections in red-spotted grouper aquaculture, even if there is no direct negative impact on the host, active deworming using the methods reported in this study and by Isshiki et al. (2007) is effective for the stable production.
We thank the staff of the culture farm for their cooperation throughout this study. We also express gratitude to Dr. Kazuya Nagasawa for sending a copy of a paper unavailable to us. We thank Dr. Tomofumi Kurobe for English editing. This study was partly funded by the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, and Fisheries of Japan.
