Blood Fluke Infection of Japanese Amberjack Seriola Quinqueradiata in Fish Farms Along the Western Coastal Area of Bungo Channel, Japan

Research article Blood Fluke Infection of Japanese Amberjack Seriola quinqueradiata in Fish Farms along the Western Coastal Area of Bungo Channel, Japan

Yutaka Fukuda1, Kazuyoshi Miyamura2, Etsuhisa Hitaka3, Keisuke Kimoto1, Yasuhiro Sanada4, Takayuki Asai3 and Kazuo Ogawa5*

1Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602, Japan 2Fisheries Management Division, Agriculture, Forestry and Fisheries Department, Oita Prefectural Government, Oita 870-8501, Japan 3Oita Prefecture Southern Region Bureau, Oita 876-0813, Japan 4Oita Prefecture Central Region Bureau, Oita 870-0021, Japan 5Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received July 31, 2017)

ABSTRACT―Blood fluke infection among diseased Japanese amberjack Seriola quinqueradiata (n = 9,470) cultured in Oita Prefecture was monitored from 1992 to 2001. Infection was confirmed by the presence of parasite eggs accumulated in the gills. Amberjack with parasite eggs were found in all culture areas except for Usuki Bay. Eggs in the gills of 0-year-old fish started to be observed from early July, with the minimum body weight (BW) of 130 g in Yonouzu Bay, whereas in Nyuzu Bay egg-positive fish was first found in late August with the minimum BW of 296 g. Amberjack were cultured in the inner part of Nyuzu Bay up to the size of 200–250 g in BW, suggesting that infection occurred when fish were later moved to the mouth of the bay for further growth. In the inner part of Nyuzu Bay, hypoxia and conspicuously high dissolved inorganic nitrogen in the water near the bottom and acid volatile sulfides in the sediment were recorded every summer in the present survey. It is speculated that such extraordinary environment is unfavorable for the propagation of the intermediate host of the causative organism in the inner part, but not at the mouth, of the bay.

Key words: Seriola quinqueradiata, Japanese amberjack, blood fluke, distribution, environmental factor, Bungo Channel

Net cage culture of Japanese amberjack Seriola and P. kampachi, while those infecting yellowtail amber- quinqueradiata in Japan started some 60 years ago. jack Seriola lalandi have not yet been identified (Ogawa Farmers use juvenile fish as culture seeds caught along and Egusa, 1986; Ogawa et al., 2015). Fish names fol- the coastal areas in May–June and culture them in float- low Froese and Pauly (2017). ing net cages for 2–3 years until fish grow to the market- Heavy accumulation of blood fluke eggs in the gills able size of about 4–7 kg in body weight (Nakada, 2008). is a direct cause of mortality after hypoxia of infected This culture regimen has not changed for many decades. fish, but even light blood fluke infection may substantially Generally in Oita Prefecture, Japanese amberjack is cul- affect physiological conditions of the host. Japanese tured to the size of 4–5 kg in 2 years. amberjack was liable to become infected with the bacte- Mortalities occur among amberjacks Seriola spp. rial pathogen, Lactococcus garvieae, under low dis- due to low blood flow impeded by accumulation of eggs solved oxygen (DO) conditions, associated with high of blood flukes in the gill capillaries (Ogawa et al., 1989; mortalities (Fukuda et al., 1997a, 1997b). It is specu- Ogawa and Fukudome, 1994; Fukuda, 1999; Shirakashi lated that mortality due to L. garvieae infection increases and Ogawa, 2016). Blood flukes infecting Japanese among amberjacks with lowered respiratory function due amberjack are identified as Paradeontacylix buri, and to accumulation of blood fluke eggs (Kumon et al., greater amberjack Seriola dumerili as P. grandispinus 2002). Despite the fact that the blood fluke infection of Japanese amberjack is an important disease, very little * Corresponding author is known about its biology and ecology to establish con- E-mail: [email protected] trol measures. 192 Y. Fukuda, K. Miyamura, E. Hitaka, K. Kimoto, Y. Sanada, T. Asai and K. Ogawa

This paper is aimed to elucidate factors determining prefecture, viz. the areas from Usuki Bay (A in Fig. 1) to the distribution of blood flukes in the Japanese amber- Kamae, Inokushi and Nagoya Bay (F in Fig. 1). jack culture areas in Oita Prefecture, based on a 10-year Generally, in Oita Prefecture, juvenile Japanese amber- diagnosis data on the host gills and on the analysis of jack caught in the adjacent waters were raised to about marine environmental data from these areas. In 2015, 10–30 g in body weight (BW) by seed farmers from May P. buri was described as a new blood fluke of Japanese to July in the inner part of Nyuzu Bay. Subsequently amberjack (Ogawa et al., 2015). However, when we they were introduced to other fish farms and cultured conducted the present survey in 1992–2001, the caus- there to marketable size. Exceptionally in Area E, they ative fluke had not yet been identified. In this paper, were cultured from wild caught juveniles to the market- the blood fluke detected in our survey was not specified able size, and in some of Area B and F farms, culture and a possibility remains that blood flukes other than P. started from larger fish of about 500–1,000 g in BW buri were included in our survey. introduced from other farms inside and outside Oita Prefecture.

Materials and Methods Survey of water and sediment quality in selected culture Fish used for blood fluke inspection sites Japanese amberjack that were introduced as juve- Water and sediment quality surveys we re con- nile, cultured in Oita Prefecture and sent to the Fisheries ducted in selected stations in the three culture areas Research Division as diseased fish for diagnosis (n = (c-1, c-2, c-3 in Area C, d-1, d-2 in Area D, e-1, e-2, e-3 9,470) were used for monitoring of blood fluke infection in Area E) once a year from mid-August to mid-Septem- from 1992–2001, and at least ten gill filaments were ber in 1992–2001, when the water underwent thermal removed from the left outermost gill of each fish, stratification. At each station, vertical distributions of mounted on a slide glass and inspected for blood fluke water temperature and salinity were measured using a eggs microscopically at a magnification of 40–100. submersible fluorometer Chlorotec ACL1180-DK (Alec Those with blood circulation impeded by the eggs in Electronics). Water samples were taken from depths of more than 90% of the gill filaments were diagnosed as 0 m, 10 m and near the bottom (1 m above the bottom), being heavily infected. and their DO and dissolved inorganic nitrogen (DIN: total of nitrate-nitrogen, nitrite-nitrogen and ammonia-nitro- Culture regimen in six study sites gen) were measured by the Winkler method and contin- Japanese amberjack culture sites (Fig. 1) in this uous flow auto-analyzer system TrAAcs 800 (Bran+ study consist of six areas from north to south of the Luebbe), respectively. Moreover, acid volatile sulfides (AVS) of the sediment, collected by a bottom mud sam- pler, were determined by a sulfides measuring kit Hydrotec-S (Gastec).

Results Occurrence of Japanese amberjack with blood fluke eggs in the gills Occurrence of Japanese amberjack with fluke eggs in each culture area was shown in Table 1. Infected fish were confirmed in all the areas except for the north- ernmost area, Area A, with BW ranging from 130 g (0-year-old) to 4,150 g (1-year-old). Egg-positive fish constituted 4.6%–7.7% of the Japanese amberjack examined. Seasonal occurrences of infected fish are shown in Figs. 2 and 3, where data in Areas A, B and F were excluded, as no infected fish were obtained in Area A and some fish were introduced in Areas B and F after rearing outside Oita Prefecture. Data from Areas C and D were summed up, as fish from these areas were cultured in a similar manner and infection occurred in a similar pattern. Fig. 1. Map of the fish sampling areas (A–F) and environment monitoring stations (●: c-1–e-3) along the south coast Infection was first confirmed by the presence of of Oita Prefecture. eggs in the 0-year fish at the beginning of July, with the Blood uke infection of Japanese amberjack 193 smallest infected fish being 149 g in BW in Area C and Table 1. Distribution of blood fluke egg positive Japanese 130 g in BW in Area D (mean BW of all fish examined in amberjack among aquaculture farms along the south coast of Oita Prefecture (1992–2001). this month: 118 g) (Fig. 2a). Proportion of egg-positive fish increased from August and fluctuated between 6.3% Number of fish Number of fish positive for Percentage of and 14.6% until June next year (Fig. 3). Infected fish Area examined eggs in gills egg-positive fish was rarely observed in July and later among 1-year fish (BW range: g) (BW range: g) (mean BW in July: 1,820 g). Frequency distributions in 170 body weight of egg-positive fish are shown in Fig. 4. A 0 0 Fish with heavily accumulated eggs in the gills were less (8–3,450) 548 34 than 800 g in BW in Areas C and D, most of which B 6.2 appeared from late August to mid-September (Fig. 4a). (18–8,150) (203–3,850) 2,560 129 In Area E, infected fish started to be observed C 5.0 among 0-year fish in late August, with the smallest (1.2–9,150) (149–4,150) 1,185 71 infected fish being 296 g in BW (mean BW in this month: D 6.0 227 g) (Fig. 2b). P roportion of egg-positive fish increased (8–5,350) (130–1,900) 4,877 224 from October and fluctuated between 3.9% and 31.1% E 4.6 until June next year (Fig. 3). Infected fish almost disap- (0.4–7,450) (296–2,450) 130 10 peared in July and later among 1-year fish (mean BW in F 7.7 July: 1,530 g). Eighty-nine percent of heavily infected (41–4,550) (350–1,750) 9,470 468 fish were less than 1,000 g in BW (Fig. 4b), most of Total 4.9 which appeared from late December to late January (0.4–9,150) (130–4,150)

Fig. 2. Monthly changes in the number of egg-positive (closed bar) and -negative (open bar) fish from Areas C and D (a) and Area E (b) in the survey conducted in 1992–2001. Circles represent body weight of egg-positive fish. Data on the body weight of infected fish were plotted separately for the first, middle and last 10 days of each month. Dotted and vertical lines indicate mean and ranges of body weight of the fish examined, respectively. 194 Y. Fukuda, K. Miyamura, E. Hitaka, K. Kimoto, Y. Sanada, T. Asai and K. Ogawa

Fig. 3. Monthly changes in the prevalence of egg-positive fish in the survey conducted in 1992–2001 (〇: fish from Areas C and D, ●: fish from Area E).

Fig. 4. Frequency distribution in body weight of egg-positive fish from Areas C and D (a) and Area E (b). ■ : fish with lightly or moderately accumulated eggs in the gills, ■: fish with heavily accumulated eggs.

(Fig. 3). Mean DO at surface was 6.4–7.2 mg/L, except for e-3, where it was higher (8.5 mg/L) and ﬂuctuated widely Water and sediment quality in Areas C, D and E (Fig. 5c). Mean DO near the bottom was 5.6–6.3 mg/L, Fig. 5 shows the total mean value of water and sedi- except for c-2 and e-3; relatively low (4.8 mg/L) at c-2 ment data in Areas C, D and E during the thermal stratiﬁ- and very low (0.58 mg/L) at e-3 (Fig. 5c). Mean DIN at cation period in summer of 1992–2001. Mean surface surface and 10 m deep ranged between 2.1 μM and 5.3 water temperature ranged from 24.3°C to 24.8°C, except μM at all stations, while DIN near the bottom tended to for c-2 and e-3, where it was higher (from 25.8°C to be high (6.2–7.5 μM) and especially high (47 μM) at e-3 25.9°C), as the two sites were located in the inner parts (Fig. 5d). of the bay (Fig. 5a). Mean water temperature near the Mean AVS of the sediment were shown in Fig. 6. bottom was from 22.5°C to 23.0°C, except for e-3, where Mean water depth ranged from 19 m at d-2 to 40 m at it was 18.7°C (Fig. 5a). Mean salinity ranged from 33.6 c-1. Mean AVS were 0.07–0.29 mg/g dry weight, with to 34.1 at 10 m deep and near the bottom in all sampling exceptionally high values at c-2 (1.1 mg/g dry weight) stations, except for surface, where it was low in several and e-3 (2.0 mg/g dry weight). stations (Fig. 5b). Blood uke infection of Japanese amberjack 195

cause of mortality in some of the infected fish (Fukuda, 1999). Streptococcosis caused by L. garvieae infection was the most serious disease of cultured Japanese amberjack in the 1990s. However, this bacterial disease had been under control by around 2002 due to the wide use of effective vaccines to juvenile fish*. For 10 years since 1992, we examined a great number of amberjacks for disease diagnosis, including inspection of the gills. Among 9,470 Japanese amberjack examined during this period, 468 (4.9%) were positive for blood fluke eggs in the gills. Of these, more than half had been infected with L. garvieae. In 1992–2001, when streptococcosis was rampant among amberjacks, diseased fish brought to us for diagnosis were mainly those with L. garvieae infection. Amberjack populations with streptococcosis had higher prevalence of blood fluke infection than those without infection, as infected fish were liable to develop streptococcosis (Kumon et al., 2002). It is, therefore, probable that the total ratio of actual proportion of egg-positive fish was lower than 4.9% in the whole populations of cultured amberjack in the study areas during this period. In Areas C and D, where juvenile amberjack introduced from seed farmers in May–June were reared to the marketable size of 4–5 kg, egg-positive fish were first found in early July, with the minimal size of 130 g BW. In the blood fluke infection of greater amberjack in Kochi Prefecture, where P. grandispinus was a domi- nant blood fluke, the infection period was thought to have started in September, demonstrated by the fish Fig. 5. Water temperature (a), Salinity (b), dissolved oxygen transfer experiments from the endemic area to a blood (c) and dissolved inorganic nitrogen (d) at the monitor- fluke-free area (Ogawa et al., 1993). Infection of ing stations in summer stratified season (□: 0 m, ■: Japanese amberjack in Oita may start earlier than that of 10 m, ■: near the bottom). Bars and vertical lines indicate the mean value and S.D. of the 10-years-data, P. grandispinus infection of greater amberjack in Kochi. respectively. In Area A, no blood fluke infection was noticed throughout the monitoring period, suggesting that the intermediate host was not distributed in this area. Furthermore, it is probable that all juveniles, introduced from the inner part of Area E, were free of blood fluke infection. In Area E, juvenile amberjack were moved to the mouth of the bay after being reared to 200–250 g in BW in the inner part of the bay. Egg-positive fish were first noticed in late August, with the minimum size of 296 g. This suggests that no infection had occurred among Fig. 6. Acid volatile sulfides in the sediments from the bottom juveniles during the period reared in the inner part of the of monitoring stations in summer stratified season. bay. Water depth is indicated in the bottom line. Bars and All known blood flukes of marine teleost use poly- vertical lines indicate the mean value and S.D. of the chaetes as their intermediate hosts (Shirakashi and 10-years-data, respectively. Ogawa, 2016). However, intermediate hosts of blood flukes infecting Seriola spp. including P. buri of Japanese amberjack are not known. Recent studies Discussion have revealed that intermediate hosts of Cardicola In Oita Prefecture, accumulation of blood fluke eggs in the gills of cultured Japanese amberjack has been * Fukuda, Y. (2016), Studies on infectious diseases control in noticed since around 1991, and was suspected to be a cultured marine fishes. Fish Pathol., 51, 137–143. (in Japanese) 196 Y. Fukuda, K. Miyamura, E. Hitaka, K. Kimoto, Y. Sanada, T. Asai and K. Ogawa opisthorchis, C. orientalis and C. forsteri, blood flukes of relationship between the intensity of infection and the bluefin tunas Thunnus spp. are all terebellid polychaetes culture density of the host in Areas C, D and E (data not (Cribb et al., 2011; Sugihara et al., 2014; Shirakashi et shown). al., 2016). These terebellids propagate among fouling Blood fluke infection is an important disease in and sessile periphytons on the floats, ropes and frames Japanese amberjack culture, directly or indirectly affect- of culture cages (Sugihara et al., 2014; Shirakashi et al., ing health of infected fish. However, no effective con- 2016). It is possible that the intermediate host in this trol method has been developed to prevent infection or study is also a terebellid which will be found among such to treat infected fish. In Pacific bluefin tuna culture, on periphytons. the other hand, praziquantel has been found to kill adult In Area E, fish were cultured both at the mouth (e-1, C. opisthorchis (Shirakashi et al., 2012; Ishimaru et al., e-2) and in the inner part (e-3) of the bay. Water condi- 2013), and its oral administration has been approved for tions at surface and 10 m deep, where culture nets were the treatment of infected tunas. It is desired to extend set, are thought to be much the same between the inner the use of this drug to the treatment of infected amber- part and the mouth part of the bay. Presumably there jacks and Psettarium-infected Japanese pufferfish is no difference in the invertebrate periphytons among Takifugu rubripes. Elucidation of the life cycles of the these sites. However, much lowered DO and higher above blood flukes and of the mechanism of the DIN in the bottom water and higher AVS in the sediment acquired immunity of infected fish would contribute to were observed in e-3 relative to that in e-1 and e-2 in the control of blood fluke infection of maricultured fish. every summer. Deterioration in the bottom water and sediment shows that e-3 is characterized by a strongly Acknowledgement enclosed environment. The lower DO limit for the survival of macrobenthos like molluscs and annelids is esti- We would like to express our gratitude to Ms. Junko mated to be 2 mL/L (2.9 mg/L), and even low-DO resis- Takano, research assistant of Fisheries Research tant animals cannot tolerate in sulfide-rich water (Shimo Division, Oita Prefectural Agriculture, Forestry and et al., 2004). 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