J. Fish. Effects Soc. of Taiwan, the Gill 2012, Parasite 39(2): Zeuxapta seriolae on the Sea Cage-cultured Amberjack Seriola dumerili (2012)

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1 J. Fish. Effects Soc. of Taiwan, the Gill 2012, Parasite 39(2): Zeuxapta seriolae on the Sea Cage-cultured Amberjack Seriola dumerili (2012) 107 Effects of the Gill Parasite Zeuxapta seriolae (Monogenea: Heteraxinidae) on the Sea Cage-cultured Amberjack Seriola dumerili (Risso, 1810) at Penghu Island (Pescadores), Taiwan Chih-Hui Lu 1 *, Chen-Chun Ku 1, Chiu-Ming Wen 2 and Shiu-Nan Chen 3 (Received, February 23, 2012; Accepted, April 26, 2012) ABSTRACT This paper reports an infestation of the cultured amberjack Seriola dumerili (Risso, 1810) with the gill fluke Zeuxapta seriola (Meserve 1938), which is thought to be the primary cause of significant mortality in the production sea cages at Penghu, Taiwan. The gross sign of the amberjack was normal. However, hypersecretion of mucus on pale gills, accompanied by large quantities of parasites, was observed in all examined fish. The flukes were morphologically identified as Z. seriolae using light and scanning electron microscopy. The parasitological analysis of 30 live amberjack collected from the infested cages showed that all the fish harboured Z. seriolae with intensities ranging between 32 and 408 parasites per fish. Histopathological examinations revealed that the parasites attached to the host by grasping one or two gill lamellae with each clamp of the haptor, which led to a hyperplastic epithelium and destruction of the lamellae. This is the first description of zeuxaptosis affecting sea cage-cultured amberjack at Penghu, Taiwan. Key words: Seriola dumerili, Zeuxapta seriolae, Penghu, Cage aquaculture. INTRODUCTION The amberjack S. dumerili is considered a suitable species for intensive aquaculture, due to its rapid growth and high commercial value. This fish has been cultured in sea cages at Penghu since 1988, with the harvest reaching 100 tons in However, the sale price went down, and a laborious freshwater bath had to be implemented every days for the control of monogenean ectoparaparasites Neobenedenia melleni (MacCallum, 1927), leading to a decline in the popularity of amberjack among Penghu cage farmers. Instead, they began to culture cobia (Rachycentron canadum, Linnaeus, 1766), which was thought to be more commercially promising. In 2002, due to the burdensome pasteurellosis caused by Photobacterium damselae subsp. piscicida which seriously affected local cobia culture (Ku et al., 2000), and the need for greater product diversity for the domestic market, some farmers began to culture amberjack again (Ku, 2002). Since then, the sea cagecultured amberjack have suffered from infection with not only the N. melleni all year round but also Z. seriolae during the winter months, causing more serious stock losses at Penghu. Z. seriolae is a heteraxinid monogenean with a wide geographical distribution and infests various species of the genus Seriola (Whittington and Chisholm, 2008). 1 Department of Aquaculture, National Penghu University of Science and Technology Makung, Penghu, Taiwan 880, R.O.C. 2 Department of Life Sciences, National Kaohsiung University, Nan-Tzu, Kaohsiung, Taiwan 811, R.O.C. 3 Department of Life Sciences, National Taiwan University, Taipei, Taiwan 10617, R.O.C. * Corresponding author. chlu@npu.edu.tw

2 108 Chih-Hui Lu, Chen-Chun Ku, Chiu-Ming Wen and Shiu-Nan Chen (2012) Fish infested by Z. seriolae presented characteristics similar to those described for Seriola quinqueradiata (Temminck and Schlegel, 1845) parasitised by Heteraxine heterocerca (Goto, 1894) in Japan (Montero et al., 2004). The gill fluke causes reduced appetite and slower growth in hosts. Signs of infection by this parasite were not obvious, as the gills are difficult to monitor and cannot be inspected closely without handling the fish. As frequent handling of sea cagecultured fish was discouraged, infection with Z. seriolae was initially detected after the observation of behavioural changes associated with advanced infections, including hyperventilation and loss of balance. Heavy infestations of Z. seriolae can also lead to anaemia, heavy mucus production, and in some instances, the loss of respiratory epithelium (Rohde, 1984; Mansell et al., 2005; Montero et al., 2004). Mild infections can be treated with hydrogen peroxide at water temperatures below 25 o C, and treatment with praziquantel or formalin might be employed for severe infections or during periods of warmer temperatures (Sharp et al., 2004; Williams et al., 2007). Hypersaline water baths are also known as an effective method of killing some monogeneans (Matsusato, 1967; Svendsen and Haug, 1991). However, these treatments in cage culture systems involve time, manpower, cost burdens and damage to the fish. It is recommended to regularly change the net in order to remove the entangled eggs ( This paper reports microscopic observations of the important monogean parasite Z. seriolae on cultured amberjack S. dumerili at Penghu, Taiwan. MATERIALS AND METHODS 1. Disease occurrence and observation by light microscopy In December 2010, amberjack (1-2 kg in body weight) cultured in sea cages (10 x 10 x 4 m) at Erkan Bay, Penghu were again infected with Z. seriolae. The amberjack were inspected (skin, gills, viscera and eyes) and parasites were observed under a light microscope (LM). Thirty amberjack were randomly sampled and anaesthetised with ice water. The gills were collected and fixed in phosphate-buffered 10% formalin for histology. The flukes on the gills were individually removed from each fish using forceps and preserved in 70% ethanol. The prevalence and intensity of infection were calculated according to the method proposed by Bush et al. (1997). 2. Scanning electron microscope observation A scanning electron microscope (SEM) study was performed on gill samples that were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline for 10 min, dehydrated in an ethanol series, critical pointdried with liquid CO 2, coated with gold, and viewed with a Hitachi S520 SEM. 3. Histological examination Gills fixed for histological studies were dehydrated in graded ethanol solution, and embedded in paraffin using routine histological techniques. Then, 6-μm sections were stained with Hematoxyln and Eosin stain. 4. Effect of hypersalinity The effect of hypersaline water baths was evaluated using 300 l tanks on the sides of the cages. Groups of amberjack (n = 6; kg) were exposed to 6% hypersaline solution for 5 and 10 min, then anaesthetised with 300 ppm 2-phenoxyethanol. The fish reactions were recorded during the exposure period. Aeration was provided in each tank. The flukes remaining on the gills were counted. RESULTS 1. Disease occurrence and observation by light microscopy The amberjack was normal with respect

3 Effects of the Gill Parasite Zeuxapta seriolae on the Sea Cage-cultured Amberjack Seriola dumerili (2012) 109 to the body coloration. No anomalies in the behaviour of affected individuals were observed, except for some slow swimming. At necropsy, hypersecretion of mucus on pale gills, accompanied by large quantities of parasites, was observed (Fig. 1A) in all examined live fish. The parasites were elongated and flat and the tegument was smooth and transparent, so the internal organs could be observed (Fig. 1B). The mean length of formalin-fixed flukes was / (range 3-8) mm (n = 50). The anterior edge presented a V-shaped head (Fig. 1C) lacking eye spots; the posterior edge presented an asymmetrical armed haptor with two rows of clamps (Fig. 1D), i.e., a longer row with clamps and a shorter one with clamps. A large number of eggs (Fig. 1E), each of them exhibiting one long polar filament (Fig. 1F), could be observed in mature parasites. These flukes were morphologically identified as Z. seriolae (Grau et al., 2003; Montero et al., 2004; Whitting and Chisholm, 2008). The parasitological analysis of the 30 live amberjack collected from the infested sea cages showed that 100% of the fish harboured Z. seriolae with intensities ranging between 32 and 408 parasites per fish. 2. Observation by SEM The SEM findings coincided with the LM observations. Fig. 2A shows three parasites attached to gill filaments. The parasites were elongated and flat, with a haptor that bore two rows of asymmetrical clamps at the posterior end (Fig. 2B and C). The clamps looked like baseball gloves aligned in a row (Fig. 2D). The head lacked eye spots (Fig. 2E). Eggs with long polar filaments were observed (Fig. 2F). 3. Histopathological examination Histopathological examination showed parasites attached to the gills by means of their haptor clamps and causing little damage to the gills (Fig. 3A). However, hyperplastic lamellae occurred in heavily infected fish (Fig. 3B). Lots of eggs were observed in the uteri of mature adults (Fig. 3C). 4. Effect of hypersalinity All Z. seriolae were detached from the gills of examined amberjack for 5 or 10 minutes in 6% hypersaline solution and no fish mortality was attributed to the treatment. DISCUSSION Z. seriolae is a blood-feedng polyopisthocotylean reported as gill parasite of Seriola spp. from different geographical area, and considered as a significant problem in aquaculture of Seriola spp. in many countries (Whittington and Chisholm, 2008). In this paper, we observed the morphology of gill fluke Z. seriolae which was thought to be the primary cause of significant mortality in the sea cage cultured amberjack S. dumerili at Penghu, Taiwan. Mechanical blockage of interlamellar spaces by parasites (Fig. 1) and fussion of gill lamellae (Fig. 3B) were to reduce or prevent water flow across the gills and effect host respiration. In addition, the pale gills indicative of anaemia were found in infested fish too. The combined effects were probably the causes leading to the mass mortality of amberjack. Similar results are also presented by Montero et al. (2004) and Mansell et al. (2005). Management of Z. seriolae in the aquaculture of the South Australian yellowtail kingfish Seriola lalandi (Valenciennes, 1833 in Cuvier and Valenciennes, 1833) relies on bathing cages of fish in hydrogen peroxide solution (Mansell et al., 2005). In Japan, a hypersaline water bath is known as an effective method of killing H. heterocerca ( fs.a.u-tokyo.ac.jp/). However, these control methods are labour-intensive, timeconsuming, weather-dependent and stressful to the fish. Presently, two oral therapeutic regimens using praziquantel have proven effective against monogenean parasites of the Seriola species, so long as suspected palatability problems are resolved (Williams et al., 2007). Farmers at Penghu regularly changed the nets in order to remove the

4 110 Chih-Hui Lu, Chen-Chun Ku, Chiu-Ming Wen and Shiu-Nan Chen (2012) Fig. 1. A. Macroscopic view of parasitised gills, which are whitish, with high hypersecretion and numerous flukes. B. Z. seriolae is elongated and flat. C. The anterior edge presented a V-shaped head lacking eye spots. D. The posterior edge presented an asymmetrical armed haptor with two rows of clamps. E. Mature Z. seriolae with a large number of eggs. F. String of eggs with a long polar filament. (A, bright field; B, C, D, E, F, dark field). entangled eggs from the net-cage, so outbreaks of zeuxaptosis seldom happened in the amberjack aquaculture. Outbreaks usually happened when management was lax due to the winter monsoon and the nets went without changing for more than 70 days. When infestation arose, farmers changed the nets immediately; however, the cumulative mortality could reach 12-20% after one month. Outbreaks of Z. seriolae have been reported in Japan (Ogawa and Yokoyama,

5 Effects of the Gill Parasite Zeuxapta seriolae on the Sea Cage-cultured Amberjack Seriola dumerili (2012) 111 Fig. 2. SEM observation of Z. seriolae. A. Three adult parasites attached to the amberjack s gills (bar = 1.20 mm). B. Frontal view of the fluke; the haptor has two rows of asymmetrical clamps at the posterior end (bar = 600 μm). C. Dorsal view of the fluke (600 μm). D. Detail of the clamps (bar = 38 μm). E. Head of the fluke (bar = 120 μm). F. Strings of eggs with a long polar filament (bar = 120 μm). 1998), the Mediterranean (Grau et al., 2003; Lia et al., 2007; Montero et al., 2004), Australia, and New Zealand (Ernst et al., 2002; Sharp et al., 2003; Tubbs et al., 2005), where the annual sea-surface temperatures fluctuate between 13 o C and 22 o C (Mazzola et al., 2000; Tubbs et al., 2005). Z. seriolae epizootics in Seriola spp. are temperature related and commonly occur with the onset of summer and increasing water temperatures in Australia (Ernst et al., 2002). Wild kingfish infected by Z. seriolae were also captured at

6 112 Chih-Hui Lu, Chen-Chun Ku, Chiu-Ming Wen and Shiu-Nan Chen (2012) Fig. 3. Histological sections of gills from amberjack infected with Z. seriolae (H&E). A. Parasites attach to the gills by means of their haptor clamps and cause little damage to the gills (bar = ~270 μm). B. Lamellar fusion occurred in heavily infected gills (bar = ~125 μm). C. Lots of eggs are observed in the uterus of the mature adult worm (bar = ~125 μm). sea surface temperatures of o C in New Zealand (Sharp et al., 2003). Tubbs et al. (2005) indicated that Z. seriolae reproduction is limited to a narrow temperature range and the parasites multiply rapidly at water temperatures between 17.5 o C and 21.0 o C. These data help explain why the outbreaks of disease due to Z. seriolae infection in

7 Effects of the Gill Parasite Zeuxapta seriolae on the Sea Cage-cultured Amberjack Seriola dumerili (2012) 113 amberjack at Penghu only occur in winter months when the sea temperature is usually at o C. REFERENCES Bush, A. O., K. D. Lafferty, J. M. Lotz and A. W. Shostak (1997). Parasitology meets ecology on its own term: Margolis et al. revisited. J. Parasitol., 83(4): Ernst, I., I. D. Whittington, S. Corneillie and C. Talbot (2002). Monogenean parasites in seacage aquaculture. Austasia Aquac., 16: Grau, A., S. Crespo, E. Pastor, P. González and E. Carbonell (2003). High infection by Zeuxapta seriolae (Monogenea: Heteraxinidae) associated with mass mortalities of amberjack Seriola dumerili Risso reared in sea cages in the Balearic Islands (western Mediterranean). Bull. Europ. Assoc. Fish Pathol., 23(3): Ku, C. C. (2002). Disease problems of sea cage cultured amberjack (Seriola dumerili) at Penghu, Taiwan. Taiwan Offshore Aquac. Q., 11: 2-4. (in Chinese) Ku, C. C., C. H. Lu, G. T. Thea and S. N. Chen (2000). Disease investigation of sea cagecultured cobia (Rachycentron canadum). FA COA Aquaculture Series No.1. Rep. Fish Dis. Res., 20: (in Chinese with English Abstract). Lia, R. P., N. Zizzo, A. Tinelli, A. Lionetti, C. C. Cantacessi and D. Otranto (2007). Mass mortality in wild amberjack (Seriola dumerili) infected by Zeuxapta seriolae (Monogenea: Heteraxinidae) in the Ionian Sea. Bull. Europ. Assoc. Fish Pathol., 27(3): Mansell, B., M. D. Powell, I. Ernst and B. F. Nowak (2005). Effects of the gill monogenean Zeuxapta seriolae (Meserve, 1938) and treatment with hydrogen peroxide on pathophysiology of kingfish, Seriola lalandi Valenciennes J. Fish Dis., 28(5): Matsusato, T. (1967). Axine (Heteraxine) heterocerca, an ectoparasitic trematode on culture yellowtail. Fish Patho., 2: (in Japanese) Mazzola, A., E. Favaloro and G. Saraă (2000). Cultivation of the Mediterranean amberjack, Seriola dumerili (Risso,1810), in submerged cages in the western Mediterranean sea. Aquaculture, 181: Montero, F. E., S. Crespo, F. Padrớs, F. De la Gándara, F. A. García and J. A. Raga (2004). Effects of the gill parasite Zeuxapta seriolae (Monogenea: Heteraxinidae) on the amberjack Seriola dumerili Risso (Teleostei: Carangidae) Aquaculture, 232: Ogawa, K. and H. Yokoyama (1998). Parasite disease of cultured marine fish in Japan. Fish Pathol., 33: Rohde, K. (1984). Diseases caused by metazoans: helminthes. In: Diseases of Marine Animals Vol. 4, Part 1 (ed. By O. Kinne), pp Pisces. Biologische Anstalt Helgoland, Hamburg. Sharp, N. J., C. W. Poortennaar, B. K. Diggles and T. J. Willis (2003). Metazoan parasites of yellowtail kingfish, Seriola lalandi lalandi, in New Zealand: Prevalence, intensity, and site preference. New Zealand J. Mar. Freshwater Res., 37(2): Sharp, N. J., B. K. Diggles, C. W. Poortenaar and T. J. Willis T. J. (2004). Efficacy of Aqui-S, formalin and praziquantel against the monogeneans, Benedenia seriolae and Zeuxapta seriolae, infecting yellowtail kingfish Seriola lalandi lalandi in New Zealand. Aquaculture, 236: Svendsen, Y. S. and T. Haug (1991). Effectiveness of formalin, benzocaine, and hypo- and hypersaline exposes against adults and eggs of Entobdella hippoglossi (Műller), an ectoparasite on Atlantic halibut (Hippoglossus hippoglossus L.). Laboratory studies. Aquaculture, 94: Tubbs, L. A., C. W. Poortenaar, M. A. Sewell and B. K. Diggles (2005). Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi. Int. J. Parasitol., 35(3): Williams, R. E., I. Ernst, C. B. Chambers and I. D. Whittington (2007). Efficacy of orally administered praziquantel against Zeuxapta seriolae and Benedenia seriolae (Monogenea) in yellowtail kingfish Seriola lalandi. Dis. Aquat. Organ., 77(3): Whittington, I. D. and C. B. Chisholm (2008). Diseases caused by Monogenea. In: Fish Diseases (ed. By J. Eiras, H. Segner, T. Wahli and B. G. Kapoor), pp Science Publishers.

8 114 Chih-Hui Lu, Chen-Chun Ku, Chiu-Ming Wen and Shiu-Nan Chen (2012) 鰓吸蟲對澎湖海上箱網養殖紅魽鰺的影響 陸知慧 1 * 古鎮鈞 1 溫秋明 2 陳秀男 3 (2012 年 2 月 23 日收件 ;2012 年 4 月 26 日接受 ) 本研究報告鰓吸蟲 (Zeuxapta seriolae) 對澎湖海上箱網養殖紅魽魚參 (Seriola dumerili) 的影響 自發病箱網中隨意捕撈 30 尾紅魽魚參, 發現盛行率為 100%, 而每尾魚受感染強度少則 32 隻, 多則達到 408 隻 這些被鰓吸蟲感染地魚隻其外觀上表現正常但其鰓絲非常蒼白且分泌大量黏液 組織病理觀察發現這些鰓吸蟲藉由其固著器上的吸鋏在鰓薄板上爬行, 導致上皮細胞增生進而使得整個鰓薄板受到破壞 經由光學顯微鏡及掃描式電子顯微鏡對鰓吸蟲進行顯微觀察後確認這些鰓吸蟲為 Z. seriolae 本文為第一篇報導 Zeuxaptosis 影響澎湖海上箱網養殖紅魽的報告 關鍵詞 : 鰓吸蟲 (Zeuxapta seriolae), 紅魽魚參 (Seriola dumerili), 澎湖, 箱網養殖 1 國立澎湖科技大學水產養殖系, 澎湖, 臺灣 88046, 中華民國 2 國立高雄大學生命科學系, 高雄, 臺灣 81148, 中華民國 3 國立臺灣大學生命科學系, 台北, 臺灣 10617, 中華民國 * 通訊作者, chlu@npu.edu.tw