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Mass mortality of hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium ocellatum (Dinoflagellida)
Aquaculture 236 (2004) 85 – 94 www.elsevier.com/locate/aqua-online
Mass mortality of hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium ocellatum (Dinoflagellida) Erlinda R. Cruz-Lacierda a,*, Yukio Maeno b, April Joy T. Pineda a, Victoria E. Matey c a
Fish Health Section, Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan 5021, Iloilo, Philippines b Seikai National Fisheries Research Institute, Fisheries Research Agency, 1551-8 Taira, Nagasaki 851-2213, Japan c Department of Biology and Center for Inland Waters, San Diego State University, San Diego, CA 92182-4614, USA
Received 27 August 2003; received in revised form 10 January 2004; accepted 13 February 2004
Abstract Outbreaks of heavy infestation by the parasitic dinoflagellate Amyloodinium ocellatum in hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused 100% mortality events in hatcheries in the Philippines. Parasites were recorded on the body surface in 14-day-old milkfish fry and on both skin and gills in 2-month-old snapper. Trophonts of A. ocellatum caused local erosions of fish skin and degeneration of epithelial cells at the sites of the parasite’s attachment to the body surface. Separation and hyperplasia of gill epithelium and fusion of secondary lamellae at the distal parts of the gill filaments were common. High pathogenicity of A. ocellatum to fish may be attributed to the severe alterations of the fish gills, the disruption of the host’s skin, and feeding of trophonts on hosts’ epithelial cells. In-vivo treatments of A. ocellatuminfested snapper with a 1 h freshwater bath and 200 ppm H2O2 showed promising results. This is the first report of A. ocellatum infestation in milkfish and mangrove red snapper in the Philippines. D 2004 Elsevier B.V. All rights reserved. Keywords: Milkfish; Chanos chanos; Mangrove red snapper; Lutjanus argentimaculatus; Parasite; Dinoflagellate; Amyloodinium ocellatum
* Corresponding author. Fax: +63-33-511-9070. E-mail address: [email protected] (E.R. Cruz-Lacierda). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.02.012
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1. Introduction Milkfish (Chanos chanos) is the most important food fish cultured in the Philippines. There is also an increasing demand for high value fish like the mangrove red snapper (Lutjanus argentimaculatus). Seed production of milkfish
Fig. 1. (a) A. ocellatum trophonts (arrows) attached to the body surface of milkfish (C. chanos) fry. 40 (fresh mount). (b) Trophonts of A. ocellatum with a short stalk (arrows) attached to the skin of milkfish (C. chanos) fry. 100. (c) Scanning electron micrograph (SEM) of A. ocellatum trophont showing the attachment organelles. R, rhizoid; st, stalk; S, stomopode. Scale bar=10 Am.
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Fig. 1 (continued).
and mangrove red snapper has been developed at the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC AQD) in Iloilo, the Philippines. The technology to produce milkfish in hatcheries in this country has been available since the early 1980s (Gapasin and Marte, 1990). Considerable progress has also been achieved in the hatchery production of snapper (Duray et al., 1996). The intensification of aquaculture in the country has led to the occurrence of infectious diseases particularly due to parasites at the hatchery stage (Duray, 1992). The present study describes the outbreaks of the disease, its causative agent and pathology, and control measures undertaken.
2. Materials and methods Mass or 100% mortality of 14-day-old hatchery-reared milkfish fry occurred at a commercial fish hatchery in Iloilo in June 2001. More recently, mass mortality of 2-month-old hatchery-reared snapper was recorded at SEAFDEC AQD hatchery in June 2003. Water conditions during the mortality events were 27– 28 jC and 30 ppt. 2.1. Collection and examination of samples The milkfish were reared in two 10-m-diameter canvas tank units while the snappers were reared in five 5-ton circular cement tank units and two 5-ton rectangular cement tank units. Moribund and freshly dead specimens of milkfish
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fry and snapper were collected from the affected hatcheries during the mortality events. Unstained and stained (Lugol’s iodine, Bouin’s fixative) whole bodies of fish (n=20/tank) were examined under dissecting and compound microscopes. Parasites attached to the fish body were counted in unstained fresh samples. Samples selected for light microscopy (LM) were fixed in 10% cold-buffered formalin, and processed using standard histological techniques. Paraffin sections were stained with haematoxylin and eosin (H&E) and examined with the light microscope (Olympus BH2). Samples for scanning electron microscopy (SEM) were fixed in a mixture of cold 8% paraformaldehyde –2.5% glutaraldehyde in 0.2 M cacodylate buffer, dehydrated in a series of ethyl alcohol, freeze-dried with t-butyl alcohol, mounted on stubs, sputtercoated with gold and examined with a scanning electron microscope, JEOL JSM5600 LV. The parasite was identified following the descriptions of Lom and Dykova (1992) and Kuperman and Matey (1999). Identification of the parasite was done by one of the authors (V. Matey). 2.2. In-vivo treatments Snapper with a mean intensity of 30 parasites on the gills were immersed for 1 h in freshwater, 200 ppm formalin and 200 ppm hydrogen peroxide (H2O2). All treatments were done using 10 fish per treatment in duplicates including the control (seawater only). Treatments were done in glass jars containing 1 l sand-filtered and aerated seawater (27 jC, 29 ppt). The fish were transferred to clean filtered and aerated seawater after the treatment period. Body surface and gills were examined for parasites under a stereomicroscope, and the number of trophonts were counted and recorded.
3. Results 3.1. Milkfish The affected milkfish fry (total length [TL]=7.5 – 8 mm; n=10) suffered 100% mortality. Fish had opaque bodies and moved sluggishly in the rearing tank before the mortality events. LM examination of fresh mounts of the whole body of moribund and dead fish revealed 100% infestation by ectoparasites that were identified as the peridinean dinoflagellate Amyloodinium ocellatum (Dinoflagellida, Blastodinida, Oodinidae). Brownish or yellowish, ovoid to pear-shaped trophonts of A. ocellatum were attached singly or in clusters of 2 –4 individuals to the fish body (Fig. 1a), fins, eyes and mouth cavity. Trophont size varied from 2018 to 8075 Am. Intensity of infestation was high with 40–
Fig. 2. (a) Local erosion (arrow) of skin epithelium of milkfish (C. chanos) fry at the site of attachment of A. ocellatum trophont. H&E. 200. (b) Scanning electron micrograph (SEM) of A. ocellatum trophont on the skin of milkfish (C. chanos) fry. Erosion of the epithelium and degeneration of epithelial cells around the sites of attachment. Imprints (arrows) of detached trophonts are recognizable. Scale bar=20 Am.
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70 parasites per fish. Trophonts were attached to the outer layer of the fish skin with a narrow basal end that formed a very short stalk or peduncle (Fig. 1b) that ended in a flattened attachment disk bearing numerous projections or rhizoids fused with the surface of epithelium, and a mobile tentacle-like stomopode (Fig. 1c). No trophonts were observed on the gills of the affected milkfish. LM and SEM studies (Fig. 2a and b) showed irritation and local erosion of fish skin and degeneration of epithelial cells at the site of parasite attachment. Several imprints of detached trophonts were seen on the body surface of the infested fish (Fig. 2b). 3.2. Mangrove snapper The affected snapper (average body weight [BW]=0.12g; average TL=19 mm; n=10) in the rectangular tanks exhibited darkening of the body, sluggish swimming movement, rubbing their head against the walls of the tank and eventually mortality of up to 100%. LM examinations showed 100% prevalence of fish infestation by A. ocellatum. From 80 to 100 trophonts were distributed on the skin and fins (Fig. 3), and 40– 50 trophonts were found on the gills of infested fish. Low mortality (V20%) was observed in snapper (average BW=0.20 g; average TL=23 mm; n=10) reared in the circular tanks stocked with fish of the same origin and age and maintained at the same stocking density, water and feeding regimes as fish in the rectangular tank. The fish in the circular tanks carried a lesser number of the parasites compared to fish from rectangular tanks. Only 2– 10 trophonts were observed on the body surface and 5– 20 trophonts on the gills. The size of trophonts that infested the fish in all tanks varied from 5050 to 9080 Am. The trophonts of A. ocellatum were revealed on the tips of filaments and between the secondary lamellae of fish gills (Fig. 4a). Distortion of the lamellae and eventual erosion of
Fig. 3. Trophonts (arrows) of A. ocellatum attached to the caudal fin of mangrove red snapper (L. argentimaculatus). 40 (fresh mount).
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Fig. 4. (a) Trophonts (P) of A. ocellatum in between gill lamellae of mangrove red snapper (L. argentimaculatus). Note erosion of lamellae (arrow) at the site of trophont’s attachment and separation of lamellar epithelium (arrow head) of surrounding lamellae. H&E. 200. (b) Severe hyperplasia and fusion (arrows) of gill lamellae of mangrove red snapper (L. argentimaculatus) caused by trophonts (P) of A. ocellatum at the distal parts of gill filaments. H&E. 200.
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Table 1 Efficacy of 1 h freshwater, 200 ppm formalin and 200 ppm hydrogen peroxide (H2O2) bath treatments against A. ocellatum infestation in mangrove red snapper, L. argentimaculatus Treatmentsa c
Control (seawater) 1 h Freshwater (0 ppt) 1 h 200 ppm formalinc 1 h 200 ppm H2O2c
Mean intensity/fish after treatmentb
Efficacy of treatment (%)
30 0 5 0
0 100 83 100
a
Mean of 10 fish/treatment; 2 replicates/treatment; 27 jC. Mean BW=0.16g; mean TL=21 mm. c 29 ppt. b
lamellar epithelium were apparent at the sites of trophont attachments. This was frequently accompanied by a separation of gill epithelium from the underlying membrane and enlargement of spaces filled with interstitial fluid (Fig. 4a). Hyperplasia and eventual fusion of adjacent gill lamellae in the distal parts of the filaments were common in heavily infested fish (Fig. 4b). 3.3. In-vivo treatments The efficacy of 1 h freshwater, 200 ppm formalin and 200 ppm H2O2 bath treatments against A. ocellatum is shown in Table 1. The 1 h freshwater and 200 ppm H2O2 treatments were effective in eliminating the parasite and did not have any adverse effect on the fish.
4. Discussion Based on the description of Lom and Dykova (1992) and Kuperman and Matey (1999), the parasites that infested milkfish and snapper at two fish hatcheries in the Philippines were identified as the trophonts of A. ocellatum (Dinoflagellida, Blastodinida, Oodinidae). The checklist of fish parasites in the Philippines did not show any record of the occurrence of A. ocellatum in milkfish or snapper (Arthur and Lumanlan-Mayo, 1997). In 1984, Baticados and Quinitio reported A. ocellatum infestation on the gills of mullet (Mugil cephalus) broodstock. Thus, milkfish and snapper are recorded as new hosts of A. ocellatum in the Philippines. Fish infestations by A. ocellatum resulted in weakening and eventual mass mortality events. The affected 14-day-old milkfish (TL=7.5– 8 mm) were smaller compared to the expected size of normal fry (TL=10– 17 mm for 7-day-old) (M. Duray, pers. comm.). Likewise, snapper in rectangular tanks with 40 –50 trophonts of A. ocellatum on the gills were smaller (average BW=0.12 g) than those fish in the circular tanks with 5– 20 trophonts of A. ocellatum on the gills (average BW=0.20 g). The dinoflagellate A. ocellatum is one of the most dangerous and destructive ectoparasites of thermophilic marine fish in aquaculture (Kabata, 1985; Noga and Levy, 1995; Colorni, 1998). Overstreet (1993) reported that A. ocellatum infestations in larval
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fishes were confined on the skin while the gills became affected in bigger fish. In the present study, trophonts of A. ocellatum were observed on the body surface only in 14day-old milkfish while in 2-month-old snapper both the skin and the gills were affected. Overstreet (1993) suggested that the mortality rate of infested fish is related to the number of parasites per gill filament. All fish in the rectangular tanks with 40– 50 trophonts of A. ocellatum on the gills eventually died, whereas only V20% of the fish died in the circular tanks with 5– 20 trophonts of A. ocellatum on the gills. Histopathological alterations of skin and gills have been recorded in several species of fish infested by this parasite worldwide (Paperna, 1980; Noga and Levy, 1995; Kuperman and Matey, 1999). In the Philippines, severe alterations of gill lamellae were found in the brood stock of mullet (M. cephalus) affected by A. ocellatum (Baticados and Quinitio, 1984). The high pathogenicity of this parasite can be attributed to the damage caused by trophont attachment to the fish skin, to the severe pathological alterations of fish gills and to the trophonts feeding on the cytoplasm of the host’s cells (Lom and Lawler, 1973; Paperna, 1980; Kuperman and Matey, 1999). It had not been excluded that A. ocellatum produces an exotoxin although this remains to be proven (Noga and Levy, 1995). Although both 1 h freshwater and 200 ppm H2O2 bath treatments were effective in eliminating the parasite, the use of freshwater will be more practical depending on the host fish’s tolerance. Freshwater has been previously reported to dislodge A. ocellatum trophonts from the affected organs (Noga et al., 1991; Overstreet, 1993; Colorni, 1998). In the present study, the remaining snappers in circular tanks that survived the infestation were maintained in flow-through water at a salinity of 20 ppt for several weeks (M. Duray, pers. com.). Under this condition, no further fish mortality was observed. Supposedly, mature trophonts of A. ocellatum can be easily detached from the host’s tissues when the composition of ambient water is changed. Subsequently, environmental manipulation through intensive water management contributed to the control of infestation when the remaining stocks were maintained in low saline flow-through water. Acknowledgements This study was done under the Regional Fish Disease Project of Government of JapanTrust Fund. We acknowledge Dr. Yasuo Inui and Dr. Kazuya Nagasawa for technical guidance, Albert Gaitan and Angelo Marte of Aquaspec Hatchery and Marietta Duray and Jhozine Damaso of SEAFDEC Fish Hatchery for providing the fish samples, the Microtechnique Service Laboratory of SEAFDEC Aquaculture Department for assistance in histological processing of the samples, and Dr. Evelyn de Jesus for editing an earlier draft of the paper. References Arthur, J.R., Lumanlan-Mayo, S., 1997. Checklist of the Parasites of Fishes of the Philippines. FAO Technical Paper No. 369, Rome. 102 pp. Baticados, M.C.L., Quinitio, G.F., 1984. Occurrence and pathology of an Amyloodinium-like protozoan parasite on gills of grey mullet, Mugil cephalus. Helgol. Meeresunters. 37, 595 – 601.
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Colorni, A., 1998. Pathobiology of marine organisms cultured in the tropics. In: De Silva, S.S. (Ed.), Tropical Mariculture. Academic Press, U.S.A, pp. 209 – 255. Duray, M., 1992. Larviculture of marine fishes at SEAFDEC/AQD. In: Villegas, C.T., Castanos, M.T., Lacierda, R.B. (Eds.), Proceedings of the Aquaculture Workshop for SEAFDEC/AQD Training Alumni. SEAFDEC, Aquaculture Department, Iloilo, Philippines, pp. 127 – 133. Duray, M., Alpasan, L.G., Estudillo, C.B., 1996. Improved hatchery rearing of mangrove red snapper, Lutjanus argentimaculatus, in large tanks with small rotifer (Brachionus plicatilis) and Artemia. Isr. J. Aquac.Bamidgeh 48, 123 – 132. Gapasin, R.SJ., Marte, C.L., 1990. Milkfish hatchery operations. Aquaculture Extension Manual No. 17. SEAFDEC, Aquaculture Department, Iloilo, Philippines. 25 pp. Kabata, Z., 1985. Parasites and Diseases of Fish Cultured in the Tropics. Taylor and Francis, Philadelphia, USA. 318 pp. Kuperman, B.I., Matey, V.E., 1999. Massive infestation by Amyloodinium ocellatum (Dinoflagellida) of fish in a highly saline lake, Salton Sea, California, USA. Dis. Aquat. Org. 39, 65 – 73. Lom, J., Dykova, I., 1992. Protozoan Parasites of Fishes. Elsevier, Amsterdam, The Netherlands. 315 pp. Lom, J., Lawler, A.R., 1973. An ultrastructural study on the attachment in dinoflagellates invading gills of Cyprinodontidae. Protistologica 9, 293 – 309. Noga, E.J., Levy, M.G., 1995. Dinoflagellida (Phylum sarcomastigophora). In: Woo, P.T.K. (Ed.), Fish Diseases and Disorders. Protozoan and Metazoan Infections, vol. 1. CAB International, Cambridge, UK, pp. 1 – 25. Noga, E.J., Smith, S.A., Landsberg, J.H., 1991. Amyloodiniosis in cultured hybrid striped bass (Morone saxatilisM. chrysops) in North Carolina. J. Aquat. Anim. Health 3, 294 – 297. Overstreet, R.M., 1993. Parasitic diseases of fishes and their relationship with toxicants and other environmental factors. Pathobiology of Marine and Estuarine Organisms. CRC Press, Boca Raton, FL, pp. 111 – 156. Paperna, I., 1980. Amyloodinium ocellatum (Brown, 1931) (Dinoflagellida) infestations in cultured marine fish at Eilat Red Sea: epizootiology and pathology. J. Fish Dis. 3, 363 – 372.
Mass mortality of hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium ocellatum (Dinoflagellida) Erlinda R. Cruz-Lacierda a,*, Yukio Maeno b, April Joy T. Pineda a, Victoria E. Matey c a
Fish Health Section, Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan 5021, Iloilo, Philippines b Seikai National Fisheries Research Institute, Fisheries Research Agency, 1551-8 Taira, Nagasaki 851-2213, Japan c Department of Biology and Center for Inland Waters, San Diego State University, San Diego, CA 92182-4614, USA
Received 27 August 2003; received in revised form 10 January 2004; accepted 13 February 2004
Abstract Outbreaks of heavy infestation by the parasitic dinoflagellate Amyloodinium ocellatum in hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused 100% mortality events in hatcheries in the Philippines. Parasites were recorded on the body surface in 14-day-old milkfish fry and on both skin and gills in 2-month-old snapper. Trophonts of A. ocellatum caused local erosions of fish skin and degeneration of epithelial cells at the sites of the parasite’s attachment to the body surface. Separation and hyperplasia of gill epithelium and fusion of secondary lamellae at the distal parts of the gill filaments were common. High pathogenicity of A. ocellatum to fish may be attributed to the severe alterations of the fish gills, the disruption of the host’s skin, and feeding of trophonts on hosts’ epithelial cells. In-vivo treatments of A. ocellatuminfested snapper with a 1 h freshwater bath and 200 ppm H2O2 showed promising results. This is the first report of A. ocellatum infestation in milkfish and mangrove red snapper in the Philippines. D 2004 Elsevier B.V. All rights reserved. Keywords: Milkfish; Chanos chanos; Mangrove red snapper; Lutjanus argentimaculatus; Parasite; Dinoflagellate; Amyloodinium ocellatum
* Corresponding author. Fax: +63-33-511-9070. E-mail address: [email protected] (E.R. Cruz-Lacierda). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.02.012
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1. Introduction Milkfish (Chanos chanos) is the most important food fish cultured in the Philippines. There is also an increasing demand for high value fish like the mangrove red snapper (Lutjanus argentimaculatus). Seed production of milkfish
Fig. 1. (a) A. ocellatum trophonts (arrows) attached to the body surface of milkfish (C. chanos) fry. 40 (fresh mount). (b) Trophonts of A. ocellatum with a short stalk (arrows) attached to the skin of milkfish (C. chanos) fry. 100. (c) Scanning electron micrograph (SEM) of A. ocellatum trophont showing the attachment organelles. R, rhizoid; st, stalk; S, stomopode. Scale bar=10 Am.
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Fig. 1 (continued).
and mangrove red snapper has been developed at the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEAFDEC AQD) in Iloilo, the Philippines. The technology to produce milkfish in hatcheries in this country has been available since the early 1980s (Gapasin and Marte, 1990). Considerable progress has also been achieved in the hatchery production of snapper (Duray et al., 1996). The intensification of aquaculture in the country has led to the occurrence of infectious diseases particularly due to parasites at the hatchery stage (Duray, 1992). The present study describes the outbreaks of the disease, its causative agent and pathology, and control measures undertaken.
2. Materials and methods Mass or 100% mortality of 14-day-old hatchery-reared milkfish fry occurred at a commercial fish hatchery in Iloilo in June 2001. More recently, mass mortality of 2-month-old hatchery-reared snapper was recorded at SEAFDEC AQD hatchery in June 2003. Water conditions during the mortality events were 27– 28 jC and 30 ppt. 2.1. Collection and examination of samples The milkfish were reared in two 10-m-diameter canvas tank units while the snappers were reared in five 5-ton circular cement tank units and two 5-ton rectangular cement tank units. Moribund and freshly dead specimens of milkfish
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fry and snapper were collected from the affected hatcheries during the mortality events. Unstained and stained (Lugol’s iodine, Bouin’s fixative) whole bodies of fish (n=20/tank) were examined under dissecting and compound microscopes. Parasites attached to the fish body were counted in unstained fresh samples. Samples selected for light microscopy (LM) were fixed in 10% cold-buffered formalin, and processed using standard histological techniques. Paraffin sections were stained with haematoxylin and eosin (H&E) and examined with the light microscope (Olympus BH2). Samples for scanning electron microscopy (SEM) were fixed in a mixture of cold 8% paraformaldehyde –2.5% glutaraldehyde in 0.2 M cacodylate buffer, dehydrated in a series of ethyl alcohol, freeze-dried with t-butyl alcohol, mounted on stubs, sputtercoated with gold and examined with a scanning electron microscope, JEOL JSM5600 LV. The parasite was identified following the descriptions of Lom and Dykova (1992) and Kuperman and Matey (1999). Identification of the parasite was done by one of the authors (V. Matey). 2.2. In-vivo treatments Snapper with a mean intensity of 30 parasites on the gills were immersed for 1 h in freshwater, 200 ppm formalin and 200 ppm hydrogen peroxide (H2O2). All treatments were done using 10 fish per treatment in duplicates including the control (seawater only). Treatments were done in glass jars containing 1 l sand-filtered and aerated seawater (27 jC, 29 ppt). The fish were transferred to clean filtered and aerated seawater after the treatment period. Body surface and gills were examined for parasites under a stereomicroscope, and the number of trophonts were counted and recorded.
3. Results 3.1. Milkfish The affected milkfish fry (total length [TL]=7.5 – 8 mm; n=10) suffered 100% mortality. Fish had opaque bodies and moved sluggishly in the rearing tank before the mortality events. LM examination of fresh mounts of the whole body of moribund and dead fish revealed 100% infestation by ectoparasites that were identified as the peridinean dinoflagellate Amyloodinium ocellatum (Dinoflagellida, Blastodinida, Oodinidae). Brownish or yellowish, ovoid to pear-shaped trophonts of A. ocellatum were attached singly or in clusters of 2 –4 individuals to the fish body (Fig. 1a), fins, eyes and mouth cavity. Trophont size varied from 2018 to 8075 Am. Intensity of infestation was high with 40–
Fig. 2. (a) Local erosion (arrow) of skin epithelium of milkfish (C. chanos) fry at the site of attachment of A. ocellatum trophont. H&E. 200. (b) Scanning electron micrograph (SEM) of A. ocellatum trophont on the skin of milkfish (C. chanos) fry. Erosion of the epithelium and degeneration of epithelial cells around the sites of attachment. Imprints (arrows) of detached trophonts are recognizable. Scale bar=20 Am.
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70 parasites per fish. Trophonts were attached to the outer layer of the fish skin with a narrow basal end that formed a very short stalk or peduncle (Fig. 1b) that ended in a flattened attachment disk bearing numerous projections or rhizoids fused with the surface of epithelium, and a mobile tentacle-like stomopode (Fig. 1c). No trophonts were observed on the gills of the affected milkfish. LM and SEM studies (Fig. 2a and b) showed irritation and local erosion of fish skin and degeneration of epithelial cells at the site of parasite attachment. Several imprints of detached trophonts were seen on the body surface of the infested fish (Fig. 2b). 3.2. Mangrove snapper The affected snapper (average body weight [BW]=0.12g; average TL=19 mm; n=10) in the rectangular tanks exhibited darkening of the body, sluggish swimming movement, rubbing their head against the walls of the tank and eventually mortality of up to 100%. LM examinations showed 100% prevalence of fish infestation by A. ocellatum. From 80 to 100 trophonts were distributed on the skin and fins (Fig. 3), and 40– 50 trophonts were found on the gills of infested fish. Low mortality (V20%) was observed in snapper (average BW=0.20 g; average TL=23 mm; n=10) reared in the circular tanks stocked with fish of the same origin and age and maintained at the same stocking density, water and feeding regimes as fish in the rectangular tank. The fish in the circular tanks carried a lesser number of the parasites compared to fish from rectangular tanks. Only 2– 10 trophonts were observed on the body surface and 5– 20 trophonts on the gills. The size of trophonts that infested the fish in all tanks varied from 5050 to 9080 Am. The trophonts of A. ocellatum were revealed on the tips of filaments and between the secondary lamellae of fish gills (Fig. 4a). Distortion of the lamellae and eventual erosion of
Fig. 3. Trophonts (arrows) of A. ocellatum attached to the caudal fin of mangrove red snapper (L. argentimaculatus). 40 (fresh mount).
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Fig. 4. (a) Trophonts (P) of A. ocellatum in between gill lamellae of mangrove red snapper (L. argentimaculatus). Note erosion of lamellae (arrow) at the site of trophont’s attachment and separation of lamellar epithelium (arrow head) of surrounding lamellae. H&E. 200. (b) Severe hyperplasia and fusion (arrows) of gill lamellae of mangrove red snapper (L. argentimaculatus) caused by trophonts (P) of A. ocellatum at the distal parts of gill filaments. H&E. 200.
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Table 1 Efficacy of 1 h freshwater, 200 ppm formalin and 200 ppm hydrogen peroxide (H2O2) bath treatments against A. ocellatum infestation in mangrove red snapper, L. argentimaculatus Treatmentsa c
Control (seawater) 1 h Freshwater (0 ppt) 1 h 200 ppm formalinc 1 h 200 ppm H2O2c
Mean intensity/fish after treatmentb
Efficacy of treatment (%)
30 0 5 0
0 100 83 100
a
Mean of 10 fish/treatment; 2 replicates/treatment; 27 jC. Mean BW=0.16g; mean TL=21 mm. c 29 ppt. b
lamellar epithelium were apparent at the sites of trophont attachments. This was frequently accompanied by a separation of gill epithelium from the underlying membrane and enlargement of spaces filled with interstitial fluid (Fig. 4a). Hyperplasia and eventual fusion of adjacent gill lamellae in the distal parts of the filaments were common in heavily infested fish (Fig. 4b). 3.3. In-vivo treatments The efficacy of 1 h freshwater, 200 ppm formalin and 200 ppm H2O2 bath treatments against A. ocellatum is shown in Table 1. The 1 h freshwater and 200 ppm H2O2 treatments were effective in eliminating the parasite and did not have any adverse effect on the fish.
4. Discussion Based on the description of Lom and Dykova (1992) and Kuperman and Matey (1999), the parasites that infested milkfish and snapper at two fish hatcheries in the Philippines were identified as the trophonts of A. ocellatum (Dinoflagellida, Blastodinida, Oodinidae). The checklist of fish parasites in the Philippines did not show any record of the occurrence of A. ocellatum in milkfish or snapper (Arthur and Lumanlan-Mayo, 1997). In 1984, Baticados and Quinitio reported A. ocellatum infestation on the gills of mullet (Mugil cephalus) broodstock. Thus, milkfish and snapper are recorded as new hosts of A. ocellatum in the Philippines. Fish infestations by A. ocellatum resulted in weakening and eventual mass mortality events. The affected 14-day-old milkfish (TL=7.5– 8 mm) were smaller compared to the expected size of normal fry (TL=10– 17 mm for 7-day-old) (M. Duray, pers. comm.). Likewise, snapper in rectangular tanks with 40 –50 trophonts of A. ocellatum on the gills were smaller (average BW=0.12 g) than those fish in the circular tanks with 5– 20 trophonts of A. ocellatum on the gills (average BW=0.20 g). The dinoflagellate A. ocellatum is one of the most dangerous and destructive ectoparasites of thermophilic marine fish in aquaculture (Kabata, 1985; Noga and Levy, 1995; Colorni, 1998). Overstreet (1993) reported that A. ocellatum infestations in larval
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fishes were confined on the skin while the gills became affected in bigger fish. In the present study, trophonts of A. ocellatum were observed on the body surface only in 14day-old milkfish while in 2-month-old snapper both the skin and the gills were affected. Overstreet (1993) suggested that the mortality rate of infested fish is related to the number of parasites per gill filament. All fish in the rectangular tanks with 40– 50 trophonts of A. ocellatum on the gills eventually died, whereas only V20% of the fish died in the circular tanks with 5– 20 trophonts of A. ocellatum on the gills. Histopathological alterations of skin and gills have been recorded in several species of fish infested by this parasite worldwide (Paperna, 1980; Noga and Levy, 1995; Kuperman and Matey, 1999). In the Philippines, severe alterations of gill lamellae were found in the brood stock of mullet (M. cephalus) affected by A. ocellatum (Baticados and Quinitio, 1984). The high pathogenicity of this parasite can be attributed to the damage caused by trophont attachment to the fish skin, to the severe pathological alterations of fish gills and to the trophonts feeding on the cytoplasm of the host’s cells (Lom and Lawler, 1973; Paperna, 1980; Kuperman and Matey, 1999). It had not been excluded that A. ocellatum produces an exotoxin although this remains to be proven (Noga and Levy, 1995). Although both 1 h freshwater and 200 ppm H2O2 bath treatments were effective in eliminating the parasite, the use of freshwater will be more practical depending on the host fish’s tolerance. Freshwater has been previously reported to dislodge A. ocellatum trophonts from the affected organs (Noga et al., 1991; Overstreet, 1993; Colorni, 1998). In the present study, the remaining snappers in circular tanks that survived the infestation were maintained in flow-through water at a salinity of 20 ppt for several weeks (M. Duray, pers. com.). Under this condition, no further fish mortality was observed. Supposedly, mature trophonts of A. ocellatum can be easily detached from the host’s tissues when the composition of ambient water is changed. Subsequently, environmental manipulation through intensive water management contributed to the control of infestation when the remaining stocks were maintained in low saline flow-through water. Acknowledgements This study was done under the Regional Fish Disease Project of Government of JapanTrust Fund. We acknowledge Dr. Yasuo Inui and Dr. Kazuya Nagasawa for technical guidance, Albert Gaitan and Angelo Marte of Aquaspec Hatchery and Marietta Duray and Jhozine Damaso of SEAFDEC Fish Hatchery for providing the fish samples, the Microtechnique Service Laboratory of SEAFDEC Aquaculture Department for assistance in histological processing of the samples, and Dr. Evelyn de Jesus for editing an earlier draft of the paper. References Arthur, J.R., Lumanlan-Mayo, S., 1997. Checklist of the Parasites of Fishes of the Philippines. FAO Technical Paper No. 369, Rome. 102 pp. Baticados, M.C.L., Quinitio, G.F., 1984. Occurrence and pathology of an Amyloodinium-like protozoan parasite on gills of grey mullet, Mugil cephalus. Helgol. Meeresunters. 37, 595 – 601.
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Colorni, A., 1998. Pathobiology of marine organisms cultured in the tropics. In: De Silva, S.S. (Ed.), Tropical Mariculture. Academic Press, U.S.A, pp. 209 – 255. Duray, M., 1992. Larviculture of marine fishes at SEAFDEC/AQD. In: Villegas, C.T., Castanos, M.T., Lacierda, R.B. (Eds.), Proceedings of the Aquaculture Workshop for SEAFDEC/AQD Training Alumni. SEAFDEC, Aquaculture Department, Iloilo, Philippines, pp. 127 – 133. Duray, M., Alpasan, L.G., Estudillo, C.B., 1996. Improved hatchery rearing of mangrove red snapper, Lutjanus argentimaculatus, in large tanks with small rotifer (Brachionus plicatilis) and Artemia. Isr. J. Aquac.Bamidgeh 48, 123 – 132. Gapasin, R.SJ., Marte, C.L., 1990. Milkfish hatchery operations. Aquaculture Extension Manual No. 17. SEAFDEC, Aquaculture Department, Iloilo, Philippines. 25 pp. Kabata, Z., 1985. Parasites and Diseases of Fish Cultured in the Tropics. Taylor and Francis, Philadelphia, USA. 318 pp. Kuperman, B.I., Matey, V.E., 1999. Massive infestation by Amyloodinium ocellatum (Dinoflagellida) of fish in a highly saline lake, Salton Sea, California, USA. Dis. Aquat. Org. 39, 65 – 73. Lom, J., Dykova, I., 1992. Protozoan Parasites of Fishes. Elsevier, Amsterdam, The Netherlands. 315 pp. Lom, J., Lawler, A.R., 1973. An ultrastructural study on the attachment in dinoflagellates invading gills of Cyprinodontidae. Protistologica 9, 293 – 309. Noga, E.J., Levy, M.G., 1995. Dinoflagellida (Phylum sarcomastigophora). In: Woo, P.T.K. (Ed.), Fish Diseases and Disorders. Protozoan and Metazoan Infections, vol. 1. CAB International, Cambridge, UK, pp. 1 – 25. Noga, E.J., Smith, S.A., Landsberg, J.H., 1991. Amyloodiniosis in cultured hybrid striped bass (Morone saxatilisM. chrysops) in North Carolina. J. Aquat. Anim. Health 3, 294 – 297. Overstreet, R.M., 1993. Parasitic diseases of fishes and their relationship with toxicants and other environmental factors. Pathobiology of Marine and Estuarine Organisms. CRC Press, Boca Raton, FL, pp. 111 – 156. Paperna, I., 1980. Amyloodinium ocellatum (Brown, 1931) (Dinoflagellida) infestations in cultured marine fish at Eilat Red Sea: epizootiology and pathology. J. Fish Dis. 3, 363 – 372.