Piscinoodinium pillulare ( SCHÄPERCLAUS , 1954 ) LOM , 1981 ( DINOFLAGELLIDA ) INFECTION IN CULTIVATED FRESHWATER FISH FROM THE NORTHEAST REGION OF SÃO PAULO STATE , BRAZIL . PARASITOLOGICAL AND PATHOLOGICAL ASPECTS

The Aquaculture Center of Unesp, Jaboticabal, SP, Brazil, received fishes for diagnosis from fish farmers reporting fish crowding at pond edges and in water inlets. Fifty-three out of 194 cases showed round to oval, immobile whitish structures, measuring up to 162 μm diameter, identified as the dinoflagellate Piscinoodinium pillulare. In 34 diagnosed cases the parasites were present in the gills, in 2 on body surface, and 9 in both. Thirty-one out of 53 were tambacu hybrids hosts; 7, Piaractus mesopotamicus; 6, Colossoma macropomum; 5, Leporinus macrocephalus; 3, Oreochromis niloticus; and 1, Prochilodus lineatus. Fish showed increased mucous production on body surface and gills, while ecchymosis in the caudal peduncle and operculum was present. The gills also showed paleness, congestion, and petechiae. Histopathology presented a great number of trophonts situated between secondary lamellae, fixed to or detached from the epithelium. Primary lamellae presented interstitial hemorrhages, severe hyperplasia of the epithelium, goblet cells, and mononuclear infiltrates. The present work is the first report of P. pillulare in Brazil and emphasizes the importance of this dinoflagellate which caused significant economic losses from 1995 through 1997.

Until now, observations related to the occurrence of and infection with this parasite were unknown in Brazil.In the present work, the authors describe histopathological and morphological aspects of P. pillulare infection in cultivated fishes from the Northeast region of São Paulo State.

MATERIAL AND METHODS
From 1995 to 1997, 265 fishes were examined after necropsy at the Laboratory Aquatic Organisms Pathology at the Aquaculture Center, Unesp, Jaboticabal, São Paulo State, Brazil.Water quality was measured with a pH-meter, a conductivimeter (Corning), dissolved oxygen with portable oxymeter (Hach), and water temperature with a mercury bulb thermometer.To search for parasites, the body surface was scraped from tail to head and pieces of gill 2-3 mm in diameter were mounted between glass microscope slide coverslips for microscopical observation.Bacteriological and fungal examinations were carried out according to Austin & Austin (1987) and Martins & Machado (1994).Parasites found were stained with lugol solution for detecting amyloid granules.A camara lucida was used to draw 39 parasite specimens.All measurements taken were transformed into mean values and are shown with their respective change intervals.For scanning electron microscopy, the infected gill filaments were excised and fixed at room temperature in a 3% glutaraldehyde solution in a 0.1 M phosphate buffer (pH 7.4).Afterwards, they were dehydrated with serial concentrations of acetone, dried with CO 2 , assembled, and coated with 20 nm gold paladium.Coated filaments were examined with a JEOL JSM-25-S2 microscope.Parasitized tissues were fixed in 10% buffered formalin and embedded in a paraffin block.Tissue sections 6 µm thick were cut and stained with haematoxilineosin for histopathological analysis.
It had been reported that fish generally gathered near the pond edges or near the water inlets.Feeding activity decreased because of appetite loss; fish became lethargic, and swam erratically with loss of equilibrium.Mortality progressively increased.According to the farmers, fish ponds were rarely cleaned.In three cases, chicken bowels were fed to the fish.
From January 1995 to August 1997, fish mortality caused by dinoflagellates resulted in 23,000 dead fish, 3,000 of which died in the period of May 1 to 15, 1996.In the same month, 4,000 fishes died in 24 hours at just one fish farm.One hundred ninetyfour cases of a total of 265 occurred during the cold season, from May to August, when the water temperature ranged between 17 and 24 o C.

Parasitology
Immobile, round to oval whitish structures on the gills and body surface of infected fish were observed.Amyloid granules in the parasites were stained by Lugol's solution.Parasite measurements were as follows: 102.7 µm (62 to 162) in diameter (Fig. 1); in the basal region of the parasites an attachment disc was seen measuring 38.6 µm (24 to 52) diameter; the cell of the attached trophont was pyriform or sac-like (Lom & Schubert, 1983) measuring 47.7 µm (36 to 70) in length and 33.5 µm (22 to 48) in width; the basal end was narrow, measuring 15.1 µm (10 to 20).Pyriform cells were observed in large number on the gills of parasitized fish.A short structure extending from the basal end, measuring 21.4 µm in length, was observed.The parasite was identified as the dinoflagellate Piscinoodinium pillulare.
Scanning electron microscopy showed trophonts attached to the gill epithelium in different developmental stages (Figs. 2 and 3).

Structural changes
On the body surface, increase in mucous production, scalelessness, suffusion, ecchymosis, petechiae, and small ulcers were observed.The gills also presented increase of mucous production besides epithelial hyperplasia, suffusion, petechiae, congestion, edema, and brownish areas (Table 1).Histopathology showed a great number of trophonts present in the secondary lamellae, sometimes with rhizocysts embedded in the epithelium which penetrated firmly into the epithelial cells.Degeneration and focal epithelial necrosis were observed.The primary lamellae showed congestion, subepithelial edema, focal interstitial hemorrhages, severe hyperplasia of epithelial and goblet cells, and inflammatory cells.The secondary lamellae were fused because of a hyperplastic process that formed cavities in which the parasites were present (Fig. 4).In addition, the extension of the hyperplastic process of the epithelial cells filled all interlamellar the space.These epithelium masses obstructed the respiratory surface of the gills (Fig. 5).

DISCUSSION
The present work showed the nonspecificity of P. pillulare that infects a great number of fish species in different regions of São Paulo State.High mortality caused by severe infection related to lack of cleaning of fish ponds; much organic matter as well as overcrowding were observed.These facts contributed to significant economic losses.Similar parasitosis associated with high levels of organic matter was described by Shaharom-Harrison et al. (1990) in Puntius gonionotus.The addition of chicken bowels to the fish diet, over population, and great quantity of food were the main causes of decreasing water quality, favoring parasitic reproduction (Martins, 1998) as observed in the present work.
Of the cases diagnosed, 73.3% occurred from May to August, demonstrating high fish susceptibility to parasites in the cold season when water temperature ranged from 17 to 24 o C. Temperatures below 24 o C or above 31.5 o C have contributed to mortality, according to Shaharom-Harrison et al. (1990).
This fact is probably related to better conditions for parasite reproduction and fish resistance decrease caused by stress.Temperatures below 10 o C inhibit inflammatory response in ectothermic animals and cause high susceptibility to infections (Bisset, 1948cited by Finn & Nielsen, 1971;Dias & Sinhorini, 1991;Matushima & Mariano, 1996).Mortality rate was higher in the hybrid tambacu than P. mesopotamicus and C. macropomum.However, tambacu and P. mesopotamicus were the most cultivated fishes in the fish farms.
Severe infections with P. pillulare in P. gonionotus cultivated in Malaysia from January 1986 through December 1987 were reported by Shaharom-Harrison et al. (1990).Some authors observed loss of appetite, lethargy, and agglomeration below the water surface (Siti & Rokiah, 1988;Shaharom-Harrison et al., 1990), corroborating the present observations.Histopathological changes described in Brazilian cultivated fish were the same as those found by Brown (1934), Jacobs (1946), Lucký (1970), Cheung et al. (1981a), andShaharom-Harrison et al. (1990) although the geographic region and breed of fish were different.
These lesions initiate a hyperplastic and edematous process in the gills causing changes in the gas and ion interchange surface with consequences for the fish metabolism (Martins & Romero, 1996).
From January 1995 to July 1997, 20% of the cases showed the dinoflagellate P. pillulare as the etiological agent.An attachment disc and rhizocysts inserted in the epithelial cells of the host (Lom, 1981) and the presence of amyloid granules (Jacobs, 1946) were used for identification.A flagellate structure situated in the basal extremity of the parasites was observed in just one case in this work, as was also reported by Lom & Schubert (1983).According to Lom (1977Lom ( , 1981)), rhizocyst characteristics, their penetration into the host cells, and presence of chloroplasts constitute the main characteristics of the genus Piscinoodinium in freshwater fishes.Nigrelli (1936) described various stages of the life cycle of O. ocellatum.When parasites are collected from the gills, rhyzocists and flagella retract.For this reason good observation of such structures was not possible in wet mounts.With the aid of scanning electronic microscopy, Cheung et al. (1981b) observed a severe epithelial irritation with hyperplastic reaction caused by O. ocellatum in the gills of Zebrassoma flavescens.These changes provoked increased mucous production and fusion of the lamellae.High organic matter content, low oxygen level, and overcrowding favoured parasite proliferation, causing stress and increasing susceptibility of fish to parasitism.
In conclusion, the present work is the first in Brazil that emphasizes the significance of dinoflagellates in cultivated fishes.The authors add that prophylaxis and water quality are essential factors in management of fish culture.
reported the occurrence of O. limneticum and O. pillularis in aquarium fishes, while Williams Jr. (1972) observed O. cyprinodontum in the gills of Fundulus similis.Later, on,