Pathological study of oysters Crassostrea gigas from culture and C. rhizophorae from natural stock of Santa Catarina Island, SC, Brazil

Aquaculture 60 (2011) 43–50

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Pathological study of oysters Crassostrea gigas from culture and C. rhizophorae from natural stock of Santa Catarina Island, SC, Brazil Rachel Costa Sabry a,⁎, Patrícia Mirella da Silva b, Tereza Cristina Vasconcelos Gesteira a, Vitor de Almeida Pontinha c, Aimê Rachel Magenta Magalhães c a

Instituto de Ciências do Mar, Universidade Federal do Ceará, Av. da Abolição, 3507, Meireles, CEP: 60165-081, Fortaleza, CE, Brazil Universidade Federal da Paraiba, Centro de Ciências Exatas e da Natureza, Departamento de Biologia Molecular, Cidade Universitária — Campus I. CEP, 58059-900, João Pessoa, PB, Brazil Universidade Federal de Santa Catarina, Departamento de Aquicultura, Núcleo de Estudos em Patologia Aquícola, Rodovia Admar Gonzaga, 1346, Itacorubi, CEP 88040-900, Florianópolis, SC, Brazil

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Article history: Received 15 April 2011 Received in revised form 31 July 2011 Accepted 4 August 2011 Available online 10 August 2011 Keywords: Oysters Oyster farming Pathogens Histopathology

a b s t r a c t This paper gives an account of the pathogens found in the cultivated Japanese oyster Crassostrea gigas and in the mangrove oyster Crassostrea rhizophorae from natural populations of two sites, Sambaqui (27° 29′18″S, 48° 32′1″W) and Ribeirão da Ilha (27° 42′51″S, 48° 34′6″W), Santa Catarina Island, Brazil. Oysters were collected in March 2008 and April 2009, 150 per site, year and condition (rocky shore and culture). For pathological study, the techniques used were: macroscopic examination, histology and tissue culture in fluid thioglycollate medium specific for Perkinsus. The results showed the presence of the polychaete Polydora sp. with high prevalence (up to 100%) in C. gigas; gametic hypertrophy in C. rhizophorae from Ribeirão da Ilha and in C. gigas from Sambaqui and Ribeirão da Ilha with low prevalence (3.3%); rickettsia-type bacteria, with greater prevalence in C. gigas (30%) than in C. rhizophorae, causing alteration in the epithelium of the stomach. It was not detected the presence of Perkinsus in any oyster sample analyzed of any site. Ciliates of genus Trichodina were observed among gill lamellae, digestive tubules, and adhered to the gills of oysters from Ribeirão da Ilha, with higher prevalence in C. rhizophorae (50%) and without causing injury. Protozoa Sphenophrya-type was found in the gills of C. gigas and C. rhizophorae, with higher prevalence in C. gigas from Sambaqui (70%), not causing changes in the gills. Protozoa of the genus Ancistrocoma were detected in the digestive tubules of C. gigas (36.7%) and C. rhizophorae (40%) from Sambaqui at low intensity and without causing apparent damage. Steinhausia-type microsporidians were observed in the cytoplasm of oocytes of C. rhizophorae and C. gigas with prevalences up to 33.3%. The intensity of infection in the animals was low, with only one oyster presenting more than 50 oocytes infected. The sporocysts of the pathogen caused alteration in the normal structure of the oocytes cytoplasm. Protozoa of genus Nematopsis were observed in the connective tissue of the gills and mantle with high prevalence (100%) in C. rhizophorae from Sambaqui, without host defense response. The observed metazoan were: turbellarians Urastoma-type, metacestode of genus Tylocephalum and copepods possibly of genus Pseudomiycola, all in low prevalence. None of the pathological occurrences found seems to cause significant damage in oysters, once they were found at low intensities. However, due to the great socioeconomic importance that the shellfish represent to the State of Santa Catarina, it's necessary to keep monitoring the health status of these populations. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The State of Santa Catarina is the biggest Brazilian producer of shellfish, contributing with 95% of national production, represented by the culture of the brown mussel Perna perna (Bivalvia: Mytilidae),

⁎ Corresponding author at: Universidade Federal do Ceará, Instituto de Ciências do Mar, Av. Abolição, 3207, Meireles, CEP: 60165-081, Fortaleza, CE, Brazil. Tel.: + 55 85 3366 7009. E-mail addresses: [email protected] (R.C. Sabry), [email protected] (P.M. da Silva), [email protected] (T.C.V. Gesteira), [email protected] (A.R.M. Magalhães). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.08.006

the Japanese oyster Crassostrea gigas (Bivalvia: Ostreidae) and scallop Nodipecten nodosus (Bivalvia: Pectinidae), which is already being grown commercially in the state. However, studies on diseases of these bivalves are scarce when compared to other parts of the world, where pathologies that affect shellfish commercially harvested are well known (Bower et al., 1994). Only in last years, researches on the health of marine bivalves from the Brazilian coast have been intensified (Boehs and Magalhães, 2004; Boehs et al., 2009; da Silva et al., 2002; Sabry and Magalhães, 2005; Sabry et al., 2007; Suárez– Morales et al., 2010) due primarily to the expansion of the cultures. The summer mass mortality of Japanese oyster (C. gigas) cultured in Santa Catarina was first-time registered in December 1987. In the

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summer of 89/90s summer mortality reached 89.5% (Silveira, 1997). The phenomenon was associated to stress caused by the breeding season, high water temperatures and perhaps by the presence of bacteria of the genus Nocardia (Silveira, 1997). Bacteria, like viruses, when at high intensities can cause mortality in shellfish (Gulka et al., 1983; Paillard, 2004; Renault and Novoa, 2004; Villalba et al., 1999), but they not cause mortality at low intensities (Carballal et al., 2001; Cremonte et al., 2005). Protozoa are important pathogens and can cause mortality in several species of shellfish in worldwide. With respect to protozoa, Nematopsis spp. (Apicomplexa: Porosporidae) use marine bivalves as intermediate hosts (Azevedo and Cachola, 1992; Carballal et al., 2001), but there is controversy regarding to its pathogenicity (Lauckner, 1983). Nematopsis sp. was described in oysters C. gigas and Crassostrea rhizophorae grown in Ponta do Sambaqui Beach, Santa Catarina Island, Brazil (Sabry and Magalhães, 2005). Trichodina (Ciliophora: Trichodinidae) can cause destruction of gill filaments and compromise weight gain of the animals (Bower et al., 1994; Figueras and Villalba, 1988). Trichodina sp. was observed in C. rhizophorae (Sabry and Magalhães, 2005) and in Anomalocardia brasiliana (Bivalvia: Veneridae) from Santa Catarina Island (Boehs and Magalhães, 2004), but without causing serious damage to the host. Recently, for oysters of this region were recorded protozoa of the genera Sphenophrya, Ancistrocoma (Ciliophora) and Steinhausia (Microspora) (Pontinha, 2009; da Silva et al., 2010). Sphenophrya can cause cellular hypertrophy in the host and form tumors called xenoma (Boehs et al., 2009; Winstead et al., 2004). However, there are no reports of mortality associated to infection by this pathogen (Bower et al., 1994; Lauckner, 1983; Winstead et al., 2004). Ancistrocoma infection also has not been associated to pathologies (Bower et al., 1994). In the case of parasitism by Steinhausia (microsporidian that infects the cytoplasm of oocytes), it is observed inflammatory or immunological reaction by the host, characterized by intense infiltration of hemocytes (Green et al., 2008). Steinhausia has already been detected in populations of Cerastoderma edule (Bivalvia: Cardiidae) from Galicia coast, Spain, without significant pathologic damage in infected animals (Carballal et al., 2001). Metazoan of genus Tylocephalum (Cestoda: Tetragonocephalidae) were observed in low prevalence and intensity of infestation in C. gigas (Sabry and Magalhães, 2005) and in A. brasiliana from Santa Catarina Island (Boehs and Magalhães, 2004). The turbellarian Urastoma cyprinae was recorded in mussels Mytilus galloprovincialis from the Black Sea (Murina and Solonchenko, 1991) and Spain (Robledo et al., 1994) and in Mytilus edulis from Portugal (dos Santos and Coimbra, 1995) causing severe alterations in the gills. Polychaetes of the genus Polydora (Polychaeta: Spionidae) are commonly found in the Japanese oyster C. gigas (da Silva et al., 2010; Ibbotson, 2002; Pontinha, 2009; Sabry and Magalhães, 2005) and in other bivalves such as the mussel P. perna (da Costa, 2007) and the cockle A. brasiliana (Boehs and Magalhães, 2004) from Santa Catarina Island. Among the copepods, Pseudomyicola spinosus (Copepoda: Myicolidae) is commonly found in marine bivalves (Cáceres-Martínez et al., 2005; Ho and Kim, 1991) and when at low prevalence and intensity does not cause pathologies (Bower et al., 1994). In Brazil, histopathological analysis in the mussel P. perna cultured in Santa Catarina, revealed that the presence of copepods of genus Monstrilla caused changes in the structure of the mantle edge with intense hemocytic infiltration (Suárez–Morales et al., 2010). None of the pathogens listed above is notifiable to the World Organization for Animal Health (OIE). Currently, according to this organization, notifiable pathogens for shellfish are the protozoa Haplosporidia: Bonamia ostreae and B. exitiosa; Paramyxea: Marteilia refringens; Perkinsozoa: Perkinsus marinus and P. Olseni; bacteria Xenohaliotis californiensis and Herpestype virus which affects abalones. Considering the socioeconomic importance of bivalves for the State of Santa Catarina, this paper gives a report of the pathogens found in the cultured oyster C. gigas and in

the mangrove oyster C. rhizophorae from cliffs next to cultures in Santa Catarina Island, SC, Brazil. 2. Materials and methods 2.1. Sampling of oysters Samples of the mangrove oyster C. rhizophorae (n = 600) and of the Japanese oyster C. gigas (n = 600) were collected at two sites of the Santa Catarina Island, in Sambaqui (North Bay, 27° 29′18″S, 48° 32′12″ W) and Ribeirão da Ilha (South Bay, 27° 42′51 ″ S, 48° 34′6 ″ W). Samples were collected in March 2008 and April 2009. In each month 150 oysters were collected from each species and site. Oysters C. rhizophorae were removed from the rocky shore, located in the intertidal zone, far 70 and 200 m from the culture of oysters C. gigas at Sambaqui and Ribeirão da Ilha, respectively. Specimens of C. gigas were collected from culture lanterns hanging at Sambaqui in longlines belonging to the Laboratory of Culture of Mollusks, Federal University of Santa Catarina, and from Ribeirão da Ilha, belonging to a shellfish farmer. The oysters had market size, corresponding to a cultivation time of 9–12 months. The oysters were measured (shell height according to Galtsoff, 1964), and opened for macroscopic observation of the shell and internal organs (mantle, gills, gonad and digestive gland), in order to verify the occurrence of pathological changes. All animals collected at each point (n = 150) were submitted individually to cultivation technique in Ray's Fluid Thioglycollate Medium (RFTM), item 2.2, and 30 individuals were randomly selected for histopathological analysis, item 2.3. The temperature and salinity of sea water were measured at each site and date of collection using a thermometer and refractometer, respectively. The water temperature in Sambaqui was 24 °C in 2008 and 2009 and in Ribeirão da Ilha, 22 and 26 °C for the years 2008 and 2009, respectively. The salinity was 32‰ (2008) and 35‰ (2009) in Sambaqui and 26‰ (2008) and 34‰ (2009) in Ribeirão da Ilha (Table 1). 2.2. Incubation of tissues in Ray's Fluid Thioglycollate Medium (RFTM) Two demibranch and the rectum of each animal were incubated in thioglycollate liquid medium for 7 days in darkness and at room temperature (±28 °C). After the incubation period, tissues were collected, macerated on a slide and stained with Lugol solution, according to the method described by Ray (1954). The preparation was observed under an optical microscope to verify the presence of Perkinsus hypnospores, which are spherical and its walls stains blue or bluish black. 2.3. Histological preparations From each oyster (n = 30 per site, specie and year), a cross section of the animal was removed, it being sampled several organs, including: gills, gonad and digestive gland. The tissues were fixed in Davidson's solution (Shaw and Battle, 1957) for 24 h, dehydrated in a graded series of alcohol up to 100%, cleared in xylene and impregnated in histological paraffin at 60 °C. Cuts of 5 μm thickness were made and stained with hematoxylin and eosin (Howard et al., 2004). The sections were analyzed under optical microscope. The prevalence of pathogens was calculated as being the number of parasitized animals over the total number of oysters collected in each collection point (Bush et al., 1997). 3. Results 3.1. Biometry The biometry of oysters C. rhizophorae (rocky shore) and C. gigas (culture) from Sambaqui and Ribeirão da Ilha aimed to verify the size

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of animals that would be submitted to pathological analysis, without, however, make comparisons on the growth of animals in the two sampled sites and not aimed to relate the size of the animals with the presence or absence of parasitism. The mean heights (mm) of oysters in 2008 and 2009 are shown in Table 1. 3.2. Macroscopic analysis Macroscopic observations did not reveal the presence of pustules or abscesses in any organs examined in both species. The prevalent macroscopical change was the parasitism by the polychaete of genus Polydora in the valves of oysters. The infestation by Polydora was characterized by the presence of horizontal and vertical tubes. Eventually the tubes drilled the tissue reaching the digestive gland. It was also observed bubbles covered by layers of conchioline occupying a representative part of the inner region of the animal valves. The highest prevalences of Polydora in C. rhizophorae were observed in the collections of 2008 in Sambaqui (81.3%) and Ribeirão da Ilha (67.3%) (Table 1). With respect to C. gigas, the prevalence reached maximum values (100%) in both study sites and in the two sampling years (Table 1). 3.3. Incubation of tissues in ray's Fluid Thioglycollate Medium (RFTM) For both species of oysters investigated for the presence of Perkinsus by the RFTM method, all results were negative, i.e., there was no presence of this protozoan. The technique of cultivation of tissues in liquid thioglycollate (RFTM) was effective for the detection of the protozoan Nematopsis. Although Nematopsis had not been stained by Lugol, it performed quite refractional and was easily observed among cells of the gills of oysters in a 20x objective. Nematopsis prevalence by RFTM in C. rhizophorae from Sambaqui was high and ranged from 90 to 96% (Table 1). 3.4. Histopathological analysis Histological analysis showed: hypertrophy of the male gametes, presence of rickettsia-type bacteria and protozoa of the genera Trichodina, Sphenophrya, Ancistrocoma, Steinhausia and Nematopsis. Among the metazoan were observed the turbellarian Urastoma, the cestode Tylocephalum and a copepod possibly of genus Pseudomiycola (Table 1).

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However, none of the histopathological changes found in this study presented risk to the health of oysters. 3.4.1. Hypertrophy of the gametes In the male gonadal follicles of both oysters species were found hypertrophied cells with a diameter ranging from 15 to 36 μm. These cells presented an acidophilic content and a basophilic peripheral region (Fig. 1A). Only three oysters showed such pathology: one oyster C. rhizophorae from Ribeirão da Ilha, collected in 2009, one oyster C. gigas from Ribeirão da Ilha, collected in 2008 and another from Sambaqui in 2009, resulting in a low prevalence (3.3%) (Table 1). 3.4.2. Rickettsia-type bacteria Rickettsia-type bacteria were seen in C. rhizophorae and C. gigas from Sambaqui and Ribeirão da Ilha in 2008 and 2009. The bacteria formed intracytoplasmic colonies of basophilic character and size ranging from 7 to 30 μm in epithelial cells of digestive tubules (Fig. 1B). In the most of the investigated oysters the intensity of infection was low (1–5 colonies). In just one oyster C. rhizophorae were also observed five colonies of these bacteria in the stomach epithelium, causing changes in epithelial cell cytoplasm (Fig. 1C). The higher prevalence of the bacteria was 30% in C. gigas from Ribeirão da Ilha, in 2009 (Table 1). 3.4.3. Protozoa Ciliates of genus Trichodina were observed adhered to the gill lamellae and free in the lumen of digestive tubules of C. rhizophorae and C. gigas, from Ribeirão da Ilha (Fig. 1D and E). In the gills, it was observed until 4 ciliates/histological section and in digestive tubules more than 5 per oyster. Trichodina appeared disk-shaped and with an evidently basophilic nucleus (Fig. 1D). In the gills, these ciliates had sizes ranging from 12 to 47 μm and in digestive tubules, 10 to 20 μm. At the ends of the pathogens were observed structures (hooks) used to anchorage in the host (Fig. 1E). The highest prevalence in C. gigas was 13.3% in 2008. In C. rhizophorae, these ciliates were only observed in 2009 in Ribeirão da Ilha, with high prevalence (50%) (Table 1). Another ciliate, Sphenophrya sp. was seen at the base of gill filaments (Fig. 1F). The ciliate had an oval or round shape, containing a basophilic macronucleus, a cytoplasm with character eosinophilic and size ranging from 9 to 17 μm. The highest prevalence of Sphenophrya was observed in C. gigas from Sambaqui in 2009 (70%) and the lowest (6.7%) in C. rhizophorae from Sambaqui in 2008 (Table 1). In general, the intensity

Table 1 Prevalence (%) of pathogens and pathological changes in the oysters Crassostrea gigas (G) and C. rhizophorae (R) at two points of the Santa Catarina Island, Sambaqui (SAM) and Ribeirão da Ilha (RIB) in March–April, 2008 and 2009. Macroscopic analysis (MA, N = 150), incubation of tissues in Ray's Fluid thioglycollate Medium (RFTM, N = 150) and histological cuts (HC, N = 30). It's also showed the average height of the oysters (mm) and standard deviation (SD). Pathogens and pathological change

2008

2009

SAM

RIB

SAM

G

R

G

R

96.7 ± 14.3

55.3 ± 10.3

84.2 ± 15.8

49.6 ± 6.4

RIB

G

R

G

R

98.1 ± 10

59.6 ± 5.1

104.9 ± 12

58.2 ± 5.4

Height (mm) ± SD

T. 24 °C; Sal. 32‰ Gamet hypertrophy Rickettsia-type bacteria Protozoa Trichodina sp. Sphenophrya sp. Ancistrocoma sp. Steinhausia sp. Nematopsis sp. (HC) Nematopsis sp.(RFTM) Metazoan Polydora sp. (MA) Urastoma sp. Tylocephalum sp. Copepod

T. 22 °C; Sal. 26‰

T. 24 °C; Sal. 35‰

T. 26 °C; Sal. 34‰

– 16.7

– 10.0

3.3 6.7

– 13.3

3.3 10.0

– 13.3

– 30.0

3.3 10.0

– 46.7 36.6 26.7 – 4.7

– 6.7 6,7 16.7 96.6 90.0

13.3 20.0 36.7 – 1.3

– 26.7 10.0 23.3 23.3 35.3

– 70.0 – 16.7 – –

– 33.3 40.0 33.3 100 96.0

3.3 13.3 6.7 3.3 – –

50.0 46.7 23.3 30.0 50.0 88.0

100 – 3.3 –

81.3 6.7 – –

100 6.7 – –

67.3 – – 3,3

100 – 3.3 –

18.7 3.3 – –

100 – – –

22.7 6.7 – –

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Fig. 1. Pathological changes and pathogens detected in tissues of Crassostrea gigas and C. rhizophorae from Sambaqui and Ribeirão da Ilha (Santa Catarina Island, Brazil) in March– April, 2008 and 2009. (A) Hypertrophied cells in the wall of male follicle (arrow); (B) Colony of Rickettsia-type bacteria (arrow) in the cytoplasm of digestive tubule epithelial cell; (C) Colonies of Rickettsia-type bacteria in the cytoplasm of epithelial cells of the stomach; (D) Trichodina sp. (arrow) in the gill filaments, showing evident nucleus; (E) Detail of fastening structures (arrow) of Trichodina; (F) Ciliated Sphenophrya sp. (arrow) in contact with the gills. Bar = 20 μm.

was low with the exception of only three animals (C. rhizophorae from Sambaqui) which presented each one more than 250 ciliates in the whole examined gill/histological cut. No change was detected in the gills neither host reactions. Ciliates of the genus Ancistrocoma were observed in the lumen of digestive tubules. The ciliates showed an oval shape, with basophilic nucleus and eosinophilic cytoplasm (Fig. 2A), with sizes ranging from 12 to 26 μm. The highest prevalences were observed in C. rhizophorae from Sambaqui in 2009 (40%) and in C. gigas from Sambaqui (36.7%) and Ribeirão da Ilha (36.7%) in 2008. The intensity of this ciliate was low, with a maximum of 4 ciliates/histological section. Steinhausia microsporidian was detected in the cytoplasm of oocytes of the oysters (Fig. 2B). The vacuoles containing the pathogen cells sometimes occupied a representative portion of the cytoplasm. The highest prevalences of these protozoa were observed in 2009, in C. rhizophorae from Sambaqui (33.3%) and Ribeirão da Ilha (30%). The intensity of infection in the most animals ranged from 1 to 35 infected oocytes/histological section. However, in one oyster C. gigas from Sambaqui, it was observed more than 50 oocytes containing the spores of the pathogen, without host defense response. Protozoa of the genus Nematopsis were observed infecting the connective tissue of various organs such as gills and mantle (Fig. 2C).

The oocysts Nematopsis densely basophilic had sizes ranging from 8 to 14 μm, hyaline wall and were found 1 to 4 oocysts/phagocyte. The found prevalences after observation of histological sections were high in C. rhizophorae from Sambaqui (96.6% in 2008 and 100% in 2009). In C. gigas, this pathogen was not detected in any of the studied locations. Despite the high prevalences of Nematopsis sp. in C. rhizophorae, it was not observed host response. The intensity of infection was low, with few oocysts/histological section. 3.4.4. Metazoan Turbellarians of the genus Urastoma were found close to the gills of the oyster C. gigas from Ribeirão da Ilha, and C. rhizophorae from Sambaqui and Ribeirão da Ilha. They presented sizes ranging from 162.5 to 245 μm (Fig. 2D). The highest observed prevalence was 6.7% (Table 1). The intensity of infestation was low, having been registered at the most two turbellarians/oyster and without causing apparent damage in the gills. Larvae of cestodes of the genus Tylocephalum were recorded only in the Japanese oysters from Sambaqui with a prevalence of 3.3% in collections from March to April of the years 2008 and 2009. The larvae were encapsulated by several layers of flattened cells. Besides the encapsulation reaction, it was not observed other change in the host tissue (Fig. 2E). A copepod, possibly of the genus Pseudomiycola, was

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Fig. 2. Pathogens observed in tissues of Crassostrea gigas and C. rhizophorae from Sambaqui and Ribeirão da Ilha (Santa Catarina Island, Brazil) in March–April, 2008 and 2009. (A) Ancistrocoma sp. (arrow) in the lumen of a digestive tubule; (B) Vacuole with parasitic spores of Steinhausia sp. (arrow) in the cytoplasm of an oocyte; (C) Oocysts of Nematopsis sp. in the digestive gland; (D) Urastoma sp. among gill filaments; (E) Tylocephalum sp. in connective tissue around digestive tubules. (F) Copepod (arrow) causing disruption of the wall of a digestive tubule. See details of the appendages (arrow). Bar = 20 μm (Figs. A, B, C); Bar = 50 μm (Figs. D, and F).

detected in the lumen of a secondary digestive tubule of the oyster C. rhizophorae from Ribeirão da Ilha (Fig. 2F). This parasite had a size of 900 μm and caused the rupture of the wall of this tubule. 4. Discussion The present study registered several pathogens with different prevalences and intensities in the two species of studied oysters. However, none of these organisms represented risk to the populations of oysters. The polychaete of genus Polydora has been present with high prevalence (100%) in C. gigas from the two studied sites. However, producers did not report mortalities during the periods of collection, showing that, despite its high prevalence, the possible damages do not lead to oyster death. For the same sites, it has already been documented similar prevalences (Ibbotson, 2002; Sabry and Magalhães, 2005) of this polychaete. Infestations by Polydora sp. are common in bivalves from several parts of the world and seem to be the biggest cause of loss for the producer, since the horizontal and vertical tubes of dark color and mud bubbles in the shell influence negatively the market value of the mollusks. In the present study, the oyster

C. rhizophorae collected in 2009 showed prevalence of Polydora below than those found in the Japanese oysters C. gigas in the same period. This might suggest that C. rhizophorae were more resistant to attack of polychaetes. However, for the year 2008 in both places (Sambaqui and Ribeirão da Ilha), the prevalences of Polydora in C. rhizophorae were high, deserving so constant monitoring to better understanding about the susceptibility of C. rhizophorae to the attack of these polychaetes. The results obtained by the technique of tissue culture in thioglycollate medium for diagnosis of Perkinsus showed that the oysters from natural beds as well as the cultured Japanese oysters are not affected by this pathogen. Studies in populations of mollusks from Santa Catarina Island, using this specific technique for diagnosis of protozoa of the genus Perkinsus are recent. The first researches with RFTM were performed in 2008 by da Silva et al. (2010), for C. rhizophorae and A. brasiliana, which also obtained negative results. In Brazil, only one study with C. rhizophorae in Bahia State refers to the use of this technique and also not detected Perkinsus sp. in the analyzed oysters (Nascimento et al., 1986). In the soft-clam (Tagelus plebeius) from Pontal da Daniela Beach, SC, it was also not detected the presence of Perkinsus sp. when this technique was used (da Silva et al., 2009).

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The hypertrophies of male gametes observed in oysters C. rhizophorae (Ribeirão da Ilha) and in C. gigas (Ribeirão da Ilha and Sambaqui) were possibly caused by a virus. The presence of hypertrophied nucleus with granules intensely basophilic in the cell periphery, suggests viruses presence of the families Papillomaviridae and Polyomarviridae that cause the disease known as Viral Gametocytic Hypertrophy (VGH), (Bower et al., 1994). This disease was recorded in bivalve mollusks, including oysters C. gigas and C. rhizophorae (Bower et al., 1994). The first report of this disease was made by Farley (1976). Since then, this disease has been documented in several mollusks (Choi et al., 2004; Garcia et al., 2006; Meyers et al., 2009; Winstead et al., 2004). The prevalence of gametocytic hypertrophy detected in oysters of the present study was low and were not observed hemocytes infiltrations or tissue damage, besides alterations of the infected gamete. In C. gigas from South Korea, the prevalences of VGH ranged from 3.3 to 7.1%, the intensity of infection was low, not being detected defense responses of the host (Choi et al., 2004). In C. gigas studied in France, the prevalence of VGH was low (3.3 to 13.3%) as well the intensity of infection. The authors observed lesions in the gonads of some animals, but with no hemocytic reaction and without lethal effects on oysters (Garcia et al., 2006). In the present study, colonies of Rikettsia-type bacteria were observed in epithelia of digestive tubules, and only in one oyster C. rhizophorae it was detected in the epithelium of the stomach. Despite the found prevalences have been up to 30%, there was no considerable damage in animals. In cockles C. edule from Spain, the infection by Rikettsia-type bacteria colonies were of low intensity and there was not observed response of the host to infection (Carballal et al., 2001), opposite to what was reported in oysters C. gigas from the Atlantic coast of France, when a severe infection caused lesions and changes in the normal gill structure (Renault and Cochennec, 1994). Rikettsia-type bacteria caused intense infections and severe damage in epithelial cells of gill, digestive gland and mantle of the oyster Crassostrea ariakensis from the Guangdong Province, China (Sun and Wu, 2004). However, the harm caused by these bacteria in host cells seem to differ among mollusks (Boehs et al., 2010; Cremonte et al., 2005; da Silva et al., 2005; Green et al., 2008; Villalba et al., 1999; Wu and Pan, 2000). Ciliates are organisms that inhabit the paleal cavity of bivalves without causing harm to the host (McGladdery and Bower, 2001). In the present study, the prevalence of the ciliate Trichodina in C. rhizophorae was high (50%) but lower than that observed in the scallop Aequipecten tehuelchus (100%) from Patagonia, Argentina, (Cremonte et al., 2005). The highest prevalence of Trichodina observed only in oysters from natural stocks of Ribeirão da Ilha, possibly may be related to the high concentration of organic matter and, in consequence, inadequate water quality conditions (Akşit et al., 2008). The occurrence of Trichodina seems to be greater in hosts of polluted areas, it being therefore, a pathogen biological indicator of pollution (Lauckner, 1983). In addition, the fact of Trichodina has been observed in a larger amount free in the digestive tubules of investigated oysters, could indicate a high amount of this protozoan in the environment, in the collection day. Ciliates of genus Trichodina occur worldwide in several bivalves mollusks (Boehs and Magalhães, 2004; Bossaïd et al., 1999; Carballal et al., 2001; Cremonte et al., 2005). However, the pathogenicity of Trichodina is controversial (Bower et al., 1994). High infections by Trichodina sp. were associated to erosions in the gills of Crassostrea angulata from France and were responsible for malfunction of this organ (Lauckner, 1983). In the scallop A. tehuelchus from natural northeastern stocks of Patagonia, Argentina (Cremonte et al., 2005) and in the cockle C. edule from the coast of Galicia, Spain (Carballal et al., 2001), Trichodina also not caused damages or responses of the hosts. However, in C. gigas very infected by Trichodina, it was detected inflammatory response and alterations in the gill epithelium (Bossaïd et al., 1999). Ciliates of genus Sphenophrya were found in the gills of C. gigas and C. rhizophorae in both studied locations. Sometimes the detected prevalences were high, reaching up to 70% in C. gigas from Sambaqui,

but the intensity was generally low and any inflammatory response was observed in affected oysters. Ciliates of this genus are found in a great variety of species of bivalves, being able to cause hypertrophy of the cell and its nucleus (xenoma) (Bower et al., 1994). In C. rhizophorae from Todos os Santos Bay, Salvador, Bahia State (BA), Sphenophrya was observed without causing any damage (Nascimento et al., 1986). However, for the same species of oyster collected in Camamu Bay, BA, it was observed the formation of xenoma (Boehs et al., 2009) and the authors concluded that this pathological effect was associated to the presence of this ciliated. In the mussel Dreissena polymorpha (Bivalvia: Mytilidae) highly infected by Sphenophrya dreissenae, was observed hyperplasia, cell hypertrophy and epithelium vacuolization (Laruelle et al., 1999). Ancistrocoma sp. is other ciliate that was observed in oysters of the present study in low intensity and caused no damages or host defense responses. Nascimento et al. (1986), also reported no damage in C. rhizophorae from Bahia affected by Ancistrocoma sp. Steinhausia sp. was observed in oocytes of C. gigas and of C. rhizophorae in both locations. The presence of the sporocysts with pathogen spores caused damage to the normal structure of the cytoplasm of oocytes, but it was not observed defense cells close to the infected oocytes. In M. galloprovincialis (Bivalvia: Mytilidae) infected with Steinhausia, it was not also observed infiltration of hemocytes (Robledo et al., 1994). Instead, the cockle C. edule infected by Steinhausia-type microsporidia showed a light hemocytic response without, however, causing significant pathologic damage to the host (Carballal et al., 2001). Oysters Saccostrea glomerata from Australia, infected with this microsporidian, showed a complete reabsorption of the oocytes with infection of germinal epithelium and high hemocytes infiltration (Green et al., 2008). The prevalences of Steinhausia observed in the present work was considered high (up 33.3%) when compared to 6.6% detected in Mytella guyanensis from Amazon River (Matos et al., 2005), 9% for C. gigas (Pontinha, 2009) and 7.5% for A. brasiliana in Santa Catarina (da Silva et al., 2010). However, the highest prevalence found in this study was similar to that recorded in Brachidontes solisianus from Santa Catarina (37.5%) (Pontinha et al., 2010). Gregarines of genus Nematopsis were detected in 100% of the oysters C. rhizophorae from Sambaqui. However, it was not observed defense response of the oysters. The protozoan Nematopsis uses bivalves as intermediate host and completes its life cycle in marine arthropods (Lauckner, 1983; McGladdery et al., 2006). The higher prevalence of Nematopsis in oysters from the rocky shore could be related to the closer proximity of crustaceans in the shores. On the other hand, the lower prevalence of this pathogen would be associated to a small amount of these animals in the cultivation lanterns. Boehs et al. (2010) found higher prevalences of Nematopsis in M. guyanensis collected in mangrove areas when compared to those found in A. brasiliana, and reported that the abundance of crustaceans in the estuary could have contributed to the differences in the found prevalences. High prevalences of Nematopsis in scallops A. tehuelchus in Argentina (100%) did not induce host defense reactions (Cremonte et al., 2005). In C. rhizophorae from Jaguaribe River, Ceará State, defense reactions were observed as evidenced by the infiltration of hemocytes (Sabry et al., 2007). High intensities of infection by Nematopsis sp. also caused changes in the morphology of the gills and mantle of M. guyanensis from the estuarine region of Cachoeira River, BA (Pinto and Boehs, 2008). In the cockle A. brasiliana from the same estuary, it was not observed tissue damage or infiltration of hemocytes in infected animals (Boehs et al., 2010). Nematopsis sp. was associated to complete destruction of gills cells and mortality in the cockle C. edule from the south region of Portugal (Azevedo and Cachola, 1992). In these cockles from Spain, the presence of Nematopsis caused mild lesions in the gill filaments and induced a light hemocytic response without, however, causing mortality (Carballal et al., 2001). It is noteworthy that in the present study

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the RFTM technique proved to be useful for diagnosing Nematopsis. The analysis of gills revealed high prevalence of this protozoan, proving to be a sensitive technique, helping the histology. Metazoan of genus Urastoma (Turbellaria) were detected free among the gill filaments of the oysters C. gigas and C. rhizophorae in this study and did not cause any apparent damage in the gills. The prevalence as well as the levels of infestation by this organism was low. In Atlantic coast of Canada, infestations by Urastoma cyprinae in oysters Crassostrea virginica were very high: up to 1,000 parasites by individuals with prevalences up to 100%. However, there was no histopathological damage in the gills (McGladdery et al., 2006). In oysters C. gigas from Sambaqui infected by a cestode larva of Tylocephalum, there was a host response, as evidenced by a layer of cells of fibrous nature, besides the infiltration of hemocytes forming encapsulation of the parasite. The host response against the parasite may vary among the affected species (Lauckner, 1983). According to Figueras and Villalba (1988), the response of C. gigas when infested by Tylocephalum seems to be less intense than in other bivalves. In bivalves of genus Pinctada, the capsule formation is not so pronounced and so it is believed that the oysters belonging to this genus are normal intermediate hosts of this parasite since the host defense response is less intense (Lauckner, 1983). In oysters C. rhizophorae from Bahia, Tylocephalum larvae were responsible for damage of mechanical nature evidenced by disruption of tissues during the penetration of the cestodes (Nascimento et al., 1986). The copepod found in the mangrove oyster C. rhizophorae from Ribeirão da Ilha, possibly belonged to the genus Pseudomyicola. This copepod caused damage to the epithelial tissue of the stomach of the oyster C. rhizophorae, probably due to its large size and the presence of the animal appendages. In the Pectinidae Argopecten ventricosus from Mexico, Cáceres-Martínez et al. (2005) also observed erosions in the epithelium of the stomach and gill, light infiltration of hemocytes and alterations in the basal membrane of ephitelium caused by the appendages of P. spinosus. The prevalence of copepods in A. ventricosus (100%) and in M. galloprovincialis (93.3%) from Mexico (Cáceres-Martínez et al., 2005; Cáceres-Martínez and Váquez-Yeomans, 1997) was quite high when compared to the prevalence of 3.3% observed in the present study. None of the pathogens found in this study seem to cause significant damage in the oysters, taking into account the low intensity of infection and infestation that were observed and, in the most animals, absence of pathological effects. Although the found pathogens are not in the list of obligatory notification of OIE, it's necessary a constant monitoring of shellfish populations in order to assess more accurately the effects of these pathogens in their hosts, mainly in stressful situations that could serve as factor for triggering non-apparent infections. Furthermore, bivalve mollusks have great socioeconomic importance for the State of Santa Catarina, deserving special attention regarding the health of these animals. Acknowledgments The authors are grateful to the National Council for Scientifical and Technological Development (CNPq) for financial support, through the annoucement of MCT/CNPq/MAPA/SDA, for the Post-Doctoral Scholarship to Patricia Mirella da Silva and to the Improvement Coordination of Personnel of Superior Level (CAPES) for the Doctoral Scholarship of the Amazônia Azul Program to Rachel C. Sabry. To Dr. Jaime F. Ferreira and to shellfish farmer Rita C. Rodrigues for the support and oysters supply. To MSc. Maximiano P. Dantas Neto for editing this English version. References Akşit, D., Falakali-Mutaf, Göçmen, B., Gürelli, G., 2008. A preliminary observations on Trichodina sp. (Ciliophora: Peritricha) on the Gills of Limpets (Patella spp.) in Antalya (Turkey). North-Western. J. Zool. 4, 295–299.

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