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This work investigated the occurrence of Perkinsus sp. in clam Anomalocardia brasiliana, oyster Crassostrea sp. and mussel Mytella falcata from the Jaguaribe River estuary, northeastern Brazil. The collection of clam (N = 300), oysters (N = 300) and mussels (N = 300) were carried out in the estuary of the Jaguaribe River, Ceará, in March and April (rainy season) and October (dry season) in 2017. The mollusks were measured in their major axis, open, and had their tissues submitted to tissue incubation techniques in Ray’s fluid thioglycollate medium (RFTM), histology, real-time polymerase chain reaction (qPCR), PCR and sequencing. The RFTM assays showed Perkinsus sp. infecting the three mollusks investigated. The prevalence of infected clams was 1.33% in both sampling periods, oysters ranged from 2.66 (rainy season) to 8% (dry period), and mussels from 0% (dry period) to 51.33% (rainy season). The intensity of infection was very light to light in clams, very soft to severe in oysters and very soft to moderate in mussels. Histological analyses showed cells of Perkinsus sp. infecting the gills and connective tissue around the digestive gland of some individuals. The qPCR generated amplicons in all positive samples in RFTM, confirming the presence of Perkinsus sp., while the sequencing evidenced high similarity (99%) with the species P. beihaiensis. In conclusion, the results obtained contribute to increasing knowledge about the occurrence of Perkinsus sp. in bivalve mollusks from northeastern Brazil.

This study investigated Perkinsus spp. infecting Crassostrea rhizophorae from the Jaguaribe River estuary, Ceara, Brazil. Fragments of gills and rectum of the oysters (n = 150) were incubated in Ray’s fluid thioglycollate medium (RFTM). Genus Perkinsus-specific PerkITS85/750 PCR assays were performed and their amplicons were sequenced by the Sanger method. The RFTM assays confirmed Perkinsus spp. The sequencing of the amplified fragments from the rDNA internal transcribed spacers (ITS) of Perkinsus spp. confirmed Perkinsus chesapeaki. Neighbor-Joining analyzes place P. chesapeaki identified in this study in a well-supported clade with other isolates of the same species. This is the first record of P. chesapeaki infecting C. rhizophorae in South America.

The mangrove oyster Crassostrea rhizophorae is an estuarine resource exploited by riverside communities in Northeast Brazil. Despite its socioeconomic importance, studies on the health status of this bivalve are scanty in this region. The purpose of the present study was to investigate the presence of the protozoan Perkinsus sp. in C. rhizophorae collected in August and September 2011 in three estuaries of the septentrional Northeast, Brazil: Jaguaribe (Ceará), Camurupim (Piauí) and Carnaubeiras (Maranhão) (n= 150 specimens/site). The samples were submitted to Ray’s fluid thioglycollate medium (RFTM), PCR and histology assays. The RFTM assay revealed spherical, blue or bluish-black hypnospores of the genus Perkinsus in 50 specimens (Jaguaribe= 17.3%, Camurupim= 5.3%, Carnaubeiras= 10.6%). The intensity of the infection ranged from very light (1-10 cells per slide) to severe (more than 40 cells in each of 10 fields of the slide) for Jaguaribe; very light for Camurupim and very light to moderate (at least 40 cells observed in each of 10 fields of the slide) for Carnaubeiras. When submitted to confirmatory PCR analysis, 6 cases were confirmed (Jaguaribe=3, Camurupim=1, Carnaubeiras=2). The histology confirmed 21 cases of infection in specimens from the three estuaries. Although local collectors have reported no mortality in oyster populations that might be attributed to infection by Perkinsus, health surveillance of oyster populations in the septentrional region of Northeast Brazil is advisable. Resumo A ostra-do-mangue Crassostrea rhizophorae é um recurso estuarino explorado por comunidades ribeirinhas do Nordeste do Brasil. Apesar de sua importância socioeconômica, estudos sobre o estado de saúde deste bivalve são escassos na região. O objetivo deste estudo foi investigar a presença do protozoário Perkinsus sp. em C. rhizophorae coletada em agosto e setembro de 2011, em três estuários da região setentrional do Nordeste brasileiro: Jaguaribe (Ceará), Camurupim (Piauí) e Carnaubeiras (Maranhão) (n = 150 espécimes/local). As amostras foram submetidas ao meio líquido de tioglicolato de Ray (RFTM), PCR e ensaios histológicos. A análise em RFTM revelou hipnósporos esféricos azuis ou preto-azulados do gênero Perkinsus em 50 espécimes (Jaguaribe= 17,3%, Camurupim= 5,3%, Carnaubeiras= 10,6%). A intensidade de infecção variou de muito leve (1-10 células por lâmina) a severa (mais de 40 células em cada um dos 10 campos da lâmina) para o Rio Jaguaribe; muito leve para o Rio Camurupim e muito leve a moderada (pelo menos 40 células observadas, em cada um dos 10 campos da lâmina) para o Rio Carnaubeiras. Quando submetidos à análise confirmatória por PCR, foram confirmados 6 casos (Jaguaribe= 3, Camurupim= 1, Carnaubeiras= 2). A histologia confirmou 21 casos de infecção em espécimes dos três estuários. Embora os coletores locais não tenham relatado nenhuma mortalidade em populações de ostras que pudesse ser atribuída à infecção por Perkinsus, é aconselhável um monitoramento sobre o estado de saúde de populações de ostras da região.

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 Ribeirao 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 Ribeirao da Ilha and in C . gigas from Sambaqui and Ribeirao 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 Ribeirao 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.

No estuario do Rio Jaguaribe, Ceara, a ostra Crassostrea gasar e um importante recurso pesqueiro sendo comumente coletado pela comunidade para alimentacao e tambem para o comercio local. Apesar da importância que esse bivalve representa para comunidade, na regiao, sao poucas as pesquisas sobre a ocorrencia de patogenos causadores de doencas, nesses animais. O objetivo desse trabalho foi investigar a ocorrencia de parasitas em C. gasar do estuario do Rio Jaguaribe, Ceara. As coletas das ostras (N=600) foram realizadas no periodo de agosto de 2014 a marco de 2016. Durante as coletas a temperatura e a salinidade da agua foram monitoradas. Todos os moluscos foram submetidos a tecnica de cultivo de tecidos em meio fluido de tioglicolato (RFTM) especifica para o protozoario do genero Perkinsus , histologia e PCR. A salinidade da agua variou de 18% o a 40% o e a temperatura de 27 a 33°C. O tamanho medio das ostras no periodo variou de 76,69 mm±10,76 a 103,97mm±30,12. As analises macroscopicas mostraram a presenca de poliquetas pertencentes ao genero Polydora . Os ensaios de RFTM detectaram Perkinsus sp. infectando as ostras investigadas com prevalencia de ate 33,3%. As analises histologicas evidenciaram virus (6,7%), bacterias (ate 65,5%), os protozoarios Nematopsis sp. e Ancistrocoma sp. com prevalencias de 80% e 20%, respectivamente e o metazoario Urastoma sp. (13,3%). Os resultados da PCR foram positivos em apenas 3 animais dos 95 positivos no RFTM. Ate o momento, nenhum dos patogenos encontrados parece representar risco potencial as ostras desse banco natural.

This is the first report of Perkinsus sp. (Bivalvia: Veneridae) infecting wild clams of the species Anomalocardia brasiliana in Brazil. The gill lamellae and rectum of 150 specimens of A. brasiliana collected in the Timonha river estuary (Ceara, Northeastern Brazil) in March 2012 were incubated in Ray's fluid thioglycollate medium (RFTM) for detection of Perkinsus sp. In RFTM, the prevalence of Perkinsus sp. was 14.7% (22/150) and the intensity of infection ranged from very light (1-10 cells across the slide) to light (12-100 cells). The presence of Perkinsus sp. was confirmed by PCR in seven (31.8%) out of 22 RFTM-positive specimens. DNA sequencing confirmed the presence of the genus Perkinsus and the phylogenetic analysis strongly indicated Perkinsus beihaiensis as the species responsible for the infection.

This study investigated the occurrence of the protozoan Perkinsus in the oyster Crassostrea rhizophorae on the coast of Bahia State, Brazil. The oysters (n=900) were collected in February–March and July–August 2010. The Ray’s fluid thioglycollate medium (RFTM) analysis of gills and rectum revealed hypnospores of Perkinsus sp. with a high mean prevalence (63%). The infection intensity varied from very light to advanced. The polymerase chain reaction confirmed Perkinsus in 87.2% of the RFTM-positive oysters. Histological analysis showed trophozoites and schizonts phagocytized by hemocytes, mainly in the intestine and the stomach epithelium.

The mangrove oyster, Crassostrea rhizophorae (Bivalvia, Ostreidae) is commonly collected by fisherwomen in the estuaries of the Ceará State (CE), Northeastern Brazil. Despite the socioeconomic importance of this natural resource, there are few studies on the health of the oysters in this region. This study aimed to survey pathological changes in the mangrove oyster C. rhizophorae in the estuary of the Pacoti River, CE. Adult oysters were collected in August 2008 (N=450) and December 2009 (N=450) at three sites of the Pacoti estuary and in 2010 (N=600) samplings were done quarterly at one site which has showed the higher prevalence de Perkinsus. Macroscopical and histological analyses were used to evaluate pathological changes, Ray’s Fluid Thioglycollate Medium (RFTM) to detect Perkinsus spp. and polymerase chain reactions (PCR) and DNA sequencing to identify Perkinsus species. In 2009, RFTM assay detected Perkinsus sp. infecting the tissues of C. rhizophorae with low prevalences of 1.3%, 6.7% e 7.3% in sites 1, 2 and 3, respectively, and in 2010, in site 3, prevalence was 2% (12 of 600 oysters). PCR did not confirm any positive case in 2009 and only 5 in 2010. The phylogenetic analyses strongly indicate that the Perkinsus species infecting oysters C. rhizophorae of this study belongs to Perkinsus beihaiensis. The histology confirmed 11 cases of Perkinsus sp. infecting the C. rhizophorae in 2009, and only two cases in 2010. Nematopsis sp. was the protozoan observed with greater prevalence (up 96.7%). Other found protozoa were: Trichodina, Sphenophrya, Ancistrocoma – like and an unknown ovarian parasite. The metazoa found were the polychaete Polydora with high prevalences, a turbellarian, possibly of the genus Urastoma, an unidentified digenean metacercariae and larvae of cestode Tylocephalum. A continuous monitoring of diseases in bivalves from this natural population is recommended, since the phylogenetic analyses indicate the occurrence of P. beihaiensis infecting oysters C. rhizophorae whose pathogenic potential is unknown.

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in several mollusk species worldwide. In the present study we describe for the first time a parasite of the genus Perkinsus infecting the mangrove oyster Crassostrea rhizophorae from the Brazilian coast. Prevalence of this parasite was low in the Pacoti River estuary (Ceara, northeast Brazil) and absent in oysters from southern Brazil. Oyster gill and rectum tissues incubated in Ray's fluid thioglycollate medium (RFTM) revealed the presence of spherical hypnospores (5 to 55 microm diam.). Histological analysis showed the occurrence of typical signet-ring trophozoites and schizonts (3 to 6 microm diam.) infecting connective tissues of several organs and digestive epithelia. PCR assays specific to the genus Perkinsus, followed by cloning and sequencing of the internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid (rRNA) gene complex, confirmed a close phylogenetic relationship between Brazilian Perkinsus sp. and P. beihaiensis infecting Chinese oysters.

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray's fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil. Fil: Silva, Patricia Mirella da. Universidade Federal de Santa Catarina; Brasil Fil: Cremonte, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina Fil: Sabry, Rachel Costa. Universidade Federal do Ceará; Brasil Fil: Rosa, Rafael Diego. Universidade Federal de Santa Catarina; Brasil Fil: Cantelli, Liege. Universidade Federal de Santa Catarina; Brasil Fil: Barracco, Margherita Anna. Universidade Federal de Santa Catarina; Brasil

Mangrove oysters Crassostrea rhizophorae were sampled monthly in the estuary of Jaguaribe River, on the east coast of Ceará State, Brazil, between August, 2000 and December, 2001, making up 170 individuals. The water temperature varied from 26 to 30 degrees C and salinity from 21 to 42. The animals' size ranged from 3.4 to 7.2 cm height. Macroscopical and histopathological analyses were carried out in the oysters' tissues. The histological exams showed protozoans and metazoans of genera Nematopsis and Tylocephalum, respectively. Nematopsis prevalence varied from 60 to 100% and it was higher in the gills and mantle. The oocysts presented a mean size of 11.5 microm (+/-1.32) length and 9.1 microm (+/-1.06) width (n = 30), up to 3 oocysts/phagocyte having been observed. Several animals presented focal hemocitical reaction. The percentage of Tylocephalum was 1.7%. In spite of the high infection prevalence by Nematopsis, infected animals did not have their reproductive cycle impaired.