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Human activities have caused climate changes and altered the salinity of the oceans. The reduction of the salinity is one of the factors that may limit the distribution and the survival of marine organisms. Coelomocytes are the immune system cells of the echinoderms and have been studied as biomarkers of stress. The aim of the present study was to investigate the effect of the salinity on the immune system cells of the tropical sea urchin Echinometra lucunter . Animals were collected in Joao Pessoa coast (Brazilian Northeast). Animals or coelomocytes were exposed to different salinities (25, 35, 45) from 4 to 24 h. Phagocytic parameters, production of reactive oxygen species (ROS), mitochondrial activity and ABC transporter activity were analyzed. The phagocytic parameters did not change when animals or cells were exposed to a salinity of 25 or 45 in any time intervals monitored. However, the coelomocytes concentration increased when animals were exposed to the lower salinity. The levels of ROS were higher when cells were incubated at a salinity of 25 but lower when cells were kept at a salinity of 45. It was observed the loss of the mitochondrial inner membrane potential when coelomocytes were incubated at a salinity of 45. The activity of ABC transporters decreased when cells were incubated at the lowest salinity and increased when cells were incubated at the highest salinity tested. The present work shows that the immune system of the tropical sea urchins Ei lucunter tolerates salinity changes from 25 to 45, and suggests two cellular parameters (ROS levels and ABC transporters activity) as potential biomarkers for the monitoring of the impact of environmental salinity changes.

SUMMARYField and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

The invasive bivalve Mytilopsis sallei caused several economic and ecological impacts in Asia. In Brazil, M. sallei was first reported in 2004, and thus far, little is known about its ecological impact on Brazilian estuaries. The present study aims to evaluate the abundance and distribution of M. sallei and to analyse the environmental factors that led to the establishment of this species in a Brazilian Northeast estuary. Sampling was carried out in six stations along the estuary, covering a broad spectrum of salinities. Results showed that M. sallei was the most abundant benthic species in this estuary, although it was not broadly distributed, occurring only in one station (S1), the most polluted and the one with the lowest salinities (mean 17.3 ± 11.56), which was the abiotic factor controlling the distribution of M. sallei. This station also had the lowest diversity of species (H′ = 0.3269). The main M. sallei microhabitat was the pneumatophores roots from mangrove trees and decomposing wood. Despite the high abundance of M. sallei in S1, this species experienced negative effect of other benthic species suggesting that its distribution was limited by the high abundance of native species in other stations. Our results suggest that the distribution of M. sallei is influenced by a set of characteristics: preference for low salinity, favourable substrate type, tolerance to pollutants, and less direct competitors. Understanding how invasive species are distributed and relate to native species will provide more robust strategies to help maintain environmental health that ultimately will prevent the expansion of M. sallei population.

WOS:000416186800021; International audience; ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraiba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes \textgreater colorless spherulocytes \textgreater vibrate cells \textgreater red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C gigas. Additionally, our results showed that C gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Blooms of the harmful alga Dinophysis acuminata, which produces okadaic acid (OA), are becoming recurrent in Santa Catarina coast, where most of the shellfish marine farms in Brazil are located. We evaluated the impact of D. acuminata blooms on various haemato-immunological parameters and on tissue integrity of cultivated oysters (Crassostrea gigas) and mussels (Perna perna). Animals were sampled during two natural algal blooms, one at Praia Alegre (PA: 2950 cells l−1) and the other at Praia de Zimbros (PZ: 4150 cells l−1). Control animals were sampled at the same sites, 30 days after the end of the bloom. The assayed parameters were: total (THC) and differential (DHC) haemocyte counts, percentage of apoptotic haemocytes (AH), phenoloxidase activity (PO), agglutinating titre (AT) and total protein concentration in haemolymph (PC). Histological analyses were carried out in oysters from PZ. The results showed that some immune parameters were modulated during the toxic blooms, but not in a consistent manner, especially in mussels that accumulated more OA (10×) than oysters. For example, mussel THC decreased significantly (54%) during the bloom at PA, whereas it augmented markedly (64%) at PZ. PO activity was significantly altered by the algal blooms in both bivalve species, while PC increased significantly (66%) only in mussels from PZ bloom. The other parameters (DHC, AH and AT) did not vary in both bivalve species. Histological analyses showed an intense haemocytic infiltration throughout the oyster digestive epithelium, particularly into the stomach lumen during the algal bloom.

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Ray's fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR-RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.

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.

Disseminated neoplasia (DN) was one of the most important pathological conditions found in cultured flat oysters (Ostrea edulis) from different geographical origins grow in Galicia (NW Spain), during a two years selective breeding programme to produce oysters less susceptible to bonamiosis. Histological characteristics observed in oysters affected by DN included intense infiltration of connective tissue of various organs (gills, stomach, digestive gland and gonad) by large undifferentiated cells, with a large nucleus and a high nucleus-to-cytoplasm ratio. The main ultrastructural features were predominance of euchromatin over heterochromatin that was arrayed in small clumps in the nucleus, prominent granular nucleolus, swollen mitochondria with few cristae and high number of free ribosomes in the cytoplasm. A seasonal pattern of DN prevalence was detected, with higher values in spring-summer, but there were no significant differences between geographic origins or families within these origins. However, the intensity of the disease was significantly different between origins; oysters originating outside of Galicia (particularly those originating from Ireland) were more susceptible to develop advanced DN. DN (8%) and bonamiosis (4.9%) were found concurrently in oysters. The nature and significance of this association warrants more investigation to determine its importance, if any.

During a parasitological survey of the brown mussel Perna perna from highly productive culture fields in the southern sector of Brazil, monstrilloid copepods were discovered in the mantle of this mytilid bivalve. Numerous specimens of endoparasitic copepods were found within nodules in the mantle of the host; they belong to an undetermined species of Monstrilla. Monstrilloid copepods were known to be endoparasitic in polychaetes and in one species of prosobranch mollusc only; their occurrence as parasites of bivalve molluscs has not been previously documented. This is also the first record of these crustaceans infecting commercial molluscs. The prevalence of Monstrilla sp. infecting the brown mussel was 25.6%. At the histological level, it was observed that the larvae induced a strong haemocytic infiltration resulting from the complete larval encapsulation within the host mantle. The effect of this monstrilloid in the cultured populations of P. perna deserves further study.

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum . Manila clams, with variable levels of infection with Perkinsus olseni , were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum . After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum ; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.

An experiment was carried out to compare the performance of various cohorts of oysters Ostrea edulis from each of four different geographic origins that were born and cultured in the same environment. This paper reports the variation of gonad condition of the oysters through on growing and analyses of the relationship between gametogenesis and infection by Bonamia ostreae. Nineteen cohorts of spat were produced in a hatchery from four oyster populations: Irish, Greek, and two Galician: Ortigueira and Coroso; the spat were transferred to a raft in the Ria de Arousa, an area affected by bonamiosis, for grow-out for two years. Significant differences were observed in the temporal pattern of gonad condition between oysters from different geographic origins. Irish oysters showed a short period of gonad activity and a long gonad resting phase. By contrast, Greek oysters had the longest period of gonad activity and the shortest gonad resting phase, as well as the highest percentage of male oysters compared to oysters from other origins. Galician oysters, from both origins, showed a pattern of gonad development intermediate between Irish and Greek, with a higher percentage of females during the second reproductive period. Significant differences were also observed between cohorts within Greek, Coroso and Irish origin, but not from Ortigueira, whose cohorts showed a similar temporal pattern of gonad condition. The differences between oysters from different origins would indicate adaptation to different environments; nevertheless the significant differences that were found between cohorts within origins would indicate that there is variability that eventually could allow adaptation to new environments. Considering sex categories, heavy infections by B. ostreae were significantly more frequent in oysters with a predominant female component; considering gonad stage, heavy infections were significantly more frequent in ripe and partially spawned oysters. The results suggest a hypothetical enhancement of progression of infection by B. ostreae associated with female maturation.

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ria de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.

This study reports the prevalence of Bucephalus sp. in Perna perna populations from a culture station of southern Brazil and its effect on the mussel reproductive tissue and immune system. The prevalence of Bucephalus sp. in P. perna (n = 1871) was considered low (3.1%) and did not seasonally vary. Histological sections of the mantle of infected mussels revealed a marked (80%) reduction of the reproductive tissue that was severe even in mussels exhibiting a moderate infection degree. The total (THC) and differential (DHC) hemocyte counts were lower in infected mussels (3.9 x 10(6) hem/ml; granular hemocytes = 33%) as compared with non-infected animals (5.5 x 10(6) hem/ml; granular hemocytes = 40%). The plasma protein concentration did not vary upon infection. Hemocyte infiltration was significantly higher only in mussels with a very heavy infection degree. The parasite sporocysts were never seen encapsulated by the host hemocytes. Our results indicate that Bucephalus sp. promotes a severe castration in its host and apparently evades the mussel immune system.

International audience; Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection ("Light-Moderate" and "Moderate-Heavy"). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index.

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.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceara state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraiba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio Sao Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

International audience; Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases.

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.

International audience; Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 +/- 1 degrees C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 +/- 1 degrees C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.

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

The present study evaluated the possible effects of a toxic dinoflagellate, Karenia selliformis , upon immunological hemocyte functions of the Manila clam Ruditapes philippinarum , and on the progression of infection by Perkinsus olseni . Clams with variable levels of perkinsosis were exposed for 6 weeks to simulated blooms of cultured the K. selliformis (10 2 and 10 3 cell ml −1 ). Samples were collected after 0, 2, 3, and 6 weeks of exposure. The following hemocyte parameters were measured by flow cytometry: percentage of dead cells, cell size and complexity, apoptosis, phagocytosis, and production of reactive oxygen species. Agglutination activities of K. selliformis on horse erythrocytes, serum protein concentration, and condition index of clams were also assessed. The harmful alga K. selliformis caused a significant decrease in hemocyte size and percentage of apoptotic cells. In contrast, P. olseni did not affect clams strongly; the only significant effect was an increase in hemocyte size in heavily infected clams. After 2 and 3 weeks, the prevalence and burden of P. olseni decreased in clams exposed to K. selliformis , but after 6 weeks, and a diminution in K. selliformis cell density in the exposure, this effect disappeared. In vitro tests exposing P. olseni to K. selliformis showed direct algal toxicity to the parasite (increased percentage of dead cells and altered morphology). Initial exposure of P. olseni -infected clams to K. selliformis appeared to modify the host–parasite interaction by causing effects in both organisms.

International audience; This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic "granulocytes" could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of "hyalinocytes", which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these "inclusions" was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a "differentiation continuum". Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.