Ray's Fluid Thioglycollate Medium Research Articles

Detecting Perkinsus-like organisms and Perkinsus marinus (Myzozoa: Perkinsidae) within new bivalve hosts in the southeastern Gulf of California.

Perkinsus marinus, a parasite myzozoa native from the Atlantic Ocean, has spread across various bivalve species in the Pacific Ocean. In the Gulf of California, clams like Larkinia grandis and Leukoma grata have become a secondary fishery, experiencing demand. During annual sampling (August 2017 - July 2018, n = 30/month), the Ray's fluid thioglycollate medium (RFTM) revealed presumptive hypnospores of Perkinsus sp. in L. grandis, with a low parasite prevalence (4.16%) and density (≤ 3.14 hypnospores g-1 tissue). From summer 2020 to summer 2021, P. marinus was seasonally identified in L. grata via staining and PCR tests, showing mean prevalence and density of 48.33% and 48 hypnospores g-1 tissue, respectively. The infection level was light in both clams. This parasite's presence in these new hosts within the southeastern Gulf of California confirms its ability to expand into non-native places, signaling a need for intervention strategies in managing and transporting mollusk species within the region.

First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray’s Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol’s iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Characterisation of common hypothetical surface peptides between protozoan parasites (Perkinsus olseni) originating from different geographical locations.

Perkinsus olseni and P. marinus are classified as notifiable pathogens by the World Organisation for Animal Health and are known to cause perkinsosis in a variety of molluscs globally. Mass mortalities due to these parasites in farms and in the wild have been a recurrent issue. Diagnosis for these protozoans is currently done using Ray's fluid thioglycollate medium method followed by optical microscopy or molecular assays. Both require a high level of skill and are time-consuming. An immunoassay method would make the diagnosis of perkinsosis quicker and cheaper. The present study used mass spectrometry-based proteomics to investigate common hypothetical surface peptides between different geographical isolates of P. olseni, which could be used to develop immunoassays in the future. Two peptides were identified: POLS_08089, which is a 42.7 kDa peptide corresponding to the 60S ribosomal subunit protein L4; and POLS_15916, which is a conserved hypothetical protein of 55.6 kDa. The identification of peptides may allow the development of immunoassays through a more targeted approach.

First record of Perkinsus marinus infecting Crassostrea sp. in Rio Grande do Norte, Brazil, using real-time PCR

A pathogen with high virulence potential in some host species, Perkinsus marinus remains a challenge for the ecological integrity of marine ecosystems and the health of bivalve molluscs. This study investigates the occurrence of P. marinus in Crassostrea sp. in estuaries of the Potengi River and the Guaraíras lagoon in Rio Grande do Norte, Brazil. A total of 203 oyster samples that tested positive for Perkinsus sp. in Ray's fluid thioglycollate medium (RFTM) were subjected to species-specific quantitiative PCR, where 61 animals (30.05 %) presented amplification graphs with a melting temperature of 80.1 ± 0.6 °C matching the positive control. This was the first record of P. marinus in oysters in these estuaries using qPCR as a diagnostic tool.

The endoparasite Perkinsus olseni affecting the Mediterranean mussels (Mytilus galloprovincialis) in the Italian and Spanish waters: A new possible threat for mussel aquaculture and wild animal population

Dinoflagellates belonging to the Perkinsus genus are OIE (World Organization for animal Health)-listed pathogens extremely virulent for clams and oysters in many marine ecosystems throughout the world. During the monitoring activities of the Mediterranean mussel (Mytilus galloprovincialis) in Campania region (Italy), the presence of typical trophozoites of Perkinsus sp. was observed in mussels from farms and natural banks. Simultaneously, following mussel mortality in the Spanish waters of Catalonia, histopathological studies revealed the presence of the same parasite. Although perkinsosis is an endemic disease in clams in Italy (with prevalence from 40 to 80%), there are no reports to date of its presence in Mediterranean mussels and of the effect on this species. For this study, histopathology, Ray’s Fluid Thioglycollate Medium (RFTM), and molecular diagnostics with conventional Polimerase Chain Reaction (PCR) and qPCR were performed. In samples from Italy, histopathology in the mussel from one farm revealed a prevalence of 26% in February 2019, 40% in February 2020, 16% in November 2020, and 23% in April 2021. In a natural bank, Perkinsus was also detected in May 2020 but in lower prevalence. In Spain, in July 2020, the presence of the parasite was 20% in one site and 10% in a second site and related to animal mortality. In both areas, Perkinsus sp. elicited multiple inflammatory capsules of different size or infiltrates at the level of the digestive gland and gonad. Molecular diagnostics of the Internal Transcriber Spacer (ITS) region of the rDNA (ITS1, 5.8S, and ITS2) showed a 97% similarity of P. olseni from Italy with samples from New Zealand, Australia, and Uruguay and in bivalves such as Pitar rostrata, Astrovenus sp., and Haliotis sp., whereas in Spain the identity was 99% samples from South Korean venerids such as Anadara granosa. Phylogenetic analysis group together P. olseni from Italian and Spanish mussels but place them distant from other P. olseni described in the clams from Europe (Italy, France, and Spain). Direct impact of transboundary animal diseases in aquaculture constitutes a serious consequence for export living animals and their products, as well for international trade. This compromises food security, also causing a high socioeconomic impact on aquaculture exporting nations. P. olseni is a generalist pathogen able to infect different bivalve species, possibly passing from clams to oysters and mussels. Recognized international organizations should take this into account in the view of possible cross-infection. Other studies are needed to define pathogen virulence in this species.

Molecular and histological identification of the protozoan parasite Perkinsus olseni in the blood cockle Anadara kagoshimensis (Tokunaga, 1906) occurring on the south coast of Korea

A recent report on co-infection by two Perkinsus species in mud arks Anadara trapezia (Bivalvia Arcidae) in Australia implies possible Perkinsus infection in other Arcidae species. Blood cockle Anadara kagoshimensis has been exploited since the 1960s on the south coast of Korea, and the landings have decreased due to mass mortality in recent years. During a histopathology survey of blood cockle on the south coast, we identified a Perkinsus-like pathogen in the visceral mass. Accordingly, we applied Perkinsus species-specific PCR and subsequent sequencing to identify the pathogen to species level using internal transcribed spacer (ITS) rDNA. Ray's fluid thioglycollate medium (RFTM) and immunofluorescence assays (IFA) were also used to quantify and visualize the infection in this novel host. Perkinsus-like organisms were mainly found in the hemal space of visceral mass connective tissue. The Bayesian inference tree based on ITS rDNA sequences is strongly affiliated with P. olseni, suggesting that the Perkinsus-like organism in A. kagoshimensis is P. olseni, commonly found in Manila clams in Korean waters. RFTM and subsequent 2 M NaOH digestion assay revealed that the infection intensity ranged from 8000 to 1,481,000 cells/g gill, with a mean of 1.2 × 105 ± 2.5 × 105, and the prevalence was higher in September (fall) with 13.5% (101/750) than in June (spring) (0.4%, 3/750). Histology revealed that heavily infected clams exhibited tissue inflammation suggesting that P. olseni infection could damage the structure and physiological function of A. kagoshimensis; although we could not find a significant difference in condition index between the infected and uninfected individuals.

Development of a simple host-free medium for efficient prezoosporulation of Perkinsus olseni trophozoites cultured in vitro.

The parasitizing stage (trophozoite) of the protozoan parasite Perkinsus olseni progresses to the dormant stage (prezoosporangium) immediately after the death of the host through physiologically and morphologically drastic changes. This development is reproducible in Ray's fluid thioglycollate medium (RFTM). In this study, supplementation with tissue extract from a host, the Manila clam, significantly improved the efficiency of development, as determined by the numbers and sizes of developed prezoosporangia. Similar results were seen following supplementation with boiled host tissue extract, which indicates that a thermally stable component of the host is required for the parasite's development. Subsequently, we found that a commercially available lipid concentrate significantly increased prezoosporulation without host tissue, suggesting that the lipids in host tissue enhance prezoosporangia development. Moreover, we determined that yeast extract, sodium thioglycollate, and sodium chloride were the only components of RFTM required for prezoosporulation. Based on these findings, we prepared a simple, host-free medium for P. olseni prezoosporulation-Lipid concentrate Yeast extract Medium (LpcYM)-consisting of yeast extract, lipid concentrate, sodium thioglycollate, and sodium chloride. We confirmed that the prezoosporangia developed in LpcYM produce zoospores that are infectious to Manila clams and that trophozoites of other Perkinsus species (P. marinus, P. honshuensis, and P. chesapeaki) also develop to prezoosporangia in this host-free medium. As LpcYM has the simplest composition of prezoosporulation media available thus far, it enables us to conduct molecular and biochemical studies examining the drastic transformation process of this parasite.

Perkinsus marinus in the pleasure oyster Crassostrea corteziensis cultivated on the southeast coast of the Gulf of California, Mexico

The protozoan Perkinsus marinus has been associated with high mortality episodes of the eastern oyster Crassostrea virginica in the USA. The presence of P. marinus on the pleasure oyster Crassostrea corteziensis cultivated in two estuaries on the southeast coast of the Gulf of California was evaluated. Oysters were collected monthly (September 2016 to September 2017) and analyzed using Ray's fluid thioglycollate medium (RFTM) and polymerase chain reaction (PCR). Water parameters and oyster biometrics were also recorded. Pathogen prevalence increased over time from 0 to 100% in oysters from La Pitahaya, and from 0 to 83.33% in those from Bacorehuis. At both oyster farms, infection intensity was light (<1×104 parasites g-1 wet tissue), pathogen prevalence and infection intensity were correlated with oyster size and weight, and there was a strong correlation between P. marinus prevalence and intensity (La Pitahaya r = 0.91; Bacorehuis r = 0.82). The oysters that resulted positive for P. marinus by RFTM also were assayed using PCR. P. marinus presence was confirmed in 98.27% (114/116) of the oysters from La Pitahaya, and 95.83% (46/48) of those from Bacorehuis. The detection of P. marinus confirms that this pathogen is well established in the area with high expression during the warmer season. Despite the light infection intensity of this parasite at both sites, health surveillance of this bivalve in the region is highly advisable.

Reporte de presuntas hipnosporas de Perkinsus sp. en Megapitaria squalida del Golfo de California con la técnica de tinción con tioglicolato

Objetivo. Detectar la presencia de presuntas hipnosporas del protozoario Perkinsus sp. en una población silvestre de la almeja chocolata mexicana (Megapitaria squalida) del sureste del Golfo de California, usando el medio fluido de tioglicolato de Ray (RFTM). Materiales y métodos. Cada mes durante un ciclo anual, se capturaron 30 especímenes con una longitud entre 56.17 y 69.04 mm. La prevalencia e intensidad de la infección y los parámetros del agua se registraron mensualmente desde septiembre 2012 a septiembre 2013. Resultados. Se detectó la presencia de presuntas hipnosporas de Perkinsus sp. en muestras de tejido de la almeja chocolata mexicana usando la prueba RFTM, por la presencia de corpúsculos redondos y oscuros que representan hipnosporas del parásito. Los muestreos mensuales revelaron un rango de prevalencia e intensidad de la infección de 0-43.33% y 1-4 (infección negativa = 0 hipnosporas/preparación, a moderada = 34 hipnosporas/preparación), respectivamente. Conclusiones. Perkinsus sp. es reportado por primera vez en una población silvestre de M. squalida en la parte más al sureste del Golfo de California. Los resultados indican que el parásito está disperso intraespecíficamente y, potencialmente, parasitaría un nuevo huésped en la región.

Perkinsus sp. infecting three important mollusks from Jaguaribe River estuary, Ceará, Brazil

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.

The effects of environmental and nutritional conditions on the development of Perkinsus olseni prezoosporangia

Perkinsus olseni is a widely distributed protozoan pathogen that infects a wide range of marine mollusks. Prezoosporulation of P. olseni trophozoites is easily observed in Ray's fluid thioglycollate medium, but in nature, trophozoites within host tissue should be able to develop into prezoosporangia without any additional artificial medium after the host dies. How this process might work in field conditions remains poorly understood, however, partly because of the lack of appropriate in vitro assays. In this study, we observed that trophozoites of P. olseni successfully developed into prezoosporangia when mixed with minced tissue of the Manila clam Ruditapes philippinarum and placed in seawater. We were thus able to establish a new method to examine the development of P. olseni to prezoosporangia under artificially simulated natural environmental conditions. Using this method, we found that low temperatures (5 °C, 15 °C) significantly suppressed prezoosporangia development. In addition, we found that prezoosporangia were developed in a wide range of salinities (10–50 practical salinity unit) and that P. olseni requires some nutrition factors from host tissue for prezoosporulation to occur. Because the transmission of P. olseni among a host population highly depends on the developmental process of prezoosporangia, which leads to production of the infective zoospore stage, these results will help further our understanding of the parasite's infection dynamics in nature.

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina, Brazil.

Perkinsus spp. have been detected in various bivalve species from north-east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C.gigas and C.gasar from São Francisco do Sul. This pathology was also detected in C.gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C.gigas and C.gasar from São Francisco do Sul and Perkinsus beihaiensis in C.gasar from Balneário Barra do Sul. This is the first report of P.marinus in C.gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.

EPIZOOTIOLOGY OF Perkinsus sp. INFESTING Crassostrea rhizophorae FROM THE SEMI-ARID REGION, BRAZIL

Abstract The coast of the semi-arid region of Brazil is known for the presence of large estuaries inhabited by numerous species of edible bivalve mollusks. The oyster C. rhizophorae is naturally found in estuarine environments along the Brazilian coast, fixed on substrates, mainly in the roots of mangroves. Protozoa of the genus Perkinsus are important parasites of marine bivalves, sometimes causing significant pathological damage throughout the world. This study reports the occurrence of parasites Perkinsus sp. in mangrove oysters from Porto de Mangue estuary (Rio Grande do Norte state) and Icapuí estuary (Ceará state), two estuaries of the semi-arid region of Brazil. In Porto do Mangue estuary, oysters were collected in December 2016, January and February 2017. In Icapuí estuary, oyster sampling occurred in September, October and November 2017. Two fragments of the gills were incubated in Ray's Fluid Thioglycollate Medium - RFTM. The ecological and parasitological parameters were calculated. Five of the ninety oysters collected in Porto do Mangue estuary were parasitized, which corresponded to a mean prevalence of 5.6%, and in Icapuí estuary 18 of the 120 oysters collected were parasitized, which corresponded to a mean prevalence of 15.0%. The intensity of infection by Perkinsus sp. ranged from very mild to mild.

Perkinsus olseni in the short neck yellow clam, Paphia malabarica (Chemnitz, 1782) from the southwest coast of India.

Parasites of the genus Perkinsus predominantly infect bivalves, and two species among them, P. olseni and P. marinus, are notifiable to OIE. P. olseni infections are known to cause extensive damage to wild as well as farmed bivalves globally with enormous implications to its fishery. Consequent to the initiation of a surveillance programme for aquatic animal diseases in India, Perkinsus infections were observed in many species of bivalves. The present paper describes P. olseni infections in the short neck yellow clam, Paphia malabarica from the southwest coast of India. Diagnosis of the parasite was carried out using Ray's Fluid Thioglycollate Medium culture, histology, in-situ hybridisation and molecular taxonomy. Pathology of infection and development of zoospores is also described. This forms the first report of a P. olseni infection in P. malabarica. High prevalence and intensity of infection of Perkinsus in clams raises concerns, as clam reserves in this geographical area sustain fisheries and the livelihoods of local fishing communities.

Parasites in two coexisting bivalves of the Patagonia coast, southwestern Atlantic Ocean: The Puelche oyster (Ostrea puelchana) and false oyster (Pododesmus rudis).

The purpose of this study was to compare the parasites of two coexisting bivalves, the edible Puelche oyster (Ostrea puelchana) and the false oyster (Pododesmus rudis) that lives attached to O. puelchana shells, and to investigate their host specificity. Samples from wild populations, 465 O. puelchana and 131 P. rudis, were collected seasonally during two years in the San José Gulf (northern Patagonia, Argentina) and were processed using standard histological techniques. To increase the natural low prevalences of Bonamia spp. and Perkinsus spp. that are present in wild populations, an in situ experiment was performed by maintaining captive sentinel bivalves at high densities inside a plastic mesh bag to enhance parasite transmission. Polymerase chain reaction (PCR) assays were used to test for apparent Bonamia sp. infections among captive sentinel O. puelchana specimens (n = 80), and Ray's fluid thioglycollate medium (RFTM) assays and histological immunoassays tested for apparent Perkinsus sp. infections among captive sentinel P. rudis specimens (n = 100). Despite histological observations that revealed the presence of microcells resembling Bonamia sp. infecting hemocytes of some Puelche oysters, PCR assays did not confirm that parasite identification. Among captive sentinel P. rudis that showed histological evidence of Perkinsus sp. infections, neither RFTM nor immunoassays confirmed such parasites. Ostrea puelchana from wild populations were occasional hosts for both Rickettsia-like organism (RLOs) and Urastoma-like turbellarians. In contrast, six parasite taxa infected P. rudis from coexisting populations, including RLOs, Urastoma-like turbellarians, an intracellular gregarine species, Nematopsis-like oocysts, an unidentified coccidian and a Perkinsus qugwadi-like protozoan. These results demonstrated specific infection patterns of the identified parasites in relation to their hosts.

Perkinsus beihaiensis (Perkinsozoa) in oysters of Bahia State, Brazil.

This study reports the pathogen Perkinsus beihaiensis in oysters of the genus Crassostrea on the coast of the State of Bahia (Brazil), its prevalence, infection intensity and correlation with salinity. Oysters (n = 240) were collected between October and December 2014 at eight sampling stations between latitudes 13°55'S and 15°42'S. The laboratory procedures included macroscopic analysis, histology, culture in Ray's fluid thioglycollate medium (RFTM), Polymerase Chain Reaction (PCR) and DNA sequencing. PCR and sequencing have been used for the genetic identification of oysters as well. Two species of oysters have been identified: Crassostrea rhizophorae and C. brasiliana. In both oyster species P. beihaiensis was the only Perkinsus species detected. In C. rhizophorae, the average prevalence was 82.8% by histology and 65.2% by RFTM. In C. brasiliana, the prevalences were 70.5% and 35.7%, respectively. The higher prevalence of P. beihaiensis in C. rhizophorae was probably influenced by salinity, with which was positively correlated (r> 0.8). In both oysters, P. beihaiensis was located mainly in the gastric epithelium. The infection was generally mild or moderate, without apparent harm to the hosts, but in cases of severe infection, there was hemocytical reaction and tissue disorganization. The generally high prevalence in the region suggests that oysters should be monitored with respect to this pathogen, especially in growing areas.

Primer registro de Perkinsus sp. (Protozoa, Apicomplexa) en el callo de hacha Atrina maura en Sinaloa, México

We evaluated the presence of the protozoan Perkinsus sp. by determining of its prevalence and level of infection in Atrina maura during 2012. Pen shells were collected per season beside an oyster farm located at Estero La Piedra, Sinaloa, Mexico, and analyzed by Ray's fluid thioglycollate medium and polymerase chain reaction. The higher prevalence (90%) and infection level (2.3) were obtained when temperature and salinity were higher. The Mackin scale showed a non-infected to moderate infection range. PCR was positive to Perkinsus sp. This is the first record of the presence of Perkinsus sp. in A. maura in Sinaloa, Mexico.

First record of Perkinsus chesapeaki infecting Crassostrea rhizophorae in South America

This study investigated Perkinsus spp. infecting Crassostrea rhizophorae from the Jaguaribe River estuary, Ceará, 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.

Perkinsus beihaiensis infectando a ostra Crassostrea rhizophorae em cultivo e em estoque natural na Baía de Camamu, Bahia, Brasil

Foi investigada a infecção de Perkinsus beihaiensis (Perkinsozoa) na ostra Crassostrea rhizophorae em um sistema de cultivo do tipo espinhel e em um estoque natural de ostras no manguezal adjacente ambos localizados no estado da Bahia, Nordeste do Brasil. As colheitas foram realizadas em outubro e novembro de 2012 e em janeiro de 2013. As ostras (n = 300) foram medidas, examinadas macroscopicamente quanto a sinais da infecção e submetidas às técnicas laboratoriais: histologia, ensaio em meio de cultivo de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento, que confirmou a identificação do patógeno. As análises histológicas e o RFTM mostraram, respectivamente, prevalência média de 93,3% e de 69%. A infecção foi geralmente leve ou muito leve. Não houve diferença significativa (p > 0,05) entre os ambientes em termos de prevalência ou severidade da infecção. Este é o primeiro registro de P. beihaiensis no estado da Bahia e o segundo em ostras do Brasil e América do Sul.

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil.

Bivalve culture is of considerable economic and social interest in northeastern (NE) Brazil. The polyculture is an alternative approach to traditional monoculture for reducing the environmental impact of shrimp farming and improving oyster culture. Perkinsus marinus and Perkinsus olseni were found infecting oysters in NE Brazil and can threaten oyster production. This study evaluated Perkinsus spp. occurrence in Crassostrea gasar during all production stages. Oyster spats were produced in a hatchery and grown in shrimp ponds in Rio Grande do Norte state. Perkinsus spp. were surveyed by Ray's fluid thioglycollate medium and confirmed by polymerase chain reaction. Prevalence and intensity of infection were determined in oysters until they reached 7 cm. Results showed that the broodstock was already infected by Perkinsus (60%), but the derived spats were Perkinsus-free. Oyster spats acquired Perkinsus infection when transferred to ponds. The prevalence gradually increased in the seven months following placement in ponds (73%), and then decreased to 17% by the tenth month. The infections were initially mild, but intensity increased at the final growth stage. In conclusion, it is possible to produce Perkinsus-free C. gasar oyster spats from infected broodstock, and their culture in shrimp ponds is feasible.