Parasitological survey of mangrove oyster, Crassostrea rhizophorae, in the Pacoti River Estuary, Ceará State, Brazil

Journal of Invertebrate Pathology 112 (2013) 24–32

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Parasitological survey of mangrove oyster, Crassostrea rhizophorae, in the Pacoti River Estuary, Ceará State, Brazil Rachel Costa Sabry a,⇑, Tereza Cristina Vasconcelos Gesteira a, Aimê Rachel Magenta Magalhães b, Margherita Anna Barracco c, Cristhiane Guertler c, Liana Pinho Ferreira a, Rogério Tubino Vianna d, Patrícia Mirella da Silva e a

Instituto de Ciências do Mar, Universidade Federal do Ceará, Av. da Abolição, 3507 – Meireles, CEP 60165-081 Fortaleza, CE, 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 c Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Departamento de Biologia Celular, Embriologia e Genética, Campus Universitário – Trindade, CEP 88040-900 Florianópolis, SC, Brazil d Universidade Federal de Santa Catarina, CEP 89520-000 Curitibanos, SC, Brazil e 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 b

a r t i c l e

i n f o

Article history: Received 11 May 2012 Accepted 17 October 2012 Available online 10 November 2012 Keywords: Crassostrea rhizophorae Histopathology Oyster Perkinsus beihaiensis

a b s t r a c t 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. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The State of Ceará, Northeastern Brazil, has several areas of mangroves inhabited by numerous species of molluscs, including man⇑ Corresponding author. Present address: Instituto Federal de Educação Ciência e Tecnologia, Campus Aracati, Rua Teófilo Pinto, 200, Farias Brito, CEP 62000-800 Aracati-CE, Brazil. E-mail addresses: [email protected], [email protected] (R.C. Sabry), [email protected] (T.C.V. Gesteira), [email protected] (A.R.M. Magalhães), [email protected] (M.A. Barracco), [email protected] (C. Guertler), [email protected] (L.P. Ferreira), [email protected] (R.T. Vianna), [email protected] (P.M. da Silva). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.10.004

grove oysters (Crassostrea spp., Ostreidae), mussels (Mytella falcata and Mytella guyanensis, Mytilidae), and cockles. In the estuary of Pacoti River, located on the east coast of Ceará, the bivalve molluscs are a resource commonly exploited by fisherwomen. This resource represents an important socioeconomic activity, contributing to the livelihoods of communities that live around the estuary. The few studies on the occurrence of pathogens that affect natural populations of bivalve molluscs in Ceará State are limited to two bivalve species from the Jaguaribe River and Pacoti River estuaries (Sabry et al., 2007; Araújo and Rocha-Barreira, 2004; Ferreira et al., 2008). The authors reported the occurrence of the protozoan parasite Nematopsis sp. (Apicomplexa, Porosporidae) infecting the

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oyster Crassostrea rhizophorae and the cockle Anomalocaria brasiliana; digenetic trematodes infecting gills and gonads of A. brasiliana, from both estuaries, metacestode Tylocephalum (Cestoda, Tetragonocephalidae) only in C. rhizophorae and Rickettsia-like organisms only in A. brasiliana. Studies on diseases in molluscs are important because some pathogens can affect and can cause massive mortality among natural or cultivated populations. The digenetic trematodes of the genus Bucephalus (Digenea, Bucephalidae) are important parasites of marine bivalves, sometimes causing significant pathological damage, mainly characterized by castration of the host (Lauckner, 1983). On the other hand, Tylocephalum larvae had already been recorded in a variety of shellfish without causing diseases (Cheng, 1967; Nascimento et al., 1986; Hine and Thorne, 2000; Boehs and Magalhães, 2004; Winstead et al., 2004; Sabry and Magalhães, 2005; Boehs et al., 2010), but at high intensities of infection they could cause physiological stress and, consequently, affect the growth and reproduction of the molluscs (McGladdery et al., 2006). Perkinsus marinus and Perkinsus olseni are included in the World Organization for Animal Health (http://www.oie.int/) list of notifiable pathogens for mollusks; these two species have caused significant impacts on populations of molluscs worldwide (Villalba et al., 2004; Choy and Park, 2010). P. marinus, the causative agent of Dermo disease, is responsible for mortalities of the American eastern oyster Crassostrea virginica along the Atlantic coast of the United States and Gulf of Mexico (Burreson and Ragone Calvo, 1996). Experimental challenges indicated that the oyster C. rhizophorae is susceptible to infection by P. marinus (Bushek et al., 2002). P. olseni caused mortalities in the abalone Haliotis rubra in Australia (Lester and Davis, 1981); in the cockles Ruditapes decussatus from Portugal (Azevedo, 1989) and was described in several bivalves worldwide (Cremonte et al., 2005; Dungan et al., 2007; Elandaloussi et al., 2009; Choy and Park, 2010). Considering the importance of bivalve molluscs as a natural resource of livelihood for the coastal communities of the Pacoti River Estuary and predicting the future development of oyster farming in Ceará State, the present work carried out a study of the pathogens found in the native oyster C. rhizophorae from the Pacoti River Estuary, Ceará.

2. Material and methods 2.1. Sampling of oysters The oysters of the species C. rhizophorae were collected from roots of the red mangrove Rhizophorae mangle in three sites of the Pacoti River Estuary (Site 1: 3°490 800 S, 38°250 900 W, near the mouth of the estuary; Site 2: 3°490 1500 S, 38°250 1000 W, located near a mangrove oyster cultivation facility and Site 3: 3°490 1900 S, 38°250 1100 W, located more distant from the mouth of the estuary (Fig. 1). The sites are about 1 km apart. The samples were collected in August 2008 (winter), December 2009 (summer) and in 2010 collections were performed only at site 3 in March, June, September and December. Each sample consisted of 150 oysters resulting in a total of 1500 individuals for analysis. In the laboratory, the oyster shell height (maximum distance from hinge to growth edge) was measured. Oysters were then opened and examined macroscopically for pathological changes on the shell and soft body (mantle, gills, gonads and digestive gland). All animals collected at each of the three sampling sites within the estuary were processed in Ray’s Fluid Thioglycollate Medium assay (RFTM, Section 2.2) and a section of the gill was fixed in 95% alcohol for further analysis in order to confirm the presence of Perkinsus by Polymerase Chain Reaction (PCR, Section 2.3). Three hundred oysters (30 from each sample) were processed for histological examination (Section 2.5).

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Fig. 1. Map highlighting the Pacoti River, Ceara, where are located the three oyster sampling points in the estuary. S1. Point near the mouth of the estuary. S2 Point located near a mangrove oyster cultivation. S3. Point away from the mouth of the river.

2.2. Incubation of tissues in Ray’s Fluid Thioglycollate Medium (RFTM) Two demibranchs and the rectum of each animal were incubated in thioglycollate liquid medium for 7 days in darkness and at room temperature. After the incubation period, tissues were collected, macerated on a slide and stained with Lugol solution (OIE, 2009) according to the method described by Ray (1954). The preparation was observed under an optical microscope to verify the presence of hypnospores of the Perkinsus genus, which are spherical and their walls stain blue or black bluish. The intensity of infection by Perkinsus in tissues of the oysters C. rhizophorae was adapted from the Mackin scale (Ray, 1954). 2.3. Perkinsus diagnosis by Polymerase Chain Reaction – PCR PCR analyses were performed on 23 animals collected in 2009 and 12 sampled in 2010. DNA was extracted from gills preserved in 95% ethanol with DNAzolÒ reagent (Invitrogen) following the manufacturer’s protocol. For PCR assays we used primers PerkITS 85/750 (Casas et al., 2002) that specifically hybridize with conserved regions of the internal transcribed spacer (ITS) of the region of the ribosomal ribonucleic acid (rRNA) gene complex that are unique to members of the Perkinsus genus (except for P. qugwadi incertae sedis). The positive controls consisted of P. olseni cells isolated in vitro from the clam R. decussatus from Galicia, Spain (provided by Dr. Antonio Villalba, Centro de Investigacións Mariñas). Negative controls were nuclease-free water instead of template DNA. PCR reactions were performed in 25 ll reactions containing 1 ll (50–100 ng) of genomic sample DNA, PCR buffer at 1 concentration, MgCl2 at 1.5 mM, nucleotides each at 0.2 mM, primers at 0.8 lM, and 1 unit of Taq DNA polymerase (Invitrogen). The protocol included denaturation of template DNA at 94 °C for 10 min; 35 amplification cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; followed by a 72 °C final extension for 10 min. PCR products were separated on a 1.5% agarose gel washed in 1 Tris–EDTA buffer (TE) and stained with ethidium bromide. 2.4. DNA sequencing and phylogenetic analysis Fresh amplified PCR products from three oysters infected by Perkinsus sp. were cloned into a pCR 2.1-TOPO vector, using a TOPO

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TA cloning kit (InvitrogenÒ). The positive recombinant clones were identified by colony PCR with the M13 vector primers (InvitrogenÒ), and one bacterial colony of each sample was sequenced in both directions. Sequencing was performed in an automated MegaBace 1000 DNA Analysis System, using the DYEnamicÒ ET Dye Terminator kit (GE Healthcare). The sequences were aligned with previously reported ITS sequences obtained from the GenBank database, using the ClustalW program (Thompson et al., 1994) implemented in Bioedit version 5.0.9 (Hall, 1999). ITS rRNA gene sequences obtained in this study (N = 03) and from GenBank (N = 49) were included in the phylogenetic analyses. The GenBank sequences were the following: P. chesapeaki (=P. andrewsi) AY876302, AY876305, AY876306, AY876311; P. olseni (=P. atlanticus) AF441207, AF441209, AF441211, EF204082, EF204083, EF204086, POU07701, PSU07698, PSU07699; P. marinus GQ861511, AY295180, AY295188,AY295194, AY295199, AY295197; P. mediterraneus AY487834, AY487835, AY487839; P. honshuensis DQ516696, DQ516697, DQ516698, DQ516699; Perkinsus beihaiensis EU068080, EU068081, EU068082, EU068083, EU068088, EU068093, EU068084, EU068085, EU068086, EU068087, EF204015, EF204068, EF526428, EF526436, from China; JN054744, JN054741, JN054739, JN054740, JN054742, JN054743 from India; P. beihaiensis – like FJ472346, FJ472347 from Brazil; and the outgroup taxon was P. qugwadii AF151528. The sequences obtained in this study were submitted to GenBank with the accession numbers: JX502840–JX502842. Based on the alignment of all sequences of P. beihaiensis, the fragment sizes of ITS region of Perkinsus sp. from the Pacoti River Estuary (this study) were established. The Maximum Parsimony analysis was made using the PAUP4.0b10 software (Swofford, 2002). The phylogen hypothesis eticusing parsimony was performed using heuristic search, 1000 replicates of random addition of taxa, 100 trees held during stepwise addition and tree-bisection-reconnection (TBR) branch swapping. Bootstrap values (50%) were calculated using the same parameters of parsimony analysis.

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 site (Bush et al., 1997). 3. Results 3.1. Biometry and macroscopic analyses The average heights of oysters and values of temperature and salinity collected in sites 1, 2 and 3 of the Pacoti River Estuary in 2008, 2009 and 2010 are shown in Table 1. Macroscopic observations of oysters C. rhizophorae collected in 2008, 2009 and 2010 did not show change other that those caused by the polychaete of genus Polydora (Polychaeta: Spionidae) in the valves of the oysters. Polydora infestation was characterized by the presence of horizontal tubes with black color. The prevalences rates of Polydora sp. were high (Table 1). 3.2. Incubation of tissues in Ray’s Fluid Thioglycollate Medium (RFTM) The results of RFTM analysis for Perkinsus in samples collected in 2008 were published by Sabry et al. (2009). In 2009 and 2010, incubation of gills and rectum of oysters C. rhizophorae in liquid thioglycolate medium showed the presence of typical Perkinsus hypnospores. The cells of the pathogen were spherical, had sizes ranging from 10 to 55 lm in diameter and were heavily black-bluish stained by lugol (Fig. 2A). The prevalence and intensities of infection in oysters infected by Perkinsus are shown in Table 2. The technique of tissue culture in RFTM also proved effective for the detection of the protozoan Nematopsis. Although Nematopsis has not been stained in black by lugol, the wall of the oocysts (gregarine spore) was refractive making the parasite easy to observe (Fig. 2B). This pathogen was detected in all samples in the two periods (2008, 2009) and in the three sites of the estuary (Table 1). In 2010, the presence of Nematopsis by RFTM was not analyzed.

2.5. Histological preparations

3.3. Histopathological analysis

A cross section from each oyster (n = 300) that included gills, gonad and digestive gland was fixed in Davidson’s solution (Shaw and Battle, 1957) for 24 h. The tissues were then dehydrated in a graded series of alcohol up to 100%, cleared in xylene and impregnated in histological paraffin at 60 °C. Sections of 5 lm thickness

3.3.1. Protozoa The protozoan of the genus Perkinsus was detected in oysters collected in 2009 and 2010 in the estuary of Pacoti River. Few trophozoites of Perkinsus sp. were found infecting the connective tissue and epithelium of the oyster digestive tubule. The cells of the

Table 1 Prevalence (%) of pathogens detected in the mangrove oyster Crassostrea rhizophorae from the Pacoti River Estuary, Ceará, in August 2008, December 2009 and in March, June, September and December 2010 at site 3. Macroscopic analysis (MA, N = 150), incubation of tissues in Ray’s Fluid Thioglycolate Medium (RFTM, N = 150) and histological section (HS, N = 30). Average height of the oysters (mm) and standard deviation (SD), not analyzed (). Year

2008

Collection Site Number Month of sampling

Site 1 August

Site 2

2009

Average height (mm) ± SD Temperatures (°C) Salinity (‰) Trichodina sp. (HS) Sphenophrya sp. (HS) Ancistrocoma sp. (HS) Steinhausia sp. (HS) Nematopsis sp. (HS) Nematopsis sp. (RFTM) Polydora sp. (MA) Urastoma sp. (HS) Metacercárias (HS) Tylocephalum sp. (HS)

58 ± 8.4 29 32 3.3 3.3 20.0 36.7 93.3 100 93.3 – – –

50 ± 5.3 30 29 – 3.3 17.2 24.1 93.1 96 70.0 3.3 – –

Site 3 49.8 ± 5.1 29 23 – – 16.7 23.3 93.3 100 78.0 – – 3.3

2010

Site 1 December

Site 2

56.7 ± 6.2 29 38 – 20.0 3.3 16.7 – 1.3 48.7 – – 3.3

52.6 ± 10 28 40 – 13.3 – 6.7 – 2.0 10.7 – 16.7 13.3

Site 3 51.8 ± 4.9 29 35 – 23.3 – 10.0 – 1.3 45.3 – 6.7 10.0

Site 3 March

June

September

December

57.1 ± 4.6 31 25 3.3 16.7 16.7 10 10  13.3 3.3 – 23.3

52.6 ± 5.4 29 30 – 30 3.3 – 80  30.0 – – –

52.6 ± 7.0 28 40 – 30 3.3 3.3 93.3  10.0 3.3 – –

50 ± 4.4 29 39 – – – – 96.7  3.3 – – 10

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Fig. 2. Pathogens detected in tissues of Crassostrea rhizophorae from the Pacoti River Estuary, Ceará, in August 2008, December 2009 and in March, June, September and December 2010. (A) Hypnospores of Perkinsus sp. (arrow) infecting the gills of C. rhizophorae after incubation in thioglycolate medium and stained with Lugol’s iodine solution. (B) Oocysts of Nematopsis sp. in thioglycolate medium. Scale bar = 50 lm (to A and B). (C). Trophozoite of Perkinsus sp. (arrow) in connective tissue near the digestive gland. Scale bar = 10 lm. (D) Trichodina sp. (arrow) in the gills. (E) Sphenophrya sp. (arrow) in contact with the gills of the oysters. (F) Ancistrocoma – like. (arrow) in the lumen of a digestive tubule. Scale bar = 20 lm (to D, E and F).

Table 2 Prevalence (%) of infection by Perkinsus sp. in the mangrove oyster Crassostrea rhizophorae from the Pacoti River Estuary in 2009 and 2010. Analyses of incubation of tissues in Ray’s Fluid Thioglycolate Medium (RFTM), histological sections (HISTO) and by Polymerase Chain Reaction (PCR). Months 2009 December

2010 March June September December a

Collection sites

Infected RFTM; n: 150

Infection intensity

Infected HISTO; n: 30

Confirmed by PCR

1 2

2 (1.3%) 10 (6.7%)

01 (3.3%) 06 (20%)a

0 0

3

11 (7.3%)

2 (Very light: 3 and 4 cells.) 8 (Very light: 1–7 cells.) 2 (Light: 15 and 50 cells.) 10 (Very light: 1–9 cells) 1 (Light: 30 cells.)

04 (13.3%)

0

3 3 3 3

6 (4%) 0 4 (2.7%) 2 (1.3%)

6 (Very light: 1–5 cells) 0 4 (Very light: 1 cell) 2 (Very light: 1 cell)

02 (6.7%) 0 0 0

04 0 0 01

2 Animals no infected by Perkinsus in RFTM.

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Fig. 3. Pathogens detected in tissues of Crassostrea rhizophorae from the Pacoti River Estuary, Ceará, in August 2008, December 2009 and in March, June, September and December 2010. (A) Steinhausia sp. (arrow) in an oocyte cytoplasm. (B) Oocysts of Nematopsis sp. (arrow) in connective tissue near the digestive tubules. Scale bar = 20 lm (to A and B). (C) Turbelarian infecting the connective tissue of the mantle. (D and E) Metacercariae of trematodes (arrows) infecting the gonadal follicles of males and females, respectively. (F) Metacestode Tylocephalum sp. (arrow) in the connective gill tissue. Note the change in the normal configuration of the gill. Scale bar = 50 lm (to C, D, E and F).

pathogen were rounded with a diameter ranging between 3 and 6 lm, (Fig. 2C). No changes were observed in the infected tissues and were not detected phagocytosed trophozoites and infiltration of haemocytes. In 2009 the histology confirmed 9 positive cases of 23 diagnosed by RFTM (Table 2) and also revealed two more cases of oysters infected with Perkinsus sp. that had not been diagnosed by the RFTM technique. The ciliate of the genus Trichodina (Ciliophora: Trichodinidae) was visualized near the gill lamellae of the oysters. It had an oval shape and measured 13 lm in its greatest diameter (Fig. 2D). Ciliated Sphenophrya-like (Ciliophora: Sphenophryidae) were detected in contact with the gill filaments of the oysters. These ciliates were oval, measuring from 5 to 18 lm in its greatest diameter and had a basophilic macronucleus and an eosinophilic cytoplasm (Fig. 2E). The intensity was low and ranged from 1 to 11 ciliates/histological section. No damage was observed in the gills of infected animals.

Other ciliate Ancistrocoma-like or Stegotricha-like was found in the lumen of digestive tubules. These ciliates presented a pearshape, with size of 10–27 lm in its greatest diameter (Fig. 2F). The intensity was low (1–4 ciliates/histological sections). Only one oyster collected in 2008 showed nine ciliates with up to 3 parasites in a single digestive tube. However, no defense reaction by the host was evidenced and no injury was observed in the epithelium of the organ. An unknown protozoan parasite was found infecting the cytoplasm of oocytes of the mangrove oyster C. rhizophorae from the Pacoti River Estuary (Fig. 3A). It was observed as a putative sporocyst, ranging in diameter from 4 to 8 lm, containing up to 9 inmature spores. The intensity of infection was low, ranging from 1 to 7 infected oocytes/histological section in 2008, from 1 to 11 in 2009 and from 1 to 25 in 2010. Protozoa of the genus Nematopsis sp. were found infecting the connective tissue near the digestive gland, gills and mantle of the

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from Brazil (Sabry et al., 2009) and China (Moss et al., 2008) and, 94–95% of identity with the sequences of P. beihaiensis from India (Sanil et al., 2012). The parsimony inference topology presented here indicated a close phylogenetic relationship among Brazilian Perkinsus sp. sequences and P. beihaiensis sequences from China and India (Fig. 5).The topology consisted of two clades supported by bootstrap values (Fig. 5). One included P. beihaiensis and its, relatives with high bootstrap value (100%); and the second clade was composed of P. olseni (=P. atlanticus) (94%), P. chesapeaki (=P. andrewsi) (100%), P. mediterraneus (100%), P. honshuensis (100%), and P. marinus (100%) (Fig. 5). All sequences of P. beihaiensis were distributed into two clades, with two sequences from this study (JX502840–JX502841) in the base of the clade. The two inclusive clades comprised as follows: one with specimens from India and Brazil, and the second clade with all Chinese and two Indian P. beihaiensis sequences. Fig. 4. Molecular diagnosis of Perkinsus sp. infecting the mangrove oyster Crassostrea rhizophorae from the Pacoti River Estuary, Ceará. Detection of the rDNA ITS region of Perkinsus sp by PCR. M: molecular size markers of 500 bp, C: negative control (water), C+: positive control (DNA isolated from P. olseni); oyster O2, O3 and O4 (Perkinsus sp.) and oyster O1, O5 and O6 (negative samples to Perkinsus).

oyster C. rhizophorae (Fig. 3B). The oocysts of Nematopsis sp. had a basophilic character and measured from 9 to 14 lm in its longest axis. The intensity of infection by Nematopsis sp. was low with few oocysts (1–6) per histological section. No lesions were observed in connective tissue, nor infiltration of haemocytes associated with the infection. 3.3.2. Metazoa A turbellarian, possibly of the genus Urastoma (Turbellaria: Urastomidae) was found in the connective tissue of the oyster mantle (440 lm in size and eosinophilic character) and also in the gill (Fig. 3C). There was no host reaction to the parasite presence. Metacercariae of trematodes were detected within the gonadal follicles of male and female oysters (Fig. 3D and E). The metacercariae ranged in size from 117.5 to 177.5 lm. The infected follicles were in the same state of gonadal development as uninfected follicles and no associated hemocytes infiltration was observed. Larvae of cestodes (termed metacestode), possibly of the genus Tylocephalum infesting oysters were detected in this study. There was a host immunological response that led to the encapsulation of the larvae by several layers of haemocytes (Fig. 3F). The metacestode measured from 80 to 145 lm in its longest axis, and were observed in the connective tissue in the region near the digestive gland and there was a case in which the metacestode was found in gill tissue. In addition to the encapsulation reaction, there was a strong modification of the architecture of the gill. 3.4. Diagnosis of Perkinsus sp. by PCR The results of PCR analysis for Perkinsus in samples collected in 2008 were published by Sabry et al. (2009). In 2009, the 23 oysters diagnosed positive by RFTM and subjected to PCR technique for confirmation of infection by Perkinsus sp. were negative. In 2010, from the 12 positive oysters in RFTM, 5 (41.7%) were positive by PCR (Table 2 and Fig. 4). 3.5. Analyses of Perkinsus sp. ITS region sequences The three Brazilian Perkinsus sp. sequences showed partial ITS1, complete 5.8S rRNA fragments, complete ITS2 and partial 28S rRNA fragments.The three Perkinsus sp. ITS rRNA gene sequences obtained in this study were submitted to BLAST tool (NCBI) and showed 91–93% of identity with the sequences of P. beihaiensis-like

4. Discussion The present study registered several parasites and commensals in the oyster C. rhizophorae with different prevalences and intensities between the two studied periods. The temperature has not presented variation and salinity has shown lower values during the rainy season. This fact could explain the distinct values of prevalence of some organisms found in the oysters of the present study. Perkinsus sp. was detected in rectum and gills of oysters C. rhizophorae collected in 2009 and 2010, after incubation in RFTM, with low prevalence, similar to those detected in 2008 (Sabry et al., 2009). In 2009, the results of histological analysis confirmed only 9 positive cases of 23 diagnosed by RFTM and also detected two additional cases of Perkinsus sp. This fact is surprising because the technique of RFTM is considered more sensitive than histology for this pathogen (OIE, 2006) and involves the use of a large amount of tissues for analysis, while the histology not. Also curious was the result of the PCR analysis which did not detect Perkinsus sp. in any of the samples positive by RFTM in 2009. In the samples of 2010, the histological analysis confirmed only 2 cases of 12 positive cases by RFTM, confirming the high sensitivity of this latter technique. The PCR assays have not confirmed 7 of the 12 positive cases in RFTM. The reason for the low sensitivity of PCR may be due to the fact that almost all animals had presented very light intensity of infection, i.e. between 10 and 50 (2009) and 1 to 5 hypnospores (2010) in the whole gill analyzed by RFTM. Burreson (2008) has discussed the occurrence of false negative by PCR analysis for detection of parasites of bivalves, and obtained the same conclusion. In histological sections from oysters C. rhizophorae collected in 2009 and 2010, the cell of protozoan Perkinsus sp. showed a vacuole occupying most of the internal volume of the cytoplasm, an eccentric nucleus and a prominent nucleoli as observed in Sabry et al. (2009). Perkinsus sp. did not cause any change in the infected tissues. In contrast, Sabry et al. (2009) detected in oysters from the same location changes in tissues and organs of the oysters infected by Perkinsus sp., and also phagocytosed trophozoites. This contradiction in the results possibly may be related to the low intensity of infection. In the present study, the phylogenetic analysis of rDNA ITS region sequences of Perkinsus sp. showed a topology in which P. beihaiensis clade was a sister group of a clade including all Perkinsus species, as observed by Moss et al. (2008). In contrast, in other topologies (Sabry et al., 2009; Sanil et al., 2012), the position of P. beihaiensis from China and Perkinsus sp. from Brazil (Sabry et al., 2009) showed a close phylogenetic relationship with P. chesapeaki. In the topology presented by Sabry et al. (2009), P. beihaiensis from China composed a sister group of the two Brazilian

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Fig. 5. Maximum Parsimony topology of DNA sequences from the ITS region of rRNA gene complex of Perkinsus spp. Bootstrap values inside the circles.

Perkinsus sp. sequences. Interestingly, our topology showed that the three sequences of Perkinsus sp. from the present study grouped with the Indian P. beihaiensis sequences, excepting two (JN054741 and JN054744) which formed a clade with Chinese sequences (Sanil et al., 2012). In addition, our sequences were included in a clade with Perkinsus sp. sequences, previously reported for the same area, the Pacoti River Estuary, Brazil (Sabry et al., 2009). Thus, our results strongly support that the Perkinsus sp. infecting oysters C. rhizophorae from this study is the species P. beihaiensis. Cilliates of genera Trichodina, Sphenophrya and Ancistrocoma – like were observed in oysters without causing any tissue damage. Ciliates are commensal organisms that usually do not affect bivalve mollusks (Bower et al., 1994). The high prevalence of Sphenophrya sp. in 2009 (23.3%) and 2010 (30%) did not cause injury in the infected animals. On the other hand, the formation of tumors known as xenomas in the tissues of C. rhizophorae from Camamu Bay, Bahia, was associated with the presence of Sphenophrya (Boehs

et al., 2009). Ancistrocoma – like also was observed in oysters Crassostrea gigas from Santa Catarina State without causing any tissue damage (Pontinha, 2009). The ovarian protozoan found in the cytoplasm of oocytes of the oysters is probably the microsporidian Steinhausia which has been already detected in the clam A. brasiliana (da Silva et al., 2012), and oysters C. rhizophorae and C. gigas from Brazil (Sabry et al., 2011). In the present study, histological analyses detected Nematopsis sp. with very high prevalence and it did not induce host response. Nematopsis, in high intensities, caused damage mainly in the gill filaments de Cerastoderma edule (Carballal et al., 2001) and in M. guyanensis from Bahia, it caused changes in the morphology of the mantle and gills (Boehs et al., 2010). Curiously in the present study the RFTM technique proved to be very sensitive for the detection of the protozoan Nematopsis sp. when compared to the histology technique. The infestation by Polydora sp. in oysters collected in August 2008, were higher than those registered in 2009 e 2010. In 2009

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and 2010, the rainy season in Ceará State, was prolonged (FUNCEME, 2009) causing mortality of the oysters (fisherwomen, personal communication) and the natural stock was renewed from the beginning of October, so the oysters certainly younger than those collected in August 2008 had a shorter exposure time to the pathogens, which explains the lower prevalences found. A turbellarian, possibly of genus Urastoma, was observed in the connective tissue of the mantle (occurrence possibly accidental) and gill of C. rhizophorae. In mussels, such turbellarians in high number have occasionally been associated with destruction of the gills (Robledo et al., 1994) but in oysters there are no reports of this nature (Brun et al., 2000). Metacercariae of unidentified trematodes were found infesting the gonads of the oysters, which was also observed in bivalves in Bahia e Santa Catarina (Boehs et al., 2010; da Silva et al., 2010). Larvae of cestodes, possibly Tylocephalum sp., were detected at low prevalences, with hemocytic reaction and a layer of cells of fibrous nature resulting in encapsulation of the parasite, corroborating the results observed in other species of bivalve molluscs from the Brazilian coast (Sabry et al., 2007; Boehs et al., 2010; da Silva et al., 2010). In the present study, the found pathogens did not cause harm to the host and, except for Nematopsis sp. and Tylocephalum sp., all are being recorded for the first time in the oyster C. rhizophorae from the Pacoti River Estuary, Ceará. However, monitoring programs in oyster diseases are recommended, especially those aimed to detect pathogens considered notifiable to the OIE, which represent high risk for natural and cultured populations.

Acknowledgments The authors are Grateful for financial support through the announcement no. 64/2008 awarded by MCT/CNPq/MAP/SDA which made this research possible and to the Coordination of Improvement of Higher Education Personnel (CAPES) for the Doctoral Scholarship of the Program Blue Amazon to Rachel C. Sabry. The authors are also grateful to Maximiano Pinheiro Dantas Neto, MSc for editing the English version.

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