Histological survey of symbionts and other conditions of pod razor clam Ensis siliqua (Linnaeus, 1758) in Galicia (NW Spain)

Journal of Invertebrate Pathology 112 (2013) 74–82

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Histological survey of symbionts and other conditions of pod razor clam Ensis siliqua (Linnaeus, 1758) in Galicia (NW Spain) Maite Ruiz a, Susana Darriba b, Rosana Rodríguez a, Carmen López a,⇑ a b

Centro de Investigación Mariñas (CIMA), Consellería do Medio Rural e do Mar, Pedras de Corón, s/n, Apdo 13, Vilanova de Arousa, 36620 Galicia, Spain Instituto Tecnolóxico para o Control do Medio Mariño de Galicia (INTECMAR), Consellería do Medio Rural e do Mar, Peirao de Vilaxoán, s/n, Vilagarcía de Arousa, 36611 Galicia, Spain

a r t i c l e

i n f o

Article history: Received 19 April 2012 Accepted 18 September 2012 Available online 28 September 2012 Keywords: Ensis siliqua Viral inclusion Prokaryotes Protozoa Metazoa Neoplasm

a b s t r a c t The aim of the present study was to carry out a survey of parasites and other conditions affecting pod razor clam populations, Ensis siliqua, in two beds from Galicia (NW Spain). In Galicia, the production of E. siliqua has increased in recent years due to the development of specific plans for its exploitation, however few and quite recent pathological studies have been carried out in this species. The results of this study showed the presence of different protozoa as the more prevalent group, especially Nematopsis sp. gregarines, unidentified branchial protozoa, renal coccidia and Trichodina sp. ciliates. Larval stages of trematodes and neoplastic disorders were also observed with lower prevalences. Furthermore, an ultrastructural analysis of two types of unidentified basophilic inclusions, both found in the digestive gland, revealed the presence of icosahedral viral particles and prokaryotic organisms, respectively. None of the parasites detected in E. siliqua from this study was notifiable to the World Organisation for Animal Health (OIE) and the majority of the symbionts and conditions observed in their tissues did not cause host damage. Nevertheless, parasites like bucephalid digenean sporocysts, viral inclusions, prokaryotic infections, disseminated neoplasm or germinoma detected in some samples could cause moderate or severe damage to the host depending on the intensity of infection. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The pod razor clam, Ensis siliqua (Linnaeus, 1758) (Pharidae, Bivalvia) is a siphon feeder which lives buried in the sand in low intertidal and infralittoral areas from Norway to Portugal and along the British coast (Darriba and Fernández-Tajes, 2011). In Galicia (NW, Spain), E. siliqua is an important marine resource, obtained by the exploitation of natural beds. The production of E. siliqua has increased in Galicia since 2005, reaching in 2011 an annual production of over 88 tons, valued at 0.65 million euros (data from Plataforma Tecnolóxica da Pesca, Consellería do Medio Rural e do Mar), due to the development of specific plans for its exploitation. In Galicia, pathological research carried out in molluscs to date has focused mainly on mussels, clams, oysters and cockles. Other groups with commercial interest such as Solenoidea superfamily have scarcely been studied (López et al., 2011). Quite recent pathological studies have been carried out in this superfamily around the world. Some are general histopathological studies (Bilei et al., 1997; Ceschia et al., 2001; Darriba et al., 2010; Fahy et al., 2002; López et al., 2011; Montes, 2011). Others are focused on a particular parasite or disease, for instance, the presence of unidentified ⇑ Corresponding author. Fax: +34 886 206372. E-mail address: [email protected] (C. López). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.09.003

intranuclear inclusions associated with high mortalities in Siliqua patula from US (Elston, 1986), Trichodina spp. and Sphenophrya solinis (Ciliophora) in Solen gracilis, Solen grandis and Sinonovacula constricta from China (Xu et al., 1999, 2011), metacercarial stages of Acanthoparyphium tyosenense (Digenea) in S. grandis in the Republic of Korea (Chai et al., 2001), germinoma and unidentified viral particles in Ensis magnus (=E. arcuatus) from Galicia (Darriba et al., 2006; Ruiz et al., 2011), as well as the detection of Marteilia sp. in Solen marginatus from Galicia (López and Darriba, 2006) and the subsequent identification, by molecular techniques, of Marteilia refringens in S. marginatus from Andalucia – South Spain (López-Flores et al., 2008). The present study reports the results of a histopathological survey performed to determine the symbionts and other conditions affecting E. siliqua populations from Galicia. 2. Materials and methods 2.1. Beds studied This study was conducted in two subtidal beds from Galicia – NW of Spain (Fig. 1), the first one located in Fisterra in the Ría of Corcubión (NW Galicia), and the second one in Barra beach in the outer of the Ría of Vigo (SW Galicia).

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Fig. 1. Map of Galicia (NW Spain) showing the location of Ensis siliqua beds sampled: Fisterra (F) in Ría de Corcubión and Barra (B) in Ría de Vigo.

2.2. Samples

2.4. Statistical analysis

Samples of 15–20 commercial pod razor clams (>100 mm in length) were taken monthly from each natural bed in different periods: between January 2003 and June 2004 in Fisterra, and from April 2009 to June 2010 in Barra.

Monthly and overall prevalence with their 95% confidence intervals were determined for each symbiont or condition in each bed. All calculations were performed using EpiCalc 2000 software Version 1.02 (Gilman and Myatt, 1998).

2.3. Processing

3. Results

In the laboratory, the valves were separated and gills, mantle and visceral mass were examined macroscopically for evidence of macroparasites, lesions, shell chambers, abnormal coloration and other malformations. A section including digestive gland, gonad, kidney, foot, mantle lobes, labial palps and gills was taken from every specimen, fixed in Davidson’s solution (Shaw and Battle, 1957) and embedded in paraffin. Paraffin blocks were sectioned at 5-lm with a microtome. Tissue sections (of about 60 mm2) were deparaffinized, stained with Harris’ hematoxylin and eosin and examined by light microscopy (Olympus BX51 coupled to Olympus DP72 and Imaging Software Cell) for parasitic and symbiotic organisms and other conditions. In the case of symbiont prevalences and histopathological alterations we established and arbitrary scale: (i) low, if prevalence is lower than 25%, (ii) moderate, when prevalence is between 25% and 50%, (iii) high, when prevalence is between 50% and 75% and (iv) very high, if prevalence is higher than 75%. For germinoma intensity we established a scale with three stages: (i) low intensity, with one or few gonadal follicles affected, (ii) moderate intensity, affecting nearly the half of follicles and (iii) high intensity, with most of the follicles affected. After the histological examination, we selected two samples showing unidentified basophilic inclusions and intracellular basophilic prokaryote-like inclusions both in epithelial cells of the digestive gland to perform an ultrastructural analysis. The portion of tissue containing the inclusions was taken out from the paraffin block. Paraffin was removed by several rinses in xylene with agitation. Tissue was placed in 2.5% glutaraldehyde, postfixed in 2% OsO4, and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a JEOL JEM 1010 transmission electron microscope at 80 kV. Furthermore, to improve the characterization of the prokaryotelike inclusions, additional staining techniques were used: Gram’s Brown and Hopps method for Gram-positive and Gram-negative bacteria, Pinkerton method for rickettsias, Ziehl-Neelsen method for Acid Fast bacteria and Feulgen picromethyl blue method for DNA (Howard and Smith, 1983).

A total of 551 pod razor clams were examined in this study: 311 from Fisterra (Ría de Corcubión) and 240 from Barra (Ría de Vigo). Histopathological analysis found the presence of different symbionts and other pathological alterations affecting to E. siliqua (Tables 1 and 2). 3.1. Viral inclusions Unidentified basophilic inclusions with irregular shape and a maximum dimension of 36.64 ± 12.18 lm (mean ± SD, n = 20) were observed, by light microscopy, in epithelial cells of digestive gland and rarely in gills, labial palps, gonad and kidney. These inclusions showed characteristic chromophilic margins (Fig. 2A), and Feulgen positive reaction indicating the presence of DNA. TEM examination showed the intranuclear position of the basophilic inclusions and the presence of viral particles inside the inclusions (Fig. 2B). The virions were unenveloped, with a rounded appearance suggesting icosahedral symmetry (Fig. 2B). The size of virions was 36.89 ± 2.07 nm in diameter (n = 30). Both empty and full capsids could be observed. The chromophilic margins, viewed by light microscopy, were identified as peripherally displaced chromatin. Overall prevalence of viral inclusions was low in both beds, as well as the intensity of infection (<10 inclusions per section) and host response was not observed. We occasionally observed intestinal epithelium cells showing a hypertrophied nucleus containing a single central eosinophilic body similar to Cowdry type A intranuclear inclusion bodies (Fig. 3). 3.2. Prokaryote Two types of intracellular prokaryote-like organisms (IPO) were detected: (i) The first one located in the epithelium of branchial filaments (bIPO), was oval in shape with a maximum dimension of 13.95 ± 3.99 lm (n = 30) (Fig. 4A). Overall prevalence was low in both beds as was the intensity of infection (<20 bIPO per section) and host response was not observed. (ii) The second type observed

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Table 1 Prevalence (%) of symbionts and other conditions of Ensis siliqua from Fisterra. Digestive gland intracellular prokaryote-like organisms (dgIPO); Branchial intracellular prokaryotelike organisms (bIPO). Date

Virus Prokaryota

Protozoa

Metazoa

dgIPO bIPO Apicomplexa

Unidentified Unidentified Trematode Germinoma I II Coccidia Nematopsis Unidentified Trichodina Unidentified Sporocysts Metacercariae sp sp

January-03 February-03 March-03 April-03 May-03 June-03 July-03 August-03 September-03 October-03 November-03 December-03 January-04 February-04 March-04 April-04 May-04 June-04

0 0 15 0 0 0 10 13 0 0 20 20 7 0 6 13 0 0

0 0 0 6 0 0 15 0 0 0 0 7 0 0 0 7 0 7

10 0 25 28 10 0 40 20 5 10 13 13 7 0 0 0 0 0

19 17 16 13 28 68 55 47 30 32 43 7 21 36 44 45 45 60

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

Ciliates

20 33 35 39 10 5 0 0 15 5 0 0 27 7 11 0 7 0

5 13 15 22 20 35 50 40 35 15 13 27 7 7 22 47 20 13

5 7 20 22 20 15 10 0 10 10 13 20 13 7 11 7 7 7

55 29 80 67 75 5 45 67 70 55 47 53 33 50 44 20 7 27

0 0 5 22 0 10 0 0 0 0 7 0 0 0 11 7 0 13

0 0 5 0 11 0 0 0 0 5 7 0 7 0 0 7 0 0

0 0 0 0 10 5 5 7 10 5 0 7 20 13 0 20 7 13

0 0 0 0 12 5 0 0 0 0 0 0 0 0 17 14 0 7

Overall prevalence 5.47 2.25

10.71 34.98

100.00

12.22

22.83

11.58

47.40

4.18

2.27

6.43

2.98

95% Confidence interval

3.32 0.99

7.59

29.49

98.48

8.89

18.37

8.34

41.73

2.34

1.00

4.07

1.46

8.77 4.78

14.85 40.89

99.97

16.51

27.98

15.79

53.14

7.22

4.81

9.91

5.78

in the epithelium of the digestive gland tubules (dgIPO), was rounded with an average diameter of 20.95 ± 3.52 lm (n = 30) and was finely granular and more dense than bIPO (Fig. 4B). The dgIPO were Gram-negative, Acid Fast negative, Feulgen positive and they took on a red color of rickettsial colonies with the Pinkerton method. Overall prevalence was low as was intensity, with <15 dgIPO per section in most of the cases, but with 20–30 dgIPO per section in a few samples. Associated haemocytic reaction was not observed. The ultrastructural analysis carried out in a sample showing moderate intensity of dgIPO confirmed the presence of prokaryotic organisms within the epithelial cells of the digestive gland (Fig. 5A). Prokaryotic organisms were pleomorphic, measured from 0.20 to 0.80 lm in length and had a cell wall with rippled appearance separated from a plasma membrane by an electron lucent zone. They showed the typical prokaryotic nucleoid and densely stained ribosomes at the periphery. Prokaryotes frequently displayed one or two lucent spherules at the periphery (Fig. 5A), and some of them appeared to contain phage-like particles inside (Fig. 5B). Fibrillar material was also observed between the individual prokaryotes (Fig. 5A). Colonies similar to the dgIPO were observed by light microscopy, in the connective tissue of gill, labial palps and pericardial gland but less frequently than in the digestive gland and host response was not observed. Ultrastructural analysis and specific stainings of bIPO and IPO in the connective tissue of different organs were not carried out because of the low intensities found. 3.3. Protozoa 3.3.1. Phylum apicomplexa: gregarines and coccidia Oocysts of gregarines belonging to Nematopsis genus were found in the connective tissue of different organs (gill, labial palps, mantle, digestive gland, pericardial gland, gonad and kidney). These oocysts were oval shape, a maximum dimension of 10.53 ± 1.33 lm (n = 30) and usually contained one sporozoite

per oocyst. Free and intrahaemocytic oocyst were usually reported showing one cyst per haemocyte, although specimens containing more than one cyst per haemocyte were also observed. Different affinities by hematoxylin and eosin stain were often detected, reporting basophilic and eosinophilic sporozoites. Overall prevalence in the sampled beds was very high (nearly 100%) throughout the survey period. Intensity was usually low although moderate intensities were also reported, but associated haemocytic reaction was not observed. An unidentified apicomplexa-like organism was mainly detected in the intestinal epithelium and less frequently in the connective tissue of the digestive gland and gills (Fig. 6). These organisms were rounded, with a diameter of 11.19 ± 3.15 lm (n = 30) and showed an evident nucleolus inside a nucleus and a refringent cytoplasm. Prevalence and intensity were low in both beds, without visible adverse effect on the host. The unidentified coccidian was mainly observed in renal tubules (Fig. 7) and rarely in gills. Four different stages of coccidian were observed: (i) macrogamonts, had rounded shape (24.70 ± 6.35 lm, n = 30), a refringent cytoplasm and an evident nucleolus within the nucleus; (ii) microgamonts, had rounded shape (21.50 ± 2.27 lm, n = 30), refringent cytoplasm and many peripheral nuclei; (iii) oocysts, had also rounded shape (26.01 ± 0.54 lm, n = 30) with enclosed sporocysts (2.86 ± 0.54 lm, n = 35) each containing two sporozoites; (iv) trophozoites, had oval shape (9.08 ± 2.93 lm, n = 30), a clear nucleolus inside a nucleus and a scarce nonrefringent cytoplasm. Macrogamonts (the most frequent stage), microgamonts and oocysts were usually observed free in the lumen of the intestinal tubules, while trophozoites were often located inside the epithelial cells of the renal tubules. Overall prevalence was moderate in all the studied beds. Intensity of infection varied throughout the study period, mostly with fewer than 30 coccidia per section, although cases with more than 200 coccidia per section were observed. In the present study, host defense was not usually detected, although in the high intensity cases we noted renal tubules occlusion (Fig. 7) and erosion of the inner surface of renal epithelium.

1.29 6.22 1.28 6.17 0.15 3.32

2.94 2.92 0.84

8.37 17.05 52.69 65.49

59.24

Fig. 2. (A) Light micrograph of a basophilic inclusion body in an epithelial cell of the digestive gland showing chromophilic margin (arrow), H and E staining. (B) Ultrathin section of viral particles suggesting icosahedral symmetry (scale bar = 200 nm).

40.25 53.19 19.75 31.06

16.74 27.53

46.67 25.00

21.67

10 20 0 15 0 15 20 – 5 – – 10 45 0 5 45 60 68 70 75 55 65 – 32 – – 30 55 85 70 75 70 35 40 55 40 20 – 10 – – 20 65 60 70 20 20 20 25 20 10 45 – 15 – – 25 35 45 20

30 30 5 15 15 25 10 – 10 – – 60 35 20 5

Trichodina sp

94.38 98.98 –: Not sampled by technical problems.

97.50 34.67

28.54 41.32 3.06 9.38

5.46

95% confidence interval

8.90 2.08

0.77 5.07

Overall prevalence

5.72 13.47

95 100 95 100 100 95 100 – 95 – – 95 95 100 100 11 35 47 47 50 33 11 – 28 – – 30 5 44 75 15 0 0 5 10 0 5 – 0 – – 5 20 5 0 5 0 5 5 10 10 5 – 0 – – 0 10 35 20 10 0 0 0 0 0 0 – 0 – – 10 0 5 0 April-09 May-09 June-09 July-09 August-09 September-09 October-09 November-09 December-09 January-10 February-10 March-10 April-10 May-10 June-10

Coccidia

Nematopsis sp

Unidentified

Unidentified

12.08

0 10 0 0 5 0 10 – 5 – – 0 0 5 0 0 5 5 0 0 0 0 – 0 – – 0 0 0 0

5 5 0 0 0 5 0 – 5 – – 5 10 0 0 Metacercariae

Trematode

Sporocysts

Metazoa

Unidentified II Unidentified I Ciliates Protozoa

Apicomplexa

Virus

bIPO

Prokaryota

dgIPO

77

Fig. 3. Light micrograph of an intestinal epithelial cell showing a central eosinophilic body in the nucleus (arrow), H and E staining.

Date

Table 2 Prevalence (%) of symbionts and other conditions of E. siliqua from Barra. Digestive gland intracellular prokaryote-like organisms (dgIPO); Branchial intracellular prokaryote-like organisms (bIPO).

Germinoma

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3.3.2. Phylum ciliophora Ciliates from the genus Trichodina (maximum dimension 35.86 ± 3.54 lm, n = 20) appeared attached to gills and labial palps as well as free in the pallial cavity (Fig. 8). Overall prevalence was low in Fisterra and moderate in Barra, and with fewer than 10 ciliates per section in most of the cases. Adverse effects on the host were not observed. Two types of unidentified ciliates were observed at the same location as Trichodina ciliates. The first type was round to oval, containing a basophilic macronucleus and had a maximum dimension of 16.71 ± 4.57 lm (n = 20); the second type was also round to oval, containing a basophilic macronucleus and was larger than the first type with a maximum dimension of 64.94 ± 24.33 lm (n = 22). Overall prevalence of unidentified ciliates was low in both beds. Intensity was low (<8 ciliates per section) and no pathological damage was noted. Furthermore, intracellular ciliates in the connective tissue of the digestive gland were observed only in one specimen; these ciliates had oval to round shape, a maximum dimension of 7.40 ± 1.19 lm (n = 10) and the nucleus was often fragmented into several micronuclei. The intensity of infection was low (around 25 ciliates per section) and host response was not observed.

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Fig. 4. (A) Light micrograph showing intracellular prokaryote-like organisms (arrow) in the epithelium of the branchial filaments. (B) Intracellular prokaryote-like organisms (arrow) in an epithelial cell of the digestive gland. H and E staining.

Fig. 5. (A) Ultrathin section of prokaryotic organisms, showing pleomorphic shape, lucent spherules (arrows) and fibrillar material between the prokaryotes (arrowheads). (B) Detail of prokaryotic organisms containing phage-like particles (arrows) (scale bar = 0.2 lm).

Fig. 7. Light micrograph showing abundant of coccidia causing occlusion of the renal tubules, H and E staining.

Fig. 6. Light micrograph of unidentified apicomplexa-like organisms in the intestinal epithelium (arrows), H and E staining.

Fig. 8. Light micrograph of Trichodina sp. ciliates (arrows) in the gill. H and E staining.

3.3.3. Unidentified protozoa Two types of unidentified protozoa were reported with different locations: (I) The first type was attached to the epithelium of the gill filaments and showed two different forms. The first one was round with an average diameter of 4.44 ± 1.26 lm (n = 30) and a well differentiated nucleolus in the nucleus (Fig. 9A), whereas the second

was larger (6.95 ± 1.05 lm, n = 30), rounded and with refringent cytoplasm (Fig. 9B). Overall prevalence of this protozoan was moderate in Fisterra and high in Barra and the intensity of infection was moderate. Haemocytic reaction was not observed, however in cases of high intensity we could see the disruption of the gill epithelium. (II) The second type of unidentified protozoan was generally observed in the epithelium of the digestive gland ducts, although it was also detected in the connective tissue of gill and labial palps,

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Fig. 9. Light micrograph of unidentified protozoa in the gill. (A) Stage I (arrows) showing oval to round shape and a well differentiated nucleolus in the nucleus. (B) Stage II (arrows) with round shape. H and E staining.

Fig. 10. Light micrograph of unidentified protozoa type II (arrows) in the epithelium of a digestive gland duct (scale bar = 20 lm). H and E staining.

Fig. 11. Light micrograph of gonad totally invaded by trematode sporocysts. H and E staining.

3.5. Other conditions and displayed a round shape, an average diameter of 9.55 ± 1.46 lm (n = 50) and a characteristic refringent cytoplasm with a prominent nucleus within (Fig. 10). Overall prevalence was low, as was the intensity, with fewer than 20 protozoa per section, and damage in the host was not observed. The cytoplasm of these protozoa showed a positive reaction to Periodic Acid Schiff (PAS) with Weigert’s hematoxylin stain (Howard and Smith, 1983) suggesting presence of neutral mucopolysaccharides or glycogen.

3.4. Metazoa Sporocysts of an unidentified trematode were observed in the gonad, digestive gland, kidney and gills. The gonad seemed to be the primary target organ of sporocysts proliferation because it always appeared heavily infected by this parasite, with complete castration in most of the cases (Fig. 11). Sporocysts prevalences were consistently low during all the period surveyed in both beds. Metacercariae larval stages of unidentified digenean trematodes were also detected encysted in different tissues: labial palps, digestive gland, pericardial gland, gonad and gills. Mean diameter of the cysts was 95.82 ± 23.82 lm (n = 21) and, normally, 1–2 cysts per histological section were observed, without host inflammatory reaction. Overall prevalence was low in both beds. A specimen showing an unidentified turbellarian flatworm in the pallial cavity and a specimen with an unidentified copepod attached to the gills, both in Fisterra bed, were found but without haemocytic reaction or pathological damage to the host.

3.5.1. Germinoma A germinoma, characterized by the presence of uncontrolled proliferation of undifferentiated germ cells forming clusters in the wall and/or lumen of follicles, was detected in male and female gonads of E. siliqua from the two beds studied (Fig. 12A). These undifferentiated cells were large (8.41 ± 1.26 lm, n = 30), eosinophilic and had a large nucleus-to-cytoplasm ratio. Nuclei were large (6.97 ± 1.14 lm, n = 30) with clumped, granular and occasionally marginated chromatin. The presence of brown cells was occasionally observed in the germinoma foci (Fig. 12B), but also in the gonadal follicles when they were in reabsorption stage and in other tissues like gills, kidney and digestive gland. Mitotic figures were found in some masses suggesting a rapid rate of growth. Neoplastic germ cells were always confined within the follicles. Rupture of follicle walls with tumor cell invasion into the interstitium was not observed. Germinoma were found in both beds at low prevalence. Most of the observed cases were classified as low or moderate intensity in both beds, whereas high intensity cases were detected in Fisterra bed only. 3.5.2. Disseminated neoplasm In May 2004, a case of disseminated neoplasm was detected in one specimen from Fisterra; this neoplasm was characterized by an abnormal proliferation of circulating cells in the connective tissue of gills (Fig. 13); foci of neoplastic cells were also observed in other organs but to a lesser extent. Neoplastic cells had a high

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Fig. 12. (A) Light micrograph of a male gonad showing germinoma foci in the gonadal follicles (arrows). (B) Light micrograph of a male gonad with germinoma (arrow) showing a focus of brown cells (asterisk). H and E staining.

bodies stained red with Gomori trichrome (Luna, 1968) indicating presence of muscle fibers. 4. Discussion

Fig. 13. Light micrograph of a disseminated neoplasm in the gill, characterized by abnormal cells with high nucleus-to-cytoplasm ratio (thick arrows). Normal cells (thin arrows). H and E staining.

Fig. 14. Unidentified eosinophilic bodies (arrows) affecting the connective tissue of the digestive gland, H and E staining.

nucleus-to-cytoplasm ratios and high frequency of mitotic figures indicating the high rate of proliferation. In the specimen showing disseminated neoplasm, we observed unidentified eosinophilic bodies in the connective tissue of different organs including the digestive gland, kidney, gills and less intensely the gonad. In the case of the digestive gland, these bodies filled the entire space of the connective tissue (Fig. 14). These unidentified eosinophilic

The present study reported several symbionts and other conditions with different prevalences and intensities in E. siliqua from Galicia (NW Spain). Viral inclusions, observed by electron microscopy, in the epithelial cells of the digestive gland resembled a virus reported in the same organ in E. magnus (=E. arcuatus) from Galicia (Ruiz et al., 2011). These viruses are also similar to the virus causing viral gametocytic hypertrophy (VGH) in oysters (Cheslett et al., 2009; Choi et al., 2004; Farley, 1976; Garcia et al., 2006; McGladdery and Stephenson, 1994; Meyers et al., 2009; Winstead and Courtney, 2003). With regard to the size and symmetry of the unenveloped virions found in oysters, these authors suggested similarities to the Papillomaviridae and Polyomaviridae families. In general, VGH has limited or no impact at population level. However, Garcia et al. (2006) commented that the virus may affect the viability of oyster gametes. Based only on ultrastructural morphological characteristics, is impossible to classify the virus found in this study into Papillomaviridae or Polyomaviridae families. Subsequent studies using molecular techniques are required to assist in the classification of these viruses. The intranuclear eosinophilic bodies occasionally found in the intestinal epithelial cells of the pod razor clams resembled those observed in other bivalves by Farley et al. (1972) and Meyers et al. (2009), named by this last author as Cowdry type A intranuclear inclusion bodies. Ultrastructural analysis of these intranuclear inclusion bodies, performed by these authors, showed arrays of virus-like particles resembling herpesvirus nucleocapsids (Farley et al., 1972; Meyers et al., 2009). In the present study, the low prevalence and intensity detected prevented us carrying out an ultrastructural analysis to confirm the presence of viral particles associated to these inclusion bodies. Intracellular basophilic inclusions similar to those observed in epithelial cells of the digestive gland and gill filaments, in the present work, were identified as rickettsia-like and/or chlamydia-like organisms in different bivalve molluscs and occasionally associated with important mortalities (Delgado et al., 2007; Fryer and Lannan, 1994; Paillard, 2004; Sun and Wu, 2004; Villalba et al., 1999; Wu and Pan, 1999). Both rickettsial and chlamydial organisms are prokaryotes, generally intracellular and Gram-negative, belonging to Rickettsiales or Chlamydiales orders. Chlamydiales are all coccoid and have a complex developmental cycle, whereas Rickettsiales are pleomorphic and they replicate by binary fission (Fryer and Lannan, 1994). In our case, characteristics observed by

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electron microscope did not allow their assignment to one of these orders, because we did not observe any stage of the complex developmental cycle of the Chlamydiales or binary fission. Prevalence of Nematopsis sp. was high in both beds, these gregarines use bivalve molluscs as intermediate hosts and complete its life cycle in marine arthropods. This genus has been described in different species of bivalve molluscs, including the superfamily Solenoidea (Ceschia et al., 2001; Conchas et al., 2001; Darriba et al., 2010; Soto et al., 1996), usually associated with a focal hemocyte infiltration, without measurable health effects (Bower et al., 1994), although there are references about associated mortalities in cockles (Azevedo and Cachola, 1992). The unidentified apicomplexa-like organisms observed in the intestinal epithelium resembled other organisms designated like unidentified coccidia in Venerupis decussata (=Ruditapes decussatus) (Joly, 1982; Navas, 2008) and unidentified gregarines in Venerupis (=Ruditapes) spp. (Bower et al., 1992; Villalba et al., 1993), E. magnus (=E. arcuatus), E. siliqua (López et al., 2011; Montes, 2011) and C. edule (Carballal et al., 2001). López (1995) put forward the difficult to distinguish between gregarines or coccidia on account of only one stage of the life cycle of this species was detected. For this reason, in this study we decided to use the term unidentified apicomplexa-like organism to designate this symbiont. The different stages of renal coccidia observed in E. siliqua in this work are equal to those detected in E. magnus (=E. arcuatus) also in Galicia (Darriba et al., 2010). These authors have tentatively assigned these coccidia to the genus Pseudoklossia on the basis that meront stages were never detected. The presence of renal coccidia in bivalves is frequent and usually harmless to the host (Bower et al., 1994), however heavy infections were reported associated with kidney damage (Carballal et al., 2001; Cremonte et al., 2005; Leibovitz et al., 1984; Morado et al., 1984). In the current study, we did not observe a defense reaction, however in the cases of high intensity of infection, occlusion of the renal tubules and erosion of the inner renal epithelium were detected. The unidentified protozoan, observed in this study, attached to the gill filaments resembled coccidian seen in the kidney. The first stage characterized by its round shape and a well differentiated nucleolus into the nucleus was similar to the coccidian trophozoite stage, whereas the second stage, larger, and showing a refringent cytoplasm resembled coccidian macrogamonts, although, both stages were smaller than the corresponding stages of the coccidia normally reported in bivalve molluscs. Further studies are needed to determine the certain position of these protozoa. In this study, inflammatory response was not observed, but disruption of the gill epithelium was detected in the high intensity cases. Ciliates observed in this study of E. siliqua have been referred in other bivalve species on the Galician coast (Villalba et al., 1997; Carballal et al., 2001; Darriba et al., 2010; López et al., 2011) and around the world (Lauckner, 1983; Xu et al., 1999, 2011; Spiers et al., 2008). Ciliates are common inhabitants of bivalve molluscs with few references about associated mortalities (Lauckner, 1983). Metazoan symbionts like turbellaria, copepods and digenean metacercariae of trematode were observed with low prevalence and intensity, these metazoa have been considered as common symbionts in bivalve molluscs (Bower et al., 1994; Lauckner, 1983). It was remarkable the presence of sporocysts of an unidentified trematode which caused castration in most of the cases. This is a very common consequence of sporocysts infections in different bivalve species (Lauckner, 1983). Sporocysts observed in E. siliqua were similar to the sporocysts reported in E. magnus (=E. arcuatus) from Galicia (Ruiz et al., 2012) which had the typical morphologic characteristics of the family Bucephalidae. Disseminated neoplasm is the most common neoplastic condition in marine invertebrate animals (Elston et al., 1992). This condition frequently progress to a fatal outcome, but some individuals

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can effect remission (Elston et al., 1992). In Solenoidea, disseminated neoplasm was reported in S. marginatus from Galicia (López et al., 2011), affecting mainly gills, and less frequently in digestive gland and heart, with overall prevalences between 17% and 25% (unpublished data). Throughout this study, we only observed one case of disseminated neoplasm in a pod razor clam from Fisterra, the specimen affected displayed unidentified eosinophilic bodies in the connective tissue of different organs. The origin of these eosinophilic bodies is unknown, as well as their possible association with the disseminated neoplasm. Germinoma is the second most frequently diagnosed neoplasm of marine bivalve molluscs (Mix, 1986), and it was first reported in quahog, Mercenaria mercenaria, by Yevich and Barry (1969) and subsequently observed in 14 other bivalve species (Peters et al., 1994). Recently, in Galicia germinoma was detected in E. magnus (=E. arcuatus) (Darriba et al., 2006). Gonadal neoplasms could cause abnormal gametogenesis and reduction in fecundity due to the progressive replacement of the normal gametes by undifferentiated germ cells (Barber, 1996; Eversole and Heffernan, 1995). In the present study, germinoma foci were detected in the follicles of E. siliqua, the morphologic characteristics of this germinoma were in concordance with the observed in Mercenaria spp. (Hesselman et al., 1988). The aetiology of neoplasms in bivalve molluscs is not well understood and different aetiological agents have been proposed (reviewed by Barber, 2004). In addition, we often observed brown cells foci into the gonadal follicles when they were in reabsorption stage, as well as in other organs like pericardial gland, digestive gland, kidney and gills but with less frequency. These cells are though to be related to the internal defense system or to the processing of biological fluids (Ruddell and Wellings, 1971; Zaroogian and Yevich, 1994) and it is established that their number increases in response to various agents, parasites and gonadal neoplasms (Lauckner, 1983; Peters et al., 1994). In conclusion, higher overall prevalences in the group of the protozoa and lower in the other symbionts and conditions were detected. Among protozoa, Nematopsis sp., and the unidentified protozoan in gills were noted for their high prevalences, together with renal coccidia and Trichodina sp. However, none were found to be detrimental to pod razor clam populations. Based on its pathogenic potential, bucephalid sporocysts, viral inclusions, germinoma and disseminated neoplasm were highlighted. However, the low prevalences observed may indicate that they do not involve a risk to the E. siliqua populations. None of the observed parasites are on the list of obligatory notification to the World Organisation for Animal Health (OIE), but it is important to monitor the health status of the exploited populations mainly in stressful situations, as Sabry et al. (2011) indicated for some oysters, that could serve as a factor for triggering non-apparent infections. Acknowledgments We thank to A. González, I. Meléndez, E. Penas, P. Rodríguez, M. de Castro, I. Santamaría, M. Miranda for the technical assistance, to the Cangas and Fisterra fisherman’s associations for providing the samples and to I. Pazos and J. Méndez for ultrastructural techniques (CACTI- Vigo University). References Azevedo, C., Cachola, R., 1992. Fine structure of the apicomplexa oocyst of Nematopsis sp. of two marine bivalve molluscs. Dis. Aquat. Org. 14, 69–73. Barber, B.J., 1996. Effects of gonadal neoplasms on oncogenesis in softshell clams, Mya arenaria. J. Invertebr. Pathol. 67, 161–168. Barber, B.J., 2004. Neoplasic disease of commercially important marine bivalves. Aquat. Living Resour. 17, 449–466.

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