Aquaculture 281 (2008) 12–16

Contents lists available at ScienceDirect Aquaculture

journal homepage: www.elsevier.com/locate/aqua-online

Infection of Nematopsis oocysts in different size classes of the farmed viridis in Thailand

Chanawat Tuntiwaranuruk a,⁎, Kashane Chalermwat b, Vanida Pongsakchat c, Ardool Meepool d, Edward Suchart Upatham d, Maleeya Kruatrachue e a Department of Biology, Faculty of Science, Burapha University, Chonburi 20131, Thailand b Department of Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131, Thailand c Department of Mathematics, Faculty of Science, Burapha University, Chonburi 20131, Thailand d Department of Medical Science, Faculty of Science, Burapha University, Chonburi 20131, Thailand e Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

ARTICLE INFO ABSTRACT

Article history: We report on the infection intensity of Nematopsis oocysts in different size classes of the green mussel Perna Received 2 August 2007 viridis farmed in Chonburi province, eastern upper Gulf of Thailand. Infection of the apicomplexan parasite Received in revised form 18 April 2008 was investigated from November 2003 to November 2004 in small (2.3±0.2 cm shell length, n=130), Accepted 17 May 2008 medium (5.6±0.3 cm, n=130) and large (8.6±0.4 cm, n=130) size classes of the green mussel. Heavy prevalence of infection was found in all size classes from November 2003 to June 2004, whereas low Keywords: Nematopsis prevalence was observed from July to October 2004. From statistical analysis, prevalence of the parasite did Perna viridis not differ between the three mussel size classes. However, infection intensity calculated per unit weight of Parasite dried gill tissue was significantly lower in the smallest size class. Protozoa From light and transmission electron microscopy, the Nematopsis oocysts were ellipsoidal 12.6±0.3 μm wide Gregarine and 17.4±0.9 μm long and 12.0±0.3 μm wide and 16.6±0.5 μm long, respectively. Oocyte wall thickness was Mussel farming between 0.5 and 0.7 μm. Oocysts were located within a parasitophorous vacuole (PV) 22–30 μm in diameter. Thailand The PVs were in turn engulfed within phagocyte sacs produced by the mussel with diameters ranging from 30–95 μm depending on the number of oocysts within. Microscopic examination suggested that this infective stage of the parasite causes severe damage to P. viridis gill tissue in conditions of heavy infection causing inflammation and disruption of gill epithelia. © 2008 Elsevier B.V. All rights reserved.

1. Introduction losses in the green mussel industry in this area have primarily been attributed to poor environmental conditions. However, infections The Apicomplexan protozoan Nematopsis is a parasite commonly by Nematopsis might play a role in the weakening of these bivalves. infecting molluscan intermediate hosts with decapod as Nematopsis sp. may be associated with mass mortalities of cockles and definitive host (Lee et al., 2000). The first species of Nematopsis was in Portugal (Azevedo and Cachola, 1992) due to their harmful discovered in the mantle of the razor Solen vagina in France, effects on the host. while the earliest nominal species referred to the genus was Nema- Our research reports upon the prevalence of Nematopsis in topsis schneideri Leger, 1903 in the gills of Mytilus edulis, also reported different size classes of the green mussel, P. viridis monitored over a in France (Sprague, 1970). Subsequently, N. schneideri Leger, 1903 13 month period and determines the infection intensity of Nematopsis became the type species and the monotype. in relation to mussel size. An account is also given of the histological Although the importance of protozoan parasites in farmed bivalves appearance of heavily infected gill tissue and ultrastructure of the is recognized, the biology and life-history of these microorganisms in infecting protozoa. Thailand's tropical waters have not been extensively investigated. Many species of bivalves in the area are heavily infected by Nematopsis 2. Materials and methods (Tuntiwaranuruk et al., 2004) including P. viridis, the primary cultivated bivalve species in Thailand in terms of landings. Annual Green mussel, P. viridis were collected from local farms in farming areas from Ang-Sila district in the Chonburi province coastal zone located at 13° 19.97ʼ N, 100° 54.94ʼ E in Thailand (Fig. 1). From ⁎ Corresponding author. November 2003 to November, 2004, 10 small, medium and large E-mail address: [email protected] (C. Tuntiwaranuruk). were collected monthly corresponding to a 13 month culture

0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.05.025 C. Tuntiwaranuruk et al. / Aquaculture 281 (2008) 12–16 13

For TEM, small pieces of heavily infected gill tissues were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.8, for 24 h at 4 °C. They were later washed for 2 h at 4 °C in the same buffer and

left overnight. Afterwards, the tissues were post-fixed in 1% OsO4 in 0.1 M sodium cacodylate buffer, pH 7.8 for 2 h at 4 °C. After washing, the samples were dehydrated through a serial concentration of ethanol and infiltrated with propylene oxide for 20 min (twice) followed by mixtures of propylene oxide and Araldite 502 resin (2:1) for 1 h and 1:2 overnight. The infiltrated samples were then em- bedded in a flat mold filled with pure Araldite 502 resin and polymerized in an incubator at 45 °C and 60 °C for 48 h each for gradual and even polymerization (Meepool et al., 2006). The specimen blocks were cut at 60 to 90 nm thicknesses using an ultramicrotome (Leica, Model Ultracut R) and placed on a meshed copper grid. The ultrathin sections were first stained in saturated alcoholic uranyl acetate followed by counterstaining in 0.1% aqueous lead citrate and observed in a TEM (TECNAI 20, FEI) operated at 70 kV. Additional semithin sections (500–1,000 µm) were also stained with 1% aqueous methylene blue for light microscope observation.

3. Results

The mean water temperature and salinity during the sampling period were 29.81 °C and 30.46 psu, respectively while the mean pH was 8.09 (Fig. 2). The mean shell length (SL) of the small, medium and large green mussels were 2.3±0.2 cm, n=130, 5.6±0.3 cm, n=130 and Fig. 1. Green mussel farms where samples were taken at Ang-Sila fishing village located 8.6±0.4 cm, n=130 respectively. A high prevalence of infection was at 13° 19.97¢ N, 100° 54.94¢ E (⁎) Chonburi Province of Thailand. found in all size classes from November 2003 to June 2004. Prevalence appears to have dropped sharply from July 2004. During the last month of sampling in November 2004 the prevalence in all size classes and harvest cycle. Small mussels were those smaller than 3 cm shell had increased to levels comparable to November 2003 (Fig. 3). From length (SL) while medium sized mussels were those between 3 and statistical analysis the prevalence profile of the Nematopsis parasite in 5.99 cm SL, large mussels were of 6 cm SL upwards. In Chonburi, green the three size categories were not significantly different. However, mussels are first harvested after 8–9 months of growout. Harvesting of infection intensity was found to be lowest in the smallest size category the annual crop usually lasts for 2–3 months. Infection intensity was (Tables 1 and 2). analyzed in relation to size and season of sampling. All mussel samples were collected from farms within a period of several hours, taken to the laboratory and immediately processed. The mussels were opened and a 16 mm2 piece of gill tissue from each individual was excised from the inner and outer demibranch on the left-hand side of the bivalve. They were squashed between pre- weighed glass coverslips, observed under a compound microscope and the total number of oocysts was enumerated utilizing a counting grid. After counting, the coverslips along with excised gill tissue were dried at 75 °C overnight in a hot air oven and re-weighed to estimate dry excised gill tissue weight. In conjunction, the remaining tissue from the inner and outer demibranch also on the left-hand side of each mussel was dissected, dried and weighed on pieces of pre- weighed aluminum foil also after drying at 75 °C for 24 h. Oocyst infection intensity and dry weight of excised tissue was then compared with total gill dry weight expressed in milligrams. An analysis of variance (ANOVA) was performed on the relation- ship between sizes of P. viridis and number of oocyst using collecting months as a blocking factor. Finally, the Duncan's multiple range test at 5% probability level was calculated for each size class when a significant difference among sizes was found.

2.1. Microscopic observation and transmission electron microscopy (TEM)

The inner and outer demibranch on the right-hand side of the mussels were dissected and prepared for histological observation and TEM. For light microscopy, the gills were placed in 10% neutral formalin at room temperature for 24 h and subsequently embedded for histological analysis following a standard protocol (Luna, 1960). Tissue sections were subsequently prepared by standard histological Fig. 2. Monthly variation of seawater temperature (A), salinity (B), and pH (C) from the methods. sampling station. 14 C. Tuntiwaranuruk et al. / Aquaculture 281 (2008) 12–16

Fig. 3. The prevalence profile of Nematopsis in three size classes of the green mussel.

Histology of gill tissue showed that the Nematopsis oocysts were undulata, a major commercial shellfisheries species in the Gulf of located in the gill lumina of each filament (Fig. 4A). The oocysts were Thailand. Tuntiwaranuruk et al. (2004) investigated the occurrence of also embedded along the epithelium of the gill filaments and Nematopsis in 7 species of commercial bivalves from Chonburi dominated the distal margins. From light microscopy the oocysts Province, Gulf of Thailand. Here we have studied Nematopsis in the were ellipsoidal 12.6±0.3 μm wide and 17.4±0.9 μm long with a thick green mussel, which is a major aquaculture species in Southeast Asia, outer surface. Within the oocyst, there was a single vermiform and the inner gulf of Thailand, in terms of landings. sporozoite enveloped by a PV whose diameter was approximately 22– Worldwide, most investigations on Nematopsis have described 30 μm. Each oocyst was in turn engulfed by phagocytes ranging from their oocyst infection stages in bivalves (Azevedo and Cachola, 1992; 30 to 95 μm in diameter depending on the number of oocysts within Canestri-Trotti et al., 1998; Azevedo and Matos, 1999; Canestri-Trotti (range from 1 to 32). There was no apparent effect on the epithelial et al., 2000). Nematopsis also occurs in crustaceans such as prawns and cells and connective tissue in areas with low infection. However, in crabs (Shanavas et al., 1989; Prema and Janardanan, 1990; Prasadan areas with heavy levels of infection, the parasite caused irregular and Janardanan, 1996; Belofastova, 1996; Jimenez et al., 2002; Fajer- arrangement and partial disruption of gill filament epithelial cells and Avila et al., 2005). also loss of cilia (Fig. 4B). The gill epithelial cells were also found to In bivalves, gregarine species including Nematopsis often show a decrease in cell height from columnar epithelial to squamous cell. In tissue preference for either mantle or gill (Sprague and Orr, 1955). Kim addition, in areas with high infection large numbers of haemocytes et al. (1998) noted that similar gregarines were common in were observed in the connective tissue compared to uninfected areas. from the southeastern United States and Gulf of Mexico and in mytilid Ultrastructurally, each thick walled Nematopsis oocyst was con- mussels from the west coast of the United States. tained within a PV of irregular outline (Fig. 4C). From electron The prevalence of Nematopsis was heavy from November 2003– microscopy the oocyst was 12.0±0.3 μm in width (n=10) and 16.6± June 2004 but low from July to October 2004, the latter coinciding 0.5 μm in length with a single uninucleated vermiform sporozoite. The with Thailand's south-west monsoon season characterized by heavy oocyst wall was 0.7 μm thick (Fig. 4D) with a circular micropyle rains and a resulting decrease in seawater salinity. In Ecuador, the approximatey1.2 μm in diameter, caped by an operculum (Op) incidence of Nematopsis infection in Litopenaeus vannamei was higher (Fig. 4E). The apical portion of the operculum was bounded to the at higher salinities while infection disappeared in lower salinity PV membrane by an amorphous mass. Numerous microfibrils regimes (Jimenez et al., 2002). This may also be the case with Nema- surrounded the PV and directly contacted the membrane of the host topsis in P. viridis. Seawater temperatures and changes in pH did not cell vacuole. appear to affect the infection profiles in this study possibly due to The sporozoite was composed of unidentified pentagonal inclu- small variations of temperature and pH in the study area. In this study sions and stained unevenly (Fig. 4F). The exterior of the oocyst wall we found a low level of prevalence during July to October for all size was surrounded by short microfibrils most dense near the oocyst wall. categories. A comparison between medium and large mussel categories 4. Discussion showed no difference in the level of infection per unit weight of dry gill tissue. Small mussels had lower Nematopsis infection intensity Recently has there been interest in the characterization and than other size groups, possibly because less water is filtered by them of protozoan infections of Thai commercial bivalves and the and therefore less Nematopsis gymnospores ingested. Alternatively, taxonomic affiliations of the protozoans involved. Leethochavalit et al. gymnospores may be acquired at the same rate by all size groups, and (2003, 2004) reported on the occurrence and genetics of the the observed differences due to cumulative acquisition. protozoan parasite Perkinsus in the undulated surf clam, Paphia High levels of infection of Nematopsis caused irregular arrange- ment of the gill epithelium and partial disruption of gill filament epithelial cells and loss of cilia. This was not observed in areas of the Table 1 Results of analysis of variance on relationship of size of green mussel and number of oocyst Table 2 Duncan's multiple range test Source Sum of squares df Mean square FP-value Size 246835.0832 2 123417.5416 8.004838a 0.002 Size Means Month 1935713.042 12 161309.4201 10.4625 0.000 1–3 cm 111.065a Error 370028.8469 24 15417.86862 3–6 cm 283.537b Total 2552576.972 38 6–9 cm 275.856b

a Level of significance=0.05. Mean; different letters show significant difference at α=0.05. C. Tuntiwaranuruk et al. / Aquaculture 281 (2008) 12–16 15

Fig. 4. A. Section of Perna viridis gill showing a sporozoite (Sz) within each oocyst (Oc) that was in turn enclosed in a parasitophorous vacuole (PV). Parasitophorous vacuoles were enclosed by a phagocyte (Pha) produced from the mussel. B. Section of P. viridis gill showing uninfected (Gf1) and infected (Gf2) gill filament. The epithelial cells (Epi) of Gf1 were covered with cilia whereas epithelial cells of Gf2 were irregular and rarely covered with cilia. In addition, the gill lumen (GL) of Gf1 was narrower and lacked the large numbers of haemocytes (Hae) as seen in Gf2. C. Ultrathin section of a sporozoite (Sz) of Nematopsis inside a thick walled (Wa) ellipsoidal oocyst. The entire oocyst was packed inside a parasitophorous vacuole (PV). D. Electron micrograph showing the wall (Wa) of the oocyst covered by microfibrils (Mf). E. Ultrathin section of the apical region of an oocyst showing an electron dense band (⁎) connected to the apical zone of one oocyst. Also visible is a view of the oocyst wall (Wa) and the operculum (Op). The matrix of the parasitophorous vacuole (PV) contains numerous anastomosed microfibrils (Mf). F. Partial ultrathin section of a sporozoite (Sz) within the oocyst wall (Wa).

gill with low levels of infection. Haemocytes accumulated in the Azevedo and Matos (1999), Padovan et al. (2003) and Azevedo and connective tissue at high infection intensity, demonstrating an Padovan (2004). Oocyst dimensions reported in this study are also inflammatory response by the bivalves. Haemocytes may also substantially larger than oocysts of a Nematosis species reported from accumulate to destroy invasive microbes at sites of epithelial damage the green-lipped mussel in New Zealand (Jones, (Moore and Lowe 1977). 1975). Heavy infection by this parasite leading to the irregular arrange- Nomination of this Nematopsis sp. must be based on TEM, ment of the mussel epithelium cell and the absence of cilia may thus molecular characterization, and all the stages of the life cycle, weaken the mussel and therefore increase their susceptibility to including those from sympatric crustaceans. The transplantation of secondary infection and adverse environmental conditions. mussels from the Chonburi area to other shellfish growing areas may Both light microscopy and TEM studies of this oocyst from P. viridis inadvertently enhance the distribution of this parasite. Shrimp revealed characteristics that are typical of the genus Nematopsis farmers in Thailand who are raising the introduced L. vannamei may (Azevedo and Cachola, 1992; Soto et al., 1996; Sprague, 1970). also take into consideration the risk associated with utilization of However, there are differences in oocyst dimensions, wall thickness, mussels to supplement the diet of their raised crop in light of evidence as well as operculum morphology of the Nematopsis oocysts reported that Nematopsis can cause significant losses to L. vannamei culture in this study and those reported by Azevedo and Cachola (1992), operations (Jimenez et al., 2002). 16 C. Tuntiwaranuruk et al. / Aquaculture 281 (2008) 12–16

Acknowledgements Leethochavalit, S., Upatham, E.S., Choi, K.-S., Sawangwong, P., Chalermwat, K., Kruatrachue, M., 2003. Ribosomal RNA characterization of non-transcribed spacer and two internal transcribed spacers with 5.8S ribosomal RNA of Perkinsus sp. We thank the Department of Biology and graduate program in Found in undulated surf clams (Paphia undulata) from Thailand. J. Shellfish Res. 22, Biological Science, Faculty of Science, Burapha University for providing 431–434. Leethochavalit, S., Upatham, E.S., Choi, K.-S., Sawangwong, P., Chalermwat, K., laboratory space and equipment. The Faculty of Science, Burapha Kruatrachue, M., 2004. Occurrence of Perkinsus sp. in undulated surf clams University also provided a research development grant to C. (Paphia undulata) from the Gulf of Thailand. Dis. Aquat. Org. 60, 165–171. Tuntiwaranuruk. We also thank the anonymous reviewers of the Luna, L.G., 1960. Manual of Histological Staining Methods of the Armed Forces Institute manuscript for their comments and suggestions. of Pathology, 3rd edition. McGraw-Hill, New York, USA. Meepool, A., Wanichanon, C., Viyanant, V., Sobhon, P., 2006. Development and roles of vitelline cells in eggshell formation in Fasciola gigantica. Invert. Reprod. Develop. References 49, 9–17. Moore, M.N., Lowe, D.M., 1977. The cytology and cytochemistry of the haemocytes of Azevedo, C., Cachola, R., 1992. Fine structure of the oocyst of Nematopsis Mytilus edulis and their responses to experimentally injected carbon particles. – sp. of two marine bivalve molluscs. Dis. Aquat. Org. 14, 69–73. J. Invertebr. Pathol. 29, 18 30. Azevedo, C., Matos, E., 1999. Description of Nematopsis mytellan. sp. (Apicomplexa), Padovan, I.P., Tavares, L.A., Corral, L.P., Padovan, A., Azevedo, C., 2003. Fine structure of parasite of the mussel Mytella guyanensis () from the Amazon Estuary and the oocyst of Nematopsis mytella (Apicomplexa, ), a parasite of the description of its oocysts. Eur. J. Protistol. 35, 427–433. mussel, Mytella falcate and of the rizophorae (, ) – Azevedo, C., Padovan, I.P., 2004. Nematopsis gigas n. sp. (Apicomplexa), a parasite of from the Northeastern Atlantic coast of Brazil. Braz. J. Morphol. Sci. 20, 141 145. Nerita ascencionis (, ) from Brazil. J. Eukaryot. Microbiol. 51, Prasadan, P.K., Janardanan, K.P., 1996. Nematopsis idella n. sp. and Uradiophora cuenoti 214–219. Mercier: two cephaline gregarines from freshwater prawns in Kerala. Acta – Belofastova, I.P., 1996. Gregarines of the genus Nematopsis (Eugregarinida: Porospor- Protozool. 35, 239 243. idae), parasites of the Black Sea molluscs. Parazitologiya 30, 159–173. Prema, S., Janardanan, K.P., 1990. Two new species of cephaline gregarines (Apicom- Canestri-Trotti, G., Baccarani, E.M., Paesanti, F., 1998. Gregarines of the genus Nema- plexa: sporozoa) from the marine prawn Penaeus indicus H. Milne Edwards. Acta – topsis (Apicomplexa: porosporidae) in bivalve molluscs of the Adriatic Sea. Boll. Soc. Protozool. 29, 365 373. It. Patol. Ittica. 10, 58–66. Shanavas, K.R., Prasadan, P.K., Janardanan, K.P., 1989. Nematopsis rosenbergii n.sp. Canestri-Trotti, G., Baccarani, E.M., Paesanti, F., Turolla, E., 2000. Monitoring of (Apicomplexa, Cephalina) from the brakishwater prawn, Macrobrachium rosenber- – infections by protozoa of the genera Nematopsis, Perkinsus and Porospora in the gii. Arch. Protistenkd. 137, 161 164. smooth venus clam from the North-Western Adriatic Sea (Italy). Dis. Soto, M., Pascual, S., Rodriguez, H., Gestal, C., Abollo, E., Arias, C., Estevez, J., 1996. Aquat. Org. 42, 157–161. Nematopsis spp. Schneider, 1892 (Apicomplexa: Gregarinida) in bivalve molluscs – Fajer-Avila, E.J., Covarrubias, M.S.M., Abad-Rosales, S., Roque, A., Meza-Bojorquez, P., off Ria de Vigo (Galicia, NW Spain). Bull. Eur. Assoc. Fish Pathol. 16, 157 160. Hernandez-Gonzalez, C., 2005. Effectiveness of oral Elancoban ™ and Avimix-STTM Sprague, V., 1970. Some protozoan parasites and hyperparasites in marine bivalve against Nematopsis (Apicomplexa: Porosporidae) gametocysts infecting the shrimp molluscs. In: Stanislas, F., Sniezko, C. (Eds.), A Symposium on Diseases of Fishes and fi Litopenaeus vannamei. Aquaculture 244, 11–18. Shell shes. . Special Publication, vol. 5. American Fisheries Society, Washington D.C, – Jimenez, R., Barniol, L., Machuca, M., 2002. Nematopsis marinus n. sp., a new septate pp. 511 526. gregarine from cultured penaeoid shrimp Litopenaeus vannamei (Boone), in Sprague, V., Orr Jr., P.E., 1955. Nematopsis ostreum and N. prytherchi (Eugregarina: Ecuador. Aquac. Res. 33, 231–240. Porosporidae) with special reference to the host-parasite relations. J. Parasitol. 41, – Jones, J.B., 1975. Nematopsis n. sp. (Sporozoa: Gregarinia) in Perna canaliculus. N.Z. 89 104. J. Mar. Freshw. Res. 9, 567–568. Tuntiwaranuruk, C., Chalermwat, K., Upathum, E.S., Kruatrachue, M., Azevedo, C., 2004. Kim, Y., Powell, E.N., Wade, T.L., Presley, B.J., Sericano, J., 1998. Parasites of sentinel Investigation of Nematopsis spp. oocysts in 7 species of bivalves from Chonburi – bivalves in the NOAA Status and Trends program: distribution and relationship to Province, Gulf of Thailand. Dis. Aquat. Org. 58, 47 53. contaminant body burden. Mar. Pollut. Bull. 37, 45–55. Lee, J.J., Leedale, G.F., Bradbury, P., 2000. An Illustrated Guide to the Protozoa, (2nd ed.). Society of Protozoologists: Allen Press, USA, pp. 190–369.