A parasitological survey of slipper-cupped oysters (Crassostrea iredalei, Faustino, 1932) in the Philippines.ABSTRACT This paper describes the first screening in the Philippines of slipper-cupped oysters (Crassostrea iredalei) for the presence of parasites. Slipper-cupped oysters were sampled at 2 sites in Ivisan, Capiz, from September to December 2007. Macroscopical and histological analyses were carried out in oyster tissues. Histological examination showed gregarine protozoan Nematopsis sp. as the most prevalent parasite (71.33% and 65.0%) at 2 sites with a moderate intensity of infection. Tylocephalum sp. cestode was found in the connective tissue around the digestive gland, with a prevalence of 60% and 52.3% in 2 sites, with a moderate intensity of infection. Digenean trematodes had a 37.80% prevalence at site 1 and a 22.45% prevalence at site 2. Ciliates were also observed with a prevalence of 18.75% (site 1) and 13% (site 2). The observed infection of oysters had no apparent effect on oyster production at these sites maybe due to low infestation levels or to the fact that the parasites have no pathological effect.
KEY WORDS: oysters, Crassostrea iredalei, parasites, Nematopsis sp., Tylocephalum sp., ciliates
The slipper-shaped oyster (Crassostrea iredalei) is a widely distributed and economically important species in the Philippines. Oyster farming has been practiced in the Philippines since the early 1900s, mainly as a small-scale family business. The reported yearly aquaculture production ranged from 14,222 MT in 2000 to 15,915 MT in 2004 (FAO 2005). Oyster is a popular bivalve delicacy because of its excellent flavor and taste. It is mostly marketed in the shell as freshly shucked meat. Oysters thrive best in brackish water to marine waters, with salinity ranging from 15 26 ppt, at 20-30[degrees]C. Most of the oyster farms are small scale and use natural spat collection. The most popular method of oyster culture in the Philippines is the hanging method, with oyster spat attached to bamboo poles that are joined together by a rope. Oysters are harvested at 10-12 mo of age, depending on size.
To date, the oyster industry in the Philippines is free of harmful parasites. There is a growing concern, however, that diseases-causing agents may appear as production densities increase. This preliminary article is based upon a detailed histological survey to determine which parasites are present in healthy stocks of cultivated oysters. This study is designed to provide baseline observations for future reference; should parasites suddenly become a problem in the oyster aquaculture region of the Philippines.
MATERIALS AND METHODS
Adult oysters were collected to ensure detection of pathogens occurring at 2% prevalence (150 oysters per site). They were obtained from 2 oyster farms in Ivisan, Capiz (27[degrees]03'S, 153 [degrees]07' E), Philippines--Barangay Cabugao (site 1) and Barangay Basiao (site 2)--from September 2007 to December 2007 and were collected every month. Water temperature and pH at the surface level of culture sites were also recorded. Samples were cleaned of encrusting organisms and mud. The oysters were measured with calipers (maximum length) and examined gross macroscopically. Shell heights were reported in millimeters. Fouling organisms were recorded. An sapproximately 5-mm-thick anterior transverse cross-section was excised that included mantle, digestive gland or gut, gonad, and labial palps; and a posterior transverse section including mantle, gonad, heart, posterior adductor muscle, and gill. Samples were fixed in Davidson's fixative and processed for pathology using standard histological techniques (Howard & Smith 1983). The samples were then embedded in paraffin, sectioned at 5 [micro]m, stained with hematoxylin--eosin stain, and examined using light microscopy at 100 x, 250 x, and 400 x magnification. Prevalence and intensity of parasites were calculated according to Bush et al. (1997).
The water temperature varied from 27-30[degrees]C and salinity ranged from 25-29 ppt. The shell length of the oysters was 61-159 mm. Table 1 summarizes parasite species, their prevalence, and intensity at 2 sites in Ivisan, Capiz.
Gross examination revealed the presence of barnacles, oyster spat, as well as artifacts and hooked mussel in the shells. Artifacts are any objects that may remain from some commensals or predatory organisms that inhabit oysters. Hooked mussels are attached to different hard surfaces, including oyster shells. They are generally 50.8-76.2 mm in length, with a dull black to gray exterior and a purple to rosy brown interior shell color. The surface of the shell is distinctly ridged and curved (Lippson & Lippson 1997).
The screening did not reveal any parasites by observation of gross morphology. Histological examination revealed the presence of a protozoan morphologically similar to Nematopsis sp. (Figs. 1 and 2), which was the most prevalent parasite (71.33% and 65.0%) at both sites, with moderate intensity of infection. It was observed in the connective tissue of the gills, mantle, gonads, and digestive glands. The oocysts of this gregarine protozoan were pyriform in shape and measured 20 [micro]m (range, 18-22 [micro]m) in length and 15[micro]m (range, 11-16 [micro]m) m in width (n = 30). Spores were found in the digestive gland, gills, and mantle. Spores were ovoid, with a marked gelatinous epispore and a solid, refractive endospore. There were 1-2 oocytes found per host cell (phagocyte), with the oocyst containing a single uninucleate sporozoite. No pathological damage was observed.
Examination of the histological sections revealed the occurrence of cestode larvae of the genus Tylocephalum (Fig. 3). They were encapsulated by a fibrous cyst wall in the connective tissue around the digestive gland, with a prevalence of 60% and 52.3%, and a moderate intensity of infection. The cyst wall is comprised of an innermost layer of dense eosinophilic fibroblastlike cells. Its outer layer is composed of loose eosinophilic fiber cells intermingled with leukocytes. No evidence of a significant pathological impact has been observed.
Encysted metacercariae of digenean trematodes were found in the connective tissues of the mantle at both sites throughout the sampling period. These parasites did not evoke any host response and did not complete their life cycle inside the oysters. A high prevalence of infection (37.80%) was found at site 1, and a 22.45% prevalence was noted at site 2.
Ciliates were observed in gills and/or the lumina of digestive diverticula of oysters from both sites. They were associated with, but did not seem to be attached to, the gill filaments of the host oyster. Prevalence was 18.75% at site 1 and 13% at site 2. No pathological changes were observed. From observation of the histological section, the length was estimated to range from 15-25 [micro]m (n = 30) and the ciliates were assumed to belong to the family Ancistrocomidae.
The slipper-cupped oyster exhibited a high prevalence of Nematopsis sp. at both collection sites; however, infections were light and did not cause any pathological damage to their hosts. Several authors (Azevedo & Cachola 1992, Winstead et al. 2004, Cremonte et al. 2005) reported that protozoans of the genus Nematopsis (Schneider, 1892) have been found in many marine bivalves, being observed in the gills and mantle of molluscs (Bower et al. 1994). Gregarines of the genus Nematopsis use marine bivalves as normal intermediate hosts, completing their life cycle in the gut of marine arthropods, and are usually associated with a focal hemocyte infiltration, without measurable effects on host health (Lauckner 1983). In the current study we observed that infected oysters did not present any damage in tissues or organs.
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No histopathological sign of focal host reaction was observed in oysters with a high number of Nematopsis sp. oocysts in this study. In contrast, Carballal et al. (2001) observed alterations of gill filaments in C. edule induced by Nematopsis sp., whereas absence of host reaction by gregarines of the genus Nematopsis in the gut epithelial cells was reported by Winstead et al. (2004) in C. virginica and by Cremonte et al. (2005) in Pitar rostrata.
Oocysts of Nematopsis sp. were present in a variable number per host cell, usually 1-2, with each oocyst containing a single uninucleate sporozoite. This was also observed by Azevedo & Matos (1999) in Mytella guyanensis. In addition, the prevalence of Nematopsis species varied among bivalve hosts in different geographic areas (Azevedo & Cachola 1992).
Larval cestodes, Tylocephalum sp., infected oysters found at both sites, causing a host response consisting of fibrous encapsulation and hemocytic infiltration. This was also observed by Lauckner (1983), Sparks (1985), and Winstead et al. (2004) in C. virginica. In addition, Hine and Thorne (2000) recorded that larval encapsulation of Tylocephalum sp. by fiber cells is a host response in the genus Pinctada.
Metacestodes of Tylocephalum sp. are common parasites in marine molluscs worldwide (Cake & Menzel 1980, Lauckner 1983, Sparks 1985). These parasites occur as adults in the digestive tract of elasmobranchs (Sindermann 1990). The elasmobranchs are the definitive hosts of Tylocephalum sp. Hence, there is a greater number of metacestodes found in oysters from the wild, rather than in hatchery-reared oysters. This probably reflects the closeness of wild oysters to definitive hosts (Butler 1987).
In this survey, there was no mortality associated with the trematode infection in oysters. Esch et al. (2001) reported that trematodes used molluscs as an obligatory first intermediate host in their life cycle. It should be also noted that larval trematodes are the main metazoan parasites of bivalves, although they have mostly no negative effect on their hosts (Lauckner 1983, Bower et al. 1994).
In the oysters examined at 2 sites in Capiz, the prevalence and intensity of ciliates were low, and no pathological changes were observed on the gills or lumina of digestive diverticula. Ciliates were considered ubiquitous and not of regulatory significance according to Moret et al. (1999).
This parasitological survey found a number of less pathogenically significant parasites, but further research will be carried out. This initial survey will be useful for planning an ongoing health monitoring program for the slipper-cupped oyster.
We acknowledge the Government of Japan Trust Fund (study code: 8001-T-FD-FH-0307). We thank Milagros Castanos for editing the manuscript and give special thanks to Fely Torreta for preparing the histological slides.
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GREGORIA ERAZO-PAGADOR *
Southeast Asian Fisheries Development Center, Aquaculture Department Tigbauan, Iloilo, 5021 Philippines
* Corresponding author: E-mail: firstname.lastname@example.org
TABLE 1. Prevalence and intensity of the different parasites in slipper-cupped oysters. Site Parasite Prevalence (%) Intensity Site l: Barangay, Nematopsis sp. 71.33 4 Cabugao; n = 150 Tylocephalum sp. 60.00 3 Ciliates 18.75 1 Trematodes 37.80 1 Site 2: Barangay, Nematopsis sp. 65.00 4 Basiao; n = 150 Tylocephalum sp. 52.30 3 Ciliates 13.00 1 Trematodes 22.45 1