Parasitic and symbiotic fauna in oysters (Crassostrea virginica) collected from the Caloosahatchee River and Estuary in Florida.

ABSTRACT Studies of oysters, Crassostrea virginica, collected from 10 sites in the Caloosahatchee River and Estuary, Florida, revealed a varied parasite and symbiotic faunae that have never been reported in this area. Organisms observed included ovacystis virus infecting gametes at four sites (prevalence <1%), ciliate protozoans Ancistrocoma sp. in the gut of a stressed oyster at one site and Sphenophrya sp. infecting the gills of animals at three sites (prevalence <1%). The gregarine protozoan Nematopsis was found at all 10 sites (prevalence 24% to 90%) and oysters at some sites had concurrent infections of Nematopsis prytherchi and Nematopsis ostrearum in connective tissue near the stomach, mantle, and gills. Light to moderate infestations of hydrozoan polyps of a species in the genus Eutima were observed in the gills of oysters at all sites (prevalence 1% to 22%). Helminths included an unidentified turbellarian (prevalence 1% to 4%) observed at three sites and the digenetic trematodes Echinostoma sp., Proctoeces maculatus and Bucephalus sp. in oysters at five (prevalence 1% to 93%), three (prevalence >1%), and six (prevalence 1% to 3%) sites respectively. The first two trematodes were found infesting the gonoducts of their hosts while sporocysts of Bucephalus sp. infected connective tissues and gonads. Metacestodes of a species in the genus Tylocephalum were found in vesicular connective tissues near the gut, mantle, and in the gills of animals at all sites (prevalence 7% to 58%). Many sites had oysters with multiple infestations/infections of the above organisms indicating a rich biotic diversity, especially at those sites least impacted by human activity.

KEY WORDS: oysters, parasite, symbiont, prevalence, diversity

INTRODUCTION

Eastern oysters (Crassostrea virginica) are highly valued as food, but their ecological significance is under-appreciated and under-studied (Coen et al. 1999a). The oyster's ability to form large biogenic reefs (Coen et al. 1999b) qualifies it as a keystone or valued ecosystem component (VEC) species. VEC species are those that sustain the ecological structure and function of dominant estuarine communities. These species provide not only food, but also the physical habitat used by other organisms for living space, refuge, and foraging sites (Volety et al. 2003a). The complex 3-dimensional reef structure it forms attracts numerous species of invertebrates and fishes. To date, over 300 species have been identified as depending, either directly or indirectly, on oyster reefs (Wells 1961, Crabtree & Dean 1982, Abbe & Breitburg 1992, Wenner et al. 1996, Coen et al. 1999a) and many of these organisms in turn serve as forage for important fishery species (Tabb & Manning 1961, Marshall 1958, Gilmore et al. 1983) and birds (Watts 1988). Using a suite of responses from oysters, studies of the effects of heavy metals, organochlorine pesticides, rainfall, and freshwater releases into the Caloosahatchee Estuary from Lake Okeechobee on these animals were carried out. In addition, the effects of rainfall and freshwater releases on species abundance, richness, biomass, and diversity in Caloosahatchee oyster reefs were investigated. Data obtained from these studies will be used to assess the anthropogenic impact on the estuary and to provide target conditions for watershed management (Volety et al. 2003a, 2003b). One aspect of these studies, histologic examination of oysters, revealed a number of parasites and symbionts living in and on collected animals. Parasites have been used as an indicator species in environmental assessments for some time. There have been a number of investigations on the effects of pollution and generalized human disturbance on parasites of fishes and mollusks to indicate anthropogenic impacts (Moller 1987, Khan & Thulin 1991, MacKenzie et al. 1995, Kuris & Lafferty 1994, Lafferty 1997). The most common approach is to compare the prevalence or intensity of parasitism among hosts sampled from a number of control and impact sites or a single site before and after the impact (Lafferty 1997). The rational being that the diversity and presence of parasites, especially larval parasites and their intermediate hosts, can be potentially useful indicators of disturbances that reduce the abundance and diversity of vertebrates that act as the parasites' final host (Lafferty 1997). Thus, information regarding the presence or absence of parasites/symbionts in the Caloosahatchee estuary should be useful in assessing anthropogenic impacts when combined with all other aspects of the study. Therefore, the objective of this study is to report, for the first time, a survey of those parasites and symbionts observed in oysters from selected collection sites in the Caloosahatchee study area and briefly discuss how their presence or absence may indicate greater biologic diversity and a measure of ecosystem health.

MATERIALS AND METHODS

Sampling Locations

Oysters were examined from two projects being conducted within the same area of the Caloosahatchee estuary (Fig. 1) during a similar time period. The first project was designed to examine the effects of season and freshwater flow on the ecologic and physiologic processes of oysters as well as the habitat use of oyster reefs by fish and crustaceans. Monthly samples, 10 oysters per site (total 239 per site), were taken from 5 collection sites between September 2000 and December 2002. Sample sites included Piney Point (PP, 4 km upstream from the river mouth), Cattle Dock (CD, 2 km upstream from the river mouth), Bird Island (BI, 4km downstream from the river mouth), Kitchel Key (KK, 6 km downstream from the river mouth), and Tarpon Bay (TB, 12 km downstream from the river mouth). The second project was designed to examine the relationship between heavy metals, PCBs, and organochlorine pesticides measured in water and oyster tissues with the physiologic and ecologic responses of sampled oysters. Samples, 10 per site (total 78 to 90 per site), were taken from 5 sites every other month between June 2001 and December 2002. Sample sites included Iona Cove (IC, 4 km upstream from the river mouth), Tarpon Point (TP, 1.5 km upstream from the river mouth), Shell Point (SP, 1.75 km upstream from the fiver mouth), Port Sanibel (PS, 5 km downstream from the river mouth), and Greg's Reef (RS, 6 km downstream from the river mouth).

[FIGURE 1 OMITTED]

Histologic Analysis

Oysters sampled for histology were shucked and transverse whole-body sections (1-2 cm) were cut from immediately behind the labial palps to obtain a representative area of mantle, gills, gonadal, and digestive organs. These tissues were fixed in Davidson's fixative (Shaw & Battle, 1957) and prepared for paraffin sectioning. Sections (7 [micro]m) were stained with Harris' hematoxylin and eosin and examined via light microscopy for parasites and gonadal reproductive stages.

Data concerning the protist parasite Perkinsus marinus and relationships of agents described in this study with the compounds and environmental factors that were measured in the second study will be presented in more detail in a subsequent manuscript.

Definition of Specific Terms

Infection/infestation: "an infection is the invasion or state resulting from an invasion by a parasite or a pathogen into a host. An infestation is an external rather than an internal invasion. If both internal and external organisms occur on one host, all the associations are collectively referred to as infections. Neither an infection nor an infestation necessarily imply disease" (Overstreet 1978).

RESULTS

A polyoma-like virus was found in gametes (eggs) of oysters from Piney Point, Kitchel Key, Tarpon Bay and Port Sanibel stations (Table 1). The viral infection produced abnormal hypertrophied, basophilic ova ranging up to 250 [micro]m in diameter with scant cytoplasm (Fig. 2). Pathologic response to the condition was limited to a light to moderate hemocytic infiltration that may have been in response to lysis of affected ova. Prevalence was low (<1%) at all stations where the virus was found (Table 1).

[FIGURE 2 OMITTED]

A ciliate, Ancistrocoma sp., was found attached to the epithelium and free in the lumen of digestive diverticulum in one oyster from the Cattle Dock Station (Table 1). This oyster appeared to be undergoing physiologic stress from Perkinsus marinus infection with a significant hemocytic response to the Perkinsus and an epithelial lesion in the stomach (Fig. 3). Another larger basophilic ciliate, Sphenophrya sp., was found attached to gills of animals collected from Bird Island, Kitchel Key, and Tarpon Bay. This ciliate formed colonies sufficient enough to cause xenomas in all affected oysters (Fig. 4). Prevalence was low at all stations (<1%) and besides the obvious xenomas, no pathologic damage was observed.

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Apicomplexan oocysts, Nematopsis sp, (Fig. 5) were found in connective tissue of the gills, mantle and digestive diverticulum at all collection sites sampled (Table 1). In some cases, oocysts found in the gills were larger than those in the mantle and digestive diverticulum of the oyster (19 x 15 [micro]m vs. 13.4 x 10 [micro]m respectively). Infections were light for the most part and no pathologic damage, other than tissue space taken up by the organism, was observed. Average prevalence was greatest at Tarpon Bay (92%, range 70% to 100%) and Piney Point stations (90%, range 20% to 100%) and lowest at Iona Cove (24%, range 0% to 70%).

[FIGURE 5 OMITTED]

Hydrozoan polyps of a species in the genus Eutima sp. (Fig. 6) were found infesting the gills of oysters at all collecting sites (Table 1). Polyps were attached to gill epithelium and most infestations appeared to be light to heavy (3-5 animals per histologic section). The hydroid's pedal disc did not appear to penetrate the oysters epithelium and no observable pathology was detected beyond possible obstruction of water flow to the gills. Highest prevalence was seen at Cattle Dock (22%, range 0% to 80%) and Shell Point (16%, range 0% to 80%)) whereas Kitchel Key and Tarpon Bay had the lowest numbers (<1%).

[FIGURE 6 OMITTED]

An unidentified turbellarian (Fig. 7) was found infesting the gills of oysters at three stations (Table 1). Worms were closely associated with gill epithelia and infestations appeared to be light (1-3 worms per histologic section). These organisms had an elongate, ciliated body 0.081 to 0.162 mm (mean, 0.107) wide by 0.112 to 0.25 mm (mean, 0.17) long. The worms illicited a moderate hemocytic response in the oysters' gills and many became engulfed in copious mucous secreted from and surrounding the worm (see Fig. 7). Prevalence was greatest at Bird Island (4%) whereas Kitchel Key and Tarpon Bay were 2.5 and 1%, respectively (Table 1).

[FIGURE 7 OMITTED]

Large numbers of encysted metacercaria of a digenetic trematode species in the genus Echinostoma (Fig. 8) were found encysted in the gonoducts of oysters from five collection sites (Table 1). Morphologic features are similar to trematodes reported in oysters (C. virginica) from Tampa Bay, Florida (Fisher et al. 1996, Winstead et al. 1998). The helminths did not appear to cause any tissue damage but localized massive hemocytic responses to necrotic worms were quite evident (see Fig. 8). Average prevalence was greatest at Tarpon Bay (93%, range 20% to 100%) and Greg's Reef sites (56%, range 0% to 100%)) and lowest (of those collection sites with the parasite) at Kitchel Key (1%, range 0% to 10%).

[FIGURE 8 OMITTED]

Unencysted metacercarial stages of another digenetic trematode species in the genus Proctoeces (Fig. 9) were found in the gonadal follicles and gonoducts of male and female oysters from Shell Point, Port Sanibel and Cattle Dock sites (Table 1). General morphologic features of the sectioned trematode are similar to Proctoeces maculatus described in oysters, C. virginica, from northern Gulf coast estuaries (Winstead & Couch 1981). Oysters showed no significant hemocytic or other cellular reaction to the worm. Also, even though the observed worms were surrounded by oyster gametes (see Fig. 9), there was no evidence of gonadal impairment to the oyster.

[FIGURE 9 OMITTED]

Sporocysts of a species of digenetic trematode in the genus Bucephalus (Fig. 10) were found in vesicular connective tissue of gills, palps, digestive diverticula, and in the gonads of male and female oysters from Piney Point, Bird Island, Kitchel Key, Iona Cove, Tarpon Point, and Port Sanibel (Table 1). Sporocysts contained numerous germ balls and developing cercaria (see Fig. 10). Many animals had heavy infections in which branching sporocysts of the parasite had completely destroyed the gonads by mechanical damage and physiologic decomposition of gametes. There was little host response to the presence of the worm except in instances where hemocytic reaction to dead or degenerate sporocysts was evident. Piney Point had the greatest prevalence (3%) whereas the other collection sites, where the parasite was observed, showed less than 1% infection.

[FIGURE 10 OMITTED]

Metacestodes of a species of cestode in the genus Tylocephalure (Fig. 11) were observed infecting vesicular connective tissue in gills, digestive diverticula, and stomach at all collection sites (Table 1). Larval worms elicited a host response, which included a thick encapsulation consisting of a fibrocyte-like inner layer and an outer hemocytic response that appeared more severe around those worms closest to the stomach (see Fig. 11). Some of the parasites appeared to be undergoing resorption by the host. Also, many oysters had multiple infections of the parasite (see Fig. 11). Average prevalence of the worm was greatest at Tarpon Bay (58%, range 30% to 90%) and Greg's Reef (53%, range 20% to 90%) whereas Iona Cove (8%, range 0% to 30%) and Shell Point (7%, range 0% to 20%) had the lowest.

[FIGURE 11 OMITTED]

Concurrent infestations in many oysters by two to five of the parasites described earlier in the discussion were observed at all collection sites. This was especially evident at Tarpon Bay and Gregs Reef (Table 1) where many oysters had Nematopsis sp., Tylocephalum and Echinostoma sp. and some at Tarpon Bay were also infested with turbellarians and hydroid polyps.

DISCUSSION

The polyoma-like virus infecting the gametes of some Caloosahatchee oysters caused massive hypertrophy of infected eggs but, other than a light to moderate hemocytic infiltration into localized areas where infected eggs were, did not appear to cause any significant pathologic response. This condition has been described in C. virginica from the Piscataqua River, Maine (Farley 1976) Long Island, New York (Meyers 1981) and the northeastern Gulf of Mexico (Winstead et al. 1998, Winstead & Courtney 2003). Similar lesions have been reported in C. gigas, C. commercialis, Ostrea lurida, and O. edulis (Farley 1978, Lauckner 1983). The condition has been termed "ovacystis disease" and is characterized by massive hypertrophy of ova and gametocytes, which contain Feulgenpositive (DNA) granular masses in the nucleus (Farley 1976, Winstead & Courtney 2003). Whereas normal ova reach a maximum of 75 [micro]m in diameter (Galtsoff 1964), hypertrophied ova can reach up to 500 ixm in diameter. Electron microscopy in the earlier cases revealed the presence of viral-like particles within the nuclei of hypertrophied cells. Prevalence of the condition was low and oysters showed little pathologic effects as was the case in the Caloosahatchee oysters.

The Ancistrocomid ciliates found in the gut of one oyster at Cattle Dock Station were probably responding to the injured oyster as secondary invaders rather than as primary pathogens. The lesion in the oyster's gut concomitant with the heavy infection of P. marinus probably weakened the oyster and made it more susceptible to these invaders. Ancistrocomid ciliates have been reported in numerous bivalves from the Atlantic, Pacific, and Gulf coasts (Lauckner 1983). In the Gulf of Mexico, what were probably ancistrocomid ciliates have been reported infecting the gut and digestive diverticula in oysters from Pascagoula Bay, Mississippi, Mobile Bay, Alabama, Pensacola Bay, Florida (Couch 1985), and Louisiana estuaries (Mackin 1962, Gauthier et ah 1990). Couch (1985) and Gauthier et al. (1990) reported little pathology associated with these infections. However, Mackin (1962) concluded that these ciliates may become a complicating factor when oysters are infected with Dermocystidium marinum (= Perkinsus marinus) and Burton (1963) reported ancistrocomid ciliates associated with an abnormal histologic appearance of the host's tissues and unusual concentrations of hemocytes in "gaper" oysters from Chesapeake Bay, Maryland. The condition of the Caloosahatchee oyster, Perkinsus infection with massive hemocytic response to the parasite and an injury, appear similar to those in oysters described by Mackin (1962) and Burton (1963). This would lend credence to Lauckner's (1983) conclusions that ancistrocomids, normally gill parasites that cause little pathology to their host, in some cases become opportunistic and invade the host's tissues under abnormal conditions of physiologic stress.

Sphenophrya sp., ciliates induced the formation of xenomas in the gills of affected oysters. Some oysters had multiple infection sites in their gills. Infected epithelial cells contained anywhere from 3 to 4 to hundreds of ciliate nuclei per site. Aside from the possibility of occluding water tubules in the gills, no observable pathology was detected. Sphenophrya-like ciliates have been reported in the gills of C. virginica from the Atlantic coast (Newman 1971, Otto et al. 1979, Meyers 1981), and in the Gulf of Mexico, similar ciliates have been observed infecting the gills in oysters from Pascagoula Bay, Mississippi, Mobile Bay, Alabama, Pensacola Bay, Florida (Couch 1985), and coastal Louisiana (Gauthier et al. 1990). Sphenophryids are unique because when they occur in large numbers they can induce a distinctive lesion known as a xenoma (Weissenberg 1922). This condition ensues after one of the organisms enters a host cell and proliferates, stimulating the host cell to undergo repeated karyokinesis and massive hypertrophy to accommodate the numerous ciliates contained therein. Xenomas range from the size of a single cell to a macroscopic cell about 3 mm long (Otto et al. 1979) and, as discussed, this condition was observed in all the Callosahatchee oysters found to be infected with the ciliate. The occurrence of these organisms, whether a single sphenophryid or in xenoma form, has not been reported to cause distress to infected oysters (Lauckner 1983).

The Caloosahatchee oysters exhibited a high prevalence of Nematopsis from most collection sites. Most infections were light and, aside from taking up tissue space in the gills or visceral mass, did not appear to cause any pathologic damage to their hosts. Many oysters had two different sizes of oocysts located either in vesicular connective tissue around the stomach and mantle or in the gills. Gregarines of the genus Nematopsis use C. virginica as a normal intermediate host and are quite common in many different species of bivalves along the Pacific, Atlantic, and Gulf coasts (see review by Lauckner 1983). More recent studies have reported the parasite in oysters from Pascagoula Bay, Mississippi; Mobile Bay, Alabama; Pensacola Bay, Florida (Couch 1985); coastal Louisiana (Gauthier et ah 1990); Apalachacola Bay, Florida (Fisher et al. 1996); and Tampa Bay, Florida (Fisher et al. 2000). The presence of different size oocysts parasitizing a number of the same oysters at different sites probably indicates a concurrent infection by two different species of this protist. The smaller size oocysts from the Caloosahatchee oysters are probably those of N. ostrearum because they are both similar in size (~10 x 14 [micro]m) and are most conspicuous in the mantle and in connective tissue around the digestive diverticula, where N. ostrearum are generally found (Sprague 1949, Lauckner 1983). Also, numerous mud crabs Panopeus sp. and Eurypanopeus depressus, which are the definitive hosts for this parasite (Lauckner 1983), were abundant at all study sites where the protist was observed (Volety et al. 2003a). The larger oocysts are probably those of Nematopsis prytherchi because they are similar in size (~19 x 16 [micro]m), occur in the gills, and have been reported to occur concurrently with N. ostrearum in C. virginica (Sprague 1949, Lauckner 1983). The definitive host for N. prytherchi is the stone crab Menippe mercenaria (Sprague 1949) which were also found at the Caloosahatchee sites sampled (Volety et al. 2003a). Reports concerning the pathogenicity of Nematopsis infections in C. virginica are inconclusive but most researchers have found no evidence to indicate the parasite is harmful to oysters (Overstreet 1978, Lauckner 1983).

The cnidarians observed infesting the gills of Caloosahatchee oysters did not seem to cause any pathologic distress to the hosts, even though large numbers of the symbiont were present in many of the collected animals. Several species of warm water hydrozoan cnidarians inhabit body folds of bivalve molluscs as inquiline symbiotes. Of these, only one North American genus, Eutima, is found in C. virginica attached to the oysters gills, mantle and palps (Mulholland & Fredl 1996, Samler 2001). Those researchers collected oysters from numerous sites along the southern Atlantic and Gulf coasts and observed hydroids only in Florida from Tampa Bay and St. Augustine southward (Samler 2001). Thus, it is probable that the hydroids observed in the Caloosahatchee oysters belong in this group. The light to heavy infestations with no observable pathology detected is not unusual because prior studies of these hydozoans reported that oysters can tolerate thousands of polyps without apparent harm (Samler 2001). Infestations appeared to have a seasonality because hydroids at those collection sites with the heaviest prevalence were observed in spring and early summer months (February to June) whereas almost none were detected in midsummer through fall. The summer decline was similar to other closely related bivalve-inhabiting cnidarians, Eugymnanthea inquilina, observed in mussels and clams from the Ionian Sea, Italy (Piraino et ah 1994) and Japanese waters (Kubota 1983). Kubota (1983) reported a summer decline in hydroids from Japanese waters noting that polyps became very small, and often degenerated, at the end of medusoid production. In addition, Samlet (2001) reported hydroid numbers in Florida waters declined rapidly following large drops in temperature or salinity after heavy summer rains. Experiments determined critical temperatures and salinities for these hydroids to be between 10 and 33[degrees]C and 8[per thousand] to 34[per thousand] respectively (Samler 2001). A drop in salinity occurred in the Caloosahatchee study sites during the summer months of the study. Salinities, which averaged 31[per thousand] during most of the study, dropped to under 10[per thousand] at most sites and to less than 5[per thousand] for weeks at each occurrence, especially at Cattle Dock and those collection sites close to it, where the hydroids were most prevalent (Volety et ah 2003a, Volety et ah 2003b).

The turbellarians found infesting the gills of Caloosahatchee oysters elicited a light to moderate hemocytic response from the gills and formed a mucus coat around themselves. However, since the prevalence was low (1% to 4%) and the numbers of individuals infesting individual oysters were few, the worms are probably more commensal than parasitic. Turbellarians are predominantly free-living predators or scavengers but certain members of the order Alloeocoels are intimately associated with marine molluscs (Lauckner 1983). Alloeocoel turbellarians have been reported in the mantle cavity and on the gills of numerous bivalves in Europe, Japan, North America, and Mexico (Lauckner 1983, Fleming 1986, Brun et al. 1999, Aguirre-Macedo & Kennedy 1999). In the Gulf of Mexico, members of the genus Urastoma ("oyster gill worm") have been observed infesting the gills of C. virginica from Tabasco State, Mexico (Aguirre-Macedo & Kennedy 1999) and the Mississippi coast (Overstreet, personal communication). These organisms have been reported most frequently as commensals in bivalves, causing very little pathology and no mortalities (Robledo et al. 1994). Brun et al.(1999) have confirmed the turbellarian has a definite attraction to oyster mucus and shows a greater preference for oysters over M. edulis and M arenaria in Atlantic Canada. This lends credence to the hypothesis of Fleming (1986) that U. cyprinae is attracted to and probably feeds on mucus produced by the oyster's gills.

Previous researchers have suggested that the copious mucous secretions produced by the worms forms a cocoon-like envelope to protect it from unfavorable environmental conditions or the negative effects of the oysters' defensive mechanisms (Bataller et al. 1999, Bataller et al. 2003). The fact that mucus surrounding the helminths, in this survey, was full of oyster hemocytes supports a defensive role for this response because mucous secretions by these worms may immobilize or make it more difficult for oyster hemocytes to attack them. It is well known that mucous production in animals plays an important role in defense mechanisms. For example, mucous secretion in marine fish gills is their chief defense against gill parasites (Khan 1987, Lafferty 1997).

Although the worms did not appear to cause significant pathology in C. virginica, studies with mussels, Mytilus galloprovincialis, from Spain have shown that Urastoma cyprinae causes serious disruption of gill filaments and significant hemocytic infiltration in the affected areas where the worms were found. This physiologic effect combined with large numbers of the flatworm was believed to significantly reduce feeding capacity in infested hosts (Robledo et al. 1994, Brunet al. 1999).

Encysted digenetic trematode metacercaria found infesting the gonoducts of oysters, beyond taking up space and eliciting localized hemocytic responses to necrotic worms, caused no significant damage to the host. These worms belong to a large group of trematodes collectively known as echinostomes. Digenetic trematodes in the family Echinostomatidae use gastropod and bivalve molluscs as intermediate hosts and have a worldwide distribution (Lauckner 1983). Metacercarial stages of this worm have been reported in C. virginica along the Gulf Coast by Little et al. (1966, 1969), Winstead et al. (1998) and Fisher et al. (2000). This group of trematodes is recognizable by the presence of a collar of spines around the oral sucker (Cheng 1973). Little et al. (1966) reported the occurrence of encysted metacercariae of another echinostome, Acanthoparyphium spinulosum, in the mantle of up to 100% of C. virginica collected near Port Isabel, Texas and on the eastern Gulf coast; 90% of oysters from one Tampa Bay, Florida site were reported to harbor encysted metacercariae of an Echinostoma sp. in their gonoducts (Winstead et al. 1998, Fisher et al. 2000). The encysted worms observed in the Caloosahatchee oysters are probably in the genus Echinostoma also, because they have the same number of collar spines and were found in the gonadal follicles just like those reported from Tampa Bay. However, unlike the trematodes reported from Texas, the worms from Florida could not be identified to species because infected oyster tissue fed to chicks and ducklings did not develop into fully mature adults (Overstreet, pets. comm.). The metacercariae found in Florida and Texas oysters caused little observable tissue reaction. Even though cercariae of this group have penetration glands and have been reported encysted in the tissues of other marine molluscs (Stunkard 1937, Cheng et al. 1966) they did not penetrate the tissues of the Caloosahatchee oysters. Instead, the trematode only infested the gonadal follicles that are, in essence, an extension of the outer environment. As such, the oysters did not have tissue "infections" and probably did not respond to the parasite because the follicle epithelium was not breached or damaged. It is also possible that the cercariae were induced to encyst before penetrating tissue by the oyster's humoral defense mechanisms. Cheng et al. (1966) reported that sera from oysters C. virginica and C. gigas could immobilize cercariae of another echinostome, Himasthla quissetensis, before they could penetrate their tissues. However, the oysters do respond to dead or dying worms with massive hemocytic reactions (Winstead et al. 1998, Fisher et al. 2000). Oysters, infested with parasites, kept in laboratory aquaria for 2 mo in Texas (Little et al. 1969) and 1 y in Florida (Winstead et al. 1998) contained up to 70% living encysted trematodes, which may indicate the oysters did not detect the worms presence and therefore did not respond. The common life cycle of these parasites involve birds, humans, and other mammals as definitive hosts and a gastropod snail as the first intermediate host. Free swimming cercariae from the snail penetrate and encyst in various bivalves or fish. These second intermediate hosts are then consumed by the definitive host to complete the life cycle (Cheng 1973). There did not appear to be any seasonality of infestation because oysters from Tarpon Bay were observed to have the parasite all months of the year.

Another trematode, Proctoeces sp., infesting the gonoducts of the Caloosahatchee oysters were unencysted, caused no pathology, occurred at low prevalences, and were associated with attached hooked mussels Ischadium recurvum (Volety, pers. comm.). Unencysted metacercariae of the fellodistomatid trematode Proctoeces maculatus have been reported in bivalves from Europe and the Atlantic coast of North America (Stunkard & Uzmann 1959, Lauckner 1983). In the Gulf of Mexico the worm has been reported in the gonadal follicles of C. virginica from Pensacola Bay, Florida; Mobile Bay, Alabama; and Pascagoula Bay, Mississippi (Winstead & Couch 1981, Couch 1985). The occurrence of unencysted trematode metacercariae in these oysters indicates the worms may be "progenetic" or precocious last-larval stages. The term "progenetic" is applied to sexually mature, ovigerous digenetic trematodes occurring in invertebrates (Aitken-Ander & Levin 1985) which, instead of using a definitive vertebrate host, can abbreviate its life cycle by maturing and completing its life cycle in a single molluscan host. This characteristic of the genus Proctoeces (i.e., to be in different stages within a mussel) is well documented and discussed by Cheng (1967). The presence of attached hooked mussels on the Caloosahatchee oysters is significant because sporocysts, cercariae and, progenetic stages of Proctoeces have been found in hooked mussels from Louisiana (Hopkins 1954a), Mississippi (Overstreet, pers. comm.), and Texas estuaries (Wardle 1980). It is possible that the attached mussels on the Caloosahatchee oysters are the source of the trematode and the worm exits the mussel as a cercariae, becoming available to either a definitive fish host or another mussel, and contacting neither, enters the oysters genital orifice by chance. From there it migrates to a gonadal follicle or duct and may either mature and complete its life cycle using the bivalves as surrogate hosts or remain in a metacercariae stage awaiting a mollusc-eating fish (Sparidae or Labridae). It is pertinent to note that pinfish, Lagodon rhomboides, and sheepshead, Archosargus probatocephalus, were both collected in the vicinity of sampling sites (Volety et al. 2003a). Seasonality of infestation could not be determined due to the low prevalence (<1%) of the trematode.

Branching sporocysts from the trematode Bucephalus sp. invaded vesicular connective tissue throughout the oyster and in heavy infections completely destroyed or "castrated" the gonads via mechanical damage. Digenetic trematode infections by the genus Bucephalus or "bucephalosis" in C. virginica is known to extend from Rhode Island to Texas, but is not continuous throughout this range (Hopkins 1954b, Sparks 1985). Bucephalus infections in Gulf coast oysters have been reported from Louisiana estuaries (Hopkins 1954a, Hopkins 1957, Menzel & Hopkins 1955, Turner 1985, Gauthier et al. 1990), Pensacola Bay, Florida (Winstead, personal observation); Apalachicola Bay, Florida (Menzel et al. 1969); and Tampa Bay, Florida (Fisher et al. 2000). Pathology of Bucephalus infections in the Caloosahatchee oysters is identical to that described in earlier studies. Trematode sporocysts invade the gonad and, via mechanical pressure, totally disrupt it's integrity. Any remaining gametes become degenerate and reduced in size, more by metabolic rather than mechanical damage, resulting in parasitic castration (Sparks 1985). In heavy infections growing sporocysts invade the connective tissue surrounding the digestive diverticula, obliterating the space they normally occupy. Later, sporocysts replace connective tissue in the mantle and invade water tubes of gill lamellae (Sparks 1985). Hemocytic response from the oyster is minimal except when sporocysts become degenerate or die. The end result is almost total replacement of oyster tissue by sporocysts, which in late stage infections probably lead to the death of the oyster, especially in adverse environmental conditions (Hopkins 1957, Sparks 1985). Except for some Louisiana bayous in which Hopkins reported 10% to 25% of oysters with Bucephalus infections, all the above studies reported low prevalence of infections (<2%) and the Caloosahatchee oysters were no exception with most sites around 1% and one with 3%. It is pertinent to note that prevalence of Bucephalus infections has been reported to be highest in low salinity and lowest in open bays with higher salinity (Hopkins 1957, Sparks 1985). This seems to be the case with the Caloosahatchee oysters; all sites with the infection had lower salinity conditions and higher salinity sites had no infections. This correlation of bucephalosis with lower salinity is probably due to the presumed definitive and second intermediate hosts (the true gar, Lepisosteus and mullet, Mugil cephalus) preference for lower salinity waters. There did not appear to be a seasonality of infection for any sites where the parasite was found.

Larval cestodes, Tylocephalum sp., infected oysters at high prevalences at most stations and caused a severe host response consisting of fibrous encapsulation and hemocytic infiltration to resorb the parasite. Host response was in accordance with the observations of Lauckner (1983) and Sparks (1985). Many of the Caloosahatchee oysters had multiple infections of the parasite. Of 60 infected C. virginica inspected by Cake and Menzel (1980), some had as many as 125 worms in their tissues but none were weak or moribund or showed any loss of body weight (Lauckner 1983). The location of encapsulated worms varied but in general they were found in loose connective tissue between the digestive diverticula and the stomach and less frequently in the connective tissue of the gills and mantle. The most common route of infection is through the stomach epithelium and there is no appreciable host response until the cestode gets into the vesicular connective tissue around the stomach, mantle or in the gills (Lauckner 1983). After the parasite gets through the stomach epithelium, connective tissue fibers begin to form around it and capsule formation begins. The end result is a thick intercellular cyst of fibrous material between the vesicular connective tissue and a hemocytic infiltration (Lauckner 1983). Encapsulated worms are gradually resorbed by oyster hemocytes, which probably means that C. virginica is not a totally compatible host for Tylocephalum (Cheng 1967). The resorption of Tylocephalum metacestode larvae has also been reported in other bivalves with the exception of the pearl oyster Pinctada spp., which is considered to be the normal intermediate host for Tylocephalum sp. (Lauckner 1983).

Tylocephalum sp. acudate glando-procercoids or metacestodes are common parasites in marine mollusks throughout the world (Cake & Menzel 1980, Lauckner 1983, Sparks 1985) and have been reported in C. virginica from numerous sites along the eastern Gulf of Mexico (Sparks 1963, Cake 1977, Cake & Menzel 1980, Couch 1985, Fisher et al. 1996, Fisher et al. 2000). Cake and Menzel (1980) reported metacestodes in oysters sampled from 22 of 27 sites between Bay St. Louis, Mississippi and Everglades City, Florida. They reported an average prevalence of 43.5% (range 0% to 100%) infection, which is similar to what other researchers have observed in Gulf coast oysters. Also, it was not unusual to find 100% of oysters infected with the parasite at some sites. The Caloosahatchee oysters also had metacestode infections at all sites and some, especially the higher salinity sites at Tarpon Bay and Gregs Reef, had prevalences of up to 90%.

The fact that many individual oysters at the Tarpon Bay and Greg's Reef sites harbored multiple species of parasites indicates a fairly rich diversity of organisms in those areas compared with the other sites. It is pertinent that those sites, where fewer parasites were observed, were closer to habitat that has been severely altered by human activity (freshwater release from Lake Okeechobee, pollution and shoreline disruption). Habitat alterations can lead to decreases in parasitism of a population by eliminating the intermediate and/or definitive hosts the parasites need to complete their life cycles (MacKenzie et al. 1995, Lafferty 1997, Lafferty & Kuris 1999). For example, Burn (1980) observed reduced diversity of species and parasite numbers in fish from Raritan Bay, which was heavily impacted by human activity, compared with Block Island Sound which was less polluted. Snails, Cerithidea californica, separated from a marsh by a parking lot and highway were completely uninfected with larval trematodes compared with a 25% prevalence in snails inhabiting the adjacent marsh. The definitive hosts for these, worms and birds, were absent in the cut off marsh area because of the habitat alteration (Lafferty 1997). Thus, a paucity or abundance of parasites, especially helminths, in a target population, in this case oysters, may be an indirect indicator of the biotic diversity in that area. If a host population harbors a large number and variety of parasites, especially metazoan parasites, it is probable that a rich assortment of free living fauna is living in or moving through that area (Cheng 1973). It is important to note, however, that there are many instances where total numbers of the same species of parasite may increase due to human activities, such as pollution (MacKenzie et al. 1995, Lafferty 1997). For example, increased Trichodinid gill ciliates in fish seems to be correlated with a toxicant's ability to impair mucus production, which is a fishes chief defense against those parasites (Khan 1987, MacKenzie et al. 1995). Therefore, as Lafferty (1997) states "it is important to take into account the relative sensitivities of each group and have a through understanding of the associations between the wide range of parasites and the impacts upon them" before one correlates anthropogenic impacts with biotic responses.

TABLE 1.

Prevalence of parasites and symbionts in oysters (Crassostrea
virginica) collected from 10 stations within the Caloosahatchee River
/Estuary study area. Values in parentheses represent prevalence (% of
parasite/symbiont presence).

                         Piney             Cattle            Bird
Parasite/Symbionte       Point             Dock             Island

Ovacystis virus        1/239 (0.4%)      0/239 (0%)        0/239 (0%)
Ancistrocorna sp.      0/239 (0%)        1/239 (0.4%)      0/239 (0%)
Sphenophrya sp.        0/239 (0%)        0/239 (0%)        1/239 (0.4%)
Nematopsis spp.       214/23 9         168/239 (70%)     158/239 (66%)
Eutima sp.            10/239 (4%)       52/239 (22%)       8/239 (3%)
Turbellaria            0/239 (0%)        0/239 (0%)       10/239 (4%)
Echinostonta sp.       0/239 (0%)        6/239 (3%)       30/239 (13%)
Proctoeces sp.         0/239 (0%)        1/239 (0.4%)      0/239 (0%)
Bucepholus sp.         8/239 (3%)        0/239 (0%)        2/239 (1%)
Tylocephalum sp.      74/239 (31%)      72/239 (30%)     115/239 (48%)

                         Kitchel            Tarpon            Iona
Parasite/Symbionte         Key               Bay              Cove

Ovacystis virus         1/238 (0.4%)      1/238 (0.4%)     0/80 (0%)
Ancistrocorna sp.       0/238 (0%)        0/238 (0%)       0/80 (0%)
Sphenophrya sp.         1/238 (0.4%)      0/238 (0.4%)     0/80 (0%)
Nematopsis spp.       172/238 (72%)     218/238 (92%)     19/80 (24%)
Eutima sp.              1/238 (0.4%)      3/238 (1%)       8/80 (10%)
Turbellaria             6/238 (2.5%)      3/228 (1%)       0/80 (0%)
Echinostonta sp.        3/238 (1%)      222/238 (93%)      0/80 (0%)
Proctoeces sp.          0/238 (0%)        0/238 (0%)       0/80 (0%)
Bucepholus sp.          2/238 (1%)        0/238 (0%)       1/80 (%)
Tylocephalum sp.       98/238 (41%)     138/238 (58%)      6/80 (8%)

                        Tarpon          Shell         Greg's
Parasite/Symbionte      Point           Point          Reef

Ovacystis virus        0/90 (0%)      0/90 (0%)      0/90 (0%)
Ancistrocorna sp.      0/90 (0%)      0/90 (0%)      0/90 (0%)
Sphenophrya sp.        0/90 (0%)      0/90 (0%)      0/90 (0%)
Nematopsis spp.       25/90 (28%)    53/90 (59%)    73/90 (81%)
Eutima sp.             8/90 (9%)     14/90 (16%)     3/90 (3%)
Turbellaria            0/90 (0%)      0/90 (0%)      0/90 (0%)
Echinostonta sp.       0/80 (0%)      0/80 (0%)     50/90 (56%)
Proctoeces sp.         0/90 (0%)      1/90 (0%)      0/90 (0%)
Bucepholus sp.         1/90 (1%)      0/90 (0%)      0/90 (0%)
Tylocephalum sp.      23/90 (26%)     6/90 (7%)     48/90 (53%)

                         Port
Parasite/Symbionte      Sanibel

Ovacystis virus        1/78 (1%)
Ancistrocorna sp.      0/78 (0%)
Sphenophrya sp.        0/78 (0%)
Nematopsis spp.       49/78 (63%)
Eutima sp.            12/78 (15%)
Turbellaria            0/78 (0%)
Echinostonta sp.       0/80 (0%)
Proctoeces sp.         1/78 (1%)
Bucepholus sp.         1/78 (1%)
Tylocephalum sp.      26/78 (33%)

ACKNOWLEDGMENTS

We thank Ms. Sharon Thurston, and Erin Rasnake for providing field assistance and Lee Courtney for providing help with figures. The authors also thank Dr. Robin Overstreet for his critical review of the manuscript.

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Funding for this study was made possible by grants from the South Florida Water Management District and the Charlotte Harbor National Estuary Program. Contribution No. 1206, of the Gulf Breeze Environmental Research Laboratory.

JAMES T. WINSTEAD, (1) ASWANI K. VOLETY (2) AND s. GREGORY TOLLEY (2)

(1) United States Environmental Protection Agency, 1 Sabine Island Dr., Gulf Breeze, Fl. 32561, (2) Florida Gulf Coast University, 10501 FGCU Blvd. South, Fort Myers, Fl. 33965

* Corresponding author. E-mail: [e-mail]