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Encapsulation of attached ectoparasitic glochidia larvae of freshwater mussels by epithelial tissue on fins of naive and resistant host fish.

Introduction

Embryonic and early development of freshwater unionoid mussels occurs within the gills of the parental mussel, which are modified to serve as brood chambers, or marsupia (Tankersley and Dimock, 1993; Schwartz and Dimock, 2001). Development culminates with the glochidium, a bivalved larval form with a single adductor muscle, sensory cells, larval mantle tissue, and the primordia for gills and other juvenile organs (Arey, 1921; Kat, 1984; Jeong et al., 1993; Pekkarinen, 1996; Fisher and Dimock, 2002). The glochidia ultimately are released from the parental mussel and, for most species, must temporarily become parasitic on fish (Arey, 1921, 1932a; Kat, 1984; Jansen et al., 2001; Wachtler et at., 2001). Once the glochidium has attached to external or branchial tissue of the fish, epidermal or branchial epithelial cells of the host ultimately encapsulate the larva, forming a cyst (Arey, 1921, 1932a, b; Fustish and Millemann, 1978; Karna and Millemann, 1978; Kat, 1984; Jeong, 1989; Waller and Mitchell, 1989; Nezlin et al., 1994; Wachtler et al., 2001). The glochidium metamorphoses into a juvenile within the cyst and subsequently emerges and assumes a benthic existence (Arey, 1932a; Kat, 1984; Wachtler et al, 2001; Fisher and Dimock, 2002).

The attachment of a glochidium to a fish host and the subsequent encapsulation of the larva occur quickly and represent a short but critical period in the life history of the mussel. According to previous studies, the cyst is composed of epithelial cells and connective tissue (Arey, 1932a, b; Waller and Mitchell, 1989). The ultrastructure of the cyst of glochidia attached to gill filaments has been described as being composed of epithelioid and necrotic cells. During cyst formation, the mitochondria of the cells become swollen, and chromatin becomes dense and sparse. These transformed epithelioid cells become necrotic and filled with connective fibers (Waller and Mitchell, 1989). Additionally, the host epidermal cells near and covering an attached glochidium were observed to have a wave-like appearance (Jeong, 1989; Nezlin et al., 1994), suggesting that the cyst is formed by migration of host cells. From the speed of the process and the few mitotic events observed in the area around the cyst, Arey (1932a) concluded that encapsulation does not involve proliferation of epithelial cells.

For cyst formation and subsequent metamorphosis of larvae into juveniles to be successful, the glochidia must attach to a suitable host fish, the specificity of which varies with the species of mussel (Zale and Neves, 1982a, b; Kat, 1984; Panha, 1993; Watters, 1994; Watters and O'Dee, 1996; O'Connell and Neves, 1999). If a glochidium attaches to a fish that is unsuitable as a host, an "abnormal" cyst may form. The formation of an abnormal cyst results in the sloughing off and death of the glochidia and is attributed to a putative natural immunity of the nonhost fish to the species of mussel (Fustish and Millemann, 1978; Karna and Millemann, 1978; Meyers et al., 1980; Waller and Mitchell, 1989).

There is increasing evidence that fish that are suitable hosts can develop resistance, or "immunity," to glochidia after multiple infections (Reuling, 1919; Arey, 1924, 1932b; Bauer and Vogel, 1987; Bauer, 1987; Claes, 1987, cited in Jansen et al., 2001; Watters and O'Dee, 1996; O'Connell and Neves, 1999; Rogers and Dimock, 2003; Dodd et al., 2005). However, the basis of this resistance is not fully understood (Rogers-Lowery, 2005). The formation of cysts on "immune" host fish was reported by Reuling (1919) and Arey (1924, 1932b), but no studies of cyst formation on resistant fish have been conducted using modern microscopy.

The goal of the current study was to compare the formation of cysts on naive and putatively resistant host fish, as well as on nonhost (unsuitable) fish, in an effort to determine potential mechanisms of host specificity and acquired resistance. The studies utilized glochidia of Utterbackia imbecillis, the paper pondshell, and, to a lesser extent, Strophitus undulatus, the creeper. The glochidia of U. imbecillis and S. undulatus have hooks on their shells that probably facilitate rapid attachment to the fins, gills, and other external surfaces of fish, where they become encapsulated and subsequently metamorphose into juveniles. We have previously shown that Lepomis macrochirus, the bluegill sunfish, develops resistance to glochidia of U. imbecillis after as few as two infections (Rogers and Dimock, 2003), and are poor hosts for S. undulatus (pers. obs.); thus this fish-mussel association is an appropriate model system to address the aforementioned objectives. In addition, encapsulation of glochidia on largemouth bass (Micropterus salmoides), goldfish (Carassius auratus), and catfish (Ictalurus punctatus) was examined to compare cyst formation on marginally suitable and nonhost fish.

Materials and Methods

Experimental organisms

Young-of-the-year specimens of Lepomis macrochirus were obtained from Foster Lake and Pond Management (Garner, Wake County, NC) and Windmill Fish Hatchery (Kernersville, Forsyth County, NC). Largemouth bass (Micropterus salmoides) were obtained from Foster Lake and Pond Management. Channel catfish (Ictalurus punctatus) were obtained from Blue Ridge Fish Hatchery (Kernersville, Forsyth County, NC), and goldfish (Carassius auratus) were obtained from retail stores. The fish were held in a temperature-controlled, filtered, recirculating aquarium system at 20-21 [degrees]C and 12h:12h light/dark photoperiod and fed commercial fish food daily (Rogers and Dimock, 2003). Since these fish had no previous exposure to mussels or glochidia of any species, they were defined as naive and held in the laboratory until use. Just prior to removal of fins for observation by time-lapse video microscopy and scanning electron microscopy (SEM), fish were anesthetized by exposing them to a solution of 100 mg/l MS-222 (tricaine; Western Chemical Inc., Ferndale, WA) for 2 min. Maintenance and manipulation of fish followed protocols approved by the Wake Forest University Animal Care and Use Committee (ACUC # A00-024 and A03-046).

Gravid Utterbackia imbecillis were collected from local ponds (in Forsyth, Rockingham, and Mecklenburg Counties, NC) as needed for experiments between November 2001 and October 2004. The mussels were maintained in 40-1 aquaria at seasonal temperatures and a natural light: dark cycle, and were fed at least every other day with an algal mixture. To procure glochidia from U. imbecillis, the outer (marsupial) demibranchs of gravid mussels were removed, teased open, and gently rinsed with a stream of water. The glochidia from two mussels were combined before exposure to fish. Gravid Strophitus undulatus were obtained from Beaver Creek (Taney County, MO) and shipped on ice to the laboratory at Wake Forest University. When warmed to room temperature, these mussels released conglutinates containing glochidia. The glochidia were separated from the matrix of the conglutinates by vigorous aeration in water. The glochidia from both species were rinsed twice with fresh water and maintained in aerated water until use.

Several experiments compared "naive" and "resistant" bluegill sunfish. Fish that had no prior exposure to glochidia of any species of mussel (i. e., hatchery-reared animals) were considered to be naive. Bluegill sunfish that were designated as resistant had previously received four infections with glochidia of U. imbecillis, over the course of 12 weeks a protocol that results in significant acquired resistance (Rogers and Dimock, 2003). Thus, in the current study, resistant fish were infected with glochidia for the fifth time.

Observation of cyst formation by time-lapse video microscopy

To follow the kinetics of cyst formation, encapsulation of glochidia by epithelial cells of fish was monitored with time-lapse video microscopy. Individual fish were placed in small containers and exposed to glochidia of U. imbecillis or S. undulatus by immersion in a vigorously aerated suspension of 100-150 larvae for 5 min. Within 10 min after initial exposure, pectoral fins, pelvic fins, or both, were removed from the fish and immediately transferred to fish Ringer's solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM Ca[Cl.sub.2], 5 mM HEPES) in a 35 X 10 mm petri dish (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ). To maintain a constant temperature during observation, the small dish was then placed in a 100 X 15 mm petri dish filled with water at room temperature. Using this setup, the temperature of the Ringer's solution in the small petri dish increased by [approximately equal to]1.5 [degrees]C over an 8-h period. Fin viability was monitored by evaluating the integrity of the epithelia; if there were signs of sloughing of cells, that fin was no longer considered viable. Fins appeared to remain viable for at least 8 h after excision, but collection of data was restricted to the first 6 h.

The fins were observed on a Nikon SMZ-2T stereomicroscope fitted with a Toshiba JK-M41A CCD color video camera connected to a Panasonic 6740 time lapse S-video cassette recorder. The analog S-VHS tape was converted to digital video using a Panasonic AG-DV2000 digital video cassette recorder. Digital images and movies were captured with Adobe Premier ver. 6.0 software running on an IBM ThinkPad A21m computer.

Observation of cyst morphology by scanning electron microscopy

To follow cyst formation beyond 6 h post-infection and to examine the morphology of cysts, fins with attached glochidia were prepared for observation by SEM. All fish were exposed to glochidia (2 ml packed volume of larvae in 21 of water) for 2 min as described for video recording. Fins were removed from naive (n=3) fish at 0.5, 1, 2, 3, 5, 6, 24, and 48 h post-infection and from resistant fish (n=3) at 0.5, 1, 2, 3, 5, and 6 h. Glochidia-infected pectoral and pelvic fins were dissected from lightly anesthetized fish and immediately transferred to fixative.

The fixation protocol was modified from Waller and Mitchell (1989) and Nezlin et al. (1994). Fins were initially fixed in 2.5% glutaraldehyde (Ted Pella, Redding, CA) and 2% paraformaldehyde (Sigma, St. Louis, MO) in 0.1 M cacodylate buffer (pH 7.2, 2 mM Ca[Cl.sub.2], 5.5% sucrose; Electron Microscopy Sciences, Fort Washington, PA), for 2 h and then rinsed in 0.1 M cacodylate buffer. Specimens were post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M cacodylate buffer for 3 h, followed by rinsing in fresh buffer. Following dehydration in a graded series to 100% ethanol, specimens were dried with a Pelco CPD-2 critical point dryer, mounted on aluminum stubs, and sputter-coated with gold-palladium using a Pelco SC-4 sputter coater. The specimens were examined on an Amray 1810 scanning electron microscope operated at 15 kV. All glochidia on the fins were examined and subjectively evaluated for the percentage of the glochidium covered by cyst tissue (% cover); scores were recorded as 0%, 1-25%, 26-50%, 51-75%, 76-99%, and 100%.

Results

Cyst formation on naive host fish

A series of still images from a representative time-lapse sequence show the progression of encapsulation of a glochidium of Ulterbackia imbecillis on a live fin of a bluegill (Fig. 1). Migration of fish epithelial cells was evident by 40 min post-infection, and the glochidium was completely encapsulated by host tissue by about 130 min after attachment. The time-lapse imaging also revealed that epithelial cells of the fin were in constant motion, even cells that were not immediately adjacent to the attached glochidia (see Video 1 and Video 2 at http://www.biolbull.org/supplemental/).

Cells of the epidermal epithelium at a sufficient distance to be unaffected by the process of cyst formation were roughly rectangular to hexagonal, with distinct sutures delineating adjacent cells (Fig. 2A). Small globular particles, presumably mucus, were observed in some of the sutures between adjacent cells. The exposed surfaces of the cells were covered by an elaborate system of microridges, forming a fingerprint-like pattern (Fig. 2A).

Formation of the cyst resulted from the migration of fish epithelial cells near the point of attachment of the glochidia, and the migration of host epidermal epithelium continued for several hours (Fig. 3). Cells at the leading edge of the migration were rounded and swollen (Fig. 2B), lacked the microridges of normal cells, and often formed nondescript masses (Fig. 2C). The cells advanced over the glochidia in an undulating, wavelike fashion (Fig. 2C). Some individual cells on or ahead of the leading edge of the migration had cytoplasmic extensions, or lamellipodia.

By 6 h post-attachment, most of the glochidia attached to fins on naive fish were completely covered by host epithelial cells (Fig. 3E), which had resumed the morphology of unaffected cells. Most 6-h cysts were not uniform in thickness, with many having thickened areas that may represent the meeting of the leading edges of tissue migration. Despite most glochidia on a fin being completely encapsulated by 6 h post-attachment on naive fish, cyst formation was not fully synchronous, and adjacent glochidia on the same fin were often at different stages in the process.

The observed times for completion of cyst formation differed between the two observation techniques. Glochidia on fins excised from fish immediately after exposure, incubated in fish Ringer's solution, and observed using time-lapse video microscopy were completely encapsulated in about 2 h after attachment (Fig. 1; Video 1 and Video 2 at http://www.biolbull.org/supplemental/). However, completion of cyst formation took 4-6 h on fins that remained attached to fish until removal just prior to preparation for observation by SEM (Fig. 3).

Comparison of cyst formation on naive and resistant fish

Both the rate of cyst formation and the quality of the resulting cyst were different on naive versus resistant fish. As determined from SEM micrographs, the percent cover of larvae by fish tissue was similar for both naive (Fig. 4A) and resistant (Fig. 4B) fish at 1 h post-exposure. Most cysts were up to 25% complete, but in both groups, 25%-30% of attached glochidia had no cyst formation. By 2 h post exposure, differences in the progress of cyst formation on naive and resistant fish were evident. On naive fish, about 47% of the glochidia had at least 25% cover and 20% were 50% complete; only 18% had no cyst formation. On resistant fish, no glochidia were more than 25% covered, and 35% still had no cyst formation. There was little change by 3 h. However, by 6 h, 48.0% of glochidia on naive fish (Fig. 4A) and only 9.6% of glochidia on resistant fish (Fig. 4B) were fully encapsulated. Additionally, only 1.6% of glochidia on naive fish had no cyst formation compared to 14.9% on live fins on resistant fish. In contrast, on live fins complete encapsulation of glochidia, as deduced from time-lapse video, took 121.0 [+ or -] 16.0 min ([bar.X] [+ or -] SD, n = 9) on naive fish and 124.0 [+ or -] 23.2 min ([bar.X] [+ or -] SD, n = 8) on resistant fish.

The morphology and pattern of formation of cysts on fins of resistant fish were different from those on naive fish. The altered (rounded-up) epidermal cells that typified the advancing tissue front on naive fish (Fig. 2B, C) were not as prominent during early cyst formation on resistant fish (Fig. 3B). On some cysts, thick, irregular aggregates of host epithelial cells were observed (Fig. 3D). During the later stages of encapsulation, the envelope of fish epithelium covering the larvae was irregular, with gaps and holes in the cyst being common and the cysts seemingly thinner than those on naive fish (Fig. 3F).

Although the cysts on resistant fish are thinner than those on naive fish at 6 h post-infection (Fig. 3F), by 48 h the cysts on resistant fish appeared to be substantially thicker and more enlarged (Fig. 5B) than those on naive fish (Fig. 5A). In addition, open shells of dead, encapsulated glochidia were frequently observed on the fins of resistant fish (Fig. 5C), a condition that was never observed on fins from naive fish.

Cyst formation on less suitable host and nonhost fish

The percent metamorphosis of glochidia of U. imbecillis has been reported to be only about 15% on largemouth bass (Micropterus salmoides; Dodd et al., 2005) and 22% on goldfish (Carassius auratus; Watters and O'Dee, 1998), in contrast to about 50% on bluegill sunfish (Rogers and Dimock, 2003). To assess whether the pattern and rate of encapsulation of glochidia of U. imbecillis on largemouth bass and goldfish differ from those on bluegill, the interaction between larvae and fish was observed with time-lapse video microscopy. A series of images from a representative time-lapse recording on largemouth bass is presented in Figure 6. Migration of epithelial cells was evident by 130 min after attachment (Fig. 6), and the cyst was completely formed by 227.0 [+ or -] 18.9 min ([bar.X] [+ or -] SD, n = 5). Cyst formation was complete at 236.4 [+ or -] 46.4 min ([bar.X] [+ or -] SD, n = 5) on goldfish.

The channel catfish, Ictalurus punctatus, is a suitable host for some species of mussels, but not for U. imbecillis (Watters, 1994). No epithelial cell migration around glochidia of U. imbecillis was evident on live fins of catfish during the standard 6-h observation with time-lapse video microscopy. However, as observed by SEM, some cyst formation occurred within 12 h (Fig. 7). The epithelial cells of catfish resembled those of bluegill sunfish by being polygonal with microridges on the exposed surface (Fig. 7A). In contrast to the condition in bluegill (Fig. 2B, C), cells adjacent to the attached glochidia did not seem to become rounded. Instead, cells on the leading edge of migration extended lamellipodia as they moved over the larval shells (Fig. 7B). By 6 h after attachment, a raised rim of cells encircled the glochidia (Fig. 7C). By 12 h, most glochidia were partially covered by a thick collar of cells (Fig. 7D), and a few were completely encapsulated (Fig. 7E). The cells that composed the complete cysts, or the collars, appeared globular, but still possessed microridges (Fig. 7F). Additionally, fibrous material was regularly observed on the cells composing the collars or the completed cysts, but not on areas of the fin distant from an attached glochidium.

Percent metamorphosis of glochidia of S. undulatus on bluegill (per. obs.) and largemouth bass (Dodd et al., 2005) is less than 2%. Time-lapse video microscopy showed that migration of epithelial cells of bluegill sunfish was evident by 80 min after attachment of S. undulatus, and the glochidium was completely encapsulated in 260 min (Fig. 8, Video 3 at http://www.biolbull.org/supplemental/). The time for completion of cyst formation on bluegill was 298.8 [+ or -] 53.8 min ([bar.X] [+ or -] SD, n = 5), which was similar to the rate of encapsulation of this species on largemouth bass (300.3 [+ or -] 30.0 min, n = 3).

Discussion

Cell motility

Migration and movement of single cells or sheets of cells plays a major role in a number of biological processes. The mechanisms of cell migration, from molecular to biophysical, have been studied in processes such as development and wound healing (Alberts et al., 2002). Fish epithelial cells, also called keratocytes, have served as a popular experimental model for studying the motility of cells, in part because of their rapid movement. Briefly, cells make focal adhesions and close contacts with the substratum at the leading margin of the cell. Binding of extracellular matrix proteins of the substratum by integrins of the membrane of the fish cells elicits contraction of the cytoskeleton (Mitchison and Cramer, 1996; Lee and Jacobson, 1997; de Beus and Jacobson, 1998). As the cytoplasm of the cell is displaced by the contraction of the cytoskeleton, new attachments are formed at the leading margin of the cell and old attachments are released at the rear. The net result is the forward "gliding" of the cells (Lee et al., 1993; Lauffenburger and Horwitz, 1996; Lee and Jacobson, 1997; Galbraith and Sheetz, 1998, 1999).

In sheets or clusters, adjacent cells are connected by desmosomes anchored by cytoskeletal elements (Kolega, 1986; Abraham et al., 2001). The contractile fibers of a cell can be realigned by tension exerted by a neighboring cell, resulting in the cell changing its direction of motility toward the stressor (Kolega, 1986). In addition, keratocytes change their migratory paths in response to an electric field (Cooper and Schliwa, 1986). Stretch-activated calcium channels (Lee et al., 1999) and voltage-gated calcium channels (Cooper and Schliwa, 1986) play a role in the control of cell motility and may also function in redirection of cell movement due to tension. This model of migration of individual and sheets of fish epithelial cells may be applied to explain the process of encapsulation of glochidia of freshwater mussels attached to fish.

Encapsulation of glochidia by host tissue

Once initiated, the process of cyst formation is rapid, and the glochidia are typically covered within 6 h (Waller and Mitchell, 1989; current study), although shorter (Arey, 1932a, b; Jeong, 1989) or longer (Nezlin et al., 1994) times have been reported for various species of mussel and fish. Initially, the cells of the epidermal epithelium immediately adjacent to the attachment site change shape by swelling and becoming rounded, resulting in the loss of their characteristic microridges (Fig. 2B). Altered cells have been observed in micrographs of cyst formation in other studies (Jeong, 1989; Nezlin et al., 1994), and a similar change in the morphology of epidermal epithelial cells has been reported during the healing of the epidermis of fish (Quilhac and Sire, 1999). The altered morphology of these cells also resembles isolated cells in studies examining the migration of individual keratocytes (Cooper and Schliwa, 1986; Lee et al., 1993; Lee and Jacobson, 1997; de Beus and Jacobson, 1998; Galbraith and Sheetz, 1999). The new shape of the cells may be more effective than the native shape for migration over new or exposed surfaces (e.g., the surface of a wound or the shell of glochidia) and may aid them in crawling or rolling as they cover the glochidium.

What initiates the rapid onset of cyst formation once a glochidium attaches to a fish is unknown. Several hypotheses have been presented, including the idea that cyst formation is the reaction of the epithelia to a wound. Superficial wound healing of fish epidermis, especially the covering of the wound site by epidermal epithelium, has been described as rapid, and it initially involves migration of existing cells and later by cell proliferation. Considering the aqueous medium in which a fish dwells, such a mechanism for rapid healing of wounds would be beneficial to prevent efflux or influx of pathogens, ions, and molecules and to prevent osmotic stress (Quilhac and Sire, 1999).

The larval shell of the glochidium is ornamented with sharp hooks ("hooked" glochidia; Arey, 1921; Jeong, 1989; Jeong et al., 1993; Kwon et al., 1993; Pekkarinen, 1996) or small teeth ("hookless" glochidia; Arey, 1921; Nezlin et al., 1994) that are utilized to adhere to the surface of a potential host. It has been suggested that when glochidia attach, they "bite" into the tissue and their sharp hooks and teeth cause small wounds (Arey, 1921, 1932a; Fustish and Millemann, 1978). Cyst formation may be the incidental byproduct of epithelial tissue healing the small wounds caused by the glochidia, a hypothesis that is consistent with the observation by Arey (1921) that aluminum or lead foil clips and small cuts on fish gills lead to the induction of cyst formation.

If cyst formation was due simply to a wound-healing response, one would predict that glochidia from different mussel species would be encapsulated at the same rate on the same species of fish. However, in bluegill sunfish, encapsulation by epithelial cells from the fins took longer with glochidia of S. undulatus (Fig. 8; Video 3 at http:// www.biolbull.org/supplemental/) than with those of U. imbecillis (Fig. 1), suggesting that larvae influence cell migration in some way. This idea is embodied in the hypothesis that a "factor" secreted by a glochidium initiates or influences migration of the epithelial cells of the host (Lefevre and Curtis, 1912; Arey, 1921; Nezlin et al., 1994). This factor has not been identified, and its existence has received little experimental support. Arey (1921) placed drops of extract from ground glochidia on the surface of fish gills but did not observe any consistent tissue response. However, his study did not take into account any potential factors (e.g., extracellular matrix proteins) present in the shell of the glochidium.

As observed in this study, the epithelial cells of the fin of a fish are in constant motion (Video 1 and Video 2 at http://www.biolbull.org/supplemental/). After the glochidium attaches to the fin, perhaps integrins of the epithelial cells in contact with the larva bind proteins associated with the shell of the glochidium and influence cell migration. The specificity of the response could be the result of glochidia from different species of mussels having shells with different compositions of proteins. Alternatively, the small wound inflicted by the shell of the glochidium clamping onto the fin or gill may affect fluxes of ions, disrupting the electrochemical potential of the epithelium. In either case, the shell would provide a new surface to which the epithelial cells could adhere and migrate over. Alteration of the shape (i.e., rounding up) would facilitate movement of the epithelial cells that contact the shell. As these cells move, they pull on neighboring cells and alter the direction of their movement, resulting in the recruitment of additional cells to the migration. The undulating appearance of the epidermis around the glochidia, which is not prominent until about 3 h after attachment (Fig. 2C), may be caused by crowding of the cells as they migrate toward and over the glochidium if the cells of the leading edge of migration are unable to move as fast as the cells that follow.

Comparison of cyst formation on naive and resistant host fish and nonhost fish

Comparison of cyst formation on naive and resistant fish has received little attention. Reuling (1919) and Arey (1932b) both compared naive and "immune" fish (referred to as "resistant" in the present study) and described the immune cyst as being bulky and irregular compared to cysts on naive fish. Arey (1932b) described the immune cysts as "irregular, malformed tumors" and observed that immune cysts and cysts from naive fish formed in approximately the same amount of time, about 4 h. Other studies have compared cysts formed on suitable host fish to those of nonhost fish and reported that the cyst on nonhost fish was also irregular and bulky compared to that on host fish (Fustish and Millemann, 1978; Meyers et at., 1980; Waller and Mitchell, 1989) although this observation has not been consistent among all studies (Karna and Millemann, 1978).

The results of the current study agree with the observations from past studies that the cysts of resistant fish were "irregular." The leading edge of most cysts on resistant fish was broken and uneven, and the cyst often had large gaps where portions of the glochidia were not covered; in contrast, the cyst on resistant fish was no thicker than that on naive fish during the first 6 h (Fig. 3D, F). We did observe aggregates of fish epithelial tissue on some cysts on resistant fish (Fig. 3D). A similar occurrence may have been described as "bulky" by Reuling (1919) and Arey (1932b). However, by 2 d after attachment, the cysts on resistant fish were noticeably thicker than those on naive fish (Fig. 5). This extra bulk may be due to inflammation and infiltration of the cyst by cells of the immune system.

Cyst formation was delayed on resistant fish (Fig. 4). Whereas 48.0% of cysts were fully developed on naive fish at 6 h, only 9.6% of cysts on resistant fish were complete. This delay is likely to have serious implications for the survival of glochidia. Rogers and Dimock (2003) found that the mortality rate of glochidia during the 3rd and 4th infections was significantly increased compared to that during the 1st and 2nd infections. Increased mortality resulted primarily from shedding of dead and live glochidia early in the infection, especially the first day. For example, during the 4th infection, 36% of all attached glochidia where shed alive and 25% dead during the first 5 days of the infection. In contrast, only 16% and 15% were shed as live and dead glochidia, respectively, from fish receiving a 2nd infection. The percent of glochidia shed on the second and third day of the infection was 2-4 times greater from fish during the 4th infection than from fish at the 2nd infection (Rogers and Dimock, 2003).

The process of cyst formation is likely to be a major factor contributing to the mortality or success of metamorphosis of glochidia on fish. Differences in length of time until complete encapsulation may contribute to acquired resistance and may be a factor that makes a particular species of fish less suitable or entirely unsuitable as a host. In the few relationships observed thus far, it takes longer for glochidia of a species of mussel to be encapsulated on a fish on which success of metamorphosis has been low. The exposure to innate immune factors in the mucus of the fish or to an unfavorable external environment for longer periods may have a negative effect on the survival of the glochidia. In a related study, we found that there are changes in glochidia-specific antibodies and protease activity in the mucus of fish during multiple infections by the larvae (Rogers-Lowery, 2005).

In summary, encapsulation of glochidia of freshwater mussels is probably due to a combination of the normal migratory activity of the fish epithelial cells and the influence of factors from the glochidia that affect cyst formation. Incompatibility with these factors or resistance, via an immune response, against them may result in delayed and abnormal cyst formation, with negative consequences for metamorphosis into juveniles.

Acknowledgments

We thank Mr. Ben Dodd and the laboratory of Dr. M. C. Barnhart at Southwestern Missouri State University for providing gravid Strophitus undulatus. Funding for this study was provided in part by a Wake Forest University Science Research Fund award to R.V.D.

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CONSTANCE L. ROGERS-LOWERY* AND RONALD V. DIMOCK, JR.

Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109

Received 5 August 2005; accepted 13 December 2005.

*To whom correspondence should be addressed, at Center for Marine Science, University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane. Wilmington, NC 28409. E-mail: rogersloweryc@uncw.edu
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Author:Rogers-Lowery, Constance L.; Dimock, Ronald V., Jr.
Publication:The Biological Bulletin
Date:Feb 1, 2006
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