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Comparative photobehavior of marine cercariae with differing secondary host preferences.


Parasitic larvae have evolved a variety of behavioral adaptations to specific microhabitats where the probability of encountering their next host is highest (Combes et al, 1994; Haas, 1994). For example, free-swimming cercariae (Trematoda) perceive and respond to exogenous factors such as light, temperature, salinity, host-derived chemicals. water flow, and gravity to orient themselves in the water column where they are more likely to find and infect an appropriate host (Combes et al, 1994; Sukhdeo and S.ukh-deo. 2004). Like other lecithotrophic larvae of marine invertebrates, cercariae experience an abbreviated dispersal phase before recruiting to their next habitat (i.e., secondary-host) (Zimmer et al 2009). Successful transmission depends on encountering a host within a limited time frame as most cercariae have life spans of 24 h or less (Holliman, 1961).

Because of their short life histories, cercariae are likely to use exogenous cues that are predictable and constant within the host's environment. An important external cue that determines the distribution of marine larvae and zooplank-ton is light (see reviews by Thorson, 1964; Forward, 1976, 1988; Cohen and Forward, 2009), and there is some indication that light influences the swimming behavior of parasitic larvae (Combes et al, 1994; Fingerut et al., 2003b), Light can act as a controlling, initiating, or orienting factor for the behavior of planktonic organisms (Bainbridge, 1961). Control refers to the light/dark adaptation state of the organism influencing its response to subsequent sensory stimuli. Initiation of a behavior by light involves photo-stimuli directly triggering a behavior, and may involve the absolute light level or, more commonly, the magnitude or rate of irradiance change (Cohen and Forward, 2009). During this behavior, light may or may not serve an orienting role to guide the direction of movement in the water column. For example, when initiated to move by light or another factor, organisms may display directed swimming toward (positive phototaxis) or away from (negative phototaxis) a light source, using the light gradient for directional information. The orientation component of light-induced swimming may passively result from non-directional changes in speed (orthokinesis) or turning rate (klinokinesis), the magnitude of which will vary in a light gradient. Alternatively, orientation may be an active directional response to a gradient in another exogenous cue such as gravity or pressure (i.e., geotaxis, barotaxis) (Fraenkel and Gunn, 1961). For marine cercariae that utilize fish as their second intermediate host, light may stimulate their ascent as they are released from benthic molluscs, their first intermediate host. Such ascent may result from increased swimming speed, decreased turning rates, or upward movement against Earth's gravitational field or in response to pressure increases. In contrast, light, gravitational, or other cues may stimulate other cercariae to swim downward to increase their contact with benthic intermediate hosts such as crabs (e.g., Fingerut et al, 2003b). The exact role of light as a controlling, initiating, or orienting factor for cercariae in marine and freshwater systems still remains unclear, and the existence of analogous photobehavioral responses in parasite species utilizing hosts in different microhabitats has not been well-studied in the marine environment.

In this study, we examined the photoresponses of two species of marine cercariae that utilize different secondary hosts. Euhaplorchis sp. (synonym Cercaria cursitans, Holliman, 1961) and Probolocoryphe lanceolata (synonym Cercaria lanceolata, Holliman, 1961; Probolocoryphe glandulosa. Heard and Sikora, 1969) are common trematodes in mangrove marshes on the Atlantic and Gulf coasts of Florida (Smith, 2001; Smith et al, 2007). Both species possess a three-host life cycle that involves the ladderhorn snail, Cerithidea scalariformis (Say, 1925), as the first intermediate host. While in the snail (usually the gonad), the trematodes castrate the host and reproduce asexually, producing vast numbers of free-swimming cercariae that are released from the infected snail to infect a second intermediate host. Cercariae of the genus Euhaplorchis (Hetero-phyidae) infect estuarine fishes (Martin, 1950). For example, cercariae of E. californiensis penetrate the skin of killifish and migrate to the brain, where they encyst as metacercariae (Martin, 1950; McNeff, 1978: Shaw et al, 2010). Infected killifish are more likely to exhibit conspicuous behaviors to attract the attention of wading birds, which in turn increases the transmission of Euhaplorchis to its avian final host (Lafferty and Morris, 1996). In contrast, cercariae of Probolocoryphe lanceolata encyst in the hepa-topancreas of fiddler crabs (Uca spp.) (Heard, 1976), which burrow in mudflats and mangrove marshes throughout Florida where they are preyed upon by birds, thus transmitting this parasite to its final host (Salmon, 1967; Smith et al, 2009).

Like most marine cercariae, Euhaplorchis cercariae are short-lived, living no more than 24 h after emerging from an infected snail (Holliman, 1961; Koprivnikar et al., 2010). Swimming is accomplished by a tail (~400 [micro]m long) attached subterminally to the body (~150 [micro]m long); the cercaria swims in rapid but intermittent zig-zag pulses as the tail lashes rapidly. The tail has an unusual combination of dorsal-ventral and lateral fins (Martin, 1950). At rest, the body is oriented downward with the tail above. Located about 1/3 the length of the body from the anterior end are two conspicuous black-pigmented "eye-spots" (Martin, 1950; Holliman, 1961). These consist primarily of melanin, a common nonvisual pigment found in sensory receptors and other cells in a wide range of marine invertebrates (Nadakal, 1960). While discrete receptor cells have yet to be characterized, the presence of photo/gravity/pressure receptors in the eye-spots or elsewhere may influence behavioral responses that affect cercarial swimming, such as the ascent of Euhaplorchis cercariae upon emergence from benthic snails toward their second intermediate host fish in the water column (Cable, 1972; Combes et al.., 1994). Indeed, emergence of Euhaplorchis cercariae from dark-adapted benthic snails is greatest immediately after light stimulation (N.F.S., unpubl. data).

Cercariae of Probolocoryphe lanceolata are also shortlived (~18 h) and possess a body (~180 [micro]m long) and tail (~195 [micro]m long), but they lack pigmented eye-spots (Holliman, 1961). At the anterior end of the body is an oral sucker and prominent stylet (~30 [micro]m long) (see Holliman, 1961, for anatomical details), which aid in penetration of secondary hosts. Upon emerging from the snail host, cercariae swim continuously with the tail moving in a figure-eight motion, but they eventually settle and creep along the bottom by extending the body and attaching to the substrate with the oral sucker (N.F.S., per. obs.). Such larval behavior may allow them to facilitate their encounter with fiddler crabs, which burrow in sediments adjacent to benthic snails (Heard, 1976; Smith et al, 2007). Probolocoryphe lanceolata cercariae have been observed to emerge from benthic snails under light and dark conditions, but emergence is greatest in darkness, immediately after the removal of light stimulation (N.F.S., unpubl. data). Negative phototaxis, positive geotaxis, positive barotaxis, or passive sinking would facilitate their dispersal and transmission to benthic second intermediate hosts such as burrowing crabs.

Thus, the present study focuses on the orientation behavior of Euhaplorchis and P. lanceolata cercariae to determine whether light plays a role as an exogenous initiating and orienting cue that may ultimately influence their distribution within the host-space environment.

Materials and Methods

Cercariae of Euhaplorchis and Probolocoryphe lanceolate were collected from infected snails (Cerithidea scalariformis) from a mangrove marsh on Weedon Island, St. Petersburg, Florida (27[degrees]50.699'N, 82[degrees]36.663'W) in May 2010. Here, snails live among salt-tolerant plants (Salicornia virginica, Batis maritima) and black mangroves (Avicennia germinans), or in open mudflats, which are exposed and dry during low tides but otherwise inundated with water reaching a depth of 0.3 m during spring tides. Snails were kept alive in the laboratory until dissected for parasites. Since cercariae are short-lived, they were removed from infected snails and immediately placed in 0.45-[micro]m filtered seawater (FSW) (seawater temperature: 24 [degrees]C; salinity: 30 psu) and tested within 2 h of emergence in the following studies.

The photoresponses of cercariae to different light intensities were tested under two types of light fields to isolate different aspects of light as a behavioral factor. First, tests conducted in a simulated natural light field were used to quantify light as an initiating factor in swimming behavior and to measure sinking speeds. Second, tests conducted with a horizontally directed light source were used to determine if light is used as an orienting cue for swimming movements.

Test of cercarial photobehavior in a simulated natural light field

To determine the role of light in initiating the swimming responses of Euhaplorchis and Probolocoryphe lanceolata, cercariae were tested in a laboratory apparatus that simulates the natural underwater distribution of angular light, which is essential for evoking normal photobehaviors in zooplankton (e.g., Forward et al, 1984). The apparatus consisted of an acrylic bath (40-cm L X 40-cm W X 15-cm H) with the inside painted flat black and filled with distilled water, ink) which was placed a small acrylic cuvette (3.9-cm L X 1.7-cm W X 5.4-cm H) containing cercariae in sea-water with the upper edge of the cuvette just above the level of the distilled water. Light from a voltage-regulated source (150-W quartz halogen, Dolan Jenner) was spectrally filtered to blue wavelengths (BG18 bandpass. Schott) that encompass the spectral responsivity maximum of larval trematodes (Wright et al, 1972), and then through neutral density filters to adjust irradiance. Filtered light was then reflected off a mirror and entered the cuvette and bath vertically from above through a white acrylic diffuser. This optical arrangement created a bright overhead light and a diffuse surrounding light as viewed from the cuvette. Irradiance at the level of the specimens in the cuvette was measured using an optometer and calibrated radiometric probe (models S471 and 260, UDT Instruments). Narrow unpainted sections of the water bath allowed for cercariae to be backlit with a custom far-red light source (>740 nm) and their swimming behavior in the X-Y plane of the cuvette captured for subsequent motion analysis by a CCTV camera (WVBP330, Panasonic) with a 5-50-mm varifocal lens (13VA550, Pelco) and a digital recorder (DN-200 Data-video).

For each test species, a group of about 100 cercariae removed from an infected snail (n = 6 snails per trematode species) and immediately placed in 0.45-[micro]m FSW in a test cuvette and dark-adapted for at least 60 min prior to the cuvette being placed in the water bath under dim red light. Once in the water bath, cercariae remained in darkness for 15 min. after which they were stimulated with a series of seven light flashes, increasing in intensity from 4 X [10.sup.11] to 4 X [10.sup.17] photons [m.sup.-2] [s.sup.-1] with 5-s flash duration and 5 min of dark-adaptation between successive stimuli. Preliminary experiments suggested that the stimulus duration, order, and recovery period were sufficient to evoke repeatable responses to an intermediate stimulus before and after the series. Digital video files were analyzed by haphazardly selecting 10 cercariae in each replicate and tracking their X-Y position, swimming speed, and direction using Max-TRAQ (ver. and MaxMATE (ver. 3.6G) software (Innovision Systems. Inc.) over a 2-s period during the middle of each stimulus, as well as over a 2-s period before stimulation (control). Problems with motion parallax were reduced by centering the 5-mm depth-of-field of the camera at the midpoint between the front and back walls of the test cuvette, and only selecting individual cercariae for analysis that were in sharp focus. Frictional wall effects were present given the small size of the test cuvette and the low Reynolds numbers for swimming cercariae in this experimental configuration (Vogel, 1994). These forces were kept relatively consistent among trials and between species by the constant positioning of the camera's field of view 1.2 cm away from the side walls and 2.1 cm from the surface/bottom. An ascending response in the natural light field was defined as movement directly upward (zenith [+or-] 45[degrees]) in the X-Y plane of the camera, while a descending response was downward movement (nadir [+ or -] 45[degrees]).

Response means (swimming speed, % ascending, % descending) were each compared to their respective controls by two-factorial repeated-measures ANOVAs (light intensity X response/control) with Tukey post hoc testing to determine the lowest light intensity that evoked a significant difference between a response during the light flash and darkness immediately prior to the flash (threshold).

Test of sinking speeds in anesthetized cercariae

To determine whether descending movements resulted from passive sinking or active swimming due to either negative phototaxis or positive geotaxis, we compared speed of movement for dark-adapted cercariae with the speed of cercariae that were anesthetized. Cercariae were anesthetized by exposing them to 0.5 mol [1.sup.-1] MgCl in FSW for at least 1 h. They were then transferred to clean FSW and gently pipetted into the top of an acrylic cuvette (5-cm L x 5-cm W X 15-cm H) filled with FSW. Sinking of anesthetized larvae was recorded digitally as described above, under dark conditions with the camera positioned in the horizontal center of the bottom quarter of the cuvette such that the camera's field of view began 1.6 cm from the side walls in order to standardize frictional wall effects on sinking cercariae. The sinking rate for an individual larva (mm [s.sup.-1]) was measured by distance moved over an interval ranging from 60 s to 2 min, using MaxTRAQ and Max-MATE software. The mean sinking rates of anesthetized Euhaplorchis (n = 7) and Probolocoryphe lanceolata (n = 7) cercariae were compared, using a Student's t-test, with the mean swimming speeds of the cercariae in darkness (n = 7 for each trematode species) from the experiment with an angular light field.

Test of light as an orienting cue for cercarial swimming

To determine if light is used as an orienting cue for swimming movement, phototactic responses of Euhap-lorchis and Proholocoryphe lanceolata cercariae were tested in a horizontal trough with a narrow, horizontally directed light source positioned at one end. The rectangular trough (11-cm L X 4-cm W X 4-cm H) was made of clear acrylic, with acrylic partitions dividing it into five chambers of equal size such that one chamber was closest to the incoming light and each successive chamber was further away from the light source, creating a light gradient along the trough with maximum quantal intensities ranging from 1.72 X [10.sup.20] photons [m.sup.-2] [s.sup.-1] to 1.5 X [10.sup.19] photons [m.sup.-2] [s.sup.-1]. The partitions were connected at their tops, allowing for their simultaneous addition or removal. The entire trough was enclosed by a large box covered in black cloth to eliminate all natural light from entering the trough. While in the darkened box, the trough was accessed for partition insertion and removal by a flap that could be opened and closed, and covered with black cloth. The only light entering the chamber was from a light source (150-W quartz halogen, Schott-Fostec) with a fiber optic cable that entered the box and emitted cold white light which then passed through the long axis of the trough. Light intensity was regulated by neutral density filters placed in the light path between the fiber optic cable and the trough.

For each test species, groups of cercariae were placed on concave microscope slides (10 larvae per slide) and dark-adapted for 30-45 min. The trough was filled with 10 ml of FSW and placed into the darkened test chamber; then with partitions inserted in the trough, 10 dark-adapted cercariae were added to the center section. Upon light stimulation, the partitions were removed, allowing the cercariae to swim in the trough. After five minutes, the partitions were inserted and the number of cercariae in each chamber was counted under a stereoscope. Cercariae found in the chamber closest to the light source were classified as positively phototactic; those in the chamber farthest from the light were considered negatively phototactic; and those in the middle chambers were counted but not used for any statistical analyses. Ten replicates of Euhaplorchis and Probolocoryphe lanceolata were tested at seven light intensities (1.46 X [10.sup.17], 1.49 X [10.sup.18], 1.37 X [10.sup.19], 4.02 X [10.sup.19], 5.90 X [10.sup.19], 1.0 X [10.sup.20], and 1.72 X [10.sup.20] photons [m.sup.-2] [s.sup.-1]), quantal values that are well within the range found in the snail-parasite habitat (see Discussion). Each cercaria was used only once. In addition to the light intensity treatments, the response of cercariae under no light stimulation (dark control, n = 10) was measured. The light intensities were measured by a cosine-corrected quantum sensor (LI-190, LI-250A, LI-COR) placed in an empty trough on the end closest to the light source. Between all experimental tests, partitions and trough were rinsed with distilled water to remove cercariae from the previous test.

Prior to statistical analyses, calculated proportion values of positively and negatively phototactic cercariae were arcsin-square-root transformed to meet assumptions of normality. Transformed means were compared to the control by a one-factorial ANOVA and a Dunnett's post hoc test (Dun-nett, 1964) to determine which phototactic responses to the seven light intensities were significantly different from the dark control.


Photobehavior in a simulated natural light field

In a simulated underwater angular light distribution, Eu-haplorchis cercariae showed an ascent response to light intensities greater than 4.0 X [10.sup.15] photons [m.sup.-2] [s.sup.-1] (Fig. 1A). This was the lowest stimulus level that evoked a significant ascending swimming response ([F.sub.5,6] = 9.593, P < 0.001; Tukey P < 0.05). No light stimulus evoked a descent response in Euhaplorchis that was significantly greater than the control (darkness) ([F.sub.5,6] = 2.344, P = 0.063; Fig. 1B).

The presence of light had a significant effect on Euhaplorchis cercariae swimming speeds ([F.sub.5,6] = 42.86, P = 0.003). Swimming speed increased significantly with increasing quantal intensity (P < 0.05), exceeding 1.5 mm s"1 under the highest light levels (Fig. 2A). The lowest light intensity to induce a significant increase in swimming speed relative to the control (darkness) was 4.0 x [10.sup.15] photons [m.sup.-2] [s.sup.-1] ([F.sub.5,6] = 18.76, P < 0.001; Tukey P < 0.05; Fig. 2A). During the 2-s interval of light stimulation, Euhaplorchis cercariae swam quickly toward the downwelling light in an upward movement, moving within [+ or -] 30[degrees] of the zenith (Fig. 3A). In contrast, cercariae exhibited less activity while in darkness, and most displayed passive sinking during the 2-s interval prior to light stimulation (Fig. 3B). Under darkness, Euhaplorchis cercariae moved an average of 0.38 mm s"1 ([+ or -]0.06 SD) and there was no significant difference in swimming speeds prior to the application of all levels of light stimuli (P > 0.05). Their swimming speeds under darkness were almost 2 times faster than their passive sinking speeds influenced by frictional wall effects (mean = 0.21 mm [s.sup.-1] SD = 0.05) (t = 5.37, P < 0.001); even without a light stimulus, Euhaplorchis cercariae were actively swimming to some degree, but they were not as active as during a light stimulus (Fig. 3A).

In contrast. Probolocoryphe lanceolata cercariae exhibited greater upward movement relative to Euhaplorchis in darkness (Fig. IC) and strong downward movement under high light intensities (Fig. ID). In darkness, a greater percentage of P. lanceolata cercariae ascended as compared to their swimming direction during light stimulation ([F.sub.5, 6] = 33.413, P = 0.002; Fig. IC). In addition, there was a significant difference in the percentage ascending among all light intensities ([F.sub.5,6] = 5.777. P < 0.001), but there was no significant interaction between the stimulus or the control and light intensity [F.sub.5,6] = 1.771. P = 0.139), indicating that the ascent response under light or in dark conditions does not depend on the level of light intensity. The percentage of cercariae descending in response to light stimuli increased as light intensity increased, with 85.6% of cercariae displaying a descent response at 4.0 X [10.sup.17] photons [m.sup.-2] [s.sup.-1] (Fig. 1D). The lowest stimulus level to evoke a significant descent response was 4.0 X [10.sup.14] photons [m.sup.-2] [s.sup.-1] ([F.sub.5,6]= 9.132, P < 0.001; Tukey P < 0.005).

Like Euhaplorchis, P. lanceolata showed swimming speeds in darkness (mean = 0.78 mm [s.sup.-1], SD = 0.03) that were significantly higher than the passive sinking rates of anesthetized cercariae influenced by frictional wall effects (mean = 0.26 mm [s.sup.-1], SD = 0.04) (t = 27.8, P < 0.001). Probolocoryphe lanceolata cercariae also swam signifi cantly faster during the light stimulus than in darkness ([F.sub.5,6] = 74.677, P < 0.001), reaching 1.08 mm [s.sup.-1] under the highest light intensity (Fig. 2B). The lowest stimulus level to evoke a significant increase in swimming speed relative to its paired control was 4.0 X [10.sup.15] photons [m.sup.-2] [s.sup.-1] ([F.sub.5,6] = 11.551, P < 0.001; Tukey P < 0.005). The swimming activity of P. lanceolata, like that of Euhaplorchis, was strongly affected by high quantal intensities (Le., [10.sup.16] photons [m.sup.-2] [s.sup.-1]); however, most P. lanceolata cercariae swam in a direct downward movement (nadir [+ or -] 10[degrees]) upon light stimulation (Fig. 3C). Under darkness, cercariae swam in variable directions (Fig. 3D) with speeds (mean = 0.78 mm [s.sup.-1] [+ or -] 0.25 SD) similar to those exhibited by cercariae subjected to low levels of light stimuli (Fig. 2B), but three times faster than anesthetized cercariae. There was no significant difference in swimming speeds during the 2-s dark interval prior to application of all levels of light stimuli (P > 0.05). Swimming speeds under conditions of light and darkness depended on levels of light intensity, as shown by the significant interaction term between stimulus/control and light intensity ([F.sub.5,6] = 12.068, P < 0.001).

Light as an orienting cue for cercarial swimming

Euhaplorchis cercariae responded to all light stimuli in the horizontal trough by swimming toward the light source (positive phototaxis) after partitions were removed ([F.sub.7] = 10.8, P < 0.001). Cercariae showed increased positive phototaxis with increasing light intensity relative to the dark control, with significantly more positive phototaxis during light stimuli than in controls, beginning at the lowest light level tested (1.46 X [10.sup.17] photons [m.sup.-2] [s.sup.-1]) (P < 0.05; Fig. 4A). Euhaplorchis cercariae rarely swam into the section of the trough furthest away from the light source; the mean number of photonegative cercariae did not differ significantly from that of the dark control treatments ([F.sub.7] = 0.67, P = 0.70) (Fig. 4A). In darkness, most cercariae (57%) were observed in the middle section of the trough, and only 3% swam into the section of the trough that indicated positive phototaxis.

in contrast, probolocoryphe lanceolata cercariae exhibited neither a negative ([F.sub.7] = 1.14, P = 0.35) nor positive ([F.sub.7] = 1.03, P = 0.42) phototactic response to light stimuli, even at the highest light intensities tested (>[10.sup.19] photons [m.sup.-2] [s.sup.-1] (Fig. 4B). The percentage of cercariae displaying positive phototactic responses ranged between 5% and 15%, which was slightly higher than the percentage of cercariae exhibiting negative phototactic responses (2%-10%). The mean percentage of photonegative and photopositive cer cariae at each light intensity was not significantly different from the control (P > 0.05; Fig. 4B), and most cercariae remained in the three middle sections of the horizontal trough at all light intensities (78%-93%) and under darkness (95%).


Light is an important environmental cue that controls the distribution of a wide range of free-living marine zooplank-ton (e.g.. Forward et al, 2000; McCarthy et al, 2002a; Ziegier et cil., 2010), and likewise, serves as an important cue for cercarial release and swimming behavior (Fingerut et al, 2003a, b). Successful transmission of cercariae, given their short life spans, depends on their ability to quickly find an appropriate second intermediate host within the host-space environment, a process that is tightly coupled to environmental cues (Sukhdeo and Sukhdeo, 2004; Zimmer et al., 2009). In this study, we found that the light-mediated behavioral responses of two species of marine cercariae that utilize different second intermediate hosts differed in ways that would facilitate their movement toward microhabitats frequented by the hosts. Furthermore, they differed in the proximate mechanisms controlling orientation during swimming.

Euhaplorchis cercariae showed positive phototaxis to all light intensities tested in the horizontal trough with a directional light source. When tested in a light field simulating the underwater angular light distribution, they also responded positively to downwelling light. These findings indicate that rather than being an artifact of a highly directional light source (e.g., Forward. 1988), positive phototaxis in Euhaplorchis cercariae is a natural behavior this species uses to move toward pelagic and surface-water habitats frequented by its fish secondary hosts. The minimum light intensity required to evoke an ascending movement was 4.0 X [10.sup.15] photons [m.sup.-2] [s.sup.-1] (6.64 X [10.sup.-3] [micro]mol photons [m.sup.-2] [s.sup.-1]). This phototactic response threshold is less sensitive than that of crustacean larvae with long planktonic durations studied using a similar experimental approach ([10.sup.11] - [10.sup.14] photons [m.sup.-2] [s.sup.-1], reviewed by Forward, 1988). Other taxa that, like Euhaplorchis, have shorter planktonic durations, on the order of hours, and less developed visual systems, such as larvae of the tunicate Polyandrocarpus zorritensis, have a similar sensitivity to light with phototaxis thresholds of about 5 X [10.sup.16] photons [m.sup.-2] [s.sup.-1] (Forward et al, 2000). Daytime intensities of surface light in the Euhaplorchis habitat (mangrove marsh) range from 3 X [10.sup.17] (shade; 0.498 [micro]mol photons [m.sup.-2] [s.sup.-1]) to 8 X [10.sup.18] photons m"2 s"1 (full sun; 13.3 [micro]mol photons [m.sup.-2] [s.sup.-1]); thus, sufficient light exists for the initiation of positive phototactic behavior during daylight.

Light also had a significant effect on the swimming speed of Euhaplorchis cercariae. As light intensity increased, their swimming speeds correspondingly increased from minimal movement to rates similar to those reported for other cercariae (e.g., Meyrowitsch et al., 1991; Fingerut et al., 2003b), with most Euhaplorchis cercariae swimming directly upward. Upon release from benthic snail hosts, Euhaplorchis cercariae would have an increased probability of encountering a fish host by fast upward movement under daytime light levels. A similar scenario occurs in freshwater Echinostoma caproni cercariae, which are initiated to ascend in response to light and gravity cues, facilitating their access to secondary intermediate snail hosts living at the margins of ponds and streams (Platt et al., 2010). Following their initial ascent in response to light, cercariae of Euhaplorchis were frequently observed drifting downward with intermittent bursts of ascent swimming. Such a swimming response is typical for cercariae that infect pelagic secondary hosts, allowing them to maintain their vertical position in the water column so they can encounter potential hosts at different depths (Loy et al, 2001; Sukhdeo and Sukhdeo, 2004).

In contrast to Euhaplorchis, Probolocoryphe lanceolata cercariae exhibited a descent response when tested in a simulated natural light field, with a light intensity threshold about one order of magnitude lower (4.0 X [10.sup.14] photons [m.sup.-2] [s.sup.-1]; 6.64 X [10.sup.-4] [micro]mol photons [m.sup.-2] [s.sup.-1] than was observed in Euhaplorchis. Whether in light or darkness, P. lanceolata cercariae exhibited mainly vertical swimming movements; horizontal swimming was rare. Similar light-mediated behavior has been reported for marine trematode larvae in California salt marshes, where emergence of cercariae from submerged snails was highest during daylight hours and decreased throughout the day as sunset approached (Fingerut et al, 2003a). For example, Himasthla rhigedana, one well-studied species in this habitat, swims downward in response to daytime and dusk light intensities (2000 and 40 [micro]mol photons [m.sup.-2] [s.sup.-1], respectively; Fingerut et al, 2003b). Such downward swimming can quickly bring both P. lanceolata and H. rhigedana cercariae to the benthic environment, facilitating their contact with crabs, the next host in their life cycles (Fingerut et al, 2003b; Smith et al, 2007). These larvae are likely to swim into deeper water or into crab burrows where light intensity is lowest. The heightened sensitivity (i.e., lower threshold) of P. lanceolata photobehavior as compared to Euhaplorchis fits with the direction of its photoresponse. While Euhaplorchis is ascending in response to light and therefore encountering a brighter environment over time, P. lanceolata is descending into darker water and would benefit from increased light sensitivity to continue its descent as light is attenuated with depth.

As opposed to their strong light-mediated descent response when tested in a simulated natural light field, Probolocoryphe lanceolata cercariae did not exhibit a directional swimming response in a shallow trough at suprathreshold light intensities (> [10.sup.17] photons [m.sup.-2] [s.sup.-1]; 0.166 [micro]mol photons [m.sup.-2] [s.sup.-1]) emanating from a highly directional source. Accordingly, although light greater than 4.0 X [10.sup.14] photons [m.sup.-2] [s.sup.-1] initiates the descent response in P. lanceolate, this species does not use the direction of light as an orienting cue for swimming movements. Rather, orientation during the descent was achieved by a cue other than light direction, such as active movements to orient swimming with the gravitational field (positive geotaxis; e.g., Pires and Wool-lacott, 1983; McCarthy

et al, 2002b) or an increasing hydrostatic pressure gradient (positive barotaxis; Stake and Sammarco, 2003). Kinetic responses such as a reduction in swimming and subsequent sinking in the presence of light, or a decrease in turning rate, are not supported by observations of P. lanceolata behavior. Swimming speeds during light-mediated descent were 3 times faster than passive sinking rates of anesthetized cercariae, and swimming paths were relatively linear in both darkness and in light. As both phototactic and geotactic behaviors are commonly reported for cercariae (Fingerut et al, 2003b; reviewed in Sukhdeo and Sukhdeo, 2004), subsequent ultrastructural studies on photoreceptors and statocysts, and neural mechanisms for their integration, are warranted. The descent response of P. lanceolata may be mediated by nonvisible photoreceptors or through a "dermal light sense" similar to that of other microphallid cercariae, which typically lack eyespots (McCarthy et al, 2002b). Furthermore, the possibility of baro-kinesis in cercariae needs to be investigated. These results with P. lanceolata underscore the importance of testing photohehavior in a natural light field, a common omission in studies of phototaxis, including most of those with cercariae (e.g., Saladin, 1982; Loy et al, 2001; Platt et al, 2009: but see Haas et al 2008).

Marine cercariae are likely to rely on a variety of exogenous cues for locating hosts (Combes et al., 1994; Haas, 1994), and uncertainty remains as to whether these responses are fixed (Sukhdeo and Sukhdeo, 2004) or plastic. Additional environmental factors, such as currents, pressure, and temperature, can complement light by inducing behavioral responses that affect cercarial swimming, potentially bringing cercariae closer to secondary hosts. For example, emergence of marine cercariae from snail hosts was highest during daylight hours, but only when snails were submerged during flood tides (Fingerut et al, 2003a). Koprivnikar and Poulin (2009) found cercarial emergence from intertidal marine snails to vary with host submersion level, reflecting differences in transmission strategies employed by different trematode species. In tidally dominated habitats where snail hosts vary in submersion time and depth, cercariae may use changes in hydrostatic pressure as a proximate cue for water level or depth prior to emergence (Mouritsen, 2002). Once released, light/gravity/pressure can initiate or orient cercarial swimming toward the appropriate host microhabitat, and tidal currents can facilitate cercarial transport and interactions with second intermediate hosts (de Montaudouin el al, 1998; Fingerut et al, 2003b). Additionally, water temperature, influenced by pholoperiod and seasonal cycles, potentially has significant effects on cercarial activity and behavior. Increases in temperature caused the swimming speeds of Himasthla rhigedana to increase, and swimming speeds correspondingly decreased with decreases in temperature (Fingerut el aL, 2003b). Similarly, cercariae of Acanthoparyphium spinudosum exhibited higher activity (swimming or crawling behaviors) when exposed to higher temperatures (Koprivnikar et al., 2010). In the mangrove marshes in Florida where Cerithidea sca-lariformis lives, water temperatures can easily exceed 45 [degrees]C in the summer (Smith and Ruiz, 2004), and such warm temperatures potentially increase cercarial swimming activity and thereby transmission to second intermediate hosts when trematode infections in C. scalariformis are the highest (N.F.S., unpubl. data). Environmentally induced upward or downward swimming can facilitate contact with pelagic or benthic secondary hosts much more quickly than passive transport alone (Fingerut et al, 2003b).

Environmental cues (i.e., light, gravity, temperature, salinity, water turbulence, etc.) that stimulate and affect cercarial swimming movements and behavior have been studied for only a few marine species, and therefore their adaptive benefits still remain uncertain. In particular, behavioral responses of marine cercariae to interactions that may occur nearby a host, such as shadow stimuli and chemical compounds including host kairomones. are especially poorly understood (Haas, 1994). Further, there may be ontogenetic changes in swimming and orientation responses to environmental cues as cercariae age, as observed in other marine invertebrate larvae (Young and Chia, 1982; Forward, 1989; McCarthy et al, 2002a). Given their short life spans and the clear host-specific postemergence behaviors that cercariae have been shown to display in the present study, it seems unlikely that such larvae would find and infect a host purely by chance. The host-recognition strategies of marine cercariae are likely to involve a suite of exogenous factors that optimize their contact with potential hosts.


We are grateful to Arya Poppema-Bannon and students in the Marine Science Freshman Research Program for their lab and field assistance, to Pinellas County Environmental Management for access to Weedon Island Preserve to collect snails, and to the anonymous reviewers for their helpful comments. J.H.C. was supported in part by grant #4710-1101-01-A from the Florida Institute of Oceanography.

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(1.) Galbraith Marine Science Laboratory, Eckerd College, 4200 54th Avenue South, St. Petersburg, Florida 33711; and (2.) College of Earth. Ocean and Environment, School of Marine Science and Policy, University of Delaware, 700 Pilottown Road, Lewes, Delaware 19958

Received 29 November 2011; accepted 10 February 2012.

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