VENUS 73 (3–4): 115–125, 2015 Re-infection by a Eulimid Gastropod ©Malacological Society of Japan115

Factors Affecting Re-infection by Hypermastus tokunagai (: ) of Its Host, the Sand Dollar Scaphechinus mirabilis (Clypeasteroida: Scutellidae)

Haruna Matsuda1,2*, Tatsuo Hamano3,4 and Kazuya Nagasawa2 1Study Support Center, Shikoku University, 123-1 Ebisuno, Furukawa, Ojin, Tokushima 771-1192, Japan 2Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 3Department of Applied Aquabiology, National Fisheries University, 2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595, Japan 4Institute of Socio-Arts and Sciences, The University of Tokushima, 1-1 Minamijosanjima-cho, Tokushima 770-8502, Japan

Abstract: Hypermastus tokunagai is a eulimid ectoparasitic on Scaphechinus mirabilis, a sand dollar mainly found in the Seto Inland Sea. This eulimid is mostly found on its host but free-living individuals are often observed in the sediment. In this study, we investigated whether solitary individuals of H. tokunagai re-infect S. mirabilis following detachment in the eld and tested the factors affecting re-infection in the laboratory. The eld experiment demonstrated that H. tokunagai is capable of re-infecting its host after detachment. The laboratory experiments suggested that H. tokunagai were not apparently attracted by chemical stimuli from their hosts. They tended to prefer light over dark regions and white over black regions, but when placed on glass beads and exposed to light, many individuals submerged and moved under a black plate. More individuals of H. tokunagai preferred dark regions when in the presence of chemical stimuli from their hosts. Our results suggest that the ability of H. tokunagai to locate its host cannot be ascribed entirely to sensory receptor response to chemical stimuli, and that they also rely on vision to approach a host-resembling object in the process of re-infection. Once in close proximity, other factors, such as olfactory and/or tactile stimuli, are likely to play a role in host recognition.

Keywords: chemical stimuli, host recognition, Hypermastus, parasitic gastropod, visual selection

Introduction

Members of the family Eulimidae Philippi, 1852 parasitize echinoderm species of all classes and either feed on the tissue of the host’s body or ingest its body uids (Warén, 1984; Jangoux, 1990). The eulimids exhibit a variety of morphological and ecological adaptations for parasitism and range in mode from temporary ectoparasites to permanent endoparasites. Among the ectoparasitic eulimids, most species are thought to parasitize the host for a limited time and to have the capability to leave voluntarily. This is supported by the fact that they are rarely collected with its host though they are parasitic (Warén, 1984; Bouchet & Warén, 1986). This detachment ability is advantageous if there is a high probability of predation on the host (Warén, 1984). However, it seems as though it would be dif cult for small eulimids to nd out another host if they were completely separated from their host and become solitary in the eld.

* Corresponding author: [email protected] 116 H. Matsuda et al.

After detaching from their host, eulimids may be vulnerable to predation by predators such as shes, crabs and polychaetes (Warén, 1984). Such solitary eulimids may also be under threat of starvation since most eulimids obtain nutrients exclusively from their host (Jangoux, 1990). Some eulimids are reported to secrete a mucus thread from the posterior pedal gland and to use it to maintain their position on the host [e.g., Crinophtheiros collinsi (identi ed as “Balcis devians” in Fretter, 1955 but subsequently cited as “ collinsi” in Bouchet & Warén, 1986), Peasistilifer nitidula (as “Mucronalia nitidula”) and Pulicicochlea calamaris] (Fretter, 1955; Hoskin & Cheng, 1970; Ponder & Gooding, 1978), whereas many eulimids are observed to detach from their host and move freely when disturbed (e.g., Curveulima dautzenbergi, Eulima bilineata, Nanobalcis nana and philippi) (Warén, 1983, 1984; Rodríguez et al., 2001a, 2001b). In temporarily parasitic eulimids, several species are observed to re-infect their host after removal from the host under arti cial conditions (Hoskin & Warén, 1983; Warén, 1984; Crossland et al., 1991). Thus, they are thought to have capability of locating and re-infecting a suitable host. There have been few studies dealing with host searching behavior of eulimids. Cabioch et al. (1978) observed “Balcis alba” [ = alba] in the laboratory and suggested that it uses sight to locate its host. Brand & Ley (1980) conducted behavioral experiments to examine whether “Balcis catalinensis” [=Melanella catalinensis] could chemically sense the presence of a host or not, and concluded that it randomly encounters its host. However, our knowledge is insuf cient to understand the mechanisms controlling re-infection by eulimids in general, especially as regards their varying morphological and ecological adaptations and biology of host species. Hypermastus tokunagai is an ectoparasite of the sand dollar Scaphechinus mirabilis and is thought to ingest the host’s external tissues (Matsuda et al., 2008). This eulimid is considered a temporary parasite since it can be easily detached by touching lightly and solitary individuals have been collected from benthic sediments (Matsuda et al., 2008). Since H. tokunagai is known to be highly host-speci c to S. mirabilis (Matsuda et al., 2010a, b), we hypothesized that they are capable of detecting and re-infecting S. mirabilis. In this study, we examined the ability of H. tokunagai to re-infect its host following detachment in the eld and tested factors affecting host recognition, focusing in the laboratory on the effects of chemical stimuli from their host and visual keys.

Materials and Methods

Re-infection ability We investigated whether solitary H. tokunagai adults were attracted to and re-infected S. mirabilis based on eld experiment. Uninfected individuals of S. mirabilis placed in the eld were monitored for infection by H. tokunagai adults. First, a total of eight polypropylene containers (12 cm in height, 60 cm in diameter) were lled with sand collected from the supratidal zone on the coast of the Seto Inland Sea at Okuni (33°52´N, 132°07´E), Hirao Town, Yamaguchi Prefecture, where H. tokunagai and S. mirabilis do not occur. Sand was collected there to exclude the possible effects of eulimids having been present in the sand. Uninfected individuals of S. mirabilis were collected from the subtidal zone 50 m away from the collecting site of the sand, and placed in the containers (5 individuals/container). The containers were each wrapped with a mesh bag (1 × 1 cm mesh) (Fig. 1), submerged 5 m apart on the sandy substrate at depths of 0.5–1.0 m based by chart datum level and xed with two stakes off Okuni on May 7, 2008. The containers were retrieved three months later (August 2, 2008). Each S. mirabilis individual was checked for the presence of H. tokunagai above water.

Factors affecting host recognition Sampling and rearing: We collected S. mirabilis individuals that were infected with H. tokunagai by hand in the subtidal zone of the Seto Inland Sea off Okuni between June 2007 and April 2009. Re-infection by a Eulimid Gastropod 117

stake sand dollar bottom surface

sand

mesh bag polypropylene container

Fig. 1. Diagrammatic representation of the experimental equipment used for monitoring re-infection ability of Hypermastus tokunagai. A polypropylene container (12 cm in height, 60 cm in diameter) lled with sand were wrapped with a mesh bag (1 × 1 cm mesh), submerged in the sandy substrate and xed using two stakes. Five uninfected individuals of S. mirabilis were placed in the container.

The specimens of H. tokunagai were placed in test tubes separately and transported immediately with a test tube rack to the nearby Tana Marine Biological Laboratory (TMBL) of the National Fisheries University. Following transport, batches of ca. 10 specimens were placed in polypropylene Grif n beakers (6 cm in height, 9 cm in diameter) covered with 1 mm mesh net and submerged in an indoor aquarium (1 m in height, 1 m in diameter). S. mirabilis was also transported to TMBL and placed in a roofed outdoor arti cial tank (476 × 145 × 90 cm). We did not measure the size of H. tokunagai specimens used for the experiments, but those collected on the same day during the monthly sample collections ranged from 3.4–6.8 (mean: 4.7, n = 28) mm in shell length (SL) (June 13, 2007), 4.2–6.6 (5.4, n = 11) mm SL (July 12, 2007), 3.8–6.6 (4.6, 4n = 1 ) mm SL (November 9, 2007), and 2.6–4.9 (3.8, n = 37) mm SL (April 24, 2009). Preference for chemical stimuli from host: The effect of chemical stimuli in the recognition of S. mirabilis by H. tokunagai was evaluated using a choice device, which allows an to approach stimuli carried by the water  ow (Fig. 2). The choice device was constructed from a circular polypropylene container (18.5 cm in diameter, 7 cm in height) (Fig. 2C). A total of 50 holes (0.2 mm in diameter) were drilled in the central circular region (4.5 cm in diameter) of the base of the container. The container had two lateral holes near the base, which were connected by polypropylene tubes to two tanks (Tank A and B, Fig. 2A, B). The tubes transferred a laminar  ow of water (1.02–1.60 ml sec–1) from Tank A and B to the choice device. We placed ve S. mirabilis individuals collected off Okuni in Tank A, 5 min before the start of the experiment. Since the water current was laminar, seawater from the two tanks was able to  ow directly into the lateral holes of the choice device and drain from the central region with minimal mixing within the container. Thus, we assumed that the seawater with chemical stimulus from Tank A is restricted to region “a” and the seawater from Tank B is restricted to region “b” (Fig. 2D). In the preliminary experiment of the choice device which traced water  ow using colored water, the water did not  ow into region “c” (designated as “other regions”). The seawater drained through the holes in the base and over owed from the outer container to regulate the  ow and water level (6 cm in depth) in the inner choice device. The seawater pumped up from Hirao Bay (in front of TMBL) was ltered for sand prior to the experiment. Ten H. tokunagai individuals were placed in the center of the choice device using a polypropylene pipe (1.5 cm in diameter). They were then presented with seawater from Tank A and B and the numbers 118 H. Matsuda et al.

A B

C

D c

Tank A a b Tank B c

Fig. 2. Diagrammatic representation of the experimental equipment used to examine the preference of Hypermastus tokunagai for chemical stimuli from Scaphechinus mirabilis. A. Tank A with S. mirabilis. B. Tank B without S. mirabilis. C. Choice device, anterolateral view. D. Choice device, top view. a, region with chemical stimuli; b, region without chemical stimuli; c, other regions. Black arrows show the direction of water ow. An arrowhead shows the position of H. tokunagai at the beginning of the experiment. of H. tokunagai in the regions “a”, “b”, and “c” were recorded every 5 min for 30 min. Their rst response when placed and any unusual behavior throughout the experiment were also recorded. Water temperature was maintained at 23.0–23.7°C, which was roughly the same as the temperature in the eld. The experiment was performed four times on June 14, 2007 using a total of 40 H. tokunagai collected on June 13, 2007. The choice device was washed with detergent and rinsed thoroughly with seawater after each exposure and was rotated by 180° each time. We conducted a similar experiment on June 25–27, 2009 using other 30 H. tokunagai that were collected on April 24, 2009 and starved for two months after detaching from their hosts. This fasting period was determined based on a previous observation that some eulimids would die but most would still be capable of moving. This experiment was repeated three times, and each group of 10 H. tokunagai was used three times. Water temperature was maintained at 25.3–25.8°C. In these experiments, position data recorded only for 10 min after starting the experiment were used for the analysis since several individuals crawled up the side walls and were trapped by the surface tension because of the extremely smooth shell. Individuals of H. tokunagai that escaped from the container by that time were excluded from the analysis. Preference for black or white substrate and dark or light condition: The role of vision in host recognition was evaluated by noting the preference for black or white substrate and dark or light condition on July 12 and 13, 2007 using 50 H. tokunagai specimens collected on July 12, 2007. The preference of H. tokunagai for black or white substrate was assessed using a circular polypropylene container (14 cm in diameter, 7 cm in height). The base of the container was divided into two equally sized regions using black and white polypropylene plates (Fig. 3A). The container was lled with seawater to a depth of 3 cm. Water temperature was maintained at 23.2–25.1°C and water surface was illuminated with the uorescent lamp at 280 Lux (SLX–1332, Sansho, Tokyo,±(4% of reading+5 digits)). Ten H. tokunagai were placed in the center of the container’s bottom surface using a polypropylene pipe and the number of H. tokunagai in each region was recorded every 5 Re-infection by a Eulimid Gastropod 119

A B a c

b d

Fig. 3. Diagrammatic representation of the experimental equipment used to examine the preference of Hypermastus tokunagai for white or black region (A) and for light or dark region (B). a and c, choice devices, anterolateral view; b and d, choice devices, top view. min for 30 min. This experiment was repeated ve times, and each group of 10 H. tokunagai was used twice. The preference of H. tokunagai for dark or light condition was assessed on the same day using the method described above with the following modi cations. The entire base of the container was white instead of half white, half black. Half of the container was covered with a black polypropylene plate to provide shade (Fig. 3B). The H. tokunagai individuals used were the same as those tested in the previous experiment. In these experiments, the position data recorded only for 10 min after starting the experiment were used for the analysis because most H. tokunagai individuals became inactive and/or escaped and were trapped with the surface tension by that time. Individuals of H. tokunagai that did not move throughout the observation were excluded from the analysis. Preference for light or dark condition in the substrate: We evaluated the visual ability of H. tokunagai to detect a host-resembling object by the preference for light or dark region in the substrate on November 9 and 10, 2007 using 40 H. tokunagai specimens collected on the rst day of the experiment. We laid glass beads (0.2 mm in diameter) on the bottom of a container (11.0 cm in diameter) then placed a black polypropylene plate (7.5 cm in diameter) in the center on top of the beads (Fig. 4A). The particle diameter of the glass beads is almost identical to the median diameter of the sediment inhabited by S. mirabilis that are infected by H. tokunagai (see Matsuda et al., 2010b). The region covered by the plate (44 cm2) was almost equivalent to the uncovered region around the plate margins. Ten H. tokunagai were placed along the margin of the container at equal intervals (Fig. 4C) and their position recorded every 15 min for 1 h, noting whether they were on the surface of the beads or submerged among the beads. Water temperature was maintained at 18.3–18.9°C and water surface was illuminated at 280 Lux. This experiment was repeated four times, and each group of 10 H. tokunagai was used twice. We then conducted a similar experiment using S. mirabilis instead of the black plate. This was done to test for the effect of chemical stimuli from the host and not for the physical presence of the host. A single S. mirabilis was placed in a smaller, transparent polypropylene container (7.5 cm in diameter) with several holes in the bottom. This smaller container was then set in the center of the larger container on the beads (Fig. 4B). The H. tokunagai individuals were the same as those used for the previous experiment. In these experiments, the position data recorded for 1 h after starting the experiment were used for the analysis. Individuals that stayed on the surface and were motionless throughout the observation were excluded from the analysis. 120 H. Matsuda et al.

A B sand dollar

glass beads

plastic plate ↓ ↓ ↓ hole

plastic container

↓ ↓ C

Fig. 4. Diagrammatic representation of the experimental equipment used to examine the preference of Hypermastus tokunagai for light or dark region in the substrate (A) and in the substrate with the presence of chemical stimuli from Scaphechinus mirabilis (B). A. A polypropylene plate placed on glass beads. B. A transparent container containing S. mirabilis placed on glass beads. C. Experimental container, top view. Arrowheads show the positions of H. tokunagai at the beginning of the experiment.

Data analysis: We compared ratios between selecting region with and without ow, and then compared ratios between selecting region with and without chemical stimuli, using generalized linear mixed models (GLMM) with binomial distribution with probit link function where “region” is the explanatory variable and “ratios” is the response variable. In the second and third experiments, we compared the number of individuals in each region based on GLMM with binomial distribution with probit link function. Trial and/or group were random effects. Effect of region was tested with Wald test for each experiment. All statistical analysis was performed using software program R (version 3.1.2) with package “lme4”.

Results

Re-infection ability Only ve (12.5%) of the 40 S. mirabilis were found infected by a total of 5 H. tokunagai (mean±S.D.: 4.81±0.80 mm SL, n = 5). All H. tokunagai were detected on the oral side of the host.

Factors affecting host recognition Preference for chemical stimuli from host: When H. tokunagai individuals were placed in the container, the majority of individuals were active in the region containing water ow. However, no signi cant difference between ratio of individuals selecting region with (region “a” + “b”) and without ow (region “c”) was detected, either immediately after (Table 1A: Wald test in GLMM, z = –0.1, P>0.05) or two months after (Table 1B: Wald test in GLMM, z = –0.98, P>0.05) being Re-infection by a Eulimid Gastropod 121

Table 1. Number of Hypermastus tokunagai in regions with and without chemical stimuli from Scaphechinus mirabilis. Experiment A was conducted four times using a total of 40 individuals. Experiment B was repeated three times using three groups (a, b, c) of a total of 30 individuals. I, II, and III indicate rst, second and third trial, respectively.

Group Samplig date Experiment date Choice Total a b c d

Region with chemical stimuli 3 3 2 3 11 Region without chemical stimuli 2 1 4 0 7 A June 13, 2007 June 14, 2007 Other regions 4 5 4 7 20

Excluded 1 1 0 0 2

Group

Samplig date Experiment date Choice a b c Total

I II III I II III I II III

Region with chemical stimuli 0 1 1 1 1 2 2 3 1 12 Region without chemical stimuli 2 1 1 6 1 0 2 1 1 15 B April 24, 2009 June 25-27, 2009 Other regions 4 5 1 3 6 1 5 6 2 33

Excluded 4 3 7 0 2 7 1 0 6 30

Table 2. Number of Hypermastus tokunagai in white and black regions (A) and light and dark regions (B). Experiment A and B was repeated ve times, respectively, using ve groups (a, b, c, d, e) of a total of 50 individuals. I and II indicate rst and second trial, respectively.

Group

Samplig date Experiment date Choice a b c d e Total

I II I II I II I II I II

White region 6 5 8 4 6 6 4 4 3 2 48 A July 12, 2007 July 12-13, 2007 Black region 1 0 0 1 1 0 1 3 1 1 9 Excluded 3 5 2 5 3 4 5 3 6 7 43

Light region 5 6 6 3 2 5 6 5 2 4 44 B July 12, 2007 July 12-13, 2007 Dark region 1 0 0 0 0 0 1 0 1 1 4 Excluded 4 4 4 7 8 5 3 5 7 5 52 separated from the host. In addition, no signi cant difference between ratio of selecting region with (region “a”) and without chemical stimuli (region “b”) was detected, either immediately after or two months after being separated from the host, respectively (Wald test in GLMM, z = 0.669, –1.562, both P>0.05). This indicates that H. tokunagai individuals are randomly distributed and the effects of water ow and chemical stimuli from the host on their behavior are not signi cant. Preference for black or white substrate and dark or light condition: In the experiments concerning preference for black or white substrate, and for light or dark condition, there were signi cant differences in number of individuals between the two regions (Wald test in GLMM, both P<0.001). The probability of selecting white and light regions was 0.842 and 0.917 respectively, indicating that the species preferred white over black regions and light over dark regions (Table 2A, B). Immediately after being placed in the container, most individuals moved rapidly toward the preferred region and stayed there. In the experiment concerning preference for the black or white 122 H. Matsuda et al.

Table 3. Number of Hypermastus tokunagai in light and dark regions in the substrate in the absence (A) or presence (B) of chemical stimuli from Scaphechinus mirabilis. Experiment A and B was repeated four times, respectively, using four groups (a, b, c, d) of a total of 40 individuals twice. I and II indicate rst and second trial, respectively.

Group

Samplig date Experiment date Choice a b c d Total

I II I II I II I II

Dark region 2 3 3 3 6 7 6 1 31 Submerged A November 9, 2007 November 9-10, 2007 Light region 4 1 3 5 3 2 2 4 24 Surface (excluded) 4 6 4 2 1 1 2 5 25

Dark region 9 6 4 3 4 4 5 4 39 Submerged B November 9, 2007 November 9-10, 2007 Light region 0 0 2 2 2 5 3 5 19 Surface (excluded) 1 4 4 5 4 1 2 1 22

substrate, H. tokunagai individuals initially placed in the white region frequently stopped at the boundary with the black region and reversed their direction back into the white region. Preference for light or dark condition in the substrate: When H. tokunagai individuals were placed in the container with glass beads and a black plate, some individuals rapidly submerged themselves among the beads, moved toward the black plate, and stayed there. Although no signi cant difference between numbers of individuals occurring in the light and the dark regions was detected (Wald test in GLMM, z = 0.862, P>0.05), more than half of them preferred the dark region (Table 3A). On the other hand, in the presence of chemical stimuli from the hosts, the probability of selecting the dark regions was 0.695 and the subjects signi cantly preferred the dark region over the light region (Wald test in GLMM, z = 2.047, P<0.05) (Table 3B).

Discussion

We experimentally demonstrated that H. tokunagai is capable of detecting and re-infecting S. mirabilis after being detached in the eld. The specimens that re-infected S. mirabilis are considered to be adult or sub-adult because most of them were above the known size at maturity (3.5 mm SL for males and 5.0 mm SL for females, respectively) (Matsuda et al., 2013). In areas containing large numbers of S. mirabilis, there is a high likelihood that H. tokunagai individuals come in close proximity to S. mirabilis as the host moves throughout its habitat. However, because adult H. tokunagai were found attached to S. mirabilis that were con ned in the eld enclosures, we hypothesize that H. tokunagai is able to detect its host at some distance. We rst evaluated whether H. tokunagai uses chemical stimuli to nd its host. The use of chemotaxis in host recognition has been reported for a range of symbiotic (Van den Spiegel et al., 1998; Eeckhaut et al., 2000; Vaïtilingon et al., 2004). Most eulimid species possess an osphradium and use it as a typical chemosensory organ (e.g., Warén, 1983; Altnöder et al., 2007) specialized for detecting odors (Hughes, 1986). However, virtually nothing is known about the effect of chemical stimuli on the behavior of eulimids. The results of this study revealed that H. tokunagai was not evidently attracted by seawater containing host-related chemicals. Their behavior did not change even after fasting for two months. Thus, chemical stimuli, if present, do not play a primary role in spotting H. tokunagai at a distance. H. tokunagai is commonly found on the aboral, shaded side of S. mirabilis (Matsuda et al., 2008). However, our results suggest that this species prefers light region to dark one. We have Re-infection by a Eulimid Gastropod 123 previously found that infection by H. tokunagai is more common in inshore regions than offshore (Matsuda et al., 2010b). They may move toward the shallower areas in the sea that receive more sunlight, after detachment. In addition, H. tokunagai individuals preferred the white region to the black region, and frequently stopped at the boundary between the two. This means that H. tokunagai may be able to detect the contrast between black and white under lighted conditions. As a number of H. tokunagai individuals submerged in the glass beads when exposed to light and moved under the black plate, they may use vision to detect the object (e.g., a host), and then approach and actively move to the underside of the object to facilitate attachment. Although we used transparent glass beads in the experiment, it is considered likely that H. tokunagai can also detect sunlight in the eld because they are only shallowly submerged in the bottom sediment. Cabioch et al. (1978) suggested that “Blacis alba” [=Melanella alba], a temporal parasite of the holothuroid Neopentadactyla mixta, was capable of detecting environmental stimuli, particularly light. These authors also reported that M. alba may move randomly relative to the direction of current and detect its host using sight. Since most eulimid species have advanced eyes with a lens (Warén, 1984) and H. tokunagai is no exception, they presumably use vision to recognize objects that resemble hosts. Van den Spiegel et al. (1998) suggested that the rst approach of the symbiotic shrimp Synalpheus stimpsoni to a host-like object is the use of visually mediated information, followed by olfaction, which enables this species to recognize an appropriate host. They also stated that host-related chemicals may not be detectable by the shrimp as they are dispersed in the water ow under natural conditions. As for H. tokunagai, it is likely that the surrounding seawater around the host population is uniformly lled with host-related chemicals since the host S. mirabilis forms beds in shallower areas (Matsuda et al., 2010b). In the present study, H. tokunagai were active under conditions containing chemical stimuli from the host S. mirabilis, and individuals of H. tokunagai signi cantly preferred a dark region in the substrate in the presence of chemical stimuli. Therefore, it is assumed that H. tokunagai move toward the black object when they sense the chemicals from the host, which will increase the possibility of host encounter. In the western Seto Inland Sea, H. tokunagai is commonly found on S. mirabilis but has never been found to parasitize other species of echinoderms (Matsuda et al., 2010a) suggesting that this eulimid is highly host-speci c. A number of other eulimid species also exhibit a high degree of host speci city (Warén, 1984). Thus, in addition to the foregoing role, sensory systems such as olfaction are likely to play a role in host detection as visual selection alone is insuf cient to mediate host speci city. While the ability of H. tokunagai to locate its host cannot be ascribed entirely to olfaction and chemosensory competence, these may play a role in nal attachment. We noted that H. tokunagai rotated upon rst contact with the spine of S. mirabilis during the laboratory experiment (Matsuda et al., unpublished), suggesting that tactile stimuli associated with the host’s spines may also play a role in host recognition by H. tokunagai. In the present study, we tested factors affecting re-infection from the viewpoint of how they recognize their host, focusing on effects of chemical stimuli and visual keys, but the interaction between individuals of H. tokunagai was not considered. As for H. tokunagai, it was suggested that males may move around, most likely to nd females for mating (Matsuda et al., 2013). When the eulimid moves to another host sand dollar, the presence of conspeci c individuals on the host may stimulate re-infection. The behavior of H. tokunagai may vary according to seasons and sex, especially in relation to mating. Similarly, the secretion intensity and chemical compositions of host-related chemicals may vary according to the seasons. Further research considering seasonality would be bene cial for elucidating mechanisms of re-infection and host-parasite association.

Acknowledgments

We are grateful to Dr. Anders Warén, Swedish Museum of Natural History, for useful advice 124 H. Matsuda et al. on the manuscript. We thank the members of the Laboratory of Aquaculture, Hiroshima University for constructive discussion. I am also grateful to Mr. Koichi Miki of the Tana Marine Biological Laboratory, National Fisheries University and the members of the Laboratory of Aquatic Biology, National Fisheries University, for assistance during the sampling. Thanks go to Mr. Minoru Saito of Tokushima University for valuable comments on the manuscript. Part of this work was supported by a Grant-in-Aid for JSPS Fellows (22–8067 to HM).

References

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(Received February 22, 2014 / Accepted June 19, 2015)

ハスノハカシパンに寄生するトクナガヤドリニナの宿主発見機構

松田春菜・浜野龍夫・長澤和也

要 約

ハナゴウナ科のトクナガヤドリニナ Hypermastus tokunagai は宿主であるハスノハカシパン Scaphechinus mirabilis の腹面に外部寄生する種であるが，宿主から離れやすく，底質中からも生きた個体が見つかって いる。そこで，宿主から離れたトクナガヤドリニナが再び寄生できるかを野外実験で確かめるとともに， どのようにして宿主を探索・認識するのかを室内実験で検討した。まず，寄生されていないハスノハカシ パンをケージに入れ，海底で一定期間置いた後に取り上げたところ，成体のトクナガヤドリニナが見出さ れ，底質中にいたトクナガヤドリニナが宿主を見つけ出して寄生できることが確かめられた。明暗の選択 性実験では，トクナガヤドリニナは明るい領域を有意に好み，白黒の選択性実験では有意に白い領域を好 む傾向を示した。白黒の境目では頻繁に留まる様子が観察されたことから，明るい環境下で白黒のコント ラストを感知する可能性が高いことが示唆された。一方で，底質上にカシパンに見立てた黒色板を置くと 潜砂して暗い領域に集まる個体が多く観察され，黒色板からカシパンの化学物質を溶出させた条件では有 意に集まる傾向を示した。ハスノハカシパンの化学刺激だけで宿主を見出すことはできなかったことか ら，宿主を見出し，近づくプロセスの中で視覚の果たす役割があると推察された。宿主特異性を有するト クナガヤドリニナにおいて，化学刺激や接触刺激の存在が最終的に宿主に寄生する上での決めてとなる可 能性が考えられる。