Function of flexible hairs

1 2 3 Functional morphology of flexible periostracal hairs in Trichotropis cancellata 4 (, ) 5 6 7 Erika V. Iyengar1,*, Michael I. Sitvarin1, Marianne Cataldo1 8 9 Biology Department, Muhlenberg College, 2400 W. Chew St., Allentown, PA 18104, USA 10 *Email: [email protected] 11 12 13 14 Corresponding author’s telephone, fax, email, and address to which proofs should be sent:

15 Dr. Erika Iyengar, Department of Biology, Muhlenberg College 16 Shankweiler Hall, 2400 W. Chew St. 17 Allentown, PA 18104, USA. 18 [email protected] 19 phone: (484) 664-3731; FAX: (484) 664-3002 20

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1 ABSTRACT 2 The marine snail Trichotropis cancellata possesses hairy projections of periostracum 3 (outer shell layer) whose function is unknown. While rigid shell projections in molluscs have 4 been extensively studied, the purpose of flexible extensions of periostracum is less clear. None 5 of the functions previously proposed for periostracum (e.g., protection from erosion and boring) 6 are promoted when it is drawn into hair-like projections. We investigated supplementary 7 functions that may be served by flexible periostracal hairs, including predator deterrence, 8 alteration of flow vectors to promote feeding or affect turbulence dynamics during freefall, and 9 camouflage. Within two crabs (Cancer productus and C. oregonensis) and one sea star 10 (Pycnopodia helianthoides), some predators consumed snails with the periostracum removed 11 more often than snails with an intact hairy periostracum. However, in all three predatory , 12 there were individuals that showed no significant preference; 10-day-long field studies showed 13 no difference in the rate of predation on hairy- versus smooth-shelled snails. The hairs did not 14 alter flow around the shells consistently in laboratory flume experiments. Additionally, hairy- 15 and smooth-shelled kleptoparasitic snails grew at rates that were statistically indistinguishable, 16 while hairy suspension feeding snails grew more slowly. The hairs did not impact the orientation 17 of a snail after a falling event or the time to righting after a fall. The presence of the hairs did 18 deter settlement by barnacles. We conclude that the hairy periostracum acts as a slight deterrent 19 to crab predators, a more powerful deterrent to sea star predators (such as Pycnopodia), and a 20 deterrent to the settlement of large calcareous epibionts, such as barnacles, that would increase 21 the weight the snail must bear and potentially increase drag. 22 23 KEY WORDS: periostracum, predator defense, suspension feeding, epibionts 24 25

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1 2 Shell morphology varies greatly among marine mollusks and the functional significance 3 of shell sculpture has been the subject of many studies. Rigid projections can deter predators 4 (e.g., Palmer 1979, Vermeij 1987, Stone 1998, Donovan et al. 1999), promote stabilization in 5 soft substrata (Palmer 1977), promote the settlement of epibionts (Feifarek 1987), and affect 6 shell trajectories during falls (Palmer 1977). Some crabs discriminate between individuals of the 7 same prey species, preferring thin- to thick-shelled snails (Palmer 1985). Blade-like shell varices 8 on foliatum, a snail sympatric with the focal of our studies, Trichotropis 9 cancellata Hinds, 1849 (Gastropoda, Capulidae; hereafter referred to as Trichotropis), 10 destabilize the snail as it falls through the water. This destabilization increases the likelihood 11 that Ceratostoma will land aperture-down, and reduces the opportunity for fish to bite the foot of 12 a dislodged snail (Palmer 1977). Opercula and feet of , a similarly-sized 13 snail that co-occurs with Trichotropis, have been found in fish guts (Palmer 1977), indicating 14 that adaptations promoting aperture-down landings may be beneficial for Trichotropis. 15 The marine snail Trichotropis cancellata lives subtidally on large rocky substrata as a 16 suspension feeder or facultative kleptoparasite, stealing food from a nearby tube-dwelling 17 polychaete worm (Pernet & Kohn 1998, Iyengar 2002, 2005). As a suspension feeder, the snail 18 draws seawater into its mantle cavity through the action of beating cilia on its gill and uses the 19 gill to trap suspended food particles in mucus (Yonge 1962). When parasitizing, the snail resides 20 on a host, extends its pseudoproboscis into the host’s mouth, and diverts food from the host 21 (Pernet and Kohn 1998). Thus kleptoparasitism allows the snail to access food that has been 22 concentrated previously by the host, important in a nutritionally dilute environment where the 23 process of suspension feeding expends measurable amounts of energy (Riisgård & Larsen 2001). 24 In the field, Trichotropis predominantly resides on or next to a tube worm in the summer, but 25 sexually mature snails leave their hosts in the winter to find mates (Iyengar 2005). The snails 26 utilize at least five different species of worm hosts from three different families (Iyengar 2005). 27 All size classes of Trichotropis grow significantly more quickly as kleptoparasites than as 28 suspension feeders (Iyengar 2002, 2004). Medium-sized snails co-occurring on a worm grow 29 more slowly than snails occurring alone on a host, suggesting that these snails compete (among 30 themselves and with the worm host) for a limited resource: the amount of food a worm host can 31 provide (Iyengar 2002). Large snails positioned in pairs, compared with those residing alone on

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1 a host, did not show a similar growth reduction, likely because these larger are able to 2 supplement their kleptoparasitic diet with effective suspension feeding (Iyengar 2002). 3 While multiple functions for rigid shell extensions have been elucidated, the function of 4 flexible periostracal extensions is less clear. The periostracum is the outer shell layer of 5 mollusks, generally thin and proteinaceous. The common name for Trichotropis cancellata is 6 “cancellate hairy shell snails,” reflecting the hair-like projections of periostracum that cover the 7 outside of the adult shell (Figure 1; but these hairs are absent from the larval shell, Iyengar 8 personal observation). Bottjer and Carter (1980) found that hair-like extensions of periostracum 9 in bivalves were common among infaunal and semi-infaunal bivalves in low-energy silty 10 environments where the potential for shell abrasion is low. While Trichotropis is often found in 11 rocky environments, shell abrasion is low for this species; the periostracum on the shell whorls is 12 often intact, even in older snails (although often abraded at the tip of the spire; Iyengar personal 13 observation). Other distantly-related marine mollusks, such as Fusitriton oregonensis 14 (Gastropoda) and many Mytilacea (Bivalvia), also possess hirsute proteinaceous projections that 15 cover the calcium carbonate shell (although in the case of the Mytilacea these hairs are probably 16 not periostracal in origin; Bottjer & Carter 1980). It is unknown whether these hair-like 17 projections are adaptations, and whether they serve the same purpose(s) across groups. No 18 experimental studies to date have investigated the function of periostracal hairs in gastropods; 19 besides Bottjer (1981, 1982), no detailed studies of the secondary functions of gastropod 20 periostracum have been performed. 21 To determine whether periostracal hairs parallel the function of some rigid shell 22 projections and deter predators, we simultaneously offered intact (hairy) snails and snails with 23 their periostracum removed (smooth snails) to a variety of potential predators in choice 24 experiments. Predator species included crabs, sea stars, snails and fish. We predicted that the 25 predator would prefer to consume smooth rather than hairy snails. For crabs, we reasoned that 26 removal of the hairs may allow the crab to gain a firmer purchase on the snail. For sea stars, fish 27 and snails, we hypothesized that the hairs may cause an uncomfortable prickly sensation during 28 predator feeding. Pycnopodia helianthoides (one of the sea star predators) digests Trichotropis 29 internally, rather than everting its cardiac stomach and digesting the snails externally, as the sea 30 star does for larger prey items (T. Gage personal communication). Nucella lapillus, a congener 31 of one of the snail predators we used, prefers mussels from which the periostracum has been

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1 removed (Harper & Skelton 1993). Other snails that bore into prey can be deterred by bivalve 2 spines (Stone 1998), and perhaps the flexible projections of Trichotropis cancellata act similarly. 3 Widely accepted hypotheses for the primary functions of the gastropod periostracum 4 include protection from acidic waters and boring organisms (Bottjer 1981) and assisting in shell 5 calcification by serving as a site for initial calcification and preventing seawater from mixing 6 with the extrapallial fluid (Bottjer & Carter 1980; Harper 1997). However, if these are the only 7 functions of the periostracum, there is no need for it to be drawn into hair-like projections, as in 8 Trichotropis. A proposed ancillary purpose of the periostracum is prevention of infestation by 9 boring epibionts (Bottjer & Carter 1980), but hirsute projections may not be necessary to prevent 10 settlers. These flexible hairs may have supplementary functions. In Trichotropis cancellata, we 11 hypothesized that the hairs may serve a variety of purposes, including perhaps promoting 12 suspension feeding or kleptoparasitism by affecting flow around the shell. In the studies 13 reported herein, we investigated whether these hairs (1) deter predators, (2) affect flow vectors 14 and thus aid in suspension feeding or kleptoparasitism, (3) promote the settlement of epibionts to 15 provide camouflage and (4) affect turbulence dynamics while falling to promote the likelihood of 16 the snail landing in a protected orientation (aperture down). 17 18 METHODS 19 Animal collection and experiments were performed near the Friday Harbor Laboratories 20 (FHL), San Juan Island, Washington state, during the summers of 2004 and 2005. We collected 21 Trichotropis cancellata (hereafter referred to as Trichotropis) from various local subtidal 22 locations using SCUBA. All subtidal field experiments were conducted at Shady Cove (48o 23 33.94’N, 123o 00.39’W) at approximately 15 meters in depth, unless noted otherwise. 24 Creating snail shell treatments. To examine the potential functions of a hairy 25 periostracum, we performed various experiments comparing the performance of intact, unaltered 26 (except for dirt and epibiont removal) “hairy” snails and “smooth” snails (those without a 27 periostracum). The entire periostracum was removed because merely clipping the hairs left 28 behind short, projecting stumps. In 2004 and 2005, we created “smooth” snails by hand-filing 29 (using a ridged metal file) the periostracum but not the underlying calcium carbonate shell. In 30 2004, we used small scissors to trim the shell at the growing edge of the aperture back to where it 31 was thickly calcified in both treatments because filing that area would destroy the thinly-

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1 calcified shell. Thus, both treatments received a (minimal) consistent amount of shell damage. 2 In 2005, rather than filing near the shell aperture, we closely trimmed the apertural hairs of the 3 hand-filed shells using scissors, leaving the aperture intact in both treatments. In 2005, we either 4 hand-filed snails (as just described) or dipped them individually in a 50% bleach solution 5 (aperture up so as not to harm the live animal) and then removed the periostracum by hand, 6 leaving the calcium carbonate shell intact. 7 Do the hairs deter predators in the laboratory? To determine whether the periostracal 8 hairs deter predators, we simultaneously offered intact (hereafter referred to as hairy) snails and 9 snails with their periostracum removed (hereafter referred to as smooth) to a variety of potential 10 predators in choice experiments. In our 2004 experiments, predators included three species of 11 crab (Cancer magister, C. oregonensis, and C. productus), three species of sea star (Pycnopodia 12 helianthoides, Dermasterias imbricata, and Orthasterias kohleri), and two species of fish 13 (striped sea perch: Embiotoca lateralis and white-spotted greenling: Hexagrammos stelleri). In 14 2005, we tested the sea star Leptasterias hexactis and the predatory snails Nucella lamellosa and 15 Lirabuccinum (formerly Searlesia) dira. The crabs and sea stars (except L. hexactis) were 16 collected using SCUBA at Shady Cove. L. hexactis, N. lamellosa and L. dira were collected at 17 various intertidal sites around San Juan Island, and the fish were collected using a beach seine at 18 Jackson’s Beach. All individuals were used in experiments within two weeks of collection and 19 returned immediately following the completion of the experiment. Every two days, the 20 individuals not currently involved in experimental trials were fed small frozen shrimp ad libitum. 21 Once a predator began a trial, it received no supplemental food for the duration of the 22 experiment. 23 Cancer productus (red rock crab) are sympatric with T. cancellata and use powerful 24 crushing claws to feed on a wide variety of snails, clams, echinoderms, and other crustaceans 25 (Carroll & Winn 1989). C. oregonensis have a similar habitat range and diet as C. productus but 26 are typically smaller, with less powerful claws. Therefore, we predicted that C. oregonensis 27 would be more selective than C. productus when choosing prey and might not be able to 28 consume the largest snails offered. C. magister are generalist feeders (Carroll & Winn 1989), 29 but feed mostly on crustaceans and clams and co-occur less often with T. cancellata as they 30 commonly inhabit mud-sand substrata.

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1 All sea star and fish species used as predators occur sympatrically with Trichotropis. We 2 chose sea stars for which diet records (Lambert 1981) indicate that they eat snails (although not 3 necessarily Trichotropis). We used this broad diet requirement rather than choosing only sea 4 stars recorded to feed on Trichotropis because diet records are often incomplete. This decision 5 should not skew the results because if a predator’s diet does not include Trichotropis, then it will 6 not eat either snail treatment. We used fish species that ate small invertebrates and possessed 7 jaws likely powerful enough to crush the shell of Trichotropis (Micheli & Halpern 2005, L. Britt 8 personal communication). 9 During each trial, crabs were placed singly in separate, plastic containers in a flow- 10 through sea table or a cold room to maintain a temperature that approximated field conditions. 11 Sea stars, except L. hexactis (see below), were placed individually in flow-through sea tables (2 12 m long × 1 m wide × 0.15m deep). The fish were maintained as a school (5 to 6 fish), separated 13 by species, in a circular flow-through tank (1 m in diameter, 0.5 m deep). 14 In June and July 2004, twenty snails (five smooth large [16-18 mm in length; measured 15 from whorl apex to the tip of the siphonal canal], five hairy large, five smooth small [10-12 mm] 16 and five hairy small) were placed in a container immediately following introduction of the 17 predator. During an experimental trial, the identity of all consumed snails was recorded twice a 18 day for seven days and consumed individuals were replaced. Consumption data were summed 19 over the seven day period. Any data interval in which a predator consumed more than four 20 individual snails of any single category (small hairy, small smooth, large hairy, large smooth) 21 between checks was removed from the analysis because the intended ratio among treatments was 22 drastically altered (there were originally only five of each choice category present). Only 23 individual predators that consumed at least ten snails over the course of the experiment were 24 considered in statistical analyses. Chi-square goodness of fit tests (with the null hypothesis that 25 there was an equal proportion of snails consumed in each category) compared the number of 26 snails eaten in each of the four categories (hairy small to smooth small to hairy large to smooth 27 large), first at the level of separate individual predators and then by summing results across 28 predators to investigate whether significant choice occurred at the species level. We also 29 performed chi-square goodness of fit tests on two categories at a time, combining data across the 30 other categories (i.e., consumption of hairy snails compared with smooth, summed over sizes or

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1 consumption of small snails compared with large, summed over snail treatment), first at the level 2 of each individual and then at the species level. 3 We performed analyses at both levels because there may be variation, with only some 4 individual predators showing strong (significant at the level of the individual) preference among 5 treatments. Individual predator preference is involved each time a snail encounters a predator. If 6 most individual predators display strong preference for smooth shells, then there is likely strong 7 selection pressure for hairy shells because, during each predatory encounter, that trait will likely 8 affect the probability of consumption. Alternatively, if a low proportion of predators shows a 9 strong preference, the selection pressure for hairy shells will likely be much less, as many of the 10 predators encountered will make meal choices based on other conditions. However, if most 11 individuals have a slight tendency to choose one snail over another, even if that tendency is not 12 significant at the level of the individual, it might result in a cumulative preference at the species 13 level. That cumulative preference could still drive the evolution of prey morphology. 14 In July 2005, we tested whether the predatory snails Nucella lamellosa and Lirabuccinum 15 dira and the sea star Leptasterias hexactis preferred smooth Trichotropis. These predators were 16 maintained for a week individually (in the case of L. hexactis) or in groups of six (in the case of 17 the snails, each species maintained separately) in flow-through containers (19 cm long × 14 cm 18 wide × 9 cm deep for the snails; 21.5 cm long × 15 cm wide × 40 mm deep for the sea star) 19 submerged in a flow-through seawater table. Each container held ten hairy and ten smooth 20 Trichotropis of medium size (7.4-23.8 mm in the L. hexactis trials, 11.8-19.0 mm in the N. 21 lamellosa trials, and 13.2-23.1 mm in the L. dira trials; there was no significant Trichotropis size 22 difference between treatments for any of the three predators tested, t test, P > 0.3 for each). Due 23 to the low number of snails eaten in these experiments, we used chi-square tests of independence 24 (using the statistical program JMP IN to calculate the Fisher’s exact test) to compare the number 25 of dead and live snails in each treatment (hairy versus smooth) at the end of the experiment. 26 Do hairs deter predation in the field? Field experiments of predator choice were 27 conducted in 2004 to confirm laboratory results. Each experimental rack was a 20.5 × 35.5 cm 28 Plexiglas sheet (placed directly on the substratum) with hairy and smooth snails in alternate 29 positions. Each rack contained either suspension feeding or kleptoparasitic snails, as the hairs 30 may differentially affect the two feeding strategies. Individual Trichotropis (13-15 mm in 31 length) were tethered using sewing thread and superglue. For the suspension feeding treatment,

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1 one end of the tether was tied around the penultimate whorl of the snail and the other end was 2 attached directly to the Plexiglas, restricting the snails to suspension feeding. For the 3 kleptoparasitic treatment, the other end of the tether was attached to a rock, next to the tube 4 opening of a Serpula columbiana (a tube-dwelling polychaete worm that is the most frequently- 5 used host of Trichotropis in this area; Iyengar 2005). Loops of latex surgical tubing held the 16 6 rocks (collected from Argyle lagoon and each with a single worm) to the racks for 7 kleptoparasitic snails. Six racks contained suspension feeding snails (5 replicates of each 8 treatment (hairy and smooth) per rack, for a total of 30 replicates per treatment); three racks 9 contained kleptoparasitic snails (8 replicates of each treatment per rack, for a total of 24 10 replicates per treatment). 11 On 13 July 2004, all racks were deployed east of Cantilever Point (offshore of FHL 12 Laboratory 6) at a depth of 18 m on a flat, silty area at the base of a rock wall with Serpula and 13 Trichotropis. Cancer productus, C. magister, C. oregonensis, Pycnopodia helianthoides, 14 Dermasterias imbricata, and Nucella lamellosa are common in this area (Iyengar personal 15 observation), and the other sea star and fish predators used in our laboratory experiments also 16 likely occur at this site. The racks were checked on 15 and 18 July and retrieved on 23 July 17 2004. During each check, we recorded mortality and whether each kleptoparasitic snail was 18 positioned next to a worm (only snails found on worms each check were considered in the 19 analysis). Snails rarely escape from their tethers and, if this does happen, they usually do not 20 move far from their initial position (Iyengar unpublished data). Therefore, during each check, 21 we carefully searched the area around the racks for any snails that may have escaped. We used 22 chi-square tests of independence (using the statistical program JMP IN to calculate the Fisher’s 23 exact test) to compare the number of dead and live snails in each treatment (hairy versus smooth) 24 at the end of the experiment. Suspension feeders and kleptoparasites were analyzed separately. 25 Do hairs affect flow around an inert shell in a laboratory flume? To test whether the 26 periostracal hairs influence ambient flow rates around the shell and thus perhaps affect the 27 efficiency of suspension feeding or kleptoparasitism, we investigated the flow rates and fluid 28 trajectories around hairy and smooth shells. In summer 2005, we froze hairy and smooth 29 Trichotropis (bleach was used as described above to create smooth shells), inserted modeling 30 clay (Sculpey, Polyform Products Co., IL) into the aperture of the shell to create an attachment 31 surface, baked the shells with clay for 15 minutes at 130oC, and then affixed the clay and shell to

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1 a wooden dowel (diameter = 4.25 mm, close to that of Serpula columbiana, a common host of 2 Trichotropis; Iyengar 2002) using superglue. Snails were glued individually onto dowels in one 3 of two positions: “side-mounted” with the shell aperture parallel to the length of the dowel and 4 perpendicular to the ground (which meant that the shell spire axis was at an angle 25o±4.4 5 [average±SD] from the dowel axis), or “top-mounted” with the shell aperture perpendicular to 6 the dowel length and horizontal to the ground (which meant that the shell spire axis was at an 7 angle 100o±5.4 from the dowel axis). The side-mounted position approximated the orientation of 8 kleptoparasitic snails in nature, as the snails are positioned next to the opening of tube worms. In 9 contrast, the top-mounted position approximated the orientation of suspension feeding snails, 10 which have their aperture flush to the substratum. Three size classes of snails were used in the 11 side- and top-mounted treatments: small (9.4-13.6 mm in length), medium (14.8-18.1 mm in 12 length) and large (19.3-23.6 mm in length). Fifteen replicate shells of each treatment (hairy or 13 smooth) were used in each of the three size classes for each of the two mounting (side, top) 14 orientations (for a total of 90 shells in each orientation; each shell was used only in one mounting 15 orientation). 16 We placed single mounted snails in a re-circulating sea water flume, each snail positioned 17 4 cm above the mounting platform and 5 cm from either side wall, and exposed them to a 18 unidirectional laminar flow rate of 8.0 cm/s. Snails in nature likely experience this moderate 19 flow rate (Eckman et al. 1989, Eckman & Duggins 1993), which is within the range of those 20 used in previous experiments investigating the effect of flow on growth rates of suspension 21 feeders (Eckman & Duggins 1993). All experiments were conducted in a cold room to maintain 22 an average water temperature of 11oC, similar to natural conditions. To visualize water flow 23 around the shell, a 1.0 mL syringe delivered a slow, continuous dye stream (0.05-0.10 mL) of 24 calcein upstream of each shell. We videotaped from the side as the dye stream moved over each 25 shell (Figure 2). Side-mounted shells were filmed with the shell upstream of the dowel, the apex 26 of the spire pointed toward the base of the flume and the shell aperture at the top of the dowel. 27 Each top-mounted shell was filmed in two different positions: with the apex of the shell parallel 28 to the current, projecting upstream or with the apex of the shell facing away from the camera, 29 perpendicular to the current (thus we videotaped currents moving over the body whorl of the 30 shell, near the opening to the mantle cavity).

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1 We used the computer programs Image J, Adobe Photoshop, and iMovie to determine the 2 flow rate and trajectory of the dye around the shell using both video and extracted still frames 3 (one extracted frame per snail replicate). All distances described refer to measurements along a 4 straight line unless we explicitly refer to an arc distance, which follows the actual dye stream 5 trajectory. From the video, we calculated the flow rate of the dye stream (one flow rate per 6 replicate) as the distance from the point where the dye stream was first affected by the shell to 7 the downstream edge of the dowel (hereafter referred to as “horizontal distance”) per unit time 8 (Figure 2). We also measured from the videotape the distance from where the dye was first 9 affected by the shell to the upstream edge of the shell. From the extracted still frames we 10 measured the maximum height reached by the dye stream arc traveling over the shell (Figure 2), 11 and also the ratio of the arc distance to the horizontal distance. The arc distance was defined as 12 the distance from the syringe to the downstream edge of the dowel, following the dye stream. 13 This ratio was calculated to quantify the shape of the arc and determine whether that shape 14 differed between treatments, potentially complicating the interpretation of the flow rate data. 15 Including distances far from the influence of the shell in one treatment and not the other could 16 affect flow rate and arc height results, so we compared the horizontal distance measured in both 17 treatments. 18 Parametric statistical analyses were performed using the statistical program DataDesk 19 (Velleman 1997). We analyzed the effects of the periostracal hairs on flow rate and arc height in 20 separate analyses using two-way ANOVAs with treatment (hairy versus smooth) and shell size 21 (small, medium, large) as the independent variables. In a few cases, the syringe introducing dye 22 was not appropriately positioned initially and so the dye stream did not solidly hit the shell and 23 move in an arc (although sometimes a slight repositioning created a dye stream in the video 24 which allowed for measurements other than flow rate from an extracted frame). These few 25 replicates (never more than two per treatment×size category) were discarded from the analysis. 26 We assessed normality and equal distribution of variance using the Kolmogorov test and 27 Levene’s test, respectively, transforming data (ln y or y2) if necessary to meet ANOVA 28 assumptions. 29 Do hairs affect the growth rate of suspension feeders or kleptoparasites in the field? 30 In field experiments in summers 2004 and 2005, we tested whether the hairs affect the growth 31 rate of snails, possibly via alterations of ambient flow rates around the snails. In 2004,

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1 kleptoparasitic snails (tethered to rocks with the worm host Serpula columbiana) were used. In 2 2005, both kleptoparasitic and suspension feeding snails were studied. Both Trichotropis and 3 Serpula naturally occur at the field site. The outer apertural lip of each snail (in both hairy and 4 smooth treatments) was marked using nail polish to assess growth rates. Cleaned snails (length 5 = 9.1-16.35 mm in 2004; 8.6-18.4 mm for suspension feeders and 11.8-19.5 mm for 6 kleptoparasites in 2005; snails did not differ significantly in size between treatments: t test, P > 7 0.3 for each) were tethered to Plexiglas experimental racks as described above (Do hairs deter 8 predation in field studies?), but each rack was attached via cable ties to a cinder block (to 9 decrease the probability of predation) rather than placed directly on the substratum. Four racks, 10 each containing eight replicates of both kleptoparasite treatments (hairy and smooth snails, 11 alternately distributed within each rack for a total of 32 replicates per treatment), were deployed 12 on 7 July 2004 and retrieved on 23 July 2004. On 2 June 2005, five similar racks were deployed 13 with kleptoparasitic snails and five additional racks with suspension feeding snails, so that each 14 of these groups had 40 replicates each of hairy and smooth snails. We standardized the tube 15 diameter of the serpulid hosts (indicative of the size of the worm inside; Iyengar 2002) across 16 kleptoparasitic treatments to prevent host attributes from differentially affecting snail growth 17 rates. The racks were checked every 7-10 days to verify that the kleptoparasitic snails remained 18 on their respective worms. As kleptoparasitism greatly affects snail growth rate (Iyengar 2002), 19 any snail not on a worm for more than one check (during the entire experiment) was discarded 20 from further analyses. We retrieved the kleptoparasites on 30 June 2005 and the suspension 21 feeders on 6 July 2005. At the end of each experiment, we measured, using an ocular 22 micrometer on a dissecting microscope, the length of Trichotropis growth (old apertural lip 23 marked with nail polish to new apertural lip, measured in the spiral direction, hereafter referred 24 to as growth). 25 We analyzed the effects of the periostracal hairs (hairy versus smooth) on growth using a 26 two-way ANCOVA in DataDesk with snail length as the covariate and rack as the blocking 27 factor. We assumed there was a common slope shared by hairy and smooth snails within a 28 feeding mode (kleptoparasite or suspension feeder) and year. We assessed normality using the 29 Kolmogorov test and equal distribution of variance using Levene’s test. 30 Do hairs affect the settlement of epibionts in the field? Boshoff (1968) proposed that 31 the periostracum of bivalves inhibits settlement of epibionts. However, Bottjer and Carter

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1 (1980) discovered that, rather than inhibiting epizoan settlement in general, hirsute projections of 2 mussel periostracum selectively deterred boring epibionts and those epibionts that preferred 3 broad attachment areas. We investigated the same question using Trichotropis. The previously 4 described 2005 growth rate experiment using suspension-feeding snails also allowed for an 5 assessment of the impact of periostracal hairs on the settlement of epibionts. In addition to those 6 five racks, an extra rack of 10 snails (5 hairy, 5 smooth) was deployed and retrieved on the same 7 dates as the growth rate experiment, for a total of 45 possible replicates of hairy and smooth snail 8 settlement substrata. On these six racks, any epibionts present were identified and enumerated as 9 they must have settled during the five week experiment. While some snails were lost during the 10 experiment (reducing the sample size to below 45), a few individuals lost the nail polish marking 11 that would indicate their amount of growth during the experiment but could still be analyzed for 12 the presence of epibionts. Therefore, the sample sizes reported for this experiment (Table 4) are 13 larger than those reported for the growth rate of suspension feeding snails in 2005 (Figure 6). 14 Epibionts in general were compared qualitatively between hairy and smooth snails. For 15 the most common macro-epibiont (Balanus sp.), the number present on hairy versus smooth 16 snails was compared using a nonparametric Mann Whitney U test (data possessed a distribution 17 that deviated from normality that could not be adjusted through transformation), with the null 18 hypothesis that the two groups possessed an equal distribution. 19 To investigate the impact of periostracal hairs on the settlement of epibionts over a longer 20 time period, on 24 June 2004 we deployed 20 replicates of each of the following four treatments: 21 live hairy, live smooth, dead (frozen for one day to determine the impact of snail behavior on 22 epibiont composition) hairy and dead smooth Trichotropis snails (sizes = 14.1-24.6 mm in 23 length). Snails were interspersed in a Latin Square Block design across 4 experimental racks 24 similar to those described above (snails were tethered directly to the racks, racks were attached to 25 the top of cinder blocks). We collected these racks on 30 May 2005 and recorded the identity 26 and number of any epibionts present on the snails. However, many replicates were lost over this 27 time period (see results). 28 Do the hairs affect freefall and promote aperture-down landings in the laboratory? 29 To determine whether the flexible periostracal hairs of Trichotropis cause hydrodynamic 30 destabilization while falling, as seen with the varices of Ceratostoma foliatum (Palmer 1977), a 31 dropping experiment (adapted from Vermeij et al. 1987) examined whether the hairs affected

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1 landing orientation and righting time. Live specimens of hairy and smooth Trichotropis were 2 held just below the water surface with the aperture flush against the vertical wall of a Plexiglas 3 tank filled with sea water to a depth of 37 cm from a brick-lined bottom (to simulate what 4 happens when a Trichotropis is dislodged from its substratum). This distance was over ten times 5 the length of any of the snails tested, a ratio previously demonstrated to allow shell shape to 6 affect landing orientation in Ceratostoma foliatum (Palmer 1977). The chosen distance (37 cm) 7 was also greater than that used to examine the landing orientation of Calliostoma ligatum 8 (Vermeij et al. 1987), a snail slightly larger than Trichotropis. The orientation of the aperture 9 upon landing was recorded as: up, down, easier righting (a relatively short, straight extension of 10 the foot to contact the substrate), or difficult righting (the foot must either be extended more than 11 half the circumference of the shell or twist itself 180o to contact the substratum). A chi-square 12 goodness of fit test (with the null hypothesis that there was an equal proportion of snails in each 13 category) was used to determine whether the presence of hairs affected landing orientation. For 14 snails not landing aperture-down, the righting time for each snail post-landing was recorded as 15 the time taken for the foot to attach to the underlying brick surface, and the time until the snail 16 repositioned the shell over its foot. Separate 1-way ANOVAs examined the effect of periostracal 17 hairs on the time elapsed from landing until foot attachment, from foot attachment until the shell 18 was repositioned, and the total righting time (from landing until the shell was repositioned), 19 using snail size (small, medium, large) as a blocking factor. Parametric statistical analyses were 20 conducted using DataDesk, and we assessed normality using the Kolmogorov test and equal 21 distribution of variance using Levene’s test. We also ran the same analyses with the inclusion of 22 the snails that landed aperture-down, attributing to each a time of 0 seconds to foot attachment 23 and shell-repositioning. The results of the analyses including aperture-down landings were 24 statistically indistinguishable from when those individuals were omitted from the analyses, and 25 so these results are not specifically reported. 26 27 RESULTS 28 The hairs deter some predators in the laboratory. No snails of either treatment were 29 eaten by the two fish species (striped sea perch (n=6) and white-spotted greenling (n= 5)), the sea 30 star Orthasterias kohleri (n = 2) or the crab Cancer magister (n=7). Three Dermasterias stars

p. 14 Function of flexible hairs

1 ate no snails and the fourth only ate two snails (one small hairy and one small smooth). 2 Therefore, these five species were discarded from further analyses. 3 All eleven Cancer productus crabs consumed Trichotropis, with nine qualifying for 4 statistical testing, as they ate more than ten snails (ranging from 14-45 snails consumed) over the 5 seven-day experiment. One crab significantly preferred smooth snails (Table 1; df = 1, χ2= 6 5.49, P < 0.05), whereas eight crabs showed no preference (df = 1, P > 0.1 for each). Two crabs 7 significantly preferred small snails (df = 1, χ2= 11.95 and 4.77, P < 0.05), while seven crabs 8 showed no preference based on size (P > 0.1 for each). One crab significantly preferred small, 9 smooth snails to any other treatment (df = 3, χ2= 14.34 , P < 0.01), but eight others did not show 10 a significant preference (P > 0.1 for each). Summing consumption data across all C. productus 11 (Table 1), the species significantly preferred small snails (df = 1, χ2= 8.35, P < 0.005), and small, 12 smooth snails (df = 3, χ2= 11.94, P < 0.01), but did not significantly discriminate between 13 smooth and hairy snails (df = 1, χ2= 3.49, 0.05 < P < 0.1). 14 Nine of the eleven Cancer oregonensis consumed Trichotropis, with five eating more 15 than ten snails (ranging from 12-35 snails consumed) over the seven-day experiment, thereby 16 qualifying for statistical analyses. None of the five crabs included in analyses discriminated 17 based on the presence of the periostracum (Table 1; df = 1, P > 0.1 for each). Three crabs 18 significantly preferred small snails (df = 1, χ2= 4.48, 6.43, and 12.5, P < 0.05 for each), while 19 two crabs showed no preference based on snail size (P > 0.1 for each). Two crabs significantly 20 preferred small, smooth snails to any other treatment (df = 3, χ2= 10.78 and 13.75, P < 0.05 for 21 each), but three others did not show a significant preference (P > 0.05 for each). Summing 22 consumption data across C. oregonensis (Table 1), the species significantly preferred smooth 23 snails (df = 1, χ2= 3.92, P > 0.05), small snails (df = 1, χ2= 26.23, P < 0.001), and small, smooth 24 snails to the other three treatments (df = 3, χ2= 30.33, P < 0.001). 25 Four of the Pycnopodia helianthoides sea stars ate more than ten Trichotropis and so 26 qualified for statistical analyses, while one Pycnopodia ate only one snail (a small smooth snail) 27 and four did not eat any snails. Three Pycnopodia significantly preferred smooth snails (df = 1, 28 χ2= 4.44, 5.59, and 11.08, P < 0.05 for each), while one showed no significant preference (df = 1, 29 P > 0.05). None of the four Pycnopodia included in analyses demonstrated significant 30 discrimination based on snail size (df = 1, P > 0.05). One sea star preferred large, smooth snails

p. 15 Function of flexible hairs

1 to the other three treatments (df = 3, χ2= 8.41, P < 0.05) and one preferred both sizes of smooth 2 snails to either size of hairy snails (df = 3, χ2= 11.22, P < 0.05), while the other two Pycnopodia 3 did not discriminate significantly among the four treatments. Summing consumption across the 4 four Pycnopodia predators (Table 1), the species preferred smooth snails (df = 1, χ2= 15.56, P < 5 0.0001), large snails (df = 1, χ2= 4.20, P < 0.05), and large and small smooth snails to either 6 large or small hairy snails (df = 3, χ2= 21.25, P < 0.0001). 7 In the 2005 trials containing Leptasterias hexactis, Nucella lamellosa and Lirabuccinum 8 dira, no more than one snail died in any container. At the end of the week, no hairy snails (n = 9 50 for L. hexactis; n = 20 for both N. lamellosa and L. dira) were dead. In the tubs containing L. 10 hexactis, two smooth snails were dead and a sea star was subduing a third smooth snail when the 11 experiment ended. In the tubs containing N. lamellosa and L. dira, two and one (respectively) 12 smooth snails were dead. None of these three predator species discriminated based on the 13 presence of the periostracum (df = 1 for each, Pearson test statistic = 3.093, Fisher’s exact P > 14 0.2 for L. hexactis; 2.105, Fisher’s exact P > 0.4 for N. lamellosa, and 1.026, Fisher’s exact P > 15 0.5 for L. dira). None of the dead snails appeared to have been drilled, indicating that the 16 predators likely killed Trichotropis by suffocation. 17 The hairs do not deter predators in the field. For both kleptoparasitic and suspension 18 feeding snails, there was no significant difference in the number of smooth or hairy snails 19 consumed (Table 2), although these results should be interpreted conservatively due to the small 20 number of snails consumed. 21 The hairs affect flow around an inert shell in a laboratory flume to a small extent. 22 The dye stream never entered the spaces between the hairs, but rather moved in a smooth arc 23 over the shell. In all three mounting orientations, there was no significant difference in the 24 average flow rate around hairy and smooth shells and no significant treatment5size interaction 25 term, but there was a significant effect of snail size in each analysis, with large shells having a 26 higher flow rate than medium and small shells in the side-mounted orientation and higher than 27 the medium shells in the two top-mounted orientations (Table 3, Figure 3). In both the side- 28 mounted orientation and the top-mounted shells with spires parallel to flow, the maximum arc 29 height was significantly higher above hairy than smooth shells and the large shells had a 30 significantly higher arc than medium or small shells (Table 3, Figure 4). In the top-mounted 31 shells with spires perpendicular to flow, there was no significant effect of treatment, but size was

p. 16 Function of flexible hairs

1 significant, with the arc above the large shells significantly higher than the other two sizes (Table 2 3, Figure 4). 3 The ratio of the arc distance to the horizontal distance (as an indication of the shape of 4 the arc) was significantly affected by treatment and size in the side-mounted shells, with hairy 5 and large shells having significantly greater ratios (Table 3, Figure 5). This result supports the 6 finding that, when significant, the arc height is greater for hairy snails. In the top-mounted shells 7 with spires perpendicular to flow, there was no significant effect of treatment, but size was 8 significant, with small shells having a significantly smaller ratio than the other two sizes (Table 9 3, Figure 5). In the top-mounted shells with spires perpendicular to flow, there was no 10 significant effect of treatment, but size was significant, with small shells having greater ratios 11 than large shells, and there was a significant treatment×size interaction term (Table 3, Figure 5). 12 In examining all possible linear contrasts, small, smooth shells had a significantly greater ratio 13 than large, smooth snails, but this was the only significant pairwise comparison. 14 The distance between the point where the dye stream was first affected by the shell and 15 the upstream edge of the shell itself was significantly greater (26% greater) for hairy shells in

16 medium and small side-mounted snails (F1, 27=8.96, P < 0.01; F1, 26=6.8, P <0.05, respectively), 17 but was not significantly different in any of the other seven mounting/orientation/size 18 combinations (P > 0.4 for each). 19 The flow rate and arc height results of the side-mounted snails were not skewed by 20 differences in the location of syringe placement or the distance used to calculate flow rate. The 21 horizontal distance was only significantly different in the small top-mounted snails with the spire

22 perpendicular to the water flow (hairy had a greater distance than smooth snails: F1, 26=6.26, P < 23 0.05) and medium top-mounted snails with the spire parallel to the water flow (smooth had a

24 greater distance than hairy snails: F1, 26=7.58, P < 0.05); in both cases, the difference between 25 treatments was less than 10% of the shorter distance measurement. None of the other seven 26 mounting/orientation/size combinations were significantly different (P > 0.1 for each). 27 Hairs do not promote growth in snails in the field. In 2004, the smooth and hairy

28 kleptoparasitic snails grew at statistically indistinguishable rates (Figure 6, F1, 29 = 0.109, P >

29 0.7) and snail length was not a significant covariate predictor (F1, 29 = 0.85, P > 0.3), but the

30 effect of rack was significant (F3, 29 = 3.03, P < 0.05). In the 2005 growth rate experiments 31 (Figure 6), hairy and smooth Trichotropis kleptoparasites did not grow at significantly different

p. 17 Function of flexible hairs

1 rates (F1, 19 = 4.26, 0.1 > P > 0.05) and neither snail length nor rack were significant predictors

2 (F1, 19 = 0.66, P > 0.4 and F4, 19 = 1.12, P > 0.3, respectively). However, in the 2005 experiment

3 that utilized suspension feeders, the smooth snails grew significantly more quickly (Figure 6, F1,

4 52 = 5.62, P < 0.05), and snail length was a significant covariate predictor (F1, 52 = 5.40, P < 0.05)

5 while rack was not a significant factor (F4, 52 = 0.38, P > 0.8). 6 Hairs affect the settlement of epibionts in the field. Periostracal hairs of Trichotropis 7 did not prevent the settlement of all epibionts, but altered the composition of epibionts found on 8 the snails. On average, smooth snails possessed a significantly larger barnacle epibiont load per 9 snail (Table 4; Mann Whitney U: smooth N = 36, hairy N = 42, z-statistic = 4.81, P < 0.0001) 10 and the prevalence of barnacle epibionts was greater on smooth snails (Table 4). Hairy snails 11 collected more dirt than did smooth snails, and dirty, smooth snails had a higher barnacle 12 epibiont load than did dirty, hairy snails (Table 4). Clean, smooth snails were more likely to 13 have barnacles than dirty, smooth snails, whereas dirty, hairy snails were more likely to have 14 barnacles than clean, hairy snails (Table 4). In addition to the barnacle epibionts and dirt, 2.8% 15 of smooth and 16.7 % of hairy snails possessed small red algal blades (most often attached to 16 hair tips in the latter case) and 2.8% of the smooth snails had small hydroid epibionts. 17 Most of the snails in the June 2004-May 2005 epibiont settlement experiment were lost 18 over the course of the winter, either due to predation, arrival of hermit crabs that broke the 19 tethers, or unknown forces. Of the 18 snails recovered in May 2005, 17 were smooth (some of 20 which had a hairy body whorl due to shell growth post-deployment) and one was hairy. Any 21 epibionts on the hairy body whorls of smooth snails will be listed as occurring on hairy snails in 22 the following discussion. The smooth snails had the following epibionts: encrusting coralline 23 algae (2 snails), encrusting non-calcified red algae (4 snails, one was a hermit crab), encrusting 24 bryozoan (7 snails, one was a hermit crab), small blades of erect red algae on the spire or edge of 25 aperture (4 snails), Pseudochitinopoma sp. (5 snails, one was a hermit crab), and a small solitary 26 tunicate (Cnemidocarpa finmarkiensis; 1 snail). The hairy shells had small blades of erect red 27 algae (3 shells) and Pseudochitinopoma sp. (2 snails). 28 Hairs do not promote certain shell orientations after freefall in the laboratory.

29 There was no significant effect of treatment (hairy versus smooth; F1,27= 1.31, P > 0.2) or snail

30 size (small, medium, and large; F2,27= 2.69, P > 0.05) on the average time to attach the foot, to

31 reorient the shell so that it rested on top of the foot (treatment: F1,24= 0.01; P > 0.9; snail size:

p. 18 Function of flexible hairs

1 F2,24= 0.09; P > 0.9), or in total righting time (treatment: F1,27= 0.99; P > 0.3; snail size: F2,27= 2 2.12; P > 0.1). There was no significant difference in the frequency of landing positions between 3 treatments (neither when all four possible positions were included: chi-square; df = 3, P > 0.5, 4 nor when the analysis only considered landings that were aperture-up or aperture-down: chi- 5 square; df = 1, P > 0.5). In all size classes, individuals in both treatments often fell in an 6 orientation other than aperture-down. Many of the snails never managed to contact the 7 substratum with their foot and finally withdrew into their shell, ceasing attempts to right 8 themselves. 9 10 DISCUSSION 11 Risk of predation. The laboratory predation experiments demonstrated that while some 12 individual predators preferred smooth snails, most Cancer productus and Cancer oregonensis 13 showed no significant preference. However, C. oregonensis’ tendency to choose smooth snails, 14 while often not significant at the level of the individual, was significant when viewed across the 15 entire species (this species-level significance was not seen in C. productus). Thus we conclude 16 that the flexible periostracal hairs of Trichotropis are slightly advantageous against some crab 17 predators, but that the efficacy of this defense varies depending on the individual crab. 18 The predatory sea star Leptasterias hexactis and the predatory snails tested (Nucella 19 lamellosa and Lirabuccinum dira) did not significantly prefer smooth snails. While a thickened 20 periostracum in some bivalves likely deters predation by drilling predators (Wright & Francis 21 1984, Harper & Skelton 1993), shells of Trichotropis are rarely found with drill holes (Iyengar, 22 personal observation). Thus, drilling predators are likely not strong selective agents for 23 Trichotropis. Indeed, neither predatory snail species (N. lamellosa and L. dira) consumed more 24 than two Trichotropis (when presented with 40) during the course of a week, despite the absence 25 of an alternative food source. While Leptasterias hexactis, N. lamellosa and L. dira did not show 26 a statistically significant preference for consuming smooth snails, the low levels of predation 27 likely complicate this issue. Interestingly, all six of the Trichotropis consumed by these three 28 predators (out of a total of 180 snails), were smooth snails. At this point it is impossible to 29 definitively distinguish whether this pattern is due to predator preference or merely reflects 30 mortality due to elevated stress levels experienced during periostracum removal. However, very 31 few smooth snails that roamed in tanks without predators died, and a Leptasterias was observed

p. 19 Function of flexible hairs

1 consuming a smooth snail in one trial (Iyengar personal observation), suggesting that the dead 2 experimental snails were killed by predators rather than dying from stress. 3 Three individuals of the sea star Pycnopodia helianthoides preferred smooth snails, and 4 one showed no significant preference. Overall, this species preferred snails with the 5 periostracum removed. However, more than half of the Pycnopodia ate only one or no 6 Trichotropis over the course of the week. Therefore, periostracal hairs may confer an advantage 7 to Trichotropis against these sea stars and snails, but these species are not likely to be major 8 predatory threats to Trichotropis. 9 Palmer (1983) demonstrated that it is energetically more demanding for gastropods to 10 produce the organic matrix in their shells than the inorganic calcium carbonate crystals. 11 Therefore, if defense were the top priority for Trichotropis, it would be energetically cheaper for 12 the snail to produce rigid, heavily calcified shell projections than the organic matrix present in 13 periostracal hairs. Perhaps the flexible projections of periostracum increase the apparent size of 14 the snail and thus deter visually-oriented, gape-limited predators (e.g., fish), without incurring 15 the cost of bearing a heavier shell. While this possibility remains untested, it is not likely the 16 primary reason for the existence of periostracal hairs. Individuals of Trichotropis are not 17 obvious in natural conditions. Many SCUBA divers overlook these snails, despite their 18 prevalence, and only recognize them in situ after forming a specific search image (Iyengar 19 personal observation). Some predators may form a search image specific to Trichotropis. 20 However, a predator that utilizes a search image is likely to have had experience handling the 21 snails. Thus, that predator would be unlikely to be fooled by an apparently large (due to 22 periostracal hairs), but in fact structurally weak, shell. 23 The adventitious hairs of the mussel Modiolus rectus may allow it to sense predators at a 24 greater distance than calcium carbonate shell material would allow (Bottjer & Carter 1980). The 25 hair-like periostracal extensions on Trichotropis may similarly extend its sensory range, but the 26 advance warning is apparently insufficient to allow the snail to launch an effective defense 27 against some of its predators: hairy and smooth snails often were eaten at indistinguishable rates 28 in predator choice experiments. The periostracal hairs also may assist a kleptoparasitic snail in 29 determining when a worm host has its tentacles extended to feed and can provide food, but hairy 30 kleptoparasites did not grow more quickly in nature than did smooth kleptoparasites. The

p. 20 Function of flexible hairs

1 possibilities that the hairs act as warning structures or notify food availability have not yet been 2 specifically investigated, but appear unlikely. 3 Flow trajectories and growth rate. Surprisingly, the presence of periostracal hairs had 4 no effect on the flow rate around the shells of Trichotropis, and only significantly elevated the 5 maximum arc height in two of the three mounting orientations (side- and top-mounted with spire 6 parallel to the current). Rather than slowing the water flow via drag, the hairs appear to increase 7 flow rates in some situations. In some shells, hairs resulted in a more steeply-sided arc, one that 8 obtained a higher zenith but covered the same horizontal distance in the same amount of time as 9 shorter arcs. The arc length:horizontal distance ratio for shells in the side-mounted orientation 10 (modeling kleptoparasitic snails) was significantly greater for hairy shells, but the rate of flow 11 was not different between treatments. However, for the top-mounted shells with their spire 12 parallel to the current, despite a significantly greater maximum arc for the hairy shells, the arc 13 length:horizontal distance ratio was not significantly different between treatments. These results 14 suggest that, for snails flat against the substratum, the hairs merely begin altering ambient 15 currents farther upstream from the shell; the overall shape of the arc trajectories between hairy 16 and smooth shells were similar, the hairy shells merely had a larger arc overall. 17 It is unclear how a snail would benefit from having ambient currents follow a higher arc 18 over its shell. While a steeper arc (as recorded for the shells orientated as kleptoparasites) may 19 bring food-ladened currents more directly to the overarching feeding tentacles of the polychaete 20 host, our growth rate data do not support this hypothesis. There was no significant difference in 21 the growth rate of hairy and smooth kleptoparasitic snails in field experiments. Furthermore, 22 suspension feeding snails with a hairy periostracum grew significantly less than smooth snails. 23 These results indicate that if the periostracal hairs affect flow rates around the shell, these 24 alterations are not adaptations for feeding. 25 Epibionts. The effect of the periostracum in Trichotropis and Fusitriton oregonensis is 26 similar: the hairs and periostracum do not prevent the settlement of epibionts in general, but alter 27 the composition of epibionts found on the snails (this also occurs in Modiolus rectus; Bottjer & 28 Carter 1980). Bottjer (1981) found that Fusitriton with thick, hairy periostracum had fewer 29 boring organisms and epibionts with large calcareous skeletons, and concluded that the 30 periostracum prevents shell weakening and saves the snail from expending energy to drag around 31 a heavy load of epibionts. However, comparative observations of shell areas with and without

p. 21 Function of flexible hairs

1 periostracum in freshly collected snails (e.g., Bottjer 1981, 1982) are necessarily limited because 2 it is unknown whether the epibiont settled on an area bare of periostracum, or the epibiont caused 3 the underlying periostracum to shear off. Our experiments are the first to experimentally remove 4 the hirsute periostracum and examine the later epibiont community. So few hairy snails 5 remained in the June 2004-May 2005 settlement experiment that concrete conclusions are 6 difficult to draw, but these findings mirror those of the shorter 2005 settlement study: species 7 settle on both hairy and smooth snails but the presence of hairy periostracum promotes a 8 different epibiont community than bare calcium carbonate shell. 9 Smooth Trichotropis had significantly more barnacles and a higher prevalence of 10 epibionts. Balanus crenatus generally survive on Fusitriton only if they settle on bare calcium 11 carbonate surface (Bottjer 1981), similar to Trichotropis. Although some small barnacles can 12 grow on the lamellar periostracum, none grew to a visible size on the hairs. This finding 13 corresponds with Bottjer and Carter (1980) who found that 100% of the barnacles encrusting 14 Modiolus rectus were located on the lamellar periostracum and none were attached to 15 adventitious hairs. Bottjer (1982) found that Balanus crenatus could settle on both periostracal 16 and hard shell surfaces of Trichotropis, although he does not specify whether the barnacles 17 attached to the hairs or the lamellar periostracum. Because our settlement racks were only 18 deployed for five weeks, we do not know whether Balanus survive differentially on hairy and 19 smooth snails. Few Trichotropis have large, calcified epibionts (Iyengar personal observation), 20 which suggests that barnacles settling on hairy periostracum are at a high risk of tearing from the 21 substratum. Unlike Fusitriton, Trichotropis is a sedentary snail (Iyengar 2002). Therefore, the 22 deterrence of epibionts does not confer large energy savings due to a smaller load during 23 movement. However, preventing the increased surface area due to large, rigid epibionts likely 24 reduces the drag on sedentary Trichotropis. For a species that relies on maintaining its position 25 next to a tube-dwelling worm host (Iyengar 2002, 2005) but lives in an area with strong currents 26 (present around the San Juan Islands), traits that reduce the probability of becoming dislodged 27 and the energy required to hold onto the substratum would be adaptive. 28 Specimens of Trichotropis in the field often are covered with a layer of sediment (Iyengar 29 personal observation). In our growth racks, even after a period as short as five weeks, large 30 amounts of sediment accumulated more on hairy snails. Bottjer (1981) noted that a thin layer of 31 sediment is often trapped among the hairs of Fusitriton and hypothesized that the sediment may

p. 22 Function of flexible hairs

1 prevent the successful establishment of borers and other epibionts. Our data are contradictory on 2 this point. Among the smooth snails, the prevalence of barnacles and the average number of 3 barnacles per shell was lower for dirt-encrusted snails (as predicted by the hypothesis), but 4 among hairy snails, the opposite was true. However, our data should be interpreted 5 conservatively because we considered a snail to be dirt-encrusted only if there were extreme 6 amounts of dirt on the shells, these experiments occurred over a relatively short time, and we had 7 very small sample sizes (six hairy snails and two smooth snails). If Bottjer’s hypothesis (1981) 8 is true, the hair-like projections effectively deter certain epibionts indirectly rather than by direct 9 action, which implies that their efficacy should differ according to local environmental 10 conditions. If there is little sediment in the area, almost no sediment will coat the snail despite 11 the presence of hairs, and epibionts should be more prevalent on both Trichotropis and 12 Fusitriton. It is clear that the hairy periostracum of these snails (and the morphologically similar 13 projections on some mussels) impact the presence of various species of epibionts. However, 14 further research into the exact mechanism of this deterrence must be performed before this 15 finding can be effectively utilized in practical applications, such as the creation of new physical 16 (rather than chemical) antifouling systems. 17 Orientation while falling. While rigid planar varices increased the likelihood that 18 Ceratostoma foliatum would land aperture down after a fall (Pamer 1977), the flexible 19 periostracal hairs of Trichotropis did not have a comparable effect. Additionally, the periostracal 20 hairs did not significantly alter the righting time of Trichotropis after dislodgement. Snails with 21 periostracal hairs removed did not significantly differ in landing orientation from snails with 22 hairs intact. Fusitriton oregonensis consistently lands aperture up if it falls a distance longer 23 than five times its body length (Palmer 1977). As the periostracal hairs apparently do not assist 24 Fusitriton to land in an aperture-down orientation, it is perhaps to be expected that they do not 25 affect the falling style of T. cancellata. 26 Hoffman (1979) noted that Odostomia columbiana, an ectoparasitic snail of Trichotropis, 27 attaches itself to the shell of Trichotropis using an elastic, resilient thread that is relatively 28 invisible underwater and prevents the snail from tumbling off its host when dislodged. 29 Trichotropis can produce similar threads (Iyengar personal observation), although we do not 30 know whether these threads are produced in the same manner or for the same function. Because 31 the mucus thread can prevent Trichotropis from falling, and instead causes the snail to swing

p. 23 Function of flexible hairs

1 back into contact with the hard vertical wall from which it was dislodged (Iyengar personal 2 observation), it is perhaps less important that the snail often land aperture-down after a falling 3 event. Further, the walls and rocky substratum which Trichotropis inhabits are usually rugose 4 (either in terms of the rock itself or due to the presence of encrusting organisms), so that a falling 5 event often involves tumbling with multiple hard substratum contacts (Iyengar unpublished 6 data). Therefore, any influence of periostracal hairs during a fall is likely negligible compared 7 with the importance of the structure of the surrounding environment and whether a mucus thread 8 can restrain the snail. Thus, it is not surprising that the presence of periostracal hairs has no 9 significant effect on the landing orientation of Trichotropis. 10 11 CONCLUSIONS 12 Our work demonstrates that the hairy periostracal projections of Trichotropis cancellata 13 do not appear to deter snail predators but do provide a slight advantage against some crabs and 14 sea stars (Pycnopodia helianthoides). The periostracal hairs have surprisingly little effect on 15 fluid flow around the shell. Further, any small effects on flow that the hairs cause do not impact 16 the growth rate of facultative kleptoparasites and, if anything, negatively impact the growth of 17 suspension feeding snails. The hairs do not appear to promote an advantageous aperture 18 orientation upon the snail’s landing after a falling event. The largest impact of the periostracal 19 hairs identified to date has been on the settlement of epibionts, as the hairs promote the 20 settlement of small, leafy red algae and deter the settlement of barnacles. However, the degree to 21 which the periostracal hairs, compared with a lamellar periostracum, reduce the settlement of 22 calcareous epibionts has not yet been tested. Whether a hirsute periostracum (compared with a 23 lamellar periostracum) increases fecundity enough to drive selection, and whether there are other 24 advantages to the flexible hair-like exterior (such as an early warning system of predator threat 25 or a notification of host feeding) remain undetermined. 26 27 Acknowledgements. We would like to thank the Director and staff of the Friday Harbor 28 Laboratories for providing us with laboratory space. We would also like to thank J. Graber, R. 29 Foley, K. Hutchinson, and P. Kitaeff for field and laboratory assistance, L. Britt for help with 30 fish collections, and D. Sitvarin for help with Figure 2. E.V.I. was funded through Muhlenberg

p. 24 Function of flexible hairs

1 College faculty summer research grants, M.I.S. and M.C. were funded by NASA and NSF grants 2 to Muhlenberg College (respectively). 3 4 LITERATURE CITED 5 Boshoff PH 1968. A preliminary study on conchological physiopathology, with special 6 references to Pelecypoda. Am Natl Mus 20:199-216. 7 Bottjer DJ 1981. Periostracum of the gastropod Fusitriton oregonensis: Natural inhibitor of 8 boring and encrusting organisms. Bull Mar Sci 31:916-921. 9 Bottjer DJ 1982. Morphology and function of projecting periostracal structures in the 10 Gastropoda (Mollusca). Third North American Paleontological Convention, Proceedings 11 1:51-56. 12 Bottjer DJ & Carter JG 1980. Functional and phylogenetic significance of projecting 13 periostracal structures in the bivalvia (Mollusca) J Paleontol 54:200-216. 14 Carroll JC & Winn RN 1989. Species profiles: life histories and environmental requirements of 15 coastal fishes and invertebrates (Pacific Southwest): brown rock crab, red rock crab, and 16 yellow crab. Biological Report 82 (11.117). Vicksburg, MS : U.S. Army Corps of 17 Engineers, Waterways Experiment Station, Coastal Ecology Group ; Washington, DC : 18 U.S. Dept. of the Interior, Fish and Wildlife Service, Research and Development, 19 National Wetlands Research Center. 20 Donovan DA, Danko JP, & Carefoot TH 1999. Functional significance of shell sculpture in 21 gastropod mollusks: test of a predator-deterrent hypothesis in Ceratostoma foliatum 22 (Gmelin). J Exp Mar Biol Ecol 236:235-251. 23 Eckman JE & Duggins DO 1993. Effects of flow speed on growth of benthic suspension 24 feeders. Biol Bull 185:28-41. 25 Eckman JE, Duggins DO, & Sewell AT 1989. Ecology of understory kelp environments. I. 26 Effects of kelps on flow and particle transport near the bottom. J Exp Mar Biol Ecol 27 129:173-187. 28 Feifarek BP 1987. Spines and epibionts as antipredator defenses in the thorny oyster Spondylus 29 americanus Hermann J Exp Mar Biol Ecol 105:39-56. 30 Harper EM 1997. The molluscan periostracum: An important constraint in bivalve evolution. 31 Palaeontology 40:71-97.

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1 Harper EM & Skelton PW 1993. A defensive value of the thickened periostracum in the 2 Mytiloidea. The Veliger 36:36-42. 3 Hoffman DL 1979. An attachment structure in an epiparasitic gastropod. The Veliger 22:75-77. 4 Iyengar EV 2002. Sneaky snails and wasted worms: Kleptoparasitism by Trichotropis 5 cancellata (Mollusca, Gastropoda) on Serpula columbiana (Annelida, Polychaeta). Mar 6 Ecol Prog Ser 244:153-162. 7 Iyengar EV 2004. Host-specific performance and host use in the kleptoparasitic marine snail 8 Trichotropis cancellata. Oecologia 138: 628-639. 9 Iyengar EV 2005. Seasonal feeding-mode changes in the marine facultative kleptoparasite 10 Trichotropis cancellata (Gastropoda, Capulidae): trade-offs between trophic strategy and 11 reproduction. Can J Zool 83:1097-1111. 12 Lambert P 1981. The sea stars of British Columbia. Royal British Columbia Museum, Victoria, 13 B.C. 14 Micheli F & Halpern BS 2005. Low functional redundancy in coastal marine assemblages. 15 Ecol Lett 8:391-400. 16 Palmer AR 1977. Function of shell sculpture in marine gastropods: Hydrodynamic 17 destabilization in Ceratostoma foliatum. Science 197:1293-1295. 18 Palmer AR 1979. Fish predation and the evolution of gastropod shell sculpture: experimental 19 and geographic evidence. Evolution 33:697-713. 20 Palmer AR 1983. Relative cost of producing skeletal organic matrix versus calcification: 21 evidence from marine gastropods. Mar Biol 75:287-292. 22 Palmer AR 1985. Adaptive value of shell variation in Thais lamellosa: effect of thick shells on 23 vulnerability to and preference by crabs. The Veliger 27:349-356. 24 Pernet B & Kohn AJ 1998. Size-related obligate and facultative parasitism in the marine 25 gastropod Trichotropis cancellata. Biol Bull (Woods Hole) 195:349-356. 26 Riisgård HU & Larsen PS 2001. Minireview: Ciliary feeding and bio-fluid mechanics 27 —present understanding and unresolved problems. Limnol Oceanogr 46: 882- 28 891. 29 Stone HMI 1998. On predator deterrence by pronounced shell ornament in epifaunal bivalves. 30 Palaeontology 41:1051-1068. 31 Velleman PF 1997. Data Desk version 6.0. Statistics guide. Data Description Inc.

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1 Ithaca, New York.

2 Vermeij GJ 1987. Evolution and escalation. An ecological history of life. Princeton University 3 Press, Princeton, N.J. 4 Vermeij GJ, Lowell RB, Walters LJ, & Marks JA 1987. Good hosts and their guests: Relations 5 between trochid gastropods and the epizoic limpet Crepidula adunca. Nautilus 101:69- 6 74. 7 Wright MM & Francis L 1984. Predator deterrence by flexible shell extensions of the horse 8 mussel Modiolus modiolus. The Veliger 27:140-142. 9 10 11

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1 Figure 1. Trichotropis cancellata (“cancellate hairy shell snail”). Scale bar is in 1 mm 2 increments. 3 4 Figure 2. Measurements performed in the flume study. Snail is in the side mounted position. 5 Water flow is from left to right. Lines indicated are: (A) arc length; (B) maximum height of the 6 arc; and (C) horizontal distance. Consistent placement of the syringe relative to the dowel was 7 verified across treatments using (D). 8 9 Figure 3. Average (+1 SE) flow rate past hairy (stippled bars) versus smooth (solid bars) shells 10 in the following orientations: (A) side mounted; (B) top mounted, oriented with the apex parallel 11 to the current; and (C) top mounted, oriented with the apex perpendicular to the current. The 12 number of replicates is indicated by the number above each bar. Statistical results are provided 13 in Table 3. 14 15 Figure 4. Average (+1SE) maximum arc height above hairy (stippled bars) versus smooth (solid 16 bars) shells in the following orientations: (A) side mounted; (B) top mounted, oriented with the 17 apex parallel to the current; and (C) top mounted, oriented with the apex perpendicular to the 18 current. The number of replicates is indicated by the number above each bar. Statistical results 19 are provided in Table 3. 20 21 Figure 5. Average (+1SE) ratio of arc distance to horizontal length for hairy (stippled bars) 22 versus smooth (solid bars) shells in the following orientations: (A) side mounted; (B) top 23 mounted, oriented with the apex parallel to the current; and (C) top mounted, oriented with the 24 apex perpendicular to the current. The number of replicates is indicated by the number above 25 each bar. Statistical results are provided in Table 3. 26 27 Figure 6. Growth of hairy and smooth Trichotropis cancellata in 2004 and 2005 field growth 28 experiments. klepto. = snails that were tethered to a Serpula columbiana worm and so allowed to 29 kleptoparasitize, susp. feeders = snails that were tethered to the Plexiglas rack and so restricted 30 solely to suspension feeding. Numbers within the bars indicate the number of replicates for that 31 treatment. There were no significant differences in the amount grown by hairy and smooth

p. 28 Function of flexible hairs

1 kleptoparasitic snails (P > 0.05), but suspension feeding smooth snails grew significantly more 2 than suspension feeding hairy snails (P < 0.05). In both 2005 experiments, the covariate snail 3 length was significant (P < 0.05).

p. 29 Function of flexible hairs Figure 1.

p. 30 Function of flexible hairs Figure 2.

A B

C D

p. 31 Function of flexible hairs Figure 3. A.

40 15 14 14 1414 15 30

Hairy shells 20 Smooth shells

Average flow rate 10

0 Small Medium Large Shell size B. 40 15 15 14 14 13 14 30

Hairy shells 20 Smooth shells

Average flow rate 10

0 Small Medium Large Shell size C. 40 15 14 13 14 13 15

30

Hairy shells 20 Smooth shells

Average flow rate 10

0 Small Medium Large Shell size p. 32 Function of flexible hairs Figure 4. A.

8 15 15 15 6

15 15 Hairy shells 4 15 Smooth shells

2

0 Average maximum arc height (in mm) Small Medium Large Shell size B.

4 15

15 3 13 14 14 15 Hairy shells 2 Smooth shells

1

0 Average maximum arc height (in mm) Small Medium Large Shell size C.

4

15 3 15 13 14 15 Hairy shells 2 14 Smooth shells

1

0

Average maximum arc height (in mm) Small Medium Large Shell size p. 33 Function of flexible hairs Figure 5. A. 1.4 15 1.35 15 15 1.3 15 1.25 15 15 Hairy shells 1.2 Smooth shells 1.15 1.1 1.05 Average distance/length ratio 1 Small Medium Large Shell size B. 1.2 14 13 14 15 15 1.15 15 Hairy shells 1.1 Smooth shells

1.05 Average distance/length ratio 1 Small Medium Large Shell size C. 1.35 15 1.3 13 15 1.25 15 14 1.2 14 Hairy shells 1.15 Smooth shells 1.1

1.05 Average distance/length ratio 1 Small Medium Large Shell size p. 34 Function of flexible hairs Figure 6.

5

Hairy Shells 4 Smooth Shells

3

2 * 1

Average snail growth (mm) Average snail growth 17 18 18 8 34 25 0 2004 2005 2005 2004, 2005, 2005, susp. klepto. klepto. feeders

Treatment

p. 35 Function of flexible hairs Table 1. Results of chi-square analyses of the laboratory predator preference experiments. Individuals of two crab species and one sea star species were tested. Displayed is the number of individuals that made a significant choice (separate analyses) and a cumulative analysis (“Across the species”) across all individuals in a particular predator species. For all analyses showing significant preference, P < 0.05. Only individuals that ate at least ten snails over the course of the experiment, and only data from intervals in which individual predators ate fewer than five snails of any single treatment, were included in the statistical analyses. Treatments No Species tested Smooth Hairy preference Across the species Cancer productus (crab) 1 0 8 No preference

Cancer oregonensis (crab) 0 0 5 Yes- preferred smooth snails Pycnopodia helianthoides (sea star) 3 0 1 Yes- preferred smooth snails

No Species tested Small Large preference Across the species Cancer productus (crab) 2 0 7 Yes- preferred small snails Cancer oregonensis (crab) 3 0 2 Yes- preferred small snails Pycnopodia helianthoides (sea star) 0 0 4 Yes- preferred large snails

Species tested Small, smooth Small, hairy Large, smooth Large, hairy No preference Across the species Yes- preferred small, Cancer productus (crab) 1 0 0 0 8 smooth snails Yes- preferred small, Cancer oregonensis (crab) 2 0 0 0 3 smooth snails Yes- preferred large and small smooth Pycnopodia helianthoides (sea star) 0.5* 0 1.5* 0 2 snails * one individual preferred both sizes of smooth snails to either size of hairy snails.

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Table 2. Field Predation Experiments. Number and percent of hairy versus smooth snails eaten during the field predation experiments (13 July to 23 July 2004). Identity of the predators could not be definitively determined, but most mortalities were likely due to Cancer sp. crabs, based on shell fragments, torn tethers and sightings of crabs in the surrounding area. Treatment Number eaten Number alive Percent of snails eaten Hairy kleptoparasites 4 18 18.18 % Smooth kleptoparasites 9 14 39.13% Pearson’s test statistic = 2.402, df = 1, Fisher’s exact P > 0.1

Hairy suspension feeders 2 28 6.67% Smooth suspension feeders 2 24 7.69% Pearson’s test statistic = 0.022, df = 1, Fisher’s exact P > 0.5

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Table 3. Statistical results of the laboratory flume experiments examining the effect of periostracal hairs on fluid flow trajectories around the shell. See Figures 3, 4, and 5 for graphical representations of the results.

Response Shell position Source Significant F test P value variable ? statistic*

flow rate side-mounted treatment no F1, 80 = 2.42 > 0.1 shell size yes F2, 80 = 7.17 < 0.01 treatment×size no F2, 80 = 0.03 > 0.5 top-mounted, spire treatment parallel to flow no F1, 80 = 1.19 > 0.2 shell size yes F2, 80 = 5.38 < 0.01 treatment×size no F2, 80 = 0.32 > 0.7 top-mounted, spire treatment perpendicular to flow no F1, 78 = 0.02 > 0.8 shell size yes F2, 78 = 5.08 < 0.01 treatment×size no F2, 78 = 0.20 > 0.8 arc height side-mounted treatment yes F1, 84= 8.28 < 0.01 shell size yes F2, 84= 11.97 < 0.0001 treatment×size no F2, 84= 0.49 > 0.6 top-mounted, spire parallel to flow treatment yes F1, 80= 4.09 < 0.05 shell size yes F2, 80= 13.07 < 0.0001 treatment×size no F2, 80= 1.58 > 0.2 top-mounted, spire perpendicular to flow treatment no F1, 80= 0.47 > 0.4 shell size yes F2, 80= 11.83 < 0.0001 treatment×size no F2, 80= 0.25 > 0.7 arc distance/ horizontal side-mounted treatment yes F1, 84= 10.87 < 0.01 shell size yes F2, 84= 9.05 < 0.001 treatment×size no F2, 84= 0.19 > 0.8 top-mounted, spire parallel to flow treatment no F1, 80= 0.02 > 0.8 shell size yes F2, 80= 4.32 < 0.05 treatment×size yes F2, 80= 4.28 < 0.05 top-mounted, spire perpendicular to flow treatment no F1, 80= 1.65 > 0.2 shell size yes F2, 80= 8.36 < 0.001 treatment×size no F2,80= 2.87 > 0.05

* Fdf = treatment, error

p. 38 Function of flexible hairs

Table 4. Balanus epibionts settling on hairy and smooth Trichotropis cancellata over a five week period (2 June–6 July 2005).

Treatment Response Hairy snail Smooth snail Total sample size 42 36 % snails with a large quantity of dirt 14.29% 5.56% % snails (dirty and clean) with barnacles 40.5% 88.89% % clean snails with barnacles 39.47% 91.18% % dirty snails with barnacles 66.67% 50% Average # (+ 1 SE) barnacles per snail (dirty and clean) 1.21 (+0.32) 5.06 (+0.78) Average # barnacles on just the dirty snails 1.67 3

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