Waterborne cues can initiate a variety of behavioral and morphological defenses. These coevolved recognitionsystems can be disrupted by abiotic factors, like wave action, or by biological invaders that share no evolutionaryhistory with native communities. In this study, we collected an intertidal whelk (Nucella lapillus) from areas inthe northeastern USA that have been long-invaded by the predatory crab Carcinus maenas and from areas inNewfoundland, Canada that were uninvaded (or only recently invaded) by the crab. In lab experiments wethen juxtaposed the behavioral and morphological responses of Nucella to waterborne cues from invasiveCarcinus and the native crab Cancer spp. We found that Nucella from both invaded and uninvaded, wave-protected sites reduced foraging in response to Carcinus, while only whelks collected from invaded, wave-protected sites expressed induced shell thickening or reduced shell growth in response to the invasive crab. Incontrast, whelks fromwave-exposed sites did not respond to Carcinus behaviorally ormorphologically. AlthoughNucella's alteration of foraging and shell weight in response to Cancer spp. resembled responses to Carcinus,whelks did not thicken shell in response to the native crab. Our results indicate that native whelks are capableof recognizing cues from the invasive crab (based on induced behaviors), but the induced morphological re-sponses are absent and perhaps latent in univaded populations. We additionally conclude that the expressionof behavioral andmorphological defenses may be locally restricted by abiotic conditions such as wave exposure.
In marine systems, predictable, physical factors like wave exposure,temperature, desiccation and upwelling often vary across large andsmall spatial scales, influencing species interactions (Bertness et al.,1999; Dethier andDuggins, 1988; Sanford, 1999) and the establishmentand evolution of biological invaders (Byers, 2002; Lee, 1999). Whilebiological invasions can have ecological and evolutionary impacts onnative communities (Cox, 2004; Phillips and Shine, 2004; Strausset al., 2006; Strayer et al., 2006), little work has delineated how the evo-lutionary impact of invaders may vary across physical gradients andlocal selection regimes. For instance, abiotic factors may mitigate theevolutionary impact of invasive species on spatial scales that are rele-vant to the invasive's local impacts. Intertidal marine organisms providean opportunity to examine invasive influences across a variety of spatialscales because they reside in a habitat with intense physical gradients.In this paper, we explore potential evolutionary impacts of an invasivecrab (Carcinus maenas) by contrasting induced responses of native,northwest Atlantic whelks, Nucella lapillus (hereafter: Nucella) acrossinvaded and uninvaded populations, and across wave-exposed andwave-protected habitats.
epartment, Adelphi University,
.
ights reserved.
In aquatic systems, prey often rely on waterborne cues to recognizethreats from predators, and predators use waterborne cues to identifypotential prey (Smee and Weissburg, 2006; Weissburg and Zimmer-Faust, 1993). When the resulting behavioral and morphological alter-ations affect other trophic levels or community members the effectscan include Non-Consumptive Effects or Trait-Mediated IndirectInteractions (NCEs or TMIIs, respectively). Biological invaders lackshared-evolutionary history with native communities and thereby cancircumvent these coevolved recognition systems and the resultingNCEs or TMIIs (Rehage et al., 2009; Sih et al., 2010). Invasive speciesthat are not recognized by native community members may also expe-rience novelty advantages if they can avoid predation or forage on naïveprey (Griffiths et al., 1998; Sih et al, 2010). A variety of factors maycontribute to the non-recognition of invasive predators, or naiveté. Forexample, naiveté to invasive predators may be more common inhabitats with persistent heterogeneous predator distributions, such afreshwater systems (Cox and Lima, 2006), however, there are severalexamples of marine native prey that appear naïve to invasive predators(Edgell and Neufeld, 2008; Freeman and Byers, 2006). Furthermore,recognition of biological invaders may change over time, due to rapidevolution, acquired predator recognition, etc. (Edgell and Hollander,2011; Edgell et al., 2009; Freeman and Byers, 2006).Whenever chemical-ly mediated recognition influences invasion success, it is important todistinguishing betweennaiveté, acquired predator recognition, evolution,and preexisting recognition.
2 A.S. Freeman et al. / Journal of Experimental Marine Biology and Ecology 452 (2014) 1–8
Induced traits (behavioral and morphological) may also be alteredby food limitation, environmental parameters and other physical prop-erties (Anholt andWerner, 1995). In marine settings, water movementorwave action can interrupt the transmission, detection and diffusion ofwaterborne cues (Smee andWeissburg, 2006). Abiotic factors may alsodirectly alter behaviors or morphologies: wave-exposed gastropodshave thinner, smaller shells and forage less than wave-protectedconspecifics (Burrows and Hughes, 1990; Etter, 1989; Trussell et al,1993); water temperature can increase foraging behaviors (Sanford,1999) and ability to allocate to shell defensive structures (Trussell,2000); and calcium limitation can alter production of shell materialfor induced morphological defenses (Rundle et al, 2004). Persistent,strong environmental gradients can lead to local adaptation, even inmarine organisms, by altering benefits and trade-offs of predator recog-nition and responses (Sanford and Kelly, 2011), and local habitats caninfluence the intensity of NCEs (Matassa and Trussell, 2011; Storferand Sih, 1998; Trussell et al., 2006).
1.1. Study system: Carcinus invasion and induced responses
The European green crab, C.maenas (hereafter:Carcinus), is native tothe eastern Atlantic (from Iceland and Norway to the MediterraneanSea). Prior to 1900, Carcinus had established invasive populations inthe Atlantic Coast of North America and southern Australia (Carltonand Cohen, 2003). The crab has since become established in Japan,South Africa, the Pacific Coast of North America, and Argentina(Carlton and Cohen, 2003; Hidalgo et al, 2005). Genetic evidence indi-cates that on the Atlantic Coast of North America, an initial Carcinusinvasion spread from the United States Maritime Canada where a sec-ond, cryptic invasion occurred in the 1980's (Roman, 2006). Possibly re-inforced by this second invasion, Carcinus has continued to spreadin Maritime Canada and in 2007 was discovered as far as southernNewfoundland (Blakeslee et al, 2010).
As Carcinus has spread on the Atlantic Coast of North America, itsimpacts on mollusks have included natural selection favoring thickershells (Vermeij, 1982; Seeley, 1986), fomenting induced defenses likeburial depth and shell thickening (Trussell, 1996; Whitlow, 2010),altered feeding behaviors (Aschaffenburg, 2008; Trussell et al, 2006),and ultimately driving canalization of behavioral or morphologicalplasticity (Edgell et al., 2009; Trussell and Nicklin, 2002). Outsidethe well-studied Northwestern Atlantic, there is mixed evidence thatwhelks from other regions invaded by Carcinus recognize the crab'scues (Edgell and Neufeld, 2008; Freeman et al, 2013). Carcinus's inva-sion and impacts may be contingent upon the naiveté of native fauna;if there is a time-lag in the recognition of Carcinus then the crab mayexperience a novelty advantage and have a greater impact on nativecommunities through direct predation. In contrast, if native faunainnately recognize Carcinus and propagate NCEs, then the invasivecrab will have a larger, immediate impact on native communities.
Snails occupyhabitats ranging in abiotic conditions, express both be-havioral and morphological responses to predators (Aschaffenburg,2008; Edgell et al, 2009; Large and Smee, 2012; Trussell et al, 2006),and can undergo local selection due to abiotic factors like wave action(Etter, 1989) and regional selection due to the invasion by Carcinus(see above). In most studies, a snail's morphological and behavioralresponses to Carcinus have been investigated separately (Edgell andHollander, 2011; but see Trussell et al, 2011), including induced shellthickening (Trussell, 1996), induced retraction into the snail's shell(Edgell et al., 2009) and altered foraging (Freeman and Hamer, 2009;Trussell et al, 2006). However, morphological and behavioral responsesmay also interact; reduced foraging in the presence of a predatormay lead to the appearance of induced shell thickening (Bourdeau,2010). Similarly, N. lapillus's induced behavioral and morphologicalresponses to Carcinus may interact with abiotic factors like waveaction (Freeman and Hamer, 2009; Large and Smee, 2012). Despiteextensive studies it is not clear whether these induced behaviors and
morphological defenses in native preymight contribute to a novelty ad-vantage for Carcinus. In this study, we determine if local environmentalfactors and/or post-invasion evolution have altered responses inNucellaby juxtaposing whether native whelks collected from wave-exposedand wave-protected areas, across invaded and uninvaded populations,express induced responses to native and invasive crabs.
2. Methods
2.1. Nucella collection and morphology
In five experiments over a 3-year periodwe evaluated the behavioralandmorphological responses ofNucella fromdifferent populations in theNWAtlantic towaterborne cues from invasive Carcinus and native crabs.Native crabs (Cancer irroratus and Cancer borealis) were included as a“positive control” and to bolster inferences about preexisting responsesto native predators. Induced foraging and induced morphology experi-ments are described in Sections 2.2 and 2.3 (respectively). In June2009, August 2010, and August 2011 we collected Nucella from 11“uninvaded” sites in Newfoundland Canada (“NFLD”) and 14 “invaded”sites in northeastern United States (“USA”) (Table 1, see Section 2.4).Based on fetch and ocean exposure, several of the sites we selected likelyexperience strong wave action during storms, including sites in NFLD(Pt. Ray, Bauline, Bryants, Grates and Broom Pt.) and in the USA (Nubble,Reid and Quoddy). The remaining sites were a priori categorized as“waveprotected” (Table 1; see Section 2.4.1). At each site we collectedNucella haphazardly within a broad size-range (12–33 mm shell length)across the whelk's intertidal distribution. After collection, Nucella fromeach site were kept in separate mesh bags in sea water and transportedin a chilled cooler to each experiment's common garden setting.Common garden experiments in 2009 were conducted at Shoals MarineLaboratory (“SML”) on Appledore Island,Maine, and in 2010 and 2011 atAdelphi University (“AU”), New York (for a comparison of experimentalconditions see Table 2). Prior to experimentswhelksweremaintained ona diet of mussels for 1–4 weeks. We wanted to examine existing mor-phological differences between Nucella populations, therefore, each ex-perimental year, we measured Nucella from the first 1–3 feeding trialswith digital calipers (±0.01 mm) for shell length and thickness (lengthn = 638 and thickness n = 636, respectively; see Palmer 1990 for shelldimensions). In 2009 and 2011, in addition to length and thickness, wemeasured the aperture length and aperture width (internal dimensions)of 279 whelks from 16 sites.
2.2. Induced foraging behavior experiments
Webroughtwhelks fromNFLD and the GOM to SML (in 2009) or AU(in 2010 & 2011) and allowed them to acclimate in non-flow throughseawater tanks. Although the Nucella we collected were representativeof the size distribution at each site, we attempted to match the Nucellaby size for each experimental trial. To size-match whelks in each trialwe selected the smallest whelks from sites with large whelks and thelargest whelks from sites with small whelks. We collected crabs usedfor predator cues (Carcinus and Cancer) and prey mussels (Mytilusedulis) from intertidal sites on Appledore Island (in 2009) and LongIsland Sound (in 2010 & 2011).
At the beginning of each feeding trial, we randomly assignedtreatments (Cancer, Carcinus, or Control (no crabs)) to mesocosms.Mesocosms had air stones andwere filled at the beginning of each feed-ing trail, but did not receive flow-through seawater. In 2009 and 2011all cages from a single treatment were housed in a single largemesocosm, while in 2010 whelk cages were housed either in shared,large mesocosms or individually in small mesocosms with a single cuecrab (see Table 2 for mesocosm details). All cue crabs were isolatedindividually in stainless steel, mesh-sided cages and not fed during theexperiments, thus cues did not include crushed conspecifics. Controlmesocosms held empty mesh-sided cage(s). During trials, any dead
Table 1Field collection sites for Nucella used in foraging experiments in 2009, 2010, and 2011, and morphological experiments in 2010 and 2011. Behavioral and morphological inductionexperiments were conducted temperature controlled aquariums at the Isles of Shoals in 2009 and at the Adelphi University campus in 2010 and 2011. 150–250 whelks were collectedsite−1 in 2009 and 2010. 120–200 whelks were collected site−1 in 2011.
Collection period Population Site Wave exposure Lat Long
a Whelks not used in morphological experiments.b Whelks not used in foraging experiments.
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cue crabs were replaced with live individuals. Fifteen Nucella from eachsite were divided among 3 mesh-sided cages (12 × 12 × 10 cm). Thuseach whelk cage contained 5 Nucella and 20 prey mussels (Table 2).Each trial ran for 5–7 days (see Table 2). At the end of each feedingtrial the number of dead and drilledmussels was counted. New, unusedcrabs, whelks and mussels were added for each trial. Between trialsmesocosms, tubing, chillers, and air stones were washed with soapywater, rinsed and wiped with paper towels to minimize carryover ofcues into subsequent trials. Cue treatments were randomly shiftedbetween mesocosms for each trial, but no single cue treatment wasrepeated in the same mesocosm. Prior to each feeding trial Nucellawere starved for 48 h.
Table 2Summary of induced behavior and morphology experiments conducted at SML (in 2009) and aNucella from all sitesweremaintained in a single largemesocosmwith cue crabs, “Small” indica“SW” indicates when mesocosms were filled with seawater, and “ASW” indicates when mesoconducted in 2009.
Year Location of experiment “Induced response” Mesocosm
2009 SML Behavior Large, SWa
2010 AU Behavior Small and Large,Morphology Small, ASWb
2011 AU Behavior Large, ASW c
Morphology Large, ASW c
a In 2009 three largemesocosms (75 × 43 × 30 cm)were filledwith unfiltered seawater (SWtreatmentmesocosm, Nucella from individual sites were isolated in site-specific mesh cages witand 47–75 mm, respectively) and were also isolated in mesh cages.
b In 2010, 24 small (3.5 l) mesocosmswere arranged in a 3 m × 1.5 m, 16 °C “cold room”. Eacandfilledwith 10 cmof InstantOceanArtifical SeaWater (ASW). PremeasuredNucella (8 replicatethe inducedmorphology experiment. Subsequent feeding trials consisted of replicates with 5 Nuc75 l of ASW, chilled to 13–1 °Cwith inline chillers. Cue crabs (Cancer and Carcinus) had overlappinmesh cages.
c In 2011, all experiments were conducted in large 230-l mesocosms (as in 2010).d In 2009 all cages were checked daily and any dead Nucellawere replaced with Nucella from t
presumed not to have been foraging during the experiment.
2.3. Induced morphology experiments
In 2010 and 2011, we conducted two experiments at AU to deter-mine if Nucella from invaded and uninvaded populations (Table 1)expressed induced morphological defenses in response to native orinvasive crabs. We collected Nucella and arranged mesocosms as de-scribed for induced foraging experiments (Tables 1 & 2). Twenty-fourwhelks from each site were divided among three treatments (Cancer,Carcinus, or Control) and whelk shell dimensions were measuredusing digital calipers (length, width, and thickness; ±0.01 mm).Immersed weight was measured using a method described by Palmer(1982); a small mesh basket was submerged in a bowl of ASW and
t AU (in 2010 and 2011). Under Mesocosm “Large” indicates that for each cue treatment,tes thatNucella andmussel prey weremaintained in individualmesocosmswith cue crabs,cosms were filled with artificial sea water. An induced morphology experiment was not
Number of trials Duration trials Dead Nucella replacedd
5 5 days YesASWb 4 7 days No
1 42 days No4 7 days No1 67 days No
) and placed in a sea table chilled by flowing seawater. Although housed in a single, large,h mussels. Cue crabs (Cancer and Carcinus) had overlapping size distributions (54–99 mm
h small mesocosmwas randomly assigned to a treatment (8 small mesocosms treatment−1)−1) from thefirst foraging trialweremaintained in these smallmesocosms for theduration ofella replicate−1 and were conducted in 3 large, 230-l mesocosms. Eachmesocosm containedg size distributions (84–200 g and 90–120 g, respectively) andwere individually isolated in
he appropriate site (5 Nucella died), but in 2010 and 2011 any dead Nucellawere noted and
Table 3ANOVA and ANCOVAs of Nucella used in morphological and foraging experiments.Population consists of invaded or uninvaded sites, Wave exposure consists ofwaveprotected or waveexposed sites, and Year consists of 2009 or 2011 (for aperture−2)and 2009, 2010 and 2011 (for length and thickness). In each analysis site (WE, Pop) wasdesignated as a random factor (“R”) causing the statistical program JMP to use thetraditional method of moments to estimate mean squares.
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suspended below a below-beam balance (Mettler-Toledo AG XS204).Individual whelks were placed in themesh basket (negating theweightof tissue) to obtain their immersed weight. To mitigate the effects of airbubbles on immersed weights each snail was gently prodded to induceretraction prior to recording immersed weight. After air-drying, whelkshells weremarked with color-coded paint dots sealed with cyanoacry-late glue, then placed in mesh-sided containers and provided an abun-dance of mussels. We attempted to size-match Nucella from thevarious sites (described above), but some differences remained be-tween sites (Fig. 1 & Table 3). Each replicate cage contained 8premeasured Nucella from a single site feeding on mussels. In additionto Nucella cages, mesocosms contained stainless steel cages housingcue crabs (Carcinus or Cancer) or empty cages for controls. Eachmesocosm was filled to 10–15 cm with ASW and aerated with an airstone. Any Nucella that died in the first week of the morphology exper-iment were replaced with premeasured Nucella of similar size from thesame site. During trials calcium, pH, salinity and temperature weremonitored every week. If pH in any tank exceeded 8.0 then it wasreturned to 7.8–8.0 by adding “pH down” (Aquarium Pharmaceuticals,Inc) to the tank according to the manufacturer directions. Upon experi-ment termination whelks were frozen, later thawed and dissected, thetissue and shell separated, dried for 72+ h in a drying oven (@ 60 °C),and then shells were remeasured for length, width, thickness andweight.
2.3.1. Statistics —site classificationWe designated each site as either “wave-exposed” or “wave-
protected” based on thepotential for intertidal animals at that site to ex-perience ocean swell or storms (Table 1). Wave exposure classifications
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Fig. 1. Shell length, aperture area2 and shell thickness ofNucella collected from sites in theGulf of Maine (GOM; invaded by Carcinus) and Newfoundland (NFLD; uninvaded byCarcinus). Aperture area and shell thickness are least square means from ANCOVAs withshell length as a covariate and therefore control for Nucella size. Shell length was greaterin invaded population than univaded. Aperture area2 was greater in exposed vs. protected(see ANOVA results, Table 2).
were done prior to analyses, and were not biased by a priori knowledgeof how whelks from each site responded to crab cues. In addition, weclassified sites as either invaded or uninvaded by Carcinus. Carcinushas become well established in southeastern NFLD (c. 2002) and hasbeen reported in western NFLD (c. 2009) (Blakeslee et al, 2010).Although the Carcinus invasion in western NFLD may have progressedsince 2009, it likely had very limited influence on Nucella from oursites for several reasons: 1) At each of our NFLD sites we searched inter-tidal benches and flotsam but we found no evidence of Carcinus.2) Carcinus populations in NFLD have established deep coves and estu-aries (McKenzie, 2011), while our whelks were collected from rockyshores. 3) Three of 4 sites sampled in 2009 were north of the crab's2009 distribution. 4) Only Cape Ray (NFLD) was within the establishedrange of Carcinus in 2009. Removing Cape Ray from our analysis did notchange our results. 5) After 2009 we sampled Nucella from sites wellnorth of the crab's distribution. We therefore designated our sites inNFLD as “uninvaded” by Carcinus and those in Maine, Massachusetts,and New York as “invaded.”
2.4. Statistics: Nucella morphology
Lengths of snails collected from all 21 sites over 3 years were thencompared by using a 3-way ANOVA with Year, Wave exposure andInvaded as fixed factors, and Site (Wave exposure, Population) as arandom nested factor. Aperture area (square-root transformed) andlip thicknesswere then compared using separate analysis of covariances(ANCOVAs) with Year, Wave exposure and Invasion as fixed factors,and length as a covariate. In all analyses interactions of fixed factors(Population, Year and Wave exposure) were first analyzed thendiscarded from the model if P N 0.20. In addition, Site (Wave exposure,Population) was designated as a random nested factor and used by thestatistical program (JMP 9) to generate denominator mean squares anddegrees of freedom for various fixed factors. For aperture area theANCOVA was conducted on transformed values, but the graph wasproduced from an ANCOVA of untransformed values.
2.4.1. Statistics: Induced foraging behavior and morphology experimentsIn each foraging experiment Nucella were offered 20 similar-sized
mussels. Initially, each replicate contained 5 Nucella as predators,however in some replicates a whelk died or, as the supply of whelks
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Fig. 2. Number of mussels consumed by Nucella in foraging experiments (±SE, musselsdead and drilled Nucella−1). Because there was no interaction between population andother factors, invaded (GOM) and uninvaded (NFLD) were combined for comparisons. Apriori contrasts (α = 0.05) indicated that at Exposed sites Control = Cancer(P = 0.2506) and Control = Carcinus (P = 0.3113), but at Protected sites Control NCancer (P b 0.0001) and Control N Carcinus (P = 0.0252; see ANOVA results, Table 3).
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fromeach sitewas depleted, some replicateswere startedwith only 3–4whelks. Mussel consumption was therefore analyzed per capita Nucella.(Nucella that died during the predation trials are presumed to have notparticipated in predation). The number of mussels dead and drilledNucella−1 was analyzed using a 3-way, split-plot ANOVA with Cue,Population andWave exposure as fixed effects, Trial (Year) as a randomblocking factor, Site as a random effect nested within Population, andSite (Population) * Cue. The latter nesting factors cause JMP to use theSatterthwaite approximation to generate the denominator meansquares resulting in fractional degrees of freedom. Analyses were con-ducted in REML, which consolidates the random nesting factors. Higherorder interactions were tested and removed from themodel if P N 0.20.A priori linear contrasts were used to compare each crab cue treatmentto controls for wave-protected and wave-exposed sites.
We also determined if morphological defenses had been induced byanalyzing final shell weight (log transformed) and final shell thicknessusing a 3-way, split-plot ANCOVAwith the same treatments as foragingexperiments, but including initial (immersed) weight and initial shellthickness as covariates (respectively). Because Year was an unexpectedlysignificant factor in morphological comparisons a priori contrasts wereinappropriate. Therefore, post-hoc comparisons (sequential Bonferroniadjusted α) were used to compare each crab cue treatment to respectivecontrols.
3. Results
3.1. Morphology of field collected Nucella
An ANOVA of whelk shell lengths indicated a significant effect ofPopulation, Year, and Site, but no effect of Wave Exposure (Table 3).Whelks from invaded populations tended to be smaller than those fromuninvaded populations (Mean ± SE: 19.56 ± 0.77, 23.20 ± 0.67,respectively; Fig. 1). Snails used in 2010 (19.19 ± 0.57) were smallerthan those in 2009 and 2011 (22.49 ± 0.53 and 22.36 ± 0.36, respec-tively). In order to accommodate site differences in size we used shelllength as a covariate when examining aperture area and shell thickness.An ANCOVA of aperture area indicated snails from waveexposed siteshad larger apertures than snails from waveprotected sites (Table 3;127.67 ± 0.35, 105.67 ± 0.21, respectively). However, Wave exposureand Population did not significantly affect Nucella shell thickness(Table 3). For the three morphological parameters, Site accounted formore variation than did Population or Wave exposure, reflecting thebroad range of habitats incorporated in site-to-site variation.
3.2. Nucella foraging behavior experiments
An ANOVA of mussels eaten by Nucella indicated a significant effectof Population: Nucella from NFLD consumed 36% fewer mussels thanNucella from the GOM (0.57 ± 0.10 vs 0.88 ± 0.10 mussels Nucella−1,respectively). Furthermore there was an interaction of Wave exposureand Cue; pooling across populations, wave-exposed Nucella did notreduce foragingwhen exposed to crab cues, but wave-protectedNucelladid reduce foragingwhen exposed to Cancer cues and, to a lesser extent,when exposed to Carcinus cues (Table 4; Fig. 2).
Table 4Results of a 3-way ANOVA comparing the number of mussels dead and drilled Nucella−1.Cue consists of Cancer, Carcinus, or Control. Random factors: Trial/Year, Site(Pop) andSite*Cue (Pop (Carc)) were included in the model, however, because JMP uses the REMLtechnique to estimate error terms, the random factors and overall error terms do notappear in our results.
Initially, Wave exposure was included in both ANCOVAs of inducedshell weight and induced shell thickness but it did not have a significanteffect on final shell weight and was removed from that model.Year affected shell weight and there was an interaction of Cue ×Population × Year. In 2010, whelks from both GOM and NFLD popula-tions had similar final shell weights and there was no effect of crabcue (Fig. 3; Table 5). However, in 2011 the populations respondeddifferently: whelks from the GOM increased shell weight in response
1.0
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Sh
ell W
Invaded (GOM) Uninvaded (NFLD)
Fig. 3. Dry shell weight of Nucella raised with waterborne cues from no crab (Control),Carcinus, or Cancer crabs. Post-hoc comparisons (Bonferroni adjusted) indicated thatin 2010 Control = Cancer and Control = Carcinus for GOM and NFLD whelks (allPs N 0.10). However, in 2011, Control b Cancer and Control b Carcinus for GOM whelks(P = 0.002 and 0.007, respectively), but Control = Cancer and Control = Carcinus forNFLD whelks (all Ps N 0.10).
Table 5Results of 3-way split plot, nested ANCOVA of final shell weight (log-transformed). In theoriginalmodelwave exposure (WE)was included, but discardedbecause itwas not signif-icant and had no interactions with other fixed factors. Population and Cue are the same asin Table 3. Year includes 2010 and 2011. Initial weight (log transformed) is the initial im-mersedweight of each liveNucella suspended in seawater and is used as a covariate. Anal-ysis excludes Nucella from Hampton, Montauk and Sandwich. Site(Pop) and Cue × Site(Pop) were included as random nested factors but because REMLwas used these randomfactors do not appear in the final ANCOVA table.
Fig. 4. Final shell thickness ofNucella raisedwith waterborne cues from no crab (Control),Carcinus, or Cancer crabs. Bonferroni adjusted post-hoc comparisons of Invaded (GOM)indicated: Protected Carcinus N Protected Control (P = 0.021) and Exposed Carcinus =Exposed Control (P = 0.053).
6 A.S. Freeman et al. / Journal of Experimental Marine Biology and Ecology 452 (2014) 1–8
to Cancer and Carcinus cues, but whelks from NFLD did not respond(Fig. 3; Table 5).
Final (induced) shell thickness did not show a significant effect ofCue, Population or Wave exposure, but did show an effect of Year andthe following interactions: Cue × Wave exposure, Pop × Cue × Waveexposure, and Pop × Year × Cue (Table 6). Interestingly if Wave expo-sure was not included as a factor, Cue, Population and interactionswerenot significant, suggesting that it is necessary to account forWave expo-sure to understand the influence of Cue and Population. Due to the dual,3-way interactions we have represented the results with all 4 factors(Fig. 4). Although 2010 exposed-shore Nucella from the GOM appearto reduce shell thickness in response to Carcinus, inclusion of 2011 re-sults render the effect of Carcinus non-significant. Thus, neither theexposed-shorewhelks nor thewhelks fromNFLDaltered shell thicknessin response to crab cues. However, whelks from protected shores in theGOM did thicken shells in response to Carcinus cues, an effect that ismore pronounced in 2011 than in 2010.
4. Discussion
Our comparisons of Nucella's induced responses across 23 sites, in-vaded and uninvaded by Carcinus, reveal several patterns that indicateconsistent spatial trends in induced responses. Whelks from bothinvaded and univaded sites reduced foraging in response to waterbornecues from Carcinus and the native crab Cancer sp. However, the degreethat these whelks reduced foraging depended on the wave exposureof their site of origin; whelks from wave-exposed sites were unlikely
Table 6Results of a 3-way split plot, nested ANCOVA of final shell thickness. Site(Pop) and Cue ×Site(Pop) were included as random nested factors but, because REML was used, theserandom factors do not appear in the final ANCOVA table. Sites are the same as in Table 4.
to respond behaviorally to either predator, but wave-protected whelksdid respond to both crabs. Population-of-origin and crab cues also af-fected shell growth, but this influence was dependent on the traitexamined; shell weight was not affected by wave exposure but shellthickness was partially affected by wave exposure. Furthermore, whileNucella increased shell weight in response to both crabs, an increase inshell thickness only occurred in response to Carcinus. Thus, even popu-lations of Nucella sharing no evolutionary history with Carcinus canrecognize the crab (based on foraging responses), however inducedmorphological defenses are only consistently expressed by Nucellafrom invaded (GOM) populations that are familiar with Carcinus, andappear to be largely latent in uninvaded, “naive” (NFLD) populations.We discuss these results in light of existing differences betweenNucellapopulations, local variation in invasion history, and interactionsbetween behavioral and morphological defense.
We found that wave-protected whelks are more likely to reduceforaging in response to crab cues and, in doing so, propagate non-consumptive effects (NCEs). The lasting effect of wave exposure weobserved may be influenced by the following: 1) Mobile predators areoften more affected by physical disturbances like wave action thansedentary prey (Menge and Sutherland, 1987), consequently predationis often viewed as more intense at wave-protected sites than at wave-exposed sites (e.g. Bourdeau, 2012). According to this view, local,predation-acclimated Nucella taken from wave-protected sites wouldcontinue to show risk-averse responses to crabs, whereas wave-exposed whelks' responses would be more risk-prone and less likelyto express NCEs in response to crabs. 2) In situ, when exposedto wave action, Nucella is forced to reduce foraging (Burrows and
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Hughes, 1990), consequentlywave-exposedNucella phenotypes tend tofeed more rapidly during low-wave periods (Menge, 1978) andimmediately after being placed in calm lab conditions (Freeman andHamer, 2009). These starved whelks may be more likely to continueforaging despite perceived threats from predators (Vadas et al., 1994).3) The rate of flow and turbulence affect transmission of predator cues(Leonard et al., 1998; Smee et al., 2010) rendering chemical cues unre-liable predictors of impending predation at wave-exposed sites. Conse-quently, recognition of crab cues may not be reinforced by selection atwave exposed sites. 4) Finally, wave-exposed whelks may alreadyhave reduced foraging enough that any further, predator-induced re-duction in foraging is not detectable.
While we did find that NFLD whelks had consistently low shellweight and shell thickness induction, the relationship of these traitschallenges expectations for several reasons. First, we expected thatNFLD whelks would deposit shell material more efficiently than GOMwhelks due to genetic predisposition to accrete shell materialmore effi-ciently (i.e. counter-gradient variation in the temperature-sensitiveprocess of shell deposition) (Freeman and Byers, 2006; Trussell,2000), however, NFLD whelks did not show substantial shell growth(Figs. 3 and 4). In addition to lower shell deposition these NFLD whelksdid not feed asmuch as GOMwhelks (Fig. 2) andmay have been limitedin ability to express induced shell thickening. The relationship betweenshell thickness and tissue growth is further complicated because shellthickness in snails is tightly coupled with tissue mass and pedal surfacearea, the latter being necessary for remaining attached to the substrateand directly affected by wave exposure (Etter, 1988; Trussell et al.,1993). Indeed, induced shell thickeningmay also be a product of contin-uous shell deposition and reduced tissue growth (Bourdeau, 2010;Kemp and Bertness, 1984). Finally, while we did find that wave-exposed whelks had larger apertures than wave-protected whelks, inspite of the expected trade-off between shell thickness and aperturearea (wave-exposed whelks have a larger pedal surface area andaperture, which requires a thinner shell; Crothers, 1985) we found nodifference in shell thickness. We suspect that Nucella's local adaptationto wave exposure has different effects on the constituents of this rela-tionship (shell deposition and tissue growth). Shell deposition is limitedby submersion time (Palmer, 1983; Richardson et al., 1981), thus whilewave-exposed whelks may be inundated by seawater more often andexperience adequate conditions for shell growth, the physical disrup-tion of wave action inhibits feeding and tissue growth (Etter, 1996).
Factors affecting induced shell thickening were not easily interpretedbut involved several interactions (Table 6). The interaction, Population ×Cue × Year, is likely an experimental artifact; in 2011 Nucella depositedshellmore effectively than in 2010 (Fig. 4). In 2011 conditionswere betterfor shell growth; calcium carbonate (coral rubble) was added to tanksand the experiment ran about 4 °C warmer than in 2010. Furthermore,in 2010, each replicate was contained in its own small bucket, potentiallyleading to Ca+ limitation and greater variability in shell growth. In addi-tion to the myriad influences of wave exposure, shell thickening is alsoaffected by interactions between wave exposure, Cue and the whelk'spopulation of origin (Population × Cue × Wave exposure). Under favor-able growth conditions in 2011, whelks from protected-shores in theGOM responded to Carcinus by thickening their shells. Yet there is noevidence of a response from exposed-shore Nucella, or NFLD Nucella, toCarcinus cues (Fig. 4). Collectively, Nucella's induced morphologicalresponses (shell thickening and shell deposition) are consistent withNFLDandwaveexposedwhelks beingnaïve or showing limited responsesto Carcinus and Cancer due to limited exposure at sites of origin. However,our experiments were about 2–6 °Cwarmer than August–September seatemperatures at our collection sites in NFLD (Fisheries and OceansCanada) and it is not possible to rule out that NFLD whelks simply didnot grow or feed well in laboratory conditions.
In addition to population of origin, crab-cue typemust be consideredwhen interpreting Nucella's induced responses. Our experimentsmanipulated cues specific to crab species and, because none of the cue
crabs were fed during the experiment, we minimized the effect of“general cues” such as crushed conspecifics (Edgell and Hollander,2011; Large and Smee, 2012). Consequently, our experiments likely un-derestimate the influence of predation threat cues on local communi-ties. There also appears to be variability in the type and hierarchy ofresponses to the different crab species: Nucella from protected sitesseems to more consistently reduce foraging in response to Cancer thanto Carcinus (Fig. 2; see also Freeman and Hamer, 2009). Because Cancerhas stronger crushing claws than Carcinus, it can crush even thickNucella shells (Freeman and Hamer, 2009; Moody and Steneck, 1993)leaving behavioral modification as Nucella's best defense. Likewise,Carcinus may trigger greater shell-thickness responses than Cancer(Fig. 4) if shell-thickening is a more effective defense from Carcinus.Finally, differences in crab cues may also occur within species; Carcinusinvading Newfoundland are the descendants of a secondary Carcinusinvasion in Nova Scotia (Blakeslee et al., 2010) and thus geneticallydistinct from members of the species in the USA and those used in ourexperiments. Although it is not clear if prey mollusks can distinguishbetween distinct crab genotypes, the significant behavioral responsesof NFLD Nucella do not suggest complete naiveté to Carcinus.
While it is clear that wave exposure influences the capacity ofNucella to respond to crab cues (Freeman and Hamer, 2009; Large andSmee, 2012) some variability may be attributable to the geographicrange sampled. Freeman and Hamer (2009) collected whelks fromonly the Isles of Shoals, all within a 2 km radius. Large and Smee(2012) collected Nucella across a large range (~230 km). In contrast,our current study collected Nucella from an even larger geographicrange (over 1500 km) encompassing substantial population variation.Such broad geographic ranges in Nucella may incur variation in preypreferences and growth in a common garden setting (Sanford andWorth, 2009). The geographical range and local variation in Nucella re-sponsesmay account for the less substantial influence ofwave exposureon existing morphological differences, compared to previous studies(Freeman and Hamer, 2009; Large and Smee, 2012).
5. Conclusions
In this study we have contrasted the responses of a common inter-tidal whelk across two abiotic habitats (wave-exposed/protected)invaded and uninvaded by Carcinus. Whelks appear to recognize andrespond to Carcinus behaviorally regardless of invasion history, butwave exposure of the origin population has an overwhelming influence.The capacity of native communities to recognize invaders, and the im-pact of invaders may be influenced by native species diversity and the(taxonomic) uniqueness of the invader (Rehage et al, 2009; Ricciardiand Atkinson, 2004; Sih et al, 2010). Clearly, behavioral andmorpholog-ical responses should be considered when assessing native species ca-pacity to respond to novel, invasive predators. Behavioral andmorphological responses to Carcinusmay have various community im-pacts, but these impacts also vary on small scales (Trussell et al, 2006).Behavioral responses are relatively rapid, require less time-lag, and arelargely reversible (Gabriel et al., 2005). Consequently behavioral re-sponses may be more readily expressed, at lower cue concentrationsand earlier in development than induced morphological defenses. Incases of rapid evolutionary responses to invasive species, behavioral re-sponses to invaders may be immediate, while morphological responsesmay lag behind (either through learning, acquired predator recognitionor evolution). Furthermore, because behavioral reductions in foragingmay lead to the appearance of shell-thickening (Bourdeau, 2010)behavioral responses to the invasive Carcinus may be an evolutionaryprecursor to an induced shell thickening response (i.e. the nascent evo-lution of inducedmorphological defense). Carcinus is a global invader inmany analogous temperate intertidal communities (Cohen et al., 1995)and many of the crab's impacts are likely mitigated by comparableabiotic and evolutionary constraints.
8 A.S. Freeman et al. / Journal of Experimental Marine Biology and Ecology 452 (2014) 1–8
Acknowledgments
Support for this researchwas provided byNH Sea Grant and AdelphiUniversity. We would like to thank the REU program at Shoals MarineLaboratory (Cornell University) for providing support for E.K. This man-uscript was greatly improved by the comments from 2 reviewers. [SE]
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