Download pdf - Generalist versus specialist strategies of plasticity: snail responses to predators with different foraging modes

Generalist versus specialist strategies of plasticity: snailresponses to predators with different foraging modes

JASON T. HOVERMAN*, RICKEY D. COTHRAN† AND RICK A. RELYEA†

*Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN, U.S.A.†Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.

SUMMARY

1. Phenotypic plasticity is a common adaptation to environmental heterogeneity, and theory predicts

that the evolution of constitutive versus plastic strategies should depend on the frequency of alterna-

tive environments, the magnitude of constraints and the costs of plasticity per se. However, it is

unclear how species should evolve when they experience more than two environments that favour

divergent phenotypes, particularly when they have absolute constraints on their morphology.

2. We examined the plasticity of three freshwater snail species (Helisoma anceps, H. campanulata and

H. trivolvis) in response to three environments: (i) no predator; (ii) shell-invading water bugs (Belos-

toma flumineum) and (iii) shell-crushing crayfish (Orconectes rusticus). We found distinct responses by

each snail species to the predator treatments. Helisoma anceps starts with a relatively low, narrow and

thick shell that becomes lower and thicker in response to crayfish but is unresponsive to water bugs.

In contrast, H. campanulata starts with a relatively high, wide and thin shell that becomes lower and

wider in response to water bugs but is unresponsive to crayfish. Helisoma trivolvis starts with a shell

of intermediate height and width while the predators induce defences in different directions.

3. These results suggest that H. trivolvis has a generalist plastic strategy while H. anceps and H. cam-

panulata have specialised plastic strategies orientated against a single type of predator at the potential

cost of being unable to respond to others.

4. We then performed predation trials to determine predator preferences using a mixture of the three

species. After 2 weeks of exposure to crayfish cues, H. anceps had higher survival than both H. trivol-

vis and H. campanulata with uncaged crayfish. After 2 weeks of exposure to water bug cues, both

H. trivolvis and H. campanulata had higher survival than H. anceps with uncaged water bugs. When

predation trials were conducted after 5 weeks of exposure to predator cues, H. trivolvis and H. cam-

panulata reached a size refuge from both predators and this shifted predation pressure to H. anceps.

5. Collectively, these results suggest that closely related prey species with different absolute con-

straints in their morphology had different defences that are either specialised or generalised to alter-

native environments.

Keywords: functional tradeoff, gastropod, inducible defence, phylogeny, selection

Introduction

Natural selection in heterogeneous environments may

lead to the evolution of phenotypic plasticity, defined as

the ability of a single genotype to produce different

phenotypes in response to different environments

(Schlichting & Pigliucci, 1998; Pigliucci, 2001).

Phenotypic plasticity exists in many species and in

response to a wide range of environmental conditions,

and substantial variation in the expression of phenotypic

plasticity can exist among closely related species

(Harvell, 1991; Kusch, 1993; Colbourne, Hebert & Taylor,

1997; Van Buskirk, 2002; Berendonk, Barraclough &

Barraclough, 2003). However, the evolution of plasticity

may be constrained by several mechanisms. For

example, absolute constraints (sensu Brakefield, 2006)

arise because the basic body plans of species are quite

‘difficult’ to change by natural selection (i.e. new traits

Correspondence: Jason T. Hoverman, Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907, U.S.A.

E-mail: [email protected]

© 2014 John Wiley & Sons Ltd 1

Freshwater Biology (2014) doi:10.1111/fwb.12332

that break the constraint may be deleterious). Addition-

ally, allocation tradeoffs can occur when resource limita-

tion constrains simultaneous investment in multiple

traits (Dewitt, 1998; Auld, Agrawal & Relyea, 2010).

These constraints, combined with the amount of envi-

ronmental heterogeneity experienced by a species over

time or space, can drive the evolution of different mean

phenotypes in closely related species (i.e. averaged

across all environments), as well as different directions

and magnitudes of phenotypic plasticity (Van Tienderen,

1991; Dewitt, Sih & Wilson, 1998; Schlichting & Pigliucci,

1998; Pigliucci, 2001; Van Kleunen & Fischer, 2007; Auld

et al., 2010). Because closely related species are likely to

be similar in biochemical, physiological and structural

constraints, comparative studies have the potential to

identify a core set of constraints that may limit the

expression of environmentally induced traits, which

may bias evolution towards ‘fixed’ solutions to environ-

mental heterogeneity (Pfennig et al., 2010).

The inducible defences of freshwater snails represent

an ideal system to assess how absolute constraints and

allocation tradeoffs may influence the divergent evolu-

tion of plasticity across species. For example, shell geom-

etry creates constraints on shell shape. The shells of

most gastropods can be described using three parame-

ters: expansion rate (W), translation (T) and distance (D)

of the generating curve from the axis of coiling (Raup,

1962; Rice, 1998). Physical relationships among these

shell parameters limit shell shape (i.e. not all regions of

morphospace can be achieved) and therefore affect the

range of options for morphological defences against var-

ious predators. For example, species with rapid shell

expansion rates (i.e. a coiling tube that rapidly increases

in diameter) produce shells with relatively large aper-

tures, which are more vulnerable to predators that enter

the shell. However, these species can invest more in

shell thickness, a defence against shell-crushing preda-

tors, because fewer coils around the central axis are

needed to increase overall body size (Raup, 1962). Alter-

natively, snails with slow expansion rates generate shells

with relatively small apertures that are difficult to enter,

although such shells are typically thinner because more

coils around the central coiling axis are required to grow

to a particular body size (Raup, 1962). In addition to

absolute constraints on shell geometry, snails also

face allocation tradeoffs (Dewitt & Langerhans, 2003;

Hoverman & Relyea, 2007a, 2009); for instance, there can

be a tradeoff between investing a limited amount of

shell material to thickness or coiling (Russell-Hunter,

1978; Kemp & Bertness, 1984; Brodersen & Madsen,

2003). As a result, snails commonly face tradeoffs in

how they respond to shell-invading versus shell-crush-

ing predators (Dewitt, 1998; Hoverman & Relyea, 2008,

2009; Bourdeau, 2009). Thus, interspecific differences in

expansion rate, coupled with constraints on shell thick-

ness, could influence patterns of phenotypic plasticity

and phenotypic diversification of snail species (Edgell &

Miyashita, 2009).

We examined the inducible defences of three closely

related planorbid snails (Helisoma trivolvis, H. anceps and

H. campanulata; Fig. 1) to determine how these snails

respond to predators with different foraging modes,

given the absolute constraints and allocation tradeoffs

that limit shell morphology. The three snail species

occur together in semipermanent to permanent water-

bodies, where they encounter a diversity of predators

including water bugs, crayfish and fish (Hoverman et al.,

2011). In a series of studies, we have explored the

responses of H. trivolvis to different predators (Hover-

man, Auld & Relyea, 2005; Hoverman & Relyea, 2007a,b,

2008, 2009). In the presence of the water bug Belostoma

flumineum, H. trivolvis invests in shell coiling but the

aperture remains relatively small because of their

moderate expansion rate; this reduces the ability of the

bug to reach the snail’s soft tissues when withdrawn

inside the shell. In contrast, H. trivolvis forms thicker

shells in the presence of the crayfish Orconectes rusticus,

which reduces the predator’s ability to crack or crush

the shell.

In contrast to the extensive research on H. trivolvis,

there appear to be no studies on the predator-induced

morphology of H. campanulata and H. anceps. Among the

three species, the main difference in shell shape is the

rate of shell expansion (Raup, 1962). Visual inspection

suggests that the expansion rate is low for H. campanula-

Fig. 1 Left side (i.e. spire) view of Helisoma anceps, H. campanulata

and H. trivolvis. These three species differ in the rate at which the

diameter of the shell increases with each rotation around the

coiling axis (termed w; Raup, 1962); w is low for H. campanulata,

intermediate for H. trivolvis and high for H. anceps, respectively. As

a consequence of variation in w, H. anceps exhibits a relatively large

aperture for a given body mass, whereas H. campanulata exhibits a

relatively small aperture.

© 2014 John Wiley & Sons Ltd, Freshwater Biology, doi: 10.1111/fwb.12332

2 J. T. Hoverman et al.

ta, intermediate for H. trivolvis and high for H. anceps.

Consequently, H. campanulata has a relatively small shell

aperture relative to its overall size, whereas H. trivolvis

has an intermediate aperture and H. anceps has a rela-

tively large aperture. Additionally, shell thickness also

appears to differ among species; shells are relatively

thick for H. anceps, intermediate for H. trivolvis, and rela-

tively thin for H. campanulata (Osenberg & Mittelbach,

1989; Brown, 1998).

These differences in basic shell geometry and thick-

ness might constrain the predator-induced defences of

each species, resulting in differences in predation risk by

shell-invading versus shell-crushing predators. For

example, because H. anceps has a thick shell and a high

shell expansion rate, it should be well defended against

shell-crushing predators but vulnerable to shell-invading

predators. Because H. campanulata has a thin shell with a

low shell expansion rate, it should be well defended

against shell-invading predators but vulnerable to crush-

ing predators. To test these hypotheses experimentally,

we assessed the phenotypic responses of the snails to

water bugs or crayfish and then examined the relative

susceptibility of each snail species to the predators.

Methods

Induction experiment

The goal of the induction experiment was to assess

predator-induced morphology in the three Helisoma spe-

cies. We collected ~100 adults of H. trivolvis, H. anceps

and H. campanulata from ponds near the University of

Pittsburgh’s Pymatuning Laboratory of Ecology (PLE) in

Linesville, PA. These ponds contain both water bugs

(Belostoma flumineum) and crayfish (Orconectes rusticus).

For each snail species, 10 individuals were placed into

each of 10 culture pools filled with 100 L of well water.

Egg deposition began immediately and continued until

the adults were removed after 2 weeks. Upon hatching,

snails were fed rabbit food ad libitum until the experi-

ment began.

In a mesocosm experiment, we examined the effects of

caged predators on the growth and morphology of each

species. We designed a completely randomised, factorial

experiment composed of three predator treatments (no

predator, caged water bug [B. flumineum], or caged cray-

fish [O. rusticus]) crossed with the three snail species.

These nine treatments were replicated eight times for a

total of 72 experimental units.

The experimental units were 90-L pools filled with

well water. We added 10 g of rabbit food and an aliquot

of pond water, containing periphyton, phytoplankton

and zooplankton, to sustain food for snails and maintain

water quality. Each pool received 100 juvenile snails of

the appropriate species. Initial mean mass � 1 SD of

H. trivolvis, H. anceps or H. campanulata was 2.0 �1.4 mg, 1.2 � 0.6 mg and 1.5 � 0.9 mg, respectively. For

each snail species, 20 snails were set aside to assess mor-

tality due to handling; 24-h survival was 100%.

After adding the snails, we placed a single predator

cage into each pool. The cages were made from corru-

gated pipes (10 cm long 9 10 cm diameter) capped with

shade cloth. For caged predator treatments, we added

one water bug or crayfish to each cage. Caged predators

emit water-borne chemical cues, which provide the

opportunity for prey to detect and respond to predators

without reducing prey density (Chivers & Smith, 1998).

The caged predators were fed 300 mg of snail biomass

(total wetmass including shells, two to five snails) of the

appropriate snail species three times per week. Based on

previous research, this amount of consumed snail bio-

mass by the predators is sufficient to elicit phenotypic

responses in H. trivolvis (Hoverman & Relyea, 2007b,

2008, 2009). The predators consumed all the snails

between feedings. To equalise disturbance, we briefly

lifted the cages in the no-predator treatment from the

water and then returned them. We placed a shade cloth

lid over each pool to prevent colonisation by insects and

amphibians.

During the experiment, we observed that the spe-

cies varied greatly in growth rates; growth was fast in

H. trivolvis, intermediate in H. campanulata and slow in

H. anceps. Because analyses of morphological plasticity

are sensitive to differences in mass, we decided to take

down the experimental units for each species at different

times so that they were similar in mass at the end of the

experiment. Although this approach resulted in differ-

ences among the species in duration of predator expo-

sure, our previous work with H. trivolvis found that the

magnitude of predator-induced morphological change is

relatively constant over ontogeny (Hoverman & Relyea,

2007a, 2009). For H. trivolvis, H. campanulata and

H. anceps, the experiment was ended after 14, 21 and

39 days, respectively. On each date, all surviving snails

were removed and preserved in 10% formalin. In one

experimental unit, all the H. campanulata died and this

was excluded from analyses. For the remaining experi-

mental units, survival was high (>95%) and did not

differ among caged predator treatments or snail species

(predator, F2,63 = 0.7, P = 0.499; species, F2,63 = 0.3,

P = 0.706; interaction, F4,63 = 1.3, P = 0.274). For each

experimental unit, 10 individuals were randomly


Defensive strategies of Helisoma snails 3

selected and dried at 60 °C for 24 h. Each individual

was then weighed to the nearest mg (total dry mass

included shell and tissue) and measured for shell width

and height, and aperture width and height using digital

imaging software (Optimas Co., Bothell, WA, U.S.A.).

We also measured the shell thickness of each snail at the

leading edge of the aperture using digital calipers.

To examine the effects of our caged predator treat-

ments on snail morphology, we began by assessing the

allometric relationship between each shell dimension

and log10-transformed mass. While there was no rela-

tionship between shell thickness and mass for each spe-

cies, the remaining shell dimensions showed positive

relationships with mass. To account for these allometric

relationships, we used analysis of covariance (ANCO-

VA) with mass as a covariate and snail species and

predator treatment as main effects. A critical assumption

in the ANCOVA procedure is that the treatments share

a common regression slope and our data met this

assumption (tests of interactions with mass all

P ≥ 0.171). From the ANCOVA, we used the estimated

marginal means and residuals from within-treatment

regressions to calculate a mass-adjusted value for each

individual. Using all the measured individuals in the

ANCOVA, we had ample power to capture the allomet-

ric relationship between shell dimensions and mass. For

each shell dimension, we then calculated the mean size-

adjusted shell dimensions for each experimental unit

and used these means as the morphological response

variables. Because shell thickness did not covary with

mass, we calculated the mean shell thickness for the

snails from each experimental unit and this served as

the response variable.

We used a multivariate analysis of variance (MANO-

VA) to analyse the effect of caged predators and snail

species on final mass, mass-adjusted shell dimensions

(shell width and height, and aperture width and height)

and shell thickness. The data were normally distributed,

and variances were equal across treatments. Significant

multivariate effects were followed by univariate tests.

When univariate tests were significant, we conducted

mean comparisons using Tukey’s HSD test. We also con-

ducted correlation analyses to examine the relationships

between the morphological traits and assess whether

allocation tradeoffs were evident across snail species

and predator treatments. For each pairwise combination

of the five morphological traits, we calculated the Pear-

son product–moment correlation coefficient across the

nine experimental treatments. We used the mean trait

value for each experimental unit in the analysis

(N = 71). We used a Bonferroni-corrected a = 0.005 to

assess the significance of the 10 correlations that were

conducted.

Predation trials

For the predation trials, the objective was to test preda-

tor preference for the different snail species after

defences in response to the cues of water bugs or cray-

fish had been induced. Pilot experiments indicated that

the two species of predators were capable of consuming

all three species of snails when they were small and of

similar mass (i.e. ~50 mg dry mass; R.D. Cothran et al.

unpublished data), but the key issue is the predation

risk when predators are given a choice of all three spe-

cies that have been exposed to predator cues for the

same amount of time. Because the species differ not

only in relative shape but also in size (i.e. they grow at

different rates), the predation trials tested how the

entire suite of traits affects relative predation risk when

a predator is given a choice among the three snail spe-

cies. In short, the predation trials were designed to

assess differences in predation risk using the typical

variation in size and shape of snails that would occur

in nature.

For each snail species, we collected 100 adult snails

from the E.S. George Reserve in Livingston County, MI,

U.S.A. The three species were collected from a single

pond (Crane pond) that contained both predators (Hov-

erman et al., 2011). For each snail species, we divided

individuals equally among five 100-L wading pools (i.e.

20 per pool) and allowed them to deposit egg masses

for 2 weeks. After hatching, the juvenile snails were fed

rabbit food ad libitum until the start of the experiment.

Water bugs and crayfish were collected from local ponds

and housed in the laboratory, where they were fed

snails weekly until used in the predation trials.

For each snail species, we created water bug- and

crayfish-induced snails by raising them in 90-L wading

pools (n = 6 for each snail species and predator combi-

nation, for a total of 36 pools). These pools contained

either a caged crayfish or a caged water bug and were

set up identically to the pools used in the induction

experiment. We added 60, 1-week-old snails of the

appropriate species to each pool.

We tested the snails for their relative susceptibility to

a given predator after being exposed to predator cues

for 2 weeks (i.e. juvenile snails) and again after 5 weeks

(i.e. adult snails). For each snail species and predator

combination, we collected 20 snails from each of the six

induction pools and combined them for a total of 120

snails that could be randomly assigned to predation



trials. We randomly selected a subsample of snails from

each treatment combination to test for species differ-

ences in mass (mg oven-dried mass including shell).

Using these induced animals, we randomly selected

10 individuals from each of the three snail species and

added the combined 30 animals to each experimental

unit. Experimental units were 10-L plastic tubs contain-

ing 7 L of well water and a predator refuge. For cray-

fish, the predator refuge was a half-piece of PVC pipe

(10.5 cm long, 4 cm high). For water bugs, the predator

refuges were two plastic ‘macrophytes’ composed of 10

pieces of polypropylene rope (10 cm long, 0.5 cm diame-

ter) bound together with a small cable tie and anchored

by a small rock. For water bugs, the size of the preda-

tors (length from tip of head to tip of abdomen) used in

the trials was 21.5 � 1.1 mm (mean � SD) and

20.8 � 0.9 mm for the 2 and 5 week trials, respectively.

For crayfish, the size of the predators (length from tip of

rostrum to tip of telson) used in the trials was

63.5 � 7.5 mm (mean � SD) and 73.4 � 4.6 mm for the

2 and 5 week trials, respectively.

The 30 snails in each tub were given 24 h to acclimate

before we added uncaged predators that had been

starved for 1 day. We set up 10 replicates containing the

mixture of snail species for each of the two predator

species. For the predation trial on snails that had been

exposed to predator cues for 2 weeks, we ended each

replicate after a predator had consumed 40–60% of the

snails. For the predation trial on snails that had been

exposed to predator cues for 5 weeks, we had to relax

this criterion because predators were not able to con-

sume many of the adult H. trivolvis and H. campanulata

(i.e. some tubs had up to 73% survival). We also

included two no-predator controls to monitor snail mor-

tality without predators and confirmed that survival

without a predator was 100%.

We used a general linear model (GLM) to compare

snail species survival after 2 and 5 weeks of exposure to

predator cues. ‘Species of snail’ was used as the within-

subject variable and ‘predator type’ (crayfish or water

bug) was used as the between-subject variable. We also

used a GLM to test for differences in size among the

snail species.

Results

Induction experiment

In the induction experiment, we found that responses to

the caged predators varied across the three snail species

(Table 1). Because of the interaction between snail

species and caged predators, we explored the effects of

caged predators within each snail species.

For H. anceps, caged predators affected all traits

(Fig. 2). Snails were 47–49% smaller with caged water

bugs compared with no predators and caged crayfish;

there was no difference in mass between the latter two

treatments. Caged crayfish induced a 3–8% reduction in

shell and aperture dimensions compared with no preda-

tors and caged water bugs. Crayfish also induced 22 and

30% thicker shells than snails reared with caged water

bugs and no predators, respectively. Compared with the

no predator treatment, water bugs induced no changes

in morphology.

For H. campanulata, caged predators affected mass and

several shell dimensions (Fig. 2). Snails had 45–73%

greater mass with caged crayfish compared with no

predators and caged water bugs; there was no difference

between the latter two treatments. Caged water bugs

induced 5–7% wider shells and 9–12% lower shells and

apertures than no predators and caged crayfish.

Compared with the no predator treatment, crayfish

induced no changes in morphology.

For H. trivolvis, caged predators affected shell width

and shell thickness (Fig. 2). Caged water bugs induced

10–13% wider shells than no predators and caged

crayfish; the latter two treatments did not differ. Caged

crayfish induced 50–52% thicker shells than no predators

and caged water bugs; the latter two treatments did not

differ.

Across the nine experimental treatments, there was

strong evidence of allocation tradeoffs between shell

thickness and shell shape. Greater investment in shell

width, shell height or aperture height was associated

with reduced investment in shell thickness (r ≤ �0.727,

Bonferroni-corrected P ≤ 0.001, n = 71; Fig. 3). There

Table 1 Results of a MANOVA (Wilks’ k with F approximations)

on the effects of snail species and caged predator species on snail

mass, shell and aperture shape, and shell thickness. Univariate tests

(P-values) are shown for both main effects and their interaction

Multivariate tests d.f. F P

Species 12,114 198.7 <0.0001Predator 12,114 18.2 <0.0001Species * Predator 24,200 5.8 <0.0001

Univariate tests Species Predator Species * Predator

Mass <0.0001 <0.0001 0.038

Shell height <0.0001 <0.0001 <0.0001Shell width <0.0001 <0.0001 <0.0001Shell thickness <0.0001 0.016 0.072

Aperture height <0.0001 <0.0001 <0.0001Aperture width <0.0001 0.467 0.011



were also positive associations between shell width,

shell height and aperture height (r ≥ 0.526, P ≤ 0.001,

n = 71). The remaining correlations were not significant

(P ≥ 0.029; Bonferroni-corrected a = 0.005).

Predation trials after 2 weeks of exposure to predator cues

We examined the susceptibility of the three Helisoma

species after being induced by water bugs or crayfish

for 2 weeks. Following 2 weeks of induction, there was

no effect of predator species on snail mass (F1,53 = 1.9,

P = 0.178), and there was no interaction between snail

species and predator species (F2,53 = 0.4, P = 0.706).

However, the three snail species did differ in mass

(F2,53 = 161.1, P < 0.001; Fig. 4a); Helisoma trivolvis was

the largest species followed by H. campanulata and

H. anceps (pairwise comparisons between H. trivolvis and

the other two species both P < 0.001; between H. anceps

and H. campanulata P = 0.053).

Survival depended on the species of snail and the

species of predator (snail species-by-predator type

interaction: F2,17 = 13.0, P < 0.001; Fig. 4b). As a result,

we split the data set by predator species to test for dif-

ferences in vulnerability among the three snail species.

Survival with uncaged water bugs differed among the

three species (F2,8 = 11.0, P = 0.005; Fig. 4b). Compared

with H. anceps, H. trivolvis and H. campanulata were

about three times more likely to survive (P = 0.003 and

P = 0.012, respectively). Survival did not differ between

H. campanulata and H. trivolvis (P = 0.678). Survival with

Fig. 2 The effects of caged predator

treatments on the dry mass, size-adjusted

shell morphology and shell thickness of

Helisoma anceps, H. campanulata and

H. trivolvis. Data are means � 1 SE. For

each species, treatments sharing lower

case letters are not significantly different

from each other based on pairwise com-

parisons using Tukey’s HSD test

(P > 0.05).



uncaged crayfish was marginally non-significant among

the three snail species (F2,8 = 3.8, P = 0.069). Helisoma

anceps was 1.5 times more likely to survive than H. cam-

panulata (P = 0.075). All other pairwise comparisons

were insignificant (all P ≥ 0.12).

Predation trials after 5 weeks of exposure to predator cues

After being induced by predators for 5 weeks, we again

compared the mass and survival of the three snail spe-

cies. In regard to mass, there was no effect of predator

(F1,54 = 1.5, P = 0.23) and no interaction between snail

species and predator species (F2,54 = 0.4, P = 0.693).

However, the three snail species differed in mass

(F2,54 = 133.28, P < 0.001; Fig. 4a); similar to the pattern

observed after 2 weeks of induction, H. trivolvis was

largest followed by H. campanulata and H. anceps (all

pairwise comparisons P < 0.001).

Snail survival depended on the predator that was

present (F2,17 = 5.1, P = 0.018; Fig. 4c); therefore, we split

the data set by predator. Survival in the presence of

(a)

(b)

(c)

Fig. 3 The association between shell thickness and shell shape

[shell width (a), shell height (b) and aperture height (c)] across the

nine experimental treatments. For each snail species (Helisoma an-

ceps, H. campanulata and H. trivolvis), the trait means are presented

for the experimental units within each predator treatment (no pred-

ator, caged water bugs and caged crayfish).

(a)

(b)

(c)

Fig. 4 Mass and survival of Helisoma anceps, H. trivolvis and

H. campanulata in the predation trials after 2 and 5 weeks of induc-

tion. The three species were either induced by caged water bugs

and then subjected to predation by lethal water bugs or induced by

caged crayfish and then subjected to predation by lethal crayfish:

(a) mass of each species of snail at the start of a predation trial

(closed symbols are 2 weeks and open symbols are 5 weeks), (b)

survival of induced snails against lethal crayfish or lethal water

bugs following 2 weeks of predator induction, (c) survival of

induced snails against lethal crayfish or lethal water bugs following

5 weeks of predator induction. Data are means � 1 SE.



uncaged water bugs differed among the three snail

species (F2,8 = 168, P < 0001). None of the H. anceps

survived, whereas survival was high for H. campanulata

and H. trivolvis (comparisons against H. anceps: both

P < 0.001; H. campanulata versus H. trivolvis: P = 1.0).

Survival with uncaged crayfish also differed among the

three snail species (F2,8 = 158, P < 0.001). Few H. anceps

survived, whereas most of the H. campanulata and H. tri-

volvis did (comparisons with H. anceps: both P < 0.001;

H. campanulata versus H. trivolvis: P = 0.486).

Discussion

We found that Helisoma trivolvis, H. anceps and H. cam-

panulata all expressed predator-induced plasticity, yet

they exhibited individual responses to the predators.

The responses of H. trivolvis to caged predators were

consistent with previous research on this species

(Hoverman et al., 2005; Hoverman & Relyea, 2007b).

When exposed to water bug cues, they formed wider

shells (i.e. coiled more) but aperture size did not change.

These responses enable H. trivolvis to withdraw into its

shell, which reduces the water bug’s ability to access the

snail’s soft tissues (Hoverman & Relyea, 2009). They

formed thicker shells when exposed to crayfish cues,

which reduces shell-crushing or chipping by crayfish

(Hoverman & Relyea, 2009). Given the adaptive value of

these responses for H. trivolvis and the co-occurrence of

H. anceps and H. campanulata with the same predators,

we predicted similar phenotypic responses across the

snail species. However, the morphology of H. anceps and

H. campanulata changed in response to one or the other

predator but not to both. Helisoma anceps responded to

crayfish by forming thicker shells, narrower shells and

apertures, and lower shells and apertures; however,

there was no response to water bugs. Helisoma campanu-

lata responded to water bugs by forming wider and

lower shells and low apertures, although there was no

response to crayfish. Although there were no

morphological responses in H. anceps and H. campanulata

when exposed to water bugs and crayfish, respectively,

mass did change. Specifically, H. anceps was smaller

with caged water bugs while H. campanulata was larger

with caged crayfish. Thus, both species did detect these

predators, but morphology did not respond. Interest-

ingly, the predator-specific morphological responses

observed in H. anceps and H. campanulata to crayfish and

water bugs, respectively, are similar to many of the

adaptive responses observed in H. trivolvis. Thus, the

responses of H. anceps to crayfish, and the responses of

H. campanulata to water bugs, might reduce risk to each

inducing predator, but the lack of response to the alter-

native predator might increase risk in the presence of

that predator (a hypothesis later tested in the predation

trials).

The differences in morphological responses to water

bugs between the species appear to reflect the geometric

constraints of shell shape. While all three species have

planospiral coiling, H. campanulata and H. trivolvis have

low expansion rates, so the diameter of their coiled tube

increases only gradually as whorls are added. In contrast,

H. anceps has a high expansion rate, so the diameter of its

coiled tube increases substantially as more whorls are

added. Consequently, H. campanulata and H. trivolvis can

respond to water bugs by increasing shell width (i.e. rap-

idly growing a longer coiled tube) because this places the

aperture of the shell further away from the soft tissue of

the snail, without substantially increasing the size of the

aperture; this makes it harder for a water bug to reach

and pierce the soft tissues. If H. anceps was to respond to

water bugs in this manner, the soft tissue would be fur-

ther from the aperture, but the aperture would also

become much larger and easier for a water bug to enter

and pierce the soft tissues. This geometric constraint of

H. anceps may explain its lack of morphological response

to water bugs.

The existence of allocation tradeoffs is an underlying

assumption of phenotypic plasticity (Schlichting & Pig-

liucci, 1998; Tollrian & Harvell, 1999; Pigliucci, 2001).

Using correlation analyses, we found strong evidence for

allocation tradeoffs across the experimental treatments;

investment in shell thickness was negatively correlated

with investment in shell shape. Thus, in addition to geo-

metric constraints on shell shape, snails face allocation

tradeoffs that constrain their ability to invest simulta-

neously in shell thickness and shape. These results are

consistent with previous work in marine snails. For

instance, Trussell & Nicklin (2002) found a negative asso-

ciation between shell length and thickness in Littorina

obtusata. This allocation tradeoff appears to be driven by

competing demands for calcium, such that thicker shells

can only be produced when investment in shell length is

reduced (Palmer, 1981; Kemp & Bertness, 1984).

As a consequence of allocation tradeoffs, snails are

faced with a fundamental tradeoff between responses to

shell-entering and shell-crushing predators. In H. trivol-

vis, the induction of wider shells, by extending the length

of the coiled tube, makes it more difficult for water bugs

to enter the aperture and pierce the soft tissue (Hoverman

& Relyea, 2009). However, this comes at the cost of

reduced investment in shell thickness, which makes the

shell more vulnerable to crushing predators such as



crayfish. Similarly, the body plan of H. campanulata is well

suited to defence against water bugs, because it can rap-

idly grow a long, thin tube that makes it difficult for water

bugs to reach the soft tissue. However, this strategy leaves

few resources for thickening the shell making H. campanu-

lata vulnerable to crushing predators such as crayfish. In

other freshwater and marine snails, research has also doc-

umented contrasting responses to predators with diver-

gent foraging modes that appear to be driven by

allocation tradeoffs (Dewitt, Robinson & Wilson, 2000;

Bourdeau, 2009). For instance, Bourdeau (2009) showed

that the marine snail, Nucella lamellosa, forms thick, round

shells when exposed to shell-crushing crabs but elongate

shells with shell-entry seastars. Thus, morphological

defences in snails appear to be driven by a fundamental

tradeoff between shell thickness and shape.

Our study focussed on morphological responses to pre-

dators, yet behavioural responses have also been

observed in H. trivolvis and other snails (Dewitt, Sih &

Hucko, 1999; Turner, Bernot & Boes, 2000; Hoverman

et al., 2005; Hoverman & Relyea, 2007b). Behavioural

responses often involve spatial avoidance, such that snails

move into areas that provide shelter (e.g. rock refuges) or

are difficult for the predator to reach (e.g. the water sur-

face). In our previous work, we did not detect behaviour-

al responses to water bugs because this predator is

capable of foraging for snails throughout the water col-

umn and under ‘refuges’ (Hoverman et al., 2005; Hover-

man & Relyea, 2007b). However, snails often move

towards the water surface or above the water line in the

presence of crayfish (Hoverman et al., 2005), although

such responses are not always observed (Hoverman &

Relyea, 2007b). As crayfish are largely benthic, this behav-

iour can reduce the probability of encounter. While

behaviour was not quantified in our experiments, it is

possible that it could play an important role in the

defences of these snails. For instance, several studies have

demonstrated trait compensation, where individuals with

poorly formed morphological defences display strong

behavioural responses to predators (Dewitt et al., 1999;

Rundle & Br€onmark, 2001; Cotton, Rundle & Smith, 2004;

Rundle et al., 2004). Thus, it is possible that H. campanula-

ta and H. anceps could display anti-predator behaviours

in the presence of crayfish and water bugs, respectively,

compensating for their vulnerable shell morphology.

Additional research is necessary to explore whether

behavioural responses are employed by these species.

The results of the predation trials conducted after

2 weeks of exposure to the cues of water bugs or

crayfish demonstrated how the phenotypes of the snails,

when all three species were combined, affected their

relative predation risk. In this trial, all three species were

within a mass range that could be consumed by either

predator (R.D. Cothran et al., unpublished data), but the

survival of each snail species with uncaged crayfish or

water bugs was strongly associated with the observed

changes in mass and morphology. When crayfish-

induced snails were exposed to crayfish, survival was

high for H. anceps, intermediate for H. trivolvis and low

for H. campanulata. This was particularly interesting

because H. anceps was the smallest of the three species,

yet it was the best defended against crayfish. When

water bug-induced snails were exposed to water bugs,

survival was low for H. anceps and high for H. trivolvis

and H. campanulata. Comparing the mass of H. trivolvis

and H. campanulata, the latter was considerably smaller,

yet its morphological defences against water bugs were

stronger. In contrast, H. campanulata and H. anceps were

more similar in mass, yet H. campanulata had better

survival, further confirming that the morphological

responses to water bug cues provided H. campanulata

with effective defences against water bugs. These results

are consistent with the argument that snails with

relatively wider shells are better defended against water

bugs and snails with thicker shells are better defended

against crayfish.

After being exposed to predator cues for 5 weeks, we

observed that H. trivolvis and H. campanulata had

very high survival with both uncaged predators while

H. anceps had very low survival. The main driver of

these contrasting outcomes of predation with time

appears to be the occurrence of size refuges from preda-

tion. We have previously shown that adult H. trivolvis

can reach a size refuge against both water bugs and

crayfish, regardless of their shell phenotype (Hoverman

& Relyea, 2009). Thus, it appears that H. trivolvis and

H. campanulata exceeded the size required to reach a ref-

uge from both predators, whereas H. anceps was still

vulnerable. As a result, it appears that the inability of

either predator to consume H. trivolvis and H. campanu-

lata shifted all of the predation pressure to H. anceps,

thereby causing much lower survival of this species. In

short, when the snails of any species are within a vul-

nerable size range, morphological defences are impor-

tant for reducing predation risk, but when a species

achieves a size refuge, the morphology of the large spe-

cies becomes unimportant to predation risk and preda-

tion can become more intense on the small species in

the community; even when the small species has a

defended phenotype. One of the limitations of our

experimental design was that we used a narrow size

range for each predator species. Although there is little



variation in the size of adult water bugs, crayfish can

attain a larger size than represented in our study. As

adults, crayfish tend to shift to more herbivorous diets,

but their greater size does allow them to feed on larger

macroinvertebrates (Lodge & Lorman, 1987; Lodge et al.,

1994). Because larger crayfish can consume larger prey,

it is possible that the results of the predation trials after

5 weeks of induction would be different if larger cray-

fish were selected for the experiment. However, if snails

were not in a size refuge from predation, we would

expect the results to be similar to the outcome observed

in the predation trials after 2 weeks of induction.

While predation is a common threat for freshwater

snails that has been hypothesised to affect patterns in

species richness, species abundance and community

structure (Lodge et al., 1987; Dillon, 2000), our

understanding of how inducible defences contribute to

patterns in species distribution and abundance remains

limited. Theory suggests that species with inducible

defences against a number of predators (i.e. generalists)

should be more broadly distributed than species that

specialise for a particular predator (Agosta & Klemens,

2008). We find support for this prediction for snail meta-

communities in Michigan, U.S.A. (Hoverman et al.,

2011). Helisoma trivolvis is the most broadly distributed

of the three species with a range that spans temporary

ponds to permanent lakes, H. campanulata is found in

semi-permanent ponds to lakes, and H. anceps is only

found in a subset of lakes. Across this habitat gradient,

there is also a gradient in predator composition; water

bugs are more frequently found in temporary and

semipermanent ponds, while crushing predators such as

crayfish and fish are more common in permanent lakes

(Hoverman et al., 2011). In addition to predation,

however, factors such as habitat size, hydroperiod and

competition can structure snail assemblages (Lodge

et al., 1987; Dillon, 2000; Hoverman et al., 2011).

Freshwater habitats also vary in abiotic conditions that

could influence the interaction between snails and their

predators. For instance, ponds with low calcium concen-

tration or low pH could interfere with shell deposition

in snails and, consequently, constrain the expression of

inducible defences (Thomas et al., 1974; Madsen, 1987;

Rundle et al., 2004). While additional research examining

the interactions between predators and different snail

species in natural populations is needed, our results

suggest that differing defences against predators may be

an important driver of species distributions in fresh-

water snails.

Our study demonstrates that three congeners have

phenotypically plastic strategies that span the full range

of possibilities: specialised defences against a shell-crush-

ing predator, specialised defences against a shell-invading

predator and generalised, flexible defences against both

predators. Theory predicts that a population should

evolve either fixed or plastic phenotypes depending on

the costs and benefits of alternative phenotypes and the

frequency of alternative environments (Pfennig et al.,

2010). However, our work suggests that species can

become somewhat specialised to a given environment, yet

still maintain some plasticity. This may reflect a condition

in which the plasticity has not yet achieved fixation for a

constitutive defence. Alternatively, it may simply be that

each specialist most commonly experiences the presence

or absence of one type of predator over space and time

(e.g. environments containing either no predators or a

shell-crushing predator) but rarely experiences the other

type of predator (e.g. a shell-invading predator). In such a

scenario, there would be little selective pressure to evolve

responses to the other type of predator. While more

research needs to be conducted on this system to under-

stand how selection shapes the evolution of phenotypic

plasticity, it is clear that an understanding of absolute

constraints on body shape, combined with knowledge of

allocation tradeoffs, can provide critical insights into the

mechanisms that produce interspecific variation in the

expression of phenotypic plasticity and patterns of

phenotypic diversification.

Acknowledgments

We thank N. Diecks, B. French, D. Jones, P. Monahan, P.

Noyes and A. Stoler for their assistance with the

experiment and S. Bagnull for measuring the snails. This

work was supported by a National Science Foundation

grant to RAR, and grants from the Conchologists of

America, the Pennsylvania Academy of Science, and

Sigma Xi to JTH.

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(Manuscript accepted 13 January 2014)

