DIFFERENTIAL GROWTH RATES AND CALCIUM-ALLOCATION
STRATEGIES IN THE GARDEN SNAIL CANTAREUS ASPERSUS
ALAN BEEBY AND LARRY RICHMOND
Department of Applied Science, London South Bank University, London SE1 0AA, UK
(Received 18 May 2006; accepted 21 December 2006)
ABSTRACT
An optimal division of a key resource between growth and reproduction is expected to produce consistent life history schedules in habitats where its supply is highly predictable. However, differential growth
rates are found between populations and within broods of Cantareus aspersus, a simultaneous hermaphrodite for which the reproductive benefits of a large body size may favour rapid growth. Although
energy is usually assumed to be the limiting resource in allocation theory, calcium limits the distribution, growth and reproduction of snails. This is a very consistent resource and populations may
have allocation strategies which reflect availability in their habitats. Three experiments compared
Ca allocation in the progeny of six populations from Ca-rich and Ca-poor habitats. In the first,
100 d-old juveniles were compared between populations for their shell/soft-tissue dry weight ratio,
their allocation of Ca to each compartment, and the variability within broods. The second measured
growth, food consumption and shell ratios in growth trials of three populations on low Ca. Thirdly,
five populations were compared on abundant or excess Ca. The relationship of shell Ca with soft-tissue
levels differs between populations, but shell ratios changed with Ca availability in all populations. Most
favoured soft-tissue growth when dietary Ca is low, but one population (LE) always had the highest
shell ratios in these trials. Ca in the parental habitat was not a good predictor of juvenile-allocation
strategies, but the consistency of LE shell ratios across several broods suggests theirs may be an inherited
trait. LE has faster growth rates and a preference for shell building, which probably represents a
strategy for early reproduction. The robustness of a snail’s shell may thus be more indicative of its
reproductive strategy rather than Ca availability in its habitat.
INTRODUCTION
Recent models of sex-allocation theory applied to simultaneous
hermaphrodites suggest body size is critical when there is competition between partners to play a preferred role (Angeloni, 2003;
Gianguzza et al., 2004). Size is also a determinant of gamete production and the storage of resources in several iteroparous
species (Leonard, 1991; Norton & Bronson, 2006), although
any reproductive strategy will be informed by the time taken
to acquire these resources, and the costs and benefits of early
maturation (Kozlowski, 1992; Stearns, 1992). Small individuals
breeding early may benefit from multiple reproductive events,
but might be at a disadvantage when size determines success
in competitive sexual encounters.
Among hermaphrodite gastropods, large individuals devote a
smaller proportion of their resources to the male role yet marine
opisthobranchs, at least, are able to flush out a competitor’s
sperm from the spermatheca of a smaller partner (Angeloni,
2003). The garden snail Cantareus aspersus (Müller) (formerly
Helix aspersa ) will store sperm from several copulations, to be
selected at fertilization (Albuquerque de Matos, 1989; Landolfa,
Green & Chase, 2001). The snail may then lay a clutch of eggs in
which large fathers are better represented in the brood (Adamo
& Chase, 1988; Landolfa, Green & Chase, 2001). Indeed, deep
and durable penetration by a reproductive dart appears to
improve sperm retention (Landolfa et al., 2001; Geoffroy,
Hutcheson & Chase, 2005). Large snails are more prolific
mothers: egg size and clutch size are positively correlated with
adult weight in several helicids (Heller, 2001). Cantareus is a
=
Correspondence: A.N. Beeby; e-mail: [email protected]
promiscuous species and a large individual is both a competitive
male and a fecund female.
There is, however, considerable variability in growth rates
within broods. High population densities or a high concentration
of slime may limit juvenile activity and feeding, though other
factors are known to influence this (Dan & Bailey, 1982; Sanz
Sampelayo, Fonolla & Gil Extremera, 1990; Jess & Marks,
1995; Cook, 2001; Hanley, Bulling & Fenner, 2003) and hatchlings raised singly still grow at different rates (Beeby & Richmond,
unpublished data). Differential growth rates may safeguard the
parental reproductive investment if mortality rates are variable
and these might be promoted by producing different sized eggs
or supplying additional nutrients. Some terrestrial gastropods
produce infertile ‘trophic’ eggs that are consumed by early
hatchlings, which then grow faster and consume more food
than their siblings (Baur, 1992; Desbuquois, 1997).
Where a key or limiting resource has a consistent supply, local
ecotypes might appear with growth strategies adapted to its
availability. The eclectic diet of most snails means they are unlikely to be energy limited. Other resources, principally water but
also N and Ca, may be constraining (Baur, 1994; Locher & Baur,
2002; Wacker & Baur, 2004) and Ca is a major determinant of
the size and distribution of many snail species (Boycott, 1934;
Fournie & Chetail, 1984; Gardenfors, 1992; Kalisz & Powell,
2003; Ondina et al., 2004). Besides reinforcing the shell, Ca is
critical to a variety of functions in soft-tissue metabolism
and reproduction (Tompa & Wilbur, 1977; Porcel, Bueno &
Almendros, 1996). Low soil levels (or high acidity) may exclude
Cantareus from some habitats or limit the fecundity of resident
populations (Crowell, 1973). While its availability can be
spatially variable, soil Ca levels do not change rapidly and this
predictability may promote local adaptation.
Journal of Molluscan Studies (2007) 73: 105–112. Advance Access Publication: 9 January 2007
# The Author 2007. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.
doi:10.1093/mollus/eym002
A. BEEBY AND L. RICHMOND
(Albuquerque de Matos, 1989; Jess & Marks, 1995) during
which they may triple their fresh weight.
The ratio of shell dry weight to soft-tissue dry weight (the
‘shell ratio’) was used as a measure of the calcium-allocation
strategy adopted by each snail. Between one-third and half the
mass of a shell is Ca (Beeby & Richmond, 2001b) and its
weight is therefore a good indication of the Ca provision to the
shell. Direct measurement of Ca in each component was also
determined in the first experiment using ICP atomic absorption
spectrometry.
Garden snails differ in their life history and calcium-allocation
strategies – both adult weight and shell size have a high heritability (Dupont-Nivet et al., 1997; Heller, 2001). Snails need to
partition resources between the shell and soft tissues, according
to availability and the costs and benefits of growth in either
component. Heller (2001) notes that heavily calcified shells are
associated with long-lived species, in which they may act as a
Ca reserve for multiple reproductive events. Adding to this
reserve increases shell strength (Tompa & Wilbur, 1977;
Porcel, Bueno & Almendros, 1996), but it also makes it
heavier and might delay soft-tissue growth. Variations in
juvenile growth rates or Ca allocation to the shell may reflect
Ca availability in the parental habitat, or possibly indicate the
strategy of a population for early or delayed maturity.
This paper examines whether variability in growth rates
might be adaptive when a key resource such as Ca is limiting,
and compares within-brood variability between populations of
Cantareus from Ca-rich and Ca-poor habitats. Additionally, a
series of growth trials measure the allocation of Ca between
the shell and soft tissues in juveniles raised on low and high Ca
diets, to establish whether allocation strategies are distinguished
by parental habitat.
Experimental series
Three experiments compared juvenile variation and the Ca-allocation strategy in populations from Ca-rich and Ca-poor sites.
The first measured variation in juvenile weights at two different
ages and assessed whether variability was consistent between
broods within a population. Calcium concentrations in the
shell and soft tissues were also determined after their rapid
growth phase. The second experiment compared growth and
shell ratios in juveniles from three populations grown singly
and fed a diet deficient in Ca and carbonate. The third experiment comprised growth trials of juveniles from five populations
fed diets with abundant and possibly excessive Ca.
The difficulty of producing hatchlings and multiple broods
from each population at the same time often constrained the
combination available for a trial. Here we report those trials
in which at least one Ca-rich and one Ca-poor population are
compared.
MATERIAL AND METHODS
Adult Cantareus aspersus (i.e. with lipped shells) were collected
from six locations in England, Wales and from the Corbières,
on the Mediterranean coast of France. These sites were chosen
for the Ca content of their soils and their distance apart
(Table 1).
The snails were washed in tap water and for each population,
20 –30 were placed in a 12-l plastic aquarium in a constant
temperature room at 228C with a 16:8 h light:dark cycle to
induce mating and egg-laying. They were fed ad libitum on
lettuce and a diet consisting of a 1:1:1 mixture of ground
oatmeal, dried milk powder and calcium carbonate (termed
the basic diet; Beeby & Richmond, 2001b). They were also provided with a 3-cm deep plastic container of soil from which they
might soil-feed and in which they could lay eggs. The soil came
from a suburban garden in a chalkland area (LE; Table 1).
Aquaria were washed twice weekly and the soil examined for
egg clutches. Each clutch was counted and transferred to a petridish with a moist filter paper until emergence. Hatchlings were
counted, transferred to a 2-l plastic container and fed initially
with lettuce and later with the basic diet. Moisture levels were
maintained by a cotton-wool wick, soaked with distilled deionized water.
A brood was allowed either to grow together in this container
until around 100 d, or, in the growth trials, 30-d juveniles were
raised individually in 250-cm3 containers until approximately
100 d old. At around 30 d (and under these culture conditions)
Cantareus juveniles enter a rapid growth phase lasting about 70 d
Experiment 1 – within-brood variation
Variation in fresh weight was measured in single broods of four
populations representing Ca-rich and Ca-poor habitats (LE,
RD, RF, SB; Table 1) and in replicate broods from RD and
RF. For most this was prior to their rapid growth phase (30 d)
but in two cases, these measurements were of older juveniles
(SB, and the two replicate broods of RF). The greater age of
the SB juveniles especially prevents direct comparison with the
other populations but these are included to allow comparison
with members of the same brood in the growth trials of Experiment 2.
Subsequently, the shell ratios of juveniles fed the basic diet
through this growth phase (beyond 100 d) were measured in
two broods from a Ca-rich site (LE) and in a single brood
from an equivalent site in southern France (RF). Both RF and
LE-a juveniles are those whose fresh weights are recorded
around 30 d. Calcium concentrations in their shell and soft
tissues were then determined using ICP-AAS and also for the
SB juveniles at 53 d.
Each brood or population was raised as a single group and
consequently an individual snail does not represent an
Table 1. Soil characteristics of the sites from which the laboratory cultures of each population were collected (n ¼ 4 for Ca analyses).
Site type
Ca-rich
Ca-poor
Label
Location
Soil description
pH
Mean Ca concentration (mg.g21)
LE
Garden, Lewes, UK
Chalky loam
7.1
108.3
RD
Allotments, Rottingdean, UK
Chalky loam
7.1
132.3
RF
Garden, Roquefort, Aude, France
Coarse sand with particulate mortar
7.7
182.1
SB
Garden, Surbiton, UK
Sandy loam
7.1
SB þ Ca
(Ca-supplemented SB)
SB with CaCO3
–
7.6
62.6
BI
Roadside, Gravelly Hill, Birmingham, UK
Clay loam
6.5
1.2
MI
Roadside, Minera, UK
Clay loam
6.5
35.4
The RD and LE populations are the closest to each other (within about 10 km).
106
CALCIUM ALLOCATION IN CANTAREUS
marked changes in shell ratios had been observed in preliminary
experiments. MI represented a site with relatively low soil Ca. A
second trial compared LE with BI (the Ca-poor site) on four
levels of Ca (Ca4 – Ca7) of which the two higher doses were in
excess of environmental levels available to any of these populations. Single broods were used throughout these trials.
There were a small number of mortalities in each experiment,
across all experiments.
independent replicate. However these conditions reflect those in
the wild during this growth phase and allow comparison
between groups at densities with the attendant factors that
might contribute to within-brood variability. Each group had
less than 30 juveniles (in a 2-l box) for most of this growth
phase, though both LE-a and SB had densities around half
those of the other populations, principally because a number
of each brood were used in subsequent growth trials. LE-b and
RF also finished with fewer than 30 because of mortalities over
the growth phase. Densities thus differed between populations,
but to mitigate this, food was always supplied in excess and
the containers washed twice weekly.
Tissue analysis
At the end of an experiment snails were starved for two days to
allow evacuation of the gut. They were then frozen and, on
thawing, the soft tissues were separated from the shell. Each
component was dried overnight at 808C and weighed prior to
digestion. Soft tissues were boiled for 1 h in 10 cm3 concentrated
HNO3, before the cooled digest was filtered (Whatman 541) and
made up to 25 cm3 with distilled deionized water. The shell was
dissolved in 5 cm3 concentrated HNO3 and made up to 10 cm3.
Glassware and filter paper blanks and also standard reference
material (Lobster hepatopancreas – ‘TORT2’; Environment
Canada) were analysed with each run. All weights were determined using a four-decimal-place Oxford A1204 balance.
Metal analyses were performed on a Thermo-Jarrell – Ash
Tracescan ICP-AAS. No values for Ca are published for
TORT2 but the uniformity of our determinations over different
runs was regarded as a check on their consistency.
Growth-trial experiments 2 and 3
In each treatment of the growth trials, five replicate juveniles of
a population were fed individually from 30 d until about 100 d
old. Where multiple broods from the same population hatched
within a few days, snails from each were randomly assigned to
each treatment. Most populations were represented by a single
brood but different broods were used in different trials.
Growth trials on Ca-poor diets
Three populations (LE, SB and RF) were compared on diets
comprising a 1:1:1 mixture of dried egg albumin, dried lettuce
and rice flour with calcium sulphate for three levels of Ca
(Ca1 – Ca3; Table 2). These dietary components ensured extremely low levels of Ca and, by using calcium sulphate, only a
limited dietary source of carbonate, to match the soils of the
Ca-poor sites. Each population was represented by a single
brood, except RF where three broods were used. SB represented
a Ca-poor site and used individuals from the same brood examined in the first experiment. This allowed calculation of the
growth rates and the addition of Ca to the shell and soft
tissues, and to express this as a proportion of the Ca consumed
on each diet. An additional trial, again using the same SB
brood, measured the effect of soil feeding on juveniles fed the
lowest Ca diet: five replicates had access either to their native
soil or their native soil with a Ca carbonate addition (Table 1).
Measurement of food consumption
In the growth trials the food was presented as a thin coating of
powder adhering to a glass microscope slide. All diets were
milled to produce a fine, well-mixed powder and a slide prepared by smearing one side with sunflower margarine, dipping
this into the powder and tapping off the excess. The margarine
provided fats and the D vitamins known to promote Ca
uptake in Cantareus (Wagge, 1952).
A fresh slide was presented to each snail every 3 or 4 days, the
container being washed prior to its replacement. Consumption
was estimated by measuring the area of slide cleared by snail
feeding after scanning the slide on an A4 flat-bed scanner. The
contrast created by the cleared area allowed this area to be
measured using image-processing software (Image J http://
rsb.info.nih.gov/ij/). The percentage area cleared was expressed
as an approximate weight of food consumed based on the
average weight of food presented on the slide. In this way the
food consumed by each snail was measured every 3/4 days. Previous work had shown that this method was quicker and more
accurate than weighing moist foods.
The weight of food presented was estimated from four replicate slides for each diet. This was consistent across all doses
with an overall mean dry weight of 0.14 g (SD ¼ 0.03 g). The
metal content was determined by analysing these slides, following extraction for 1 h in 10 ml of boiling 50% HNO3, filtering
and making up to 25 cm3 (Table 2).
Growth trials on Ca-rich diets
The Ca-rich diet used equal quantities of ground oatmeal and
dried milk powder with one of four levels of calcium carbonate
(Ca4 – Ca7; Table 2) to provide abundant and possibly excessive
Ca. This comprised two trials: in the first, four populations (LE,
RD, RF and MI) were fed Ca4 and Ca5, diets covering the
range of soil levels of the Ca-rich sites and between which
Table 2. Mean (+SE; n ¼ 4) Ca and Mg concentrations in the diets
used in the growth-trial experiments.
Growth trial
Diet
Ca concentration
(mg.g21)
Ca-poor
Ca-rich
Mg concentration
(mg.g21)
Ca1
1.4 (0.1)
0.9 (0.04)
Ca2
2.5 (0.1)
0.9 (0.06)
Ca3
10.6 (0.1)
0.8 (0.15)
Ca4
70.4 (2.6)
1.4 (0.08)
Ca5
175.9 (7.4)
0.9 (0.01)
Ca6
235.2 (6.9)
1.2 (0.03)
Ca7
305.3 (3.6)
1.0 (0.03)
Statistical analysis
Treatment and population differences were analysed principally
by analysis of variance and regression analysis using MINITABw. Analyses of variance (general linear model) compared
responses to the various diets, having checked the normality of
the data (Anderson – Darling test) and the equality of variances
between subsets. Pairwise comparisons between treatment or
population means used Tukey’s method. Testing of differences
between coefficients of variation used Miller’s tests (Zar,
1996). Comparisons of fresh weights found no significant
Mg is used as an indicator of the consistency in the non-Ca components of the
different diets and is itself a significant component of the soft tissues and the
shell.
107
A. BEEBY AND L. RICHMOND
mass of Ca in the RF shell correlates with the logarithm of softtissue Ca (Fig. 1). In contrast, this relationship is best described
by a linear regression for both LE broods, and these share similar
regression coefficients (LE-a: b ¼ 62, r ¼ 0.96, P , 0.001,
n ¼ 16; LE-b: b ¼ 59, r ¼ 0.94, P , 0.001, n ¼ 23). The consistency between these two broods, both in this relationship and in
their shell ratios (Table 4), indicates that LE makes a larger proportionate investment of Ca in their shell than RF.
SB were measured at around half the age of the other populations, close to the start of their rapid growth phase. Neither
here or subsequently in the growth trails (below) does any SB
snail achieve a shell ratio above 0.7, even when soil-feeding on
a Ca-supplemented soil. Nor is there a significant relationship
between their soft-tissue and shell Ca levels at this age.
Table 3. Variation in mean total fresh weight between populations and
between broods where two broods are measured together (n ¼ 30 or (SB
only) 36).
Population
Number of
Age
Mean fresh
Coefficient
broods
(days)
weight (mg)
of variation
LE
1
29
104
37
RD
2
32
106
51
RF
1
33
140
48
RF
2
42
114
29
SB
1
53
101
37
Juveniles were measured close to the start of their rapid growth phase.
differences in the initial fresh weights of snails allocated to different treatments for any population in any of the growth trials.
Ca-poor growth trials
These experiments compared populations for their growth and
Ca allocation on low or Ca-deficient diets during the rapid
growth phase. In each trial, food consumed was measured and
percentage assimilation calculated using estimated initial and
measured final dry weights for each juvenile.
Comparisons of three RF broods show that brood is not a significant factor determining shell ratios (F ¼ 1.46, df ¼ 2, 21).
Differences between broods were not detected in either LE or
RF populations in these or the previous experiment, suggesting
that shell ratios are consistent within these populations.
Ratios do differ between the three populations (F ¼ 7.53,
P ¼ 0.001, df ¼ 2, 46; Fig. 2) and LE again has a higher ratio
than RF (t ¼ 3.82, P ¼ 0.001, df ¼ 3, 46) and SB (t ¼ 2.79,
P ¼ 0.02, df ¼ 3, 46). Like RF, SB favour soft tissue over shell
growth; relative to their siblings at 54 d (Table 4) the threefold growth in their mean shell weight at 100 d is outpaced by
a seven-fold increase in soft-tissue weight.
Across the populations, shell ratios are lower on these diets
than those of the first experiment, and there is a significant
response to increasing Ca (F ¼ 8.11, P ¼ 0.001, df ¼ 2, 46),
most pronounced in LE (Fig. 2). Shell weights generally increase
with Ca (F ¼ 8.41, P ¼ 0.001, df ¼ 2, 46) with Ca3 shells
heavier than either Ca1 (t ¼ 3.31, P ¼ 0.005, df ¼ 3, 46) or
Ca2 (t ¼ 3.83, P ¼ 0.001, df ¼ 3, 46). Neither total dry weight
nor overall growth differs between populations or treatments,
so the effect of Ca on shell ratios is due to differential growth
in the two compartments. For example, all three RF broods
have their lowest ratio on Ca2 because here soft-tissue growth
dominates. Thus Ca-allocation strategies change with availability, at least in LE and RF.
Food consumption varies considerably between siblings, but
there is no indication of higher feeding rates on the lower Ca
diets. Consuming more Ca does increase the mass of Ca in the
soft tissues, but their rapid growth means concentrations in
this component actually fall. This may indicate some regulation
of assimilation, even at these low doses. Again, there are
RESULTS
Within-brood variation
Prior to their rapid growth phase, juveniles from different populations have relatively uniform fresh weights and the scale of
their variation does not differ significantly (Table 3; Miller –
Feltz test: x 2 ¼ 8.87 ns; df ¼ 3). Variability is no greater when
measuring two broods rather than one in the RF population.
Broods from two neighbouring chalkland sites (LE and RD)
show no consistent difference from the other populations. Thus
no population or habitat-type shows more within-brood variability than any other in juveniles close to the start of their
rapid growth phase.
Based on a comparison of their fresh weights (Table 3) and
their later dry weights (Table 4), the same group of LE-a and
RF individuals become more variable as they age. Additionally,
the older of two LE broods is heavier than and twice as variable
as the other. Of the two groups closest in age, the RF brood is
not significantly more variable than LE-b (Miller’s test:
Z ¼ 1.72 ns) despite its larger coefficient of variation.
Although both originate from Ca-rich sites, LE-b snails build
more robust shells than RF and their shell ratio is significantly
higher (Table 4; t ¼ 12.28, P , 0.001; df ¼ 40): nearly all LE
snails in this experiment have ratios greater than 1, whereas
no RF snail does. The difference is due to the soft tissues –
they share similar mean shell weights but RF grows more soft
tissues than LE-b (t ¼ 2.065, P ¼ 0.04; df ¼ 35). RF have the
widest range of total weights and include several very large
individuals (one of which almost reaches 1 g total dry weight
at 105 d).
Larger RF juveniles devote a smaller fraction of their Ca
to the shell, even though Ca is not limiting. Shell concentrations
decline logarithmically as soft-tissue dry weight rises and there is
a close relationship between Ca in the two compartments – the
Table 4. Mean (+SE) soft-tissue dry weight, shell dry weight and their ratio (and associated coefficients of variation) in juveniles from three
populations.
Population
n
Age
Mean soft-tissue
Mean shell dry
Coefficient of variation
Mean shell/ soft-tissue
Coefficient of variation
(days)
dry weight (mg)
weight (mg)
in total dry weight (%)
dry weight ratio
in shell ratio (%)
Range of shell ratio
LE-a
16
130
143 (33)
161 (38)
94
1.13 (0.04)
16
LE-b
24
108
86 (9.8)
107 (12.2)
55
1.26 (0.04)
16
0.83 –1.42
0.98 –1.48
RF
27
105
138 (23.1)
94 (18.5)
93
0.67 (0.02)
20
0.49 –0.97
SB
14
53
8.8 (0.7)
4.5 (0.3)
24
0.54 (0.05)
36
0.32 –1.11
Individuals from each brood or population were raised collectively on a diet which included abundant Ca. LE is represented by two broods and although one is more
variable in size, their shell ratios are highly consistent.
108
CALCIUM ALLOCATION IN CANTAREUS
Figure 1. Regression analysis of shell Ca mass with the logarithm of softtissue Ca in a single brood of RF juveniles raised together until 105 d old
(n ¼ 23; y ¼ 3.1 log x þ 10.3; r 2 ¼ 0.74, P , 0.001).
Figure 3. Mean percentage growth and shell ratio (+SE; n ¼ 5) for SB
juveniles fed a Ca-deficient diet (Ca1), either with no access to soil (open
boxes), access to their native soil (light boxes) or their native soil with a
Ca supplement (dark boxes).
population differences: compared to the populations from the
Ca-rich sites SB retains a larger proportion of its consumed Ca
for each dose (for example, means for Ca1: SB 233%; RF
93%; LE 59%).
Soil feeding increases SB juvenile growth but has no effect on
the shell ratio (Fig. 3). However, supplementing their native soil
with CaCO3 (Table 1) causes this ratio to increase to three times
that of the other treatments. The coefficients of variation in total
dry weight also rise with soil feeding, from 31% (Ca1; no soil
feeding) to 69% (with the Ca-enriched soil). Again, variation
between the juveniles increased with Ca supply and, with abundant Ca, SB is also shown to change its allocation strategy.
Ca5, while RD alone shows a significant increase in its ratio
on this diet (t ¼ 2.25, P ¼ 0.03, df ¼ 8).
In the growth trial with excessive Ca, LE juveniles have lower
mean growth rates than the same treatment in the previous trial
(Ca4: 744 vs. 1237%; Ca5: 1054 vs. 1286%) possibly because
these juveniles had already commenced the rapid growth
phase. Initial fresh weights in both LE and BI were almost
twice those starting the previous experiment, despite being the
same age. Possible reasons for the larger initial weights were
not investigated, but maternal shell size and season are known
to affect egg size in Arianta (Baur & Baur, 1997). It may be
that physiological age, rather than chronological age, determines the onset of the rapid growth phase.
Both LE and BI show the same response to diets above Ca4: to
grow soft tissues without matching shell growth at Ca5, but
thereafter to add to their shell mass (Fig. 5). Total final
weights are highest in each population on Ca6 when growth in
both components peak, so the most rapid growth is between
Ca5 and Ca6, with dietary Ca between 170 and 230 mg.g21.
Soft-tissue growth is checked on Ca7 (Fig. 6). Food consumption did not change in either population with this treatment,
though BI consistently consumed around one-third of the food
presented, while LE took only 20%. Increased food consumption leads to a greater increase in total weight in each population
on each treatment and consequently there is also a significant
positive correlation with Ca consumed, for each diet (Fig. 7).
LE has a significantly faster increase in total dry weight
Ca-rich growth trials
Of four populations LE has the fastest growth rate on both Ca4
and Ca5 diets (mean growth rate for both diets: LE 1261%; MI
818%; RD 746%; RF 645%). A two-way analysis of variance
demonstrates significant differences between populations in
growth (F ¼ 6.89, P ¼ 0.001, df ¼ 3, 32). However, neither
LE nor RD respond to the higher Ca diet, whereas both MI
and RF grow larger on this treatment, principally by increasing
their soft-tissue weight.
Shell ratios are equivalent for the juveniles from the three Carich sites on Ca4, but are significantly higher for the Ca-poor
site, MI (Fig. 4; F ¼ 3.34, P ¼ 0.03, df ¼ 3, 32). However,
growth in their soft tissues means the MI ratio is smaller on
Figure 2. Mean shell ratios (+SE; n 4) in juveniles of three populations on three low Ca diets. LE (open boxes) shows consistently
higher ratios than either RF (dark boxes) or SB (light boxes) though
this difference is only significant on Ca3 (F ¼ 5.29, P ¼ 0.02; df ¼ 2,13)
Figure 4. Mean (+SE; n ¼ 5) shell/soft-tissue dry weight ratio of 106 dold juveniles from four populations fed two high Ca diets–Ca4 (open
boxes) or Ca5 (filled boxes).
109
A. BEEBY AND L. RICHMOND
Figure 5. Mean (+SE; n ¼ 5) shell ratio of 103 d-old juveniles from a
Ca-rich site (LE light boxes) and Ca-poor site (BI dark boxes) raised
on diets with high and potentially excessive Ca levels.
Figure 7. The increase in total dry weight with Ca consumed for LE
juveniles on each of the higher Ca diets, shown as a regression line for
each diet. These run consecutively from left to right (Ca4– Ca7) and
all are significant (with correlation coefficients greater than 0.96,
n ¼ 5). The same pattern of declining growth with dietary Ca occurs
in BI, with similar regression coefficients.
with consumed Ca on Ca4 (LE-b ¼ 6.4; BI-b ¼ 3.8; t ¼ 3.723,
P , 0.01, df ¼ 6), but these rates decline on successive diets in
both populations and on Ca 7 their regression coefficients are
indistinguishable (LE-b ¼ 2.1; BI-b ¼ 1.8). This suggests that
physiological costs are incurred when high levels of Ca are
consumed, reducing soft tissue and latterly shell growth in
both populations.
Despite their different origins these populations show considerable uniformity in their shell ratios. It seems that differences
in Ca-allocation strategies emerge between populations when
their demand is not being met and disappear when Ca is abundant. At low levels, three LE broods had consistently higher shell
ratios since soft-tissue growth was favoured in other populations
as more Ca became available.
(Fearnley, 1996) and here (and in previous work; Beeby &
Richmond, 2001a) LE juveniles typically grow faster than
other populations when Ca is available.
Land snails are typically long-lived, iteroparous species,
whereas slugs are fast-growing, short-lived and semelparous
(Heller, 1990). Rapid growth and reproduction may only be
possible without a large shell, and slugs may have foregone
shelled protection to exploit Ca-poor habitats. An iteroparous
gastropod may need a shell if its reproductive Ca has to be
acquired during brief periods of activity or if there are physiological constraints on the supply from the soft tissues. Calcium
is shown here to be a key factor determining growth rates in
Cantareus and perhaps the reproductive strategy of different
populations.
Preferential allocation of Ca to the juvenile shell is not characteristic of either the Ca-rich or Ca-poor populations and all shift
their allocation according to Ca availability. Nearly all give softtissue growth priority when Ca becomes available, presumably
to support early maturation, and this may be optimal if resources
take a long time to acquire or if being large confers a competitive
advantage. Exceptionally, LE, from a Ca-rich site, consistently
reinforces the shell at the apparent expense of its soft tissues,
even when Ca is in short supply. Possibly LE has a less adaptable
Ca-allocation strategy, one closely tied to the high availability in
its native habitat, building a Ca reserve quickly, perhaps to
benefit from multiple copulations at the earliest opportunity.
The robust shells of lipped adults are characteristic of Ca-rich
habitats and the reinforcement that comes with age, but juvenile
growth strategies must be informed by climatic regimes and
reproductive opportunities. Very large garden snails, some
with shell weights in excess of 3 g, are found in Mediterranean
and southern European habitats, perhaps representing a subspecies (‘maxima’; Madec & Daguzan, 1993). There are also
indications of genetically determined reproductive traits in
populations from France and Portugal (Guéméné & Daguzan,
1983; Albuquerque de Matos, 1989). Some populations of
Cantareus with a large adult size show high locomotor activity
and a high propensity to mate, probably to promote dispersal
and outbreeding (Fearnley, 1996). This may be critical for
Cantareus which suffers infertility within three to four generations
of sib –sib matings (Albuquerque de Matos, 1989). A staggered
emergence, with trophic eggs to nourish hatchlings, would
promote rapid growth of a few, and it may be that fast
growing juveniles become the most dispersive and promiscuous
progeny. Although size does not appear to determine partner
DISCUSSION
The trade-offs in any reproductive strategy seek to optimize
traits for reproductive success, minimizing the risks in an unpredictable world (Stearns & Hoekstra, 2005) according to resource
supply and the time available to meet demand. Freshwater snail
species with high adult mortality typically invest more in reproduction, at the expense of somatic growth (Norton & Bronson,
2006). Delayed maturity is optimal when mortality rates are
low, and then large individuals retain most of their somatic
tissues from one season to the next. Their growth is sigmoidal
which, in a determinate species like Cantareus, is an indication
that resources are increasingly allocated to reproduction with
age (Kozlowski, 1992). However, population differences in
growth and reproductive activity are known for Cantareus
=
Figure 6. Mean (+SE; n ¼ 5) soft-tissue dry weight of 103 d-old juveniles of two populations (LE light boxes, BI darker boxes) raised on diets
with increasingly high Ca levels.
110
CALCIUM ALLOCATION IN CANTAREUS
and soft tissues in Cantareus may, in part, explain the variation
in growth and activity rates between siblings and between populations. Further experimentation might examine how changes in
the Ca supply affects time to reproduction and clutch size in
different populations. If the size of the shell does influence a
snail’s capacity to fulfil both male and female roles, any selective
advantage of a being large may simply follow from the size of its
Ca reserve.
choice in several other species of land snail (Locher & Baur,
2002; Jordaens, Pincel & Backeljau, 2005), a large maternal
shell in Arianta does allow for larger eggs (Baur & Baur, 1997)
and possibly faster juvenile growth in their offspring.
Variation within a brood develops as Cantareus juveniles pass
through their rapid growth phase (Sanz Sampelayo, Fonolla &
Gil Extremera, 1990) and this is shown here to be comparable
between populations. High variability is known for other terrestrial pulmonates and this has some genetic component (Jordaens,
Pincel & Backeljau, 2006). Size differences in Cantareus only
appear several weeks after emergence, with or without soilfeeding (Daguzan, 1982). Thereafter hatchlings which soil-feed
quickly outgrow other siblings, and a high level of exchangeable
Ca is a key factor (Gomot et al., 1989). Food consumption by
Arianta increases on diets with low Ca levels (Wacker & Baur,
2004), but here consumption was highly consistent between
diets and did not decline in Cantareus even on the higher, possibly
detrimental, Ca doses.
On Ca-poor diets LE and SB broods became more variable as
dietary Ca increased though no individuals utilized all the available resources. This variability is not due to a shortage of food or
inhibition from siblings, but from differences in activity. In each
trial some juveniles fed sporadically and grew slowly. Whether
their access to trophic eggs, prior to their separation, or some
other form of maternal influence has determined their activity
is not known. In a previous experiment MI juveniles hatched
over a shorter period, but came from smaller clutches with a
higher emergence rate than RD (Beeby & Richmond, 2001b),
and these together may limit the scope for egg cannibalism.
The larger clutches of RD hatched over a longer period,
which, with their lower emergence rate, perhaps indicated
cannibalism. Overall RD produced more hatchlings and
made twice the Ca investment of MI in a brood. Even after
oviposition, RD had larger Ca reserves than MI because of
their heavier shells (Beeby & Richmond, 2001b).
All parents hedge their reproductive investment against
various risks and differential growth within a brood may anticipate uncertainty in aestivation or hibernation periods. Unpredictable seasons lead to variable growth rates and hedging of
the parental investment in a range of animal groups (Lampert
& Linsenmair, 2002). In Arianta limited food reduces the frequency of copulations (Locher & Baur, 2002) and eggs from
larger snails tend to have higher hatching success (Jordaens,
Pincel & Backeljau, 2006). Perhaps the preference of RF snails
for soft-tissue growth allows for earlier reproduction, possible
in the mild winters of the Mediterranean Basin, and possibly a
necessity before the enforced aestivation of its dry summers.
The populations of Cantareus studied here show no distinct
growth or Ca-allocation strategy associated with Ca-rich or
Ca-poor habitats. All change their allocation according to availability, with most limiting their shell growth on low dietary
levels. Only LE favours shell growth at all but the highest
levels. The consistency between its broods suggests this is an
inherited strategy, and a response to persistent local conditions,
and not simply the availability of Ca.
The significance of Ca to terrestrial gastropod molluscs is
perhaps unique in the animal kingdom. Few other animals
have their ecology so closely tied to a single element which,
directly or indirectly, determines their distribution, their
response to adverse environmental conditions, their capacity to
aestivate, hibernate and mate, their growth rate and their reproductive capacity. High dietary levels do produce more robust
shells, but the Ca-rich growth trials suggest that very high
uptake may incur physiological costs which can inhibit softtissue growth.
Its shell allows Cantareus to be iteroparous. Larger shells
support larger clutch sizes and may reduce the refractory
period between clutches. The allocation of Ca between shell
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