/ MolL Stud. (1996), 62, 315-325
© The Malacological Society of London 1996
STRONTIUM LABELLING OF THE SHELL OF THE
ANTARCTIC LIMPET NACELLA CONCINNA (STREBEL,
1908)
LLOYD S. PECK1*, ANDREW C. BAKER2 and LUCY 2. CONWAY1
2
'British Antarctic Survey, High Cross, Madmgley Road, Cambridge CB3 OET, UK
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ
(Received 24 October 1995, accepted 13 December 1995)
ABSTRACT
Nacella concinna (Strebel, 1908) held in seawater
enriched with Sr, partially replaced Ca in their shells
with Sr The bands laid down were clearly visible In
back-scattered, scanning electron
microscope
images, and such bands could be used to mark carbonate skeletons in field investigations of growth Sr
directly replaced Ca in the carbonate shells,
although not entirely, and newly secreted shell material always contained less Sr than would be expected
from the levels used in treatments. Specimens held
in water with atomic Ca;Sr ratios of 114:1 (control
seawater, no added Sr), 4.4:1, 2.2:1 and 1.1.1 produced shell ratios of ca. 635:1, 11.0.1, 3.1:1, and 8.1:1
respectively These values were between 1.4 and 7 4
tunes higher than expected, indicating that, when
shell was laid down Sr was discriminated against by a
factor of around 4. Growth rates were similar before
and after placing specimens in experimental treatments and feeding rates were not significantly different between treatments and controls, indicating that
the enhanced Sr regimes caused little stress for the
limpets. Limpets were exposed to the experimental
treatments for 12 days. However, in specimens
where growth bands were visible, it was only possible to distinguish between 5 and 8 micro-growth
lines. In specimens removed after 3 days exposure,
there was no detectable area of enhanced Sr in the
shell. These two observations were interpreted as
either meaning that it takes around 5 days for Sr to
enter the extra-pallial fluids which are used for laying down shell, or that the limpets did not produce
any shell growth during that period. Experiments
placing Sr in food and not in the treatment water,
and vice-versa, suggested that Sr taken directly from
the water may be more important than uptake from
food.
INTRODUCTION
Estimating growth in wild populations of animals has many associated problems, and a
variety of techniques have been used to mea•Coimpondencr UK Dr US. Peck, Bntnh Anureuc Survey. Hijh
Cnw, Mutinito Rd. Cambndjt CB3 OET. Tet 01223 231603
sure growth rates. These include analysing
size-frequency distributions, the analysis of
growth checks or rings in skeletal material, and
the repeated measurement of tagged or
marked individuals. Size-frequency analysis
often suffers from difficulties in detecting
peaks in distributions and overlaps in the size
of different age classes (Grant, Morgan &
Olive, 1987; Peck & Culley, 1990, Gage &
Tyler, 1991); growth checks and rings can be
caused by external stresses and not be related
to obvious environmental periodicities (Seed,
1976); and analyses using marked individuals
often suffer from problems of low numbers of
catch returns and stresses caused by the marking technique, which may significantly affect
the growth and survival of the experimental
animals (Nielsen, 1988; Allison & Brand,
1995).
Repeated measurements of tagged individuals or placing internal marks in the skeletons
of experimental specimens has the advantage
of allowing fine scale analysis of growth rates
over defined periods. It also allows more
detailed studies of individual variations in
growth rate, and for those variations to be
assessed in relation to other factors. Using
internal shell markers avoids many of the
problems associated with external tagging,
such as the possibility of making experimental
specimens more visible to predators, or affecting the physiology of those specimens by causing trauma on application or at later periods
by providing an energetically costly burden
(Berg, 1986; Tulonen, 1989). Internal shell
marks do, however, suffer from the problems
that, although marking may be repeated during an experiment, the measurement of growth
increments almost always involves the destruction of the specimens in the study.
The range of methods used to place marks
in skeletal structures of ectotherms includes
L.S. PECK ETAL.
316
tetracycline, which has been used to label fish
(eg. Panfili & Xime'nes, 1994) and invertebrates (eg Gage, 1992; Ebert & Russel, 1993)
and the fluorescent marker calcein which has
been used to mark the shells of brachiopods
(Rowley & Mackinnon, in press). In gastropod
molluscs tetracycline has been used to mark
shells (Pirker & Schiel, 1993), and calcein has
been used in a study of limpet growth (A.
Clarke, pen. comm.). Notches etched on leading edges of shells (Ekaratne & Crisp, 1982)
and temperature shocks (Richardson, 1987;
Richardson & Liu, 1994) have also been used
for the same purpose. It is clear that direct
shocks stress the organisms under investigation
to a greater or lesser extent. However, this is
also true of methods employing antibiotics,
and some applications have been shown to
have powerful stress effects in the abalone
Haliotis ins (Pirker & Schiel, 1993).
These difficulties led Hurley, Odense,
O'Dor & Dawe (1985) to choose strontium to
label statoliths of the short finned squid Ilex
illecebrosus. Strontium has been shown to be
essential for the normal development of
cephalopod statoliths, with concomitant effects
on the normal development of swimming
behaviour (Hanlon, Bidwell & Tait, 1989).
Strontium has previously been used to mark
fish scales (Ophel & Judd, 1968; Yamada, Mulligan & Fairchild, 1979), and statoliths of the
scyphomedusan Aurelia aurita (Spangenberg,
1979).
Nacella concinna is a patellid gastropod
which is widely distributed in Antarctic waters.
It is locally abundant and can be common on
hard substrata in intertidal and shallow subtidal zones, into which it migrates during the
summer from the deeper sites where winter
ice-bound periods are spent (Walker, 1972). N.
concinna has been the subject of considerable
study in recent years, and there are data on
growth, faecal production, metabolism, reproduction and mucus production (Shabica, 1976;
Picken, 1980, Houlihan & Allan, 1982; Peck,
1989; Clarke, 1990; Peck, Prothero-Thomas &
Hough, 1993). This paper reports on experiments investigating the substitution of calcium
with strontium in the shell of N. concinna, and
assesses the effects of enhanced strontium on
the behaviour and physiology of the limpets.
MATERIAL AND METHODS
Nacella concinna used in this investigation were collected by SCUBA divers from depths between 3 m
and 15 m at sites close to the British Antarctic Survey station on Signy Island, South Orkney Islands
(60°43'S, 45°38'W). They were collected in February
and March 1992 and transported in April and May
1992 to the UK by ship, where they were held in
cooled aquaria at temperatures between -1.0°C and
+0.5-161>C, and fed on algal films growing on the
sides of the tanks.
Algal culture and experimental regime
An experimental regime was set up based on 10
tanks (each 200 x 200 x 300 mm) providing a range
of holding condiuons in terms of the availability of
Sr to the limpets, both in the seawater in tanks, and
in the algae cultured on tank surfaces as a food
source. Algae were grown on the sides of 9 of the 10
tanks, and each of these was initially filled with 8
litres of seawater, enriched with f/2 (Guulard, 1975)
medium, which is similar to Erdschreiber medium. It
was added at 50% of the concentration suggested by
GuiUard (1975). Small amounts of algal material
were removed from the sides of the holding aquarium and added to each of the 9 experimental tanks
to enhance the establishment of epihthic growth on
the sides. The establishment of epilithic material was
also aided by maintaining cultures at +15°C in a
high light environment. Media in tanks were aerated
and the whole system left to develop for 20 days.
After 20 days Sr was added as SrCl2.6H2O according to the regime shown in Table 1. It was added at a
concentration which was isotonic with seawater of 35
%> salinity Final concentrations in tanks were in proportion to the naturally occurring level of Ca in seawater of 10 mM (F0yn, 1969; Kalle, 1971; Brown,
Colling, Park, Philips, Rothery & Wnght, 1989)
After a further 3 days of incubation, to allow for Sr
incorporation into the algae, tanks were moved to a
controlled temperature room set to give water temperatures between -0_5°C and +0-5°C Following a
further 2 days of incubation for equilibration to the
low temperatures the culture medium was replaced
in each tank with filtered seawater containing Sr at
identical concentrations. During this process tank
surfaces were exposed to air for a maximum of 3
mins and disturbance to the algae was minimised
When each tank was empty, representative samples
of algae were removed from the sides and bottom
using a razor blade and checks made to ensure that
areas sampled were completely cleared of algae.
Ash-free dry mass (AFDM) was obtained from the
difference between the dry mass (obtained after drying at 60°C for 24 h) and ash remaining after ignition
at 475°C for 24 h. Measurements of areas sampled
were combined with the AFDM assessments to provide estimates of algal cover and food available to
the limpets.
Two trials (treatments 7 and 8 in Table 1) were
made to investigate the site of uptake of Sr by N.
concinna. In treatment 7, algae were cultured in a
medium with an atomic Ca;Sr ratio of 2.2:1 However, when the water was changed prior to placing
the limpets in tanks the replacement water used con-
Sr LABELLING OF LIMPET SHELL
tamed no added Sr. This resulted in a tank containing Sr in the algae but not in the water. Treatment 8
was the reverse of this During the algal culture
period no Sr was added, and prior to limpets being
introduced the tank was filled with seawater with a
Ca:Sr atomic ratio of 2.2:1. This tank, therefore contained algae with normal Sr levels, but water with an
enhanced Sr content.
Feeding assessments and Sr incorporation
For 7 days pnor to being placed in the experimental
system, 60 N. concinna were taken from the holding
facility and held in cleaned tanks with no algae present This brought all the animals used to a similar
nutritional status and ensured that as many as possible would begin feeding upon being placed in the
experimental treatments. They were then carefully
transferred to the experimental regime indicated in
Table 1, and over the ensuing 12 h regular feeding
observations made in terms of area of tank surface
cleared. Areas cleared by each limpet were converted to AFDM consumed using measurements of
algal biomass from the tank holding that individual.
Limpets were left in experimental tanks for 12
days to allow Sr incorporation into their shells. This
period was chosen following the analysis of specimens sampled at 3, 5, 7 and 10 days exposure to the
experimental regime which indicated that 12 days
would allow good Sr incorporation in the shells of
the experimental animals. Throughout the trials the
light regime was 16 h light 8 h dark.
After 12 days all N. concinna in the treatments
were removed, the tissues excised and dried and
AFDM determined. Shell length, breadth and height
were measured to the nearest 0 1 mm using vemier
callipers, and shell mass measured to the nearest 1
mg. Shells were then embedded in Epofix polyester
EM resin and cut in half along the mid line of the
shell length using an Extec labcut 1010 low speed
saw Where necessary, after sectioning, blocks were
317
cleaned with a detergent in a Branson 1200 ultrasonic bath. They were then polished to provide a
smooth surface for elemental analysis, using silicon
carbide papers between 220 and 4000 gnt, followed
by a final polish with 004 ujn colloidal silica on a
polishing doth.
Shell surfaces were observed on a Leica Cambridge Steroscan 360 scanning electron microscope
using back-scattered electron images The energy of
a back-scattered electron is proportional to the density of the material from which it is reflected. Areas
observed with higher average atomic number, therefore, appear brighter. Zones of high Sr incorporation
appeared brighter under this view compared with
the normal shell because of the higher atomic mass
of Sr (87.6) compared with Ca (40.0). Sr and Ca concentrations in shell were measured with an Oxford
Instruments energy dispersive X-ray analyser
(E D X.), and calibrations made using a calcite standard (Ca = 40 0% by mass) for Ca measurements,
and strontium fluoride (Sr = 70.0% by mass) for Sr
assessments.
RESULTS
In the treatment containing the highest Sr concentration (Table 1, treatment 6, Ca:Sr =
1:1.8) a white crystalline precipitate appeared
during the first 48 h after adding SrCl2.6H2O.
This precipitate became thicker when the
water was changed prior to introducing the
experimental animals. Windowless E.D.X.
analysis showed the precipitate to be crystals
of strontium sulphate (SrSO4). Because of the
large quantities of precipitate present the N
concinna used in this treatment were excluded
from further analysis. There was no visible
precipitate in any of the other treatments.
Table 1. Nacella concinna: experimental regime with treatments providing a range of
availabilities of Sr to the experimental animals. In all treatments ratios are quoted in
atomic units. In treatments 3-6, algae were cultured in enhanced Sr conditions and
Sr was added to the water limpets were held in.
Treatment
conditions
Ca:Sr ratio
1. Control
2. low ration level
3. Sr enriched (1)
4. Sr enriched (2)
5. Sr enriched (3)
6. Sr enriched (4)
7. Source trial (1)
114:1
2.2:1
4.4:1
2.2:1
1.1:1
1:1.8
2.2:1
8. Source trial (2)
2.2:1
no. of tanks
no algae present
Sr added to
algae, not water
Sr added to
water, not algae
no. of
individuals
other
1
1
1
3
1
1
1
5
5
6
24
5
5
5
1
5
L.S. PECK ETAL.
318
Q. 100
CO
I
"co
~
60
CO
To
40-
e
o
'c
f
20-
s>o
°c"
I
r
CO
1..JL
TRACE Sr
1_L
Ca:Sr= 2.2:1
Ca:Sr= 4.4:1
Ca:Sr=1.1:1
L|
Ca:Sr=1:1.8
Treatment
Figure 1. The inorganic content of algal material cultured on tank surfaces from treatments in Table 1. Error
bars indicate standard errors.
Feeding and algal organic content
Mean algal density on tank sides prior to placing limpets in the treatments was 0.052 mg
AFDM cm"2 (SD = 0.046), while on the bottom surfaces the density was 0.699 mg AFDM
cm~2 (SD = 0.127). There were strong variations in the inorganic content of algae in the
different treatments (Fig. 1). The lowest inorganic content (approx. 20% of the dry mass)
was in trials with no added Sr. At Ca:Sr ratios
of 4.4:1, 2.2:1 and 1.1:1, algal inorganic content
was between 50 and 65%. In the treatment
with Ca:Sr at 1:1.8 this value rose to nearly
90%. Thus, in treatments with enhanced Sr,
where no precipitate was visible the inorganic
content was around x 2.5 to x 3 that of normal conditions, and in the highest Sr treatment, where a precipitate was visible the
inorganic content was x 4.5 that of normal
conditions.
Overall 7 specimens did not consume algae
during the experiments. The distribution of
these limpets amongst trials was not significantly different from an even spread (Chi
square = 2253, P = 0.81), which indicated that
there was no large effect of Sr concentration
on inhibition of feeding.
In an attempt to measure maximal feeding
rates, amounts eaten across treatments were
assessed in relation to limpet AFDM (Fig. 2).
Only data from specimens which fed continuously for at least 6 h of the first 12 h of exposure to food were used and animals which did
not settle rapidly in the new conditions were
excluded from the analysis. Specimens which
did not feed at all during the first or last 4 h of
observations were also excluded, on the
grounds that they may have been stressed during the transfer from the starvation tank.
Residuals analysis indicated that a straight
line was not a good fit to the data in Fig. 2. A
two line model was, therefore, utilised. The
point of intersection of the two lines, for best
fit to the data, was obtained by iterating the
model to minimise residual variance. This
point was at a tissue mass of 0.123 g AFDM.
Sr incorporation into shell
Strontium incorporation into shells produced
distinct bright bands on back-scattered electron images (Fig. 3). Sr incorporation was visible in shells from treatments 2, 3, 4, 5 and 8,
but not treatments 1 and 7. In treatment 2,
where the Ca:Sr ratio in the water was 2.2:1
but no food was provided, 2 of the 5 specimens
produced bright bands. Of the total of 40 shells
Sr LABELLING OF LIMPET SHELL
£..3
-
A
1
A
Q
u_
^^
o>
A
A
1-
/
0.5-
A
rat
o
D)
C
T3
CD
CD
LL
A^
^
V
X
A _
A
A
A A
A
A
>AA
0^5-
A
j
A
/
A
/
/
0.1 0.025
0.05
0.1
0.25
0.5
Tissue A.F.D.M. (g)
Figure 2. Nacella concinrur. maximum observed feeding rate. Feeding rate is plotted against limpet AFDM,
note logarithmic axes. At tissue AFDM less than 0.123 g the relationship between feeding rate and AFDM
was- log. feeding rate = -4.17 + 129 log. AFDM; above this size the relationship was: log. feeding rate =
-6.73 + 0.068 log. AFDM.
Figure 3. Nacella concinnar. back-scattered electron image of a longitudinal shell section. The area shown is at
the middle of the antenor edge of the shell. The zone of high Sr incorporation at the growing tip is much
brighter than the older shell laid down in normal Sr conditions. Growth bands are clearly visible in the Sr
enhanced portion of the shell. Scale bar = 20 pjn
320
L.S. PECK ETAL.
Figure 4. Nacella concmna: back-scattered electron image of a longitudinal section of a shell showing irregular, or patchy incorporation of Sr into the shell matrix. Bnght zones indicate areas of high Sr incorporation.
Scale bar •» 20 \isn.
studied from the other treatments where incorporation was noted, only 7 individuals did not
show a Sr enhanced band in the shell. These 7
specimens were also those individuals
excluded from the feeding rate assessment
because of low or very intermittent periods of
food consumption. A further 8 specimens did
not produce growth bands similar to that
shown in Fig. 3, but showed irregular incorporation of Sr (Fig. 4). In the source trials (treatments 7 and 8), there was incorporation of Sr
in the treatment where Sr was added to the
water, but not the algae, and there were no
enhanced Sr bands in specimens from the
treatment with enhanced Sr levels in the algae,
but not in the water.
Measurements of Ca:Sr ratios in areas of
enhanced Sr in shells, and controls laid down
in normal Sr environments showed progressive
replacement of Ca by Sr in the shell valves
(Fig. 5). Measurements were made using an
EDX analyser, and results were in terms of
percentage mass. On this basis if shells were
composed entirely of CaCO3 with no organic
matrix or other elements present the expected
proportion as Ca would be 40.0%, and under
the same criteria for Sr the proportion would
be 59.3%. The slope of the line in Fig. 5 under
these conditions should be -0.67. The actual
values obtained were 36.7% (SE: 0.93), 48.6%
(SE: 3.08) and -0.755 (SE: 0.076), respectively. A model II (Sokal & Rohlf, 1981), or
geometric mean regression (Ricker, 1973), was
used here, because Ca and Sr levels in shells
were not independent of each other. The proportions of Ca and Sr in shells in the absence
of the other element estimated from the intercepts, are both significantly less than the proportions calculated for CaCO3 (t = 3.55, P <
0.01) and SrCO, (t = 3.47, P < 0.01), but the
slope of the relationship is not significantly different from 0.67 (t = 1.12, P > 0.10).
Comparing Ca:Sr ratios in the shells with
those of the treatments in which they were
held produced no clear correlation (Table 2).
All of the shell Ca:Sr ratios were, however,
higher than the relevant treatment ratios, and
in 3 of the 4 treatments the differences were
highly significant. Two values have been
quoted for measurements on control areas of
shell. This is because Sr levels in these areas
were close to the detection limits of the EDX
Sr LABELLING OF LIMPET SHELL
321
38H
°co
O
28-
18-
Sr%
Figure 5. Nacella concinna: Ca and Sr contents of shells from individuals held in a range of conditions. A
model II regression was fitted to the data, because the X and Y values clearly interacted. Ca =• 36.68 - 0.755
Sr (SE slope. 0.076). Values quoted were measured on a mass, not atomic basis.
Table 2. Nacella concinna: Ca/Sr atomic ratios in shells and treatments.
Control measurements were obtained from shell laid down prior to specimens being placed in treatments; t tests compare values from shells with
the relevant treatment ratios. In controls shell ratios were tested against a
value of 114.1, the mean of the ratio of Ca:Sr quoted by Kalle (1971) and
Brewer (1975). Sr levels in controls were close to the detection limit of the
EDX, and 2 controls are therefore quoted, one including zero points (a) and
one excluding them (b). All values quoted were means with SE in parentheses.
Ca.Sr ratio
Treatment
Shell
Control a
Control b
4.41
2,2:1
1.1:1
852.1
413.5
10.9
3.0
8.1
(141.0)
(22.25)
(1.10)
(2.04)
(4.39)
system, and some zero values were obtained.
These clearly underestimated the true value.
However, eliminating areas of low Sr content
would bias the data towards lower Ca;Sr ratios.
In Table 2 one of the controls included zero
values (control a), while the other excluded
them (control b). These two ratios, therefore,
form the upper and lower limits for Ca:Sr
ratios in N. concihna shell under normal condi-
n
t
P(<)
12
12
5.23
13.93
5.90
0.40
8.89
0.001
0.001
0.01
NS
0.001
4
3
6
tions, and the real value lies between 413 and
852.
Shell growth
Micro-growth bands were visible in some
back scattered electron images (Fig. 3). In
these specimens the width of the bands were
different between individuals, but bands laid
LS PECKETAL.
down prior to the limpets being placed in high 852:1 for control areas of shell. Thus the range
Sr conditions were not significantly different of Ca:Sr ratios found here encompasses the
values quoted by Dodd (1967) for calcitic
from those produced during the trials (t =
1.38, n = 8, P > 0.10). Where bands were bivalve molluscs, and is significantly lower than
levels quoted for all other taxonomic groups,
detectable it was possible to distinguish
between 5 and 8 in the bright Sr enhanced with the exception of the Brachiopoda, showing an enhanced ability to discriminate against
region.
Sr compared with other marine invertebrates.
In control areas of shell Sr incorporation
DISCUSSION
was between 3.6 and 7.5 times levels of Sr in
Sr is a natural minor component of seawater, seawater. In trials with enhanced Sr atomic
usually at concentrations around 90 |j.M (8 mg Ca:Sr ratios measured in shells were, on averkg"1), a level approximately 114 times less than age, 4.47 (SE = 1.26, n = 5, Table 2) times
that of Ca in seawater (Kalle, 1971; Brewer, higher than in the corresponding treatments,
1975). It is incorporated in invertebrate skele- and this may also indicate discrimination
tons when they are deposited, as a substitution against Sr by the limpets. Other factors may,
for Ca. During this process many factors have however, have affected this observed reducbeen implicated as important. These include tion in Sr incorporation in the limpet shells. In
water chemistry, shell mineralogy, animal the highest Sr treatment (atomic Ca:Sr ratio
1:1.8) large quantities of SrSO4 precipitated
physiology, and environmental temperature
and salinity (Dodd, 1965, 1967; Rosenberg, out of solution, and the trial was abandoned.
1980, 1990; Dodd & Crisp, 1982). Mineralogy No similar precipitates were observed in other
is clearly important in this context, as inorgani- treatments, but the algal inorganic contents in
cally precipitated aragonite from aqueous solu- 4.4:1, 2.2:1 and 1.1:1 treatments were all signifitions contains around 6 to 7 times as much Sr cantly higher than in control algae (Fig. 1).
as does calcite from the same solutions (Mcln- This may have been due to precipitation of
trye, 1963; Holland Kirsipu, Huebner & Oxen- SrSO4 on the algae, but the trend in inorganic
burgh, 1964). This is because the Sr2* ion (1.12 content of algae in different treatments runs
contrary to this, as inorganic levels declined
angstrom) has a larger diameter than the Ca2+
ion (0.99 angstrom), and the unit spaces avail- with increasing Sr concentration across treatable in the crystal lattice of aragonite favour ments, when the Ca:Sr ratio 1:1.8 trial is
substitutions by ions with larger diameters excluded.
than does the calcite lattice. N. concinna has a
Any precipitation of SrSO< would lower Sr
predominantly foliated calcitic shell structure, concentrations in the treatment seawater,
with small amounts of aragonite present (Mac- reducing Sr availability to the limpets. HowClintock, 1967). An accurate calculation of any ever, if the precipitate was ingested with the
mineralogical effect is not possible, in an inves- food and absorbed Sr levels in shells could be
tigation of skeletal carbonate deposition by enhanced by such precipitation. The source trianimals, without an exact knowledge of the als (treatments 7 and 8) were an attempt to
proportions of calcite and aragonite being distinguish which of these routes was being
deposited.
used to provide Ca and Sr for shell growth.
Comparisons with other taxonomic groups Incorporation of Sr in the shells of limpets
do, however, suggest that molluscs discrimi- when Sr was added to the water but not algae,
nate against Sr during shell deposition. Sr con- and no incorporation in the reverse treatment
tents of gastropod and bivalve molluscan shells suggested that Sr and Ca were taken directly
are low in comparison with other invertebrate from the seawater and not from food. This
taxa, especially those with aragonitic skeletons supports the idea that the depression of Sr lev(Dodd, 1967). Similar data are not available els in shells in some trials could have been due
for gastropods with calcitic shells. However, to reduced Sr in the water because of precipiDodd (1967) found that Ca.Sr atomic ratios for tation. However, the source trial results could
bivalves with calcitic skeletons were all greater also have been produced by Sr entering the
than 500:1, which was between x2 and x5 the limpets with ingested material, if the equilibravalues for coelenterates, sponges, annelids, tion of Sr between the algae and environment
arthropods and echinoderms. These data were was rapid. Rapid equilibration in treatment 7
all calculated on an atomic basis, and compare (Sr in algae but not water) would have
removed Sr from the algae prior to its ingeswith ratios for N. Concinna between 414:1 and
322
323
Sr LABELLING OF LIMPET SHELL
tion by the limpets. As the mass and volume of
concinna would suggest that several days may
algae present was between 0.025 and 0.012 indeed be necessary for Sr to be incorporated
times that of the seawater, the final concentra- into the extrapailial fluids at the site of shell
tion in treatment 7 would have been approxi- deposition, however, the most likely explanamately double that in controls. Bands with tion of our data is that significant shell growth
this level of enhancement may not have does not occur until around 5 days after a feedbeen detected. Conversely, in treatment 8 ing event. It is interesting to note here that the
rapid equilibration would have lead to 2.2:1 post-prandial rise in metabolic rate also peaks
Ca.Sr levels in the algae before they were con- approximately 5 days after a feeding event in
sumed, thus producing the observed bright N concinna (Peck & Veal, unpublished obserbands.
vation).
A few specimens in this study did not proSr uptake from food with artificially
enhanced Sr contents has been reported for duce clear uniform bands of Sr enriched matethe short-finned squid Ilex illecebrosus. How- rial at the growing edges of the shell. Erratic or
ever, most investigations ignore this route, and patchy inclusions of high Sr content similar to
concentrate on the relationships between that shown in Fig. 4 were observed. These
skeletal and water Sr contents (Dodd, 1967, areas probably indicate zones of shell repair
Dodd & Stanton, 1981; Robertson, 1982; Repairs of molluscan shell can take the form of
Mann, 1992). Dietary effects on shell Sr con- plates, tablets or spherules of calcite or aragonite laid down around areas of damage. Repair
tents could explain some of the variability seen
previously, and also differences between mechanisms also take precedence over, and
juvenile and adult shell concentrations, as proceed more rapidly than, normal shell
diets often change from juvenile to adult growth (Watabe, 1983). In wild populations
stages. Gearly more work is needed to eluci- slight damage removing small shell fragments
date the importance of each pathway for Sr is relatively common (Nolan, 1991a, b). The
transfer of the N. concinna into the experimenuptake.
The observation that between 5 and 8 bands tal treatments used in this study may have lead
could be distinguished in areas of enhanced to minor shell damage, followed by the inclushell Sr combined with finding no bright bands sive areas of high Sr content observed.
in specimens processed after 3 days, and only
The strong Sr enriched bands produced in
small amounts after 5 days suggested several the shells of N. concinna clearly demonstrate
days might be necessary for Sr to be incorpo- that Sr will replace Ca in carbonate skeletons
rated into the Ca pools used for shell growth. of invertebrates and indicates its potential for
It could also, however, be explained by growth use as a permanent marker in investigations of
being halted in the N. concinna by the 7 days growth in field populations. Data indicating
of starvation prior to being placed in treat- that growth rates were similar prior to and
ments, and around 5 days being necessary for after placing limpets in high Sr conditions, and
the resumption of shell growth when food was that feeding rates were similar in treatments
again available. In cold water marine inverte- and controls show that raising Sr concentrabrates growth is often associated with periods tions to high levels in the surrounding seawater
of food supply, and resource limitations associ- can only have produced very minor stress
ated with low growth rates (Clarke, 1983,1988, effects.
1991; Clarke & Peck, 1991). Significant incorporation of ^Ca into shells of brachiopods and
bivalve molluscs has also been shown to occur
within 24 h of exposure to the isotope
ACKNOWLEDGEMENTS
(Wheeler, Blackwelder & Wilbur, 1975; Pan &
Watabe, 1988). This would suggest that the The authors would like to thank Andrew
loss of 4 to 7 bands was due to a cessation of
Clarke for helpful discussion and comment
growth. However, there are examples of shell
production in the absence of feeding (Palmer, during the course of this study. Ken Robinson
1981), and a recent study of the bivalve Yoldia provided a wealth of advice and assistance in
eightsi at Signy Island found substantial shell the production of SEM images and EDX analgrowth during periods of low food supply ysis of shells. Limpets were kindly collected by
(Peck & Colman, unpublished). Shell growth Simon Brockington and Rob Wood in Signy
was also decoupled from periods of tissue Island, and were maintained in the Cambridge
aquanum system prior to use in the experiAFDM increase. Similar abilities in A'
ments by Jon Ward.
324
L.S. PECK ETAL.
REFERENCES
ALLISON, E.H., & BRAND, A.R. 1995. A mark-recap-
ture experiment on queen scallops, Aequipecten
operculans, on a north Irish Sea fishing ground.
Journal of the Marine Biological Association of the
U.K., 75: 323-336
BERG, R. 1986. Field studies on eel (Anguilla
anguilla) in Lake Constance: tagging effects
caused retardation of growth. Vie et Milieu, 36:
285-286.
BREWER, P.G. 1975. Minor elements in seawater. In:
Chemical oceanography (J.P. Riley & G. Skirrow,
eds), 1:415-496. Academic Press, London.
BROWN, J., COLLING, A., PARK, D., PHILLIPS, J ,
ROTHERY, D & WRIGHT, J. 1989 Seawater: Us
composition, properties and behaviour. Pergamon
Press, Oxford.
CLARKE, A. 1983. Life in cold water the physiological ecology of polar marine ectotherms. Oceanography and Marine Biology Annual Reviews, 21:
341-453.
CLARKE, A. 1988. Seasonality in the Antarctic
marine environment Comparative
Biochemistry
and Physiology, 90B: 461-473.
CLARKE, A. 1990. Faecal egestion and ammonia
excretion in the Antarctic limpet Nacella concmna
(Strebel, 1908). Journal of Experimental Marine
Biology and Ecology, 138: 227-246.
CLARKE, A. 1991. What is cold adaptation and how
should we measure it? American Zoologist, 31: 8192.
CLARKE, A. & PECK, L.S. 1991. The physiology of
polar marine zooplankton. In: Proceedings of the
pro mare symposium on polar marine ecology
Trondheim (Sakshaug, E, Hopkins, C.C.E. & 0 n t sland, N.A., eds), 355-369. Polar Research, 10(2).
DODD, J.R. 1965. Environmental control of strontium and magnesium in Mytilus. Ceochimica et
Cosmochimica Acta, 29: 385-398.
DODD, J.R. 1967. Magnesium and Strontium m calcareous skeletons: a review. Journal of palaeontology, 41:1313-1329.
D O D D , J.R. & CRISP, E L. 1982. Non-linear variation
with salinity of Sr/Ca and Mg/Ca ratios in water
and aragonitic bivalve shells and implications for
paleosaUnity studies. Palaeogeography, Palaeoclimatology, Palaeoecology, 38: 45-56.
D O D D , J.R. & STANTON, R J . 1981.
concepts and applications.
New York.
Paleoecology:
John Wiley & Sons,
EBERT, T A. & RUSSEL, M.P. 1993. Growth and mor-
tality of subtidal red sea urchins (Strongylocentrotus franciscanus) at San Nichols Island, California,
USA; problems with models. Marine Biology, 117:
79-89.
EKARATNE, S.U.K. & CRISP, D J . 1982. Tidal microgrowth bands in gastropod shells, with an evaluation of band-dating techniques. Proceedings of the
Royal Society, London, B214: 305-323.
FBYN, E. 1969. The composition of seawater and the
significance of the chemical components of the
marine environment In: Chemical oceanography.
An introduction (R. Lange ed.), 11-34 Uiuversitetsforlaget, Oslo.
GAGE, J.D 1992. Natural growth bands and growth
variability in the sea urchin Echinus esculentusr.
results from tetracycline tagging. Marine Biology,
114:607-617.
GAGE, J.D. & TYLER, P.A. 1991. Deep sea biology: a
natural history of organisms at the deep sea floor.
Cambridge University Press, Cambridge.
GRANT, A., MORGAN, PJ. & OLIVE, P J W
1987.
Use made in marine ecology of methods for estimating demographic parameters from size-frequency data. Marine Biology, 95- 201-208.
GUILLARD, R.R.L. 1975. Culture of phytoplankton
for feeding marine invertebrates, in: Culture of
marine invertebrate animals (W.L. Smith & M.H.
Chanley eds), 29-60. Plenum, New York.
HANLON, R.T., BIDWELL, J.P. & TAIT, R. 1989
Strontium is required for statohth development
and thus normal swimming behaviour of hatchling
cephalopods. Journal of Experimental
Biology,
141:187-195.
HOLLAND, H.D., KIRSIPU, T.V, HUEBNER, J.S. &
OXBURGH, U.M. 1964. On some aspects of the
chemical evolution of cave waters. Journal of
Geology, Tt 36-67.
HOULIHAN, D.F. & ALLAN, D. 1982. Oxygen con-
sumption of some Antarctic and British gastropods: an evaluation of cold adaptation.
Comparative Biochemistry and Physiology, 73A
383-387
HURLEY,
G.V.,
ODENSE,
P.H.,
O'DOR,
R.K.
&
DAWE, E.G. 1985. Strontium labelling for verifying daily growth increments in the statohth of the
short finned squid (Ilex Ulecebrosus). Canadian
Journal of Fisheries and Aquatic Science, 42. 380383.
KALLE, K. 1971. Salinity, general introduction. In:
Marine Ecology: a comprehensive, integrated treatise on life in the oceans and coastal waters, VoL 1,
pt 2, (O. Kinne, ed.), 4, 683-688. Wiley, London.
MACCUNTOCK, C. 1967. Shell structure of patelloid
and bellerophontoid gastropods (Mollusca). Bulletin of the Peabody Museum of Natural History,
22:1-140.
MANN, K.O. 1992. Physiological, environmental and
mineralogical controls on Mg and Sr concentrations in Nautilus. Journal of Paleontology, 66. 620636.
MCINTYRH, W.L. 1963. Trace element partition coefficients—a review of theory and applications to
geology. Ceochimica et Cosmochimica Acta, 27.
1209-1264.
NIELSEN, J. 1988. A review on marking and tagging
methods applied to eel (Anguilla anguilla \l~)).
FAO EIFAC Occasional Papers, 2L 459-470.
NOLAN, C.P. 1991a. Size, shape and shell morphology in the Antarctic limpet Nacella condnna at
Signy Island, South Orkney Islands. Journal of
Molluscan Studies. 57: 225-238.
NOLAN, C.P. 1991b. Environmental aspects of growth
in the Antarctic molluscs Nacella condnna (Patellidae) and Yoldia eightsi (Nuculanidae) at Signy
Sr LABELLING OF LIMPET SHELL
Island, South Orkney Islands. PhD thesis, University of Hull
OPHEL, I.L. & JUDD, J.D. 1968. markingfishwith stable strontium. Journal of the Fisheries Research
Board of Canada,2S 1333-1337
PALMER, A.R. 1981. Do carbonate skeletons limit
the rate of body growth? Nature, 292:150-152.
PAN, C-M & WATABE, N. 1988. Uptake and transport of shell minerals in Glottidia pyramidata
Stimpson (Brachiopoda: Inarticulata). Journal of
Experimental Marine Biology and Ecology, 118.
257-268.
PANHLI, J & XiMENES, M-C. 1994. Sources of variation in growth of the european eel (Anguilla
anguilla) estimated from otoliths. Canadian Journal of Fisheries and Aquatic Science, 51: 506-515.
PECK, L.S. 1989 Temperature and basal metabolism
in two Antarctic marine herbivores. Journal of
Experimental Marine Biology and Ecology, 127- 112.
325
(Nancollas, G H , ed.), 5-21 Springer-Verlag,
Berlin.
ROSENBERG, G.D 1980. An ontogeneac approach to
the environmental significance of bivalve shell
chemistry. In. Skeletal growth of aquatic organisms
(Rhoads, D C. & Lutz, R.A., eds), 133-168.
Plenum Press, New York.
ROSENBERG, G.D 1990. The 'vital effect' on skeletal
trace element content as exemplified by magnesium. In. Skeletal biomineralization. patterns,
processes and evolutionary trends (Carter,
J G., ed.), 567-577 Van Nostrand Reinhold, New
York.
ROWLEY, RJ. & MACKINNON, D.I. (in press). Use of
the fluorescent marker calcein in biomineralisation studies of brachiopods and other marine
organisms. Proceedings of the 7th International
Biomineraksation symposium. November 1993,
Monaco.
SEED, R 1976. Ecology In. Marine mussels: their
ecology and physiology (B L. Bayne, ed.), 13-65
PECK, L.S. & CULLEY, M B 1990. Structure and denCambridge University Press, Cambridge
sity of Halions tuberculata populations around the
coasts of Jersey, Channel Isles. Journal of the SHABICA, SV 1976. The natural history of the
Marine Biological Association of the l/.K., 70- 67Antarctic limpet Patinigera polaris (Hombron <4
75.
Jacqumot). PhD thesis, University of Oregon,
Eugene.
PECK, L.S., PROTHEROTHOMAS, E. & HOUGH, N
1993. Pedal mucus production by the Antarctic
SOKAL, R.R. & ROHLF, FJ. 1981. Biometry (second
limpet Nacella concinna (Strebel, 1908). Journal of
edition). Freeman & Co San Francisco.
Experimental Marine Biology and Ecology, 174: SPANGENBERG, D B. 1979 Statolith development in
177-192.
aurelia metamorphosing in strontium and low calcium-contairung sea water Scanning Electron
PICKEN, G.B. 1980. The distribution, growth and
Microscopy, 1979(2): 432-438.
reproduction of the Antarctic limpet, Nacella
(Patinigera) concinna (Strebel, 1908). Journal of TULONEN, J. 1989 Growth and sex ratio of eels
Experimental Marine Biology and Ecology, 42: 71- (Angudla anguilla) of known age in four small
85.
lakes in southern Finland. EIFAC Working Party
on Eel, Porto, Portugal 15pp.
PIRKER, J.G. & SCHTEL, D R. 1993. Tetracyclme as a
WALKER, AJ.M. 1972. Introduction to the ecology
fluorescent shell-marker in the abalone Halions
of the Antarctic limpet Patinigera polaris (Homins. Marine Biology, 116: 81-86.
bron & Jacquinot) at Signy Island, South Orkney
RICHARDSON, C A 1987 Tidal bands in the shell of
Islands Bulletin of the British Antarctic Survey, 28
the clam Tapes philippinarum (Adams & Reeve
49-71
1850). Proceedings of the Royal Society of
London, B230: 367-387
WATABE, N. 1983. Shell Repair. Chapter 7, In: The
Mollusca, volume 4, Physiology part 1 (A.S M
RICHARDSON, CA. & Liu, J.H 1994 Tidal
Saleuddin & K.M. Wilbur, eds), 289-316 Acamicrogrowth bands in the shell of the intertidal
demic Press, New York.
limpet Cellana toreuma (Reeve, 1885) from the
shores of Cape d'Aguilar, Hong Kong In. The WHEELER, A.P, BLAOCWELDER, P.L. & WILBUR,
malacofauna of Hong Kong and Southern China
K.M. 1975. Shell growth in the scallop Argopecten
III (B. Morton, ed.), 445-465. University of Hong
irradians I. Isotope incorporation with reference
Kong Press, Hong Kong
to diurnal growth. Biological Bulletin, 148: 472482.
RICKER, W E. 1973. Linear regressions in fishery
research Journal of the Fisheries Research Board YAMADA, S B., MULLIGAN, T J . & FAIRCHILD, SJ
of Canada, 30- 409-434.
1979 Strontium marking of hatchery-reared coho
salmon (Oncorhynchus kisutch, Walbaum). JourROBERTSON, WG.D. 1982. The solubility concept.
nal of Fish Biology, 14.267-275
In: Biological mineralization and deminerahzauon