Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

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Journal of Experimental Marine Biology and Ecology

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Antarctic intertidal limpet ecophysiology: A winter–summer comparison

Birgit E. Obermüller, Simon A. Morley ⁎, Melody S. Clark, David K.A. Barnes, Lloyd S. PeckBritish Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 0ET, UK

⁎ Corresponding author.E-mail address: smor@bas.ac.uk (S.A. Morley).

0022-0981/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.jembe.2011.04.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 December 2010Received in revised form 4 April 2011Accepted 6 April 2011Available online 10 May 2011

Keywords:Antarctic intertidalFeedingMetabolic activityNacella concinnaSeasonal comparisonThermal limits

The Antarctic intertidal zone is one of the world's most extreme marine environments. As well as having atypically high annual temperature variation (~30 °C), it is affected by frequent ice scouring in summer and iceencasement inwinter, aswell as substantial salinity fluctuations during tidal cycles. Formany years the Antarcticintertidal was believed to host only migratory species during summer, however, recent studies have foundseveral permanently residentmacrofaunal species, including the limpetNacella concinna. Herewepresent resultsof the first seasonal comparison of different ecophysiological parameters in this species collected from theintertidal in bothwinter (August and September) and summer (January and February) on Adelaide Island (WestAntarctic Peninsula). There was clear evidence of seasonal acclimatisation with a shift in thermal windowbetween winter and summer limpets. The seasonal change in metabolic rate did not show increased costs inwinter (cf metabolic cold adaptation) and the seasonal increase in oxygen consumption was within the rangeexpected due to the physical effects of temperature alone. O:N ratios indicated that the animals were using thesamemetabolic substrate (mainly protein) all year round. There was no significant difference in condition factorbetween winter and early summer individuals. However comparisons with subtidal N. concinna showed thatthose from the intertidal had a lower condition factor than those permanently immersed.Whilst remaining in theice-encased intertidal during winter may give access to ice-algae and microphytobenthos in the shallows andprovide a feeding advantage early in the season, there are clearly extra costs to living in the intertidalper se. HenceN. concinna may not derive any obvious fitness advantage but may simply be occupying an available niche andsurviving the physical challenges in the shallows.

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1. Introduction

Intertidal zones are typically highly variable in disturbance andstress compared to deeper subtidal habitats due to the effects of waveexposure, temperature and salinity fluctuations, desiccation andimmersion, and anthropogenic influences during tidal cycles (reviewedin Raffaelli and Hawkins, 1996; Menge and Branch, 2001). Manyhabitats in the intertidal zone have been intensively studied intemperate and tropical regions but polar intertidal zones remain littlestudied and their faunas poorly characterised (Barnes and Brockington,2003; Waller et al., 2006a). Unlike elsewhere in the world, most of theAntarctic coast line is coveredwith ‘permanent’ ice sheets but where icefree the littoral zone appears to be a particularly extreme and stressfulenvironment (e.g. see Peck et al., 2006). Antarctic shores are frequentlyscoured by brash ice and icebergs in summer and encased by ice inwinter when fast ice connects to the shore and an ice foot builds upwhichcan last for severalmonths(Barnes, 1999; Smaleet al., 2007).Alsothe Antarctic intertidal zonehas extreme salinityfluctuations (b5 to 40)in summer due to ice melt and glacial freshwater runoff, but also saltenriched conditions due to leakage from brine channels during winter

and spring (Davenport, 2001). In addition there are wide diurnalfluctuations in temperature over tidal cycles (N5 °C; extremes up to18 °C) as well as large seasonal temperature differences (−15 to+17 °C; Waller et al., 2006a, 2006b); also compared to the more stablesubtidal, which typically exhibits a very small annual variation (−1.9 to+1.8 °C; Clarke et al., 2008). Such extreme physical conditions initiallyled to the Antarctic intertidal being considered as largely devoid ofmacro-organisms and to merely host a few transitory species duringsummer rather than permanent residents (reviewed in Peck et al.,2006).However,Waller et al. (2006b)demonstrated species presence inintertidal habitats adjacent to Rothera Research Station (68°34′20″S,68°07′50″W) during winter. The twelve species recorded includedmultiannual (3–4 years old) bryozoancolonies on rocks, showing that atleast some species are capable of permanent residency in thesupposedly harsh intertidal zone (see also Barnes and Brockington,2003). Sampledmacrofauna also comprised severalmobile taxa such assea urchins, nemertean worms, crustaceans, and limpets.

Limpets are conspicuous members of near-shore habitats world-wide. They colonise tidal rocky shores from inter- to deeper subtidaldepths. The Antarctic limpet Nacella concinna (Strebel 1908) is aubiquitous and abundant near-shore gastropod along the AntarcticPeninsula and sub-Antarctic Islands (Picken, 1980). N. concinnaoccurring in the intertidal had originally been thought to be migratory,moving seasonally between inter- and subtidal depths (downwards in

Table 1Summary of environmental parameters during sampling of Nacella concinna in winter2008 and summer 2009 at Adelaide Island (West Antarctic Peninsula). n=number ofspecimens collected.

CollectionDate

Lowtide

Intertidal watertemperature

Salinity Airtemperature

n

22/08/08 0.49 m −2.1 to −1.9 °C 45.0 −19.4 °C 1518/09/08 0.37 m −1.2 to −1.1 °C 42.0 −2.1 °C 4010/01/09 0.50 m +0.6 to +0.7 °C 34.5 +1.0 °C 4005/02/09 0.46 m −1.2 to −1.3 °C 35.0 −2.2 °C 40

40 B.E. Obermüller et al. / Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

winter, upwards in summer) (Walker, 1972; Brêthes et al., 1994).However, some individuals have been found to remain in intertidalduring winter (Rakusa-Suszczewski, 1992; Waller et al., 2006b).

Despite its wide distribution in the shallows along the AntarcticPeninsula and sub-Antarctic region N. concinna faces severe physio-logical challenges. It is sensitive to the extremes of salinity andtemperature that occur in the intertidal zone. For example, themedian lethal salinity of 20.9 for N. concinna (Davenport, 2001) is wellabove the minimum salinity that has been recorded during summertidal cycles. However, to overcome some of these challenges intertidalN. concinna has evolved certain behavioural responses such as theearly detachment of air exposed specimens and congregation at thebottom of tidal pools rather than higher up on the shore (Davenport,2001; Weihe and Abele, 2008). With regards to the cold, N. concinnahas been shown to be sufficiently freeze tolerant to survive summerlow temperatures with supercooling points (SCPs) measured inspecimens collected in summer ranging between −2.9 and −9.3 °C(Waller et al., 2006b). They are also known to have other mechanismsto reduce the risk of freezing. N. concinna secretes mucus to isolateand protect tissues from freezing (Hargens and Shabica, 1973). Mucussecretion was effective in prolonging survival down to −20 °C withmortality increasing from −10 °C onwards.

Looking at these constraints the questions arise as to why animalscolonise the intertidal and whether remaining there, especially duringthe adverse winter period, provides any advantages? The limpet N.concinna is the ideal model to study the response of macrofauna tooverwintering in the Antarctic intertidal zone, as it is the best studiedspecies described from this habitat to date and its ecology is wellunderstood. The aim of this study was to establish if N. concinna thatremain in the intertidal duringwinter were dormant. To achieve this anecophysiological comparison was carried out, measuring conditionfactor, faecal egestion (feeding activity), oxygen consumption, nitrogenexcretion, O:N ratio, righting ability, and upper lethal temperature limitduring the winter and summer seasons. The following questions wereaddressed: Are there ecophysiological differences between winter andsummer limpets? Is there physiological evidence for (energetic)advantages (e.g. with regards to food availability) in the intertidal thatcould outweigh the costs of higher stress impacts compared to deepersubtidal habitats? Or is the Antarctic intertidal just an available niche inwhich N. concinna is able to tolerate physical challenges and survive?

2. Material and methods

2.1. Intertidal limpets in South Cove in winter 2008

Seasonal sampling of intertidal N. concinna during winter wascarried out in August and September 2008 and limpets were collectedduring low tide at a shore site in South Cove (67°34′11″S, 68°08′89″W)near Rothera Research Station, Adelaide Island, Antarctica. As outlinedbelow, ice and snow cover and weather conditions differed markedlybetween these two months.

In August 2008 South Cove was mostly covered by sea ice (25–30 cm) with a solid ice edge, which had a thick cover of snow,amounting to an approximately 1.5 to 2 m thick layer covering theintertidal sampling site. After the snow was cleared a hole was cut intothe ice edge. The ice foot reached down right onto the intertidal rocksand boulders. Limpets were observed to sit on the sides of these rocksand near or in cracks and crevices, thus relatively sheltered from icescouring. The followingday, ice slushwhichhadaccumulatedduring thetidal cycle overnight was cleared away before sampling. Watertemperature, salinity, and air temperature were measured directlybefore the sampling (Table 1). 15 limpets (not encased in ice) werecollectedduring low tide from the rocks directly below thewater and icesurface. Faecal egestion of this first group of winter animals (W. Gr. I)was measured during the first 48 h after collection, followed bymeasurements of routine oxygen consumption and nitrogen excretion

(see below for protocols). Thereafter, the righting ability (capability ofindividuals to turn back over after being turned onto their shell) of theselimpetswas tested over a period of 24 h. Allmeasurementswere carriedout at a temperature of −1.5 to −1.4 °C, which was the watertemperature of theflow-through in the aquariumandhereafter referredto as winter ambient.

In September 2008 South Covewas ice-free andmost of the ice edge,which had covered the intertidal sampling site before, had broken offand left the sampling site ice-free. As before,water temperature, salinity,and air temperatureweremeasured directly before sampling at low tide(Table 1). 21 limpets of this second group of winter animals (W. Gr. II)were heated up by 1 °C per day and their righting ability tested at+2.5 °C over 24 h. +2.5 °C was chosen because a previous studyshowed that this was the temperature where the subtidal Antarcticlimpet N. concinna had a 50% failure of this essential biological function(Peck et al., 2004). As a final measurement the limpets' upper lethaltemperature limit was determined by increasing the temperature by1 °C per day until individuals failed to respond to tactile stimulus of thefoot and were deemed ‘dead’ as described in Peck et al. (2009). Datawere expressed as % mortality. At the end of the experiment limpetmorphometrics were determined (see below).

2.2. Intertidal limpets in South Cove in summer 2009

Seasonal sampling of intertidal N. concinna during summer wascarried out in January and February 2009 and limpets were collectedduring low tide at the same location in South Cove as in winter (seeabove). Water temperature, salinity, and air temperature weremeasured directly before the sampling in both months (Table 1).

In January 2009 faecal egestion, routine oxygen consumption, andnitrogen excretion were measured a first group of limpets (S. Gr. I). Allmeasurements were carried out at ambient temperature of +0.1 to0.2 °C (i.e. aquariumflow-through). Thereafter, the animalswere cooleddown to−1.5 °C (at 1 °C per day) and their righting ability was testedover a 24 h period. This was followed by a measurement of rightingability at +2.5 °C (temperature raised by 1 °C per day). Righting abilitywas measured again in a second group of summer limpets (S. Gr. II)collected at the same date as S. Gr. I to test if the order of temperaturechange had an effect on righting ability; measurements started at thehigher temperature of +2.5 °C followed by tests at −1.5 °C (temper-ature changed by 1 °C per day).

In February 2009 righting ability was also tested at the ambientsummer temperature (+0.6 °C) in summer groups III and IV, whereafter these limpets were either cooled down to −1.5 °C (S. Gr. III) orheated up to +2.5 °C (S. Gr. IV) and their righting ability was testedagain. As a final measurement upper lethal temperature limits weredetermined in summer Gr. IV. Starting at +2.5 °C the limpets wereheated up by 1 °C per day and checked daily for mortality (see above).

2.3. Experimental protocols

2.3.1. Condition factorTo assess the general physiological condition of winter and summer

limpets Fulton's condition factor (CF=M⁎L−3; see Nash et al., 2006)wasmodified. As thebodymass (M)ofN. concinnawasbetter correlated

Season of sampling

winter summer winter summer

Co

nd

itio

n f

acto

r (g

WM

V-1

)

0.8

0.9

1.0

1.1

1.2

1.3

1.4F

aeca

l eg

esti

on

(g

FD

M d

-1)

corr

ecte

d f

or

stan

dar

d m

ass

limp

et

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16*

Fig. 1. Condition factor (CFm) expressed as g tissue wet mass (WM) per internal shellvolume (V) (filled circles), and faecal egestion rate expressed asmg faecesdrymass (FDM)per 24 h corrected to a standard mass limpet of 175.52 mg DM (filled triangles), inindividualwinter and summer intertidalNacella concinna. * indicates significant differencebetween winter and summer data.

41B.E. Obermüller et al. / Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

with shell height andwidth, than length (L) an internal shell volume (V)was calculated, using the formula for anelliptical cone. This shell volumewas substituted for length cubed and tissuewetmass (WM)wasused inthe calculation of the modified condition factor:

CFm = WM⁎V−1:

In addition, condition factors were calculated from data collected inthe preceding summer 2008 for a population genetics study of intertidaland subtidal N. concinna from Rothera Point (see Hoffman et al., 2010).

2.3.2. Faecal egestionFaecal egestion was measured during the first 48 h after collection.

Immediately after collection the limpets were placed individually inbuckets with a mesh bottom containing filtered sea water (53 μm). Thewater was changed after 24 h and faeceswere collected over a period of48 h, briefly rinsed indistilledwater, and faeces drymass (FDM) and ashfree dry mass (AFDM) were measured in accordance to the protocolused in Clarke (1990) and Fraser et al. (2002). Faecal egestion wasexpressed as mg FDM per 24 h corrected to a standard mass limpet of175.52 mg tissueDM(using anexponent of 0.82; see Clarke et al., 1994).

2.3.3. Oxygen consumption and nitrogen excretionWhole animal oxygen consumption and nitrogen excretion rates

weremeasured simultaneously in the same limpetsmonitored for faecalegestion. Metabolic rates were assessed two days after collection tominimise the effects of handling stress whilst still measuring routinemetabolic rates as close to field values as possible. Closed bottletechniques were used in accordance to the protocol described inObermüller et al. (2010). Oxygen consumption was expressed as μmolO2 h−1 corrected to a standard mass limpet (see above). At the end ofeach experiment water samples were taken from each chamber foranalyses of ammonia content. Control chambers (blanks not containinganimals) were run in parallel and all rates were corrected for ‘blank’respiration and background levels of ammonia. At the end of each trialwhole animal volume and WM (tissue plus shell) were determined aswell as WM of tissue and shell only. Tissue DM was measured afterdrying to a constant mass at 60 °C, and ash mass (AM) was obtained byincineration in a muffle furnace at 500 °C for 1000 min. AM wassubtracted from DM to gain AFDM. Shell length, width and height werealso measured.

Ammonia was assayed with o-phthalaldehyde (OPA) and fluo-rometry, using the method as described by Holmes et al. (1999) withminor modifications (see Obermüller et al., 2010). Ammoniaexcretion was expressed as nmol NH3 h−1 corrected for a standardlimpet of 175.52 mg DM.

O:N ratios were calculated on an atomic basis from standardisedrates (converted into ng atoms per hour), including ammonia nitrogenonly (sensu Fraser et al., 2002).

2.3.4. Statistical analysisAll data were expressed as mean±standard deviation (SD).

Statistical analysis was carried out usingMinitab version 15.1 (Minitab,Pennsylvania State University, USA) with a threshold for significance atp=0.05. Normally distributed data, tested with the Ryan-Joiner test,were analysed for seasonal (winter vs. summer) differences using atwo-sample t-test. If data were not normally distributed the non-parametricMann–Whitney test was used. These seasonal differences inmetabolic rates were assessed by comparing Q10 temperature co-efficients of the winter–summer-change in oxygen consumption andnitrogen excretion. For analysis of monthly differences (more than twogroups) in CFs a one-way ANOVA followed by a post hoc Tukey'smultiple comparison test was used for normally distributed data. Non-normally distributed data were analysed for monthly differences withthe non-parametric Kruskal–Wallis test. Chi-square tests were per-formed to detect differences in righting ability between groups (winter

and summer months) and treatments (experimental temperatures).When groups were not significantly different data for different monthswithin the same season were pooled.

3. Results

3.1. Condition factor and faecal egestion

There was no significant difference in CFm values between winterlimpets collected in August 2008 and summer specimens collected inJanuary 2009 (S. Gr. I) or within the winter season between August andSeptember (Fig. 1, Table 2). However, CFm values were significantlyhigher later in the summer (February 2009) than in early January(Mann–Whitney test, W=217.0, p=0.02) and higher than duringwinter (Mann–Whitney test, W=192.0, p=0.015; Table 2).

Summer CFm values calculated for intertidal N. concinna fromJanuary 2008 (Hoffman et al., 2010) were higher (1.447±0.17) thanthose recorded in the current study (Table 2) in winter (August andSeptember 2008) (two-sample t-test: T=9.46, pb0.001) and earlysummer (January 2009) (two-sample t-test: T=10.20, pb0.001).Intertidal CFm values in January 2008 (two-sample t-test: T=−4.57,pb0.001) and 2009 (T=12.42, pb0.01) were also significantly lowerthan the subtidal CFm in January 2008 (1.705±0.257).

Intertidal N. concinna mean faecal egestion (corrected to a standardmass limpet) was significantly higher in winter (93.36±30.58 mgFDMday−1) than in summer (56.98±27.87 mg FDMday−1) (two-sample t-test, T=3.45, p=0.002, DF=28; Fig. 1).

Limpet shell length (L),width (W), and height (H)were significantlylower inwinter (W. Gr. I) than in summer animals (S. Gr. I) analysed forCF, faecal egestion, oxygen consumption, and nitrogen excretion(Table 2).

3.2. Oxygen consumption and nitrogen excretion

Winter limpets had significantly lower oxygen consumption thansummer specimens (two-sample t-test, T=−3.61, p=0.002,DF=22; Fig. 2). Mean values were 0.84±0.18 μmol O2 h−1 in wintercompared to 1.04±0.11 μmol O2 h−1 in summer (both corrected to astandard mass limpet). The Q10 temperature coefficient betweenwinter and summer was 3.5.

Nitrogen excretion (measured as ammonia) in N. concinna wassignificantly lower inwinter (228.18±53.09 nmol NH3 h−1) comparedto summer (336.84±60.24 nmol NH3 h−1), both corrected to astandard mass limpet (two-sample t-test, T=−5.34, pb0.001,DF=28; Fig. 2). The Q10 between winter and summer was 9.9.

Table 2Summary of shell length (L), width (W), height (H), tissue wet mass (WM), dry mass (DM), and condition factor (CFm, V= internal shell volume) from all sampled Nacella concinna.

Collection date Length (mm) Width (mm) Height (mm) Tissue WM (g) Tissue DM (g) CFm (WM⁎V−1)

22/08/08 19.4±3.0a 13.2±2.1a 5.9±1.1a 0.462±0.284a 0.103±0.069a 1.066±0.099b

18/09/08 20.8±4.7 12.6±4.5c 5.6±2.6c 0.619±0.429 n.d. 1.063±0.140b

10/01/09 23.1 ±5.2 16.2±4.0 7.9±2.4 0.993±0.774 0.243±0.192 1.077±0.075 S. Gr. I1.153±0.092 S. Gr. IId

05/02/09 21.8±5.1 15.1±3.9 7.0±2.3 0.866±0.643 n.d. 1.172±0.109

a indicates that L, W, H, WM, and DM were significantly lower in winter (August 2008) than in summer limpets (January 2009): Mann–Whitney tests, L: W=187.5, p=0.040;W: W=183.5, p=0.027; H: W=175.5, p=0.011; WM: W=176.5, p=0.013; DM: W=166.0, p 0.004.

b indicates that CFm was significantly lower in August and September 2008 than in February 2009: Mann–Whitney tests: W=192.0, p=0.015 and W=256.0, p=0.009.c indicates thatW and Hwere significantly lower in winter (September 2008) than in summer limpets (January 2009): Mann–Whitney tests, W:W=381.5, p=0.018; H:W=387.0,

p=0.011.d indicates that CFm was significantly higher in January summer group (S. Gr.) II than in winter, August 2008: Mann–Whitney test W=201.0, p=0.022 and also higher than in

September 2008: Mann–Whitney test W=271.0, p=0.020. Other measurements were not significantly different.

42 B.E. Obermüller et al. / Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

Winter limpets had significantlyhighermeanO:N ratios (7.46±1.26)than summer specimens (6.35±1.30) (two-sample t-test: T=2.43,p=0.022, DF=28).

40

50

3.3. Righting ability

There were two main experiments performed: righting ability ofboth winter and summer animals at two set temperatures (−1.5 °Cand +2.5 °C) and righting ability of summer limpets over a range oftemperatures (−1.5 °C to +2.5 °C). In the first experiment there wasa significant seasonal difference between the righting of winter andsummer animals. At−1.5 °C righting ability was significantly lower inboth summer-acclimatised groups sampled and tested in January2009 (S. Gr. I 13%, Chi-Sq=4.386, p=0.036, DF=1; S. Gr. II 11%, Chi-Sq=5.625, p=0.018, DF=1) than in W. Gr. I limpets (47%) sampledand tested in August 2008 (Fig. 3). At +2.5 °C, very few animals fromany of the groups righted within 24 h, this figure was only 5% for W.Gr. II (comparisonW. Gr. I: Chi-Sq=8.890, p=0.003, DF=1), 15% forS. Gr. II, whilst no S. Gr. I specimens righted within 24 hrs at +2.5 °C(Fig. 3) (0% righting not included in statistical analyses). All othermeasurements were not significantly different and data points werethus grouped at each treatment temperature (see boxes in Fig. 3).

In the second experiment, righting abilities of both summergroups, S. Gr. III and S. Gr. IV sampled and tested in February 2009, didnot differ significantly when tested at the ambient temperature of+0.6 °C (35% of S. Gr. III and 26% of S. Gr. IV righted, respectively) andat the lower (26% of S. Gr. III righted at −1.5 °C) or higher (11% of Gr.IV righted at +2.5 °C) temperatures. Overall, when groups werepooled within each season righting ability was higher at the

Season of sampling

corr

ecte

d f

or

stan

dar

d li

mp

et

0.00

0.25

0.50

0.75

1.00

1.25

1.50

0

100

200

300

400

500* *

winter summer winter summer

Fig. 2.Oxygenconsumption expressedasmicromoles of oxygenper hour (left y axis;filledcircles) and ammonia excretion expressed as nanomoles NH3 per hour (right y axis; filledtriangles), both corrected to a standardmass limpet of 175.52 mgDM, in individualwinterand summer intertidalNacella concinna. * indicates significant differences betweenwinterand summer data.

respective seasonal ambient temperature; this was, however, signif-icant only in winter but not in summer N. concinna.

3.4. Upper lethal temperature limit

There was a significant difference in upper lethal temperature limitsbetween winter and summer limpets (two-sample t-test: T=−2.15,p=0.042, DF=25; Fig. 4). 50%mortalitywas reached at 11.8±0.7 °C inwinter and 12.4±0.1 °C in summer N. concinna. Mean shell lengths ofthese limpets were 20.8±4.7 mm in W. Gr. II (September 2008) and21.8±5.1 mm in S. Gr. IV (February 2009) and were not significantlydifferent (Table 2).

4. Discussion

This study is the first to present ecophysiological data acquiredduring a consecutive winter and summer season in a gastropodcollected in one of the world's most extreme environments, theAntarctic intertidal zone. Contrary to some reports (e.g. EsperanzaBay, Brêthes et al., 1994; Signy Island, Picken, 1980) but in agreementwith others (e.g. Rakusa-Suszczewski, 1992; Waller et al., 2006b) ourdata indicate that at Adelaide Island some N. concinna remain in theintertidal under the ice during winter and are not dormant. It should benoted that the animals obtained from the rocks directly underneath the

Temperature (°C)

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Rig

hti

ng

(%

)

0

10

20

30 **ssd

*ste

Fig. 3. Temperature related righting ability of intertidal Nacella concinna in both winter(W) and summer (S). Data are shown as proportion of limpets righting within 24 h at therespective test temperature. W. Gr. I (n=15) at ambient−1.5 °C (open circles). W. Gr. II(n=21) heated to +2.5 °C (grey circles). S. Gr. I (n=16) 1st cooled to −1.5 °C, 2ndheated to +2.5 °C (filled squares). S. Gr. II (n=20) 1st heated to +2.5 °C, 2nd cooled to−1.5 °C (filled triangles). *ste indicates a significant difference between winter limpets(W. Gr. I vs. W. Gr. II; open vs grey circles). **ssd indicates a significant seasonal differencebetweenwinter and summer limpets (W. Gr. I vs. both S. Gr. I and S. Gr. II; open circles vsfilled squares and triangles). All other measurements were not significantly different anddata points were thus grouped at each treatment temperature (boxes).

Temperature (°C)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Mo

rtal

ity

(%)

0

10

20

30

40

50

60

70

80

90

100

Fig. 4. Upper lethal temperature limits in winter (n=21; open circles) and summer(n=18; crosses) intertidal Nacella concinna shown as proportion of mortality at therespective test temperature. Each group was heated up from +2.5 °C by 1 °C per day.

43B.E. Obermüller et al. / Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

ice surface in the winter were at the small end of the size rangecompared with animals obtained when conditions were ice-free. Thismight be because smaller limpets provide less resistance to scouring iceand can more likely seek shelter in rock crevices than larger limpets,which may migrate to subtidal depths. Obtaining such animals waslogistically difficult and all data, except CFm, comparing winter andsummer animals have been size corrected for a standard animal.Colonising theAntarctic intertidal zoneall year round, these limpets facesevere challenges imposed by scouring sea ice, fluctuating temperatureand salinity. The aim of this studywas to understand the effects of theseconditions on their seasonal physiology using a number of relatedmetrics.

4.1. Condition factor and faecal egestion

One of the main costs in winter is likely to result from the greaterneed for cold protection during winter months. N. concinna has beendemonstrated to produce and secrete mucus to isolate and protecttissues from freezing (Hargens and Shabica, 1973), although, noestimate exists for the energy expenditure of this form of freezeprotection. However, the costs of mucus production for locomotion andattachment are high, accounting for between 10 and 30% of energyconsumed (Kideys and Hartnoll, 1991; Peck et al., 1993). Alteration ofSCPsmaybea secondpossible source for increased energydemands, butit remains to be investigated whether N. concinna is able to seasonallyup- and down-regulate its SCPs in the way that some temperateintertidalmolluscs alter their cold hardiness (Ansart andVernon, 2003).Comparisons of intertidal and subtidal N. concinna in summer showedthat intertidal specimens had significantly lower SCPs and higher ratesof survival after cooling than subtidal limpets (Waller et al., 2006b) andsince these comprise a homogeneous population (Hoffman et al., 2010),it is possible to infer an ability to regulate SCPs according to theenvironmental temperature regime. The means by which this isachieved is unknown as the same SCP study did not identify anythermal hysteresis proteins in N. concinna (Waller et al., 2006b).

A comprehensive population genetics-based investigation of N.concinna from Rothera Point (Hoffman et al., 2010) found significantlydifferent shell morphometrics, particularly the length to heightrelationship, between limpets from different depths. CFm calculationsusing shell volume account for depth differences better than lengthalone. A comparison of CFm data from the present study from winter2008 and summer 2009 and the Hoffman et al. (2010) study fromsummer 2008 suggests that condition factor for intertidal N. concinnaincreases fromwinter and early summer to themiddle of summer. Thishas previously been found for subtidal N. concinna at Rothera (Fraser

et al., 2002); but condition factor of intertidal always remains lower thanthat of subtidal N. concinna. Higher stress impact from ice scouring,fluctuations in salinity and temperature during tidal cycles as well asdesiccation may increase the cost of living for intertidal limpets (Weiheand Abele, 2008; Weihe et al., 2010). In addition, reduced feedingopportunities during low water emersion contribute to less energybeing available for somatic growth. However, there might be otherfactors driving the differences in shell volume to tissue mass, andtherefore CFm, between intertidal and subtidal limpets. For examplesubtidal N. concinna from the South Shetland Islands devoted moreinternal space to free water, as a means of avoiding hypoxia duringemersion and this could affect the amount of wet tissue (Weihe andAbele, 2008).

Brêthes et al. (1994) assessed the potential microalgal foodavailability for N. concinna at Esperanza Bay (North-East AntarcticPeninsula) throughout the year measuring standing stocks of micro-phytobenthos, ice-algae, and phytoplankton. Microphytobenthospeaked inMarchbutwas present for severalmonths (Feb–Jun)whereasice-algae started and peaked in growth in August, decreasing rapidlyafter ice melt. Phytoplankton showed maxima in December to Januarybut was highly variable throughout the season. Brêthes et al. (1994)concluded that benthic microalgae were the major food source forintertidal limpets around Esperanza Bay, however, at Rothera N.concinna which remain in the intertidal during winter could takeadvantage of ice-algae inhabiting the underside of the ice-foot and fastice. This would explain the higher faecal production observed in thepresent study in winter limpets. Shortly after spring ice break-upbenthic microalgae may be more productive in shallow intertidal areasthan in the deeper subtidal zone due to greater light availability. Thesecombined factors may prove a nutritional advantage for intertidalcompared to subtidal limpets.

4.2. Oxygen consumption and nitrogen excretion

Both oxygen consumption and nitrogen excretionwere significantlylower in winter than in summer intertidal N. concinna. These seasonaldifferences can be assessed by comparingQ10 values for each parameterbetween winter and summer. Calculating the Q10 temperaturecoefficient is a useful way to express the temperature dependence ofphysiological functions. Whole animal systems generally exhibit Q10

values between 2 and 3 for purely temperature driven effects (Clarke,1983; Hochachka, 1991). Thus, a Q10 of 3.5 for the 24% increase inoxygen consumption measured in the present study suggests mainlytemperature effects caused by the rise from−1.5 °C to+0.2 °C. Rakusa-Suszczewski (1992) also found mainly temperature driven increases inlimpet respiration when monitoring oxygen consumption in intertidalN. concinna sampled from Admiralty Bay (South Shetland Islands) inwinter and summer, with Q10 values ranging between 2.3 and 3.7.

The Q10 for ammonia excretion between winter and summer was9.9, far more than can be explained by temperature alone. Thesignificantly lower ammonia excretion measured in the present studyin winter N. concinna in combination with higher feeding activity issomewhat unusual, as in most species ammonia excretion increases inresponse to feeding anddecreases during starvation (Clarke, 1990;Peck,1998). The recent nutritional history might have influenced theammonia excretion rates as these also depend on the composition ofthe food being eaten and high ammonia excretion rates together withlow O:N ratios indicate a high metabolic protein turnover (Peck andVeal, 2001) (see below).

In contrast to oxygen consumption and nitrogen excretion, O:Nratios were significantly higher in winter than in summer N. concinna.O:N ratiosmaybeused asametabolic index to elucidatewhichsubstratefuels metabolic activities. Pure protein utilisation is characterised byratios of 3 to 16, whilst ratios of 50 to 60 are indicative of a morebalanced catabolism of protein as well as lipid and carbohydrate(Mayzaud and Conover, 1988). In the present study mean O:N ratios

44 B.E. Obermüller et al. / Journal of Experimental Marine Biology and Ecology 403 (2011) 39–45

fromboth seasonswere below10,which is in linewith previous studies,showing that for most of the year subtidal N. concinna relies heavily onproteins to fuel metabolic demands (Fraser et al., 2002). Even duringacute food deprivation of up to four weeks in the laboratory theselimpets donot catabolise lipids frombody storage butmaintain protein-dominated energy use, as pre-feeding O:N ratios of around 2 indicated(Peck andVeal, 2001).O:N ratiosnot onlydependondifferentmetabolicdemands, determining which substrate is catabolised or used indifferent pathways (e.g. lipids in gonad maturation during reproduc-tion), which can have a clear seasonal component (Brêthes et al., 1994).O:N ratios are also influenced by the dietary composition of the foodsource and a difference in O:N might indicate a change in food type(Mayzaud and Conover, 1988). Benthic microalgae can have highprotein content, whereas ice-algae such as centric and pennate diatomsare rich in lipids (Whitaker and Richardson, 1980; Fahl and Kattner,1993). This supports our findings of higher O:N ratios in N. concinnameasured in winter, possibly feeding on ice-algae (see above), whereasspecimens sampled in summer utilised mainly benthic microalgae.Towards the end of the summer in February and March O:N ratios mayincrease again up to ~20 as limpets switch food type and take advantageof sedimenting phytoplankton algae and detritus from the summerbloom (Fraser et al., 2002).

4.3. Seasonal changes in righting ability and upper lethal temperaturelimits

If a limpet gets scraped off a rock in the field, e.g. by ice scouring, itis essential that it manages to flip itself over onto the foot and re-attach quickly to minimise opportunistic predation (Shabica, 1976).Both winter and summer N. concinna generally had better rightingabilities at their ambient temperatures and reduced righting at higherand lower test temperatures (significant only in winter limpets). Thisstrengthens the idea that as the stresses in the intertidal vary duringthe year N. concinna alters its physiology and acclimatises to changingconditions. This is exemplified by the fact that winter limpets in thepresent study had better performance at −1.5 °C compared tosummer limpets. There was also a significant difference in upperlethal temperature limits between N. concinna measured in winterand in summer, with higher upper lethal temperature limits (LT50) inthe latter group. This likely reflects the different thermal histories ofthe two groups as summer limpets had been exposed to higherseasonal temperatures for several months in their natural environ-ment and had adjusted vital physiological processes to that highertemperature envelope. This result is in line with previous findings inthe marine environment (Buckley et al., 2001; Somero, 2002;Tomanek, 2002; Osovitz and Hofmann, 2005).

Thermal adjustments of biochemical and metabolic processes mayimpose considerable physiological costs on intertidal species (Som-ero, 2002). An organism living in a more variable environment mightbe expected to be more limited in its ability to warm and coldacclimate as temperature changes approach thermal tolerance limits(e.g. seasonal temperature maximum) than a congener/conspecificliving rather within the “normal” or optimal thermal range (Stillmanand Somero, 1996). In this context the lower overall condition andthermal performance of righting of intertidal limpets in the presentstudy compared to subtidal N. concinna tested previously (Peck et al.,2004; Morley et al., 2010) confirms the increased costs of coping withthe variable temperature, salinity and ice scouring events in theintertidal (Waller et al., 2006a).

It should be noted that the different nutritional history may alsohave contributed to the higher thermal tolerance in summer limpetsmeasured in this study. Although the actual feeding activity waslower, the food supply in general (quantity and quality) is expected tobe better in summer compared to winter, resulting in greater energyreserves and better overall fitness (see higher CFm in February) and

potential to deal with the effects of heat stress (Brêthes et al., 1994;Fraser et al., 2002).

5. Conclusions

This study established that N. concinna do remain in the intertidalduring winter at Adelaide Island (West Antarctic Peninsula) and thephysiological activities tested do not indicate winter dormancy. Therewas clear evidence for seasonal acclimatisation of temperature limits,with winter N. concinna having a lower temperature limit for survivaland better activity performance atwinter temperatures than summerN.concinna. However, the seasonal change in metabolic rate (oxygenconsumption)waswithin the range expected due to the physical effectsof temperature alone, with no evidence of increased costs in winter (cfmetabolic cold adaptation). Although O:N ratios were higher in winterthan summer the animals were still using the same, mainly protein,metabolic substrate. There was no significant difference in conditionfactor between winter and early summer animals, so remaining in theice-encased intertidal zone during winter may give access to ice-algaeand microphytobenthos in the shallows, providing a feeding advantageearly in the season. However, living in the Antarctic intertidal comeswith extra costs throughout the year — extra energy is required forfreeze protection (e.g. mucus production) and enhanced constitutiveproduction of stress proteins (Clark et al., 2008; Clark and Peck, 2009).There are also the costs of accommodating seasonal acclimatisation andsurviving the rigours of regular emersion, which at the cellular levelresults in higher levels of damaged ubiquitinated proteins (Hofmannand Somero, 1995, 1996; Roberts et al., 1997). Any one or all of thesemay be the cause of the lower condition factor in intertidal limpetsthroughout the year. N. concinnamay thus not have any obvious fitnessadvantage from life in the intertidal but may simply be occupying anavailable niche, merely withstanding and surviving the physicalchallenges.

Acknowledgements

Thanks go to Catherine Waller, Jason Coventry and Rob Webster,and to all members of the Rothera Boating and Diving Team forsupporting work in the intertidal and providing samples. This workwas carried out within the British Antarctic Survey Q4 BIOREACH/BIOFLAME core programmes and the current Ecosystems coreprogramme. These experiments comply with current UK legislation.[SS]

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