gy and Ecology 352 (2007) 78–88www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolo

Responses of the limpet, Cellana grata (Gould 1859), tohypo-osmotic stress during simulated tropical, monsoon rains

David Morritt a, Kenneth M.Y. Leung b, Maurizio De Pirro c, Cynthia Yau b,Tak-Cheung Wai b, Gray A. Williams b,⁎

a School of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UKb The Swire Institute of Marine Science, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road,

Hong Kong SAR, PR Chinac Dipartimento di Biologia Animale e Genetica, Università degli Studi di Firenze, Firenze, Italy

Received 22 February 2007; received in revised form 4 July 2007; accepted 4 July 2007

Abstract

Although heat stress is often cited as the dominant physical stress on tropical shores, intertidal organisms in regions withmonsoonal climates are also regularly exposed to prolonged periods of heavy rainfall. Such events are predicted to have adversephysiological effects on individuals and may result in mortality. In a series of laboratory experiments, the impact of simulatedmonsoonal rains was investigated on the patellid limpet, Cellana grata. Sub-lethal responses in terms of body water content, bodyfluid osmolality and heart rate were measured in two different size cohorts maintained on horizontal and vertical substrata. Limpetswere unable to achieve any effective behavioural isolation, and exposure to either simulated rainfall or diluted seawater resulted inboth large and small C. grata gaining water with subsequent dilution of mantle water and haemolymph osmolalities. Withincreased duration of rainfall, dilution of body fluids increased with little difference between individuals on horizontal and verticalsurfaces. Body fluids generally showed proportional dilution during prolonged rain, but in some individuals there was evidence forregulation of the haemolymph relative to the mantle fluid. Overall, smaller limpets were more susceptible to prolonged rainfall thanlarge animals in terms of swelling of soft tissues and detachment and also had higher heart rates than large limpets. Both cohortsreduced heart rates with prolonged rainfall, suggesting a degree of metabolic depression, especially on horizontal surfaces. In smalllimpets, no difference in heart rate was found with substratum orientation, whereas large limpets had elevated heart rates on verticalas compared to horizontal substrata, when exposed to either simulated rainfall or washed with dilute seawater. This may reflect theincreased energetic costs required to maintain a relatively larger body on a vertical surface under stressful conditions. Monsoonalrainfall during emersion, and subsequent dilution of seawater, therefore, have sub-lethal physiological and possible lethal effects onintertidal limpets. This influence has been largely overlooked, but coupled with the possible synergistic effects of thermal stress,monsoon rains are likely to play an important role in community dynamics on tropical shores.© 2007 Elsevier B.V. All rights reserved.

Keywords: Freshwater; Limpet; Monsoon; Physiology; Rain; Tropical shores

⁎ Corresponding author. Tel.: +852 2809 2179; fax: +852 2517 6082.E-mail address: hrsbwga@hkucc.hku.hk (G.A. Williams).

0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2007.07.002

79D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

1. Introduction

Physical stresses experienced by intertidal organismsduring emersion periods play an important role instructuring rocky shore communities; limiting speciesdistribution patterns on spatial (e.g. with tidal height)and temporal scales (e.g. between seasons, e.g. Connell,1961; Kaehler and Williams, 1996; Helmuth andHofmann, 2001). Previous work has largely concentrat-ed on sub-lethal and lethal effects of high summertemperatures and resulting thermal stress (e.g. Wolcott,1973; Garrity, 1984; Williams and Morritt, 1995;Helmuth and Hofmann, 2001). These stresses areparticularly prevalent on tropical shores, where the hotsummer represents an especially stressful environmentfor intertidal organisms and heavy mortality events mayoccur (e.g. Williams, 1994; Williams and Morritt, 1995;Chan and Williams, 2003; Chan et al., 2006).

Although heat stress is themost obvious environmentalstress experienced on tropical shores, many tropical areasare affected by monsoonal rains (e.g. W. Africa, Lawson,1957; India, Rao and Sundaram, 1974; Hong Kong,Kaehler andWilliams, 1996). Heavy rainfall brings threatsof dislodgement due to the impact and run-off from rain(Oghaki, 1988) and also imposes severe hypo-osmoticstress to intertidal organisms, especially when rainfalls during periods of emersion. Hong Kong shoresexperience such a climate, which is dictated by seasonal,monsoon, patterns.Winter is generally cool and dry (meanair temperature 15 °C, sea temperature 17 °C) whereassummer is hot and wet (mean air temperature 28 °C, seatemperature 27 °C, see Kaehler and Williams, 1996). Insummer, tides are lower than in winter (by ∼0.5 m aboveChart Datum (CD), maximum tidal range∼2.5m) and fallduring the afternoon when rock temperatures can exceed50 °C (Williams, 1994). During the summer monsoonseason, N500 mm of rain can fall in a day with intensitiesreaching N100 mm per hour (Hong Kong Observatory)and in the ensuing downpours organisms emersed on theshore are often flooded by rainwater (pers. obs.). Midshore species may, therefore, be subjected to periods ofhypo-osmotic stress in excess of 7 h during summer lowtides before they are washed by seawater as the tide rises(Ng, 2007). Even then, species may experience continuedhypo-osmotic stress as the flood tide may have reducedsalinity due to freshwater run-off in the immediate vicinity(e.g. 14 psu following heavy monsoon rain as comparedwith a normal value of 28– 33 psu, unpublished data fromCape d'Aguilar, Hong Kong).

Typically molluscs demonstrate little, if any, ability toregulate their blood osmolality. To cope with moderatesalinity changes, euryhaline molluscs generally rely on

cell volume regulation involving changes in intracellularamino acid and inorganic ion concentrations (e.g.Shumway and Freeman, 1984; Taylor and Andrews,1988; Gainey, 1994) and associated changes in rates ofoxygen consumption (see Kinne, 1971; Findley et al.,1978). If, however, molluscs are presented with largeand/or prolonged salinity changes, they often resort to‘behavioural osmoregulation’ whereby they isolate theirsoft tissues from the osmotically stressful environment.Bivalves achieve this by shutting their valves and somegastropods by withdrawal into their shell and closing theaperture with the operculum. In patellid limpets, the onlybehavioural option is to clamp tightly to the rock in orderto reduce contact of the soft tissues with freshwater. Suchbehavioural isolation can limit oxygen uptake, and maylead to depression of aerobic metabolism and heart rate(Marshall and McQuaid, 1992, 1993; Chelazzi et al.,2001) and, whilst generally successful in the short-term,longer term isolation may lead to potentially lethalphysiological effects (Michaelidis and Beis, 1990;Sokolova et al., 2000).

In Hong Kong, aspects of the physiological ecologyof the limpet, Cellana grata, have been described inrelation to behavioural and physiological responses toheat stress (see Williams and Morritt, 1995; Williamsand McMahon, 1998; Chelazzi et al., 1999; Williamset al., 2005). This limpet forages whilst awash, movingup the shore on the flood tide and then returning downthe shore with the ebb tide to rest between 1.5-2.0 mabove CD on semi-exposed to exposed shores. C. gratais inactive during the ensuing low tide period, showing apreference for certain microhabitats (crevices andvertical rock surfaces) which ameliorate thermal stress(Williams and Morritt, 1995). As such, this species maybe emersed for N7 h during the summer when monsoonrains occur and, as a non-homing limpet, is unable toseal its shell tightly against the substratum resulting inunavoidable contact with rainwater. Horizontal surfaces,where rainwater may pool around the foot of the animalare, therefore, predicted to be more stressful osmoticenvironments than vertical surfaces. The degree of stresswill also vary with animal size. Smaller animals, whichhave a relatively larger mantle edge circumference tobody volume ratio, may be more susceptible to hypo-osmotic stress than large animals.

In the present study, a rain simulator was used to testthe potentially stressful effects of monsoonal rains onCellana grata during summer. Specifically, sub-lethalresponses (variation in apparent water content, osmo-lality of mantle water, haemolymph and heart rate) wereused to test whether (i) hypo-osmotic stress increasesover time during heavy rainfall; (ii) small limpets suffer

80 D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

more from this stress than large limpets; and (iii) if thiseffect is greater on horizontal than vertical surfaces. Theindirect effects of heavy rainfall, via dilution of coastalseawater, were also tested by investigating the responsesof limpets awash with diluted seawater as compared tothose awash with full strength seawater.

2. Materials and methods

2.1. Effects of rainfall duration on large and smalllimpets

This experiment was conducted on two differentdays, for two cohorts of Cellana grata (see Williamset al., 2005) with shell length ranges 24-30 mm (17-19 g wet weight, ∼1 yr old representing the 2004

Fig. 1. Temporal profiles of (a) net change in body weight, (b) heart beat rain large (closed circle; solid line) and small (open circle; dashed line) limpsurfaces. Lines indicate significant linear regressions. For the net change in bopb0.001) and Y=0.002X–0.134 (r2=0.478, pb0.001) for large and small limY=-0.0008X+0.0397 (r2=0.578, pb0.0001) and Y=0.0010X+0.1616 (r2=

cohort) and 34-38 mm (20-24 g wet weight, ∼2 yr oldrepresenting the 2003 cohort), subsequently referred toas ‘small’ and ‘large’ limpets respectively. 30 indivi-duals in each size class were removed from south facingshores at Cape d'Aguilar Marine Reserve (Hong Kong22°N, 114°E) whilst awash (see Williams et al., 2005).Animals were placed on individual Perspex tiles(4.5×4.5 cm) which were fixed into holes drilled intoa Perspex base plate and transported back to The SwireInstitute of Marine Science (SWIMS, a distance of∼400 m). Limpets on the base plates were placed undera steady spray of seawater (33 psu) in the aquarium toallow them to settle, adhere to their tiles, and regainmantle water (see Williams et al., 2005 for details).Following this period (30 min at 28±1 °C) the platewas removed, animals were allowed to dry and the wet

te, (c) osmolality of mantle water and (d) osmolality of haemolymphets, Cellana grata, during simulated rainfall exposure on horizontaldy weight, the regression equations are: Y=0.002X+0.124 (r2=0.257,pets, respectively. For the heart beat rate, the regression equations are:0.561, pb0.0001) for large and small limpets, respectively.

Fig. 2. The relationship between mantle water and haemolymphosmolality in large (closed circles) and small (open circles) Cellanagrata during simulated rainfall exposure on horizontal surfaces. Dottedline indicates the iso-osmotic line.

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weight of the limpet plus tile determined (±0.0001 g).Infrared sensors (seeWilliams et al., 2005) were then gluedto their shells in a position above the heart and 25individuals, on their individual tiles, were haphazardlyrelocated on the base plate.

Animals were then placed horizontally at the bottom ofthe experimental chamber in an Armfield rain simulator(see Hill et al., 2002). The machine was set to mimicmonsoonal rain conditions of ∼50 mm h−1, which is areasonable rate for monsoon conditions in Hong Kong(Hill et al., 2002). Prior to rainfall (0 min), and then after30, 90, 150 and 240 min under simulated rainfall, 5individuals were randomly chosen and heart rate, wetweight, mantle water and haemolymph osmolalityrecorded (Williams et al., 2005 for details; also seebelow). Heart rate (HR, beats s−1) was monitored whenthe rain was turned off in the simulator, using the non-invasive method developed by Depledge and Andersen(1990) (see Chelazzi et al., 1999 for details). Immediatelyfollowing heart rate measurements, the animal on its tilewas removed from the base plate and the wet weightdetermined after removal of the sensor. The animal wasthen removed from the tile and qualitative observationsmade on the degree of swelling of the soft tissues visiblefrom the ventral surface of the animal. Mantle water wascollected by placing a filter paper disc (Wescor Inc, Utah,USA) between the foot and mantle of the animal andallowing the disc to saturate. Osmolality was subsequent-ly determined using a vapour pressure osmometer(Wescor 5520, Wescor Inc., Utah, USA). The remainingmantle water was removed using a finely drawn glasscapillary and then a haemolymph sample was taken bydirect puncture of the pallial vein or heart (see WilliamsandMorritt, 1995 for details). Haemolymph samples wereheld in closed Eppendorf tubes over ice until osmolality of10 μl aliquots was measured as described above. Theosmometer was regularly calibrated against NaCl stan-dards (Wescor Optimole). To allow estimation of apparentchange ofwater content in individuals, animals were driedto constant weight in an oven at 60 °C and flesh and shellweights recorded (±0.0001 g). During the experiment, airtemperatures were also monitored in the aquarium andexperimental chamber.

2.2. Effects of rainfall on large and small limpets atdifferent orientations

Following a similar protocol, limpets of the twocohorts were established (again on different days) ontwo separate base plates (each with 10 animals), oneplaced in a vertical orientation (N60°) and the other in ahorizontal orientation (b10°). Five animals from each

plate were sampled prior to rainfall and then 2.5 h aftersimulated rainfall for small animals and 4 h for largeanimals. The shorter exposure duration was selected forsmall limpets because of their apparent lower toleranceto hypo-osmotic stress. In addition, a further 5 animalsof each cohort were also placed on the horizontal baseplate and, instead of being sampled at the end of the rainexposure, were transferred to the aquarium and sprayedwith seawater to mimic the incoming tide for 1 h prior tobeing sampled as described above.

2.3. Variation in physiology of large and small limpetswhen washed by diluted seawater

Limpets were collected just as they were beingwashed by the rising tide to begin their natural awashphase. At this point, 5 small and 5 large animals hadmantle water samples collected on the shore, stored inEppendorf tubes and held over ice. These animals wereimmediately transported to SWIMS (within 5 min) onPetri dishes where a sample of haemolymph was takenfrom each individual. The osmolality of mantle waterand haemolymph were determined as described above.A further 10 small and 10 large limpets were collected,transported back to SWIMS and 5 large and 5 smallanimals were sprayed with either 14 psu seawater (Caped'Aguilar seawater, diluted with distilled water) or33 psu seawater in a re-circulating experimental spraysystem. 14 psu was selected as an appropriate dilution of

82 D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

natural seawater as this salinity has been recorded inLobster Bay (adjacent to the study site) following heavymonsoonal rain; whereas 33 psu was the normal salinityat high tide during the experimental period. Animalswere placed on individual Perspex tiles (as describedabove) fitted on base plates and placed under a steadyspray of the appropriate salinity in the aquarium. All

Fig. 3. Mean (+1SD, n=5) of the heart beat rate, osmolality of haemolymphgrata on vertical (V) and horizontal (H) surfaces at different times of expos

base plates were positioned vertically with a slope ofN60°. Limpets were sprayed for 4 h (the duration of theawash period varies depending on tidal conditionsbetween ∼3-8 h, Davies et al., 2006). During thisperiod behaviour and mortality were recorded andsurviving animals were sampled for physiologicalmeasurements as described above.

and mantle water of large (left panel) and small (right panel) Cellanaure to simulated rainfall.

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2.4. Statistical analyses

Analysis of covariance (ANCOVA) was used tocompare the temporal pattern of each of the measuredparameters (i.e. water content, heart rate and osmolality)between small and large limpets on horizontal surfaces,using time as a covariate. The significance of therelationship between mantle and haemolymph osmolalitywas tested using linear regression analysis. As differentexposure durations were applied to the large and smalllimpets in the experiment comparing the effects of differentslopes on the two size classes, two separate two-wayanalyses of variance (ANOVA) were used to test the effectof time (before and after exposure, a fixed factor) andorientation (horizontal and vertical, a fixed factor) on eachof the measured parameters. Student's t-tests for unpairedsamples were used to compare heart rate and osmolalitybetween the treatment and the recovery groups.

To investigate the influence of different salinityseawater spray, a two-way ANOVA was used to testvariation in heart rates between large and small animals(size) and the two seawater salinities (14 psu and 33 psu,both as fixed factors). As the second stage of theexperiment involved comparisons between animals ini-tially sprayed with natural seawater and then divided intodifferent treatments before a recovery treatment, groupingswere uneven so it was not possible to test for interactionterms. As a result, one-way ANOVA was used to in-vestigate differences between all treatment groups. Alldata were checked for homogeneity of variances usingLevene's test before running ANCOVA or ANOVA usingSPSS (SPSS version 14.0 for Windows).

Table 1Variation in heart rate (beats sec−1), mantle water and haemolymph osmolalilimpets were exposed to simulated rainfall for 2.5 and 4 h respectivel(∑n=2×2×5=20)

Factor df Heart rate

MS F

Small LimpetsTime 1 0.184 7.44Orientation 1 0.083 3.343Time×Orientation 1 0.006 0.243Error 16 0.025Total 20

Large LimpetsTime 1 0.160 1.43Orientation 1 0.454 41.58Time×Orientation 1 0.591 54.2Error 16 0.011Total 20

Significant effects (pb0.05) are indicated in bold. Data for mantle and haemolhomogeneity of variances despite attempts at transformation. For these data

3. Results

3.1. Effects of rain duration on large and small limpets

During exposure to simulated rainfall, both limpet co-horts showed a significant increase in apparent watercontent with time (Fig. 1a; ANCOVA: time effect,F1,46=21.4, pb0.001). The slopes for both groups wereidentical, but their elevations differed (ANCOVA: sizeeffect, F1,46=4.6, pb0.05) indicating that larger animalsconsistently holdmorewater than small animals. Both largeand small limpets showed a significant decrease in heartrate (HR) with increasing duration of exposure to rainfall(Fig. 1b, ANCOVA: time effect: F1,43=56.6, pb0.001).Rainfall had a similar effect on limpets of either size(similar slopes, ANCOVA: pN0.05). There was, however,a significant difference in elevation (ANCOVA: significantsize effect: F1,43=17.7, pb0.001) demonstrating thatsmaller animals had faster heart rates than largerindividuals.

Although there was considerable variability in osmo-lality between individuals within both size classes, therewas a significant decrease in osmolality of both mantlewater and haemolymph with duration of rain (Fig. 1c, d;ANCOVA: time effect, Mantle water F1,46 =42.8,pb0.001; Haemolymph F1,45=41.8, pb0.001). Thelarge between-individual variation in osmolality meantthat no size differences were detected in either the mantlewater or haemolymph, nor was there any interaction withsize (ANCOVA: pN0.05). There was, however, a strongpositive relationship between mantle water and haemo-lymph osmolality for both size classes (Fig. 2). In the

ty (mOsm kg−1) before and after (Time, a fixed factor) large and smally on horizontal and vertical surfaces (Orientation, a fixed factor)

Mantle Haemolymph

MS F MS F

1104970 185.8 920205 288.56230 1.05 8160 2.568040 0.26 10857 3.405947 3190

922351 222 851193 185.412054 2.90 7683 1.677722 1.86 6845 1.494153 4591

ymph osmolality in both large and small limpets failed Levene's test forsignificant effects are therefore set at pb0.01.

Fig. 4. Mean (+1SD, n=5) of the heart beat rate, osmolality ofhaemolymph and mantle water of large (open bar) and small (shadedbar) Cellana grata exposed for a 4 h “awash” phase to two differentsalinity spray regimes on vertical surfaces.

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majority of individuals, mantle water and haemolymphosmolality showed a proportional decrease with time. Insome animals exposed to rainfall for longer periods,however, mantle water osmolality was diluted tob500 mOsm.kg−1, whilst haemolymph osmolality wasmaintained above this concentration (Fig. 2).

3.2. Effects of rainfall on large and small limpets atdifferent orientations

In general, HR was faster in small than in large limpets(Fig. 3). Although there was no significant differencebetween the two treatment groups of small limpets, the HRof small limpets on both horizontal and vertical orientationsdecreased significantly after 2.5 h exposure to rain (Table 1,Fig. 3). The HR of small limpets exposed to rain for 2.5 hwas similar to those of animals that had been exposed for2.5 h and then undergone a 1 h recovery period in seawaterspray (t-test: pN0.05). Large limpets were exposed to alonger period of rain (4 h, Fig. 3) and showed a significantinteraction between time and treatment (Table 1). Initially(0 h), both limpets on horizontal and vertical treatmentsshowed a similar HR (SNK tests, t0: V=H), but after 4 h,large limpets on vertical surfaces had significantly fasterHRs than those on horizontal surfaces (SNK tests, t4:VNH). The HR of the large limpets on horizontal surfacesexposed to rain for 4 h significantly increased after therecovery period (t-test: t=6.53, df=7, pb0.001).

Haemolymph osmolality of small limpets were verysimilar between the two orientations (Table 1, Fig. 3,Power=32%). Haemolymph osmolalities, however, sig-nificantly decreased after exposure to rain in bothtreatments (Table 1, Fig. 3) but significantly increasedafter the recovery period (t-test with unequal variances:t=19.15, df=4.007, pb0.001). Similarly, no differencewas recorded in haemolymph osmolality of large limpetsbetween the two treatments (Table 1; Power=23%) but itdecreased significantly after exposure to rain for 4 h inboth treatments (Table 1). As with small limpets,haemolymph osmolality of large limpets also increasedsignificantly after the recovery period (t-test with unequalvariances: t=7.094, df=4.02, pb0.01). Changes inmantle water osmolality for the different treatmentsmirrored those for haemolymph osmolality (Table 1,Fig. 3).

3.3. Variation in physiology of limpets of two sizeclasses when washed by seawater of different salinities

There was a significant interaction in heart rate betweenanimal size and salinity treatment (Fig. 4, Two-wayANOVA: F1, 16=4.8, pb0.05 although SNK tests were

unable to further resolve differences between means). Indilute seawater (14 psu), small limpets showed a relativelylower heart rate than large individuals whereas, in contrast,small limpets had a faster heart rate at 33 psu. Limpets ofboth sizes in dilute seawater had lower haemolymph andmantle water osmolalities during the experimental period

85D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

(Fig. 4; Data failed homogeneity of variance tests despiteattempts at transformation, as a result significance levelswere adjusted to pb0.001. One-Way ANOVA: Haemo-lymph, F5, 24=134.3, pb0.001;Mantlewater, F5, 24=86.2,pb0.001). Limpets that received natural seawater sprayhad similar levels to those animals sampled directly fromthe shore (Fig. 4, SNK tests following ANOVA).

4. Discussion

Exposure to simulated rainfall resulted in both largeand small Cellana grata taking on water and subsequentswelling of soft tissues and dilution of mantle andhaemolymph osmolalities. With prolonged duration,heart rates decreased, wet weights increased, andconsequently dilution of body fluids also increased.Overall, smaller limpets were more susceptible tosimulated prolonged rain than large animals. Whilstsmall and large limpets gained weight (presumably byosmosis) at a comparable rate these data were quitevariable. A number of individuals also demonstratedadverse effects in terms of swollen soft tissues,especially at the mantle edge, which affected the capacityto form a tight seal between the edge of the shell and thesubstratum. Detachment eventually resulted under thesecircumstances, especially in smaller individuals.

Both groups of animals demonstrated a reduction inthe concentration of haemolymph and mantle waterosmolalities with increased exposure to rainfall. Thesetwo fluid compartments generally exhibit a very strongpositive correlation, as would be expected in animals withlimited osmoregulatory capacity (e.g. Shumway andFreeman, 1984; Taylor and Andrews, 1988; Gainey,1994). Therewas, however, some evidence for a deviationin this relationship in a few individuals where thehaemolymph was maintained at a higher concentrationthan would be predicted from the concentration of themantle fluid. Thismay hint at a limited capacity for hyper-osmotic regulation under hypo-osmotic stress.

This provides an interesting contrast with previous datafor Cellana grata (Williams et al., 2005) and othergastropods (e.g. Emson et al., 2002 for Cenchritismuricatus) which suggest that these species have a limitedcapacity for hypo-osmotic regulation associated withdesiccation (and thus hyper-osmotic) stress. Chew andothers (1999) suggested that the pulmonate, Onchidiumtumidium was better able to cope with hyper-osmotic thanhypo-osmotic stress via a degree of cell volume regulation.Under hypo-osmotic conditions, higher rates of amino acidcatabolism were implicated, as supported by higherammonia content in body wall and internal organs andhigher rates of ammonia excretion in specimens exposed to

salinity of 10 psu. Slightly higher (but not significantly so)mantle water concentration than the haemolymph osmoticconcentration was also recorded during exposure to hypo-osmotic stress in the present study. This could be partlyexplained by increased excretion of ammonia due to aminoacid catabolism (c.f. Chew et al., 1999), although this willbe affected by the degree of ionisation to ammonium,whichwill be dependent, amongst other things, on pH. It isknown that C. grata exhibits marked acidosis duringprolonged exposure to desiccating / emersed conditions,possibly as a consequence of impaired CO2 elimination(Williams and McMahon, 1998). Whether a similaracidosis occurs during behavioural isolation in C. gratawhen exposed to hypo-osmotic conditions is unknown,but this is unlikely as there is continued contact betweenmantle water (and haemolymph) and the externalenvironment which will facilitate elimination of CO2.

Accompanying the decrease in the osmotic concen-tration of the haemolymph, there was a concomitantdecrease in the heart rate of both size classes of Cellanagrata, suggesting metabolic rate depression, which isknown to occur during emersion in some intertidalgastropods (McMahon, 1988). Similarly, Stickle andSabourin (1979) described a reduction in heart rate in thebivalve, Mytilus edulis, and the chiton, Katherinatunicata, in response to relatively rapidly changes insalinity during fluctuating salinity exposures. Thisrelationship was not, however, seen in longer term, step-wise acclimated animals. Importantly the same slowing ofheart rate (bradycardia) and even acardia has beenpreviously described in limpets during behaviouralisolation in response to environmental stresses such aslow salinity (Marshall andMcQuaid, 1993; De Pirro et al.,1999; Chelazzi et al., 2001) and exposure to pollutants(Marshall et al., 2004; De Pirro andMarshall, 2005). Bothsmall and large animals in the present study apparentlyreduce heart rate to the same degree but, as has beendescribed previously, smaller animals generally havehigher baseline heart rates (Chelazzi et al., 1999;Williamset al., 2005). A similar difference in heart rates betweensmall and large limpets was recorded when consideringthe effect of substratum orientation, small animalsshowing a higher rate which was not affected bysubstratum orientation but decreased significantly duringprolonged (2.5 h) exposure to rain.

Large limpets, in contrast, showed a greaterdifference in HR response dependent on substratumorientation. On horizontal surfaces, large individualsshowed a bradycardic response associated with expo-sure to simulated rain whereas individuals on verticalsurfaces exhibited elevated heart rates when exposed tosimulated rainfall for 4 h (comparable to rates normally

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seen in small animals). Heart rate is known to belinearly related to oxygen consumption in a number oflimpet species (e.g. Marshall and McQuaid, 1992;Santini et al., 1999) and thus is a reasonable proxy ofenergetic requirement. Increased heart rates in indivi-duals on vertical surfaces thus suggest that there maybe an extra energetic cost to large limpets, possiblyassociated with maintaining position and avoidingdetachment. In contrast, animals on horizontal surfacesdo not encounter this problem, and the downward forceexperienced in smaller limpets on vertical surfaces willbe proportionately lower.

The effects of rainfall on limpet haemolymph andmantle water at different substratum orientations werealmost identical. Haemolymph and mantle waterdemonstrated a significant decrease in osmolality forboth small limpets exposed for 2.5 h and large animalsexposed for 4 h. It is important to notice, however, thatwhilst the decrease from ∼1000 mOsm.kg− 1 to∼600 mOsm.kg−1 (vertical) and b600 mOsm.kg−1

(horizontal) was similar for each size class, largeranimals were exposed for 1.5 h longer, which furtherillustrates the increased susceptibility of smaller limpetsto hypo-osmotic stress.

In all cases, subsequent exposure for 1 h to seawaterfollowing exposure to prolonged simulated rainfallresulted in rapid and significant increases in bothhaemolymph and mantle water osmolality and heartrate. Cellana grata, especially small animals, show afairly rapid change in haemolymph osmolality whensubjected to changing external osmotic conditionssuggesting that ‘behavioural osmoregulation’ is notparticularly well developed. In contrast, Sokolova et al.(2000) described the effects of hypo-osmotic stress onthree littorinid species from theWhite Sea and concludedthat the rate of salt and water exchange between extra-visceral fluid and water was rather slow (duringbehavioural isolation). They proposed that osmoticshock was not the primary reason for mortality oflittorinids during freshwater exposure and demonstratedthat such exposure resulted in anaerobiosis, as indicatedby a significant accumulation of succinate in the foottissue. High levels of succinate have also been recordedin the limpet, Patella caerulea (Michaelidis and Beis,1990), and the intertidal pulmonate, Onchidium tumi-dium (Lim et al., 1996), during anaerobiosis. Thebradycardic response generally recorded in the presentstudy for hypo-osmotically stressed C. grata (with theexception of large animals on vertical surfaces) maysuggest that a similar anaerobiosis is likely to occur. Therapid increase in heart rate when sprayed with seawatermay be a response to overcome an accumulated oxygen

debt during hypo-osmotic stress, as suggested byChelazzi et al. (2001).

Haemolymph and mantle water osmotic concentra-tions of both size classes were significantly reducedduring exposure to diluted seawater (i.e. being sprayedwith seawater at low salinity to mimic the effect ofsurface runoff from heavy rainfall). Large limpetsexposed to 14 psu seawater (on inclined verticalsurfaces) showed elevated heart rates compared withanimals exposed to 33 psu, similar to the large limpetson vertical orientations exposed to rainfall for 4 h. Incontrast, smaller limpets showed a faster heart rate in33 psu seawater spray, again demonstrating metabolicrate depression when exposed to hypo-osmotic stress.The elevated body weight of larger animals, andassociated increased cost (relative to small animals) inmaintaining position on vertical orientations whilstunder hypo-osmotic stress (in this case 14 psu), mayagain explain the higher heart rate. In these cases, it isproposed that the immediate energetic requirements tomaintain position on the shore override the generalmetabolic rate depression which would otherwiseappear to be the norm.

The effects of exposure to high temperature duringdaytime emersion and the importance of refuges havebeen described previously in Cellana grata (Williamsand Morritt, 1995), as has the adaptive significance ofthe ‘mushrooming’ response (lifting the shell to exposethe foot) recorded during extreme heat stress (Williamset al., 2005). Monsoonal rain conditions regularly occurat the same time of year as conditions of extreme heatstress, and it is therefore pertinent to consider how thedifferent, and indeed contrasting, stresses interact andaffect limpets on the shore. Whilst vertical surfacesrepresent a less stressful microhabitat to C. grata duringperiods of heat stress as compared to horizontal surfaces(Williams and Morritt, 1995), in contrast there is littleevidence to suggest that the two orientations offervarying costs/benefits to the limpets during monsoonalrain. Indeed, for large animals, vertical surfaces mayrepresent more stressful environments during prolongedrain (and during the final mushrooming stage of heatstress) due to gravitational considerations. Furthermorethe hyper-osmotic stress (due to evaporative water loss)experienced during prolonged emersion in hot weatheris the exact opposite to the hypo-osmotic stressexperienced during exposure to monsoonal rain and/orsubsequent immersion by diluted seawater. The relativecontributions of prolonged heat stress, periodic hypo-osmotic stress, and their synergistic effects are,therefore, likely to play a major role in the ecology ofboth sessile and mobile species on tropical rocky shores.

87D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

Acknowledgements

We are grateful to the Agriculture, Fisheries andConservation Department, the Hong Kong SAR Govern-ment for permission towork in the Cape d'AguilarMarineReserve. DM acknowledges financial support from theoffice of theDean of Science, RoyalHolloway, Universityof London and The University of Hong Kong. TCW waspartially supported by the Area of Excellence Scheme(Project No. AoE/P-04/2004), the University GrantsCommittee of the Hong Kong Special AdministrativeRegion, China. Many thanks to Ms Cecily Law and MrAlbert Au for excellent technical assistance and Profs. RDHill and AW Jaywardena for help and advice with the rainsimulator. [SS]

References

Chan, B.K.K., Williams, G.A., 2003. The impact of physical stress andmolluscan grazing on the settlement and recruitment of Tetraclitaspecies (Cirripedia: Balanomorpha) on a tropical shore. J. Exp.Mar. Biol. Ecol. 284, 1–23.

Chan, B.K.K., Morritt, D., De Pirro, M., Leung, K.M.Y.,Williams, G.A.,2006. Summermortality: effects on the distribution and abundance ofthe acorn barnacle Tetraclita japonica on tropical shores. Mar. Ecol.Prog. Ser. 328, 195–204.

Chelazzi, G., Williams, G.A., Gray, D.G., 1999. Field and laboratorymeasurement of heart rate in a tropical limpet, Cellana grata.J. Mar. Biol. Assoc. UK 79, 749–751.

Chelazzi, G., De Pirro, M., Williams, G.A., 2001. Cardiac responses toabiotic factors in two tropical limpets, occurring at different tidallevels. Mar. Biol. 139, 1079–1085.

Chew, S.F., Ho, S.Y., Ip, Y.K., 1999. Free amino acids andosmoregulation in the intertidal pulmonate Onchidium tumidium.Mar. Biol. 134, 735–741.

Connell, J.H., 1961. Effects of competition, predation by Thaislapillus, and other factors on natural populations of the barnacleBalanus balanoides. Ecol. Monogr. 31, 61–104.

Davies, M.S., Edwards, M., Williams, G.A., 2006. Movement patternsof the limpet Cellana grata (Gould) observed over a continuousperiod through a changing tidal regime. Mar. Biol. 149, 775–787.

Depledge, M.H., Andersen, B.B., 1990. A computer-aided physiolog-ical monitoring system for continuous long-term recording ofcardiac activity in selected invertebrates. Comp. Biochem.Physiol., A 96, 474–477.

De Pirro, M., Marshall, D.J., 2005. Phylogenetic differences in cardiacactivity, metal accumulation and mortality of limpets exposed tocopper: a prosobranch–pulmonate comparison. J. Exp. Mar. Biol.Ecol. 322, 29–37.

De Pirro, M., Santini, G., Chelazzi, G., 1999. Cardiac responses tosalinity variations in two differently zoned Mediterranean limpets.J. Comp. Physiol. B 169, 501–506.

Emson, R.H., Morritt, D., Andrews, E.B., Young, C.M., 2002. Life ona hot dry beach: behavioural, physiological and ultrastructuraladaptations of the littorinid gastropod Cenchritis (Tectarius) mur-icatus. Mar. Biol. 140, 723–732.

Findley, A.M., Belisle, B.W., Stickle, W.B., 1978. Effects of tidalfluctuations of salinity on the respiration rate of the southern oyster

drill, Thais haemostoma and the blue crab, Callinectes sapidus.Mar. Biol. 49, 59–67.

Gainey, L.F., 1994. Volume regulation in three species of marinemussels. J. Exp. Mar. Biol. Ecol. 181, 201–211.

Garrity, S.D., 1984. Some adaptations of gastropods to physical stresson a tropical rocky shore. Ecology 65, 559–574.

Hong Kong Observatory Monthly meteorological normals andextremes for Hong Kong. http://www.hko.gov.hk/wxinfo/climat/normals.htm.

Helmuth, B.S.T., Hofmann, G.E., 2001. Microhabitats, thermalheterogeneity, and patterns of physiological stress in the rockyintertidal zone. Biol. Bull. 201, 374–384.

Hill, R.D., Nagarkar, S., Jayawardena, A.W., 2002. Cyanobacterialcrust and soil particle detachment: a rain-chamber experiment.Hydrol. Process. 16, 2989–2994.

Kaehler, S., Williams, G.A., 1996. Distribution of algae on tropicalrocky shores: spatial and temporal patterns of non-corallineencrusting algae in Hong Kong. Mar. Biol. 125, 177–187.

Kinne, O., 1971. In: Kinne, O. (Ed.), Salinity: Animals: Invertebrates.Marine Ecology, London, vol. 1, pp. 821–996.

Lawson, G.W., 1957. Seasonal variation in intertidal zonation on thecoasts of Ghana in relation to tidal factors. J. Anim. Ecol. 45,831–860.

Lim, C.B., Low, W.P., Chew, S.F., Ip, Y.K., 1996. Survival of theintertidal pulmonate Onchidium tumidium during short term andlong term anoxic stress. Mar. Biol. 125, 707–713.

Marshall, D.J., McQuaid, C.D., 1992. Comparative aerial metabolismand water relations of the intertidal limpets Patella granularis L.(Mollusca: Prosobranchia) and Siphonaria oculus Kr. (Mollusca:Pulmonata). Physiol. Zool. 65, 1040–1056.

Marshall, D.J., McQuaid, C.D., 1993. Effects of hypoxia andhyposalinity on the heart beat of intertidal limpets Patellagranularis (Prosobranchia) and Siphonaria capensis (Pulmonata).Comp. Biochem. Physiol., A 106, 65–68.

Marshall, D.J., Ryan, P., Chown, S.L., 2004. Regulated bradycardia inthe pulmonate limpet Siphonaria (Gastropoda: Mollusca) duringpollutant exposure: implication for biomarker studies. Comp.Biochem. Physiol. A 139, 309–316.

McMahon, R.F., 1988. Respiratory response to periodic emergence inintertidal molluscs. Am. Zool. 28, 97–114.

Michaelidis, B., Beis, I., 1990. Studies on the anaerobic energymetabolism in the foot muscle of the marine gastropod Patellacaerulea. Comp. Biochem. Physiol., B 95, 493–500.

Ng, J.S.S., 2007. Resource partitioning and coexistence of molluscangrazers on Hong Kong rocky shores. Unpublished PhD thesis. TheUniversity of Hong Kong, Hong Kong.

Oghaki, S.I., 1988. Rain and the distribution of Nodilittorina exigua(Dunker) (Gastropoda: Littorinidae). J. Exp. Mar. Biol. Ecol. 122,213–223.

Rao, K.S., Sundaram, K.S., 1974. Ecology of intertidal molluscs of theGulf of Mannar and Palk Bay. Ind. Nat. Sci. Acad. Bull. 47,462–474.

Santini, G., De Pirro, M., Chelazzi, G., 1999. In situ and laboratoryassessment of heart rate in a Mediterranean limpet using a non-invasive technique. Physiol. Biochem. Zool. 72, 198–204.

Shumway, S.E., Freeman, R.F.H., 1984. Osmotic balance in a marinepulmonate Amphibola crenata. Mar. Behav. Physiol. 11, 157–183.

Sokolova, I.M., Bock, C., Pörtner, H.-O., 2000. Resistance tofreshwater exposure in White Sea Littorina spp. I: Anaerobicmetabolism and energetics. J. Comp. Physiol., B 170, 91–103.

Stickle, W.B., Sabourin, T., 1979. Effects of fluctuations of salinity onthe respiration and heart rate of the common mussel,Mytilus edulis

88 D. Morritt et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 78–88

L. and the black chiton Katherina tunicata (Wood). J. Exp. Mar.Biol. Ecol. 41, 257–268.

Taylor, P.M., Andrews, E.B., 1988. Osmoregulation in the intertidalgastropod Littorina littorea. J. Exp. Mar. Biol. Ecol. 122, 35–46.

Williams, G.A., 1994. The relationship between shade and molluscangrazing in structuring communities on a moderately-exposedtropical rocky shore. J. Exp. Mar. Biol. Ecol. 178, 79–95.

Williams, G.A., Morritt, D., 1995. Habitat partitioning and thermaltolerance in a tropical limpet, Cellana grata. Mar. Ecol. Prog. Ser.124, 89–103.

Williams, G.A., McMahon, B.R., 1998. Haemolymph pH and oxygenlevels in a naturally stressed tropical limpet, Cellana grata. In:

Morton, B. (Ed.), The Marine Biology of the South China Sea.Proceedings of the Third International Conference on the MarineBiology of the South China Sea. Hong Kong University Press,Hong Kong, pp. 239–245.

Williams, G.A., De Pirro, M., Leung, K.M.Y., Morritt, D., 2005.Physiological responses to heat stress on a tropical shore: thebenefits of mushrooming behaviour in the limpet Cellana grata.Mar. Ecol. Prog. Ser. 292, 213–224.

Wolcott, T.G., 1973. Physiological ecology and intertidal zonation inlimpets (Acmaea): a critical look at ‘limiting factors’. Biol. Bull.145, 389–422.