Huddling up in a dry environment: the physiological benefits of aggregation in an intertidal gastropod

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(1)Mar Biol (2013) 160:1119–1126 DOI 10.1007/s00227-012-2164-6. ORIGINAL PAPER. Huddling up in a dry environment: the physiological benefits of aggregation in an intertidal gastropod José M. Rojas • Simón B. Castillo • Joan B. Escobar Jennifer L. Shinen • Francisco Bozinovic. •. Received: 21 June 2012 / Accepted: 22 December 2012 / Published online: 6 January 2013 Ó Springer-Verlag Berlin Heidelberg 2013. Abstract In many intertidal gastropods, the formation of aggregations and closing of the opercular opening are behaviors commonly assumed to be associated with water conservation and maintenance of body temperature during tidal emersion periods. In the laboratory, we quantified the relationship between these two behaviors in a littorinid snail common to the north-central shores of Chile, Echinolittorina peruviana, and evaluated any benefit of these behaviors during desiccating conditions. We predicted that solitary individuals would maintain their opercula open for less time than aggregated snails when exposed to drier conditions due, at least in part, to differences in evaporative water loss. In laboratory trials, where relative humidity was manipulated, we observed that aggregated snails maintained their opercula open for longer periods of time than solitary snails under increasingly drier conditions. These results, together with observations of body temperature, suggest that aggregated animals may able to maintain gaseous exchange with their environment for longer Communicated by M. G. Chapman. J. M. Rojas (&) S. B. Castillo J. B. Escobar J. L. Shinen F. Bozinovic Departamento de Ecologı́a, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 6513677 Santiago, Chile e-mail: jmrojas@bio.puc.cl J. B. Escobar J. L. Shinen Estación Costera de Investigaciones Marinas (ECIM) and Center for Marine Conservation, Pontificia Universidad Católica de Chile, 6513677 Santiago, Chile F. Bozinovic Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, 6513677 Santiago, Chile. periods of time than solitary individuals in response to desiccation stress. Our results suggest an interactive effect of two behaviors that confer physiological benefits when confronted with extreme physical conditions experienced during periods of emersion.. Introduction With few exceptions, intertidal invertebrates face dramatic cyclical change between marine conditions during high tide and terrestrial conditions during low tide. These tidal oscillations can generate steep gradients in immersion and emersion time over only a few meters, exposing marine organisms to terrestrial conditions that can persist for hours, days or even up to a week for certain organisms that live at the upper vertical limit of the shore (Finke et al. 2009; Marshall and McQuad 2011). Increases in the aerial temperature at many coastal locations around the world have been predicted from current global warming models, and these predictions have awakened a renewed interest in how supralitoral organisms confront desiccation and thermal stress during exposure to an aerial environment (Scavia et al. 2002; Finke et al. 2007; Firth et al. 2011; Urian et al. 2011). Specific consequences may include effects at a community level such as changes in the trophic dynamics due to climate-induced changes in growth, mortality, distribution and behavior of organisms (Garrity 1984; Helmuth et al. 2006, 2011; Finke et al. 2007; Kordas et al. 2011). A great deal of ecological work has focused on the physiological challenges intertidal organisms face, which has in turn led to improvement advances in our understanding of the process of metabolic depression, commonly experienced by organisms living at high temperatures. 123.

(2) 1120. during tidal emersion periods. More specifically, acknowledging the functions of several factors that modulate the expression of heat-shock protein genes, involved in ameliorating the effects of overheating, has allowed researchers to suggest that many intertidal organisms are living at or close to their thermal limits (Hofman 1999; Helmuth et al. 2006; Somero 2002; Tomanek 2010; Dong et al. 2011; Marshall and McQuad 2011). Among the aforementioned responses to thermal stress, behavioral adjustments are mentioned recurrently in the literature, particularly social behaviors such as aggregation of individuals or individual isolation from surroundings and conspecifics. Although these behaviors have often been assumed to be associated with water conservation and the regulation of body temperature, a consensus is still lacking on the purpose behind these behavioral adjustments or the mechanisms linking the physiological responses to specific behaviors as described above (Garrity 1984; Chapman and Underwood 1996; Muñoz et al. 2005; Chapperon and Seuront 2011a, b; Stafford et al. 2012). Thus, one of the current challenges is to improve our understanding of the physiological and behavioral capabilities of organisms to adapt to changing physical conditions if we are to predict how organisms will adapt to larger climatic changes (Helmuth et al. 2005; Bozinovic et al. 2011). Intertidal gastropods, a largely mobile group of marine invertebrates, exhibit certain physiological, morphological and, in particular, behavioral traits which allow them to cope with thermal stress and desiccation risk during emersion (Garrity 1984; Somero 2002; Helmuth et al. 2006; Lee and Lim 2009; Chapperon and Seuront 2011a, b; Miller and Denny 2011). One common behavior is the closing of the opercular opening using a lid or ‘‘trapdoor’’ attached to the muscular foot, enclosing the soft body tissues within the calcareous shell. When the operculum is closed, gastropods are also anchored to the substrata hermetically with a mucus seal, effectively reducing water loss by evaporation, cutting off gas exchange with the surrounding environment (Gendron 1977; Garrity 1984; Hughes 1986; Bates et al. 2005; Denny et al. 2006). A second behavior commonly observed is the formation of dense aggregations of individuals near rock crevices or scars. It is generally thought that by joining an aggregation, individuals experience reduced thermal stress by collectively forming a wet microclimate, allowing for greater water conservation and maintenance of cooler body temperature when exposed to warm terrestrial conditions (Hughes 1986; Chapman 1995; Chapman and Underwood 1996; Rojas et al. 2000; Chapperon and Seuront 2011a, b). Although field studies have examined the relationship between thermal stress amelioration and snail aggregations, conclusive results suggesting that microclimatic effects could promote aggregation behavior are still lacking. 123. Mar Biol (2013) 160:1119–1126. (Chapman 1995; Chapman and Underwood 1996; Rojas et al. 2000; Stafford et al. 2012) or even convey an antipredator defense (Coleman et al. 1999; Coleman 2010). Identifying the physiological tradeoffs that occur during tidal emersion is critical in resolving the relationship between aggregation behavior and the amelioration of thermal stress. With the operculum closed, isolated snails (nonaggregated individuals) may limit their water loss due to desiccation, but as a consequence may also reduce their oxygen supply due to limited gas exchange. This, in turn, would result in a loss of efficiency in the physiological machinery of the heat-shock protein system that is critical in combating thermal stress (Dahlhoff et al. 2001; Somero 2002; Tomanek 2002; Dong et al. 2011; Judge et al. 2011, Canals and Bozinovic 2011). In contrast, individuals that are part of aggregations may be able to delay the closure of their opercula in response to the humid microclimate created by aggregation, thereby maintaining gas exchange with the environment for longer periods of time. If considered at the scale of an individual snail, the relative humidity and temperature of the air surrounding an individual sets the limits to which the water loss by evaporation occurs. For example, among animals that are exposed to high aerial temperatures, those which are subject to lower relative humidity will experience greater water loss at lower thermal differences between body and air than individuals exposed to greater relative humidity (Porter and Gates 1969; Helmuth 1998). This prediction is consistent with the lack of movement reported in snails during dry periods (Emson et al. 2002, Judge et al. 2009). Thus, for a snail forming part of an aggregation, the establishment of a humid microclimate may contribute not only to water conservation but may also reduce its exposure to the dry environment. Here, we evaluate the relationship between the behavioral responses to desiccation stress in a littorinid gastropod, Echinolittorina peruviana, common to the high intertidal south east Pacific shores from Panama to central Chile. At mid latitudes, the temperature of the air and rock substrata often reaches maximal temperatures of over 30 and 38 °C, respectively, during warm summer months (Rojas et al. 2000; Finke et al. 2009; Mellado 2003). Furthermore, during summer, it is common to register two low tide events in the daylight hours, which may prolong the exposure of snails to aerial conditions (Rojas et al. 2000; Finke et al. 2007). Through a series of laboratory trials, tidal emersion conditions with varied relative humidity were simulated, we examined the relationship between two behaviors in E. peruviana, opercular closure and aggregation, while evaluating any benefit of these behaviors during conditions of thermal and desiccation stress. While this approach might not represent a faithful reproduction of the natural environment, it allowed us to recreate, at least in.

(3) Mar Biol (2013) 160:1119–1126. part, the microclimatic environment experienced by a littorinid during tidal emersion periods. We hypothesized that solitary snails should keep their opercula open for less time than aggregated snails when exposed to thermal stress, but that this relationship should decrease with increasing relative humidity. Rates of water loss and increase in body temperature should be less among aggregated snails in comparison with solitary individuals, although both should vary as a function of environmental humidity.. Materials and methods For each trial, approximately 100 individuals of E. peruviana with similar body size (11.0 ± 3.0 mm) were collected during low tide periods from the high intertidal zone at the wave exposed shore of Las Cruces, Chile (33°350 S; 71°38 W). Isolated and aggregated individuals were chosen randomly from horizontal platforms with similar topographic features. A total 4 collections were carried out between April and June of 2011. Once collected, individuals were transported to the laboratory and acclimated for 3 days in tanks (50 L) in a temperature controlled room (21 °C) and exposed to a 12:12 photoperiod prior to the experiments. In an effort to better simulate the natural environment, in each tank we placed stone plates forming an irregular horizontal surface surrounded by a pool of aerated seawater. The seawater was changed daily, and the emergent stone surface was moistened once a day with a hand sprayer. In the experimental trials, snails were exposed to simulated tidal emersion conditions for a period of 4 h in one of two growth cabinets (Intelligent Artificial Climate box Model MPA-100; Winpark Electronics Co.). The cabinets were maintained with a constant aerial temperature (Ta) of 30 °C; we selected this value considering. Fig. 1 Microclimatic simulation of the daily variation in the relative humidity of the air for the Las Cruces on rock surface (3 cm high), during the summer months. 1121. the mean maximum daily temperatures registered at the collection site (Rojas et al. 2000; Finke et al. 2009). Relative humidity (RH) was held constant at 30, 50, 65, 80 or 90 %. Extreme humidity values used corresponded to the results of simulations of microclimatic conditions that may be experienced by a snail on the upper shores of Las Cruces during a typical midday low tide during the summer months (Fig. 1). The simulations were performed using Niche Mapper software (Natori and Porter 2007), using data collected during the last 10 years by the meteorological station (Campbell CR10X) located in the marine research station of ECIM (Estación Costera de Investigaciones Marinas), Las Cruces, Chile. Within each cabinet, we placed 8 Petri plates (60 mm diameter) into which snails were transferred from the acclimation tanks. Individuals selected for each trial were active (opened operculum) and out of water. Prior to transfer, each Petri plate was moistened with seawater, with the objective that each snail began the study under similar environmental conditions. A Petri plate with one snail was defined as the solitary treatment (60 total individuals) and a plate with six individuals as the aggregated treatment (300 total individuals). Individual snails were used in only once in a single trial. Once snails were placed, experimental units were exposed to five levels of RH (30, 50, 65, 80 or 90 %) following an orthogonal two-way ANOVA design, resulting in 12 replicates by treatment combination for solitary individuals and 10 replicates for aggregated treatments. Within aggregated treatments, only one individual per plate was used as the study subject at time of evaluation. These focal snails were selected only if the individual was surrounded by at least 3 conspecifics by the end of the experimental trial (Fig. 2). For each replicate, we measured the time that focal snails (both solitary and aggregated treatments) maintained their opercula open. The close of the operculum was monitored constantly over the course of the trials using a webcam placed inside the cabinet underneath each group of experimental units. Body temperature (Tb) was recorded during the first hour of the experiment for the two extreme humidity conditions (30 and 90 %), every 9 min, using an infrared thermographic camera (Flir model i40; Fig. 2). Preliminary data collected prior to experimental trials showed that snails reached a stable body temperature after 1 h of exposure to 30 °C, without consequences for survival. This last observation offers further support to the appropriateness of the air temperature selected and the feasibility of estimating rates of temperature change of during the first hour of our experimental trials. The rate of water loss experienced by individuals was then estimated from the difference between initial and final body mass standardized by the initial weight (obtained with a Chyo, JK-180 analytic balance; with precision ± 0.0001 g) and time that the operculum remained open.. 123.

(4) 1122. Mar Biol (2013) 160:1119–1126. Prior to the fitting procedure, the normality assumption was tested using D’Agostino and Pearson omnibus K2 test, and the independence of error was evaluated with a visual inspection of residuals for each data series. As mentioned previously, results were compared by performing a twoway ANOVA, considering aggregation condition (solitary or aggregated individuals) and RH level as fixed factors. We used a type II model in order to reduce the potential effects of having an unbalanced design (10 solitary vs. 12 aggregated) on the estimation of sums of squares for ANOVA (Langsrud 2003). Prior to all analyses, we tested differences in the body mass at the start of the study among experimental groups (aggregated or solitary individuals) using a one-way ANOVA. A slope homogeneity test was performed to evaluate the possible influence of the number of neighbors on the response variables, using the number of conspecifics surrounding the focal snail over the duration of the trial as a covariate. Paired comparisons were performed using the Fisher’s least significant difference test. Assumptions of each test were evaluated before analysis. The time that opercula remained open was transformed using loge(x), and the water loss rate was transformed using the arcsin (Hp). Unless otherwise stated, all statistical analyses were performed using the STATISTICA software (Statsoft v5.0).. A B. Fig. 2 Thermal image of a typical experimental group after 40 min of exposure to an ambient temperature of 30 °C and 30 % relative humidity. The spatial arrangement solitary (a) and aggregated (b) snails is a consequence of the natural displacement of individuals during the treatment. The kinetics of the heat gain in solitary and aggregated individuals exposed to 30 or 90 % RH was characterized by fitting a single exponential model of association with three parameters (y = Y0 ? a(1-exp(-K*x)) (Table 1 for details) using least square mean method (GraphPad Prism 5.0). To test whether solitary and aggregated individuals followed the same heat gain kinetic, we evaluated whether the same parameters fit the best curve for the data points of solitary snails and aggregate within the same RH level using the extra sum of squares F test (GraphPad Prism 5.0).. Results Solitary and aggregated snails exposed to extreme RH values (30 or 90 %) showed differences in kinematic warming (Fig. 3; Table 1). Maximum Tb values were slightly higher among solitary than aggregated individuals; however, differences of the same order were shown in the initial Tb between the two treatment groups, thereby limiting any conclusions as to the effect of aggregation on total. Table 1 Fitted parameters to curve warming of snails exposed to 30 and 90 % relative humidity Parameters. 30 % Solitary. 90 % Aggregate. Solitary. Aggregate. Y0. 20.7 ± 0.2. 19.9 ± 0.2. 20.2 ± 0.2. 19.9 ± 0.2. a. 30.1 ± 0.2. 29.5 ± 0.2. 29.7 ± 0.1. 28.7 ± 0.2. K. 0.08 ± 0.07. 0.05 ± 0.04. 0.08 ± 0.01. 0.06 ± 0.01. Span. 9.4 ± 0.2. 9.6 ± 0.3. 9.4 ± 0.2. 8.8 ± 0.3. Half-time. 8.5. 15.3. 8.5. 10.2. R. 0.96. 0.95. 0.96. 0.94. df. 93. 77. 93. 77. Series comparisons. F (3,170) = 125. p \ 0.0001. F (3,72) = 43.7. p \ 0.0001. 2. F and p values for the statistic F and significance from extra sum of square test used to compare the data series Y0 initial average body temperature, a mean maximal temperature reached, K constant, Span increment in temperature, Half-time time to reach half temperature maximum observed. 123.

(5) Mar Biol (2013) 160:1119–1126. 1123 Table 2 Results of the two-way analysis of variance for the effect of the aggregation (Ag) and the percentage of relative humidity (RH) of environment on the opercular exposure time (time spent open) and weight loss rate in the snail E. peruviana Response variable. Effects. Weight loss rate (mg g-1 min-1). RH. 4. 0.00. 2.00. 0.091. Ag. 1. 0.00. 33.00. 0.001. RH 9 Ag. 4. 0.00. 5.00. 0.001. 100. 0.00. Exposure time (min). df. MS. F. p. RH. 4. 0.13. 1.27. 0.293. Ag. 1. 3.24. 32.12. 0.001. 4. 0.74. 5. 46. 0.001. 100. 0.10. RH 9 Ag Error. Fig. 3 Body temperature warming curves for solitary and aggregated snails exposed to a 30 % and b 90 % relative humidity. Ambient temperature was held constant at 30 °C. See text for details. Tb gain (Table 1). Nevertheless, at 30 % RH, solitary individuals exhibited greater rates of Tb increase than aggregated snails (Table 1; Fig. 3). A similar result was also observed, albeit to a lesser extent, in snails exposed to 90 % RH (Table 1; Fig. 3). Solitary and aggregated snails exhibited differences in the rate of water loss when exposed to simulated tidal emersion conditions with the magnitude of this difference dependent on the prevailing RH (Table 2). Solitary individuals lost water at a greater rate when exposed to lower RH (30 and 50 %) than aggregated snails (Fig. 4). No differences in water loss were observed between solitary or aggregated snails exposed to greater RH (65, 80 and 90 %). Among solitary snails, those exposed to 80 and 90 % RH lost water at a similar rate, but less than snails exposed to 30 and 50 % RH. Solitary snails exposed to 65 % RH experienced less weight loss than snails exposed at 80 % RH (Fig. 4), but similar to loss rates of snails exposed to 30, 50 and 90 % RH. Snails exposed to 30 and 50 % RH no showed significant difference between them (LSD Fisher test, p [ 0.05, Fig. 4). Among aggregated snails,. Fig. 4 a Mean (±SE) loss of body mass due to evaporative water loss and b average time that kept open the operculum of both solitary and aggregated individuals exposed to different levels of relative humidity. Changes in mass are standardized by the initial weight and opened time. Asterisks indicate significant differences (p \ 0.05) between aggregated and solitary individuals exposed to similar relative humidity conditions. (See text to other paired comparisons). differences in water loss were observed only between snails exposed to RH 80 and 90 % (LSD Fisher test, p \ 0.05; Fig. 4), where snails exposed to 80 % RH experienced a slightly greater water loss. Solitary and aggregated individuals also exhibited differences in the time that their opercula remained open. 123.

(6) 1124. under varying RH conditions (Table 2). The opercula of solitary snails remained open for less time than aggregated snails when they were exposed to 30, 50 or 65 % RH (Fig. 4). For 80 and 90 % RH, solitary and aggregated snails did not exhibit differences in the time that the opercula remained open. Among solitary snails, individuals at 30 % RH kept their opercula open for less time compared with snails at 65, 80 or 90 % RH (LSD Fisher test, p \ 0.05; Fig. 4). Solitary snails held at 50 % RH maintained their opercula open for less time than snails held at 80 % RH (LSD Fisher test, p \ 0.05, Fig. 4). No differences were observed in time that solitary individuals maintained their opercula open when exposed to 65, 80 and 90 % RH (LSD Fisher test, p [ 0.05; Fig. 4). Among aggregated snails, open time of the opercula was significantly higher in snails held at 30, 50 and 65 % RH in comparison with snails held at 80 and 90 % RH (LSD Fisher test, p \ 0.05, Fig. 4). No differences were observed among aggregated snails held at 30, 50 and 65 % RH (Fig. 4) or among snails exposed to 80 and 90 % RH (Fig. 4). The number of neighbors that surrounded the focal snail in the aggregated treatment had no effect on the time that the opercula were kept open nor on the water loss rate (exposure time: F (4;40) = 0.37, p = 0.82; water loss: F (4;40) = 0.21, p = 0.93). Finally, initial body mass was similar among all snails (Interaction RH x aggregation treatment: F (4;100) = 0.25, p = 0.91), reducing bias associated with differences in the rate of water loss on initial snail condition.. Discussion Previous research has suggested that the formation of aggregations among littorinid snails may be the result of individual behaviors in response to risk of desiccation, starvation, or predation (Chapman 1995; Stafford et al. 2007; Coleman 2010). During tidal emersion periods, dry substrata restrict the movement of snails and prompt the closing of their operculum (Emson et al. 2002; Judge et al. 2009). Hence, it is possible to assume that for snails exposed to drier, aerial conditions, the amount of time the operculum remains open should be less than when conditions are more wet or humid. In this study, aggregated snails maintained their opercula open for more time than solitary individuals when exposed to drier conditions. Moreover, aggregated individuals held at low RH maintained lower body temperatures and sustained less water loss than solitary individuals. Our findings suggest that while huddling up together, snails apparently are less susceptible to harsh environmental conditions, being able to maintain gaseous exchange with the environment for more time than solitary snails under periods of desiccation stress.. 123. Mar Biol (2013) 160:1119–1126. In addition to the challenges of desiccation stress, the body temperature of snails in the upper intertidal fringe is subjected to daily oscillations in air temperature, imposing unfavorable conditions and negative consequences for physiological and locomotory performance of ectotherm organisms. For littorinids, there is a described suppression of metabolic rate in response to heating, which allows entry to short-term metabolic diapause, thus conserving energy at higher temperatures to offset lifelong constraints on energy gain due to daily thermal oscillations (McMahon et al. 1995; Marshall and McQuad 2011). As part of this metabolic adjustment, the performance curve follows a bimodal shape with temperature (Marshall and McQuad 2011). Field observations and laboratory experimentation indicate that snails are not static within aggregations, but move around among individuals with potential displacement only halted when the snails have closed their opercula (Chapman 1995; Rojas et al. 2000). In our study, an open operculum is not synonymous with locomotory activity; hence, we cannot estimate the energetic gain associated with forming part of an aggregation. Instead, our results suggest that aggregation behavior in snails may aid individuals to cope with the climatic variability typical of tidal emersion periods. Furthermore, our findings should promote further debate and research on the potential importance of aggregation for metabolic performance. For instance, a reduction in the heat transfer rate between a snail and its environment related to a more humid microclimate, together with the potential benefit for the heat-shock protein system of a prolonged oxygen supply, could be two interesting points of interaction in the physiology–behavior relationship for thermal control. However, whether the increase in time that opercula are kept open has a direct, positive consequence for physiological performance during low tide requires further study. Indeed, higher levels of heat-shock proteins (Hsp70) were described in hydrated snails in comparison with snails on dry substrata in the field (Judge et al. 2011). Historically, evidence espousing the role of aggregation behavior in water conservation has been largely anecdotal, but what is clear is the size of aggregations in intertidal communities and the frequency with which they occur vary greatly in space and time (Chapman 1995; Chapman and Underwood 1996; Rojas et al. 2000). Although aggregations are clearly beneficial under thermally stressful conditions characterized by low RH, snails held at higher RH experienced increasingly similar rates of water loss and body temperature increases, regardless of whether or not they were surrounded by conspecifics. Along the northcentral coast of Chile, where E. peruviana are frequently observed as both solitary individuals and as part of dense aggregations, the tendency to aggregate may be a function of RH during low tide or specific microclimate. This does not necessarily imply that the aggregations of gastropods.

(7) Mar Biol (2013) 160:1119–1126. conform to a group behavior. Indeed, these aggregations are commonly observed near rock cracks and crevices; thus, the observation of aggregations may be simply the natural consequence of a common preference of snails to avoid dry substrata and to congregate where substrata topography creates a humid microclimate (Chapman 1994; Stafford and Davies 2005; Jackson 2010; Chapperon and Seuront 2011a, b; Stafford et al. 2012). Aggregations may also increase competition for food among conspecifics, thus snails may only be compelled to congregate when either food supply is abundant (Mak and Williams 1999) or thermal or desiccation stress is particularly harsh. Future field studies of aggregation behavior might also incorporate the potential effects of specific behavioral cues or individual risk factors involved in promoting and maintaining aggregations. Finally, the effects of topographic features and their influence on microhabitat conditions are likely to influence the distribution and behavior of snails (Underwood and Chapman 1992; Jackson 2010). In conclusion, our results suggest a complementary role of two behaviors that confer physiological benefits when confronted with extreme physical conditions experienced during emersion periods. We concur with Kearney et al. (2009), who emphasize the importance of behavior in buffering the impacts of global climate change, arguing that behavior is an important and missing element from models of climatic change and predictions of impacts on biodiversity. 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