Heat-shock response of the upper intertidal barnacle Balanus glandula: thermal stress and acclimation

In the intertidal zone in the Pacific Northwest, body temperatures of sessile marine organisms can reach 35 degrees C for an extended time during low tide, resulting in potential physiological stress. We used immunochemical assays to examine the effects of thermal stress on endogenous Hsp70 levels in the intertidal barnacle Balanus glandula. After thermal stress, endogenous Hsp70 levels did not increase above control levels in B. glandula exposed to 20 and 28 degrees C. In a separate experiment, endogenous Hsp70 levels were higher than control levels when B. glandula was exposed to 34 degrees C for 8.5 h. Although an induced heat-shock response was observed, levels of conjugated ubiquitin failed to indicate irreversible protein damage at temperatures up to 34 degrees C. With metabolic labeling, we examined temperature acclimation and thermally induced heat-shock proteins in B. glandula. An induced heat-shock response of proteins in the 70-kDa region (Hsp70) occurred in B. glandula above 23 degrees C. This heat-shock response was similar in molting and non-molting barnacles. Acclimation of B. glandula to relatively higher temperatures resulted in higher levels of protein synthesis in the 70-kDa region and lack of an upward shift in the induction temperature for heat-shock proteins. Our results suggest that B. glandula may be well adapted to life in the high intertidal zone but may lack the plasticity to acclimate to higher temperatures.     

Heat-shock protein 70 (Hsp70) expression in four limpets of the genus Lottia: interspecific variation in constitutive and inducible synthesis correlates with in situ exposure to heat stress

Limpets of the genus Lottia occupy a broad vertical distribution on wave-exposed rocky shores, a range that encompasses gradients in the frequency and severity of thermal and desiccation stress brought on by aerial emersion. Using western blot analysis of levels of heat-shock protein 70 (Hsp70), we examined the heat-shock responses of four Lottia congeners: Lottia scabra and L. austrodigitalis, which occur in the high-intertidal zone, and L. pelta and L. scutum, which are restricted to the low- and mid-intertidal zones. Our results suggest distinct strategies of Hsp70 expression in limpets occupying different heights and orientations in the rocky intertidal zone. In freshly field-collected animals and in specimens acclimated at ambient temperature ( approximately 14 degrees C) for 14 days, the two high-intertidal species had higher constitutive levels of Hsp70 than the low- and mid-intertidal species. During aerial exposure to high temperatures, the two low-shore species and L. austrodigitalis exhibited an onset of Hsp70 expression at 28 degrees C; no induction of Hsp70 occurred in L. scabra. Our findings suggest that high-intertidal congeners of Lottia employ a "preparative defense" strategy involving maintenance of high constitutive levels of Hsp70 in their cells as a mechanism for protection against periods of extreme and unpredictable heat stress.     

Effects of environmental stress on intertidal mussels and their sea star predators

Consumer stress models of ecological theory predict that predators are more susceptible to stress than their prey. Intertidal mussels, Mytilus californianus, span a vertical stress gradient from the low zone (lower stress) to the high zone (higher thermal and desiccation stress), while their sea star predators, Pisaster ochraceus, range from the low zone only into the lower edge of the mussel zone. In summer 2003, we tested the responses of sea stars and mussels to environmental stress in an experiment conducted on the Oregon coast. Mussels were transplanted from the middle of the mussel bed to cages in the low and high edges of the mussel bed. Sea star predators were added to half of the mussel cages. Mussels and sea stars were sampled between June and August for indicators of sublethal stress. Mussel growth was measured, and tissues were collected for heat shock protein (Hsp70) analyses and histological analyses of reproduction. Sea stars were weighed, and tissues were sampled for Hsp70 analyses. Mussels in high-edge cages had higher levels of total Hsp70 and exhibited spawning activity earlier in the summer than mussels in the low-edge cages. Sea stars suffered high mortality in the high edge, and low-edge sea stars lost weight but showed no differences in Hsp70 production. These results suggest that stress in the intertidal zone affected the mobile predator more than its sessile prey, which is consistent with predictions of consumer stress models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interactive effects of food availability and aerial body temperature on the survival of two intertidal Mytilus species

Intertidal organisms must episodically contend with the rigors of both the terrestrial and the marine environments. While body temperatures during high tide are driven primarily by water temperature, aerial body temperatures are driven by multiple environmental factors such that temperature of an organism during low tide is usually quite different from air temperature. Thus, whereas decades of research have investigated the effects of water temperature on intertidal species, considerably less is known about the physiological impacts of temperature during aerial exposure at low tide, especially with regard to the interaction of aerial body temperature with other stressors. We examined the interactive effects of aerial body temperature and food supply on the survival of two intertidal blue mussels, Mytilus galloprovincialis and Mytilus trossulus. Survival was monitored for nine weeks using a simulated tidal cycle, with two levels of food and three levels of aerial body temperature (30, 25, and 20 °C). Decreased food supply significantly reduced the survival of mussels, but only under the 30 °C treatment. In the other two thermal regimes there were no significant effect of food on survival. When aerial body temperatures are high, food availability may have a greater effect on intertidal organisms. Decreases in ocean productivity have been linked to increased in ocean temperatures, thus intertidal organisms may become more susceptible to thermal stress as climates shift.     

Morphological and ecological determinants of body temperature of Geukensia demissa, the Atlantic ribbed mussel, and their effects on mussel mortality

Measurements of body temperatures in the field have shown that spatial and temporal patterns are often far more complex than previously anticipated, particularly in intertidal regions, where temperatures are driven by both marine and terrestrial climates. We examined the effects of body size, body position within the sediment, and microhabitat (presence or absence of Spartina alterniflora) on the body temperature of the mussel Geukensia demissa. We then used these data to develop a laboratory study exposing mussels to an artificial "stressful" day, mimicking field conditions as closely as possible. Results suggested that G. demissa mortality increases greatly at average daily peak temperatures of 45 degrees C and higher. When these temperatures were compared to field data collected in South Carolina in the summer of 2004, our data indicated that mussels likely experienced mortality due to high-temperature stress at this site during this period. Our results also showed that body position in the mud is the most important environmental modifier of body temperature. This experiment suggested that the presence of marsh grass leads to increases in body temperature by reducing convection, overwhelming the effects of shading. These data add to a growing body of evidence showing that small-scale thermal variability can surpass large-scale gradients.     

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r² = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.     

Daily stress protein (hsp70) cycle in chitons (Acanthopleura granulata Gmelin, 1791) which inhabit the rocky intertidal shoreline in a tropical ecosystem

Stress protein (heat shock protein, hsp70) response is involved in protecting organisms from the detrimental effects of environmental stressors, such as radiation and high temperatures. Tropical chitons can briefly tolerate high temperatures. However, they minimize the effects of elevated temperature during daylight hours and periods of tidal air exposure by remaining in rocky intertidal microhabitats along the shoreline of tropical waters. To study the natural variability of the hsp70 level, individuals of the polyplacophoran species Acanthopleura granulata Gmelin, 1791 were sampled every 4 h on two days in spring of 1999. Hsp70 levels were separately measured in the supernatant of the intestinal tract and foot muscle homogenates with a standardized immunoassay. The hsp70 level in the intestinal tract was highest in the early morning, decreased during the mid-morning hours and dropped to a comparatively low level in the afternoon, before increasing again during the night. The stress protein level in the foot muscle followed the daily air temperature curve with a time delay of a few hours, reaching the highest level in the afternoon and the lowest level in the early morning. The stress protein response can be interpreted as a sign of heat tolerance development and may play a role in allowing A. granulata to tolerate the temperature variability typical of its intertidal habitat.     

Microhabitats choice in intertidal gastropods is species-, temperature- and habitat-specific

Understanding how behavioural adaptations can limit thermal stress for intertidal gastropods will be crucial for climate models. Some behavioural adaptations are already known to limit desiccation and thermal stresses as shell-lifting, shell-standing, towering, aggregation of conspecifics or habitat selection. Here we used the IRT (i.e. infrared thermography) to investigate the thermal heterogeneity of a rocky platform, with four different macrohabitats (i.e. bare rock, rock with barnacles, mussels and mussels incrusted by barnacles) over four thermally contrasted months. We investigated the body temperature of Littorina littorea and Patella vulgata found on this platform and the temperature of their microhabitat (i.e. the substratum within one body length around of each individual). We also considered the aggregation behaviour of each species and assessed the percentage of thermal microhabitat choice (i.e choice for a microhabitat with a temperature different than the surrounding substrate). We did not find any aggregation of L. littorea on the rocky platform during the four studied months. In contrast, P. vulgata were found in aggregates in all the studied periods and within each habitat, but there was no difference in body temperature between aggregated and solitary individuals. These two gastropods species were preferentially found on rock covered by barnacles in the four studied months. The presence of a thermal microhabitat choice in L. littorea and P. vulgata is habitat-dependent and also season-dependent. In June, July and November the choice was for a microhabitat with temperatures lower than the temperatures of the surrounding substrate whereas in December, individuals choose microhabitats with higher temperatures than the temperatures of their substratum. Taken together, these results suggest that gastropods species are able to explore their environment to find sustainable thermal macrohabitats and microhabitats and adapt this behaviour in function of the conditions of temperatures.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Heat-shock protein 70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (genus: Tegula)

Although previous studies have demonstrated that heat-shock protein 70 (Hsp70) can be induced by environmental stress, little is known about natural variation in this response over short time scales. We examined how Hsp70 levels varied over days to weeks in two intertidal snail species of the genus Tegula: Sampling was conducted both under naturally changing environmental conditions and in different vertical zones on a rocky shore. The subtidal to low-intertidal T. brunnea was transplanted into shaded and unshaded mid-intertidal cages to assess temporal variation in Hsps under conditions of increased stress. For comparison, the low to mid-intertidal T. funebralis was transplanted into mid-intertidal cages, within this species' natural zone of occurrence. Snails were sampled every 3 to 4 days for one month, and endogenous levels of two Hsp70-kDa family members (Hsp72 and Hsp74) were quantified using solid-phase immunochemistry. Following periods of midday low tides, levels of Hsps increased greatly in transplanted T. brunnea but not in T. funebralis. Levels of Hsps increased less in T. brunnea transplanted to shaded cages than to unshaded cages, suggesting that prolonged emersion and reduction in feeding time per se are factors that are only mildly stressful. Upregulated levels of Hsps returned to base levels within days. In unmanipulated snails collected from their natural zones, Hsp levels showed little change with thermal variation, indicating that these species did not experience thermally stressful conditions during this study. However, under common conditions in the mid-intertidal zone, Hsp70 levels reflected the different thermal sensitivities of the physiological systems of these two species.     

Thermal physiology and vertical zonation of intertidal animals: optima, limits, and costs of living

Temperature's pervasive effects on physiological systems arereflected in the suite of temperature-adaptive differences observedamong species from different thermal niches, such as specieswith different vertical distributions (zonations) along thesubtidal to intertidal gradient. Among the physiological traitsthat exhibit adaptive variation related to vertical zonationare whole organism thermal tolerance, heart function, mitochondrialrespiration, membrane static order (fluidity), action potentialgeneration, protein synthesis, heat-shock protein expression,and protein thermal stability. For some, but not all, of thesethermally sensitive traits acclimatization leads to adaptiveshifts in thermal optima and limits. The costs associated withrepairing thermal damage and adapting systems through acclimatizationmay contribute importantly to energy budgets. These costs arisefrom such sources as: (i) activation and operation of the heat-shockresponse, (ii) replacement of denatured proteins that have beenremoved through proteolysis, (iii) restructuring of cellularmembranes ("homeoviscous" adaptation), and (iv) pervasive shiftsin gene expression (as gauged by using DNA microarray techniques).The vertical zonation observed in rocky intertidal habitatsthus may reflect two distinct yet closely related aspects ofthermal physiology: (i) intrinsic interspecific differencesin temperature sensitivities of physiological systems, whichestablish thermal optima and tolerance limits for species; and(ii) ‘cost of living’ considerations arising fromsub-lethal perturbation of these physiological systems, whichmay establish an energetics-based limitation to the maximalheight at which a species can occur. Quantifying the energeticcosts arising from heat stress represents an important challengefor future investigations.     

Love Thy Neighbour: Group Properties of Gaping Behaviour in Mussel Aggregations

By associating closely with others to form a group, an animal can benefit from a number of advantages including reduced risk of predation, amelioration of environmental conditions, and increased reproductive success, but at the price of reduced resources. Although made up of individual members, an aggregation often displays novel effects that do not manifest at the level of the individual organism. Here we show that very simple behaviour in intertidal mussels shows new effects in dense aggregations but not in isolated individuals. Perna perna and Mytilus galloprovincialis are gaping (periodic valve movement during emersion) and non-gaping mussels respectively. P. perna gaping behaviour had no effect on body temperatures of isolated individuals, while it led to increased humidity and decreased temperatures in dense groups (beds). Gaping resulted in cooler body temperatures for P. perna than M. galloprovincialis when in aggregations, while solitary individuals exhibited the highest temperatures. Gradients of increasing body temperature were detected from the center to edges of beds, but M. galloprovincialis at the edge had the same temperature as isolated individuals. Furthermore, a field study showed that during periods of severe heat stress, mortality rates of mussels within beds of the gaping P. perna were lower than those of isolated individuals or within beds of M. galloprovincialis, highlighting the determinant role of gaping on fitness and group functioning. We demonstrate that new effects of very simple individual behaviour lead to amelioration of abiotic conditions at the aggregation level and that these effects increase mussel resistance to thermal stress.     

Response of heat shock protein genes of the oriental fruit moth under diapause and thermal stress reveals multiple patterns dependent on the nature of stress exposure

Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.     

Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii: acclimation-influenced variations in the induction temperatures for Hsp70

The intracellular build-up of thermally damaged proteins following exposure to heat stress results in the synthesis of heat shock proteins (Hsps). In the present study, the upper thermal tolerance and expression of heat shock protein 70 (Hsp70) were examined in juveniles of the freshwater prawn Macrobrachium malcolmsonii that had been acclimated at two different temperatures, i.e. 20 degrees C (group A) and 30 degrees C (group B), in the laboratory for 30 days. Upper thermal tolerance was determined by a standard method. For heat-shock experiments, prawns in groups A and B were exposed to various elevated temperatures for 3 h each, followed by 1 h recovery at the acclimation temperature. Endogenous levels of Hsp70 were determined in the gill, heart, hepatopancreas and skeletal muscle tissues by Western blotting analysis of one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The critical thermal maximum (CT max) for prawns in groups A and B was 37.7+/-0.27 degrees C and 41.41+/-0.16 degrees C, respectively. In general, Western blotting analysis for Hsp70 revealed one band at the 70 kDa region, containing both constitutive (Hsc70) and inducible (Hsp70) isoforms, in the gill and heart tissues; these were not detected in the hepatopancreas and skeletal muscle tissues. The onset temperature for Hsp70 induction in both gill and heart tissues was 30 degrees C for prawns in group A and 34 degrees C for those in group B. The optimum induction temperatures (at which Hsp70 induction was maximum) were found to be 34 degrees C and 32 degrees C, respectively, in the gill and heart tissues of group A prawns, and 38 degrees C and 36 degrees C, respectively, for group B prawns. These results suggest that the temperature at which acclimation occurs influences both upper thermal tolerance and Hsp70 induction in M. malcolmsonii.     

How do we Measure the Environment? Linking Intertidal Thermal Physiology and Ecology Through Biophysics

Recent advances in quantifying biochemical and cellular-levelresponses to thermal stress have facilitated a new explorationof the role of climate and climate change in driving intertidalcommunity and population ecology. To fruitfully connect thesedisciplines, we first need to understand what the body temperaturesof intertidal organisms are under field conditions, and howthey change in space and time. Newly available data logger technologymakes such an exploration possible, but several potential pitfallsmust be avoided. Body temperature during aerial exposure isdriven by multiple, interacting climatic factors, and extremesduring low tide far exceed those during submersion. Moreover,because of effects of body size and morphology, two organismsexposed to identical climatic conditions can display very differentbody temperatures, which can also be substantially differentfrom the temperature of the surrounding air. These same factorsdrive the temperature recorded by data loggers, and one loggertype is unlikely to serve as an effective proxy for all organismsat a site. Here I describe the difficulties involved in quantifyingpatterns of body temperature in intertidal organisms, and explorethe implications of this complexity for intertidal physiologicalecology. I do so using data from temperature loggers designedto mimic the thermal characteristics of the mussel Mytilus californianus,and deployed at multiple sites along the West Coast of the UnitedStates. Results indicate a highly intricate pattern of thermalstress, where the interaction of climate with the dynamics ofthe tidal cycle determines the timing and magnitude of temperatureextremes, creating a unique "thermal signal" at each site.     

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

Although heat stress is often cited as the dominant physical stress on tropical shores, intertidal organisms in regions with monsoonal climates are also regularly exposed to prolonged periods of heavy rainfall. Such events are predicted to have adverse physiological effects on individuals and may result in mortality. In a series of laboratory experiments, the impact of simulated monsoonal rains was investigated on the patellid limpet, Cellana grata. Sub-lethal responses in terms of body water content, body fluid osmolality and heart rate were measured in two different size cohorts maintained on horizontal and vertical substrata. Limpets were unable to achieve any effective behavioural isolation, and exposure to either simulated rainfall or diluted seawater resulted in both large and small C. grata gaining water with subsequent dilution of mantle water and haemolymph osmolalities. With increased duration of rainfall, dilution of body fluids increased with little difference between individuals on horizontal and vertical surfaces. Body fluids generally showed proportional dilution during prolonged rain, but in some individuals there was evidence for regulation of the haemolymph relative to the mantle fluid. Overall, smaller limpets were more susceptible to prolonged rainfall than large animals in terms of swelling of soft tissues and detachment and also had higher heart rates than large limpets. Both cohorts reduced heart rates with prolonged rainfall, suggesting a degree of metabolic depression, especially on horizontal surfaces. In small limpets, no difference in heart rate was found with substratum orientation, whereas large limpets had elevated heart rates on vertical as compared to horizontal substrata, when exposed to either simulated rainfall or washed with dilute seawater. This may reflect the increased energetic costs required to maintain a relatively larger body on a vertical surface under stressful conditions. Monsoonal rainfall during emersion, and subsequent dilution of seawater, therefore, have sub-lethal physiological and possible lethal effects on intertidal 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.     

Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin

Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 +/- 0.3 degrees C), and to ambient Pacific Ocean temperatures (13.6 +/- 1.2 degrees C), for 4-5 weeks. Both groups were subjected to an acute thermal stress of 13.0 degrees C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25-2 hours) and after several recovery periods (2-48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.     

Adaptations of the Ribbed Mussel, Modiolus demissus (Dillvvyn), to the Intertidal Habitat

SYNOPSIS. This is a review of the behavioral, biochemical-physiological,and morphological adaptations of Modiolus demissus (Dillwyn)to the high intertidal habitat. The ribbed mussel, Modiolus,has fully exploited the behavioral adaptations within the limitsimposed by the bivalve body plan. Air-gaping is undoubtedlya significant behavioral adaptation which permits aerial respirationand penetration of the high intertidal zone. Modiolus fullyutilizes physiological-biochemical adaptations also. The musselis both eurythermal and euryhaline: the thermal range is atleast 56 °C and the salinity range is at least 70%, bothimpressive ranges for an organism which conforms to these environmentalparameters. The two primary adaptations made by Modiolus arean extensive tolerance to dehydration and a very high enzymethermostability. These two adaptations are resjxmsible for Modiolus'tolerance toward desiccation, variation in salinity, thermalstress, and possibly anaerobic conditions. There is no obviousmorphological adaptation. Thus, the physiological adaptations,coupled with air-gaping, are responsible for the vertical penetrationof Modiolus into the intertidal zone and its horizontal migrationover an extensive geographic range.