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This study evaluated the effect of dietary taurine levels on growth, serum biochemical parameters, salinity adaptability, and antioxidant activity of rainbow trout (Oncorhynchus mykiss). Four diets were formulated with taurine supplements at 0, 0.5, 1, and 2% w/v (abbreviated as T(0), T(0.5), T(1), and T(2), respectively). Rainbow trouts (initial weight of 80.09 ± 4.72 g) were stocked in tanks (180 L capacity), and were fed these diets for six weeks and subsequently underwent salinity acclimation. Physiological indicators were determined before salinity acclimation at 1, 4, 7, and 14 days afterwards. Results showed that there were no significant differences in growth performance (final mean weight ranged from 182.35 g to 198.48 g; percent weight gain was between 127.68% and 147.92%) of rainbow trout in freshwater stage, but dietary taurine supplement significantly increased serum-free taurine content. After entering seawater, the Na(+)-K(+)-ATPase activity of T(2) group returned to its freshwater levels, and the serum cortisol content was significantly higher than T(0) and T(0.5) groups. At the end of this experiment, the liver superoxide dismutase activity in the T(0) and T(0.5) groups was significantly lower than in the T(1) and T(2) groups, and the liver catalase in the T(0) group was the lowest whereas that in the T(2) group was the highest. Muscle malondialdehyde content was the highest in the T(0) group, and the lowest in the T(2) group. Based on the results of this study, supplement of dietary taurine (0.5–2%) enhanced the salinity tolerance in rainbow trout, which increased with the higher taurine concentration.

The predictability of high temperatures is important for intertidal species for coping with thermal stress. To investigate the roles of high temperature and its predictability on the population abundance and reproductive output of an intertidal rocky shore limpet, Siphonaria japonica, we monitored the operative temperature, recorded the population abundance of egg ribbons and adults, and quantified the expression of heat shock protein 70 (hsp70) mRNA on two warm-temperate rocky shores with different thermal environments. Abundances of limpets and egg ribbons in the hotter but more predictable (HP) habitats were higher than those in the relatively benign and unpredictable (BU) habitats. In the HP habitats, there was a strong positive correlation between habitat temperature and population abundance. For limpets living in the HP habitats, the expression of hsp70 exhibited a smaller increase with rising body temperature than for BU limpets, indicating the existence in HP limpets of a preparatory strategy in cellular stress response against thermal stress. Our results demonstrate that the predictability of operative temperature can affect physiological responses and population dynamics. The importance of predictability should be considered in analyses of the ecological consequences of climate warming.

Realistic assessments of the impacts of global warming on population extinction risk are likely to require an integrated analysis of the roles of standing genetic variation, microhabitat thermal complexity, and the inter-individual variation of heat tolerance due to both genetic differences and seasonal acclimatization effects. Here, we examine whether balancing selection and microhabitat temperature heterogeneity can interact to enhance the population persistence to thermal stress for the black mussel Septifer virgatus. We deployed biomimetic data loggers on the shore to measure the microhabitat-specific thermal variation from June 2014 to April 2016. Thermal tolerance of specimens was indexed by measuring effects of temperature on heart rate. Genotyping of specimens was performed using double digestion restriction association RADSeq (ddRADseq). Our results show that inter-individual variations in thermal tolerance correlate significantly with genetic differences at some specific gene loci, and that heterozygotes have higher thermal tolerances than homozygotes. The observed seasonal changes in genotype frequency suggest that these loci are under balancing selection. The ability of thermally resistant heterozygotes to survive in sun-exposed microhabitats acts to balance the loss of homozygotes during summer and enable the persistence of genetic polymorphisms. Population persistence of the mussel is also facilitated by the micro-scale variation in temperature, which provides refugia from thermal stress. Our results emphasize that inter-individual variation in thermal tolerance and in microhabitat heterogeneity in temperature are important for the persistence of populations in rocky shore habitats.

Elucidating the physiological mechanisms that underlie thermal stress and discovering how species differ in capacities for phenotypic acclimatization and evolutionary adaptation to this stress is critical for understanding current latitudinal and vertical distribution patterns of species and for predicting their future state in a warming world. Such mechanistic analyses require careful choice of study systems (species and temperature-sensitive traits) and design of laboratory experiments that reflect the complexities of in situ conditions. Here, we critically review a wide range of studies of intertidal molluscs that provide mechanistic accounts of thermal effects across all levels of biological organization - behavioural, organismal, organ level, cellular, molecular, and genomic - and show how temperature-sensitive traits govern distribution patterns and capacities for coping with thermal stress. Comparisons of congeners from different thermal habitats are especially effective means for identifying adaptive variation. We employ these mechanistic analyses to illustrate how species differ in the severity of threats posed by rising temperature. Counterintuitively, we show that some of the most heat-tolerant species may be most threatened by increases in temperatures because of their small thermal safety margins and minimal abilities to acclimatize to higher temperatures. We discuss recent molecular biological and genomic studies that provide critical foundations for understanding the types of evolutionary changes in protein structure, RNA secondary structure, genome content, and gene expression capacities that underlie adaptation to temperature. Duplication of stress-related genes, as found in heat-tolerant molluscs, may provide enhanced capacity for coping with higher temperatures. We propose that the anatomical, behavioural, physiological, and genomic diversity found among intertidal molluscs, which commonly are of critical importance and high abundance in these ecosystems, makes this group of animals a highly appropriate study system for addressing questions about the mechanistic determinants of current and future distribution patterns of intertidal organisms.

Climate-driven adaptive genetic variation is one of the most important ways for organisms to tolerate environmental change and succeed in altered environments. To understand rapid climate-driven evolution, and how this evolution might shift biogeographic distributions in response to global change, we measured the adaptive genetic variation to the local environment of a marine invasive species Mytilus galloprovincialis. The genetic structure of eight populations from the Mediterranean Sea, northeastern Atlantic, northeastern Pacific, and northwestern Pacific were determined using genome-wide screens for single nucleotide polymorphisms. The relationships of genetic variation to environmental (seawater and air) temperature were analyzed using redundancy analysis and BayeScEnv analysis to evaluate the impacts of temperature on the genetic divergences among these eight populations. We found that the genetic compositions were significantly different among populations and the adaptive genetic variation was associated with temperature variables. Further, we identified some genetic markers exhibiting signatures of divergent selection in association with environmental features that can be used in the future to closely monitor adaptive variation in this species. Our results suggest that divergent climatic factors have driven adaptive genetic variation in M. galloprovincialis over the past century. The rapid evolutionary adaptation has played a pivotal role in enabling this species to invade a wide range of thermal habitats successfully. Species like M. galloprovincialis that possess high levels of genetic variation may not only be especially capable of invading new habitats with different environmental conditions, but also poised to cope rapidly and successfully with rising global temperatures.

The periwinkle snail Echinolittorina malaccana, for which the upper lethal temperature is near 55°C, is one of the most heat-tolerant eukaryotes known. We conducted a multi-level investigation – including cardiac physiology, enzyme activity, and targeted and untargeted metabolomic analyses – that elucidated a spectrum of adaptations to extreme heat in this organism. All systems examined showed heat intensity-dependent responses. Under moderate heat stress (37–45°C), the snail depressed cardiac activity and entered a state of metabolic depression. The global metabolomic and enzymatic analyses revealed production of metabolites characteristic of oxygen-independent pathways of ATP generation (lactate and succinate) in the depressed metabolic state, which suggests that anaerobic metabolism was the main energy supply pathway under heat stress (37–52°C). The metabolomic analyses also revealed alterations in glycerophospholipid metabolism under extreme heat stress (52°C), which likely reflected adaptive changes to maintain membrane structure. Small-molecular-mass organic osmolytes (glycine betaine, choline and carnitine) showed complex changes in concentration that were consistent with a role of these protein-stabilizing solutes in protection of the proteome under heat stress. This thermophilic species can thus deploy a wide array of adaptive strategies to acclimatize to extremely high temperatures.

Anticipatory changes in organismal responses, triggered by reliable environmental cues for future conditions, are key to species' persistence in temporally variable environments. Such responses were tested by measuring the physiological performance of a tropical high-shore oyster in tandem with the temporal predictability of environmental temperature. Heart rate of the oyster increased with environmental temperatures until body temperature reached ∼37°C, when a substantial depression occurred (∼60%) before recovery between ∼42° and 47°C, after which cardiac function collapsed. The sequential increase, depression, and recovery in cardiac performance aligned with temporal patterns in rock surface temperatures, where the risk of reaching temperatures close to the oysters' lethal limit accelerates if the rock heats up beyond ∼37°C, coinciding closely with the body temperature at which the oysters initiate metabolic depression. The increase in body temperature over a critical threshold serves as an early-warning cue to initiate anticipatory shifts in physiology and energy conservation before severe thermal stress occurs on the shore. Cross-correlating the onset of physiological mechanisms and temporal structures in environmental temperatures, therefore, reveals the potential role of reliable real-time environmental cues for future conditions in driving the evolution of anticipatory responses.

The terrestrial radiation of vertebrates required changes in skin that resolved the dual demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Using the amphibious killifish Kryptolebias marmoratus, we found that transcriptional regulation of skin morphogenesis was quickly activated upon air exposure (1h). Rapid regulation of cell-cell adhesion complexes and pathways that regulate stratum corneum formation was consistent with barrier function and mechanical reinforcement. Unique blood vessel architecture and regulation of angiogenesis likely supported cutaneous respiration. Differences in ionoregulatory transcripts and ionocyte morphology were correlated with differences in salinity acclimation and resilience to air exposure. Evolutionary analyses reinforced the adaptive importance of these mechanisms. We conclude that rapid plasticity of barrier, respiratory, and ionoregulatory functions in skin evolved to support K. marmoratus’ amphibious lifestyle; similar processes may have facilitated the terrestrial radiation of other contemporary and ancient fishes.

Populations at the edge of their species' distribution ranges are typically living at the physiological extreme of the environmental conditions they can tolerate. As a species' response to global change is likely to be largely determined by its physiological performance, subsequent changes in environmental conditions can profoundly influence populations at range edges, resulting in range extensions or retractions. To understand the differential physiological performance among populations at their distribution range edge and center, we measured levels of mRNA for heat shock protein 70 (hsp70) as an indicator of temperature sensitivity in two high-shore littorinid snails, Echinolittorina malaccana and E. radiata, between 1°N to 36°N along the NW Pacific coast. These Echinolittorina snails are extremely heat-tolerant and frequently experience environmental temperatures in excess of 55 °C when emersed. It was assumed that animals exhibiting high temperature sensitivity will synthesize higher levels of mRNA, which will thus lead to higher energetic costs for thermal defense. Populations showed significant geographic variation in temperature sensitivity along their range. Snails at the northern range edge of E. malaccana and southern range edge of E. radiata exhibited higher levels of hsp70 expression than individuals collected from populations at the center of their respective ranges. The high levels of hsp70 mRNA in populations at the edge of a species' distribution range may serve as an adaptive response to locally stressful thermal environments, suggesting populations at the edge of their distribution range are potentially more sensitive to future global warming.

Salmonid are farmed far offshore in a submersible cage in China, and air supplementation in the swim bladder is an important issue while the fish are confined in the submersed cage. The objective of this study is to test the effects of different types of air supplementation on the growth, energy budget, and fatty acid composition of the rainbow trout, Oncorhynchus mykiss, confined underwater. The fish were divided into four groups with four replicates (63.84 ± 2.03 g, 13 fish per tank): The control group fish (Ctrl) were cultured in a glass fiber tank with airstone; The mesh group (ME) used a net in a tank to keep the experimental fish from the water surface, and an airstone above the net; The airstone group (AM) added an airstone under the net; And the airdome group (DM) added an airdome under the net. At the end of a 35-day trial, the final mean weight (70.00 ± 1.63 g), percent weight gain (7.09 ± 0.96%) and survival rate (76.92 ± 0.00%) of the fish in ME group were significantly lower than those in Ctrl group (91.54 ± 5.19 g, 43.49 ± 10.35% and 100.00 ± 0.00%, respectively). The final mean weight of AM group was significantly lower than that of Ctrl group, whereas there were no significant differences in other growth indexes between control group, DM and AM group. The proportion of feeding energy allocated to growth in ME group was the lowest, the proportion in AM was significantly higher than ME group and lower than Ctrl group, and there was no significant difference between those in DM and Ctrl groups. The muscle saturated fatty acid level in Ctrl group was significantly lower than those in other groups. In conclusion, the growth of rainbow trout confined underwater for 35 days will slow down due to lower proportion of feeding energy allocated to growth and higher proportion to respiration. The addition of an airdome or airstone can partially compensate for the growth of the fish confined underwater long time. Rainbow trout confined underwater can partially compensate for the loss of buoyancy through increasing the muscle saturated fatty acid content.

Research on the interaction of primary producers and consumers is crucial for understanding trophic transfer in intertidal food webs. This study explores the association between epilithic and planktonic microalgae, and gut contents of two targeted intertidal gastropods, the periwinkle Echinolittorina radiata (splash zone) and the limpet Cellana toreuma (mid-intertidal zone). With the application of gut fluorescence technique and metabarcoding, this study investigates the quantity and composition of two different sources of microalgae (epilithic and planktonic) and the food ingested by the gastropods. The results suggest the following findings: 1) The planktonic microalgae have higher compositional similarity to the gut contents of grazing gastropods. 2) Increased gut pigment content in C. toreuma is observed with increasing abundance of epilithic and planktonic microalgae. However, there was no such pattern observed for E. radiata. This difference could be attributed to potentially divergent foraging behaviours of the two species that inhabit different shore heights.

The Yangtze (Changjiang) River enters the East China Sea with huge annual freshwater and sediment deposits. This outflow, known as the Changjiang Diluted Water (CDW), causes striking ecological gradients that potentially shape coastal species’ distribution and differentiation. The CDW has long been rendered as a marine biogeographic boundary separating cold-temperature and warm-water faunas, but it remains unclear whether and to what extent it acts as an intraspecific barrier. Here we synthesize published phylogeographic studies related to the CDW to address these issues. We find that the influence of the CDW on population differentiation is taxonomically variable, and even congeneric species may respond differently. In studies that claim the CDW is a phylogeographic barrier, the underlying assumptions explaining observed genetic breaks are sometimes incorrect, and some may have misinterpreted results due to conceptual confusion or insufficient geographical sampling. After excluding these studies, the remaining ones generally show shallow genetic divergence but significant population structure for coastal species across the CDW, suggesting that the CDW has not been a historically persistent barrier, but rather has acted as a filter within some species recently, probably after the last glacial maximum. This article is protected by copyright. All rights reserved.

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41–44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis–Menten constant of the cofactor NADH, KMNADH) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Understanding physiological adaptations of organisms to temperature changes that characterize their habitat is the first step in predicting the putative effects of global climate change on population dynamics. Mudflats are an important part of the intertidal zone and experience extreme and fluctuating temperatures. Therein, species would be potentially susceptible to global warming. The present study explored physiological adaptations of burrowing species to life in an intertidal mudflat by analyzing the potential operative temperatures in the mudflat, and assessing cardiac performance and the transcriptional response to thermal stress by a typical burrowing bivalve, the razor clam Sinonovacula constricta in different thermal environments, mimicking conditions during low tides. Clams showed higher thermal sublethal limits in mud with overlying air than in mud with overlying water, indicating an adaptation to rapidly changing ambient temperatures and thermal environments during emersion. This sublethal limit was far above the maximum operative temperature in the actual habitat site and suggests a potential buffer zone to ensure survival under unexpected high temperatures, that could occur with global warming. In response to high temperature, S. constricta exhibited the common heat stress response by up-regulating expression of the Bcl2-associated athanogene 3 (BAG3) and heat shock proteins to cope with the adverse effects of high temperature on protein homeostasis. Increased expression of key genes, including molybdenum cofactor synthesis 3 (MOCS3), oligoribonuclease (REX2), and NFκappaB inhibitor alpha (NFIA) may further remit the effect of thermal stress during the emersion period and delay a situation where clams reach their thermal sublethal limit, thereby helping to endure high temperature during low tide. These results clearly illustrate significant adaptations of a burrowing bivalve to life in intertidal mudflats at both physiological and molecular levels and can provide insights into potential physiological or evolutionary responses that could aid survival of mudflat species in a changing global climate.

The Devonian radiation of vertebrates from aquatic into terrestrial habitats required behavioral, physiological, and morphological adaptations. Changes to skin structure and function were likely crucial, but adaptations were needed to resolve contrasting demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Little is known of the mechanisms that underlie skin plasticity and adaptation between water and air. We performed experiments using two isogenic lineages of an amphibious killifish (Kryptolebias marmoratusfrom brackish and freshwater habitats) and used transcriptional and morphological data to reveal mechanisms recruited to resolve the dual challenges of skin providing both a barrier and an exchange interface during terrestrial acclimation. Transcriptional regulators of skin morphogenesis were quickly activated upon emersion. Regulation of cell-cell adhesion complexes, coupled with pathways homologous with those that regulate stratum corneum formation, was consistent with barrier function and mechanical reinforcement. Cutaneous respiration was associated with regulation of angiogenesis pathways and with blood vessel architecture that facilitated extremely short diffusion distances and direct delivery to ionocyotes. Evolutionary analyses revealed directional selection operating on proteins involved in barrier and respiratory functions, reinforcing the importance of these mechanisms for enabling the amphibious lifestyle ofK. marmoratus. Fish from brackish niches were more resilient to emersion and also differed from freshwater fish in ionoregulatory responses to emersion. We conclude that plasticity of barrier, respiratory, and ionoregulatory functions in skin evolved to support the amphibious lifestyle ofK. marmoratus; similar processes may have facilitated the terrestrial radiation of ancient fishes.Significance statementThe transition of vertebrate life from water to land coincided with solving multiple physiological challenges including avoiding drying out while also exchanging gases and ions with the environment. Though changes in the skin were likely important, little is known of the mechanisms that underlie skin flexibility and adaptation between water and air. We performed air exposure experiments with an amphibious killifish; gene expression profiling, microscopy, and evolutionary analysis of proteins revealed cell structures, proteins, and molecular pathways that support skin flexibility and adaptations during air exposure, and ion regulation contributed to differences in killifish abilities to adjust to air. Amphibious killifish are useful models to help us understand changes that enable water to air transitions in contemporary and ancient fishes.

Temperature stress is one of the major threats to the economic and ecological sustainability of marine fisheries and aquaculture. The abalone aquaculture in China has been in development for more than twenty years, while summer mortality for Pacific abalone ( Haliotis discus hannai ) is still the most serious issue for its aquaculture in southern China. Therefore, an effective and accurate measurement for evaluating the abalone's thermal tolerance is crucial for both scientific researchers and farmers. In this study, we evaluate the thermal tolerance of two abalone species: H. discus hannai (DD), H. gigantea (GG) and their offspring H. gigantea ♀ × H. discus hannai ♂ (GD) using Arrhenius break temperatures (ABT) of cardiac performance. These three groups possessed different thermal limits, as indicated by ABT, GD (32.50 ± 0.71 °C) > GG (31.30 ± 1.52 °C) > DD (30.00 ± 1.23 °C). The results were similar to further assessments of lethal temperature, Kaplan–Meier cumulative survival curves and critical thermal maximum. These results indicate that cardiac performance is an effective method to assess heat tolerance and can represent the different physiological statuses of abalones. Moreover, the results also suggested that global warming may have a profound influence on abalone aquaculture in China in the near future. Statement of relevance The results of the present study may facilitate in better understanding the difference on the thermal tolerance ability of H. discus hannai , H. gigantea and their offspring H. gigantea ♀ × H. discus hannai ♂ using Arrhenius break temperatures (ABT) of cardiac performance. The results were similar to further assessments of lethal temperature, Kaplan–Meier cumulative survival curves and critical thermal maximum. These findings indicate that cardiac performance is an effective method to assess heat tolerance and can represent the different physiological statuses of abalone. The ABT of cardiac performance could afford an efficient method to predict abalone thermal performance, which would be greatly beneficial in aquaculture and breeding projects. Moreover, it may have potentially broad application in the study of thermal tolerance of abalone and other commercially aquaculture species.

Thermal performance curves (TPCs), which quantify how an ectotherm's body temperature (T-b) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key - but often biologically unreasonable - assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced T-b. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with T-b, to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.

Atlantic salmon (Salmo salar) are migratory salmonids and high-end seafood, and are widely cultivated. Its growth and development process needs to experience from freshwater to seawater phases. Salinity, as a key environmental factor, affects metabolism of Atlantic salmon, which in turn greatly affects its volatile organic compound (VOC) content and taste. We used headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and electronic tongue techniques combined with multivariate statistical analysis to detect VOCs and determine changes in the overall taste of Atlantic salmon in five different growth stages (freshwater: 8 months old; seawater: 15, 18, 21, and 24 months old; M1, M2, M3, M4, and M5, respectively). In addition, the content of free amino acids (FAAs) that affect flavor was determined. The fingerprint of Atlantic salmon from HS-GC-IMS identified ten potential markers, including one and three biomarkers in the freshwater and seawater phases, respectively, and showed the potential markers in each growth stage. The electronic tongue effectively distinguished the overall taste of Atlantic salmon in the freshwater and seawater phases. The total FAA content in the freshwater phase was significantly lower than that in the seawater phase, and the umami FAA level in M3 was the highest. Therefore, flavor compounds can be used as potential markers for Atlantic salmon culturing in freshwater and seawater environments and for their different growth stages. Our research will provide reference data for further improving its aquaculture technology and studying the changes of physiological and biochemical indicators during migration of Atlantic salmon from freshwater to seawater.

Rainbow trout are a widely consumed fish species. However, the effects of fish size on flavor have not been fully elucidated. Here, the flavor qualities of the dorsal, tail, and abdomen of four sizes of rainbow trout (average weights 0.25, 1.14, 2.26, and 2.89 kg, respectively) were investigated by measuring fatty acids, nonvolatile compounds, and volatile compounds and by performing electronic tongue analysis. 5′-Nucleotide levels were higher in the dorsal and tail regions than in the abdomen of fish. Additionally, the heaviest fish had the highest contents of lactic acid and succinic acid, whereas fish weighing 1.14 kg had the highest levels of total free amino acids. Additionally, 0.25 and 1.14 kg fish had higher concentrations of aldehydes. t-Distributed stochastic neighborhood embedding and correlation analyses revealed that the overall flavor profiles were reflected by electronic-tongue analysis. Overall, our findings showed that fish size affected flavor in different parts of rainbow trout.

Temperature plays a major role in controlling species' distributions, and small-scale variation in the thermal environment are potentially an important factor that governs distributions on a local scale. For untangling the roles of behavioral and physiological adaptations on species' distribution at a small-scale level, we carried out a comparative study of two mudflat snails (genus Cerithidea) by determining these congeners' burying behavior, lethal temperature, cardiac performance and heat-shock protein (hsp70) gene expression. These two sympatric snails occupy different microhabitats on the upper intertidal mudflat. During periods of emersion, C. cingulata inhabits the open mudflat and C. largillierti usually aggregates around small rocks on the upper intertidal mudflat. Our results indicate that the two Cerithidea congeners show different behavioral and physiological responses to high temperature. Compared to C. largillierti, C. cingulata prefers to bury into the mud, has a higher thermal limit and a higher level of inducible expression of hsp70 mRNA, implying important roles of behavioral and physiological adaptations to the harsh thermal environment on the open mudflat. Furthermore, results of generalized additive modelling (GAM) analysis of cardiac performance and coefficient of variation (CV) of hsp70 mRNA expression showed high inter-individual variation in C. cingulata. These results highlight the importance of behavioral and physiological adaptions in sympatric species' distributions on the mudflat and help to shed light on the mechanisms of how small-scale differences in the thermal environment shape sympatric species’ distributions.

The theory for thermal acclimation of ectotherms suggests that (1) heat tolerance is traded-off for thermal acclimation in thermophilic species and that (2) plasticity is constrained in tropically-distributed ectotherms, which commonly experience relatively thermally-stable environments. We observed substantial heat tolerance plasticity in a test of this theory using tropical, thermophilic marine intertidal snails that inhabit extremely hot and highly-variable thermal environments. The implication of these results is that plasticity selection is largely driven by habitat temperature conditions irrespective of basal heat tolerance or latitude. However, heat tolerance of field-fresh snails was comparable with that of laboratory warm-acclimated snails, suggesting that snails in the field may often be unable to improve heat-hardening with further environmental warming. These findings suggest that field-referencing is crucial to using laboratory-measured acclimation capacity when inferring climate warming vulnerability in ectotherms, and overall they question how well current thermal biology theory predicts the outcomes of global change in intertidal environments.

High levels of biodiversity and phylogeographic structure in marine species in Southeast Asia are strongly linked to Quaternary sea-level fluctuations and complex oceanographic conditions. Cellana toreuma is a common limpet on intertidal rocky shores and is widely distributed in the Western Pacific. Analyses of partial mitochondrial COI gene sequences from Southeast Asia, combined with previously published sequences from East Asia and Indonesia, revealed the existence of five well-supported clades with high genetic divergences (between 1.4 and 7.6%), namely the East Asia clade, the eastern Southeast Asia clade, the western Southeast Asia clade, the Pelabuan Ratu (Java) clade and the Ogasawara clade. The geographical distribution of the five clades is likely related to the history of glaciations and rapid postglacial population expansions. Analyses of pairwise ΦST and hierarchical analysis of molecular variance shows significant population structure among collections in East and Southeast Asia. These results suggest that historical events have had strong effects on the phylogeographic structure of C. toreuma. In addition, present environmental factors, such as unsuitable habitats and ocean currents, have also affected the genetic footprints of past environments.

Summary For rocky intertidal species that experience changes in a number of potential stressors seasonally and during the tidal cycle, sensing cellular energy status and modulating it adaptively may be crucial for responding to stressor effects. However, the responses of energy metabolism of intertidal species to multiple sublethal stressors are still unclear. Here, we examined gene expression profiles of biomarkers related to sensing of cellular energy status and regulation of catabolism and energy expenditure in a mid-intertidal limpet Cellana toreuma for elucidating the species’ cellular energy responses stresses from high temperature, desiccation and rainfall. Expression levels of genes encoding metabolic regulators [two subunits of AMP-activated protein kinase, ampkα, ampkβ; Fu gene inhibition axis formation, axin; two sirtuins, NAD-dependent deacetylase sirtuin-1 (sirt1); NAD-dependent deacetylase sirtuin-5 (sirt5)], metabolic enzymes (hexokinase, hk; pyruvate kinase, pk; isocitrate dehydrogenase, idh) and heat shock protein 70 (hsp70) were quantified in specimens exposed to different temperatures and aerial/freshwater spray conditions. Based on the gene expression patterns, all individuals could be divided into three groups with divergent cellular energy status, indicating that the selected target genes are appropriate indicators of cellular metabolism. The divergent gene expression patterns indicated a sequence in which individuals from group 1, group 2 and group 3 were faced with increasing energy stress. The frequency distributions of individuals in the three groups were different among different time points and treatments, indicating that high temperature, desiccation, and rainfall, singly or in combination, could cause energy stress. Compared to the high percentage (100%) of individuals placed in the highest-stress group (group 3), after 2 h of freshwater spray at 18 °C, the lower percentage (77·8%) of individuals in group 3 after 2 h of freshwater spray at 30 °C indicated the existence of interactive effects of high temperature and rain; high temperature resulted in a lower response of cellular energy metabolism to rainfall. Sublethal environmental stresses from single stressors such as temperature or osmotic challenges can lead to cellular energy stress. Interactions among stressors may lead to a complex overall effect on cellular energy status in intertidal species.

Warming of the world’s oceans is predicted to have many negative effects on organisms as they have optimal thermal windows. In coastal waters, however, both temperatures and pCO2 (pH) exhibit diel variations, and biological performances are likely to be modulated by physical and chemical environmental changes. To understand how coastal zooplankton respond to the combined impacts of heat shock and increased pCO2, the benthic copepod Tigriopus japonicus were treated at temperatures of 24, 28, 32 and 36 °C to simulate natural coastal temperatures experienced in warming events, when acclimated in the short term to either ambient (LC, 390 μatm) or future CO2 (HC, 1000 μatm). HC and heat shock did not induce any mortality of T. japonicus, though respiration increased up to 32 °C before being depressed at 36 °C. Feeding rate peaked at 28 °C but did not differ between CO2 treatments. Expression of heat shock proteins (hsps mRNA) was positively related to temperature, with no significant differences between the CO2 concentrations. Nauplii production was not affected across all treatments. Our results demonstrate that T. japonicus responds more sensitively to heat shocks rather than to seawater acidification; however, ocean acidification may synergistically act with ocean warming to mediate the energy allocation of copepods.

Potential future distribution shifts of intertidal invertebrates along the Asian coast in the face of global change remain unclear. Integrative study that comprise environmental temperature monitoring and comparative physiological study of thermal adaptation among different geographical populations of species are important to identify population-related differences in thermal ecology that could affect the persistence of species in their present distribution ranges. In the present study, in situ operative temperatures were recorded continuously for 5 mo from August 2011 to January 2012, and measurements of lethal temperature, cardiac performance and gene expression were carried out in 3 geographical populations of the limpet Cellana toreuma (tropical shore: Sichang Island, Thailand; subtropical shore: Nanding Island, Zhangzhou, China; temperate shore: Dagong Island, Qingdao, China). Compared to limpets on the tropical shore in Sichang Island with high mean temperature (28.28°C) and narrow thermal range (19.13 to 46.56°C), limpets on the subtropical shore in Nanding Island have to cope with a thermal environment characterized by a wider temperature range (6.42 to 44.98°C). The absence of differences in lethal temperature (LT50) of limpets from all the locations indicates that limpets currently suffer from intensive heat stress across their biogeographic range. Although the populations lacked differences in upper thermal limit, variations were noted among populations in transcriptional responses in genes linked to energy metabolism. Limpets on the subtropical shore in Nanding Island live closer to their upper thermal limits and thus will be very sensitive to future temperature increases.

The littorinid gastropod Littoraria flammea has been widely quoted as one of only three or four marine molluscs believed to have become extinct in historic times. It is easily recognized by its delicate, elongate, striped shell. We review the literature and available museum material to show that its only recorded locality was Shanghai and that it has not been collected since about 1855. We report the rediscovery of this species in salt marshes near Shanghai, where the habitat is under threat from invasive Spartina. For the first time we describe the penis, pallial oviduct and radula. These are the same as those of L. melanostoma, previously recorded only from Fujian province and southwards, where its shell is different from that of L. flammea. We now report a population of L. aff. melanostoma from between Fujian and Shanghai, with shells of intermediate form and colour. Analyses of sequences of COI, 12S and 28S genes do not support separation of these two Littoraria species, in terms of either reciprocal monophyly or genetic distance. Present evidence suggests that L. flammea could be only a distinctive morphological form or extreme of a cline, at the northern limit of the range of L. melanostoma.

The physiological performance of a mid-intertidal limpet Cellana toreuma was determined to study the physiological adaptation of intertidal animals to rapid changes and extreme temperatures during emersion. The relationship between the Arrhenius breakpoint temperature (ABT) and in situ operative body temperature was studied to predict the possible impact of climate change on the species. The temperature coefficient (Q10) of emersed animals was higher than that of submersed animals and the ratio of aerial: aquatic heart rate rose with increasing temperature. The ABTs of submersed and emersed animals were 30.2 and 34.2°C, respectively. The heart rate and levels of molecular biomarkers (hsps, ampkα, ampkβ and sirt1 mRNA) were determined in 48 h simulated semi-diurnal tides. There were no obvious changes of heart rate and gene expression during the transition between emersion and submersion at room temperature, although expressions of hsp70 and hsp90 were induced significantly after thermal stress. These results indicate that C. toreuma can effectively utilize atmospheric oxygen, and the higher Q10 and ABT of emersed animals are adaptations to the rapid change and extreme thermal stress during emersion. However, the in situ operative body temperature frequently exceeds the aerial ABT of C. toreuma, indicating the occurrence of large-scale mortality of C. toreuma in summer, and this species should be sensitive to increasing temperature in the scenario of climate change.

The different life-history stages of organisms may have divergent thermal limits and thus respond differently to global warming. For comprehensive understanding of the ecological impacts of global warming, it is crucial to determine thermal limits among different life stages and to understand how close an organism lives to its thermal limits at different life stages. Here, we measured the effect of temperature on hatching and the lethal temperature (LT50, lethal temperature of 50%) of larvae to explore effects of high temperature on the encapsulated and planktonic stages of the pulmonate limpet Siphonaria japonica. We measured cardiac performance to determine adult limpets’ thermal limit. At the encapsulated stage, heat shock at 34.5 °C affected larval hatching significantly. Pelagic larvae showed a high LT50 (45.03 °C), indicating a high thermal tolerance for the pelagic larval stage. For the adults, the Arrhenius break temperature and flatline temperature were 38.49 ± 1.27 and 44.27 ± 1.08 °C, respectively. The integrated study of environmental temperatures and thermal sensitivities at different life stages of S. japonica indicates that the egg ribbon and adult stages are potentially most sensitive to the future temperature increases on subtropical shores.

Cardiac activity (Arrhenius breakpoint temperatures and Q10 relationships) and heat shock response (hsp70 expression) were measured in the congeneric limpets Patella rustica, P. caerulea and P. ulyssiponensis in order to test the relationship between their vertical distribution and physiological thermal tolerance. These species exhibit different vertical distributions along Mediterranean shores and despite the narrow tidal range in the Mediterranean, they experience different environmental conditions and consequently had specific thermal windows. Cardiac activity of the upper zoned P. rustica was maintained at higher temperatures than its mid- or low shore counterparts, P. caerulea and P. ulyssiponensis. P. rustica had the highest Arrhenius breakpoint temperature (37.9 ± 2.1 °C, mean ± SD), followed by P. caerulea (35.9 ± 2.6 °C), and finally the low-zoned P. ulyssiponensis (32.2 ± 2.3 °C). The same pattern was found for Q10 relationships. Expression of hsp70 increased at 34 °C and kept increasing with temperature in P. rustica. In P. caerulea, expression reached a maximum at 36 °C and decreased at 38 °C, suggesting that hsp70 expression in P. rustica provides a more efficient defence against thermal stress than in P. caerulea. As summer environmental temperatures in the Mediterranean regularly reach 35 to 38 °C, performances of these limpets suggest that they are already living at the edges of their thermal window, and further temperature changes may have large-scale consequences for these keystone species.

To identify the Nipponacmea limpets along the coast of China, their taxonomy was investigated with three molecular markers (one mitochondrial gene, COI; two nuclear markers, 28S rDNA and H3). Three species (N. radula, N. fuscoviridis and N. nigrans) were found among 274 individuals collected from 14 sites. Intraspecific variation was far less than interspecific variation and obvious barcoding gaps existed. These results indicate that the three Nipponacmea species can be efficiently identified by DNA barcoding. The phylogeographic patterns of the three species were also analysed using COI sequences. There was clear biogeographic separation between the northern N. radula and the southern two species (N. fuscoviridis and N. nigrans), with the Yangtze River estuary as a barrier. In the southern N. fuscoviridis, there was a star-shaped haplotype network and the dominant haplotype was detected in all populations. In the northern N. radula, there were five main haplotypes; some adjacent populations showed no significant difference according to the pairwise Fst values. The southern N. nigrans showed two main haplotypes. The phylogeographic break between the Nipponacmea species is possibly due to the local and regional hydrographic conditions in the Yangtze River estuary, the large salt marsh in the river delta and difference of temperature between northern and southern China.

Intertidal invertebrates develop effective physiological adaptations to cope with the rapidly changing thermal environment in the intertidal zone. In the present study, the temporal patterns of heart rate, protein carbonyl groups, and genes encoding heat shock proteins (hsp70 and hsp90) and metabolic sensors (ampkα, ampkβ and sirt1) were measured to study the effect of sublethal heat stress on the cardiac function, oxidative stress, heat shock response and cellular metabolism of an intertidal limpet Cellana toreuma. All the physiological parameters are sensitive to temperature and duration of heat stress. Spearman correlation analysis revealed that the correlations between heart rate and levels of heat shock proteins mRNA and metabolic sensors mRNA were statistically significant. These results further suggest that cardiac function plays crucial roles in cellular energy metabolism and heat shock responses. The significant increase of protein carbonyl groups at 34°C after 4h exposure indicated that the failure of cardiac function and the increase of anaerobic metabolism partly leads to the increase of protein carbonyl groups. Generally, the physiological responses to heat stress are sensitive to temperature and are energy-consumptive, as indicated by the upregulation of metabolic sensors mRNA. However, the upregulation of heat shock proteins and metabolic sensors at the post-transcriptional level and related functions need to be confirmed in further experiments.

The combined effects of acute temperature and salinity on osmolality, expressions of heat shock proteins mRNA (hsp70, hsp90a and hsp90b) and superoxide dismutase mRNA (sod) were investigated in the sea cucumber Apostichopus japonicus Selenka. There were 12 treatments (combinations of temperature at 16, 20, 24 and 28 °C and salinity at 22, 27 and 32 ppt). In low salinity environments, the osmolality of the sea cucumber’s coelomic fluid decreased immediately and reached osmotic balance within 6 h. The decline of osmolality after 2 h of hypo-osmotic stress was faster at high temperatures (28 °C) than that at low temperatures (16 and 20 °C). Cellular level stress was indicated by up-regulation of hsp70, hsp90s and sod mRNA, and the maximal expression of all genes occurred at 6 h after stresses. The up-regulation of hsps and sod mRNA indicated the emergence of protein denaturation and oxidative damage and also suggested an increase in energy consumption at high temperature and low salinity. These results indicated that high temperature and low salinity could change biochemical pathways and energy budgets and then potentially impair the osmoregulation of the sea cucumber. Therefore, effective ways should be taken (e.g., draining off the upper freshwater, exchanging water and adding man-made sea water) to prevent the damage to sea cucumber culture caused by low salinity induced by rainstorms, especially at high temperature.

This study deals with the mortality and related physiological responses of aestivating sea cucumber Apostichopus japonicus to acute salinity decrease. Aestivating and active sea cucumbers were exposed to a decrease in salinity (from 30 to 20 psu) at a rate of 2.5 psu every 6 h, and then maintained at 20 psu for 96 h. The mortality of aestivating sea cucumbers was ~30%, which was significantly higher than that of active sea cucumbers (~10%). This result indicated that sea cucumbers in aestivation were more susceptible to hypo-salinity stress. To elucidate the underlying physiological mechanisms, the osmotic pressure in coelomic fluid and the levels of hsp70 and hsp90 mRNA in aestivating and active sea cucumbers were measured. No significant difference in osmoregulation was observed between the two groups. The osmotic pressure of coelomic fluid in both groups changed with decrease in ambient salinity. There were significant differences in the time course and magnitude of hsp70 and hsp90 expression between the two groups. After exposure to decreased salinity, aestivating sea cucumbers showed a delayed up-regulation of hsp70 and hsp90 expression compared with animals in active state, and these levels decreased rapidly to control values. The expression of hsp70 and hsp90 in aestivating sea cucumbers were significantly lower than those in active sea cucumbers after salinity change. The differences in hsp70 and hsp90 expression between the states may partly explain the higher mortality of sea cucumbers in aestivation when exposed to low salinity.

Natural Science Funds for Distinguished Young Scientists of Shandong Province [JQ201009]; Great Project of Scientific and Technical Supporting Programs [2011BAD13B03]; National Natural Science Foundation of China [30771661]; Public Science and Technology Research Funds Projects of Ocean, State Oceanic Administration of the People's Republic of China [200905020]; Program for New Century Excellent Talents in University [NCET-08-0503]; 111 Project, China Ministry of Education [B08049]

Chinese National Science Foundation [30400333]; National Key Technologies RD Reward Research Foundation for Talented Young and Middle Aged Scientists of Shandong, China [BS2009NY019]

Chinese National Science Foundation [30400333]; National Key Technologies RD Reward Research Foundation for Talented Young and Middle Aged Scientists of Shandong, China [BS2009NY019]

The effects of heat-shock during pelagic stages on growth and survival of juvenile sea cucumber Apostichopus japonicus were investigated to test the possibility to acquire high thermal tolerance individuals after the heat-shock selection. Larvae at the stages of blastula, gastrula, and auricularia were heat-shocked at selected temperatures (21.5, control; 26, 28, and 30°C) for 45 min and returned to 21.5°C for continuous rearing. There was a significant difference in thermal sensitivity among different developmental stages, and thermal tolerance of the larvae was correlated with the expression of heat shock protein 70 (Hsp70). Juveniles after the heat-shock selection at pelagic stages showed higher induced thermotolerance than those without heat-shock. Therefore, heat-shock application at early pelagic development stages is potentially a useful way to select high-thermotolerance variety of the sea cucumbers.

Understanding variation in physiological adaptations to thermal stress is vital when investigating intertidal species’ distribution patterns. The thermal sensitivities of two limpets, Cellana grata and C. toreuma, differed in accordance with their vertical distributions. Cardiac performance was maintained at higher temperatures (~47°C) for the high-zone C. grata than the mid-zone C. toreuma (~42°C). At 40°C, C. grata maintained regular heart function for ~4 h, while heart function of C. toreuma decreased rapidly. Heat shock protein expression revealed that C. toreuma had two constitutive isoforms, Hsp77 and Hsp72, and C. grata one inducible form, Hsp75, which was upregulated at 40°C, suggesting C. grata has a more effective heat shock response than C. toreuma. The temperature-adaptive differences in cardiac thermal tolerance and Hsp expression match observed differences in thermally induced mortalities with the onset of summer and may help predict differential effects of climate change on the two congeners.

Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates (Vo2) and biochemical compositions under laboratory conditions of low (3°C), normal (17°C) and high (24°C) temperature. The results show that the characteristics of A. japonicus in dormancy (hibernation and estivation) states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates (128.90±11.70 μg/g h) of estivating sea cucumbers were notably higher (107.85±6.31 μg/g h) than in starving sea cucumbers at 17°C, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.

We studied thermal tolerance limits, heat-hardening, and Hsp70 to elucidate the difference in thermotolerance between two color variants of the sea cucumber Apostichopus japonicus. Green and Red variants occupy different habitats and have different aestivation responses to high temperature in summer. In the absence of heat-hardening, the variants showed no difference in the temperature at which 50% of the individuals died: Green, 31.49 degrees C; Red, 31.39 degrees C. However, Green specimens acquired higher thermotolerance than Red after a prior sublethal heat exposure. After 72 h of recovery from a heat-hardening treatment (30 degrees C for 2 h), the survival of Green variants was more than 50% and that of Red was less than 5% when they were treated at 33 degrees C for 2 h. Levels of mRNA and protein for Hsp70 were significantly higher in Green than Red after the heat shock of 30 degrees C, and the stability of hsp70 mRNA of Green was significantly higher than that of Red. Our findings suggest that within the same species, different variants that have similar thermal limits in the absence of heat-hardening can acquire different thermotolerances after a prior sublethal heat shock. The difference in induced thermotolerance between Green and Red is closely related to the expression pattern of Hsp70, which was partly governed by the stability of hsp70 mRNA.

The purpose of this study was to investigate the growth and physiological status of Litopenaeus vannamei subjected to one constant temperature (25°C) and four cyclical temperature change regimes (25 ± 1°C, 25 ± 2°C, 25 ± 3°C and 25 ± 4°C). The growth rates of shrimp at 25 ± 2°C or 25 ± 3°C were significantly higher than that at a constant temperature of 25°C. On the other hand, the growth rate in 25 ± 4°C regime was significantly lower than those in other regimes. The daily feed intake rate of shrimp at 25 ± 4°C was the lowest, and the food conversion efficiency was also significantly lower than those at 25 ± 2°C and 25 ± 3°C, respectively. The food conversion efficiency at 25 ± 2°C or 25 ± 3°C was significantly higher than those in other regimes. Thus, it can be inferred that the growth enhancement in the test shrimp at the suitable diel fluctuating temperatures was due to high food conversion efficiency. Studies of the physiological parameters showed that at 25 ± 4°C, the hemolymph glucose content of the test shrimp was the lowest, while the activity of PK in hepatopancreas was the highest, which indicated that the test shrimp at 25 ± 4°C was in a stressed condition. The hemolymph glucose content of the test shrimp at 25 ± 3°C was the highest, and the activity of HK in hepatopancreas was the lowest. These results indicated that the test shrimps at 25 ± 3°C were not in a stressed condition. Compared with the constant temperature regime, the expression of HSP70 in any of the four cyclical temperature change regimes was not significantly increased. The reason for this might be that the fluctuation amplitude of ± 4°C did not induce the increased expression of HSP70.

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.

Thermal history can affect organisms' thermal resistance, however, effects of thermal history on thermal resistance and roles of heat shock protein 70 (Hsp70) in sea cucumber Apostichopus japonicus are not well understood. In the present study, the lethal thermal limits and expressions of Hsp70 of sea cucumbers that were acclimated at different temperatures (22 °C or 12 °C) for 30 days were studied. The upper lethal temperatures (ULTs 50 ) for sea cucumbers acclimated at 22 and 12 °C were 31.8 °C (31.5–32.1 °C) and 30.9 °C (30.6–31.3 °C), respectively. In heat shock experiments, sea cucumbers acclimated at different temperatures (22 °C or 12 °C) were exposed to several high temperatures (25, 27, 29, 31 and 32 °C) for 2 h, followed by 2 h recovery at 17 °C, and the levels of hsp70 were measured using semi-quantitative RT-PCR and western blotting. The maximum induction temperatures for sea cucumbers in 22 °C and 12 °C acclimation treatments were 31 °C and 29 °C, respectively. These results showed that the temperature acclimation could affect sea cucumbers' upper thermal limit, which was related to the changed expression pattern of Hsp70 in the sea cucumber. In practice, sea cucumbers that overwinter in southern China could acquire higher thermal resistance than those reared in the north.

Optimal stocking densities were investigated for the sea cucumber Apostichopus japonicus Selenka under feed-supplement and non-feed-supplement regimes in net enclosures for 333 d. Substantial weight loss occurred during the aestivation phase (AE). Decreased growth rates were also observed during the winter phase (WT). In contrast, sea cucumbers showed rapid growth during the spring (SP) and autumn (AU) phases. Feeding regimes considerably influenced the growth performance, i.e., sea cucumbers grew faster under feed-supplement regime than under non-feed-supplement regime (P < 0.05). The average survival rates of sea cucumbers under feed-supplement regime were higher than those under non-feed-supplement regime for both the autumn phase and spring phase, but the differences were only significant for the latter phase (P < 0.05). The fitted B-N curves showed that the optimal stocking densities, in terms of net production, were 22.3 ind. m-2 for feed-supplement regime and 14.1 ind. m-2 for non-feed-supplement regime.

SUMMARY We characterized functional and structural properties of cytoplasmic malate dehydrogenases (cMDHs) from six limpets of the genus Lottia that have different vertical and latitudinal distributions. Particular attention was given to the cryptic species pair Lottia digitalis (northern occurring) and L. austrodigitalis (southern occurring) because of recent contraction in the southern range of L. digitalis and a northward range extension of L. austrodigitalis. As an index of adaptation of function, we measured the effects of temperature on the apparent Michaelis–Menten constant (Km) of the cofactor NADH(KmNADH). KmNADHvalues of cMDHs from the mid- to high-intertidal, low-latitude species L. scabra and L. gigantea were less sensitive to high temperature than those of cMDHs from the low- and mid-intertidal, high-latitude species L. scutum and L. pelta. cMDH of L. digitalis was more sensitive to high temperatures than the cMDH ortholog of L. austrodigitalis. Thermal stability (rate of loss of activity at 42.5°C) showed a similar pattern of interspecific variation. Comparison of the deduced amino acid sequences showed that interspecific differences ranged from one to as many as 17 residues. Differences in KmNADH and thermal stability between orthologs of L. digitalis and L. austrodigitalis result from a single amino acid substitution. At position 291, the glycine residue in cMDH of L. digitalis is replaced by a serine in cMDH of L. austrodigitalis, a change that favors additional hydrogen bonding and reduced conformational entropy. This difference between closely related congeners demonstrates the role of minor alterations in protein sequence in temperature adaptation and suggests that such variation is important in governing shifts in biogeographic range in response to climate change.

article i nfo Aestivation is an adaptation of the sea cucumber Apostichopus japonicus Selenka to high temperature, however, the causations and physiological responses of aestivation are not well understood. This study deals with the relationship between temperature and aestivation. Sea cucumbers were allocated into four treatments. In two treatments of temperature elevation, the ambient temperature gradually was increased from 16 °C to 26 °C linearly (treatment FA) or by a fluctuating temperature profile (treatment FB). Two control treatments maintained constant temperatures of 16 °C and 26 °C, and were designated as optimum temperature of growth and threshold of aestivation, respectively. During the 40-day experiment, body weight, oxygen consumption, daily food intake, catalase (CAT) and superoxide dimutase (SOD) activities and heat shock protein 70 (Hsp70) levels were determined periodically. When the temperature gradually increased from 16 °C to 26 °C, the body weight of the tested sea cucumbers decreased gradually. After the ambient temperature reached 26 °C, the tested sea cucumbers in treatments of FA and FB were reared at 26 °C for an additional twenty days. During this period, symptoms of aestivation appeared in the tested sea cucumbers. Activities of antioxidases and Hsp70 levels increased when the ambient temperature increased from 16 °C to 26 °C, and decreased when the temperature was kept at 26 °C. These results indicate that aestivation in A. japonicus is an adaptive strategy to reduce the production of reactive oxygen species (ROS) and denatured proteins which were induced at high temperature.

The effects of four modes of diel temperature-fluctuation with two designated fluctuating temperatures (15 ± 3°C and 18 ± 3°C) on the growth and energy budget of young sea cucumber, Apostichopus japonicus Selenka, were studied to develop a highly efficient temperature-control scheme for aquaculture of the species. Sea cucumbers with a mean wet body weight of 8.0 ± 1.2 g (mean ± SD) were allocated to each treatment randomly with five replicates. After a 38-day trial, specific growth rate (SGR) and food conversion efficiency (FCE) decreased with increasing temperature in constant-temperature treatments. Among the four modes of temperature fluctuation, SGR of sea cucumbers reared under a mode which simulated the natural fluctuation of the temperature (mode C) of seawater was significantly higher than that of sea cucumbers reared at the corresponding constant temperatures. This enhancement of growth rate by use of mode C was attributed to higher FCE and lower energy allocated to respiration and feces. In large-scale culture, a temperature-control mode designed based on mode C could enhance not only growth but also efficiency of food utilization by the young sea cucumber.