Linking biogeography to physiology: Evolutionary and acclimatory adjustments of thermal limits

Temperature-adaptive physiological variation plays important roles in latitudinal biogeographic patterning and in setting vertical distributions along subtidal-to-intertidal gradients in coastal marine ecosystems. Comparisons of congeneric marine invertebrates reveal that the most warm-adapted species may live closer to their thermal tolerance limits and have lower abilities to increase heat tolerance through acclimation than more cold-adapted species. In crabs and snails, heart function may be of critical importance in establishing thermal tolerance limits. Temperature-mediated shifts in gene expression may be critical in thermal acclimation. Transcriptional changes, monitored using cDNA microarrays, have been shown to differ between steady-state thermal acclimation and diurnal temperature cycling in a eurythermal teleost fish (Austrofundulus limnaeus). In stenothermal Antarctic notothenioid fish, losses in capacity for temperature-mediated gene expression, including the absence of a heat-shock response, may reduce the abilities of these species to acclimate to increased temperatures. Differences among species in thermal tolerance limits and in the capacities to adjust these limits may determine how organisms are affected by climate change

Temperature adaptation of cytosolic malate dehydrogenases of limpets (genus Lottia): differences in stability and function due to minor changes in sequence correlate with biogeographic and vertical distributions

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 (K(m)) of the cofactor NADH (K(m)(NADH)). K(m)(NADH) values 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 degrees 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 K(m)(NADH) 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

Heat-Shock Protein 70 (Hsp70) Expression in Four Limpets of the Genus \u3cem\u3eLottia\u3c/em\u3e: Interspecific Variation in Constitutive and Inducible Synthesis Correlates With \u3cem\u3ein situ\u3c/em\u3e 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 (≈14 °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 °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

Thermal adaptation of enzymes

董云伟团队在潮间带生物生化适应机制的研究过程中，结合分子动力学模拟和实验调控手段，发现极端高温下，耐热滨螺能够通过增强代谢关键酶的作用，避免蛋白质的解链，保持微结构完整和功能维持；通过对原位体温跨度达60℃的12种软体动物的研究，定量了cMDH结构柔性的温度适应性变化程度，揭示了氨基酸温度适应性进化的关键位点，阐述了蛋白质结构稳定性与生物地理分布的内在联系。将海洋软体动物生化适应研究从单一的定性实验，拓展到了基于计算生物学的定量研究，揭示了海洋软体动物细胞质苹果酸脱氢酶（cMDH）结构稳定性和功能适应性的趋同进化模式，建立了基于代谢关键酶的“酶促动力学—蛋白合成—模拟计算”的生化适应机制的创新性研究模式。基于上述基础，课题组进一步拓展研究的深度与广度，比较分析了从南极洲半致死温度仅为4℃的扇贝，到中国沿海可耐受60℃以上高温的滨螺等26种海洋软体动物 cMDH 的温度耐受性，提出了蛋白质不同区域氨基酸的温度适应性变化模式，通过分子动力学分析揭示了具有重要功能的区域及其作用机制。这一系列研究成果加深了对海洋生物蛋白质温度适应机制的认识，为该领域提供了新的研究模式与思路，对于查明环境温度对生物分布的影响及其机制，预测气候变暖的生态学效应具有重要意义。董云伟团队致力于潮间带生态学研究，重点研究潮间带生物对复杂环境条件的响应特征和时空规律，及其适应机制。近年来研究主要集中在潮间带生物生化适应机制、生理调节策略及地理格局变化等方面。【Abstract】Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9℃ (Antarctica) to～55℃ (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some, but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally, but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.This work was supported by National Natural Science Foundation of China Grants 41776135 and 41476115 (to Y.-w.D.) and Nature Science Funds for Distin-guished Young Scholars of Fujian Province, China Grant 2017J07003 (to Y.-w.D.). Work in the collection of the Antarctic specimens was supported by National Science Foundation Grant 1245703 (to Donal Manahan, co-PI).该成果得到国家自然科学基金项目（项目编号：41776135、41476115）和福建省杰出青年基金（2017J07003）资助

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 (approximate to 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

滨螺卓越的耐高温能力，温度耐受上限竟高达55℃——潮间带 Echinolittorina 属滨螺细胞质苹果酸脱氢酶高温耐受机制的研究

研究团队以分布于高潮间带，具有高耐热性的两种滨螺为研究对象，通过生理生化学实验及计算机模拟手段，发现耐热的软体动物通过增强代谢关键酶的作用，避免了在高温条件下的解链；研究团队还提出，局部柔性的增长使得酶在低温条件下能够发挥其催化功能，同时能在极端高温条件下，起到稳定蛋白质的作用，确保了蛋白质功能的维持。这些发现对于查明环境温度对生物分布的影响及其机制，预测气候变暖的生态学效应将具有重要意义。 滨螺广泛分布于潮间带高潮区，是潮间带垂直分布区划分的标志性物种。塔结节滨螺（Echinolittorina malaccana）和粒结节滨螺（E. radiata）是我国岩相潮间带高潮区常见物种，其温度耐受上限高达 55-60°C。董云伟教授团队与美国斯坦福大学 George Somero 教授，以及华侨大学张光亚教授团队合作，进行了一系列研究，探索了高热耐受性滨螺在极端高温条件下，仍旧保持体内蛋白质微结构的完整、功能的维持的奥妙所在。【Abstract】Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41–44℃ in summer and withstand temperatures up to at least 55℃. 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.This research was substantially supported by grants from National Natural Science Foundation of China (41476115), Program for New Century Excellent Talents of Ministry of Education, China, Nature Science Foundation for Distinguished Young Scholars of Fujian Province (2017J07003), China and the State Key Laboratory of Marine Environmental Science Internal Program, Xiamen University (MELRI1501)

New Frontiers for Organismal Biology

Understanding how complex organisms function as integrated units that constantly interact with their environment is a long-standing challenge in biology. To address this challenge, organismal biology reveals general organizing principles of physiological systems and behavior—in particular, in complex multicellular animals. Organismal biology also focuses on the role of individual variability in the evolutionary maintenance of diversity. To broadly advance these frontiers, cross-compatibility of experimental designs, methodological approaches, and data interpretation pipelines represents a key prerequisite. It is now possible to rapidly and systematically analyze complete genomes to elucidate genetic variation associated with traits and conditions that define individuals, populations, and species. However, genetic variation alone does not explain the varied individual physiology and behavior of complex organisms. We propose that such emergent properties of complex organisms can best be explained through a renewed emphasis on the context and life-history dependence of individual phenotypes to complement genetic data.Organismic and Evolutionary Biolog

Time Course and Magnitude of Synthesis of Heat-Shock Proteins in Congeneric Marine Snails (Genus \u3cem\u3eTegula\u3c/em\u3e) from Different Tidal Heights

The time course and magnitude of the heat-shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30°C for 2.5 h, followed by 50 h recovery at 13°C) that induced the heat-shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S-labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat-shock proteins (hsp’s) of size classes 90, 77, 70, and 38 kDa. In the low- to mid-intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30°C during emersion, synthesis of hsp’s commenced immediately after heat stress, reached maximal levels 1–3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low-intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30°C represents a near lethal heat stress, synthesis of hsp’s commenced 2–14 h after heat stress; reached maximal levels after 15–30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30°C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress