Living on the Edge: Physiological and Kinetic Trade-Offs Shape Thermal Tolerance in Intertidal Crabs From Tropical to Sub-Antarctic South America.

Temperature is an important abiotic factor that drives the evolution of ectotherms owing to its pervasive effects at all levels of organization. Although a species' thermal tolerance is environmentally driven within a spatial cline, it may be constrained over time due to differential phylogenetic inheritance. At the limits of thermal tolerance, hemolymph oxygen is reduced and lactate formation is increased due to mismatch between oxygen supply and demand; imbalance between enzyme flexibility/stability also impairs the ability to generate energy. Here, we characterized the effects of lower (LL50) and upper (UL50) critical thermal limits on selected descriptors of aerobic and anaerobic metabolism in 12 intertidal crab species distributed from northern Brazil (≈7.8°S) to southern Patagonia (≈53.2°S), considering their phylogeny. We tested for (i) functional trade-offs regarding aerobic and anaerobic metabolism and LDH kinetics in shaping thermal tolerance; (ii) influence of shared ancestry and thermal province on metabolic evolution; and (iii) presence of evolutionary convergences and adaptive peaks in the crab phylogeny. The tropical and subtropical species showed similar systemic and kinetic responses, both differing from the sub-Antarctic crabs. The lower UL50's of the sub-Antarctic crabs may reflect mismatch between the evolution of aerobic and anaerobic metabolism since these crabs exhibit lower oxygen consumption but higher lactate formation than tropical and subtropical species also at their respective UL50's. LDH activity increased with temperature increase, while Km Pyr remained fairly constant; catalytic coefficient correlated negatively with thermal niche. Thermal tolerance may rely on a putative evolutionary trade-off between aerobic and anaerobic metabolism regarding energy supply, while temperature compensation of kinetic performance is driven by thermal habitat as revealed by the LDH affinity/efficiency equilibrium. The overall physiological evolution revealed two homoplastic adaptive peaks in the sub-Antarctic crabs with a further shift in the tropical/subtropical clade. The physiological traits at UL50 have evolved in a phylogenetic manner while all others were more plastic. Thus, shared inheritance and thermal environment have driven the crabs' thermal tolerance and metabolic evolution, revealing physiological transformations that have arisen in both colder and warmer climes, especially at higher levels of biological organization and phylogenetic diversity.

Microplastics and copper effects on the neotropical teleost Prochilodus lineatus: Is there any interaction?

Microplastics (MP) are emerging contaminants widely found in aquatic ecosystems. In addition to MP toxicity itself, there is increasing concern about the MP adsorption capacity and the interactive effects with other contaminants, such as copper. The objective of this research was to investigate the effects of polyethylene microplastic and its association with copper (Cu) in genotoxic, biochemical, and physiological biomarkers of the neotropical teleost Prochilodus lineatus. Fish were exposed for 24 and 96 h to MP (20 µg L-1) and Cu (10 µg L-1) and MP + Cu. The results showed that MP and Cu, both isolated and in combination, promoted DNA damage in erythrocytes (96 h) and liver cells (24 and 96 h) indicating that MP and Cu are genotoxic. Fish exposed only to Cu (96 h) showed a decrease in lipid peroxidation in the liver despite of the decrease in glutathione content, indicating the efficiency of other antioxidant defenses. Brain acetylcholinesterase was inhibited in the animals from all the treatments. Although MP did not influence on Cu accumulation in tissues, decreased plasma Na+ and Ca2+ (24 h) occurred after the exposure to MP and Cu, isolated and combined. Exposure to MP and MP + Cu resulted in decreased activity of Ca2+-ATPase (24 h). Taken altogether, these results showed that MP and Cu depicted genotoxic, neurotoxic, and physiological effects on P. lineatus, both alone and combined. An interaction between Cu and MP was observed in plasma Ca2+, where the combination of both contaminants caused a greater effect than the contaminants alone.

Life-time exposure to waterborne copper III: Effects on the energy metabolism of the killifish Poecilia vivipara.

Copper ions (Cu) are essential to life maintenance, nonetheless, elevated concentrations can be hazardous. Acute and sub-chronic toxic effects of this metal are well known and are usually related to enzymatic inhibition, elevated ROS production and dysfunction of energy metabolism. Despite that, chronic studies are extremely rare. Therefore, the aim of this study was to assess the effects of chronic exposure to 5, 9 and 20 µg/L Cu (28 ad 345 days) on the energy metabolism and survival of the killifish Poecilia vivipara. To accomplish that, we evaluated the activity of enzymes related to aerobic (pyruvate kinase (PK); citrate synthase (CS)) and anaerobic metabolism (lactate dehydrogenase (LDH)) in whole-body (28 days) or in gills, liver and muscle (345 days) of exposed fish. Additionally, whole-body oxygen consumption was evaluated in fish exposed for 28 days and hepatic and muscular expression of genes involved in mitochondrial metabolism (cox I, II and III and atp5a1) was assessed in animals exposed for 345 days. Finally, final survival was evaluated. Following 28 days, Cu did not affect survival neither enzyme activities. However, increased whole-body oxygen consumption was observed in comparison to control condition. After 345 days, 76.8%, 63.9%, 60.9% and 0% survival were observed for control, 5, 9 and 20 µg/L groups, respectively. Animals exposed to 5 and 9 µg/L had a significant reduction in branchial and muscular LDH activity and in hepatic PK activity. Also, exposure to 9 µg/L significantly increased hepatic CS activity. For gene expression, Cu down-regulated muscular cox II (9 µg/L) and III (5 and 9 µg/L), and up-regulated hepatic atp5a1 (9 µg/L). Findings reported in the present study indicate that chronic exposure to Cu induces tissue-specific responses in key aspects of the energetic metabolism. In gills and muscle, Cu leads to reduced energy production through inhibition of anaerobic pathways and mitochondrial respiratory chain. This effect is paralleled by an increased ATP consumption in the liver, characterized by the augmented CS activity and atp5a1 expression. Finally, reduced PK activity indicate that oxidative stress may be involved with the observed outcomes.

Impaired regulation of divalent cations with acute copper exposure in the marine clam Mesodesma mactroides.

The mechanism of copper (Cu) toxicity in marine invertebrates remains unclear. Therefore, marine clams (Mesodesma mactroides) were exposed (96h) to a concentration of dissolved Cu (1.6µmolL(-1)) inducing 10% mortality in sea water (30ppt). After in vivo exposure, tissue Cu accumulation (hemolymph, gill and digestive gland); hemolymph ionic (Na(+), K(+), Mg(2+) and Ca(2+)) and osmotic concentrations; tissue (gill and digestive gland) ionic concentration, enzyme (Na(+),K(+)-ATPase and carbonic anhydrase) activity, and oxygen consumption; and whole-body oxygen consumption were analyzed. Succinate dehydrogenase activity was evaluated in mitochondria isolated from gills and digestive gland and exposed (1h) in vitro to different concentrations of dissolved Cu (0.8, 7.7 and 78.7µmolL(-1)). In vivo exposure induced Cu accumulation in hemolymph, gills and digestive gland; increased Mg(2+) and decreased Ca(2+) concentration in hemolymph; decreased Mg(2+) concentration, increased Na(+),K(+)-ATPase activity and reduced carbonic anhydrase activity in gills; decreased Mg(2+) concentration, increased Ca(2+) concentration and increased Na(+),K(+)-ATPase activity in digestive gland; and reduced gill, digestive gland and whole-body oxygen consumption. Succinate dehydrogenase activity was inhibited after in vitro exposure to 78.7µmolL(-1) Cu. These findings indicate that Cu is an ionoregulatory toxicant in the marine clam M. mactroides. However, toxicity is related to disturbances in regulation of divalent cations (Mg(2+) and Ca(2+)) without effect on regulation of major monovalent cations (Na(+) and K(+)), as opposed to that observed in osmoregulating invertebrates exposed to Cu. However, other mechanism(s) of toxicity cannot be ruled out. Future studies must be performed to evaluate the consequence of the Cu-induced respiratory disturbances observed in M. mactroides.

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities.

The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.

Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation.

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.