Evolutionary trade-offs in osmotic and ionic regulation and expression of gill ion transporter genes in high latitude, cold clime Neotropical crabs from the 'end of the world'.

Osmoregulatory findings on crabs from high Neotropical latitudes are entirely lacking. Seeking to identify the consequences of evolution at low temperature, we examined hyperosmotic/hypo-osmotic and ionic regulation and gill ion transporter gene expression in two sub-Antarctic Eubrachyura from the Beagle Channel, Tierra del Fuego. Despite sharing the same osmotic niche, Acanthocyclus albatrossis tolerates a wider salinity range (2-65‰ S) than Halicarcinus planatus (5-60‰ S); their respective lower and upper critical salinities are 4‰ and 12‰ S, and 63‰ and 50‰ S. Acanthocyclus albatrossis is a weak hyperosmotic regulator, while H. planatus hyperosmoconforms; isosmotic points are 1380 and ∼1340 mOsm kg-1 H2O, respectively. Both crabs hyper/hypo-regulate [Cl-] well with iso-chloride points at 452 and 316 mmol l-1 Cl-, respectively. [Na+] is hyper-regulated at all salinities. mRNA expression of gill Na+/K+-ATPase is salinity sensitive in A. albatrossis, increasing ∼1.9-fold at 5‰ compared with 30‰ S, decreasing at 40-60‰ S. Expression in H. planatus is very low salinity sensitive, increasing ∼4.7-fold over 30‰ S, but decreasing at 50‰ S. V-ATPase expression decreases in A. albatrossis at low and high salinities as in H. planatus. Na+/K+/2Cl- symporter expression in A. albatrossis increases 2.6-fold at 5‰ S, but decreases at 60‰ S versus 30‰ S. Chloride uptake may be mediated by increased Na+/K+/2Cl- expression but Cl- secretion is independent of symporter expression. These unrelated eubrachyurans exhibit similar systemic osmoregulatory characteristics and are better adapted to dilute media; however, the expression of genes underlying ion uptake and secretion shows marked interspecific divergence. Cold clime crabs may limit osmoregulatory energy expenditure by hyper/hypo-regulating hemolymph [Cl-] alone, apportioning resources for other energy-demanding processes.

Ecotoxicological impacts of the Fundão dam failure in freshwater fish community: Metal bioaccumulation, biochemical, genetic and histopathological effects.

This study investigates the ecotoxicological impacts of the Fundão dam rupture, one of the major environmental disaster that occurred in Brazil and in the world mining industry history, through multi-biomarkers responses and metals bioaccumulation in the fish community of different trophic levels. Specimens of the fishes (omnivorous/herbivorous and carnivorous) were collected along the Doce River channel and its affluent Guandú River, and in different lakes and coastal lagoons adjacent to the river channel, in the Espirito Santo State, Southeast of Brazil. Four sampling collections were carried out over two years (2018 to 2020, during dry and rainy seasons). For both trophic groups the biomarkers responses indicated physiological alterations related to metals exposure and showed strong seasonal variations. The principal component analysis and integrated biomarker response index showed that DNA damage and lipid peroxidation were more associated with dry season 2 (Sep/Oct 2019) and the oxidative damage in proteins, metallothioneins concentration and the activity of superoxide dismutase in the gills showed a greater association with rainy season 2 (Jan/Feb 2020). On the other hand, the enzymes of energy metabolism, catalase and histological damage in the liver and the gills, were more associated with the dry and rainy campaigns of the first year of monitoring. The multivariate approach also suggested a temporal intensification in the bioaccumulation of metals and biological effects in the lacustrine environments. Thus, these results demonstrate that the release of mineral residues from the rupture of the Fundão mine dam affects the health status of the fish from the Doce River basin, provoking metals bioaccumulation, hepatic and branchial damage in the fish besides inducing of enzyme activity related to metal contamination, even four years after the rupture.

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes.

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.

Immunocytochemical localization of V-H(+) -ATPase, Na(+) /K(+) -ATPase, and carbonic anhydrase in gill lamellae of adult freshwater euryhaline shrimp Macrobrachium acanthurus (Decapoda, Palaemonidae).

Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps.