A systematic evaluation on the relationship between hypo-osmoregulation and hyper-osmoregulation in decapods of different habitats.

Decapods occupy all aquatic, and terrestrial and semi-terrestrial environments. According to their osmoregulatory capacity, they can be osmoconformers or osmoregulators (hypo or hyperegulators). The goal of this study is to gather data available in the literature for aquatic decapods and verify if the rare hyporegulatory capacity of decapods is associated with hyper-regulatory capacity. The metric used to quantify osmoregulation was the osmotic capacity (OC), the gradient between external and internal (hemolymph) osmolalities. We employ phylogenetic comparative methods using 83 species of decapods to test the correlation between hyper OC and hypo OC, beyond the ancestral state for osmolality habitat, which was used to reconstruct the colonization route. Our analysis showed a phylogenetic signal for habitat osmolality, hyper OC and hypo OC, suggesting that hyper-hyporegulators decapods occupy similar habitats and show similar hyper and hyporegulatory capacities. Our findings reveal that all hyper-hyporegulators decapods (mainly shrimps and crabs) originated in estuarine waters. Hyper OC and hypo OC are correlated in decapods, suggesting correlated evolution. The analysis showed that species which inhabit environments with intense salinity variation such as estuaries, supratidal and mangrove habitats, all undergo selective pressure to acquire efficient hyper-hyporegulatory mechanisms, aided by low permeabilities. Therefore, hyporegulation can be observed in any colonization route that passes through environments with extreme variations in salinity, such as estuaries or brackish water.

Strategies of Invertebrate Osmoregulation: An Evolutionary Blueprint for Transmuting into Fresh Water from the Sea.

Early marine invertebrates like the Branchiopoda began their sojourn into dilute media some 500 million years ago in the Middle Cambrian. Others like the Mollusca, Annelida, and many crustacean taxa have followed, accompanying major marine transgressions and regressions, shifting landmasses, orogenies, and glaciations. In adapting to these events and new habitats, such invertebrates acquired novel physiological abilities that attenuate the ion loss and water gain that constitute severe challenges to life in dilute media. Among these taxon-specific adaptations, selected from the subcellular to organismal levels of organization, and constituting a feasible evolutionary blueprint for invading freshwater, are reduced body permeability and surface (S) to volume (V) ratios, lowered osmotic concentrations, increased osmotic gradients, increased surface areas of interface epithelia, relocation of membrane proteins in ion-transporting cells, and augmented transport enzyme abundance, activity, and affinity. We examine these adaptations in taxa that have penetrated into freshwater, revealing diversified modifications, a consequence of distinct body plans, morpho-physiological resources, and occupation routes. Contingent on life history and reproductive strategy, numerous patterns of osmotic regulation have emerged, including intracellular isosmotic regulation in weak hyper-regulators and well-developed anisosmotic extracellular regulation in strong hyper-regulators, likely reflecting inertial adaptations to early life in an estuarine environment. In this review, we address osmoregulation in those freshwater invertebrate lineages that have successfully invaded this biotope. Our analyses show that across 66 freshwater invertebrate species from six phyla/classes that have transmuted into freshwater from the sea, hemolymph osmolalities decrease logarithmically with increasing S:V ratios. The arthropods have the highest osmolalities, from 300 to 650 mOsmoles/kg H2O in the Decapoda with 220-320 mOsmoles/kg H2O in the Insecta; osmolalities in the Annelida range from 150 to 200 mOsmoles/kg H2O, and the Mollusca showing the lowest osmolalities at 40-120 mOsmoles/kg H2O. Overall, osmolalities reach a cut-off at ∼200 mOsmoles/kg H2O, independently of increasing S:V ratio. The ability of species with small S:V ratios to maintain large osmotic gradients is mirrored in their putatively higher Na+/K+-ATPase activities that drive ion uptake processes. Selection pressures on these morpho-physiological characteristics have led to differential osmoregulatory abilities, rendering possible the conquest of freshwater while retaining some tolerance of the ancestral medium.

Osmo-ionic regulation and carbonic anhydrase, Na+/K+-ATPase and V-H+-ATPase activities in gills of the ancient freshwater crustacean Aegla schmitti (Anomura) exposed to high salinities.

Aeglidae anomuran crabs originated in the sea, but invaded and diversified in southern South American freshwater (FW) streams. We here aimed at examining their tolerance of increased salinity, after a long time of evolution in FW (~33 million years). Aegla schmitti were exposed to FW and dilute seawater of salinities 15, 20, and 25‰ for 1, 5 and 10 days. Mortality in 35‰ was also assessed. Hemolymph osmolality, Na+, K+, Cl-, and Mg2+ ions, and hydration levels of the abdominal muscle were assayed. The activities of the Carbonic Anhydrase (CA), Na+/K+-ATPase (NKA) and V-H+-ATPase (VHA) were also assayed in the gills. A. schmitti preserves osmoregulatory mechanisms of its marine ancestors. It is able to survive in high salinities (25‰) for at least 10 days. Mortality in 35‰ was of 56% after 1 day, and of 100% after 7 days. In 25‰, NaCl is apparently hyporegulated at all times, while hemolymph osmolality rises after 5 days. CA and NKA activities remained unchanged in all experimental conditions, while VHA activity decreased after 10 days in 25‰. Hemolymph NaCl data was compatible with either hyporegulation and/or putative influx of NaCl into cells for regulatory volume increase (RVI). Further studies should deepen the understanding of the roles of low permeabilities and saturation of high affinity uptake systems in truly FW decapods, in their responses to high salinities. Moreover, the fate of extracellular NaCl as secretion in true hypo-regulation and/or influx into cells for RVI should also be investigated.

Thermal Imaging Reveals Changes in Body Surface Temperatures of Blacktip Sharks (Carcharhinus limbatus) during Air Exposure.

Fish physiology is significantly affected by temperature variability. During fisheries interactions, fish are often exposed to air and subjected to rapid temperature changes. Fish thermal dynamics during such exposure, and the possible outcomes to their physiology, depend on how heat is distributed across their bodies, the speed at which their body temperatures change, and the size of the individual. Nevertheless, such thermal patterns remain unknown for sharks. This study employed a novel application of thermal imaging to evaluate external body temperature profiles of blacktip sharks (Carcharhinus limbatus) above-water exposure after capture. We found that above-water exposure duration, shark total length, and air temperature on the day of capture significantly influenced body surface temperatures of the analyzed sharks ([Formula: see text]). Body surface temperature significantly increased with increasing exposure; however, thermal profiles of immature sharks (<140 cm) were significantly warmer than those of mature sharks. Moreover, blacktip surface body temperatures were significantly higher during days when air temperatures were at least 2.5°C warmer than water temperatures. We discuss these results as they relate to the ecology of blacktip sharks and their potential vulnerability to fisheries capture due to such changes in peripheral body temperature.

Histological alterations in gills of Macrobrachium amazonicum juveniles exposed to ammonia and nitrite.

Aquaculture has shown great growth in the last decades. Due to the restrictions on water use, production systems are becoming increasingly more intensive, raising concerns about the production water quality. Macrobrachium amazonicum is among the freshwater prawn species with favorable characteristics for production and possibility of intensification. Nitrogen compounds such as ammonia and nitrite affect the health of aquatic organisms since they quickly reach toxic concentrations. These compounds can also cause damage to the gill structure, leading to hypoxia in tissues, affecting acid-base balance, osmoregulation (salt absorption) and ammonia excretion, decreasing the immune capacity of the animal and, in extreme cases, cause death. The aim of this study was to assess histological changes in the gills of Macrobrachium amazonicum juveniles subjected to different concentrations of total ammonia and nitrite. The prawns were subjected to different concentrations of those compounds and their gills were removed and preserved for histological analysis. The gills were assessed for changes according to the Organ Index (Iorg) and, for each change, an importance factor (w) was attributed according to the degree of reversibility and applied according to the degree of extension or frequency of the damage. The damage to the gills in the treatments with 100% mortality, both for ammonia and nitrite, corresponded to the high occurrence of progressive, regressive, circulatory, and inflammation damages. The other treatments (which caused less mortality) had mainly inflammation and regressive damages, whose occurrence increased according to the increase in ammonia and nitrite concentration. The histological analysis confirmed that the higher the total ammonia and nitrite concentrations, the larger the damages caused to the gill structure and that lower nitrite concentrations caused similar damages to those caused by higher total ammonia concentrations, which reflects the lower capacity M. amazonicum has to tolerate nitrite.

Talking to the dead: using Post-mortem data in the assessment of stress in tiger sharks (Galeocerdo cuvier) (Péron and Lesueur, 1822).

Sharks are very sensitive to stress and prone to a high mortality rate after capture. Since approximately 50 million of sharks are caught as bycatch every year, and current recommendations to reduce the impact of commercial fishing strongly support immediate release, it is imperative to better understand post-release mortality caused by the stress of capture and handling. Blood samples allow the assessment of stress levels which are valuable tools to reduce mortality in commercial, recreational and scientific fishing, being essential for the improvement in those conservation measures. Biochemical analyses are widely used for sharks as stress indicators, with secondary plasma parameters (lactate, glucose and ions) being the most often employed assays. However, it is virtually impossible to determine baseline plasma parameters in free-ranging sharks, since blood withdrawal involves animal capture and restrain, which are stressful procedures. This study aims at analyzing secondary parameters of five healthy tiger sharks captured with circular hooks and handlines in Fernando de Noronha (Northeastern Brazil) and comparing them with secondary parameters of three dead tiger sharks caught off Recife (also Northeastern Brazil). The results showed that the analysis of some plasma constituents in dead animals may be an efficient tool to assess stress and lethality. However, traditional parameters such as glucose and calcium, need to be used with caution. The results also demonstrated the extreme importance of urea and phosphorus for assessing stress response and mortality in tiger sharks, both parameters frequently neglected and of utmost importance for shark's homeostasis.

Early time course of variation in coelomic fluid ionic concentrations in sea urchins abruptly exposed to hypo- and hyper-osmotic salinity challenges: Role of size and cross-section area of test holes.

Echinoderms are restricted to the marine environment and are osmoconformer invertebrates. However, some species live in unstable environments. Especially those species, and those of larger body size, tend to show variable, albeit transient, ionic gradients between their coelomic fluid and external seawater. In order to further examine how sea urchin size relates to apparent ionic permeability of their body wall/epithelia, specimens of Echinometra lucunter, Lytechinus variegatus, Paracentrotus gaimardi, and Arbacia lixula-A. lixula of two distinct populations, Rio de Janeiro and Santa Catarina-were abruptly transferred from 35 psu to either 25 or 45 psu. Sodium, chloride, magnesium, and potassium concentrations were assayed in their coelomic fluids after 0, 1, 2, and 3 hr of exposure. Relative area of putative permeable (i.e., cross section areas of soft tissues, or test holes) surfaces (PPS) was estimated in empty tests as the sum of the peristomial area (oral hole in the empty test) and the total cross-section area of ambulacral holes, divided by the total volume (TV) of the test. L. variegatus and E. lucunter, the largest species, had PPS/TV values similar to that of the much smaller P. gaimardi. A. lixula was the "most putatively-permeable and conformer" among them all, especially urchins from the Santa Catarina population. Internal ionic levels equilibrated faster with external water in 45 than in 25, and differences among ions were observed. Body size is relevant, among many other factors, to aid conformers such as sea urchins to dwell in intertidal unstable habitats.

Acute Toxicity of Nitrite to Various Life Stages of the Amazon River Prawn, Macrobrachium amazonicum, Heller, 1862.

This study determined the effects of nitrite on different life stages of the Amazon river prawn Macrobrachium amazonicum. Prawns of each life stage (postlarvae, juveniles and adults) were stocked in 24 experimental units (n = 10 prawns), under a complete randomized design. Individuals were exposed to nitrite (0, 1, 2, 4, 8 and 16 mg L-1). The median lethal concentration after 96 h (96 h LC50) was calculated through the Weibull I. The mortality results showed that M. amazonicum is slightly less tolerant to nitrite than other species of Macrobrachium. The 96 h LC50 for postlarvae, juveniles and adults of M. amazonicum were of 1.49, 2.36 and 2.34 mg nitrite/L, respectively. Nitrite intoxication risk quotient suggest moderated risk to low risk to the species. Usually in production systems nitrite values are lower than safe levels suggested in this study (0.1 mg L-1 to postlarvae and 0.2 mg L-1 nitrite to juvenile and adults), which makes our results appropriate for the production of this species.

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.

Skin extract from Rhamdia quelen (Siluriformes: Heptapteridae) does not promote stress in conspecifics

Chemical communication is widely used in aquatic environments, where visual or auditory signals may not be always effective. Fish of the superorder Ostariophysi are known to display epidermal cells (club cells) that produce and store alarm substances, which are released to the water when the skin is damaged. Responses to alarm substances range widely, between active searches for refuge to a complete stop in any locomotor activity. In this study a large number of binucleated club cells (average density of 11 cells /5m2) were histologically observed in the skin of the catfish Rhamdia quelen (known as jundia). Skin extract (2, 5, and 10% w/v) applied for 15 minutes to conspecifics elicited increase in swimming activity and in the area visited by the fish inside the tank. However, exposure to the epithelial alarm cue did not evoke any stress response: plasma osmolality, ions (sodium, chloride, magnesium, and potassium), glucose and cortisol remained unchanged. In conclusion, the conspecific alarm cue of the jundia induces behavioral responses but not an acute stress response upon short-term exposure, compatible with its role in fostering physical integrity without representing major stress activation. Considering that in the natural environment such stimuli must quickly disappear due to dilution and that rapid protection responses may be necessary upon the possibility of an approaching predator, a faster mechanism to assure survival may come into play, such as sympathetic nervous system activation. Comunicagco qummica i amplamente utilizada por animais que vivem em ambiente aquatico, onde sinais visuais e auditivos nem sempre sco facilmente identificados. Os Ostariophysi sco conhecidos por apresentarem cilulas club na epiderme, as quais produzem e estocam substbncia de alarme que sco liberadas para o ambiente quando a pele i lesionada. As respostas dos peixes a substbncia de alarme variam entre exploragco ativa por refzgios ati a parada completa de atividade locomotora. Neste estudo, grande nzmero de cilulas club binucleadas (densidade midia de 11 cilulas/5m2) foram histologicamente observadas na epiderme do jundia, Rhamdia quelen. Peixes expostos a extrato de pele de conspecmficos (2, 5, e 10% peso/vol) por 15 minutos apresentaram aumento da atividade locomotora e da area de dispersco. No entanto, essa exposigco nco promoveu nenhuma resposta de estresse - osmolalidade plasmatica, mons (ssdio, cloreto, magnisio e potassio), glicose e cortisol nco sofreram alteragco. Conclummos que a exposigco aguda a extrato de pele de conspecmficos promovem respostas comportamentais de fuga, que essa espicie apresenta grande concentragco de cilulas club, as quais devem estar envolvidas nessas respostas e que a exposigco aguda ao estmmulo nco promoveu respostas bioqummicas indicativas de estresse. Considerando que no ambiente natural tais estmmulos devem desaparecer rapidamente dados a diluigco do meio e que respostas rapidas de protegco devem ser desencadeadas frente ` possibilidade de presenga de predador, vias rapidas de suporte a essas respostas, como sistema nervoso simpatico, por exemplo, devem estar envolvidos.