Viviane Prodocimo

Muscle water control in crustaceans and fishes as a function of habitat, osmoregulatory capacity, and degree of euryhalinity

This study aimed at detecting possible patterns in the relationship between Anisosmotic Extracellular Regulation (AER) and Isosmotic Intracellular Regulation (IIR) in crustaceans and teleost fish from different habitats and evolutionary histories in fresh water (FW), thus different osmoregulatory capabilities, and degrees of euryhalinity. Crustaceans used were the hololimnetic FW Aegla schmitti, and Macrobrachium potiuna, the diadromous FW Macrobrachium acanthurus, the estuarine Palaemon pandaliformis and the marine Hepatus pudibundus; fishes used were the FW Corydoras ehrhardti, Mimagoniates microlepis, and Geophagus brasiliensis, and the marine-estuarine Diapterus auratus. The capacity for IIR was assessed in vitro following wet weight changes of isolated muscle slices incubated in anisosmotic saline (~50% change). M. potiuna was the crustacean with the highest capacity for IIR; the euryhaline perciforms G. brasiliensis and D. auratus displayed total capacity for IIR. It is proposed that a high capacity for IIR is required for invading a new habitat, but that it is later lost after a long time of evolution in a stable habitat, such as in the FW anomuran crab A. schmitti, and the Ostariophysian fishes C. ehrhardti and M. microlepis. More recent FW invaders such as the palaemonid shrimps (M. potiuna and M. acanthurus) and the cichlid G. brasiliensis are euryhaline and still display a high capacity for IIR.

Effects of the herbicide atrazine in neotropical catfish (Rhamdia quelen)

The exposure to a world-wide used herbicide atrazine (ATZ) (96h exposure to 2, 10, and 100μgL(-1)), was investigated on the freshwater fish Rhamdia quelen through a multi biomarker approach. Liver histopathology revealed leukocyte infiltration, hepatocyte vacuolization like steatosis and necrosis areas, leading to raised lesion index levels in all tested concentrations. The increase of free melanomacrophage numbers was observed. Gill filaments revealed considerable loss of the microridges on pavement cells at 10 and 100μgL(-1) of ATZ, and a significantly increased of chloride cell (CC) number and density on apical surface area at 100μgL(-1) of ATZ. CAT, GST, GPx, and GR activities were inhibited by all tested concentrations. GSH levels were reduced in individuals exposed to 100µgL(-1). Osmoregulatory function was also disturbed. We observed an increase of plasma magnesium concentrations at 10µgL(-1). Additionally the inhibition of branchial carbonic anhydrase activity was observed at 100µgL(-1). In the kidney, carbonic anhydrase activity decreased only in the group exposed to 2µgL(-1). These results suggest that ATZ, represents a potential ecotoxicological hazard and can be hepatotoxic and nephrotoxic even low concentrations. The current study was the first to show the nephrotoxic effect of ATZ in fish. Besides, in Brazil, the environmental protection agency (CONAMA) establishes that the maximum allowed level of dissolved ATZ in water is 2μgL(-1), but the present results showed that this concentration may cause histopathological, biochemical and physiological changes in R. quelen.

Unidirectional Na+ and Ca2+ fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na+ and Ca2+ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na+ and Ca2+, whole body Na+ and Ca2+, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na+ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na+ balance was restored by the time killifish reached 100% SW. Whole body Na+ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na+ influx (50-fold relative to FW values), but not Na+ efflux between 40 and 60% SW, resulting in a large net loading of Na+ at higher salinities (60–100% SW), and increases in plasma Na+ and whole body Na+ at 100% SW. Killifish were in negative Ca2+ balance at all salinities, whereas trout were in positive Ca2+ balance throughout. Ca2+ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca2+ efflux. Ca2+ flux rates were affected to a larger degree in trout, with twofold increases in Ca2+ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca2+ efflux with salinity, so plasma Ca2+ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.

Risks of waterborne copper exposure to a cultivated freshwater Neotropical catfish (Rhamdia quelen)

As it is the case in all animal food production systems, it is often necessary to treat farmed fish for diseases and parasites. Quite frequently, fish farmers still rely on the aggressive use of copper to control bacterial infections and infestations by ecto-parasites, and to manage the spread of diseases. The susceptibility of the neotropical fish Rhamdia quelen to copper was here evaluated at different waterborne copper concentrations (2, 7 or 11 μg Cu L(-1)) for 96 h, through a multi biomarkers approach. Liver histopathological findings revealed leukocyte infiltration, hepatocyte vacuolization and areas of necrosis, causing raised levels of lesions upon exposure to 7 and 11 μg Cu L(-1). Decreased occurrence of free melano-macrophages and increased densities of melano-macrophage centers were noted upon exposure to 11 μg Cu L(-1). Gills showed damages on their secondary lamellae already at 2 μg Cu L(-1); hypertrophy and loss of the microridges of pavement cells at 7 and 11 μg L(-1), and increased in chloride cell (CC) apical surface area (4.9-fold) and in CC density (1.5-fold) at 11 μg Cu L(-1). In the liver, catalase (CAT), glutathione peroxidase activities (GPx) and glutathione concentration (GSH) remained unchanged, compared to the control group. However, there was inhibition of 7-ethoxyresorufin-O-deethylase (EROD) at all copper concentrations tested. Glutathione reductase activity (GR) was reduced and levels of lipid peroxidation (LPO) were increased at 11 μg Cu L(-1). Glutathione S-transferase activity (GST) at 7 μg Cu L(-1) and superoxide dismutase activity (SOD) at both 7 and 11 μg Cu L(-1) were reduced. However, copper exposure did not alter brain and muscle acetylcholinesterase (AChE) activity. Osmoregulatory function was also disturbed, in agreement with the above-mentioned changes noted in the gills, as detected by plasma osmolality reduction in the group exposed to 11 μg Cu L(-1), and plasma chloride reduction at 2 μg Cu L(-1). These concentrations also, coherently, lead to inhibition of branchial carbonic anhydrase activity. In the kidney, increased carbonic anhydrase activity was measured in the groups exposed to 2 and 7 μg Cu L(-1). When these effects are compared to data available in the literature for other freshwater fish, also for 96 h of exposure, R. quelen appears as a relatively sensitive species. In addition, the concentrations employed here were quite low in comparison to levels used for disease control in real culture practices (ranging from 4 μg Cu L(-1) used against bacteria to 6000 μg Cu L(-1) against fungal infections). We can conclude that the concentrations frequently employed in aquaculture are in fact not safe enough for this species. Such data are essential for the questioning and establishment of new policies to the sector.

Physiological tools to predict invasiveness and spread via estuarine bridges: tolerance of Brazilian native and worldwide introduced freshwater fishes to increased salinity

Non-native freshwater fishes may spread via estuaries, invading nearby basins. The Brazilian natives Rhamdia quelen and Geophagus brasiliensis, and the worldwide introduced Clarias gariepinus, Ictalurus punctatus, Oreochromis niloticus and Cyprinus carpio were acutely exposed (6h) to salinities 15 and 30. Hypothetically, the introduced species display greater physiological plasticity than do the natives. Exposure to salinity 30 was lethal after 1.5-3h to all species except for O. niloticus and G. brasiliensis. Increase in plasma osmolality was inversely related to muscle water content, mainly in salinity 30 for all species. R. quelen and C. gariepinus displayed increased expression of heat-shock protein 70 (HSP70) on salinity increase; differently, I. punctatus, O. niloticus and C. carpio showed high constitutive levels already in freshwater. Species with high constitutive expression of HSP and/or high degree of euryhalinity (cichlids) could potentially use estuaries as bridges, especially through areas of salinity ,15. Additionalkeywords: biologicalinvasions,environmentalmatching,heat-shockprotein,musclewater,osmoregulation, physiological tolerance.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Regulation of muscle hydration upon hypo- or hyper-osmotic shocks: differences related to invasion of the freshwater habitat by decapod crustaceans.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Estuarine pufferfishes (Sphoeroides testudineus and S. Greeleyi) submitted to sea water dilution during ebb tide: a field experiment

The pufferfishes Spheroides testudineus and S. greeleyi are very abundant in estuaries along the Brazilian coast, with S. greeleyi restricted to areas of higher salinity than S. testudineus. Their osmoregulatory behaviour was tested with the fish inside fixed cages in the estuary, forced to endure sea water dilution during ebb tide. Fish were sampled when sallinity naturally decreased to 29.5‰, 14‰, and 4.5‰, along 5–6 hours. Plasma osmolality and chloride concentration were well regulated by the fish S. greeleyi displayed higher values than S. testudineus, and showed mortality at the lowest salinity, compatible with the ecological observation of the more frequent presence of S. testudineus in very dilute sea water.

Critical thermal maxima and minima of the platyfish Xiphophorus maculatus Günther (Poecillidae, Cyprinodontiformes): a tropical species of ornamental freshwater fish

Temperature as an environmental factor has been a frequent subject of study, since it affects either directly or indirectly all living organisms. The determination of thermal limits (critical thermal minima - CTmin and maxima - CTmax) for the tropical ornamental freshwater teleost Xiphophorus maculatus Gunther, 1866 (platyfish) was performed after their acclimation to the following temperatures: 15, 20, 25, and 30oC, for seven days. After this period, the water temperature was elevated or reduced at a rate of 0.125oC/min until CTmax and CTmin could be determined as the temperature at which 50% of the animals had lost equilibrium. Mean values for CTmax and CTmin for the acclimation temperatures of 15, 20, 25, and 30oC were respectively: 39.8,39.8,40.4,41.5oC (CTmax), and 9.6, 12.8, 13.1, 16.0oC (CTmin). CTmax and CTmin for X. maculatus were thus affected by acclimation temperature. This tropical species is more heat- than cold- tolerant and would not resist the typical low winter temperatures of southern Brazil. Platyfish can adapt to natural environments in regions of mean annual temperatures around 20-25oC or be kept in aquaria with other ornamental species that accordingly prefer this temperature range.

Physiological biomarkers in a resident and a non-resident estuarine teleosts species: a comparison between fish from an industrially impacted site and a non-impacted site

Physiological biomarkers were compared in two estuarine fishes: an estuarine resident (Atherinella brasiliensis) and a marine/estuarine transient (Sphoeroides testudineus). These fish were sampled from two estuarine systems: one with an active harbor (impacted site), and another which receives mostly domestic urban runoffs and is much less impacted (reference site). The resident fish displayed higher plasma osmolality and sodium (1.13 and 1.35-fold, respectively), and the transient fish displayed higher plasma osmolality (1.05-fold), potassium (1.17-fold), and magnesium levels (1.84-fold) in the impacted site, when compared to the reference site. The resident fish showed increased renal carbonic anhydrase activity and branchial expression of HSP70 (3.3-fold) in the impacted site, when compared to the reference site. Osmoregulatory parameters and HSP70 expression are strong candidates as physiological biomarkers for environmental assessment of industrial impact on estuarine teleost fishes.