Carolina A. Freire

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.

Revisiting the potential conservation value of non-native species.

JEAN RICARDO SIMOES VITULE,∗ CAROLINA A. FREIRE,† DIEGO P. VAZQUEZ,‡ MARTIN A. NUNEZ,§ AND DANIEL SIMBERLOFF§ ** ∗Laboratorio de Ecologia e Conservacao, Departamento de Engenharia Ambiental, Setor de Tecnologia, Universidade Federal do Parana, 81531, 980, Curitiba, Parana, Brazil †Departamento de Fisiologia, Setor de Ciencias Biologicas, Centro Politecnico, Universidade Federal do Parana, 81531, 980, Curitiba, Parana, Brazil ‡Instituto Argentino de Investigaciones de las Zonas Aridas, CONICET; Instituto de Ciencias Basicas, Universidad Nacional de Cuyo, Mendoza, Argentina §Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996, 1610, U.S.A.

An assessment of acute biomarker responses in the demersal catfish Cathorops spixii after the Vicuña Oil Spill in a harbour estuarine area in Southern Brazil

The Vicuña oil tanker exploded in Paranaguá Bay (South of Brazil), during methanol unloading operations in front of Paranaguá Harbour, on November 15th, 2004, releasing a large amount of bunker oil and methanol. Two weeks after the accident, the acute effects of the Vicuña Oil Spill (VOS) were evaluated in the demersal catfish Cathorops spixii, comparing a contaminated (at the spill site) and a reference site inside the Bay. Data were compared to previous measurements, taken before the accident, in the same species, from the same sites. The physiological biomarkers were the ones that best reflected acute effects of the spill: plasma osmolality, chloride, calcium, magnesium, and potassium. Morphological (liver and gill histopathology) and genetic (piscine micronucleus and DNA strand breaks) biomarkers revealed that damage was already present in fishes from both reference and contaminated sites inside the Bay. Thus, the reference site is not devoid of contamination, as water circulation tends to spread the contaminants released into other areas of the Bay. Acute field surveys of oil spill effects in harbour areas with a long history of contamination should thus be viewed with caution, and whenever possible previous evaluations should be considered for proper appraisal of biomarker sensitivity, especially in mobile bioindicators such as fish.

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.

Responses of free radical metabolism to air exposure or salinity stress, in crabs (Callinectes danae and C. ornatus) with different estuarine distributions.

The swimming crabs Callinectes danae and C. ornatus are found in bays and estuaries, but C. danae is more abundant in lower salinities, while C. ornatus remains restricted to areas of higher salinity. Experimental crabs of both species were submitted to: air exposure (Ae, 3h), reimmersion in 33‰ (control) sea water (SW) (Ri, 1h) following air exposure; hyposaline (Ho, 10‰ for 2h) or hypersaline (He, 40‰ for 2h) SW, then return to control 33‰ SW (RHo and RHe, for 1h). Hemolymph was sampled for osmolality and chloride determinations. Activity of antioxidant enzymes [glutathione peroxidase (GPX), catalase, glutathione-S-transferase] and levels of carbonyl proteins and lipid peroxidation (TBARS) were evaluated in hepatopancreas, muscle, anterior and posterior gills. In Ho groups, hemolymph concentrations were lower in both species, compared to He groups. C. danae displayed higher control activities of GPX (hepatopancreas and muscle) and catalase (all four tissues) than C. ornatus. C. ornatus presented increased activities of catalase and GPX in Ae, Ri, and He groups. Increased TBARS was seen in C. ornatus tissues (He group). The more euryhaline species displayed higher constitutive activities of antioxidant enzymes, and the less euryhaline species exhibited activation of these enzymes when exposed to air or hyper-salinity.

Do osmoregulators have lower capacity of muscle water regulation than osmoconformers? A study on decapod crustaceans

Decapod crustaceans occupy various aquatic habitats. In freshwater they are osmoregulators, while marine species are typically osmoconformers. Freshwater crustaceans are derived from marine ancestors. The hypothesis tested here was that osmoregulators, which can rely on salt transport by interface epithelia to prevent extracellular disturbance, would have a lower capacity of tissue water regulation when compared with osmoconformers. Four species of decapod crustaceans (the marine osmoconformer crab Hepatus pudibundus, and three osmoregulators of different habitats) have been exposed in vivo to a salinity challenge, for up to 24 hr. Osmoregulators were: the estuarine shrimp Palaemon pandaliformis, the diadromous freshwater shrimp Macrobrachium acanthurus, and the hololimnetic red crab Dilocarcinus pagei. H. pudibundus displayed hemolymph dilution already after 0.5 hr in 25 per thousand, reaching approximately 30% reduction in osmolality, but its muscle degree of hydration did not increase. To make the different in vivo salinity challenges directly comparable, the ratio between the maximum change in muscle hydration with respect to the control value measured for the species and the maximum change in hemolymph osmolality was calculated (x 1,000): H. pudibundus (25 per thousand, 8.1% kg H(2)O/mOsm x 10(3))>P. pandaliformis (2 per thousand, 9.2)>M. acanthurus (30 per thousand, 12.6)>P. pandaliformis (35 per thousand, 16.7)>D. pagei (7 per thousand, 60.4). Muscle slices submitted in vitro to a 30% osmotic challenge confirmed in vivo results. Thus, the estuarine/freshwater osmoregulators displayed a lower capacity to hold muscle tissue water than the marine osmoconformer, despite undergoing narrower variations in hemolymph osmolality.

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.

Distinct patterns of water and osmolyte control between intertidal (Bunodosoma caissarum) and subtidal (Anemonia sargassensis) sea anemones

Anemones are frequently found in rocky intertidal coasts. As they have highly permeable body surfaces, exposure to the air or to salinity variations inside tidal pools can represent intense osmotic and ionic challenges. The intertidal Bunodosoma caissarum has been compared with the subtidal Anemonia sargassensis concerning their response to air exposure or salinity changes. B. caissarum maintains tissue hydration through mucus production and dome-shape formation when challenged with air exposure or extreme salinities (fresh water or hypersaline seawater, 45 psu) for 1-2h. Upon exposure to mild osmotic shocks for 6h (hyposmotic: 25 psu, or hyperosmotic: 37 psu), B. caissarum was able to maintain its coelenteron fluid (CF) osmolality stable, but only in 25 psu. A. sargassensis CF osmolality followed the external medium in both salinities. Isolated cells of the pedal disc of B. caissarum showed full capacity for calcium-dependent regulatory volume decrease (RVD) upon 20% hyposmotic shock, at least partially involving the release of KCl via K(+)-Cl(-) cotransport, and also of organic osmolytes. Aquaporins (HgCl(2)-inhibited) likely participate in this process. Cells of A. sargassensis showed partial RVD, after 20 min. Cells from both species were not capable of regulatory volume increase upon hyperosmotic shock (20%). Whole organism and cellular mechanisms allow B. caissarum to live in the challenging intertidal habitat, frequently facing air exposure and seawater dilution.

Differences in ion regulation in the sea urchins Lytechinus variegatus and Arbacia lixula (Echinodermata: Echinoidea)

The regular sea urchin Lytechinus variegatus , a species previously reported from areas of reduced salinities, and Arbacia lixula , a species unreported from diluted waters, were submitted to seawater dilution or seawater dilution in magnesium-supplemented waters. Seawater (35 psu) was either proportionally diluted with filtered dechlorinated tap water (30 psu, 25 psu), or diluted and supplemented with magnesium as MgCl 2 (30+Mg, 25+Mg), up to full-strength seawater Mg 2+ levels (35 psu, ~54 mM Mg 2+ ). Magnesium supplementation was intended to verify the interfering effect of magnesium on osmo-ionic concentrations of the coelomic fluid (CF) of two ecologically distinct species of sea urchins. After 6 h in control (35 psu) or experimental seawater, CF samples were withdrawn by puncturing through the peristomial membrane. Coelomic fluid osmolality ([Osm]), and concentrations of ([Na + ]), ([Cl - ]), ([Mg 2+ ]) and ([K + ]) were measured for both species. Under all conditions, L. variegatus displayed higher CF osmolality, [Na + ], and [K + ] values than the water (and A. lixula ). Comparatively, L. variegatus is designated as a‘hyper-conformer’, while A. lixula is an ‘iso-conformer’. The CF [Mg 2+ ] showed no evidence of being controlled by either species. Mg 2+ supplementation in diluted seawater affected Mg 2+ and Cl - levels only. Na + appears to be taken up actively by L. variegatus , rendering its CF mostly hyper-ionic for Na + (and hyperosmotic) relative to external seawater. The different gradients observed with the different ions suggest selective permeabilities or ion regulation by L. variegatus .