In situ experiment to evaluate biochemical responses in the freshwater mussel Diplodon chilensis under anthropogenic eutrophication conditions.

An in-situ experiment was performed to study metabolic responses of the freshwater mussel Diplodon chilensis to water contaminated by leachates from an open dump and cattle activity, in order to analyze both the effects of those contaminants on aquatic environments and the potential use of a native bivalve to evaluate the effects of anthropic influence and eutrophication. Bivalves from a reference site were cage-transplanted to a control site (site A) and to a temporal water pond (site B) over 30 and 60 periods. Water quality analyses revealed that the site B was affected by anthropogenic influence. Mussel's hemocytes from site B showed 50% lower reactive oxygen species production and 130% higher lysosomal membrane stability in the site B mussels. In addition, no oxidative stress was evident in gills, despite the elevated copper and iron concentrations recorded in the site B water samples (CuB = 0.3350 ± 0.0636 mg. L-1vs. CuA = 0.0045 ± 0.0007 mg. L-1; FeB = 3.8650 ± 0.4031 mg. L-1vs. FeA = 0.0365 ± 0.0049 mg. L-1). In contrast, the adductor muscle accumulated more Fe (~10-20-fold) than the gills and showed signs of oxidative stress, e.g. superoxide dismutase activity and TBARS levels were increased by 10% were 34%, respectively, in the site B compared with the site A after 60 days of exposure. Additionally, the adductor muscle showed signs of anaerobic metabolism activation. Cu is accumulated in gills from both sites' individuals, at 60 days, in concordance with the increase in the activity of the cu-containing enzyme cytochrome-c-oxidase. There was a reduction in the overall condition and digestive gland index in bivalves exposed at site B, associated with diminished levels of lipid and protein contents. Metal-pollution and eutrophication affects D. chilensis metabolism and is associated to tissue-specific exposure, anaerobic metabolism and general energetic condition depletion.

Crude extract of cyanobacteria (Radiocystis fernandoi, strain R28) induces liver impairments in fish.

Radiocystis fernandoi R28 strain is a cyanobacterium which produces mostly the RR and YR microcystin variants (MC-RR and MC-YR, respectively). The effects of crude extract of the R. fernandoi strain R28 were evaluated on the protein phosphatases and on the structure and ultrastructure of the liver of the Neotropical fish, Hoplias malabaricus, after acute and subchronic exposure. Concomitantly, the accumulation of the majority of MCs was determined in the liver and muscle. The fish were exposed to 120.60 MC-RR+MC-LR kg-fish-1 (=100µg MC-LReq kg-fish-1) for 12 and 96h (one single dose, acute exposure) and 30days (one similar dose every 72h, subchronic exposure). MCs did not accumulate in the muscle but, in the liver, MC-YR accumulated after acute exposure and MC-RR and MC-YR accumulation occurred after subchronic exposure. Protein phosphatase 2A (PP2A) activity was inhibited only after subchronic exposure. Acute exposure induced liver hyperemia, hemorrhage, changes in hepatocytes and cord-like disorganization. At the ultrastructural level, the decreasing of glycogen and lipid levels, the swelling of mitochondria and whirling of endoplasmic reticulum suggested hepatocyte necrosis. Subchronic exposure resulted in a complete disarrangement of cord-like hepatocytes, some recovery of mitochondria and whirling endoplasmic reticulum and extensive connective tissues containing fibrous materials in the liver parenchyma. Despite microcystin toxicity and liver alterations, no tumor was induced by MCs. In conclusion, the increased algal mass of R. fernandoi in tropical freshwater, producing mainly MC-RR and MC-YR variants, results in fish liver impairments.

Possible role of carbonic anhydrase, V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion transport across the gills of the euryhaline crab Chasmagnathus granulatus.

We studied the participation of carbonic anhydrase (CA), V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion absorption through the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2 per thousand, 10 per thousand, 30 per thousand (about seawater), and 45 per thousand salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from crabs acclimated to either 2 per thousand or 10 per thousand with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2 per thousand and perfused with 20 per thousand saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (V(te)) by 47%, further addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30 per thousand saline (iso-osmotic for seawater acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V-H(+)-ATPase and Cl(-)/HCO3-) reduced V(te) by 15-16% in gills perfused with normal 20 per thousand saline, and by 77% and 45%, respectively when they were applied in Na-free 20 per thousand saline, suggesting the participation of those transporters and cytosolic CA in electrogenic ion absorption.

Oxidative stress generated by diesel seawater contamination in the digestive gland of the Antarctic limpet Nacella concinna.

The aim of this work was to investigate the activity of antioxidant enzymes and oxidative damage in the digestive gland of the limpet Nacella concinna, and their suitability as biomarkers for hydrocarbon pollution in Antarctic coasts. Three groups of 30 individuals each were kept in seawater containing 0%, 0.05% or 0.1% diesel. Superoxide dismutase, catalase, glutathione S transferase and glutathione peroxidase activities, as well as lipid peroxidation and protein oxidation were studied in 18 animals of each group after 24, 48 and 168 h of exposure. The activity levels of most enzymes were increased by diesel in a dose-dependent manner. Glutathione peroxidase showed the most clear effect; its activity significantly increased in the 0.1% diesel group respect to the control. Lipid peroxidation and protein oxidation were significantly increased by diesel after 168 h. Both variables were higher in the group exposed to the lowest dose.

Modulation of ion uptake across posterior gills of the crab Chasmagnathus granulatus by dopamine and cAMP.

Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10 per thousand salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na(+) influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose-response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 micromol l(-1). When the effects of forskolin and DA on the Na(+)/K(+)-ATPase activity were tested, they correlated well with the Vte and Na(+) influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism.

Toxic effects of microcystins in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae).

Microcystins are toxins produced by cyanobacteria, being toxic to aquatic fauna. It was evaluated alternative mechanisms of microcystins toxicity, including oxidative stress and histopathology in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Microcystins was administered to crabs (MIC group) over 1 week, whereas the control (CTR group) received the saline from cyanobacteria culture medium. At day 7, catalase activity was higher in the MIC than in the CTR group, although a decrease of activity was verified in both groups with respect to time 0. Glutathione-S-transferase activity augmented in MIC with respect to CTR, suggesting a higher conjugation rate of the toxins with glutathione. No differences were detected in the superoxide dismutase activity. Lipid peroxidation remained stable in both groups. Histopathological analyses showed that the number of B cells decreased significantly in the CTR as a possible effect of starvation, while no significant change was observed in the MIC group. The hepatopancreas from the MIC group exhibited some necrotic tubules and melanin-like deposits. Overall, results showed that some enzymes of the antioxidant defense system were activated after microcystins exposure, this response being able to maintain lipid peroxidation levels, but insufficient to completely prevent histological damage.

Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations.

In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (V(te)) of isolated and perfused gills increased significantly when hyposmotic saline (699 mosmol l(-1)) was used instead of isosmotic solution (1045 mosmol l(-1)). A reduction of the concentration of Na(+) or Cl(-) at constant osmolarity did not produce any change in V(te). Transepithelial short-circuit current (I(sc)) and conductance (G(te)), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (I(sc): from -89.0+/-40.8 microA cm(-2) to -179.3+/-37.0 microA cm(-2); G(te): from 40.5+/-16.9 mS cm(-2) to 47.3+/-15.8 mS cm(-2)). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The activity of the Na(+)/K(+)-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73+/-6.35 micromol P(i) h(-1) mg(-1) to 41.84+/-14.54 micromol P(i) h(-1) mg(-1). Theophylline maintained part of the elevated V(te) induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na(+)/K(+)-ATPase and by changing a conductive pathway, probably at the apical membrane.

Transepithelial potential differences and Na(+) flux in isolated perfused gills of the crab Chasmagnathus granulatus (Grapsidae) acclimated to hyper- and hypo-salinity.

We studied the transepithelial potential difference (TEPD) and (22)Na flux across isolated perfused gills (anterior pair 5 and posterior pairs 6-8) of the crab Chasmagnathus granulatus acclimated to either hypo- or hyper-osmotic conditions. The gills of crabs acclimated to low salinity, perfused and bathed with 10 per thousand saline solutions, produced the following TEPDs (hemolymph side with respect to bath side): 0.4+/-0.7, -10.2+/-1.6, -10.8+/-1.3 and -6.7+/-1.3 mV for gills 5, 6, 7 and 8, respectively. Gills 6, 7 and 8 did not differ significantly. Reducing the saline concentration of bath and perfusate from 30 per thousand to 20 per thousand or 10 per thousand increased significantly the TEPDs of these gills. TEPDs of gill 6 (representative of posterior gills) were reduced by 69+/-5 % and 60+/-5 % after perfusion with ouabain or BaCl(2) (5 mmol l(-1) each), respectively. The same gill showed a net ouabain-sensitive Na(+) influx of 1150+/-290 microequiv g(-1) h(-1). Gill 6 of crabs acclimated to high salinity produced TEPDs of -1.5+/-0.1 and -1.3+/-0.09 mV after perfusion with 30 per thousand or 40 per thousand salines, respectively. Perfusion with ouabain or BaCl(2) reduced TEPDs by 76+/-7 % and 86+/-4 %, respectively. A net ouabain-sensitive Na(+) efflux of 2282+/-337 microequiv g(-1) h(-1) was recorded in gill 6 perfused with 38 per thousand saline.

The morphometric changes in the gills of the estuarine crab Chasmagnathus granulatus under hyper- and hyporegulation conditions are not caused by proliferation of specialised cells.

Chasmagnathus granulatus is a hyper-hyporegulating crab that inhabits changing habitats of salinity in Brazil, Uruguay and Argentina. Since the gills are the main sites for active ion transport in crabs, the adaptive changes in the gill epithelium occurring under different conditions of salinity were studied by means of morphological and morphometric analysis, and immunohistochemical identification of cell proliferation (BrdU technique). In anterior (1-3) gills the epithelium thickness from crabs acclimatised to 12, 34 and 44 g/l ranged from 1.27 to 2.46 microm, with no significant change during acclimatisation, thus denoting a respiratory function. Medial (4-5) gill epithelium was slightly thicker in extreme salinities, but these differences were not statistically significant. In contrast, epithelial thickness of the posterior (6-8) gills increased significantly up to 8.10 microm (dorsal zone of gill 8) both in hyper- and hyposaline media compared with seawater. The dark areas measured in gill 8 treated with AgNO3 revealed putative ion transporting tissue, especially at 12 and 44 g/l, corresponding to the zones of higher epithelial thickness. Hence these areas seem to participate both in hyper- and hyporegulation. Proliferating cells labelled with BrdU almost never occurred in the gills/salinity combinations studied during the initial 48 h of transfer from seawater to hyperconcentrated or diluted media, thus suggesting an increase in cell size rather than cell proliferation.

Bimodal breathing in the estuarine crab Chasmagnathus granulatus Dana 1851--physiological and morphological studies.

Chasmagnathus granulatus is an estuarine crab which actively moves from subtidal to supratidal areas. To elucidate the possible existence of extrabranchial sites for aerial gas exchange, we measured respiratory and acid-base variables in animals with and without branchial water (controls and experimental crabs, respectively) during air exposure. An histological study of the branchiostegite was also performed. Throughout 4 h of emergence C. granulatus did not suffer venous hypoxia, even without branchial water. The rate of oxygen uptake (M(O(2))) was similar in both groups. The rate of carbon dioxide excretion (M(CO(2))) and the gas exchange ratio (R) significantly decreased during emergence in both groups, with R significantly lower for experimental crabs. Consequently, CO(2) was accumulated in the hemolymph. This variable stabilized after 90 min in control animals, but experimental crabs continued accumulating CO(2). Histological study of the branchiostegites demonstrated the presence of an attenuated and greatly perfused epithelium facing the branchial chamber lumen, with a shortest diffusion distance of 0.5 microm. Simple folds and lobulated projections increase the respiratory surface area. These results suggest that C. granulatus is a bimodal breathing crab, active both in water and air. When emerged, this species extract oxygen directly from air through branchiostegal lungs, but relies on branchial exchange to eliminate carbon dioxide.