The development of oil and gas production together with the fruit production in nearby areas of N... more The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors' regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 μg L − 1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L − 1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT− PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8-and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF− CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2-3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.
Antarctic ®sh have been isolated for over several million years in an environment with a very low... more Antarctic ®sh have been isolated for over several million years in an environment with a very low and constant temperature and high oxygen concentration. In such conditions the oxidative stress might be an important factor aecting their metabolic adaptive strategies. Activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), vitamin E levels and total antioxidant capacity (TRAP) were measured in liver, gill, heart and muscle homogenates of red-blooded (Nototheniidae) and white-blooded (Channichthyidae) Antarctic ®sh. SOD activity was also measured in blood samples. Gill SOD activity was threefold higher in channichthyids than in nototheniids while CAT and GPx were signi®cantly higher in the gills of channichthyids. The increased SOD activity of channichthyids probably re¯ects the large PO 2 gradient across their gills. The H 2 O 2 produced seems to be preferentially eliminated by diffusion, according to the low levels of CAT and GPx found in the gills of these species. In contrast, blood SOD was about ®vefold higher in the latter group, which possesses erythrocytes and thus a much higher oxygencarrying capacity. CAT activity was always higher in nototheniids except in muscle. However, vitamin E did not show clear dierences between families except for the pattern observed in muscle. The higher content of vitamin E in this tissue shown in channichthyids is related to the higher volume density of mitochondria reported for this group, since vitamin E is responsible for preventing membrane lipid peroxidation. Accordingly, TRAP (representative of hydrosoluble antioxidant capacity) was also higher in muscle of channichthyids. This is probably related to the role of ascorbic (a hydrosoluble compound) acid in regenerating vitamin E.
The aim of this work was to investigate the activity of antioxidant enzymes and oxidative damage ... more 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.
We studied Na + /K +-ATPase activity and ultrastructure in gills of the hyper-hypo-regulating cra... more We studied Na + /K +-ATPase activity and ultrastructure in gills of the hyper-hypo-regulating crab Chasmagnathus granulatus Dana, 1851 acclimated to different salinities: 10, 30 and 45&, known to be hypo-, iso-, and hyper-osmotic to the hemolymph, respectively. After centrifugation of homogenates at 11,000 g, Na + / K +-ATPase activity was almost entirely found in the pellets from the posterior (6-8) and anterior (3-5) gills, whereas very little was detected in the supernatant liquid. Specific activity of gill 6 was 41.3, 30.2, and 28.2 lmol Pi h-1 mg prot-1 for crabs acclimated to 10, 30, and 45&, respectively, the result for 10& being significantly higher than those at 30 and 45&. Although the concentration of sodium at which the reaction rate is half-maximal (K M) was similar in the three acclimation salinities, only the enzyme from crabs acclimated to 10& was inhibited by high sodium concentration. Specific activity of gill 5 increased with the increment in external salinity (10.1, 15, and 18.1 lmol Pi h-1 mg prot-1 for 10, 30, and 45&, respectively), the only significant difference being that between the extreme salinities. The epithelium thickness of the dorsal portion of gill 6 showed a variation among salinities: 21.7, 15.8 and 17.2 lm for 10, 30 and 45&, respectively. There were significant differences in epithelium thickness between the 10& and the other salinities. In all three salinities, the ultrastructure of gill 6 epithelium showed a high density of mitochondria, estimated by their volume fraction (Vv m =0.307-0.355). These mitochondria were packed between extensive basolateral membrane interdigitations in ionocytes and pillar cells. Gill 5 showed three cell types: pillars which possess mitochondria packed between membrane folds only in their interdigitations with neighbouring cells; type-I cells 8.0 lm thick with low density of mitochondria (Vv m =0.088), and type-II cells, 9.9 lm thick and rich in mitochondria (Vv m =0.423), but lacking basolateral interdigitations. Vv m of type-I cells of gill 5 was significantly lower than those of type-II cells of the same gill and the ionocytes of gill 6. No significant difference in Vv m was detected between the latter cell types.
Chasmagnathus granulatus is a hyper-hyporegulating crab that inhabits changing habitats ... more 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 1n27 to 2n46 µm, 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 8n10 µm (dorsal zone of gill 8) both in hyper-and hyposaline media compared with seawater. The dark areas measured in gill 8 treated with AgNO $ 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.
GILL MORPHOLOGY OF THE INTERTIDAL ESTUARINE CRAB CHASMAGNATHUS GRANULATUS DANA, 1851 (DECAPODA, G... more GILL MORPHOLOGY OF THE INTERTIDAL ESTUARINE CRAB CHASMAGNATHUS GRANULATUS DANA, 1851 (DECAPODA, GRAPSIDAE) IN RELATION TO HABITAT AND RESPIRATORY HABITS ... CARLOS M. LUQUET1'2), GABRIEL A. ROSA1), CARINA C. FERRARI1), GRISELDA ...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2004
Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills o... more Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10x 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 Amol l À1. 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.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002
We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the c... more We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the crab Chasmagnathus granulatus as a first step to establish this enzyme as a possible biomarker for environmental contamination. We performed a comparative study of crab UroD with the enzyme UroD present in Wistar rat liver, which is known as a useful indicator of intoxication by polyhalogenated aromatic hydrocarbons (PAHs). The final products were the same in crab and rat UroD: the remaining substrate (8-carboxyl-porphyrinogen), the final product Coproporphyrinogen (4-COOH) and intermediate compounds with 7-, 6-and 5-COOH. The elimination of the second carboxyl group seems to be the rate-limiting step in this multiple decarboxylation, because large amounts of 7-COOH porphyrinogen are accumulated. The V yK ratio was 100-fold higher for rat liver UroD than for crab hepatopancreas UroD, suggesting max m a higher efficiency of the rat enzyme. Optimum pH for enzyme activity was 7.2 and 6.8 for crab and rat, respectively. Although both systems showed the same optimum temperature (47 8C), the activation energy was clearly different, 51.5 kJymol for C. granulatus and 5.4 kJymol for Rattus norvegicus (Wistar strain). Superdex 75 gel chromatography yielded a single symmetrical peak with an apparent molecular mass of 48"3 kDa for crab hepatopancreas UroD, suggesting the existence of only one enzymatic species in C. granulatus.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2008
Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused wit... more Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3−] and pH. Elevating [HCO3−] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference ( Vte), a measure of ion transport. The elevation in [HCO3−] also induced a switch from acid secretion (−43.7 ± 22.5 μequiv·kg−1·h−1) in controls to base secretion (84.7 ± 14.4 μequiv·kg−1·h−1). The HCO3−-induced Vteincrease was inhibited by basolateral acetazolamide (200 μmol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vteresponse to HCO3−did not take place in Cl−-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 ± 18.4 μequiv kg−1h−1. ...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2000
Chasmagnathus granulatus is an estuarine crab which actively moves from subtidal to supratidal ar... more 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 mm. 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.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 1999
The passage of a fish meal (15% BaSO,) was followed through the gut system of the blue crab, Call... more The passage of a fish meal (15% BaSO,) was followed through the gut system of the blue crab, Callinectes sapidus, by x-raying crabs at set intervals. Food started to move out of the stomach into the gut within l-2 hr. The stomach was completely emptied between 8-9 br and the gut was emptied at 18 hr. Oxygen uptake was monitored using a flow through respirometer. There was a three fold increase in oxygen uptake immediately upon feeding, and it remained elevated for over 24hr. Heart rate and haemolymph flow through each arterial system of C. sapidus, was measured using a pulsed-Doppler flowmeter, allowing calculation of stroke volume and cardiac output. Heart rate and cardiac output increased during feeding and remained elevated for up to 12 hr after feeding, whilst stroke volume of the heart was unchanged. There was differential haemolymph flow, with the majority being directed to the chelae and mouthparts via the sternal artery, with a smaller increase in flow rates through the anterolateral and hepatic arteries which supply the stomach and digestive gland. Haemolymph flow through the anterior and posterior aortae was more variable. These changes in physiology are discussed in relation to the time course of food passage through the gut system.
The adaptation of Chasmagnathus granulata to air breathing depends on two types of physiological ... more The adaptation of Chasmagnathus granulata to air breathing depends on two types of physiological mechanisms: (1) Biochemical adjustments, comprising the achievement of new steady state values for partial pressure of carbon dioxide (P CO 2) and total carbon dioxide concentration (C CO 2). The initial increase in hemolymph bicarbonate is stabilized by dehydration to CO 2 presumably catalyzed by carbonic anhydrase (CA) at the basolateral side of the gill epithelium. Thus, an adequate transbranchial gradient of P CO 2 is restored. Inhibition of CA with acetazolamide (Az) reduces the rate of CO 2 excretion and elevates P CO 2. The respiratory acidosis caused by increased P CO 2 is compensated by increase in the difference between Na + and Cl − concentrations. Az does not affect hemolymph ionic concentrations. (2) Ventilatory control: untreated animals show a significant decrease in scaphognathite frequency (F SC) during emersion, while Az treated crabs show a slight increase of this variable. F SC of Az crabs tends to decrease with hemolymphatic CO 2 , presumably by clearance of the CA inhibitor from hemolymph. These results suggest that C. granulata possess a ventilatory control based on a primary oxygen-dependent stimulus and a secondary one dependent on CO 2 .
Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clam... more Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clams (Dipolodon chilensis), from a sewage-polluted (SMA) and a clean site, were compared. Trophic markers FA were analyzed in clams and sediment. Saturated FA (SAFA), and bacteria and sewage markers were abundant in SMA sediments, while diatom markers were 50% lower. Proportions of SAFA, branched FA, 20:5n-3 (EPA) and 22:6n-3 (DHA) were higher in SMA clams. Chronic exposure of D. chilensis to increasing eutrophication affected its lipid and FA composition. The increase in EPA and DHA proportions could be an adaptive response, which increases stress resistance but could also lead to higher susceptibility to lipid peroxidation TBARS, lipofuscins (20-fold) and GSH concentrations were higher in SMA clams. FA markers indicated terrestrial plant detritus and bacteria are important items in D. chilensis diet. Anthropogenic input in their food could be traced using specific FA as trophic markers.
We studied the transepithelial potential difference (TEPD) and (22)Na flux across isolated perfus... more 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 c...
We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells o... more We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along w...
SUMMARYUsing quantitative real-time PCR, the expression of mRNAs encoding three transport-related... more SUMMARYUsing quantitative real-time PCR, the expression of mRNAs encoding three transport-related proteins and one putative housekeeping protein was analyzed in anterior and posterior gills of the euryhaline crab Chasmagnathus granulatus following transfer from isosmotic conditions (30‰salinity) to either dilute (2‰) or concentrated (45‰) seawater. Modest changes were observed in the abundance of mRNAs encoding the housekeeping protein arginine kinase and the vacuolar-type H+-ATPase B-subunit, both of which were highly expressed under all conditions. By contrast, the expression of Na+/K+-ATPaseα-subunit mRNA and Na+/K+/2Cl-cotransporter mRNA was strongly responsive to external salinity. During acclimation to dilute seawater, cotransporter mRNA increased 10-20-fold in posterior gills within the first 24 h while Na+/K+-ATPase α-subunit mRNA increased 35-55-fold. During acclimation to concentrated seawater, cotransporter mRNA increased 60-fold by 96 h and Na+/K+-ATPase α-subunit increa...
The development of oil and gas production together with the fruit production in nearby areas of N... more The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors' regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 μg L − 1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L − 1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT− PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8-and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF− CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2-3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.
Antarctic ®sh have been isolated for over several million years in an environment with a very low... more Antarctic ®sh have been isolated for over several million years in an environment with a very low and constant temperature and high oxygen concentration. In such conditions the oxidative stress might be an important factor aecting their metabolic adaptive strategies. Activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), vitamin E levels and total antioxidant capacity (TRAP) were measured in liver, gill, heart and muscle homogenates of red-blooded (Nototheniidae) and white-blooded (Channichthyidae) Antarctic ®sh. SOD activity was also measured in blood samples. Gill SOD activity was threefold higher in channichthyids than in nototheniids while CAT and GPx were signi®cantly higher in the gills of channichthyids. The increased SOD activity of channichthyids probably re¯ects the large PO 2 gradient across their gills. The H 2 O 2 produced seems to be preferentially eliminated by diffusion, according to the low levels of CAT and GPx found in the gills of these species. In contrast, blood SOD was about ®vefold higher in the latter group, which possesses erythrocytes and thus a much higher oxygencarrying capacity. CAT activity was always higher in nototheniids except in muscle. However, vitamin E did not show clear dierences between families except for the pattern observed in muscle. The higher content of vitamin E in this tissue shown in channichthyids is related to the higher volume density of mitochondria reported for this group, since vitamin E is responsible for preventing membrane lipid peroxidation. Accordingly, TRAP (representative of hydrosoluble antioxidant capacity) was also higher in muscle of channichthyids. This is probably related to the role of ascorbic (a hydrosoluble compound) acid in regenerating vitamin E.
The aim of this work was to investigate the activity of antioxidant enzymes and oxidative damage ... more 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.
We studied Na + /K +-ATPase activity and ultrastructure in gills of the hyper-hypo-regulating cra... more We studied Na + /K +-ATPase activity and ultrastructure in gills of the hyper-hypo-regulating crab Chasmagnathus granulatus Dana, 1851 acclimated to different salinities: 10, 30 and 45&, known to be hypo-, iso-, and hyper-osmotic to the hemolymph, respectively. After centrifugation of homogenates at 11,000 g, Na + / K +-ATPase activity was almost entirely found in the pellets from the posterior (6-8) and anterior (3-5) gills, whereas very little was detected in the supernatant liquid. Specific activity of gill 6 was 41.3, 30.2, and 28.2 lmol Pi h-1 mg prot-1 for crabs acclimated to 10, 30, and 45&, respectively, the result for 10& being significantly higher than those at 30 and 45&. Although the concentration of sodium at which the reaction rate is half-maximal (K M) was similar in the three acclimation salinities, only the enzyme from crabs acclimated to 10& was inhibited by high sodium concentration. Specific activity of gill 5 increased with the increment in external salinity (10.1, 15, and 18.1 lmol Pi h-1 mg prot-1 for 10, 30, and 45&, respectively), the only significant difference being that between the extreme salinities. The epithelium thickness of the dorsal portion of gill 6 showed a variation among salinities: 21.7, 15.8 and 17.2 lm for 10, 30 and 45&, respectively. There were significant differences in epithelium thickness between the 10& and the other salinities. In all three salinities, the ultrastructure of gill 6 epithelium showed a high density of mitochondria, estimated by their volume fraction (Vv m =0.307-0.355). These mitochondria were packed between extensive basolateral membrane interdigitations in ionocytes and pillar cells. Gill 5 showed three cell types: pillars which possess mitochondria packed between membrane folds only in their interdigitations with neighbouring cells; type-I cells 8.0 lm thick with low density of mitochondria (Vv m =0.088), and type-II cells, 9.9 lm thick and rich in mitochondria (Vv m =0.423), but lacking basolateral interdigitations. Vv m of type-I cells of gill 5 was significantly lower than those of type-II cells of the same gill and the ionocytes of gill 6. No significant difference in Vv m was detected between the latter cell types.
Chasmagnathus granulatus is a hyper-hyporegulating crab that inhabits changing habitats ... more 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 1n27 to 2n46 µm, 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 8n10 µm (dorsal zone of gill 8) both in hyper-and hyposaline media compared with seawater. The dark areas measured in gill 8 treated with AgNO $ 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.
GILL MORPHOLOGY OF THE INTERTIDAL ESTUARINE CRAB CHASMAGNATHUS GRANULATUS DANA, 1851 (DECAPODA, G... more GILL MORPHOLOGY OF THE INTERTIDAL ESTUARINE CRAB CHASMAGNATHUS GRANULATUS DANA, 1851 (DECAPODA, GRAPSIDAE) IN RELATION TO HABITAT AND RESPIRATORY HABITS ... CARLOS M. LUQUET1'2), GABRIEL A. ROSA1), CARINA C. FERRARI1), GRISELDA ...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2004
Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills o... more Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10x 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 Amol l À1. 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.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002
We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the c... more We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the crab Chasmagnathus granulatus as a first step to establish this enzyme as a possible biomarker for environmental contamination. We performed a comparative study of crab UroD with the enzyme UroD present in Wistar rat liver, which is known as a useful indicator of intoxication by polyhalogenated aromatic hydrocarbons (PAHs). The final products were the same in crab and rat UroD: the remaining substrate (8-carboxyl-porphyrinogen), the final product Coproporphyrinogen (4-COOH) and intermediate compounds with 7-, 6-and 5-COOH. The elimination of the second carboxyl group seems to be the rate-limiting step in this multiple decarboxylation, because large amounts of 7-COOH porphyrinogen are accumulated. The V yK ratio was 100-fold higher for rat liver UroD than for crab hepatopancreas UroD, suggesting max m a higher efficiency of the rat enzyme. Optimum pH for enzyme activity was 7.2 and 6.8 for crab and rat, respectively. Although both systems showed the same optimum temperature (47 8C), the activation energy was clearly different, 51.5 kJymol for C. granulatus and 5.4 kJymol for Rattus norvegicus (Wistar strain). Superdex 75 gel chromatography yielded a single symmetrical peak with an apparent molecular mass of 48"3 kDa for crab hepatopancreas UroD, suggesting the existence of only one enzymatic species in C. granulatus.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2008
Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused wit... more Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3−] and pH. Elevating [HCO3−] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference ( Vte), a measure of ion transport. The elevation in [HCO3−] also induced a switch from acid secretion (−43.7 ± 22.5 μequiv·kg−1·h−1) in controls to base secretion (84.7 ± 14.4 μequiv·kg−1·h−1). The HCO3−-induced Vteincrease was inhibited by basolateral acetazolamide (200 μmol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vteresponse to HCO3−did not take place in Cl−-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 ± 18.4 μequiv kg−1h−1. ...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2000
Chasmagnathus granulatus is an estuarine crab which actively moves from subtidal to supratidal ar... more 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 mm. 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.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 1999
The passage of a fish meal (15% BaSO,) was followed through the gut system of the blue crab, Call... more The passage of a fish meal (15% BaSO,) was followed through the gut system of the blue crab, Callinectes sapidus, by x-raying crabs at set intervals. Food started to move out of the stomach into the gut within l-2 hr. The stomach was completely emptied between 8-9 br and the gut was emptied at 18 hr. Oxygen uptake was monitored using a flow through respirometer. There was a three fold increase in oxygen uptake immediately upon feeding, and it remained elevated for over 24hr. Heart rate and haemolymph flow through each arterial system of C. sapidus, was measured using a pulsed-Doppler flowmeter, allowing calculation of stroke volume and cardiac output. Heart rate and cardiac output increased during feeding and remained elevated for up to 12 hr after feeding, whilst stroke volume of the heart was unchanged. There was differential haemolymph flow, with the majority being directed to the chelae and mouthparts via the sternal artery, with a smaller increase in flow rates through the anterolateral and hepatic arteries which supply the stomach and digestive gland. Haemolymph flow through the anterior and posterior aortae was more variable. These changes in physiology are discussed in relation to the time course of food passage through the gut system.
The adaptation of Chasmagnathus granulata to air breathing depends on two types of physiological ... more The adaptation of Chasmagnathus granulata to air breathing depends on two types of physiological mechanisms: (1) Biochemical adjustments, comprising the achievement of new steady state values for partial pressure of carbon dioxide (P CO 2) and total carbon dioxide concentration (C CO 2). The initial increase in hemolymph bicarbonate is stabilized by dehydration to CO 2 presumably catalyzed by carbonic anhydrase (CA) at the basolateral side of the gill epithelium. Thus, an adequate transbranchial gradient of P CO 2 is restored. Inhibition of CA with acetazolamide (Az) reduces the rate of CO 2 excretion and elevates P CO 2. The respiratory acidosis caused by increased P CO 2 is compensated by increase in the difference between Na + and Cl − concentrations. Az does not affect hemolymph ionic concentrations. (2) Ventilatory control: untreated animals show a significant decrease in scaphognathite frequency (F SC) during emersion, while Az treated crabs show a slight increase of this variable. F SC of Az crabs tends to decrease with hemolymphatic CO 2 , presumably by clearance of the CA inhibitor from hemolymph. These results suggest that C. granulata possess a ventilatory control based on a primary oxygen-dependent stimulus and a secondary one dependent on CO 2 .
Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clam... more Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clams (Dipolodon chilensis), from a sewage-polluted (SMA) and a clean site, were compared. Trophic markers FA were analyzed in clams and sediment. Saturated FA (SAFA), and bacteria and sewage markers were abundant in SMA sediments, while diatom markers were 50% lower. Proportions of SAFA, branched FA, 20:5n-3 (EPA) and 22:6n-3 (DHA) were higher in SMA clams. Chronic exposure of D. chilensis to increasing eutrophication affected its lipid and FA composition. The increase in EPA and DHA proportions could be an adaptive response, which increases stress resistance but could also lead to higher susceptibility to lipid peroxidation TBARS, lipofuscins (20-fold) and GSH concentrations were higher in SMA clams. FA markers indicated terrestrial plant detritus and bacteria are important items in D. chilensis diet. Anthropogenic input in their food could be traced using specific FA as trophic markers.
We studied the transepithelial potential difference (TEPD) and (22)Na flux across isolated perfus... more 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 c...
We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells o... more We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along w...
SUMMARYUsing quantitative real-time PCR, the expression of mRNAs encoding three transport-related... more SUMMARYUsing quantitative real-time PCR, the expression of mRNAs encoding three transport-related proteins and one putative housekeeping protein was analyzed in anterior and posterior gills of the euryhaline crab Chasmagnathus granulatus following transfer from isosmotic conditions (30‰salinity) to either dilute (2‰) or concentrated (45‰) seawater. Modest changes were observed in the abundance of mRNAs encoding the housekeeping protein arginine kinase and the vacuolar-type H+-ATPase B-subunit, both of which were highly expressed under all conditions. By contrast, the expression of Na+/K+-ATPaseα-subunit mRNA and Na+/K+/2Cl-cotransporter mRNA was strongly responsive to external salinity. During acclimation to dilute seawater, cotransporter mRNA increased 10-20-fold in posterior gills within the first 24 h while Na+/K+-ATPase α-subunit mRNA increased 35-55-fold. During acclimation to concentrated seawater, cotransporter mRNA increased 60-fold by 96 h and Na+/K+-ATPase α-subunit increa...
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