Participation of Na+/K+-ATPase and aquaporins in the uptake of water during moult processes in the shrimp Palaemon argentinus (Nobili, 1901).

Due to the presence of the exoskeleton, the moult cycle is a required event in the life of crustaceans. In order for the exoskeleton to be replaced, it is necessary for these animals to uptake water from the environment for their body tissues during the late pre-moult, ecdysis and in the early post-moult for the expansion of the new cuticle. The mechanisms and organs used to uptake water in these events are not yet completely clear. In this study, we investigated the participation of aquaporins and Na+/K+-ATPase in cells of two potential organs responsible for the uptake of water (gills and gut) at three different stages of the moult cycle in freshwater shrimp Palaemon argentinus. We showed the participation of these two proteins with different functional patterns in gills and intestinal cells as water uptake pathways for moult and early post-moult. Our results indicate that Na+/K+-ATPase promotes the necessary osmotic gradient in the gills for water uptake through the gut cells during the pre-moult. This process, in turn, remains active during the post-moult stage with the addition of water influx through the gill cells.

Low salinity negatively affects early larval development of Nile tilapia, Oreochromis niloticus: insights from skeletal muscle and molecular biomarkers.

The present study evaluated the effects of low salinity on the early larval development of Oreochromis niloticus, specifically histological damage to white muscle, morphology of the yolk-sac surface and trunk area, and molecular expression of apoptosis and cell proliferation biomarkers. Newly hatched larvae were submitted to four salinity treatments for a period of 48 or 72 h, in duplicate: (S0) freshwater, (S2) 2 g l-1, (S4) 4 g l-1, and (S6) 6 g l-1NaCl. Larval development was examined using histology, electron microscopy, enzyme-linked immunosorbent assay (ELISA), and morphometry. At the yolk-sac surface, larvae of S4 and S6 displayed alterations to the apical opening of chloride cells that may be related to osmotic expenditure caused by the increased salinity. Caspase-3 expression did not differ significantly among treatments, however significantly lower proliferating cell nuclear antigen (PCNA) expression (P < 0.05) suggested minor cell proliferation in larvae of S4 and S6 compared with S0 and S2. Furthermore, there was a significant reduction in both trunk area and percentage of normal white muscle fibres (WF) in larvae of S4 and S6. Vacuolated areas and myofibrils concentrated at the cell periphery and found in the white muscle from larvae exposed to saline environments suggested disturbance to muscle development. Oedema and mononuclear infiltrate were also observed in the white muscle of S4 and S6 larvae. Together these results indicated that treatments with 4 and 6 g l-1 NaCl may cause osmoregulation expenditure, morphological alterations to the yolk-sac surface and histological damage to skeletal muscle that negatively affected the early larval development of O. niloticus.

Morphofunctional description of mucous cells in the gills of the Arapaimidae Arapaima gigas (Cuvier) during its development.

The gill structure of the Amazonian fish Arapaima gigas (Cuvier 1829) shows ontogenetic changes during development, particularly due the transition from the aquatic to the obligatory air breathing mode of respiration. However, three main cell types can be found in the gills: mitochondrial rich cells, pavement cells and mucous cells (MCs). The MCs are involved in the secretory pathway. The functions of the secreted molecules include mechanical protection of epithelia, protection against parasites and bacterial infection, and role on ion regulation. In this study, we analysed mucous cell location and mucous cell type, based on pH, during the development of A. gigas. Using samples obtained from the environment, gills were collected and fixed in buffered solution. Histological techniques for the identification of MCs were performed Alcian Blue (AB) and periodic acid-Schiff (PAS). The results showed the presence of PAS+ and AB+ cells in the whole filament in all examined fish. In animals less than 50 g, few MCs were present, and no differences were observed in AB+ and PAS+ cells. In animals weighing close to 500 g, more PAS+ cells than AB+ cells were observed, and in animals that weighed more than 1,000 g, more AB+ cells than PAS+ cells were observed. These observations may be a result of the ontogenetic changes in the gill epithelia, which can change the osmorespiratory compromise in ion regulation functions as well the glycosaminoglycans secreted by PAS cells, which in large animals can play a role in the protection against parasites and bacterial infection.

Mitochondria-rich cells changes induced by nitrite exposure in tambaqui (Colossoma macropomum Cuvier, 1818)

ABSTRACT The gill mitochondria-rich cells of the juvenile Amazonian fish Colossoma macropomum were analyzed using light and scanning and transmission electron microscopy after 96 h exposure to 0.04 and 0.2 mM nitrite. Although the number of mitochondria-rich cells decreased significantly in the lamellar epithelium, no decrease was found in the interlamellar region of the gill filament. Nitrite exposure caused significant reduction on the apical surface area of individual mitochondria-rich cells (p < 0.05), with a resulting reduction of the fractional area of these cells in both the lamellar and filament epithelium. Swelling of endoplasmic reticulum cisternae, nuclear envelope and mitochondria were the main changes found in the mitochondria-rich cells. Cristae lysis and matrix vacuolization characterized the mitochondrial changes. The overall ultrastructural changes indicated cellular functional disruption caused by exposure to nitrite. The changes observed in the gill indicate that the cellular structures involved in the process of energy production become severely damaged by exposure to nitrite indicating irreversible damage conducting to cell death.

Mitochondria-rich cells changes induced by nitrite exposure in tambaqui (Colossoma macropomum Cuvier, 1818).

The gill mitochondria-rich cells of the juvenile Amazonian fish Colossoma macropomum were analyzed using light and scanning and transmission electron microscopy after 96 h exposure to 0.04 and 0.2 mM nitrite. Although the number of mitochondria-rich cells decreased significantly in the lamellar epithelium, no decrease was found in the interlamellar region of the gill filament. Nitrite exposure caused significant reduction on the apical surface area of individual mitochondria-rich cells (p < 0.05), with a resulting reduction of the fractional area of these cells in both the lamellar and filament epithelium. Swelling of endoplasmic reticulum cisternae, nuclear envelope and mitochondria were the main changes found in the mitochondria-rich cells. Cristae lysis and matrix vacuolization characterized the mitochondrial changes. The overall ultrastructural changes indicated cellular functional disruption caused by exposure to nitrite. The changes observed in the gill indicate that the cellular structures involved in the process of energy production become severely damaged by exposure to nitrite indicating irreversible damage conducting to cell death.

Characterization of ethoxyresorufin O-deethylase activity (EROD) in oyster Crassostrea brasiliana.

Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30°C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32µM for gills and 5.56µM for digestive gland. EROD Vmax was 337.3fmol·min-1·mg of protein-1 in gills and 297.7fmol·min-1·mg of protein-1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100µM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

Histopathological alterations in the gills of Nile tilapia exposed to carbofuran and multiwalled carbon nanotubes.

Carbofuran is a nematicide insecticide with a broad spectrum of action. Carbofuran has noxious effects in several species and has been banned in the USA and Europe; however, it is still used in Brazil. Aquatic organisms are not only exposed to pesticides but also to manufactured nanoparticles, and the potential interaction of these compounds therefore requires investigation. The aim of this study was to examine the histopathological alterations in the gills of Nile tilapia (Oreochromis niloticus) to determine possible effects of exposure to carbofuran, nitric acid-treated multiwalled carbon nanotubes (HNO3-MWCNTs) and the combination of carbofuran with nanotubes. Juvenile fish were exposed to different concentrations of carbofuran (0.1, 0.5, 2.0, 4.0 and 8.0mg/L), different concentrations of HNO3-MWCNTs (0.5, 1.0 and 2.0mg/L) or different concentrations of carbofuran (0.1, 0.5, 2.0, 4.0 and 8.0mg/L) with 1.0mg/L of HNO3-MWCNTs. After 24h of exposure, the animals were removed from the aquarium, the spinal cord was transversely sectioned, and the second gill arch was removed for histological evaluation. Common histological changes included dislocation of the epithelial cells, hyperplasia of the epithelial cells along the secondary lamellae, aneurism, and dilation and disarrangement of the capillaries. All the groups exposed to carbofuran demonstrated a dose-dependent correlation in the Histological Alteration Index; the values found for carbofuran and carbon nanotubes were up to 25% greater than for carbofuran alone. This result indicates an interaction between these toxicants, with enhanced ecotoxic effects. This work contributes to the understanding of the environmental impacts of nanomaterials on aquatic organisms, which is necessary for the sustainable development of nanotechnologies.

Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.

Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 µg L(-1). Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-π-like), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock protein-like (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 µg L(-1). A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COX-like, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 µg L(-1) for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 µg L(-1)) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.

Genotoxic effects of the herbicide Roundup Transorb and its active ingredient glyphosate on the fish Prochilodus lineatus.

Roundup Transorb (RT) is a glyphosate-based herbicide and despite its wide use around the world there are few studies comparing the effects of the active ingredient with the formulated product. In this context the purpose of this study was to compare the genotoxicity of the active ingredient glyphosate with the formulated product RT in order to clarify whether the active ingredient and the surfactant of the RT formula may exert toxic effects on the DNA molecule in juveniles of fish Prochilodus lineatus. Erythrocytes and gill cells of fish exposed to glyphosate and to RT showed DNA damage scores significantly higher than control animals. These results revealed that both glyphosate itself and RT were genotoxic to gill cells and erythrocytes of P. lineatus, suggesting that their use should be carefully monitored considering their potential impact on tropical aquatic biota.

Zebrafish and mouse TASK-2 K(+) channels are inhibited by increased CO2 and intracellular acidification.

TASK-2 is a K2P K(+) channel considered as a candidate to mediate CO2 sensing in central chemosensory neurons in mouse. Neuroepithelial cells in zebrafish gills sense CO2 levels through an unidentified K2P K(+) channel. We have now obtained zfTASK-2 from zebrafish gill tissue that is 49 % identical to mTASK-2. Like its mouse equivalent, it is gated both by extra- and intracellular pH being activated by alkalinization and inhibited by acidification. The pHi dependence of zfTASK-2 is similar to that of mTASK-2, with pK 1/2 values of 7.9 and 8.0, respectively, but pHo dependence occurs with a pK 1/2 of 8.8 (8.0 for mTASK-2) in line with the relatively alkaline plasma pH found in fish. Increasing CO2 led to a rapid, concentration-dependent (IC50 ~1.5 % CO2) inhibition of mouse and zfTASK-2 that could be resolved into an inhibition by intracellular acidification and a CO2 effect independent of pHi change. Indeed a CO2 effect persisted despite using strongly buffered intracellular solutions abolishing any change in pHi, was present in TASK-2-K245A mutant insensitive to pHi, and also under carbonic anhydrase inhibition. The mechanism by which TASK-2 senses CO2 is unknown but requires the presence of the 245-273 stretch of amino acids in the C terminus that comprises numerous basic amino acids and is important in TASK-2 G protein subunit binding and regulation of the channel. The described CO2 effect might be of importance in the eventual roles played by TASK-2 in chemoreception in mouse and zebrafish.

Histopatologia das brânquias deTilápia do Nilo Oreochromis Niloticus, provenientes da Represa Billings, área de proteção ambiental Bororé-Colônia / Histopathology of Nile Tilapia gills Oreochromis niloticus from Billings Dam in the environmental protection area Bororé-Colônia

O presente trabalho teve como objetivo evidenciar alterações morfológicas nas brânquias de Tilápia do Nilo (Oreochromis niloticus) da Represa Billings. Foram utilizados dez animais, sendo cinco provenientes da Represa Billings e cinco provenientes dos tanques comerciais da Royal Fish. Prepararam-se lâminas histológicas para análise morfométrica e quantitativa por meio do Índice de Alteração Histológica (I.A.H.). O I.A.H. apresentou diferença estatística significativa (p<0,05) entre os grupos, o que permitiu a conclusão de que os poluentes afetaram histologicamente as brânquias dos animais provenientes da Represa Billings e que esses animais estão parcialmente adaptados ao ambiente.

The aim of this study is to show morphological changes in the gills of Nile Tilapia (Oreochromis niloticus) at Billings Dam. Ten specimens were used, five of which came from the Billings Dam and five from Royal Fish commercial aquaculture tanks. Slides were prepared for morphometric and quantitative analysis of histological sections. There was a significant statistical difference (p<0.05) in the histological alteration index, which led us to conclude that pollutants are affecting Tilapia gills histologically and these animals are partially adapted to the environment.

Histopatologia das brânquias deTilápia do Nilo Oreochromis Niloticus, provenientes da Represa Billings, área de proteção ambiental Bororé-Colônia / Histopathology of Nile Tilapia gills Oreochromis niloticus from Billings Dam in the environmental protection area Bororé-Colônia

O presente trabalho teve como objetivo evidenciar alterações morfológicas nas brânquias de Tilápia do Nilo (Oreochromis niloticus) da Represa Billings. Foram utilizados dez animais, sendo cinco provenientes da Represa Billings e cinco provenientes dos tanques comerciais da Royal Fish. Prepararam-se lâminas histológicas para análise morfométrica e quantitativa por meio do Índice de Alteração Histológica (I.A.H.). O I.A.H. apresentou diferença estatística significativa (p<0,05) entre os grupos, o que permitiu a conclusão de que os poluentes afetaram histologicamente as brânquias dos animais provenientes da Represa Billings e que esses animais estão parcialmente adaptados ao ambiente.(AU)

The aim of this study is to show morphological changes in the gills of Nile Tilapia (Oreochromis niloticus) at Billings Dam. Ten specimens were used, five of which came from the Billings Dam and five from Royal Fish commercial aquaculture tanks. Slides were prepared for morphometric and quantitative analysis of histological sections. There was a significant statistical difference (p<0.05) in the histological alteration index, which led us to conclude that pollutants are affecting Tilapia gills histologically and these animals are partially adapted to the environment.(AU)

Mutagenicity and genotoxicity in gill erythrocyte cells of Poecilia reticulata exposed to a glyphosate formulation.

Poecilia reticulata were exposed to herbicide Roundup Transorb(®) for micronucleus test, nuclear abnormalities and comet assay. The exposure-concentrations were based on CL50-96 h following 0, 1.41, 2.83, 4.24 and 5.65 µL L(-1) for 24 h. Micronucleus and comets were significantly increased in the gill erythrocyte cells after herbicide exposure compared with the non-exposed group. Results showed a gradual increase in the number of damaged cells, indicating a concentration-dependent effect and that this herbicide was mutagenic and genotoxic to P. reticulata and this effect could be attributed to a combination of compounds contained in the formulation with the active ingredient glyphosate.

Calcium transport in gill cells of Ucides cordatus, a mangrove crab living in variable salinity environments.

Crustaceans show discontinuous growth and have been used as a model system for studying cellular mechanisms of calcium transport, which is the main mineral found in their exoskeleton. Ucides cordatus, a mangrove crab, is naturally exposed to fluctuations in calcium and salinity. To study calcium transport in this species during isosmotic conditions, dissociated gill cells were marked with fluo-3 and intracellular Ca(2+) change was followed by adding extracellular Ca(2+) as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5mM), together with different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with Vmax=0.137±0.001 ∆Ca(2+)i (µM×22.10(4)cells(-1)×180s(-1); N=4; r(2)=0.99); Km=0.989±0.027mM. The use of different inhibitors for gill cells showed that amiloride (Na(+)/Ca(2+) exchange inhibitor) inhibited 80% of Ca(2+) transport in gill cells (Vmax). KB-R, an inhibitor of Ca influx in vertebrates, similarly caused a decrease in Ca(2+) transport and verapamil (Ca(2+) channel inhibitor) had no effect on Ca(2+) transport, while nifedipine (another Ca(2+) channel inhibitor) caused a 20% decrease in Ca(2+) affinity compared to control values. Ouabain, on the other hand, caused no change in Ca(2+) transport, while vanadate increased the concentration of intracellular calcium through inhibition of Ca(2+) efflux probably through the plasma membrane Ca(2+)-ATPase. Results show that transport kinetics for Ca(2+) in these crabs under isosmotic conditions is lower compared to a hyper-regulator freshwater crab Dilocarcinus pagei studied earlier using fluorescent Ca(2+) probes. These kinds of studies will help understanding the comparative mechanisms underlying the evolution of Ca transport in crabs living in different environments.

Organochlorines and metals induce changes in the mitochondria-rich cells of fish gills: an integrative field study involving chemical, biochemical and morphological analyses.

Through integrating chemical, biochemical and morphological analyses, this study investigated the effects of multiple pollutants on the gill mitochondria-rich cells (MRCs) in two fish species, Astyanax fasciatus and Pimelodus maculatus, collected from five sites (FU10, FU20, FU30, FU40 and FU50) in the Furnas Hydroelectric Power Station reservoir. Water analyses revealed aluminum, iron and zinc as well as organochlorine (aldrin/dieldrin, endosulfan, heptachlor/heptachlor epoxide and metolachlor) contamination at all of the sites, with the exception of FU10. Copper, chrome, iron and zinc were detected in the gills of both species, and aldrin/dieldrin, endosulfan and heptachlor/heptachlor epoxide were detected in the gills of fish from all of the sites, with the exception of FU10. Fish collected at FU20, FU30 and FU50 exhibited numerous alterations in the surface architecture of their pavement cells and MRCs. The surface MRC density and MRC fractional area were lower in fish from FU20, FU30, FU40 and FU50 than in those from the reference site (FU10) in the winter, and some variability between the sites was observed in the summer. The organochlorine contamination at FU20 and FU50 was associated with variable changes in the MRCs and inhibition of Na(+)/K(+)-ATPase (NKA) activity, especially in P. maculatus. At FU30, the alterations in the MRCs were associated with the contaminants present, especially metals. A multivariate analysis demonstrated a positive association between the biological responses of both species and environmental contamination, indicating that under realistic conditions, a mixture of organochlorines and metals affected the MRCs by inhibiting NKA activity and inducing morphological changes, which may cause an ionic imbalance.

Lead hampers gill cell volume regulation in marine crabs: stronger effect in a weak osmoregulator than in an osmoconformer.

Hepatus pudibundus is a strictly marine osmoconformer crab, while Callinectes ornatus inhabits estuarine areas, behaving as a weak hyper-osmoregulator in diluted seawater. Osmoconformers are expected to have higher capacity for cell volume regulation, but gill cells of a regulator are expected to display ion transporters to a higher degree. The influence of lead nitrate (10 µM) on the ability of isolated gill cells from both species to volume regulate under isosmotic and hyposmotic conditions were here evaluated. Without lead, under a 25% hyposmotic shock, the gill cells of both species were quite capable of cell volume maintenance. Cells of C. ornatus, however, had a little swelling (5%) during the hyposmotic shock of greater intensity (50%), while cells of H. pudibundus were still capable of volume regulation. In the presence of lead, even under isosmoticity, the gill cells of both species showed about 10% volume reduction, indicating that lead promotes the loss of water by the cells. When lead was associated with 25% and 50% hyposmotic shock, C. ornatus cells lost more volume (15%), when compared to isosmotic conditions, while H. pudibundus cells showed volume regulation. We then analyzed the possible ways of action of lead on the mechanisms of cell volume regulation in the two species. Verapamil (100 µM) was used to inhibit Ca²âº channels, ouabain (100 µM) to inhibit Na⁺/K⁺-ATPase, and HgCl2 (100 µM) to inhibit aquaporins. Our results suggest that: (1) Ca²âº channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus, being the target of lead action in these cells; (2) aquaporins are much more relevant for water flux in H. pudibundus; and (3) the Na⁺/K⁺-ATPase is much more relevant for volume regulation in C. ornatus. Osmoregulators may be more susceptible to metal contamination than osmoconformers, especially in situations of reduced salinity, for two basic reasons: (1) lower capacity of volume regulation and (2) putative higher uptake of Pb²âº through ionic pathways that operate in salt absorption, such as, for example, the Na⁺/K⁺-ATPase.

The influence of lead on different proteins in gill cells from the freshwater bivalve, Corbicula fluminea, from defense to repair biomarkers.

The objective of this study was to evaluate the influence of lead (Pb) on regulatory proteins linked to mechanisms of animal adaptation to polluted environments (using in vivo and in vitro tests) and to validate the in vitro assay as a tool for environmental assessment. Specimens of the bivalve Corbicula fluminea were exposed to nominal concentrations of Pb 5 mg l(-1) for 96 h. Isolated gill cells were exposed to three concentrations (1, 10, and 100 µM) for 5 h. Metal toxicity was evaluated by cell viability (trypan blue exclusion). We also analyzed Na+/K+ adenosine triphosphatase (ATPase) and carbonic anhydrase activity. Additionally, the multixenobiotic-resistance (MXR) phenotype was evaluated by the accumulation of rhodamine B (RB). Immunolabeling was used to quantify the expression of P-glycoproteins (C219) and proteins involved in ion transport, water movement, and cellular repair using antibodies against Na+/K+ ATPase, aquaporin 1, and heat-shock protein 70 (Hsp70). Pb was shown to be toxic in both in vivo and in vitro tests, in which cellular viability significantly decreased by approximately 25%. Cellular viability in the in vivo assays was determined by gill cell isolation after the entire animal was exposed to Pb. We observed that Na+/K+ ATPase activity was inhibited by 70%. Also, the expression of the MXR phenotype significantly increased in our in vivo tests. A statistically significant difference was observed in the expression of all proteins in the in vitro assays, whereas only Hsp70 increased in vivo. Employing these analyses, we could validate the sensitivity of the in vitro tests and can propose our in vitro model as a possible tool for environmental assessment.

New aspects concerning to the characterization and the relationship with the immune response in vivo of the spiny lobster Panulirus argus nitric oxide synthase.

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.

Separation and viability of gill and hepatopancreatic cells of a mangrove crab Ucides cordatus.

The gills contain essential cells for respiration and osmoregulation, whereas the hepatopancreas is the site of digestion, absorption, and nutrients storage. The aim of this work was to separate and characterize gill and hepatopancreatic cells of the mangrove crab, Ucides cordatus. For gills, the methodology consisted of an enzymatic cellular dissociation using Trypsin at 0.5%, observation of cellular viability with Tripan Blue, and separation of cells using discontinuous sucrose gradient at concentrations of 10%, 20%, 30%, and 40%. The hepatopancreatic cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at the same concentrations above. For gills, a high cellular viability was observed (92.5±2.1%), with hemocyte cells in 10% sucrose layer (57.99 ± 0.17%, *P < 0.05), principal cells in the 20% sucrose layer (57.33 ± 0.18, *P < 0.05), and thick cells and pillar cells in the 30% and 40% sucrose layers, respectively (39.54 ± 0.05%, *P < 0.05; and 41.81 ± 0.04%, *P < 0.05). The hepatopancreatic cells also showed good viability (79.22 ± 0.02%), with the observation of embryonic (E) cells in the 10% sucrose layer (67.87 ± 0.06%, **P < 0.001), resorptive (R) and fibrillar (F) cells in the 20% and 30% sucrose layers (44.71 ± 0.06%, **P < 0.001, and 43.25 ± 0.01%, *P < 0.05; respectively), and blister (B) cells in the 40% sucrose layer (63.09 ± 0.03%, **P < 0.001). The results are a starting point for in vitro studies of heavy metal transport in isolated cells of the mangrove crab U. cordatus, subjected to contamination by metals in the mangrove habitat where they are found.