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Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes ( aggR , aatA , aaiC , and aap ). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet , pic , sat , sepA , and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli . The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl 2 -induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Correa, Freire, Dibo, Huerta-Cantillo, Navarro-Garcia, Barbosa, Elias and Moraes.)

Decapods occupy all aquatic, and terrestrial and semi-terrestrial environments. According to their osmoregulatory capacity, they can be osmoconformers or osmoregulators (hypo or hyperegulators). The goal of this study is to gather data available in the literature for aquatic decapods and verify if the rare hyporegulatory capacity of decapods is associated with hyper-regulatory capacity. The metric used to quantify osmoregulation was the osmotic capacity (OC), the gradient between external and internal (hemolymph) osmolalities. We employ phylogenetic comparative methods using 83 species of decapods to test the correlation between hyper OC and hypo OC, beyond the ancestral state for osmolality habitat, which was used to reconstruct the colonization route. Our analysis showed a phylogenetic signal for habitat osmolality, hyper OC and hypo OC, suggesting that hyper-hyporegulators decapods occupy similar habitats and show similar hyper and hyporegulatory capacities. Our findings reveal that all hyper-hyporegulators decapods (mainly shrimps and crabs) originated in estuarine waters. Hyper OC and hypo OC are correlated in decapods, suggesting correlated evolution. The analysis showed that species which inhabit environments with intense salinity variation such as estuaries, supratidal and mangrove habitats, all undergo selective pressure to acquire efficient hyper-hyporegulatory mechanisms, aided by low permeabilities. Therefore, hyporegulation can be observed in any colonization route that passes through environments with extreme variations in salinity, such as estuaries or brackish water., (© 2023 Wiley Periodicals LLC.)

Hepatus pudibundus ("flecked box crab") is a stenohaline osmoconfomer, and restricted to marine habitats. Callinectes danae ("swimming crab Dana") lives in coastal/estuarine waters and is a weak hyper regulator. There is no consensus on which strategy is more expensive metabolically face salinity challenges: conformation with higher dependence on cell volume regulation, or hyper regulation, alleviating the need for intense cell volume regulation. Crabs were probed for their acute response to dilute seawater through exposures to salinities 35‰, 30‰, 25‰, and 20‰ for 2, 4, and 6 h. Hemolymph osmolality, lactate, and ions (chloride, sodium, magnesium, potassium) were assayed, as well as muscle water content. Water dissolved oxygen, ammonia, and pH levels were also measured. H. pudibundus conformed for osmolality and displayed increase in muscle hydration along the decrease in salinity down to 25‰, while C. danae efficiently maintained hemolymph osmo ionic stability, consumed more oxygen, acidified more the water, and released more ammonia. In 25‰, both species spent energy: H. pudibundus putatively controlling cell volume, and C. danae regulating hemolymph concentrations. In 20‰, H. pudibundus closed itself, avoiding the contact of the interface epithelia with the external environment and producing much lactate, whereas C. danae spent more energy (aerobic) in extracellular osmo ionic stability. Under these conditions, anisosmotic extracellular regulation (together with additional cell volume regulation) is more oxygen consuming than osmoconformation with a putatively more intense challenge to cell volume. The exposure to hyposalinity limits the occupation of estuarine environments by H. pudibundus in short and middle term., (© 2023 Wiley Periodicals LLC.)

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in children and adults worldwide. This pathotype is phenotypically characterized by the aggregative-adherence (AA) pattern in HEp-2 cells and genetically associated to the presence of the aatA gene. EAEC pathogenesis relies in different virulence factors. At least, three types of adhesins have been specifically associated with EAEC strains: the five variants of the aggregative adherence fimbriae (AAF), the aggregative forming pilus (AFP) and more recently, a fibrilar adhesin named CS22. Our study aimed to evaluate the presence of AAF, AFP and CS22-related genes among 110 EAEC strains collected from feces of children with diarrhea. The presence of aggR (EAEC virulence regulator) and genes related to AAFs ( aggA, aafA , agg3A , agg4A , agg5A and agg3/4C ), AFP ( afpA1 and afpR ) and CS22 ( cseA ) was detected by PCR, and the adherence patterns were evaluated on HeLa cells. a ggR -positive strains comprised 83.6% of the collection; among them, 80.4% carried at least one AAF-related gene and presented the AA pattern. aggA was the most frequent AAF-related gene (28.4% of aggR + strains). cseA was detected among aggR + (16.3%) and aggR - strains (22.2%); non-adherent strains or strains presenting AA pattern were observed in both groups. afpR and afpA1 were exclusively detected among aggR - strains (77.8%), most of which (71.4%) also presented AA pattern. Our results indicate that AAF- and AFP-related genes may contribute to identify EAEC strains, while the presence of cseA and its importance as an EAEC virulence factor and genotypic marker needs to be further evaluated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Freire, Rodrigues, Elias and Abe.)

Early marine invertebrates like the Branchiopoda began their sojourn into dilute media some 500 million years ago in the Middle Cambrian. Others like the Mollusca, Annelida, and many crustacean taxa have followed, accompanying major marine transgressions and regressions, shifting landmasses, orogenies, and glaciations. In adapting to these events and new habitats, such invertebrates acquired novel physiological abilities that attenuate the ion loss and water gain that constitute severe challenges to life in dilute media. Among these taxon-specific adaptations, selected from the subcellular to organismal levels of organization, and constituting a feasible evolutionary blueprint for invading freshwater, are reduced body permeability and surface (S) to volume (V) ratios, lowered osmotic concentrations, increased osmotic gradients, increased surface areas of interface epithelia, relocation of membrane proteins in ion-transporting cells, and augmented transport enzyme abundance, activity, and affinity. We examine these adaptations in taxa that have penetrated into freshwater, revealing diversified modifications, a consequence of distinct body plans, morpho-physiological resources, and occupation routes. Contingent on life history and reproductive strategy, numerous patterns of osmotic regulation have emerged, including intracellular isosmotic regulation in weak hyper-regulators and well-developed anisosmotic extracellular regulation in strong hyper-regulators, likely reflecting inertial adaptations to early life in an estuarine environment. In this review, we address osmoregulation in those freshwater invertebrate lineages that have successfully invaded this biotope. Our analyses show that across 66 freshwater invertebrate species from six phyla/classes that have transmuted into freshwater from the sea, hemolymph osmolalities decrease logarithmically with increasing S:V ratios. The arthropods have the highest osmolalities, from 300 to 650 mOsmoles/kg H2O in the Decapoda with 220-320 mOsmoles/kg H2O in the Insecta; osmolalities in the Annelida range from 150 to 200 mOsmoles/kg H2O, and the Mollusca showing the lowest osmolalities at 40-120 mOsmoles/kg H2O. Overall, osmolalities reach a cut-off at ∼200 mOsmoles/kg H2O, independently of increasing S:V ratio. The ability of species with small S:V ratios to maintain large osmotic gradients is mirrored in their putatively higher Na+/K+-ATPase activities that drive ion uptake processes. Selection pressures on these morpho-physiological characteristics have led to differential osmoregulatory abilities, rendering possible the conquest of freshwater while retaining some tolerance of the ancestral medium., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Climate change is causing habitat salinity to transform at unprecedented rates across the globe. While much of the research on climate change has focused on rapid shifts in temperature, far less attention has focused on the effects of changes in environmental salinity. Consequently, predictive studies on the physiological, evolutionary, and migratory responses of organisms and populations to the threats of salinity change are relatively lacking. This omission represents a major oversight, given that salinity is among the most important factors that define biogeographic boundaries in aquatic habitats. In this perspective, we briefly touch on responses of organisms and populations to rapid changes in salinity occurring on contemporary time scales. We then discuss factors that might confer resilience to certain taxa, enabling them to survive rapid salinity shifts. Next, we consider approaches for predicting how geographic distributions will shift in response to salinity change. Finally, we identify additional data that are needed to make better predictions in the future. Future studies on climate change should account for the multiple environmental factors that are rapidly changing, especially habitat salinity., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Freire, Silva, Ruiz, Pimenta, Bryant, Henderson, Barbosa and Elias.)

Decapod crustaceans show variable degrees of euryhalinity and osmoregulatory capacity, by responding to salinity changes through anisosmotic extracellular regulation and/or cell volume regulation. Cell volume regulatory mechanisms involve exchange of inorganic ions between extra- and intra-cellular (tissue) compartments. Here, this interplay of inorganic ions between both compartments has been evaluated in four decapod species with distinct habitats and osmoregulatory strategies. The marine/estuarine species Litopenaeus vannamei (Lv) and Callinectes danae (Cd) were submitted to reduced salinity (15‰), after acclimation to 25 and 30‰, respectively. The freshwater Macrobrachium acanthurus (Ma) and Aegla schmitti (As) were submitted to increased salinity (25‰). The four species were salinity-challenged for both 5 and 10 days. Hemolymph osmolality, sodium, chloride, potassium, and magnesium were assayed. The same inorganic ions were quantified in muscle samples. Muscle hydration (MH) and ninhydrin-positive substances (NPS) were also determined. Lv showed slight hemolymph dilution, increased MH and no osmotically-relevant decreases in muscle osmolytes; Cd displayed hemolymph dilution, decreased muscular NaCl and stable MH; Ma showed hypo-regulation and steady MH, with no change in muscle ions; As conformed hemolymph sodium but hypo-regulated chloride, had stable MH and increased muscle NPS and ion levels. Hemolymph and muscle ions (especially chloride) of As were highly correlated (Pearson, +0.83). Significant exchanges between hemolymph and muscle ionic pools were more evident in the two species with comparatively less AER regulatory power, C. danae and A. schmitti. Our findings endorse that the interplay between extracellular and tissue ionic pools is especially detectable in euryhaline species with relatively lower osmoregulatory strength., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ray-finned fishes of the superorder Ostariophysi are primarily freshwater (FW), and normally stenohaline. Differently, fishes of the superorder Acanthopterygii are essentially marine, and frequently euryhaline, with some secondary FW. Na + /K + -ATPase-immunoreactive ionocytes were localized in the branchial epithelia of 4 species of Ostariophysi and 3 of Acanthopterygii. The Ostariophysi grass carp (Ctenopharyngodon idella, Cypriniformes), twospot Astyanax (Astyanax bimaculatus) and piracanjuba (Brycon orbignyanus), Characiformes, and the jundiá (Rhamdia quelen, Siluriformes), all from FW, displayed ionocytes in the filament plus secondary lamellae (F + SL). In their turn, all the three species of Acanthopterygii showed immunoreactive ionocytes in the filaments only (F). They were the Nile tilapia (Oreochromis niloticus, Cichliformes) in FW, the dog snapper (Lutjanus jocu, Perciformes) in seawater (SW), and the green puffer (Sphoeroides greeleyi, Tetraodontiformes) in SW. Ionocytes normally extend their distribution to the secondary lamellae (F + SL) in Ostariophysi. In Acanthopterygii, we find more plasticity: ionocytes are more frequently restricted to the filament in SW, but also spread to SL in FW. It may be that the occurrence of ionocytes in SL is the ancestral condition, but some euryhaline acanthopterygians rely on the space of the SL for placement of additional ionocytes when in FW absorbing salt. Our study contributed to the identification of the pattern of ionocyte distribution in gills of Ostariophysi in respect to that of Acanthopterygii., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The term "allostasis", meaning the assumption that homeostasis may not be as static as the term implies, has been vastly employed for mammals, and other vertebrates, for which the degree of internal stability is maximal, according to their higher complexity. We have here investigated how these states of homeostasis, allostasis, and allostatic overload could be diagnosed in decapod crustaceans, upon acute salinity challenges. Decapods of distinct lineages and habitats have been submitted to 3 salinity levels for 6 and 12 h. The first salinity was the habitat salinity (control), considered as the one that allows the homeostatic condition. The next salinity represented a mild challenge, that would potentially lead to allostasis, and the third salinity was intended to represent an overload, albeit not lethal. Species used were: the marine crab Hepatus pudibundus (Hp, osmoconformer, salinities 33, 25, and 20‰), the marine/estuarine swimming crab Callinectes danae (Cd, weak regulator, salinities 30, 20, and 10‰), and the diadromous freshwater prawn Macrobrachium acanthurus (Ma, strong regulator, salinities <0.5, 15, and 30‰). These 3 species follow a sequence of growing regulatory capacity (Hp

Polychaete worms are widespread and diverse in marine and estuarine habitats subject to varying salinity, in areas influenced by tides, demanding physiological adjustment for internal homeostasis. They are typically considered and reported to be osmoconformers, but they are not often studied for their osmoregulation. Here, three species of polychaete worms from distinct coastal habitats have been investigated: the spionid Scolelepis goodbody (intertidal in saline, exposed sandy beaches), the nereidid Laeonereis culveri (estuarine polyhaline), and the nephtyid Nephtys fluviatilis (estuarine oligohaline). The general objective here was to relate ecological aspects and physiology of the studied species. Constitutive whole body osmolality and carbonic anhydrase activity (CAA, relevant for osmoregulation, acid-base balance and respiration) have been assayed. In addition, cell volume regulatory capacity (from whole body cell dissociation) was challenged under hypoosmotic and hyperosmotic shocks (50% intensity), with respect to isosmotic control. S. googdbody and L. culveri, the two species from most saline environments (marine/estuarine), showed higher CAA than N. fluviatilis, which, in turn, displayed a hyperosmotic gradient to water of salinity 15. Cells from S. goodbody and L. culveri showed regulatory volume decrease upon swelling, with S. goodbody showing the largest volume increase. As in other more studied marine invertebrate groups, polychaetes also show variability in their osmoregulatory physiology, related to distinct saline challenges faced in their coastal habitats., (© 2020 Wiley Periodicals, Inc.)

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Aeglidae anomuran crabs originated in the sea, but invaded and diversified in southern South American freshwater (FW) streams. We here aimed at examining their tolerance of increased salinity, after a long time of evolution in FW (~33 million years). Aegla schmitti were exposed to FW and dilute seawater of salinities 15, 20, and 25‰ for 1, 5 and 10 days. Mortality in 35‰ was also assessed. Hemolymph osmolality, Na + , K + , Cl - , and Mg 2+ ions, and hydration levels of the abdominal muscle were assayed. The activities of the Carbonic Anhydrase (CA), Na + /K + -ATPase (NKA) and V-H + -ATPase (VHA) were also assayed in the gills. A. schmitti preserves osmoregulatory mechanisms of its marine ancestors. It is able to survive in high salinities (25‰) for at least 10 days. Mortality in 35‰ was of 56% after 1 day, and of 100% after 7 days. In 25‰, NaCl is apparently hyporegulated at all times, while hemolymph osmolality rises after 5 days. CA and NKA activities remained unchanged in all experimental conditions, while VHA activity decreased after 10 days in 25‰. Hemolymph NaCl data was compatible with either hyporegulation and/or putative influx of NaCl into cells for regulatory volume increase (RVI). Further studies should deepen the understanding of the roles of low permeabilities and saturation of high affinity uptake systems in truly FW decapods, in their responses to high salinities. Moreover, the fate of extracellular NaCl as secretion in true hypo-regulation and/or influx into cells for RVI should also be investigated., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study aimed to characterize morphological aspects related to abortion through a non-lethal approach in the shortnose guitarfish Zapteryx brevirostris, an endemic and threatened species commonly caught by artisanal fisheries. Two females with signs of abortion and one female exhibiting external signs of pregnancy were purchased alive at a fish market during the period when this species has developing embryos in southern Brazil. Scans were conducted using a portable ultrasound. Females with signs of abortion revealed an absence of embryos despite having a similar morphology of the uterus when compared to the pregnant female. Examination of the pregnant female revealed the presence of two embryos, measuring c. 100 mm, each detected through their midline. This study presents new data on uterine macromorphology following successive abortive events in a viviparous elasmobranch species and validates ultrasonography as a diagnostic tool for the species. Ultrasonography is an effective, non-lethal and less-invasive methodology that is recommended for use in future studies of abortion and other reproductive events in elasmobranchs., (© 2018 The Fisheries Society of the British Isles.)

The use of anesthetic in fish farming is a traditional practice which aims to reduce the stress caused by transport and handling. However, anesthesia-induction protocols are commonly established and implemented without proper physiological/behavioral evaluation. Additionally, concentration and time of exposure to the anesthetic are often set without considering species-specific responses. The fat snook (Centropomus parallelus) is a fish with great potential for aquaculture. Given its remarkable euryhalinity, it can grow in fresh- or seawater. Most studies on fat snook anesthesia tested natural compounds (essential oils) instead of traditional anesthetics. However, the use of benzocaine is much more common in the commercial sector, as it is easy to obtain and of relatively low cost. The present study aimed at analyzing the effects benzocaine exposure on glucose and cortisol plasma levels (two traditional stress markers in teleost fish), as well as on plasma osmolality, chloride and magnesium, (indicators of osmo-ionic allostasis) in animals acclimated to different salinities. Results showed that while osmo-ionic allostasis was strictly maintained across the treatments, time of anesthesia had a strong positive relationship to plasma cortisol and glucose, regardless the salinity of exposure and acclimation. The results are discussed as they relate to anesthesia protocols and how stress response generated by time of anesthesia may challenge farming flexibility., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Fish physiology is significantly affected by temperature variability. During fisheries interactions, fish are often exposed to air and subjected to rapid temperature changes. Fish thermal dynamics during such exposure, and the possible outcomes to their physiology, depend on how heat is distributed across their bodies, the speed at which their body temperatures change, and the size of the individual. Nevertheless, such thermal patterns remain unknown for sharks. This study employed a novel application of thermal imaging to evaluate external body temperature profiles of blacktip sharks (Carcharhinus limbatus) above-water exposure after capture. We found that above-water exposure duration, shark total length, and air temperature on the day of capture significantly influenced body surface temperatures of the analyzed sharks ([Formula: see text]). Body surface temperature significantly increased with increasing exposure; however, thermal profiles of immature sharks (<140 cm) were significantly warmer than those of mature sharks. Moreover, blacktip surface body temperatures were significantly higher during days when air temperatures were at least 2.5°C warmer than water temperatures. We discuss these results as they relate to the ecology of blacktip sharks and their potential vulnerability to fisheries capture due to such changes in peripheral body temperature.

Some Macrobrachium shrimps (Caridea, Palaemonidae) are diadromous; freshwater adults are truly euryhaline, while larvae need saline water for development. Branchial Na + /K + -ATPase (NKA) and carbonic anhydrase (CA) are involved in NaCl absorption in freshwater. This study aimed at verifying the time course of the osmoregulatory response of adult Macrobrachium acanthurus to high salinity brackish water (20‰), from the first 30min to 5days. The goal was to detect possible transition from hyper- to hyporegulation, the putative involvement of branchial NKA and CA, or the induction of muscular HSP70 expression. Hemolymph osmotic and ionic concentrations remained relatively stable and close to control levels until ~9h of exposure, but later increased consistently (~50%). A fast reduction in NKA activity (3-6h) was observed; these shrimps seem to shut off salt absorption already in the first hours. Later on, especially after 24h, hemolymph concentrations rise but HSP70 expression is not induced, possibly because constitutive levels are already sufficient to prevent protein damage. Time-dependent response mechanisms effective in high salinity brackish water, resulting in salt loading avoidance and suggestive of hyporegulation should be further investigated in decapods that evolutionary invaded freshwater., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Increase in industrial growth, urban and agricultural pollution, with consequent impacts on aquatic ecosystems are a major focus of research worldwide. Still, not many studies assess the impacts of contamination through in situ studies, using native species, also considering the influence of seasonality on their responses. This study aimed to evaluate the water quality of the basin of the Upper Iguaçu River, the main source of water supply to Curitiba, a major capital of Southern Brazil, and its Metropolitan area. Several biomarkers were evaluated after in situ exposure of the native catfish Rhamdia quelen inside cages for 7 days. Ten study sites were chosen along the basin, based on a diffuse gradient of contamination, corresponding to regions upstream, downstream, and within "great Curitiba". In each site, fish were exposed in Summer and Winter. The complex mixture of contaminants of this hydrographic basin generated mortality, and ion-, osmoregulatory and respiratory disturbances in the catfish as, for example, reduction of plasma osmolality and ionic concentrations, increased hematocrit levels and gill water content, altered branchial and renal activities of the enzyme carbonic anhydrase, as well as raised levels of plasma cortisol and glucose. Biomarkers were mostly altered in fish exposed in Great Curitiba and immediately downstream. There was a notable influence of season on the responses of the jundiá. A multivariate redundancy analysis revealed that the best environmental variables explained 30% of the variation in biomarkers after controlling for spatial autocorrelation. Thus, this approach and the chosen parameters can be satisfactorily used to evaluate contamination environments with complex mixtures of contaminants, in other urban basins as well., (Copyright © 2017. Published by Elsevier Ltd.)

Echinoderms are restricted to the marine environment and are osmoconformer invertebrates. However, some species live in unstable environments. Especially those species, and those of larger body size, tend to show variable, albeit transient, ionic gradients between their coelomic fluid and external seawater. In order to further examine how sea urchin size relates to apparent ionic permeability of their body wall/epithelia, specimens of Echinometra lucunter, Lytechinus variegatus, Paracentrotus gaimardi, and Arbacia lixula-A. lixula of two distinct populations, Rio de Janeiro and Santa Catarina-were abruptly transferred from 35 psu to either 25 or 45 psu. Sodium, chloride, magnesium, and potassium concentrations were assayed in their coelomic fluids after 0, 1, 2, and 3 hr of exposure. Relative area of putative permeable (i.e., cross section areas of soft tissues, or test holes) surfaces (PPS) was estimated in empty tests as the sum of the peristomial area (oral hole in the empty test) and the total cross-section area of ambulacral holes, divided by the total volume (TV) of the test. L. variegatus and E. lucunter, the largest species, had PPS/TV values similar to that of the much smaller P. gaimardi. A. lixula was the "most putatively-permeable and conformer" among them all, especially urchins from the Santa Catarina population. Internal ionic levels equilibrated faster with external water in 45 than in 25, and differences among ions were observed. Body size is relevant, among many other factors, to aid conformers such as sea urchins to dwell in intertidal unstable habitats., (© 2018 Wiley Periodicals, Inc.)

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs., (Copyright © 2017. Published by Elsevier Inc.)

Aquaculture has shown great growth in the last decades. Due to the restrictions on water use, production systems are becoming increasingly more intensive, raising concerns about the production water quality. Macrobrachium amazonicum is among the freshwater prawn species with favorable characteristics for production and possibility of intensification. Nitrogen compounds such as ammonia and nitrite affect the health of aquatic organisms since they quickly reach toxic concentrations. These compounds can also cause damage to the gill structure, leading to hypoxia in tissues, affecting acid-base balance, osmoregulation (salt absorption) and ammonia excretion, decreasing the immune capacity of the animal and, in extreme cases, cause death. The aim of this study was to assess histological changes in the gills of Macrobrachium amazonicum juveniles subjected to different concentrations of total ammonia and nitrite. The prawns were subjected to different concentrations of those compounds and their gills were removed and preserved for histological analysis. The gills were assessed for changes according to the Organ Index (I org ) and, for each change, an importance factor (w) was attributed according to the degree of reversibility and applied according to the degree of extension or frequency of the damage. The damage to the gills in the treatments with 100% mortality, both for ammonia and nitrite, corresponded to the high occurrence of progressive, regressive, circulatory, and inflammation damages. The other treatments (which caused less mortality) had mainly inflammation and regressive damages, whose occurrence increased according to the increase in ammonia and nitrite concentration. The histological analysis confirmed that the higher the total ammonia and nitrite concentrations, the larger the damages caused to the gill structure and that lower nitrite concentrations caused similar damages to those caused by higher total ammonia concentrations, which reflects the lower capacity M. amazonicum has to tolerate nitrite., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sharks are very sensitive to stress and prone to a high mortality rate after capture. Since approximately 50 million of sharks are caught as bycatch every year, and current recommendations to reduce the impact of commercial fishing strongly support immediate release, it is imperative to better understand post-release mortality caused by the stress of capture and handling. Blood samples allow the assessment of stress levels which are valuable tools to reduce mortality in commercial, recreational and scientific fishing, being essential for the improvement in those conservation measures. Biochemical analyses are widely used for sharks as stress indicators, with secondary plasma parameters (lactate, glucose and ions) being the most often employed assays. However, it is virtually impossible to determine baseline plasma parameters in free-ranging sharks, since blood withdrawal involves animal capture and restrain, which are stressful procedures. This study aims at analyzing secondary parameters of five healthy tiger sharks captured with circular hooks and handlines in Fernando de Noronha (Northeastern Brazil) and comparing them with secondary parameters of three dead tiger sharks caught off Recife (also Northeastern Brazil). The results showed that the analysis of some plasma constituents in dead animals may be an efficient tool to assess stress and lethality. However, traditional parameters such as glucose and calcium, need to be used with caution. The results also demonstrated the extreme importance of urea and phosphorus for assessing stress response and mortality in tiger sharks, both parameters frequently neglected and of utmost importance for shark's homeostasis.

Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study determined the effects of nitrite on different life stages of the Amazon river prawn Macrobrachium amazonicum. Prawns of each life stage (postlarvae, juveniles and adults) were stocked in 24 experimental units (n = 10 prawns), under a complete randomized design. Individuals were exposed to nitrite (0, 1, 2, 4, 8 and 16 mg L -1 ). The median lethal concentration after 96 h (96 h LC 50 ) was calculated through the Weibull I. The mortality results showed that M. amazonicum is slightly less tolerant to nitrite than other species of Macrobrachium. The 96 h LC 50 for postlarvae, juveniles and adults of M. amazonicum were of 1.49, 2.36 and 2.34 mg nitrite/L, respectively. Nitrite intoxication risk quotient suggest moderated risk to low risk to the species. Usually in production systems nitrite values are lower than safe levels suggested in this study (0.1 mg L -1 to postlarvae and 0.2 mg L -1 nitrite to juvenile and adults), which makes our results appropriate for the production of this species.

In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present., (Copyright © 2015. Published by Elsevier Inc.)

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

Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps., (© 2015 Wiley Periodicals, Inc.)

Purpose: To evaluate the 18-month clinical performance of four adhesive strategies in posterior composite restorations placed in private practice., Materials and Methods: Upon approval by an institutional review board, 144 restorations were inserted in 45 subjects (average age = 32.6) to treat caries lesions or to replace existing restorations in vital molars and premolars. The adhesives OptiBond FL (three-step etch-and-rinse), OptiBond SOLO Plus (two-step etch-and-rinse), OptiBond XTR (two-step self-etch), and OptiBond All-in One (one-step self-etch) were applied as per manufacturer's (Kerr Co.) instructions followed by a nanofilled resin composite (Filtek Z350XT, 3M ESPE) under rubber dam isolation. Restorations were evaluated at baseline and at 18 months using United States Public Health Service (USPHS)-modified criteria and high-resolution digital photographs. Statistical analyses included the McNemar and the Mann-Whitney non-parametric tests (p < 0.05)., Results: A total of 137 restorations were evaluated after 18 months. The number of alfa ratings did not change significantly from baseline to 18 months for any of the adhesion strategies. When the 18-month evaluation criteria were pooled by pairs of adhesives, none of the adhesives resulted in a significantly different number of alfa ratings for any of the criteria compared with the other adhesives., Conclusion: Bonding strategy did not influence the clinical performance of posterior composite restorations under the clinical conditions used in this study., Clinical Significance: When used in ideal clinical conditions, the composition of current dentin adhesives may be more clinically relevant than their adhesion strategy., (© 2015 Wiley Periodicals, Inc.)

Intertidal pools are intensely challenging environments, due to rapid and extreme fluctuations in water conditions during the tidal cycle. Emersion is another challenge intertidal fishes may face. Mechanisms of ammonia excretion and ion regulation were studied in the resident amphibious blennid Lipophrys pholis. The ammonia transporters Rhcg1 and Rhcg2 were cloned and characterized. Fish were challenged for 24h to 1) emersion, 2) fresh water (FW), and 3) high environmental ammonia (HEA; 1mM NH4Cl), or 4) ammonia loading (1.5μmol/g NH4HCO3). When air exposed, L. pholis maintained aquatic ammonia excretion rates (JAmm) while branchial Na(+)/K(+)-ATPase (NKA) activity increased, but no changes at the protein or mRNA levels of transporters were noted. In FW, JAmm decreased and osmotic problems were encountered. Skin NKA activity decreased, branchial Rhcg2, and skin Rhcg1 and Rhcg2 increased. Exposure to HEA only increased branchial Rhcg2 levels. Although internal ammonia loading only led to a modest non-significant increase in JAmm, skin NKA (activity and α-subunit), carbonic anhydrase protein levels, and branchial Rhcg1 levels increased. In summary, variable responses were observed involving both gill and skin but given the instability of its habitat, the constitutive expression of transporters is likely also of importance., (© 2013.)

Elasmobranchs are essentially marine, but ~15% of the species occur in brackish or freshwater. The Brazilian marine coastal skate Zapteryx brevirostris, non-reported in nearby estuaries, was submitted to 35, 25, 15, and 5 psu, for 6 or 12h (n=6). Plasma was assayed for osmolality, urea, and ions (Na(+), Cl(-), K(+), Mg(2+)). Muscle water content was determined, and the rectal gland, kidney and gills were removed for carbonic anhydrase (CA) and Na(+),K(+)-ATPase (NKA) activities. The skate survived to all treatments. Plasma osmolality and urea levels decreased respectively by 27% and 38% after 12h in 5 psu (with respect to levels when in seawater), but plasma Na(+), Cl(-), and Mg(2+) were well regulated. Plasma K(+) showed some conformation after 12h. Muscle hydration was maintained. Branchial CA and NKA did not respond to salinity. Rectal gland NKA decreased upon seawater dilution, while renal NKA increased. This skate was shown to be partially euryhaline. The analysis of plasma urea of elasmobranchs in brackish and freshwater versus salinity and time-allied to the widespread occurrence of some euryhalinity in the group-led us to revisit the hypothesis of a brackish water habitat for elasmobranch ancestors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The exposure to a world-wide used herbicide atrazine (ATZ) (96h exposure to 2, 10, and 100μgL(-1)), was investigated on the freshwater fish Rhamdia quelen through a multi biomarker approach. Liver histopathology revealed leukocyte infiltration, hepatocyte vacuolization like steatosis and necrosis areas, leading to raised lesion index levels in all tested concentrations. The increase of free melanomacrophage numbers was observed. Gill filaments revealed considerable loss of the microridges on pavement cells at 10 and 100μgL(-1) of ATZ, and a significantly increased of chloride cell (CC) number and density on apical surface area at 100μgL(-1) of ATZ. CAT, GST, GPx, and GR activities were inhibited by all tested concentrations. GSH levels were reduced in individuals exposed to 100µgL(-1). Osmoregulatory function was also disturbed. We observed an increase of plasma magnesium concentrations at 10µgL(-1). Additionally the inhibition of branchial carbonic anhydrase activity was observed at 100µgL(-1). In the kidney, carbonic anhydrase activity decreased only in the group exposed to 2µgL(-1). These results suggest that ATZ, represents a potential ecotoxicological hazard and can be hepatotoxic and nephrotoxic even low concentrations. The current study was the first to show the nephrotoxic effect of ATZ in fish. Besides, in Brazil, the environmental protection agency (CONAMA) establishes that the maximum allowed level of dissolved ATZ in water is 2μgL(-1), but the present results showed that this concentration may cause histopathological, biochemical and physiological changes in R. quelen., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages., (Copyright © 2013 Wiley Periodicals, Inc.)

Echinoderms are considered marine osmoconforming invertebrates. However, many are intertidal or live next to estuaries, tolerating salinity changes and showing extracellular gradients to dilute seawater. Three species of echinoids - Lytechinus variegatus, which can occur next to estuarine areas, the rocky intertidal Echinometra lucunter, and the mostly subtidal Arbacia lixula - were submitted to a protocol of stepwise (rate of 2-3 psu/h) dilution, down to 15 psu, or concentration, up to 45 psu, of control seawater (35 psu). Coelomic fluid samples were obtained every hour. The seawater dilution experiment lasted 8h, while the seawater concentration experiment lasted 6h. Significant gradients (40-90% above value in 15 psu seawater) for osmolality, sodium, magnesium, and potassium were shown by L. variegatus and E. lucunter. A. lixula showed the smallest gradients, displaying the strongest conforming behavior. The esophagus of the three species was challenged in vitro with 20 and 50% osmotic shocks (hypo- and hyperosmotic). A. lixula, the most "conforming" species, showed the highest capacity to avoid swelling of its tissues upon the -50% hyposmotic shock, and was also the species less affected by salinity changes concerning the observation of spines and ambulacral feet movement in the whole-animal experiments. Thus, the most conforming species (A. lixula) displayed the highest capacity to regulate tissue water/volume, and was also the most euryhaline among the three studied species. In addition, tissues from all three species swelled much more than they shrank under osmotic shocks of same magnitude. This distinct trend to gain water, despite the capacity to hold some gradients upon seawater dilution, helps to explain why echinoderms cannot be fully estuarine, or ever enter fresh water., (Copyright © 2012 Elsevier Inc. All rights reserved.)

As it is the case in all animal food production systems, it is often necessary to treat farmed fish for diseases and parasites. Quite frequently, fish farmers still rely on the aggressive use of copper to control bacterial infections and infestations by ecto-parasites, and to manage the spread of diseases. The susceptibility of the neotropical fish Rhamdia quelen to copper was here evaluated at different waterborne copper concentrations (2, 7 or 11 μg Cu L(-1)) for 96 h, through a multi biomarkers approach. Liver histopathological findings revealed leukocyte infiltration, hepatocyte vacuolization and areas of necrosis, causing raised levels of lesions upon exposure to 7 and 11 μg Cu L(-1). Decreased occurrence of free melano-macrophages and increased densities of melano-macrophage centers were noted upon exposure to 11 μg Cu L(-1). Gills showed damages on their secondary lamellae already at 2 μg Cu L(-1); hypertrophy and loss of the microridges of pavement cells at 7 and 11 μg L(-1), and increased in chloride cell (CC) apical surface area (4.9-fold) and in CC density (1.5-fold) at 11 μg Cu L(-1). In the liver, catalase (CAT), glutathione peroxidase activities (GPx) and glutathione concentration (GSH) remained unchanged, compared to the control group. However, there was inhibition of 7-ethoxyresorufin-O-deethylase (EROD) at all copper concentrations tested. Glutathione reductase activity (GR) was reduced and levels of lipid peroxidation (LPO) were increased at 11 μg Cu L(-1). Glutathione S-transferase activity (GST) at 7 μg Cu L(-1) and superoxide dismutase activity (SOD) at both 7 and 11 μg Cu L(-1) were reduced. However, copper exposure did not alter brain and muscle acetylcholinesterase (AChE) activity. Osmoregulatory function was also disturbed, in agreement with the above-mentioned changes noted in the gills, as detected by plasma osmolality reduction in the group exposed to 11 μg Cu L(-1), and plasma chloride reduction at 2 μg Cu L(-1). These concentrations also, coherently, lead to inhibition of branchial carbonic anhydrase activity. In the kidney, increased carbonic anhydrase activity was measured in the groups exposed to 2 and 7 μg Cu L(-1). When these effects are compared to data available in the literature for other freshwater fish, also for 96 h of exposure, R. quelen appears as a relatively sensitive species. In addition, the concentrations employed here were quite low in comparison to levels used for disease control in real culture practices (ranging from 4 μg Cu L(-1) used against bacteria to 6000 μg Cu L(-1) against fungal infections). We can conclude that the concentrations frequently employed in aquaculture are in fact not safe enough for this species. Such data are essential for the questioning and establishment of new policies to the sector., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 Ca²⁺ channels, ouabain (100 μM) to inhibit Na⁺/K⁺-ATPase, and HgCl₂ (100 μM) to inhibit aquaporins. Our results suggest that: (1) Ca²⁺ 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 Pb²⁺ through ionic pathways that operate in salt absorption, such as, for example, the Na⁺/K⁺-ATPase., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The swimming crabs Callinectes danae and C. ornatus are found in bays and estuaries, but C. danae is more abundant in lower salinities, while C. ornatus remains restricted to areas of higher salinity. Experimental crabs of both species were submitted to: air exposure (Ae, 3h), reimmersion in 33‰ (control) sea water (SW) (Ri, 1h) following air exposure; hyposaline (Ho, 10‰ for 2h) or hypersaline (He, 40‰ for 2h) SW, then return to control 33‰ SW (RHo and RHe, for 1h). Hemolymph was sampled for osmolality and chloride determinations. Activity of antioxidant enzymes [glutathione peroxidase (GPX), catalase, glutathione-S-transferase] and levels of carbonyl proteins and lipid peroxidation (TBARS) were evaluated in hepatopancreas, muscle, anterior and posterior gills. In Ho groups, hemolymph concentrations were lower in both species, compared to He groups. C. danae displayed higher control activities of GPX (hepatopancreas and muscle) and catalase (all four tissues) than C. ornatus. C. ornatus presented increased activities of catalase and GPX in Ae, Ri, and He groups. Increased TBARS was seen in C. ornatus tissues (He group). The more euryhaline species displayed higher constitutive activities of antioxidant enzymes, and the less euryhaline species exhibited activation of these enzymes when exposed to air or hyper-salinity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Anemones are frequently found in rocky intertidal coasts. As they have highly permeable body surfaces, exposure to the air or to salinity variations inside tidal pools can represent intense osmotic and ionic challenges. The intertidal Bunodosoma caissarum has been compared with the subtidal Anemonia sargassensis concerning their response to air exposure or salinity changes. B. caissarum maintains tissue hydration through mucus production and dome-shape formation when challenged with air exposure or extreme salinities (fresh water or hypersaline seawater, 45 psu) for 1-2h. Upon exposure to mild osmotic shocks for 6h (hyposmotic: 25 psu, or hyperosmotic: 37 psu), B. caissarum was able to maintain its coelenteron fluid (CF) osmolality stable, but only in 25 psu. A. sargassensis CF osmolality followed the external medium in both salinities. Isolated cells of the pedal disc of B. caissarum showed full capacity for calcium-dependent regulatory volume decrease (RVD) upon 20% hyposmotic shock, at least partially involving the release of KCl via K(+)-Cl(-) cotransport, and also of organic osmolytes. Aquaporins (HgCl(2)-inhibited) likely participate in this process. Cells of A. sargassensis showed partial RVD, after 20 min. Cells from both species were not capable of regulatory volume increase upon hyperosmotic shock (20%). Whole organism and cellular mechanisms allow B. caissarum to live in the challenging intertidal habitat, frequently facing air exposure and seawater dilution., (Copyright © 2011 Elsevier Inc. All rights reserved.)

An oil tanker loaded with methanol and bunker oil has exploded in November 2004 in Paranaguá Bay, in front of Paranaguá Harbor, southern Brazil. In order to investigate the chronic effects of an oil spill on a resident estuarine fish, the Brazilian silverside Atherinella brasiliensis was sampled 1, 4, and 7 months after the spill, from 2 sites inside Paranaguá Bay, and also from a reference site inside nearby Guaratuba Bay, non-affected by the spill. Increases in plasma osmolality (reaching ~525 mOsm/kg H₂O, or ~70% above values in reference fish) and chloride (reaching 214 mM in site C, or ~51% above values in reference fish) were detected 4 months after the spill, in parallel with branchial carbonic anhydrase inhibition (to 56% of the activity measured in reference fish) in silversides obtained from the contaminated sites. Plasma cortisol concentration increased progressively in samples from fish obtained 4 (462 ng/mL) and 7 (564-650 ng/mL) months after the spill, when compared to values in reference fish (192 ng/mL). Osmoregulation of a resident estuarine fish is still affected by an oil spill, months after the accident. It is, thus, a sensitive tool for the evaluation of the chronic effects of oil spills inside tropical estuarine systems, and A. brasiliensis is proposed as an adequate sentinel species for monitoring protocols., (Copyright © 2010. Published by Elsevier B.V.)

Decapod crustaceans occupy various aquatic habitats. In freshwater they are osmoregulators, while marine species are typically osmoconformers. Freshwater crustaceans are derived from marine ancestors. The hypothesis tested here was that osmoregulators, which can rely on salt transport by interface epithelia to prevent extracellular disturbance, would have a lower capacity of tissue water regulation when compared with osmoconformers. Four species of decapod crustaceans (the marine osmoconformer crab Hepatus pudibundus, and three osmoregulators of different habitats) have been exposed in vivo to a salinity challenge, for up to 24 hr. Osmoregulators were: the estuarine shrimp Palaemon pandaliformis, the diadromous freshwater shrimp Macrobrachium acanthurus, and the hololimnetic red crab Dilocarcinus pagei. H. pudibundus displayed hemolymph dilution already after 0.5 hr in 25 per thousand, reaching approximately 30% reduction in osmolality, but its muscle degree of hydration did not increase. To make the different in vivo salinity challenges directly comparable, the ratio between the maximum change in muscle hydration with respect to the control value measured for the species and the maximum change in hemolymph osmolality was calculated (x 1,000): H. pudibundus (25 per thousand, 8.1% kg H(2)O/mOsm x 10(3))>P. pandaliformis (2 per thousand, 9.2)>M. acanthurus (30 per thousand, 12.6)>P. pandaliformis (35 per thousand, 16.7)>D. pagei (7 per thousand, 60.4). Muscle slices submitted in vitro to a 30% osmotic challenge confirmed in vivo results. Thus, the estuarine/freshwater osmoregulators displayed a lower capacity to hold muscle tissue water than the marine osmoconformer, despite undergoing narrower variations in hemolymph osmolality., ((c) 2009 Wiley-Liss, Inc.)

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

Osmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills, together with the excretory organs. In addition to their role in gas exchange, the gills constitute organs of active, transepithelial, ion transport, an activity of major importance that underlies many essential physiological functions like osmoregulation, calcium homeostasis, ammonium excretion and extracellular pH regulation. This review focuses on structure-function relationships in crustacean gills and excretory effectors, from the organ to molecular levels of organization. We address the diversity of structural architectures encountered in different crustacean gill types, and in constituent cell types, before examining the physiological mechanisms of Na(+), Cl(-), Ca(2+) and NH(4)(+) transport, and of acid-base equivalents, based on findings obtained over the last two decades employing advanced techniques. The antennal and maxillary glands constitute the principal crustacean excretory organs, which have received less attention in functional studies. We examine the diversity present in antennal and maxillary gland architecture, highlighting the structural similarities between both organ types, and we analyze the functions ascribed to each glandular segment. Emphasis is given to volume and osmoregulatory functions, capacity to produce dilute urine in freshwater crustaceans, and the effect of acclimation salinity on urine volume and composition. The microanatomy and diversity of function ascribed to gills and excretory organs are appraised from an evolutionary perspective, and suggestions made as to future avenues of investigation that may elucidate evolutionary and adaptive trends underpinning the invasion and exploitation of novel habitats.

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

Oysters have been largely employed as bioindicators of environmental quality in biomonitoring studies. Crassostrea rhizophorae was selected to evaluate the health status of three estuarine areas impacted by anthropogenic activities along the Brazilian coast, in three estuarine complexes, ranging in latitude from 7 to 25 degrees S. In each estuary three sites were sampled in Winter and in Summer: a site considered as reference, and two sites next to contamination sources. Condition index was similar at all sites and estuaries, with the highest values found for Itamaracá oysters in Summer. Necrosis, hyperplasia, mucocyte hypertrophy and fusion of ordinary filaments were the main histopathological lesions observed. Muscle cholinesterase activity was overall similar, but with a strong seasonal effect. Inhibition or activation of branchial total ATPase and Na,K-ATPase activities at the contaminated sites was observed. The health status of these estuarine areas is quite similar, and the combined use of biomarkers is recommended.

Na(+) and Ca(2+) regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na(+) and Ca(2+), whole body Na(+) and Ca(2+), and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na(+) influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na(+) balance was restored by the time killifish reached 100% SW. Whole body Na(+) did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na(+) influx (50-fold relative to FW values), but not Na(+) efflux between 40 and 60% SW, resulting in a large net loading of Na(+) at higher salinities (60-100% SW), and increases in plasma Na(+) and whole body Na(+) at 100% SW. Killifish were in negative Ca(2+) balance at all salinities, whereas trout were in positive Ca(2+) balance throughout. Ca(2+) influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca(2+) efflux. Ca(2+) flux rates were affected to a larger degree in trout, with twofold increases in Ca(2+) influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca(2+) efflux with salinity, so plasma Ca(2+) concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.