Posterior Gills Research Articles

Effects of salinity on short-term waterborne zinc uptake, accumulation and sub-lethal toxicity in the green shore crab (Carcinus maenas)

Waterborne zinc (Zn) is known to cause toxicity to freshwater animals primarily by disrupting calcium (Ca) homeostasis during acute exposure, but its effects in marine and estuarine animals are not well characterized. The present study investigated the effects of salinity on short-term Zn accumulation and sub-lethal toxicity in the euryhaline green shore crab, Carcinus maenas. The kinetic and pharmacological properties of short-term branchial Zn uptake were also examined. Green crabs (n=10) were exposed to control (no added Zn) and 50μM (3.25mgL−1) of waterborne Zn (∼25% of 96h LC50 in 100 seawater) for 96h at 3 different salinity regimes (100%, 60% and 20% seawater). Exposure to waterborne Zn increased tissue-specific Zn accumulation across different salinities. However, the maximum accumulation occurred in 20% seawater and no difference was recorded between 60% and 100% seawater. Gills appeared to be the primary site of Zn accumulation, since the accumulation was significantly higher in the gills relative to the hepatopancreas, haemolymph and muscle. Waterborne Zn exposure induced a slight increase in haemolymph osmolality and chloride levels irrespective of salinity. In contrast, Zn exposure elicited marked increases in both haemolymph and gill Ca levels, and these changes were more pronounced in 20% seawater relative to that in 60% or 100% seawater. An in vitro gill perfusion technique was used to examine the characteristics of short-term (1–4h) branchial Zn uptake over an exposure concentration range of 3–12μM (200–800μgL−1). The rate of short-term branchial Zn uptake did not change significantly after 2h, and no difference was recorded in the rate of uptake between the anterior (respiratory) and posterior (ion transporting) gills. The in vitro branchial Zn uptake occurred in a concentration-dependent manner across different salinities. However, the rate of uptake was consistently higher in 20% seawater relative to 60% or 100% seawater – similar to the trend observed with tissue Zn accumulation during in vivo exposure. The short-term branchial Zn uptake was found to be inhibited by lanthanum (a blocker of voltage-independent Ca channels), suggesting that branchial Zn uptake occurs via the Ca transporting pathways, at least in part. Overall, our findings indicate that acute exposure to waterborne Zn leads to the disruption of Zn and Ca homeostasis in green crab, and these effects are exacerbated at the lower salinity.

Salinity-induced changes in gene expression from anterior and posterior gills of Callinectes sapidus (Crustacea: Portunidae) with implications for crustacean ecological genomics

Decapods represent one of the most ecologically diverse taxonomic groups within crustaceans, making them ideal to study physiological processes like osmoregulation. However, prior studies have failed to consider the entire transcriptomic response of the gill – the primary organ responsible for ion transport – to changing salinity. Moreover, the molecular genetic differences between non-osmoregulatory and osmoregulatory gill types, as well as the hormonal basis of osmoregulation, remain underexplored. Here, we identified and characterized differentially expressed genes (DEGs) via RNA-Seq in anterior (non-osmoregulatory) and posterior (osmoregulatory) gills during high to low salinity transfer in the blue crab Callinectes sapidus, a well-studied model for crustacean osmoregulation. Overall, we confirmed previous expression patterns for individual ion transport genes and identified novel ones with salinity-mediated expression. Notable, novel DEGs among salinities and gill types for C. sapidus included anterior gills having higher expression of structural genes such as actin and cuticle proteins while posterior gills exhibit elevated expression of ion transport and energy-related genes, with the latter likely linked to ion transport. Potential targets among recovered DEGs for hormonal regulation of ion transport between salinities and gill types included neuropeptide Y and a KCTD16-like protein. Using publically available sequence data, constituents for a “core” gill transcriptome among decapods are presented, comprising genes involved in ion transport and energy conversion and consistent with salinity transfer experiments. Lastly, rarefication analyses lead us to recommend a modest number of sequence reads (~10–15M), but with increased biological replication, be utilized in future DEG analyses of crustaceans.

Characterization and expression analysis of B Cell receptor accessory molecule CD79 gene in humphead snapper (Lutjanus sanguineus)

CD79, a key component of the B cell antigen receptor complex, is composed of CD79α(Igα) and CD79β(Igβ) encoded by mb-1 and B29 respectively, and plays an important role in B cell signaling. In this study, we isolated and characterized mb-1 and B29 from humphead snapper (Lutjanus sanguineus). Their tissue distribution and expression profiles after stimulations in vitro and in vivo were also investigated. The humphead snapper mb-1 and B29 contain open reading frames of 684 bp and 606 bp, encoding 227 amino acids and 201 amino acids, respectively. Both CD79α and CD79β possess signal peptide, extracellular Ig domain, transmembrane region and immunoreceptor tyrosine kinase activation motif (ITAM). Mb-1 is highly expressed in lymphoid organs (thymus, posterior kidney and spleen) and mucosal-associated lymphoid tissues (gill and intestine), while B29 is mainly detected in posterior kidney, spleen, gill and skin. Furthermore, transcription of mb-1 and B29 in head kidney leucocytes was up-regulated following lipopolysaccharide (LPS), pokeweed mitogen (PWM), and polyinosinic-polycytidylic acid (PolyI:C) stimulation, respectively, and their expression level in anterior kidney and spleen was also increased after challenged with formalin-inactived Vibrio harveyi. These results indicated that humphead snapper CD79 molecule might play an important role in immune response to pathogen infection.

The role of an ancestral hyperpolarization-activated cyclic nucleotide-gated K+ channel in branchial acid-base regulation in the green crab, Carcinus maenas.

Numerous electrophysiological studies on branchial K(+) transport in brachyuran crabs have established an important role for potassium channels in osmoregulatory ion uptake and ammonia excretion in the gill epithelium of decapod crustaceans. However, hardly anything is known of the actual nature of these channels in crustaceans. In the present study, the identification of a hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN) in the transcriptome of the green crab Carcinus maenas and subsequent performance of quantitative real-time PCR revealed the ubiquitous expression of this channel in this species. Even though mRNA expression levels in the cerebral ganglion were found to be approximately 10 times higher compared with all other tissues, posterior gills still expressed significant levels of HCN, indicating an important role for this transporter in branchial ion regulation. The relatively unspecific K(+)-channel inhibitor Ba(2+), as well as the HCN-specific blocker ZD7288, as applied in gill perfusion experiments and electrophysiological studies employing the split gill lamellae revealed the presence of at least two different K(+)/NH4(+)-transporting structures in the branchial epithelium of C. maenas. Furthermore, HCN mRNA levels in posterior gill 7 decreased significantly in response to the respiratory or metabolic acidosis that was induced by acclimation of green crabs to high environmental PCO2 and ammonia, respectively. Consequently, the present study provides first evidence that HCN-promoted NH4(+) epithelial transport is involved in both branchial acid-base and ammonia regulation in an invertebrate.

Osmotic/ionic status of body fluids in the euryhaline cephalopod suggest possible parallel evolution of osmoregulation.

Acclimation from marine to dilute environments constitutes among the dramatic evolutionary transitions in the history of life. Such adaptations have evolved in multiple lineages, but studies of the blood/hemolymph homeostasis mechanisms are limited to those using evolutionarily advanced Deuterostome (chordates) and Ecdysozoa (crustaceans). Here, we examined hemolymph homeostasis in the advanced Lophotrochozoa/mollusc, the other unexplored taxa, and its possible regulation by the vasopressin/oxytocin superfamily peptides known to be implicated in fluid homeostasis in Chordata and Arthropoda. The hemolymph osmotic and ionic status in the euryhaline cephalopod (Octopus ocellatus) following transfer from 30-ppt normal seawater to 20 ppt salinity indicate hyperosmo- and hyperionoregulatory abilities for more than 1 week, as in crustaceans and teleost fish. While ventilation frequency decreased by 1 day, Na+/K+-ATPase activity, which has been generally implicated in ion transport, was induced in two of the eight posterior gills after 1 week. In addition, the octopuses were intravenously injected with 1 or 100 ng/g octopressin or cephalotocin, which are Octopus vasopressin/oxytocin orthologs. After 1 day, octopressin, but not cephalotocin, decreased the hemolymph osmolality and Ca concentrations, as well as urinary Na concentrations. These data provide evidence for possible parallel evolution in hyperionoregulatory mechanisms and coordination by conserved peptides.

Low salinity enhances NI-mediated oxidative stress and sub-lethal toxicity to the green shore crab (Carcinus maenas)

Nickel (Ni) is a metal of environmental concern, known to cause toxicity to freshwater organisms by impairing ionoregulation and/or respiratory gas exchange, and by inducing oxidative stress. However, little is known regarding how nickel toxicity is influenced by salinity. In the current study we investigated the salinity-dependence and mechanisms of sub-lethal Ni toxicity in a euryhaline crab (Carcinus maenas). Crabs were acclimated to three experimental salinities – 20, 60 and 100% seawater (SW) – and exposed to 3mg/L Ni for 24h or 96h. Tissues were dissected for analysis of Ni accumulation, gills were taken for oxidative stress analysis (catalase activity and protein carbonyl content), haemolymph ions were analysed for ionoregulatory disturbance, and oxygen consumption was determined in exercised crabs after 96h of Ni exposure. Total Ni accumulation was strongly dependant on salinity, with crabs from 20% SW displaying the highest tissue Ni burdens after both 24 and 96-h exposures. After 96h of exposure, the highest accumulation of Ni occurred in the posterior (ionoregulatory) gills at the lowest salinity, 20% SW. Posterior gill 8 exhibited elevated protein carbonyl levels and decreased catalase activity after Ni exposure, but only in 20% SW. Similarly, decreased levels of haemolymph Mg and K and an increased level of Ca were recorded but only in crabs exposed to Ni for 96h in 20% SW. Oxygen consumption after exercise was also inhibited in crabs exposed to Ni in 20% SW. These data show for the first time the simultaneous presence of all three modes of sub-lethal Ni toxicity in exposed animals, and indicate a strong salinity dependence of sub-lethal Ni toxicity to the euryhaline crab, C. maenas, a pattern that corresponded to tissue Ni accumulation.

Gill-specific (Na(+), K(+))-ATPase activity and α-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura).

We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.

Making sense of nickel accumulation and sub-lethal toxic effects in saline waters: Fate and effects of nickel in the green crab, Carcinus maenas

In freshwater, invertebrates nickel (Ni) is considered an ionoregulatory toxicant, but its mechanism of toxicity in marine settings, and how this varies with salinity, is poorly understood. This study investigated Ni accumulation and physiological mechanisms of sub-lethal Ni toxicity in the euryhaline green crab Carcinus maenas. Male crabs were exposed to 8.2μg/L (the US EPA chronic criterion concentration for salt waters) of waterborne Ni (radiolabelled with 63Ni) at three different salinities, 20%, 60% and 100% SW for 24h. Whole body Ni accumulation in 20% SW was 3–5 fold greater than in 60% or 100% SW, and >80% of accumulated Ni was in the carapace at all salinities. Ni also accumulated in posterior gill 8, which showed a higher accumulation in 20% SW than in other salinities, a pattern also seen at higher exposure concentrations of Ni (500 and 3000μg/L). Gill perfusion experiments revealed that Ni was taken up by both anterior and posterior gills, but in 20% SW the posterior gill 8, which performs ionoregulatory functions, accumulated more Ni than the anterior gill 5, which primarily has a respiratory function. The sub-lethal consequences of Ni exposure were investigated by placing crabs in Ni concentrations of 8.2, 500, and 3000μg/L at 20, 60 or 100% SW for 24h. In 20% SW, haemolymph Ca levels were significantly decreased by exposure to Ni concentrations of 8.2μg/L or higher, whereas Na concentrations were depressed only at 3000μg/L. Na+/K+-ATPase activity was inhibited at both 500 and 3000μg/L in gill 8, but only in 20% SW. Haemolymph K, Mg, and osmolality were unaffected throughout, though all varied with salinity in the expected fashion. These data suggest that Ni impacts ionoregulatory function in the green crab, in a gill- and salinity-dependent manner.

Serotonin effects in the crab Neohelice granulata: Possible involvement of two types of receptors in peripheral tissues

In crustaceans, serotonin (5-HT) controls various physiological processes, such as hormonal secretion, color changes, reproduction, and metabolism. Since 5-HT injections cause hyperglycemia, this study was designed to further investigate this action of 5-HT in the crab Neohelice granulate, fed with a high-carbohydrate (HC) or a high-protein (HP) diet. The effects of pre-treatment with mammalian 5-HT receptor antagonists, cyproheptadine and methiothepin, were also investigated. A series of in vivo experiments with 3H-5-HT was carried out in order to investigate the presence of putative receptors in peripheral tissues. Since gills were the tissue with the highest labeling in in vivo experiments, in vitro studies with isolated anterior and posterior gills were also conducted. Cyproheptadine blocked the hyperglycemic effect of 5-HT in HP-fed crabs. Methiothepin reduced glycogen levels in the anterior gills of HP crabs and partially blocked the 5-HT-like posture. The injection of 3H-5-HT identified specific binding sites in all the tissues studied and revealed that the binding can be influenced by the type of diet administered to the crabs. Incubation of the anterior and posterior gills with 3H-5-HT and 5-HT confirmed the specificity of the binding sites. Both antagonists inhibited 3H-5-HT binding. In conclusion, this study highlights the importance of serotonin in the control of glucose homeostasis in crustaceans and provides evidences of at least two types of 5-HT binding sites in peripheral tissues. Further studies are necessary to identify the structure of these receptors and their signaling pathways.

Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress.

Effects of salinity on the accumulation of hemocyte aggregates and bacteria in the gills of Callinectes sapidus, the Atlantic blue crab, injected with Vibrio campbellii

In addition to respiration and ion regulation, crustacean gills accumulate and eliminate injected particles, along with hemocyte aggregates that form in response to those particles. Here we report that the dose of Vibrio campbellii previously shown to induce a decrease in respiration and hemolymph flow across the gill in the Atlantic blue crab, Callinectes sapidus, also triggered the formation of aggregates containing four or more hemocytes in the gills, compared with saline-injected controls. More bacteria were trapped and rendered non-culturable per unit weight by anterior respiratory gills than posterior gills specialized for ion regulation. Further, more bacteria accumulated in the anterior gills of animals held at 30ppt than those at 10ppt. Thus, the role of the gills in immune defense comes at an energetic cost to this and likely to other crustaceans; this cost is influenced by acclimation salinity and the position and specialized function of individual gills.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus

Membrane pathway for intracellular cadmium (Cd2+) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd2+ transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd2+ concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd2+ transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl2 and different inhibitors. Addition of extracellular calcium (Ca2+) to the cells affected little the fluorescence of FluoZin, confirming that Cd2+ was the main ion increasing intracellular fluorescence. Ca2+ channels blockers (nimodipine and verapamil) decreased Cd2+ influx as well as vanadate, a Ca2+-ATPase blocker. Chelating intracellular Ca2+ (BAPTA) decreased Cd2+ influx in gill cells, while increasing intracellular Ca2+ (caffeine) augmented Cd influx. Cd2+ and ATP added at different temporal conditions were not effective at increasing intracellular Cd2+ accumulation. Ouabain (Na+/K+-ATPase inhibitor) increased Cd2+ influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd2+ influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.

<strong>Toxicity and bioaccumulation of fluoride ion on <em>Branchiura</em> <em>sowerbyi</em>, Beddard, (Oligochaeta, Tubificidae)</strong>

Fluoride concentrations are increasing significantly in many aquatic ecosystems as a consequence of human activities (agrochemicals, pharmaceuticals, refrigerants, pesticides, surfactant compounds). Several investigations have revealed that sensitivity to fluorides and safe concentrations vary greatly within classes, families and genera. Aquatic oligochaetes have often been used for pollution assessment and accumulation testing, but no information has been given about tolerance to fluoride ion. Among endobenthic tubificids Branchiura sowerbyi is easily identifiable (evident posterior gills, large size) and particularly useful for tissue requirements in chemical analysis. The purpose of this study was to examine the tolerance of this tubificid to fluoride ion and its bioaccumulation capacity by performing short (LC50 96h) and long-term (18 day) experiments at different temperatures (17°C and 22 °C). LC50 values (91.3 and 61.7 mg/L for 17°C and 22°C respectively), especially in the presence of sediment (267.6 and 80.1 mg/L for 17°C and 22°C respectively) showed that B. sowerbyi is more resistant to fluoride than other freshwater invertebrates. Fluoride became more toxic with increased temperature, demonstrating that seasonal temperature changes could influence the sensitivity of this freshwater tubificid. Bioaccumulation was lower when the organisms were exposed to sodium fluoride in the absence of sediment, indicating that this animal also accumulates fluoride by ingesting sediment.

Growth, osmoregulatory responses and changes to the lipid and fatty acid composition of organs from the mud crab, Scylla serrata, over a broad salinity range

Aquatic animals can often undergo substantial physiological responses to salinity; however, associated lipid/fatty acid alterations to their various tissues have received little attention. To investigate this, we measured the growth of mud crab, Scylla serrata, juveniles over two moults (duration of 23–60 days) at salinities of 4, 12, 20, 28, 36 and 44‰ (30 replicates/treatment). After the second moult, 6-day post moult crabs were sampled for hepatosomatic index (HSI), haemolymph osmolality, Na+, K+ and Ca2+ levels, anterior/posterior gill Na+/K+-ATPase activity as well as the lipid/fatty acid composition of the anterior and posterior gills, hepatopancreas and muscle. High salinities of 36 and 44‰ significantly lowered crab growth and HSI (p<0.01). S. serrata strongly hyper-regulated haemolymph osmolality and ions likely due to significantly enhanced posterior gill Na+/K+-ATPase activity. At decreasing salinities, eicosapentaenoic acid, arachidonic acid, n-3 and long chain polyunsaturated fatty acid (PUFA) significantly increased (p<0.05), likely to maintain Na+/K+-ATPase activity via increased membrane fluidity. Muscle and hepatopancreas n-3/n-6 PUFA ratios significantly decreased (p<0.05) at increasing salinities indicating possible fatty acid metabolic modifications. Results indicate that S. serrata juveniles are well adapted to low salinities, with higher salinities likely reducing their metabolism, reflected by lower growth, HSI and higher posterior gill and hepatopancreatic triacylglycerol.

Characterization of copper transport in gill cells of a mangrove crab Ucides cordatus

The branchial epithelium of crustaceans is exposed to the environment and is the first site affected by metal pollution. The aim of this work was to characterize copper (Cu) transport using a fluorescent dye, Phen Green, in gill cells of a hypo-hyper-regulator mangrove crab Ucides cordatus. The results showed that added extracellular CuCl2 (0, 0.025, 0.150, 0.275, 0.550 and 1.110μM) showed typical Michaelis–Menten transport for Cu in anterior and posterior gill cells (Vmax for anterior and posterior gills: 0.41±0.12 and 1.76±0.27 intracellular Cu in μM×22.104cells−1×300s−1 respectively and Km values: 0.44±0.04 and 0.32±0.13μM, respectively). Intracellular Cu was significantly higher for posterior gill cells compared to anterior gill cells, suggesting differential accumulation for each gill type. Extracellular Ca at 20mM decreased cellular Cu transport for both anterior and posterior gill cells. Nifedipine and verapamil, calcium channel inhibitors from plasma membrane, decreased Cu transport and affected Km for both gills. These results could be due to a competition between Cu and Ca. Amiloride, a Na/Ca exchanger inhibitor, as well as bafilomycin, a proton pump inhibitor, caused a decrease of intracellular Cu compared to control. Ouabain and KB-R 7943, acting on Na homeostasis, similarly decreased intracellular Cu in both gill cells. Besides that, gill cells exposed to ATP and Cu simultaneously, showed an increase in intracellular copper, which was inhibited by vanadate, an inhibitor of P-type ATPase. These results suggest either the presence of a Cu-ATPase in crab gill cells, responsible for Cu influx, or the effect of a change in electrochemical membrane potential that could also drive Cu to the gill cell interior. Caffeine increased intracellular Cu, suggesting that intracellular Ca could be affecting Cu uptake. Overall the results show that copper uptake in gill cells of crabs is regulated by intracellular Ca, Ca channels and by Na exchangers. This is the first report of Cu transport characterization in whole gill cells of crabs.

Deep-water prawn Pandalus borealis displays a relatively high pH regulatory capacity in response to CO2-induced acidosis

Deep-water prawn Pandalus borealis displays a relatively high pH regulatory capacity in response to CO2-induced acidosis

Synergistic stimulation by potassium and ammonium of K+-phosphatase activity in gill microsomes from the crab Callinectes ornatus acclimated to low salinity: Novel property of a primordial pump

We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg2+, Na+, K+, Rb+, NH4+ and pH of gill microsomal K+-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21‰). The synergistic stimulation by K+ and NH4+ of the K+-phosphatase activity is a novel finding, and may constitute a species-specific feature of K+/NH4+ interplay that regulates crustacean gill (Na+, K+)-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2±2.8nmolPimin−1mg−1 with K0.5=2.3±0.1mmolL−1, obeying cooperative kinetics (nH=1.7). Stimulation by Mg2+ (V=70.1±3.0nmolPimin−1mg−1, K0.5=0.88±0.04mmolL−1), K+ (V=69.6±2.7nmolPimin−1mg−1, K0.5=1.60±0.07mmolL−1) and NH4+ (V=90.8±4.0nmolPimin−1mg−1, K0.5=9.2±0.3mmol L−1) all displayed site-site interaction kinetics. In the presence of NH4+, enzyme affinity for K+ unexpectedly increased by 7-fold, while affinity for NH4+ was 28-fold greater in the presence than absence of K+. Ouabain partially inhibited K+-phosphatase activity (KI=320±14.0μmolL−1), more effectively when NH4+ was present (KI=240±12.0μmolL−1). We propose a model for the synergistic stimulation by K+ and NH4+ of the K+-phosphatase activity of the (Na+, K+)-ATPase from C. ornatus posterior gill tissue.

Hemolymph osmotic, ionic status, and branchial Na+/K+-ATPase activity under varying environmental conditions in the intertidal grapsid crab, Gaetice depressusd

Abstract Osmo- and ionoregulatory abilities were examined in the intertidal grapsid crab, Gaetice depressus, transferred from normal seawater (30 ppt) to low (10 ppt) or high (50 ppt) salinities for 2 and 10 days, in addition to animals kept out of water for 2 days. The results of the hemolymph osmotic and ionic status indicate that G. depressus is able to adapt for more than 10 days in these salinities and for 2 days under terrestrial conditions. Especially, the free Ca2+ concentration was relatively maintained compared with concentrations of monovalent ions and osmolality values in 10 and 50 ppt, partly using the complexed calcium (total minus free calcium) as an internal reserve in the hemolymph. In 10 ppt, complexed calcium disappeared from the hemolymph after 10 days, indicating that all the hemolymph calcium was ionized. In 50 ppt, free Ca2+ was regulated to lower levels than concentrations in the medium, while total calcium increased to higher levels after 2 days. Examination of Na+/K+-ATPase activity, which has been implicated in ion transport in many crustaceans, revealed that induction of high Na+/K+-ATPase activity varies among the posterior gills in response to salinities. Ten-ppt salinity induces activity in two of the posterior gills (gill numbers 6 and 7, eight in total), albeit with differing degrees of response. In contrast, 50-ppt salinity stimulates the activity primarily in gill number 8, suggesting that this gill may be associated specifically with ion excretion in G. depressus. As a euryhaline amphibious crab, this abundant species around Japan will serve as a model to study the osmotic/ionic regulatory mechanisms which operate in crustaceans.

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities

The estuarine crab Neohelice granulata was exposed (96h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2ppt salinity, 1mg Cu/L; isosmotic crabs: 30ppt salinity, 5mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.