Biology subjects

McNamara, J. C.

Publications and source records attributed to McNamara, J. C..

9 recordsLinked to original sources

bioRxiv2022

Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the blue crab Callinectes danae (Decapoda, Brachyura)

To evaluate the crustacean gill (Na+, K+)-ATPase as a molecular marker for toxic contamination by heavy metals of estuarine and coastal environments, we provide a comprehensive analysis of the effects of Co2+ in vitro on modulation of the K+-phosphatase activity of a gill (Na+, K+)-ATPase from the blue crab Callinectes danae. Using p-nitrophenyl phosphate as a substrate, Co2+ can act as both stimulator and inhibitor of K+-phosphatase activity. Without Mg2+, Co2+ stimulates K+-phosphatase activity similarly but with a {approx}4.5-fold greater affinity than with Mg2+. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Substitution of Mg2+ by Co2+ slightly increases enzyme affinity for K+ and NH4+. Independently of Mg2+, ouabain inhibition is unaffected by Co2+. Mg2+ displaces bound Co2+ from the Mg2+-binding site in a concentration dependent mechanism. However, at saturating Mg2+ concentrations, Co2+ does not displace Mg2+ from its binding site even at elevated concentrations. Saturation by Co2+ of the Mg2+ binding site does not affect pNPP recognition by the enzyme. Given that the interactions between heavy metal ions and enzymes are particularly complex, their toxic effects at the molecular level are poorly understood. Our findings elucidate partly the mechanism of action of Co2+ on a crustacean gill (Na+, K+)-ATPase. HighlightsO_LIWithout Mg2+, cobalt ions stimulate the gill (Na+, K+)-ATPase C_LIO_LICo2+ has a 4.5-fold greater affinity for the gill (Na+, K+)-ATPase than does Mg2+ C_LIO_LIMg2+ displaces Co2+ from the Mg2+-binding site in a concentration dependent manner C_LIO_LIOuabain inhibition with Co2+ or Mg2+ is identical C_LIO_LISaturation by Co2+ of Mg2+-binding sites does not affect substrate recognition C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/516930v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@100edcforg.highwire.dtl.DTLVardef@1fce3d0org.highwire.dtl.DTLVardef@1d096bdorg.highwire.dtl.DTLVardef@1586bd7_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract (synopsis)Using a crab gill (Na+, K+)-ATPase, we demonstrate that Co2+ inhibits K+-phosphatase activity with Mg2+, which is stimulated without Mg2+. Mg2+ displaces Co2+ from the Mg2+-binding site but Co2+ cannot displace Mg2+. Ouabain inhibition is unaffected by Co2+, independently of Mg2+. The molecular mechanism of Co2+ toxicity is partly elucidated.

pharmacology and toxicology↗

bioRxiv2022

Can hyper/hypo-osmoregulating fiddler crabs from the Atlantic coast of South America mobilize intracellular free amino acids as osmotic effectors during salinity challenge?

Weakly osmoregulating crustaceans use intracellular free amino acids (FAA) to attenuate cell volume changes consequent to alterations in hemolymph osmolality. Whether semi-terrestrial, strong hyper/hypo-osmoregulators exhibit this ability is not known. We investigate FAA mobilization in muscle tissue of ten fiddler crabs from the genera Minuca, Leptuca and Uca distributed along the Atlantic coast of South America. Crabs were subjected to severe hypo- or hyper-osmotic challenge at their upper or lower critical salinity limits for five days; control crabs were held in isosmotic media. Hemolymph osmolality was measured, chela muscle FAA were identified and quantified, and percent contribution to intracellular osmolality (%FAA) was calculated. At isosmoticity, total FAA were nominally 2-fold higher in Minuca species ({approx}116 mmol/kg wet mass) than in Uca ({approx}60 mmol/kg wet mass). Glycine, alanine, arginine and taurine constituted >80% of total FAA. On hyper-osmotic challenge, hemolymph osmolalities ranged from 843 to 1,282 mOsm/kg H2O. FAA increased, although %FAA remained unaltered. Hypo-osmoregulating crabs thus can mobilize FAA, likely owing to a lesser ability to secrete salt near their upper critical limits. On hypo-osmotic challenge, osmolalities were more tightly regulated, between 475 and 736 mOsm/kg H2O. Total FAA and %FAA showed little change, probably due to the crabs strong hyper-osmotic extracellular regulation, FAA consequently playing a diminished role in isosmotic intracellular regulation. Total FAA responses to hyper/hypo-osmotic challenge are thus asymmetrical. There was no effect of crab genus on total FAA or on %FAA at isosmoticity or on either osmotic challenge, reinforced by the absence of phylogenetic signal.

physiology↗

bioRxiv2022

Evolutionary trade-offs in osmotic and ionic regulation and the expression of gill ion transporter genes in high latitude, Neotropical cold clime crabs from the 'end of the world'

Seeking to identify consequences of evolution at low temperature, we examine hyper/hypo-osmotic and ionic regulation and gill ion transporter gene expression in two sub-Antarctic Eubrachyura from the Beagle Channel, Tierra del Fuego. Despite sharing the same osmotic niche, Acanthocyclus albatrossis tolerates a wider salinity range (2-65 {per thousand}S) than Halicarcinus planatus (5-60 {per thousand}S); respective lower and upper critical salinities are 4 and 12 {per thousand}S, and 63 and 50 {per thousand}S. Acanthocyclus albatrossis is a weak hyperosmotic regulator, while H. planatus hyper-osmoconforms; isosmotic points are 1,380 and {approx}1,340 mOsm kg-1 H2O. Both crabs hyper/hypo-regulate [Cl-] well with iso-chloride points at 452 and 316 mmol L-1 Cl-, respectively. [Na+] is hyper-regulated at all salinities. mRNA expression of gill Na+/K+-ATPase is salinity-sensitive in A. albatrossis, increasing {approx}1.9-fold at 5 {per thousand}S compared to 30 {per thousand}S, decreasing at 40 to 60 {per thousand}S. Expression in H. planatus is very low salinity-sensitive, increasing {approx}4.7-fold over 30 {per thousand}S, but decreasing at 50 {per thousand}S. V(H+)-ATPase expression decreases in A. albatrossis at low and high salinities as in H. planatus. Na+-K+-2Cl- symporter expression in A. albatrossis increases 2.6-fold at 5 {per thousand}S, but decreases at 60 {per thousand}S compared to 30 {per thousand}S. Chloride uptake may be mediated by increased Na+-K+-2Cl- expression but Cl- secretion is independent of symporter expression. These unrelated eubrachyurans exhibit similar systemic osmoregulatory characteristics and are better adapted to dilute media; however, the gene expressions underlying ion uptake and secretion show marked interspecific divergences. Cold clime crabs may have limited energy expenditure by regulating hemolymph [Cl-] alone, apportioning resources for other metabolic processes. Summary statementSub-Antarctic crabs may skimp on osmoregulatory capabilities to apportion energy for metabolic processes. They regulate chloride but not sodium or osmolality. Transporter gene expressions diverge markedly. Adaptive, differential ion regulation may characterize cold clime crabs.

physiology↗

bioRxiv2022

Strategies of invertebrate osmoregulation: an evolutionary blueprint for transmuting into fresh water from the sea

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, are reduced body permeability and surface (S) to volume (V) ratios, lowered 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 adaptations in taxa that have penetrated into fresh water, 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. Our analyses show that across sixty-four freshwater invertebrate species from six phyla/classes, 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 to 320 mOsmoles/kg H2O in the Insecta; osmolalities in the Annelida range from 150 to 200 mOsmoles/kg H2O, the Mollusca showing the lowest osmolalities at 40 to 120 mOsmoles/kg H2O. Overall, osmolalities reach a cut-off at {approx}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 fresh water while retaining some tolerance of the ancestral medium.

evolutionary biology↗

bioRxiv2020

Survival on a semi-arid island: submersion and desiccation tolerances of fiddler crabs from the Galapagos Archipelago

During tidal cycles, semi-terrestrial fiddler crabs are subject to alternating periods of submersion and desiccation. Here, we compare physiological and biochemical adjustments to forced submersion and desiccation in two fiddler crabs from the Galapagos archipelago: the indigenous Leptuca helleri, and Minuca galapagensis. We examine ecological distributions and habitat characteristics using transect analysis; survival after 6 h forced submersion at different salinities (0, 21 and 42 {per thousand}S), and after 6 or 12 h desiccation challenge, including alterations in hemolymph osmolality; and, oxidative stress responses in the gills and hepatopancreas, accompanying glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) activities, and lipid peroxidase (LPO). We provide an integrated biomarker response (IBR) index for each species based on oxidative stress in each tissue and condition. Our transect study revealed that L. helleri occupies an intertidal niche while M. galapagensis is supralittoral, L. helleri being less resistant to submersion and desiccation. After 6 h submersion, L. helleri survived only at 21 {per thousand}S while M. galapagensis survived at all salinities. Hemolymph osmolality decreased at 0 {per thousand}S in M. galapagensis. After 6 h desiccation, osmolality decreased markedly in L. helleri but increased in M. galapagensis. Enzyme assays were not performed in L. helleri owing to high mortality on submersion/desiccation challenge. After submersion in M. galapagensis, hepatopancreas GPx activities decreased in 0 and 21 {per thousand}S while GR activity was strongly inhibited at all salinities. Gill LPO decreased in 42 {per thousand}S. On desiccation in L. helleri, GPx activity was inhibited in the hepatopancreas but increased in the gills. GST activity increased while LPO decreased in both tissues. After desiccation in M. galapagensis, hepatopancreas GPx activity increased. Both hepatopancreas and gill GST and GR activities and LPO were strongly inhibited. The IBR indexes for L. helleri were highest in fresh caught crabs, driven by gill and hepatopancreas LPO. For M. galapagensis, submersion at 21 {per thousand}S contributed most to IBR, LPO in both tissues responding markedly. Leptuca helleri appears to be a habitat specialist adapted to a narrow set of niche dimensions while M. galapagensis survives over a much wider range, exhibiting little oxidative stress. The species physiological flexibilities and limitations provide insights into how fiddler crabs might respond to global environmental change on semi-arid islands.

physiology↗

bioRxiv2020

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes

Palaemonid shrimps include species from distinct osmotic niches that hyper-regulate hemolymph osmolality and ionic concentrations in dilute media but hypo-regulate in saline media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. Using a palaemonid series from niches including marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium), we established their critical upper salinity limits (UL50) and examined their short-(24 h) and long-term (120 h) hypo-regulatory abilities at salinities corresponding to 80% of the UL50s (80%UL50). We tested for phylogenetic correlations between gill NKCC gene and protein expression and hemolymph Cl- hypo-regulatory capability, and evaluated whether niche salinity might have driven gill NKCC expression. The Palaemon species from saline habitats showed the highest UL50s and greatest hypo-regulatory capabilities compared to the Macrobrachium species among which UL50s were higher in the diadromous than in the hololimnetic species. While basal gill NKCC mRNA transcription rates differed among species, expressions were unaffected by exposure time or salinity, suggesting post-transcriptional regulation of protein synthesis. Unexpectedly, hemolymph Cl- hyper-regulatory capability correlated with gill NKCC gene expression, while gill NKCC protein synthesis was associated with hyper-regulation of hemolymph osmolality at the 80%UL50s of almost all Macrobrachium species, suggesting a role for the gill NKCC symporter in salt uptake. The evolutionary history of osmoregulation in these palaemonid shrimps suggests that, while some molecular and systemic mechanisms have accompanied cladogenetic events during radiation into different osmotic niches, others may be driven by salinity.

physiology↗

bioRxiv2020

Effect of salinity on modulation by ATP, protein kinases and FXYD2 peptide of gill (Na+, K+)-ATPase activity in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae)

The gill (Na+, K+)-ATPase is the main enzyme that underpins osmoregulatory ability in crustaceans that occupy biotopes like mangroves, characterized by salinity variation. We evaluated osmotic and ionic regulatory ability in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. We also analyzed modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+- calmodulin-dependent kinase of (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35 {per thousand}S. Cl- was well hyper-/hypo- regulated although Na+ much less so, becoming iso-natremic at high salinity. (Na+, K+)- ATPase activity was greatest in isosmotic crabs (26 {per thousand}S), diminishing progressively from 18 and 8 {per thousand}S ({approx}0.5 fold) to 2 {per thousand}S (0.04-fold), and decreasing notably at 35 {per thousand}S (0.07-fold). At low salinity, the (Na+, K+)-ATPase exhibited a low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18 {per thousand}S, an additional, high affinity ATP-binding site, corresponding to 10-20% of total (Na+, K+)-ATPase activity appeared. Activity is stimulated by exogenous pig kidney FXYD2 peptide, while endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase all inhibit activity. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. Curiously, hyper-osmoregulation in U. cordatus shows little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of osmoregulatory and enzymatic adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/058297v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@19a35f2org.highwire.dtl.DTLVardef@14bd0d3org.highwire.dtl.DTLVardef@7adaa5org.highwire.dtl.DTLVardef@1a88e10_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGill (Na+, K+)-ATPase activity is greatest in isosmotic crabs, diminishing in lower and higher salinities. C_LIO_LIA high affinity ATP-binding site (10-20% of total activity) is exposed above 18 {per thousand}S. C_LIO_LIExogenous FXYD2 peptide stimulates activity; endogenous PKA, PKC and CaMK inhibit activity. C_LIO_LIFirst demonstration of inhibitory phosphorylation of crustacean (Na+, K+)-ATPase by CaMK. C_LIO_LIHyper-osmoregulation shows little dependence on (Na+, K+)-ATPase activity. C_LI

biochemistry↗

bioRxiv2020

Tissue accumulation and the effects of long-term dietary copper contamination on osmoregulation in the mudflat fiddler crab Minuca rapax (Crustacea, Ocypodidae)

We examined copper accumulation in the hemolymph, gills and hepatopancreas, and hemolymph osmolality, Na+ and Cl- concentrations, together with gill Na+/K+-ATPase and carbonic anhydrase activities, after dietary copper delivery (0, 100 or 500 {micro}g Cu/g) for 12 days in a fiddler crab, Minuca rapax. In contaminated crabs, copper concentration decreased in the hemolymph and hepatopancreas, but increased in the gills. Hemolymph osmolality and gill Na+/K+-ATPase activity increased while hemolymph [Na+] and [Cl-] and gill carbonic anhydrase activity decreased. Excretion likely accounts for the decreased hemolymph and hepatopancreas copper titers. Dietary copper clearly affects osmoregulatory ability and hemolymph Na+ and Cl- regulation in M. rapax. Gill copper accumulation decreased carbonic anhydrase activity, suggesting that dietary copper affects acid-base balance. Elevated gill Na+/K+-ATPase activity appears to compensate for the ion-regulatory disturbance. These effects of dietary copper illustrate likely impacts on semi-terrestrial species that feed on metal contaminated sediments.

physiology↗

bioRxiv2020

Osmotic and ionic regulation by hololimnetic freshwater crustaceans: a molecular role for gill ion transporters

Owing to their extraordinary niche diversity, the Crustacea are ideal for comprehending the evolution of osmoregulation. The processes that effect systemic hydro-electrolytic homeostasis maintain hemolymph ionic composition via membrane transporters located in highly specialized gill ionocytes. We evaluated physiological and molecular hyper- and hypo-osmoregulatory mechanisms in two phylogenetically distant, freshwater crustaceans, the crab Dilocarcinus pagei and the shrimp Macrobrachium jelskii, when osmotically challenged for up to 10 days. When in distilled water, hemolymph osmolality and [Cl-] increased briefly in D. pagei, stabilizing at initial values, while [Na+] decreased continually. Gill V(H+)-ATPase, Na+/K+-ATPase and Na+/K+/2Cl- gene expressions were unchanged. In M. jelskii, hemolymph osmolality, [Cl-] and [Na+] decreased continually for 12 h, the shrimps no longer surviving. Gill transporter gene expressions increased 2- to 5-fold. After 10-days exposure to brackish water, D. pagei was isosmotic, iso-chloremic and iso-natriuremic. Gill V(H+)-ATPase expression decreased while Na+/K+-ATPase and Na+/K+/2Cl- expressions were unchanged. In M. jelskii, the hemolymph was hypo-regulated, particularly [Cl-]. Transporter expressions initially increased 3- to 12-fold, declining to control values. Gill V(H+)-ATPase expression underlies the ability of D. pagei to survive in fresh water while Na+/K+-ATPase and Na+/K+/2Cl- expressions enable M. jelskii to deal with osmotic challenge. These findings reveal divergent responses in two unrelated crustaceans habiting a similar osmotic niche. While D. pagei has maintained the capacity to tolerate elevated cellular isosmoticity despite its inability to secrete salt, M. jelskii displays clear hypo-osmoregulatory ability. Each species has developed distinct strategies at the transcription and systemic levels during adaptation to fresh water. Summary statementDuring their evolutionary adaptation to fresh water, unrelated hololimnetic crustaceans have developed physiological strategies like tolerating elevated cellular isosmoticity or regulating hypo-osmoregulatory ability at the gene transcription level.

physiology↗