Low salinity‐induced alterations in epithelial ultrastructure, Na⁺/K⁺‐ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab,Clibanarius vittatus(Anomura, Diogenidae)

Abstract

Fresh caught Clibanarius vittatus [SW, 31‰ salinity (S)] were acclimated to a dilute medium (15‰ S) for 10 days, employing silver staining to locate gill ion transporting tissue, immunofluorescence to localize the Na/K-ATPase α-subunit in the lamellae, and electron microscopy to portray ultrastructural changes in the gill epithelia. Na/K-ATPase activity was characterized kinetically in a gill microsomal fraction, including synergistic stimulation by NH plus K. Silver staining revealed that all 26 phyllobranchiate arthro- and pleurobranchiae participate in ion transport. Na/K-ATPase α-subunit staining was weak in SW crabs and distributed exclusively and irregularly within the intralamellar septal cells, particularly at the septal-pillar cell body junctions, and septal cell cytoplasm facing the hemolymph space. In 15‰ S crabs, α-subunit localization was intense, occupying the entire thickened septum. Pillar cells and flanges did not stain. Mitochondria and membrane foldings increased in the pillar cell flanges and intralamellar septal cells, greatly amplifying surface area. Only a single ATP binding site (V = 130.8 ± 10.5 nmol min mg protein; K = 55.3 ± 1.7 μmol l) obeying Michaelis-Menten kinetics was disclosed. Na/K-ATPase activity was modulated by Mg, Na, and NH, exhibiting site-site interactions; K modulation showed Michaelis-Menten kinetics. K plus NH synergistically stimulated activity ≈ 1.7-fold. Ouabain inhibited total ATPase activity by ≈ 70% (K = 220-300 μmol l), revealing phosphohydrolytic activities other than the Na/K-ATPase. Despite ample phylogenetic separation, the phyllobranchiate lamellae of the Anomura and Caridea share many ultrastructural features, that is, an intralamellar septum and opposed abutting pillar cells, similar Na/K-ATPase distribution, and comparable kinetic characteristics. These findings suggest either convergent evolution at the structural and biochemical levels, or preservation of traits present in a remote common ancestor.

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)

“…(Na + , K + )-ATPase isoforms showing high and low affinity ATP-binding sites are present in many crustacean gill epithelia (Masui et al, 2002; Lucu and Towle, 2003; Leone et al, 2017; Farias et al, 2017). The non-exposure of the high affinity site after acclimation of U. cordatus to dilute media is similar to findings for the hermit crab C. symmetricus (Faleiros et al, 2018; and Antunes et al, 2017 as C. vittatus ), the blue crab Callinectes danae (Masui et al, 2009) and the rock crab Cancer pagurus (Gache et al, 1976). ATP is considered to play both a catalytic and an allosteric role in the (Na + , K + )-ATPase reaction cycle (Beaugé et al, 1997; Krumscheid et al, 2004), and high and low affinity ATP- binding sites on the (Na + , K + )-ATPase are well known (Glynn, 1985; Ward and Cavieres, 1998).…”

“…Gill (Na + , K + )-ATPase activity also decreased progressively and markedly with acclimation to lower salinities (18, 8 and 2 ‰S, see Fig. 3), which is unusual, since activities generally increase at low salinities, counterbalancing passive Na + efflux (Lucu and Towle, 2003; Luquet et al, 2005; Garçon et al, 2009; Antunes et al, 2017; Faleiros et al, 2018). This decrease may derive from our use of crabs acclimated under emerged rather than submerged conditions, with free access to their experimental media.…”

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)

“…(Na + , K + )-ATPase isoforms showing high and low affinity ATP-binding sites are present in many crustacean gill epithelia (Masui et al, 2002; Lucu and Towle, 2003; Leone et al, 2017; Farias et al, 2017). The non-exposure of the high affinity site after acclimation of U. cordatus to dilute media is similar to findings for the hermit crab C. symmetricus (Faleiros et al, 2018; and Antunes et al, 2017 as C. vittatus ), the blue crab Callinectes danae (Masui et al, 2009) and the rock crab Cancer pagurus (Gache et al, 1976). ATP is considered to play both a catalytic and an allosteric role in the (Na + , K + )-ATPase reaction cycle (Beaugé et al, 1997; Krumscheid et al, 2004), and high and low affinity ATP- binding sites on the (Na + , K + )-ATPase are well known (Glynn, 1985; Ward and Cavieres, 1998).…”

“…Gill (Na + , K + )-ATPase activity also decreased progressively and markedly with acclimation to lower salinities (18, 8 and 2 ‰S, see Fig. 3), which is unusual, since activities generally increase at low salinities, counterbalancing passive Na + efflux (Lucu and Towle, 2003; Luquet et al, 2005; Garçon et al, 2009; Antunes et al, 2017; Faleiros et al, 2018). This decrease may derive from our use of crabs acclimated under emerged rather than submerged conditions, with free access to their experimental media.…”

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss)

“…For example, ATP binds NKA with an affinity constant of approximately 200 µM, while the equivalent value for H + -ATPase is in the range of 8 to 40 µM (Jorgensen & Pedersen, 2001;Nakano et al, 2008;Tirtom et al, 2013). Although binding affinity depends significantly on experimental conditions and organism physiology (Antunes et al, 2017), the ATP binding affinities derived for fish NKA are similar to other values in the literature (210-540 µM; Morrison et al, 2006). Assuming that binding affinities of ATP to H + -ATPase are also similar, this suggests that ATP has a greater affinity for H + -ATPase than for NKA.…”

Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition‐dependent manner

“…NKA, Na + /K + ATPase; NKCC, Na + /K + /2Cl − cotransporter; 1.2 MCK, 30.5 mmol/L MgCl 2 , 11.7 mmol/L CaCl 2 , and 13.4 mmol/L KCl including NKA (Apell et al, 2017). In fact, activity of NKA in gills of crab is modulated by ambient Mg 2+ (Antunes et al, 2017;Masui et al, 2005Masui et al, , 2009. Moreover, Ca 2+ plays an important role in various biological processes, particularly in stabilizing biological membranes, increasing the tightness of intracellular tight junctions, and thereby controlling ion permeability across the gill epithelium in fish (McDonald & Milligan, 1988;McDonald et al, 1983).…”

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)

“…(Na + , K + )-ATPase isoforms showing high and low affinity ATP-binding sites are present in many crustacean gill epithelia (Masui et al, 2002; Lucu and Towle, 2003; Leone et al, 2017; Farias et al, 2017). The non-exposure of the high affinity site after acclimation of U. cordatus to dilute media is similar to findings for the hermit crab C. symmetricus (Faleiros et al, 2018; and Antunes et al, 2017 as C. vittatus ), the blue crab Callinectes danae (Masui et al, 2009) and the rock crab Cancer pagurus (Gache et al, 1976). ATP is considered to play both a catalytic and an allosteric role in the (Na + , K + )-ATPase reaction cycle (Beaugé et al, 1997; Krumscheid et al, 2004), and high and low affinity ATP- binding sites on the (Na + , K + )-ATPase are well known (Glynn, 1985; Ward and Cavieres, 1998).…”

“…Gill (Na + , K + )-ATPase activity also decreased progressively and markedly with acclimation to lower salinities (18, 8 and 2 ‰S, see Fig. 3), which is unusual, since activities generally increase at low salinities, counterbalancing passive Na + efflux (Lucu and Towle, 2003; Luquet et al, 2005; Garçon et al, 2009; Antunes et al, 2017; Faleiros et al, 2018). This decrease may derive from our use of crabs acclimated under emerged rather than submerged conditions, with free access to their experimental media.…”

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss)

“…For example, ATP binds NKA with an affinity constant of approximately 200 µM, while the equivalent value for H + -ATPase is in the range of 8 to 40 µM (Jorgensen & Pedersen, 2001;Nakano et al, 2008;Tirtom et al, 2013). Although binding affinity depends significantly on experimental conditions and organism physiology (Antunes et al, 2017), the ATP binding affinities derived for fish NKA are similar to other values in the literature (210-540 µM; Morrison et al, 2006). Assuming that binding affinities of ATP to H + -ATPase are also similar, this suggests that ATP has a greater affinity for H + -ATPase than for NKA.…”

Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition‐dependent manner

“…NKA, Na + /K + ATPase; NKCC, Na + /K + /2Cl − cotransporter; 1.2 MCK, 30.5 mmol/L MgCl 2 , 11.7 mmol/L CaCl 2 , and 13.4 mmol/L KCl including NKA (Apell et al, 2017). In fact, activity of NKA in gills of crab is modulated by ambient Mg 2+ (Antunes et al, 2017;Masui et al, 2005Masui et al, , 2009. Moreover, Ca 2+ plays an important role in various biological processes, particularly in stabilizing biological membranes, increasing the tightness of intracellular tight junctions, and thereby controlling ion permeability across the gill epithelium in fish (McDonald & Milligan, 1988;McDonald et al, 1983).…”

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)

“…(Na + , K + )-ATPase isoforms showing high and low affinity ATP-binding sites are present in many crustacean gill epithelia (Masui et al, 2002; Lucu and Towle, 2003; Leone et al, 2017; Farias et al, 2017). The non-exposure of the high affinity site after acclimation of U. cordatus to dilute media is similar to findings for the hermit crab C. symmetricus (Faleiros et al, 2018; and Antunes et al, 2017 as C. vittatus ), the blue crab Callinectes danae (Masui et al, 2009) and the rock crab Cancer pagurus (Gache et al, 1976). ATP is considered to play both a catalytic and an allosteric role in the (Na + , K + )-ATPase reaction cycle (Beaugé et al, 1997; Krumscheid et al, 2004), and high and low affinity ATP- binding sites on the (Na + , K + )-ATPase are well known (Glynn, 1985; Ward and Cavieres, 1998).…”

“…Gill (Na + , K + )-ATPase activity also decreased progressively and markedly with acclimation to lower salinities (18, 8 and 2 ‰S, see Fig. 3), which is unusual, since activities generally increase at low salinities, counterbalancing passive Na + efflux (Lucu and Towle, 2003; Luquet et al, 2005; Garçon et al, 2009; Antunes et al, 2017; Faleiros et al, 2018). This decrease may derive from our use of crabs acclimated under emerged rather than submerged conditions, with free access to their experimental media.…”

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss)

“…For example, ATP binds NKA with an affinity constant of approximately 200 µM, while the equivalent value for H + -ATPase is in the range of 8 to 40 µM (Jorgensen & Pedersen, 2001;Nakano et al, 2008;Tirtom et al, 2013). Although binding affinity depends significantly on experimental conditions and organism physiology (Antunes et al, 2017), the ATP binding affinities derived for fish NKA are similar to other values in the literature (210-540 µM; Morrison et al, 2006). Assuming that binding affinities of ATP to H + -ATPase are also similar, this suggests that ATP has a greater affinity for H + -ATPase than for NKA.…”

Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition‐dependent manner

“…NKA, Na + /K + ATPase; NKCC, Na + /K + /2Cl − cotransporter; 1.2 MCK, 30.5 mmol/L MgCl 2 , 11.7 mmol/L CaCl 2 , and 13.4 mmol/L KCl including NKA (Apell et al, 2017). In fact, activity of NKA in gills of crab is modulated by ambient Mg 2+ (Antunes et al, 2017;Masui et al, 2005Masui et al, , 2009. Moreover, Ca 2+ plays an important role in various biological processes, particularly in stabilizing biological membranes, increasing the tightness of intracellular tight junctions, and thereby controlling ion permeability across the gill epithelium in fish (McDonald & Milligan, 1988;McDonald et al, 1983).…”