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 crabUcides 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.…”

“…Euryhaline crabs exhibit adjustments in gill (Na + , K + )-ATPase activity and α-subunit mRNA expression in response to salinity change (Lovett et al, 2006a; Serrano et al, 2007; Masui et al, 2009; Garçon et al, 2009; Faleiros et al, 2018). The effects of reduced salinity on gill (Na + , K + )-ATPase activities have been investigated in the blue crabs Callinectes ornatus (Garçon et al, 2009; Leone et al, 2015), C. danae (Masui et al, 2009) and C. sapidus (Lovett et al, 2006b; Serrano et al, 2007), the hermit crab C. symmetricus (Faleiros et al, 2018; and Lucena et al, 2012; Antunes et al, 2017 as C. vittatus ) and the estuarine crab Neohelice granulata (Castilho et al, 2001; Genovese et al, 2004; Luquet et al, 2002; 2005). However, it is not clear whether the consequent increases in (Na + , K + )-ATPase activity result from enzyme activation, synthesis and recruitment of new protein to the cell membrane (Henry et al, 2002) or from adjustment of transport activity through regulatory phosphorylation (Silva et al, 2012).…”

Estudo bioquímico comparativo da (Na+ , K+)-ATPase branquial de Macrobrachium amazonicum (Heller, 1862) habitante de regiões continentais

“…Entretanto a população de M. amazonicum que habita uma represa em Sertãozinho apresenta duas famílias de sítios para a ligação de ATP (Santos et al, 2007), bem como a enzima de D. pagei (Furriel et al, 2010), Goniopsis cruentata (Moraes, 2019), Callinectes danae (Masui et al, 2002), C. symmetricus (Gonçalves et al, 2006;Lucena et al, 2012;Faleiros et al, 2018), e Ucides cordatus (Lucena et al, 2019b). Curiosamente estas últimas três espécies quando aclimatadas em baixa salinidade não apresentam o sítio de alta afinidade apresentando uma única curva estimulação (Masui et al, 2009;Antunes et al, 2017;Faleiros et al, 2018;Lucena et al, 2019b).…”

“…Dentro da família Varunidae de caranguejos de água salgada, o Eriocher sinensis e o H. sanguineus apresentam uma diminuição de atividade da (Na + , K + )-ATPase ao serem aclimatados em baixas salinidades, enquanto a espécie Helice formosensis apresenta um aumento da atividade (Tsai & Lin, 2007). Crustáceos que habitam o estuário e o mangue apresentam diferentes relações entre a atividade da (Na + , K + )-ATPase e salinidade de aclimatação (Gonçalves et al, 2006;Lucena et al, 2012;Antunes et al, 2017;Farias, 2017;Faleiros et al, 2018;Lucena et al, 2019b;Moraes, 2019).…”