A Multi-Species Comparison and Evolutionary Perspectives on Ion Regulation in the Antennal Gland of Brachyurans.

Brachyurans inhabit a variety of habitats and have evolved diverse osmoregulatory patterns. Gills, antennal glands and a lung-like structure are important organs of crabs that maintain their homeostasis in different habitats. Species use different processes to regulate ions in the antennal gland, especially those with high terrestriality such as Grapsoidea and Ocypodoidea. Our phylogenetic generalized least square (PGLS) result also suggested that there is a correlation between antennal gland NKA activity and urine-hemolymph ratio for Na+ concentration in hypo-osmotic environments among crabs. Species with higher antennal gland NKA activity showed a lower urine-hemolymph ratio for Na+ concentration under hypo-osmotic stress. These phenomenon may correlate to the structural and functional differences in gills and lung-like structure among crabs. However, a limited number of studies have focused on the structural and functional differences in the antennal gland among brachyurans. Integrative and systemic methods like next generation sequencing and proteomics method can be useful for investigating the differences in multi-gene expression and sequences among species. These perspectives can be combined to further elucidate the phylogenetic history of crab antennal glands.

Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction.

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9-Asn28; α3, His34-Gly38; and α5, Glu62-Arg72), and a π-helix (π4, Cys43-Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, and Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

Ion regulation in the antennal glands differs among Ocypodoidea and Grapsoidea crab species.

Gills and the antennal gland are ion-regulatory organs in crabs. Previous studies have suggested that the differences in the morphology and ion regulation of gills and accessory respiratory organs between ocypodid and grapsid species are related to their distinct evolutionary transition to land habitats. In addition, Na+, K+-ATPase (NKA) activity and Na+ and NH4+ regulation in the antennal gland differ between ocypodid and grapsid species, which had different terrestrial adaptation trajectories. This study used five Ocypodoidea species and three Grapsoidea species from the intertidal and supratidal zones to further investigate the differences in ion regulation and NKA activity in the antennal gland between these crab families in different habitats. Crabs were transferred to 5 practical salinity unit (PSU) water, and osmolality, Na+ and Cl- concentrations in the urine and hemolymph, and NKA activity in the antennal gland were examined. Phylogenetic ANOVA results showed that the NKA activity in the antennal gland was higher in the ocypodid than grapsid groups, and Moran's I autocorrelation analysis also indicated that NKA activity in the antennal gland was phylogenetically correlated among crabs. K-means clustering showed a difference among the crabs in the crabs' Na+ and Cl- concentrations in the urine/hemolymph, NKA activities in the antennal gland and gill 6, and number of pairs of gills. Crabs with relatively high antennal gland NKA activity were found not only in the Ocypode species, which are better adapted to terrestrial environments, but also in two intertidal species of Gelasiminae. In conclusion, part of the Ocypodidae lineage may have a) the ability to reabsorb Na+ and b) higher NKA activity in the antennal gland than other families, and this phenomenon is phylogenetically correlated in Ocypodoidea and Grapsoidea. The physiological diversity in osmoregulation among intertidal and costal species provides a base to further investigate their ecological niches and guilds.

Functional anatomy and ion regulatory mechanisms of the antennal gland in a semi-terrestrial crab, Ocypode stimpsoni.

Brachyuran crabs from diverse habitats show great differences in their osmoregulatory processes, especially in terms of the structural and physiological characteristics of the osmoregulatory organs. In crustaceans, the antennal glands are known to be important in osmoregulation, and they play a functional role analogous to that of the vertebrate kidney. Nevertheless, the detailed structure and function of the antennal glands in different species have rarely been described. The aim of this study is to investigate the role of the antennal gland in ion regulation by examining the ultrastructure of the cells and the distribution of the ion regulatory proteins in each cell type in the antennal gland of a semi-terrestrial crab. The results showed that Na(+), K(+)-ATPase activity significantly increased in the antennal gland after a 4-day acclimation in dilute seawater and returned to its original (day 0) level after 7 days. Three major types of cells were identified in the antennal gland, including coelomic cells (COEs), labyrinthine cells (LBRs) and end-labyrinthine cells (ELBRs). The proximal tubular region (PT) and distal tubular region (DT) of the antennal gland consist of LBRs and COEs, whereas the end tubular region (ET) consists of all three types of cells, with fewer COEs and more ELBRs. We found a non-uniform distribution of NKA immunoreactivity, with increasing intensity from the proximal to the distal regions of the antennal gland. We summarise our study with a proposed model for the urine reprocessing pathway and the role of each cell type or segment of the antennal gland.

V-type H+-ATPase and Na+,K+-ATPase in the gills of 13 euryhaline crabs during salinity acclimation.

Because of their diverse habitats, crabs are excellent experimental species to study owing to the morphological changes and physiological adaptation that occur during their terrestrial invasion. Their hemolymphic osmoregulation in brackish water is crucial for a successful terrestrial invasion. Crabs can actively uptake or excrete ions upon salinity change, and the gills play a major role among the osmoregulatory organs. Several enzymes are involved in the osmoregulatory process, including Na+, K+-ATPase and V-type H+-ATPase (V-H+-ATPase). Na+, K+-ATPase is the driving force in establishing an ion gradient across the epithelial cell membrane in marine crabs. It has been reported that the osmoregulatory mechanisms in freshwater crabs are different from those in marine ones, suggesting that the driving force may come from V-H+-ATPase by generating the H+ ion gradient to facilitate the ion flow. Thirteen crab species from two families were used in this study. These crabs lived in five different habitats, including marine, intertidal, bimodal, freshwater and terrestrial habitats. The distribution of V-H+-ATPase in the 13 euryhaline crabs was revealed by histochemistry. V-H+-ATPase was localized in the apical region in crabs that could survive in the freshwater environment. We found that the freshwater and terrestrial crabs with stable Na+, K+-ATPase activity during salinity changes tended to have an apical V-H+-ATPase, whereas the intertidal ones with varying Na+, K+-ATPase activity showed a cytoplasmic V-H+-ATPase distribution. Finally, in Uca formosensis, a crab that had stable Na+, K+-ATPase activity, a significant difference in V-H+-ATPase activity between salinities was found. In conclusion, the hypothesis that V-H+-ATPase plays a crucial role in the freshwater adaptation of crabs is supported by our systemic investigation on 13 euryhaline crabs.