Two isoforms of the enzyme carbonic anhydrase (CA) from the gills of the euryhaline green crab were sequenced and identified; these were found to match the cytoplasmic (CAc) and membrane-associated (CAg) isoforms known from other species. The mRNA of the membrane-associated isoform is present in significantly higher levels of abundance in gills of crabs acclimated to 32 ppt, at which the crab is an osmotic and ionic conformer. Upon transfer to low salinity (15 ppt), in which the crab is an osmoregulator, however, the cytoplasmic isoform undergoes a rapid 100-fold increase in abundance in the posterior gills, becoming the dominant isoform. CAg increases 3-fold initially and then remains elevated through 14 days of low salinity acclimation. The induction of CAc mRNA is believed to be the molecular basis for the 20 fold increase in CA protein-specific activity during low salinity acclimation. The initial increase in CAc mRNA takes place at 6 h, and maximal levels of expression are achieved by 24 h; this precedes the induction of CA activity and is within the time in which hemolymph osmotic and ionic concentrations stabilize at new acclimated levels. The increase in expression of the CAg isoform is believed to be more closely related to changes in the population of branchial chloride cells. Changes in the relative abundance of mRNA for the α-subunit of the Na+/K+-ATPase were smaller in magnitude than those for CAc, but the timing was similar. There were no changes in expression of a control gene, arginine kinase (AK) in posterior gills, and there were no significant changes in expression in anterior gills for any of the genes measured here. These results support the use of a control tissue (anterior gills) in addition to a control gene for expression studies.

Ablation/ligation, injection and gill perfusion experiments of different neuroendocrine centers such as the X-organ/sinus gland complex (XO/ SG) in the eyestalk, central nervous system, thoracic ganglia and the pericardial organs have demonstrated a neuroendocrine control of hydro-mineral homeostasis in crustaceans. The thoracic ganglia and pericardial organs contain mainly biogenic amines that have respectively effects on water movements and Na+ uptake via the activation of Na+/K+-ATPase with cAMP as second messenger. The eyestalk XO/SG complex synthesizes and stores a family of neuropeptides among which the Crustacean Hyperglycemic Hormone (CHH) appears as an important neuroendocrine factor influencing osmo- and iono-regulatory mechanisms. Recent investigations have revealed different CHH isoforms in crustaceans. In Astacidae, two CHH enantiomers are present, a levogyre (L-CHH) and a dextrogyre (D-Phe3-CHH) form, but only the D-isomer has a stimulating effect on osmoregulatory parameters. In Brachyura, different CHH-related peptides are stored in and released from different endocrine organs and they seem to act on ion homeostasis and water balance in several target organs depending on environmental or physiological stimuli.

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na(+)/K(+)-ATPase alpha-subunit, and one putative housekeeping gene, beta-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na(+)/K(+)-ATPase alpha-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). beta-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na(+)/K(+)-ATPase alpha-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 mum), and one in embryos just before hatching (at EI=420 mum), continuing in the freshly hatched juveniles. The Na(+)/K(+)-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.

SUMMARY Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill,CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill,and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the α-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative `housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity,but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

The structures of crustacean hyperglycemic hormones (CHH) were investigated in two crabs, the coastal euryhaline crab Pachygrapsus marmoratus11The sequences of PamCHHA XO, PamCHHB XO and PamCHHB PO have been included in the GenBank sequence database with accession numbers AY180333, AY180334 and AY180335 respectively. and the fresh water crab Potamon ibericum.22The sequences of PoiCHH XO and PoiCHH PO have been included in the GenBank sequence database with accession numbers DQ176431 and DQ176432 respectively. The neuropeptide mRNAs were extracted from pericardial and X-organs (PO and XO), and the sequences of the cDNA encoding the hormones’ precursors were determined. The X-organ preprohormones are composed of 29 and 28 amino acid signal peptides in P. marmoratus and P. ibericum respectively, followed by 43 and 41 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP) flanking the 72 amino acid crustacean hyperglycemic hormones. A similar organization is reported for pericardial preprohormones with identical sequences for the signal peptide, the CPRP and the N-terminal sequences of CHH (1–40), but remaining sequences (41–72 and 41–71) differing considerably. In P. marmoratus two CHH cDNAs were characterized from XO and evidences were obtained for the existence of at least two forms in the PO. From our results and by comparison with other known sequences, a consensus pattern for crab pericardial CHH could be pointed out. Analysis of the data presented in this article using phylogenetic methods reveals that the two crab species studied are much closer than previously predicted.

We studied the ontogeny of the eyestalk structure and of the l-CHH and d-Phe3-CHH synthesis in the X-organ/sinus gland (XO/SG) complex by light microscopy and immunocytochemistry in the freshwater crustacean Astacus leptodactylus. The optic ganglia start to differentiate in embryos at EI 190 μm (EI: eye index; close to 410 μm at hatching). At EI 270 μm, the three medullae (externa, interna, and terminalis) and the lamina ganglionaris are present and are organized as in the adult eyestalk. The l-CHH was localized in perikarya of neuroendocrine cells, in their tracts, and in SG from the metanauplius stage to the adult. The d-Phe3-CHH was visualized in XO perikarya, in their tracts and in SG of embryos from EI 350 μm and in all later studied stages. Co-localization of both CHH stereoisomers always occurred in the d-Phe3-CHH-producing cells. These results show that the synthesis of CHH enantiomers starts during the embryonic life in A. leptodactylus, and that the d-isomer is synthesized later than its l-counterpart. We discuss the post-translational isomerization as a way to generate hormonal diversity and the putative relation between d-Phe3-CHH synthesis and the ability to osmoregulate, occurring late during the embryonic life of Astacus leptodactylus.

This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na+ concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)related peptides, including stereoisomers (L-CHH and D-Phe(3) CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe(3) CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe(3) CHH fraction significantly increased the hemolymph osmolality and Na+ content 24h after injection. Two other CHH-related peptides caused a smaller increase in Na+ concentration. No significant variation was observed in hemolymph Cl-concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe(3) CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.