Effects of photoperiod on gluconeogenic activity and total lipid concentration in organs of crabs, Neohelice granulata, challenged by salinity changes

Abstract. This study assessed the effects of long (LD) or short (SD) days on the conversion of [¹⁴C]-glycerol to [¹⁴C]-glucose and total lipid concentration in organs of the crab Neohelice granulata challenged by a change in external salinity. In the 20‰-acclimated crabs, no difference was found in the concentration of total lipids in the muscle, hepatopancreas, gills, or hemolymph between crabs acclimated to SD or LD. In SD crabs, the total lipid levels in the anterior and posterior gills did not decrease during an osmotic challenge. Only in the posterior gills did the total lipid levels decrease during acclimation to the 34‰ medium in LD animals. The total lipid concentration in the hemolymph decreased after 1 d of osmotic stress in SD, and increased in the hepatopancreas. In LD crabs, the lipid contents decreased gradually in muscle, and in the hepatopancreas on day 3 after transfer to 34‰ medium. In 20‰-acclimated crabs, the gluconeogenesis activity in both sets of gills was higher in LD than in SD animals. The gluconeogenesis capacity decreased in both sets of gills on the first day of osmotic challenge in SD, and in the posterior gills on the third day in LD crabs. These results suggest that in organs of N. granulata , photoperiod affects the metabolic adjustments to an osmotic challenge.     

Hepatopancreas gluconeogenesis during hyposmotic stress in crabs Chasmagnathus granulata maintained on high-protein or carbohydrate-rich diets

The incorporation of [14C]-alanine or [14C]-lactate into glucose was measured in hepatopancreas fractions from Chasmagnathus granulata crabs adapted to a high protein or a carbohydrate-rich diet and submitted or not (control group) to hyposmotic stress. Gluconeogenic capacity and phosphoenolpyruvate carboxykinase (PEPCK) activity increased during acclimation to a dilute medium in C. granulata hepatopancreas. In intact animals, high hemolymph urea levels occurred for the high-protein regimen and for crabs fed both diets and submitted to hyposmotic stress. It could be that the amino acids released during hyposmotic stress are deaminated in the hepatopancreas, and that the carbon chains are used as substrate for gluconeogenesis. Hepatopancreas gluconeogenesis seems to be one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress in C. granulata.     

Metabolism and growth of juveniles of Litopenaeus vannamei: effect of salinity and dietary carbohydrate levels

The present study was designed to understand how carbohydrate (CBH) and protein metabolism are related in the penaeid shrimp Litopenaeus vannamei. With this information, we obtained a comprehensive schedule of the protein-carbohydrate metabolism including enzymatic, energetic, and functional aspects. We used salinity to determine its role as a modulator of the protein-carbohydrate metabolism in shrimp. Two experiments were designed. The first experiment evaluated the effect of CBH-salinity combinations in growth and survival, and hemolymph glucose, protein, and ammonia levels, digestive gland glycogen, osmotic pressure, and glutamate dehydrogenase (GDH) of L. vannamei juveniles acclimated during 18 days at a salinity of 15 per thousand and 40 per thousand. The second experiment was done to evaluate the effect of dietary CBH level on pre- and postprandial oxygen consumption, ammonia excretion, and the oxygen-nitrogen ratio (O/N) of juvenile L. vannamei in shrimps acclimated at 40 per thousand salinity. We also evaluated the ability of shrimp to carbohydrate adaptation. We made phosphoenolpyruvate carboxykinase (PECPK) and hexokinase activity measurements after a change in dietary carbohydrate levels at different times during 10 days. The growth rate depended on the combination salinity-dietary CBH-protein level. The maximum growth rate was obtained in shrimps maintained at 15 per thousand salinity and with a diet containing low CBH and high protein. The protein in hemolymph is related to the dietary protein levels; high dietary protein levels produced a high protein concentration in hemolymph. This suggests hemolymph is able to store proteins after a salinity acclimation. Depending on the salinity, the hemolymph proteins could be used as a source of osmotic effectors or as metabolic energy. The O/N values obtained show that shrimp used proteins as a source of energy, mainly when shrimps were fed with low CBH. The role played by postprandial nitrogen excretion (PPNE) in apparent heat increase (AHI) (PPNE/AHI ratio) is lower in shrimps fed diets containing high CBH in comparison with shrimps fed diets containing low CBH levels. These results confirm that the metabolism of L. vannamei juveniles is controlled by dietary protein levels, affecting the processes involved in the mechanical and biochemical transformations of ingested food. A growth depression effect was observed in shrimps fed with low-CBH protein diets and maintained in 40 per thousand salinity. In these shrimps, the hemolymph ammonia concentration (HAC) was significantly higher than that observed in shrimps fed with low CBH and maintained in 15 per thousand salinity. That high HAC level coincided with lower growth rate, which suggests that this level might be toxic for juveniles of L. vannamei. Results obtained for GDH activity showed this enzyme regulated both HAC and hemolymph protein levels, with high values in shrimps fed with low CBH levels and maintained in 40 per thousand salinity, and lower in shrimps fed with high CBH and maintained in 15 per thousand salinity. These differences mean that shrimp with a high-gill GDH activity might waste more energy in oxidation of the excess proteins and amino acids, reducing the energy for growth. It was evident that L. vannamei can convert protein to glycogen by a gluconeogenic pathway, which permitted shrimp to maintain a minimum circulating glucose of 0.34 mg/ml in hemolymph. A high PECPK activity was observed in shrimps fed a diet containing low CBH level indicating that the gluconeogenic pathway is activated, as in vertebrates by low dietary CBH levels. After a change in diet, we observed a change in PEPCK; however, it was lower and seems to depend on the way of adaptation, because it occurred after 6 days when adapting to a high-CBH diet and with little change for the low-CBH diet.     

Hepatopancreas gluconeogenesis during anoxia and post-anoxia recovery in Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets

C. granulata is a semiterrestrial crab that lives in the mesolittoral and the supralittoral zones of estuaries and faces hypoxia and anoxia when exposed to atmospheric air. The carbohydrate or protein content of the diets administered to the crabs induced different metabolic adjustments during anoxia and post-anoxia recovery period. During the first hour in anoxia a marked increase in L-lactate concentration in hemolymph was induced, followed by a reduction in its levels accompanied by two peaks in hepatopancreas gluconeogenic capacity. Anoxia exposure did not induce a reduction in the hepatopancreas phosphoenolpyruvate carboxykinase activity in either dietary group. Our results suggest that in anaerobiosis this crab uses the conversion of lactate to glucose in hepatopancreas to maintain the acid-base balance and the glucose supply. In post-anoxia recovery, the fate of L-lactate is the hepatopancreas gluconeogenesis in high protein maintained crabs. On the other hand, in the crabs maintained on carbohydrate-rich diet the L-lactate levels decreased gradually in the hemolymph during the post-anoxia recovery; however, the hepatopancreas gluconeogenesis did not increase. In both dietary groups, an increase in the gluconeogenic capacity of hepatopancreas occurred at 30 h of post-anoxia recovery.     

The effects of rapid salinity change on in vivo arginine kinase flux in the juvenile blue crab,Callinectes sapidus

The effect of acclimation salinity and salinity changes on the concentration of high-energy phosphate metabolites and arginine kinase (AK) flux was examined in vivo in juvenile blue crabs using 31P-nuclear magnetic resonance (NMR). Crabs were acclimated for 7 days to a salinity of 5 or 35 per thousand and then placed in a flow apparatus that could sustain the animals while NMR spectra were acquired. Crabs were subjected to either hyperosmotic salinity changes, where an animal acclimated to 5 per thousand was exposed to a salinity of 35 per thousand, or hyposmotic changes, which involved the reciprocal exchange. Neither acclimation salinity nor salinity change had a significant effect on the concentrations of arginine phosphate, inorganic phosphate or ATP. 31P-NMR saturation transfer experiments were used to determine the effect of salinity on the forward and reverse flux of the AK reaction. There was no significant effect of acclimation salinity or salinity change on the flux rate through this reaction. This is in contrast to previous results, which showed that AK flux in isolated muscle was sensitive to prevailing osmotic conditions (Holt and Kinsey, J. Exp. Biol. 205 (2002) 1775-1785). The present study indicates that the integrated osmoregulatory capacity of the intact animal is sufficient to preserve cellular energy status and enzyme function during acute salinity changes.     

Possible role of carbonic anhydrase, V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion transport across the gills of the euryhaline crab Chasmagnathus granulatus

We studied the participation of carbonic anhydrase (CA), V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion absorption through the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2 per thousand, 10 per thousand, 30 per thousand (about seawater), and 45 per thousand salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from crabs acclimated to either 2 per thousand or 10 per thousand with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2 per thousand and perfused with 20 per thousand saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (V(te)) by 47%, further addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30 per thousand saline (iso-osmotic for seawater acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V-H(+)-ATPase and Cl(-)/HCO3-) reduced V(te) by 15-16% in gills perfused with normal 20 per thousand saline, and by 77% and 45%, respectively when they were applied in Na-free 20 per thousand saline, suggesting the participation of those transporters and cytosolic CA in electrogenic ion absorption.     

Ontogeny of osmoregulation in the crayfish Astacus leptodactylus

Osmoregulation was studied during the postembryonic development of Astacus leptodactylus Eschscholtz 1823 in juvenile stages 1-8 and in adults. Juveniles hatch and later stages develop in freshwater or in moderately saline waters. The time of acclimation from freshwater to a saline medium increased from early juveniles to adults. At all stages, it was longer than in comparable stages of marine crustaceans, reflecting the high impermeability of the teguments to water and ions. All stages were able to hyperisoosmoregulate. In freshwater, the ability to hyperosmoregulate was established at hatching and increased during development. The hemolymph osmolality increased from 286 mosm kg-1 in stage 1 juveniles to 419 mosm kg-1 in adults. All stages also hyperregulated at low salinities (7 per thousand and 13 per thousand salinity) and were osmoconformers at higher salinities up to 21 per thousand salinity. The lowest isosmotic salinity tended to increase with the developmental stages. The ability to osmoregulate at hatch and throughout postembryonic development is probably a key physiological adaptation in this and other freshwater crayfish.     

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.     

Hydromineral regulation in the hydrothermal vent crab Bythograea thermydron.

This study investigates the salinity tolerance and the pattern of osmotic and ionic regulation of Bythograea thermydron Williams, 1980, a brachyuran crab endemic to the deep-sea hydrothermal vent habitat. Salinities of 33 per thousand-35 per thousand were measured in the seawater surrounding the captured specimens. B. thermydron is a marine stenohaline osmoconformer, which tolerates salinities ranging between about 31 per thousand and 42 per thousand. The time of osmotic adaptation after a sudden decrease in external salinity is about 15-24 h, which is relatively short for a brachyuran crab. In the range of tolerable salinities, it exhibits an iso-osmotic regulation, which is not affected by changes in hydrostatic pressure, and an iso-ionic regulation for Na(+) and Cl(-). The hemolymph Ca(2+) concentration is slightly hyper-regulated, K(+) concentration is slightly hyper-hypo-regulated, and Mg(2+) concentration is strongly hypo-regulated. These findings probably reflect a high permeability of the teguments to water and ions. In addition to limited information about salinity around hydrothermal vents, these results lead to the hypothesis that B. thermydron lives in a habitat of stable seawater salinity. The osmoconformity of this species is briefly discussed in relation to its potential phylogeny.     

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea--Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs.     

Combined effect of temperature and salinity on the Thermotolerance and osmotic pressure of juvenile white shrimp litopenaeus vannamei (Boone)

Thermotolerance (CTMax) was determined in L. vannamei in three salinities and five acclimation temperatures 20, 23, 26, 29 and 32 °C. In white shrimp, the CTMax was not significantly affected by salinity (P>0.05). A direct relationship was obtained between CTMax and acclimation temperature. The end point of the CTMax in L. vannamei exposed to different combinations of temperature and salinity was defined as the loss of the righting response (LRR). The acclimation response ratio (ARR) for the juveniles of white shrimp ranged from 0.42 to 0.49; values in agreement with other crustaceans from tropical and sub tropical climates. The osmotic pressure of the hemolymph was measured in control organisms and in organisms exposed to CTMax; significant differences were found in organisms maintained in 10 and 40 psu, but there were no significant differences in hemolymph osmotic pressure in those that were acclimated to 26 psu.     

Ontogeny of osmoregulation and salinity tolerance in a mangrove crab, Sesarma curacaoense (Decapoda: Grapsidae)

The grapsid crab Sesarma curacaoense is believed to represent the closest saltwater relative to the ancestor which gave rise to an adaptive radiation of endemic freshwater and terrestrial species on the island of Jamaica. Living in mangrove swamps with variable salinity conditions and showing semiterrestrial behaviour, S. curacaoense exhibits ecological adaptations to non-marine conditions. In laboratory experiments, we studied the salinity tolerance during development from hatching to the end of the first juvenile stage. Successful development through metamorphosis occurred in the full salinity range tested (15-32 per thousand), although mortality was significantly enhanced and development delayed at 15 per thousand. In another series of experiments, we studied the ontogeny of the capability for osmoregulation, which is considered as the physiological basis of osmotic stress tolerance. Our results show that S. curacaoense is from hatching a fairly strong hyperosmoregulator in dilute media. This capability increased gradually from hatching throughout the larval and juvenile development. In seawater (32 per thousand) and at an enhanced salt concentration (44 per thousand), the zoeal stages remained hyperosmoconformers. The capability for hypoosmoregulation in concentrated media appeared first in the megalopa stage and increased thereafter. Adult crabs were observed to be strong hyper-hypo-osmoregulators in a salinity range from at least 1 per thousand to 44 per thousand. The unusually early appearance of strong regulatory capabilities, particularly in dilute media, is interpreted as a physiological preadaptation that should have facilitated the evolutionary process of adaptive radiation in non-marine environments on Jamaica.     

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.)

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.     

Respiratory and osmoregulatory responses of the hermit crab, Clibanarius vittatus (Bosc), to salinity changes

Intertidal hermit crabs were stepwise acclimated to 10, 20, and 30‰ salinity (S) and 21 ± 1 °C. Hemolymph osmolality, sodium, chloride, and magnesium were isosmotic (isoionic) to ambient sea water at 30‰ and hyperosmotic (hyperionic) at 20 and 10‰ S, while hemolymph potassium was significantly hyperionic in all acclimation salinities. Total body water did not differ significantly at any acclimation salinity. Oxygen uptake rates were higher in summer-than winter-adapted crabs. No salinity effect on oxygen consumption occurred in winter-adapted individuals. Summer-adapted, 30‰ acclimated crabs had a significantly lower oxygen consumption rate than those acclimated 10 and 20‰ S. Crabs exposed to 30 10 30‰ and 10 30 10‰ semidiurnal (12 h) and diurnal (24.8 h) fluctuating salinity regimes showed variable osmoregulatory and respiratory responses. Hemolymph osmolality followed the osmolality of the fluctuating ambient sea water in all cases, but was regulated hyperosmotically. Hemolymph sodium, chloride, and magnesium concentrations were similar to hemolymph osmolality changes. Sodium levels fluctuated the least. Hemolymph potassium was regulated hyperionically during all fluctuation patters, but corresponded to sea water potassium only under diurnal conditions. The osmoregulatory ability of Clibanarius vittatus (Bosc) resembles that reported for several euryhaline brachyuran species. The time course of normalized oxygen consumption rate changed inversely with salinity under semidiurnal and diurnal 10 30 10‰ S fluctuations. Patterns of 30 10 30‰ S cycles had no effect on oxygen consumption rate time course changes. The average hourly oxygen consumption rates during both semidiurnal fluctuations were significantly lower than respective control rates, but no statistical difference was observed under diurnal conditions.     

Enzymatic and feeding behaviour of Argopecten purpuratus under variation in salinity and food supply

Argopecten purpuratus is a bivalve filter feeder inhabiting protected areas of the Peruvian coastline and northern and central Chile. The species is commercially valuable and the natural stocks are over-exploited in Chile, mainly in the central region. Expansion of aquaculture farms to the geographically protected coastline of southern Chile is a potential solution to this problem. So, the objective of the present study was to determine the acute feeding and digestive response of A. purpuratus in relation to short acclimation periods (1, 3 and 7 days) and exposure to variable salinity (24 per thousand, 27 per thousand and 30 per thousand) with two different diets. The feeding behaviour of A. purpuratus follows that described for other species of bivalves whereby the decrease in salinity produces a reduction in the clearance and ingestion rates. This behaviour is not affected by the diet type or the 7 day acclimation period. Lower values of absorption efficiency are associated with increasing food concentration. The response of the AE at 24 per thousand salinity and low organic content diet indicates that the interaction of low salinity and low organic content diet reduces the AE to values approaching zero. The enzymatic investment of A. purpuratus at low salinity (24 per thousand) is similar for both diets and more reduced than for 30 per thousand and 27 per thousand salinity. The results show that osmotic stress leads to a reduction of the enzymatic response capacity. The multiple regression analysis indicates that different responses are observed as a function of the enzymatic activity and the organ. A reduction of the carbohydrase activities in the digestive gland was observed to low salinity (24 per thousand), specifically amylase and celullase. The results of this study are ecophysiologically relevant and highlight a relationship between digestive enzymatic activity and salinity, and may explain the possible differences in the energetic balance of A. purpuratus in response to changes in salinity.     

The effect of seawater composition and osmolality on hemolymph levels of methyl farnesoate in the green crab Carcinus maenas

Green crabs, Carcinus maenas, exposed to dilute seawater (e.g., 5 ppt salinity, approximately 150 mOsm/kg) have hemolymph levels of methyl farnesoate (MF) that are up to 10-fold higher than animals in isosmotic seawater (27 ppt, approximately 800 mOsm/kg). In this paper, we examine aspects of osmotic and ionic stress to identify factors involved in elevating MF levels. MF levels did not rise after exposure to concentrated seawater, so only hypoosmotic stress elevates MF. MF levels rose in animals exposed to dilute seawater containing mannitol to make it isosmotic, indicating that the hypoosmotic rise in MF is due to decreased ion concentrations. Individual ions were investigated by exposing crabs either to isosmotic seawater with low concentrations of an ion or to dilute seawater with high concentrations of an ion. Ca(2+) and Mg(2+) in combination affected MF levels. Finally, we found that the increase in MF levels was accelerated when hemolymph osmolality was precociously lowered by partially replacing hemolymph with deionized water prior to transferring animals to dilute seawater. Thus, the 6-8 h delay between exposing crabs to dilute sea water and observing an increase in MF appears to reflect the time needed for specific hemolymph ions to decrease below a threshold concentration.     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.