The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na(+)-K(+)-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na(+)-K(+)-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.     

Effects of prolactin-releasing peptide on tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in estrogen-treated female rats

Both systemic and central effects of a newly discovered prolactin (PRL)-releasing factor (PRF), prolactin-releasing peptide (PrRP), were determined in this study. Systemic injection of PrRP (1 and 10 microg/rat, i.v.) stimulated PRL secretion in ovariectomized, estrogen-treated rats similar to the effect of another PRF, thyrotropin-releasing hormone (TRH). Pretreatment with a dopamine D2 receptor antagonist, sulpiride (1 microg/rat, i.v.), potentiated the stimulatory effect of both PrRP and TRH on PRL secretion. Using the double-labeling immunohistochemical method, PrRP-immunoreactive terminals were found in close contact with tyrosine-hydroxylase-immunoreactive neurons in the hypothalamic arcuate nucleus. Central administration of PrRP (0.1-1,000 ng/rat, i.c.v.) stimulated tuberoinfundibular but not nigrostriatal dopaminergic neuronal activity in 15 min. Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence and striatum were used as indices for tuberoinfundibular dopaminergic (TIDA) and nigrostriatal dopaminergic neuronal activities, respectively. The serum PRL level, however, was not significantly changed. Similar treatment with TRH (10 ng/rat, i.c.v.) stimulated and inhibited TIDA neuronal activity and serum PRL, respectively, at 30 min. In summary, PrRP may play a role in both the central and peripheral control of PRL secretion.     

Wide-gene expression analysis of lipid-relevant genes in nutritionally challenged gilthead sea bream (Sparus aurata)

Disturbances of lipid metabolism are a major problem in livestock fish and the present study analysed the different tissue expression patterns and regulations of 40 lipid-relevant genes in gilthead sea bream. Nineteen sequences, including fatty acid elongases (4), phospholipases (7), acylglycerol lipases (8) and lipase-maturating enzymes (1), were new for gilthead sea bream (GenBank, JX975700, JX975701, JX975702, JX975703, JX975704, JX975705, JX975706, JX975707, JX975708, JX975709, JX975710, JX975711, JX975712, JX975713, JX975714, JX975715, JX975716, JX975717 and JX975718). Up to six different lipase-related enzymes were highly expressed in adipose tissue and liver, which also showed a high expression level of Δ6 and Δ9 desaturases. In the brain, the greatest gene expression level was achieved by the very long chain fatty acid elongation 1, along with relatively high levels of Δ9 desaturases and the phospholipase retinoic acid receptor responder. These two enzymes were also expressed at a high level in white skeletal muscle, which also shared a high expression of lipid oxidative enzymes. An overall down-regulation trend was observed in liver and adipose tissue in response to fasting following the depletion of lipid stores. The white skeletal muscle of fasted fish showed a strong down-regulation of Δ9 desaturases in conjunction with a consistent up-regulation of the “lipolytic machinery” including key enzymes of tissue fatty acid uptake and mitochondrial fatty acid transport and oxidation. In contrast, the gene expression profile of the brain remained almost unaltered in fasted fish, which highlights the different tissue plasticity of lipid-related genes. Taken together, these findings provide new fish genomic resources and contribute to define the most informative set of lipid-relevant genes for a given tissue and physiological condition in gilthead sea bream.     

Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Serum levels of prolactin during the ovarian cycle,pregnancy, and lactation in the common marmoset (Callithrix jacchus)

A homologous double-antibody radioimmunoassay developed for humans was used to measure serum prolactin, progesterone, and estradiol in common marmosets. In the ovarian cycle of common marmosets, serum progesterone began to increase after an estradiol surge, attained a peak level, and then declined before the ensuing pre-ovulatory rise in estradiol. During the luteal phase, the change in serum concentrations of estradiol was synchronized with that of progesterone. During the ovarian cycle there was no consistent change in serum prolactin concentrations. During the last 75 days of pregnancy the prolactin level was higher than during the ovarian cycle and the first 70 days of pregnancy. Moreover, during lactation, mothers with suckling twin infants had a higher prolactin level than during the final stage of pregnancy.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Effects of low-salinity on the growth and development of spotted halibut Verasper variegatus in the larva-juvenile transformation period with reference to pituitary prolactin and gill chloride cells responses

Low-salinity adaptability was investigated in a flatfish spotted halibut Verasper variegatus during the period from late metamorphic larvae to early juveniles by a 20-day rearing experiment under different salinity regimes (1, 4, 8, 16 and 32 ppt). Effects of low-salinity on growth and development were examined and the changes in the prolactin (PRL) production level in the pituitary and the gill chloride cell morphology were examined as physiological backgrounds for low salinity adaptation. PRL cells and chloride cells were identified by immunocytochemistry with a specific antiserum for PRL₁₈₈ and Na⁺,K⁺-ATPase. Most of the fish exposed to over 4 ppt survived for 20 days, but all the fish exposed to 1 ppt died within 5 days. Fish kept in intermediate salinities (8, 16 ppt) grew significantly better than those in the control group (32 ppt). Fish exposed to 4 ppt attained almost the same body length as the control group at 20 days after transfer, although these fish showed an abnormally dark body color as well as delayed development. These results suggested that spotted halibut has a high-adaptability to low-salinity environments and prefers an intermediate salinity near iso-osmolality (about 12 ppt) from the late metamorphic larval stage, but does not completely adapt to a hypoosmotic of 4 ppt salinity or less than half of the osmolality. The percentage of PRL-cell volume to pituitary volume was significantly higher at 4 ppt than in the control group. The chloride cells in gill filaments were significantly larger at 4 ppt than in the control group. These results suggest that juveniles could adapt to a low-salinity environment due to the activation of PRL production and enlargement of chloride cells. These laboratory findings suggest that late metamorphic larvae and early juveniles of spotted halibut may utilize a low salinity environment such as estuarine tidal flats or very shallow coastal areas as their nursery grounds in the sea.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Physiological impact of salinity increase at organism and red blood cell levels in the European flounder (Platichthys flesus)

Blood respiratory, acid-base, and ionic changes in response to hyperosmotic shock were studied in vivo and in vitro in the European flounder. One primary aim was to evaluate regulatory changes in red blood cell (RBC) volume and its interrelationship with blood O₂ transporting properties. An acute increase in the ambient salinity from 10 to 30 ppt caused small but significant increases in extracellular osmolality (<20 mosM kg⁻¹), [Na⁺], and [Cl⁻], which were corrected within 48 h. RBC volume was not significantly changed 3 h after the in vivo exposure to elevated salinity. A small metabolic acidosis was fully developed within 3 h, and this acidosis seemed responsible for a modest decrease in blood O₂ affinity (i.e., increased P₅₀-O₂ tension at 50% O₂ saturation). RBC organic phosphates were unchanged. In vitro elevation of whole blood extracellular osmolality by 60 mosM kg⁻¹ caused immediate RBC shrinkage. The subsequent regulatory volume increase (RVI) showed a graded dependency on blood O₂ saturation (S_O2). At S_O2 values of 0% and 20%, there were full RBC volume recoveries within 120 min, RVI was partial at S_O2 values of 45% and 55%, and RVI was absent at a S_O2 of 100%. S_O2 and P₅₀ did not change significantly during RBC shrinkage and RVI. Thus, the up-concentration of cellular haemoglobin and organic phosphates in hyperosmotically shrunken RBCs had minimal influence on blood O₂ transporting properties. The degree of cell shrinkage and time needed for RVI were positively correlated with the magnitude of the rise in extracellular osmolality. The RVI proceeded via elevation of cellular [Na⁺], [Cl⁻], and to some extent also [K⁺]. Cell volume regulatory mechanisms are only needed to correct minor volume disturbances in vivo, because changes in extracellular osmolality were limited by an efficient osmotic regulation at the epithelial interface between extracellular compartment and environment.     

Characteristics of the antitumor activities in tumor cells and modulation of the inflammatory response in RAW264.7 cells of a novel antimicrobial peptide, chrysophsin-1, from the red sea bream (Chrysophrys major)

The antimicrobial peptide, chrysophsin-1, exhibits antimicrobial activities with similar efficiencies for both gram-negative and gram-positive bacteria. In this study, we examined the antitumor activity and modulation of the inflammatory response of a synthetic chrysophsin-1 peptide. In vitro results showed that chrysophsin-1 had greater inhibitory effects against human fibrosarcoma (HT-1080), histiocytic lymphoma (U937), and epithelial carcinoma (HeLa) cells. LDH release by HeLa cells was comparable to that of an MTS assay after treatment with 1.5-3 μg/ml chrysophsin-1 for 24 h. Under SEM and TEM observations, we found no intact cell membranes after chrysophsin-1 treatment of HeLa cells for 8 h. The suggested mechanism of the cytotoxic activity of chrysophsin-1 was disruption of cancer cell membranes. In addition, we also examined caspase-3, -8, and -9 activities by Western blotting; the results excluded the participation of apoptosis in chrysophsin-1's effect on HeLa cells. Stimulation by lipopolysaccharide induced tumor necrosis factor (TNF)-α which was able to modulate chrysophsin-1 treatment of RAW264.7 cells and inhibited endogenous TNF-α release but did not block its secretion. With data from this study, we demonstrate that chrysophsin-1 has antimicrobial and antitumor activities and modulates the inflammatory response in RAW264.7 cells.     

Metabolic and osmoregulatory changes in response to reduced salinities in the red grouper, Epinephelus akaara (Temminck & Schlegel), and the black sea bream, Mylio macrocephalus (Basilewsky)

Red groupers (Epinephelus akaara Temminck & Schlegel) and black sea breams (Mylio macrocephalus Basilewsky) were transferred from 30‰ into 3, 7, 12, 20, and 30‰ salinity. Fish were sampled at 0, 6, 24, 96, 168 and 336h after transfer. Serum osmolality, glucose, protein, Na⁺, K⁺, Ca²⁺, liver glycogen, liver protein, muscle water and haematocrit were determined. In general, transient disturbances in these variables were observed after transfer. For both species, no tissue hydration was observed upon acclimation to different salinities, whereas a progressive increase in haematocrit value was found as salinity decreased. Liver glycogen of both species, however, was higher in hypo-osmotic salinities. Serum Na⁺ of the red groupers declined upon acclimation to 7‰ salinity but the opposite was found for the black sea breams. The results indicate that both species are extremely euryhaline, and physiological stress is unlikely to occur within the salinity regime of 7 to 30‰ Comparatively, the black sea bream appears to be a more efficient osmoregulator.     

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.     

The use of metabolism to evaluate the toxicity of dodecil benzen sodium sulfonate (LAS-C12) on the Mugil platanus (mullet) according to the temperature and salinity

The effects of watering exposition to different concentrations of LAS-C12 (1, 2.5 and 5 mg l⁻¹) about the Mugil platanus routine metabolism were evaluated. The metabolic rates were estimated through experiments accomplished in each of the twelve possible combinations of three temperatures (25, 20 and 15 °C) and three salinities (35, 20 and 5). The results show that the oxygen consumption increases according to the LAS-C12 concentration in all temperatures and salinities studied. At the highest concentration employed (5 mg l⁻¹), and the salinity 5 in the temperatures 25 and 20 °C, oxygen consumption increases 80% in relation to the control. In general, the pollutant effects on oxygen consumption were more pronounced at the highest temperatures and salinities 5 and 35.     

The influence of salinity on fertilization and larval development of Nereis virens (Polychaeta, Nereidae) from the White Sea

Effect of salinity on fertilization and early development of the polychaeta Nereis virens (Sars) from the White Sea was examined in laboratory experiments. The comparison of salinity resistance of different developmental stages of N. virens showed gradual increase of euryhalinity during ontogenesis—from fertilized eggs to juveniles.Successful fertilization and effective development (≥70-75%) was possible in narrow salinity range 22-34‰. The salinity range of successful development for trochophore and nectochaete larvae reached 14-45‰. This increase of the limits of salinity tolerance in trochophore and nectochaete larvae probably was due to the formation of protonephridium system.Rate of metamorphosis of N. virens was tested under temperature 5, 10, 17 and 23 °C and salinity 22-14‰. The highest rate of metamorphosis was marked at the temperature of 23 °C in salinities higher than 14‰.Our data confirms that N. virens originates from warm seas with oceanic salinity.     

The nature of the adhesion of tentacles to shells during shell-climbing in the sea anemone Calliactis parasitica (Couch)

Phase contrast microscopy and scanning electron microscopy show that during the response of the symbiotic sea anemone Calliactis parasitica (Couch) to shells of Buccinum undatum (L.) three times as many spirocysts as nematocysts are discharged. Observations indicate that spirocysts are responsible for the adhesion of tentacles to shells.Discharge levels are not significantly influenced by the nature of the substratum to which the anemones are attached. The reported observation that fewer tentacles adhere to shells when anemones are settled on shells than when they are fixed on a different substratum is re-interpreted in terms of a new model for the control of spirocyst discharge.     

The morphology and ultrastructure of the peripheral olfactory organ in newly metamorphosed coral-dwelling gobies, Paragobiodon xanthosomus Bleeker (Gobiidae, Teleostei)

We examined the peripheral olfactory organ in newly metamorphosed coral-dwelling gobies, Paragobiodon xanthosomus (SL=5.8mm+/-0.8mm, N=15), by the aid of electron microscopy (scanning and transmission) and light microscopy. Two bilateral olfactory placodes were present in each fish. They were oval-shaped and located medio-ventrally, one in each of the olfactory chambers. Each placode had a continuous cover of cilia. The placode epithelium contained three different types of olfactory receptor neurons: ciliated, microvillous and crypt cells. The latter type was rare. Following a pelagic larval phase, P. xanthosomus settle to the reef and form an obligate association with one species of coral, Seriatopora hystrix. Their well-developed olfactory organs likely enable larvae of P. xanthosomus to detect chemical cues that assist in navigating towards and selecting appropriate coral habitat at settlement. Our findings support past studies showing that the peripheral olfactory organ develops early in coral reef fishes.     

Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Pagellus bogaraveo.The spermatozoon of P. bogaraveo belongs, like that of the other sparid fish, to the teleostean “type I” spermatozoon with the flagellar axis insert perpendicular to the nuclear fossa. It has an ovoidal head, a short, cylindrically shaped midpiece and a long tail region. The nucleus reveals a deep invagination (nuclear fossa), in which the centriolar complex is located, and a satellite nuclear notch shaped like a golf club. The two centrioles are perpendicular to each other and show a conventional “9+0” pattern. The distal centriole is attached to the nuclear envelope by means of basal feet and radial fibers made of electron-dense material. Below the basal plate, plasma membrane pinches in, and the necklace, a specialized connection joining axonemal doublets to the plasma membrane, is visible. The short midpiece houses one mitochondrion. The flagellum is perpendicularly and eccentrically with respect to the nucleus and contains the conventional “9+2” axoneme.     

Fuel use and corticosterone dynamics in hatchling green sea turtles (Chelonia mydas) during natal dispersal

During their natal dispersal hatchling sea turtles depart their nest, beach and inshore areas quickly to move into offshore developmental habitat using their finite energy stores. Patterns of fuel use and endocrine responses that could facilitate hatchling sea turtle dispersal activity are poorly understood. This study, examined aspects of intermediary metabolism by measuring plasma fuel use and an endocrine response of hatchling green turtles (Chelonia mydas) during terrestrial and aquatic activity coinciding with natal dispersal. Specifically, we measured plasma concentrations of glucose, non-esterised free fatty acids and protein to gauge the contributions of carbohydrate, lipid and protein metabolism for fuelling natal dispersal. In addition, we measured plasma levels of the steroid hormone corticosterone (CORT) a hormone implicated in regulating a number of metabolic events associated with migration and energy use in vertebrates. During terrestrial activity, hatchlings ascended through the sand from their nests and exhibited significant increases in plasma CORT and lactate indicating intense periods of anaerobic activity. During swimming, all plasma metabolites, with the exception of plasma protein, peaked between 1 and 4 h post-beginning swimming activity. Plasma CORT peaked at between 3 and 5 h of swimming activity. These plasma concentrations are consistent with intensive activity inducing catabolism of carbohydrate, lipid and protein stores to support prolonged activity. These results are similar to other vertebrates and suggest a relatively uniform cascade of physiological processes during such arduous migratory events.     

Sexual and seasonal variations in osmoregulation and ionoregulation in the estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda)

The estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda, Brachyura) inhabits salt marshes along the South Atlantic coast from Rio de Janeiro (Brazil) to Patagonia (Argentina). In the present study, salinity tolerance (0-45‰; 16-1325 mOsm/kg H₂O) and hemolymph osmotic and ionic (Na⁺, Cl⁻, and K⁺) regulation in both female and male C. granulatus were analyzed in summer and winter. Results showed that both female and male C. granulatus are euryhaline. Mortality was only observed in extremely low salinity (0‰; 16 mOsm/kg H₂O) for both sexes. For females, the LT₅₀ at 0‰ salinity was similar in summer (20.1 h) and winter (17.4 h). Males were more tolerant to salinity than females in both seasons, and mortality was observed only in summer (LT₅₀ = 50.9 h). Results from freshly collected crabs or long-term (16-day) osmotic and ionic regulation experiments in the laboratory showed that male C. granulatus is a better hyper-osmoregulator than female in summer and winter. However, a hypo-osmoregulatory ability was only observed in females experimentally subjected to salinity 40‰ (1176 ± 11 mOsm/kg H₂O) in both seasons. In both sexes, hyper-osmotic regulation was achieved by hyper-regulating hemolymph Na⁺, Cl⁻, and K⁺ concentration. In females, hypo-osmotic regulation was achieved by hypo-regulating hemolymph Na⁺ and Cl⁻ concentration. Long-term (16-day) osmotic and ionic regulations in different salinities were similar in males or females collected and tested in summer and winter. Despite this lack of a seasonal effect on hemolymph osmoregulatory and ionoregulatory patterns in males or females, a marked seasonal difference in the dynamics of these processes was observed for both sexes. In the first 2 days after hypo-osmotic shock (20‰→5‰; 636→185 mOsm/kg H₂O), variations in female osmolality and ion (Na⁺ and Cl⁻) concentration were larger and faster in winter than in summer, while in males the opposite was observed. Furthermore, a seasonal effect on the crab response to hyper-osmotic shock (20‰→40‰; 636→1176 mOsm/kg H₂O) was only observed in males. A new osmolality and ion (Na⁺ and Cl⁻) concentration steady state was faster achieved in winter than in summer. Regarding sexual differences, females showed a better capacity to hypo-regulate the hemolymph osmolality and Na⁺ concentration than males, even after a sudden increase in salinity (hyper-osmotic shock) in both seasons. On the other hand, males showed a better capacity to hyper-regulate the hemolymph osmolality and Na⁺ concentration than females, even after a sudden decrease in salinity (hypo-osmotic shock), especially in winter. Taken together, results reported in the present study suggest the need to consider both sex and collection season as important factors in future osmotic and ionic regulation studies in estuarine crabs.