Anatomy of the olfactory system and potential role for chemical communication in the sound-producing Lusitanian toadfish, Halobatrachus didactylus.

The current study investigated the structure and function of the olfactory system of the Lusitanian toadfish, Halobatrachus didactylus, using histology and electrophysiology (electro-olfactogram [EOG]), respectively. The olfactory system consists of a digitated anterior peduncle, of unknown function, containing the inhalant nostril. This then leads to a U-shaped olfactory chamber with the olfactory epithelium-identified by Gαolf-immunoreactivity-on the ventral surface. A large lacrimal sac is connected to this tube and is likely involved in generating water movement through the olfactory chamber (this species is largely sedentary). The exhalent nostril lies by the eye and is preceded by a bicuspid valve to ensure one-way flow of water. As do other teleosts, H. didactylus had olfactory sensitivity to amino acids and bile acids. Large-amplitude EOG responses were evoked by fluid from the anterior and posterior testicular accessory glands, and bile and intestinal fluids. Anterior gland and intestinal fluids from reproductive males were significantly more potent than those from non-reproductive males. Male urine and skin mucus proved to be the least potent body fluids tested. These results suggest that chemical communication-as well as acoustic communication-may be important in the reproduction of this species and that this may be mediated by the accessory glands and intestinal fluid.

Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

Environmental salinity modulates olfactory sensitivity in the euryhaline European seabass, Dicentrarchus labrax, acclimated to seawater and brackish water.

The olfactory epithelium of fish is - of necessity - in intimate contact with the surrounding water. In euryhaline fish, movement from seawater to freshwater (and vice versa) exposes the epithelium to massive changes in salinity and ionic concentrations. How does the olfactory system function in the face of such changes? The current study compared olfactory sensitivity in seawater- (35‰) and brackish water-adapted seabass (5‰) using extracellular multi-unit recording from the olfactory nerve. Seawater-adapted bass had higher olfactory sensitivity to amino acid odorants when delivered in seawater than in freshwater. Conversely, brackish water-adapted bass had largely similar sensitivities to the same odorants when delivered in seawater or freshwater, although sensitivity was still slightly higher in seawater. The olfactory system of seawater-adapted bass was sensitive to decreases in external [Ca2+], whereas brackish water-adapted bass responded to increases in [Ca2+]; both seawater- and brackish water-adapted bass responded to increases in external [Na+] but the sensitivity was markedly higher in brackish water-adapted bass. In seawater-adapted bass, olfactory sensitivity to l-alanine depended on external Ca2+ ions, but not Na+; brackish water-adapted bass did respond to l-alanine in the absence of Ca2+, albeit with lower sensitivity, whereas sensitivity was unaffected by removal of Na+ ions. A possible adaptation of the olfactory epithelium was the higher number of mucous cells in brackish water-adapted bass. The olfactory system of seabass is able to adapt to low salinities, but this is not immediate; further studies are needed to identify the processes involved.

Assessment of male reproductive traits in endangered leuciscids from the Iberian Peninsula: first attempts to store gametes both at short- and long-term.

During the spring of 2022, several endangered leuciscid species (Anaecypris hispanica, Squalius aradensis, Anachondrostoma Occidentale, and Iberochondrostoma lusitanicum) were sampled both at the Vasco da Gama aquarium facilities and in some rivers of the Algarve region, Portugal. Sperm samples were extracted by gentle abdominal pressure and sperm motion parameters were assessed for the first time in four species, using a computerized analysis system. The results obtained showed that spermatozoa kinetic patterns were similar for all 4 species, with high motility and velocity values after the sperm activation time and with a marked decrease after 20. On the other hand, sperm longevity was highly variable between species, with short longevities (around 40 s) for A. hispanica and S. aradensis, and longer longevities (100-120 s) for A. occidentale and I. lusitanicum, which could indicate a latitudinal pattern in terms of sperm longevity. At the same time, morphometric analysis was carried out for the four target species, revealing that spermatozoa showed similar sizes and shapes to other external fertilizers belonging to Leuscididae, with small spherical heads, uniflagellate, and without acrosomes. In addition, a short-term gamete storage trail was performed by diluting sperm in 1:9 (sperm:extender) and storing them at 4ºC. Although the results obtained were uneven among the species studied, the dilution and extender used generated motilities above 40% up to day 4 of storage in S. aradensis and I. lusitanicum, and up to days 1-2 in A. hispanica and A. occidentale, respectively. Finally, gamete cryopreservation trials were also carried out on these threatened species. Although cryopreserved samples showed significantly lower motility than fresh samples, some protocols generate acceptable percentages of viability, DNA integrity, and sperm motility in some species such as I. lusitanicum and A. occidentale. The data revealed that the protocol based on 10% DMSO plus 7.5% egg yolk generated the best results.This study is the first to assess the reproductive traits of wild and captive populations of endangered leuciscids endemic from the Iberian Peninsula, describing the spermatozoa kinetics and developing protocols for managing male gametes both in short- and long-term storage. Outcomes will provide new and useful tools to complement the management and conservation of ex situ breeding programs that are being developed for these four endangered species.

Toll-Like Receptor Evolution: Does Temperature Matter?

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

Environmental Salinity Modifies Mucus Exudation and Energy Use in European Sea Bass Juveniles.

The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p < 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p < 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

Freshening effect on the osmotic response of the Antarctic spiny plunderfish Harpagifer antarcticus.

Global warming is having a significant impact around the world, modifying environmental conditions in many areas, including in zones that have been thermally stable for thousands of years, such as Antarctica. Stenothermal sedentary intertidal fish species may suffer due to warming, notably if this causes water freshening from increased freshwater inputs. Acute decreases in salinity, from 33 down to 5, were used to assess osmotic responses to environmental salinity fluctuations in Antarctic spiny plunderfish Harpagifer antarcticus, in particular to evaluate if H. antarcticus is able to cope with freshening and to describe osmoregulatory responses at different levels (haematological variables, muscle water content, gene expression, NKA activity). H. antarcticus were acclimated to a range of salinities (33 as control, 20, 15, 10 and 5) for 1 week. At 5, plasma osmolality and calcium concentration were both at their lowest, while plasma cortisol and percentage muscle water content were at their highest. At the same salinity, gill and intestine Na+ -K+ -ATPase (NKA) activities were at their lowest and highest, respectively. In kidney, NKA activity was highest at intermediate salinities (15 and 10). The salinity-dependent NKA mRNA expression patterns differed depending on the tissue. Marked changes were also observed in the expression of genes coding membrane proteins associated with ion and water transport, such as NKCC2, CFTR and AQP8, and in the expression of mRNA for the regulatory hormone prolactin (PRL) and its receptor (PRLr). Our results demonstrate that freshening causes osmotic imbalances in H. antarcticus, apparently due to reduced capacity of both transport and regulatory mechanisms of key organs to maintain homeostasis. This has implications for fish species that have evolved in stable environmental conditions in the Antarctic, now threatened by climate change.

Evaluation of an Acute Osmotic Stress in European Sea Bass via Skin Mucus Biomarkers.

European sea bass is a marine teleost which can inhabit a broad range of environmental salinities. So far, no research has studied the physiological response of this fish to salinity challenges using modifications in skin mucus as a potential biological matrix. Here, we used a skin mucus sampling technique to evaluate the response of sea bass to several acute osmotic challenges (for 3 h) from seawater (35‱) to two hypoosmotic environments, diluted brackish water (3‱) and estuarine waters (12‱), and to one hyperosmotic condition (50‱). For this, we recorded the volume of mucus exuded and compared the main stress-related biomarkers and osmosis-related parameters in skin mucus and plasma. Sea bass exuded the greatest volume of skin mucus with the highest total contents of cortisol, glucose, and protein under hypersalinity. This indicates an exacerbated acute stress response with possible energy losses if the condition is sustained over time. Under hyposalinity, the response depended on the magnitude of the osmotic change: shifting to 3‱ was an extreme salinity change, which affected fish aerobic metabolism by acutely modifying lactate exudation. All these data enhance the current scarce knowledge of skin mucus as a target through which to study environmental changes and fish status.

STC1 and PTHrP Modify Carbohydrate and Lipid Metabolism in Liver of a Teleost Fish.

Stanniocalcin 1 (STC1) and parathyroid hormone-related protein (PTHrP) are calciotropic hormones in vertebrates. Here, a recently hypothesized metabolic role for these hormones is tested on European sea bass treated with: (i) teleost PTHrP(1-34), (ii) PTHrP(1-34) and anti-STC1 serum (pro-PTHrP groups), (iii) a PTHrP antagonist PTHrP(7-34) or (iv) PTHrP(7-34) and STC1 (pro-STC1 groups). Livers were analysed using untargeted metabolic profiling based on proton nuclear magnetic resonance (1H-NMR) spectroscopy. Concentrations of branched-chain amino acid (BCAA), alanine, glutamine and glutamate increased in pro-STC1 groups suggesting their mobilization from the muscle to the liver for degradation and gluconeogenesis from alanine and glutamine. In addition, only STC1 treatment decreased the concentrations of succinate, fumarate and acetate, indicating slowing of the citric acid cycle. In the pro-PTHrP groups the concentrations of glucose, erythritol and lactate decreased, indicative of gluconeogenesis from lactate. Taurine, trimethylamine, trimethylamine N-oxide and carnitine changed in opposite directions in the pro-STC1 versus the pro-PTHrP groups, suggesting opposite effects, with STC1 stimulating lipogenesis and PTHrP activating lipolysis/ß-oxidation of fatty acids. These findings suggest a role for STC1 and PTHrP related to strategic energy mechanisms that involve the production of glucose and safeguard of liver glycogen reserves for stressful situations.

Effects of different slipping methods on the mortality of sardine, Sardina pilchardus, after purse-seine capture off the Portuguese Southern coast (Algarve).

The effects of two different slipping methods on the survival, physical and physiological response of sardines, Sardina pilchardus, captured in a purse-seine fishery were investigated in southern Portugal. Sardines were collected and transferred into holding tanks onboard a commercial fishing vessel after being captured, crowded and deliberately released using two slipping procedures: standard and modified. The standard slipping procedure aggregated fish at high densities and made them "roll over" the floatline, while the modified procedure aggregated the fish at moderate densities and enabled them to escape through an opening created by adding weights to the floatline. Both slipping methods were compared with minimally harmed non-slipped sardines (sardines collected from the loose pocket of the purse seine). Survival rates were monitored in captivity over 28 days using three replicates for each treatment. The estimated survival of sardines was 43.6% for the non-slipped fish, 44.7% for the modified slipping and 11.7% for the standard slipping treatments. Scale loss indicated the level of physical impact experienced, with dead fish from the non-slipped and modified slipping technique showing significantly lower scale loss than those fish from the standard slipping treatment within the same period. Of the physiological indicators of stress measured, cortisol, glucose, lactate and osmolality attained peak values during slipping and up to the first hours after introduction to captivity. This work indicates that although delayed mortality after release may be substantial, appropriately modified slipping techniques significantly enhance survival of slipped sardines.

Conservation physiology of marine fishes: state of the art and prospects for policy.

The state of the art of research on the environmental physiology of marine fishes is reviewed from the perspective of how it can contribute to conservation of biodiversity and fishery resources. A major constraint to application of physiological knowledge for conservation of marine fishes is the limited knowledge base; international collaboration is needed to study the environmental physiology of a wider range of species. Multifactorial field and laboratory studies on biomarkers hold promise to relate ecophysiology directly to habitat quality and population status. The 'Fry paradigm' could have broad applications for conservation physiology research if it provides a universal mechanism to link physiological function with ecological performance and population dynamics of fishes, through effects of abiotic conditions on aerobic metabolic scope. The available data indicate, however, that the paradigm is not universal, so further research is required on a wide diversity of species. Fish physiologists should interact closely with researchers developing ecological models, in order to investigate how integrating physiological information improves confidence in projecting effects of global change; for example, with mechanistic models that define habitat suitability based upon potential for aerobic scope or outputs of a dynamic energy budget. One major challenge to upscaling from physiology of individuals to the level of species and communities is incorporating intraspecific variation, which could be a crucial component of species' resilience to global change. Understanding what fishes do in the wild is also a challenge, but techniques of biotelemetry and biologging are providing novel information towards effective conservation. Overall, fish physiologists must strive to render research outputs more applicable to management and decision-making. There are various potential avenues for information flow, in the shorter term directly through biomarker studies and in the longer term by collaborating with modellers and fishery biologists.

Stanniocalcin 1 effects on the renal gluconeogenesis pathway in rat and fish.

The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from (14)C-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from (14)C-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from (14)C-acid lactic, while STC1-A increased gluconeogenesis from (14)C-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes.

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids.

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17ß-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function.

Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium.

Dynamics of scale regeneration in seawater- and brackish water-acclimated sea bass, Dicentrarchus labrax.

Scale loss is a common occurrence in both wild fish and those in aquaculture production systems, and regeneration of scales has been described in several freshwater species. Relatively little information exists about this process in marine fish, and in the present study, the chronology of scale regeneration was characterized in juvenile sea bass Dicentrarchus labrax, maintained in full seawater (SW; 36‰, 11.2 mM Ca(2+)) or brackish water (BW; 3.5‰, 1.1 mM Ca(2+)). Despite the significant differences in plasma osmolality, plasma calcium (Ca) and phosphorus (P) were similar between SW and BW. Removal of scales (2.25 cm(2)) did not modify plasma osmolality but a significant reduction in plasma Ca occurred in sea bass in BW during early phases of scale regeneration. Scale Ca and P contents changed during regeneration in SW and BW fish, and in fully regenerated scales, their concentration was comparable to ontogenic scales which had a Ca/P ratio around 1.0. Regenerating scales were first visible at 3 days after removal, and by 6 days, the descaled area was covered with new scales in both SW and BW fish. From this stage onwards, while scale area and mass rose gradually, a rapid accretion of calcium crystals was observed over the basal plate, especially in SW, and Ca and P content per scale increased at a very high rate, decreasing only at the later stages, and between 21 and 30 days the newly formed scales had already ontogenic-like morphometric features. Scale tartrate-resistant acid phosphatase (TRAP) or alkaline phosphatase (ALP) activity was initially similar in SW and BW. However, during regeneration, TRAP was significantly higher in scales of sea bass in SW compared to those in BW where ALP predominated; these differences persisted at 30 days.

Differential expression of PTHrP and its receptor in pituitary gland and gills in estradiol-treated gilthead sea bream (Sparus auratus, L.).

In the gilthead sea bream (Sparus auratus) 17ß-estradiol (E2) plays an important role in the synthesis of vitellogenin. During vitellogenesis, vitellogenin as a nutritional precursor protein is loaded with calcium, which requires elevated plasma calcium levels. This is accomplished via E2-dependent processes. Reports have shown that hypercalcemic effects of E2 are possibly mediated by another hypercalcemic factor, viz. parathyroid hormone related protein (PTHrP). To further investigate the possibility of PTHrP as a mediator of E2-induced hypercalcemia, we investigated the local expression levels of the pthrp mRNA and of the gene coding for the PTHrP receptor, PTH1R (pth1r) in two tissues involved in the calcium regulation (gills, pituitary gland) of the sea bream treated with E2. Compared to control, treatment with E2 resulted in: significantly increased total calcium and plasma PTHrP levels (P<0.01), a down-regulation of pthrp mRNA in the pituitary gland (P<0.01), and up-regulation of expression levels for both pthrp and pth1r in the branchial system (P<0.05). These findings provide direct evidence for a mediating role of PTHrP in E2 induced hypercalcemia, and in addition support the idea for the presence of two independent systems, an endocrine pituitary PTHrP system and a peripheral paracrine branchial PTHrP system.

Piscine PTHrP regulation of calcium and phosphate transport in winter flounder renal proximal tubule primary cultures.

Multiple factors control calcium (Ca(2+)) and inorganic phosphate (P(i)) transport in the fish nephron, and the recently discovered members of the piscine parathyroid hormone-like protein family are likely participants in such regulatory mechanisms. The effects of an NH(2)-terminal peptide (amino acids 1-34) of Takifugu rubripes parathyroid hormone-related protein, (1-34)PTHrP, on Ca(2+) and P(i) transport were investigated in winter flounder (Pseudopleuronectes americanus) proximal tubule cells in primary culture (fPTCs). RT-PCR performed on RNA extracted from fPTCs and from intact kidney tissue indicated that expression of PTHrP and types 1 and 3 PTH/PTHrP receptors occurred both in vivo and in vitro and that circulating levels of PTHrP measured by specific radioimmunoassay averaged 2.5 +/- 0.13 ng/ml. fPTC monolayers were mounted in Ussing chambers, and under neutral electrochemical conditions, addition of 10 nM (1-34)PTHrP to the basolateral side induced a slight increase in Ca(2+) transport rate from luminal to peritubular side, significantly stimulating net Ca(2+) reabsorption. (1-34)PTHrP also significantly increased the P(i) secretory flux, and slightly reduced P(i) reabsorption, evoking a significant increase in P(i) net secretion. This stimulatory effect was partially inhibited by bisindolylmaleimide, an inhibitor of protein kinase C. Incubation of ex vivo flounder renal tubules with (1-34)PTHrP resulted in apparent reduction of Na(+)-P(i) cotransporter type II (NaP(i)-II) protein in tubule membranes. PTHrP seems therefore to participate in the modulation of Ca(2+) and P(i) homeostasis by fish kidney.

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge.

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p<0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p<0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p<0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na(+)/K(+)-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.

A PTH/PTHrP receptor antagonist blocks the hypercalcemic response to estradiol-17beta.

Estradiol (E(2)) increases circulating calcium and phosphate levels in fish, thus acting as a hypercalcemic and hyperphosphatemic factor during periods of high calcium requirements, such as during vitellogenesis. Since parathyroid hormone (PTH)-related protein (PTHrP) has been shown to be calciotropic in fish, we hypothesized that the two hormones could be mediating the same process. Sea bream (Sparus auratus) juveniles receiving a single intraperitoneal injection of piscine PTHrP(1-34) showed an elevation in calcium plasma levels within 24 h. In contrast, injections of the PTH/PTHrP receptor antagonist PTHrP(7-34) decreased circulating levels of calcium in the same period. Intraperitoneal implants of estradiol-17beta (E(2); 10 microg/g) evoked significant increases of circulating plasma levels of calcium and phosphorus and a sustained increases of circulating plasma levels of PTHrP. However, a combined treatment of E(2) and PTHrP(7-34) evoked a markedly lower calcium response compared with E(2) alone. We conclude that PTHrP or a related peptide that binds the PTH/PTHrP receptor mediates, at least in part, the hypercalcemic effect of E(2) in calcium and phosphate balance in fish.

Regulation of calcium balance in the sturgeon Acipenser naccarii: a role for PTHrP.

Calcium regulation in sturgeon is of special interest because they are a representative of the ancient fishes possessing mainly cartilaginous skeletons and a supposedly low calcium demand. The present study aimed to characterize the effect of a chronic absence of dietary calcium and the effect of parathyroid hormone-related protein (PTHrPA) (1-34) (7) on calcium balance in juvenile sturgeon (Acipenser naccarii). At rest, sturgeon juveniles are in net positive calcium balance, since whole body calcium uptake is significantly higher than efflux and calcium accumulates in the body. To study the importance of dietary calcium, the sturgeon were kept on a calcium-free diet for 8 wk. This manipulation impaired growth as measured by failure to gain weight or increase in length and indicates that dietary calcium is important for growth in sturgeon. An increased whole body calcium uptake partially compensated dietary calcium deficiency and was associated with increased gill chloride cell number in lamellae and filaments in parallel with increased gill Na(+)K(+)-ATPase activity. In addition, a single injection of piscine PTHrP(1-34) significantly increased whole body calcium uptake and decreased whole body calcium efflux. Administration of PTHrP significantly increased circulating plasma calcium 4-24 h postinjection. The increase in net calcium transport and increased plasma levels of calcium is consistent with the actions of a hypercalcemic factor. It would appear that the sturgeon rely on calcium for growth and tightly regulate calcium transport. The action in calcium balance is consistent with PTHrP acting as a hypercalcemic factor in sturgeon.