Do reproductive hormones control Gulf toadfish pulsatile urea excretion?

Gulf toadfish (Opsanus beta) can excrete the majority of their nitrogenous waste as urea in distinct pulses across their gill. Urea pulses are controlled by cortisol and serotonin (5-HT) and are believed to contain chemical signals that may communicate reproductive and/or social status. The objectives of this study were to determine if reproductive hormones are involved in controlling pulsatile urea excretion, and if toadfish respond to prostaglandins as a chemical signal. Specifically, 11-ketotestosterone (11-KT), estradiol (E2), and the teleost pheromone prostaglandin E2 (PGE2) were investigated. Castration during breeding season did not affect pulsatile urea excretion but serial injections of 11-KT outside of breeding season did result in a 48% reduction in urea pulse size in fish of both sexes. Injections of E2 and PGE2, on the other hand, did not alter urea excretion patterns. Toadfish also did not pulse urea in response to waterborne exposure of PGE2 suggesting that this compound does not serve as a toadfish pheromone alone. Toadfish have significantly higher plasma 5-HT during breeding season compared to the months following breeding season. Future research should focus on the composition of the chemical signal in toadfish and the potential importance of seasonal changes in plasma 5-HT in toadfish pulsatile urea excretion and teleost reproduction in general.

Extrinsic nerves are not involved in branchial 5-HT dynamics or pulsatile urea excretion in Gulf toadfish, Opsanus beta.

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance.

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth, and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and that RBC Hb concentration is often close to its solubility limit throughout vertebrates, similar mechanisms may operate in nonhuman vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible, and without any signs of hemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in nonhuman vertebrates, show enhanced aggregation during hemoparasite infections, and can be uncovered in vivo by splenectomy. We discuss the working hypothesis that widespread Hb aggregation within several vertebrate groups may be the result of ongoing or past selection pressure against RBC parasites. Further comparative studies of these evolutionary old systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals but also into human malaria and sickle cell disease.

Glucocorticoid and androgen signaling pathways diverge between advertisement calling and non-calling fish.

Behavioral and neuroendocrine mechanisms of social vocalization in teleost fish are influenced by the glucocorticoid cortisol and the androgen 11-ketotestosterone (11kT). The relative abundance of both 11kT, which binds to androgen receptors (ARα, ARß), and cortisol, which binds to glucocorticoid receptors (GR-1, GR-2), is regulated by 11ß-hydroxylase (11ßH) that converts 11-deoxycortisol to cortisol and testosterone to 11ß-OH-testosterone, and 11ß-hydroxysteroid dehydrogenase (11ßHSD) that converts cortisol to the inactive metabolite cortisone and 11ß-OH-testosterone to 11kT. In midshipman fish, we tested the hypothesis that plasma steroid levels, mRNA abundance for 11ßH and 11ßHSD in the vocal muscle and testis (known site of 11kT synthesis), and mRNA abundances for ARs and GRs in vocal muscle, would differ between males that did or did not recently produce 'hum' advertisement calls. Quantitative real-time PCR demonstrated that non-calling male vocal muscle had significantly higher mRNA levels for all receptors except ARα, and a strong trend for higher 11ßHSD; 11ßH was similar to that in calling males. Calling males had higher plasma and testis 11kT, but lower plasma cortisol, levels. Testis enzyme levels did not differ between male groups, although calling males showed a positive linear correlation between plasma 11kT and testis 11ßHSD mRNA levels, consistent with testis being the main source of plasma 11kT. We propose that higher vocal muscle 11ßHSD levels in non-calling males reflect increased local conversion of elevated cortisol to cortisone, providing protection from cortisol-related toxicity, while increased receptor expression in non-calling males functions as a preparatory mechanism for meeting the physiological demands of future vocalization.

Seasonal plasticity of auditory hair cell frequency sensitivity correlates with plasma steroid levels in vocal fish.

Vertebrates displaying seasonal shifts in reproductive behavior provide the opportunity to investigate bidirectional plasticity in sensory function. The midshipman teleost fish exhibits steroid-dependent plasticity in frequency encoding by eighth nerve auditory afferents. In this study, evoked potentials were recorded in vivo from the saccule, the main auditory division of the inner ear of most teleosts, to test the hypothesis that males and females exhibit seasonal changes in hair cell physiology in relation to seasonal changes in plasma levels of steroids. Thresholds across the predominant frequency range of natural vocalizations were significantly less in both sexes in reproductive compared with non-reproductive conditions, with differences greatest at frequencies corresponding to call upper harmonics. A subset of non-reproductive males exhibiting an intermediate saccular phenotype had elevated testosterone levels, supporting the hypothesis that rising steroid levels induce non-reproductive to reproductive transitions in saccular physiology. We propose that elevated levels of steroids act via long-term (days to weeks) signaling pathways to upregulate ion channel expression generating higher resonant frequencies characteristic of non-mammalian auditory hair cells, thereby lowering acoustic thresholds.

Individual, temporal, and population-level variations in circulating 11-ketotestosterone and 17beta-estradiol concentrations in the oyster toadfish Opsanus tau.

Sex steroid hormones are important for reproduction in all vertebrates, but few studies examine inter-individual, temporal, and population-level variations, as well as environmental influences on circulating steroid levels within the same species. In this study we analyzed plasma 11-ketotoestosterone (11-KT) and 17beta-estradiol (E(2)) levels in the oyster toadfish to test for 1) individual and temporal variations by serially sampling the same individuals during the reproductive and post-reproductive period, 2) variations in steroid levels among toadfish obtained from different sources or maintained under different holding conditions, and 3) correlations with environmental parameters. Results from serial sampling showed marked inter-individual variations in male 11-KT levels in two separate groups of toadfish, but no temporal differences from June to September. Females also showed inter-individual variations in E(2) concentrations, but most had elevated levels late in the reproductive season coincident with oocyte growth prior to winter quiescence. E(2) concentration, but not 11-KT, was positively correlated with water temperature, and negatively correlated with daylength and lunar phase. Maricultured toadfish held under constant conditions had elevated levels of E(2) and 11-KT that should be considered when using these fish for experimentation. This study provides important comparative information on the relationship between individual variations in steroid levels, and how they relate to physiological and environmental correlates in a model marine teleost.

Pulsatile urea excretion in Gulf toadfish: the role of circulating serotonin and additional 5-HT receptor subtypes.

The neurochemical serotonin (5-HT) is involved in stimulating pulsatile urea excretion in Gulf toadfish (Opsanus beta) through the 5-HT2A receptor; however, it is not known if (1) the 5-HT signal originates from circulation or if (2) additional 5-HT receptor subtypes are involved. The first objective was to test whether 5-HT may be acting as a hormone in the control of pulsatile urea excretion by measuring potential fluctuations in circulating 5-HT corresponding with a urea pulse, which would suggest circulating 5-HT may be involved with urea pulse activation. We found that plasma 5-HT significantly decreased by 38% 1 h after pulse detection when branchial urea excretion was significantly elevated and then returned to baseline. This suggests that 5-HT is removed from the circulation, possibly through clearance or excretion, and may be involved in the termination of pulsatile urea excretion. There appeared to be no pulsatile release of 5-HT from peripheral tissues to trigger a urea pulse. The second objective was to determine if additional 5-HT receptor subtypes, such as an additional 5-HT2 receptor (5-HT2C receptor) or the 5-HT receptors that are linked to cAMP (5-HT4/6/7 receptors), played a role in the stimulation of urea excretion. Intravenous injection of 5-HT2C, 5-HT4, 5-HT6, and 5-HT7 receptor agonists did not result in a urea pulse, suggesting that these receptors, and thus cAMP, are not involved in stimulating urea excretion. The involvement of circulating 5-HT and the 5-HT2A receptor in the regulation of pulsatile urea excretion may provide insight into its adaptive significance.

The impact of acute PAH exposure on the toadfish glucocorticoid stress response.

The objective of the present study was to determine whether the polycyclic aromatic hydrocarbons (PAHs) associated with the Deepwater Horizon (DWH) oil spill impacted the stress response of teleost fish. The hypothesis was that intraperitoneal (IP) treatment with PAHs associated with the DWH oil spill or waterborne exposure to DWH oil high energy water-accommodated fraction (HEWAF) would result in the downregulation of the stress response of Gulf toadfish, Opsanus beta, a benthic marine teleost fish that resides in the Gulf of Mexico. In vivo plasma cortisol levels and adrenocorticotropic hormone (ACTH)-mediated cortisol secretion by in vitro isolated kidney tissue were measured. Toadfish at rest IP-treated with naphthalene had higher plasma cortisol compared to fluorene-treated and control fish; phenanthrene-treated fish tended to have higher plasma cortisol levels that fluorene-treated and controls. When subjected to an additional crowding stress, naphthalene and phenanthrene-treated fish were no longer able to mount a stress response compared to fluorene-treated and control fish, suggesting exhaustion of the stress response. Supporting this in vivo data, there tended to be less cortisol released by the kidney in vitro from naphthalene and phenanthrene-treated fish in response to ACTH compared to controls. In contrast, toadfish at rest exposed to 3% Slick A HEWAF did not have significantly different plasma cortisol levels compared to controls. But, exposed fish did have significantly less cortisol released by the kidney in vitro in response to ACTH. When toadfish were subjected to an additional stress, there were no significant differences in plasma cortisol or ACTH, suggesting the action of a secondary secretagogue to maintain plasma cortisol in vivo. Combined, these data suggest that in response to acute PAH exposure, there may be internalization or downregulation of the melanocortin 2 receptor (MC2R) that mediates the action of ACTH.

Reproductive success in the Lusitanian toadfish: Influence of calling activity, male quality and experimental design.

Acoustic signals are sexual ornaments with an established role on mate choice in several taxa, but not in fish. Recent studies have suggested that fish vocal activity may signal male quality and influence male's reproductive success but experimental evidence is lacking. Here we made two experiments to test the hypothesis that vocal activity is essential for male breeding success in a highly vocal fish, the Lusitanian toadfish. We first compared the reproduction success between muted and vocal males. In a second experiment we related male reproduction success with acoustic activity and male quality, including biometric, condition and physiological features. As a proxy for reproductive success we tallied both total number and number of sired eggs, which were correlated. Muting experiments showed that successful mating was dependent on vocalizing. In addition, the number of eggs was positively associated with the male's maximum calling rate. In the second experiment male's reproductive success was positively associated with male condition and negatively related with circulating androgen levels and relative gonad mass, but was not associated with vocal activity. Differences in results may be related with nest design which could have influenced mate choice costs and intra-sexual competition. In the muting experiment nests had a small opening that restrained the large nest-holder but allowed smaller fish, such as females, to pass while in the second experiment fish could move freely. These experiments suggest that a combination of factors, including vocal activity, influence reproductive success in this highly vocal species.

Higher levels of aggression are observed in socially dominant toadfish treated with the selective serotonin reuptake inhibitor, fluoxetine.

The following study set out to test the hypothesis that acute treatment with the selective serotonin reuptake inhibitor, fluoxetine, would result in a rise in circulating 5-HT levels and consequently a decrease in territorial aggression in the Gulf toadfish, Opsanus beta. Size-matched pairs of toadfish were implanted intraperitoneally with the same dose of fluoxetine (0, 10 or 25 µg g⁻¹). After a social interaction between a pair of fish, circulating levels of serotonin (5-HT; 5-hydroxytryptamine) and cortisol were measured and relative mRNA expression of the 5-HT(1A) receptor in the toadfish brain was determined using quantitative (real-time) PCR (qPCR). Behavioral endpoints such as the number of aggressive acts and swimming activity were also quantified so that dominant and subordinate fish could be identified. Fluoxetine treatment resulted in an increase in circulating levels of 5-HT, regardless of social status. Circulating cortisol concentrations were unaffected by fluoxetine, but were significantly higher in subordinate individuals when compared to dominant fish. Toadfish brain 5-HT(1A) receptor mRNA expression was not affected by treatment or social status. Lastly and contrary to our predictions, fluoxetine treatment resulted in an increase in the number of aggressive acts made by dominant individuals, with no differences in the level of aggression or swimming activity of subordinate fish. This study is the first to describe elevated aggression in a teleost fish with elevated circulating levels of 5-HT.

Diel patterns of nitrogen excretion, plasma constituents, and behavior in the gulf toadfish (Opsanus beta) in laboratory versus outdoor mesocosm settings.

Nitrogen excretion by the gulf toadfish (Opsanus beta) is of interest because of its high proportion of urea excretion compared with that of other teleosts. To better understand the factors influencing the timing of nitrogen excretion, the ratio of excreted urea∶ammonia, and the effector molecules regulating these processes, gulf toadfish were subjected to a series of experiments that moved them progressively from internal laboratory to outdoor mesocosm settings while assessing their behavior, nitrogen excretion patterns, levels of plasma hormones/effectors, and other parameters. In confined flux chambers in both laboratory and outdoor settings, toadfish nitrogen excretion was largely observed as urea pulses, with no apparent diel patterns to the pulses. Unrestrained toadfish in mesocosms exhibited distinctly nocturnal behavior, remaining exclusively in shelters during the day but taking several forays out into the mesocosm at night. In contrast to nitrogen excretion patterns in chambers, urea and ammonia were coexcreted in mesocosms and ratios for urea∶ammonia were very close to 1∶1 for both fed and fasted toadfish. The majority of measured excretion (and corresponding declines in plasma urea levels) occurred during two distinct periods of pulsing during daylight hours (0600-1000 and 1600-1800 hours). The declines in plasma urea associated with excretion were preceded by/coincided with declines in plasma cortisol. No day/night or hourly patterns in plasma serotonin (5-hydroxytryptamine [5-HT]) were observed, but there was a strong positive correlation among all samples between plasma urea and 5-HT. There was also a negative correlation between plasma cortisol and 5-HT. As expected for a nocturnally active species, plasma melatonin was significantly lower in daylight hours. A variety of enzyme activities (glutamine synthetase, glutaminase) and mRNA levels (glutamine synthetase, urea transporter, and Rhesus proteins) showed no significant variation over a diel cycle. Unlike prior laboratory studies, our results show that gulf toadfish in a natural setting have a distinctly diurnal pattern of nitrogen excretion and that ammonia and urea are coexcreted. The decline in plasma cortisol associated with urea pulses noted in prior laboratory studies was not as evident in the natural setting.

Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish.

Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1)). Fluoxetine treatments of 25 and 50 microgg(-1) were sub-lethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25 microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.

Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish.

Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1). Fluoxetine treatments of 25 and 50 microgg(-1) were sublethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25g microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.