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Abstract: Endosulfan and flutamide, a widely used pesticide and a prostate cancer/infertility drug, respectively, have an increased risk of causing endocrine disruption if they reach water bodies. Though many studies are available on neurotoxicity/bioaccumulation of endosulfan and receptor antagonism of flutamide, only little is known about their impact on testicular steroidogenesis at molecular level. Sex steroids play an important role in sex differentiation of lower vertebrates including fishes. Hence, a small change in their levels caused by endocrine disruptors affects the gonadal development of aquatic vertebrates significantly. The aim of this study was to evaluate the effects of endosulfan and flutamide on testis-related transcription factor and steroidogenic enzyme genes with a comparison on the levels of androgens during critical period of catfish testicular development. We also analyzed the correlation between the above-mentioned genes and catfish gonadotropin-releasing hormone (cfGnRH)-tryptophan hydroxylase2 (tph2). The Asian catfish, Clarias batrachus males at 50 days post hatch (dph) were exposed to very low dose of endosulfan (2.5μg/L) and flutamide (33μg/L), alone and in combination for 50 days. The doses used in this study were far less than those used in the previous studies of flutamide and reported levels of endosulfan in surface water and sediments. Sampling was done at end of the treatments (100dph) to perform testicular germ cell count (histology), measurements of testosterone (T) and 11-ketotestosterone (11-KT) by enzyme immunoassay and transcript quantification by quantitative real-time PCR. In general, treatments decreased the expression of several genes including testis-related transcription factors (dmrt1, sox9a and wt1), steroidogenic enzymes (11β-hsd2, 17β-hsd12 and P450c17), steroidogenic acute regulatory protein and orphan nuclear receptors (nr2c1 and Ad4BP/SF-1). In contrast, the transcripts of cfGnRH and tph2 were elevated in the brain of all treated groups with maximum elevation in the endosulfan group. However, combination of endosulfan and flutamide (E+F) treatment showed minor antagonism in a few results of transcript quantification. Levels of T and 11-KT were elevated after flutamide and E+F treatments while no change was seen in the endosulfan group signifying the effect of flutamide as an androgen receptor antagonist. All the treatments modulated testis growth by decreasing the progression of differentiation of spermatogonia to spermatocytes. Based on these results, we suggest that the exposure to endosulfan and flutamide, even at low doses, impairs testicular development either directly or indirectly at the level of brain. [Copyright &y& Elsevier]

Abstract: The diverse cichlid species flocks of the East African lakes provide a classical example of adaptive radiation. Territorial aggression is thought to influence the evolution of phenotypic diversity in this system. Most vertebrates mount hormonal (androgen, glucocorticoid) responses to a territorial challenge. These hormones, in turn, influence behavior and multiple aspects of physiology and morphology. Examining variation in competition-induced hormone secretion patterns is thus fundamental to an understanding of the mechanisms of phenotypic diversification. We test here the hypothesis that diversification in male aggression has been accompanied by differentiation in steroid hormone levels. We studied two pairs of sibling species from Lake Victoria belonging to the genera Pundamilia and Mbipia. The two genera are ecologically differentiated, while sibling species pairs differ mainly in male color patterns. We found that aggression directed toward conspecific males varied between species and across genera: Pundamilia nyererei males were more aggressive than Pundamilia pundamilia males, and Mbipia mbipi males were more aggressive than Mbipia lutea males. Males of both genera exhibited comparable attack rates during acute exposure to a novel conspecific intruder, while Mbipia males were more aggressive than Pundamilia males during continuous exposure to a conspecific rival, consistent with the genus difference in feeding ecology. Variation in aggressiveness between genera, but not between sibling species, was reflected in androgen levels. We further found that M. mbipi displayed lower levels of cortisol than M. lutea. Our results suggest that concerted divergence in hormones and behavior might play an important role in the rapid speciation of cichlid fishes. [Copyright &y& Elsevier]

Abstract: The evidence for androgens having a pivotal role in the functioning of the female reproductive axis – such as initiating puberty or vitellogenesis – is mounting. However, the use of aromatizable androgens and the tissue-specific focus of most studies often make it unclear if androgenic effects throughout the axis proceed via androgen or estrogen signalling mechanisms. In this study, we assessed the effects of 11-ketotestosterone (11KT, a non-aromatizable androgen) on the pituitary and ovary of previtellogenic (PV) freshwater eels Anguilla australis, comparing them with eels naturally undergoing early vitellogenesis (EV). We found that 11KT treatment produces molecular and morpho-physiological phenotypes that were generally intermediate between PV and EV. Most notably, we demonstrated that 11KT induces effects on follicle-stimulating hormone (FSH) signalling in the pituitary and ovaries that are in opposition to each other. Thus, 11KT significantly reduced fshβ subunit expression in the pituitary. At the same time, 11KT dramatically increased mRNA levels of ovarian FSH receptor and plasma levels of estradiol-17β, very likely sensitizing the previtellogenic follicle to the FSH signal. Androgens therefore may be important in facilitating puberty in the eel. [Copyright &y& Elsevier]

Abstract: Much is known about the role of hormones in the regulation of vertebrate mating behavior, including receptivity, and several components of mate choice. Hormones may modulate reproductive behavior in such a way to increase or decrease the individual''s motivation, and therefore hormones may be important in mediating behavior associated with reproductive isolation. The mating complex of the all female gynogenetic Amazon mollies, Poecilia formosa, and their parental species (sailfin mollies, P. latipinna, and Atlantic mollies, P. mexicana) is a model system for studying ultimate mechanisms of species recognition. However, proximate mechanisms, such as variation in hormone levels, and the effect of hormones on sperm production have not been extensively examined. We predict that one or more of the sex steroid hormones in teleost fish (11-ketotestosterone (KT), testosterone (T), and estradiol (E)) will play a role in species recognition (during mate choice and/or sperm priming) for Atlantic mollies (the maternal parental species) that are sympatric with Amazon mollies. We sequentially paired male Atlantic mollies with female conspecifics and Amazon mollies and obtained water-borne hormone samples before and after mating for all fish. We measured circulating KT, T, and E from the water samples. Although we did not find an overall KT response to mating with conspecifics as has been found previously in sailfin mollies, male Atlantic mollies that mated more with conspecific females had lower postmating T levels. Additionally, males attempted to mate more with conspecific females that had lower postmating E levels, but attempted to mate more with Amazon mollies that had higher postmating KT levels. We also examined the effect of KT on sperm priming (a mechanism of premating mate choice), and found that KT levels of male Atlantic mollies prior to mating are correlated with the sperm priming response when males were paired with conspecific females, but this correlation was not found when males were paired with Amazon mollies. Our results indicate that male mating behavior is affecting or responding to both male and female hormones, but that the hormones alone are not playing a role in species recognition. Male Atlantic mollies may not discriminate against Amazon mollies as strongly as male sailfin mollies because Amazon mollies resemble their maternal parental species more than their paternal species. [Copyright &y& Elsevier]

Abstract: The proximate mechanisms that underlie the evolution of within-sex variation in mating behavior, sexual characters and reproductive investment patterns are still poorly understood. Species exhibiting alternative reproductive tactics (ARTs) are ideal model systems to examine these mechanisms. Chinook salmon (Oncorhynchus tshawytscha) exhibits two distinct ARTs: hooknoses, which are large males that establish spawning dominance hierarchies via intense male–male competition and jacks, which are smaller precocious sneaking males that steal fertilizations via sperm competition. In this study, we examine plasma testosterone (T), 11-ketotestosterone (11-KT) and maturation-inducing steroid (MIS; 17α,20β-dihydroxy-4-pregnen-3-one) profiles of spawning hooknoses and jacks. Furthermore, we examine relationships between androgens and primary (gonad mass, gonadosomatic index and sperm traits) and secondary (total mass, body size, hump depth and kype length) sexual characters. Relationships between MIS and sperm traits are also examined. We found that hooknoses and jacks did not significantly differ in terms of plasma T, 11-KT or MIS concentrations. Moreover, we found significant positive relationships between levels of both androgens within each ART. There were no significant relationships between androgens, MIS and sperm traits. T and 11-KT concentrations co-varied positively with gonad investment and kype length in jacks. In hooknoses, 11-KT concentration was positively related to total mass, hump depth and condition factor. Overall, these findings suggest that there are differential androgen effects for each of the ARTs in Chinook salmon. [Copyright &y& Elsevier]

Alternations of reproductive physiology were studied in the male goldfish (Carassius auratus L.) exposed to environmentally relevant concentrations (0.6, 4.5 and 11.0μg/L) of bisphenol A (BPA) at days 10, 20 and 30 after exposure. Significant effects of BPA concentration, exposure time and their interactions were observed on testosterone (T), 11-ketotestosterone (11-KT) and sperm motility and velocity, but gonadosomatic index (GSI), hepatosomatic index (HSI) and 17β-estradiol (E2) were not affected. Vitellogenin (VTG) was only affected by BPA concentration. The T and 11-KT levels were significantly decreased in the BPA-treated groups after 20 or 30 days. Sperm motility was significantly decreased at 15, 30, 60 and 90s post-activation in the BPA-treated groups after 20 or 30 days. But, significant decrease in sperm velocity was observed at 30, 60 and 90s post-activation in the BPA-treated groups at all exposure times. The VTG was significantly increased in the males exposed to 11.0μg/L at day 30 after exposure. The GSI, HSI and E2 did not differ between the BPA-treated groups and control. The present study shows that the decrease of sperm quality is concurrent with the decrease of androgens and increase of VTG. The results suggest adverse effects of BPA on sperm motility and velocity via modifications of testicular steroidogenesis that might correspond to alternation in sperm maturation. [Copyright &y& Elsevier]

Abstract: Androgens are known to be involved in reproductive behaviours including courtship and aggression. According to the Challenge Hypothesis, androgen activity upregulates male reproductive behaviour seasonally and also modulates short term adaptation of these behaviours in response to social context. In the weakly electric fish, Apteronotus leptorhynchus, 11-ketotestosterone (11-KT) has been previously implicated in the regulation of electrocommunication behaviours that are believed to have roles in both aggression and courtship. Changes in male 11-KT levels were quantified using a non-invasive measurement technique alongside changes in electrocommunication behaviour following environmental cues that simulated the onset of the breeding season. Males showed an increase in mean electric organ discharge frequency (EODf), which is consistent with earlier results showing a female preference for high EODf. A subset of males with high initial EODfs showed increases in both 11-KT and EODf, which provides support for an EODf-based dominance hierarchy in this species. Males housed in social conditions and exposed to breeding conditioning also showed higher overall electric organ discharge frequencies and 11-KT compared to males housed in isolation. Evidence is presented that another type of electrocommunication signal previously implicated in courtship may also serve as an inter-male signal of submission. Our results are consistent with earlier observations that electrocommunication signals produced during inter-male aggression serve in deterring attacks, and their pattern of production further suggested the formation of a dominance hierarchy. [Copyright &y& Elsevier]

Behavioural endocrinologists have shown that stressors including competition for resources can affect an individual’s circulating level of cortisol, whereas agonistic interactions typically affect androgen levels. Conservation biologists have used such data to facilitate the restoration and management of biodiversity by monitoring social interactions and stress among individuals and species. Here, we examined whether competition and agonistic interactions with non-native salmonids is hindering restoration of native Atlantic salmon, Salmo salar, in Lake Ontario. Using semi-natural streams, we examined the effects of competition with non-native brown trout, Salmo trutta, and rainbow trout, Oncorhynchus mykiss, on plasma cortisol and 11-ketotestosterone (11-KT) concentrations in juveniles from three Atlantic salmon strains, as well as the relationship between these hormones and social dominance and growth. Basal hormone levels and the hormonal response to the presence of the trouts varied among the strains. Cortisol increased in the presence of the trouts for two strains and was associated with lowered aggression and food consumption. Conversely, the presence of the non-native species had little influence on overall concentrations of 11-KT in the Atlantic salmon, although unexpectedly, there was a negative relationship between 11-KT and initiated aggression when either one of the trouts was present. Interestingly, elevated 11-KT levels were associated with declines in both food consumption and growth. Overall, our results suggest that competition with non-native salmonids negatively impacts Atlantic salmon. We discuss how our results can improve poststocking success and restoration of Atlantic salmon in Lake Ontario. [Copyright &y& Elsevier]

Abstract: Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression. [Copyright &y& Elsevier]

Abstract: Fibrates are pharmaceuticals commonly used to control hypercholesterolemia in humans and they are frequently detected in the freshwater environment. Since cholesterol is the precursor of all steroid hormones, it is suspected that low cholesterol levels will impact steroidogenesis. However, the effect of fibrates on fish reproductive endocrinology is not clear; therefore the aim of the present study was to evaluate the effect of bezafibrate (BZF) on gonadal steroidogenesis and spermatogenesis of zebrafish (Danio rerio). For this purpose, adult males were exposed orally to 1.7, 33 and 70mgBZF/g food for 21 days. Blood and gonads were collected after 48h, 7 days and 21 days to evaluate plasma cholesterol and plasma 11-ketotestosterone (11-KT). The expression of gonadal genes involved in the steroidogenesis was quantified to determine a potential mechanism of action, likewise the effect on spermatogenesis was evaluated by examining gonadal histopathology. A time dependent monotonic decrease in the plasma cholesterol concentration was observed in fish exposed to BZF. Plasma 11-KT decreased significantly after 21 days of exposure in fish exposed to the high concentration of BZF. Different gene expression patterns were observed: down-regulation in ppara and pparg mRNA levels was observed in fish exposed to the higher concentrations after 48h; however, the expression of pparg increased after 21 days. After 21 days an increase in the star and cyp17a1 mRNA expression was observed in fish exposed to 70mgBZF/g food. Sampling time and bezafibrate concentration explained 52.4% and 20%, respectively, of the gene expression variability. Gonadal histology revealed the presence of germ cell syncytia in the tubular lumen of fish exposed to bezafibrate and also an increased number of cysts containing spermatocytes, which indicate testicular degeneration. The study shows that bezafibrate exerts a hypocholesterolemic effect in adult male zebrafish and its potential as an endocrine disruptor due to its effect on the gonadal steroidogenesis and spermatogenesis. [Copyright &y& Elsevier]

Abstract: The innate immune response in fishes includes both cellular (phagocytes) and humoral (complement system mainly) components. In fish, as in mammals, reactive oxygen metabolites (ROM) are involved in the respiratory burst of phagocytes and three pathways of complement activation can be discerned. The aim of this study was to analyze the innate immune response of fish using parameters such as respiratory burst of phagocytes and the complement activity of plasma of the common carp (Cyprinus carpio, Cyprinidae). Samples from a total of 160 individuals were collected in five periods of the year (early summer, late summer, autumn, winter and spring). Respiratory burst activity of a constant blood volume was measured luminometrically and also calculated per phagocyte number. A trend of negative relation between respiratory burst activity and water temperature was observed, thus the respiratory activity reached the lowest values in summer. Total complement activity of plasma was determined as bacteriolytic activity against bioluminescent bacteria. The highest total complement activity was observed in autumn, it decreased in summer and winter and the lowest activity was detected in spring. The highest activity of alternative pathway of complement activation was detected in spring, which decreased in autumn and the lowest values were found in winter and in summer. To evaluate the effect of steroid hormones, the level of 11-ketotestosterone was analyzed in males and the maximum was found in spring. A negative correlation was found between 11-ketotestosterone and both respiratory burst and total complement activity. Our results indicate that the measured parameters of innate immunity in the common carp are strongly affected by seasonal changes. Moreover, we confirmed that the innate immune response is immuno-suppressed by 11-ketotestosterone in spring. [Copyright &y& Elsevier]

To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells.These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Dihydrotestosterone (DHT) is one of the most physiologically important androgens in many male vertebrates, with the exception of teleost fish, in which 11-ketotestosterone (KT) is generally considered the major circulating male androgen. In the present study, we investigated the effects of KT and DHT on fathead minnow juveniles (Pimephales promelas), with the aim to compare the effects of the two androgens on critical physiological processes, such as somatic growth, male secondary sexual characteristics expression, and gonad maturation. Juvenile fish (60days post-hatch) were exposed to 20 and 200ng/L of KT and DHT for 45days. Exposure to both androgens significantly stimulated somatic growth in both males (20 and 200ng/L) and females (200ng/L). Nuptial tubercle formation was induced by both KT and DHT, but only the latter, at 200ng/L, caused the appearance of dorsal fin spot in 92% of males and 75% of females. Circulating plasma T concentrations showed a sex-specific response; a significant increase was recorded in exposed males and a decrease in females. Both androgens induced a significant advancement of the spermatogenic processes in males at 200ng/L. In contrast, only DHT caused a severe disruption of ovarian physiology and morphology in females, inducing the development of spermatogenic tissue (intersex). These results show that in fathead minnow juveniles, DHT had in vivo androgenic potency comparable to KT in males, and higher than KT in females, suggesting a potential involvement of DHT in the mediation of fathead minnow androgenic responses. [Copyright &y& Elsevier]

Abstract: In the bluebanded goby, Lythrypnus dalli, removal of the male from a social group results in a rapid behavioral response where one female becomes dominant and changes sex to male. In a previous study, within hours of male removal, aromatase activity in the brain (bAA) of dominant females was almost 50% lower than that of control females from a group in which the male had not been removed. For those females that displayed increased aggressive behavior after the male was removed, the larger the increase in aggressive behavior, the greater the reduction in bAA. To investigate whether decreased bAA leads to increased aggression, the present study used a more rapid time course of behavioral profiling and bAA assay, looking within minutes of male removal from the group. There were no significant differences in bAA between control females (large females from groups with the male still present), females that doubled their aggressive behavior by 10 or 20min after male removal, or females that did not double their aggressive behavior within 30min after male removal. Further, individual variation in bAA and aggressive behavior were not correlated in these fish. Whole brain decreases in aromatase activity thus appear to follow, rather than precede, rapid increases in aggressive behavior, which provides one potential mechanism underlying the rapid increase in androgens that follows aggressive interactions in many vertebrate species. For fish species that change sex from female to male, this increase in androgens could subsequently facilitate sex change. [ABSTRACT FROM AUTHOR]

Abstract: In order to elucidate how androgens may mediate their effects on ovarian growth, we investigated the mRNA levels of two subtypes of androgen receptor (ara and arb) in the ovary of feminized Japanese eel (Anguilla japonica) during artificially induced ovarian development by quantitative real-time reverse transcriptase polymerase chain reaction and in situ hybridization. Ara mRNA levels were high from the late oil droplet stage to the late vitellogenic stage, whereas arb mRNA levels were high from the late oil droplet stage to the midvitellogenic stage. Both ar mRNAs were predominantly observed in the follicle cells and the epithelial cells of the ovigerous lamellae in all stages. In the oil droplet stage, oogonia exhibited intense signals for ar mRNAs. There was no obvious difference in localization pattern between ara and arb in all ovaries examined, irrespective of maturational stage. It was difficult to identify the follicle cell types that were positive for ar mRNA during ovarian development. Only in post-ovulatory follicles could theca and granulosa cells be clearly identified, and ar signals were observed in both layers. The predominant localization of ar mRNA in the follicle cells suggests that androgens play important roles in oocyte growth by acting on these cells in this species. We have shown the expression profile and localization of ar mRNA during ovarian development for the first time in an oviparous vertebrate. [ABSTRACT FROM AUTHOR]

Abstract: The weakly electric fish from the main channel of the Amazon river, Sternarchogiton nattereri, offers a striking case of morphological variation. Females and most males are toothless, or present only few minute teeth on the mandible, whereas some males exhibit exaggerated, spike-like teeth that project externally from the snout and chin. Androgens are known to influence the expression of sexually dimorphic traits, and might be involved in tooth emergence. In this study we assess the relationship in S. nattereri between morphological variation, 11 ketotestosterone (11-KT) and testosterone (T). We also examine relationships of morphology and androgen levels with electric organ discharge (EOD) frequency, reproductive condition, and seasonality. Our main finding is that male morph categories differed significantly in plasma concentrations of 11-KT, with toothed males showing higher levels of 11-KT than toothless males. By contrast, we did not detect statistical differences in T levels among male morph categories. Reproductive condition, as measured by gonadosomatic indexes (GSI), differed across two sample years, increased as the season progressed, and was higher in toothed males than in non-toothed males. EOD frequency was higher in toothed males than in either toothless males or females. Taken together, our findings suggest that S. nattereri male sexual characters are regulated by 11-KT levels, and that both morphology and androgens interact with reproductive condition and EOD frequency in ways that vary within and across reproductive seasons. [ABSTRACT FROM AUTHOR]

Abstract: Proximate mediators of reproductive behaviors in vertebrates have a long history of study. In fishes, relatively few studies have focused on hormonal control of parental care, despite a comprehensive background on the general physiology of fishes, and the frequent occurrence of parental care behaviors. Studies on this taxon have repeatedly found that the relationships between androgens and paternal care do not follow the predictions made in the avian and mammalian literature. Glucocorticoids may also have a role in mediating parental behaviors, possibly through their role as regulators of metabolism. As such, we investigated the role of 11-ketotestosterone (11-KT) and cortisol in mediating parental effort of male smallmouth bass (Micropterus dolomieu) by manipulating hormone titers in wild fish. In smallmouth bass, males spawn annually with a single female and defend a single brood for up to 30days. Treatment of parental fish with cyproterone acetate (CYA; an androgen receptor antagonist) resulted in a decrease in nest defense in response to a simulated brood predator; however, no changes in nest success, nest tending or biochemical indicators of nutritional status were detected. Treatment with exogenous cortisol did not change parental behavior, but did increase the rate of nest failure, possibly owing to the energetic cost of chronically elevated cortisol concentrations. We discuss these findings in the context of resource-driven trade-offs and highlight life history as an important factor controlling parental effort in species with costly parental care behaviors. [ABSTRACT FROM AUTHOR]

Abstract: The effects of 11-ketotestosterone (11KT) and dietary fishmeal on growth of tilapia (Oreochromis mossambicus) were examined. Juvenile tilapia, weighing about 8g, were reared for 50days with isonitrogenous and isocaloric diets containing fishmeal or all plant proteins, each with or without supplementation of 11KT. Fish fed fishmeal-based (FM) diets grew significantly faster than those fed soybean meal-based (SM) diets. Addition of 11KT (10mg/kg) to the FM diet significantly augmented growth, whereas no significant effect of 11KT was observed in fish fed SM diet. In accord with the accelerated growth, specific growth rates of fish fed FM diets were significantly greater than those of fish fed SM diets, although addition of 11KT to FM or SM diet had no effect on this parameter. Males showed significantly higher plasma levels of insulin-like growth factor-I (IGF-I) than did females; however, there was no consistent effect of fishmeal or 11KT on plasma IGF-I levels within each sex. Plasma levels of 11KT were significantly higher in males than in females. In males, plasma 11KT levels were significantly higher in fish fed FM diets than in those fed SM diet. No significant effect of fishmeal or 11KT was observed on plasma levels of 11KT or 17β-estradiol (E2) in females. Low but significant levels of vitellogenin (Vg) were found in male plasma. Plasma Vg levels were significantly lower in fish fed SM diets than in fish fed FM diets in both males and females. These results indicate that addition of 11KT to the fishmeal-based diet stimulated growth, minimally affecting plasma levels of IGF-I, 11KT, E2 and Vg. The absence of effect of 11KT in fish fed the soybean meal-based diet may indicate that the estrogenic activities or some components in the SM diet are likely to interfere with the growth-promoting effects of 11KT. [Copyright &y& Elsevier]

Abstract: In many vertebrates, reproduction is regulated by social interactions in which dominant males control access to females and food. Subordinate males that displace dominant individuals must rapidly adopt behavioral and physiological traits of the higher rank to gain reproductive success. To understand the process of phenotypic plasticity during social ascent, we analyzed the temporal expression pattern of dominance behaviors and circulating androgen levels when socially-suppressed males of an African cichlid fish Astatotilapia burtoni ascended in status. These experiments tested a prediction of the ‘challenge hypothesis’ that, during periods of social instability, male androgen levels are higher than during socially stable times. We found that socially and reproductively suppressed males perform territorial and reproductive behaviors within minutes of an opportunity to ascend in status, and that animals switch from initial expression of territorial behaviors to more reproductive behaviors during territory establishment. Following this rapid response, social stability may be achieved within 1–3days of social ascent. Consistent with predictions of the ‘challenge hypothesis’, circulating 11-ketotestosterone (11-KT) levels were elevated within 30min following social opportunity, coincident with increased aggressive behavior. However, territorial behaviors and serum 11-KT levels were then dissociated by 72h after social ascent, suggesting either rapid social stability and/or increased physiological potential for androgen production. This behavioral and physiological plasticity in male A. burtoni suggests that perception of social opportunity triggers a suite of quick changes to facilitate rapid transition towards reproductive success, and reveals important features of social ascent not previously recognized. [Copyright &y& Elsevier]

Abstract: Male and female teleost seabream (Pagrus major) were examined for seasonal variation of eumelanin, pheomelanin, 11-ketotestosterone (11KT, fish androgen), lightness (L* value) and Gonad Somatic Index (GSI: gonad mass/body mass×100). In males, levels of pyrrole-2,3,5-tricarboxylic acid (a marker of eumelanin), 11KT and the GSI increased sharply from September and plateaued in March and April when the fish are sexually mature. These results are consistent with the lightness of their body color. Using the data from males, a high correlation was observed for all combinations of those four variables (PTCA, 11KT, lightness and GSI). In females, little change was observed in those variables except for the GSI. 4-Amino-3-hydroxyphenylalanine (a marker of pheomelanin) was also analyzed, but it was below the detection limit at all times. Oral treatment of juvenile red seabream with synthetic androgen methyl-testosterone for 2months induced eumelanin accumulation about 3 times higher than the control. These data show that there is a close relationship between androgen levels and eumelanin accumulation in teleosts. This is the first report that androgen affects melanin accumulation in a dose-dependent manner. [Copyright &y& Elsevier]

Abstract: The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman’s two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11β-hydroxylase (11βH) and 11β-hydroxysteroid dehydrogenase (11βHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11βH and 11βHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11βHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology. [Copyright &y& Elsevier]

Abstract: A full-length cDNA encoding 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11β-HSD2 was then transfected to COS-7 cells, which converted 11β-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD+, 11β-HSD2 from testicular microsomes oxidized 11-OHT with apparent K m 56±4nM and V max 55±6pmol/h/mgprotein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11β-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11β-HSD2 transcript at 4h followed by gradual increase at 8h, 12h and peaking at 24h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11β-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11β-HSD2 during testicular differentiation and seasonal testicular cycle in catfish. [Copyright &y& Elsevier]

Abstract: Hormones play a critical role in the regulation of vertebrate mating behavior, including receptivity, and several components of mate choice. However, less is known about the role of these chemical messengers in mediating behavior associated with premating reproductive isolation. The bisexual–unisexual mating complex of sailfin mollies, Poecilia latipinna, and Amazon mollies, Poecilia formosa (sexual parasites of sailfins) has been a model system for studying ultimate mechanisms of species recognition. However proximate mechanisms, such as variation in hormone levels, have not been examined. We paired male sailfin mollies with either female conspecifics or Amazon mollies and obtained water-borne hormone samples before and after mating for all fish. We measured 11-ketotestosterone, testosterone, and estradiol from the water samples. As expected from previous studies, males mated with conspecifics more frequently than with Amazon mollies. 11-Ketotestosterone production by males increased when they mated with female sailfin mollies who themselves also showed elevated production of 11-ketotestosterone. This increase in male and female 11-ketotestosterone levels was not seen when males mated with Amazon mollies. This unique endocrine interaction represents a potential proximate mechanism for species recognition by male sailfin mollies. We found no significant change in testosterone or estradiol under these conditions suggesting that a single hormone mediates bidirectional interactions between males and females during courtship. [Copyright &y& Elsevier]

The aim of the present study is to assess aquatic ecosystem contamination using selected biochemical markers: cytochrome P450, ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), tripeptide glutathione, vitellogenin, and 11-ketotestosterone in chub (Leuciscus cephalus L.). Seven locations on the Svitava and Svratka rivers (in the Brno conurbation, Czech Republic) were assessed. The results were compared with the levels of the most important inductors of these biomarkers: organic pollutants hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), DDT and its metabolites, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in bottom sediment, fish muscle, and semipermeable membrane devices (SPMDs) and metals in sediment. The highest levels of pollutants were observed at sites situated downstream from Brno, especially at Modřice and Rajhradice. Significant positive correlations (p < 0.05) were found between EROD activity and HCH concentration in SPMDs, and also between GST and EROD activity with HCB concentration in muscle, after adjusting for age. Environ. Toxicol. Chem. 2010;29:541–549. © 2009 SETAC [ABSTRACT FROM AUTHOR]

Abstract: Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common α subunit non-covalently associated with a hormone-specific β subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone–receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species. [Copyright &y& Elsevier]

Abstract: Many aquatic species, such as teleosts, release into the water and detect multiple bioactive substances to assist in schooling, migration, alarm reactions, and to stimulate behavioral and physiological responses during reproduction and in parent–offspring interactions. Understanding the complex relationship between hormones, behavior and their function in communication requires the simultaneous examination of multiple circulating hormones. However, repeated blood sampling within a short time period is not possible in smaller animals without impacting the very behaviors under investigation. The non-invasive technique of collecting and measuring hormone values in holding water using either radioimmunoassay (RIA) or enzyme immunoassay (EIA) is becoming widely used in teleost research. Commercial assay kits in particular enable rapid and reliable data generation, yet their assay buffers are often specific and potentially incompatible with each other, which can hinder measuring multiple hormones from the same sample. We present here the validation and application of a “nested” elution technique we developed that allows for repeated sampling of multiple reproductive hormones – testosterone (T), 17β-estradiol (E2), progesterone (P), prostaglandin F2α (PGF) and 11-ketotestosterone (11KT) – from individual samples of animal holding water by using commercial EIA systems. Our results show that when using appropriate controls to account for possible technical and biological confounds, this technique provides a powerful new tool for research in aquatic endocrinology and physiology. [Copyright &y& Elsevier]

Measurement of steroids that are released into the water via the gills has previously been shown to be an effective way of studying the reproductive endocrinology of the male three-spined stickleback Gasterosteus aculeatus without having to kill the fish. In the present paper, a previous observation on the existence of a compound other than 11-ketotestosterone (11-KT) in water, which cross-reacted in the 11-KT radioimmunoassay was repeated. The amounts of this compound, however, were not sufficient to warrant a separation step prior to carrying out assay. The lack of association between androstenedione levels in water and those in plasma was also confirmed. For the first time, the amounts of testosterone released into the water were shown to be positively correlated with the amounts in plasma, the sampling procedure (placing the fish for 30 min in 50 ml water) had no effect on the rate of release of cortisol but caused a rapid drop in the rate of release of 11-KT (which means that the fish should not be sampled twice in short succession), physical interaction between two nesting males (which was accompanied by aggression) significantly increased the rate of release of 11-KT, androstenedione and testosterone (but not of cortisol) and the rate of release of 11-KT was at its maximum between 2 and 4 h after exposure. [ABSTRACT FROM AUTHOR]

Abstract: In arid regions of the southwestern United States, municipal wastewater treatment plants commonly discharge treated effluent directly into streams that would otherwise be dry most of the year. A better understanding is needed of how effluent-dependent waters (EDWs) differ from more natural aquatic ecosystems and the ecological effect of low levels of environmentally persistent organic wastewater compounds (OWCs) with distance from the pollutant source. In a controlled experiment, we found 26 compounds common to municipal effluent in treatment raceways all at concentrations <1.0μg/L. Male bonytail chub (Gila elegans) in tanks containing municipal effluent had significantly lower levels of 11-ketotestosterone (p =0.021) yet higher levels of 17β-estradiol (p =0.002) and vitellogenin (p =0.036) compared to control male fish. Female bonytail chub in treatment tanks had significantly lower concentrations of 17β-estradiol than control females (p =0.001). The normally inverse relationship between primary male and female sex hormones, expected in un-impaired fish, was greatly decreased in treatment (r =0.00) versus control (r =−0.66) female fish. We found a similar, but not as significant, trend between treatment (r =−0.45) and control (r =−0.82) male fish. Measures of fish condition showed no significant differences between male or female fish housed in effluent or clean water. Inter-sex condition did not occur and testicular and ovarian cells appeared normal for the respective developmental stage and we observed no morphological alteration in fish. The population-level impacts of these findings are uncertain. Studies examining the long-term, generational and behavioral effects to aquatic organisms chronically exposed to low levels of OWC mixtures are needed. [Copyright &y& Elsevier]

The article presents a field study which assesses the impact of diel hypoxia in the reproductive capacity of estuarine fishes captured in Alabama and Florida. The method of the study employs the acetylcholinesterase-based competitive enzyme-linked immunosorbent assay (ELISA) to analyze the hormones and vitellogenin (VTG) of sampled gulf killifish Fundulus grandis coming from sites with different dissolved oxygen cycles and different rate of diel hypoxia. It also analyzes the differences in condition factor, relative gonad size, steroid sex hormones and VTG via stepwise forward regression statistics. The result shows that testes and ovaries of gulf killifish coming from the site with severe and moderate diel hypoxia are significantly smaller.

The relationship between whole-body concentrations of 11-ketotestosterone (11-KT) and sexual function was examined in the coral goby Gobiodon erythrospilus, a bi-directional sex-changing fish. 11-KT occurred in both female and male G. erythrospilus, but levels were not always higher in males than in females within heterosexual pairs, and were not related to the stage of gonadal development of individual fish. These results suggest that comparable 11-KT levels in both sexes may allow serial adult sex change to take place in bi-directional sex-changing species, such as Gobiodon spp. [ABSTRACT FROM AUTHOR]

In maximizing reproductive success, individuals face a trade-off between parental care for their current offspring and investing in androgen-dependent sexual traits to produce further offspring. It has thus been proposed that parental effort would suppress androgen levels. Here, we studied parental effort by manipulating brood size in the peacock blenny, Salaria pavo, a littoral fish species in which males show long periods of paternal care. We focused on the effects of brood size on female spawning rate (measured as number of eggs received in the nest by focal males) and androgen levels. We found a positive linear relationship between brood size and the number of eggs received subsequently. Accordingly, spawning rate increased for males with experimentally enlarged broods while it decreased for males in which brood size was reduced. However, over a longer time interval, brood sizes of both treatments returned to the nonmanipulated brood size, suggesting an effect of additional factors such as male quality. Brood size did not show the expected negative relationship with levels of testosterone and 11-ketotestosterone. On the contrary, testosterone levels were positively correlated with brood size. However, in agreement with the prediction, changes in the level of parental care by manipulation of brood size showed an inverse relationship with testosterone levels. As with brood size, given time, testosterone levels tended to return to nonmanipulated levels. Such changes suggest that males adjust their brood size to an individual value through androgen modulation of courtship or other traits influencing female spawning rate. [Copyright &y& Elsevier]

Abstract: The Ca2+-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca2+ and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80μM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80μM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20α-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11β-hydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca2+-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca2+-dependent CaM and that possibly Ca2+-CaM determines the shift in steroidogenesis from C21 steroids to androgens. [Copyright &y& Elsevier]

Abstract: A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50ng/ml), limits of detection (0.1–0.2ng/ml) and quantification (0.2–0.5ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA). [Copyright &y& Elsevier]

Growth performances and sexual growth dimorphism were compared in two pure species of tilapia, Oreochromis niloticus niloticus (OO) and Sarotherodon melanotheron melanotheron (SS), and their reciprocal intergeneric hybrids (male O. n. niloticus × female S. m. melanotheron [OS] and male S. m. melanotheron × female O. n. niloticus [SO]). Fish obtained from artificial reproduction were reared on artificial diets over 10 weeks at 25 ± 2 °C, under light regimes of 12 h light:12 h darkness. Growth was measured on a weekly basis. Social interactions were recorded with a video camera. Pure O. n. niloticus achieved the fastest growth rates (Mean Specific Growth Rate (SGR) of 2.7 ± 0.6% d-1 for males and 2.3 ± 0.4% d-1 for female) and S. m. melanotheron the slowest (1.3 ± 0.3% d-1 for males and 1.4 ± 0.3% d-1 for females). The SGR of the intergeneric hybrids fell between that of the two pure strains. OS females grew faster (1.7 ± 0.4% d-1) than SO females (1.3 ± 0.2% d-1), whereas no difference was observed between males. Aggressive behaviour emerged fi rst among faster-growing fi sh (O. niloticus and SO). The role of parental components in behavioural and physiological traits of tilapia is discussed. [ABSTRACT FROM AUTHOR]

Abstract: In most vertebrates, aggression and dominance are tightly linked to circulating testosterone. Fish, however, have two androgens (testosterone, T and 11-ketotestosterone, 11KT) that influence aggression and dominance. To date, few studies have compared the relationship between androgen levels and the outcome of aggressive contests in both females and males of the same species. To investigate sex differences in androgens we staged size-matched, limited-resource (territory) contests with 14 female–female and 10 male–male pairs of the highly social cichlid Neolamprologus pulcher. We then examined androgen levels in recently established dominants, who won the contest and subsequently acquired a territory (for 3h), and subordinates, who lost and did not acquire a territory. Newly dominant females had higher plasma T but similar 11KT levels to newly subordinate females. In contrast, newly dominant males had higher 11KT but similar T levels to subordinate males. The ratio of 11KT to T, which demonstrates physiological importance of T conversion to 11KT, was positively correlated with submissive behavior in female winners, and correlated weakly with aggressive behavior in male winners (p =0.05). These findings provide support for the hypothesis that different androgens play equivalent roles in female versus male dominance establishment, and suggest that relative levels of 11KT and T are implicated in female dominance behavior and perhaps behavior of both sexes. [Copyright &y& Elsevier]

Abstract: Thyroid hormones are important regulators of vertebrate growth and development, and are under the control of the hypothalamic–pituitary–thyroid axis. Nuclear thyroid receptors (TRs), which act as inducible transcription factors, mediate cellular functions of thyroid hormones. The molecular structure of several subtypes of TRs have been elucidated in vertebrate species, including N-terminal truncations as well as C-terminal variations in the domain responsible for binding hormone. In this paper, we review current information on the thyroid receptors studied in the vertebrate species with emphasis on recent findings in goldfish concerning functional significance of the thyroid receptor subtypes. [Copyright &y& Elsevier]

Czech Republic. The effect of three major chemical plants located on the Elbe River (at Pardubice, Neratovice, and Usti nad Labem) on fish was studied in 2004. Health status, chemical concentrations (Hg, PCB, DDT, HCH, HCB, OCS, 4-tert-nonylphenols, 4-tert-octylphenol) in muscle, and biomarkers (hepatic ethoxyresorufin-O-deethylase (EROD), plasma vitellogenin, and plasma 11-ketotestosterone) were assessed in male chub (Leuciscus cephalus L.). Differences between localities upstream (US) and downstream (DS) from the monitored source of pollution were identified. Fish from DS sampling sites showed significantly higher levels of contaminants than fish from US sampling sites. Generally, the concentrations of pollutants in fish from the Elbe sites were significantly higher compared to the reference site. Reduced gonad size, decreased plasma levels of 11-ketotestosterone, EROD and vitellogenin induction, and histopathologies of male gonads indicated harmful effects of aquatic pollution in fish from the Czech portion of the Elbe River. [Copyright &y& Elsevier]

Male Atlantic salmon (Salmo salar) display different sexual strategies, maturing either as parr during the freshwater phase (as sneaky spawners), or as post smolts following one or several years at sea. First sexual maturation (puberty) occurs at different times depending on environmental and genetic factors. To improve our knowledge on the timing (age and season) of first sexual maturation in Atlantic salmon male parr, we investigated pubertal activation in second generation farmed salmon from the Norwegian river Figgjo, reared under natural conditions of photoperiod and water temperature. Histological analysis, in combination with morphometric measurements, plasma androgen levels and pituitary gonadotropin gene expression analysis revealed that, as previously reported, some male parr initiated early sexual maturation in spring at one year of age. Interestingly, some male parr were observed to initiate sexual maturation already in autumn, six months after hatching (under-yearlings), much earlier than reported in previous studies. One-year old maturing males showed a low induction in gonadotropin levels, while under-yearling maturing males displayed a significant increase in fshb transcripts as compared to immature/early-maturing fish. Plasma testosterone, detectable also in immature/early-maturing males, increased constantly during testes development, while 11-ketotestosterone, undetectable in immature/early-maturing and early maturing males, increased during more advanced stages of maturation. A mild feminization of the testes (ovotestes) was detected in a subset of samples. This study brings new knowledge on the little investigated field of sexually maturing under-yearlings in Atlantic salmon. This is also the first study comparing the physiology of under-yearling vs one-year old maturing male parr, thus bringing new insights to the remarkable plasticity of Atlantic salmon puberty. • Time of puberty was investigated in farmed Atlantic salmon male parr. • Some males mature in autumn (under-yearling), others in spring (one-year old). • Mild symptoms of feminization (ovotestis) were detected in a subset of male parr. • First study to compare under-yearlings vs one-year old maturing male parr. [ABSTRACT FROM AUTHOR]

Agonistic behavior governs the settlement of conflicts among conspecifics for limiting resources. Sex steroids play a critical role in the regulation of agonistic behavior which in turn may produce modulations in hormone titres. In this study we analyzed the association of androgens and estrogens with agonistic behavior in the annual fish Austrolebias reicherti. This native species inhabits temporary ponds that dry out completely during summer, having one of the shortest lifespans among vertebrates. They are highly sexually dimorphic and have a single breeding season during which they reproduce continuously. Here we measured plasma levels of 11-ketotestosterone (11KT) and 17β-estradiol (E 2) in adult males after the resolution of a social conflict and assessed the role of the aromatase conversion of testosterone (T) to E 2 in male aggression. Winners had higher levels of 11KT than losers yet; winner 11KT levels did not differ from those of males not exposed to a social challenge. E 2 levels did not show differences among winners, losers or control males. However, fights under the aromatase inhibitor Fadrozole were overall less aggressive than control fights. Our results suggest an androgen response to losing a conflict and that the conversion of T to E 2 is involved in the regulation of aggressive behavior. Annual fish extreme life history may give new insights on hormone-behavior interactions. • Post-fight androgen levels are unrelated to aggression. • Losers have lower androgen levels than winners. • Losers and winners have similar estrogen levels. • Aromatase inhibition significantly affects male-male aggression. [ABSTRACT FROM AUTHOR]

Endocrine disrupting chemicals, such as bisphenol A (BPA) and ethinylestradiol (EE2), are detected in the marine environment from plastic waste and wastewater effluent. However, their impact on reproduction in sexually labile coral reef fish is unknown. The objective of this study was to determine impacts of environmentally relevant concentrations of BPA and EE2 on behavior, brain gene expression, gonadal histology, sex hormone profile, and plasma vitellogenin (Vtg) levels in the anemonefish, Amphiprion ocellaris. A. ocellaris display post-maturational sex change from male to female in nature. Sexually immature, male fish were paired together and fed twice daily with normal food (control), food containing BPA (100 μg/kg), or EE2 (0.02 μg/kg) (n = 9 pairs/group). Aggression toward an intruder male was measured at 1, 3, and 6 months. Blood was collected at 3 and 6 months to measure estradiol (E2), 11-ketotestosterone (11-KT), and Vtg. At the end of the study, fish were euthanized to assess gonad morphology and to measure expression of known sexually dimorphic genes in the brain. Relative to control, BPA decreased aggression, altered brain transcript levels, increased non-vitellogenic and vitellogenic eggs in the gonad, reduced 11-KT, and increased plasma Vtg. In two BPA-treated pairs, both individuals had vitellogenic eggs, which does not naturally occur. EE2 reduced 11-KT in subordinate individuals and altered expression of one transcript in the brain toward the female profile. Results suggest BPA, and to a lesser extent EE2, pollution in coral reef ecosystems could interfere with normal reproductive physiology and behavior of the iconic sexually labile anemonefish. • BPA alters brain gene expression and aggressive behavior of anemonefish. • BPA dramatically reduces circulating 11-KT, and decreases % testicular tissue. • BPA increases non-vitellogenic and vitellogenic eggs in the gonads of anemonefish. • BPA can produce pairs of female anemonefish, which does not naturally occur. • EE2 has a lesser effect, but both could interfere with anemonefish reproduction. [ABSTRACT FROM AUTHOR]

• Androgens, but not glucocorticoids (GC), increase during pre-breeding in hellbenders. • Hellbenders are likely associated breeders. • Pre-breeding shifts in plasma androgen concentrations are comparable to other salamanders. • Testosterone and DHT are the predominant androgens in hellbenders. • Cortisol, not corticosterone, is the major GC in hellbenders. Seasonally breeding species exhibit cyclical changes in circulating steroid hormone profiles that correspond with changes to their reproductive behavior and ecology. Such information is critical to the conservation of imperiled and data-deficient species, such as the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis). We determined changes in plasma testosterone (T), dihydrotestosterone (DHT), 11-ketotestosterone (11-KT), 11-ketoandrostenedione (11-KA), dehydroepiandrosterone (DHEA), cortisol, corticosterone, and progesterone (P 4) during a four-month period preceding breeding in adult male and female eastern hellbenders. This pre-breeding period is characterized by increased diel movement and aggression by both sexes, follicular development and yolk production in females, and sperm production, territoriality, and nest site establishment in males. In both males and females, we observed a progressive increase in circulating T and DHT during the pre-reproductive season, both peaking in August (17 days before breeding), but concentrations of both hormones were higher in males. Conversely, 11-KT was higher in females, but did not vary significantly by date. These results suggest that T and DHT are the predominant androgens in eastern hellbenders and are likely important regulators of reproductive processes in both males and females. The detection of significant quantities of DHT and 11-KT in females is particularly interesting, considering that unlike T, neither of these androgens can be converted to estrogens. Therefore, it seems possible that aggression or some aspect of reproduction in the female eastern hellbender may be directly mediated by androgen signaling. Baseline cortisol did not vary throughout the pre-breeding period but was higher in females than males, and also became highly variable in females leading up to breeding. Progesterone, 11-KA, DHEA, and corticosterone were rarely or never detected, and thus, do not appear to be important during the pre-reproductive season. This study provides a physiological framework for future studies of hellbender reproductive biology, which could ultimately be important for their conservation. [ABSTRACT FROM AUTHOR]

Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the ‘static’ sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release — and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort. [ABSTRACT FROM AUTHOR]

Abstract: Within species, color morphs may enhance camouflage, improve communication and/or confer reproductive advantage. However, in the male cichlid Astatotilapia burtoni, body color may also signal a behavioral strategy. A. burtoni live in a lek-like social system in Lake Tanganyika, Africa where bright blue or yellow territorial (T) males (together ~10–30% of the population) are reproductively capable and defend territories containing food with a spawning site. In contrast, non-territorial (NT) males are smaller, cryptically colored, shoal with females and have regressed gonads. Importantly, males switch between these social states depending on their success in aggressive encounters. Yellow and blue morphs were thought to be adaptations to particular habitats, but they co-exist both in nature and in the laboratory. Importantly, individual males can switch colors so we asked whether color influences behavioral and hormonal profiles. When pairing territorial males with opposite colored fish, yellow males became dominant over blue males significantly more frequently. Moreover, yellow T males had significantly higher levels of 11-ketotosterone than blue T males while only blue NT males had higher levels of the stress hormone cortisol compared to the other groups. Thus color differences alone predict dominance status and hormone profiles in T males. Since T males can and do change color, this suggests that A. burtoni may use color as a flexible behavioral strategy. [Copyright &y& Elsevier]

The reproductive behaviour in the male threespine stickleback, Gasterosteus aculeatus is under androgenic control, in which 11-ketotestosterone (11KT) is the most important androgen. Previous studies have shown that castration removes reproductive behaviour, whereas treatment with 11-ketoandrostenedione (11KA), which is converted to 11KT extra-testicularily, restores all aspects of it, at least qualitatively. In this study, we investigated whether 11KT and/or testosterone (T) could restore the full magnitude of some of the more pronounced reproductive behaviours, including gluing (nest-building behaviour), zig-zagging (courtship behaviour) and displacement fanning in castrated male sticklebacks. Non-breeding males were castrated and implanted with either an empty Silastic capsule (castrated control), or a capsule containing 11KA, T or a combination of both capsules. Sham-operated males also received an empty capsule. While castrated males showed low or non-detectable plasma levels of both T and 11KT, castrated males given T or 11KA implants showed elevated levels of T and 11KT respectively, levels being within the physiological range found in intact nest-building males. Castrated males given both implants showed elevated levels of both T and 11KT. While all sham-operated males actively displayed all reproductive behaviours, these behaviours were either completely abolished or greatly reduced by castration. 11KA treated fish displayed more zig-zags and fanning than castrated fish, and unlike castrated males, built nests. Overall, 11KA or 11KA + T were more effective than T in restoring the expression of reproductive behaviours in castrated males. However, sham-operated fish displayed more gluing compared to 11KA, T and 11KA + T treated fish, and more fanning than 11KA or T treated fish. Our results indicate that 11KT and/or T cannot mediate the entire testicular effect on reproductive behaviours in the male stickleback and suggest that other testicular hormones also play a role. [ABSTRACT FROM AUTHOR]

Abstract: Given the dramatic behavioral effects of winning and losing contests, and pronounced changes in stress and sex steroid hormones post-fight, it is reasonable to suppose that these hormones also dictate future behavior. We sampled water-borne cortisol, testosterone (T), and 11-ketotestosterone (KT) before and after contests in the mangrove killifish, Kryptolebias marmoratus, to determine how endogenous steroid hormone levels might predict and respond to contest dynamics or success. Pre-fight cortisol related negatively, and pre-fight T related positively to contest initiation and winning, particularly in the smaller opponent. In the pairs where a larger fish won the contest, winners with higher pre-fight T and lower pre-fight cortisol delivered more attacks to the losers. Contest duration and escalation influenced post-fight hormone concentrations primarily in losers. Escalation significantly increased post-fight cortisol, T, and KT for losers but not for winners. However, winners that attacked losers at higher rates had higher levels of post-fight cortisol. Losers also demonstrate the most consistent post-fight hormone responses, particularly to contest escalation and duration. Despite the bidirectional relationship between hormones and contest behavior, we found no overall mean differences in pre- or post-fight cortisol, T, or KT between eventual winners and losers. Thus, it is evident that the categorical states of winner and loser cannot alone reveal the complex, reciprocal associations between endocrine systems and social behavior. [Copyright &y& Elsevier]

Among taxonomically widespread cooperatively breeding vertebrates, those with non-breeding helpers-at-the-nest provide an excellent opportunity to understand the proximate mechanisms underlying care and allocare. In this study, we examined androgen levels in relation to care behavior in a cooperatively breeding cichlid fish, Neolamprologus pulcher, from Lake Tanganyika. We concentrated on androgens, as these hormones have been linked to the defense behavior, and the defense of young is a common form of parental care in fishes. N. pulcher dominant female breeders performed the most care and also displayed the highest levels of plasma testosterone (T) compared with other individuals within the social group. We also found that dominant male breeders provided a similar amount of care as did the subordinate helpers, but breeding males had the highest levels of 11-ketotestosterone (11KT), an important androgen in fish. Breeders had higher levels of both androgens ( T and 11KT) compared to helpers. There was a weak but significant positive correlation between T levels and the frequency of care regardless of sex and status. Our results suggest that androgens may promote defense of young and are in contrast to the commonly reported trade-off between androgen and parental care. [ABSTRACT FROM AUTHOR]

Abstract: The primary goals of this study were to evaluate diurnal patterns of and sex differences in the levels of cortisol, 11-ketotestosterone, testosterone, and 17β-estradiol in the sex-changing bluebanded goby Lythrypnus dalli. Steroid hormones were collected from water samples and analyzed by enzyme immunoassay. During the breeding season, hormones were sampled from both males and females at seven time points between 0600 and 2000h. When comparing each time point separately, there were significant overall time effects for cortisol and 17β-estradiol. Cortisol concentrations were lowest at the 0800–1000h sampling point and showed a qualitative peak in late morning (1000–1200h). Concentrations of 17β-estradiol were elevated at the last sampling point (1800–2000h). Broader temporal trends were revealed for testosterone and 11-ketotestosterone concentrations, both of which were elevated in the morning. There were no sex differences in overall hormone concentrations or temporal profiles for cortisol, 11-ketotestosterone, or testosterone. Males and females showed similar diurnal patterns of 17β-estradiol but females had significantly higher water-borne 17β-estradiol levels than males. The results show the presence of diurnal changes in steroid hormone levels in male and female bluebanded gobies. The lack of sex differences in androgens suggests that males of this species, and perhaps other bi-directional sex-changing species in which males do not exhibit prominent secondary sexual characteristics, do not require persistent elevations in 11-ketotestosterone or testosterone to maintain the male phenotype. Although the role of 17β-estradiol in maintaining sex differences in sexually plastic species is unclear, our results suggest that, of the hormones measured, 17β-estradiol has the greatest potential for future studies interested in this question. [Copyright &y& Elsevier]

Abstract: Human nutrient input has significantly altered dissolved oxygen (DO) cycles in coastal waters such that summertime hypoxia (DO <2 mg/L) and anoxia of bottom water are common worldwide. Prolonged hypoxia usually reduces metabolic rate in fish and potentially reduces reproduction, particularly in a spring and summer spawning species such as the Gulf killifish, Fundulus grandis. To evaluate the effects of long term hypoxia on reproduction, Gulf killifish were subjected to either normoxia (6.68±2.1 mg/L DO) or hypoxia (1.34±0.45 mg/L DO) for one month. Fecundity, growth, gonadosomatic index (GSI), circulating sex steroids (testosterone, T; 11-ketotestosterone, 11KT; and estradiol-17β, E2), and egg yolk protein (vitellogenin, VTG) were measured. Hypoxia significantly reduced growth and reproduction. E2 was 50% lower in females and 11KT was 50% lower in males, although the precursor hormone T was unchanged in either sex after hypoxic exposure. Hypoxia-exposed females produced significantly fewer eggs and initiated spawning later than control fish. Plasma VTG concentration was unchanged, suggesting that hypoxia may delay VTG uptake by oocytes. Long term laboratory exposure clearly suppressed reproductive capacity in Gulf killifish. Wild populations experience cyclic hypoxia which could have equivalent effects if daily hypoxic periods are long and frequent — a potential consequence of anthropogenic nutrient enrichment in marsh systems. [Copyright &y& Elsevier]

The immunocompetence handicap hypothesis (ICHH) provides a functional explanation for how sexual ornaments can provide honest signals of male quality. A key aspect of this hypothesis is that testosterone (T) has a bimodal effect: a higher T level enhances the expression of ornaments (increasing mating success and, ultimately, fitness); however, at the same time, it suppresses immune function. Tests of the latter assumption, which have focused mainly on aspects of adaptive immunity in birds, led to equivocal results. We performed a hormone-implant experiment in male three-spined sticklebacks (Gasterosteus aculeatus) to test the key assumptions of the ICHH in a fish, where the dominant circulating androgen is 11-ketotestosterone (11kT) rather than T. Males were implanted with 11-ketoandrostenedione, which is a natural precursor of 11kT. Each individual's circulating 11kT level, ornamentation, and immunocompetence were measured 2 weeks later. In addition, we quantified oxidative tissue damage because the ICHH has been hypothesized to work via oxidative stress. We found that the males' 11kT levels correlated positively with ornamentation but negatively with immunocompetence, in particular, measures of innate immunity. Moreover, there was a trend for fish with high 11kT levels to suffer more from oxidative stress. Thus, our data provide support for the ICHH. [ABSTRACT FROM AUTHOR]

Abstract: Measurement of fish steroids in water provides a non-invasive alternative to measurement in blood samples, offering the following advantages: zero or minimal intervention (i.e. no anaesthetic, bleeding or handling stress); results not being biased by sampling stress; repeat measurements on the same fish; the possibility of making non-lethal measurements on small and/or rare fish; integrating the response of many (or of single) fish; and allowing concurrent monitoring of behaviour or physiology. The procedure is relatively new and, although applications are still fairly limited, there are several themes and potential problem areas that are worthy of review. [Copyright &y& Elsevier]