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The Challenge Hypothesis (Wingfield et al. Am. Nat. 136, 829-846) aims to explain the complex relationship between androgens and social interactions. Despite its well acceptance in the behavioral endocrinology literature, several studies have failed to found an androgen response to staged social interactions. Possible reasons for these inconsistencies are the use of single sampling points that may miss the response peak, and the occurrence of inter-individual variability in the androgen response to social interactions. In this study we addressed these two possible confounding factors by characterizing the temporal pattern of the androgen response to social interactions in the African cichlid, Oreochromis mossambicus, and relating it to inter-individual variation in terms of the individual scope for androgen response (i.e. the difference between baseline and maximum physiological levels for each fish) and behavioral types. We found that the androgen response to territorial intrusions varies between individuals and is related to their scope for response. Individuals that have a lower scope for androgen response did not increase androgens after a territorial intrusion but were more aggressive and exploratory. In contrast males with a higher scope for response had fewer aggressive and exploratory behaviors and exhibited two peaks of KT, an early response 2-15 min after the interaction and a late response at 60-90 min post-interaction. Given that the pharmacological challenge of the Hypothalamic-Pituitary-Gonad axis only elicits the late response, we suggest that these two peaks may be regulated by different physiological mechanisms, with the early response being mediated by direct brain-gonad neural pathways. In summary, we suggest that determining the temporal pattern of the androgen response to social interactions and considering inter-individual variation may be the key to understanding the contradictory results of the Challenge Hypothesis., (Copyright © 2020. Published by Elsevier Inc.)

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Androgens respond to social challenges and this response has been interpreted as a way for males to adjust androgen-dependent behavior to social context. However, the androgen responsiveness to social challenges varies across species and a conceptual framework has been developed to explain this variation according to differences in the mating system and parental care type, which determines the regimen of challenges males are exposed to, and concomitantly the scope (defined as the difference between the physiological maximum and the baseline levels) of response to a social challenge. However, this framework has been focused on territorial species and no clear predictions have been made to gregarious species (e.g. shoaling fish), which although tolerating same-sex individuals may also exhibit intra-sexual competition. In this paper we extend the scope of this conceptual framework to shoaling fish by studying the endocrine response of zebrafish (Danio rerio) to social challenges. Male zebrafish exposed to real opponent agonistic interactions exhibited an increase in androgen levels (11-ketotestosterone both in Winners and Losers and testosterone in Losers). This response was absent in Mirror-fighters, that expressed similar levels of aggressive behavior to those of winners, suggesting that this response is not a mere reflex of heightened aggressive motivation. Cortisol levels were also measured and indicated an activation of the hypothalamic-pituitary-interrenal axis in Winners of real opponent fighters, but not Losers or in Mirror-fighters. These results confirm that gregarious species also exhibit an endocrine response to an acute social challenge., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social and reproductive behaviors in fishes. However, the question remains, as to whether there is a link between the distribution of active nonapeptides across brain regions and fishes specific behavioral phenotypes. To explore this link we compared the nonapeptides' profile across the brains of fishes representing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis australis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and brain stem, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We showed that the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to the development of sexual dimorphism, which occurs only in this non-cleaner species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Photoperiod is a major environmental cue that signals breeding conditions in animals living in temperate climates. Therefore, the activity of the reproductive (i.e. hypothalamic-pituitary-gonadal, HPG) axis and of the expression of reproductive behaviors, including territoriality, is responsive to changes in day length. However, at low latitudes the seasonal variation in day length decreases dramatically and photoperiod becomes less reliable as a breeding entraining cue in tropical species. In spite of this, some tropical mammals and birds have been found to still respond to small amplitude changes in photoperiod (e.g. 17min). Here we tested the effect of 2 photoperiod regimes, referred to as long-day (LD: 16L:08D) and short-day (SD: 08L:16D), on the activity of the HPG axis, on aggressive behavior and in the androgen response to social challenges in males of the tropical cichlid fish Tilapia rendalli. For each treatment, fish were transferred from a pre-treatment photoperiod of 12L:12D to their treatment photoperiod (either LD or SD) in which they were kept for 20days on stock tanks. Afterwards, males were isolated for 4days in glass aquaria in order to establish territories and initial androgen levels (testosterone, T; 11-ketotestosterone, KT) were assessed. On the 4th day, territorial intrusions were promoted such that 1/3 of the isolated males acted as residents and another 1/3 as intruders. Territorial intrusions lasted for 1h to test the effects of a social challenge under different photoperiod regimes. Photoperiod treatment (either SD or LD) failed to induce significant changes in the HPG activity, as measured by androgen levels and gonadosomatic index. However, SD increased the intensity of aggressive behaviors and shortened the time to settle a dominance hierarchy in an androgen-independent manner. The androgen responsiveness to the simulated territorial intrusion was only present in KT but not for T. The percent change in KT levels in response to the social challenge was different between treatments (SD>LD) and between male types (resident>intruder). The higher androgen response to a social challenge in residents under SD may be explained by the time course of the androgen response that due to the long time it takes to fight resolution under LD, might have been delayed. This result illustrates the importance of incorporating time response data in social endocrinology studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Gonads are the main source of sex steroids, which have been implicated in the regulation of sexually differentiated behavior, such as reproductive and aggressive displays. In the Mozambique tilapia (Oreochromis mossambicus) territorial males have higher androgen levels than non-territorials, express reproductive behavior and use a urine-borne pheromone to signal their social status towards conspecifics. Here we investigated the effects of gonadectomy on the circulating levels of androgens and cortisol, and on the expression of aggressive and reproductive behavior (nest building, courtship behavior, and nuptial coloration). Males were either castrated, urine bladder damaged, or sham-operated and visually exposed to a group of females during 8 consecutive days and subsequently to a male on day 9. The urine bladder damaged treatment was included in the experimental design because a full castration procedure in this species causes quite often damage to the urine bladder. Gonadectomy lowers dramatically the circulating levels of androgens measured at 4 and 8days post-castration and abolishes the expression of nest building, courtship behavior and nuptial coloration, but has no effect on the expression of aggressive behavior. These results confirm the gonads as the main source of androgens in this species and show that androgens are necessary for the expression of reproductive behaviors. However, the expression of aggressive behavior seems to be decoupled from gonadal steroids, namely androgens, suggesting the action of independent central mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent empirical research, mostly done on humans, recognizes that individuals' physiological state affects levels of cooperation. An individual's internal state may affect the payoffs of behavioural alternatives, which in turn could influence the decision to either cooperate or to defect. However, little is known about the physiology underlying condition dependent cooperation. Here, we demonstrate that shifts in cortisol levels affect levels of cooperation in wild cleaner wrasse Labroides dimidiatus. These cleaners cooperate by removing ectoparasites from visiting 'client' reef fishes but prefer to eat client mucus, which constitutes cheating. We exogenously administrated one of three different compounds to adults, that is, (a) cortisol, (b) glucocorticoid receptor antagonist mifepristone RU486 or (c) sham (saline), and observed their cleaning behaviour during the following 45min. The effects of cortisol match an earlier observational study that first described the existence of "cheating" cleaners: such cleaners provide small clients with more tactile stimulation with their pectoral and pelvic fins, a behaviour that attracts larger clients that are then bitten to obtain mucus. Blocking glucocorticoid receptors led to more tactile stimulation to large clients. As energy demands and associated cortisol concentration level shifts affect cleaner wrasse behavioural patterns, cortisol potentially offers a general mechanism for condition dependent cooperation in vertebrates., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Androgens are known to respond to social challenges and to control the expression of social behavior and reproductive traits, such as gonadal maturation and sperm production, expression of secondary sex characters and reproductive behaviors. According to the challenge hypothesis variation in androgen levels above a breeding baseline should be explained by the regime of social challenges faced by the individual considering the trade-offs of androgens with other traits (e.g. parental care). One prediction that can be derived from the challenge hypothesis is that androgen levels should increase in response to social instability. Moreover, considering that a tighter association of relevant traits is expected in periods of environmental instability, we also predict that in unstable environments the degree of correlations among different behaviors should increase and hormones and behavior should be associated. These predictions were tested in a polygamous cichlid fish (Mozambique tilapia, Oreochromis mossambicus) with exclusive maternal care. Social instability was produced by swapping dominant males among groups. Stable treatment consisted in removing and placing back dominant males in the same group, in order to control for handling stress. Cortisol levels were also measured to monitor stress levels involved in the procedure and their relation to the androgen patterns and behavior. As predicted androgen levels increased in males in response to the establishment of a social hierarchy and presence of receptive females. However, there were no further differential increases in androgen levels over the social manipulation phase between social stable and social unstable groups. As predicted behaviors were significantly more correlated among themselves in the unstable than in the stable treatment and an associated hormone-behavior pattern was only observed in the unstable treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Studies on birds and mammals indicate that sexual traits may signal superior health because active immunity, like inflammatory responses to infections, is suppressive to the production of androgens that facilitate the expression of these traits. Here we test this possible pathway for honest signaling in a teleost species, Sarotherodon galilaeus, by activating the immune system with sheep red blood cells (SRBC), which is a non-pathogenic T- and B-cell stimulating antigen. Two weeks after the start of treatment adult males injected with SRBC showed a significant increase in antibody production in comparison with control males. The variation in specific antibody production was negatively related with variation in both testosterone and 11-ketotestosterone levels. This suggests that investment in immune protection is incompatible with increased activity of the hypothalamic-pituitary-gonadal axis. However, opposite to our expectation no difference in androgen levels was found between placebo and SRBC treatment suggesting that immune activation did not cause androgen suppression in our studied species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The challenge hypothesis proposes that testosterone (T) elevation above what is needed for breeding is associated with social factors, and males possibly modulate their hormonal response to variations in population density and sex ratio. We investigated the role of social environment in altering testosterone levels and aggression in a tropical, seasonally breeding grassquit (Volatinia jacarina). We exposed males to three social conditions during 1 year: all-males treatment (six males), mixed treatment (three males-three females), and paired treatment (one male-one female). We quantified aggressiveness among males and T plasma concentration for each individual in each treatment monthly. We found that more aggressive interactions occurred in the all-males treatment than in the mixed treatment. The data also revealed that, coincident with these behavioral changes, the patterns of T variation through time in each treatment were markedly different. The all-males treatment exhibited an early increase in T concentration, which was sustained for a lengthy period with two distinctive peaks, and subsequently declined sharply. The mixed treatment presented an intermediate pattern, with more gradual increase and decrease in T levels. At the other extreme, the paired treatment presented a later rise in T concentration. We conclude that the more competitive environment, with higher density of males, caused the early and higher elevation in T level, thus the presence of competitors may influence the decision of how much a male should invest in reproduction. We suggest that the male's perception of his social environment ultimately mediates hormonal production and alters his reproductive strategy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Interactions between the bluestreak cleaner wrasse Labroides dimidiatus and its client reef fish are a textbook example of interspecific mutualism. The fact that clients actively visit cleaners and invite inspection, together with evidence that cleaners eat many client ectoparasites per day, indeed strongly suggests a mutualistic relationship. What remains unknown is how parasite removal affects the physiology of clients and thereby their body condition, health, and immune function. Here we addressed these issues in a field study in Ras Mohammed National Park, Egypt. In our study area, small reef patches are inter-spaced with areas of sandy substrate, thereby preventing many species (i.e., residents, including cleaner wrasses) from travelling between the reef patches. This habitat structure leads to a mosaic of resident clients with and without access to bluestreak cleaner wrasses, further referred to as "cleaner access", on which we focused our study. We found that residents with cleaner access had higher body condition than residents without cleaner access. However, indicators of stress like variation in cortisol levels corrected for handling time and various immune parameters were apparently unaffected by cleaner access. In fact antibody responses were significantly higher in fishes without cleaner access. This suggests that cleaner access decreases the need for active immunity and that this releases resources that might be allocated to other functions such as somatic growth and reproduction., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Morphology and endocrinology were studied in two populations of the peacock blenny Salaria pavo, with different regimes of sexual selection imposed by differences in nest site availability. The peacock blenny is a small, sexually dimorphic benthic fish that presents exclusive paternal care of the clutch and inhabits rocky shores of the Mediterranean and adjacent Atlantic areas. In a population from the Gulf of Trieste (Northern Adriatic sea) inhabiting rocky shores where nest sites are abundant, male-male competition for nests is low, males court females and a low frequency of alternative reproductive tactics (small, parasitic female-mimicking sneaker males that change tactic into nest holders in subsequent breeding seasons) occurs. Conversely at Ria Formosa, a coastal lagoon in Southern Portugal, where nest sites are scarce and highly aggregated, male-male competition for nests is very high, there is sex-role reversal with female courtship and a high frequency of alternative reproductive tactics is observed. Concomitantly, at Ria Formosa nest holder males are larger and present more developed secondary sex characters and higher levels of 11KT than at the Gulf of Trieste. However, the gonads of nest holders and parasitic males were larger in the Gulf of Trieste population. Competition for nests at Ria Formosa seems to promote more developed secondary sex characters in nest site scarcity conditions, while competition for females at the Gulf of Trieste seems to be spurring sperm competition among males in populations where nest sites are more abundant. 11KT was thus associated with the development and expression of secondary sex characters in contrasting environments. These results exemplify how the modulation of behavioral plasticity and secondary sex characters by the social environment can be mediated by androgens., (Copyright 2009 Elsevier Inc. All rights reserved.)

Aromatase, the key enzyme in the conversion of androgens to estrogens, regulates the availability of these hormones in tissues and controls many physiological and behavioral processes. In fish and other vertebrates, the regulation of aromatase expression in the brain has been implicated in the modulation of male sexual and aggressive behaviors. Here, the pattern of mRNA expression of the brain aromatase isoform (encoded by the CYP19A2 gene also referred as CYP19b) was quantified at the peak of spawning season in brain macroareas from males and females of the blenny Salaria pavo originated from two populations displaying male alternative reproductive tactics but differing in their mating systems. In Trieste (Adriatic) nesting males aggressively defend nests and take the initiative in courtship and perform sexual displays more often than females while in Ria Formosa (Southern Portugal) the pattern is reversed as a result of shortage of appropriate nesting sites. Nesting males from Ria Formosa had overall higher levels of brain aromatase mRNA expression than nesting males from Trieste, suggesting a higher brain estrogen synthesis in these males. Since in some fish species exogenous estradiol administration has been shown to decrease sexual and agonistic behaviors, the higher levels of brain aromatase in Ria Formosa nesting males may explain their reduced expression of sexual and aggressive displays when compared with nesting males from Trieste. Alternatively, the higher brain aromatase levels in nesting males from Ria Formosa could be a mechanism to decrease the putative androgen-induced activation of aggressive and sexual displays by reducing the local availability of androgens through their metabolization into estrogens. Although females and parasitic female-like males also differ in their displays between populations, the interpopulational pattern of brain aromatase mRNA expression was similar, suggesting that other neuroendocrine agents mediate the expression of female and female-like behaviors. In conclusion, brain aromatase availability seems like a probable mechanism to regulate the effects of steroids on the brain circuits underlying the expression of sexual and agonistic displays in S. pavo., (Copyright 2009 Elsevier Inc. All rights reserved.)

In the peacock blenny Salaria pavo large males with well-developed secondary sexual characters establish nests and attract females while small "sneaker" males mimic female sexual displays in order to approach the nests of larger males and parasitically fertilize eggs. These alternative reproductive tactics are sequential, as sneakers irreversibly switch into nesting males. This transition involves major morphologic and behavioral changes and is likely to be mediated by hormones. This study focuses on the role of aromatase, an enzyme that catalyses the conversion of androgens into estrogens, in the regulation of male sexual polymorphism in S. pavo. For this, sex steroid plasma levels and aromatase activity (AA) in gonads, whole brain and brain macroareas were determined in sneakers, transitional males (i.e. sneakers undergoing the transition into nesting males), nesting males and females collected in the field. AA was much higher in ovarian tissue than in testicular tissue and accordingly circulating estradiol levels were highest in females. This supports the view that elevated AA and estradiol levels are associated with the development of a functional ovary. Transitional males are in a non-reproductive phase and had underdeveloped testes when compared with sneakers and nesting males. Testicular AA was approximately 10 times higher in transitional males when compared with sneakers and nesting males, suggesting high AA has a suppressive effect on testicular development. Nesting males had significantly higher plasma levels of both testosterone (T) and 11-ketotestosterone when compared with the other male morphs and previous studies demonstrated that these androgens suppress female-like displays in sneakers. In the brain, AA was highest in macroareas presumably containing hypothalamic nuclei traditionally associated with the regulation of reproductive behaviors. Overall, females presented the highest levels of brain AA. In male morphs AA increased from sneakers, to transitional males, to nesting males in all brain macroareas. These results suggest that the transition into the nesting male tactic is accompanied both by an increase in testicular androgen production and by a higher conversion of androgens into estrogens in the brain. The increase in androgen production is likely to mediate the development of male secondary sexual characters while the increase in brain AA may be related to the behavioral changes associated with tactic transition.

The effects of castration and sex steroid manipulations on the expression of sexual behavior were investigated in a small fish, the peacock blenny, Salaria pavo. In this species, large males defend nests and attract females while small "sneaker" males reproduce by imitating the female morphology and courtship behavior in order to approach nests during spawning events and parasitically fertilize eggs. Sneakers switch into nest holders in their second breeding season, thus displaying both male and female-like sexual behavior during their lifetime. We tested the effects of castration and of an aromatase inhibitor (Fadrozole, F), testosterone (T) or 17beta-estradiol (E(2)) implants on the expression of male and female-like behavior in sneakers. Sneakers were either sham-operated, castrated or castrated and implanted with vehicle, F, T+F or E(2)+F. Seven days after the treatment, sneakers were placed in a tank with a nesting male, two ripe females and an available nest. Castrated fish had lower levels of circulating T and increased the time spent displaying female typical nuptial coloration. T implants had the opposite effect, inhibiting the expression of female-like behavior and coloration. E(2) implants had no significant effect on the display of sexual behavior but the frequency of aggressive displays decreased. The results agree with previous findings in sneakers of S. pavo that demonstrated an inhibition of female-like behavior by 11-ketotestosterone (11-KT). The reported increase in T and 11-KT production when sneakers change into nest holders may thus contribute to behaviorally defeminize sneakers. Contrarily, both T and E(2) failed to promote male-like behavior, suggesting that behavioral masculization during tactic switching depends on other neuroendocrine mechanisms or that the time length of the experiment was insufficient to induce male-like behavioral changes in sneakers.

We tested whether subordinate helper males of the Lake Tanganyika cooperatively breeding cichlid Neolamprologus pulcher show elevated excretion levels of the stress hormone cortisol and reduced levels of 11-ketotestosterone and testosterone when living in groups with a small breeder male, compared to similar helper males living in groups with a large breeder male, in a full-factorial repeated measures experimental design. We also measured the same hormones in breeder males with and without helper males. Previous research showed that the size difference between large male helpers and male breeders in groups of this species influences behaviour and growth decisions. Contrary to our expectation, no effect of the size-difference between helper males and breeder males on helper hormone levels was detected. Furthermore, helper males had similar hormone excretion levels to those of size-matched breeder males without helpers, and to small breeder males. There was no influence of egg laying on breeder male and helper hormone levels during the experiment. Interestingly, all three hormone levels were significantly lower in helpers showing elevated levels of submissive behaviour towards the breeders, independently of the size of the breeder males. The low cortisol levels suggest that helper males can successfully reduce stress by appeasing breeder males through submission. Furthermore, helper males showing a high level of submissive behaviour had lower levels of androgens than less submissive helpers, suggesting a lower reproductive potential in submissive helpers. We propose that helper submission may be used as an honest signal of reduced interest in reproduction towards the breeder male in this species.

The occurrence of intra-sexual variation in reproduction is a widespread phenomenon in teleosts. One such form of variation consists in the occurrence of alternative male types: males that invest resources in mate attraction and males that exploit the investment of the former males, by trying to sneak fertilizations during spawning. These alternative reproductive tactics can be classified according to their plasticity during the life span of the individuals (i.e., fixed vs. sequential vs. reversible). Furthermore, the differences between morphs within a given species may involve a set of different traits, including reproductive behavior, the differentiation of male morphological traits, and the patterns of gonad tissue allocation and the differentiation of gonadal accessory glands. In this paper, we review the available data on four species exhibiting different types of intra-sexual plasticity in reproduction that have been studied in our lab. The data on the proximate mechanisms, androgens and forebrain arginine-vasotocin (AVT), underlying these alternative tactics suggest that between-morph differences in androgen levels, especially in 11-ketotestosterone, are especially present in species where the alternative male types have evolved morphological traits that are tactic-specific (i.e., sexual polymorphisms) and that differences in AVT appear to be related to between-morph differences in the expression of courtship behavior. Therefore, this comparative approach leads us to propose that the different endocrine systems are involved in the differentiation of different sets of traits that make up alternative phenotypes, and that the differentiation of alternative tactics is not controlled by a single endocrine system (e.g., androgens).

The Challenge Hypothesis postulates that androgen levels are a function of the social environment in which the individual is living. Thus, it is predicted that in polygynous males that engage in social interactions, androgen levels should be higher than in monogamous animals that engage in parental care. In this study, we tested this hypothesis at the intra-specific level using a teleost species, Sarotherodon galilaeus, which exhibits a wide variation in its mating system. Experimental groups of individually marked fish were formed in large ponds with different operational sex-ratios (OSR) to study the effects of partner availability on blood plasma levels of sex steroids [11-ketotestosterone (11-KT), testosterone (T), and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)] and gonadosomatic index (GSI). Polygyny mostly occurred in the female biased OSR groups. 17,20beta-P and gonadosomatic index did not differ among OSR groups. However, 11-KT was high in male biased OSR and positively correlated with aggressive challenges, thereby supporting the central postulate of the Challenge Hypothesis. The results of T were the inverse of those of 11-KT, probably because 11-KT is metabolized from T. 11-KT levels of polygynous males did not differ neither from those of monogamous males, nor from those of males that participated in parental care. These results do not support the expected relationships between polygyny, parental care, and androgen levels. The differences from expectations for 11-KT may be related to the fact that in S. galilaeus, the mating and the parenting phase are not clearly separated and thus, males may still fight and court while they are brooding.

In the common Azorean rock-pool blenny, Parablennius parvicornis, males exhibit alternative reproductive morphologies: (1) larger males defend nest sites, provide parental care, have anal glands (involved in pheromone release), testicular glands, and low gonad:body weight ratio (GSI) and (2) smaller, younger, males do not defend nests, have reduced glands and high GSI. These smaller non-nesting males behave as satellites (associated with nests) or sneakers (moving among nests), attempting to achieve parasitic fertilizations via sperm competition. In non-mammals, arginine vasotocin (AVT) is a key hypothalamic peptide involved in the control of reproductive behavior and physiology, and several fish species that exhibit alternative male reproductive morphs show polymorphism in AVT brain chemistry. We conducted an immunocytochemical study to generate comparative data on this intertidal blenny. Our analysis showed no difference in AVT-immunoreactive cell number or size between the male morphs, which is consistent with studies on other fish, including blennies. The number of AVT cells was positively correlated to fish body mass, while cell size showed no such relation. If corrected for body mass, the smaller non-nesting males have significantly more cells than the large nesting males. Our data suggest that the size and number of forebrain AVT cells develops initially to allow for reproduction in the young non-nesting males and this pattern does not appear to change when males take on the nesting morphotype later in life. This result appears to be consistent in many fishes with alternative male morphotypes.

Male androgen responses to social challenges have been predicted to vary with mating system, male-male aggressiveness, and the degree of paternal investment in birds ("challenge hypothesis," Am. Nat. 136 (1990), 829). This study focused on the interspecific predictions of the challenge hypothesis. Comparative methods were used to control for effects of the phylogenetic relatedness among the sampled species. Male androgen data of 84 bird species were collected from literature records on seasonal androgen patterns. From these, the androgen responsiveness (AR) was calculated as described in the original challenge hypothesis (i.e., maximum physiological level/breeding baseline). Scatterplots of AR versus mating strategy, male-male aggressiveness, and the degree of paternal care confirmed the expected interspecific patterns. When phylogenetic analyses were performed among all of the sampled species, the effects of paternal investment disappeared while the AR remained covarying to a high degree with mating system and male-male aggressiveness. Although these mechanisms may be different at the intraspecific level, this suggests that interspecific differences of AR in male birds may have evolved in response to changes of mating strategies, rather than in response to altered paternal duties. However, control for phylogeny among the subsample of 32 passerine species revealed that if any paternal investment contributed to the observed variance in AR, then the change from "no male incubation" to "male shares incubation duties" represented the most effective, whereas the male's contribution to feeding offspring did not explain the observed variation of AR.