Can estrogens be considered as key elements of the challenge hypothesis? The case of intrasexual aggression in a cichlid fish.

Territorial aggression has been widely studied in males and it has been historically suggested that androgens are key mediators of this behavior. However, more recent evidence suggests that it is the aromatization to estrogens, rather than androgens themselves, that is key to regulating this behavior. Females also display aggressive behaviors, but the physiological regulation of female aggression is still understudied when compared to males. In this context, the challenge hypothesis postulates that male-male aggressive interactions stimulate the production of androgens in males in periods of social instability. Here we determine plasma sex steroid levels in Cichlasoma dimerus to assess whether estrogens are related to aggressive behavior and to test the challenge hypothesis in both males and females. We set-up challenge trials as intrasexual dyadic encounters and determined androgen and estrogen levels before and after the trial in both winners and losers. Even though there were no differences in initial estradiol-17ß plasma levels between male winners and losers, initial levels were higher (p = .046) in female winners than in losers, while there were no differences in testosterone or 11-ketotestosterone levels. After trials, both males and females showed elevated levels of estradiol-17ß and both androgens, but only males exhibited a significant 1.45, 5.42 and 3.2-fold increase in estradiol-17ß, testosterone and 11-ketotestosterone, respectively (p = .023, p = .016, p = .018). Moreover, changes in circulating levels of estradiol-17ß in females after the trials do not depend on their reproductive status or on the outcome of the contest. We suggest that female aggression is associated with initial levels of estradiol-17ß, and that the challenge hypothesis, originally defined for androgens, could also be extended to estrogens.

Fighting cichlids: Dynamic of intrasexual aggression in dyadic agonistic encounters.

Aggression is an extremely complex behaviour and female aggression is understudied when compared to males. Despite the fact that it has been suggested that conflict among females may be more frequently resolved peacefully, in many species females show high levels of aggression. We used Cichlasoma dimerus to describe dynamics and conflict outcome in intrasexual agonistic encounters. We performed encounters of two sex-matched animals in a neutral arena and we recorded agonistic interactions during one hour. All aggressive and submissive behaviours were described and quantified to perform the ethogram. Encounters followed three phases: pre-contest, contest and post-resolution. Latency, time of resolution and frequency of aggressive displays did not differ between sexes. Relative variations in size between female opponents better explained aggression outcome in each contest, since higher levels of aggression occurred in dyads of more similar fish. However, this was not observed in males, suggesting that probably morphological characteristics could be less relevant in male conflict resolution. Altogether these results suggest that in this ethological context, C. dimerus females are as aggressive as males and that they have similar motivation towards territorial aggression, emphasizing the need of deepening the study of aggression in females and not only in males.

A game of two? Gene expression analysis of brain (cyp19a1b) and gonadal (cyp19a1a) aromatase in females of a Neotropical cichlid fish through the parental care period and removal of the offspring.

For many species parental behavior is essential for the survival of the offspring. While the ultimate causes of teleost parental behavior have been widely studied, comparatively little is known about its proximate causes. The aim of this study was to analyze the yet unexplored, potential dual role of brain and gonadal aromatases, the enzymes responsible for the conversion of androgens to estrogens in the brains and gonads of teleosts, respectively, on the different stages of the maternal care period of the biparental cichlid Cichlasoma dimerus, locally known as chanchita. By immunohistochemistry we analyzed the neural distribution of brain aromatase and observed it exclusively within the forebrain, including areas involved in the regulation of parental behavior. We next analyzed the gene expression of brain aromatase in the brain, and gonadal aromatase in the ovary, of female chanchitas through the parental care period. To further characterize the physiological environment associated to maternal care, we also evaluated sex steroid levels (17ß-estradiol, testosterone and 11-ketotestoterone) and ovarian follicle percentage. The onset of parental behavior specifically downregulated sex steroids synthesis and the rate of ovarian maturation, as denoted by a more than 10-fold decrease in steroid levels and delayed detection of mature follicles in females with offspring, compared to females which eggs were removed. Gene expression levels of both aromatases were independent of maternal care at the evaluated time points, even though they varied during the parental care period.

From molecule to behavior: Brain aromatase (cyp19a1b) characterization, expression analysis and its relation with social status and male agonistic behavior in a Neotropical cichlid fish.

The enzyme aromatase, responsible for the conversion of C19 androgens to C18 estrogens, exists as two paralogue copies in teleost fish: Cyp19a1a mostly expressed in the gonads, referred as gonadal aromatase, and Cyp19a1b, mostly expressed in the brain, accordingly known as brain aromatase. The neural localization of Cyp19a1b is greatly contained within the social behavior network and mesolimbic reward system in fish, suggesting a strong role of estrogen synthesis in the regulation of social behavior. In this work we aimed to analyze the variation in cyp19a1b expression in brain and pituitary of males of a highly social cichlid, Cichlasoma dimerus (locally known as chanchita), and its relation with inter-individual variability in agonistic behavior in a communal social environment. We first characterized chanchita's cyp19a1b mRNA and deduced amino acid sequence, which showed a high degree of conservation when compared to other teleost brain aromatase sequences, and its tissue expression patterns. Within the brain, Cyp19a1b was solely detected at putative radial glial cells of the forebrain, close to the brain ventricles. We then studied the relative expression levels of cyp19a1b by Real Time PCR in the brain and pituitary of males of different social status, territorial vs. non-territorial, and its relationship with an index of agonistic behavior. We found that even though, brain aromatase expression did not differ between types of males, pituitary cyp19a1b expression levels positively correlated with the index of agonistic behavior. This suggests a novel role of the pituitary in the regulation of social behavior by local estrogen synthesis.

The pineal complex: a morphological and immunohistochemical comparison between a tropical (Paracheirodon axelrodi) and a subtropical (Aphyocharax anisitsi) characid species.

Cardinal neon Paracheirodon axelrodi and bloodfin tetra Aphyocharax anisitsi are two species of characids with high trade value as ornamental fish in South America. Although both species inhabit middle water layers, cardinal neon exhibits a tropical distribution and bloodfin tetra a subtropical one. In this work, we carried out an anatomical, histological and immunohistochemical study of the pineal complex of P. axelrodi and A. anisitsi. In both species, the pineal complex consisted of three components, the pineal and parapineal organs and the dorsal sac (DS). The pineal organ was composed of a short, thin pineal stalk (PS), vertically disposed with respect to the upper surface of the telencephalon, and a pineal vesicle (PV), located at the distal end of the PS and attached to the skull by connective tissue. The pineal window (PW), a site in the skull where the luminal information accesses the pineal organ, appeared just above the latter structures. In the epidermis of P. axelrodi's PW, club cells were identified, but were not observed in the epidermis of A. anisitsi's one. With respect to the DS, it appeared to be folded on itself, and was bigger and more folded in A. anisitsi than in P. axelrodi. Immunohistochemical assays revealed the presence of cone opsin-like and rod opsin-like photoreceptor cells in the PS and PV. These results provide a first insight into the morphological assembly of the pineal complex of both species, and contribute to a better understanding of the integration and transduction of light stimuli in characids. J. Morphol. 277:1355-1367, 2016. © 2016 Wiley Periodicals, Inc.

Serotonergic outcome, stress and sexual steroid hormones, and growth in a South American cichlid fish fed with an L-tryptophan enriched diet.

Reared animals for edible or ornamental purposes are frequently exposed to high aggression and stressful situations. These factors generally arise from conspecifics in densely breeding conditions. In vertebrates, serotonin (5-HT) has been postulated as a key neuromodulator and neurotransmitter involved in aggression and stress. The essential amino acid L-tryptophan (trp) is crucial for the synthesis of 5-HT, and so, leaves a gateway for indirectly augmenting brain 5-HT levels by means of a trp-enriched diet. The cichlid fish Cichlasoma dimerus, locally known as chanchita, is an autochthonous, potentially ornamental species and a fruitful laboratory model which behavior and reproduction has been studied over the last 15years. It presents complex social hierarchies, and great asymmetries between subordinate and dominant animals in respect to aggression, stress, and reproductive chance. The first aim of this work was to perform a morphological description of chanchita's brain serotonergic system, in both males and females. Then, we evaluated the effects of a trp-supplemented diet, given during 4weeks, on brain serotonergic activity, stress and sexual steroid hormones, and growth in isolated specimens. Results showed that chanchita's brain serotonergic system is composed of several populations of neurons located in three main areas: pretectum, hypothalamus and raphe, with no clear differences between males and females at a morphological level. Animals fed with trp-enriched diets exhibited higher forebrain serotonergic activity and a significant reduction in their relative cortisol levels, with no effects on sexual steroid plasma levels or growth parameters. Thus, this study points to food trp enrichment as a "neurodietary'' method for elevating brain serotonergic activity and decreasing stress, without affecting growth or sex steroid hormone levels.

A multidisciplinary study on social status and the relationship between inter-individual variation in hormone levels and agonistic behavior in a Neotropical cichlid fish.

Social animals with hierarchal dominance systems are particularly susceptible to their social environment. There, interactions with conspecifics and hierarchal position can greatly affect an individual's behavior, physiology and reproductive success. Our experimental model, Cichlasoma dimerus, is a serially-monogamous Neotropical cichlid fish with a hierarchical social system, established and sustained through agonistic interactions. In this work, we aimed to describe C. dimerus social structure and its association with hormonal profiles and testicular cellular composition. We recorded and quantified agonistic interactions from the territorial pair, i.e. the top ranked male and female, and the lowest ranked male of stable social groups. Plasma levels of 11-ketotestosterone (11-KT), testosterone, 17ß-estradiol (E2) and cortisol were measured by ELISA. Results show that territorial pairs cooperatively guarded the territory, but rarely attacked in synchrony. Territorial males had higher testosterone and 11-KT plasma levels than non-territorial males, while E2 and an index of its metabolization from testosterone were higher in non-territorial males. No difference was observed in cortisol levels. Plasma 11-KT and an index of the conversion of testosterone to 11-KT, positively correlated with the frequency of aggressiveness, while E2 showed the opposite pattern. Territorial males had a higher gonadosomatic index than non-territorial males. The quantification of testicular cellular types revealed that the percentage of spermatocytes and spermatids was higher in non-territorial males, while territorial males showed a greater percentage of spermatozoa. Thus, C. dimerus male social position within a stable hierarchy is associated with distinct behaviors, steroid levels and testicular degree of development.

The endocrine regulation of cichlids social and reproductive behavior through the eyes of the chanchita, Cichlasoma dimerus (Percomorpha; Cichlidae).

Sociobiology, the study of social behavior, calls for a laboratory model with specific requirements. Among the most obvious is the execution of social interactions that need to be readily observable, quantifiable and analyzable. If, in turn, one focuses on the neuroendocrinological basis of social behavior, restrictions grow even tighter. A good laboratory model should then allow easy access to its neurological and endocrine components and processes. During the last years, we have been studying the physiological foundation of social behavior on what we believe fits all the aforementioned requirements: the so called "chanchita", Cichlasoma dimerus. This Neotropical cichlid fish exhibits biparental care of the eggs and larvae and presents a hierarchical social system, established and sustained through agonistic interactions. The aim of the current article is to review new evidence on chanchita's social and reproductive behavior.

The interrenal gland in males of the cichlid fish Cichlasoma dimerus: relationship with stress and the establishment of social hierarchies.

In teleosts, cortisol is the primary glucocorticoid secreted by the steroidogenic cells of the interrenal gland and an increase in its plasma concentration is a frequent indicator of stress. Cortisol has been postulated as an endogenous mediator involved in the regulation of reproduction and aggression related to social dynamics. The cichlid fish Cichlasoma dimerus, is a monogamous species that exhibits complex social hierarchies; males appear in one of two basic alternative phenotypes: non-territorial and territorial males. In this work, we postulated as a general hypothesis that the morphometry of the interrenal gland cells and the plasma levels of cortisol and 11-ketotestosterone (11-KT) are related to the social rank in adult males of C. dimerus. First, the location and distribution of the interrenal gland with respect to its context - the kidney - was studied. Plasma levels of cortisol and 11-KT in territorial and non-territorial males were established by ELISA. Finally, a morphometric analysis of steroidogenic and chromaffin cells of the interrenal gland was performed. Results showed that the interrenal gland was exclusively located in the posterior portion of the cephalic kidney. Non-territorial males presented a greater nuclear area of their steroidogenic cells. Additionally, plasma cortisol and 11-KT levels were lower and higher, respectively, in territorial males. Finally, plasma cortisol levels positively correlated with the nuclear area of interrenal steroidogenic cells. Thus, the interrenal gland, by means of one of its products, cortisol, may be fulfilling an important role in the establishment of social hierarchies and their stability.

Arginine-vasotocin expression and participation in reproduction and social behavior in males of the cichlid fish Cichlasoma dimerus.

In non-mammalian vertebrates, the nonapeptide arginine-vasotocin (AVT) is involved in the regulation of social behavior related to reproduction and aggression. The cichlid fish Cichlasoma dimerus is a monogamous species with complex social hierarchies. Males are found in one of two basic alternative phenotypes: Non-territorial and territorial males. In this work we characterize the vasotocinergic system in males of C. dimerus in relation to social status with particular emphasis on the various putative sites of action of AVT across the hypothalamic-pituitary-gonad (HPG) axis, and its effects on reproductive and social behavior. The location and distribution of vasotocinergic neurons in the brain was studied, highlighting a morphometric analysis of AVT producing neurons in males of different social status. The effect of AVT on pituitary gonadotropin secretion was analyzed by single pituitary culture while expression of AVT in peripheral organs was studied by RT-PCR using specific primers. Finally, the role of AVT on testicular androgen release was assessed by in vitro incubation of testis. Results showed a positive effect of AVT on gonadotropin secretion, where ß-LH showcased a triphasic response under increasing AVT concentration, while ß-FSH's response was dose-dependent and directly proportional. AVT showed a positive and concentration-dependent effect over testicular androgens synthesis and secretion in vitro. Vasotocin expression was observed in testicular somatic tissue located in the interstitial compartment. Thus, the AVT system in C. dimerus appears to be of high complexity, with multiple sites of action in the hypothalamus-pituitary-gonadal axis.