Fighting cichlids: An integrated multimodal analysis to understand female and male aggression in Cichlasoma dimerus.

Aggression has been historically linked to males and androgen levels and, even if females from different species also display aggressive behavior, female aggression is still widely understudied. The aim of the present work is to disentangle how sex differences in social plasticity can be explained by sex steroid hormone levels, gonadal state and/or morphometric characteristics. In this context, we performed intrasexual dyadic encounters to identify social plasticity after acquiring a winner or loser status in males and females of Cichlasoma dimerus. This integral analysis suggests that the reproductive and hormonal variables analyzed explain the behavioral variation among winner and loser males and females, and that there are significant differences between sexes and contest outcome when individual morphometric variables are excluded from the analysis. Interestingly, there are no sex differences in aggressive and submissive behaviors, and clustering into winners and losers is mainly explained by specific behavioral displays, such as bites, chases, approaches, passive copings, and escapes. Correlation heatmaps show a positive correlation between estradiol with aggression and a negative correlation with submission, suggesting estrogens may have a dual role regulating agonistic behavior. Finally, these results suggest that size difference can help to understand aggression in females but not in males, and that assessment of the opponent's body size is important to understand aggression also before the initiation of the contest in both sexes. Overall, this study constitutes an integral approach adding insights into the importance of reproductive and hormonal variables to understand social plasticity in males and females.

Social control of spermatogenesis and steroidogenesis in cichlid fish: a comparative approach.

Social animals with hierarchical dominance systems are susceptible to changes their environment. Interactions with conspecifics can greatly affect individual's behavior and reproductive success. This review will show how social behavior modulates gonadal steroidogenesis and spermatogenesis in African and Neotropical cichlid fish with different social systems and how this modulation regulates reproductive capacity. Social behavior and aggressiveness are strongly linked to sex steroids, glucocorticoids and neuropeptides. The challenge hypothesis suggests that behavioral interactions increase androgen levels in response to social instability, but there is little evidence regarding estradiol levels. It has been recently demonstrated that in male Cichlasoma dimerus, a Neotropical cichlid fish, the challenge hypothesis could also be extended to estrogens. In C. dimerus, dominant males have higher gonadosomatic index than subordinated; the percentage of spermatocytes and spermatids is higher in subordinates, while dominants show a greater percentage of spermatozoa. In other species of African cichlids, socially suppressed subordinate males are not reproductively incompetent maintaining some activity at every level of their reproductive axis. Axis reactivation upon social ascent is similar to the initiation of puberty in mammals, as well as the reoccurrence of puberty observed in seasonally breeding animals. In conclusion, social behavior and reproductive strategies in females cichlids are still understudied, and Neotropical cichlids still constitute a group that deserves more attention, considering cichlids' diversity in mating systems, reproductive behavior and parental care. This review highlights the importance of performing further studies and additional research in these two areas, which still remain to be addressed.

Effect of previous fighting on the dynamic of agonistic encounters in zebrafish males.

Aggression among individuals which compete for resources such as food or territory, or to establish dominance relationships, can cause injuries that may be risky for the contenders. In this way, individuals of many species have strategies to resolve conflicts reducing levels of aggression. Thus, if individuals are able to recognize each other and remember previous agonistic experiences and the result of the fight, they will resolve the subsequent encounter with lower levels of aggression. Here, we evaluated the effect of previous fighting experiences (24 h interval) on agonistic behaviors of subsequent encounters in zebrafish (Danio rerio) males. Specifically, we examined if any reduction in aggression is because of an individual's ability to remember other individuals from previous interactions, or if reductions come directly from winning or losing a fight. We found that when a pair of opponents (of the same size) had fought, and 24 h later the same dyad fought again, the number of bites decreases 85% and the duration of aggression decreases 73% in the second encounter, and this effect persisted in a third encounter (a decrease of 90% and 95%, respectively). To evaluate if the effect of previous experience on agonistic behaviors depended on facing the same opponent, in the second fight of a new experiment the opponent was changed. In this case, no decreases in the number of bites or in the duration of aggression was observed. In conclusion, pairs of zebrafish males resolve a conflict with lower levels of aggression when having previously fought with that particular opponent, but not with an unknown opponent. As a whole, these results suggest that zebrafish males are capable of recognizing the opponent, remembering previous experiences and changing their fighting strategies accordingly, but not only as a consequence of the result of a previous encounter.

Vasotocinergic control of agonistic behavior told by Neotropical fishes.

The hypothalamic neuropeptides of the vasopressin-oxytocin family (and their homologs for non-mammalian species) are key modulators of the Social Brain Network, acting via specific receptors reported in all the nuclei of this network. Different conclusive examples have proven the context-dependency actions of hypothalamic nonapeptides on social behavior in several vertebrate taxa. Teleost fishes provide endless possibilities of experimental model systems to explore the underlying mechanisms of nonapeptide actions on social behavior given that they are the most diverse group of vertebrates. Although it has been difficult to identify commonalities of nonapeptide actions across species, indisputable evidence in many teleost species have demonstrated a clear role of vasotocin in the modulation of aggressive and sexual behaviors. Though Neotropical South American fish contribute an important percentage of teleost diversity, most native species remain unexplored as model systems for the study of the neuroendocrine bases of social behavior. In this review, we will revise recent data on the two model systems of Neotropical fish, South American cichlids and weakly electric fish that have contributed to this issue.

Characterization of the gonadotropin-releasing hormone system in the Neotropical teleost, Steindachneridion parahybae during the annual reproductive cycle in captivity.

This study evaluated by immunohistochemical and Western blot methods, the distribution of two distinct gonadotropin-releasing hormones (GnRHs), corresponding to catfish GnRH (cfGnRH or GnRH1) and chicken-II GnRH (cGnRH-II or GnRH2), in Steindachneridion parahybae females in captivity, focusing these analyses on the reproductive cycle by semi-quantification of optical density (OD). Further, we found that the GnRH neuronal systems co-localized with their respective GnRH-associated peptides (GAPs). A group of neurons immunoreactive (ir) to GnRH1 were identified along the ventral region of the olfactory bulb (vOB) in the telencephalon (vTel) and in the main areas of the diencephalon (especially the medial basal hypothalamus, HBM), including fibers extending into the pituitary gland. In contrast, GnRH2 neurons were confined to the midbrain tegmentum, close to the ventricular surface, without projections to the pituitary gland. Moreover, a cfGAP (GnRH1)-specific band (9 kDa) was identified in the brain and pituitary gland, while a cGAP-II (GnRH2)-specific band (26 kDa) was observed only in the brain extract. During the reproductive cycle, GnRH1-ir presented greater OD values at the vitellogenic and regression stages than at the previtellogenic stage and after artificially induced to spawn. Larger GnRH2-ir neurons were observed during the reproductive cycle, but a higher OD was identified only in the regression stage compared with the other maturation stages. Finally, GnRH1 axons were found to be directed towards the pituitary, and this GnRH type, which is probably the hypophysiotropic form, can contribute to the reproductive dysfunction that occurs in S. parahybae females in captivity, whereas GnRH2 may act as a neuromodulator and/or neurotransmitter.

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.

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.

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.

Reproductive and parental care physiology of Cichlasoma dimerus males.

The South American cichlid fish Cichlasoma dimerus presents a high breeding frequency and biparental care of the eggs and larvae. The male parental care period was divided in four different phases according to the developmental degree of the offspring: pre-spawning activity (MP, day 0), guarding eggs (ME, one day after fertilization (1 DAF)), guarding hatched larvae (MHL, 3 DAF), and guarding swimming larvae (MSL, 8 DAF). The aim of this study was to characterize male reproductive physiology by measuring steroid hormone plasma levels and analyzing testes cellular composition. Males exhibiting pre-spawning activity showed 8.4 times higher 11-ketotestosterone and 5.63 times higher testosterone levels than MHL. No differences were observed in estradiol and cortisol levels among the different phases. The cellular composition of the testes varied during the reproductive and parental care periods. Testes of MP were composed of 50% of spermatozoa, whereas spermatogonia type B and spermatocytes were predominant in the subsequent parental phases. A morphometric analysis of Leydig cells nuclear area revealed that MP and ME's Leydig cells averaged 1.27 times larger than that those of MHL and MSL and was positively correlated with circulating 11-KT and T levels. Hence, C. dimerus males showed important changes in its hormonal profiles and testicular cellular composition throughout the reproductive and parental care period.

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.

Endosulfan affects GnRH cells in sexually differentiated juveniles of the perciform Cichlasoma dimerus.

Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxicity towards non-target organisms such as fish. It has been demonstrated that ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the alterations produced by this pesticide on the reproductive axis of fish prior to sexual maturity, as well as possible modes of action have hardly been studied. This study aimed at assessing the effect of waterborne exposure to the pesticide ES on the reproductive axis during sexual differentiation of juveniles of the South American freshwater cichlid fish Cichlasoma dimerus. No mortality was observed due to ES subchronic exposure (90 days post-fertilization). Exposure to ES did not affect body weight nor morphometric parameters, indicating that larvae nutritional state was not affected. Timing of sexual differentiation, gonadal morphology and sex ratio were likewise not altered by ES. However, ES acted as an endocrine disrupting chemical in this species as the morphometry of gonadotropin-releasing hormones (GnRH) producing cells was altered. Exposure to ES altered nuclear area, cell area and nucleus/cytoplasm ratio of GnRH II neurons, and cell and nuclear area and diameter of GnRH III neurons. Interestingly, in our previous study, exposure before sex differentiation (30 day exposure) caused no alteration to GnRH II and III, and did alter GnRH I and FSH cells. These alterations could lead to changes in circulating hormone levels, especially when fish are exposed for prolonged periods, ultimately impairing reproductive fitness. C. dimerus juveniles can be an interesting biological model to perform toxicological studies with the intent to assess early disruption endpoints in the reproductive axis during development.

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.

Ultrastructural and immunohistochemical characteristics of the olfactory organ cardinal tetra, Paracheirodon axelrodi (Characiformes: Characidae).

The cardinal tetra Paracheirodon axelrodi belongs to the family Characidae, an economically important and morphologically diverse family of fishes. Information on the olfactory system of this species is scattered and scarce. Among teleost fishes, differences exist in the shape, number, and arrangement of the olfactory lamellae, in the distribution of the sensory and nonsensory epithelium, as well as in the abundance of various receptor cell types. Here, an anatomical and morphological description of the olfactory system was carried out using light microscopic histology, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. P. axelrodi is a ditremous and isosmat species. It has an arrow-shaped olfactory rosette arrangement. The olfactory epithelium is covering the 12-14 lamellae of the olfactory rosette and, using scanning electron microscopy, we observed that the apical surface of the olfactory epithelium carries a dense layer of mucus. Based on the histological, immunohistochemical, and ultrastructural descriptions, all characteristic sensory and nonsensory cell types of the olfactory epithelium of teleost fish were identified. Three types of olfactory receptor neurons were identified: ciliated, microvilli, and crypt cells. The distribution of sensory and nonsensory cell types is like that described in Aphyocharax anisitsi, another species of the Characidae family. A. anisitsi inhabits slow-flowing water bodies with high-density vegetation such as P. axelrodi.

Effect of the organochlorine pesticide endosulfan on GnRH and gonadotrope cell populations in fish larvae.

Endocrine-disrupting chemicals can influence the hypothalamus-pituitary-gonad axis and possibly affect reproduction in vertebrates. We analyzed the effect of 30-day endosulfan (ES) exposure in sexually undifferentiated larvae of the cichlid fish Cichlasoma dimerus. The number, area, mean cytoplasmic and nuclear diameter, and mean cytoplasmic optical density of gonadotropin-releasing hormone (GnRH) I, II, and III immunoreactive (ir-) neurons and ß follicle-stimulating hormone (ßFSH) ir-cells were measured. Animals exposed to the highest ES concentration (0.1 µg/l) showed a decrease in GnRH I nucleus/cytoplasm area ratio upon exposure. Nuclear area and mean nuclear diameter of ßFSH ir-cells was higher in ES treated fish. ßFSH nucleus/cytoplasm area ratio was high in exposed animals, and animals exposed to 0.1 µg/l ES showed smaller mean cytoplasmic optical density. These findings suggest that ES affects GnRH I and ßFSH protein synthesis/release. However, these responses seem to be insufficient to affect gonadal differentiation at this stage of development.

Sexual Dimorphism in Aggression: Sex-Specific Fighting Strategies Across Species.

Aggressive behavior is thought to have evolved as a strategy for gaining access to resources such as territory, food, and potential mates. Across species, secondary sexual characteristics such as competitive aggression and territoriality are considered male-specific behaviors. However, although female-female aggression is often a behavior that is displayed almost exclusively to protect the offspring, multiple examples of female-female competitive aggression have been reported in both invertebrate and vertebrate species. Moreover, cases of intersexual aggression have been observed in a variety of species. Genetically tractable model systems such as mice, zebrafish, and fruit flies have proven extremely valuable for studying the underlying neuronal circuitry and the genetic architecture of aggressive behavior under laboratory conditions. However, most studies lack ethological or ecological perspectives and the behavioral patterns available are limited. The goal of this review is to discuss each of these forms of aggression, male intrasexual aggression, intersexual aggression and female intrasexual aggression in the context of the most common genetic animal models and discuss examples of these behaviors in other species.

Effects of intraspecific chemical cues on the behaviour of the bloodfin tetra Aphyocharax anisitsi (Ostariophysi: Characidae).

Chemical communication can induce a multitude of behaviours when detected by fish olfactory systems, from parental care, predation and alarm signalling, to foraging, schooling, reproduction, and migration. Chemical cues provide information that visual traits cannot and fish can respond to chemical cues without any additional sensory cue. In this way, pheromones play an essential role in the fitness of fishes. Given that Aphyocharax anisitsi inhabits environments characterized by cloudy and highly vegetated waters, it is interesting to evaluate the olfactory contribution in their communication. Here, we investigated the relevance of chemical cues in the types of behaviours triggered in A anisitsi by two experimental contexts: 1) non-social and olfactory context (conspecific-chemical cues), and 2) social context (conspecific female or male presence). Non-social context experiments suggest that males of A. anisitsi respond to both male and female-chemical cues even in the absence of other sensory inputs. The high olfactory sensitivity of characids in general and of A. anisitsi, in particular, could facilitate vital functions, such as foraging and conspecific recognition in habitats that impose severe restrictions on the visual system.

Evidence to suggest glutamic acid involvement in Bisphenol A effect at the hypothalamic level in prepubertal male rats.

OBJECTIVES: The aim of present paper was to study the probable role of glutamic acid (GLU) as a mediator of bisphenol A (BPA) effect at the hypothalamic level and its effects on the reproductive axis of prepubertal male rats. METHODS: Mated Wistar rats were treated with either 0.1% ethanol (control group, n=10) or BPA (BPA group, n=10) in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight/day of BPA. At the prepubertal stage (35 days of age), the male rats were sacrificed and Gn-RH and glutamic acid (GLU) release, an amino acid involved in Gn-RH secretion, were measured in hypothalamic samples containing medio basal and anterior preoptic area (MBH-APOA), by RIA and HPLC respectively. LH, FSH serum levels were measured by RIA and testosterone by EQLIA. RESULTS: Gn-RH and GLU release decreased significantly in animals exposed to BPA (p<0.001, p<0.01). LH, FSH and testosterone serum levels were also decreased by treatment (p<0.0001). CONCLUSION: Present results provide evidence that BPA may act at the hypothalamic level to decrease GLU release which in turn may modify Gn-RH secretion altering the normal function of the axis.

Morphology and immunohistochemistry of the olfactory organ in the bloodfin tetra, Aphyocharax anisitsi (Ostariophysi: Characidae).

Among teleost fishes, differences exist in the shape, number, and arrangement of the olfactory lamellae, the distribution of the sensory and non-sensory epithelium, as well as, the abundance of various receptor cells. The objective of this work was to describe the morphology, immunohistochemistry, and scanning electron microscopy ultrastructure of the olfactory epithelium of the bloodfin tetra, Aphyocharax anisitsi. This is the first complete description including the anatomy, histology, and immunohistochemistry of the peripheral olfactory organ from a Characiformes. Based on the external morphology of the olfactory organ, A. anisitsi was classified as a ditermous species, with an olfactory cavity containing two openings divided by a skin flap that separates the anterior and posterior nostril. This species belongs to the group of isosmates, since the presence of accessory olfactory sacs was not observed, and non-sensory ciliated cells were identified. A. anisitsi has an olfactory rosette with an arrow-shaped arrangement, with differences in length between the anterior and posterior lamellae. In the olfactory epithelium, three types of olfactory receptor neurons were identified using histology and confirmed by immunohistochemistry, that is, ciliated olfactory receptor neurons in the basal region of the epithelium, microvillar olfactory receptor neurons in the middle region; and Crypt cells, in smaller numbers compared to the other neuronal types, present in the apical region. Sensory and non-sensory areas were scattered and mixed along the lamellar lateral surface but the nasal cavity and the midline raphe lacked olfactory receptor neurons. The presence of abundant kinocilia in the non-sensory cells could be related in A. anisitsi with ventilation and quality control of water entering the olfactory cavity. The spatial organization of the sensory and non-sensory areas in A. anisitsi was similar to that observed in other species that also inhabit still and slow-flowing bodies of water with high-density vegetation.

Presence of beta-follicle-stimulating hormone and beta-luteinizing hormone transcripts in the brain of Cichlasoma dimerus (Perciformes: Cichlidae): effect of brain-derived gonadotropins on pituitary hormone release.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for beta-FSH and beta-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary beta-LH, beta-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in beta-LH release, dose-dependent increases in beta-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.