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.

Cloning, phylogenetic analysis and expression of somatolactin and its receptor in Cichlasoma dimerus: their role in long-term background color acclimation.

Somatolactin (SL) and SL receptor (SLR) belong to the growth hormone and cytokine type I receptor superfamilies, respectively. However, further research is required to define the duplications and functions of SL and its receptors in basal vertebrates including environmental background color adaptation in fish. In the present study, we cloned and sequenced SL and its putative receptor (SLR), classified and compared the sequences phylogenetically, and determined SL and SLR mRNA expression levels during long-term background color exposure in Cichlasoma dimerus, a freshwater South American cichlid. Our results show that C. dimerus SL and SLR share high sequence similarity with homologous from other perciform fish. Phylogenetic analysis indicates that C. dimerus SL belongs to the SLα clade sub-group. C. dimerus SLR is clearly a member of the GHR1 receptor subgroup, which includes the experimentally validated SLR from salmonids. Higher transcript levels of SLα in the pituitary and SLR in the epidermis and dermis cells of fish scales were observed in fish following long-term black background color exposure compared to those exposed to a white background. A higher number of melanophores was also observed in fish exposed for 10days to a black background compared to those exposed to a white background. These changes were concomitant to differences in SL or SLR transcript levels found in fish exposed to these two different background colors. Our results suggest, for the first time, that SLR is expressed in fish scales, and that there is an increase in SL in the pituitary and the putative SLR in likely target cells, i.e., melanophores, in long-term black background exposure in C. dimerus.

Social and reproductive physiology and behavior of the Neotropical cichlid fish Cichlasoma dimerus under laboratory conditions

In this work we describe for the first time the social and reproductive behavior of the Neotropical fish Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endemic to the Paraná River basin, using a comprehensive-integral approach, including morphological and physiological features. This substrate breeding fish has biparental care of the fry and presents a dominance hierarchy that determines access to breeding territories among males, and to males with territories among females. Gregarious behavior associated with a pale body color, was observed before reproductive behaviors started. Afterwards, a dominance hierarchy was established through aggressive interactions. Territorial individuals had bright body color patterns and non territorial an opaque grey one. Black ventral coloration was associated with reproductive individuals. Courtship displays, which were similar to threatening displays, had the common effect of increasing the visible area of the individual. The dominant male was always the largest one suggesting that size is probably a major factor determining the hierarchy establishment and that these intra-sexually selected traits may have been reinforced by inter-sexual selection. Reproductive males had higher pituitary levels of β-follicle stimulating hormone (β-FSH) and somatolactin (SL) than non reproductive ones, while no differences were found among females. No differences were found among male gonadosomatic indexes. Non reproductive individuals had higher plasma cortisol levels for both sexes. It is possible that dominant reproductive individuals may be inhibiting reproduction of subordinate fish through physical contact, increasing their cortisol levels and diminishing FSH and SL pituitary content. However, this was not reflected as an inhibition at the gonadal level in our experimental design.

En este trabajo se describen por primera vez el comportamiento social y reproductivo del pez cíclido neotropical Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endémico de la cuenca del Paraná, desde un enfoque integral y abarcador, incluyendo características morfológicas y fisiológicas. Éste pez incubador de substrato, tiene cuidado biparental de las crías y presenta una jerarquía de dominancia que determina el acceso a territorios reproductivos entre los machos, y a machos con territorios entre las hembras. Se observó un comportamiento gregario con una coloración corporal pálida característica, antes que comenzaran los comportamientos reproductivos. Luego, una jerarquía de dominancia se estableció a través de interacciones agresivas. Los individuos territoriales presentaron patrones de coloración corporal brillantes y los individuos no territoriales uno gris opaco. Una coloración ventral oscura fue observada asociada a individuos reproductivos. Los despliegues de cortejo fueron similares a los de amenaza y tuvieron la característica común de aumentar el área visible de los peces. El macho dominante fue siempre el más grande, sugiriendo que probablemente la fuerza (tamaño) es un factor preponderante determinando el establecimiento de las jerarquías y que éstas características seleccionadas intrasexualmente pueden haber sido reforzadas por selección intersexual. Los machos reproductivos presentaron un mayor contenido hipofisario de β-FSH y SL que aquellos no reproductivos, mientras que no se encontraron diferencias entre las hembras. No se encontraron diferencias entre los índices gonadosomáticos de los machos. Los individuos no reproductivos presentaron niveles plasmáticos mayores de cortisol para ambos sexos. Aunque los individuos reproductivos dominantes podrían estar inhibiendo la reproducción de los peces menos dominantes a través de interacciones de contacto físico, aumentando sus niveles de cortisol y disminuyendo el contenido hipofisario de FSH y SL, esto no se vería reflejado a nivel gonadal en nuestro diseño experimental.

Social and reproductive physiology and behavior of the Neotropical cichlid fish Cichlasoma dimerus under laboratory conditions

In this work we describe for the first time the social and reproductive behavior of the Neotropical fish Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endemic to the Paraná River basin, using a comprehensive-integral approach, including morphological and physiological features. This substrate breeding fish has biparental care of the fry and presents a dominance hierarchy that determines access to breeding territories among males, and to males with territories among females. Gregarious behavior associated with a pale body color, was observed before reproductive behaviors started. Afterwards, a dominance hierarchy was established through aggressive interactions. Territorial individuals had bright body color patterns and non territorial an opaque grey one. Black ventral coloration was associated with reproductive individuals. Courtship displays, which were similar to threatening displays, had the common effect of increasing the visible area of the individual. The dominant male was always the largest one suggesting that size is probably a major factor determining the hierarchy establishment and that these intra-sexually selected traits may have been reinforced by inter-sexual selection. Reproductive males had higher pituitary levels of β-follicle stimulating hormone (β-FSH) and somatolactin (SL) than non reproductive ones, while no differences were found among females. No differences were found among male gonadosomatic indexes. Non reproductive individuals had higher plasma cortisol levels for both sexes. It is possible that dominant reproductive individuals may be inhibiting reproduction of subordinate fish through physical contact, increasing their cortisol levels and diminishing FSH and SL pituitary content. However, this was not reflected as an inhibition at the gonadal level in our experimental design.(AU)

En este trabajo se describen por primera vez el comportamiento social y reproductivo del pez cíclido neotropical Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endémico de la cuenca del Paraná, desde un enfoque integral y abarcador, incluyendo características morfológicas y fisiológicas. Éste pez incubador de substrato, tiene cuidado biparental de las crías y presenta una jerarquía de dominancia que determina el acceso a territorios reproductivos entre los machos, y a machos con territorios entre las hembras. Se observó un comportamiento gregario con una coloración corporal pálida característica, antes que comenzaran los comportamientos reproductivos. Luego, una jerarquía de dominancia se estableció a través de interacciones agresivas. Los individuos territoriales presentaron patrones de coloración corporal brillantes y los individuos no territoriales uno gris opaco. Una coloración ventral oscura fue observada asociada a individuos reproductivos. Los despliegues de cortejo fueron similares a los de amenaza y tuvieron la característica común de aumentar el área visible de los peces. El macho dominante fue siempre el más grande, sugiriendo que probablemente la fuerza (tamaño) es un factor preponderante determinando el establecimiento de las jerarquías y que éstas características seleccionadas intrasexualmente pueden haber sido reforzadas por selección intersexual. Los machos reproductivos presentaron un mayor contenido hipofisario de β-FSH y SL que aquellos no reproductivos, mientras que no se encontraron diferencias entre las hembras. No se encontraron diferencias entre los índices gonadosomáticos de los machos. Los individuos no reproductivos presentaron niveles plasmáticos mayores de cortisol para ambos sexos. Aunque los individuos reproductivos dominantes podrían estar inhibiendo la reproducción de los peces menos dominantes a través de interacciones de contacto físico, aumentando sus niveles de cortisol y disminuyendo el contenido hipofisario de FSH y SL, esto no se vería reflejado a nivel gonadal en nuestro diseño experimental.(AU)

Photoperiodic modulation of reproductive physiology and behaviour in the cichlid fish Cichlasoma dimerus.

Timing of breeding to an optimal season is a requirement for a successful reproductive outcome in seasonally breeding species. Photoperiodic signals are capable of modifying the reproductive behaviour and reproductive systems in several vertebrate species. The cichlid fish Cichlasoma dimerus shows highly organized breeding activities and different social status. The aim of this study is to test whether C. dimerus reproductive behaviour (male aggressive behaviour and female choice) and reproductive physiology (GnRH3 morphometric parameters, pituitary hormones content and organ-somatic indexes) are modulated by photoperiod. Before spawning, dominant pairs were isolated and kept in opposite tanks of 20 l for one week, so they could see each other but not physically interact. Afterwards, a group was exposed for four weeks to a short photoperiod (8h light:16 h dark) (short photoperiod exposed animals: SP) while another group was exposed to a long photoperiod (14 h light:10h dark) (long photoperiod exposed animals: LP). Temperature was maintained constant. Behavioural experiments showed that male aggression related to territory selection and its defence is reduced in SP males. Further, SP females were never chosen. At the brain level we demonstrated that GnRH3 neuronal optical density of staining was reduced. Finally, at the pituitary level we showed that SP males showed low levels of beta-LH, PRL and GH in the pituitary, and that SP females showed no significant differences in the pituitary content of any hormone. Taken all together these results suggest that in C. dimerus the photoperiod is a relevant environmental cue related to reproductive behaviour and physiology.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae).

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.(AU)

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.

Effect of salmon melanin-concentrating hormone and mammalian gonadotrophin-releasing hormone on somatolactin release in pituitary culture of Cichlasoma dimerus.

We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1-10 microM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P<0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1-10 microM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P<0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.

Involvement of somatolactin in background adaptation of the cichlid fish Cichlasoma dimerus.

Somatolactin (SL) is a pituitary hormone present exclusively in fish that is involved in different physiological processes. The role of SL was evaluated in Cichlasoma dimerus (Teleostei, Perciformes) exposed for 10 days to a black and white background (BB and WB). Changes in alpha-melanophore stimulating hormone (alphaMSH) and melanin concentrating hormone (MCH) cells were also analyzed for comparison with SL. A melanin dispersing effect was observed in fish exposed to a BB, while a concentrating one was observed in those exposed to a WB. By Western blot, three SL-immunoreactive (ir) bands (32, 28 and 23.5 kD) were evidenced. Pituitary SL-ir levels were 2.66- and 2.67-fold greater in the 32 Kd and 28 kD bands, respectively, in BB fish compared with those of WB fish. The SL-ir 23.5 Kd band was not included in the analysis because of its unknown identity. In addition, SL-ir cell number and area were significantly higher in the BB condition (BB 22.73+/-1.46, WB 7.37+/-0.54 and BB 27.39+/-1.00 microm2; WB: 16.61+/-0.65 microm2). No significant differences were observed in the number of the hypothalamic MCH-ir neurons. However, a significant difference was observed in their nuclear area (BB 11.61+/-0.42 microm2, WB 17.80+/-0.84 microm2). alphaMSH-ir cells showed a marked increased in number (BB 35.96+/-1.22, WB 24.36+/-1.04), but no significant differences were observed in the cell area. In conclusion, this study presented clear evidence towards a possible involvement of SL in the adaptation to background colors in teleost together with alphaMSH and MCH.