The saccus vasculosus of the neotropical cichlid fish Cichlasoma dimerus: characterization, developmental studies and its response to photoperiod.

The saccus vasculosus is an organ present in gnathostome fishes, located ventral to the hypothalamus and posterior to the pituitary gland, whose structure is highly variable among species. In some fishes, this organ is well-developed; however, its physiological function is still under debate. Recently, it has been proposed that this organ is a seasonal regulator of reproduction. In the present work, we examined the histology, ultrastructure, and development of the saccus vasculosus in Cichlasoma dimerus. In addition, immunohistochemical studies of proteins related to reproductive function were performed. Finally, the potential response of this organ to different photoperiods was explored. C. dimerus presented a well-developed saccus vasculosus consisting of a highly folded epithelium, composed of coronet and supporting cells, closely associated with blood vessels, and a highly branched lumen connected to the third ventricle. Coronet cells showed all the major characteristics described in other fish species. In addition, some of the vesicles of the globules were positive for thyrotropin beta subunit, while luteinizing hormone beta subunit immunostaining was observed at the edge of the apical processes of some coronet cells. Furthermore, neuropeptide Y and gonadotropin inhibitory hormone innervation in the saccus vasculosus of C. dimerus were shown. Finally, animals exposed to the long photoperiod showed lower levels of thyrotropin beta and common alpha subunits expression in the saccus compared to those of animals exposed to short photoperiod. All these results support the hypothesis that the saccus vasculosus is involved in the regulation of reproductive function in fish.

Anatomy and histology of the digestive tract and immunolocalization of Npy in the fish Cichlasoma dimerus (Cichliformes: Cichlidae) / Anatomía e histología del tracto digestivo e inmunolocalización del Npy (péptido orexigénico) en el pez Cichlasoma dimerus (Cichliformes: Cichlidae)

Abstract Introduction: The digestive tract of Neotropical cichlids has not been deeply studied, and it is a fundamental topic for understanding fish physiology, nutrition, trophic associations, and evolution. Objective: To describe anatomically and histologically the digestive tract of the Neotropical cichlid fish Cichlasoma dimerus and to immunolocalize the orexigenic peptide (Npy) along the intestine. Methods: We euthanized 14 adult individuals and fixed the organs in Bouin´s solution; we stained 7 μm thick paraffin sections for general description and with Alcian Blue (pH = 2.5, AB) and Periodic acid-Schiff (PAS) to identify acid or neutral glycoconjugates, respectively. Additionally, we performed immunohistochemistry for Npy in 3 adult individuals. We manually counted PAS- and AB-positive cells, and Npy-immunoreactive cells per fold. Results: There is a short oesophagus, a sac-like stomach, and a tubular intestine with two loops. The oesophagus has a stratified epithelium with a high density of PAS- and AB-positive goblet cells and striated muscle fibers in the tunica muscularis. The stomach mucosa is formed by simple columnar epithelium. The intestine has a simple columnar epithelium, with brush border and interspersed PAS- and AB-positive goblet cells, and Npy-immunoreactive cells. There is an ileorectal valve in the transition between the posterior intestine and the rectum. This last gut portion has goblet cells and a thicker tunica muscularis. Conclusions: C. dimerus shares features with other Neotropical cichlids, but the goblet cells and gastric glands distribution seems to be unique for the species. To our understanding, this is the first work to describe Npy-immunoreactive cells distribution in the intestine of a Neotropical cichlid fish.

Resumen Introducción: El tracto digestivo de los cíclidos neotropicales no ha sido profundamente estudiado y es un tema fundamental para entender la fisiología, nutrición, asociaciones tróficas y evolución de los peces. Objetivo: Describir anatómica e histológicamente el tracto digestivo del pez cíclido neotropical Cichlasoma dimerus e inmunolocalizar el péptido orexigénico (Npy) a lo largo del intestino. Métodos: Sacrificamos 14 individuos adultos y fijamos los órganos en solución de Bouin; teñimos secciones de parafina de 7 μm de espesor para una descripción general y con azul alcián (pH = 2.5, AB) y ácido periódico-Schiff (PAS) para identificar glicoconjugados ácidos o neutros, respectivamente. Además, en 3 individuos adultos se realizaron inmunohistoquímicas contra Npy. Contamos manualmente las células PAS y AB positivas, y las células inmunorreactivas a Npy por pliegue. Resultados: Hay un esófago corto, un estómago en forma de saco y un intestino con dos vueltas. El esófago tiene un epitelio estratificado con una alta densidad de células caliciformes PAS- y AB- positivas y fibras esqueléticas estriadas en las capas musculares. La mucosa del estómago está revestida por epitelio simple cilíndrico. El epitelio intestinal es simple cilíndrico con chapa estriada y células caliciformes PAS- y AB- positivas intercaladas, y células inmunorreactivas a Npy. Hay una válvula ileorrectal en la transición entre el intestino posterior y el recto. Esta última porción intestinal tiene células caliciformes y una túnica muscular más gruesa. Conclusiones: C. dimerus comparte características con otros cíclidos neotropicales, pero la distribución de las células caliciformes y las glándulas gástricas, serían rasgos propios de esta especie. A nuestro entender, este es el primer trabajo que describe la distribución de células inmunorreactivas a Npy en el intestino de un pez cíclido neotropical.

Post-spawning feed deprivation effects on testicular and ovarian maturation in the neotropical cichlid fish Cichlasoma dimerus.

Many teleost fishes can withstand long feed deprivation periods, either due to an eventual lack of food or because of their behavior during reproduction and/or parental care. In this work, the effects of total food restriction on the oogenesis, spermatogenesis, and reproductive hormones of the neotropical cichlid fish Cichlasoma dimerus were studied. Specifically, different pairs were isolated after having a spawning event and were feed-deprived or daily fed for 3 weeks. After that period, gonadal histology, messenger levels of genes related to reproduction (gonadotropin-releasing hormone 1, gonadotropins, and insulin-like growth factor 1) and 11-ketotestosterone plasma levels were evaluated in both groups. Food restriction did not affect the reproductive axis in females since follicular maturation and gene expression showed no differences with respect to controls. However, in males, food restriction showed a stimulatory effect on the reproductive axis, reflected in a greater number of spermatozoa in their seminiferous lobes and spermatic ducts, and in an increase in follicle stimulating hormone messenger expression. Despite the negative effect reported for many fish species, C. dimerus seems to redirect their energy reserves towards gonadal development when faced with to a feed deprivation period.

Changes in digestive enzyme activities during the early ontogeny of the South American cichlid (Cichlasoma dimerus).

Cichlasoma dimerus is a neotropical cichlid that has been used as a biological model for neuroendocrinology studies. However, its culture is problematic in terms of larval feeding to allow having enough fry quantity and quality. Larviculture requires full knowledge about the digestive system and nutrition; therefore, this study was intended to assess the digestive enzymes' changes at different ages during the early ontogeny. Acid protease activity was detectable from the first day after hatching (dah), increasing to its maximum peaks on 9 dah. In contrast, alkaline proteases had low activity in the first days of life but reached their maximum activity on 17 dah. Chymotrypsin, L-aminopeptidase, and carboxypeptidase A activities increased at 6 dah, while trypsin activity was first detected on 13 dah and reached its maximum activity on 17 dah. Lipase and α-amylase activity were detectable at low levels in the first days of life, but the activity fluctuated and reaching its maximum activity at 21 dah. Alkaline phosphatase continued to oscillate and had two maximum activity peaks, the first at 6 dah and the second at 19 dah. Zymograms of alkaline proteases on day 6 dah six revealed four activity bands with molecular weights from 16.1 to 77.7 kDa. On 13 dah, two more activity bands of 24.4 and 121.9 kDa were detected, having a total of six proteases. The enzymatic activity analyzes indicate the digestive system shows the low activity of some enzymes in the first days after hatching, registering significant increases on 6 dah and the maximum peaks of activities around at 17 dah. Therefore, we recommend replacing live food with dry feed and only providing dry feed after day 17 dah.

Morphological colour adaptation during development in fish: involvement of growth hormone receptor 1.

Morphological background adaptation is both an endocrine and a nervous response, involving changes in the amount of chromatophores and pigment concentration. However, whether this adaptation takes place at early developmental stages is largely unknown. Somatolactin (Sl) is a pituitary hormone present in fish, which has been associated to skin pigmentation. Moreover, growth hormone receptor type 1 (Ghr1) has been suggested to be the Sl receptor and was associated with background adaptation in adults. In this context, the aim of this work was to evaluate the ontogeny of morphological adaptation to background and the participation of ghr1 in this process. We found in larval stages of the cichlid Cichlasoma dimerus that the number of head melanophores and pituitary cells immunoreactive to Sl was increased in individuals reared with black backgrounds compared with that in fish grown in white tanks. In larval stages of the medaka Oryzias latipes, a similar response was observed, which was altered by ghr1 biallelic mutations using CRISPR/Cas9. Interestingly, melanophore and leucophore numbers were highly associated. Furthermore, we found that somatic growth was reduced in ghr1 biallelic mutant O. latipes, establishing the dual function of this growth hormone receptor. Taken together, these results show that morphological background adaptation is present at early stages during development and that is dependent upon ghr1 during this period.

GH, IGF-I and GH receptors mRNA expression in response to growth impairment following a food deprivation period in individually housed cichlid fish Cichlasoma dimerus.

Cichlasoma dimerus is a social cichlid fish capable of growing at high rates under laboratory conditions, but knowledge on somatic growth regulation is still unclear. Growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is the key regulator of somatic growth in vertebrates. Two types of growth hormone receptors have been described in teleost fish, named GH receptor type 1 (GHR1) and type 2 (GHR2). In addition, isoforms of these receptors lacking part of the intracellular region have been described. The aim of this study was to evaluate the somatic growth, liver histology and changes in the GH/IGF-I axis after 4 weeks of food deprivation in C. dimerus. Four-week fasted fish showed reductions in specific growth rates in body weight (p < 0.001) and standard length (p < 0.001). Additionally, the hepatosomatic index (p < 0.001) and hepatocyte area (p < 0.001) decreased in fasted fish, while no changes in glucose levels were detected in plasma. The starvation protocol failed to induce changes in GH mRNA levels in the pituitary and IGF-I mRNA levels in liver. In contrast, IGF-I mRNA levels in muscle decreased in fasted fish (p = 0.002). On the other hand, GHR2 (detected with primer sets designed over the extracellular and intracellular region) was upregulated by starvation both in liver and muscle (p < 0.05), while GHR1 remained unchanged. These results show that a fasting period reduced somatic growth both in length and body weight concomitantly with alterations on liver and muscle GHR2 and muscle IGF-I mRNA expression.

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.