Environmental pollution affects molecular and biochemical responses during gonadal maturation of Astyanax fasciatus (Teleostei: Characiformes: Characidae).

Endocrine disrupting compounds (EDCs) have the potential to alter fish reproduction at various levels of organization. The aim of this study was to assess the impact of a natural environment with heavily anthropogenic influence on the physiological processes involved in reproduction in the freshwater fish lambari (Astyanax fasciatus) using different biomarkers. Adult males and females were collected in different seasons from two distinct sites in the same watershed: Ponte Nova Reservoir (PN) considered a pristine or small anthropogenic influence reference point; and Billings Reservoir (Bil), subjected to a large anthropogenic impact. Biological indices, such as hepatosomatic index and gonadosomatic index (GSI), gonadal histomorphology, fecundity, and biomarkers such as plasma levels of estradiol (E2) as well as hepatic gene expression of its alfa nuclear receptor (ERα), were analyzed. Hepatic vitellogenin (VTG) gene expression was evaluated in both sexes, as an indicator of xenoestrogen exposure. Females collected at PN presented a typical annual variation reflected in GSI, whereas for those sampled at Bil the index did not change through the seasons. The higher concentration of E2 in males collected at Bil during spring/2013, together with the detection of VTG gene expression, suggest the presence of EDCs in the water. These EDCs may have also influenced fecundity of females from Bil, which was higher during winter and spring/2013. Gene expression of ERα and ovarian morphology did not differ between fish from both sites. Water conditions from Bil reservoir impacted by anthropic activity clearly interfered mainly with biomarkers of biological effect such as plasma E2 levels and absolute and relative fecundity, but also altered biomarkers of exposure as VTG gene expression. These facts support the notion that waterborne EDCs are capable of causing estrogenic activity in A. fasciatus.

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.

Post-feeding Molecular Responses of Cobia (Rachycentron canadum): RNA-Sequencing as a Tool to Evaluate Postprandial Effects in Hepatic Lipid Metabolism.

We used transcriptome sequencing to investigate the hepatic postprandial responses of Rachycentron canadum (cobia), an important commercial fish species. In total, 150 cobia juveniles (50 per tank, triplicate) were fed ad libitum with a commercial diet for 7 days, fasted for 24 h, and fed for 10 min. The liver was sampled 10 min prior to feeding and 30 min, 1, 2, 4, 8, 12, and 24 h after the feeding event. Each sample was evaluated in terms of liver fatty acid profile and gene expression. Differential gene expressions were evaluated, focusing on fatty acid synthesis and oxidation pathways. In general, the liver fatty acid profile reflected diet composition. Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) levels increased at 8 to 12 h but decreased at 24 h after the feeding event. A high number of differentially expressed genes (DEGs) were observed comparing fish that fasted for 8 h with those fasted for 30 min and 24 h, while a reduced number of DEGs was observed comparing individuals who fasted for 30 min compared with those who fasted for 24 h. Similarly, the main differences in the expression of genes related to the fatty acid biosynthesis and oxidation pathways were noticed in individuals who fasted for 8 h compared with those who fasted for 30 min and 24 h. The results suggested that the adequate time to sample the individuals ranged between 8 and 12 h after the meal since, apparently, after 24 h, differential gene expression was not necessarily influenced by food intake.

Long-Chain Polyunsaturated Fatty Acids n-3 (n-3 LC-PUFA) as Phospholipids or Triglycerides Influence on Epinephelus marginatus Juvenile Fatty Acid Profile and Liver Morphophysiology.

Phospholipids (PL) are membrane components composed of fatty acids (FA), while triglycerides (TG) are a main source of energy and essential FA. Polyunsaturated FA (PUFA), such as docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), are essential for marine carnivorous fish; thus, an 8-week experiment was performed to evaluate the influence of DHA and EPA, provided as PL and TG, on the morphophysiology of Epinephelus marginatus juveniles. A basal diet was manufactured, and DHA and EPA in PL form (PL1-low amount PL2-high amount) and TG form (TG1-low amount; TG2-high amount) were added. Dusky grouper juveniles were equally distributed in 12 tanks of 20 animals each, and liver and muscle were sampled for metabolic analysis. The total hepatic lipids in PL1 and PL2 were higher when compared to the initial, TG1 and TG2 groups. Total lipids in muscle were higher in PL2 and TG1 than PL1 and TG2, respectively. Diets rich in DHA and EPA in PL and TG resulted in higher deposition of these FA in the muscle polar fraction. However, fish fed diets containing lower amounts of DHA and EPA in PL and TG stored those in the muscle neutral fraction and liver, centralizing the storage of DHA and EPA.

Fatty Acid Composition of Tropical Fish Depends on Reservoir Trophic Status and Fish Feeding Habit.

Eutrophication results in a deficiency of n-3 LC-PUFA (long-chain polyunsaturated fatty acids) in aquatic food chains, affecting fish nutrition and physiology. The trophic transfer of FA (fatty acids) to fish species of different feeding habits was investigated in two reservoirs in southeast Brazil-the mesotrophic Ponte Nova Reservoir (PN) and the hypereutrophic Billings Reservoir (Bil). Total FA profile of stomach contents and adipose tissue, triacylglycerols (TAG), and phospholipids (PL) from liver and muscle of the omnivorous Astyanax fasciatus and the carnivorous Hoplias malabaricus were analyzed by gas chromatography. A prevalence of n-6PUFA, as 18:2n-6 (linoleic acid) and 20:4n-6 (arachidonic acid, ARA) was observed in the stomach contents and in the tissues of A. fasciatus from the PN reservoir. In contrast, n-3 LC-PUFA, as 20:5n-3 (eicosapentaenoic acid, EPA) was accumulated in fish tissues from Bil, resulting in higher n3/n6 and EPA/ARA ratios, compared to fish from PN. This differential FA accumulation was also observed for H. malabaricus, but differences were slightly minor, and no changes were observed in the EPA/ARA ratios between fish from both reservoirs. Regardless reservoir, FA profiles of TAG resembled that of their diet, whereas FA profiles of PL were more conservative and mainly comprised by LC-PUFA. We conclude that reservoir trophic status affected the FA composition of food resources available to these fish species, resulting in differential allocation of n-3 and n-6 FA. As expected, FA profile of the investigated fish species also reflected their feeding habit and physiological demands.

Inappropriate management conditions, especially for the regressed class, are related to sperm quality in Prochilodus lineatus.

The aims of this study were to evaluate the characteristics of the reproductive classes and semen quality in curimbatá (Prochilodus lineatus) breeders maintained in two different rearing systems. To achieve this goal, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively. The fish were maintained under the experimental conditions for 18 months. During this period, males were randomly sampled every 2 months for biometric analysis (n = 30 per sample) and for an evaluation of selected characteristics of the testes (n = 5 per sample). After this period, males maintained in EPs and males maintained in Cs (CMs) were evaluated in induced breeding experiments. We observed that rearing P. lineatus in a C at a high stocking density for the long 18-month period of study produced reductions in growth, testis development, gonadosomatic index values, and sperm quality in the fish. We found differences between the groups in all the reproductive classes examined, especially in the regression class, which showed a pronounced accumulation of immature germ cells in the CMs. In this group, we also noted a less intense transition from a continuous to discontinuous germinal epithelium, with an extended and abnormal but less intense spermatogenic period resulting in decreases in semen volume and sperm concentration in the breeding season. Together, such dysfunctions resulted in the production of low-quality sperm in the CMs, as demonstrated by lower-quality DNA (as evaluated by the comet assay), low fertilization success, and low hatching success. In conclusion, to ensure high-quality semen in P. lineatus, appropriate management conditions must be provided throughout the reproductive cycle, especially for the regressed class, even in winter, two seasons before the breeding season.

Influence of spawning procedure on gametes fertilization success in Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae): Implications for the conservation of this species

Artificial reproduction and gamete fertilization were evaluated in Salminus hilarii wild and domesticated broodstocks. Wild and domesticated broodstocks were artificially induced to reproduction using a carp pituitary treatment. Four groups were considered: Group 1 (G1), fish caught in the wild maintained for three years in the same conditions as the domesticated broodstocks and spawned naturally; Group 2 (G2), broodstock born and raised in captivity and spawned naturally; Group 3 (G3), wild broodstocks, which were manually stripped for gamete collection and dry fertilization; and Group 4 (G4), domesticated males and females, also manually stripped. Oocytes, eggs, and larvae were sampled at different time intervals throughout embryonic development. Yolk sac absorption occurred approximately 24-29 h after hatching. Twenty-six h after hatching, the larvae mouths opened. Cannibalism was identified just 28-30 h after hatching. There was no morphological difference in embryonic development among all groups. The number of released eggs per gram of female was: G1: 83.3 ± 24.5 and G2: 103.8 ± 37.4; however, the fertilization success was lower in G2 (42.0 ± 6.37 percent) compared with G1 (54.7 ± 3.02 percent) (P = 0.011). Hand-stripping of oocytes was not successful and the fertilization rate was zero. The reproduction of this species in captivity is viable, but it is necessary to improve broodstock management to enhance fertilization rates and obtain better fingerling production for restocking programs.

A reprodução artificial e fertilização dos gametas foram avaliados em reprodutores selvagens e de cativeiro de Salminus hilarii. Reprodutores selvagens e de cativeiro foram induzidos artificialmente à reprodução utilizando hipófise de carpa. Quatros grupos foram considerados: Grupo 1 (G1), peixes capturados na natureza, mantidos por três anos nas mesmas condições de reprodutores de cativeiro e desovados naturalmente; Grupo 2 (G2), reprodutores nascidos e criados em cativeiro e desovados naturalmente; Grupo 3 (G3), reprodutores selvagens que foram extrusados manualmente para a coleta de gametas e fertilização a seco; e Grupo 4 (G4), com machos e fêmeas domesticadas, também extrusados manualmente. Oócitos, ovos e larvas foram amostrados em diferentes intervalos de tempo ao longo do desenvolvimento embrionário. A absorção do saco vitelínico ocorreu aproximadamente 24-29 h após a eclosão. Vinte e seis h após a eclosão, as larvas abriram a boca. O canibalismo foi identificado apenas 28-30 h após a eclosão. Não houve diferença morfológica no desenvolvimento embrionário entre todos os grupos. O número de ovos liberados por grama de fêmea foi: G1: 83,3 ± 24,5 e G2: 103,8 ± 37,4; embora, o sucesso na fertilização tenha sido menor no G2 (42,0 ± 6,37 por cento) em comparação com G1 (54,7 ± 3,02 por cento) (P = 0,011). A extrusão manual dos oócitos não foi bem sucedida e a taxa de fertilização foi zero. A reprodução em cativeiro desta espécie é viável, mas é necessário um melhor manejo dos reprodutores para aumentar as taxas de fertilização, visando a obtenção de uma melhor produção de alevinos para os programas de repovoamento.