Physiological responses on the reproductive, metabolism and stress endpoints of Astyanax lacustris females (Teleostei: Characiformes) after diclofenac and ibuprofen exposure.

Diclofenac (DCF) and ibuprofen (IBU) are pharmaceutical compounds frequently detected in aquatic compartments worldwide. Several hazard effects including developmental abnormalities and redox balance impairment have been elucidated in aquatic species, but multiple endocrine evaluations are scarce. Therefore, the present study aimed to assess the disruptive physiological effects and toxicity of DCF and IBU isolated and combined, using females of the native freshwater teleost Astyanax lacustris. In regards to NSAIDs bioavailability, the results showed absence of degradation of IBU and DCF after 7 days of exposure. IBU LC50 for A. lacustris was 137 mgL-1 and females exposed to IBU isolated increased thyroxine (T4) concentration at 24 h and decreased after 96 h; DCF exposure decreased triiodothyronine (T3) concentration at 96 h. Circulating levels of 17ß-estradiol (E2), cortisol (F) and testosterone (T) were not affected by any treatment. HPG and HPI axis genes fshß, pomc and vtg were upregulated after 24 h of IBU exposure, and dio2 was downregulated in DCF fish exposed group after 96 h compared to the mixture. Protein concentration was reduced in muscle and increased in the liver by DCF and mixtures exposures at 24 h; while liver lipids were increased in the mixture groups after 96 h. The study point out the capacity of NSAIDs to affect endocrine endpoints in A. lacustris females and induce changes in energetic substrate content after acute exposure to isolated and mixed NSAIDs treatments. Lastly, the present investigation brings new insights into the toxicity and endocrine disruptive activity of NSAIDs in Latin America teleost species and the aquatic environment.

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.

Effects of vitamin A supplementation on ovarian development of Astyanax lacustris (Teleostei: Characidae) during the non-breeding season.

Vitamin A (VA) is essential for fish reproduction, however, knowledge of VA requirement of broodstock fish is scarce. We investigated the physiological role of VA supplementation in the ovarian maturation of Astyanax lacustris throughout the non-breeding season. Adult females were distributed in five nutritional treatments, which differed by the amount of VA supplemented in a plant-based diet: T1 = 0, T2 = 1800, T3 = 3600, T4 = 7200, and T5 = 14,400 IU kg-1 of VA for 60 days after a period of 16 days feeding on a diet poor in VA to reduce body VA content. Plasma total lipids (TL), lipoproteins, and 17ß-estradiol (E2) levels were measured after 30 and 60 days. Somatic indexes and morphophysiological ovarian parameters were calculated and VA concentration was analyzed in the ovaries and liver after 60 days. VA supplementation did not increase the concentration of this nutrient in the liver and ovaries. E2 plasma level increased in animals fed on 3600 IU kg-1 of VA (T3) after 60 days and females from T5 group presented a higher RF after 60 days. Ovarian development increased after 60 days compared to that after 30 days, confirmed by the higher gonadosomatic index (all groups). The supplementation with the highest VA level increased RF and decreased plasma TL (after 30 days) and low-density lipoprotein (LDL) (after 60 days) concentrations, showing a potential to improve reproduction even during the non-breeding season; however, its nutritional requirements of VA could be higher.

Transcriptomic Profiling and Microsatellite Identification in Cobia (Rachycentron canadum), Using High-Throughput RNA Sequencing.

Cobia (Rachycentron canadum) is a marine teleost species with great productive potential worldwide. However, the genomic information currently available for this species in public databases is limited. Such lack of information hinders gene expression assessments that might bring forward novel insights into the physiology, ecology, evolution, and genetics of this potential aquaculture species. In this study, we report the first de novo transcriptome assembly of R. canadum liver, improving the availability of novel gene sequences for this species. Illumina sequencing of liver transcripts generated 1,761,965,794 raw reads, which were filtered into 1,652,319,304 high-quality reads. De novo assembly resulted in 101,789 unigenes and 163,096 isoforms, with an average length of 950.61 and 1,617.34 nt, respectively. Moreover, we found that 126,013 of these transcripts bear potentially coding sequences, and 125,993 of these elements (77.3%) correspond to functionally annotated genes found in six different databases. We also identified 701 putative ncRNA and 35,414 putative lncRNA. Interestingly, homologues for 410 of these putative lncRNAs have already been observed in previous analyses with Danio rerio, Lates calcarifer, Seriola lalandi dorsalis, Seriola dumerili, or Echeneis naucrates. Finally, we identified 7894 microsatellites related to cobia's putative lncRNAs. Thus, the information derived from the transcriptome assembly described herein will likely assist future nutrigenomics and breeding programs involving this important fish farming species.

Experimental effects of cadmium on physiological response of Callinectes danae (Crustacea, Portunidae) from environments with different levels of Cd contamination.

Cadmium (Cd) can adversely affect aquatic life, altering reproductive and molting processes in crustaceans. The objective of this study was to evaluate the influence of Cd on reproduction and molting in the crab Callinectes danae. Adult females were obtained from environments with different levels of pollution: low (LC), medium (MC), and high contaminated (HC) areas. Animals from LC, MC, and HC areas were exposed to 0, 0.5, and 2 mg L-1 of CdCl2 for 3 h. Cd bioaccumulation, oxidative stress (evaluated by antioxidant enzymes activity), and lipid peroxidation (LPX) were analyzed in mature ovaries (stage II), gills, and hepatopancreas. The expression levels of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) genes were quantified in the eyestalks, while 17ß-estradiol (E2) and melatonin concentration were measured in the hemolymph. Cd bioaccumulated mainly in the hepatopancreas and gills, with increased E2, LPX, and antioxidant enzymes in HC compared to the LC region. Decreased CHH and MIH transcripts were observed in the animals from HC regions compared to LC and MC areas. Physiological differences were recorded, especially for bioaccumulation, oxidative stress, and hormone levels, in animals sampled in HC areas compared to LC and MC regions. In conclusion, the physiological damage triggered by Cd could be reduced due to higher levels of melatonin and antioxidant enzymes in HC areas.

Integrated use of biomarkers to evaluate the reproductive physiology of Astyanax fasciatus and Hoplias malabaricus males (Teleostei: Characiformes) in polluted reservoirs.

The reproductive physiology of fish can be changed by the presence of pollutants in the water, which act as endocrine disrupting compounds (EDC). We evaluated the impacts of water contaminants in polluted reservoirs acting as possible EDC on the reproductive physiology of Astyanax fasciatus and Hoplias malabaricus males. We used biomarkers with different levels of biological organization. Hoplias malabaricus adult males were collected in the summer and winter at five different sites in the Tietê River Basin: the Ponte Nova reservoir (PN), considered a reference site due to the low anthropogenic influence; the Billings reservoir (BIL) at two different branches; and the Guarapiranga reservoir (GUA) at two different branches. Astyanax fasciatus adult males were collected at PN and BIL. BIL and GUA are subjected to great anthropogenic action. We analyzed gonadal histomorphology, testosterone (T), 11-ketotestosterone (11-KT), estradiol (E2) plasma levels, and gene expression of hepatic vitellogenin (vtgA) and pituitary follicle stimulating hormone (fshß). In the PN reservoir (reference), the biomarkers analyzed in both species did not differ between the periods analyzed. This is an evidence that the animals keep the same reproductive activity during both seasons. The changes in the plasma concentration of gonadal steroids in both species in polluted reservoirs suggest the presence of EDC compounds in the water and/or adjusts of the physiological setpoint to allow the reproduction in such adverse conditions. The use of vtgA as biomarker suggests the presence of estrogenic compounds, mainly in BIL, but with a more evident response of H. malabaricus. However, even considering physiological changes, both species present testes during the maturation phase that allow the reproduction in an environment with a high degree of pollution.

Molecular characterization of different preproGnRHs in Astyanax altiparanae (Characiformes): Effects of GnRH on female reproduction.

Gonadotropin-releasing hormone (GnRH) is a key molecule in the initiation of the hypothalamic-pituitary-gonadal axis. Thus, knowledge about GnRH may contribute to the effectiveness of species reproduction. Using a Neotropical tetra Astyanax altiparanae as a fish model species, the GnRH forms were characterized at the molecular level and the role of injected GnRHs in vivo was evaluated. The full-length complementary DNA (cDNA) sequences of preproGnRH2 (612 bp) and preproGnRH3 (407 bp) of A. altiparanae were obtained, and the GnRH1 form was not detected. The cDNA sequences of preproGnRH2 and preproGnRH3 were found to be conserved, but a change in the amino acid at position 8 of the GnRH3 decapeptide of A. altiparanae was observed. All the injected GnRHs stimulated lhß messenger RNA (mRNA) expression but not fshß mRNA expression, and only GnRH2 was able to increase maturation-inducing steroid (MIS) levels and possibly stimulate oocyte release. Furthermore, only GnRH2 was able to start the entire reproductive hormonal cascade and induce spawning.

Effects of biological traits on capture-induced parturition in a freshwater stingray and perspectives for species management.

Elasmobranchs are particularly vulnerable to overexploitation and population depletion, especially due to their life-history traits, such as low reproductive output and slow growth. Given that capture-induced parturition (abortion or premature birth) is a common consequence of fisheries in elasmobranchs, but still little studied, we investigated how the abortion/premature birth process varies in response to reproductive traits in a freshwater stingray, Potamotrygon amandae. Our results revealed that capture-induced parturition was affected by reproductive traits, such as litter size (one to seven) and gestation stage. The event occurred faster in pregnant females with high litter size during late pregnancy. Also, as found in other elasmobranchs, litter size was positively correlated with maternal size. These findings indicate that larger pregnant females in late pregnancy are more vulnerable to capture-induced parturition. This study improves our understanding of the capture-induced parturition process in stingrays, and provides useful information for management strategies and future recommendations for elasmobranch conservation.

Endocrine disruptive action of diclofenac and caffeine on Astyanax altiparanae males (Teleostei: Characiformes: Characidae).

Diclofenac (DCF) and caffeine (CAF) are persistent pharmaceuticals that occur in mixtures in the aquatic ecosystems causing effects in the reproductive physiology of aquatic organisms. This study evaluated the physiological reproductive responses of Astyanax altiparanae males exposed to nominal concentrations of DCF (3.08 mg L-1) and CAF (9.59 mg L-1) separately and combined, for 96 h. The steroids profile, estrogenic biomarker vitellogenin (vtgA), testes and liver morphology, and also mortality of males were assessed. DCF and CAF degradation was 5% of the initial concentration for 24 h. The LC50 of the DCF and CAF were 30.8 mg L-1 and 95.9 mg L-1, respectively. Males exposed to DCF and CAF exhibited a reduction of 17ß-Estradiol (E2) concentration compared to control (CTL). Similarly, testosterone (T) was also reduced in the DCF treatment, but this response was not observed in 11-Ketotestosterone (11-KT). Males exposed to DCF + CAF combined did not exhibit differences in T, E2 and 11-KT steroids. The vtgA gene expression and the sperm concentration did not change among the treatments. Moreover, acute exposure revealed a hypertrophy of hepatocytes cells in the DCF and DCF + CAF treatments. In conclusion, DCF and CAF, isolated, exhibit an endocrine disruptive activity in A. altiparanae male, an opposite response observed with the mixture of both compounds that abolishes the endocrine disruptive effects. DCF seems to be more toxic for this species, altering also hepatocytes morphology.

Characterization of lipid metabolism genes and the influence of fatty acid supplementation in the hepatic lipid metabolism of dusky grouper (Epinephelus marginatus).

Dusky grouper is an important commercial fish species in many countries, but some factors such as overfishing has significantly reduced their natural stocks. Aquaculture emerges as a unique way to conserve this species, but very little biological information is available, limiting the production of this endangered species. To understand and generate more knowledge about this species, liver transcriptome sequencing and de novo assembly was performed for E. marginatus by Next Generation Sequencing (NGS). Sequences obtained were used as a tool to validate the presence of key genes relevant to lipid metabolism, and their expression was quantified by qPCR. Moreover, we investigated the influence of supplementing different dietary fatty acids on hepatic lipid metabolism. The results showed that the different fatty acids added to the diet dramatically changed the gene expression of some key enzymes associated with lipid metabolism as well as hepatic fatty acid profiles. Elongase 5 gene expression was shown to influence intermediate hepatic fatty acid elongation in all experimental groups. Hepatic triglycerides reflected the diet composition more than hepatic phospholipids, and were characterized mainly by the high percentage of 18:3n3 in animals fed with a linseed oil rich diet. Results for the saturated and monounsaturated fatty acids suggest a self-regulatory potential for retention and oxidation processes in liver, since in general the tissues did not directly reflect these fatty acid diet compositions. These results indicated that genes involved in lipid metabolism pathways might be potential biomarkers to assess lipid requirements in the formulated diet for this species.

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.

Use of a nursery area by cownose rays (Rhinopteridae) in southeastern Brazil

Using non-lethal methods for data collection of individuals and participatory monitoring by fishermen, we provide the first empirical evidence of the use of a nursery area by neonate and young-of-the-year cownose rays Rhinoptera bonasus and R. brasiliensis in southeastern Brazil. Two methods were used to collect data: (1) information provided by fishermen (reports, pictures, and videos) and (2) field sampling by researchers. A total of 746 cownose rays were captured; 113 have been identified as R. bonasus, 15 as R. brasiliensis, and 618 were reported by fishermen and could not be identified to the species. Records of newborns were made only in late spring and summer in 2015, 2016, and 2017, which suggests an annual reproductive cycle, with birth in late spring, extending to summer. A repeated use of this area by R. bonasus suggests that it is potentially important to the reproduction of this species. However, R. brasiliensis requires more studies. Small increases in mortality, resulting from increased fishing or other anthropogenic stressors, can have a disproportionately large effect on population viability. Thus, management of areas used during critical stages of the life cycle of rays is crucial to their conservation.(AU)

Usando métodos não-letais para levantamento de dados dos indivíduos e o monitoramento participativo dos pescadores fornecemos a primeira evidência empírica do uso de uma área de berçário por neonatos e jovens do ano de raias Ticonha Rhinoptera bonasus e R. brasiliensis no sudeste do Brasil. Dois métodos foram utilizados para coletar dados: (1) informações fornecidas pelos pescadores (relatórios, fotos e vídeos) e (2) amostragem de campo pelos pesquisadores. Um total de 746 raias Ticonha foram capturadas; 113 foram identificadas como R. bonasus, 15 como R. brasiliensis e 618 foram registrados pelos pescadores e não puderam ser identificadas em nível específico. Os registros de recém-nascidos foram feitos apenas no final da primavera e no verão de 2015, 2016 e 2017, o que sugere um ciclo reprodutivo anual, com nascimento no final da primavera, se estendendo até o verão. O uso repetido desta área por R. bonasus sugere sua potencial importância para a reprodução destas espécies. No entanto, R. brasiliensis requer mais estudos. Pequenos aumentos na mortalidade, resultantes do aumento da pesca ou outros estressores antropogênicos, podem ter um efeito desproporcionalmente grande na viabilidade populacional. Assim, o gerenciamento das áreas usadas durante os estágios críticos do ciclo de vida das raias é crucial para sua conservação.(AU)

Mobilization of energetic substrates in the endangered catfish Steindachneridion parahybae (Siluriformes: Pimelodidae): changes in annual reproductive cycle in captivity

This study aimed at analyzing the energetic substrate (ES) in the main storage tissues of Steindachneridion parahybae, throughout the reproductive cycle in captivity. Differently from wild, in captivity, feeding is not interrupted during the reproductive period, the females do not spawn spontaneously, and they are sedentary. Adult females were sampled monthly and based on their histology and gonadosomatic index (GSI), ovaries were classified into: previtellogenic (PRV), vitellogenic (VTG), and regression (REG) stages. Ovaries at the VTG stage showed higher protein and lipids levels than at the PRV stage with a positive correlation between these substrates and the GSI. Muscle was the main source of proteins transferred to the ovaries, according to the negative correlation between these organs. Lipids remained unchanged in the liver, which is an important supplier in vitellogenesis, a pattern that probably occurs due to the continuous feeding. Muscular glycogen levels were higher at the VTG and REG than at the PRV stages. Plasma triglycerides were also higher during REG, while glucose levels were more elevated during the VTG stage. These results suggest that with constant food supply, the pattern of deposition of ES in S. parahybae is different from that described for other wild potamodromous species.(AU)

O objetivo deste estudo foi analisar a composição do substrato energético (SE) nos principais tecidos de armazenamento de Steindachneridion parahybae, durante todo o ciclo reprodutivo em cativeiro. Diferentemente do ambiente natural, em cativeiro, a alimentação desses animais não é interrompida durante o período reprodutivo, as fêmeas não desovam espontaneamente, e são sedentárias. Fêmeas adultas foram amostradas mensalmente e baseada na histologia e no índice gonadossomático (IGS), os ovários foram classificados: estádios pré-vitelogênico (PRV), vitelogênico (VTG) e regressão (REG). Os ovários no estádio VTG apresentaram uma maior concentração de lipídeos e proteínas em relação ao estágio PRV. Esses substratos correlacionaram-se positivamente com o IGS. O músculo foi a principal fonte de proteína transferida aos ovários, como foi confirmado pela análise de correlação negativa entre esses órgãos. Os lipídeos mantiveram-se inalterados no fígado, considerado um importante órgão fornecedor de lipídeos para a vitelogênese, padrão que possivelmente ocorreu devido à contínua alimentação. A concentração do glicogênio muscular foi mais elevada durante os estágios VTG e REG em relação ao PRV. A concentração de triglicerídeos plasmática apresentou maiores valores no estádio REG enquanto a concentração de glicose no plasma foi maior durante os estádios VTG. Esses resultados sugerem que com alimentação constante, as fêmeas de S. parahybae apresentam um distinto padrão de mobilização dos substratos energéticos em relação ao que já foi descrito para outras espécies potamódromas de ambiente natural.(AU)

This study aimed at analyzing the energetic substrate (ES) in the main storage tissues of Steindachneridion parahybae, throughout the reproductive cycle in captivity. Differently from wild, in captivity, feeding is not interrupted during the reproductive period, the females do not spawn spontaneously, and they are sedentary. Adult females were sampled monthly and based on their histology and gonadosomatic index (GSI), ovaries were classified into: previtellogenic (PRV), vitellogenic (VTG), and regression (REG) stages. Ovaries at the VTG stage showed higher protein and lipids levels than at the PRV stage with a positive correlation between these substrates and the GSI. Muscle was the main source of proteins transferred to the ovaries, according to the negative correlation between these organs. Lipids remained unchanged in the liver, which is an important supplier in vitellogenesis, a pattern that probably occurs due to the continuous feeding. Muscular glycogen levels were higher at the VTG and REG than at the PRV stages. Plasma triglycerides were also higher during REG, while glucose levels were more elevated during the VTG stage. These results suggest that with constant food supply, the pattern of deposition of ES in S. parahybae is different from that described for other wild potamodromous species.(AU)

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.

Gonadotropin subunits of the characiform Astyanax altiparanae: Molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity.

To better understand the endocrine control of reproduction in Characiformes and the reproductive dysfunctions that commonly occur in migratory fish of this order when kept in captivity, we chose Astyanax altiparanae, which has asynchronous ovarian development and multiple spawning events, as model species. From A. altiparanae pituitary total RNA, we cloned the full-length cDNAs coding for the follicle-stimulating hormone ß subunit (fshb), the luteinizing hormone ß subunit (lhb), and the common gonadotropin α subunit (gpha). All three sequences showed the highest degree of amino acid identity with other homologous sequences from Siluriformes and Cypriniformes. Real-time, quantitative PCR analysis showed that gpha, fshb and lhb mRNAs were restricted to the pituitary gland. In situ hybridization and immunofluorescence, using specific-developed and characterized polyclonal antibodies, revealed that both gonadotropin ß subunits mRNAs/proteins are expressed by distinct populations of gonadotropic cells in the proximal pars distalis. No marked variations for lhb transcripts levels were detected during the reproductive cycle, and 17α,20ß-dihydroxy-4-pregnen-3-one plasma levels were also constant, suggesting that the reproductive dysfunction seen in A. altiparanae females in captivity are probably due to a lack of increase of Lh synthesis during spawning season. In contrast, fshb transcripts changed significantly during the reproductive cycle, although estradiol-17ß (E2) levels remained constant during the experiment, possibly due to a differential regulation of E2 synthesis. Taken together, these data demonstrate the putative involvement of gonadotropin signaling on the impairment of the reproductive function in a migratory species when kept in captivity. Future experimental studies must be carried to clarify this hypothesis. All these data open the possibility for further basic and applied studies related to reproduction in this fish model.

Physiological differences in the crab Ucides cordatus from two populations inhabiting mangroves with different levels of cadmium contamination.

Crustaceans found in metal-contaminated regions are able to survive, and the authors investigated the physiological mechanisms involved by comparing populations from contaminated and noncontaminated areas. The objective of the present study was to measure the cellular transport of a nonessential metal (cadmium [Cd]) in gills and hepatopancreas of Ucides cordatus, together with cell membrane fluidity, metallothionein levels, and lipid peroxidation. The 2 populations compared were from a polluted and a nonpolluted mangrove area of São Paulo State, Brazil. The authors found, for the first time, larger Cd transport in gills and hepatopancreatic cells from crabs living in polluted mangrove areas. The cells also had lower plasma membrane fluidity, increased lipid peroxidation and less metallothionein compared to those from nonpolluted regions. The authors also found larger amounts of Cd in intracellular organelles of gills, but not in the hepatopancreas, from crabs in polluted regions. Therefore, in polluted areas, these animals showed higher Cd transport and lower plasma membrane fluidity and storage of Cd intracellularly in gill cells, whereas hepatopancreatic cells used metallothionein as their main line of defense. The findings suggest that crabs from polluted areas can accumulate Cd more easily than crabs from nonpolluted areas, probably because of an impairment of the regulatory mechanisms of cell membrane transport. Environ Toxicol Chem 2017;36:361-371. © 2016 SETAC.

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.

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season.

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.

A case of intersex occurrence in Steindachneridion parahybae (Steindachner, 1877) (Siluriformes: Pimelodidae) under captivity condition: a cytogenetic and morphological study

Little is known about reproductive biology of endangered Steindachneridion parahybae , a gonochoristic teleost species inhabiting the Paraíba do Sul River Basin, and herein is the first description of intersex in S. parahybae juvenile. The normal appearance of ovaries and testes in juvenile from the same lot of breeding were also described for comparison, even as cytogenetic analysis was performed in these juveniles. One specimen was a priori classified as female due to the macroscopic characteristic of ovaries, with small yellow oocytes, without fringes (a main characteristic of catfish male), and larger than testes; however the microscopic analysis revealed the presence of ovotestes, including the complete spermatogenesis. S. parahybae had diploid number, 2n = 56 chromosomes with no evidence of differentiated sex chromosomes or supernumerary chromosomes among them. These findings may be due to the result of exposure to endocrine disrupting compounds or may also be influenced by environmental conditions. The possibility of intersexes might also happen spontaneously and it cannot be ruled out. Therefore, the functional significance and reproductive consequences of this anomaly remain to be determined, suggesting that this species may be susceptible to endocrine disruption. These results contribute to gain expertise about reproductive biology of an endangered species in captivity.(AU)

Poco se sabe sobre la biología reproductiva de Steindachneridion parahybae , una especie de teleósteo gonocorístico en peligro de extinción que habita la cuenca del río Paraíba do Sul y en éste trabajo se describe por primera vez la aparición de individuo intersexo en juvenil de S. parahybae . También se describió el aspecto normal de los ovarios y de los testículos de individuos juveniles procedentes del mismo lote de cría para su comparación; se realizó además el análisis citogenético. Un espécimen fue clasificado a priori como hembra debido a las características macroscópicas de los ovarios, con pequeños oocitos amarillos, sin flecos (característica principal de los bagres macho) y más grande que los testículos; sin embargo el análisis microscópico reveló la presencia de un ovotestis, incluyendo una espermatogénesis completa. S. parahybae presentó un número diploide, 2n = 56 cromosomas, sin evidencia de cromosomas sexuales diferenciados o supernumerarios entre ellos. Estos hallazgos pueden deberse al resultado de la exposición de los individuos a desorganizadores endocrinos o estar influenciados por las condiciones ambientales. Sin embargo no se puede descartar la posibilidad de la presencia de intersexos de forma espontánea. Por lo tanto, la importancia funcional y las consecuencias reproductivas de estas anomalías permanecen aún sin ser determinadas, sugiriendo que esta especie puede ser susceptible a los disruptores endocrinos. Estos resultados contribuyen a ampliar el conocimiento de la biología reproductiva de esta especie en peligro de extinción en condiciones de cautiverio.(AU)