A biomarker approach to study the effects of polluted Brazilian urban reservoirs in a native fish.

Two of the largest water reservoirs in the Metropolitan Region of São Paulo, Brazil (MRSP), named Billings and Guarapiranga, are facing high levels of anthropic impact. This is evidenced by the presence of contaminants and pollutants, which are deteriorating their water quality. Therefore, this study evaluated antioxidant defense enzymes, lipoperoxidation and genotoxicity, in adult females of a native species, Astyanax altiparanae from the Guarapiranga and Billings reservoirs. The study also aimed to evaluate these biomarkers during two different periods of the year, the rainy (summer) and dry (winter) seasons. The oxidative stress was evaluated by the activity of enzymes such as glutathione peroxidase, glutathione S-transferases, superoxide dismutase, and catalase in the gills and liver, and the occurrence of lipoperoxidation was also evaluated in both organs. The genotoxicity was assessed by performing comet assay, micronucleus, and nuclear abnormality tests on blood samples. The results showed that fish from both reservoirs are subjected to oxidative stress and genotoxic damage, mainly during winter, but fish living in Billings showed greater alterations than fish from Guarapiranga. Likewise, the results of the principal component analysis suggested that caffeine, nitrogenous compounds, and some metals might be triggering these toxic effects in fish.

Alterations in Gene Expression and the Fatty Acid Profile Impact but Do Not Compromise the In Vitro Maturation of Zebrafish (Danio rerio) Stage III Ovarian Follicles after Cryopreservation.

The vitrification of ovarian follicles is a strategic tool that may contribute to advances in aquaculture and the conservation of many important species. Despite the difficulties inherent to the cryopreservation of oocytes, some successful protocols have been developed for different species, but little is known about the capacity of oocytes to develop after thawing. Therefore, the profiles of the reproductive pathway genes and fatty acid membrane composition during the initial stages of development were analyzed in fresh ovarian follicles and follicles after the vitrification process. There were differences in the expression of the hypothalamic-pituitary-gonad axis genes during the follicular development in the control group as well as in the vitrified group. Similarly, alterations in the composition of fatty acids were observed after vitrification. Despite this, many alterations were observed in the vitrified group; more than half of the stage III ovarian follicles were able to grow and mature in vitro. Therefore, the vitrification of ovarian follicles may impact them at molecular and membrane levels, but it does not compromise their capability for in vitro maturation, which indicates that the technique can be a strategic tool for aquaculture.

Influence of Docosahexaenoic and Eicosapentaenoic Acid Ratio and Temperature on the Growth Performance, Fatty Acid Profile, and Liver Morphology of Dusky Grouper (Epinephelus marginatus) (Teleostei: Serranidae) Juveniles.

An 8-week trial was performed to evaluate the influence of docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) ratios and temperature on the fatty acid (FA) metabolism and liver morphology of Epinephelus marginatus. A basal diet was manufactured, and DHA (D1:0.5%; D2:0.75%; D3:1%; and D4:1.5%) and EPA (D1:1.0%; D2:0.75%; D3:0.5%; and D4:0%) levels were added. E. marginatus were kept in twelve tanks with a lower temperature (LT, 23.17 ± 0.12 °C) and twelve with a higher temperature (HT, 28.63 ± 0.06 °C). The DHA/EPA ratio did not affect performance, regardless of the temperature, but the animals fed with the D4 diet showed better growth at HT. A higher lipid deposition and a large lipid vacuole area in the hepatocytes at HT were observed, regardless of the diet. Triacylglycerol (TG) in reflected the diet FA profile mainly in the muscle and liver, whereas the phospholipid (PL) was less influenced by the diet. The same DHA content in the TG fraction of muscle (D3 and D4) were observed at LT; however, only the DHA/EPA ratio of D4 could be differentially stored in the TG fraction of muscle (HT). Monounsaturated and polyunsaturated FA increased in the PL of the eyes at HT, whereas saturated FA was reduced in the TG and PL fractions at LT. These results evidence the importance of temperature and a balanced DHA/EPA ratio in the diet of marine fish.

Dynamics of ovarian maturation during the annual reproductive cycle of the endangered fish Steindachneridion parahybae (Siluriformes: Pimelodidae) in captivity.

To characterize the female reproductive biology of the endangered species Steindachneridion parahybae in captivity, the authors used the concentration of gonadal steroids and the oocyte development during the annual reproductive cycle (RC) and after artificial induced spawning (AIS) until 48 h. Three stages of gonadal maturation were identified, based on morphological and physiological features: early maturation or previtellogenic (PRV) oocyte, advanced maturation or vitellogenic (VTG) oocyte and regression (REG) or follicular atresia. They identified and characterized the following stages of germ cells: oogonia, perinucleolar and cortical alveoli, and VTG and atretic oocytes during RC. The oestradiol levels were higher in PRV than those in VTG and REG during the RC, whereas androgens showed higher levels of oestradiol in VTG than those in PRV and REG. The progestogen levels remained unchanged during the whole RC. The final oocyte maturation (FOM) was achieved after AIS and postovulatory follicles (POF) were identified. Plasma concentration of progestogens (17α,20ß-dihydroxy-pregnen-3-one and 17α-hydroxyprogesterone) increased considerably after AIS, remaining high up to 6 h after AIS, and progressively decreased over time after AIS. During RC, the lack of FOM and POFs reveals that captivity negatively impacts S. parahybae reproduction. Nonetheless, the VTG stage of oocytes, reached during RC, is suitable for ovulation induction with artificial hormone manipulation, enabling the reproduction of this species in captivity and being essential for the success of fish farming and/or fish conservation programmes (conservationist aquaculture).

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.

Physiological markers suggest energetic and nutritional adjustments in male sharks linked to reproduction.

Energetic condition is one of the most important factors that influence fitness and reproductive performance in vertebrates. Yet, we lack evidence on how energetic states change in response to reproduction in large marine vertebrates. In the present study, we used a non-lethal approach to assess relationships among reproductive stage, circulating steroid hormones (testosterone and relative corticosteroid levels), plasma fatty acids, and the ketone body ß-hydroxybutyrate in male sharks of two species with divergent ecologies, the benthic nurse shark (Ginglymostoma cirratum) and the epipelagic blacktip shark (Carcharhinus limbatus). We found higher relative corticosteroid levels in adult nurse sharks during the pre-mating period and in blacktip sharks during the mating period. Higher levels of ß-hydroxybutyrate were found in adult nurse sharks during the mating period, but concentrations of this ketone body did not significantly vary across reproductive stages in blacktip sharks. We also detected reduced percentages of essential fatty acids during the mating period of both nurse and blacktip sharks. Taken together, our findings suggest that nurse and blacktip sharks differ in their energetic strategy to support reproduction, however, they likely rely on physiologically important fatty acids during mating, to support spermatogenesis.

Eutrophication effects on fatty acid profiles of seston and omnivorous fish in tropical reservoirs.

It has been postulated that eutrophication causes replacement of n3 highly unsaturated fatty acids (n3 HUFA) rich taxa, such as Bacillariophyta, Cryptophyta and Dinophyta, with taxa poor in these fatty acids (FA), such as Chlorophyta and Cyanobacteria. Such a change in community composition at the basis of the food web may alter the FA composition of consumer tissues. Here, we investigated the effects of eutrophication on phytoplankton composition and FA profiles of seston and muscle of two omnivorous fish species (Astyanax fasciatus and Astyanax altiparanae) from reservoirs of different trophic status in Southeast Brazil. The phytoplankton composition and seston FA profiles reflected the degree of eutrophication at most of the studied sites. Three of the five most eutrophic sites were dominated by cyanobacteria and had the highest saturated fatty acid (SFA) and lowest polyunsaturated fatty acid (PUFA) relative contents among all sites. In contrast, the remaining two sites presented a higher phytoplankton diversity and higher relative contribution of sestonic PUFAs with 18 carbons (C18) and HUFAs than less eutrophic systems. However, there were no clear effects of sestonic FA profiles on the FA profiles of muscle of both fish species. A higher percentage of n3 HUFAs was found in the fish samples from a hypereutrophic and cyanobacteria dominated reservoir than in those from sites with a more diverse phytoplankton community in which fish mainly showed higher percentages of C18 PUFA. These results suggest a lack of a direct relationship between the degree of eutrophication and the percentage of n3 HUFAs in both fish species, which can be caused by specific characteristics of the reservoirs that may modulate eutrophication effects. Therefore, consumer FA biochemistry seemed to be dictated by their ability to select, accumulate, and modify dietary FAs, rather than by the eutrophication degree of the studied tropical reservoirs.

Aluminum bioconcentration in female Nile tilapia Oreochromis niloticus (Perciformes: Cichlidae) and the effects on pituitary gonadotropins.

In this study, we measured aluminum (Al) bioconcentration in the brain, ovaries, and liver of Oreochromis niloticus females, and analyzed the effects of exposure to Al and acidic pH on the gene expression of follicle-stimulating hormone (ßfsh) and luteinizing hormone (ßlh) in these animals. Mature females were divided into 4 groups, thus being maintained for 96 h in one of the following conditions: control at neutral pH (Ctr); Al at neutral pH (Al); acidic pH (Ac), and Al at acidic pH (Al-Ac). pH alone did not influence Al bioconcentration in the brain. The animals from the Al-Ac group bioconcentrated more Al in the ovaries than those from the Al group, while no differences were observed in the liver. Aluminum bioconcentration was higher in the brain than in the liver and ovaries in Al-exposed animals (Al and Al-Ac), and higher in the brain than in the ovaries in the Ctr and Ac groups. The liver bioconcentrates more Al than the ovaries in the females from the Ctr and Ac groups. Aluminum and/or acidic pH did not alter ßfsh gene expression, while ßlh gene expression decreased in females from the Al group. Aluminum acted as an endocrine disruptor, suggesting deleterious effects in reproduction that could result in ovulation failure. Aluminum can act directly and/or indirectly in the pituitary, affecting ovarian steroidogenesis and altering the reproductive endocrine axis of mature O. niloticus females in an acute period of exposure.

Aluminum at environmental concentrations affects the sperm quality of the freshwater teleost Astyanax altiparanae: An in vitro study.

Aluminum (Al) is present in rivers and reservoirs in concentrations above that is allowed by regulatory agencies (e.g. 0.5 mg L-1 Al), which can impair fish reproduction. The present study evaluated the in vitro effects on the sperm of Astyanax altiparanae upon Al exposure at different concentrations (0, 0.05, 0.1, 0.3, and 0.5 mg L-1) with various exposure periods (50 s, 10 min, and 30 min). The following biomarkers were evaluated: membrane vitality, DNA fragmentation, morphology, kinetics (10 s and 30 s after sperm activation), and sperm mitochondrial activity. Al damages the membrane vitality of gametes at 0.3 and 0.5 mg L-1 after 50 s of exposure. After 30 min of exposure, there was a decrease in membrane vitality at 0.1 and 0.5 mg L-1, and the membrane vitality decreased with increased exposure time. Within 30 s after sperm activation, Al (0.3 and 0.5 mg L-1) reduced sperm motility by more than 50% at the longest exposure time, while at 0.1 and 0.5 mg L-1, Al exposure reduced motility over time. The average path speed (VAP; 10 s post-sperm activation) was reduced at longer exposure times at 0.05 and 0.5 mg L-1 of Al. Increased exposure time had deleterious effects on mitochondrial activity at the highest concentrations tested. Al did not damage DNA and sperm morphology. In conclusion, Al negatively influences the sperm quality of A. altiparanae with a potential effect of exposure time and increasing concentrations.

Aluminum bioconcentration in female nile tilapia Oreochromis niloticus (Perciformes: Cichlidae) and the effects on pituitary gonadotropins

In this study, we measured aluminum (Al) bioconcentration in the brain, ovaries, and liver of Oreochromis niloticus females, and analyzed the effects of exposure to Al and acidic pH on the gene expression of follicle-stimulating hormone (βfsh) and luteinizing hormone (βlh) in these animals. Mature females were divided into 4 groups, thus being maintained for 96 h in one of the following conditions: control at neutral pH (Ctr); Al at neutral pH (Al); acidic pH (Ac), and Al at acidic pH (Al-Ac). pH alone did not influence Al bioconcentration in the brain. The animals from the Al-Ac group bioconcentrated more Al in the ovaries than those from the Al group, while no differences were observed in the liver. Aluminum bioconcentration was higher in the brain than in the liver and ovaries in Al-exposed animals (Al and Al-Ac), and higher in the brain than in the ovaries in the Ctr and Ac groups. The liver bioconcentrates more Al than the ovaries in the females from the Ctr and Ac groups. Aluminum and/or acidic pH did not alter βfsh gene expression, while βlh gene expression decreased in females from the Al group. Aluminum acted as an endocrine disruptor, suggesting deleterious effects in reproduction that could result in ovulation failure. Aluminum can act directly and/or indirectly in the pituitary, affecting ovarian steroidogenesis and altering the reproductive endocrine axis of mature O. niloticus females in an acute period of exposure.

Spawning induction and embryonic development of Salminus hilarii (Characiformes: Characidae).

The present study aimed to evaluate two different methods of artificial reproduction induction, and characterize the embryonic development of Salminus hilarii. Different than observed for other tropical fish species, artificial reproduction induction followed by hand-stripping of gametes was considered unfeasible for S. hilarii, as no gamete fertilization was observed. However, females that were induced and spawned naturally presented a fertilization rate of 65.64 ± 0.54%. With a mean temperature of 26.20 ± 0.90ºC it was possible to clearly distinguish a large perivitelline space at 14 min after fertilization (AF) and at 49 min AF more than 50% of the embryos presented two blastomeres, and these cleavages occurred until 1 h 54 min AF. The gastrula phase was characterized at 3 h 56 min AF, and blastopore closure was observed at 8 h 31 min AF. At 9 h 1 min, organogenesis started, with a clear distinction of the yolk mass, embryonic axis, cephalic and caudal regions; at 11 h 51 min AF the embryos already had advanced segmentation and a free tail. Total hatching occurred at 21 h 17 min and after opening the mouth, which occurred at 33 h 9 min, the larvae of S. hilarii presented a strong and characteristic cannibalism. This information can be considered fundamental to improving S. hilarii production in captivity and for collaboration with a conservation programme in the upper Tietê river basin.

Fisiologia reprodutiva aplicada ao cultivo de peixes neotropicais / Reproductive physiology applied to neotropical fish rearing

A reprodução dos peixes teleósteos é regulada pela interação dos sistemas nervoso e endócrino, e essa interação é realizada pelo eixo hipotálamo-hipófise-gônadas (H-H-G). No entanto, sabe-se que os sinais ambientais (fotoperíodo, temperatura, pluviosidade entre outros fatores), são responsáveis por modular este processo. O eixo H-H-G, sintetiza e libera fatores internos (neuro-hormônios e neurotransmissores) e hormônios hipotalâmicos, hipofisários e gonadais que permitem a sincronização dos reprodutores aptos, que com condições ambientais adequadas permitem a reprodução em momento propício, com consequentemente maior sobrevivência da prole. O entendimento da fisiologia do eixo H-H-G tem sido muito importante para compreender os mecanismos regulatórios do controle neuroendócrino da reprodução em peixes teleósteos, possibilitando entender as razões do bloqueio da reprodução em cativeiro (piscicultura), ou quando a interrupção da migração reprodutiva (peixes reofílicos) por barragens é bloqueada, o que pode levar à extinção de espécies endêmicas. Esta revisão abordará o controle fisiológico do eixo H-H-G, com ênfase nas espécies neotropicais nacionais (espécies marinhas e dulciaquícolas) e discutirá as disfunções reprodutivas observadas nestes animais, quando em cativeiro. Neste contexto, a partir deste conhecimento teórico na fisiologia reprodutiva, a aplicação e/ou sugestões de tecnologias com o objetivo de obter sucesso na reprodução de espécies ameaçadas de extinção em cativeiro também serão abordados. É importante salientar que esta revisão não pretende cobrir todo o conhecimento sobre a fisiologia reprodutiva dos peixes teleósteos.

Teleost fish reproduction is regulated by the interaction of the nervous and endocrine systems, and this interaction is performed by the hypothalamic-pituitary-gonadal axis (H-H-G). However, it is known that environmental signals (photoperiod, temperature, rainfall, among other factors), are responsible to modulate this entire process. The H-H-G axis, synthesizes and releases internal factors (neurohormones and neurotransmitters) and hypothalamic, pituitary, and gonadal hormones that allow the synchronization of the able broodstocks and that, with adequate environmental conditions allow reproduction in the right time, and consequently, with a greater offspring survival. Understanding the physiology of the H-H-G axis has been important to understand the regulatory mechanisms of neuroendocrine control of reproduction in teleost fish, making it possible to understand the reasons for blocking reproduction in captivity (fish farming), or when the interruption of reproductive migration (reophilic fish) by dams is blocked, taking to the extinction of endemic species. This review will address the physiological control of the H-H-G axis, with an emphasis on National neotropical species (marine and freshwater species) and will discuss the reproductive dysfunctions observed in these animals when they are in captivity. In this context, from this theoretical knowledge about reproductive physiology, the application and/or suggestions of technologies with the objective of obtaining success in the reproduction of endangered species in captivity will also be considered. It is important to note that this review does not intend to cover all knowledge about the reproductive physiology of teleost fish.

Fisiologia reprodutiva aplicada ao cultivo de peixes neotropicais / Reproductive physiology applied to neotropical fish rearing

A reprodução dos peixes teleósteos é regulada pela interação dos sistemas nervoso e endócrino, e essa interação é realizada pelo eixo hipotálamo-hipófise-gônadas (H-H-G). No entanto, sabe-se que os sinais ambientais (fotoperíodo, temperatura, pluviosidade entre outros fatores), são responsáveis por modular este processo. O eixo H-H-G, sintetiza e libera fatores internos (neuro-hormônios e neurotransmissores) e hormônios hipotalâmicos, hipofisários e gonadais que permitem a sincronização dos reprodutores aptos, que com condições ambientais adequadas permitem a reprodução em momento propício, com consequentemente maior sobrevivência da prole. O entendimento da fisiologia do eixo H-H-G tem sido muito importante para compreender os mecanismos regulatórios do controle neuroendócrino da reprodução em peixes teleósteos, possibilitando entender as razões do bloqueio da reprodução em cativeiro (piscicultura), ou quando a interrupção da migração reprodutiva (peixes reofílicos) por barragens é bloqueada, o que pode levar à extinção de espécies endêmicas. Esta revisão abordará o controle fisiológico do eixo H-H-G, com ênfase nas espécies neotropicais nacionais (espécies marinhas e dulciaquícolas) e discutirá as disfunções reprodutivas observadas nestes animais, quando em cativeiro. Neste contexto, a partir deste conhecimento teórico na fisiologia reprodutiva, a aplicação e/ou sugestões de tecnologias com o objetivo de obter sucesso na reprodução de espécies ameaçadas de extinção em cativeiro também serão abordados. É importante salientar que esta revisão não pretende cobrir todo o conhecimento sobre a fisiologia reprodutiva dos peixes teleósteos.(AU)

Teleost fish reproduction is regulated by the interaction of the nervous and endocrine systems, and this interaction is performed by the hypothalamic-pituitary-gonadal axis (H-H-G). However, it is known that environmental signals (photoperiod, temperature, rainfall, among other factors), are responsible to modulate this entire process. The H-H-G axis, synthesizes and releases internal factors (neurohormones and neurotransmitters) and hypothalamic, pituitary, and gonadal hormones that allow the synchronization of the able broodstocks and that, with adequate environmental conditions allow reproduction in the right time, and consequently, with a greater offspring survival. Understanding the physiology of the H-H-G axis has been important to understand the regulatory mechanisms of neuroendocrine control of reproduction in teleost fish, making it possible to understand the reasons for blocking reproduction in captivity (fish farming), or when the interruption of reproductive migration (reophilic fish) by dams is blocked, taking to the extinction of endemic species. This review will address the physiological control of the H-H-G axis, with an emphasis on National neotropical species (marine and freshwater species) and will discuss the reproductive dysfunctions observed in these animals when they are in captivity. In this context, from this theoretical knowledge about reproductive physiology, the application and/or suggestions of technologies with the objective of obtaining success in the reproduction of endangered species in captivity will also be considered. It is important to note that this review does not intend to cover all knowledge about the reproductive physiology of teleost fish.(AU)

Ploidy influences on metabolic substrate deposition of rainbow trout

Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.(AU)

Ploidy influences on metabolic substrate deposition of rainbow trout

Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.

Resource partitioning between two young-of-year cownose rays Rhinoptera bonasus and R. brasiliensis within a communal nursery inferred by trophic biomarkers.

Although interspecific trophic interactions plays a principal role within elasmobranch communal nurseries, little is known over variation in foraging strategies adopted by young-of-year of sympatric species. To test the hypothesis of dietary resource partitioning between batoids within a communal nursery, we investigated two cownose ray species, Rhinoptera bonasus and R. brasiliensis, which occur in heterospecific groups, a strategy predicted to increase survival and foraging success. Using two biochemical tracers, fatty acids (FA) and stable isotopes (δ15 N and δ13 C), the combined effects of maternal investment and the formation of heterospecific groups implying competition for, or partitioning of available food resources were investigated. Through univariate and multivariate analyses of biochemical tracers in several tissues (fin clip, muscle, liver, red blood cells; RBC) and plasma, our results revealed significant interspecific differences in tracers between the two species. Total FAs (∑saturated FA, ∑monounsaturated FA and ∑polyunsaturated FA) and trophic biomarkers (i.e., docosahexaenoic acid, arachidonic acid, oleic acid and δ15 N) were the principle tracers responsible for the differences detected. These data revealed that R. brasiliensis was less enriched in physiologically important essential FAs than R. bonasus. Our findings suggest that these congeneric species differ in maternal investment strategy and moderately partition food resources over relatively fine spatial scales within a single nursery habitat to limit competition. These results provide further knowledge on the foraging strategies adopted by batoids in communal nursery areas, information that is required for improving spatial conservation and management planning.

Development and characterization of 20 polymorphic microsatellite markers for Epinephelus marginatus (Lowe, 1834) (Perciformes: Epinephelidae) using 454 pyrosequencing.

The dusky grouper, Epinephelus marginatus, is a well-known and widespread marine fish assessed as endangered by the International Union for the Conservation of Nature. Analyzing the genetic diversity of this species is, therefore, of utmost importance and necessary for conservation purposes. Microsatellites are molecular tools with advantages that are ideal for population analyses. This study provides the first set of species-specific microsatellite loci for E. marginatus that can be applied when assessing both intra- and interpopulation genetic variation. Twenty microsatellite loci were isolated and characterized for the dusky grouper by genotyping 20 individuals obtained from the North Eastern Atlantic Ocean (n = 4) and from the South Western Atlantic Ocean (n = 16). The number of alleles per locus varied from 2 to 11, while the observed and expected heterozygosities ranged from 0.25 to 0.94 and 0.34 to 0.89, respectively. The polymorphic information content varied from moderately to highly informative. This suite of markers provides the first specific nuclear tools for E. marginatus and, thus, allows to assess with more specificity different populations' structures.

Gonadal steroids and energy availability during ovarian maturation stages of the Amazonian pirarucu Arapaima gigas (Teleostei: Osteoglossidae) in the wild.

The Amazonian pirarucu, Arapaima gigas, belongs to a phylogenetically basal group of teleosts (family Osteoglossidae). The commercial importance and the overexploited status of natural stocks of A. gigas brought the interest to rear it in the Amazon region, but rearing techniques are not fully understood to obtain reproduction success. Gonadal steroids (plasma and cephalic liquid), lipids and proteins (muscles, liver and ovary) of wild A. gigas females were analyzed during one year on Mexiana Island (Amazon Basin, Brazil) and ovarian maturation stages were described. The sexual gender determination method based on 11-ketostestosterone (11KT) and estradiol (E2) ratio was applied to check if the value is in agreement of the sex previously determined by direct observation. Adult females were classified into four reproductive stages according to ovary development stage: previtellogenic, maturation, mature, and regression/spawning. Immature females were also analyzed. All mature females were sampled during the rainy season. Lipids stored in the ventrecha (belly) are a possible source of this substrate for the developing ovary. Plasma steroid profile throughout A. gigas female reproductive stages is similar to that found in most teleosts, but plasma 11KT levels peaked at the mature stage, suggesting an important role of this androgen in female ovulation. The 11KT/E2 method was 100% efficient when sexing juveniles and adults at the beginning of their reproductive cycle and can successfully be applied to identify the sexual gender of animals. The steroid profile of the cephalic liquid suggests a possible role of this fluid as a pheromone, due to the high level of 17α-hydroxyprogesterone at the final stages of the reproductive cycle.

Monitoring whole blood, plasma and serum variables of Nile tilapia during 24 hours, after capture stress / Monitoramento das variáveis do sangue total, plasma e soro de Tilápia-do-Nilo, durante 24 horas, após estresse de captura

Stress is know as responsible for the decrease in immunity and reduced survival, growth, and reproductive capacity of animals. Besides, organisms respond to certain biological rhythms, such as circadian, to which animals naturally adjust. This study aimed to monitor the plasma (lactate and total protein), serum (cortisol and cholesterol) and whole blood (glucose) variables in tilapia in a 24 h cycle. A set of 49 aquariums with 5 individuals each were used. All of them, at the beginning of the experiment, were subjected to the same stress of capture and handling. The animals from the first aquaria were sampled after 15 min of stress. Blood samples from animals of the remaining aquarium were sequentially drawn at 30-min intervals. Radar charts were used to visualize the hourly change in the measured variables. Significant differences of each analysis between the measured variables by aquarium and the phases of the circadian cycle (light-dark) and morning-afternoon-night-dawn intervals, were evaluated using the Mann-Whitney and Kruskal-Wallis tests, respectively. A positive relationship between cortisol and glucose levels was found. Most analysis, except cholesterol and lactate did not show any significant difference between light and dark periods, and the intensity and frequency of peaks observed were not deleterious to individuals.

O estresse é conhecidamente responsável pela diminuição da imunidade e redução da sobrevivência, crescimento e capacidade reprodutiva dos animais. Além disso, os organismos respondem a certos ritmos biológicos, como o circadiano, aos quais se ajustam naturalmente. Este estudo teve como objetivo monitorar as variáveis plasmática (lactato e proteína total), soro (cortisol e colesterol) e sangue total (glicose) na tilápia em um período de 24 h. Quarenta e nove aquários com 5 indivíduos cada foram utilizados. Todos eles, no início do experimento, foram submetidos ao mesmo estresse de captura e manuseio. Os animais do primeiro aquários foram amostrados após 15 min do estresse. Os demais As amostras de sangue de indivíduos de cada aquário foram sequencialmente em intervalos de 30 min. Somente os indivíduos do primeiro aquário foram analisados após 15 minutos de estresse. Os gráficos de radar foram usadas para visualizar a variação horária nas variáveis medidas. Diferenças estatisticamente significativas entre a média dos indivíduos de cada aquário, para as fases do ciclo circadiano (luz-escuro) e os intervalos manhã-tarde-noite-madrugada foram respectivamente avaliadas utilizando os testes Mann-Whitney e Kruskal-Wallis. Observou-se relação positiva entre os níveis de cortisol e glicose. As análises, com exceção de colesterol e lactato, não mostraram diferença significativa entre períodos de luz e sombras, e a intensidade e a freqüência dos picos observados não foram prejudiciais aos indivíduos.