Neurotoxic, biotransformation, oxidative stress and genotoxic effects in Astyanax altiparanae (Teleostei, Characiformes) males exposed to environmentally relevant concentrations of diclofenac and/or caffeine.

The present study evaluated neurotoxic, biotransformation, genotoxic and antioxidant responses to relevant environmental concentrations of diclofenac (0.4 µg L-1) and caffeine (27.5 µg L-1), separate and combined, in adult males of the freshwater fish Astyanax altiparanae after a subchronic exposure (14 days). Fish exposed to diclofenac and caffeine, both separate and combined, revealed a neurotoxic effect through the inhibition of acetylcholinesterase activity in the muscle, while diclofenac alone and in combination caused cyclooxygenase inhibition. Caffeine alone produces genotoxicity on this species but, when combined with diclofenac, it potentiates hepatic lipoperoxidation and the inhibition of oxidative stress enzymes, while diclofenac alone or in combination produces a general inhibition of important enzymes. This study suggests that aquatic contamination produced by these pharmaceuticals has the potential to affect homeostasis and locomotion in A. altiparanae and compromise their immune system and general health.

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.

Nonsteroidal anti-inflammatory drugs act as endocrine disruptors in Astyanax lacustris (Teleostei: Characidae) reproduction: An ex vivo approach.

Pharmaceutical products can act as endocrine-disrupting compounds (EDCs), affecting the physiological processes of animals, such as development or reproduction. This study aimed to investigate the influence of different concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac (DCF) and ibuprofen (IBU) alone and mixed (MIX) on gonadotropin gene expression and gonadal steroid release using Astyanax lacustris pituitary and testes explant systems, respectively. The explant organs were maintained for 12 h in Leibovitz (L-15) medium supplemented with 0, 0.1, 1, 10, 100, and 1000 ng L-1 of DCF, IBU, and MIX (ratio 1:1 of the same concentrations of DCF and IBU alone) and gonadotropin releasing-hormone (cGnRH2) stimulation in pituitary explants and human chorionic gonadotropin (hCG) stimulation in testes explants. The pituitary glands and the media from the testicular explants were collected for gene expression analysis including the ß subunit of the follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) and secreted gonadal steroid concentration analysis, respectively. Both DCF and IBU (alone and mixed) decreased pituitary gene expression of fshß and lhß and this inhibitory effect was evident even at low concentrations. In the testes, DCF and IBU did not change the levels of estradiol, and both pharmaceuticals increased the release of 11-ketotestosterone at low doses, while only IBU decreased the levels of testosterone in all concentrations. IBU's inhibitory effect in the testes was not triggered by the mixture of the two drugs. These results suggest that NSAIDs, may interfere in fish reproduction by acting as EDCs, thereby negatively affecting A. lacustris spermatogenesis and maturation.

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.

Diclofenac and caffeine inhibit hepatic antioxidant enzymes in the freshwater fish Astyanax altiparanae (Teleostei: Characiformes).

Although concentrations of pharmaceutical compounds in aquatic ecosystems are low, they can cause toxic effects on organisms. The aim of this study was to evaluate the effects of diclofenac (DCF), a non-steroidal anti-inflammatory drug, and caffeine (CAF), a central nervous system stimulant, both alone or combined, in Astyanax altiparanae males under acute exposure (96 h), measuring neurotoxicity biomarkers, antioxidant response and damage at biochemical and cellular levels. DCF concentration in water, separated and combined, was 3.08 mg L-1 and that of CAF was 9.59 mg L-1. To assess neurotoxicity, brain and muscle acetylcholinesterase (AChE) activities were measured. To evaluate oxidative stress, the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST), as well as lipoperoxidation (LPO), were analyzed in liver and gills. Activity of hepatic cyclooxygenase (COX) was also evaluated. Genotoxicity was assessed in blood using comet assay and micronucleus test, as well as nuclear abnormalities. DCF and CAF, alone or combined, had neither effect on AChE activity, nor in the activity of SOD, CAT, GPx and GST in gills. In liver, DCF inhibited SOD and GPx activity, CAF inhibited CAT activity, the mixture inhibited SOD and GST activity; although only fish exposed to CAF showed increased hepatic LPO. Under these experimental conditions, no effect on COX activity was observed, nor cytotoxic and genotoxic damage. The most pronounced effects were caused by the drugs separately, since both compounds altered the enzymes, but only CAF triggered LPO, showing more harmful effects.

Paternal exposure to aluminum, acidity, and temperature affect fatty acid seminal profile, embryonic and larval development of Astyanax altiparanae.

We investigated the effects of water acidity, temperature, and aluminum (Al) on the fatty acid (FA) seminal profile, reproductive parameters (fertilization and hatching) and embryonic development of Astyanax altiparanae. We treated males with different experimental treatments, corresponding to the combination of water temperature (20 °C; 25 °C), pH (neutral - 7.0; acidic - 5.5), and the absence or presence of Al (0.5 mg L-1). After 96 h, we analyzed the FA profile of semen and performed artificial fertilization in activating medium with neutral pH or activating medium in the same experimental conditions of the males (neutral pH, acidic pH, and Al) to evaluate fertilization and hatching rates and to monitor embryonic development. Polyunsaturated FA percentage decreased in semen of fish from the neutral group, while monounsaturated FA increased in all groups maintained at 20 °C compared to 25 °C. Aluminum exposure decreased the percentage of C20:4n6 and increased the percentage of C22:5n3 at 20 °C. Males exposed to acidic pH and Al showed lower fertilization and hatching rates, as well as increased mortality of embryos and larvae. Moreover, Al favoured a higher percentage of abnormal larvae. Fertilization in Al activating medium harmed the embryos and larvae since fertilization and hatching rates decreased. Finally, temperature influenced fertilization time, hatching rate, and the morphology of embryos and larvae. Males exposed to Al had lower fertilizing capacity, which negatively affected the embryonic development of the species. Furthermore, Al activating medium reduced the number of fertilized oocytes, hatched embryos, and normal larvae. All events were temperature dependent.

Effects of GnRH and the dual regulatory actions of GnIH in the pituitary explants and brain slices of Astyanax altiparanae males.

The pituitary gonadotropins, Fsh (follicle-stimulating hormone) and Lh (luteinizing hormone), regulate testicular development and functions in all vertebrates. At the pituitary, different signaling systems regulate the synthesis and secretion of the gonadotropins, such as the hypothalamic neuropeptides GnRH (gonadotropin-releasing hormone) and GnIH (gonadotropin-inhibitory hormone). While GnRH exerts stimulatory roles, the actions of GnIH remain controversial for many teleost species. Therefore, the aim of this study was to evaluate the in vitro effects of chicken GnRH2 (cGnRH2) and zebrafish GnIH-3 (zGnIH-3) on the male gonadotropin and GnRH system expression using pituitary explants and brain slices from a neotropical species with economical and ecological relevance, Astyanax altiparanae. Our results showed that in males, cGnRH2 increased fshb and lhb mRNA levels in the pituitary explants. Interestingly, zGnIH-3 has no effect on basal gonadotropin expression, however zGnIH-3 decreased the cGnRH2-induced fshb and lhb transcripts in male pituitary explants. In the male brain slices, zGnIH-3 showed stimulatory effects, increasing gnrh2 mRNA levels. Overall, our results suggested that GnIH seems to have dual regulatory actions on gonadotropin and GnRH2 expression of A. altiparanae males. This study provided basic information on endocrine regulation of A. altiparanae reproduction, and the obtained results will expand our knowledge, improving the reproductive management of this economically important freshwater species.