Histopathology and changes in the expression of metallothioneins, heat shock proteins and inducible nitric oxide synthase in Prochilodus costatus from a neotropical river contaminated by heavy metals.

The most recent dam rupture in Brazil released tons of mining tailings into the upper course of the Paraopeba River, affecting this river in an unprecedented way. The present study aimed to evaluate the influence of heavy metals on Prochilodus costatus, an important commercial species in Brazil, four years after the dam colapse. To this end, biomarkers of heavy metals, oxidative stress, and environmental stress were analyzed, and histological analyses of target organs were performed. The results demonstrated critical contamination of fish from the Paraopeba River. Increased expression of Metallothioneins - MTs, Heat Shock Protein - HSP70, and inducible nitric oxide synthase - iNOS, as well as greater rates of histological changes in the liver, spleen, and gonads, were observed in P. costatus. These findings demonstrate that, despite past contamination, the metals present in mining tailings have significantly increased the contamination of the Paraopeba River basin.

Reproductive toxicity induced by lead exposure: Effects on gametogenesis and sex steroid signaling in teleost fish.

Lead (Pb) is an emerging contaminant widely distributed in aquatic environments, which has serious effects on human and animal health. In this study, we determined whether Pb exposure affects gametogenesis, sex steroids, estrogen (ERα and ERß), and androgen (AR) receptors. Adult specimens of Astyanax bimaculatus were exposed in duplicate to 15, 50, and 100 µg/L of lead acetate, whereas the control group was not exposed. After 28 days of exposure, fish were euthanized and samples of the gonads, liver, and blood were collected for analysis. The results indicated a reduction in the gonadosomatic index as well as the diameters of the vitellogenic follicles and seminiferous tubules in the exposed groups. Morphometry of gametogenesis revealed inhibition of the secondary oocyte growth and a reduction in the number of spermatozoa in the 50 and 100 µg/L Pb-treated groups. In females, plasma 17ß-estradiol (E2) increased following 15 and 50 µg/L Pb treatment, whereas males exhibited an increase in E2 and 11-ketotestosterone following treatment with 15 and 100 µg/L Pb, respectively. Vitellogenin was significantly reduced in females exposed to 100 µg/L Pb, but metallothionein levels were unchanged. ERα, ERß, and AR were immunolocalized in the somatic and germ cells, with increased ovarian expression of ERα and Erß in the 100 µg/L Pb-treated group, but no significant difference in AR among the groups. In males, only ERα increased in the 100 µg/L Pb-treated group. These results indicate that Pb exposure impairs gametogenesis, disrupts estrogen receptor signaling, and affects the expression of major reproductive biomarkers in A. bimaculatus.

Adverse effects of herbicides in freshwater Neotropical fish: A review.

Although herbicides have been developed to act on the physiological processes of plants, they are responsible for causing deleterious effects on animals. These chemical compounds are widely used throughout the world, but especially in countries that export agricultural products such as Central and South America, their use has increased in recent years. Aquatic environments are natural reservoirs of herbicides, which after being applied on crops, run off through the soil reaching rivers, lakes, and oceans. Fish are among the many organisms affected by the contamination of aquatic environments caused by herbicides. These animals play an important ecological role and are a major source of food for humans. However, few studies address the effects of herbicides on fish in this region. Thus, in the present review we discuss the morphophysiological and molecular consequences of herbicide exposure in Neotropical fish systems as well as how the environmental and land use characteristics in this region can influence the toxicity of these pollutants. A toxicity pathway framework was developed summarizing the mechanisms by which herbicides act and endpoints that need to be further investigated.

Morphological analysis of Prochilodus argenteus ovaries in successful and unsuccessful hypophysation-induced reproduction.

Pampus argenteus is a species of economic importance for professional fishing and aquaculture. To analyse the quality of oocytes of P. argenteus, 10 females were kept in tanks of 200 m2 with an average depth of 1 m in the Integrated Center of Fishing Resources and Aquaculture of Três Marias - CODEVASF. Specimens were submitted to hypophysation-induced reproduction with crude extract of common carp pituitary (EBHC). Females received two doses (0.8-1.0 and 5.0-6.6 mg of EBHC/kg body weight, respectively), with a 12-h interval between doses. For males, a single dose (2.7 mg/kg body weight) was applied at the same time as the females' second dose. Oocytes were extruded manually 8 h after the second hormonal dose at 26°C. It was observed that seven females responded positively to the procedure while the other three released bloody and lumpy oocytes. For histological analysis, ovarian fragments were fixed in Bouin's liquid for 8-12 h and submitted to routine histological techniques. The protocol was considered successful for 70% of females and 100% of males. The fertilization rate of the females from the unsuccessful group was very low, and histological analyses showed that most of their oocytes were in follicular atresia, suggesting a delay in hormonal administration or extrusion could have occurred. Despite the hypophysation protocol being considered adequate for the induced reproduction of P. argenteus, complementary studies are necessary to evaluate the possible causes of this degenerative process.

Comparative analysis of the small and large intestines of Acará Geophagus brasiliensis (Quoy & Gaimard, 1824) (Pisces: Cichlidae).

Geophagus brasiliensis, popularly known as acará, is a common fish in lentic freshwater environments in South America. This species has a detritivorous-iliophagous or omnivorous feeding habit, with high food plasticity; however, there are no studies describing its intestinal tract histologically. Therefore, the present study analysed through histological and histochemical techniques the intestines of the acará. Adult specimens were collected with gillnets, anaesthetized and euthanized. Then, the fish were submitted to biometry and dissection to remove fragments of intestines. The samples were fixed in Bouin liquid for 12 hours and subjected to histological and histochemical techniques. Histologically, all samples of intestines were organized into four layers: mucosa, submucosa, muscular and serosa. The small intestine (foregut and midgut) was characterized by the presence of intestinal villi covered by simple prismatic epithelium with a striated border and goblet cells supported by the connective lamina propria. In the large intestine (hindgut), there was an absence of villi and an abundance of goblet cells. Positive reaction to Periodic Acid-Schiff (PAS) and Alcian Blue (AB) pH 2.5 reactions were detected in goblet cells, indicating the presence of mucosubstances. No lipids were detected in the intestine cells due to the negative reaction to the Sudan Black B. The results of the present study provide subsidies for a better understanding of the intestinal morphology of teleosts and provide valuable information for phylogenetic studies.

Biomarker responses induced by bisphenol A on spermatogenesis in a Neotropical teleost fish are temperature-dependent.

Bisphenol A (BPA) is an organic synthetic compound used in the plastic industry with endocrine disrupting activity. Although it is frequently found in surface waters, few studies have investigated its impact on fish gametogenesis, particularly when associated with natural stressors. In this regard, the present study evaluated BPA toxicity on spermatogenesis in the lambari Astyanax bimaculatus under controlled conditions and its interactive effects with water temperature. Adult specimens were exposed in duplicate to 40 µg/L and 400 µg/L BPA at 23 °C and 28 °C for 21 days; the control group did not receive BPA. Testicular samples were collected and analyzed using different cellular and molecular techniques. The results showed a significant reduction in the gonadosomatic index in the BPA-treated groups at both temperatures. A decrease in the testicular levels of 11-ketotestosterone was observed in the 400 µg/L BPA group at 23 °C, 17ß-estradiol increased significantly in the treated groups at 28 °C, and vitellogenin showed no difference between the treatments. The morphometric analysis of spermatogenesis revealed a significant increase in the proportion of spermatogonia, spermatocytes, and Sertoli cells in the treated groups, with a higher proportion at 23 °C than at 28 °C. Otherwise, the proportion of spermatozoa was significantly lower in the BPA-treated groups, with a greater reduction at 23 °C. In addition, BPA also stimulated spermatogonial proliferation in the treated groups, but apoptosis was significantly increased in spermatids at 23 °C. Testis-ova, cell degeneration, and chromatin alterations in spermatids and Sertoli cells were observed in the germinal epithelium of the BPA-treated groups. The integrated biomarker response (IBR) index revealed that the analyzed endpoints are suitable for assessing estrogenic contamination. Taken together, our results indicate that the interactive effects of BPA and temperature contribute to the impairment of spermatogenesis in A. bimaculatus with more severe effects observed on sperm production at 23 °C than at 28 °C.

Heavy metals affecting Neotropical freshwater fish: A review of the last 10 years of research.

The expansion of urban centers, the increase in the use of chemicals in agriculture, and the significant expansion of industry has dumped increasing amounts of heavy metals into aquatic ecosystems, directly and indirectly affecting the entire food web in freshwater environments. In freshwater fish, many studies conducted worldwide have shown that these pollutants can cause alterations from the molecular level to behavioral, in all stages of life, including in future generations. Despite the great diversity of freshwater fish in the Neotropical region, few studies addressing the influence of metals on native species have been carried out. The last decade of studies showed that the effects of metals in neotropical species can be from the molecular to the behavioral level. Metals like Arsenic, Cadmium, Copper, Lead, Mercury, Chromium and Zinc have often been detected in environmental studies and evaluated in experimental approaches. Metabolically active organs showed higher levels of accumulation. In addition, some environmental studies have shown that ecologic and trophic conditions can be determinant in the level of exposure to metals. Oxidative stress, genotoxicity and several histopathological alterations were the most recurrent effects detected in target organs.

Influence of temperature on larval ontogenesis of Geophagus brasiliensis (Quoy & Gaimard, 1824) (Pisces: Cichlidae).

Studies on the larval development of fish are essential for conservation and improvements in cultivation techniques. Geophagus brasiliensis popularly known as Cará has potential as a fish of interest in ornamental aquaculture. Wild adults of G. brasiliensis were kept in an aquarium for spontaneous reproduction. Newly hatched larvae were transferred to 5-litre aquaria at 22, 26 and 30°C until total yolk sac resorption. Histological slides were used for biometric analysis and monitoring of larval ontogenesis at different temperatures. Histologically, from the first to the fourth days it was possible to identify myomeres, optic vesicle, yolk syncytial layer, brain, heart and differentiation of the eye layers. From the fourth to the seventh days, it was possible to identify mandibular and gill cartilages, swim bladder, liver, prismatic epithelium with striated border in intestine and renal epithelium. All biometric measurements increased over the days, except height and length of the yolk sac, which gradually decreased until the complete resorption of the yolk sac that occurred on the fifth day at a temperature of 30°C, the sixth day at 26°C and the seventh day at 22°C. Morphological events at 30°C such as the reabsorption of the yolk sac, the appearance of cartilage in the branchial arches and differentiation of the layers of the eyes occurred faster compared with the other temperatures tested. Opening of the mouth and digestive tract occurred at a similar time on the fourth day in all temperatures.

Evaluation of the oestrogenic potential of oestrone and bisphenol-A on the reproduction of Astyanax bimaculatus males after subacute exposure.

In the last decades, oestrogenic compounds have often been reported in environmentally relevant concentrations in aquatic environments around the world. Most laboratory studies of oestrogens try to understand the effects of a single contaminant, but in natural environments, the effects may be quite different due to interactions with other compounds. The present study aimed to compare the action of oestrone (E1) and bisphenol-A (BPA), acting singularly and in combination, on the spermatogenesis of Astyanax bimaculatus. After exposure to 100 ng/L of E1, BPA and a mixture of the two for 15 days, our results showed that E1 and the E1 + BPA mixture significantly altered the number of spermatogenic cells. BPA presented high cytotoxicity when compared to other treatments. Analysis of the two oestrogenic compounds suggests that the E1 + BPA mixture has no additive or synergistic effects. Together, the results of the present study indicate that endocrine-disrupting chemicals (EDCs) analysed alone may behave differently than when administered with other substances.

Induced reproduction and early development in dourado, Salminus franciscanus Lima & Britski, 2007 (Pisces: Characiformes).

Our study aimed to establish the response of Salminus franciscanus to hypophysation and describe the main morphological events of its embryonic process. Wild fish were captured in São Francisco River and selected broodstock (females: 66.4 ± 11.1 cm and 4.04 ± 2.32 kg; males: 58.3 ± 10.2 cm and 3.62 ± 1.12 kg) were kept at 26.1 ± 0.6°C for induction of final maturation/gamete release via the hypophysation technique. In females, two doses (0.8 and 5.6 mg/kg body weight) of crude pituitary extract of common carp (Cyprinus carpio) were administered with a 14 h interval. For males, a single dose (2.7 mg/kg body weight) of crude pituitary extract was applied at the same time as the females' second dose. Oocytes and sperm were manually stripped 8 h after a females' second hormonal dose. Fertilization was carried out using the dry method. Eggs were kept in funnel-type 60 L incubators at 24.3 ± 0.3°C and were analyzed and photographed every 10 min. After hormonal induction, 60% of females and 100% of males reacted positively and no broodstock mortality was recorded. The females released an average of 385.2 ± 78.4 g of oocytes and the fertilization rate observed was 50.4 ± 12.3%. The blastopore closure occurred at 7.5 h, somite formation at 12 h and hatching at 20 h post-fertilization. In general, the results of this study improve the understanding of the reproductive biology of dourado and confirm its potential for fish farming in the neotropical region.

Heavy metal contamination in a highly consumed Brazilian fish: immunohistochemical and histopathological assessments.

Due to industrial, rural, and domestic waste disposal, heavy metals such as cadmium (Cd), chromium (Cr), lead (Pb), zinc (Zn), copper (Cu), and iron (Fe) continually infiltrate aquatic environments. These pollutants do not degrade naturally and, thus, have a high capacity for bioaccumulation in tissues and organs. The present study uses histological and immunohistochemical analyses to evaluate the contamination status of Salminus franciscanus, a large and economically important fish. Levels of Cd, Cr, Pb, Zn, Cu, and Fe were evaluated by atomic absorption spectrometry in the liver and muscle of fish sampled from two tributaries of the upper São Francisco River Basin, Brazil: the Abaeté and Paraopeba Rivers. In addition, histopathological alterations and expressions of three environmental biomarkers were assessed: metallothionein (MT), heat shock protein-70 (HSP70), and cytochrome P450-1A (CYP1A). The results show that fish from the Paraopeba River are unsuitable for human consumption, with several metals being detected above the safe limits established by the World Health Organization. Histopathological alterations in the liver and spleen were also significantly more frequent in fish from the Paraopeba River than in those from the Abaeté River (P < 0.05). Significant differences in the expressions of environmental biomarkers were observed between the rivers. Fish from the Abaeté River presented significantly higher values of the gonadosomatic index (GSI) and lower levels of metal contamination in the liver and muscle.

Reproduction of pimelodus maculatus (siluriformes: pimelodidae) in three section of grande river basin, downstream Porto Colombia dam, south-eastern Brazil

The alterations to the hydrologic regime downstream from hydroelectric dams may cause an impact on the reproductive success of fishes. This study aimed to analyse the influence of the physical and chemical parameters of the water of the Grande and Pardo Rivers on gonadal maturation, oocyte diameter, follicular atresia and biological indices of Pimelodus maculatus collected from three river sections: Grande River, downstream from the Porto Colômbia dam (S1), Grande River, downstream from the confluence with the Pardo River (S2) and in the Pardo River channel (S3). Males and females captured in S1 presented significantly higher average values for total length and body weight than those captured in S2 and S3. The gonadosomatic index values were significantly higher in fish collected in S3 and the Fulton condition factor did not show significant differences in fish collected from the three sections. The oocyte diameter, the follicular cells height and the zona pellucida thickness did not show any statistical differences between the sections. Conductivity presented a significant difference between S1 and S3 and during the reproductive period, water transparency presented similar values in the two sampling sections of the Grande River, but a much lower value in the Pardo River. A low frequency of fish with reproductive activity was registered in S1, whereas in S2 and S3 higher frequencies were recorded, emphasising the need of preserving the tributaries for the reproductive success of P. maculatus of the Grande River in south-eastern Brazil.

A jusante das barragens hidrelétricas ocorrem alterações no regime hidrológico que podem causar impactos sobre o processo reprodutivo dos peixes. O objetivo do presente estudo foi analisar a influência de parâmetros físico-químicos da água do rio Grande e rio Pardo sobre a maturação gonadal, diâmetro ovocitário, atresia folicular e índices biológicos de Pimelodus maculatus coletados em três trechos: a jusante do reservatório de Porto Colômbia (P1), a jusante da confluência do rio Grande com rio Pardo (P2) e na calha do rio Pardo, a aproximadamente 100 km de P2 (P3). Machos e fêmeas capturados no ponto 1 apresentaram valores médios de comprimento total e peso corporal significativamente maiores do que aqueles capturados nos pontos 2 e 3. Os valores de IGS foram significativamente maiores nos peixes coletados no ponto 3 e o fator de condição de Fulton não apresentou diferenças significativas entre os peixes coletados nos três pontos. O diâmetro ovocitário, a altura das células foliculares e a espessura da zona pelúcida dos ovócitos vitelogênicos não mostraram diferenças estatísticas entre os pontos. A condutividade apresentou diferença significativa entre os pontos 1 e 3 e a transparência da água durante o período reprodutivo apresentou valores próximos nos dois pontos amostrais do rio Grande, e valor bem menor no ponto amostral do rio Pardo. Em P1 registrou-se baixa frequência de peixes em atividade reprodutiva, e em P2 e P3 maiores frequências de peixes reproduzindo. Os resultados encontrados enfatizam a necessidade de conservação de tributários para o sucesso reprodutivo de P. maculatus, do rio Grande no sudeste do Brasil.

Profiles of sex steroids, fecundity and spawning of a migratory characiform fish from the Paraguay-Paraná basin: a comparative study in a three-river system.

This study investigated for the first time the reproductive biology of Prochilodus lineatus in a system of rivers in southeastern Brasil, relating it to the role of tributary rivers in the reproductive success of this important commercial fish in the Upper Paraná River basin, where a cascade of hydroelectric dams were deployed. Specimens were caught bimonthly in three river sites: (S1) Grande River, downstream from the Porto Colômbia dam; (S2) Pardo River; and (S3) Mogi Guaçu River. Sex steroid plasma levels, fecundity, follicular atresia, oocyte diameter and gonadosomatic index (GSI) were compared among sites. In S1, fish exhibited changes in the reproductive parameters: lower GSI, oocyte diameter and fecundity and higher follicular atresia index, when compared to S2 and S3. Frequency of maturing fish was higher in S3 and spawning was only registered in S3. In sites S2 and S3, plasma concentrations of testosterone and 17ß-estradiol in females and testosterone in males showed wide variations following gonadal maturation. Fish from S1 showed few significant variations in sex steroid concentrations throughout the gonadal cycle. These results indicate that P. lineatus does not reproduce in Grande River (S1), but probably uses the Pardo River (S2) as a migratory route towards the Mogi Guaçu River (S3) where they complete gonadal maturation and spawning. Our findings contribute for understanding the reproductive biology of P. lineatus and to highlight the importance of tributaries in impounded rivers as a favourable environment for migration and spawning of fish.