Different response of females and males Neotropical catfish (Rhamdia quelen) upon short-term temperature increase.

Climate change has been one of the most discussed topics in the world. Global warming is characterized by an increase in global temperature, also in aquatic environments. The increased temperature can affect aquatic organisms with lethal and sublethal effects. Thus, it is necessary to understand how different species respond to temperature. This study aimed to evaluate how the Neotropical catfish species Rhamdia quelen responds to temperature increases. The fish were exposed to temperatures of 25 °C (control) and 30 °C after gradual temperature increase for 7 days. After 96 h in each temperature, the fish were anesthetized, blood was collected, and after euthanasia, brain, liver, posterior kidney, gills, muscle, and gonads were collected. The gonads were used for sexing, while other tissues were used for the hematological, biochemical, genotoxic, and histopathological biomarkers analysis. Hepatic proteomic analysis with a focus on energy production was also carried out. Blood parameter changes in both sexes, including an increase in glucose in males, leukopenia in females, and genotoxicity in both sexes. Hepatic proteins related to energy production were altered in both sexes, but mainly in males. Others biomarker alterations, such as histopathological, were not observed in other tissues; however, the antioxidant system was affected differently between sexes. These showed that R. quelen juveniles, at temperatures higher than its optimum temperature such as 30 °C, has several sublethal changes, such as hematological alterations, antioxidant system activation, and energetic metabolism alteration, especially in males. Thus, short-term temperature rise can affect females and males of R. quelen differently.

Molecular and biochemical evaluation of effects of malathion, phenanthrene and cadmium on Chironomus sancticaroli (Diptera: Chironomidae) larvae.

In-vitro effects of sub-lethal concentrations of malathion, phenanthrene (Phe) and cadmium (Cd) were tested on Chironomus sancticaroli larvae in acute bioassays by measuring biochemical and molecular parameters. Malathion was evaluated at 0.001, 0.0564 and 0.1006 mg L-1; Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 0.001, 3.2 and 7.4 mg L-1. The recovery test carried out at the highest concentration of each compound showed that survival of larvae exposed to Phe ranged from 4% to 5%, while the effects of malathion and Cd were irreversible, not allowing the emergence of adults. Results showed that malathion and Cd inhibited AChE, EST-α and ES-ß activities at the two highest concentrations. Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 3.2 and 7.4 mg L-1 inhibited glutathione S-transferase activity. Oxidative stress was exclusively induced by the lowest concentration of malathion considering SOD activity once CAT was unaffected by the stressors. Lipid peroxidation was registered exclusively by malathion at the two highest concentrations, and total hemoglobin content was only reduced by Cd at the two highest concentrations. The relationship among biochemical results, examined using the PCA, evidenced that malathion and Cd concentrations were clustered into two groups, while Phe only formed one group. Four hemoglobin genes of C. sancticaroli were tested for the first time in this species, with Hemoglobin-C being upregulated by malathion. The toxicity ranking was malathion > Phe > Cd, while biochemical and molecular results showed the order malathion > Cd > Phe. Our results highlight the importance of combining different markers to understand the effects of the diverse compounds in aquatic organisms.

Sublethal effects of microcystin-LR in the exposure and depuration time in a neotropical fish: Multibiomarker approach.

Eutrophication is an ecological process that results in cyanobacterial blooms. Microcystin-LR is the most toxic variant of microcystins and may cause toxic effects in the organisms, mainly in hepatic tissues. The aims of this study were to use multiple biomarkers in order to evaluate the sublethal effects of a low concentration of MC-LR (1 µg/L) in fish Geophagus brasiliensis by waterborne exposure; and evaluate the depuration of this toxin during 15 days. A group of 30 fish was exposed to 1 µg/L of MC-LR solution for 96 h in a static bioassay. After this time, blood, brain, muscle, liver, gonad and gills were collected from half of the exposed fish group in order to evaluate chemical, biochemical, histological and genotoxic biomarkers. The rest of the fish group was submitted to the depuration experiment with free MC-LR water for 15 days. After this time the same tissues were collected and evaluated using biomarkers analysis. Toxic effects were found mostly in the fish liver from depuration time as alterations on the antioxidant system and histopathologies. The results showed that even low concentrations can cause sublethal effects to aquatic organisms, and cyanotoxins monitoring and regulation tools are required.

Cytotoxicity and enzymatic biomarkers as early indicators of benthic responses to the soluble-fraction of diesel oil.

Xenobiotics from oil tanker leaks and industrial discharges are amongst the main human impacts to confined coastal areas. We assessed the genotoxic responses to the water-soluble fraction of diesel oil in the polychaete Laeonereis culveri and the bivalve Anomalocardia flexuosa, two widespread benthic species in subtropical estuaries from the Southwestern Atlantic. We hypothesized that the highest responsiveness would be expressed by significantly different biomarkers responses between control and oil-impacted treatments. Responsiveness to diesel oil was investigated using an experimental design with two fixed factors (contaminant percentages and times of exposure). After exposure, we monitored the responses of the oxidative stress enzymes and performed micronuclei tests. Results were congruent for both species. Antioxidant defense of glutathione S-transferase and the induction of micronuclei and nuclear buds, the latter just for the bivalve, were significantly affected by polycyclic aromatic hydrocarbons, with significant increases on the seventh day of exposure and in the higher concentrations, compared to controls groups. We assessed the benefits and drawbacks of using each biomarker in laboratory experiments. Both species are indicators of early, and rapid responses to genotoxic contaminants in subtropical estuarine habitats. We suggest that the micronuclei frequency in A. flexuosa is a simple, fast and cheap test for genotoxicity in oil-impacted areas. Such early biomarkers are needed to develop better protocols for impact assessment and monitoring under real field conditions.

How does temperature rise affect a freshwater catfish Rhamdia quelen? A proteomic approach.

Outside of scientific circles, climate change is a hotly debated topic due to all its consequences. Changes in the temperature can affect aquatic organisms and it is important to understand and to detect earlier signals. This study aimed to analyze how a Neotropical fish species responds to temperature increases, using proteomic analysis as a tool. For this, fish of the species Rhamdia quelen, male and female, were exposed to two temperatures: 25 °C and 30 °C. After 96 h, the animals were anesthetized, euthanized and the liver was collected for proteomic analysis. Using freely available online software and databases (e.g. MetaboAnalyst, Gene Ontology and UniProt), we define the altered proteins in both sexes: 42 in females and 62 in males. Data are available via ProteomeXchange with identifier PXD046475. Differences between the two temperatures were observed mainly in the amino acid metabolic pathways. The cellular process and the immune response was altered, indicating that effects at lower levels of biological organization could serve as a predictor of higher-level effects when temperature rise affects wildlife populations. Thus, we conclude that the increase in temperature is capable of altering important cellular and physiological processes in R. quelen fish, with this response being different for males and females.

Alterations in neurotransmitters, steroid hormones, vitellogenin, and antioxidant system induced by di-n-butyl phthalate and di-isopentyl phthalate on catfish Rhamdia quelen.

Phthalates, such as di-n-butyl phthalate (DBP) and di-isopentyl phthalate (DiPeP), are pollutants with a high potential for endocrine disruption. This study aimed to evaluate parameters of endocrine disruption in specimens of the Neotropical fish Rhamdia quelen exposed to DBP and DiPeP through their food. After 30 days of exposure, the fish were anesthetized and then euthanized, and blood, hypothalamus, liver, and gonads were collected. DBP caused statistically significant alterations in the serotoninergic system of males (5 and 25 ng/g) and females (5 ng/g) of R. quelen and it increased testosterone levels in females (25 ng/g). DiPeP significantly altered the dopaminergic system in females, reduced plasma estradiol levels (125 ng/g) and hepatic vitellogenin expression (25 ng/g), and changed the antioxidant system in gonads (125 ng/g). The results suggest that DBP and DiPeP may have different response patterns in females, with the former being androgenic and the latter being anti-estrogenic. These findings provide additional evidence regarding the molecular events involving DBP and DiPeP in the endocrine disruption potential in juvenile specimens of Rhamdia quelen.

Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish.

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Salvinia molesta phytoremediation capacity as a nature-based solution to prevent harmful effects and accumulation of ciprofloxacin in Neotropical catfish.

Phytoremediation has been a potential solution for the removal of pharmaceuticals from water. Here, we evaluated the toxicological safety of ciprofloxacin-contaminated water treated by 96 h with Salvinia molesta. The Neotropical catfish Rhamdia quelen was used as a model, and the potential of the phytoremediation technique for mitigating the drug accumulation in the fishes was also studied. Fish exposed to Cipro (1 and 10 µg·L-1) in untreated water showed toxic responses (alteration of hematological, genotoxicity, biochemical, and histopathological biomarkers) and accumulated Cipro in their muscles at concentrations high for human consumption (target hazardous quotient > 1). Fish exposed to water treated with S. molesta showed no toxic effect and no accumulation of Cipro in their tissues. This must be related to the fact that S. molesta removed up to 97% of Cipro from the water. The decrease in Cipro concentrations after water treatment with S. molesta not only prevented the toxic effects of Cipro on R. quelen fish but also prevented the antimicrobial accumulation in fish flesh, favouring safe consumption by humans. For the very first time, we showed the potential of phytoremediation as an efficiently nature-based solution to prevent environmental toxicological effects of antimicrobials to nontarget organisms such as fish and humans. The use of S. molesta for Cipro-removal from water is a green technology to be considered in the combat against antimicrobial resistance.

Sublethal biochemical, histopathological and genotoxicological effects of short-term exposure to ciprofloxacin in catfish Rhamdia quelen.

Ciprofloxacin (Cipro) is commonly detected in water worldwide, however, the ecotoxicological effects to aquatic biota is still not fully understood. In this study, using multiple biomarkers, it was investigated sublethal effects of short-term exposure to Cipro concentrations (1, 10 and 100 µg.L-1) in the Neotropical catfish Rhamdia quelen compared to non-exposure treatment (Control). After 96 h of exposure, the fishes were anesthetized for blood collection to hematological and genotoxicity biomarkers analysis. After euthanasia, the brain and muscle were sampled for biochemical biomarkers analyses. Gills, liver and posterior kidney for genotoxicity, biochemical and histopathological biomarkers analysis and anterior intestine for histopathological biomarkers analysis. Genotoxicity was observed in all tissues, regardless of the Cipro concentrations. Hematological alterations, such as reduction of the number of erythrocytes and leucocytes, as well as in hematocrit concentration and histopathological damages, such as reduction of microridges in gill epithelium and necrosis in liver and posterior kidney, occurred mainly at 100 µg.L-1. In addition, at 100 µg.L-1, Cipro increased antioxidant system activity (Catalase in liver and posterior kidney). These results demonstrated that under short-term exposure, Cipro causes toxic effects in R. quelen that demands attention and surveillance of environmental aquatic concentrations of this antibiotic.

Effects of cadmium on the female reproductive axis of a Neotropical fish.

Metals are one of the contaminants released from the increase of anthropic activities. They can be classified as endocrine disruptors once they can affect the reproductive parameters of different organisms. The aim of the study was to evaluate the potential effects of cadmium on regulatory reproduction axis (Hypothalamic-Pituitary-Gonadal-Liver, the HPGL axis) in females of Rhamdia quelen exposed to nominal concentrations of 0.1; 1; 10 and 100 µg.L-1 of cadmium. After 15 days, tissues were collected for hormonal quantification, brain aromatase (cyp19a1b), hepatic vitellogenin (vtg) gene expression, and biomarkers analysis. Cadmium was quantified in water, gonad and liver samples. The plasma levels of estradiol, testosterone and gonad and hepatosomatic indexes did not changed after Cd exposure. The cyp19a1b was not different among the groups. Cadmium was detected at higher concentrations in the liver compared to the gonads. No genotoxicity was observed, only erythrocytes nuclear alterations. Metallothionein was reduced at 10 µg.L-1 in the liver and 10 and 100 µg.L-1 in the gonad. Hepatic superoxide dismutase activity increased and this can lead to a hydrogen peroxide increase, one of reactive oxygen species. This increase without a compensation of other enzymes of the antioxidant system can lead to lipoperoxidation, as occurred at 100 µg.L-1. Hepatic vitellogenin gene expression increased as well as the injury index at 0,1 and 100 µg.L-1. The tested cadmium concentrations have been found in the freshwater ecosystems and can affect the female reproductive regulation axis HPGL of the Neotropical species R. quelen.

Multiple biomarkers response in a Neotropical fish exposed to paralytic shellfish toxins (PSTs).

The Alagados Reservoir (Southern Brazil) is used as water supply, and since 2002 there have been reports with a presence of cyanobacterial blooms and cyanotoxins. In order to assess the water quality and the ecological integrity of the reservoir, we evaluated biochemical, genotoxic and osmoregulatory biomarkers in the freshwater cichlid fish (Geophagus brasiliensis) that were exposed to PSTs. The fish were sampled in the Alagados Reservoir in February 2016 (Summer) and were divided in three groups: 1) Reservoir group (RES): fish were collected immediately after sampling; 2) Depuration group (DEP): fish were submitted to the depuration experiment for 90 days in the laboratory; and 3) Reproduction group (REP): fish were kept in the laboratory until the fertilization and the chemical analyses were performed on the offspring (F1 generation). In the RES and DEP the blood, brain, muscle, liver and gills were collected for biochemical, genotoxic and osmoregulatory biomarkers analysis. Our results showed that the fish from the Alagados Reservoir (RES) presented oxidative stress and DNA damage; and after 90 days (DEP), the antioxidant system and DNA damage were recovered. Although PSTs were considered a risk to the ecological integrity of this water body; PSTs concentrations were not found in the tissues of the F1 generation. In addition, the biomarkers used were useful tools to evaluate the effects of environment contamination. Therefore, it is necessary to develop new technologies and monitoring programs in order to reduce cyanobaterial blooms, cyanotoxins and human activities that cause the contamination in aquatic environments.

Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae)

ABSTRACT Changes in physiology of the nervous system and metabolism can be detected through the activity of acetylcholinesterase (AChE), alpha esterase (EST-a) and beta esterase (EST-ß) in chironomids exposed to pollutants. However, to understand the real effect of xenobiotics on organisms, it is important to investigate how certain factors can interfere with enzyme activity. We investigated the effects of different temperatures, food stress and two steps of the enzymatic protocol on the activity of AChE, EST-a and EST-ß in Chironomus sancticaroli. In experiment of thermal stress individuals from the egg stage to the fourth larval instar were exposed to different temperatures (20, 25 and 30 °C). In food stress experiment, larvae were reared until IV instar in a standard setting (25 °C and 0.9 g weekly ration), but from fourth instar on they were divided into groups and offered different feeding regimes (24, 48 and 72 h with/without food). In sample freezing experiment, a group of samples was processed immediately after homogenization and another after freezing for 30 days. To test the effect of centrifugation on samples, enzyme activity was quantified from centrifuged and non-centrifuged samples. The activity of each enzyme reached an optimum at a different temperature. The absence of food triggered different changes in enzyme activity depending on the period of starvation. Freezing and centrifugation of the samples significantly reduced the activity of three enzymes. Based on these results we conclude that the four factors studied had an influence on AChE, EST-a and EST-ß and this influence should be considered in ecotoxicological approaches.

Morpho-histological characterization of immature of the bioindicator midge Chironomus sancticaroli Strixino and Strixino (Diptera, Chironomidae)

ABSTRACTChironomidae immature are used as bioindicators of sediment quality in aquatic ecosystems and ecotoxicological assays. Histological descriptions for this family are outdated and limited and there are no studies with Neotropical species. The aim of this study was to describe the tissue architecture of several organs of the larva of Chironomus sancticaroli. For the description of the histological pattern, the larvae were fixed in Duboscq solution for insects at 56 °C, followed by routine histologic processing, infiltration in paraffin, and the sections were stained with Hematoxylin–Eosin. After examining the slides, the tube digestive, salivary gland, excretory, nervous, endocrine, circulatory, and integumentary systems and fat body were histologically characterized. The histology allows evaluation of cell morphology, and for being not expensive and easily accessible can be routinely used in biomonitoring. In addition, is a useful tool in ecotoxicological assays and allow to evaluate biomarkers at tissue and cell levels.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.