Assessing testicular morphofunctionality under Roundup WG® herbicide exposure in zebrafish.

Glyphosate-based herbicides, like Roundup WG® (RWG) used for a range of crops, such as corn, soybean, coffee, sugarcane, rice, apple, and citrus, can reach aquatic ecosystems and impact non-target organisms like fish. Thus, the fish were exposed to three RWG concentrations plus one negative control, which represents the concentration allowed for inland Brazilian waters and concentrations found in surface water worldwide (0.0, 0.065, 0.65, and 6.5 mg a.i./L) for 7 and 15 days. Morphological analysis revealed significant alterations in the testicular structure, particularly in Sertoli cell extensions and cytoplasmic bridges between germ cells. Subcellular compartments also displayed alterations, including dilated mitochondria and the loss of electron density and autophagic vesicles. Gene transcript levels related to autophagy and steroidogenic regulation were upregulated in exposed fish. Germ cell quality was also affected, increasing ROS (reactive oxygen species) production and DNA fragmentation. The study highlighted the RWG reproductive toxicity, providing valuable insights into understanding the morphofunctional alterations in somatic and germ cells of Danio rerio. In conclusion, the environmental relevant concentrations used in this study were toxic to male somatic and germ cells, which raises a concern about the concentrations considered safe for human and animal use.

Pyriproxyfen exposure induces DNA damage, cell proliferation impairments and apoptosis in the brain vesicles layers of chicken embryos.

Larvicide pyriproxyfen (PPF), used in drinking water reservoirs to control Aedes mosquitoes, has already been shown as a possible cause of congenital anomalies in the central nervous system. However, the neurotoxic effects of PPF on the development of vertebrate embryos are still underexplored. Thus, the aim of this study was to investigate the effects of PPF on the morphometric parameters of the head and brain, as well as on the cell layers of the forebrain and midbrain, using embryos of Gallus domesticus as a model. Two sublethal PPF concentrations (0.01 mg/L and 10 mg/L), as defined by a survival curve, were tested. Analysis of the biometry of embryos showed significant reduction in body and brain mass and also in measurements of the head and brain. A reduction in cell layer thickness of the forebrain and midbrain was observed, accompanied by a reduction in the numerical density of cells per area. Changes in brain and head sizes and in the thickness of the cell layers of the forebrain and midbrain were significant at 10 mg/L PPF. Notably, PPF caused DNA doublestrand breaks and induced apoptosis in embryos exposed to 10 mg/L, which were accompanied by a reduction in cell proliferation. Regarding neuronal and glial differentiation, no changes were observed in the number of neurons and glial cells on the analyzed layers. Furthermore, PPF did not impact the head ossification process. These findings reveal that PPF is a strong stressor for neurodevelopment, causing damage to the cell architecture of brain vesicles.

Inorganic mercury effects on biomarker gene expressions of a freshwater amphipod at two temperatures.

Mercury (Hg) is a global contaminant resulting of both natural processes and human activities. In aquatic environments, studies conducted on vertebrates highlighted changes of gene expression or activity of antitoxic and oxidative enzymes. However, although Hg is a highly toxic compound in aquatic environments, only a few studies have evaluated the lethal and sublethal effects of inorganic Hg on Gammarus sp. Therefore, this study aimed at evaluating the effects of inorganic Hg (HgCl2) on the expression of 17 genes involved in crucial biological functions or mechanisms for organisms, namely respiration, osmoregulation, apoptosis, immune and endocrine system, and antioxidative and antitoxic defence systems. The study was performed in males of the freshwater amphipod Gammarus pulex exposed to two environmentally relevant concentrations (50 and 500 ng/L) at two temperature regime fluctuations (16 °C and 20 °C +/-2 °C) for 7 and 21 days. Results showed that G. pulex mortality was dependent on Hg concentration and temperature; the higher the concentration and temperature, the higher the mortality rate. In addition, the Integrated Biomarker Response emphasized that HgCl2 toxicity was dependent on the concentration, time and temperature of exposure. Overall, antioxidant and antitoxic defences, as well as the endocrine and immune systems, were the biological functions most impacted by Hg exposure (based on the concentration, duration, and temperature tested). Conversely, osmoregulation was the least affected biological function. The results also demonstrated a possible adaptation of G. pulex after 21 days at 500 ng/L, regardless of the exposure temperature. This study allowed us to show that Hg deregulates many crucial biological functions after a short exposure, but that during a long exposure, an adaptation phenomenon could occur, regardless of temperature.

Reproductive toxicity of Roundup WG® herbicide: impairments in ovarian follicles of model organism Danio rerio.

Glyphosate-based herbicides are widely used in global agriculture, and their effects on different non-target animal organisms have been the focus of many toxicological studies. Regarding the potential role of glyphosate-based herbicides as an endocrine disruptor, the present study aims to investigate the effects of the herbicide Roundup WG® (RWG) on female reproduction, specifically on the ovarian maturation of Danio rerio. Adult females were exposed to low concentrations of RWG (0.065, 0.65, and 6.5 mg L-1) for 15 days, and then the ovaries were submitted to structural and morphometric procedures, accompanied by analysis of the vitellin protein content. Our results showed an increase of initial ovarian follicle numbers, decrease of late ovarian follicles, and smaller diameter of ovarian follicles in fish exposed to 0.065 and 6.5 mg L-1. The thickness of vitelline envelope was reduced, and the vitellin protein content was increased in the ovarian follicle in the two highest concentrations. Ultrastructural changes in the ovarian follicular component were evident and expressed by the cell index; vacuolization in follicular cells, increase of perivitelline space, and impaired mitochondria in oocytes were observed. Therefore, RWG adversely affects the ovarian maturation in D. rerio, and these changes can lead to reproductive toxicity, compromising population dynamics.

Roundup® disrupts chitinolytic enzyme activity and ecdysteroid concentration in Macrobrachium potiuna.

The endocrine system of crustaceans regulates the molt cycle with ecdysteroid hormones, mainly the 20-hydroxyecdysone (20-HE). Moreover, the molt process requires the action of chitinolytic enzymes (e.g., chitinase, chitobiase) to break down the old cuticle. However, endocrine disrupting compounds (EDC) are capable of altering their normal functioning. Glyphosate-based herbicides (GBH), such as Roundup®, the most widely used herbicides, are found in freshwater environments and have been considered EDC for many aquatic organisms. Therefore, this study examined the effects of environmentally relevant GBH concentrations (0.0065, 0.065, and 0.28 mg L-1) on the 20-HE concentration and chitobiase activity in the decapod prawn Macrobrachium potiuna exposed for 14 days. Additionally, lipid peroxidation, a biomarker of membrane lipid degradation, was evaluated in hepatopancreas to assess cellular damage. Results showed that GBH decreased the 20-HE concentration in females at the two highest concentrations tested, while an increase was observed in males exposed to the highest GBH concentration. In addition, GBH also decreased chitobiase activity in males (all concentrations) and females (the two highest concentrations). Finally, GBH caused increased lipid peroxidation in males, indicating cellular damage in the hepatopancreas. In conclusion, this work suggests that GBH is an EDC for crustaceans by disrupting molting, which could lead to altered reproduction and thus population dynamics. Graphical abstract Decrease in the 20-HE concentration and chitobiase activity in muscle of males and females of the freshwater prawn Macrobrachium potiuna exposed to the herbicide Roundup® for 14 days.

Ultraviolet-B radiation induces transcriptional modulation of components associated with the extracellular matrix in embryos of decapod Macrobrachium olfersii.

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.

Modulation of antioxidant gene expressions by Roundup® exposure in the decapod Macrobrachium potiuna.

Glyphosate-based herbicides (GBH), including Roundup®, are the most used herbicides in agricultural and non-agricultural areas, which can reach aquatic environments through drift during application or surface runoff. Some studies, mostly in fish, demonstrated that GBH caused oxidative stress in non-target animals. However, only few information is available on the GBH effects in the antioxidant and stress proteins of many other organisms, such as freshwater crustaceans. Thus, we aimed to investigate the effects of environmentally relevant GBH concentrations on the relative transcript expression (RTE) of the superoxide dismutase (sod1), catalase (cat), selenium-dependent glutathione peroxidase (gpx), glutathione-S-transferase (gst), thioredoxin (txn), heat shock protein (hsp70 and hsp90) in the hepatopancreas of the ecologically important freshwater prawn Macrobrachium potiuna. Moreover, this study aimed to assess the gender-differences responses to GBH exposure. Male and female prawns were exposed to three Roundup WG® concentrations (0.0065, 0.065 and 0.28 mg of glyphosate/L) and a control group (0.0 mg/L) for 7 and 14 days. In general, males had an under-expression of the studied genes, indicating an oxidative stress and possible accumulation of ROS in the hepatopancreas. In the opposite, females had an overexpression of the same genes, indicating a more robust antioxidant system, in order to cope with the possible ROS increase after Roundup WG® exposure. Therefore, results confirmed that gender could be a confounding factor in ecotoxicological assessment of GBH effects. Additionally, this work highlights that sod1, cat, gpx, gst, txn, hsp70 and hsp90 gene expressions seem to be useful biomarkers to investigate the oxidative stress caused by Roundup WG® in Macrobrachium sp.

Effects of low glyphosate-based herbicide concentrations on endocrine-related gene expression in the decapoda Macrobrachium potiuna.

Glyphosate-based herbicides (GBH) are the most used herbicides worldwide and are considered as endocrine-disrupting compounds (EDC) for non-target organisms. However, effects of GBH on their endocrine systems remain poorly understood. Thus, the aim of this study was to assess the effects of low concentrations of Roundup WG® on growth and reproduction process molecules in both males and females of the decapod crustacean Macrobrachium potiuna, by the relative transcript expression levels of the ecdysteroid receptor (EcR), the molt-inhibiting hormone (MIH), and the vitellogenin (Vg) genes. Prawns were exposed to three concentrations of GBH (0.0065, 0.065, and 0.28 mg L-1) for 7 and 14 days. The results revealed that only in males the three genes transcript levels were influenced by the GBH concentration, time of exposure, and the interaction between the concentrations and time of exposure, suggesting that males were more sensitive to GBH than females. For males, after 7 days of exposure at 0.065 mg L-1, EcR and MIH were over-expressed, while the Vg expression was only over-expressed after 14 days. The present study highlighted that GBH impacted endocrine systems of M. potiuna. Moreover, EcR and MIH gene expressions could be promising EDC biomarkers of exposure in crustaceans. These results also indicate that GBH concentrations, considered secure by regulatory agencies, should be reviewed to minimize the effects on non-target organisms. Potential effects of glyphosate-based herbicides on the endocrine system of decapods Macrobrachium sp.

Histopathological and ultrastructural indices for the assessment of glyphosate-based herbicide cytotoxicity in decapod crustacean hepatopancreas.

Glyphosate-based herbicides (GBH), including Roundup, are the most widely used pesticides in the world. Glyphosate residues have been detected in surface and groundwater, in food, and in human blood and urine. The effects of this herbicide on different levels of biological organization are an important concern that needs to be investigated. In general, the toxicity of GBH in invertebrates is poorly understood, and it is the motivation of this study. Thus, the aim of this study was to evaluate cellular responses of the hepatopancreas, an organ involved in the detoxification process in invertebrates, after exposure to environmentally relevant concentrations of GBH, using prawn Macrobrachium potiuna as a model. Prawns were exposed to three concentrations of GBH (0.0065, 0.065 and 0.28 mg L-1) for 7 or 14 days. Alterations in the morphology of the hepatopancreas and in subcellular components of R cells, which are responsible for the detoxification process, were analyzed, and an index for subcellular alterations was standardized. GBH exposure induced tissue commitments on the hepatopancreas, as well as important impairments of R cells that could compromise the normal functioning of the cells, especially in the detoxification processes. The major cellular impairments were intense vacuolization, dilatation of the cisterns of the rough endoplasmic reticulum and Golgi bodies, increase of perinuclear space, necrosis, concentric membrane formation and mitochondria crest loss. Our data contribute to the knowledge of the cytotoxic effects of low GBH concentrations on aquatic invertebrates, specifically their effects on the hepatopancreas, an important organ for the metabolism of crustaceans. These results also indicate that concentrations considered safe by regulatory agencies should be reviewed to minimize the effects on non-target organisms. This study also contributes to the standardization of an ultrastructure index for the assessment of GBH in palaemonids, which could be used for the assessment of contaminants in crustaceans and other species with hepatopancreas.