A Pilot Study of Gene Expression Modulation from Antioxidant System of Killifish Austrolebias charrua After Exposure to Roundup Transorb®.

Roundup Transorb® (RDT) is the most popular glyphosate-based herbicide (GHB) used in agriculture, and its impact extends to non-target organisms. The annual killifish Austrolebias charrua is an endangered species endemic to southern South America and inhabits temporary ponds. This study evaluates the effects of RDT concentrations (0.065 and 5 mg/L GAE) on A. charrua exposed for 96 h. Gene expression of cat, sod2, gstα, gclc, and ucp1 was evaluated on the liver and gills. Highlighting that even at low concentrations permitted by Brazilian legislation, the RDT can have adverse effects on A. charrua.

Roundup® induces premature senescence of mouse granulosa cells via mitochondrial ROS-triggered NLRP3 inflammasome activation.

Roundup, a glyphosate-based herbicide widely used in agriculture, has raised concerns regarding its potential impact on human health due to the detection of its residues in human urine and serum. Granulosa cells are essential for oocyte growth and follicle development. Previous research has shown that Roundup could affect steroid synthesis, increases oxidative stress, and induces apoptosis in granulosa cells. However, little is known about the effects of Roundup on NLRP3 (nucleotide binding oligomerization domain-like receptor family pyrin-containing domain protein 3) inflammasome activation and cellular senescence in granulosa cells. Here, we provided evidence that exposure to Roundup induced premature senescence in mouse granulosa cells through the activation of NLRP3 inflammasome triggered by mitochondrial ROS. Our findings demonstrated that Roundup significantly reduced the viability of granulosa cells under in vitro culture conditions. It also disrupted mitochondrial function and induced oxidative stress in these cells. Subsequent investigations showed that NLRP3 inflammasome was activated in treated granulosa cells, as evidenced by the upregulation of inflammasome-related genes and the processing of inflammatory cytokines IL-1ß and IL-1α into their mature forms. Consequently, premature cellular senescence occurred in response to the challenge posed by Roundup. Notably, direct inhibition of NLRP3 inflammasome with MCC950 does not alleviate mitochondrial damage and oxidative stress. However, supplementation of resveratrol, which has been known to attenuate mitochondrial damage and oxidative stress, effectively mitigated the inflammatory response and the expression of senescence-related markers, and prevented the senescence in granulosa cells. These results suggested that mitochondrial function and oxidative homeostasis might play pivotal roles as upstream regulators of NLRP3 inflammasome. In summary, our findings indicated that the premature senescence of granulosa cells caused by mitochondrial ROS-triggered NLRP3 inflammasome activation might contribute to the ovarian toxicity of Roundup, in addition to its known effects on steroidogenesis and apoptosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-024-00229-0.

Glyphosate resistance and biodegradation by Burkholderia cenocepacia CEIB S5-2.

Glyphosate is a broad spectrum and non-selective herbicide employed to control different weeds in agricultural and urban zones and to facilitate the harvest of various crops. Currently, glyphosate-based formulations are the most employed herbicides in agriculture worldwide. Extensive use of glyphosate has been related to environmental pollution events and adverse effects on non-target organisms, including humans. Reducing the presence of glyphosate in the environment and its potential adverse effects requires the development of remediation and treatment alternatives. Bioremediation with microorganisms has been proposed as a feasible alternative for treating glyphosate pollution. The present study reports the glyphosate resistance profile and degradation capacity of the bacterial strain Burkholderia cenocepacia CEIB S5-2, isolated from an agricultural field in Morelos-México. According to the agar plates and the liquid media inhibition assays, the bacterial strain can resist glyphosate exposure at high concentrations, 2000 mg·L-1. In the degradation assays, the bacterial strain was capable of fast degrading glyphosate (50 mg·L-1) and the primary degradation metabolite aminomethylphosphonic acid (AMPA) in just eight hours. The analysis of the genomic data of B. cenocepacia CEIB S5-2 revealed the presence of genes that encode enzymes implicated in glyphosate biodegradation through the two metabolic pathways reported, sarcosine and AMPA. This investigation provides novel information about the potential of species of the genus Burkholderia in the degradation of the herbicide glyphosate and its main degradation metabolite (AMPA). Furthermore, the analysis of genomic information allowed us to propose for the first time a metabolic route related to the degradation of glyphosate in this bacterial group. According to the findings of this study, B. cenocepacia CEIB S5-2 displays a great glyphosate biodegradation capability and has the potential to be implemented in glyphosate bioremediation approaches.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

The exposure in ovo to glyphosate on the integrity of intestinal epithelial tight junctions of chicks.

Glyphosate is an ingredient widely used in various commercial formulations, including Roundup®. This study focused on tight junctions and the expression of inflammatory genes in the small intestine of chicks. On the sixth day of embryonic development, the eggs were randomly assigned to three groups: the control group (CON, n = 60), the glyphosate group (GLYP, n = 60), which received 10 mg of active glyphosate/kg egg mass, and the Roundup®-based glyphosate group also received 10 mg of glyphosate. The results indicated that the chicks exposed to glyphosate or Roundup® exhibited signs of oxidative stress. Additionally, histopathological alterations in the small intestine tissues included villi fusion, complete fusion of some intestinal villi, a reduced number of goblet cells, and necrosis of some submucosal epithelial cells in chicks. Genes related to the small intestine (ZO-1, ZO-2, Claudin-1, Claudin-3, JAM2, and Occludin), as well as the levels of pro-inflammatory cytokines (IFNγ, IL-1ß, and IL-6), exhibited significant changes in the groups exposed to glyphosate or Roundup® compared to the control group. In conclusion, the toxicity of pure glyphosate or Roundup® likely disrupts the small intestine of chicks by modulating the expression of genes associated with tight junctions in the small intestine.

Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Mutagenic effects of a commercial glyphosate-based herbicide formulation on the soil filamentous fungus Aspergillus nidulans depending on the mode of exposure.

Glyphosate-based herbicides (GBH) are the most used pesticides worldwide. This widespread dissemination raises the question of non-target effects on a wide range of organisms, including soil micro-organisms. Despite a large body of scientific studies reporting the harmful effects of GBHs, the health and environmental safety of glyphosate and its commercial formulations remains controversial. In particular, contradictory results have been obtained on the possible genotoxicity of these herbicides depending on the organisms or biological systems tested, the modes and durations of exposure and the sensitivity of the detection technique used. We previously showed that the well-characterized soil filamentous fungus Aspergillus nidulans was highly affected by a commercial GBH formulation containing 450 g/L of glyphosate (R450), even when used at doses far below the agricultural application rate. In the present study, we analysed the possible mutagenicity of R450 in A. nidulans by screening for specific mutants after different modes of exposure to the herbicide. R450 was found to exert a mutagenic effect only after repeated exposure during growth on agar-medium, and depending on the metabolic status of the tested strain. The nature of some mutants and their ability to tolerate the herbicide better than did the wild-type strain suggested that their emergence may reflect an adaptive response of the fungus to offset the herbicide effects. The use of a non-selective molecular approach, the quantitative random amplified polymorphic DNA (RAPD-qPCR), showed that R450 could also exert a mutagenic effect after a one-shot overnight exposure during growth in liquid culture. However, this effect was subtle and no longer detectable when the fungus had previously been repeatedly exposed to the herbicide on a solid medium. This indicated an elevation of the sensitivity threshold of A. nidulans to the R450 mutagenicity, and thus confirmed the adaptive capacity of the fungus to the herbicide.

Toxicity assessment and DNA repair kinetics in HEK293 cells exposed to environmentally relevant concentrations of Glyphosate (Roundup® Control Max).

Glyphosate is a systemic, non-selective, pre and post-emergence wide range herbicide. In 2015, IARC classified Glyphosate as "a probable carcinogenic agent for humans". The aim of this study was to evaluate the cytotoxicity and genotoxicity of the commercial formulation of glyphosate (Roundup® Control Max) at environmentally relevant concentrations and measure the potential effect of this herbicide over the cell capacity to repair DNA damage. HEK293 cells were exposed to 5 concentrations of Roundup® Control Max equivalent to 0.7; 7; 70; 700 and 3,500 µg/L glyphosate acid, for 1, 4 and 24 h. Cytotoxicity was quantified by the Trypan Blue staining method and by the MTT assay, while genotoxicity and evaluation of DNA damage repair kinetics were analyzed through the alkaline comet assay. In all treatments, cell viability was higher than 80%. The three highest glyphosate concentrations-70 µg/L, 700 µg/L, and 3,500 µg/L-increased levels of DNA damage compared to the control at the three exposure times tested. Finally, concerning the kinetics of DNA damage repair, cells initially exposed to 3,500 µg/L of glyphosate for 24 h were unable to repair the breaks in DNA strands even after 4 h of incubation in culture medium. The present study demonstrated for the first time that Roundup® Control Max may induce genetic damage and cause alterations in the DNA repair system in human embryonic kidney cells even at concentrations found in blood and breast milk of people exposed through residues of the herbicide in food, which values have been poorly assessed or not studied yet according to the existent literature.

Hormesis of glyphosate on ferulic acid metabolism and antifungal volatile production in rice root biocontrol endophyte Burkholderia cepacia LS-044.

Glyphosate (GP, N-phosphonomethyl glycine) is one of the most popular organophosphate herbicides widely used in agricultural practices worldwide. There have been extensive reports on the biohazard attributes and hormetic impacts of GP on plant and animal systems. However, the effects of GP on plant growth-promoting microbes and its ecological relevance remain unknown. Here, we show that GP does exert a hormetic impact on Burkholderia cepacia LS-044, a rice (Oryza sativa ssp. japonica cv. Tainung 71) root endophytic isolate. We used increasing doses of ferulic acid (FA, 1-25 mM) and GP (0.5-5 mM) to test the growth and antifungal volatile production in LS-044 by electrochemical, liquid chromatographic, gas chromatographic and spectrophotometric means. GP treatment at a low dose (0.5 mM) increased FA utilization and significantly (P < 0.0001) enhanced antifungal volatile activity in LS-044. Although FA (1 mM) was rapidly utilized by LS-044, no chromatographically detectable utilization of GP was observed at tested doses (0.5-5 mM). LS-044 emitted predominant amounts of tropone in addition to moderate-to-minor amounts of diverse ketones and/or their derivatives (acetone, acetophenone, 2-butanone, 1-propanone, 1-(2-furanyl-ethanone, 1-phenyl-1-propanone and 1-(3-pyridinyl)-1-propanone), d-menthol, 2-methoxy-3-(1-methylethyl)-pyrazine, dimethyl disulfide, pyridine and ammonium carbamate when grown under GP supplement. GP hormesis on LS-044 induced phenotypic variations in O. sativa ssp. japonica cv. Tainan 11 as evident through seed germination assay. Genes involved in the transformation of FA, and a key gene encoding 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) with Gly-94 and Tyr-95 residues localized at active site most likely rendering EPSPS sensitivity to GP, were detected in LS-044. This is the first report on the GP hormesis influencing morphological and metabolic aspects including volatile emission in a biocontrol bacterium that could modulate rice plant phenotype.

Effects of Chronic Roundup Exposure on Medaka Larvae.

The use of glyphosate-based herbicides is increasing yearly to keep up with the growing demands of the agriculture world. Although glyphosate-based herbicides target the enzymatic pathway in plants, the effects on the endocrine systems of vertebrate organisms, mainly fish, are widely unknown. Many studies with glyphosate used high-exposure concentrations (mg/L), and the effect of environmentally relevant or lower concentrations has not been clearly understood. Therefore, the present study examined the effects of very low, environmentally relevant, and high concentrations of glyphosate exposure on embryo development and the thyroid system of Japanese medaka (Oryzias latipes). The Hd-rR medaka embryos were exposed to Roundup containing 0.05, 0.5, 5, 10, and 20 mg/L glyphosate (glyphosate acid equivalent) from the 8 h post-fertilization stage through the 14-day post-fertilization stage. Phenotypes observed include delayed hatching, increased developmental deformities, abnormal growth, and embryo mortality. The lowest concentration of glyphosate (0.05 mg/L) and the highest concentration (20 mg/L) induced similar phenotypes in embryos and fry. A significant decrease in mRNA levels for acetylcholinesterase (ache) and thyroid hormone receptor alpha (thrα) was found in the fry exposed to 0.05 mg/L and 20 mg/L glyphosate. The present results demonstrated that exposure to glyphosate formulation, at a concentration of 0.05 mg/L, can affect the early development of medaka larvae and the thyroid pathway, suggesting a link between thyroid functional changes and developmental alteration; they also showed that glyphosate can be toxic to fish at this concentration.

Editors' Roundup: June 2023.

This commentary article represents the latest edition of the Biophysical Reviews 'Editors' Roundup' Series - a platform made available to the editorial board members of any journal with a genuine interest in promoting biophysical content. Each journal associated editor is able to submit a short description of up to five articles recently appearing in their journals with an explanation of why these articles are of interest. This edition (Vol. 15 Issue 3 June 2023) carries contributions from editorial members associated with Biophysics and Physicobiology (Biophysical Society of Japan), Biophysics (Russian Academy of Sciences), Cell Biochemistry and Biophysics (Springer), and Biophysical Reviews (IUPAB-International Union for Pure and Applied Biophysics).

Mapping the key characteristics of carcinogens for glyphosate and its formulations: A systematic review.

Glyphosate was classified as a probable human carcinogen (Group 2A) by the International Agency for Research on Cancer (IARC) partially due to strong mechanistic evidence in 2015. Since then, numerous studies of glyphosate and its formulations (GBF) have emerged. These studies can be evaluated for cancer hazard identification with the newly described ten key characteristics (KC) of carcinogens approach. Our objective was to assess all in vivo, ex vivo, and in vitro mechanistic studies of human and experimental animals (mammals) that compared exposure to glyphosate/GBF with low/no exposure counterparts for evidence of the ten KCs. A protocol with our methods adhering to PRISMA guidelines was registered a priori (INPLASY202180045). Two blinded reviewers screened all in vivo, ex vivo, and in vitro studies of glyphosate/GBF exposure in humans/mammals reporting any KC-related outcome available in PubMed before August 2021. Studies that met inclusion criteria underwent data extraction conducted in duplicate for each KC outcome reported along with key aspects of internal/external validity, results, and reference information. These data were used to construct a matrix that was subsequently analyzed in the program R to conduct strength of evidence and quality assessments. Of the 2537 articles screened, 175 articles met inclusion criteria, from which we extracted >50,000 data points related to KC outcomes. Data analysis revealed strong evidence for KC2, KC4, KC5, KC6, KC8, limited evidence for KC1 and KC3, and inadequate evidence for KC7, KC9, and KC10. Notably, our in-depth quality analyses of genotoxicity (KC2) and endocrine disruption (KC8) revealed strong and consistent positive findings. For KC2, we found: 1) studies conducted in humans and human cells provided stronger positive evidence than counterpart animal models; 2) GBF elicited a stronger effect in both human and animal systems when compared to glyphosate alone; and 3) the highest quality studies in humans and human cells consistently revealed strong evidence of genotoxicity. Our analysis of KC8 indicated that glyphosate's ability to modulate hormone levels and estrogen receptor activity is sensitive to both exposure concentration and formulation. The modulations observed provide clear evidence that glyphosate interacts with receptors, alters receptor activation, and modulates the levels and effects of endogenous ligands (including hormones). Our findings strengthen the mechanistic evidence that glyphosate is a probable human carcinogen and provide biological plausibility for previously reported cancer associations in humans, such as non-Hodgkin lymphoma. We identified potential molecular interactions and subsequent key events that were used to generate a probable pathway to lymphomagenesis.

Roundup and immune challenge have different effects on a native field cricket and its introduced competitor.

Animals face many natural challenges, and humans have added to this burden by applying potentially harmful herbicides and unintentionally introducing competitors. We examine the recently introduced Velarifictorus micado Japanese burrowing cricket which shares the same microhabitat and mating season as the native Gryllus pennsylvanicus field cricket. In this study, we assess the combined effects of Roundup (glyphosate-based herbicide) and a lipopolysaccharide (LPS) immune challenge on both crickets. In both species, an immune challenge reduced the numbers of eggs that the female laid; however, this effect was much larger in G. pennsylvanicus. Conversely, Roundup caused both species to increase egg production, potentially representing a terminal investment strategy. When exposed to both an immune challenge and herbicide, G. pennsylvanicus fecundity was harmed more than V. micado fecundity. Furthermore, V. micado females laid significantly more eggs than G. pennsylvanicus, suggesting that introduced V. micado may have a competitive edge in fecundity over native G. pennsylvanicus. LPS and Roundup each had differing effects on male G. pennsylvanicus and V. micado calling effort. Overall, introduced male V. micado spent significantly more time calling than native G. pennsylvanicus, which could potentially facilitate the spread of this introduced species. Despite the population-level spread of introduced V. micado, in our study, this species did not outperform native G. pennsylvanicus in tolerating immune and chemical challenge. Although V. micado appears to possess traits that make this introduced species successful in colonizing new habitats, it may be less successful in traits that would allow it to outcompete a native species.

Involvement of glyphosate in disruption of biotransformation P450 enzymes and hepatic lipid metabolism in chicken.

The current study investigated the potentially harmful consequences of pure glyphosate or Roundup® on CYP family members and lipid metabolism in newly hatched chicks. On the sixth day, 225 fertilized eggs were randomly divided into three treatments: (1) the control group injected with deionized water, (2) the glyphosate group injected with 10 mg pure glyphosate/Kg egg mass and (3) the Roundup group injected 10 mg the active ingredient glyphosate in Roundup®/Kg egg. The results of the study revealed a reduction in hatchability in chicks treated with Roundup®. Moreover, change of Lipid concentration in serum and the liver-treated groups. Additionally, increased liver function enzymes and increased oxidative stress in the glyphosate and Roundup® groups. Furthermore, liver tissues showed histological changes and several lipid deposits in glyphosate-treated groups. Hepatic CYP1A2 and CYP1A4 expressions were significantly increased (p < .05) after glyphosate exposure, and suppression of CYP1C1 mRNA expression was significant (p < .05) after Roundup® exposure. The pro-inflammatory cytokines genes IFN-γ and IL-1ß expression were significantly increased (p < .05) after Roundup® exposure. In addition, there were significant differences in the levels of expression genes which are related to lipid synthesis or catabolism in the liver. In conclusion, in ovo glyphosate exposure caused disruption of biotransformation, pro-inflammatory and lipid metabolism in chicks.

The herbicide glyphosate inhibits hippocampal long-term potentiation and learning through activation of pro-inflammatory signaling.

Background: Glyphosate, a herbicide marketed under the trade name Roundup, is now widely used, in part because genetically modified organism plants that are resistant to this agent have been developed. Environmental or dietary exposure to glyphosate is omnipresent and there are concerns this exposure could impair cognitive function in addition to carcinogenicity. Methods: Using hippocampal slices from juvenile male rats, we investigated whether glyphosate alters synaptic transmission and induction of long-term potentiation (LTP), a cellular model of learning and memory. Our hypothesis is that glyphosate alters neuronal function and impairs LTP induction via activation of pro-inflammatory processes, because increases in pro-inflammatory cytokines and neuroinflammation have been reported following glyphosate exposure. LTP was induced by delivery of 100 Hz x 1 sec high frequency stimulation (HFS) of the Schaffer collateral pathway and excitatory synaptic potentials (EPSPs) were monitored 60 min after HFS. Resulsts: We first tested effects of Roundup on basal synaptic function and LTP induction. Roundup depressed EPSPs in a dose-dependent manner. Basal synaptic transmission was completely suppressed by 2000 ppm. At concentrations ≤ 20 ppm Roundup did not affect basal transmission, but 4 ppm Roundup administered 30 min before HFS inhibited LTP induction. We also observed that acute administration of 10-100 µM glyphosate inhibits LTP induction. Minocycline, an inhibitor of microglial activation, and TAK-242, an inhibitor of toll-like receptor 4 (TLR4), both overcame the inhibitory effects of 100M glyphosate. Similarly, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) overcame the inhibitory effects. In addition, ISRIB (integrated stress response inhibitor) and quercetin, an inhibitor of endoplasmic reticulum stress, allowed LTP induction in the presence of glyphosate. We also observed that in vivo glyphosate injection (16.9 mg/kg i.p.) impaired one-trial inhibitory avoidance learning. This learning deficit was overcome by TAK-242. Conclusion: While Roundup inhibits LTP induction, these observations indicate that glyphosate alone, the major ingredient of Roundup, can impair cognitive function through pro-inflammatory signaling in microglia. Manipulation of pro-inflammatory signaling could be a useful strategy to prevent cognitive impairment after exposure to a glyphosate-based herbicide (GBH).

Contrasting effects of fungicide and herbicide active ingredients and their formulations on bumblebee learning and behaviour.

Fungicides and herbicides are two of the most heavily applied pesticide classes in the world, but receive little research attention with regards to their potential impacts on bees. As they are not designed to target insects, the mechanisms behind potential impacts of these pesticides are unclear. It is therefore important to understand their influence at a range of levels, including sublethal impacts on behaviours such as learning. We used the proboscis extension reflex (PER) paradigm to assess how the herbicide glyphosate and the fungicide prothioconazole affect bumblebee olfactory learning. We also assessed responsiveness, and compared the impacts of these active ingredients and their respective commercial formulations (Roundup Biactive and Proline). We found that learning was not impaired by either formulation but, of the bees that displayed evidence of learning, exposure to prothioconazole active ingredient increased learning level in some situations, while exposure to glyphosate active ingredient resulted in bumblebees being less likely to respond to antennal stimulation with sucrose. Our data suggest that fungicides and herbicides may not negatively impact olfactory learning ability when bumblebees are exposed orally to field-realistic doses in a lab setting, but that glyphosate has the potential to cause changes in responsiveness in bees. As we found impacts of active ingredients and not commercial formulations, this suggests that co-formulants may modify impacts of active ingredients in the products tested on olfactory learning without being toxic themselves. More research is needed to understand the mechanisms behind potential impacts of fungicides and herbicides on bees, and to evaluate the implications of behavioural changes caused by glyphosate and prothioconazole for bumblebee fitness.

The effects of short-term glyphosate-based herbicide exposure on insect gene expression profiles.

Glyphosate-based herbicides (GBHs) are the most frequently used herbicides worldwide. The use of GBHs is intended to tackle weeds, but GBHs have been shown to affect the life-history traits and antioxidant defense system of invertebrates found in agroecosystems. Thus far, the effects of GBHs on detoxification pathways among invertebrates have not been sufficiently investigated. We performed two different experiments-1) the direct pure glyphosate and GBH treatment, and 2) the indirect GBH experiment via food-to examine the possible effects of environmentally relevant GBH levels on the survival of the Colorado potato beetle (Leptinotarsa decemlineata) and the expression profiles of their detoxification genes. As candidate genes, we selected four cytochrome P450 (CYP), three glutathione-S-transferase (GST), and two acetylcholinesterase (AChE) genes that are known to be related to metabolic or target-site resistances in insects. We showed that environmentally relevant levels of pure glyphosate and GBH increased the probability for higher mortality in the Colorado potato beetle larvae in the direct experiment, but not in the indirect experiment. The GBHs or glyphosate did not affect the expression profiles of the studied CYP, GST, or AChE genes; however, we found a large family-level variation in expression profiles in both the direct and indirect treatment experiments. These results suggest that the genes selected for this study may not be the ones expressed in response to glyphosate or GBHs. It is also possible that the relatively short exposure time did not affect gene expression profiles, or the response may have already occurred at a shorter exposure time. Our results show that glyphosate products may affect the survival of the herbivorous insect already at lower levels, depending on their sensitivity to pesticides.

Glyphosate-based herbicide use affects individual microbial taxa in strawberry endosphere but not the microbial community composition.

AIMS: In a field study, the effects of treatments of glyphosate-based herbicides (GBHs) in soil, alone and in combination with phosphate fertilizer, were examined on the performance and endophytic microbiota of garden strawberry. METHODS AND RESULTS: The root and leaf endophytic microbiota of garden strawberries grown in GBH-treated and untreated soil, with and without phosphate fertilizer, were analyzed. Next, bioinformatics analysis on the type of 5-enolpyruvylshikimate-3-phosphate synthase enzyme was conducted to assess the potential sensitivity of strawberry-associated bacteria and fungi to glyphosate, and to compare the results with field observations. GBH treatments altered the abundance and/or frequency of several operational taxonomic units (OTUs), especially those of root-associated fungi and bacteria. These changes were partly related to their sensitivity to glyphosate. Still, GBH treatments did not shape the overall community structure of strawberry microbiota or affect plant performance. Phosphate fertilizer increased the abundance of both glyphosate-resistant and glyphosate-sensitive bacterial OTUs, regardless of the GBH treatments. CONCLUSIONS: These findings demonstrate that although the overall community structure of strawberry endophytic microbes is not affected by GBH use, some individual taxa are.

Roundup Original DI® and thermal stress affect survival, morphology and thermal tolerance in tadpoles of Boana faber (Hylidae, Anura).

In amphibians, stressful environments can lead to accelerated metamorphosis at the expense of total length, resulting in the occurrence of morphological abnormalities. Many studies have linked the occurrence of these phenomena to the pollution of habitats by pesticides and thermal stress. Here, we assessed how exposure to Roundup Original DI® and higher constant temperatures affect the survival of Boana faber tadpoles and estimate the CL5096hs for the population. In addition, we evaluated how exposure to Roundup affects larval growth, morphology and thermal tolerance. Our findings suggest that even at sublethal doses, Roundup Original DI® may affect the survival of Boana faber larvae. There also appears to be an additive effect between Roundup and temperature increase on larval survival, however, we need to further explore this point to determine a pattern, proving to be a promising issue to be investigated in the future. We observed effects of chronic exposure to the herbicide formulation on the morphology and growth of the tadpoles, resulting in a reduction in total length and differences in the shape of the larvae. Although we did not recover any direct effects of herbicide exposure on CTMax, we did observe an upward trend in CTMax for tadpoles exposed to Roundup. Understanding how anthropogenic changes affect anuran persistence is fundamental for the management and conservation of the species and can be considered an initial step toward the formulation of legislations that regulate the use of herbicides.

Effect of glyphosate, its metabolite AMPA, and the glyphosate formulation Roundup® on brown trout (Salmo trutta f. fario) gut microbiome diversity.

Glyphosate is used worldwide as a compound of pesticides and is detectable in many environmental compartments. It enters water bodies primarily through drift from agricultural areas so that aquatic organisms are exposed to this chemical, especially after rain events. Glyphosate is advertised and sold as a highly specific herbicide, which interacts with the EPSP synthase, an enzyme of the shikimate metabolism, resulting in inhibition of the synthesis of vital aromatic amino acids. However, not only plants but also bacteria can possess this enzyme so that influences of glyphosate on the microbiomes of exposed organisms cannot be excluded. Those influences may result in subtle and long-term effects, e.g., disturbance of the symbiotic interactions of bionts with microorganisms of their microbiomes. Mechanisms how the transformation product aminomethylphosphonic acid (AMPA) of glyphosate might interfere in this context have not understood so far. In the present study, molecular biological fingerprinting methods showed concentration-dependent effects of glyphosate and AMPA on fish microbiomes. In addition, age-dependent differences in the composition of the microbiomes regarding abundance and diversity were detected. Furthermore, the effect of exposure to glyphosate and AMPA was investigated for several fish pathogens of gut microbiomes in terms of their gene expression of virulence factors associated with pathogenicity. In vitro transcriptome analysis with the fish pathogen Yersinia ruckeri revealed that it is questionable whether the observed effect on the microbiome is caused by the intended mode of action of glyphosate, such as the inhibition of EPSP synthase activity.