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.

Does Glyphosate Affect the Human Microbiota?

Glyphosate is the world's most widely used agrochemical. Its use in agriculture and gardening has been proclaimed safe because humans and other animals do not have the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, increasing numbers of studies have demonstrated risks to humans and animals because the shikimate metabolic pathway is present in many microbes. Here, we assess the potential effect of glyphosate on healthy human microbiota. Our results demonstrate that more than one-half of human microbiome are intrinsically sensitive to glyphosate. However, further empirical studies are needed to determine the effect of glyphosate on healthy human microbiota.

Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

Glyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (5-enolpyruvylshikimate-3-phosphate, EPSP) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in bacteria, fungi and plants. Our results reveal that bacteria may easily become resistant to glyphosate through changes in the 5-enolpyruvylshikimate-3-phosphate synthase active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

Glyphosate-based herbicides influence antioxidants, reproductive hormones and gut microbiome but not reproduction: A long-term experiment in an avian model.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. GBH residues in the wild, in animal and human food may expose non-target organisms to health risks, yet the developmental and cumulative effects of GBHs on physiology and reproduction remain poorly understood. We present the first long-term study on the effects of subtoxic GBH exposure (160 mg/kg) on multiple key physiological biomarkers (cellular oxidative status and neurotransmitters), gut microbiome, reproductive hormones, and reproduction in an avian model. We experimentally exposed in Japanese quail females and males (Coturnix japonica) to GBHs and respective controls from the age of 10 days-52 weeks. GBH exposure decreased hepatic activity of an intracellular antioxidant enzyme (catalase), independent of sex, but did not influence other intracellular oxidative stress biomarkers or neurotransmitter enzyme (acetylcholinesterase). GBH exposure altered overall gut microbiome composition, especially at a younger age and in females, and suppressed potentially beneficial microbes at an early age. Many of the microbial groups increased in frequency from 12 to 28 weeks under GBH exposure. GBH exposure decreased male testosterone levels both at sexual maturity and at 52 weeks of exposure, but did not clearly influence reproduction in either sex (maturation, testis size or egg production). Future studies are needed to characterize the effects on reproductive physiology in more detail. Our results suggest that cumulative GBH exposure may influence health and reproduction-related traits, which is important in predicting their effects on wild populations and global poultry industry.

Glyphosate-based herbicide has soil-mediated effects on potato glycoalkaloids and oxidative status of a potato pest.

Glyphosate is the most used herbicide worldwide, targeting physiological pathways in plants. Recent studies have shown that glyphosate can also cause toxic effects in animals. We investigated the glyphosate-based herbicide (GBH)-induced changes in potato (Solanum tuberosum) plant chemistry and the effects of a GBH on the survival rate and oxidative status of the Colorado potato beetle (Leptinotarsa decemlineata). The beetles were reared on potato plants grown in pots containing soil treated with a GBH (Roundup Gold, 450 g/l) or untreated soil (water control). The 2nd instar larvae were introduced to the potato plants and then collected in 2 phases: as 4th instar larvae and as adults. The main glycoalkaloids of the potato plants, α-solanine and α-chaconine, were measured twice during the experiment. The α-solanine was reduced in potato plants grown in GBH-treated soil, which can be detrimental to plant defenses against herbivores. GBH treatment had no effect on the survival rate or body mass of the larvae or the adult beetles. In the larvae, total glutathione (tGSH) concentration and the enzyme activity of catalase (CAT), superoxide dismutase, and glutathione-S-transferase were increased in the GBH treatment group. In the adult beetles, CAT activity and tGSH levels were affected by the interactive effect of GBH treatment and the body mass. To conclude, environmentally relevant concentrations of a GBH can affect the potato plant's glycoalkaloid concentrations, but are not likely to directly affect the survival rate of the Colorado potato beetle, but instead, modify the antioxidant defense of the beetles via diet.

Effects of parental exposure to glyphosate-based herbicides on embryonic development and oxidative status: a long-term experiment in a bird model.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. GBH residues are detected in soil, water, crops, and food products, potentially exposing non-target organisms to health risks; these organisms include wildlife, livestock, and humans. However, the potential for GBH-related parental effects are poorly understood. In the case of birds, GBHs may be transferred directly from mothers to eggs, or they may indirectly influence offspring performance by altered maternal resource allocation to eggs. We experimentally exposed a parental generation of Japanese quails (Coturnix japonica) to GBHs (200 mg/kg feed) or respective controls. Glyphosate residues were found in eggs (ca 0.76 kg/mg). Embryonic development tended to be poorer in the eggs of GBH-exposed parents (76% of eggs showed normal development) compared to control parents (89% normal eggs). Embryonic brain tissue from GBH-exposed parents tended to express more lipid damage (20% higher), yet other biomarkers showed no apparent differences. We detected no differences in egg quality (egg, yolk, or shell mass, egg hormone concentration) across the treatment groups. Given this is the first long-term study testing parental effects of GBHs with birds, more studies are needed characterizing GBH-associated changes in maternal allocation and for example epigenetic programming.

Glyphosate-based herbicide affects the composition of microbes associated with Colorado potato beetle (Leptinotarsa decemlineata).

Here, we examined whether glyphosate affects the microbiota of herbivores feeding on non-target plants. Colorado potato beetles (Leptinotarsa decemlineata) were reared on potato plants grown in pots containing untreated soil or soil treated with glyphosate-based herbicide (GBH). As per the manufacturer's safety recommendations, the GBH soil treatments were done 2 weeks prior to planting the potatoes. Later, 2-day-old larvae were introduced to the potato plants and then collected in two phases: fourth instar larvae and adults. The larvae's internal microbiota and the adults' intestinal microbiota were examined by 16S rRNA gene sequencing. The beetles' microbial composition was affected by the GBH treatment and the differences in microbial composition between the control and insects exposed to GBH were more pronounced in the adults. The GBH treatment increased the relative abundance of Agrobacterium in the larvae and the adults. This effect may be related to the tolerance of some Agrobacterium species to glyphosate or to glyphosate-mediated changes in potato plants. On the other hand, the relative abundances of Enterobacteriaceae, Rhodobacter, Rhizobium and Acidovorax in the adult beetles and Ochrobactrum in the larvae were reduced in GBH treatment. These results demonstrate that glyphosate can impact microbial communities associated with herbivores feeding on non-target crop plants.

Female Preference and Adverse Developmental Effects of Glyphosate-Based Herbicides on Ecologically Relevant Traits in Japanese Quails.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. An increasing number of studies have identified GBH residues in soil, water, and even human food that may expose nontarget organisms including wildlife, livestock, and humans to health risks. After a heated debate, the European Union allowed the use of GBHs to continue until 2022, after which their risks will be re-evaluated. Thus, decision makers urgently need scientific evidence on GBH residues and their possible effects on ecosystems. An important, yet neglected, aspect is to assess whether animals show preference or avoidance for GBH-contaminated food, as it can influence the likelihood of adverse health effects in wildlife. Here, using Japanese quails (Coturnix japonica) as our model, we show that females preferred GBH-contaminated food compared to control food. In females, exposure to GBHs caused delayed plumage development, and GBH residues were present in eggs, muscles, and liver. These results indicate that female preference is not adaptive, potentially exposing nontarget animals to greater risk of adverse effects of GBHs in natural and agricultural environments. Our results on tissue residues suggest that further studies are needed to understand the risks of such residues in the food chain.

Effects of a glyphosate-based herbicide on survival and oxidative status of a non-target herbivore, the Colorado potato beetle (Leptinotarsa decemlineata).

Glyphosate is the globally most used herbicide against a wide range of weeds. Glyphosate has been considered safe to animals as it mainly targets physiological pathways in plants. However, recent toxicological studies have revealed that glyphosate can cause various toxic effects also on animals. In this study, we investigated the direct toxic effects of a glyphosate-based herbicide (GBH, Roundup® Bio) on 1) survival and 2) oxidative status of a non-target herbivore by using Colorado potato beetles (Leptinotarsa decemlineata), originating from Poland and USA, as model species. Larvae were randomly divided into three groups: 1) high concentration (100% Roundup Bio, 360 g/l), 2) low concentration (1.5% Roundup Bio) and 3) control group (water). Larvae were exposed to Roundup for different time periods: 2 h, 24 h, 48 h, 72 h and 96 h. Larval survival decreased in the group treated with high concentration of GBH compared to controls, whereas the low concentration group did not differ from the control group. GBH treatment had no association with oxidative status biomarkers (i.e. catalase, superoxide dismutase, glutathione-S-transferase, glutathione and glutathione related enzymes), but increased lipid hydroperoxide levels after 2 h exposure, suggesting increased oxidative damage soon after the exposure. Larvae of different origin also differed in their oxidative status, indicating population-dependent differences in antioxidant defence system. Environmentally relevant concentrations of GBH are not likely to affect larval survival, but high concentrations can reduce survival and increase oxidative damage of non-target herbivores. Also, populations of different origin and pesticide usage history can differ in their tolerance to GBH.

Temporal trends in metal pollution: using bird excrement as indicator.

Past mining and smelting activities have resulted in metal polluted environments all over the world, but long-term monitoring data is often scarce, especially in higher trophic levels. In this study we used bird (Parus major and Ficedula hypoleuca) excrement to monitor metal pollution in the terrestrial environment following 16 years of continuously reduced emissions from a copper/nickel smelter in Finland. In the early 1990s, lead and cadmium concentrations dropped significantly in excrement, but the reduction did not directly reflect the changes in atmospheric emission from the smelter. This is likely due to a continuous contribution of metals also from the soil pool. We conclude that bird excrement can be used to assess changes in the environment as a whole but not specifically changes in atmospheric emission. Inter-annual variation in excrement concentration of especially copper and nickel demonstrates the importance of long-term monitoring to discern significant trends.

Effects of early-life lead exposure on oxidative status and phagocytosis activity in great tits (Parus major).

Lead is a highly poisonous metal with a very long half-life, distributing throughout the body in blood and accumulating primarily in bones and kidney. We studied the short and long-term effects of early-life lead exposure on antioxidant defense and phagocytosis activity in a small passerine bird, the great tit (Parus major) by manipulating dietary lead levels of the nestlings. We had three experimental groups, exposed to environmentally relevant lead concentrations; high (4 µg/g body mass), low (1 µg/g body mass) and control (0 µg/g body mass) group. As a comparison, a great tit population breeding in the vicinity of a metal smelter was included to the experimental set-up. We measured glutathione, the ratio of reduced and oxidized glutathione, and the antioxidant enzymes: glutathione peroxidase, glutathione-S-transferase, catalase and superoxide dismutase together with protein carbonylation and phagocytosis activity to study the effects of lead on the oxidative status and immune function of birds. We found differences in enzyme activities between the study groups, but in most cases the smelter group differed from the other groups. Despite the differences observed in antioxidant enzymes, our results indicate only minor short-term effects of lead exposure on oxidative status, since either glutathione ratio or protein carbonylation were not affected by lead. Phagocytosis activity was not linked to higher lead concentrations either. Interestingly, protein carbonylation was positively associated with enzyme activities and glutathione level. Our results did not show major long-term effects of lead on the oxidative status of great tits.

Oxidative status in nestlings of three small passerine species exposed to metal pollution.

Antioxidant defense has an important role in the protection of organisms against oxidative stress caused by reactive oxygen species (ROS). Many metals are capable of generating ROS and inducing oxidative damage, and may therefore lead to changes in oxidative regulation. We studied species-specific variation in the oxidative status of great tit (Parus major), blue tit (Cyanistes caeruleus) and pied flycatcher (Ficedula hypoleuca) nestlings in a vicinity of a non-ferrous smelter. Non-enzymatic (glutathione [tGSH], GSH:GSSG ratio, and carotenoids) and enzymatic (glutathione peroxidase [GP], glutathione-S-transferase [GST], superoxide dismutase [SOD], and catalase [CAT]) antioxidants were evaluated to determine the effects of metal exposure on the oxidative status of the birds. We found strong evidence of interspecific variation in CAT and SOD activities, whereas less variation was observed in parameters related to glutathione metabolism. Oxidative state (in terms of tGSH and GSH:GSSG) did not vary between species, suggesting that different species may employ different antioxidant pathways to achieve the same oxidative state. Oxidative status was only weakly related to metal exposure, and these associations were further obscured by species-specific environmental effects. Our results indicate that effects on oxidative status observed in one species cannot be generalized to other ones. Future work should attempt to incorporate species-specific biology and environmental context into assessments of contaminant impacts on oxidative regulation of passerine birds.

Decreased metal accumulation in passerines as a result of reduced emissions.

Technological advances in industrial processes have resulted in reduced atmospheric emissions from metal industries all over the globe, but the response of the environment is not well known. The authors studied metal (As, Cd, Cu, Ni, Pb, and Se) accumulation in passerine birds (pied flycatcher, Ficedula hypoleuca, and great tit, Parus major) following almost 20 years of reduced metal emissions from the largest nonferrous smelter in Finland. Close to the industry, emission reductions resulted in reduced exposure to several of the elements and, more importantly, reduced accumulation by 58 to 95% in liver tissue. Albeit showing significant tissue reductions, nestlings had elevated concentrations of arsenic, cadmium, lead, and selenium close to the industry. Single-element concentrations were below critical levels associated with subclinical effects, but the mixture of toxic elements indirectly affected health and reproduction. Our study indicates that in environments with moderate duration of industrial activity, impact, and soil pollution, metal accumulation in organisms can decrease relatively rapidly when atmospheric emissions are reduced.

Variation of basal EROD activities in ten passerine bird species--relationships with diet and migration status.

Inter-specific differences in animal defence mechanisms against toxic substances are currently poorly understood. The ethoxyresorufin-O-deethylase (EROD) enzyme plays an important role in defence against toxic chemicals in a wide variety of animals, and it is an important biomarker for environmental contamination. We compared basal hepatic EROD activity levels among ten passerine species to see if there is inter-specific variation in enzyme activity, especially in relation to their diet and migration status. Migratory insectivores showed higher EROD activity compared to granivores. We hypothesize that the variable invertebrate diet of migratory insectivores contains a wider range of natural toxins than the narrower diet of granivores. This may have affected the evolution of mixed function oxidases (MFO) system and enzyme activities. We further tested whether metabolic rates or relative liver size were associated with the variation in detoxification capacity. We found no association between EROD activity and relative (per mass unit) basal metabolic rate (BMR). Instead, EROD activity and relative liver mass (% of body mass) correlated positively, suggesting that a proportionally large liver also functions efficiently. Our results suggest that granivores and non-migratory birds may be more vulnerable to environmental contaminants than insectivores and migratory birds. The diet and migration status, however, are phylogenetically strongly connected to each other, and their roles cannot be fully separated in our analysis with only ten passerine species.