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

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

IntroductionGlyphosate (N-[Phosphonomethyl]glycine)-based herbicides (GBHs) are the most frequently used herbicides globally and also one of the most controversial agrochemicals1. Evidence is accumulating with regard to the potentially negative effects of glyphosate on the development, phenotype, and fitness of most non-target animal taxa from invertebrates to vertebrates, yet, exposure levels (natural exposure load vs. levels used in experimental studies) need to be carefully accounted for2,3,4. Non-target organisms are commonly exposed to GBH residues in the food chain because residues can persist in soil, water, and plants5,6. In particular, the estimated amount of glyphosate introduced into the food chain through genetically modified, i.e. glyphosate tolerant crops (such as soybeans) add up to several thousands of metric tonnes yearly7. Consequently, different regulatory authorities heatedly debate the effects of GBH in our ecosystems.Organisms in early developmental stages are generally more susceptible to external stress compared to adults. In the case of environmental toxins, this may be related to disturbed ontogeny or undeveloped detoxification metabolism in juveniles8. In aquatic animals, embryos can be directly exposed to GBHs via the surrounding water. Glyphosate and commercial products (e.g. RoundUp) made with glyphosate have been repeatedly reported to cause embryo mortality and deformations in fish (zebrafish 10uM to 1 mM pure glyphosate9, 10 mg/L RoundUp or pure glyphosate10), and aquatic amphibians (Xenopus 0.3–1.3 mg/L RoundUp11, 500 pg/egg pure glyphosate12). In contrast, mammal and bird embryos and fetuses are exposed to glyphosate residues only via maternal transfer of the chemicals, which may result in malformations, altered sex ratios, and low sperm quality in rodent models (doses: 500 mg/kg RoundUp13, 5 g/L pure glyphosate14, 50–450 mg/kg RoundUp15). Such effects are referred to as (transmissive) maternal effects sensu16. Furthermore, recent studies suggest that effects of GBHs on the next generation can be mediated via epigenetic paternal effects, for example via alterations of paternal sperm(17, parental generation, a dose of 25 mg/kg BW pure glyphosate in rats18).However, the true maternal and paternal effects of GBH are poorly understood because the majority of the studies are involving direct embryo manipulations with high doses of GBHs. The authors of future studies should take into account that GBHs may influence the quality of the resources allocated to eggs/embryos and thus offspring development, phenotype, and fitness indirectly. Prenatal environmental conditions, and for example hormonal signals from the mother are known to have crucial importance for offspring development and even lasting effects into adulthood19,20,21.In this study we used birds as a model to study the parental and developmental effects of GBHs. Birds are highly underrepresented in studies testing the adverse effects of GBH residues on non-target taxa2, although they have recently been suggested as a key group for biomonitoring with regard to the effects of GBHs22. The importance of poultry in food production also calls for more attention on residues and the effects of GBHs in birds. In the two available studies of poultry and GBH-related maternal effects, a direct injection of a relatively high concentration of RoundUp (10 mg/kg glyphosate) in eggs was found to decrease hatchability, induce oxidative stress and cause damage to lipids in the exposed chicks, as compared to the control group23,24, potentially via the disruption of retinoid acid signaling12.To understand the potential for GBH-induced parental effects, we studied parental exposure of GBHs on embryo development and key physiological biomarkers—embryonic brain oxidative status in a bird model. To our knowledge, this is the first long-term study on parental effects of GBHs in bird taxa. Oxidative stress refers to the imbalance between reactive oxygen species (ROS) and antioxidants: If antioxidants are not able to neutralize ROS, oxidative damage to cell components (proteins, lipids, and DNA) will occur, which then has negative consequences on cell functions25. GBHs have been previously found to induce oxidative stress and damage in a variety of organisms and tissues, including embryos reviewed in2. We quantified glyphosate residues in eggs, but also maternal allocation to eggs (egg, yolk, and shell mass and yolk thyroid hormone concentration) to account for potential indirect GBH effects. Prenatal thyroid hormones (THs) (thyroxine, T4 and triiodothyronine, T3) play a key role in coordinating embryo development20,26, especially brain development27. Embryo THs have been reported to vary with maternal GBH exposure in rats (dose: RoundUp 5 and 50 mg/kg/day28), but generally the effects of GBHs on THs are poorly understood. Japanese quails were selected as the model species because the results can be applied to both wild birds feeding on GBH-contaminated food in the field and to poultry farming. We experimentally exposed parental bird generation to GBHs (ca 200 mg/kg feed) or respective controls from 10 days of age to 12 months. The egg samples were collected at 4 and 12 months to examine the potential cumulative effects of long-term exposure. We measured the potential effects on (1) on egg quality (egg, yolk, and shell mass as well as egg thyroid hormones); (2) on embryo development; and (3) embryo oxidative stress and damage.ResultsWe detected 0.76 mg/kg (S.D. ± 0.16) of glyphosate residue in eggs see also29, which is above the levels reported in the previous literature30. Egg mass from GBH and control parents did not differ after 4 or 12 months of exposure (treatment F1, 17,1 = 0.12, p = 0.73, treatment*exposure duration F1, 270 = 0.02, p = 0.89, Table 1) but was generally larger at 12 months of age (duration: F1, 271 = 8.8, p = 0.003). No differences between GBH exposed and control females in yolk mass, shell mass, or egg T3 and T4 concentrations were detected (Table 1, Suppl Fig. 1).Table 1 Quality of the eggs (egg, yolk, and shell mass; thyroid hormone concentrations: T3 = triiodothyronine, T4 = thyroxine, average ± SD) from GBH (glyphosate based herbicide)-exposed and control females. The egg mass was averaged over all eggs (4 and 12 months of exposure). The other parameters were measured after 4 months of exposure. See text for power analysis.Full size tableEmbryo development was normal in 89% of control eggs, while 76% of GBH eggs had normally developed embryos when pooling data from 3-day and 10-day embryos. The lower percentage of normal development in GBH eggs tended to be statistically significant (treatment F1, 22 = 3.08, p = 0.09) and the trend was similar at both 4 and 12 months of parental exposure (treatment*duration F1, 312 = 0.6, p = 0.43, Fig. 1). The eggs with no or little development were distributed across pairs and for none of the pairs were all eggs classified as undeveloped. Brain mass did not differ between embryos from GBH-exposed and control parents (mean ± SD in mg; GBH: 67.1 ± 12.5, control 68.1 ± 15.5; F1,31 = 0.04, p = 0.84). Brain oxidative status at 12 months of parental exposure was measured from 19 control and 16 GBH embryos. We measured ca 20% higher lipid damage in the GBH embryos than controls. This difference tended to be statistically significant (F1, 16.8 = 3.2, p = 0.088, Table 2, Suppl Fig. 2), yet there were no differences in the activity of antioxidant enzymes (GST, GP or CAT) between the two groups (Table 2, Suppl Fig. 2).Figure 1Embryonic status in relation to glyphosate-based herbicide exposure and duration of the exposure. GBH = glyphosate exposed, CO = controls. The bars are drawn separately for GBH and control eggs and after 4 and 12 months of exposure: we assessed 3-day-old embryos at 4 months and at 10-day-old embryos 12 months. Sample sizes are indicated in parentheses.Full size imageTable 2 Average (±SD) of glutathione-S-transferase (GST), glutathione peroxidase (GP), catalase (CAT) activity, and damage to lipids (MDA) in 10-day-old Japanese quail embryos exposed to maternally-derived glyphosate-based herbicide (GBH) or unexposed embryos (control). Associate statistics from linear mixed models (LMMs) are reported below. See text for power analysis.Full size tableDiscussionOur results indicate that parental exposure to GBHs may lead to weak negative effects on embryo development and physiology. We detected no evidence for changes in egg quality (egg, yolk, shell mass, or egg hormone concentration), suggesting no indirect effects via the altered allocation of resources or hormones to eggs and embryos.The tendency for poorer embryo development in eggs of GBH-exposed parents may be explained by GBH-related effects via either a paternal or maternal route, or both. We did not appear to observe cumulative effects of GBH exposure on embryo viability, as longer exposure (12 vs 4 months) did not lead to less viability. The eggs with no visible development (to the naked eye) could have been completely infertile or showing developmental arrest at an early stage. There are multiple potential mechanisms underlying such effects: For example, early embryos of fish showed developmental anomalies (disproportional head and body size) and increased heart rate (medeka, 100–500 mg/L RoundUp31, neurotoxic effects zebrafish, up to 50 mg/L RoundUp32) and Xenopus showed craniofacial deformities12. Poultry embryos and mice oocytes expressed increased oxidative stress (500 mM pure glyphosate33) and lower hatchability (10 mg/kg egg RoundUp<a title="Fathi, M. A. et al. Effect of in ovo glyphosate injection on embryonic development, serum

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.</p

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

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

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.</p

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

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 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.peerReviewe

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

Abstract 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