Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles: II: agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory conditions

Glyphosate-based herbicides are currently the most commonly used herbicides in the world. They have been shown to affect survival, growth, development and sexual differentiation of tadpoles under chronic laboratory exposures but this has not been investigated under more environmentally realistic conditions. The purpose of this study is (1) to determine if an agriculturally relevant exposure to Roundup WeatherMax®, a relatively new and understudied formulation, influences the development of wood frog tadpoles (Lithobates sylvaticus) through effects on the mRNA levels of genes involved in the control of metamorphosis; (2) to compare results to the well-studied Vision® formulation (containing the isopropy-lamine salt of glyphosate [IPA] and polyethoxylated tallowamine [POEA] surfactant) and to determine which ingredient(s) in the formulations are responsible for potential effects on development; and (3) to compare results to recent field studies that used a similar experimental design. In the present laboratory study, wood frog tadpoles were exposed to an agriculturally relevant application (i.e., two pulses) of Roundup WeatherMax® and Vision® herbicides as well as the active ingredient (IPA) and the POEA surfactant of Vision®. Survival, development, growth, sex ratios and mRNA levels of genes involved in tadpole metamorphosis were measured. Results show that Roundup WeatherMax® (2.89 mg acid equiv-alent (a.e.)/L) caused 100% mortality after the first pulse. Tadpoles treated with a lower concentration of Roundup WeatherMax® (0.21 mg a.e./L) as well as Vision® (2.89 mg a.e./L), IPA and POEA had an increased condition factor (based on length and weight measures in the tadpoles) relative to controls at Gosnerstage (Gs) 36/38. At Gs42, tadpoles treated with IPA and POEA had a decreased condition factor. Alsoat Gs42, the effect on condition factor was dependent on the sex of tadpoles and significant treatment effects were only detected in males. In most cases, treatment reduced the normal mRNA increase of key genes controlling development in tadpoles between Gs37 and Gs42, such as genes encoding thyroid hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail. We conclude that glyphosate-based herbicides have the potential to alter mRNA profiles during metamorphosis. However, studies in natural systems have yet to replicate these negative effects, which highlight the need for more ecologically relevant studies for risk assessment

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frogs (Lithobates sylvaticus) tadpoles: I, Chronic laboratory exposures to VisionMax®

The purpose of this study was to determine if chronic exposure to the glyphosate-based herbicideVisionMax®affects the survival, development, growth, sex ratios and expression of specific genesinvolved in metamorphosis of wood frog tadpoles (Lithobates sylvaticus). We hypothesized that expo-sure to this herbicide will affect developmental rates by disrupting hormone pathways, sex ratios and/orgonadal morphology. Tadpoles were chronically exposed in the laboratory from Gosner developmen-tal stage 25 to 42 to four different concentrations of VisionMax® (ranging from 0.021 to 2.9 mg acidequivalents/L). Chronic exposures to VisionMax® had direct effects on the metamorphosis of L. sylvaticustadpoles by decreasing development rates, however, there was a decrease in survival only in the group exposed to the highest dose of VisionMax® (2.9 mg a.e./L; from approximately 96% in the control groupto 77% in the treatment group). There was a decrease in the number of tadpoles reaching metamor-phic climax, from 78% in the control group to 42% in the VisionMax®(2.9 mg a.e./L) group, and a 7-daydelay to reach metamorphic climax in the same treatment group. No effects of exposure on sex ratiosor gonadal morphology were detected in tadpoles exposed to any of the concentrations of VisionMax®tested. Gene expression analyses in brain and tail tissues demonstrated that exposure to VisionMax® alters the expression of key genes involved in development. Results showed significant interaction (two-way ANOVA, P < 0.05) between developmental Gosner stage and treatment in brain corticotropin-releasingfactor, deiodinase type II (dio2) and glucocorticotiroid receptor (grII) and tail dio2 and grII. This demon-strates that mRNA levels may be differently affected by treatment depending on the developmental stageat which they are assessed. At the same time there was a clear dose–response effect for VisionMax®toincrease thyroid hormone receptor ˇ in tadpole brain (F(2,69)= 3.475, P = 0.037) and tail (F(2,69)= 27.569,P < 0.001), regardless of developmental stage. Interestingly, delays in development (or survival) wereonly observed in the group exposed to 2.9 mg a.e./L of VisionMax®, suggesting that tadpoles need to be exposed to a “threshold” concentration of glyphosate-based herbicide to exhibit phenotypic observable effects. We suggest that the up regulation of genes that trigger metamorphosis following VisionMax® herbicide exposure might result from a compensatory response for the delays in development observed. Further studies are needed to determine if disruption of expression of these key genes leads to long-termeffects when metamorphs reach adult stages

Sexing frogs by real-time PCR : using aromatase (cyp19) as an early ovarian differentiation marker

Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, Lithobates sylvaticus and Silurana tropicalis tadpoles were sampled at different developmental stages covering the entire process of sex differentiation.Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase (cyp19), forkhead box L2 (foxl2) and the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17). Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of cyp17, cyp19 and foxl2 in L. sylvaticus adult gonads and gonad-mesonephros complex(GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S.tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future