Exposure to a fungicide for a field-realistic duration does not alter bumble bee fecal microbiota structure.

Social bees are frequently exposed to pesticides when foraging on nectar and pollen. Recent research has shown that pesticide exposure not only impacts social bee host health but can also alter the community structure of social bee gut microbiotas. However, most research on pesticide-bee gut microbiota interactions has been conducted in honey bees; bumble bees, native North American pollinators, have received less attention and, due to differences in their ecology, may be exposed to certain pesticides for shorter durations than honey bees. Here, we examine how exposure to the fungicide chlorothalonil for a short, field-realistic duration alters bumble bee fecal microbiotas (used as a proxy for gut microbiotas) and host performance. We expose small groups of Bombus impatiens workers (microcolonies) to field-realistic chlorothalonil concentrations for 5 days, track changes in fecal microbiotas during the exposure period and a recovery period, and compare microcolony offspring production between treatments at the end of the experiment. We also assess the use of fecal microbiotas as a gut microbiota proxy by comparing community structures of fecal and gut microbiotas. We find that chlorothalonil exposure for a short duration does not alter bumble bee fecal microbiota structure or affect microcolony production at any concentration but that fecal and gut microbiotas differ significantly in community structure. Our results show that, at least when exposure durations are brief and unaccompanied by other stressors, bumble bee microbiotas are resilient to fungicide exposure. Additionally, our work highlights the importance of sampling gut microbiotas directly, when possible.IMPORTANCEWith global pesticide use expected to increase in the coming decades, studies on how pesticides affect the health and performance of animals, including and perhaps especially pollinators, will be crucial to minimize negative environmental impacts of pesticides in agriculture. Here, we find no effect of exposure to chlorothalonil for a short, field-realistic period on bumble bee fecal microbiota community structure or microcolony production regardless of pesticide concentration. Our results can help inform pesticide use practices to minimize negative environmental impacts on the health and fitness of bumble bees, which are key native, commercial pollinators in North America. We also find that concurrently sampled bumble bee fecal and gut microbiotas contain similar microbes but differ from one another in community structure and consequently suggest that using fecal microbiotas as a proxy for gut microbiotas be done cautiously; this result contributes to our understanding of proxy use in gut microbiota research.

Seasonal dynamics in the mammalian microbiome between disparate environments.

Host-associated bacterial microbiomes can facilitate host acclimation to seasonal environmental change and are hypothesized to help hosts cope with recent anthropogenic environmental perturbations (e.g., landscape modification). However, it is unclear how recurrent and recent forms of environmental change interact to shape variation in the microbiome. The majority of wildlife microbiome research occurs within a single seasonal context. Meanwhile, the few studies of seasonal variation in the microbiome often restrict focus to a single environmental context. By sampling urban and exurban eastern grey squirrel populations in the spring, summer, autumn, and winter, we explored whether seasonal rhythms in the grey squirrel gut microbiome differed across environments using a 16S amplicon sequencing approach. Differences in the microbiome between urban and exurban squirrels persisted across most of the year, which we hypothesize is linked to anthropogenic food consumption, but we also observed similarities in the urban and exurban grey squirrel microbiome during the autumn, which we attribute to engrained seed caching instincts in preparation for the winter. Host behaviour and diet selection may therefore be capable of maintaining similarities in microbiome structure between disparate environments. However, the depletion of an obligate host mucin glycan specialist (Akkermansia) during the winter in both urban and exurban squirrels was among the strongest differential abundance patterns we observed. In summary, urban grey squirrels showed different seasonal patterns in their microbiome than squirrels from exurban forests; however, in some instances, host behaviour and physiological responses might be capable of maintaining similar microbiome responses across seasons.

An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

Pesticide-induced disturbances of bee gut microbiotas.

Social bee gut microbiotas play key roles in host health and performance. Worryingly, a growing body of literature shows that pesticide exposure can disturb these microbiotas. Most studies examine changes in taxonomic composition in Western honey bee (Apis mellifera) gut microbiotas caused by insecticide exposure. Core bee gut microbiota taxa shift in abundance after exposure but are rarely eliminated, with declines in Bifidobacteriales and Lactobacillus near melliventris abundance being the most common shifts. Pesticide concentration, exposure duration, season and concurrent stressors all influence whether and how bee gut microbiotas are disturbed. Also, the mechanism of disturbance-i.e. whether a pesticide directly affects microbial growth or indirectly affects the microbiota by altering host health-likely affects disturbance consistency. Despite growing interest in this topic, important questions remain unanswered. Specifically, metabolic shifts in bee gut microbiotas remain largely uninvestigated, as do effects of pesticide-disturbed gut microbiotas on bee host performance. Furthermore, few bee species have been studied other than A. mellifera, and few herbicides and fungicides have been examined. We call for these knowledge gaps to be addressed so that we may obtain a comprehensive picture of how pesticides alter bee gut microbiotas, and of the functional consequences of these changes.

Thyroid Disruptors: Extrathyroidal Sites of Chemical Action and Neurodevelopmental Outcome-An Examination Using Triclosan and Perfluorohexane Sulfonate.

Many xenobiotics are identified as potential thyroid disruptors due to their action to reduce circulating levels of thyroid hormone, most notably thyroxine (T4). Developmental neurotoxicity is a primary concern for thyroid disrupting chemicals yet correlating the impact of chemically induced changes in serum T4 to perturbed brain development remains elusive. A number of thyroid-specific neurodevelopmental assays have been proposed, based largely on the model thyroid hormone synthesis inhibitor propylthiouracil (PTU). This study examined whether thyroid disrupting chemicals acting distinct from synthesis inhibition would result in the same alterations in brain as expected with PTU. The perfluoroalkyl substance perfluorohexane sulfonate (50 mg/kg/day) and the antimicrobial Triclosan (300 mg/kg/day) were administered to pregnant rats from gestational day 6 to postnatal day (PN) 21, and a number of PTU-defined assays for neurotoxicity evaluated. Both chemicals reduced serum T4 but did not increase thyroid stimulating hormone. Both chemicals increased expression of hepatic metabolism genes, while thyroid hormone-responsive genes in the liver, thyroid gland, and brain were largely unchanged. Brain tissue T4 was reduced in newborns, but despite persistent T4 reductions in serum, had recovered in the PN6 pup brain. Neither treatment resulted in a low dose PTU-like phenotype in either brain morphology or neurobehavior, raising questions for the interpretation of serum biomarkers in regulatory toxicology. They further suggest that reliance on serum hormones as prescriptive of specific neurodevelopmental outcomes may be too simplistic and to understand thyroid-mediated neurotoxicity we must expand our thinking beyond that which follows thyroid hormone synthesis inhibition.

Towards a catalogue of biodiversity databases: An ontological case study.

Biodiversity informatics depends on digital access to credible information about species. Many online resources host species' data, but the lack of categorisation for these resources inhibits the growth of this entire field. To explore possible solutions, we examined the (now retired) Biodiversity Information Projects of the World (BIPW) dataset created by the Biodiversity Information Standards (TDWG); this project, which ran from 2007-2015 (officially removed from the TDWG website in 2018) was an attempt at organising the Web's biodiversity databases into an indexed list. To do this, we applied a simple classification scheme to score databases within BIPW based on nine data categories, to characterise trends and current compositions of this biodiversity e-infrastructure. Primarily, we found that of 600 databases investigated from BIPW, only 315 (~53%) were accessible at the time of this writing, underscoring the precarious nature of the biodiversity information landscape. Many of these databases are still available, but suffer accessibility issues such as link rot, thus putting the information they contain in danger of being lost. We propose that a community-driven database of biodiversity databases with an accompanying ontology could facilitate efficient discovery of relevant biodiversity databases and support smaller databases - which have the greatest risk of being lost.

Thyroid Hormone Disruption in the Fetal and Neonatal Rat: Predictive Hormone Measures and Bioindicators of Hormone Action in the Developing Cortex.

Adverse neurodevelopmental consequences remain a primary concern when evaluating the effects of thyroid hormone (TH) disrupting chemicals. Though the developing brain is a known target of TH insufficiency, the relationship between THs in the serum and the central nervous system is not well characterized. To address this issue, dose response experiments were performed in pregnant rats using the goitrogen propylthiouracil (PTU) (dose range 0.1-10 ppm). THs were quantified in the serum and brain of offspring at gestational day 20 (GD20) and postnatal day 14 (PN14), two developmental stages included in OECD and EPA regulatory guideline/guidance studies. From the dose response data, the quantitative relationships between THs in the serum and brain were determined. Next, targeted gene expression analyses were performed in the fetal and neonatal cortex to test the hypothesis that TH action in the developing brain is linked to changes in TH concentrations within the tissue. Results show a significant reduction of T4/T3 in the serum and brain of the GD20 fetus in response to low doses of PTU; interestingly, very few genes were significantly different at any dose tested. In the PN14 pup significant reductions of T4/T3 in the serum and brain were also detected; however, twelve transcriptional targets were identified in the neonatal cortex that correlated well with reduced brain THs. These results show that serum T4 is a good predictor of brain THs, and offer several target genes that could serve as pragmatic readouts of T4/T3 dysfunction within the PN14 cortex.

Atrazine does not induce pica behavior at doses that increase hypothalamic-pituitary-adrenal axis activation and cause conditioned taste avoidance.

Previous work has shown that a single oral administration of atrazine (ATR), a chlorotriazine herbicide, causes rapid increases in plasma adrenocorticotropic hormone (ACTH), serum corticosterone (CORT) and progesterone. The mechanism for these effects is unknown. To test whether administration of ATR causes hypothalamic-pituitary-adrenal (HPA) axis activation through the production of a generalized stress response resulting from gastrointestinal distress, we conducted both conditioned taste avoidance (CTA) and pica behavior experiments. Body temperature data were also collected to detect the presence of stress-induced hyperthermia. Adult male Wistar rats were given a single oral dose of ATR (0, 5, 25, 50, 100, or 200 mg/kg) or the primary ATR metabolite diamino-s-chlorotriazine (DACT; 135 mg/kg). Increases were observed in ACTH (LOEL, 12.5 mg/kg), CORT (LOEL, 5 mg/kg) and progesterone (LOEL, 5 mg/kg) 15 min following a single dose of ATR. DACT (135 mg/kg) increased ACTH (1.3-fold), CORT (2.9-fold) and progesterone (1.9-fold) above vehicle control concentrations, but the magnitude of the responses was much lower than that observed for an equal molar dose of ATR (200 mg/kg; 7.0, 9.0 and 11.0-fold above ACTH, CORT, progesterone controls, respectively). CTA results demonstrated conditioned taste avoidance to ATR, with a NOEL of 5 mg/kg. Animals dosed with DACT developed avoidance responses comparable to the highest dose of ATR. In the pica experiment, lower doses (5-50 mg/kg) of ATR had no effect on pica behavior, as measured 6 and 24 h post-dosing, nor did DACT. However, the highest dose of ATR (200 mg/kg) did induce pica behavior at both time points. No differences in body temperature were observed. Overall, results indicate that increases in ACTH and CORT secretion following administration of ATR occur at doses that are without effect on the display of pica behavior, indicating that the HPA-axis activation caused by ATR is not likely the result of gastrointestinal distress.

Gestational atrazine exposure: effects on male reproductive development and metabolite distribution in the dam, fetus, and neonate.

Few studies have investigated the long-term effects of atrazine (ATR) following in utero exposure. We evaluated the effects of gestational exposure of Sprague Dawley dams to ATR (0, 1, 5, 20, or 100mg/kg-d) on the reproductive development of male offspring. We also quantified the distribution of ATR and its chlorinated metabolites in maternal, fetal, and neonatal fluid and tissue samples following gestational and/or lactational exposure. Dose-dependent levels of chlorotriazines, primarily diamino-s-chlorotriazine, were present in most samples analyzed, including fetal tissue. In utero exposure to 1-20mg/kg-d ATR did not alter testosterone production, the timing of puberty, play behavior, or other androgen-dependent endpoints of male offspring. Significant maternal toxicity and postnatal mortality were observed at 100mg/kg-d. We conclude that, although levels of chlorotriazines within the fetus were considerable, gestational exposures of 1-20mg/kg-d do not lead to alterations in the measures of male development examined in this study.

Understanding the effects of atrazine on steroidogenesis in rat granulosa and H295R adrenal cortical carcinoma cells.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) was introduced in the 1950s as a broad spectrum herbicide, and remains one of the most widely used herbicides in the United States. Several studies have suggested that atrazine modifies steroidogenesis and may disrupt reproductive function and development in a variety of species. A primary concern has been whether atrazine increases the synthesis of estrogens, perhaps by enhancing aromatase gene expression and activity. In this study, the effect of atrazine was compared in cultures using primary granulosa cells and H295R adrenal cortical carcinoma cells. Atrazine (10 µM), but not its metabolite, 2-chloro-4,6-diamino-1,2,5-triazine (DACT), significantly increased estradiol production and aromatase activity in granulosa cell cultures only when measured for 1-h following 24h of exposure. In H295R cells, atrazine (10 µM) increased estradiol and estrone production. Importantly, atrazine (10 µM) increased progesterone production from both cell types suggesting a broader effect of atrazine on steroidogenesis.

Chlorotriazine herbicides and metabolites activate an ACTH-dependent release of corticosterone in male Wistar rats.

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

Effects of altered food intake during pubertal development in male and female wistar rats.

The U.S. Environmental Protection Agency is currently validating assays that will be used in a Tier I Screening Battery to detect endocrine disrupting chemicals. A primary concern with the Protocols for the Assessment of Pubertal Development and Thyroid Function in Juvenile Male and Female Rats is that a nonspecific reduction in body weight (BWT) during the exposure period may potentially confound the interpretation of effects on the endocrine endpoints. Wistar rats were underfed 10, 20, 30, or 40% less than the ad libitum food consumed by controls from postnatal days (PNDs) 22 to 42 (females) or PNDs 23 to 53 (males). Terminal BWT of females and males were 2, 4, 12, and 19% and 2, 6, 9, and 19% lower than controls, respectively. In the females, neither the age of pubertal onset nor any of the thyroid hormone endpoints were affected by food restriction (FR) that led to a 12% decrease in BWT. Similarly, none of the male reproductive endpoints examined were altered by FR that led to a 9% BWT decrease. However, decreased triiodothyronine and thyroxin was observed in FR males with a 9% reduced BWT. While these data support the use of the maximum tolerated dose for BWT (10%) for the female protocol, effects on the male thyroid endpoints indicate that a slightly lower limit (