The herbicidal action of diquat dibromide (DD) on plant cells is due primarily to the initiation of reactive oxygen species (ROS) formation, lipoperoxidation, and apoptotic cell death. It has been demonstrated that oxidative stress also occurs in animal cells exposed to high concentrations of DD; however, observations of DD’s effects on animal cells at concentrations below the reported ROS-initiation threshold suggest that some of these effects may not be attributable to ROS-induced cell death. Our results suggest that DD causes disruption of the Wnt pathway, calcium dysregulation, and cytoskeletal damage during development. Using embryos of the pond snail Lymnaea palustris as our model organism, we observed increased mortality, developmental delay and abnormality, altered motility, calcium dysregulation, decreased heart rate, and arrhythmia in embryos exposed to DD. Sperm extracted from adult snails that were exposed to DD exhibit altered motility, increased abundance, and high mortality. Effects were quantified via real-time imaging, heart rate assessment, flow cytometry, and mortality scoring. We propose that there are two models for the mechanism of DD’s action in animal cells: at low concentrations (≤28 µg/L), apoptotic cell death does not occur, but cytoskeletal elements, calcium regulation, and Wnt signaling are compromised, causing irreversible damage in L. palustris embryos; such damage is partially remediated with antioxidants or lithium chloride. At high concentrations of DD (≥44.4 µg/L), calcium dysregulation may be triggered, leading to the establishment of an intracellular positive feedback loop of ROS formation in the mitochondria, calcium release from the endoplasmic reticulum, calcium efflux, and apoptotic cell death. Permanent cellular damage occurring from exposure to sublethal concentrations of this widespread herbicide underscores the importance of research that elucidates the mechanism of DD on nontarget organisms.

How tree species perform across the breadth of their realized niche is essential for understanding their response to changing environmental conditions. This is especially relevant in topographically heterogeneous landscapes, where changes in elevation, exposure, slope and aspect all interact to determine how a particular species will perform at any location.  We focused on growth performance in Quercus rubra (red oak) in Shenandoah National Park. We established 4 transects that spanned >700 meters of elevation change. Each transect encompassed an exposure gradient from a stream-side to a ridgetop location, and were positioned on opposing aspects. Along each transect we cored five mature red oak trees at six equally spaced points. Tree cores were prepared using standard dendroecological methods. Temperature and precipitation data from 1945 to 2016 were obtained from National Climatic Data Center for nine weather stations surrounding Shenandoah National Park. Elevational lapse rates were calculated for temperature data to adjust regional temperature to accurately represent coring locations. Ring width was modelled against climatic and topographic variables to determine which variables were important to red oak growth.Results showed spring and autumn precipitation and temperature from both current and previous year were associated with growth performance. Red oak growth was maximized at low elevations at near to intermediate distances from stream, however as elevation increased red oak performed better at greater distances from streams. As climate continues to warm, higher elevations may receive more precipitation due to evapotranspiration and red oak will be found further from streams at higher elevations.

Diquat dibromide (DD), an active ingredient in the herbicide Roundup, is known to elicit oxidative stress in plant tissues leading to lipoperoxidation and apoptotic cell death. While exposure to DD has been demonstrated to cause ROS formation in animal tissues as well, its dose-dependent effects have been minimally characterized. Using embryos of the model organism Lymnaea palustris, we observe and quantify DD-induced effects including increased mortality, abnormal motility, heartrate arrhythmias, impaired shell formation in juvenile snails, and cellular dissociation using real-time imaging, heartrate assessment, and immunohistochemistry of cytoskeletal proteins. We have also demonstrated that calcium regulation is compromised in the presence of DD, causing a myriad of developmental and cytoskeletal defects, some of which are lessened by antioxidants. We suspect that the observed calcium dysregulation induced by high concentrations of DD (≥ 44.4 µg/L) may be one of the initial steps leading to the establishment of an intracellular positive feedback loop of ROS formation in the mitochondria and calcium release from the SER, leading to calcium efflux and apoptotic cell death. At low concentrations of DD (≤ 28 µg/L), however, apoptotic cell death does not occur, though cytoskeletal elements and calcium regulation are clearly impaired, suggesting that ROS formation may not occur at low DD concentrations and that the observed phenotypes result from ion dysregulation and disruption of cytoskeletal elements which negatively affect motility and heartrate. Ongoing research is being conducted to delineate the effects of DD on embryonic cytoskeletal elements.

It is well established that tree species grow within a realized niche. However, within that niche tree performance may not be equal. Therefore, we sought to quantify growth differentiation along elevational and climatic gradients in central Appalachia. A major issue of modelling tree growth is that regional weather stations are unable to capture fine-scale variation in temperature patterns along ecological gradients. To account for this variation, a linear regression model of temperature as a function of elevation was developed using daily temperature and precipitation data obtained from the National Oceanic and Atmospheric Administration for nine different weather stations in and around Shenandoah National Park, from which fine-scale temperature patterns of specific stations could be plotted and predicted.            The elevational range of our study spanned 294-1022 meters, with an average lapse rate of -0.007 °C/m for maximum temperature, and -0.005 °C/m for minimum temperature. Models of red oak ring width against climate and topographic variables found that predominantly spring and autumn maximum and minimum temperature, and precipitation from both the current and previous year, most strongly influenced growth performance. It was also discovered that while spring maximum temperatures have been increasing at higher elevations, they remain relatively stable at lower elevations. Conversely, spring minimum temperatures are increasing at lower elevations and not at high elevations, indicating that the temperature envelope is expanding at high elevations and constricting at lower elevations. This may have significant implications for how tree species will respond to changing climate differentially along ecological gradients.

We have established that the herbicide Roundup and its constituents are capable of causing developmental abnormalities and death in the pond snail Lymnaea palustris. Diquat dibromide (DD) has been identified as the Roundup constituent most consistently associated with developmental delays, embryonic abnormalities and lethality. However, the mechanism of DD action on live embryos remains to be discovered.   DD-treated embryos experience delayed development, loss of cell-cell adhesion, and irregular spinning patterns, possibly resulting from calcium dysregulation in ciliary motility. Notably, adult snails treated with DD exhibit weakening of their shell, also a calcium-dependent structure. Concurrent treatments of varying concentrations of calcium together with DD were conducted in order to determine how altered levels of calcium availability in the medium influence embryonic progression.  It was found that DD-treated embryos in low-calcium media experienced less phenotypic abnormalities compared to high-calcium and calcium ionophore treated embryos. Additionally, embryos in calcium-free medium containing DD exhibited more normal development overall compared to embryos in calcium-free medium without DD.   These results strongly suggest that calcium dysregulation plays a role in the developmental abnormalities and death observed in embryos treated with DD.  Experiments are being conducted to determine if DD exerts an increase in intracellular calcium through influx, inhibition of calcium release, or upregulation of calcium release from intracellular stores. Alternatively, there may be a different role of DD in modulating calcium regulation.   This work was funded in part by the Research Challenge Fund through a Summer Undergraduate Research Experience Grant from the West Virginia HEPC Division of Science and Research