Factors of global change affecting plants act at different levels of the ecological hierarchy.

Plants and ecosystems worldwide are exposed to a wide range of chemical, physical, and biological factors of global change, many of which act concurrently. As bringing order to the array of factors is required in order to generate an enhanced understanding of simultaneous impacts, classification schemes have been developed. One such classification scheme is dedicated to capturing the different targets of global change factors along the ecological hierarchy. We build on this pioneering work, and refine the conceptual framework in several ways, focusing on plants and terrestrial systems: (i) we more strictly define the target level of the hierarchy, such that every factor typically has just one target level, and not many; (ii) we include effects above the level of the community, that is, there are effects also at the ecosystem scale that cannot be reduced to any level below this; (iii) we introduce the level of the landscape to capture certain land use change effects while abandoning the level below the individual. We discuss how effects can propagate along the levels of the ecological hierarchy, upwards and downwards, presenting opportunities for explaining non-additivity of effects of multiple factors. We hope that this updated conceptual framework will help inform the next generation of plant-focused global change experiments, specifically aimed at non-additivity of effects at the confluence of many factors.

Studying interactions among anthropogenic stressors in freshwater ecosystems: A systematic review of 2396 multiple-stressor experiments.

Understanding the interactions among anthropogenic stressors is critical for effective conservation and management of ecosystems. Freshwater scientists have invested considerable resources in conducting factorial experiments to disentangle stressor interactions by testing their individual and combined effects. However, the diversity of stressors and systems studied has hindered previous syntheses of this body of research. To overcome this challenge, we used a novel machine learning framework to identify relevant studies from over 235,000 publications. Our synthesis resulted in a new dataset of 2396 multiple-stressor experiments in freshwater systems. By summarizing the methods used in these studies, quantifying trends in the popularity of the investigated stressors, and performing co-occurrence analysis, we produce the most comprehensive overview of this diverse field of research to date. We provide both a taxonomy grouping the 909 investigated stressors into 31 classes and an open-source and interactive version of the dataset (https://jamesaorr.shinyapps.io/freshwater-multiple-stressors/). Inspired by our results, we provide a framework to help clarify whether statistical interactions detected by factorial experiments align with stressor interactions of interest, and we outline general guidelines for the design of multiple-stressor experiments relevant to any system. We conclude by highlighting the research directions required to better understand freshwater ecosystems facing multiple stressors.

Winter is coming: Interactions of multiple stressors in winter and implications for the natural world.

Winter is a key driver of ecological processes in freshwater, marine and terrestrial ecosystems, particularly in higher latitudes. Species have evolved various adaptive strategies to cope with food limitations and the cold and dark wintertime. However, human-induced climate change and other anthropogenic stressors are impacting organisms in winter in unpredictable ways. In this paper, we show that global change experiments investigating multiple stressors have predominantly been conducted during summer months. However, effects of anthropogenic stressors sometimes differ between winter and other seasons, necessitating comprehensive investigations. Here, we outline a framework for understanding the different effects of anthropogenic stressors in winter compared to other seasons and discuss the primary mechanisms that will alter ecological responses of organisms (microbes, animals and plants). For instance, while the magnitude of some anthropogenic stressors can be greater in winter than in other seasons (e.g. some pollutants), others may alleviate natural winter stress (e.g. warmer temperatures). These changes can have immediate, delayed or carry-over effects on organisms during winter or later seasons. Interactions between stressors may also vary with season. We call for a renewed research direction focusing on multiple stressor effects on winter ecology and evolution to fully understand, and predict, how ecosystems will fare under changing winters. We also argue the importance of incorporating the interactions of anthropogenic stressors with winter into ecological risk assessments, management and conservation efforts.

Rapid evolution generates synergism between multiple stressors: Linking theory and an evolution experiment.

Global change encompasses many co-occurring anthropogenic stressors. Understanding the interactions between these multiple stressors, whether they be additive, antagonistic or synergistic, is critical for ecosystem managers when prioritizing which stressors to mitigate in the face of global change. While such interactions between stressors appear prevalent, it remains unclear if and how these interactions change over time, as the majority of multiple-stressor studies rarely span multiple generations of study organisms. Although meta-analyses have reported some intriguing temporal trends in stressor interactions, for example that synergism may take time to emerge, the mechanistic basis for such observations is unknown. In this study, by analysing data from an evolution experiment with the rotifer Brachionus calyciflorus (~35 generations and 31,320 observations), we show that adaptation to multiple stressors shifts stressor interactions towards synergism. We show that trade-offs, where populations cannot optimally perform multiple tasks (i.e. adapting to multiple stressors), generate this bias towards synergism. We also show that removal of stressors from evolved populations does not necessarily increase fitness and that there is variation in the evolutionary trajectories of populations that experienced the same stressor regimes. Our results highlight outstanding questions at the interface between evolution and global change biology, and illustrate the importance of considering rapid adaptation when managing or restoring ecosystems subjected to multiple stressors under global change.

Mechanisms underpinning nonadditivity of global change factor effects in the plant-soil system.

Plant-soil systems are key for understanding the effects of factors of global change. Recent work has highlighted the general importance of considering the simultaneous incidence of some factors or stressors. To help mechanistically dissect the possible interactions of such factors, we here propose three broad groups of mechanisms that may generally lead to nonadditivity of responses within a plant-soil system: direct factor interactions (that is one factor directly changing another), within-plant information processing and crosstalk, and effects of factors on groups of soil biota interacting with plants. Interactions are also possible within and across these groups. Factor interactions are very likely to be present in experiments, especially when dealing with an increasing number of factors. Identifying the nature of such interactions will be essential for understanding and predicting global change impacts on plants and soil.

Towards a unified study of multiple stressors: divisions and common goals across research disciplines.

Anthropogenic environmental changes, or 'stressors', increasingly threaten biodiversity and ecosystem functioning worldwide. Multiple-stressor research is a rapidly expanding field of science that seeks to understand and ultimately predict the interactions between stressors. Reviews and meta-analyses of the primary scientific literature have largely been specific to either freshwater, marine or terrestrial ecology, or ecotoxicology. In this cross-disciplinary study, we review the state of knowledge within and among these disciplines to highlight commonality and division in multiple-stressor research. Our review goes beyond a description of previous research by using quantitative bibliometric analysis to identify the division between disciplines and link previously disconnected research communities. Towards a unified research framework, we discuss the shared goal of increased realism through both ecological and temporal complexity, with the overarching aim of improving predictive power. In a rapidly changing world, advancing our understanding of the cumulative ecological impacts of multiple stressors is critical for biodiversity conservation and ecosystem management. Identifying and overcoming the barriers to interdisciplinary knowledge exchange is necessary in rising to this challenge. Division between ecosystem types and disciplines is largely a human creation. Species and stressors cross these borders and so should the scientists who study them.

Heatwaves and carbon dioxide enrichment impact invertebrate drift and insect emergence patterns across time in experimental streams.

Climate change and human land use are considered key threats to freshwater invertebrates. Heatwaves can impact the phenology of insects and population dynamics, yet have been largely ignored in experiments compared to mean temperature changes. Another major anthropogenic stressor driving invertebrate community changes is deposited fine sediment; therefore, effects of key climate-change drivers on invertebrate drift and insect emergence rates may differ between sediment-impacted and non-impacted streams. However, this has never been tested in a realistic outdoor experiment. We investigated the individual and combined effects of two 7-day heatwaves, CO2 enrichment, flow velocity variability (periods of fast and slow) and fine sediment on stream drift and emergence responses, sampled four times during a 7-week experiment in 128 flow-through stream mesocosms. We examined invertebrate drift and insect emergence responses to the four stressors, and used these responses to help explain the benthic invertebrate community responses already assessed (sampled at the end of the experiment). Heatwave 1 strongly increased emergence (dominated by Chironomidae), causing an earlier emergence peak, an effect not repeated during heatwave 2, seven days later. During heatwave 1, emerged chironomids were larger in heated channels, but smaller in heated channels afterwards, suggesting a different effect on body size of short-term heatwaves to previous constant warming experiments. CO2 enrichment reduced drifting EPT and total and Chironomidae emergence on three sampling occasions each. After heatwave 1, total drift and total emergence were strongly reduced by heating in ambient-CO2 channels, whereas no reduction occurred in CO2-enriched channels. During heatwave 2, total drift increased in channels without sediment but not in channels with added sediment. Overall, our findings suggest heatwaves can shift the timing of stream insect emergence, regardless of longer-term mean temperatures. They also show that heatwaves, raised CO2, and fine sediment can modulate each others' effects on drift and emergence dynamics.

Flow cytometry and laser scanning cytometry, a comparison of techniques.

OBJECTIVE: Flow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications. METHODS: A Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting. RESULTS: Both techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer's disease. CONCLUSIONS: Understanding the differences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capabilities with the cellular characteristics they wish to measure delineating molecular and cellular events occurring in health and disease. Discovery of mechanisms in cells using FCM and LSCM provide evidence needed to guide future treatment and interventions.

Detection of thromboxane A(2) receptor mRNA in rabbit nodose ganglion neurons.

Thromboxane A(2) (TXA(2)) is an arachidonic acid metabolite that is released during tissue trauma and elicits platelet aggregation and vascular smooth muscle contraction. Previous research has shown that TXA(2) stimulates pulmonary and cardiac vagal afferent neurons. Therefore, we hypothesized that the presence of the TXA(2) receptor (TP) in vagal neurons would allow for stimulation or modulation of these neurons by TXA(2). To test this hypothesis, single cell RT-PCR was employed using neurons obtained from primary cell cultures of nodose ganglia excised from adult rabbits. Since the sequence for the rabbit TP gene was unknown, a portion of the rabbit TP cDNA was first amplified, cloned, and sequenced. Primer sets for TP were then designed based on this sequence and used in conjunction with a neuronal marker, medium weight neurofilament (NFM), in multiplex RT-PCR reactions. Ninety-three cells were isolated from culture and RT-PCR was carried out on individual cells. Using an aliquot from the initial RT-PCR reaction, a second round of PCR was then employed in which the NFM and TP primer sets were split up into separate reactions. Twenty-three of the 82 cells that were positive for NFM were also positive for TP. Therefore, we conclude that the presence of TP mRNA in a subset of cultured nodose ganglion neurons allows for the possibility that TXA(2) may directly stimulate or modulate vagal afferent neurons.

Gastric reflux: association with aspiration and oral secretion pH as marker of reflux: a descriptive correlational study.

BACKGROUND: Gastric reflux leading to pulmonary aspiration is a frequent event in mechanically ventilated, gastric-fed patients, which can lead to ventilator-associated complications and pneumonia. OBJECTIVES: The objectives of this study were to determine the association between gastric reflux and aspiration using the presence of pepsin in oral or tracheal secretions as a marker of reflux or aspiration and to determine the association between the pH (range, 0-14) and the presence of pepsin in oral secretions. METHODS: A descriptive correlational study was conducted in mechanically ventilated surgical or medical patients receiving gastric tube feedings. Oral secretions were suctioned hourly and tracheal secretions every 2 to 3 hours for 12-hour periods over 1 to 2 days in 15 patients. RESULTS: There were 142 paired samples of oral tracheal secretions. A majority of samples (60%) had the same results, with 32% both pepsin-positive and 27% both pepsin-negative. The range of pH measurements was 4 to 8, with a mean of 6.3 ± 0.05. Ninety oral specimens had a pH of 4 to 6. Forty-seven of the oral specimens with pH measures between 4 and 6 (52%) were pepsin-positive. The correlation of pH percent pepsin-positive oral secretions was not significant. CONCLUSION: Aspiration events were more frequent than reflux events. Measurement of actual pepsin concentration to detect new reflux and aspiration events is recommended in future studies. Bedside pH measures of oral secretions are not a valid marker of gastric reflux.

Characterization of thromboxane A2 receptor and TRPV1 mRNA expression in cultured sensory neurons.

Thromboxane A(2) (TxA(2)) is an arachidonic acid metabolite that stimulates platelet aggregation and vasoconstriction when released from platelets and other cell types during tissue trauma. More recent research has demonstrated that TxA(2) can also stimulate vagal and spinal sensory nerves. The purpose of this study was twofold. One, we compared the expression of the TxA(2) receptor (TxA2R) in neurons from two sensory ganglia: the nodose ganglion (NG) containing cell bodies of vagal afferent nerves and the thoracic dorsal root ganglion (DRG) containing cell bodies of spinal afferent nerves. Two, we determined if TxA2R co-localizes with mRNA for the nociceptive marker, TRPV1, which is the receptor for the noxious substance capsaicin. We found a greater percentage of neurons in the NG that are positive for TxA2R expression than in the DRG. We also found that there was no correlation of expression of TxA2R with TRPV1. These data suggest that while TxA2R is expressed in both vagal and spinal neurons, TxA(2) may elicit stronger vagal or parasympathetic reflexes in the rabbit when released during tissue trauma depending on the location of release. Our data also indicate that TxA(2) is likely to stimulate both nociceptive and non-nociceptive neurons thereby broadening the types of neurons and reflexes that it may excite.

Thromboxane A2-induced arrhythmias in the anesthetized rabbit.

Experiments were conducted in the anesthetized rabbit to investigate mechanisms for arrhythmias that occur after left atrial injection of the thromboxane A(2) (TxA(2)) mimetic U-46619. Arrhythmias were primarily of ventricular origin, dose dependent in frequency, and TxA(2) receptor mediated. The response was receptor specific since arrhythmias were absent after pretreatment with a specific TxA(2) receptor antagonist (SQ-29548) and did not occur in response to another prostaglandin, PGF(2alpha). Alterations in coronary blood flow were unlikely the cause of these arrhythmias because coronary blood flow (as measured with fluorescent microspheres) was unchanged after U-46619, and there were no observable changes in the ECG-ST segment. In addition, arrhythmias did not occur after administration of another vasoconstrictor (phenylephrine). The potential involvement of autonomic cardiac efferent nerves in these arrhythmias was also investigated because TxA(2) has been shown to stimulate peripheral nerves. Pretreatment of animals with the beta-adrenergic receptor antagonist propranolol did not reduce the frequency of these arrhythmias. Pretreatment with atropine or bilateral vagotomy resulted in an increased frequency of arrhythmias, suggesting that parasympathetic nerves may actually inhibit the arrhythmogenic activity of TxA(2). These experiments demonstrate that left atrial injection of U-46619 elicits arrhythmias via a mechanism independent of a significant reduction in coronary blood flow or activation of the autonomic nervous system. It is possible that TxA(2) may have a direct effect on the electrical activity of the heart in vivo, which provides significant implications for cardiac events where TxA(2) is increased, e.g., after myocardial ischemia or administration of cyclooxygenase-2 inhibitors.

Thromboxane A(2) mimetic evokes a bradycardia mediated by stimulation of cardiac vagal afferent nerves.

Injections of the thromboxane A(2) mimetic U-46619 (10 and 20 microg) into the left atrium of anesthetized rabbits evoked decreases in heart rate (HR) and arterial blood pressure (ABP) followed by an increase in ABP. Bilateral, cervical vagotomy abolished the U-46619-induced bradycardia and attenuated the hypotension. Injections of U-46619 into the ascending aorta did not evoke the bradycardia and hypotension but did cause arterial hypertension. To further define the origin of the vagal reflex, recordings of nerve impulses were made from 11 chemosensitive cardiac vagal afferent nerves. Impulse frequency increased in all 11 fibers in response to left atrial injections of phenylbiguanide (20-30 microg) and U-46619 (5-10 microg). Onset time of nerve activity induced by U-46619 correlated with the onset time of bradycardia. We conclude that U-46619 injections into the left heart elicit decreases in HR and ABP via a vagal reflex that originates from the heart similar to the coronary chemoreflex described for other agents.