Impacts of soil temperature,phenology and plant community composition on invertebrate herbivory in a natural warming experiment

Species and community-level responses to warming are well documented, with plants and invertebrates known to alter their range, phenology or composition as temperature increases. The effects of warming on biotic interactions are less clearly understood, but can have consequences that cascade through ecological networks. Here, we used a natural soil temperature gradient of 5–35°C in the Hengill geothermal valley, Iceland, to investigate the effects of temperature on plant community composition and plant–invertebrate interactions. We quantified the level of invertebrate herbivory on the plant community across the temperature gradient and the interactive effects of temperature, plant phenology (i.e. development stage) and vegetation community composition on the probability of herbivory for three ubiquitous plant species, Cardamine pratensis, Cerastium fontanum and Viola palustris. We found that the percentage cover of graminoids and forbs increased, while the amount of litter decreased, with increasing soil temperature. Invertebrate herbivory also increased with soil temperature at the plant community level, but this was underpinned by different effects of temperature on herbivory for individual plant species, mediated by the seasonal development of plants and the composition of the surrounding vegetation. This illustrates the importance of considering the development stage of organisms in climate change research given the variable effects of temperature on susceptibility to herbivory at different ontogenetic stages.     

Niche partitioning of avian predators in northern grasslands amended by biosolids

Many food webs are affected by bottom‐up nutrient addition, as additional biomass or productivity at a given trophic level can support more consumers. In turn, when prey are abundant, predators may converge on the same diets rather than partitioning food resources. Here, we examine the diets and habitat use of predatory and omnivorous birds in response to biosolids amendment of northern grasslands used as grazing range for cattle in British Columbia, Canada. From an ecosystem management perspective, we test whether dietary convergence occurred and whether birds preferentially used the pastures with biosolids. Biosolids treatments increased Orthoptera densities and our work occurred during a vole (Microtus spp.) population peak, so both types of prey were abundant. American Kestrels (Falco sparverius) consumed both small mammals and Orthoptera. Short‐eared Owls (Asio flammeus) and Long‐eared owls (Asio otus) primarily ate voles (>97% of biomass consumed) as did Northern Harriers (Circus hudsonius, 88% vole biomass). Despite high dietary overlap, these species had minimal spatial overlap, and Short‐eared Owls strongly preferred pastures amended with biosolids. Common Ravens (Corvus corax), Black‐billed Magpies (Pica hudsonia), and American Crows (Corvus brachyrhynchos) consumed Orthoptera, Coleoptera, vegetation, and only a few small mammals; crows avoided pastures with biosolids. Thus, when both insect and mammalian prey were abundant, corvids maintained omnivorous diets, whereas owls and Harriers specialized on voles. Spatial patterns were more complex, as birds were likely responding to prey abundance, vegetation structure, and other birds in this consumer guild.     

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1–3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4–8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and label-free quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS)¹ has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9–12). On the other hand, label-free quantitation has gained momentum in recent years (13–15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quantitation. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides. Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples (20). One solution to large-scale quantitation without any isotope labeling is pseudo internal standard approach (21), which selects endogenous house-keeping proteins as the internal standard so that no spike-in reagent is required. However, finding a good pseudo internal standard remains a challenge for phosphoproteome samples. Spike-in experiments are an alternative way to improve normalization profile. Some internal standard peptides such as MassPREP^TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22–24). There are two major normalization procedures. In one approach, sample peptides were normalized to the total peak intensity of spike-in peptides (25). Alternatively, the digested peptides were compared at first and the normalization factor was determined in different ways such as the median (26) or average of ratios (27). However, spiking an identical amount of standard proteins into phosphoproteomic samples before protein digestion may not be compatible with phosphoproteomic analyses which typically require a phosphopeptide enrichment step. Spectral counting has been extensively applied in large sets of proteomic samples because of its simplicity but the method is often not reliable for the quantitation of phosphoproteins, which are typically identified by single phosphopeptides with few spectra (28–30). Many software packages have been implemented to support the wide variety of those quantitation techniques, including commercial platforms such as Progenesis LC-MS^TM, Mascot Distiller^TM, PEAKS Q^TM, etc., as well as open-source software packages including MaxQuant (31), PEPPeR (32), Skyline (33), etc.In this study, we have devised a novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency (Fig. 1). The approach employs synthetic peptide libraries as the internal standard. These peptides were prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides were designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity is performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (34). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide.Open in a separate windowFig. 1.Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R²) and accuracy (%Acc).Water deficit and salinity causes osmotic stress, which is a major environmental factor limiting plant agricultural productivity. Osmotic stress rapidly changes the metabolism, gene expression and development of plant cells by activating several signaling pathways. Several protein kinases have been characterized as key components in osmotic stress signaling. Arabidopsis histidine kinase AHK1 can complement the histidine kinase mutant yeast, which can act as the osmosensor in yeast (35). Overexpression of AHK1 gene increases the drought tolerance of transgenic plants in Arabidopsis (36). Similar to yeast, the MAPK kinase cascade is also involved in osmotic stress response in plants. It is reported that AtMPK3, AtMPK6, and tobacco SIPK can be activated by NaCl or mannitol, and play positive roles in osmotic signaling (37, 38). MKK7 and MKKK20 may act as the up-stream kinase in the kinase cascade (39). Involvement of some calcium-dependent protein kinases, such as AtCPK21, AtCPK6, and OsCPK7 (CDPK) in osmotic stress signaling has also been reported (40–42). Another kinase family, SNF1-related protein kinase (SnRK) 2, also participates in osmotic stress response. In Arabidopsis, there are ten members in the SnRK2 family. Five from the ten SnRK2s, SnRK2, 3, 6, 7, and 8, can be activated by abscisic acid (ABA) and play central roles in ABA-receptor coupled signaling (43, 44). Furthermore, all SnRK2s except SnRK2.9 can be activated by NaCl or mannitol treatment (43). The decuple mutant of SnRK2 showed a strong osmotic hypersensitive phenotype (45). It is proposed that protein kinases including MAPK and SnRK2s have a critical function in osmotic stress (46), but the detailed mechanism and downstream substrates or target signal components are not yet clarified. We applied, therefore, the LAXIC approach with a self-validating method (47) to profile the osmotic stress-dependent phosphoproteome in Arabidopsis by quantifying phosphorylation events before and after mannitol treatment. Among a total of over 2000 phosphopeptides, more than 400 of them are dependent on osmotic stress. Those phosphoproteins are present on enzymes participating in signaling networks that are involved in many processes such as signal transduction, cytoskeleton development, and apoptosis. Overall, LAXIC represents a powerful tool for label-free quantitative phosphoproteomics.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

The Ozone-Iodine-Chlorate Clock Reaction

This work presents a new clock reaction based on ozone, iodine, and chlorate that differs from the known chlorate-iodine clock reaction because it does not require UV light. The induction period for this new clock reaction depends inversely on the initial concentrations of ozone, chlorate, and perchloric acid but is independent of the initial iodine concentration. The proposed mechanism considers the reaction of ozone and iodide to form HOI, which is a key species for producing non-linear autocatalytic behavior. The novelty of this system lies in the presence of ozone, whose participation has never been observed in complex systems such as clock or oscillating reactions. Thus, the autocatalysis demonstrated in this new clock reaction should open the possibility for a new family of oscillating reactions.     

Can Winter-Active Bumblebees Survive the Cold? Assessing the Cold Tolerance of Bombus terrestris audax and the Effects of Pollen Feeding

There is now considerable evidence that climate change is disrupting the phenology of key pollinator species. The recently reported UK winter activity of the bumblebee Bombus terrestris brings a novel set of thermal challenges to bumblebee workers that would typically only be exposed to summer conditions. Here we assess the ability of workers to survive acute and chronic cold stress (via lower lethal temperatures and lower lethal times at 0°C), the capacity for rapid cold hardening (RCH) and the influence of diet (pollen versus nectar consumption) on supercooling points (SCP). Comparisons are made with chronic cold stress indices and SCPs in queen bumblebees. Results showed worker bees were able to survive acute temperatures likely to be experienced in a mild winter, with queens significantly more tolerant to chronic cold temperature stress. The first evidence of RCH in any Hymenoptera is shown. In addition, dietary manipulation indicated the consumption of pollen significantly increased SCP temperature. These results are discussed in the light of winter active bumblebees and climate change.