Microplastics in human urine: Characterisation using µFTIR and sampling challenges using healthy donors and endometriosis participants.

Microplastics (MPs) are found in all environments, within the human food chain, and have been recently detected in several human tissues. The objective herein was to undertake an analysis of MP contamination in human urine samples, from healthy individuals and participants with endometriosis, with respect to their presence, levels, and the characteristics of any particles identified. A total of 38 human urine samples and 15 procedural blanks were analysed. MPs were characterised using µFTIR spectroscopy (size limitation of 5 µm) and SEM-EDX. In total, 123 MP particles consisting of 22 MP polymer types were identified within 17/29 of the healthy donor (10 mL) urine samples, compared with 232 MP particles of differing 16 MP polymer types in 12/19 urine samples from participants with endometriosis. Healthy donors presented an unadjusted average of 2589 ± 2931 MP/L and participants with endometriosis presented 4724 ± 9710 MP/L. Polyethylene (PE)(27%), polystyrene (PS)(16%), resin and polypropylene (PP)(both 12%) polymer types were most abundant in healthy donor samples, compared with polytetrafluoroethylene (PTFE) (59%), and PE (16%) in samples from endometriosis participants. The MP levels within healthy and endometriosis participant samples were not significantly different. However, the predominant polymer types varied, and the MPs from the metal catheter-derived endometriosis participant samples and healthy donors were significantly smaller than those observed in the procedural blanks. The procedural blank samples comprised 62 MP particles of 10 MP polymer types, mainly PP (27%), PE (21%), and PS (15%) with a mean ± SD of 17 ± 18, highlighting the unavoidable contamination inherent in measurement of MPs from donors. This is the first evidence of MP contamination in human urine with polymer characterisation and accounting for procedural blanks. These results support the phenomenon of transport of MPs within humans, specifically to the bladder, and their characterisation of types, shapes and size ranges identified therein.

Loss of 15-Lipoxygenase in Retinodegenerative RCS Rats.

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

A new Rothamsted long-term field experiment for the twenty-first century: principles and practice.

Agriculture faces potentially competing societal demands to produce food, fiber and fuel while reducing negative environmental impacts and delivering regulating, supporting and cultural ecosystem services. This necessitates a new generation of long-term agricultural field experiments designed to study the behavior of contrasting cropping systems in terms of multiple outcomes. We document the principles and practices of a new long-term experiment of this type at Rothamsted, established at two contrasting sites in 2017 and 2018, and report initial yield data at the crop and system level. The objective of the Large-Scale Rotation Experiment was to establish gradients of system properties and outcomes to improve our fundamental understanding of UK cropping systems. It is composed of four management factors-phased rotations, cultivation (conventional vs reduced tillage), nutrition (additional organic amendment vs standard mineral fertilization) and crop protection (conventional vs smart crop protection). These factors were combined in a balanced design resulting in 24 emergent cropping systems at each site and can be analyzed at the level of the system or component management factors. We observed interactions between management factors and with the environment on crop yields, justifying the systems level, multi-site approach. Reduced tillage resulted in lower wheat yields but the effect varied with rotation, previous-crop and site. Organic amendments significantly increased spring barley yield by 8% on average though the effect again varied with site. The plowed cropping systems tended to produce higher caloric yield overall than systems under reduced tillage. Additional response variables are being monitored to study synergies and trade-offs with outcomes other than yield at the cropping system level. The experiment has been established as a long-term resource for inter-disciplinary research. By documenting the design process, we aim to facilitate the adoption of similar approaches to system-scale agricultural experimentation to inform the transition to more sustainable cropping systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s13593-023-00914-8.

Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

Quantitative Pharmacodynamic Characterization of Resistance versus Heteroresistance of Colistin in E. coli Using a Semimechanistic Modeling of Killing Curves.

Heteroresistance corresponds to the presence, in a bacterial isolate, of an initial small subpopulation of bacteria characterized by a significant reduction in their sensitivity to a given antibiotic. Mechanisms of heteroresistance versus resistance are poorly understood. The aim of this study was to explore heteroresistance in mcr-positive and mcr-negative Escherichia coli strains exposed to colistin by use of modeling killing curves with a semimechanistic model. We quantify, for a range of phenotypically (susceptibility based on MIC) and genotypically (carriage of mcr-1 or mcr-3 or mcr-negative) different bacteria, a maximum killing rate (Emax) of colistin and the corresponding potency (EC50), i.e., the colistin concentrations corresponding to Emax/2. Heteroresistant subpopulations were identified in both mcr-negative and mcr-positive E. coli as around 0.06% of the starting population. Minority heteroresistant bacteria, both for mcr-negative and mcr-positive strains, differed from the corresponding dominant populations only by the maximum killing rate of colistin (differences for Emax by a factor of 12.66 and 3.76 for mcr-negative and mcr-positive strains, respectively) and without alteration of their EC50s. On the other hand, the resistant mcr-positive strains are distinguished from the mcr-negative strains by differences in their EC50, which can reach a factor of 44 for their dominant population and 22 for their heteroresistant subpopulations. It is suggested that the underlying physiological mechanisms differ between resistance and heteroresistance, with resistance being linked to a decrease in the affinity of colistin for its site of action, whereas heteroresistance would, rather, be linked to an alteration of the target, which will be more difficult to be further changed or destroyed.

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality.

BACKGROUND: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. RESULTS: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. CONCLUSIONS: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

Application of the hollow fibre infection model (HFIM) in antimicrobial development: a systematic review and recommendations of reporting.

OBJECTIVES: This systematic review focuses on the use of the in vitro hollow fibre infection model (HFIM) for microbial culture. We summarize the direction of the field to date and propose best-practice principles for reporting of the applications. METHODS: Searches in six databases (MEDLINE®, EMBASE®, PubMed®, BIOSIS®, SCOPUS® and Cochrane®) up to January 2020 identified 129 studies meeting our inclusion criteria. Two reviewers independently assessed and extracted data from each publication. The quality of reporting of microbiological and technical parameters was analysed. RESULTS: Forty-seven out of 129 (36.4%) studies did not report the minimum pharmacokinetic parameters required in order to replicate the pharmacokinetic profile of HFIM experiments. Fifty-three out of 129 (41.1%) publications did not report the medium used in the HFIM. The overwhelming majority of publications did not perform any technical repeats [107/129 (82.9%)] or biological repeats [97/129 (75.2%)]. CONCLUSIONS: This review demonstrates that most publications provide insufficient data to allow for results to be evaluated, thus impairing the reproducibility of HFIM experiments. Therefore, there is a clear need for the development of laboratory standardization and improved reporting of HFIM experiments.

Agricultural intensification and climate change have increased the threat from weeds.

Weeds represent a significant threat to crop yields and global food security. We analysed data on weed competition from the world's longest running agricultural experiment to ask whether potential yield losses from weeds have increased in response to management and environmental change since the advent of the Green Revolution in the 1960s. On plots where inorganic nitrogen fertiliser has been applied, potential yield losses from weeds have consistently increased since 1969. This was explained by a warming climate, measured as air temperature averaged over the growing season for the weeds, and a shift towards shorter crop cultivars. Weeds also reduced yield proportionally more on plots with higher rates of nitrogen which had higher yields when weeds were controlled; the relative benefit of herbicides was, therefore, proportional to potential crop yield. Reducing yield losses from weed competition is increasingly challenging because of the evolution of herbicide resistance. Our results demonstrate that weeds now represent a greater inherent threat to crop production than before the advent of herbicides and integrated, sustainable solutions to weed management are urgently needed to protect the high yield potential of modern crop genotypes.

Determination of colistin in luminal and parietal intestinal matrices of chicken by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Justification for continued use of colistin in veterinary medicine, for example medicated water, relies on pharmacokinetic/pharmacodynamic (PK/PD) studies that require accurate measurement of colistin content in the digestive tract. A method for the detection and quantification of colistin in poultry intestinal material was developed and validated. Colistin is not absorbed after oral administration, and the biophase is the gastrointestinal tract. Extraction of colistin from the matrix was achieved using solid-phase extraction with a methanol:water (1:2; v/v) solution. Polymyxin B was used as an internal standard. Colistin A and colistin B, the main components of colistin, were separated, detected and measured using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated for linearity/quadraticity between 1.1 (LOQ) and 56.7 mg/kg. Mean accuracy was between 82.7% and 107.7% with inter- and intra-day precision lower than 13.3% and 15% respectively. Freeze-thaw, long-term and bench storage were validated. Incurred samples following colistin treatment in poultry at the approved clinical dose of 75,000 IU/kg in drinking water and oral gavage were quantifiable and in line with expected intestinal transit times. The method is considered appropriately accurate and precise for the purposes of pharmacokinetic analysis in the gastrointestinal tract.

Mechanical Characteristics of Ultrafast Zebrafish Larval Swimming Muscles.

Zebrafish (Danio rerio) swim within days of fertilization, powered by muscles of the axial myotomes. Forces generated by these muscles can be measured rapidly in whole, intact larval tails by adapting protocols developed for ex vivo muscle mechanics. But it is not known how well these measurements reflect the function of the underlying muscle fibers and sarcomeres. Here, we consider the anatomy of the 5-day-old, wild-type larval tail, and implement technical modifications to measuring muscle physiology in intact tails. Specifically, we quantify fundamental relationships between force, length, and shortening velocity, and capture the extreme contractile speeds required to swim with tail-beat frequencies of 80-100 Hz. Therefore, we analyze 1000 frames/s videos to track the movement of structures, visible in the transparent tail, which correlate with sarcomere length. We also characterize the passive viscoelastic properties of the preparation to isolate forces contributed by nonmuscle structures within the tail. Myotomal muscles generate more than 95% of their maximal isometric stress (76 ± 3 mN/mm2) over the range of muscle lengths used in vivo. They have rapid twitch kinetics (full width at half-maximal stress: 11 ± 1 ms) and a high twitch/tetanus ratio (0.91 ± 0.05), indicating adaptations for fast excitation-contraction coupling. Although contractile stress is relatively low, myotomal muscles develop high net power (134 ± 20 W/kg at 80 Hz) in cyclical work loop experiments designed to simulate the in vivo dynamics of muscle fibers during swimming. When shortening at a constant speed of 7 ± 1 muscle lengths/s, muscles develop 86 ± 2% of isometric stress, whereas peak instantaneous power during 100 Hz work loops occurs at 18 ± 2 muscle lengths/s. These approaches can improve the usefulness of zebrafish as a model system for muscle research by providing a rapid and sensitive functional readout for experimental interventions.

Investigating the effects of inter-annual weather variation (1968-2016) on the functional response of cereal grain yield to applied nitrogen, using data from the Rothamsted Long-Term Experiments.

The effect of weather on inter-annual variation in the crop yield response to nitrogen (N) fertilizer for winter wheat (Triticum aestivvum L.) and spring barley (Hordeum vulgare L.) was investigated using yield data from the Broadbalk Wheat and Hoosfield Spring Barley long-term experiments at Rothamsted Research. Grain yields of crops from 1968 to 2016 were modelled as a function of N rates using a linear-plus-exponential (LEXP) function. The extent to which inter-annual variation in the parameters of these responses was explained by variations in weather (monthly summarized temperatures and rainfall), and by changes in the cultivar grown, was assessed. The inter-annual variability in rainfall and underlying temperature influenced the crop N response and hence grain yields in both crops. Asymptotic yields in wheat were particularly sensitive to mean temperature in November, April and May, and to total rainfall in October, February and June. In spring barley asymptotic yields were sensitive to mean temperature in February and June, and to total rainfall in April to July inclusive and September. The method presented here explores the separation of agronomic and environmental (weather) influences on crop yield over time. Fitting N response curves across multiple treatments can support an informative analysis of the influence of weather variation on the yield variability. Whilst there are issues of the confounding and collinearity of explanatory variables within such models, and that other factors also influence yields over time, our study confirms the considerable impact of weather variables at certain times of the year. This emphasizes the importance of including weather temporal variation when evaluating the impacts of climate change on crops.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

Differential susceptibility to tetracycline, oxytetracycline and doxycycline of the calf pathogens Mannheimia haemolytica and Pasteurella multocida in three growth media.

For clinical isolates of bovine Mannheimia haemolytica and Pasteurella multocida, this study reports minimum inhibitory concentration (MIC) differences for tetracycline, oxytetracycline and doxycycline between cation-adjusted Mueller-Hinton broth (CAMHB), foetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) medium. MICs were determined according to CLSI standards and additionally using five overlapping sets of twofold dilutions. Matrix effect: (a) free drug MICs and minimum bactericidal concentrations (MBC) for all drugs were significantly higher in FBS than in CAMHB for both pathogens (p < 0.001); (b) MICs and MBCs were higher for CAMHB and FBS compared to RPMI for P. multocida only. Net growth rate for P. multocida in CAMHB was significantly slower than in FBS and higher than in RPMI, correlating to MIC and MBC ranking. Drug effect: doxycycline MICs and MBCs were significantly lower (p < 0.001) in both CAMHB and FBS than tetracycline and oxytetracycline for both pathogens. Only for M. haemolytica were oxytetracycline MIC and MBC significantly lower than tetracycline, precluding the use of tetracycline to predict oxytetracycline susceptibility in this species. Determining potencies of tetracyclines in a physiological medium, such as FBS, is proposed, when the objective is correlation with pharmacokinetic data for dosage determination.

Beyond the one-way ANOVA for 'omics data.

BACKGROUND: With ever increasing accessibility to high throughput technologies, more complex treatment structures can be assessed in a variety of 'omics applications. This adds an extra layer of complexity to the analysis and interpretation, in particular when inferential univariate methods are applied en masse. It is well-known that mass univariate testing suffers from multiplicity issues and although this has been well documented for simple comparative tests, few approaches have focussed on more complex explanatory structures. RESULTS: Two frameworks are introduced incorporating corrections for multiplicity whilst maintaining appropriate structure in the explanatory variables. Within this paradigm, a choice has to be made as to whether multiplicity corrections should be applied to the saturated model, putting emphasis on controlling the rate of false positives, or to the predictive model, where emphasis is on model selection. This choice has implications for both the ranking and selection of the response variables identified as differentially expressed. The theoretical difference is demonstrated between the two approaches along with an empirical study of lipid composition in Arabidopsis under differing levels of salt stress. CONCLUSIONS: Multiplicity corrections have an inherent weakness when the full explanatory structure is not properly incorporated. Although a unifying 'single best' recommendation is not provided, two reasonable alternatives are provided and the applicability of these approaches is discussed for different scenarios where the aims of analysis will differ. The key result is that the point at which multiplicity is incorporated into the analysis will fundamentally change the interpretation of the results, and the choice of approach should therefore be driven by the specific aims of the experiment.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.

Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield.

Improving photosynthesis is a major target for increasing crop yields and ensuring food security. Phenotyping of photosynthesis in the field is critical to understand the limits to crop performance in agricultural settings. Yet, detailed phenotyping of photosynthetic traits is relatively scarce in field-grown wheat, with previous studies focusing on narrow germplasm selections. Flag leaf photosynthetic traits, crop development, and yield traits were compared in 64 field-grown wheat cultivars in the UK. Pre-anthesis and post-anthesis photosynthetic traits correlated significantly and positively with grain yield and harvest index (HI). These traits included net CO2 assimilation measured at ambient CO2 concentrations and a range of photosynthetic photon flux densities, and traits associated with the light response of photosynthesis. In most cultivars, photosynthesis decreased post-anthesis compared with pre-anthesis, and this was associated with decreased Rubisco activity and abundance. Heritability of photosynthetic traits suggests that phenotypic variation can be used to inform breeding programmes. Specific cultivars were identified with traits relevant to breeding for increased crop yields in the UK: pre-anthesis photosynthesis, post-anthesis photosynthesis, light response of photosynthesis, and Rubisco amounts. The results indicate that flag leaf longevity and operating photosynthetic activity in the canopy can be further exploited to maximize grain filling in UK bread wheat.

A virulent strain of deformed wing virus (DWV) of honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission.

The globally distributed ectoparasite Varroa destructor is a vector for viral pathogens of the Western honeybee (Apis mellifera), in particular the Iflavirus Deformed Wing Virus (DWV). In the absence of Varroa low levels DWV occur, generally causing asymptomatic infections. Conversely, Varroa-infested colonies show markedly elevated virus levels, increased overwintering colony losses, with impairment of pupal development and symptomatic workers. To determine whether changes in the virus population were due Varroa amplifying and introducing virulent virus strains and/or suppressing the host immune responses, we exposed Varroa-naïve larvae to oral and Varroa-transmitted DWV. We monitored virus levels and diversity in developing pupae and associated Varroa, the resulting RNAi response and transcriptome changes in the host. Exposed pupae were stratified by Varroa association (presence/absence) and virus levels (low/high) into three groups. Varroa-free pupae all exhibited low levels of a highly diverse DWV population, with those exposed per os (group NV) exhibiting changes in the population composition. Varroa-associated pupae exhibited either low levels of a diverse DWV population (group VL) or high levels of a near-clonal virulent variant of DWV (group VH). These groups and unexposed controls (C) could be also discriminated by principal component analysis of the transcriptome changes observed, which included several genes involved in development and the immune response. All Varroa tested contained a diverse replicating DWV population implying the virulent variant present in group VH, and predominating in RNA-seq analysis of temporally and geographically separate Varroa-infested colonies, was selected upon transmission from Varroa, a conclusion supported by direct injection of pupae in vitro with mixed virus populations. Identification of a virulent variant of DWV, the role of Varroa in its transmission and the resulting host transcriptome changes furthers our understanding of this important viral pathogen of honeybees.