Climate change disrupts the seasonal coupling of plant and soil microbial nutrient cycling in an alpine ecosystem.

The seasonal coupling of plant and soil microbial nutrient demands is crucial for efficient ecosystem nutrient cycling and plant production, especially in strongly seasonal alpine ecosystems. Yet, how these seasonal nutrient cycling processes are modified by climate change and what the consequences are for nutrient loss and retention in alpine ecosystems remain unclear. Here, we explored how two pervasive climate change factors, reduced snow cover and shrub expansion, interactively modify the seasonal coupling of plant and soil microbial nitrogen (N) cycling in alpine grasslands, which are warming at double the rate of the global average. We found that the combination of reduced snow cover and shrub expansion disrupted the seasonal coupling of plant and soil N-cycling, with pronounced effects in spring (shortly after snow melt) and autumn (at the onset of plant senescence). In combination, both climate change factors decreased plant organic N-uptake by 70% and 82%, soil microbial biomass N by 19% and 38% and increased soil denitrifier abundances by 253% and 136% in spring and autumn, respectively. Shrub expansion also individually modified the seasonality of soil microbial community composition and stoichiometry towards more N-limited conditions and slower nutrient cycling in spring and autumn. In winter, snow removal markedly reduced the fungal:bacterial biomass ratio, soil N pools and shifted bacterial community composition. Taken together, our findings suggest that interactions between climate change factors can disrupt the temporal coupling of plant and soil microbial N-cycling processes in alpine grasslands. This could diminish the capacity of these globally widespread alpine ecosystems to retain N and support plant productivity under future climate change.

Citizen science monitoring reveals links between honeybee health, pesticide exposure and seasonal availability of floral resources.

We use a national citizen science monitoring scheme to quantify how agricultural intensification affects honeybee diet breadth (number of plant species). To do this we used DNA metabarcoding to identify the plants present in 527 honey samples collected in 2019 across Great Britain. The species richness of forage plants was negatively correlated with arable cropping area, although this was only found early in the year when the abundance of flowering plants was more limited. Within intensively farmed areas, honeybee diets were dominated by Brassica crops (including oilseed rape). We demonstrate how the structure and complexity of honeybee foraging relationships with plants is negatively affected by the area of arable crops surrounding hives. Using information collected from the beekeepers on the incidence of an economically damaging bee disease (Deformed Wing Virus) we found that the occurrence of this disease increased where bees foraged in agricultural land where there was a high use of foliar insecticides. Understanding impacts of land use on resource availability is fundamental to assessing long-term viability of pollinator populations. These findings highlight the importance of supporting temporally timed resources as mitigation strategies to support wider pollinator population viability.

Impacts of Life-Time Exposure of Arsenic, Cadmium and Fluoranthene on the Earthworms' L. rubellus Global DNA Methylation as Detected by msAFLP.

This study reports on the effects of long-term exposure to the metals arsenic (As), cadmium (Cd) and the polycyclic aromatic hydrocarbon fluoranthene on the survival, growth, development and DNA methylation status of the earthworm Lumbricus rubellus. Exposures to the three chemicals were conducted over their whole juvenile developmental period from egg to adult. Significant effects on one or more measured endpoints were found for all three chemicals. Arsenic had no effect on survival, but had a significant effect on growth rates at concentrations of 36 mg/kg or higher and also slowed the rate of maturation. Cadmium significantly reduced juvenile survival at 500 mg/kg, juvenile growth at 148 mg/kg and maturation rates at all tested concentrations. Fluoranthene had no effect on survival or the developmental period, but did significantly reduce growth rates at 800 mg/kg. Effects at these concentrations are consistent with the known effects of these three chemicals on earthworms from previous studies conducted mainly with Eisenia fetida. Both As and Cd had no effect on DNA methylation patterning in earthworms measured at the end of the exposure. Fluoranthene was shown, for the first time. to have an effect on a species' DNA methylation levels. These results suggest that apical phenotypic changes for As and Cd are not necessarily associated with changes in DNA methylation profiles. However, exposure to the organic chemical fluoranthene influenced DNA methylation patterns, suggesting wider remodelling of the epigenome for this chemical.

Shrub expansion modulates belowground impacts of changing snow conditions in alpine grasslands.

Climate change is disproportionately impacting mountain ecosystems, leading to large reductions in winter snow cover, earlier spring snowmelt and widespread shrub expansion into alpine grasslands. Yet, the combined effects of shrub expansion and changing snow conditions on abiotic and biotic soil properties remains poorly understood. We used complementary field experiments to show that reduced snow cover and earlier snowmelt have effects on soil microbial communities and functioning that persist into summer. However, ericaceous shrub expansion modulates a number of these impacts and has stronger belowground effects than changing snow conditions. Ericaceous shrub expansion did not alter snow depth or snowmelt timing but did increase the abundance of ericoid mycorrhizal fungi and oligotrophic bacteria, which was linked to decreased soil respiration and nitrogen availability. Our findings suggest that changing winter snow conditions have cross-seasonal impacts on soil properties, but shifts in vegetation can modulate belowground effects of future alpine climate change.

Integration of DNA extraction, metabarcoding and an informatics pipeline to underpin a national citizen science honey monitoring scheme.

Worldwide honeybees (Apis mellifera L.) are one of the most widely kept domesticated animals, supporting domestic and commercial livelihoods through the production of honey and wax, as well as in the delivery of pollination services to crops. Quantifying which plant species are foraged upon by honeybees provides insights into their nutritional status as well as patterns of landscape scale habitat utilization. Here we outline a rapid and reproducible methodology for identifying environmental DNA (eDNA) originating principally from pollen grains suspended within honey. The process is based on a DNA extraction incorporating vacuum filtration prior to universal eukaryotic internal transcribed spacer 2 region (ITS2) amplicon generation, sequencing and identification. To provide a pre-cursor to sequence phylotyping, we outline systems for error-corrected processing amplicon sequence variant abundance tables that removes chimeras. This methodology underpins the new UK National Honey Monitoring Scheme.•We compare the efficacy and speed of centrifugation and filtration systems for removing pollen from honey samples as a precursor to plant DNA barcoding.•We introduce the 'HONEYPI' informatics pipeline, an open access resource implemented in python 2.7, to ensure long-term reproducibility during the process of amplicon sequence variant classification.

Accumulation of polybrominated diphenyl ethers and microbiome response in the great pond snail Lymnaea stagnalis with exposure to nylon (polyamide) microplastics.

Microplastics attract widespread attention, including for their potential to transport toxic chemicals in the form of plasticisers and associated hydrophobic organic chemicals, such as polybrominated diphenyl ethers (PBDEs). The aims of this study were to investigate how nylon (polyamide) microplastics may affect PBDE accumulation in snails, and the acute effects of nylon particles and PBDEs on survival, weight change and inherent microbiome diversity and community composition of the pond snail Lymnaea stagnalis. Snails were exposed for 96 h to BDEs-47, 99, 100 and 153 in the presence and absence of 1% w/w nylon microplastics in quartz sand sediment. No mortality was observed over the exposure period. Snails not exposed to microplastics lost significantly more weight compared to those exposed to microplastics. Increasing PBDE concentration in the sediment resulted in an increased PBDE body burden in the snails, however microplastics did not significantly influence total PBDE uptake. Based on individual congeners, uptake of BDE 47 by snails was significantly reduced in the presence of microplastics. The diversity and composition of the snail microbiome was not significantly altered by the presence of PBDEs nor by the microplastics, singly or combined. Significant effects on a few individual operational taxonomic units (OTUs) occurred when comparing the highest PBDE concentration with the control treatment, but in the absence of microplastics only. Overall within these acute experiments, only subtle effects on weight loss and slight microbiome alterations occurred. These results therefore highlight that L. stagnalis are resilient to acute exposures to microplastics and PBDEs, and that microplastics are unlikely to influence HOC accumulation or the microbiome of this species over short timescales.

Genetic, epigenetic and microbiome characterisation of an earthworm species (Octolasion lacteum) along a radiation exposure gradient at Chernobyl.

The effects of exposure to different levels of ionising radiation were assessed on the genetic, epigenetic and microbiome characteristics of the "hologenome" of earthworms collected at sites within the Chernobyl exclusion zone (CEZ). The earthworms Aporrectodea caliginosa (Savigny, 1826) and Octolasion lacteum (Örley, 1881) were the two species that were most frequently found at visited sites, however, only O. lacteum was present at sufficient number across different exposure levels to enable comparative hologenome analysis. The identification of morphotype O. lacteum as a probable single clade was established using a combination of mitochondrial (cytochrome oxidase I) and nuclear genome (Amplified Fragment Length Polymorphism (AFLP) using MspI loci). No clear site associated differences in population genetic structure was found between populations using the AFLP marker loci. Further, no relationship between ionising radiation exposure levels and the percentage of methylated loci or pattern of distribution of DNA methylation marks was found. Microbiome structure was clearly site dependent, with gut microbiome community structure and diversity being systematically associated with calculated site-specific earthworm dose rates. There was, however, also co-correlation between earthworm dose rates and other soil properties, notably soil pH; a property known to affect soil bacterial community structure. Such co-correlation means that it is not possible to attribute microbiome changes unequivocally to radionuclide exposure. A better understanding of the relationship between radionuclide exposure soil properties and their interactions on bacterial microbiome community response is, therefore, needed to establish whether these the observed microbiome changes are attributed directly to radiation exposure, other soil properties or to an interaction between multiple variables at sites within the CEZ.

Helminth burden and ecological factors associated with alterations in wild host gastrointestinal microbiota.

Infection by gastrointestinal helminths of humans, livestock and wild animals is common, but the impact of such endoparasites on wild hosts and their gut microbiota represents an important overlooked component of population dynamics. Wild host gut microbiota and endoparasites occupy the same physical niche spaces with both affecting host nutrition and health. However, associations between the two are poorly understood. Here we used the commonly parasitized European shag (Phalacrocorax aristotelis) as a model wild host. Forty live adults from the same colony were sampled. Endoscopy was employed to quantify helminth infection in situ. Microbiota from the significantly distinct proventriculus (site of infection), cloacal and faecal gastrointestinal tract microbiomes were characterised using 16S rRNA gene-targeted high-throughput sequencing. We found increasingly strong associations between helminth infection and microbiota composition progressing away from the site of infection, observing a pronounced dysbiosis in microbiota when samples were partitioned into high- and low-burden groups. We posit this dysbiosis is predominately explained by helminths inducing an anti-inflammatory environment in the proventriculus, diverting host immune responses away from themselves. This study, within live wild animals, provides a vital foundation to better understand the mechanisms that underpin the three-way relationship between helminths, microbiota and hosts.

Soil pH effects on the interactions between dissolved zinc, non-nano- and nano-ZnO with soil bacterial communities.

Zinc oxide nanoparticles (ZnO NPs) are used in an array of products and processes, ranging from personal care products to antifouling paints, textiles, food additives, antibacterial agents and environmental remediation processes. Soils are an environment likely to be exposed to manmade nanoparticles due to the practice of applying sewage sludge as a fertiliser or as an organic soil improver. However, understanding on the interactions between soil properties, nanoparticles and the organisms that live within soil is lacking, especially with regards to soil bacterial communities. We studied the effects of nanoparticulate, non-nanoparticulate and ionic zinc (in the form of zinc chloride) on the composition of bacterial communities in soil with a modified pH range (from pH 4.5 to pH 7.2). We observed strong pH-dependent effects on the interaction between bacterial communities and all forms of zinc, with the largest changes in bacterial community composition occurring in soils with low and medium pH levels (pH 4.8 and 5.9). The high pH soil (pH 7.2) was less susceptible to the effects of zinc exposure. At the highest doses of zinc (2500 mg/kg dw soil), both nano and non-nano particulate zinc applications elicited a similar response in the soil bacterial community, and this differed significantly to the ionic zinc salt treatment. The results highlight the importance of considering soil pH in nanotoxicology studies, although further work is needed to determine the exact mechanisms controlling the toxicity and fate and interactions of nanoparticles with soil microbial communities.

Rearing and foraging affects bumblebee (Bombus terrestris) gut microbiota.

Bumblebees are ecologically and economically important as pollinators of crop and wild plants, especially in temperate systems. Species, such as the buff-tailed bumblebee (Bombus terrestris), are reared commercially to pollinate high-value crops. Their highly specific gut microbiota, characterized by low diversity, may affect nutrition and immunity and are likely to be important for fitness and colony health. However, little is known about how environmental factors affect bacterial community structure. We analysed the gut microbiota from three groups of worker bumblebees (B. terrestris) from distinct colonies that varied in rearing and foraging characteristics: commercially reared with restricted foraging (RR); commercially reared with outside foraging (RF); and wild-caught workers (W). Contrary to previous studies, which indicate that bacterial communities are highly conserved across workers, we found that RF individuals had an intermediate community structure compared with RR and W types. Further, this was shaped by differences in the abundances of common operational taxonomic units (OTUs) and the diversity of rare OTUs present, which we propose results from an increase in the variety of carbohydrates obtained through foraging.

Catchment-scale biogeography of riverine bacterioplankton.

Lotic ecosystems such as rivers and streams are unique in that they represent a continuum of both space and time during the transition from headwaters to the river mouth. As microbes have very different controls over their ecology, distribution and dispersion compared with macrobiota, we wished to explore biogeographical patterns within a river catchment and uncover the major drivers structuring bacterioplankton communities. Water samples collected across the River Thames Basin, UK, covering the transition from headwater tributaries to the lower reaches of the main river channel were characterised using 16S rRNA gene pyrosequencing. This approach revealed an ecological succession in the bacterial community composition along the river continuum, moving from a community dominated by Bacteroidetes in the headwaters to Actinobacteria-dominated downstream. Location of the sampling point in the river network (measured as the cumulative water channel distance upstream) was found to be the most predictive spatial feature; inferring that ecological processes pertaining to temporal community succession are of prime importance in driving the assemblages of riverine bacterioplankton communities. A decrease in bacterial activity rates and an increase in the abundance of low nucleic acid bacteria relative to high nucleic acid bacteria were found to correspond with these downstream changes in community structure, suggesting corresponding functional changes. Our findings show that bacterial communities across the Thames basin exhibit an ecological succession along the river continuum, and that this is primarily driven by water residence time rather than the physico-chemical status of the river.

Weekly flow cytometric analysis of riverine phytoplankton to determine seasonal bloom dynamics.

Understanding the relative role of anthropogenic and environmental drivers on the timing, magnitude and composition of algal and cyanobacterial blooms is vitally important for the effective management of river catchments. Whilst taxonomic identification and enumeration of algal species can provide valuable insights, the time and specialist skills needed for this approach makes it prohibitive for high frequency and multiple-site studies. Other proxies for phytoplankton, such as total chlorophyll concentration provide little information on community composition. Here we demonstrate the use of flow cytometry (FCM) as a viable alternative approach for monitoring the changing seasonal patterns of abundance, composition and biovolume of phytoplankton in rivers. A FCM assay was set up and calibrated using a range of pure algal cultures and then applied to a year-long, weekly sampling campaign on the River Thames at Wallingford, UK. Ten groups of phytoplankton representing diatoms, chlorophytes, cryptophytes and cyanobacteria were monitored over the course of the year and examined in relation to river physiochemical parameters. Major diatom blooms occurred in spring and autumn, correlating with depletion of soluble reactive phosphorus and dissolved silicon concentrations and we also observed a significant and sustained cyanobacteria bloom between July and October. Pico-chlorophytes (0.2-2.0 µm in diameter) dominated the community throughout the summer period but were not detected using traditional colorimetric chlorophyll analysis, suggesting underestimates of actual phytoplankton standing stocks by traditional methods. We demonstrate high resolution sampling and FCM as a sensitive method for river ecosystem monitoring and that FCM data may be used as an indicator of riverine health.

Marine bacterial communities are resistant to elevated carbon dioxide levels.

It is well established that the release of anthropogenic-derived CO2 into the atmosphere will be mainly absorbed by the oceans, with a concomitant drop in pH, a process termed ocean acidification. As such, there is considerable interest in how changes in increased CO2 and lower pH will affect marine biota, such as bacteria, which play central roles in oceanic biogeochemical processes. Set within an ecological framework, we investigated the direct effects of elevated CO2, contrasted with ambient conditions on the resistance and resilience of marine bacterial communities in a replicated temporal seawater mesocosm experiment. The results of the study strongly indicate that marine bacterial communities are highly resistant to the elevated CO2 and lower pH conditions imposed, as demonstrated from measures of turnover using taxa­time relationships and distance­decay relationships. In addition, no significant differences in community abundance, structure or composition were observed. Our results suggest that there are no direct effects on marine bacterial communities and that the bacterial fraction of microbial plankton holds enough flexibility and evolutionary capacity to withstand predicted future changes from elevated CO2 and subsequent ocean acidification.

Using boreholes as windows into groundwater ecosystems.

Groundwater ecosystems remain poorly understood yet may provide ecosystem services, make a unique contribution to biodiversity and contain useful bio-indicators of water quality. Little is known about ecosystem variability, the distribution of invertebrates within aquifers, or how representative boreholes are of aquifers. We addressed these issues using borehole imaging and single borehole dilution tests to identify three potential aquifer habitats (fractures, fissures or conduits) intercepted by two Chalk boreholes at different depths beneath the surface (34 to 98 m). These habitats were characterised by sampling the invertebrates, microbiology and hydrochemistry using a packer system to isolate them. Samples were taken with progressively increasing pumped volume to assess differences between borehole and aquifer communities. The study provides a new conceptual framework to infer the origin of water, invertebrates and microbes sampled from boreholes. It demonstrates that pumping 5 m(3) at 0.4-1.8 l/sec was sufficient to entrain invertebrates from five to tens of metres into the aquifer during these packer tests. Invertebrates and bacteria were more abundant in the boreholes than in the aquifer, with associated water chemistry variations indicating that boreholes act as sites of enhanced biogeochemical cycling. There was some variability in invertebrate abundance and bacterial community structure between habitats, indicating ecological heterogeneity within the aquifer. However, invertebrates were captured in all aquifer samples, and bacterial abundance, major ion chemistry and dissolved oxygen remained similar. Therefore the study demonstrates that in the Chalk, ecosystems comprising bacteria and invertebrates extend from around the water table to 70 m below it. Hydrogeological techniques provide excellent scope for tackling outstanding questions in groundwater ecology, provided an appropriate conceptual hydrogeological understanding is applied.

The response of marine picoplankton to ocean acidification.

Since industrialization global CO(2) emissions have increased, and as a consequence oceanic pH is predicted to drop by 0.3-0.4 units before the end of the century - a process coined 'ocean acidification'. Consequently, there is significant interest in how pH changes will affect the ocean's biota and integral processes. We investigated marine picoplankton (0.2-2 µm diameter) community response to predicted end of century CO(2) concentrations, via a 'high-CO(2) ' (∼ 750 ppm) large-volume (11 000 l) contained seawater mesocosm approach. We found little evidence of changes occurring in bacterial abundance or community composition due to elevated CO(2) under both phytoplankton pre-bloom/bloom and post-bloom conditions. In contrast, significant differences were observed between treatments for a number of key picoeukaryote community members. These data suggested a key outcome of ocean acidification is a more rapid exploitation of elevated CO(2) levels by photosynthetic picoeukaryotes. Thus, our study indicates the need for a more thorough understanding of picoeukaryote-mediated carbon flow within ocean acidification experiments, both in relation to picoplankton carbon sources, sinks and transfer to higher trophic levels.