The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients.

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Rapid Response to Experimental Warming of a Microbial Community Inhabiting High Arctic Patterned Ground Soil.

The influence of climate change on microbial communities inhabiting the sparsely vegetated patterned ground soils that are widespread across the High Arctic is poorly understood. Here, in a four-year experiment on Svalbard, we warmed patterned ground soil with open top chambers and biannually irrigated the soil to predict the responses of its microbial community to rising temperatures and precipitation. A 1 °C rise in summertime soil temperature caused 44% and 78% increases in CO2 efflux and CH4 consumption, respectively, and a 32% increase in the frequency of bacterial 16S ribosomal RNA genes. Bacterial alpha diversity was unaffected by the treatments, but, of the 40 most frequent bacterial taxa, warming caused 44-45% reductions in the relative abundances of a Sphingomonas sp. and Ferruginibacter sp. and 33-91% increases in those of a Phenylobacterium sp. and a member of the Acetobacteraceae. Warming did not influence the frequency of fungal internal transcribed spacer 2 copies, and irrigation had no effects on the measured variables. Our study suggests rapid changes to the activities and abundances of microbes, and particularly bacteria, in High Arctic patterned ground soils as they warm. At current rates of soil warming on Svalbard (0.8 °C per decade), we anticipate that similar effects to those reported here will manifest themselves in the natural environment by approximately the mid 2030s.

Arctic soil respiration and microbial community structure driven by silicon and calcium.

Global warming is most pronounced in the Arctic region. Greenhouse gas (GHG) release from Arctic soils increase due to global warming. By this, the Arctic may change from currently being a carbon sink to a future source. To improve accurate predictions of future GHG release from Arctic soils, it is important to unravel factors controlling both the microbial community structure and activity. Soil microbial activity is important for Arctic greenhouse gas production, but depends on soil conditions such as salinity being increased by calcium (Ca) and decreased by amorphous silica (Si) potentially enhancing water availability. In the Arctic, climate changes may alter salinity by changing Si and Ca concentrations upon permafrost thaw as a result of global warming with Si potentially decreasing and Ca potentially increasing salinity. Here, we show that higher Si concentration increased and higher Ca concentrations decreased the microbial CO2 production for both a salt-poor and a salt-rich soil from Greenland. In the salt-rich soil, Si amendment increased CO2 production and the abundance of gram-negative bacteria. However, the bacterial community became dominated by spore-forming gram-positive Firmicutes and Actinobacteria. The CO2 release from soils was directly affected by the abundance of bacteria and fungi, and their community structure. Our results highlight the importance of the soil Si and Ca concentration on organic carbon turnover by strongly changing microbial abundance and community structure, with consequences for CO2 release in the Arctic. Consequently, Ca and Si and their relation to Arctic soil microbial community structure has to be considered when estimating pan-Arctic carbon budgets.

Unexpected high carbon losses in a continental glacier foreland on the Tibetan Plateau.

Closely related with microbial activities, soil developments along the glacier forelands are generally considered a carbon sink; however, those of continental glacier forelands remain unclear. Continental glaciers are characterized by dry conditions and low temperature that limit microbial growth. We investigated the carbon characteristics along a chronosequence of the Laohugou Glacier No. 12 foreland, a typical continental glacier on the Tibetan Plateau, by analyzing soil bacterial community structure and microbial carbon-related functional potentials. We found an unexpected carbon loss in which soil organic carbon decreased from 22.21 g kg-1 to 10.77 g kg-1 after receding 50 years. Structural equation modeling verified the important positive impacts from bacterial community. Lower carbon fixation efficiency along the chronosequence was supported by less autotrophic bacteria and carbon fixation genes relating to the reductive tricarboxylic acid cycle. Lower carbon availability and higher carbon requirements were identified by an increasing bacterial copy number and a shift of the dominant bacterial community from Proteobacteria and Bacteroidetes (r-strategists) to Actinobacteria and Acidobacteria (K-strategists). Our findings show that the carbon loss of continental glacier foreland was significantly affected by the changes of bacterial community, and can help to avoid overestimating the carbon sink characteristics of glacier forelands in climate models.

Soil microbial legacies differ following drying-rewetting and freezing-thawing cycles.

Climate change alters frequencies and intensities of soil drying-rewetting and freezing-thawing cycles. These fluctuations affect soil water availability, a crucial driver of soil microbial activity. While these fluctuations are leaving imprints on soil microbiome structures, the question remains if the legacy of one type of weather fluctuation (e.g., drying-rewetting) affects the community response to the other (e.g., freezing-thawing). As both phenomenons give similar water availability fluctuations, we hypothesized that freezing-thawing and drying-rewetting cycles have similar effects on the soil microbiome. We tested this hypothesis by establishing targeted microcosm experiments. We created a legacy by exposing soil samples to a freezing-thawing or drying-rewetting cycle (phase 1), followed by an additional drying-rewetting or freezing-thawing cycle (phase 2). We measured soil respiration and analyzed soil microbiome structures. Across experiments, larger CO2 pulses and changes in microbiome structures were observed after rewetting than thawing. Drying-rewetting legacy affected the microbiome and CO2 emissions upon the following freezing-thawing cycle. Conversely, freezing-thawing legacy did not affect the microbial response to the drying-rewetting cycle. Our results suggest that drying-rewetting cycles have stronger effects on soil microbial communities and CO2 production than freezing-thawing cycles and that this pattern is mediated by sustained changes in soil microbiome structures.

Low Turnover of Soil Bacterial rRNA at Low Temperatures.

Ribosomal RNA (rRNA) is used widely to investigate potentially active microorganisms in environmental samples, including soil microorganisms and other microbial communities that are subjected to pronounced seasonal variation in temperature. This raises a question about the turnover of intracellular microbial rRNA at environmentally relevant temperatures. We analyzed the turnover at four temperatures of RNA isolated from soil bacteria amended with 14C-labeled uridine. We found that the half-life of recently produced RNA increased from 4.0 days at 20°C to 15.8 days at 4°C, and 215 days at -4°C, while no degradation was detected at -18°C during a 1-year period. We discuss the implications of the strong temperature dependency of rRNA turnover for interpretation of microbiome data based on rRNA isolated from environmental samples.

AgNO3 Sterilizes Grains of Barley (Hordeum vulgare) without Inhibiting Germination-A Necessary Tool for Plant-Microbiome Research.

To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant-microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H2O2, NaClO, and AgNO3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO3 by testing several concentrations of AgNO3 and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO3, and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO3 can be used successfully for experiments on plant-microbiome interactions.

Bacterial and protozoan dynamics upon thawing and freezing of an active layer permafrost soil.

The active layer of soil overlaying permafrost in the Arctic is subjected to annual changes in temperature and soil chemistry, which we hypothesize to affect the overall soil microbial community. We investigated changes in soil microorganisms at different temperatures during warming and freezing of the active layer soil from Svalbard, Norway. Soil community data were obtained by direct shotgun sequencing of total extracted RNA. No changes in soil microbial communities were detected when warming from -10 to -2 °C or when freezing from -2 to -10 °C. In contrast, within a few days we observed changes when warming from -2 to +2 °C with a decrease in fungal rRNA and an increase in several OTUs belonging to Gemmatimonadetes, Bacteroidetes and Betaproteobacteria. Even more substantial changes occurred when incubating at 2 °C for 16 days, with declines in total fungal potential activity and decreases in oligotrophic members from Actinobacteria and Acidobacteria. Additionally, we detected an increase in transcriptome sequences of bacterial phyla Bacteriodetes, Firmicutes, Betaproteobacteria and Gammaproteobacteria-collectively presumed to be copiotrophic. Furthermore, we detected an increase in putative bacterivorous heterotrophic flagellates, likely due to predation upon the bacterial community via grazing. Although this grazing activity may explain relatively large changes in the bacterial community composition, no changes in total 16S rRNA gene copy number were observed and the total RNA level remained stable during the incubation. Together, these results are showing the first comprehensive ecological evaluation across prokaryotic and eukaryotic microbial communities on thawing and freezing of soil by application of the TotalRNA technique.

Fast response of fungal and prokaryotic communities to climate change manipulation in two contrasting tundra soils.

BACKGROUND: Climate models predict substantial changes in temperature and precipitation patterns across Arctic regions, including increased winter precipitation as snow in the near future. Soil microorganisms are considered key players in organic matter decomposition and regulation of biogeochemical cycles. However, current knowledge regarding their response to future climate changes is limited. Here, we explore the short-term effect of increased snow cover on soil fungal, bacterial and archaeal communities in two tundra sites with contrasting water regimes in Greenland. In order to assess seasonal variation of microbial communities, we collected soil samples four times during the plant-growing season. RESULTS: The analysis revealed that soil microbial communities from two tundra sites differed from each other due to contrasting soil chemical properties. Fungal communities showed higher richness at the dry site whereas richness of prokaryotes was higher at the wet tundra site. We demonstrated that fungal and bacterial communities at both sites were significantly affected by short-term increased snow cover manipulation. Our results showed that fungal community composition was more affected by deeper snow cover compared to prokaryotes. The fungal communities showed changes in both taxonomic and ecological groups in response to climate manipulation. However, the changes were not pronounced at all sampling times which points to the need of multiple sampling in ecosystems where environmental factors show seasonal variation. Further, we showed that effects of increased snow cover were manifested after snow had melted. CONCLUSIONS: We demonstrated rapid response of soil fungal and bacterial communities to short-term climate manipulation simulating increased winter precipitation at two tundra sites. In particular, we provide evidence that fungal community composition was more affected by increased snow cover compared to prokaryotes indicating fast adaptability to changing environmental conditions. Since fungi are considered the main decomposers of complex organic matter in terrestrial ecosystems, the stronger response of fungal communities may have implications for organic matter turnover in tundra soils under future climate.

Long-term soil metal exposure impaired temporal variation in microbial metatranscriptomes and enriched active phages.

BACKGROUND: It remains unclear whether adaptation and changes in diversity associated to a long-term perturbation are sufficient to ensure functional resilience of soil microbial communities. We used RNA-based approaches (16S rRNA gene transcript amplicon coupled to shotgun mRNA sequencing) to study the legacy effects of a century-long soil copper (Cu) pollution on microbial activity and composition, as well as its effect on the capacity of the microbial community to react to temporal fluctuations. RESULTS: Despite evidence of microbial adaptation (e.g., iron homeostasis and avoidance/resistance strategies), increased heterogeneity and richness loss in transcribed gene pools were observed with increasing soil Cu, together with an unexpected predominance of phage mRNA signatures. Apparently, phage activation was either triggered directly by Cu, or indirectly via enhanced expression of DNA repair/SOS response systems in Cu-exposed bacteria. Even though total soil carbon and nitrogen had accumulated with increasing Cu, a reduction in temporally induced mRNA functions was observed. Microbial temporal response groups (TRGs, groups of microbes with a specific temporal response) were heavily affected by Cu, both in abundance and phylogenetic composition. CONCLUSION: Altogether, results point toward a Cu-mediated "decoupling" between environmental fluctuations and microbial activity, where Cu-exposed microbes stopped fulfilling their expected contributions to soil functioning relative to the control. Nevertheless, some functions remained active in February despite Cu, concomitant with an increase in phage mRNA signatures, highlighting that somehow, microbial activity is still happening under these adverse conditions.

Catch Crop Residues Stimulate N2O Emissions During Spring, Without Affecting the Genetic Potential for Nitrite and N2O Reduction.

Agricultural soils are a significant source of anthropogenic nitrous oxide (N2O) emissions, because of fertilizer application and decomposition of crop residues. We studied interactions between nitrogen (N) amendments and soil conditions in a 2-year field experiment with or without catch crop incorporation before seeding of spring barley, and with or without application of N in the form of digested liquid manure or mineral N fertilizer. Weather conditions, soil inorganic N dynamics, and N2O emissions were monitored during spring, and soil samples were analyzed for abundances of nitrite reduction (nirK and nirS) and N2O reduction genes (nosZ clade I and II), and structure of nitrite- and N2O-reducing communities. Fertilization significantly enhanced soil mineral N accumulation compared to treatments with catch crop residues as the only N source. Nitrous oxide emissions, in contrast, were stimulated in rotations with catch crop residue incorporation, probably as a result of concurrent net N mineralization, and O2 depletion associated with residue degradation in organic hotspots. Emissions of N2O from digested manure were low in both years, while emissions from mineral N fertilizer were nearly absent in the first year, but comparable to emissions from catch crop residues in the second year with higher precipitation and delayed plant N uptake. Higher gene abundances, as well as shifts in community structure, were also observed in the second year, which were significantly correlated to NO 3 - availability. Both the size and structure of the nitrite- and N2O-reducing communities correlated to the difference in N2O emissions between years, while there were no consistent effects of management as represented by catch crops or fertilization. It is concluded that N2O emissions were constrained by environmental, rather than the genetic potential for nitrite and N2O reduction.

Warming, shading and a moth outbreak reduce tundra carbon sink strength dramatically by changing plant cover and soil microbial activity.

Future increases in temperature and cloud cover will alter plant growth and decomposition of the large carbon pools stored in Arctic soils. A better understanding of interactions between above- and belowground processes and communities of plants and microorganisms is essential for predicting Arctic ecosystem responses to climate change. We measured ecosystem CO2 fluxes during the growing season for seven years in a dwarf-shrub tundra in West Greenland manipulated with warming and shading and experiencing a natural larvae outbreak. Vegetation composition, soil fungal community composition, microbial activity, and nutrient availability were analyzed after six years of treatment. Warming and shading altered the plant community, reduced plant CO2 uptake, and changed fungal community composition. Ecosystem carbon accumulation decreased during the growing season by 61% in shaded plots and 51% in warmed plots. Also, plant recovery was reduced in both manipulations following the larvae outbreak during the fifth treatment year. The reduced plant recovery in manipulated plots following the larvae outbreak suggests that climate change may increase tundra ecosystem sensitivity to disturbances. Also, plant community changes mediated via reduced light and reduced water availability due to increased temperature can strongly lower the carbon sink strength of tundra ecosystems.

Legacy Effects on the Recovery of Soil Bacterial Communities from Extreme Temperature Perturbation.

The type and frequency of disturbances experienced by soil microbiomes is expected to increase given predicted global climate change scenarios and intensified anthropogenic pressures on ecosystems. While the direct effect of multiple disturbances to soil microbes has been explored in terms of function, their effect on the recovery of microbial community composition remains unclear. Here, we used soil microcosm experiments and multiple model disturbances to explore their short-term effect on the recovery of soil microbiota after identical or novel stresses. Soil microcosms were exposed to a heat shock to create an initial effect. Upon initial community recovery (25 days after stress), they were subjected to a second stress, either a heat or a cold shock, and they were monitored for additional 25 days. To carefully verify the bacterial response to the disturbances, we monitored changes in community composition throughout the experiment using 16S rRNA gene transcript amplicon sequencing. The application of a heat shock to soils with or without the initial heat shock resulted in similar successional dynamics, but these dynamics were faster in soils with a prior heat shock. The application of a cold shock had negligible effects on previously undisturbed soils but, in combination with an initial heat shock, caused the largest shift in the community composition. Our findings show that compounded perturbation affects bacterial community recovery by altering community structure and thus, the community's response during succession. By altering dominance patterns, disturbance legacy affects the microbiome's ability to recover from further perturbation within the 25 days studied. Our results highlight the need to consider the soil's disturbance history in the development of soil management practices in order to maintain the system's resilience.

Autogenic succession and deterministic recovery following disturbance in soil bacterial communities.

The response of bacterial communities to environmental change may affect local to global nutrient cycles. However the dynamics of these communities following disturbance are poorly understood, given that they are often evaluated over macro-ecological time scales and end-point measurements. In order to understand the successional trajectory of soil bacterial communities following disturbances and the mechanisms controlling these dynamics at a scale relevant for these organisms, we subjected soil microcosms to a heat disturbance and followed the community composition of active bacteria over 50 days. The disturbance imposed a strong selective pressure that persisted for up to 10 days, after which the importance of stochastic processes increased. Three successional stages were detected: a primary response in which surviving taxa increased in abundance; a secondary response phase during which community dynamics slowed down, and a stability phase (after 29 days), during which the community tended towards its original composition. Phylogenetic turnover patterns indicated that the community experienced stronger deterministic selection during recovery. Thus, soil bacterial communities, despite their extreme diversity and functional redundancy, respond to disturbances like many macro-ecological systems and exhibit path-dependent, autogenic dynamics during secondary succession. These results highlight the role of autogenic factors and successional dynamics in microbial recovery.

Potential microbial contamination during sampling of permafrost soil assessed by tracers.

Drilling and handling of permanently frozen soil cores without microbial contamination is of concern because contamination e.g. from the active layer above may lead to incorrect interpretation of results in experiments investigating potential and actual microbial activity in these low microbial biomass environments. Here, we present an example of how microbial contamination from active layer soil affected analysis of the potentially active microbial community in permafrost soil. We also present the development and use of two tracers: (1) fluorescent plastic microspheres and (2) Pseudomonas putida genetically tagged with Green Fluorescent Protein production to mimic potential microbial contamination of two permafrost cores. A protocol with special emphasis on avoiding microbial contamination was developed and employed to examine how far microbial contamination can penetrate into permafrost cores. The quantity of tracer elements decreased with depth into the permafrost cores, but the tracers were detected as far as 17 mm from the surface of the cores. The results emphasize that caution should be taken to avoid microbial contamination of permafrost cores and that the application of tracers represents a useful tool to assess penetration of potential microbial contamination into permafrost cores.

Long-term and realistic global change manipulations had low impact on diversity of soil biota in temperate heathland.

In a dry heathland ecosystem we manipulated temperature (warming), precipitation (drought) and atmospheric concentration of CO2 in a full-factorial experiment in order to investigate changes in below-ground biodiversity as a result of future climate change. We investigated the responses in community diversity of nematodes, enchytraeids, collembolans and oribatid mites at two and eight years of manipulations. We used a structural equation modelling (SEM) approach analyzing the three manipulations, soil moisture and temperature, and seven soil biological and chemical variables. The analysis revealed a persistent and positive effect of elevated CO2 on litter C:N ratio. After two years of treatment, the fungi to bacteria ratio was increased by warming, and the diversities within oribatid mites, collembolans and nematode groups were all affected by elevated CO2 mediated through increased litter C:N ratio. After eight years of treatment, however, the CO2-increased litter C:N ratio did not influence the diversity in any of the four fauna groups. The number of significant correlations between treatments, food source quality, and soil biota diversities was reduced from six to three after two and eight years, respectively. These results suggest a remarkable resilience within the soil biota against global climate change treatments in the long term.

Enhanced summer warming reduces fungal decomposer diversity and litter mass loss more strongly in dry than in wet tundra.

Many Arctic regions are currently experiencing substantial summer and winter climate changes. Litter decomposition is a fundamental component of ecosystem carbon and nutrient cycles, with fungi being among the primary decomposers. To assess the impacts of seasonal climatic changes on litter fungal communities and their functioning, Betula glandulosa leaf litter was surface-incubated in two adjacent low Arctic sites with contrasting soil moisture regimes: dry shrub heath and wet sedge tundra at Disko Island, Greenland. At both sites, we investigated the impacts of factorial combinations of enhanced summer warming (using open-top chambers; OTCs) and deepened snow (using snow fences) on surface litter mass loss, chemistry and fungal decomposer communities after approximately 1 year. Enhanced summer warming significantly restricted litter mass loss by 32% in the dry and 17% in the wet site. Litter moisture content was significantly reduced by summer warming in the dry, but not in the wet site. Likewise, fungal total abundance and diversity were reduced by OTC warming at the dry site, while comparatively modest warming effects were observed in the wet site. These results suggest that increased evapotranspiration in the OTC plots lowered litter moisture content to the point where fungal decomposition activities became inhibited. In contrast, snow addition enhanced fungal abundance in both sites but did not significantly affect litter mass loss rates. Across sites, control plots only shared 15% of their fungal phylotypes, suggesting strong local controls on fungal decomposer community composition. Nevertheless, fungal community functioning (litter decomposition) was negatively affected by warming in both sites. We conclude that although buried soil organic matter decomposition is widely expected to increase with future summer warming, surface litter decay and nutrient turnover rates in both xeric and relatively moist tundra are likely to be significantly restricted by the evaporative drying associated with warmer air temperatures.

Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition.

Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406. IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with humans, animals, and the environment. Such insight could further the development of innovative ecosystem management approaches for the protection of our natural resources and the design of more effective and sustainable solutions to prevent and control infectious diseases. Genome sequence information is an organism (pathogen)-independent language that can be used across sectors, space, and time. Harmonized standards, protocols, and workflows for sample processing and analysis can facilitate the generation of such actionable information. In this study, we assessed several procedures for the isolation of DNA for next-generation sequencing. Our study highlights several important aspects to consider in the design and conduct of sequence-based analysis of microbiomes. We provide a standard operating procedure for the isolation of DNA from a range of biological specimens particularly relevant in clinical diagnostics and epidemiology.