The development of terrestrial ecosystems emerging after glacier retreat.

The global retreat of glaciers is dramatically altering mountain and high-latitude landscapes, with new ecosystems developing from apparently barren substrates1-4. The study of these emerging ecosystems is critical to understanding how climate change interacts with microhabitat and biotic communities and determines the future of ice-free terrains1,5. Here, using a comprehensive characterization of ecosystems (soil properties, microclimate, productivity and biodiversity by environmental DNA metabarcoding6) across 46 proglacial landscapes worldwide, we found that all the environmental properties change with time since glaciers retreated, and that temperature modulates the accumulation of soil nutrients. The richness of bacteria, fungi, plants and animals increases with time since deglaciation, but their temporal patterns differ. Microorganisms colonized most rapidly in the first decades after glacier retreat, whereas most macroorganisms took longer. Increased habitat suitability, growing complexity of biotic interactions and temporal colonization all contribute to the increase in biodiversity over time. These processes also modify community composition for all the groups of organisms. Plant communities show positive links with all other biodiversity components and have a key role in ecosystem development. These unifying patterns provide new insights into the early dynamics of deglaciated terrains and highlight the need for integrated surveillance of their multiple environmental properties5.

The generality of cryptic dietary niche differences in diverse large-herbivore assemblages.

Ecological niche differences are necessary for stable species coexistence but are often difficult to discern. Models of dietary niche differentiation in large mammalian herbivores invoke the quality, quantity, and spatiotemporal distribution of plant tissues and growth forms but are agnostic toward food plant species identity. Empirical support for these models is variable, suggesting that additional mechanisms of resource partitioning may be important in sustaining large-herbivore diversity in African savannas. We used DNA metabarcoding to conduct a taxonomically explicit analysis of large-herbivore diets across southeastern Africa, analyzing ∼4,000 fecal samples of 30 species from 10 sites in seven countries over 6 y. We detected 893 food plant taxa from 124 families, but just two families-grasses and legumes-accounted for the majority of herbivore diets. Nonetheless, herbivore species almost invariably partitioned food plant taxa; diet composition differed significantly in 97% of pairwise comparisons between sympatric species, and dissimilarity was pronounced even between the strictest grazers (grass eaters), strictest browsers (nongrass eaters), and closest relatives at each site. Niche differentiation was weakest in an ecosystem recovering from catastrophic defaunation, indicating that food plant partitioning is driven by species interactions, and was stronger at low rainfall, as expected if interspecific competition is a predominant driver. Diets differed more between browsers than grazers, which predictably shaped community organization: Grazer-dominated trophic networks had higher nestedness and lower modularity. That dietary differentiation is structured along taxonomic lines complements prior work on how herbivores partition plant parts and patches and suggests that common mechanisms govern herbivore coexistence and community assembly in savannas.

Dynamics and drivers of mycorrhizal fungi after glacier retreat.

The development of terrestrial ecosystems depends greatly on plant mutualists such as mycorrhizal fungi. The global retreat of glaciers exposes nutrient-poor substrates in extreme environments and provides a unique opportunity to study early successions of mycorrhizal fungi by assessing their dynamics and drivers. We combined environmental DNA metabarcoding and measurements of local conditions to assess the succession of mycorrhizal communities during soil development in 46 glacier forelands around the globe, testing whether dynamics and drivers differ between mycorrhizal types. Mycorrhizal fungi colonized deglaciated areas very quickly (< 10 yr), with arbuscular mycorrhizal fungi tending to become more diverse through time compared to ectomycorrhizal fungi. Both alpha- and beta-diversity of arbuscular mycorrhizal fungi were significantly related to time since glacier retreat and plant communities, while microclimate and primary productivity were more important for ectomycorrhizal fungi. The richness and composition of mycorrhizal communities were also significantly explained by soil chemistry, highlighting the importance of microhabitat for community dynamics. The acceleration of ice melt and the modifications of microclimate forecasted by climate change scenarios are expected to impact the diversity of mycorrhizal partners. These changes could alter the interactions underlying biotic colonization and belowground-aboveground linkages, with multifaceted impacts on soil development and associated ecological processes.

Local climate modulates the development of soil nematode communities after glacier retreat.

The worldwide retreat of glaciers is causing a faster than ever increase in ice-free areas that are leading to the emergence of new ecosystems. Understanding the dynamics of these environments is critical to predicting the consequences of climate change on mountains and at high latitudes. Climatic differences between regions of the world could modulate the emergence of biodiversity and functionality after glacier retreat, yet global tests of this hypothesis are lacking. Nematodes are the most abundant soil animals, with keystone roles in ecosystem functioning, but the lack of global-scale studies limits our understanding of how the taxonomic and functional diversity of nematodes changes during the colonization of proglacial landscapes. We used environmental DNA metabarcoding to characterize nematode communities of 48 glacier forelands from five continents. We assessed how different facets of biodiversity change with the age of deglaciated terrains and tested the hypothesis that colonization patterns are different across forelands with different climatic conditions. Nematodes colonized ice-free areas almost immediately. Both taxonomic and functional richness quickly increased over time, but the increase in nematode diversity was modulated by climate, so that colonization started earlier in forelands with mild summer temperatures. Colder forelands initially hosted poor communities, but the colonization rate then accelerated, eventually leveling biodiversity differences between climatic regimes in the long term. Immediately after glacier retreat, communities were dominated by colonizer taxa with short generation time and r-ecological strategy but community composition shifted through time, with increased frequency of more persister taxa with K-ecological strategy. These changes mostly occurred through the addition of new traits instead of their replacement during succession. The effects of local climate on nematode colonization led to heterogeneous but predictable patterns around the world that likely affect soil communities and overall ecosystem development.

Metabarcoding data reveal vertical multitaxa variation in topsoil communities during the colonization of deglaciated forelands.

Ice-free areas are expanding worldwide due to dramatic glacier shrinkage and are undergoing rapid colonization by multiple lifeforms, thus representing key environments to study ecosystem development. It has been proposed that the colonization dynamics of deglaciated terrains is different between surface and deep soils but that the heterogeneity between communities inhabiting surface and deep soils decreases through time. Nevertheless, tests of this hypothesis remain scarce, and it is unclear whether patterns are consistent among different taxonomic groups. Here, we used environmental DNA metabarcoding to test whether community diversity and composition of six groups (Eukaryota, Bacteria, Mycota, Collembola, Insecta, and Oligochaeta) differ between the surface (0-5 cm) and deeper (7.5-20 cm) soil at different stages of development and across five Alpine glaciers. Taxonomic diversity increased with time since glacier retreat and with soil evolution. The pattern was consistent across groups and soil depths. For Eukaryota and Mycota, alpha-diversity was highest at the surface. Time since glacier retreat explained more variation of community composition than depth. Beta-diversity between surface and deep layers decreased with time since glacier retreat, supporting the hypothesis that the first 20 cm of soil tends to homogenize through time. Several molecular operational taxonomic units of bacteria and fungi were significant indicators of specific depths and/or soil development stages, confirming the strong functional variation of microbial communities through time and depth. The complexity of community patterns highlights the importance of integrating information from multiple taxonomic groups to unravel community variation in response to ongoing global changes.

Double-digest RAD-sequencing: do pre- and post-sequencing protocol parameters impact biological results?

Next-generation sequencing technologies have opened a new era of research in population genetics. Following these new sequencing opportunities, the use of restriction enzyme-based genotyping techniques, such as restriction site-associated DNA sequencing (RAD-seq) or double-digest RAD-sequencing (ddRAD-seq), has dramatically increased in the last decade. From DNA sampling to SNP calling, the laboratory and bioinformatic parameters of enzyme-based techniques have been investigated in the literature. However, the impact of those parameters on downstream analyses and biological results remains less documented. In this study, we investigated the effects of sevral pre- and post-sequencing settings on ddRAD-seq results for two biological systems: a complex of butterfly species (Coenonympha sp.) and several populations of common beech (Fagus sylvatica). Our results suggest that pre-sequencing parameters (i.e., DNA quantity, number of PCR cycles during library preparation) have a significant impact on the number of recovered reads and SNPs, on the number of unique alleles and on individual heterozygosity. In the same way, we found that post-sequencing settings (i.e., clustering and minimum coverage thresholds) influenced loci reconstruction (e.g., number of loci, mean coverage) and SNP calling (e.g., number of SNPs; heterozygosity) but had only a marginal impact on downstream analyses (e.g., measure of genetic differentiation, estimation of individual admixture, and demographic inferences). In addition, replication analyses confirmed the reproducibility of the ddRAD-seq procedure. Overall, this study assesses the degree of sensitivity of ddRAD-seq data to pre- and post-sequencing protocols, and illustrates its robustness when studying population genetics.

Effects of soil preservation for biodiversity monitoring using environmental DNA.

Environmental DNA (eDNA) metabarcoding is becoming a key tool for biodiversity monitoring over large geographical or taxonomic scales and for elusive taxa such as soil organisms. Increasing sample sizes and interest in remote or extreme areas often require the preservation of soil samples and thus deviations from optimal standardized protocols. However, we still ignore the impact of different methods of soil sample preservation on the results of metabarcoding studies and there is no guideline for best practices so far. Here, we assessed the impact of four methods of soil sample preservation that can be conveniently used also in metabarcoding studies targeting remote or difficult to access areas. Tested methods include: preservation at room temperature for 6 hr, preservation at 4°C for 3 days, desiccation immediately after sampling and preservation for 21 days, and desiccation after 6 hr at room temperature and preservation for 21 days. For each preservation method, we benchmarked resulting estimates of taxon diversity and community composition of three different taxonomic groups (bacteria, fungi and eukaryotes) in three different habitats (forest, river bank and grassland) against results obtained under ideal conditions (i.e., extraction of eDNA immediately after sampling). Overall, the different preservation methods only marginally impaired results and only under certain conditions. When rare taxa were considered, we detected small but significant changes in molecular operational taxonomic units (MOTU) richness of bacteria, fungi and eukaryotes across treatments, but MOTU richness was similar across preservation methods if rare taxa were not considered. All the approaches were able to identify differences in community structure among habitats, and the communities retrieved using the different preservation conditions were extremely similar. We propose guidelines on the selection of the optimal soil sample preservation conditions for metabarcoding studies, depending on the practical constraints, costs and ultimate research goals.

Modelling technical and biological biases in macroinvertebrate community assessment from bulk preservative using multiple metabarcoding markers.

DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programmes. Here, we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring programme, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy versus metabarcoding from storage ethanol using three markers (COI-M19BR2, 16S-Inse01 and 18S-Euka02) and a multimarker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI-M19BR2 and the multimarker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalized linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotized exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multimarker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimization efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.

Comparison of markers for the monitoring of freshwater benthic biodiversity through DNA metabarcoding.

Metabarcoding of bulk or environmental DNA has great potential for biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater bioassessment. Nevertheless, few studies have so far assessed markers for metabarcoding of freshwater macrobenthos. Here we combined in silico and laboratory analyses to test the performance of different markers amplifying regions in the 18S rDNA (Euka02), 16S rDNA (Inse01) and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic macroinvertebrates of France and Europe, with a particular focus on key insect orders (Ephemeroptera, Plecoptera and Trichoptera). Analyses on 1,514 individuals representing different taxa of benthic macroinvertebrates showed very different amplification success across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. BF1_BR2-COI showed the highest resolution, while the resolution of Euka02 was often limited. By combining our data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different primers highlights the complexity in identifying the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity.

Morphological vs. DNA metabarcoding approaches for the evaluation of stream ecological status with benthic invertebrates: Testing different combinations of markers and strategies of data filtering.

Macroinvertebrate assemblages are the most common bioindicators used for stream biomonitoring, yet the standard approach exhibits several time-consuming steps, including the sorting and identification of organisms based on morphological criteria. In this study, we examined if DNA metabarcoding could be used as an efficient molecular-based alternative to the morphology-based monitoring of streams using macroinvertebrates. We compared results achieved with the standard morphological identification of organisms sampled in 18 sites located on 15 French wadeable streams to results obtained with the DNA metabarcoding identification of sorted bulk material of the same macroinvertebrate samples, using read numbers (expressed as relative frequencies) as a proxy for abundances. In particular, we evaluated how combining and filtering metabarcoding data obtained from three different markers (COI: BF1-BR2, 18S: Euka02 and 16S: Inse01) could improve the efficiency of bioassessment. In total, 140 taxa were identified based on morphological criteria, and 127 were identified based on DNA metabarcoding using the three markers, with an overlap of 99 taxa. The threshold values used for sequence filtering based on the "best identity" criterion and the number of reads had an effect on the assessment efficiency of data obtained with each marker. Compared to single marker results, combining data from different markers allowed us to improve the match between biotic index values obtained with the bulk DNA versus morphology-based approaches. Both approaches assigned the same ecological quality class to a majority (86%) of the site sampling events, highlighting both the efficiency of metabarcoding as a biomonitoring tool but also the need for further research to improve this efficiency.

Body size determines soil community assembly in a tropical forest.

Tropical forests shelter an unparalleled biological diversity. The relative influence of environmental selection (i.e., abiotic conditions, biotic interactions) and stochastic-distance-dependent neutral processes (i.e., demography, dispersal) in shaping communities has been extensively studied for various organisms, but has rarely been explored across a large range of body sizes, in particular in soil environments. We built a detailed census of the whole soil biota in a 12-ha tropical forest plot using soil DNA metabarcoding. We show that the distribution of 19 taxonomic groups (ranging from microbes to mesofauna) is primarily stochastic, suggesting that neutral processes are prominent drivers of the assembly of these communities at this scale. We also identify aluminium, topography and plant species identity as weak, yet significant drivers of soil richness and community composition of bacteria, protists and to a lesser extent fungi. Finally, we show that body size, which determines the scale at which an organism perceives its environment, predicted the community assembly across taxonomic groups, with soil mesofauna assemblages being more stochastic than microbial ones. These results suggest that the relative contribution of neutral processes and environmental selection to community assembly directly depends on body size. Body size is hence an important determinant of community assembly rules at the scale of the ecological community in tropical soils and should be accounted for in spatial models of tropical soil food webs.

Diet shifts by adult flightless dung beetles Circellium bacchus, revealed using DNA metabarcoding, reflect complex life histories.

Life history changes may change resource use. Such shifts are not well understood in the dung beetles, despite recognised differences in larval and adult feeding ability. We use the flightless dung beetle Circellium bacchus to explore such shifts, identifying dung sources of adults using DNA metabarcoding, and comparing these with published accounts of larval dung sources. C. bacchus is traditionally considered to specialise on the dung of large herbivores for both larval and adult feeding. We successfully extracted mammal DNA from 151 adult C. bacchus fecal samples, representing 16 mammal species (ranging from elephants to small rodents), many of which are hitherto undescribed in the diet. Adult C. bacchus showed clear dung source preferences, especially for large herbivores inhabiting dense-cover vegetation. Our approach also confirmed the presence of cryptic taxa in the study area, and we propose that this may be used for biodiversity survey and monitoring purposes. Murid rodent feces were the most commonly fed-upon dung source (77.5%) for adult C. bacchus, differing markedly from the large and megaherbivore dung sources used for larval rearing. These findings support the hypothesis of life history-specific shifts in resource use in dung beetles, and reveal a hitherto unsuspected, but ecologically important, role of these dung beetles in consuming rodent feces. The differences in feeding abilities of the larval and adult life history stages have profound consequences for their resource use and foraging strategies, and hence the ecological role of dung beetles. This principle and its ecological consequences should be explored in other scarabaeids.

Gene expression patterns and sequence polymorphisms associated with mosquito resistance to Bacillus thuringiensis israelensis toxins.

BACKGROUND: Despite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain suggest that evolving resistance to Bti is costly. The aim of the present study was to identify transcription level and polymorphism variations associated with resistance to Bti toxins in the dengue vector Aedes aegypti. We used RNA sequencing (RNA-seq) for comparing a laboratory-selected strain showing elevated resistance to Bti toxins and its parental non-selected susceptible strain. As the resistant strain displayed two marked larval development phenotypes (slow and normal), each phenotype was analyzed separately in order to evidence potential links between resistance mechanisms and mosquito life-history traits. RESULTS: A total of 12,458 genes were detected of which 844 were differentially transcribed between the resistant and susceptible strains. Polymorphism analysis revealed a total of 68,541 SNPs of which 12,571 SNPs exhibited more than 40% frequency difference between the resistant and susceptible strains, affecting 2,953 genes. Bti resistance is associated with changes in the transcription level of enzymes involved in detoxification and chitin metabolism. Among previously described Bti-toxin receptors, four alkaline phosphatases (ALPs) were differentially transcribed between resistant and susceptible larvae, and non-synonymous changes affected the protein sequence of one cadherin, six aminopeptidases (APNs) and four α-amylases. Other putative Cry receptors located in lipid rafts, such as flotillin and glycoside hydrolases, were under-transcribed and/or contained non-synonymous substitutions. Finally, immunity-related genes showed contrasted transcription and polymorphisms patterns between the two developmental resistant phenotypes, suggesting the existence of trade-offs between Bti-resistance, life-history traits and immunity. CONCLUSIONS: The present study is the first to analyze the whole transcriptome of Bti-resistant mosquitoes by RNA-seq, shedding light on the importance of studying both transcription levels and sequence polymorphism variations to get a comprehensive view of insecticide resistance.

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

BACKGROUND: Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. RESULTS: After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. CONCLUSIONS: The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides.

Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence.

BACKGROUND: Despite numerous studies suggesting that amphibians are highly sensitive to cumulative anthropogenic stresses, the role pollutants play in the decline of amphibian populations remains unclear. Amongst the most common aquatic contaminants, polycyclic aromatic hydrocarbons (PAHs) have been shown to induce several adverse effects on amphibian species in the larval stages. Conversely, adults exposed to high concentrations of the ubiquitous PAH, benzo[a]pyrene (BaP), tolerate the compound thanks to their highly efficient hepatic detoxification mechanisms. Due to this apparent lack of toxic effect on adults, no studies have examined in depth the potential toxicological impact of PAH on the physiology of adult amphibian livers. This study sheds light on the hepatic responses of Xenopus tropicalis when exposed to high environmentally relevant concentrations of BaP, by combining a high throughput transcriptomic approach (mRNA deep sequencing) and a characterization of cellular and physiological modifications to the amphibian liver. RESULTS: Transcriptomic changes observed in BaP-exposed Xenopus were further characterized using a time-dependent enrichment analysis, which revealed the pollutant-dependent gene regulation of important biochemical pathways, such as cholesterol biosynthesis, insulin signaling, adipocytokines signaling, glycolysis/gluconeogenesis and MAPK signaling. These results were substantiated at the physiological level with the detection of a pronounced metabolic disorder resulting in a possible insulin resistance-like syndrome phenotype. Hepatotoxicity induced by lipid and cholesterol metabolism impairments was clearly identified in BaP-exposed individuals. CONCLUSIONS: Our data suggested that BaP may disrupt overall liver physiology, and carbohydrate and cholesterol metabolism in particular, even after short-term exposure. These results are further discussed in terms of how this deregulation of liver physiology can lead to general metabolic impairment in amphibians chronically exposed to contaminants, thereby illustrating the role xenobiotics might play in the global decline in amphibian populations.

Chemical and biological insecticides select distinct gene expression patterns in Aedes aegypti mosquito.

Worldwide evolution of mosquito resistance to chemical insecticides represents a major challenge for public health, and the future of vector control largely relies on the development of biological insecticides that can be used in combination with chemicals (integrated management), with the expectation that populations already resistant to chemicals will not become readily resistant to biological insecticides. However, little is known about the metabolic pathways affected by selection with chemical or biological insecticides. Here we show that Aedes aegypti, a laboratory mosquito strain selected with a biological insecticide (Bacillus thuringiensis israelensis, Bti) evolved increased transcription of many genes coding for endopeptidases while most genes coding for detoxification enzymes were under-expressed. By contrast, in strains selected with chemicals, genes encoding detoxification enzymes were mostly over-expressed. In all the resistant strains, genes involved in immune response were under-transcribed, suggesting that basal immunity might be a general adjustment variable to compensate metabolic costs caused by insecticide selection. Bioassays generally showed no evidence for an increased susceptibility of selected strains towards the other insecticide type, and all chemical-resistant strains were as susceptible to Bti as the unselected parent strain, which is a good premise for sustainable integrated management of mosquito populations resistant to chemicals.

Effect of DNA extraction and sample preservation method on rumen bacterial population.

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.