In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.

• MeSH
• digoxigenin metabolismus   MeSH
• hybridizace in situ fluorescenční metody   MeSH
• messenger RNA genetika   metabolismus   MeSH
• RNA sondy metabolismus   MeSH
• Schistosoma mansoni cytologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards.IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.

• MeSH
• Bacteria klasifikace   MeSH
• Eukaryota klasifikace   MeSH
• fluorescenční mikroskopie MeSH
• fylogeneze MeSH
• mikrobiota * MeSH
• mořská voda mikrobiologie   MeSH
• reprodukovatelnost výsledků MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• statistické modely MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02-14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. CONCLUSIONS: A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract.

• MeSH
• Bacteria MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• jezera mikrobiologie   MeSH
• kardiopulmonální resuscitace * MeSH
• metagenom * genetika   MeSH
• metagenomika MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake.

• MeSH
• amoniak metabolismus   MeSH
• Archaea klasifikace   genetika   metabolismus   MeSH
• biodiverzita * MeSH
• DNA archebakterií chemie   genetika   MeSH
• fylogeneze MeSH
• geologické sedimenty mikrobiologie   MeSH
• hybridizace in situ fluorescenční MeSH
• jezera mikrobiologie   MeSH
• molekulární sekvence - údaje MeSH
• oxidace-redukce MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Niche specialization of nitrifying prokaryotes is usually studied with tools targeting molecules involved in the oxidation of ammonia and nitrite. The ecological significance of diverse CO2 fixation strategies used by nitrifiers is, however, mostly unexplored. By analyzing autotrophy-related genes in combination with amoA marker genes based on droplet digitial PCR and CARD-FISH counts targeting rRNA, we quantified the distribution of nitrifiers in eight stratified lakes. Ammonia oxidizing (AO) Thaumarchaeota using the 3-hydroxypropionate/4-hydroxybutyrate pathway dominated deep and oligotrophic lakes, whereas Nitrosomonas-related taxa employing the Calvin cycle were important AO bacteria in smaller lakes. The occurrence of nitrite oxidizing Nitrospira, assimilating CO2 with the reductive TCA cycle, was strongly correlated with the distribution of Thaumarchaeota. Recently discovered complete ammonia-oxidizing bacteria (comammox) belonging to Nitrospira accounted only for a very small fraction of ammonia oxidizers (AOs) present at the study sites. Altogether, this study gives a first insight on how physicochemical characteristics in lakes are associated to the distribution of nitrifying prokaryotes with different CO2 fixation strategies. Our investigations also evaluate the suitability of functional genes associated with individual CO2 assimilation pathways to study niche preferences of different guilds of nitrifying microorganisms based on an autotrophic perspective.

• MeSH
• amoniak metabolismus   MeSH
• Archaea klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• autotrofní procesy MeSH
• bakterie fixující dusík klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• dusitany metabolismus   MeSH
• jezera mikrobiologie   MeSH
• koloběh uhlíku * genetika   MeSH
• oxidace-redukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5-3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.

• MeSH
• Archaea genetika   izolace a purifikace   klasifikace   MeSH
• biodiverzita MeSH
• Chiroptera mikrobiologie   MeSH
• DNA archebakterií genetika   MeSH
• ekosystém MeSH
• feces chemie   mikrobiologie   MeSH
• financování organizované MeSH
• fylogeneze MeSH
• molekulární sekvence - údaje MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Geografické názvy
• Slovenská republika MeSH

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• Cryptophyta mikrobiologie   MeSH
• fylogeneze MeSH
• heterotrofní procesy MeSH
• hybridizace in situ fluorescenční MeSH
• plankton mikrobiologie   MeSH
• potravní řetězec MeSH
• roční období MeSH
• sladká voda mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• heptachlor škodlivé účinky   toxicita   MeSH
• rizikové faktory MeSH
• vystavení vlivu životního prostředí MeSH

• Konspekt
• Chemie. Mineralogické vědy
• NLK Obory
• chemie, klinická chemie
• environmentální vědy
• pracovní lékařství
• NLK Publikační typ
• publikace WHO

• MeSH
• hodnocení rizik MeSH
• tetrachlorethylen škodlivé účinky   toxicita   MeSH
• vystavení vlivu životního prostředí MeSH
• Publikační typ
• zprávy MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• toxikologie
• chemie, klinická chemie
• NLK Publikační typ
• publikace WHO

• MeSH
• dehet uhelný toxicita   škodlivé účinky   MeSH
• hodnocení rizik MeSH
• kreosot toxicita   MeSH
• nebezpečné látky MeSH
• pracovní expozice MeSH
• vystavení vlivu životního prostředí MeSH

• Konspekt
• Veřejné zdraví a hygiena
• NLK Obory
• environmentální vědy
• toxikologie
• hygiena
• NLK Publikační typ
• publikace WHO