Wohlrab, S. | Search bioRxiv

bioRxiv2024

A CRISPR-Cas9 assisted analysis of single-cell microbiomes for identifying rare bacterial taxa in phycospheres of diatoms

Primary production in aquatic systems is governed by interactions between microalgae and their associated bacteria. Most of our knowledge about algal microbiomes stems from natural mixed communities or isolated algal monocultures, which therefore does neither address the role of genotypic diversity among the algal host cells nor do they reveal how this host diversity impacts the assembly process of associated bacteria. To overcome this knowledge gap, we developed a single-cell 16S sequencing approach in combination with CRISPR-Cas9 guided depletion of host 16S contaminations from the chloroplast. The validity of this novel method was tested by comparing bacterial communities of 144 single-cells across three genotypes of the Arctic marine diatom Thalassiosira gravida grown under different environmental conditions. From these, 62 single-cells were additionally sequenced after CRISPR-Cas9 treatment. Due to the improved sequencing depth, bacterial richness associated with individual diatom cells was increased by up to 56%. By applying this CRISPR-Cas9 treatment we not only revealed intraspecific host-genotype associations but also rare bacterial taxa that were not detected by standard 16S rRNA gene metabarcoding. Thus, the CRISPR-Cas9 assisted single-cell approach developed in this study advances our understanding on how the intraspecific diversity among algal hosts impacts the assembly process of their associated bacteria. This knowledge is essential to understand the co-evolution and adaptation of species in algal microbiomes.

microbiology↗

bioRxiv2023

Acclimation Capacity to Global Warming of Amphibians and Freshwater Fishes: Drivers, Patterns, and Data Limitations

Amphibians and fishes play a central role in shaping the structure and function of freshwater environments. These organisms have a limited capacity to disperse across different habitats and the thermal buffer offered by freshwater systems is small. Understanding determinants and patterns of their physiological sensitivity across life history is, therefore, imperative to predicting the impacts of climate change in freshwater systems. Based on a systematic literature review including 345 experiments with 998 estimates on 96 amphibian (Anura/Caudata) and 93 freshwater fish species (Teleostei), we conducted a quantitative synthesis to explore phylogenetic, ontogenetic, and biogeographic (thermal adaptation) patterns in upper thermal tolerance (CTmax) and thermal acclimation capacity (Acclimation Response Ratio, ARR) as well as the influence of the methodology used to assess these thermal traits using a conditional inference tree analysis. We found globally consistent patterns in CTmax and ARR, with phylogeny (taxa/order), experimental methodology, climatic origin, and life stage as significant determinants of thermal traits. The analysis demonstrated that CTmax does not primarily depend on the climatic origin but on experimental acclimation temperature and duration, and life stage. Higher acclimation temperatures and longer acclimation times led to higher CTmax values, whereby Anuran larvae revealed a higher CTmax than older life stages. The ARR of freshwater fishes was more than twice that of amphibians. Differences in ARR between life stages were not significant. In addition to phylogenetic differences, we found that ARR also depended on acclimation duration, ramping rate, and adaptation to local temperature variability. However, the amount of data on early life stages is too small, methodologically inconsistent, and phylogenetically unbalanced to identify potential life cycle bottlenecks in thermal traits. We therefore propose methods to improve the robustness and comparability of CTmax/ARR data across species and life stages, which is crucial for the conservation of freshwater biodiversity under climate change.

ecology↗