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Background Understanding the influence of methodology on results is an essential consideration in experimental design. In the expanding field of fish microbiology, many best practices and targeted techniques remain to be refined. This study aimed to compare microbial assemblages obtained from Atlantic salmon (Salmo salar) gills by swabbing versus biopsy excision. Results demonstrate the variation introduced by altered sampling strategies and enhance the available knowledge of the fish gill microbiome. Results The microbiome was sampled using swabs and biopsies from fish gills, with identical treatment of samples for 16S next generation Illumina sequencing. Results show a clear divergence in microbial communities obtained through the different sampling strategies, with swabbing consistently isolating a more diverse microbial consortia, and suffering less from the technical issue of host DNA contamination associated with biopsy use. Sequencing results from biopsy-derived extractions, however, hint at the potential for more cryptic localisation of some community members. Conclusions Overall, results demonstrate a divergence in the obtained microbial community when different sampling methodology is used. Swabbing appears a superior method for sampling the microbiota of mucosal surfaces for broad ecological research in fish, whilst biopsies might be best applied in exploration of communities beyond the reach of swabs, such as sub-surface and intracellular microbes, as well as in pathogen diagnosis. Most studies on the external microbial communities of aquatic organisms utilise swabbing for sample collection, likely due to convenience. Much of the ultrastructure of gill tissue in live fish is, however, potentially inaccessible to swabbing, meaning swabbing might fail to capture the full diversity of gill microbiota. This work therefore also provides valuable insight into partitioning of the gill microbiota, informing varied applications of different sampling methods in experimental design for future research.

Over the past 150 million years, the hyperarid core of the Atacama Desert has been transformed by geologic and atmospheric conditions into one of the most unique and inhospitable landscapes on the planet. This makes it an ideal Mars analog that has been explored for decades as preliminary studies on the space life discovery. However, two heavy rainfalls that occurred in the Atacama in 2015 and 2017 provide a unique opportunity to study the response of resident extremophiles to rapid environmental change associated with excessive water and salt shock. Here we combine geochemical analyses with molecular biology to study the variations in salts and microbial communities along an aridity gradient, and to examine the reshuffling of hyperarid microbiomes before and after the two rainfall events. Analysis of microbial community composition revealed that soils within the southern desert were consistently dominated by Actinobacteria, Proteobacteria, Acidobacteria, Planctomycetes, Chloroflexi, Bacteroidetes, Gemmatimonadetes, and Verrucomicrobia; soils within the hyperarid sites were dominated by Aquificae and Deinococcus-Thermus before heavy rainfalls, while these organisms almost totally diminished after rainfall, and the hyperarid microbial consortia and metabolisms transformed to a more southern desert pattern along with increased biodiversity. Salts at the shallow subsurface were dissolved and leached down to a deeper layer, both benefitting and challenging indigenous microorganisms with the excessive input of water and ions. Microbial viability was found to change with aridity and rainfall events but correlated with elevation, pH, conductivity, chloride, nitrate, sulfate, and soil organic matters (SOM). Metagenomic functional pathways related to stressor responses also increased in post-rainfall hyperarid soils. Our findings contribute to the primary goal of Atacama Mars analog research for understanding the microbial community structure and adaptations: this study sheds light on the structure of xerophilic, halophilic, and radioresistant microbiomes in hyperarid environments, and their response to changes in water availability.

Funding: This work was funded by the John Templeton Foundation Grant 60501, “Putting the Extended Evolutionary Synthesis to the Test”. It also received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland, (https://www.masts.ac.uk)) and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. DMP received funding from MASTS, funded by the Scottish Funding Council (grant reference HR09011), and AJW received funding under the MASTS small grant scheme (grant reference SG433). Intertidal systems are complex and dynamic environments with many interacting factors influencing biochemical characteristics and microbial communities. One key factor are the actions of resident fauna, many of which are regarded as ecosystem engineers because of their bioturbation, bioirrigation and sediment stabilising activities. The purpose of this investigation was to elucidate the evolutionary implications of the ecosystem engineering process by identifying, if any, aspects that act as selection pressures upon microbial communities. A mesocosm study was performed using the well characterised intertidal ecosystem engineers Corophium volutator, Hediste diversicolor, and microphytobenthos, in addition to manual turbation of sediments to compare effects of bioturbation, bioirrigation and stabilisation. A range of sediment functions and biogeochemical gradients were measured in conjunction with 16S rRNA sequencing and diatom taxonomy, with downstream bacterial metagenome function prediction, to identify selection pressures that incited change to microbial community composition and function. Bacterial communities were predominantly Proteobacteria, with the relative abundance of Bacteroidetes, Alphaproteobacteria and Verrucomicrobia being partially displaced by Deltaproteobacteria, Acidobacteria and Chloroflexi as dissolved oxygen concentration and redox potential decreased. Bacterial community composition was driven strongly by biogeochemistry; surface communities were affected by a combination of sediment functions and overlying water turbidity, and subsurface communities by biogeochemical gradients driven by sediment reworking. Diatom communities were dominated by Nitzschia laevis and Achnanthes sp., and assemblage composition was influenced by overlying water turbidity (manual or biogenic) rather than direct infaunal influences such as grazing. Publisher PDF