DNA based sequencing technology has revolutionized the field of microbial ecology and environmental studies. However, biases can be introduced at all experimental steps and, thus, affect the interpretation of microbial community. So far, previous studies on the biases introduced from the key steps of DNA extraction and primer sets mainly focused on the bacterial communities in soil or sediment samples, while little is known about the effect on the eukaryotic microbial communities. Here, we studied the effects of three different DNA extraction kits on both prokaryotic and micro-eukaryotic communities by 16S and 18S rRNA gene amplicon sequencing, and further disentangled the influence of primer choice on the micro-eukaryotic communities. Our results showed that the FastDNA SPIN Kit for Soil and DNeasy PowerSoil Kit produced much higher DNA yield with good reproducibility, and observed more eukaryotic OTUs compared to the MinkaGene DNA extraction kit, but all three kits exhibited comparable ability in recovering bacterial alpha diversity. Of the two primer sets, both targeting the V4 region of the 18S rRNA gene, the TAR primer set detected higher number of unique OTUs than the EK primer set, while the EK primer set resulted in longer amplicons and better reproducibility between replicates. Based on our findings, we recommend using the DNeasy PowerSoil Kit with the EK primer set to capture the abundant micro-eukaryotic taxa from freshwater sediment samples. If a more complete picture of the eukaryotic microbial community is desired, the TAR primer set in combination with the FastDNA SPIN Kit is more efficient in this study.

Freshwater lakes are globally significant sources of potent greenhouse gases (GHGs), but how their GHGs emissions respond to changing nutrient levels remains unclear. Here, we demonstrated that nitrous oxide (N₂O) production pathways in lake sediments are tightly linked to trophic state, whereas methane (CH₄) production appears to be multifactorial Through global metagenomics and controlled batch experiments. In eutrophic sediments, N₂O is efficiently removed through complete denitrification, with nitrification serving as the main production pathway, whereas oligotrophic sediments produce N₂O primarily via incomplete denitrification. By simulating nutrient transitions using an innovative cross-inoculation experiment, we further revealed that lake sediments systematically shift between these N₂O production pathways as their trophic state changes, from denitrification-driven to nitrification-dominated during eutrophication, with the inverse pattern during oligotrophication. Consequently, N₂O emissions can be effectively mitigated by inhibiting nitrification in eutrophic lakes and restricting incomplete denitrification in oligotrophic ones. Our findings establish trophic status as a key driver of N₂O production sources in lake sediments.

The accelerating spread of antimicrobial resistance in natural ecosystems, driven principally by the dissemination of antibiotic resistance genes (ARGs), represents an escalating challenge for both environmental integrity and public health security. Aquatic systems contaminated with ARGs alongside associated virulence factors (VFs) and metal resistance elements (MRGs) have emerged as critical reservoirs of resistance propagation. This study employed metagenomic approaches to analyze microbial communities and functional diversity in the Chishui River, which spans three distinct regions under significant anthropogenic influence. The results revealed that microbial communities exhibit distinct spatiotemporal variations predominantly governed by temperature, DO, TP, and TN. In addition, variations in land use types across different regions also directly shaped microbial diversity patterns, subsequently exerting direct and indirect effects on mobile genetic elements (MGEs), ARGs, and VFs, ultimately leading to the enrichment and dissemination of high-risk resistance genes. Both microbial communities and ARGs exhibited short-distance migration patterns. Notably, a synergistic covariation pattern was observed between antibiotic resistance genes (ARGs) and dissimilatory nitrate reduction to ammonium (DNRA) functional genes, indicating a potential ecological linkage between these two genetic traits. A total of 138 metagenome-assembled genomes have been identified as potential vectors for ARG dissemination. We further revealed a novel synergistic link between ARG abundance and the DNRA process, and the class Gammaproteobacteria was identified as the primary vector of resistance dissemination, functioning as dominant co-hosts for ARGs, MRGs, VFs, and DNRA genes in the Chishui River. These findings offer new insights into river ecosystems, underscoring the importance of monitoring the fate of ARGs to enhance our understanding of how river ecosystems respond to human activities.

The EARLY MATURITY 8 (EAM8) gene of barley is homologous to the EARLY FLOWERING 3 (ELF3) gene in Arabidopsis, as loss-of-function mutations in this circadian clock gene promote rapid flowering. A previous study demonstrated that the early flowering phenotype of a hulless barley, Lalu, was due to allele eam8.l carrying an alternative splicing mutation in intron 3 that led to intron retention. In the present study, we verified that eam8.l encoded a truncated protein. Although EAM8 was expressed at a higher level in Lalu than in other barley lines with a longer growth period, it did not negatively regulate flowering time. This result further proved that the eam8.l protein was nonfunctional in regulating flowering in barley. The early flowering phenotype of Lalu plants was strongly dependent on the biosynthesis of gibberellin (GA). The eam8.l mutation stopped the suppression of GA biosynthesis, and Lalu accumulated excessive GA, especially in leaves. This was achieved through the upregulated expression of genes in the GA pathway, including GA20ox2, LFY1, SOC1, PAP2, and FPF3. The mutation of the EAM8 gene also abolished the inhibition of FLOWERING LOCUS T-like (FT1) gene expression at night. During the night, expression levels of the FT1 gene were higher than those during the day in Lalu. However, the GA-dependent pathway and FT1 gene mechanism are two independent pathways that promote flowering in Lalu. Alleles of EAM8, therefore, demonstrated an important breeding value in barley, which is probably effective in many other day-length-sensitive crop plants as well; thus, they could be used to tune adaptation in different geographic regions and climatic conditions, a critical issue in times of global warming.

Vitamin B12 (VB12) is essential for human health, and its deficiency can lead to various health issues, including anemia and neurological problems. Additionally, the presence of antibiotic resistance genes in various environments raises concerns about the spread of antibiotic resistance. This study aims to explore potential connections between these two functional gene categories in freshwater environments, which are closely intertwined with human health. We conducted a comprehensive metagenomic sequencing analysis across 23 sampling points from five different eutrophic urban lakes. The results highlight the dominance of the precorrin-2 synthesis pathway in the microbial synthesis of VB12 within urban lakes. Eutrophication may potentially enhance the precorrin-2 synthesis pathway while inhibiting others. The risk of antibiotic resistance is reduced in the anaerobic pathway of VB12 synthesis, while the abundance of metal resistance genes is increased. Binning analysis reveals that 26 metagenome-assembled genomes (MAGs) actively participate in VB12 synthesis, with at least 4 MAGs showing resistance during the VB12 synthesis process and demonstrating pathogenicity. These findings provide critical insights into the VB12 synthesis process and its implications for human health in terms of resistance risks.