Selection of aquatic microbiota exposed to the herbicides flufenacet and metazachlor.

Herbicides are important, ubiquitous environmental contaminants, but little is known about their interaction with bacterial aquatic communities. Here, we sampled a protected natural freshwater habitat and characterised its microbiome in interaction with herbicides. We evolved the freshwater microbiomes in a microcosm assay of exposure (28 days) to flufenacet and metazachlor at environmental concentrations of 0.5, 5 and 50 µg L-1 . Inhibitory effects of herbicides were exemplarily assessed in cultured bacteria from the same pond (Pseudomonas alcaligenes, Paenibacillus amylolyticus and Microbacterium hominis). Findings were compared to long-term concentrations as provided by local authorities. Here, environmental concentrations reached up to 11 µg L-1 (flufenacet) and 76 µg L-1 (metazachlor). Bacteria were inhibited at minimum inhibitory concentrations far above these values; however, concentrations of 50 µg L-1 of flufenacet resulted in measurable growth impairment. While most herbicide-exposed microcosm assays did not differ from controls, Acidobacteria were selected at high environmental concentrations of herbicides. Alpha-diversity (e.g., taxonomic richness on phylum level) was reduced when aquatic microbiomes were exposed to 50 µg metazachlor or flufenacet. One environmental strain of P. alcaligenes showed resistance to high concentrations of flufenacet (50 g L-1 ). In total, this study reveals that ecologic imbalance due to herbicide use significantly impacts aquatic microbiomes.

Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples.

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony-forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α- and ß-diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

Effector and regulator: Diverse functions of C. elegans C-type lectin-like domain proteins.

In C. elegans, 283 clec genes encode a highly diverse family of C-type lectin-like domain (CTLD) proteins. Since vertebrate CTLD proteins have characterized functions in defense responses against pathogens and since expression of C. elegans clec genes is pathogen-dependent, it is generally assumed that clec genes function in C. elegans immune defenses. However, little is known about the relative contribution and exact function of CLEC proteins in C. elegans immunity. Here, we focused on the C. elegans clec gene clec-4, whose expression is highly upregulated by pathogen infection, and its paralogs clec-41 and clec-42. We found that, while mutation of clec-4 resulted in enhanced resistance to the Gram-positive pathogen Bacillus thuringiensis MYBt18247 (Bt247), inactivation of clec-41 and clec-42 by RNAi enhanced susceptibility to Bt247. Further analyses revealed that enhanced resistance of clec-4 mutants to Bt247 was due to an increase in feeding cessation on the pathogen and consequently a decrease in pathogen load. Moreover, clec-4 mutants exhibited feeding deficits also on non-pathogenic bacteria that were in part reflected in the clec-4 gene expression profile, which overlapped with gene sets affected by starvation or mutation in nutrient sensing pathways. However, loss of CLEC-4 function only mildly affected life-history traits such as fertility, indicating that clec-4 mutants are not subjected to dietary restriction. While CLEC-4 function appears to be associated with the regulation of feeding behavior, we show that CLEC-41 and CLEC-42 proteins likely function as bona fide immune effector proteins that have bacterial binding and antimicrobial capacities. Together, our results exemplify functional diversification within clec gene paralogs.