Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in Michigan and its associated state fish hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein we describe a diverse assemblage of Flavobacterium and Chryseobacterium spp. isolates recovered from diseased as well as apparently healthy wild, feral, and farmed fish of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 were identified as Flavobacterium spp., whereas 43 were identified as Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Although F. psychrophilum and F. columnare were indeed associated with multiple fish mortality events, many previously uncharacterized flavobacteria were recovered from systemically infected fish showing overt signs of disease, and in vitro protease assays demonstrated that these isolates were highly proteolytic to multiple substrates that comprise host tissues. Indeed, the majority of the isolates either (1) were most similar to recently described fish-associated Flavobacterium and Chryseobacterium spp. that have never before been reported in North America (e.g., F. oncorhynchi, F. araucananum, C. viscerum, C. piscicola, and C. chaponense) or (2) did not cluster with any described species and most likely represent novel flavobacterial taxa. This study highlights the extreme diversity of flavobacteria that are potentially associated with flavobacteriosis in Michigan.

We investigated microbial succession on lake sturgeon (Acipenser fulvescens) egg surfaces over the course of their incubation period as a function of simulated stream flow rate. The primary objective was to characterize the microbial community assembly during succession and to examine how simulated stream flow rate affect the successional process. Sturgeon eggs were reared under three flow regimes; high (0.55 m/s), low (0.18 m/s), and variable (0.35 and 0.11 m/s alternating 12 h intervals). Eggs were collected from each flow regime at different egg developmental stages. Microbial community DNA was extracted from egg surface and the communities were examined using 16S rRNA gene-based terminal restriction fragment length polymorphism and 454 pyrosequencing. Analysis of these datasets using principal component analysis revealed that microbial communities were clustered by egg developmental stages (early, middle, and late) regardless of flow regimes. 454 pyrosequencing data suggested that 90-98 % of the microbial communities were composed of the phyla Proteobacteria and Bacteroidetes throughout succession. β-Protebacteria was more dominant in the early stage, Bacteroidetes became more dominant in the middle stage, and α-Proteobacteria became dominant in the late stage. A total of 360 genera and 5,826 OTUs at 97 % similarity cutoff were associated with the eggs. Midway through egg development, the egg-associated communities of the low flow regime had a higher diversity than those communities developed under high or variable flow regimes. Results show that microbial community turnover occurred during embryogenesis, and stream flow rate influenced the microbial succession processes on the sturgeon egg surfaces.

Characterization of interactions within a host-associated microbiome can help elucidate the mechanisms of microbial community formation on hosts and can be used to identify potential probiotics that protect hosts from pathogens. Microbes employ various modes of antagonism when interacting with other members of the community. The formation of biofilm by some strains can be a defense against antimicrobial compounds produced by other taxa. We characterized the magnitude of antagonistic interactions and biofilm formation of 25 phylogenetically diverse taxa that are representative of isolates obtained from egg surfaces of the threatened fish species lake sturgeon (Acipenser fulvescens) at two ecologically relevant temperature regimes. Eight isolates exhibited aggression to at least one other isolate. Pseudomonas sp. C22 was found to be the most aggressive strain, while Flavobacterium spp. were found to be one of the least aggressive and the most susceptible genera. Temperature affected the prevalence and intensity of antagonism. The aggressive strains identified also inhibited growth of known fish pathogens. Biofilm formations were observed for nine isolates and were dependent on temperature and growth medium. The most aggressive of the isolates disrupted biofilm formation of two well-characterized isolates but enhanced biofilm formation of a fish pathogen. Our results revealed the complex nature of interactions among members of an egg associated microbial community yet underscored the potential of specific microbial populations as host probiotics.

Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.