Despite progress understanding microbial communities involved in terrestrial vertebrate decomposition, little is known about the microbial decomposition of aquatic vertebrates from a functional and environmental context. Here, we analyzed temporal changes in the “necrobiome” of rainbow darters, which are common North American fish that are sensitive indicators of water quality. By combining 16S rRNA gene and shotgun metagenomic sequence data from four time points, we studied the progression of decomposers from both taxonomic and functional perspectives. The 16S rRNA gene profiles revealed strong community succession, with early decomposition stages associated with Aeromonas and Clostridium taxa and later stages dominated by members of the Rikenellaceae (i.e., Alistipes/Acetobacteroides genera). These results were reproducible and independent of environmental perturbation, given that exposure to wastewater treatment plant effluent did not substantially influence the necrobiome composition of fish or the associated water sample microbiota. Metagenomic analysis revealed significant changes throughout decomposition in degradation pathways for amino acids, carbohydrates/glycans, and other compounds, in addition to putrefaction pathways for production of putrescine, cadaverine, and indole. Binning of contigs confirmed a predominance of Aeromonas genome assemblies, including those from novel strains related to the pathogen Aeromonas veronii. These bins of Aeromonas genes also encoded known hemolysin toxins (e.g., aerolysin) that were particularly abundant early in the process, potentially contributing to host cell lysis during decomposition. Overall, our results demonstrate that wild-caught fish have a reproducible decomposer succession and that the fish necrobiome serves as a potential source of putative pathogens and toxigenic bacteria. IMPORTANCE The microbial decomposition of animal tissues is an important ecological process that impacts nutrient cycling in natural environments. We studied the microbial decomposition of a common North American fish (rainbow darters) over four time points, combining 16S rRNA gene and shotgun metagenomic sequence data to obtain both taxonomic and functional perspectives. Our data revealed a strong community succession that was reproduced across different fish and environments. Decomposition time point was the main driver of community composition and functional potential; fish environmental origin (upstream or downstream of a wastewater treatment plant) had a secondary effect. We also identified strains related to the putative pathogen Aeromonas veronii as dominant members of the decomposition community. These bacteria peaked early in decomposition and coincided with the metagenomic abundance of hemolytic toxin genes. Our work reveals a strong decomposer succession in wild-caught fish, providing functional and taxonomic insights into the vertebrate necrobiome.

The effluent from municipal wastewater treatment plants is a major point source of contamination in Canadian waterways. The improvement of effluent quality to reduce contaminants, such as pharmaceuticals and personal care products, before being released into the environment is necessary to reduce the impacts on organisms that live in the river downstream. Here, we aimed to characterize the metabolic and gill physiological responses of rainbow (Etheostoma caeruleum), fantail (Etheostoma flabellare), and greenside (Etheostoma blennioides) darters to the effluent in the Grand River from the recently upgraded Waterloo municipal wastewater treatment plant. The routine metabolism of darters was not affected by effluent exposure, but some species had increased maximum metabolic rates, leading to an increased aerobic scope. The rainbow darter aerobic scope increased by 2.2 times and the fantail darter aerobic scope increased by 2.7 times compared to the reference site. Gill samples from effluent-exposed rainbow darters and greenside darters showed evidence of more pathologies and variations in morphology. These results suggest that darters can metabolically adjust to effluent-contaminated water and may also be adapting to the urban and agricultural inputs. The modification and damage to the gills provide a useful water quality indicator but does not necessarily reflect how well acclimated the species is to the environment due to a lack of evidence of poor fish health.