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Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline’s effect, OTU threshold and rarefaction had a minimal impact on all measurements. 

ABSTRACT Corals owe their ecological success to their symbiotic relationship with dinoflagellate algae (family Symbiodiniaceae). While the negative effects of heat stress on this symbiosis are well studied, how heat stress affects the onset of symbiosis and symbiont specificity is less explored. In this work, we used the model sea anemone, Exaiptasia diaphana (commonly referred to as Aiptasia), and its native symbiont, Breviolum minutum , to study the effects of heat stress on the colonization of Aiptasia by algae and the algal cell-surface glycome. Heat stress caused a decrease in the colonization of Aiptasia by algae that were not due to confounding variables such as algal motility or oxidative stress. With mass spectrometric analysis and lectin staining, a thermally induced enrichment of glycans previously found to be associated with free-living strains of algae (high-mannoside glycans) and a concomitant reduction in glycans putatively associated with symbiotic strains of algae (galactosylated glycans) were identified. Differential enrichment of specific sialic acid glycans was also identified, although their role in this symbiosis remains unclear. We also discuss the methods used to analyze the cell-surface glycome of algae, evaluate current limitations, and provide suggestions for future work in algal-coral glycobiology. Overall, this study provided insight into how stress may affect the symbiosis between cnidarians and their algal symbionts by altering the glycome of the symbiodinian partner. IMPORTANCE Coral reefs are under threat from global climate change. Their decline is mainly caused by the fragility of their symbiotic relationship with dinoflagellate algae which they rely upon for their ecological success. To better understand coral biology, researchers used the sea anemone, Aiptasia, a model system for the study of coral-algal symbiosis, and characterized how heat stress can alter the algae's ability to communicate to the coral host. This study found that heat stress caused a decline in algal colonization success and impacted the cell surface molecules of the algae such that it became more like that of nonsymbiotic species of algae. This work adds to our understanding of the molecular signals involved in coral-algal symbiosis and how it breaks down during heat stress. 

The Asian clam Corbicula fluminea (Family: Cyneridae) has aggressively invaded freshwater habitats worldwide, resulting in dramatic ecological changes and declines of native bivalves such as freshwater mussels (Family: Unionidae), one of the most imperiled faunal groups. Despite increases in our knowledge of invasive C. fluminea biology, little is known of how intrinsic and extrinsic factors, including co-occurring native species, influence its microbiome. We investigated the gut bacterial microbiome across genetically differentiated populations of C. fluminea in the Tennessee and Mobile River Basins in the Southeastern United States and compared them to those of six co-occurring species of native freshwater mussels. The gut microbiome of C. fluminea was diverse, differed with environmental conditions and varied spatially among rivers, but was unrelated to host genetic variation. Microbial source tracking suggested that the gut microbiome of C. fluminea may be influenced by the presence of co-occurring native mussels. Inferred functions from 16S rRNA gene data using PICRUST2 predicted a high prevalence and diversity of degradation functions in the C. fluminea microbiome, especially the degradation of carbohydrates and aromatic compounds. Such modularity and functional diversity of the microbiome of C. fluminea may be an asset, allowing to acclimate to an extensive range of nutritional sources in invaded habitats, which could play a vital role in its invasive success. 

Freshwater mussels perform essential ecosystem functions, yet we have no information on how their microbiomes fluctuate over time. In this study, we examined temporal variation in the microbiome of six mussel species (Lampsilis ornata, Obovaria unicolor, Elliptio arca, Fusconaia cerina, Cyclonaias asperata, and Tritogonia verrucosa) sampled from the same river in 2016 and 2019. We examined the taxonomic, phylogenetic, and inferred functional (from 16S rRNA sequences) facets of their microbiome diversity. Significant differences between the two years were identified in five of the six species sampled. However, not all species that exhibited a temporally variable microbiome were functionally distinct across years, indicating functional redundancy within the mussel gut microbiome. Inferred biosynthesis pathways showed temporal variation in pathways involved in degradation, while pathways involved in cellular metabolism were stable. There was no evidence for phylosymbiosis across any facet of microbiome biodiversity. These results indicate that temporal variation is an important factor in the assembly of the gut microbiomes of freshwater mussels and provides further support that the mussel gut microbiome is involved in host development and activity. 

Neotropical wood‐eating catfishes (family Loricariidae) can occur in diverse assemblages with multiple genera and species feeding on the same woody detritus. As such, they present an intriguing system in which to examine the influence of host species identity on the vertebrate gut microbiome as well as to determine the potential role of gut bacteria in wood digestion. We characterized the gut microbiome of two co‐occurring catfish genera and four species: Panaqolus albomaculatus , Panaqolus gnomus , Panaqolus nocturnus, and Panaque bathyphilus , as well as that of submerged wood on which they feed. The gut bacterial community did not significantly vary across three gut regions (proximal, mid, distal) for any catfish species, although interspecific variation in the gut microbiome was significant, with magnitude of interspecific difference generally reflecting host phylogenetic proximity. Further, the gut microbiome of each species was significantly different to that present on the submerged wood. Inferring the genomic potential of the gut microbiome revealed that the majority of wood digesting pathways were at best equivalent to and more often depleted or nonexistent within the catfish gut compared to the submerged wood, suggesting a minimal role for the gut microbiome in wood digestion. Rather, these fishes are more likely reliant on fiber degradation performed by microbes in the environment, with their gut microbiome determined more by host identity and phylogenetic history.