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Microeukaryotes are a diverse and often overlooked group of microbes that are important in food webs and other ecological linkages. Little is known about microeukaryotes associated with aquatic invertebrates, although filter feeders such as mussels are likely to take in and potentially retain microeukaryotes in their gut while feeding. Microeukaryotes such as apicomplexans have been reported in marine mussel species, but no studies have examined the presence of these microorganisms in freshwater mussels or how they relate to mussel host species or environmental conditions. In this study, microbial community DNA was extracted from the gut tissue of over 300 freshwater mussels, representing 22 species collected from rivers in the southeastern USA. Microeukaryote DNA was detected using PCR amplification, followed by the sequencing of positive amplicons. Microeukaryotes were found in 167 individual mussels (53%) of those tested. Amplicons included dinoflagellates/algae that differed between mussel species and are likely food sources that were distinct from those found in water and sediment samples analyzed concurrently. A total of 5% of the positive amplicons were non-photosynthetic alveolates that could represent parasitic microeukaryotes. Understanding the distribution of microeukaryotes in the freshwater mussel gut microbiome could further our understanding of the ongoing decline of mussel populations. 

Abstract In freshwater ecosystems, consumers can play large roles in nutrient cycling by modifying nutrient availability for autotrophic and heterotrophic microbes. Nutrients released by consumers directly supportgreen food websbased on primary production andbrown food websbased on decomposition. While much research has focused on impacts of consumer driven nutrient dynamics on green food webs, less attention has been given to studying the effects of these dynamics on brown food webs.Freshwater mussels (Bivalvia: Unionidae) can dominate benthic biomass in aquatic systems as they often occur in dense aggregations that create biogeochemical hotspots that can control ecosystem structure and function through nutrient release. However, despite functional similarities as filter‐feeders, mussels exhibit variation in nutrient excretion and tissue stoichiometry due in part to their phylogenetic origin. Here, we conducted a mesocosm experiment to evaluate how communities of three phylogenetically distinct species of mussels individually and collectively influence components of green and brown food webs.We predicted that the presence of mussels would elicit a positive response in both brown and green food webs by providing nutrients and energy via excretion and biodeposition to autotrophic and heterotrophic microbes. We also predicted that bottom‐up provisioning of nutrients would vary among treatments as a result of stoichiometric differences of species combinations, and that increasing species richness would lead to greater ecosystem functioning through complementarity resulting from greater trait diversity.Our results show that mussels affect the functioning of green and brown food webs through altering nutrient availability for both autotrophic and heterotrophic microbes. These effects are likely to be driven by phylogenetic constraints on tissue nutrient stoichiometry and consequential excretion stoichiometry, which can have functional effects on ecosystem processes. Our study highlights the importance of measuring multiple functional responses across a gradient of diversity in ecologically similar consumers to gain a more holistic view of aquatic food webs. 

Freshwater mussels are important indicators of the overall health of their environment but have suffered declines that have been attributed to factors such as habitat degradation, a loss of fish hosts, climate change, and excessive nutrient inputs. The loss of mussel biodiversity can negatively impact freshwater ecosystems such that understanding the mussel’s gut microbiome has been identified as a priority topic for developing conservation strategies. In this study, we determine whether ethanol-stored specimens of freshwater mussels can yield representative information about their gut microbiomes such that changes in the microbiome through time could potentially be determined from museum mussel collections. A short-term preservation experiment using the invasive clam Corbicula fluminea was used to validate the use of ethanol as a method for storing the bivalve microbiome, and the gut microbiomes of nine native mussel species that had been preserved in ethanol for between 2 and 9 years were assessed. We show that ethanol preservation is a valid storage method for bivalve specimens in terms of maintaining an effective sequencing depth and the richness of their gut bacterial assemblages and provide further insight into the gut microbiomes of the invasive clam C. fluminea and nine species of native mussels. From this, we identify a “core” genus of bacteria (Romboutsia) that is potentially common to all freshwater bivalve species studied. These findings support the potential use of ethanol-preserved museum specimens to examine patterns in the gut microbiomes of freshwater mussels over long periods. 

Research on the microbiomes of animals has increased substantially within the past decades. More recently, microbial analyses of aquatic invertebrates have become of increased interest. The storage method used while collecting aquatic invertebrates has not been standardized throughout the scientific community, and the effects of common storage methods on the microbial composition of the organism is unknown. Using crayfish and dragonfly nymphs collected from a natural pond and crayfish maintained in an aquarium, the effects of two common storage methods, preserving in 95% ethanol and freezing at −20 °C, on the invertebrate bacterial microbiome was evaluated. We found that the bacterial community was conserved for two sample types (gut and exoskeleton) of field-collected crayfish stored either in ethanol or frozen, as was the gut microbiome of aquarium crayfish. However, there were significant differences between the bacterial communities found on the exoskeleton of aquarium crayfish stored in ethanol compared to those that were frozen. Dragonfly nymphs showed significant differences in gut microbial composition between species, but the microbiome was conserved between storage methods. These results demonstrate that preserving field-collected specimens of aquatic invertebrates in 95% ethanol is likely to be a simple and effective sample preservation method for subsequent gut microbiome analysis but is less reliable for the external microbiome. 

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. 

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. 

Insects that undergo metamorphosis from juveniles to adults provide an intriguing opportunity to examine the effects of life stage, species, and the environment on their gut microbiome. In this study, we surveyed the gut microbiomes of 13 species of dragonfly collected from five different locations subject to different levels of human impact. Juveniles were collected as nymphs from aquatic habitats while airborne adults were caught at the same locations. The gut microbiome was characterized by next generation sequencing of the bacterial 16S rRNA gene. Life stage was an important factor, with the gut microbiomes of dragonfly nymphs differing from those of adult dragonflies. Gut microbiomes of nymphs were influenced by sample site and, to a lesser extent, host species. Neither sample location nor host species had a strong effect on the gut microbiome of dragonfly adults. Regardless of life stage, gut microbiomes were dominated by members of the Proteobacteria, with members of the Bacteroidetes (especially in adults), Firmicutes, and Acidobacteria (especially in nymphs) also being proportionally abundant. These results demonstrate that different life stages of metamorphosing insects can harbor very different gut microbiomes and differ in how this microbiome is influenced by the surrounding environment.