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Abstract Freshwater ecosystems are complex, diverse, and face multiple imminent threats that have led to changes in both structure and function. It is urgent that we develop and standardize monitoring tools that allow for rapid and comprehensive assessment of freshwater communities to understand their changing dynamics and inform conservation. Environmental DNA surveys offer a means to inventory and monitor aquatic diversity, yet most studies focus on one or a few taxonomic groups because of technical challenges. In this study, we (1) create an eDNA metabarcoding dataset (natural water bodies) with 14 validated primer pairs; (2) create a free online, user‐friendly tool for primer selection that can be used for any metabarcoding data (SNIPe); and (3) using SNIPe, explore our dataset to derive subsets of informative, cost‐effective primer pairs that maximize detection of freshwater diversity. We first evaluated the completeness of public reference sequence databases and the efficiency of 14 primer pairs in silico, in vitro on five mock communities (mix of DNA from tissues of select taxa), in vivo on water samples from aquarium samples with known taxonomic composition, and finally in vivo on water samples from freshwater systems in Eastern Canada. Results from analyses using SNIPe revealed that 13 or 14 primer pairs are necessary to recover 100% of the species in water samples (natural systems), but that four primer pairs are sufficient to recover almost 75% of taxa with little overlap. Our work highlights the power of eDNA metabarcoding for reconstructing freshwater communities, including prey, parasite, pathogen, invasive, and declining species. It also emphasizes the importance of marker choice on species resolution, and primer characteristics and filtering parameters on detection success and accuracy of biodiversity estimates. Together, these results highlight the usefulness of eDNA for freshwater monitoring and should prompt more studies of tools to survey all communities.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Background Climate change has driven shifts in breeding phenology of many amphibians, causing phenological mismatches (e.g., predator-prey interactions), and potentially population declines. Collecting data with high spatiotemporal sensitivity on hibernation emergence and breeding times can inform conservation best practices. However, monitoring the phenology of amphibians can be challenging because of their cryptic nature over much of their life cycle. Moreover, most salamanders and caecilians do not produce conspicuous breeding calls like frogs and toads do, presenting additional monitoring challenges. Methods In this study, we designed and evaluated the performance of an environmental DNA (eDNA) droplet digital PCR (ddPCR) assay as a non-invasive tool to assess the breeding phenology of a Western Chorus Frog population (Pseudacris maculata mitotype) in Eastern Ontario and compared eDNA detection patterns to hourly automatic acoustic monitoring. For two eDNA samples with strong PCR inhibition, we tested three methods to diminish the effect of inhibitors: diluting eDNA samples, adding bovine serum albumin to PCR reactions, and purifying eDNA using a commercial clean-up kit. Results We recorded the first male calling when the focal marsh was still largely frozen. Chorus frog eDNA was detected on April 6th, 6 days after acoustic monitoring revealed this first calling male, but only 2 days after males attained higher chorus activity. eDNA signals were detected at more sampling locales within the marsh and eDNA concentrations increased as more males participated in the chorus, suggesting that eDNA may be a reasonable proxy for calling assemblage size. Internal positive control revealed strong inhibition in some samples, limiting detection probability and quantification accuracy in ddPCR. We found diluting samples was the most effective in reducing inhibition and improving eDNA quantification. Conclusions Altogether, our results showed that eDNA ddPCR signals lagged behind male chorusing by a few days; thus, acoustic monitoring is preferable if the desire is to document the onset of male chorusing. However, eDNA may be an effective, non-invasive monitoring tool for amphibians that do not call and may provide a useful complement to automated acoustic recording. We found inhibition patterns were heterogeneous across time and space and we demonstrate that an internal positive control should always be included to assess inhibition for eDNA ddPCR signal interpretations.

Cyanobacteria in the genus Microcystis are dominant components of many harmful algal blooms worldwide. Their pelagic–benthic life cycle helps them survive periods of adverse conditions and contributes greatly to their ecological success. Many studies on Microcystis overwintering have focused on benthic colonies and suggest that sediment serves as the major inoculum for subsequent summer blooms. However, the contemporaneous overwintering pelagic population may be important as well but is understudied. In this study, we investigated near-surface and near-bottom pelagic population dynamics of both microcystin-producing Microcystis and total Microcystis over six weeks in winter at Dog Lake (South Frontenac, ON, Canada). We quantified relative Microcystis concentrations using real-time PCR. Our results showed that the spatiotemporal distribution of overwintering pelagic Microcystis was depth dependent. The abundance of near-bottom pelagic Microcystis declined with increased depth with no influence of depth on near-surface Microcystis abundance. In the shallow region of the lake (Microcystis was found near the lake bottom (>90%). However, the proportion of near-surface Microcystis rose sharply to over 60% as the depth increased to approximately 18 m. The depth-dependent distribution pattern was found to be similar in both microcystin-producing Microcystis and total Microcystis. Our results suggest the top of the water column may be a more significant contributor of Microcystis recruitment inoculum than previously thought and merits more attention in early CHAB characterization and remediation.