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Environmental managers need a rapid and cost‐effective monitoring tool for tracking the spread of invasive species, particularly at the onset of introduction. The macroalgaeCaulerpa proliferais considered an invasive species outside its native range, colonizing large patches of seafloor, reducing native species, and altering ecosystem functioning. Here, we developed a droplet digital PCR assay for detection ofC. proliferafrom environmental DNA seawater samples using the internal transcribed spacer (ITS) region. While the assay itself was confirmed to be highly efficient, we discovered concentrations ofC. proliferaeDNA were present below detectable levels in the water column surrounding an outbreak. To understand why, we conducted tank‐based experiments for two California invasive algae species,Caulerpa proliferaandSargassum horneri. The steady‐state eDNA concentration (eDNA copies/ gram of biomass detected) ofC. proliferawas found to be two orders of magnitude lower thanS. horneri. A meta‐analysis of steady‐state concentrations reported in the literature showed a remarkable range from ~10⁴–10¹¹(copies/g), revealing C. proliferato have the lowest recorded steady‐state concentrations of eDNA of any known species. We attributeC. prolifera'slow steady‐state eDNA concentration to its unique biology as a unicellular macroscopic algae which reduces the possible modes of eDNA release compared to similarly sized multicellular organisms. Critically our results demonstrate the potential limits of eDNA approaches, the influence of shedding rates in the reliability of species detections, and the vital importance of benchmarking and validating eDNA assays in both field and laboratory settings.

 

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.