Accurate detection and quantification of seasonal abundance of American bullfrog (Lithobates catesbeianus) using ddPCR eDNA assays

Everts, Teun; Halfmaerten, David; Neyrinck, Sabrina; Regge, Nico De; Jacquemyn, Hans; Brys, Rein

doi:10.1038/s41598-021-90771-w

Cited by 21 publications

(35 citation statements)

References 72 publications

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“…The laboratory protocol and the primer/probe assays used were comprehensively described and validated in Everts et al (2021). Briefly, 1 ml of a lysis buffer was added via the inlet of each filter ( n = 71).…”

Section: Methodsmentioning

confidence: 99%

“…A volume of 150 µl of this DNA extract was then purified with Qiagen's DNeasy PowerClean Cleanup Kit and was finally eluted in 100 µl Tris–EDTA. The concentration of both bullfrog eDNA and IPC was simultaneously quantified by ddPCR in duplicate technical measurements (once undiluted and once 1:2 diluted) following Everts et al (2021). Biological replicates of experimental ponds 1 and 2 at the abundance of 25 tadpoles, and experimental pond 3 at the abundance of 1 and 5 tadpoles got lost during the workflow, resulting in a total of 68 eDNA samples that were finally analyzed.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we used droplet digital PCR (ddPCR) in eDNA surveys to estimate American bullfrog (Lithobates catesbeianus [Shaw, 1802]; hereafter referred to as bullfrog) abundance in natural lentic systems in Belgium and investigated how quantitative eDNA analyses can be integrated in monitoring and eradication programs of this invasive species. Previous research has shown that eDNA concentrations were closely related to bullfrog tadpole abundances under controlled conditions (Everts et al, 2021), but the extent to which this relation holds in natural water bodies remains unclear.…”

Section: Introductionmentioning

confidence: 99%

“…Because all living organisms leave traces of genetic material enclosed in gametes, metabolic waste, or shed tissue in their environment, the capture of this environmental DNA (eDNA) and subsequent amplification of genetic markers can reveal their presence, which eliminates the need for visual observation (Ficetola et al, 2008). In addition to eDNA‐based species detection, abundance estimates can also be inferred from quantitative eDNA analyses (Brys, Halfmaerten, et al, 2021; Everts et al, 2021; Spear et al, 2021). Even though eDNA concentrations are strongly related to species abundance in experimental systems, a considerable proportion of variance remains unaccounted for in field conditions (Yates et al, 2019), which can be attributed to both abiotic (temperature, UV, pH, suspended organic matter, hydrodynamics, etc.)…”

Section: Introductionmentioning

confidence: 99%

“…Following overnight incubation at 56°C, the lysis solution was collected from the filters, and DNA was extracted with Qiagen's DNeasy Blood & Tissue Kit.All eDNA samples were eluted in 200 µl Tris-EDTA. A volume of 150 µl of this DNA extract was then purified with Qiagen's DNeasy PowerClean Cleanup Kit and was finally eluted in 100 µl Tris-EDTA.The concentration of both bullfrog eDNA and IPC was simultaneously quantified by ddPCR in duplicate technical measurements (once undiluted and once 1:2 diluted) followingEverts et al (2021).…”

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confidence: 99%

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Using quantitative eDNA analyses to accurately estimate American bullfrog abundance and to evaluate management efficacy

et al. 2022

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Biological invasions contribute now more than ever to the global homogenization of fauna and flora. Large‐scale monitoring programs are, therefore, needed to detect incipient invasions and to evaluate management interventions. As conventional monitoring methods are constrained by large costs, environmental DNA (eDNA)‐based methods are increasingly recognized as valuable monitoring tools. However, accurately estimating species abundance from eDNA concentrations in natural systems remains challenging and consequently hinders their integration in management applications. Here, we used droplet digital PCR (ddPCR) in eDNA surveys to estimate the abundance of invasive American bullfrogs (Lithobates catesbeianus). We first introduced bullfrog tadpoles in natural ponds to assess the relationship between abundances and eDNA concentrations under field conditions. Next, we combined eDNA sampling with fyke netting in naturally colonized ponds to investigate whether bullfrog eDNA concentrations can estimate bullfrog capture success and conventional abundance measures obtained via depletion sampling. Finally, we evaluated eradication measures by comparing bullfrog eDNA concentrations before and after fyke netting. We found a strong linear relationship between the numbers of introduced tadpoles and eDNA concentrations (r2 = 0.988). Bullfrog eDNA concentrations were not only linearly related to the catch‐per‐unit‐effort (r2 = 0.739), but also to conventional abundance estimates (r2 = 0.716), particularly when eDNA concentrations were standardized for pond area (r2 = 0.834) and volume (r2 = 0.888). Bullfrog tadpoles were only captured when eDNA concentrations exceeded 1.5 copies µl−1, indicating that quantitative eDNA analyses enable the localization of breeding ponds. We found a significant reduction in eDNA concentrations after fyke netting proportional to the number of captured bullfrogs. These results demonstrate that eDNA quantification is a reliable tool that accurately estimates bullfrog abundance in natural lentic systems. We show that quantitative eDNA analyses can complement the toolbox of natural resource managers and facilitate the coordination of eradication campaigns targeting alien invasive species.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Using quantitative eDNA analyses to accurately estimate American bullfrog abundance and to evaluate management efficacy

et al. 2022

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Using eDNA to survey amphibians: Methods, applications, and challenges

Sun,

Guo,

Gao

et al. 2023

Biotech & Bioengineering

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In recent years, environmental DNA (eDNA) has received attention from biologists due to its sensitivity, convenience, labor and material efficiency, and lack of damage to organisms. The extensive application of eDNA has opened avenues for the monitoring and biodiversity assessment of amphibians, which are frequently small and difficult to observe in the field, in areas such as biodiversity survey assessment and detection of specific, rare and threatened, or alien invasive species. However, the accuracy of eDNA can be influenced by factors such as ambient temperature, pH, and false positives or false negatives, which makes eDNA an adjunctive tool rather than a replacement for traditional surveys. This review provides a concise overview of the eDNA method and its workflow, summarizes the differences between applying eDNA for detecting amphibians and other organisms, reviews the research progress in eDNA technology for amphibian monitoring, identifies factors influencing detection efficiency, and discusses the challenges and prospects of eDNA. It aims to serve as a reference for future research on the application of eDNA in amphibian detection.

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Phenological mismatches mitigate the ecological impact of a biological invader on amphibian communities

Everts,

Van Driessche,

Neyrinck

et al. 2024

Ecological Applications

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Horizon scans have emerged as a valuable tool to anticipate the incoming invasive alien species (IAS) by judging species on their potential impacts. However, little research has been conducted on quantifying actual impacts and assessing causes of species‐specific vulnerabilities to particular IAS due to persistent methodological challenges. The underlying interspecific mechanisms driving species‐specific vulnerabilities therefore remain poorly understood, even though they can substantially improve the accuracy of risk assessments. Given that interspecific interactions underlying ecological impacts of IAS are often shaped by phenological synchrony, we tested the hypothesis that temporal mismatches in breeding phenology between native species and IAS can mitigate their ecological impacts. Focusing on the invasive American bullfrog (Lithobates catesbeianus), we combined an environmental DNA (eDNA) quantitative barcoding and metabarcoding survey in Belgium with a global meta‐analysis, and integrated citizen‐science data on breeding phenology. We examined whether the presence of native amphibian species was negatively related to the presence or abundance of invasive bullfrogs and whether this relationship was affected by their phenological mismatches. The field study revealed a significant negative effect of increasing bullfrog eDNA concentrations on native amphibian species richness and community structure. These observations were shaped by species‐specific vulnerabilities to invasive bullfrogs, with late spring‐ and summer‐breeding species being strongly affected, while winter‐breeding species remained unaffected. This trend was confirmed by the global meta‐analysis. A significant negative relationship was observed between phenological mismatch and the impact of bullfrogs. Specifically, native amphibian species with breeding phenology differing by 6 weeks or less from invasive bullfrogs were more likely to be absent in the presence of bullfrogs than species whose phenology differed by more than 6 weeks with that of bullfrogs. Taken together, we present a novel method based on the combination of aqueous eDNA quantitative barcoding and metabarcoding to quantify the ecological impacts of biological invaders at the community level. We show that phenological mismatches between native and invasive species can be a strong predictor of invasion impact regardless of ecological or methodological context. Therefore, we advocate for the integration of temporal alignment between native and IAS's phenologies into invasion impact frameworks.

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Accurate detection and quantification of seasonal abundance of American bullfrog (Lithobates catesbeianus) using ddPCR eDNA assays

Cited by 21 publications

References 72 publications

Using quantitative eDNA analyses to accurately estimate American bullfrog abundance and to evaluate management efficacy

Using quantitative eDNA analyses to accurately estimate American bullfrog abundance and to evaluate management efficacy

Using eDNA to survey amphibians: Methods, applications, and challenges

Phenological mismatches mitigate the ecological impact of a biological invader on amphibian communities

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