Improved detection sensitivity using an optimal eDNA preservation and extraction workflow and its application to threatened sawfishes

Cooper, Madalyn K.; Huerlimann, Roger; Edmunds, Richard C.; Budd, Alyssa M.; Port, Agnès Le; Kyne, Peter M.; Jerry, Dean R.; Simpfendorfer, Colin A.

doi:10.1002/aqc.3591

Cited by 15 publications

(23 citation statements)

References 97 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There is little information available on the relative decay rates of different genes, although some eDNA studies have shown that the frequencies of positives can differ depending on the target locus (Zhan et al, 2014; Wood et al, 2019). Primers were recently published to target a small fragment of the 12S ribosomal RNA ( 12S ) locus in P. pectinata (Cooper et al, 2021), although additional cross‐testing in closely related, co‐occurring species (e.g. guitarfish) is needed before this assay can be incorporated into eDNA surveys in the Atlantic Ocean.…”

Section: Discussionmentioning

confidence: 99%

“…ddPCR™ assays offer greater sensitivity (Baker et al, 2018; Lehman et al, 2020; Drymon et al, 2021) and more precise quantification of target DNA when compared to conventional PCR (e.g. Simpfendorfer et al, 2016; Bonfil et al, 2021) or quantitative PCR approaches (see Wood et al, 2019; Cooper et al, 2021), making them ideal for ‘early detection’ surveys targeting highly threatened species.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Environmental DNA evidence of the Critically Endangered smalltooth sawfish, Pristis pectinata, in historically occupied US waters

Lehman

Poulakis

Scharer

et al. 2021

Aquatic Conservation

View full text Add to dashboard Cite

1. Formerly common in tropical and subtropical waters of the Atlantic Ocean, the Critically Endangered smalltooth sawfish, Pristis pectinata, underwent severe declines over the past century, restricting population(s) to south and south-west Florida in the US, and Bahamian waters.2. Anecdotal evidence (e.g. encounter reports from the public) suggests that P. pectinata have recently been observed in historically occupied US waters; however, no directed surveys have been conducted to verify their extent of occupancy.3. Here, environmental DNA (eDNA) surveys were used to investigate the occurrence of P. pectinata in three formerly occupied estuaries in US waters.Water samples were collected in the summer from the Indian River Lagoon and

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Environmental DNA evidence of the Critically Endangered smalltooth sawfish, Pristis pectinata, in historically occupied US waters

Lehman

Poulakis

Scharer

et al. 2021

Aquatic Conservation

View full text Add to dashboard Cite

show abstract

“…In the case of filtration, where it is recommended to maximize collection of trace eDNA through use of a small pore size (Minamoto et al, 2015;Turner et al, 2014) or increase filtrate volume (Sepulveda et al, 2019), highly turbid or productive environments will cause rapid filter clogging (Robson et al, 2016). Filtering water in turbid environments could be considered one of the most widespread, yet undesirable methodological challenge (Cooper et al, 2021;Ip et al, 2021;Robson et al, 2016;Sanches & Schreier, 2020), but may be compensated with the use of larger pore size filters (e.g., 1.2-20 µm, as in this study; Barnes et al, 2020), prefiltration (Takahara et al, 2013), or multiple filter replicates (Hunter et al, 2019). The downside of these options would be the increase in cost and time for field and laboratory processing of additional replicates (Sepulveda et al, 2019).…”

Section: Methods Sensitivity and Efficiencymentioning

confidence: 99%

“…Evidence suggests that the majority of macro-organismal eDNA is efficiently captured by filter pore sizes between 1 and 10 µm (Turner et al, 2014). Additionally, studies in turbid waters have proven that target species eDNA can be effectively captured using 20 µm filter pore sizes (Cooper et al, 2021;Robson et al, 2016). Therefore, we tested filter pore sizes ranging from 0.45 to 20 µm.…”

Section: Particle Size Fractionationmentioning

confidence: 99%

“…A partial fragment of the largetooth sawfish 12S ribosomal RNA was amplified using a QuantStudio 5 quantitative real-time PCR machine (Life Technologies, ThermoFisher Scientific Pty Ltd) with a previously optimized primer and TaqMan probe assay (Cooper et al, 2021; Table 2). To estimate copy number, triplicate 12-point standard curves were run in adjacent wells on the same qPCR plate.…”

Section: Quantitative Pcr Analysismentioning

confidence: 99%

See 1 more Smart Citation

Practical eDNA sampling methods inferred from particle size distribution and comparison of capture techniques for a Critically Endangered elasmobranch

et al. 2022

Self Cite

View full text Add to dashboard Cite

Environmental DNA (eDNA) methods are increasingly applied in the marine environment to identify species and community structure. To establish widely applicable eDNA techniques for elasmobranchs, we used the Critically Endangered largetooth sawfish (Pristis pristis Linnaeus, 1758) as a model species for: (1) assessing eDNA particle size distribution; (2) assessing the efficiency of long-term preservation of water samples; and (3) comparing the efficiency and detection sensitivity of filtration and precipitation methods. Water samples (1 L) collected from a tank containing one largetooth sawfish specimen were sequentially filtered through five filter membranes of decreasing pore size (20, 10, 5, 1.2, and 0.45 μm). The proportion of sawfish eDNA retained within each size class was determined through quantitative real-time PCR (qPCR) using a species-specific TaqMan probe assay. A linear mixed-effects model (lme) showed that the 1.2 and 20 µm filters captured most of the eDNA particles present in the sampled water. Additionally, whole water samples (0.375 L) were preserved in Longmire's buffer, stored at tropical ambient temperatures (26.3°C ± 3.0 SD) and extracted at five time points: immediately, one, two, and three months after collection, as well as frozen and extracted three months later, to assess the preservation efficiency of Longmire's buffer via qPCR analysis. A linear mixed-effects model showed that samples maintained maximal eDNA yield for at least three months after collection at ambient storage. Lastly, when comparing the filtration and precipitation methods, filtration using 0.45 µm pore size was more sensitive to capture of largetooth sawfish eDNA than filtration with 20 µm filter or water precipitation. However, water precipitation was more efficient when accounting for volume of water processed. These results provide options for best capture and preservation of elasmobranch eDNA.

show abstract

An environmental DNA assay for the detection of Critically Endangered angel sharks (Squatina spp.)

Faure,

Manel,

Macé

et al. 2023

Aquatic Conservation

View full text Add to dashboard Cite

The three sympatric angel shark species occurring in the Mediterranean – Squatina squatina (the angelshark), Squatina aculeata (the sawback angelshark), and Squatina oculata (the smoothback angelshark) – are all classed as Critically Endangered on the International Union for Conservation of Nature (IUCN) Red List. There is a clear need to better quantify their current status, using appropriate non‐destructive methods, to help inform future conservation measures. This study introduces an environmental DNA (eDNA) assay able to detect and distinguish S. aculeata, S. oculata, and S. squatina in the Mediterranean Sea by combining probe‐based quantitative polymerase chain reaction (qPCR) technology and Sanger sequencing. The assay targets a 173‐bp barcode in the mitochondrial cytochrome c oxidase I (COI) gene. It was tested in silico, in vitro on tissue‐extracted DNA, and on eDNA extracted from filtration samples. This genus‐specific assay was applied to detect the presence of S. squatina in eDNA samples collected in Corsica, France. The target barcode was found in seven of 76 eDNA samples, revealing the presence of S. squatina in north‐western Corsica, where the shark has never been observed, and confirming its existence on the eastern coast. The study also demonstrates that using eDNA sampling, based on 30 L of seawater filtered close to the substrate with a waterproof peristaltic pump, it was possible to detect the eDNA of this rare benthic species. The results of detection can help identify critical areas for angel shark conservation and facilitate the development of local public awareness initiatives. This novel qPCR assay should be used for future applications in the Mediterranean and eastern Atlantic targeting angel sharks to better identify the remaining populations. In this study the qPCR assay was applied for S. squatina eDNA, but application to S. aculeata and S. oculata still needs to be validated in the field.

show abstract

Improved detection sensitivity using an optimal eDNA preservation and extraction workflow and its application to threatened sawfishes

Cited by 15 publications

References 97 publications

Environmental DNA evidence of the Critically Endangered smalltooth sawfish, Pristis pectinata, in historically occupied US waters

Environmental DNA evidence of the Critically Endangered smalltooth sawfish, Pristis pectinata, in historically occupied US waters

Practical eDNA sampling methods inferred from particle size distribution and comparison of capture techniques for a Critically Endangered elasmobranch

An environmental DNA assay for the detection of Critically Endangered angel sharks (Squatina spp.)

Contact Info

Product

Resources

About