Using environmental eDNA for early detection of Bd
@Zerina Rahic
Replacing traditional method
Traditional techniques for detecting Bd include swabbing the keratinized portion of skin on frogs and tadpoles, and then analyzing the skin swabs for Bd zoospores using PCR techniques (Kriger et al. 2006). This method is dependent on finding and handling host individuals. Environmental DNA (eDNA) is a non-invasive alternative technique that has been used to accurately detect Bd through the collection and extraction of Bd DNA found in water samples (Kirshtein et al. 2007, Walker et al. 2007, Hyman & Collins 2012, Chestnut et al.2014).
Materials and methods
Water filter eDNA samples were collected from 13 restoration sites in Sequoia Kings Canyon National Park (National ParkScientific Research and Collecting permit # SEKI2015-SCI-0011). To accommodate variance in lake size, a 50 ml water sample has been collected every 40 m along the shoreline. Every 5 water samples were combined into a 250 ml composite sample (see Table 1). Each 250 ml composite sample was filtered using 1.2 μm polycarbonate track-etched (PCTE) filter membrane (EMD Millipore) with a single-use 47 mm diameter filter funnel (Thermo Fisher Scientific). Scientists used 1.2 μm PCTE filter membranes because our lake sampling effort was part of an eDNA sampling study where particle size distribution was being evaluated (Kamoroff 2016, C. Kamoroff & C. S. Goldberg unpubl. data), and PCTE filter membranes provide a fixed pore size (Turner et al. 2014). They filtered all water samples in the field using a polypropylene vacuum flask with a rubber stopper fixed to a hand pump. Furthermore, they used forceps soaked in a 50% bleach solution for at least 1 min and rinsed in distilled water to remove the filter membrane after filtration. All personnel wore single use disposable latex gloves during sample collection and changed gloves prior to handling filter membranes. All equipment used to collect water samples were single-use items and were properly disposed of after sample collection to prevent spread of Bd. During each sampling event, scientists filtered 250 ml of distilled water as a field negative control after all sampling was completed. Prior to DNA extraction, filter membranes have been stored in 95% molecular-grade ethanol, at room temperature away from any light sources (Minamoto et al. 2016). They extracted samples within 6 mo of collection. Due to the nature of our eDNA collection methods in which the sampler was required to walk the perimeter of the lake collecting water samples, a visual encounter survey at each site has been opportunistically conducted. In the laboratory, each filter has been cut in half, used half the filter for DNA extraction, and stored the other half in 95% ethanol as a reserve. A QIAshredder/Qiagen DNeasy Blood has been used and Tissue DNA extraction protocol (Goldberg et al. 2011) to extract DNA from filters in a limited-access clean room. The clean room is managed such that no high-quality DNA extraction or PCR product are handled in it, and personnel who have been exposed to PCR product or high-quality DNA are required to shower and change clothes before entering.
Scientists analyzed samples using the assay of Boyle et al. (2004). To quantify the initial DNA copy number for Bd eDNA samples, a standard curve has been created by using a 4-point serial dilution (1 000 000 to 1000 copies) of a synthesized gene (gBlocks; Integrated DNA Technologies) designed using genomic sequences from Bd. Scientists chose the range of the standard curve (1 000 000 to 1000 copies) because large quantities of Bd DNA generally persist during chytridiomycosis outbreaks. It is only possible to detect quantities of DNA above or below the range set by the standard curve, but exact quantification cannot be determined if the quantities are outside the range. They used 3 μl of DNA extract in each reaction and ran each quantitative PCR (qPCR) reaction in triplicate (3 technical replicates) using 1× QuantiTect Multiplex PCR Mix (Qiagen) with 0.2 μM of each primer and 0.2 μM of the probe. To run all reactions, scientists used a cycle of 15 min at 95°C followed by 50 cycles at 94°C for 60 s and 60°C for 60 s. Most eDNA studies use reactions with high PCR cycle numbers (50 to 55) to detect low quantities of DNA that may be present in the sample (Kirshtein et al. 2007, Goldberg et al. 2011). All wells included an exogenous internal positive control to insure no qPCR inhibition had occurred (IPC; Applied Bio systems). Finally, scientists created and analyzed negative extraction and qPCR controls with every extraction batch and plate.
Results
Bd has been detected using eDNA techniques at all 3 sites that experienced a Bd die-off (Table 1). Samples collected from SEKI-3 and SEKI-2 had at least 2 samples test positive for Bd (i.e. all technical replicates detected Bd DNA). Very low quantities of Bd DNA have been detected from all 3 samples collected from SEKI-1, and those samples yielded equivocal results because only 1 or 2 of the 3 technical replicates returned a positive detection of Bd DNA (see Hyatt et al. 2007). All technical replicates indicated a positive detection (e.g. beginning of an amplification curve) at or below a 40 cycle threshold. After results have been analysed, no Bd has been detected at any of the 4 sites where yellow-legged frogs were present and no die-off was observed, as expected. All of the negative control samples, extraction negatives, and qPCR negatives tested negative. The standard curve for the Bd assay had an efficiency of 93.9% and R2 > 0.99.
Discussion
Environmental DNA is a promising, non-invasive alternative to skin swabs. Our sampling techniques accurately detected the presence of Bd at sites that experienced Bd related die-offs, and unlike swabs, did not require the capture or handling of target animals. Using non-invasive techniques such as eDNA is ideal when researching federally or state listed species because it may shorten or avoid lengthy research permitting processes. Environmental DNA techniques are also useful when working with species or habitats sensitive to human impacts.In addition to being non-invasive, our sampling techniques detected Bd at a critical time for management.
Statistical analysis devices to accurately measure detection probability
@Michelle Anne Hughes
Methodology
"In 2010, we sampled 20 boreal chorus frog (Pseudacris maculata) breeding ponds for the presence of eDNA from the fungal pathogen Batrachochytrium dendrobatidis (Bd). Ponds were located in Coconino National Forest, Arizona, USA. For eDNA detection, we filtered approximately 600 mL of pond water from each pond at four different time points: (T1) when amphibian host breeding was initiated (March); (T2) 1–2 weeks postbreeding initiation (March–April); (T3) 3–4 weeks postbreeding initiation, when tadpoles were present (April–May); and (T4) 10 weeks postbreeding initiation when metamorphosed froglets began to emerge (June–July). We collected one filter from each pond at each time point, unless ponds had dried, in which case no filters were collected. Water was filtered and extracted following Kirshtein et al. (2007). The presence of Bd DNA in filter extracts was determined by qPCR analysis using internal positive controls (IPC; Hyatt et al. 2007) and bovine serum albumin (BSA; Garland et al. 2010) to control for and reduce PCR inhibition. Each sample was run in duplicate on a single qPCR plate. Each duplicate well contained BSA, but only one of each duplicate well contained IPC (see Hyman & Collins 2012)."
Data analysis (more information and equations in the link)
"We fitted two variants of a site occupancy model to our data. The first model was the classical, two‐level model of MacKenzie et al. (2002) and Tyre et al. (2003), which we fitted to an aggregated version of our data (see below) using both the program presence 3.1 (Hines 2006) and also the Bayesian modelling software winbugs (Lunn et al. 2000). In this model, we only distinguished between occupied and unoccupied sites and a single observation process; thus, the stochasticity due to the repeated water sampling and the error in PCR analysis was not separately modelled (nor were the parameters governing these processes). Hence, in a second analysis, we used winbugs to fit the three‐level occupancy model of Nichols et al. (2008) and Mordecai et al. (2011), which distinguishes all three stochastic levels of the process underlying our observed data: (i) the occupancy process leading to some ponds being occupied and others being unoccupied; (ii) the water sampling process resulting in samples that contain the DNA of the respective species and others that do not; and (iii) the PCR sampling process, which results in a detection of a species or not given that DNA was available for detection in the water sample (see McClintock et al. 2010 for discussion of such hierarchical sampling schemes for disease surveillance)."
Discussion
"New statistical methods are necessary if we want to take full advantage of eDNA data (Yoccoz 2012). The analysis and results presented here show that eDNA data can easily be analysed in a statistically sound way using site occupancy models if multiple samples per pond are taken (MacKenzie et al. 2002; Tyre et al. 2003; Royle & Dorazio 2008). Our re‐analysis of the data presented and analysed in Hyman & Collins (2012) showed that Bd occurred in more ponds than estimated by Hyman & Collins (2012) using methods that did not explicitly account for imperfect detection. Hyman & Collins (2012) used two methods to detect Bd in ponds: eDNA and swabs of individual frogs. With both methods, they detected Bd in 17 of 20 ponds. There were, however, only 16 ponds where they detected Bd with both methods. Based on the results of the site occupancy analysis, there is a high probability that Bd was present in at least 19 ponds, suggesting a small underestimation of pond occupancy by Hyman & Collins (2012). Per‐sample detection probability using eDNA from water samples was low [0·45 (95% CRI: 0·32, 0·58)], but cumulative detection probabilities after four water samples were high (Fig. 2). This explains the small difference between the estimate of the number of ponds with Bd obtained by Hyman & Collins (2012) and our new results. Imperfect detection of species by means of eDNA seems to be the rule rather than an exception."
"Per‐visit availability probability in water samples was estimated at 0·45 (95% CRI 0·32, 0·58) and per‐PCR detection probability at 0·85 (95% CRI 0·74, 0·94), and six water samples from a pond were necessary for a cumulative detection probability >95%. A simulation study showed that when using site occupancy analysis, researchers need many fewer samples to reliably estimate presence and absence of species than without use of site occupancy modelling."
Seasonal detection rates of Bd by citizen scientists
@Michelle Anne Hughes
The full text has to be loaned from Bobst Library Interlibrary Loan — let me know if insight is valuable enough to warrant
Bd eDNA detection probability fluctuates seasonally. PI and volunteers show similiar probabilities of capturing and detecting eDNA, making volunteers a potential viable option for increased sample collections.
"We trained volunteers from conservation organizations to collect environmental DNA (eDNA) from 21 ponds with amphibian communities that had a history of Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) infections. Volunteers were given sampling kits to filter pond water and preserve eDNA on filter paper, as were the principal investigators (PIs), who made independent collections within 48 h of volunteer collections. Using multi-scale occupancy modeling, we found no evidence to suggest the observer who collected the water sample (volunteer or PI) influenced either the probability of capturing eDNA on a filter or the probability of detecting extracted eDNA in a quantitative PCR (qPCR) reaction. The cumulative detection probability of Bd eDNA at a pond decreased from May through July 2017 because there was a decrease in the probability of detecting eDNA in qPCR reactions. In contrast, cumulative detection probability increased from May to July for Rv due to a higher probability of capturing eDNA on filters later in the year. Our models estimate that both pathogens could be detected with 95% confidence in as few as 5 water samples taken in June or July tested with either 4 or 3 qPCR reactions, respectively. Our eDNA protocols appeared to detect pathogens with 95% confidence using considerably fewer samples than protocols which typically recommend sampling ≥30 individual animals. In addition, eDNA sampling could reduce some biosecurity concerns, jurisdictional and institutional permitting, and stress to biota at ponds."
eDNA for early detection of Bd
A study found eDNA can be used to detect Bd presence in sites with Bd-susceptible amphibians prior to die-offs occurring. This means it is a viable early detection tool.
"Amphibian chytridiomycosis caused by the fungus Batrachochytrium dendrobatidis (Bd) is an emerging infectious disease that has been associated with mass mortality and extinctions of amphibians worldwide. Environmental DNA (eDNA) techniques have been used to detect the presence of Bd in the environment, but not to detect Bd prior to an amphibian die-off. We collected eDNA using filtered water samples from 13 lakes across Sequoia Kings Canyon National Park. Seven of those sites had populations of mountain yellow-legged frogs, an amphibian highly susceptible to chytridiomycosis, and 3 of those populations experienced a Bd related die-off 1 mo post-eDNA sampling. We detected Bd in eDNA samples that were collected 1 mo prior to the observed Bd-caused die-off at all 3 sites affected by Bd, and we did not detect Bd at the other sites where no die-off was observed. Our study indicates the potential to use eDNA techniques for early detection of Bd in the environment."
** many protocols followed the work by "Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water" http://www.int-res.com/abstracts/dao/v77/n1/p11-15/
References
| (Author, year) | Link | Comment |
|---|---|---|
| James T. Julian, Gavin W. Glenney, Christopher Rees (2019) | https://www.int-res.com/abstracts/dao/v134/n1/p15-24 | "Evaluating observer bias and seasonal detection rates in amphibian pathogen eDNA collections by citizen scientists" — full text by loan exchange program |
| Colleen Kamoroff, Caren S. Goldberg, (2017) | http://www.int-res.com/abstracts/dao/v127/n1/p75-79/ | "Using environmental DNA for early detection of amphibian chytrid fungus Batrachochytrium dendrobatidis prior to a ranid die-off" — full text by loan exchange program |
| Benedikt R. Schmidt, Marc Kéry, Sylvain Ursenbacher, Oliver J. Hyman, James P. Collins (2013) | https://besjournals.onlinelibrary.wiley.com/doi/10.1111/2041-210X.12052 | "Site occupancy models in the analysis of environmental DNA presence/absence surveys: a case study of an emerging amphibian pathogen" |