DOI: 10.5061/dryad.jh9w0vtj8
title: Data from: Optimizing detectability of the endangered fan mussel using eDNA and ddPCR

description: Spatial and temporal monitoring of species threatened with extinction is
 of critical importance for conservation and ecosystem management. On the
 Mediterranean coast, the fan mussel (Pinna nobilis) is listed as
 critically endangered after suffering from a mass mortality event since
 2016, leading to 100% mortality in most marine populations. Conventional
 monitoring for this macroinvertebrate is done using scuba, which is
 challenging in dense meadows or with low visibility. Here we developed an
 environmental DNA assay targeting the fan mussel and assessed the
 influence of several environmental parameters on the species detectability
 in situ. We developed and tested an eDNA molecular marker and collected 48
 water samples in two sites at the Thau lagoon (France) with distinct fan
 mussel density, depths, and during two seasons (summer and autumn). Our
 marker can amplify fan mussel DNA but lacks specificity since it also
 amplifies a conspecific species (Pinna rudis). We successfully amplified
 fan mussel DNA from in situ samples with 46 positive samples (out of 48)
 using ddPCR, although the DNA concentrations measured were low over almost
 all samples. Deeper sampling depth slightly increased DNA concentrations,
 but no seasonal effect was found. We highlight a putative spawning event
 on a single summer day with much higher DNA concentration compared to all
 other samples. We present an eDNA molecular assay able to detect the
 endangered fan mussel and provide guidelines to optimize the sampling
 protocol to maximize detectability. Effective and non-invasive monitoring
 tools for endangered species are promising to monitor remaining
 populations and have the potential for ecological restoration or habitat
 recolonisation following a mass mortality event.
description: Assay development 
 Reference sequences on the mitochondrial genome were downloaded
 for <em>P.nobilis</em> and co-occurring related species of the
 same family (<em>Pinna rudis</em> and <em>Atrina
 fragilis</em>) from EMBL (Kanz et al. 2005), and aligned using
 Geneious Prime 2020 (https://www.geneious.com/). Primer selection was done by maximizing specificity on the binding sites for the target species while maximizing the number of mismatches of ligation sites of closely related species. Primers were designed manually with the assistance of the primer3 algorithm on Geneious and amplified a sequence insert of ~202 bp on the mitochondrial COI gene for <em>P.nobilis (Supl Mat Table S1)</em>, the full amplified sequence being ~243 pb. The selected primer pair (PN_COI_M15; forward-TCAGCTTTTGTAGAGGGCGG; reverse- AGAGACTACCAACAGCACAGC) was also tested on the entire NCBI database using in-silico PCR with the ecoPCR software (Boyer et al., 2016) allowing up to 3 mismatches on each primer (so 6 in total), to verify the absence of unrelated species cross-amplification. Additionally, a probe (PN_COIM15-Probe; FAM-5’ TGGATTTGTTCCCTTGGGCTGTTC 3’- BHQ1) was designed to enhance specificity using the Primer3Plus software (Untergasser et al. 2012). Sampling for eDNA DNA sampling aimed to first collect live fan mussel from an aquarium and then sample water in real field condition in a Mediterranean lagoon with known populations of fan mussel. We sampled water in the field with known presence and densities of fan mussels in the Thau lagoon (Sète, France) (Foulquie et al. 2020), one of the last known locations to harbor healthy mussel populations in France. Sampling sites were chosen from the study of Foulquie et al. (2020) which assessed fan mussel densities in several sites around the lagoon 3 months prior to our sampling. We selected sites with at least 3m of depth and varying densities: the Barrou (~9 ind/100m<sup>2</sup>) and the Sete Canal (~4 ind/100m<sup>2</sup>) (Fig. 1). Maximum depth of those sites was ~2.5m. We filtered water from a boat using the same pump and settings as for the aquaria samples but made linear transects of ~300m over the site area, for a total of 30L per sample. Transects were made at low speed (5 knots) and by going back and forth to remain in the area, with one pump on each side of the boat and using disposable tubing and gloves. Surface samples were done at ~0.5m of the surface with short tubes, and deeper samples were done to target the benthos using 3m-long weighted tubes. We chose to sample seasonally during Summer and Autumn to encompass various environmental conditions and test a potential effect of the reproduction period, known to occur during the summer months. We collected a total of 48 samples, with 24 samples over two days in summer in July (2020-07-27 and 2020-07-30), and 24 samples over two days in autumn in October (2020-10-20 and 2020-10-21). Each day, 12 samples were collected spanning both sites and two sampling depths (bottom and surface), so that three replicates were obtained for each site-depth combination. eDNA extraction and amplification by ddPCR <strong>DNA extraction</strong> DNA extraction was performed at SPYGEN (Le Bourget du Lac, France) following the protocol described in Polanco Fernández et al., (2020), in a dedicated lab for eDNA extraction with UV treatment and positive air pressure. Briefly, each capsule was agitated for 15 min on an S50 shaker (cat Ingenieurbüro™) at 800 rpm. The buffer was then emptied into two 50-ml tubes before being centrifuged for 15 min at 15,000 g. The supernatant was removed with a sterile pipette, leaving 15 ml of liquid at the bottom of each tube. Then, 33 ml of ethanol and 1.5 ml of 3 M sodium acetate were added to each 50-ml tube and stored for at least one night at −20°C. The tubes were centrifuged at 15,000 g for 15 min at 6°C, and the supernatants were discarded. After this step, 720 μl of ATL buffer from Qiagen Blood and Tissue Kit(Qiagen GmbH) was added to each tube. Each tube was then vortexed, and the supernatant was transferred to a 2-ml tube containing 20 μl of Proteinase K. The tubes were finally incubated at 56°C for 2 hr. Subsequently, DNA extraction was performed using NucleoSpin® Soil (MACHEREY-NAGEL GmbH & Co.) starting from step 6 and following the manufacturer's instructions, and two DNA extractions were carried out per filtration capsule. The elution was performed by adding 100 μl of SE buffer twice. The two DNA samples were pooled before the amplification step. After the DNA extraction, the samples were tested for inhibition by qPCR (Biggs et al. 2015). If the sample was considered inhibited, it was diluted 5-fold before amplification. <strong>Amplification with ddPCR</strong> ddPCRs were run with a BioRad QX200 Droplet Digital PCR system™ (Bio-Rad, Temse, Belgium). Each 22 μl ddPCR reaction mix contained 1× Bio-Rad ddPCR supermix for probes (no dUTP), 900 nM forward primer, 900 nM reverse primer, 250 nM probe, 2,5 μl template, and 3,99 μl H20. ddPCR reaction was placed in a QX200 Droplet Generator to generate approximately 20000 droplets in which independent PCR reactions occur.PCR was performed with the following thermal conditions: 95°C for 10 min followed by 40 cycles of 95°C for 30s and 58°C for 1 min; and 98°C for 10 min and 4°C for 30 min. Optimal annealing temperature (58°C) was determined based on an initial thermal gradient experiment testing temperatures from 54 to 64°C. Droplets were then read on a QX200 droplet reader (Bio-Rad). Each run included 3 PCR positive and 3 PCR negative controls and samples were tested in triplicate (N=3). QuantaSoft software was used to count the PCR-positive and PCR-negative droplets and to provide absolute quantification of target DNA. The baseline threshold for separating positive and negative droplets was manually chosen per run, based on the distribution of the negative droplets from the negative control wells. The quantification measurements of each target were expressed as the copies number per 1 μl of reaction. Analysis Amplification results from ddPCR were analyzed both considering the number of positive replicates among the 3 replicates per sample and quantitatively using the number of copies per µL. Repeatability by sample between the PCR replicates was assessed with the R package rptR (Stoffel et al. 2017). The average number of copies per µL measured with ddPCR were related to site, depth and season using a General Linear Model (GLM). We used a Poisson distribution to model the average number of copies per µL among the 3 replicates for each sample. We added a dummy variable representing a putative reproduction event on a particular summer sampling day.
description: # Optimizing detectability of the endangered fan mussel using eDNA and
 ddPCR This repository contains data and code to reproduce the analyses and
 produce the figures from Marques et al. (2023) Optimizing detectability of
 the endangered fan mussel using eDNA and ddPCR ## Content ### Data
 Contains all data needed to reproduce analyses and figures * ?
 **data/data_pinna_clean.csv**: contains field and ddPCR data for all
 samples considered in the analysis Spygen_ID: is the individual code for
 the eDNA sample Site: site name Depth: Depth of sampling [m] Replicat:
 Replicat of sample ncopies.ddPCR[1-3]: number of copies detected for each
 of the three replicates of ddPCR [copies/uL] date_clean: date in format
 DD.MM.YYYY Month: Month number season: Season Sampling: Sampling period
 (either 1 or 2, for summer or autumn) ddPCR_count_sum: number of
 detections over the three ddPCR replicates * ? **data/v_COI_M15.ecopcr**:
 contains the output of an *in silico* PCR done with ecoPCR on the EMBL
 database, allowing up to 3 mismatches per primer. This contains all
 sequences theoretically amplified by the primer par COI_M_15. * ?
 **data/ncbi/**: contains NCBI taxonomic information ### Scripts * ?
 *make.R*: generates all figures and analyses from the scripts in
 *scripts/* * *eval_primers.R* uses the in silico PCR done on EMBL to
 assess the theoretical specificity of the designed assay *
 *plots_description.R* create figures to describe the dataset, to correlate
 the number of DNA copies detected with site and season * *model.R* uses
 GLMs to link environmental and technical variables to the ddPCR detections
 * *repeatability.R* execute the ddPCR repeatability analysis using the
 **rptR** package among ddPCR replicates ## Links Paper: [Optimizing
 detectability of the endangered fan mussel using eDNA and
 ddPCR](doi.org/10.1002/ece3.10807)