An application of PCR-RFLP species identification assay for environmental DNA detection

Water tank experiment

To characterize basic performance of our PCR-RFLP assay for eDNA detection, we firstly conducted model experiments by preparing eight different water tank culture setups with the following animals: (1) 40 Rana japonica tadpoles for 2 days, (2) 40 Rana japonica tadpoles for 5 days, (3) nine Rana japonica and one Rana ornativentris adults for 1 day, (4) one Rana japonica and nine Rana ornativentris adults for 1 day, (5) five Rana japonica and five Rana ornativentris adults for 1 day, (6) one Rana tagoi tagoi and nine Rana japonica adults for 1 day, (7) one Rana tagoi tagoi and nine Rana ornativentris adults for 1 day, and (8) negative control (to check the specificity of our method)—no animals for 1 day (but water was used previously for other frog species (Rhacophorus arboreus)). The tadpoles of Rana japonica were collected from a pond in Hiroshima University campus in Higashi-Hiroshima city (Hiroshima Prefecture) in April 2016 just before the experiment and were classified as growth stage 33 according to Tahara (1974) (equivalent to stage 37 in Gosner (1960)). The adult frogs of Rana japonica, Rana ornativentris, and Rana tagoi tagoi were collected from Higashi-Hiroshima City in 2014, Kita-Hiroshima-cho (Hiroshima Prefecture) in 2015, and Kisa, Miyoshi City (Hiroshima Prefecture) in 2011, respectively, and reared in the Amphibian Research Center, Hiroshima University, until the start of experiments. We transferred and kept animals in plastic tanks (450 × 650 ×150 mm) filled with 8 L aged tap water (water level about 30 mm which immersed half body of adults). The animals were not fed during the experiment and were held at room temperature around 18 °C under 12 h:12 h light-dark cycle. We collected a 500 mL water sample from each tank after the specific periods according to the treatments using DNA-free 500 mL bottles, and samples were immediately transferred to a −30 °C freezer. These experiments were conducted in April 2016 for tadpoles (tanks 1 and 2) and September 2016 for adults (tanks 3–8); all animals were returned to the original habitat or enclosures under healthy conditions soon after the experiments.

All procedures were approved by the Hiroshima University Animal Research Committee (Approval number: G17-4) and were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Hiroshima University Animal Research Committee.

Field survey and experiments

To check the performance and sensitivity of PCR-RFLP assay for eDNA from the field environment, we collected water samples from ponds and rivers at 28 sites (Fig. 1). For validation of applicability to monitoring studies in various spatial scales, we collected samples in three hierarchical levels: (A) regional samples covering 60 km from FNPP (sample no. 1–20), (B) local area samples from a single body of water in Hiroshima University campus, Higashi-Hiroshima (sample no. 21–26), and (C) microhabitat level samples from a single breeding site of Rana japonica in Etajima Island, Hiroshima Prefecture (N 34.27067, E 132.47679) (sample no. 27 and 28). We collected 500 mL by filling sodium hypochlorite-treated 500 mL bottles (i.e., DNA-free bottles). Field survey was conducted in spring and summer because the true Rana species are active in these seasons. Samples from Fukushima (A) were collected during two periods, May 12, 2015 to May 14, 2015 and August 11, 2015 to August 13, 2015. Samples from Hiroshima University (B) and Etajima (C) were collected on 11 April and 17 April in 2016, respectively. To prevent contamination by a field sampler, we began collecting at downstream sites, and moved upstream as subsequent samples were collected. Sampled water bottles were transported on ice in a cooling box to the laboratory and stored at −30 °C. The subsequent procedure of filtering water samples that were stored frozen was performed within 3 weeks.

In addition, we recorded the presence and absence of frogs and/or tadpoles by visual observation, exploring the vicinity of the field sampling sites. Basically, three or four people conducted field observation for more than 20 min at each site. For sampling in Fukushima, permission to enter the survey area was obtained from the local governments (Iitate Vilage (approval number: 2705-01 and 2707-0) and Namine Town (approval number: 755 and 1959)). Notification of the field survey was accepted by the Iwaki District Forest Office, and permits were obtained from private landowners as required for each site.

DNA extraction and PCRs

To avoid contamination, we followed a unidirectional lab flow, whereby we performed all PCR protocols, including preparation/addition of the standards and qPCR cycling, in two separate rooms (rooms 1 and 2, respectively). To prevent carry-over contamination, no equipment or samples were returned from room 2 to room 1. In both rooms, laboratory benches were decontaminated using commercial bleach.

The water samples stored at −30 °C were thawed and vacuum-filtered through glass microfiber filters (GF/F; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with 0.7 μm mesh size, which were used in other amphibian studies (Katano et al., 2017; Iwai, Yasumiba & Takahara, 2019). Filter funnels and tweezers used in the filtration treatment were sterilized with 10% commercial bleach (ca. 0.6% hypochlorous acid) for 5 min (i.e., sodium hypochlorite treatment), flushed with a large amount of tap water, and then rinsed with DNA-free distilled water between samples to avoid cross contamination. Filtering controls (i.e., 500 mL of distilled water) in laboratory experiment controls filtered on each day of sample filtration. The filter papers were wrapped in new aluminum foil (i.e., DNA-free), placed in plastic bags, and stored at −30 °C. The eDNA was extracted from the filters according to the methods of Uchii, Doi & Minamoto (2016), using a Salivette tube (Sarstedt, Nümbrecht, Germany) and a DNeasy Blood and Tissue Kit for DNA purification (Qiagen, Hilden, Germany). The filters were incubated by submersion in a mixed buffer (400 μL buffer AL and 40 μL Proteinase K; Qiagen) using a Salivette tube in a dry oven at 56 °C for 30 min. The tubes with filters were centrifuged at 5,000×g for 5 min at room temperature. Then, 220 μL of TE (Tris–EDTA) buffer (pH: 8.0; 10 mM Tris–HCl and one mM EDTA) was added to the filters, and tubes were centrifuged again at 5,000×g for 5 min. Buffer AL (200 μL) and 100% ethanol (600 μL) were then added to each filtrate and mixed by pipetting. The mixture was applied to a DNeasy Mini spin column and centrifuged at 6,000×g for 1 min. This step was repeated until the mixture was completely processed. We followed the manufacturer’s instructions for further steps, and eDNA was eluted from each sample solution with a final volume of 100 μL Buffer AE.

The eDNA samples were then used for PCR-RFLP assay to detect DNA from the three target Japanese brown frog species based on Igawa et al. (2015). This method utilizes species-specific restriction enzyme (SpeI and HphI) digestion sites in a partial nucleotide fragment of mitochondrial 16S rRNA amplified by PCR. However, the oligonucleotide primers used by Igawa et al. (2015) could amplify 16S rRNA fragments originating from other vertebrate species including humans which showed similar banding patterns. Therefore, we altered the primers to amplify a shorter region in which only the three target frog species have restriction sites. Specifically, we modified the method of Igawa et al. (2015) by changing the primers: F96 5′-GTCCAGCCTGCCCAGTGAYAAA-3′ and R19 5′-GTTGAACAAACGAACCATTGGT-3′. These new primers were designed from an internal region of the previous study and amplify shorter fragments (Rana japonica: 514–517 bp, Rana ornativentris: 516–517 bp, and Rana tagoi tagoi: 514–516 bp).

For higher efficiency of PCR amplification, we also modified the PCR protocol described by Igawa et al. (2015). In this study, PCR was conducted using KOD FX Neo (TOYOBO, Osaka, Japan). The 20 µL total volume of reaction solution included 10 µL of 2 × PCR Buffer for KOD FX Neo, 2 µL of 2 mM dNTPs, 10 pmole of each primer, 3 µL of eDNA solution and 0.4 U of KOD FX Neo. Thermal cycling was performed using two-step PCR cycling: 95 °C for 3 min followed by 35 cycles of 98 °C for 10 s and 65 °C for 30 s. Following PCR amplification, independent digestion using two restrictions enzymes (SpeI and HphI) and electrophoresis were conducted in the same manner as Igawa et al. (2015). Then, 5 µl of the original PCR product or 15 µl of each PCR product digested with the two restriction enzymes were electrophoresed on a 2% agarose gel for 30 min at 100 V and visualized. We performed the experiment several times to confirm the reproducibility.

For Rana japonica, the amplified fragments are expected to have different digestion patterns between the western (including Hiroshima) and northern part (including Fukushima) of the Japanese mainland (Igawa et al., 2015). In all Rana japonica populations, amplicons digested with SpeI result in 255 and 259 bp subfragments. However, after Hph1 digestion, Rana japonica frogs from the western part have no subfragments, while those from the northern part have 301 and 220 bp subfragments (Fig. 2A). For Rana ornativentris and Rana tagoi tagoi, amplified fragments are commonly digested only with HphI, resulting in 231, 199, and 86 bp subfragments and 313 and 200 bp subfragments, respectively (Fig. 2A). To confirm the specificity of eDNA detection by our PCR-RFLP method, PCR products of sample no. 1, 10, 11, 14, 17, 19, and 20 were directly sequenced using the same primers and BigDye Terminator ver 3.1 (Life Technologies, Carlsbad, CA, USA) after PEG precipitation. The obtained nucleotide sequences were annotated by NCBI blastn (https://blast.ncbi.nlm.nih.gov).

qPCR assay

To compare sensitivity between the methods, we conducted real-time qPCR assay for samples from Fukushima to compare the sensitivity of eDNA detection for Rana tagoi tagoi. At first, we defined regions to design the primers and TaqMan MGB probes for the qPCR assays for this species using PrimerExpress 3.0.1 (Thermo Fisher Scientific, Carlsbad, CA, USA), for amplification of 106 bp fragments: Rtag16SrRNA-F, 5′-AGAAGGAACTCGGCAAACCTT-3′; Rtag16SrRNA-R, 5′-CCGCGGCCGTTGAAT-3′; Rtag16SrRNA-Pr, 5′-[FAM]-CCAGCCTGCCCAGTG-[NFQ]-[MGB]-3′. To prove that the primers did not amplify other sympatric frog species, we confirmed no amplification when the primers were applied to DNA extracted from Rana japonica and Rana ornativentris tissues. However, in addition to Rana tagoi tagoi, three closely related species (i.e., Rana sakuraii, Rana tagoi okiensis, and Rana tagoi yakushimensis) were also detected in an in silico specificity screen, which was performed using Primer-BLAST, with the setting “nr” for database and “vertebrates” for organisms parameters (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Rana sakuraii may inhabit sympatrically with Rana tagoi tagoi in our field survey. In contrast, Rana tagoi okiensis and Rana tagoi yakushimensis never share distribution with our target species because they are only found on Oki island in Shimane Prefecture and the Yakushima islands in Kagoshima Prefecture, respectively. Thus, in this study, the qPCR primers we developed are specific for Rana tagoi tagoi/Rana sakuraii.

Environmental DNA was quantified using a StepOnePlus™ Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). Each TaqMan reaction contained 10 µL of TaqMan^® Environmental Master Mix 2.0 (Thermo Fisher Scientific, Carlsbad, CA, USA), 1 µL of the primer (900 nM)/probe (125 nM) mix, 7 µL of distilled water, and 2 µL of eDNA extract. The PCR cycles were as follows: 2 min at 50 °C, 10 min at 95 °C, then 55 cycles of 15 s at 95 °C, and 60 s at 60 °C. In order to produce standard DNA for the qPCR, a target amplicon was inserted into a pMD20-T vector (Takara Bio, Shiga, Japan), and the vector was digested with BamHI. A dilution series of the plasmid containing 1 × 10¹ to 1 × 10⁴ copies was amplified as standards in duplicates in all qPCR assays. The qPCR for each sample was performed in eight wells, and the mean was used as the concentration of eDNA (copies/L). If any of the eight replicates of each sample yielded a positive result, the sample was designated as containing Rana tagoi tagoi/Rana sakuraii eDNA. As a negative control, each qPCR assay included eight wells that contained no template (2 μL of DNA-free water) (i.e., no template control (NTC)). In addition, using the primer-probe set of this species, qPCR amplicons were sequenced directly from a positive PCR of the field samples (N = 6) after treatment with ExoSAP-IT (USB Corporation, Cleveland, OH, USA) or DNA fragment isolation with FastGene™ Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) following agarose gel electrophoresis. Products were sequenced by a commercial sequencing service (Takara Bio, Shiga, Japan) or by our experiment using ABI3130xl automated sequencer (Thermo Fisher Scientific, Carlsbad, CA, USA).

The range of qPCR efficiency across the entire study, calculated from the slope of standard curves, was 91.937–94.716%, and the R² value for standard curve was 0.997. Since we could detect two copies of DNA in at least one of the three replicates, we defined the limit of detection (LOD, the lowest concentration where all triplicate samples registered a Ct value) for DNA from Rana tagoi tagoi using qPCR assay as two copies.

Water tank experiments

For the eDNA solutions from water tank experiments, we successfully amplified 16S rRNA fragments from all samples, each showing a single band following gel electrophoresis (Fig. 2B). The band patterns of digested fragments in each sample were completely matched with the species that were held in each tank, except for the negative control tank without animals (Fig. 2B). Notably, for the samples from water tanks in which two species were cultured, we could simultaneously amplify eDNA from both species whereby both band patterns specific to two species were displayed.

Field survey and experiments

In the survey in Fukushima (A), field observation of Rana tagoi tagoi adults were recorded in 15 of 20 sites (Fig. 1; Table S1). Among the sites where Rana tagoi tagoi adults were observed, adults of Rana ornativentris were also observed sympatrically in five sites (Table S1). However, we could not identify presence of either Rana tagoi tagoi or Rana ornativentris by PCR-RFLP analyses of eDNA from any of the samples from Fukushima (Fig. S1). Subsequently, we applied the more sensitive Rana tagoi tagoi/Rana sakuraii-specific qPCR which could still only confirm presence of Rana tagoi tagoi in five of the 15 sites with field observations of adults of the species (Fig. 1; Table S1). Our direct sequences of the qPCR amplicons (106 bp product size) were almost identical to haplotypes of Rana tagoi tagoi deposited in DNA Data Bank of Japan (DDBJ). In addition, no amplification was observed in any control samples (i.e., filtering controls and NTC).

We could obtain clear sequence data of PCR products from site no. 1 and 21, while the others showed mixed chromatograms that could not be further interpreted. In Fukushima site no. 1, we also reported field observation of Bufo japonicus tadpoles, a species outside of our target species. PCR amplification of eDNA from the same site resulted in an approximately 600 bp fragment; subsequent sequencing and BLAST search confirmed a match of nucleotide sequence with partial sequence of B. japonicus (= B. j. formosus) 16S rRNA (AB159565, a haplotype from Atsumi, Yamagata Pref. (Igawa et al., 2006)).

From field experiment (B) and (C), field observation of Rana japonica tadpoles was recorded for three sites (no. 21, 22, and 27; Fig. 1; Table S1). This was consistent with our PCR-RFLP which showed Rana japonica-specific banding patterns following SpeI restriction digestion in the same samples (Fig. 2C). We also confirmed the sequence from site no. 21 had BLAST identity with partial sequence of 16s rRNA from Rana japonica (LC014155, a haplotype from Hiroshima University (Igawa et al., 2015)). In addition, PCR-RFLP analyses of site no. 28 supports the presence of Rana japonica, despite no field observation of tadpoles at this small drainage site. We also confirmed amplification of a fragment that may be amplified from non-target species in site no. 21, 22, 27, and 28. These fragments were digested with HphI and showed weak bands around 400 and/or 300 bp that were different from the target species.