Study area
We conducted our study at Nopporo Forest Park in Hokkaido, Japan (43° 03′ N, 141° 32′ E) (Fig. 1). This forest is a semi-isolated 2,053 ha area surrounded by residential areas and farmland. There have been concerns about raccoon impacts on this ecosystem since they were first detected in 199234.
Animals
We captured raccoons using box traps (Havahart Large Collapsible Pro Cage Model 1089, Woodstream Corp., Lititz, PA, USA) from May to July in 2018 and April to August in 2019. Traps were placed at 86 sites (Fig. 1). Trapping points within 250 m of the forest boundary line facing the farmland were defined as ‘around the farmland’ (31 points), and other points were defined as ‘in the forest’ (55 points) using QGIS version 3.1023. Captured raccoons were anesthetised with butorphanol tartrate (Vetorphale 5 mg, 1.2 mg/kg; Meiji Seika, Tokyo, Japan), medetomidine hydrochloride (Dolbene, 40 µg/kg; Kyoritsu, Tokyo, Japan), and midazolam (Dormicum injection 10 mg, 0.2 mg/kg; Astellas, Tokyo, Japan) by intramuscular injection and euthanised by potassium chloride injection into the heart. We captured 48 raccoons (34 around the farmland, 14 in the forest) in 2018, and 27 (12 around the farmland, 15 in the forest) in 2019 (Fig. 1). We collected thigh muscle samples, rectal faeces, and gastric contents from captured raccoons. Samples were stored at − 20 °C until analysis.
DNA metabarcoding
Sample preparation
Rectal faeces and gastric contents that were dominated by plant materials and groomed hair were excluded from analyses. In total, 18 rectal faeces and six gastric content samples that contained animal materials or whose contents were unknown were selected. Rapidly digested food resources such as amphibians cannot be confirmed visually in gastrointestinal contents, and even DNA may not be detected. Therefore, multiple samples were pooled and analysed because the target DNA may not be detected otherwise. The rectal faeces and gastric contents were divided into five (S1–S5) and two groups (G1–G2), respectively, based on the capture site and date of raccoons (Table 1). Each group was mixed and stored at − 20 °C until DNA extraction. Subsequent DNA analyses were performed at Bioengineering Lab. Co., Ltd. (Kanagawa, Japan).
DNA extraction
Samples (about 100 mg each) were lyophilised using a VD-250R lyophiliser freeze dryer (TAITEC, Saitama, Japan) and ground using a ShakeMaster NEO homogeniser (Bio Medical Science, Tokyo, Japan). Crude DNA was extracted from each group; then, DNA was purified using the MPure-12 Automated Nucleic Acid Purification System (MP Biomedicals, California, USA) with a MPure Bacterial DNA Extraction Kit (MP Biomedicals).
Library preparation and sequencing
Each library was prepared using two-step tailed PCR. We used the gSalamander primer, which amplifies salamander and newt 12S rRNA, as a specific primer to detect Hokkaido salamander, and the gInsect primer, which amplifies arthropod 16S rRNA, to detect Japanese crayfish (Table 5). All primers were designed by Bioengineering Lab. Co., Ltd.
The first PCR amplified the target region using gSalamander and gInsect. These reactions were conducted in a final volume of 10 μl, comprising 2 μl DNA template, 0.5 μl each primer (10 μM), 0.1 μl Ex Taq HS (5 U/µl) (Takara Bio Inc., Shiga, Japan), 1.0 μl 10 × Ex Taq buffer, 0.8 μl dNTP mixture (2.5 mM), and 5.1 μl sterile distilled water. The PCR conditions were: first denaturation for 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C, and final extension for 5 min at 72 °C.
The second PCR used the first PCR products as the template with index primers (2ndF and 2ndR). These reactions were conducted in a final volume of 10 μl, as described for the first PCR. The PCR conditions were: first denaturation for 2 min at 94 °C, followed by 12 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, and final extension for 5 min at 72 °C. At each step, PCR products were purified using the Agencourt AMPure XP system (Beckman Coulter, Inc., California, USA). The library concentrations were measured with a Synergy H1 microplate reader (BioTek) and a QuantiFluor dsDNA System (Promega). Library quality was assessed using a Fragment Analyser (Advanced Analytical Technologies, Iowa, USA) with a dsDNA 915 Reagent Kit (Agilent, California, USA). Paired-end sequencing (2 × 300 bp) was conducted on the Illumina MiSeq platform (Illumina, California, USA).
Data analysis
Reads that began with a sequence that completely matched the primer used were extracted using the fastq_barcode_splitter tool in the FASTX-Toolkit; then, the primer sequence was trimmed. The reads were trimmed and filtered using the Sickle tool with a quality value of 20; then, trimmed and paired-end reads with fewer than 150 bases were discarded. The remaining reads were merged using the FLASH paired-end merge script35 under the following conditions: fragment length after merge, 300 bases; read fragment length, 230 bases; and minimum overlap length, 10 bases. The UCHIME2 algorithm within USEARCH was used to check all filtered sequences for chimeric sequences36. All sequences that were not judged to be chimeras were used for further analysis. The UPARSE algorithm within USEARCH was used for OTU creation and taxonomic assignments. The constructed OTUs were subjected to Basic Local Alignment Search Tool (BLASTN) searches. More than 100 reads and the top BLAST hit with a sequence identity of ≥ 97% were used to assign species (target length: about 300 bp) to each representative sequence37.
Additional analyses of COI region
S–2, S–3, G–1, and G–2 sample group DNA was successfully extracted using gSalamander or gInsect primers (Table 1) and analysed by PCR using COI and blocking primers for raccoon (Table 5). The library was prepared using two-step tailed PCR. The first PCR amplification using the primer set 1st-IntF and 1st-HCOmR was conducted in a final volume of 10 μl, comprising 2 μl DNA template, 5 μl of each primer (10 μM) (forward primer 0.5 µl, reverse primer 0.5 µl, blocking primer 4 µl), 0.08 μl Ex Taq HS (5 U/µl), 1.0 μl 10 × Ex Taq buffer, 0.8 μl dNTP mixture (2.5 mM), and 1.12 μl DDW. The PCR conditions were as follows: first denaturation for 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 15 s at 67 °C, and 30 s at 52 °C and 30 s at 72 °C, and final extension for 5 min at 72 °C. Subsequent methods were as described above, except for the FLASH paired-end merge script (fragment length after merge, 310 bases; read fragment length, 225 bases).
Stable isotope analysis
Stable isotope ratios of muscle tissue reflect the diet over the previous few weeks to one month38,39. We used the muscles of raccoons captured from April to August and assumed that the stable isotope ratios in raccoon muscle samples reflected their diet from March to July, i.e. late winter to early summer, in Hokkaido.
Raccoon muscle and potential prey item samples were dried at 60 °C for > 24 h and then ground with a mortar and pestle. Potential food items (such as amphibians and crustaceans) were collected from the forest. The raccoon muscle and potential prey item samples were rinsed with a 2:1 chloroform: methanol solution to remove lipids and then dried at 60 °C for at least 24 h40. Each sample (1.0–3.0 mg) was enclosed in a tin cup and combusted in an elemental analyser (Vario MICRO cube, Elementar Gmbh, Hanau, Germany) interfaced with an isotope ratio mass spectrometer (IsoPrime100, Elementar Gmbh). We determined the δ13C, δ15N, and δ34S values for each sample. The results are reported as parts per thousand of the isotopes relative to a standard. For δ13C, δ15N, and δ34S values, Vienna Pee Dee Belemnite, air, and Vienna Cañon Diablo Triolite were used as standards, respectively. We used L-alanine (Shoko Science Co., Ltd., Tokyo, Japan) and sulfanilamide (Elementar GmbH) as working standards. A working standard, sulfanilamide (δ34S value, − 1.92‰), was calibrated against IAEA (International Atomic Energy Agency, Vienna, Austria) silver sulfides, IAEA-S-1, IAEA-S-2, IAEA-S-3, and was used as a working standard for δ34S.
Statistical analysis
We performed two-way analysis of variance to examine the interactions between two independent variables, season (spring: April–June vs. summer: July–August) and capture site (around the farmland vs. in the forest), and their relationship with the dependent variables (stable isotope ratio; δ13C, δ15N, and δ34S). After examining interactions between two independent variables (season and capture site), a Mann–Whitney U test was conducted. Differences were considered statistically significant at P < 0.05.
We estimated the contribution of each food resource to the raccoon diet using IsoWeb22. This analysis was performed with the trophic enrichment factors of the δ13C, δ15N, and δ34S values set to 1.1‰, 3.4‰, and 0.5‰, respectively41,42.
SPSS Statistics 20.0 (IBM, Tokyo, Japan) and R (R Core Development Team, R Foundation for Statistical Computing, Vienna, Austria) were used for all statistical analyses.
Ethical approval
All procedures were conducted in accordance with the Guidelines for Animal Care and Use of Hokkaido University and were approved by the Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (Permit Number: 18-0001). Permission to capture raccoons was obtained from the Ministry of the Environment, as part of the feral raccoon control program.
Source: Ecology - nature.com
