Bats are sentinels for invasive pest surveillance based on DNA metabarcoding

Insectivorous bats have been identified as important agents for biological pest control in the agroecosystems and impose strong top-down pressure on pests (Ramirez-Francel et al., 2022). Without bats, it is estimated that the United States would spend more than $3 billion a year on pesticides alone (Boyles et al., 2011). Thus, bats are expected to be natural pest collectors (Kunz, 2011). However, it is still unproven assumption that bats can be used to efficiently monitor invasive pests in agroecosystems. Due to the wide distribution of bats, their high foraging efficiency, large activity range, and because most bats return to the same habitat and cluster in roosts together after foraging, collecting fecal samples is feasible and convenient. In our preliminary experiments, multiple bat species were found to normally consume FAW, which provides an excellent opportunity for verifying the practicability of DNA metabarcoding in monitoring the invasion of this pest.

Methods
DNA was extracted from three to five faeces (dry weight 15-20 mg) per bat using the QIAamp DNA Stool Mini Kit (Qiagen, UK) principally following the manufacturer’s protocol (2016 version). Extracts were amplified in three replicates using two primer pairs, referred to as LCO (LCO‐1490: 5′‐GGTCAACAAATCATAAAGATATTGG‐3′; ZBJ‐ArtR2c: WACTAATCAATTWCCAAATCCTCC) (Chang et al., 2019; Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994) and Epp (Coleop_16Sc: TGCAAAGGTAGCATAATMATTAG; Coleop_16Sd: TCCATAGGGTCTTCTCGTC) (Epp et al., 2012). The primer sets are hereafter referred to as LCO and Epp primers, respectively. The products of three separate PCR replicates were mixed and extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) and quantified using QuantiFluor-ST (Promega) according to the protocol of manufacturer. During extraction, PCR and library preparation steps, two negative controls that reactions with no DNA were included to assess the incidence of cross‐-contamination (Alberdi et al., 2019). The DNA samples were Paired-end sequencing on an Illumina MiSeq PE300 platform (Illumine Inc., San Deigo, CA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).