Metabarcoding for the parallel identification of several hundred predators and their preys: application to bat species diet analysis

Abstract

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two-step PCR protocol enabling the ‘all at once’ taxonomic identification of bats and their arthropod preys for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity, and amplification biases. Our parallel identification strategy of predators and preys reduces the risk of mis-assigning preys to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost-effective screening tool for addressing evolutionary ecological issues or developing ‘chirosurveillance’ and conservation strategies.

One‐locus‐several‐primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies

“…Lepidoptera were the primary prey while Diptera, Araneae, and Coleoptera constituted secondary prey. This pattern is similar to the prey composition estimated by Galan et al () for a similar set of bat species.…”

“…However, the high variability that makes the COI a powerful marker consequently makes truly universal primer sets hard to design. Yet, several recent attempts, that involved highly degenerated primers, appeared very promising (Elbrecht & Leese, 2017a;Vamos et al, 2017;Galan et al, 2018; see also Figure 1), and comparative in vivo tests (sensu Alberdi et al, 2018) would be desirable to confirm the broad applicability of these highly degenerated primers for exhaustively recovering the biodiversity in environmental samples.…”

“…Although false negatives often occur for rare taxa (e.g., Ficetola et al, 2015), they are also produced when the affinity between primer and primer-binding sites during polymerase chain reaction (PCR) is low (e.g., Elbrecht & Leese, 2017a;Vamos, Elbrecht, & Leese, 2017), which in turn will determine amplification success and hence the taxonomic coverage of a given primer set. Although there is growing use of control samples to assess levels of false positives (De Barba et al, 2014;Beng et al, 2016;Corse et al, 2017;Galan et al, 2018), no post hoc bioinformatic procedures can identify false negatives. Two primary strategies have been adopted to maximize the taxonomic coverage of primer sets used in metabarcoding studies.…”

Bats as potential suppressors of multiple agricultural pests: A case study from Madagascar

“…The DNA metabarcoding of bat faecal pellets can offer valuable insights into the dietary preferences of bats and their potential role as pest suppressors (Bohmann et al, 2014;Swift et al, 2018). Recent diet analyses of multiple bat species have detected a wide range of arthropods in bat populations (Galan et al, 2017) including several economically important pest species (Taylor et al, 2017).…”

First isolation and genetic characterization of Puumala orthohantavirus strains from France

“…300ng of viral RNA from supernatant of Vero cells and 1500ng from lung tissue of infected rodents were reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. The reverse transcription reaction was performed with 2µM and with 50ng of random hexamers provided in the kit.The sequencing libraries were prepared using a two-step PCR strategy adapted from Galan et al 2018(14) (see supporting information S2) and combined with a unique dual indexes (UDI) multiplex sequencing approach (15): each 9-bp i5 and i7 dual index were used only for one PCR sample without combinatorial indexing, to make sure that libraries were sequence and demultiplex with the highest accuracy without problem of "leak" between libraries(16). The libraries were sequenced on a MiSeq platform (GenSeq, Montpellier, France) with a 500-cycle reagent kit v2 (Illumina).…”