Primer biases in the molecular assessment of diet in multiple insectivorous mammals

Samuel S Browett,Rachael E Antwis,Denise B O’Meara,Allan D Mcdevitt,Thomas G Curran,Andrew P Harrington,David O’Neill,Naiara Guimarães Sales

doi:10.1007/s42991-021-00115-4

Abstract

Our understanding of trophic interactions of small insectivorous mammals has been drastically improved with the advent of DNA metabarcoding. The technique has continued to be optimised over the years, with primer choice repeatedly being a vital factor for dietary inferences. However, the majority of dietary studies examining the effect of primer choice often rely on in silico analyses or comparing between species that occupy an identical niche type. Here, we apply DNA metabarcoding to empirically compare the prey detection capabilities of two widely used primer sets when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus hipposideros) and two ground-dwelling insectivores (greater white-toothed shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R. hipposideros primarily rely on two prey orders (Lepidoptera and Diptera), the unique taxa detected by each primer shows that a combination of primers may be the best approach to fully describe bat trophic ecology. However, random forest classifier analysis suggests that one highly degenerate primer set detected the majority of both shrews’ diet despite higher levels of host amplification. The wide range of prey consumed by ground-dwelling insectivores can therefore be accurately documented from using a single broad-range primer set, which can decrease cost and labour. The results presented here show that dietary inferences will differ depending on the primer or primer combination used for insectivores occupying different niches (i.e., hunting in the air or ground) and demonstrate the importance of performing empirical pilot studies for novel study systems.

Highlights

In a constantly changing environment, knowledge of complex food webs is vital for our understanding of ecosystem functioning and biodiversity conservation
The advent of Next-Generation Sequencing (NGS) technology has revolutionised the analyses of trophic interactions (Deagle et al 2019; Browett et al 2020), with DNA metabarcoding of faecal samples or gut contents becoming widely adopted for describing diets (Pompanon et al 2012)
The MiSeq sequencing run produced 18,527,116 sequence reads; 48.4% associated with bat samples and 49.5% associated with shrew samples

Summary

Introduction

In a constantly changing environment, knowledge of complex food webs is vital for our understanding of ecosystem functioning and biodiversity conservation. To capture the expected wide range of invertebrate taxonomic groups, highly degenerative (nonspecific) primers can be used, but studies comparing their efficiency have largely been restricted to analyses performed in silico (Piñol et al 2018) or using bulk samples and/or mock communities (Elbrecht et al 2019). While these are essential steps in primer design and have led to the ability to detect a wide range of invertebrate species, they may not account for some of the potential biases within a dietary context (i.e., predator/host amplification; Zeale et al 2011). The broader the taxonomic range of the primers, the more likely the chance of amplifying non-target taxa and reducing the amount of information on a species diet

Methods

Results

Conclusion