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

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.

One Bat’s Waste is Another Man’s Treasure: A DNA Metabarcoding Approach for the Assessment of Biodiversity and Ecosystem Services Using Bat Faeces

“…Here, we further explored the diet of a bat species, R. hipposideros, and described the range of arthropods found in its diet, with a particular focus on the effects of roost location and sex and explored the ecosystem services provided by the species in the form of pest species consumption. This and the earlier work by Browett et al 2021). In McAney and Fairley (1989), arthropods were only identified to family level, whereas here we have been able to identify arthropod species predated upon by R. hipposideros, something not normally achievable via hard-part analysis.…”

“…Using a combination of COI primers aids in maximising amplification and assessment of diversity within the diet. Extended details regarding PCR reaction mixes, multiplexing, thermocycling conditions, library preparation, sequencing, and bioinformatic steps required to generate Molecular Operational Taxonomic Units (MOTUs) are provided in Browett et al (2021).…”

“…DNA metabarcoding can be described as the simultaneous and parallel identification of multiple taxa using a standardised region of DNA. It is a useful technique not only to address questions related to the diet of a species, but it can also be used as an ecosystem approach to detect and track trophic interactions at spatio-temporal scales (Bohmann et al 2014) with significant developments in the analysis of mammalian diets being made over the last decade (Pompanon et al 2012;Shokralla et al 2014;Tournayre et al 2020;Browett et al 2020Browett et al , 2021Tournayre et al 2021).…”

“…A degree of functional flexibility was also evident within the trophic niche of R. euryale, as it consumed a wide range Lepidoptera which varied in their energy content throughout the season (Arrizabalaga-Escudero et al 2019). Browett et al (2021) optimised a dual primer approach for the DNA metabarcoding of bat diet using DNA previously extracted from non-invasively collected faeces, previously identified to species, sex and individual level using real-time PCR and microsatellite genotyping (Harrington 2018;Harrington et al 2019). This approach uses proven good quality and quantity DNA, and excludes low quality samples, and facilitates the inclusion of questions related to sex and individual level dietary preferences.…”

“…Browett et al (2021) are the first studies in Ireland or Great Britain to use a DNA metabarcoding approach to examine the diet of R. hipposideros.McAney and Fairley (1989) used traditional hard-part…”