From feces to data: A metabarcoding method for analyzing consumed and available prey in a bird‐insect food web

Abstract

Diets play a key role in understanding trophic interactions. Knowing the actual structure of food webs contributes greatly to our understanding of biodiversity and ecosystem functioning. The research of prey preferences of different predators requires knowledge not only of the prey consumed, but also of what is available. In this study, we applied DNA metabarcoding to analyze the diet of 4 bird species (willow tits Poecile montanus, Siberian tits Poecile cinctus, great tits Parus major and blue tits Cyanistes caeruleus) by using the feces of nestlings. The availability of their assumed prey (Lepidoptera) was determined from feces of larvae (frass) collected from the main foraging habitat, birch (Betula spp.) canopy. We identified 53 prey species from the nestling feces, of which 11 (21%) were also detected from the frass samples (eight lepidopterans). Approximately 80% of identified prey species in the nestling feces represented lepidopterans, which is in line with the earlier studies on the parids' diet. A subsequent laboratory experiment showed a threshold for fecal sample size and the barcoding success, suggesting that the smallest frass samples do not contain enough larval DNA to be detected by high‐throughput sequencing. To summarize, we apply metabarcoding for the first time in a combined approach to identify available prey (through frass) and consumed prey (via nestling feces), expanding the scope and precision for future dietary studies on insectivorous birds.

Assessing insect biodiversity with automatic light traps in Brazil: Pearls and pitfalls of metabarcoding samples in preservative ethanol

“…Failure in PCR amplification can be due to several causes, from sample preservation and DNA quality to primer design and thermocycler parameters (Taberlet et al, ). Although a number of DNA extraction approaches are available for environmental samples such as soil (Dopheide, Xie, Buckley, Drummond, & Newcom, ), feces (Rytkönen et al, ), and water (Brannock & Halanych, ), little attention has been given to preservative ethanol. Excepting, Shokralla et al () that successfully amplified COI fragments from a single Lepidoptera larva directly from a preservative medium containing 95% ethanol but also mescal solution, followed by first generation sequencing (but see Ritter, Häggqvist, et al, for a recent method).…”

“…The OTUs obtained from the negative controls were excluded from the analyses. (a) Data set including OTUs taxonomically assigned at a ≥97% similarity level excluding singletons, doubletons, and tripletons (SDTs); (b) the same data set using a ≥98% similarity level; (c and d) data set using OTUs assigned at a ≥97% similarity level and excluding SDTs; (e) the same data set including SDTs; (f) normalized data set using OTUs assigned at a ≥97% similarity level, and excluding SDTs, obtained after standardizing the number of sequences in the subsamples to 15,840 sequences (see Section 2 for details) as soil (Dopheide, Xie, Buckley, Drummond, & Newcom, 2019), feces (Rytkönen et al, 2018), and water (Brannock & Halanych, 2015), little attention has been given to preservative ethanol. Excepting, Shokralla et al (2010) The total number of OTUs evidences that the light traps used in our field survey sampled highly diverse biological communities, nonetheless approximately 82% of the OTUs remained taxonomically unassigned (Table1, Figure 2).…”

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

“…This approach allows the simultaneous identification of multiple taxa in environmental samples, bypassing the need to isolate organisms prior to identification (Clare, 2014; Taberlet, Coissac, Hajibabaei, & Rieseberg, 2012). eDNA metabarcoding is of particular interest in dietary analysis of rare or elusive species, and this approach has now been applied to a large spectrum of organisms (Clare, Fraser, Braid, Fenton, & Hebert, 2009; Corse et al., 2017; Kartzinel & Pringle, 2015; Rytkönen et al., 2019; Shehzad et al., 2012). Compared to the traditional microscopic study of undigested fragments in fecal remains, eDNA metabarcoding has three key advantages for dietary analysis: (a) a finer resolution (potentially to the species level), (b) the simultaneous processing and sequencing of several hundred samples, and (c) the detection of species that could not be detected using previous techniques such as visual recognition of morphological features (e.g., soft‐bodied species; Galan et al., 2018; Nielsen, Clare, Hayden, Brett, & Kratina, 2018).…”

Threats from the air: Damselfly predation on diverse prey taxa

“…Recently, DNA‐barcoding techniques have opened new ways for studying which prey species–predatory species are eating in the wild (Vesterinen, Lilley, Laine, & Wahlberg, ; Vesterinen et al, ). Furthermore, these new DNA‐based tools for diet analysis offer scope for identifying the full diet of focal predators (Eitzinger et al, ; Kaunisto, Roslin, Saaksjarvi, & Vesterinen, ; Rytkönen et al, ; Vesterinen, Puisto, Blomberg, & Lilley, ). Now that we can identify the taxonomic distribution of prey species, we can begin to assess which species and species assemblages are likely affected by predation.…”