Introduction
Understanding ecosystems require construction and modelling of complex
networks that represent various species interactions, and abiotic
factors. In such ecosystems, small invertebrates form important
components as consumers, prey, decomposers, pollinators and ecosystem
engineers, and are thus critical to include (Kellert, 1993), but large
and charismatic animals like birds and mammals have to date garnered the
bulk of trophic ecologists attention (Eisenhauer & Hines, 2021).
Traditionally, dietary studies have been conducted through direct
observation of feeding behavior, or morphological identification of prey
species in regurgitate, stomach or fecal contents (Pompanon et al.,
2012; Sousa et al., 2019; Symondson, 2002), but these approaches are
highly impractical for a large portion of invertebrates whose small
bodies complicate stomach content extraction and produce comparatively
small fecalia.
Microscopy has helped identify small prey from small consumers, but is
laborious and biased in favor of big, well-preserved prey (Berg, 1979),
and demands morphological and taxonomic expertise (Pompanon et al.,
2012). Conversely, experimental monitoring of communities over time can
detect trophic interactions, and even allow quantifying ingestion rates,
but struggles in reproducing the natural variability. The species
assortment would typically be limited to prey expected in advance, or
that co-occurred with the consumer if a natural sample was used as the
starting point. Prey metabarcoding has become popular because it allows
identification of diverse prey from complex and partly digested
material, and does not require considerable a priori knowledge of
prey, or taxonomical or morphological knowledge from the researcher
(Casper et al., 2007). In broad strokes, metabarcoding includes
extraction of DNA from dietary material – most often regurgitate, feces
or stomach content, PCR amplification of target DNA (the marker gene or
barcode), sequencing of PCR amplicons, and culminates with taxonomic
identification by comparing obtained sequences to those in a reference
database (Santoferrara, 2019).
Deciding on a dietary material is an important step that depends on the
logistics and ethics of sampling, or the nature of the species being
studied, such as its size or tendency for violence (Pompanon et al.,
2012). Medium-sized crustaceans like northern shrimp (Pandalus
borealis ) may be suitable for excision of stomach content (Urban et
al., 2022), but small invertebrates (< 1 mm) are challenging
to dissect, and may require whole body extraction (e.g. Novotny et al.,
2021; Zamora-Terol et al., 2020). This comes at a cost, however, because
the majority of DNA in the sample will naturally stem from the consumer
itself (Piñol et al., 2014, 2015). An overabundance of consumer DNA may
also be a challenge when other materials (e.g., feces or gut content)
are sampled (Kohn & Wayne, 1997), but its concentration becomes
severely exalted in extracts of whole bodies (Piñol et al., 2014).
Hence, the DNA of interest are in minority, while the unexciting
consumer DNA will compose a competitive majority.
Conceptually different approaches have been developed to enable prey
studies from such “mixed” DNA samples. Nowadays most popular approach
was spurred when Nielsen et al., (1991) researched synthetic analogs to
DNA. A polymer with peptide instead of a sugar-phosphate backbone showed
particular promise, because it formed stable hybrid duplexes with DNA
(Nielsen et al., 1991). So-called peptide nucleic acids (PNAs) had
higher melting temperatures than DNA (Egholm et al., 1993), went
unrecognized by DNA polymerases, and could not initiate amplification by
PCR (Orum et al., 1993). PNAs could be introduced prior to PCR to
hybridize irreversibly with a target sequence and thereby suppress its
amplification (Orum et al., 1993). Comparatively rare but interesting
sequences would thus be allowed to replicate to detectable abundances
(e.g. eukaryote parasites of blue crab, Troedsson et al., 2008). Other
variants of blocking primers have also been put to the test, such as
oligonucleotides modified with inhibitory C3 spacers (Deagle et al.,
2009; Vestheim & Jarman, 2008). Since then, blocking primers have been
used to study the prey of many different animals and with sample
material ranging from whole body extracts of copepods (Cleary et al.,
2016, 2017; Durbin & Casas, 2014; Novotny et al., 2021; Ray et al.,
2016; Zamora-Terol et al., 2020), to dragonflies and apex canine
predators (Morrill et al., 2021; Shi et al., 2021).
Although blocking primers have enabled many prey studies there are
issues that warrant attention. Like universal PCR primers typically used
in metabarcoding, blocking primers can introduce bias during
amplification (Elbrecht & Leese, 2015; Leray & Knowlton, 2017; Piñol
et al., 2015). With universal primers, primer-template mismatches and
stochasticity may result in skewed relative abundances of species of
interest (Sipos et al., 2007). Blocking primer bias also relates to
primer-template mismatches, but it is rather the lack of them that leads
to unreliable results. A blocking primer should have zero mismatches
with the consumer, and as many as possible mismatches with prey to limit
off-target interactions. Piñol et al., (2015) showed that a blocking
primer with four and five mismatches to interesting prey decreased their
relative abundances. Hence, PNAs and blocking oligonucleotides may
introduce strong taxonomic biases during amplification (Piñol et al.,
2015, 2019). Furthermore, production of specific and ultimately
successful blocking primers remains an expensive chemical procedure, and
relies on both consumer and prey sequences before the design can begin.
We have explored a simple, cost-efficient, and versatile approach to
prey metabarcoding of small invertebrate consumers. By sequencing deep
to offset for the overabundance of consumer DNA, we show that one can
obtain ample prey reads from mixed whole-body copepod extracts, while at
the same time avoiding the costs and laborious design of potentially
biased blocking primers. Conceptually, we argue that the brute force
method holds a lot of promise because current sequencing and computation
enable acquiring and processing large amounts of data, and continuous
development will only improve these capabilities (Lightbody et al.,
2019). Through two sequencing runs (a pilot and a full-scale) using two
commercially available NGS platforms (Illumina HiSeq4000 and Illumina
NovaSeq6000), we tested the brute force approach for the first time with
marine invertebrates. We report on the effect of sequencing depth for
resolving prey composition and discuss advantages and caveats of the
brute force methodology for prey studies of small consumers.