Curation of prey
To obtain datasets with putative
prey only, the assigned reads were subjected to a two-step curation
process in R studio (v. 4.1.3). First, the reads were manually filtered
based on taxonomy using ‘tidyverse’ functions (Wickham et al., 2019).
All Maxillopoda reads were discarded to remove consumer DNA. We
acknowledge that maxillopods may compose a food-source for the species
studied but the short read-length used to capture prey from partly
digested materials, did not allow for distinguishing DNA from maxillopod
prey and consumer. Taxa known to interact with copepods (any Copepoda)
in symbiosis (parasitism, commensalism and mutualism) were recorded from
current literature (Bass et al., 2021; Cleary et al., 2017; Cleary &
Durbin, 2016; Zamora-Terol et al., 2021) and used to discard likely
non-dietary interactions. By inspection, we discarded several zOTUs
assigned to unlikely prey including seed-plants, insects and mammals.
Putative contaminants from the marine environment were likewise
discarded, notably large gelatinous organisms (Cnidaria, Ctenophora). We
acknowledge that also these taxa may have a dietary origin, but we
consider it more plausible that most of the sequences originated from
the batch sample from which the copepods were picked. Gelatinous
organisms are sticky and fragile, and have been suspected of
contaminating other studies of copepods using similar methodologies
(Cleary et al., 2017). Decontam was used to identify and discard
remaining contaminants by comparing the prevalence of putative
contaminant zOTUs in real samples and extraction negatives (the
‘prevalence method’, Davis et al., (2018)). Relatively few contaminants
(2 and 38 for pilot and full datasets, respectively) were identified and
discarded at this stage. The remaining zOTUs were considered putative
prey, and were stored with metadata as phyloseq-objects (McMurdie &
Holmes, 2013).