Experimental feeding validates nanofluidic array technology for DNA detection of ungulate prey in wolf scats

The study of carnivores' diet is a key component to enhance knowledge
on the ecology of predators and their effect on prey populations. Although
molecular approaches to detect prey DNA in carnivore scats are improving,
the validation of their accuracy, a prerequisite for reliable applications
within ecological frameworks, is still lagging behind the methodological
advances. Indeed, variation in detection probability among prey species
can occur, representing a potentially insidious source of bias in
food-habit studies of carnivores. Calibration of DNA-based methods
involves the optimization of specificity and sensitivity and, whereas
priority is usually given to the former to avoid false positives,
sensitivity is rarely investigated so that false negatives may be largely
overlooked. We conducted feeding trials with captive wolves (Canis lupus)
to validate a nanofluidic array technology recently developed for
detection of multiple prey species in scats. Using 371 scat samples from
12 wolves fed with a single-prey diet, the sensitivity of our nanofluidic
array method varied between 0.45 and 0.95 for the six main ungulate prey
species. The method sensitivity was enhanced by using multiple markers per
species and by a relatively low threshold for the number of amplifying
markers required to confirm a detection. Yet, at least two markers should
be used to avoid false positives. By acknowledging sources of bias in
sensitivity to reliably interpret results of DNA-based dietary methods,
our study highlights the relevance of feeding experiments to optimally
calibrate the relative thresholds to define a positive detection and
investigate occurrence and extent of biases in sensitivity.