DNA barcoding in diet assessment

A major benefit of using DNA barcoding in diet assessment is the ability to provide high taxonomic resolution of consumed species.^[10] Indeed, when compared to traditional morphological analysis, DNA barcoding enables a more reliable separation of closely related taxa reducing the observed bias.^[11][12] Moreover, DNA barcoding enables to detect soft and highly digested items, not recognisable through morphological identification.^[13] For example, Arachnids feed on pre-digested bodies of insects or other small animals and their stomach content is too decomposed and morphologically unrecognizable using traditional methods such as microscopy.^[14]

When investigating herbivores diet, DNA metabarcoding enables detection of highly digested plant items with a higher number of taxa identified compared to microhistology and macroscopic analysis.^[15][16] For instance, Nichols et al. (2016) highlighted the taxonomic precision of metabarcoding on rumen contents, with on average 90% of DNA-sequences being identified to genus or species level in comparison to 75% of plant fragments recognised with macroscopy. Moreover, another empirically tested advantage of metabarcoding compared to traditional time-consuming methods, involves higher cost efficiency.^[17] Finally, with its fine resolution, DNA barcoding represents a crucial tool in wildlife management to identify the feeding habits of endangered species and animals that can cause feeding damages to the environment.^[18]

With DNA barcoding it is not possible to retrieve information about sex or age of prey species, which can be crucial. This limitation can anyway be overcome with an additional step in the analysis by using microsatellite polymorphism and Y-chromosome amplification.^[19][20] Moreover, DNA provides detailed information of the most recent events (e.g. 24–48 hr) but it is not able to provide a longer dietary prospect unless a continuous sampling is conducted.^[21] Additionally, when using generic primers that amplify 'barcode' regions from a broad range of food species, the amplifiable host DNA may largely outnumber the presence of prey DNA, complicating prey detection. However, a strategy to prevent the host DNA amplification can be the addition of a predator-specific blocking primer.^[22][23][24] Indeed, blocking primers for suppressing amplification of predator DNA allows the amplification of the other vertebrate groups and produces amplicon mixes that are predominately food DNA.^[22][25]

Despite the improvement of diet assessment via DNA barcoding, secondary consumption (prey of the prey, parasites, etc.) still represents a confounding factor. In fact, some secondary prey may result in the analysis as primary prey items, introducing a bias. However, due to a much lower total biomass and to a higher level of degradation, DNA of secondary prey might represent only a minor part of sequences recovered compared to primary prey.^[26]

The quantitative interpretation of DNA barcoding results is not straightforward.^[12] There have been attempts to use the number of sequences recovered to estimate the abundance of prey species in diet contents (e.g. gut, faeces). For example, if the wolf ate more moose than wild boar, there should be more moose DNA in their gut, and thus, more moose sequences are recovered. Despite the evidence for general correlations between the sequence number and the biomass, actual evaluations of this method have been unsuccessful.^[5][27] This can be explained by the fact that tissues originally contain different densities of DNA and can be digested differently.^[28]