DNA barcoding in diet assessment is the use of
DNA barcoding to analyse the
diet of organisms.[1][2] and further detect and describe their
trophic interactions.[3][4] This approach is based on the identification of consumed
species by characterization of
DNA present in dietary samples,[5] e.g. individual food remains, regurgitates, gut and fecal samples, homogenized body of the host organism, target of the diet study (for example with whole body of
insects[6]).
The
DNA sequencing approach to be adopted depends on the
diet breadth of the target consumer. For organisms feeding on one or only few species, traditional
Sanger sequencing techniques can be used. For
polyphagous species with diet items more difficult to identify, it is conceivable to determine all consumed species using
NGS methodology.[5]
The
barcodemarkers utilized for amplification will differ depending on the diet of the target organism. For
herbivore diets, the standard DNA barcode
loci will differ significantly depending on the plant
taxonomic level.[7] Therefore, for identifying
plant tissue at the taxonomic
family or
genus level, the markers
rbcL and
trn-L-intron are used, which differ from the loci
ITS2,
matK,
trnH-psbA (noncoding intergenic spacer) used to identify diet items to genus and
species level.[7] For animal prey, the most broadly used DNA barcode markers to identify diets are the mitochondrial cytochrome C oxydase (
COI) and cytochrome b (
cytb).[8] When the diet is broad and diverse, DNA
metabarcoding is used to identify most of the consumed items.[9]
Advantages
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. Morevoer, 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]
Challenges
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]
Examples
Mammals
Mammals diet is widely studied using
DNA barcoding and
metabarcoding. Some differences in the methodology can be observed depending on the feeding strategy of the target mammal species, i.e. whether it is
herbivore,
carnivore, or
omnivore.
For herbivore mammal species, DNA is usually extracted from faeces samples[29][16][30][31] or
rumen contents collected from road kills or animals killed during regular hunting.[15] Within DNA barcoding, the trnL approach can be used to identify plant species by using a very short but informative
fragment of
chloroplast DNA (P6 loop of the
chloroplast trnL (UAA) intron).[32] Potentially, this application is applicable to all herbivorous species feeding on
angiosperms and
gymnosperms[32] Alternatively to the trnL approach, the markers
rbcL,
ITS2,
matK,
trnH-psbA can be used to amplify plant species.
When studying small herbivores with a cryptic life style, such as
voles and
lemmings, DNA barcoding of ingested plants can be a crucial tool giving an accurate picture of food utilization.[16] Additionally, the fine resolution in plant identification obtained with DNA barcoding allows researchers to understand change in diet composition over time and variability among individuals, as observed in the
alpine chamois (Rupicapra rupicapra).[33] Between October and November, by analyzing the faeces composition via DNA barcoding, the alpine chamois showed a shift in diet preferences. Also, different diet categories were observed amongst individuals within each month.[33]
For carnivores, the use of
non-invasive approaches is crucial especially when dealing with elusive and
endangered species. Diet assessment through DNA barcoding of faeces can have a greater efficiency in prey species detection compared to traditional diet analysis, which mostly rely upon the morphological identification of undigested hard remains in the faeces.[23] Estimating the vertebrate diet diversity of the
leopard cat (Prionailurus bengalensis) in Pakistan, Shehzad et al. (2012) identified a total of 18 prey taxa using DNA barcoding on faeces. Eight distinct bird taxa were reported, while previous studies based on conventional methods did not identify any bird species in the leopard cat diet.[23] Another example is the use of DNA barcoding to identify soft remains of prey in the stomach contents of predators e.g.
grey seals (Halichoerus grypus) and
harbour porpoises (Phocoena phocoena).[34]
DNA metabarcoding is a game changer for the study of complex diets, such as for
omnivores predators, feeding on many different species with both plants and animal origin.[35][36] This methodology does not require knowledge about the food consumed by animals in the habitat they occupy.[35] In a study on
brown bear (Ursus arctos) diet, DNA metabarcoding allowed accurate reconstruction of a wide range of taxonomically different items present in faecal samples collected in the field.[35]
Birds
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Fish
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Arthropods
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adding to it. (September 2020)
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