Removing external DNA contamination from arthropod predators destined for molecular gut-content analysis

Ecological research requires large samples for statistical validity, typically hundreds or thousands of individuals, which are most efficiently gathered by mass-collecting techniques. For the study of interspecific interactions, molecular gut-content analysis enables detection of arthropod predation with minimal disruption of community interactions. Field experiments have demonstrated that standard mass-collection methods, such as sweep netting, vacuum sampling and foliage beating, sometimes lead to contamination of predators with nontarget DNA, thereby compromising resultant gut-content data. We deliberately contaminated immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of Leptinotarsa decemlineata by topically applying homogenate of the alternate prey Leptinotarsa juncta. We then attempted to remove contaminating DNA by washing in ethanol or bleach. A 40-min wash with end-over-end rotation in 80% EtOH did not reliably reduce external DNA contamination. Identical treatment with 2.5% commercial bleach removed most externally contaminating DNA without affecting the detectability of the target prey DNA in the gut. Use of this bleaching protocol, perhaps with minor modifications tailored to different predator-prey systems, should reliably eliminate external DNA contamination, thereby alleviating concerns about this possible source of cross-contamination for mass-collected arthropod predators destined for molecular gut-content analysis.     

Unnecessary roughness? Testing the hypothesis that predators destined for molecular gut‐content analysis must be hand‐collected to avoid cross‐contamination

Molecular gut‐content analysis enables detection of arthropod predation with minimal disruption of ecosystem processes. Mass‐collection methods, such as sweep‐netting, vacuum sampling and foliage beating, could lead to regurgitation or rupturing of predators along with uneaten prey, thereby contaminating specimens and compromising resultant gut‐content data. Proponents of this ‘cross‐contamination hypothesis’ advocate hand‐collection as the best way to avoid cross‐contamination. However, hand‐collection is inefficient when large samples are needed, as with most ecological research. We tested the cross‐contamination hypothesis by setting out onto potato plants immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of either Leptinotarsa decemlineata or Leptinotarsa juncta, or unfed individuals of these predator species along with L. decemlineata larvae. The animals were then immediately re‐collected, either by knocking them vigorously off the plants onto a beat cloth and capturing them en masse with an aspirator (‘rough’ treatment) or by hand‐searching and collection with a brush (‘best practice’). Collected predators were transferred in the field to individual vials of chilled ethanol and subsequently assayed by PCR for fragments of cytochrome oxidase I of L. decemlineata and L. juncta. Ten to 39 per cent of re‐collected fed predators tested positive by PCR for DNA of both Leptinotarsa species, and 14–38% of re‐collected unfed predators contained L. decemlineata DNA. Overall levels of cross‐contamination in the rough (31%) and best‐practice (11%) samples were statistically different and supported the cross‐contamination hypothesis. A pilot study on eliminating external DNA contamination with bleach prior to DNA extraction and amplification gave promising results.     

The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?

Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed “the predator problem”. We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.