A tale of two fluids: does storing specimens together in liquid preservative causeDNAcross‐contamination in molecular gut‐content studies?

Abstract

The study of food webs and trophic interactions increasingly relies on PCR-based molecular gutcontent analysis. However, this technique may be prone to error from contamination of minute quantities of DNA; i.e., simply storing specimens together in a liquid medium may lead to cross-contamination. In this study, we used PCR to determine the contamination rate when (1) specimens were stored together in 95% ethanol for various time periods, and (2) predators fall into ethylene glycol-filled pitfall traps where the dying predator may inadvertently consume prey DNA-contaminated liquid. We designed experiments and PCR primers to quantify the risk of contamination for both situations and found no contamination by storing specimens together in 95% ethanol. Furthermore, zero predators contained prey DNA in their gut contents from imbibing prey DNA-contaminated ethylene glycol. These results support the use of mass sampling techniques, like wet pitfall traps, for molecular gut-content analysis.

Insights into aphid prey consumption by ladybirds: Optimising field sampling methods and primer design for high throughput sequencing

“…Taking these findings together, they suggest that sticky trap-sampling likely gives more representative insights into ladybird diet by considerably reducing sampling bias towards the sampled local vegetation. A further advantage of sticky trap-sampling is the reduction in potential cross-contamination, reported to be problematic in methods allowing interactions of trapped insects in the sampling container, such as in combi traps, sweep-netting, beating or vacuum sampling [ 12 – 14 ]. Thus, there are some limitations for this method mainly imposed by trade-offs between sampling effectiveness, DNA recovery rate and sampling effort.…”

“…Furthermore, collecting large numbers of individual insect predators, from which prey DNA can be isolated and contamination avoided, is difficult. For example, widely-used approaches such as pitfall trapping, vacuum sampling or sweep-netting may ensure collection of insects in sufficient numbers and in satisfactory condition for DNA analysis [ 8 – 11 ], but these sampling methods can introduce cross-contamination through interaction of insects in the sampling containers [ 12 – 14 ]. Moreover, the resource-use patterns found in studies using such sampling methods are often prone to an “observer bias”, i.e.…”

Molecular detection of field predation among larvae of two ladybird beetles is partially predicted from laboratory experiments

“…gossypii . Sample cross-contamination during collection with aspirator might be avoided if the individuals are readily frozen then preserved in EtOH 58 , 59 . In addition, before extractions, all larvae were rinsed with 0.1% NaClO solution to avoid possible carry-on of surface contamination (similar to ref.…”

Overcoming the pitfalls of merging dietary metabarcoding into ecological networks

“…There are many aspects of best practice that can mitigate these issues. Appropriately sterile approaches to fieldwork that limit cross‐contamination, for example sterilisation of any tools and individual collection of samples, can reduce contamination prior to DNA extraction (Athey et al., 2017; King et al., 2012). Stringent use of both negative and positive controls throughout the experimental process, and implementation of additional safeguards like spatial separation of pre‐ and post‐PCR samples and oil sealing of reactions (e.g.…”