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Title: Quantification of predation using qPCR: Effect of elapsed time, chaser diet, quantity of prey eggs, and sample preservation

Author
item Weber, Donald
item Lundgren, Jonathan

Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2008
Publication Date: 6/18/2009
Citation: Weber,D.C., and Lundgren, J.G., 2009. Detection of predation using qPCR: effect of prey quantity, elapsed time, chaser diet, and sample preservation on detectable quantity of prey DNA. Journal of Insect Science. 9(41). Available http://www.insectscience.org/9.41.

Interpretive Summary: Predatory insects such as ladybeetles eat many insect pests, non-pests, and non-insect foods. To find out how valuable a predator is as a beneficial biological control, entomologists want to know how many pest insects a predator eats in the field, but this can be difficult and time-consuming to observe directly. Entomologists are now using the molecular technique known as PCR, to determine whether a pest-specific DNA marker is present or not in the predator gut. We applied a more advanced quantitative PCR method (qPCR) to see if we could improve application of the PCR method to predation detection. The aim was to see what factors affect the amount of marker DNA (from the pest insect, Colorado potato beetle) detectable in the predator (pink-spotted lady beetle). Our results show that qPCR quantification of target prey DNA varies according to size of meal and time since the meal (as expected), but also according to what else the predator eats afterwards, and very importantly according to how the sample is preserved for analysis. This research will help scientists to develop qPCR as a superior technique to determine where and when specific predators are valuable for biological control of pests.

Technical Abstract: Using quantitative PCR with a prey-specific mtDNA 214 bp amplicon from COI and cds mitochondrial genes of Colorado potato beetle, we fed prey eggs of known age and number to larvae of the generalist coccinelid predator Coleomegilla maculata, to elucidate the effects of time and diet since consumption, number and age of prey eggs, and methods for sample fixation and preservation, on prey marker DNA quantity detected. Signal was strongly attenuated at t=0 minutes after cessation of feeding, and immediate freezing of the sample at -20C. However, marker quantity detected was significantly related to amount of prey (number of eggs) consumed, elapsed time, and chaser diet. Decrease in detected prey DNA was consistent with a negative binomial model; marker disappeared from starved predators more slowly than those fed potato aphids, whereas those fed C. maculata eggs were intermediate in prey marker degradation. We demonstrated that fixative protocols are of critical importance in proper use of the qPCR technique. Among 7 methods tested, 70% ethanol prechilled to -20C yielded the highest amount of marker, 22.8% of that recovered directly from a single intact prey egg. Samples frozen without solvent at -80C and -20C yielded only 6.0% and 2.3% respectively, and room temperature ethanol and ethylene glycol-based antifreeze averaged below 1% signal recovery, nevertheless with 80% or more positive detects each. Predators killed and held at room temperature for 4h or 5 days yielded no prey marker DNA in 18 of 20 cases. This is the first demonstration of a quantitative effect on ingested prey DNA of subsequent diet in a predator. It is also the first report to compare common protocols for fixation and preservation of arthropod predators on quantification of consumed prey DNA. Results emphasize both the value and the complexities for application of the qPCR technique to field predation studies. Using the same marker(s), a properly-executed qPCR analysis of predators will always yield more information than conventional PCR.