title: Detection of predation using qPCR: effect of prey quantity, elapsed time, chaser diet, and sample preservation on detectable quantity of prey DNA.
abstract: Using quantitative PCR that amplified a prey-specific mtDNA 214 bp amplicon from the COI mitochondrial gene of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), prey eggs of known age and number were fed to larvae of the generalist predator lady beetle Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae), to elucidate the effects of time and diet since consumption, number of prey eggs, and methods for sample fixation and preservation, on the quantity of target DNA detected. Signal was strongly attenuated directly after cessation of feeding, even when predators were immediately frozen at -20 degrees C. However, the quantity of target detected was significantly related to the number of eggs consumed and the time elapsed since eating. Decrease in detected prey DNA was consistent with a negative exponential model. The target DNA sequence disappeared from starved predators (quantitative half-life estimate of 59 min) more slowly than those fed potato aphids after consuming the target prey eggs (half-life estimate 16 min), whereas those fed C. maculata eggs as a chaser were intermediate in the rate at which they degraded the target prey DNA sequence. Fixative protocols are of critical importance in proper use of the qPCR technique. Among seven methods tested, storing the predator immediately in 70% ethanol prechilled to -20 degrees C yielded the highest amount of target sequence, 22.8% of that recovered directly from a single intact prey egg. Samples frozen without solvent at -80 degrees C and -20 degrees C yielded only 6.0% and 2.3% of the target DNA respectively, and room temperature ethanol and ethylene glycol-based antifreeze averaged below 1% recovery of target DNA. Nevertheless, target prey was detected in more than 80% of antifreeze-stored predators. Predators killed and held at room temperature for 4 h or 5 days yielded no target prey DNA in 18 of 20 cases. These results emphasize both the value and the complexities of application of the qPCR technique to field predation studies.
authors.affiliation: USDA Agricultural Research Service, Invasive Insect Biocontrol and Behavior Laboratory, BARC-West 11 1A, Beltsville, MD 20705 USA. Don.Weber@ars.usda.gov
authors.forename: Donald C
authors.initials: DC
authors.lastname: Weber
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Lundgren
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000502:physiology*; D004247:DNA / Q000235:genetics*; D004032:Diet; D010063:Ovum; D016133:Polymerase Chain Reaction / Q000379:methods*; D011235:Predatory Behavior / Q000502:physiology*; D013997:Time Factors
chemical_list: D004247:DNA