Diet, predator, and molecular marker properties influence dietary DNA detection in ladybird beetles (Coleoptera: Coccinellidae) during digestion.

DNA-based gut content analysis is a powerful tool for quantifying predation, yet dietary DNA detectability is influenced by multiple factors. Here, we systematically evaluated the effects of diet, predator traits (species, life stage, and sample type), and molecular marker properties (marker length and type) on dietary DNA detectability in ladybird beetles (Coleoptera: Coccinellidae), using Cryptolaemus montrouzieri Mulsant, 1853 as a representative species alongside other ladybird species. Dietary DNA detectability varied markedly among food types. The 50% detection success (DS50) of Ephestia kuehniella Zeller, 1879 (Lepidoptera: Pyralidae) egg DNA was extremely short, indicating that some diet items may be difficult to detect because of rapid degradation. In contrast, Brassica rapa L., 1753 (Brassicales: Brassicaceae) pollen DNA exhibited DS50 values comparable to those of the mealybug Planococcus citri Risso, 1813 (Hemiptera: Pseudococcidae) and the aphid Megoura crassicauda Mordvilko, 1919 (Hemiptera: Aphididae). Propylea japonica Thunberg, 1781 (Coleoptera: Coccinellidae) consistently exhibited shorter DS50 values than C. montrouzieri when fed on the same prey, demonstrating strong predator-dependent variation in dietary DNA detectability. Fourth-instar larvae maintained nearly 100% detection rates before pupation, and gut tissues yielded the longest DS50 among sample types. Molecular marker properties also influenced detectability: shorter mitochondrial COI fragments showed longer detection windows than longer fragments, whereas nuclear markers generally exhibited longer DS50 values than mitochondrial markers. These findings demonstrate that dietary DNA detectability is jointly shaped by diet, predator traits, and molecular marker properties, providing practical guidance for interpreting molecular dietary data and improving trophic interaction studies and biological control assessments.