Detection of herbivory: eDNA detection from feeding marks on leaves

Abstract

Many techniques have been developed to investigate the interactions between plants and herbivorous insects in natural environments and are generally used to determine either (a) which plant species are eaten by a specific herbivorous insect or (b) which herbivorous insect species are herbivores of a specific plant. The former problem is usually addressed by the direct observation of feeding and microscopic observation of gut contents and excrements, as well as the application of DNA‐barcoding techniques. However, the latter problem has typically been addressed using time‐consuming methods, such as direct observation and rearing. Therefore, more efficient techniques are needed for identifying and quantifying the interactions of plants with herbivorous insects. The present study demonstrates that the environmental DNA (eDNA) of herbivorous insects can be recovered from leaves with external foliage feeding marks. Mitochondrial DNA fragments of herbivorous insects were detected from insect‐exposed leaves using primer sets that amplified the DNA of target species. The amplification rate of the herbivorous insect DNA was positively associated with the rim length of feeding marks, which suggests that most of the insect DNA came from the feeding marks. Additionally, we showed that this method has the potential to detect eDNA from field‐collected leaves. This time‐efficient approach will contribute to the detection of plant–insect herbivore interactions.