A Protocol to Identify the Host of Parasitoids by DNA Barcoding of Vestigial Tissues

Many parasitoid wasps parasitize herbivorous insect larvae growing within plant organs (e.g., fruits and leaves). As it is hard to identify the insect host species directly, one approach to deal with this issue is to identify it by means of molecular analysis from puparia left within plant organs after wasps emerge. Unfortunately, current barcoding methods are either too expensive or too inefficient for mass species identification. Here, we present a protocol that is comparatively inexpensive and rapid. It includes two major modifications in the barcoding process. One is to use a modified Chelex DNA extraction method, which performed best in PCR amplification and was the least costly and time-consuming among four candidate methods. The other is to use general PCR primers for the host taxon, which had the highest sequencing success rate when coupled with the Chelex DNA extraction method. Our protocol proved to be successful in identifying the hosts (i.e., tephritid fly species) of parasitoid wasps in a Tibetan alpine meadow. The protocol can be widely used for mass identification of insect host species from puparia tissues to facilitate the studies on host–parasitoid interactions.