New internal primers targeting short fragments of the mitochondrial COI region for archival specimens from the subfamily Aphidiinae (Hymenoptera, Braconidae)

Abstract

Archival specimens are a great resource for molecular research in population biology, taxonomy and conservation. A primary goal for researchers is to preserve specimens from collections by improving noninvasive methods for DNA extraction and to achieve successful amplification of the short fragments of a target gene in the event of DNA fragmentation. We tested the suitability of a noninvasive method of DNA extraction and amplification of the barcoding region of the mitochondrial gene cytochrome c oxidase subunit I from archival specimens of aphid parasitoids belonging to the genera Aphidius, Lysiphlebus and Praon (Aphidiinae, Braconidae, Hymenoptera). Using a commercial kit as a noninvasive method, we successfully extracted DNA from dry 7 to 41 year old samples of 26 different parasitoid species. However, amplification of the barcoding region failed using the standard primer pair LCO1490/HCO2198. In order to reconstruct DNA barcodes we designed internal genus-specific degenerative primers and a new amplification protocol to target the short fragments within the mitochondrial region. Novel primers were designed using as a template the reference sequences from congeners retrieved from the public database. The combination of standard primers with internal primers, in direct and nested amplification reactions, produced short overlapping subsequences, concatenated to recover long barcoding sequences. Additional analyses also confirmed that primers initially designed for Aphidius, Lysiphlebus and Praon can be combined in a mixture, and successfully used to obtain short fragments of disintegrated DNA from archival specimens of several other braconid species from the genera Ephedrus and Monoctonus.