DNA Extraction and PCR Protocol
Genomic DNA extractions were performed using the DNeasy Blood and Tissue kit (QIAGEN) following the manufacturer’s protocol. DNA was extracted from Immature beetles and parasitoids via destructive sampling, while DNA from adult parasitoids were extracted non-destructively from the right midleg as the extraction medium with the remainder of the wasp retained as a voucher and deposited at the University of Central Florida Collection of Arthropods (UCFC). All specimens for which DNA was extracted were assigned a unique voucher code consisting of the letters RDR followed by three numbers (e.g. RDR001). For the identification of destructively sampled specimens, the barcode region of Cytochrome oxidase c subunit 1 (CO1 ) was amplified using universal primers (Forward: 5’- GGT CAA CAA ATC ATA AAG ATA TTG G - 3’; Reverse: 5’ - TAA ACT TCA GGG TGA CCA AAA AAT CA - 3’) (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994). Polymerase chain reactions were performed using 1 µL of template DNA, 0.2 mM dNTP solution (New England Biolabs, (NEB)), 4 mM MgSO4, 1X standard Taq buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2) (NEB), 400 nM of each primer (Integrated DNA Technologies), 1 unit of Taq DNA polymerase (NEB), and PCR grade water to bring the reaction to a volume of 25 µL. Thermocycler settings were as follows: initial denaturation at 95°C for 1 minute, 34 cycles of denaturation at 95°C for 15 seconds, annealing at 49°C for 15 seconds, and extension at 72°C for 45 seconds. PCR products were visualized using 5 µL of PCR product mixed with 1 µL of dye, loaded into a 1% agarose gel, and imaged after separation via gel electrophoresis. Samples observed with faint bands were re-run using 2 µL of template DNA and 1.25 units of Taq DNA polymerase (NEB) for 38 cycles. PCR products were processed using magnetic bead clean-up and sequenced on an Applied Biosystems 3730x 96 capillary sequencer at the UK Healthcare Genomic Core Laboratory. Forward and reverse reads were trimmed, assembled into contigs, and then edited for quality using Geneious Prime 2020.04 (https://www.geneious.com). Sequences were uploaded to GenBank under accession numbers MT246305-MT246448.