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.