Sample collection and DNA isolation
Surveys for associated medfly parasitoids were carried out between June and July in 2021 in Adana (37°03′ 98.21′′N, 35°36′ 05.73′′E), Turkey. These specimens were collected from loquat fruits. Surveys for C. capitata parasitoids were conducted on both infested fruits and pupae that dropped into the soil. The infested fruits and pupae samples were transferred to the Citrus Pest Laboratory at Cukurova University. Infested fruits were maintained in plastic trays at 24 ± 1°C and 65–80% RH until the emergence of adult C. capitata and parasitoids. The emerged parasitoids were stored in 96% ethanol for preservation. DNA was isolated from C. capitata parasitoids using the standard extraction kit and protocol provided by DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany).
Approximately 630 bp of the barcoding region of the mtCOI gene was amplified using the primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer et al., 1994). Amplification was conducted in 50 µl with the containing 2x Master Mix (containing Tris-HCl pH 8.5, (NH4)2S04, 3 mM MgCl2, 0.2% Tween 20, 0.4 mM of each dNTP, Taq DNA Polymerase), 10 µM primer, 50 ng DNA template and added PCR grade H2O to a final volume. The thermocycler conditions were: 5 min at 94°C for pre-denaturation, followed by 35 cycles at 94°C for 1 min, 50°C for 1 min, at 72°C for 1 min, and a final period at 72°C for 7 min. All the PCR products were confirmed electrophoretically using 1% agarose gel, stained with ethidium bromide, and viewed with a gel imaging system before two-way sequencing by a commercial company (Macrogen, Amsterdam, Netherlands).
The sequences were edited using FinchTV (version 1.4.0) and aligned by CLUSTAL W that had been integrated with MEGA7 software (Tamura et al., 2013). The MEGA 7 software was also used to create maximum likelihood (ML) trees, with 1000 bootstrap replicates performed to assess branch support. The sequences were compared to the closest available reference sequences in the NCBI nucleotide collection in GenBank (http://blast.ncbi.nlm.nih.gov/Blast). To root the phylogenetic tree, the sequence of Diachasmimorpha longicaudata Ashmead (Hymenoptera: Braconidae) was used as the out-group.