Investigations were conducted in northern Tunisia from 2013 to 2018. A total of 1060 larvae of Orgyia trigotephras were collected (with 926 larvae from Jebel Abderrahmane (Cap-Bon, alt. 432 m; 36°52′N, 10°48′E) and 134 larvae from Dam Ziatine (Sejnane, alt. 48 m; 37°11′N, 9°11′E). Each larva was kept individually in a plastic box (30 × 70 ml) at 25 ± 2 °C and reared on fresh leaves of its host plant, Quercus coccifera (Fagaceae). A periodic check of rearing boxes was carried out, and we noticed the emergence of parasitoids from all larval instars. Adult flies were stored in ethanol (95%) until identification. The morphological characters of adult flies were described using keys of Shima (1997); Tschorsnig and Herting (1994); Tschorsnig and Richter (1998).
For specific final recognition, molecular analysis of 1–3 legs of the 8 specimens of flies was carried out by amplification of 650pb fragment of the mitochondrial cytochrome c oxidase gene subunit 1 (COX1) using the universal primers LCO1490/HCO2198 (Folmer et al. 1994). DNA extraction was carried out using the Syngen DNA Mini Kit (Syngen, Poland) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification mix was prepared in 25 μl contained 1 × PCR Buffer (Taq PCR Core Kit, QIAGEN), 1.5 mM MgCl2, 0.4 mM of each dNTP, 0.2 µM of each primer, 1 U of Taq polymerase and 10–20 ng of template DNA. Thermal cycling was executed on a T gradient thermal cycler (Bio-Rad) with an initial denaturation at 96 °C for 3 min, followed by 40 cycles of denaturation at 96 °C for 30 s, annealing at 48 °C for 56 s, extension at 72 °C for 1 min and 20 s, and a final extension at 72 °C for 10 min. Amplicons were analyzed by electrophoresis, visualized in a 1% agarose gel stained with the GelRed® dye (Biotium, USA) and purified using the CleanUp Kit (A&A Biotechnology, Poland). Sequencing of the purified amplification products was performed at Genomed Company (Warsaw, Poland). The obtained sequences were analyzed using the Basic Local Alignment Search Tool Nucleotide (BLASTN) searches at GenBank (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) in order to determine the closest matches and confirm the morphological identification. To perform phylogenetic analysis, the generated sequences were supplemented with additional sequences of Compsilura concinnata specimens obtained from GenBank and other species from Tachindae family used as outgroups. The phylogenetic tree was carried out by using the Maximum Likelihood Method based on the Kimura 2-parameter model (Kimura 1980). The analysis involved 18 nucleotide sequences and was conducted in MEGA v.6.