Encarsia agona, Otim and Polaszek, 2025

Encarsia agona and Bemisia tabaci-complex partial cox1 gene characterisation

We developed the PCR/Sanger sequencing primers for our target Encarsia species using the mtDNA COI (cox1/mtCOI) gene sequence of E. agona assembled from our Illumina HTS platform below), and included also publicly available Encarsia spp. cox1 partial genes from GenBank (KF KF778400 View Materials ; KF778464 View Materials ; KF778433 View Materials ; KF778411 View Materials ). Sequences were aligned within Geneious 8.1.9 (Biomatters Auckland, NZ) using MAFFT v. 7.490 ( Katoh et al. 2002; Katoh and Standley 2013) with default settings algorithm, scoring matrix = 200PAM/K = 2; gap open penalty = 1.53; offset value = 0.123) to identify most-conserved regions on the 5’ and 3’ partial cox1 gene. Potential forward and reverse primer sites identified were assessed and primers (Black-COI-F01: 5’-GGWGGAGGWGAYCCAATTTTATATCA-3 COI-R01: 5’-CCACTTAAACCTAAGAAATGTTGAGG-3’) were developed using the Oligo 7 Primer Software ( Rychlik 2007).

The PCR reaction for each sample included 0.2 µM of forward and reverse primers, 0.2 mM dNTPs, extracted gDNA, and 0.25 units of DNA polymerase (Phusion Taq, NEB), using the PCR conditions of 5 95°C for 1 cycle, 35 cycles of 95°C (60 s)–47°C (30 s)–72°C (30 s), and a final template extension step of

5 min (1 cycle). PCR amplification of parasitised Bemisia puparia used the primers MED-F1/R1 conditions as reported in Elfekih et al. (2018b). Presence of amplicons of the expected size (635 parasitoid, 759–788 bp for Bemisia ) were visualised by loading 1 µL 6x Loading Dye (NEB Cat. #) to 5 µL reaction onto a 1.5% 1x TBE agarose gel and run for 90 min at 75 V, stained with GelRed Nucleic Stain (Biotium, Fremont California) following the manufacturer’s recommendation.