The codling moth (Cydia pomonella L.) is a widely distributed harmful pest in Kazakhstan that affects agricultural crops, primarily fruits such as apples. Despite its economic significance, its molecular identification had not been conducted in the country before this study.

A total of 91 fruit samples infested with C. pomonella larvae were collected in orchards across Almaty and Zhetysu (Kazakhstan), as well as the Jalal-Abad regions (Kyrgyzstan). In orchards, fruit pests were misidentified as G. molesta due to the lack of molecular identification, distorting quarantine pest statistics.

Genomic DNA was isolated using the CTAB method. Partial sequences of the 16S rRNA and cytochrome c oxidase subunit I (COI) genes were amplified and sequenced using Oxford Nanopore Technology (MinION Mk1B). Mapping to reference 16S rRNA sequences NC_020003.2 (C. pomonella) and KT004682.1 (G. molesta), as well as to reference COI sequences PP991409.1 (C. pomonella) and PQ876920.1 (G. molesta), was performed using Minimap2, and post-alignment metrics were calculated with SAMtools. The distribution of these metrics was visualized using ggplot2 in R.

Sequencing data from 91 samples exhibited a mean sequence identity of 93.31% to the reference sequences of C. pomonella and 90.5% to G. molesta. Mapping quality scores ranged from 40 to 60, with an average sequencing depth of ~1,300× and a mean coverage of 99.83%. The high sequence identity to C. pomonella reference sequences across both mitochondrial markers provides strong molecular confirmation that all collected specimens belonged to C. pomonella rather than G. molesta. These findings underscore the need to revise pest identification methods, as the widespread misclassification of pests can impact quarantine measures and pest management strategies. These results highlight the importance of implementing accurate molecular diagnostic techniques to ensure reliable pest monitoring and control.

This research was funded by the Ministry of Agriculture of the Republic of Kazakhstan (Program No. BR22887230).