Non-lethal genotyping in Drosophila suzukii, Zeugodacus cucurbitae, Bactrocera dorsalis, and Aedes aegypti for functional genomics and genetic control

Non-lethal genotyping has advanced ecological and genetic research by enabling molecular analyses without sacrificing specimen viability. Concurrently, genome-editing tools such as the CRISPR/Cas9 system have spurred innovations in pest genetic control. A rapid, efficient, non-invasive genotyping method is therefore essential for functional genomics and genetic control studies. We evaluated tissue-specific genotyping in three economically important fruit flies – Drosophila suzukii, Zeugodacus cucurbitae, and Bactrocera dorsalis – and the public-health pest Aedes aegypti. PCR amplicon yield and quality were compared across up to three tissue types; genotyping accuracy was confirmed by PCR and Sanger sequencing; and effects on survival and reproduction were recorded. Pupal shells provided sufficient DNA for PCR and sequencing without any impact on insect survival or fecundity, whereas other tissues showed trade-offs between genotyping reliability and fitness. Based on these results, we propose optimized non-lethal genotyping protocols that minimize fitness costs, facilitating strain development for pest management and CRISPR-based research.