Molecular monitoring of insecticide resistance in <i>Aphis gossypii</i> Glover (Hemiptera: Aphididae) from different crops in Greece, using novel ddPCR diagnostics

Abstract Aphis gossypii Glover is a highly polyphagous pest causing significant economic losses worldwide. In Greece, while the species is a primary threat to various crops, comprehensive molecular data regarding its insecticide resistance status remain scarce. This study, the first time in Greece, developed and implemented droplet digital PCR (ddPCR) to detect and quantify the frequency of key resistance mutations (R81T, S431F, A302S, L1014F/M918L, and A2666V – resistance to nicotinic acetylcholine receptor competitive modulators, dimethyl carbamates, organophosphates and carbamates, pyrethroids, and keto–enols, respectively) in field populations from citrus, cotton, and cucurbits plantations between 2021 and 2025. Our results identified four out of the six target mutations (L1014F and A2666V mutations were not found in any of the samples). Cucurbit populations exhibited the highest resistance levels, with S431F being fixed in seven populations and reaching a total resistance allele frequency (RAF) of 75%. The mutations A302S and M918L were also prevalent in cucurbits (total RAF of 40% and 49%, respectively), while the neonicotinoid-associated R81T mutation was detected at lower frequencies (total RAF of 9%). In contrast, citrus and cotton populations showed lower resistance diversity, with citrus populations possessing only the S431F mutation. The high resistance frequencies in cucurbits likely reflect intense selection pressure from specific insecticides used in these crops. Given the predominantly asexual reproduction of A. gossypii in Greece, the persistence of resistant genotypes poses a significant challenge. The ddPCR diagnostics developed here provide highly sensitive tools for monitoring target-site resistance, supporting effective insecticide resistance management.