Due to their nutritional value and sustainability, edible insect-based foods are gaining popularity in Europe. Their use is regulated by EU legislation, which defines authorised species and sets labelling requirements. Molecular tools are being developed to authenticate such products. In this study, yellow mealworm (Tenebrio molitor) larvae authorised for human consumption were fed wheat flour-based diets containing varying proportions of house cricket (Acheta domesticus) flour for 21 days. This was followed by a 48 h starvation period to assess the persistence of insect DNA in the digestive tract. Two novel, species-specific, single-copy markers were designed: ampd gene for the Acheta domesticus and MyD88 gene for the Tenebrio molitor. These were applied using qPCR and ddPCR. Both methods successfully detected cricket DNA in the guts of starved larvae. Linear regression analysis revealed a strong, statistically significant correlation between the proportion of Acheta domesticus flour in the diet and the normalised relative quantity of DNA. ddPCR proved to be more sensitive than qPCR, particularly in the detection of low DNA levels. These results suggest that the presence of DNA from undeclared insect species in edible insects may be indicative of their diet rather than contamination or adulteration. This highlights the importance of contextual interpretation in food authenticity testing.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Food Science Czech Agrifood Research Center Drnovská 507 73 161 00 Prague Czech Republic

Department of Genetics and Breeding Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague Czech Republic

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