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. 2025 Jul 28;16(8):776.
doi: 10.3390/insects16080776.

Detection and Quantification of House Crickets (Acheta domesticus) in the Gut of Yellow Mealworm (Tenebrio molitor) Larvae Fed Diets Containing Cricket Flour: A Comparison of qPCR and ddPCR Sensitivity

Pavel Vejl  1 , Agáta Čermáková  1 , Martina Melounová  1 , Daniela Čílová  1 , Kamila Zdeňková  2 , Eliška Čermáková  2   3 , Jakub Vašek  1
Affiliations

Detection and Quantification of House Crickets (Acheta domesticus) in the Gut of Yellow Mealworm (Tenebrio molitor) Larvae Fed Diets Containing Cricket Flour: A Comparison of qPCR and ddPCR Sensitivity

Pavel Vejl et al. Insects. .

Abstract

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.

Keywords: ddPCR; edible insects; food authentication; gut content analysis; house cricket (Acheta domesticus); qPCR; yellow mealworm (Tenebrio molitor).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Representative results of A. domesticus quantification in the digestive tract of non-starved T. molitor larvae fed a diet containing 50% A. domesticus flour. (a) Amplification curves from the qPCR assay. (b) Melting temperature analysis from the qPCR assay. (c) Copy number of the ampd gene (A. domesticus) determined by ddPCR. (d) Copy number of the MyD88 gene (T. molitor) determined by ddPCR.
Figure 2
Figure 2
Comparison of ddPCR and qPCR methods via regression and correlation analysis (a) and starvation effect (b). In both (a) and (b), groups A, B, C, and D represent different proportions of A. domesticus flour in the diet of T. molitor: A = 25%, B = 50%, C = 75%, and D = 100%.
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