2.4. Gene Expression Analysis

The internal physiological state of hives bees and larvae was assessed with a set of molecular biomarkers of the detoxifying and immune response. Changes in the transcription of this gene set are signs of chemical stress associated with the intake of herbicides in food [36,41,42].

Total RNA was extracted from each set of pooled midguts and larvae with a TRIzol extraction protocol (Invitrogen Life Technologies, Waltham, MA, USA). Total RNA was treated with DNase, before reverse transcription (RT) reactions. The cDNA was synthesized (Revertaid RT, Thermo Fisher Scientific, Waltham, MA, USA) with an input of 1 µg of total RNA.

The qRT-PCR reactions were performed using three technical replicates for each biological replicate to assess the expression level of antimicrobial peptide Abaecin (AB) and P450 cytochromes CYP6AS2, CYP6AS3, CYP6AS4, CYP6BD1 and CYP9Q3, involved in phase I of the xenobiotic detoxification pathways in honeybees [47], using previously validated primers (Table 1). The comparative qRT-PCR analysis was performed using the Applied Biosystems 7500 software [56] following the manufacturer’s protocol for the ∆∆Ct method. For the correct use of this method, the efficiencies of the primers for both targets and housekeeping were calculated and validated (Table S1). Melting curve analysis was used to ensure amplification specificity. Rpl8 was used as the endogenous control [57,58] and samples of the first sampling moment were taken as a control to compare how different the relative expression of our selected targets after the post-emergence weed control was.

Primer models. Sequence, amplified size product and melting temperature for the constitutive gene (Rpl8) and target genes Abaecin, CYP6BD1, CYP9Q3, CYP6AS2, CYP6AS3, CYP6AS4.