Figure 2.

Apis mellifera Xbp1 non-spliceosomal intron splicing in worker bees is unaffected by thiamethoxan, carbendazim and glyphosate. (A) Gene structure of Apis mellifera Xbp1 depicting the tRNA-type spliced intron and primers used to analyse its splicing (top). (B) To resolve similar sized spliced and unspliced isoforms, the RT-PCR product was digested with PstI, which only cuts the unspliced RT-PCR product. The size of the smaller fragment for return primer Xbp1 R1 is 91 bp (not shown). (C) Agarose gel showing the alternative splicing pattern of Xbp1 amplified with primers Xbp1 F3 and R2 by digestion of the RT-PCR product with PstI (P) at different time-points after injection of 2 µl 20 mM DTT. (D) Quantification of the changes in Xbp1 splicing shown in (C) as mean with the standard error from three replicates of the ration of spliced to unspliced. Only the large fragment of unspliced was used for quantification. (E) Agarose gel showing the alternative splicing pattern of Xbp1 amplified with primers Xbp1 F3 and R1 by digestion of the RT-PCR product with PstI (P) compared to undigested input (I) in control bees dissected immediately after collection (Control 1), control bees fed with water and sucrose for 24 h (Control 2) and control bees injected with water (Control 3) compared to bees injected with thiamethoxam (1 µM) and bees injected with a mixture of thiamethoxam (1 µM, T), carbendazim (2 mM, C) and glyphosate (32 mM, G) 24 h prior dissection. Samples were run on a 3% agarose gel. Ma: DNA marker. The undigested PCR product is shown at the bottom.