Fig 4.

(A and B) Expression levels of the AMP genes in symbiotic organs of fifth-instar nymphs of R. pedestris. AMP expression levels were compared between M4B regions from symbiotic and aposymbiotic insects (A) and between the M4B region and the M4 region of symbiotic insects (B). To calculate the normalized fold expression (2^−ΔΔCT), EF1α was used as a reference gene, and the expression levels were normalized to the expression of the thanatin-like peptide gene in symbiotic insects, set as 1. Means and standard deviations are shown (n = 3). The asterisk indicates a statistically significant difference (unpaired t test; *, P < 0.05). (C) Antimicrobial activity test of M4B lysates treated at different temperatures. M4B lysate (0.2 μg/ml in PB) was temperature treated prior to testing the antimicrobial activity against the symbiotic Burkholderia strain. Statistically significant differences between the bacterial CFU before (PB) and after incubation with M4B lysate are indicated by asterisks (unpaired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3). NS, not significant.