Fig 1.
Summary of the different experimental approaches used to characterize TGIP mechanisms in T. molitor.
Table 1.
Statistics of our transcriptome of T. molitor compared to those from other coleopteran reported in previous studies.
Fig 2.
2D-gel electrophoresis highlights spots differentially abundant between priming conditions.
Left panels: two-dimensional difference gel electrophoresis (2D-DiGE) with the three fluorescent channels corresponding to the three CyDyes (Cy5, Cy3 and Cy2) merged. Right panels: 2D-SDS-PAGE stained by mass spectrometry-compatible silver staining protocol are shown for eggs sampled in ovaries (top gels) or sampled within 16 h post-laying (bottom gels). Eggs were collected 3 days after female priming. Spots significantly (p < 0.05, Mann-Whitney test) 1.5 differentially abundant in eggs from B. thuringiensis compared to control, S. entomophila compared to control, both B. thuringiensis and S. entomophila compared to control and B. thuringiensis compared to S. entomophila are represented by green, red, yellow and purple circles, respectively. The same color code is used in Table 2 presenting a summary of proteins identified. A full list of the proteins identified and associated statistics is available in S3 Table. Six biological replicates per condition, each containing 70–74 eggs, for a total of 433 eggs from 117 different females were included in the 2D-DiGE analysis.
Table 2.
2D-DiGE identifies differentially abundant proteins between the three conditions tested.
Table 3.
Top-down nano-LC-MS/MS identifies key candidate AMPs differing between the three conditions tested in freshly laid eggs (sampled within 16 h after laying) 3-day post-priming.
Fig 3.
AMPs condition the number of eggs protected but not the level of protection of protected eggs.
Antimicrobial activity was measured in 3 days old eggs originated from females that were primed and injected with either PBS (PBS), dsRNA from non-relevant GFP (dsGFP) or dsRNA targeting the five candidate AMPs (dsAMP). Eggs were laid 5 days after priming. A. The zone of inhibition around eggs disposed on A. globiformis-covered agar plates was measured. Eggs that did not exhibit antimicrobial activity (null zone of inhibition) were not considered. B. The percentage of eggs exhibiting antibacterial activity is represented for the same three samples. A star ‘*’ above the bar indicates a significant difference between the dsAMP condition and the PBS and dsGFP samples (p < 0.05; Fisher’s exact test).
Fig 4.
Mothers and eggs exhibit desynchronized candidate gene expression patterns.
Expression of the 11 candidate immune genes was measured in adult females (panels A to C) and in 7 days-old eggs (panels D to F) from mothers 1, 5 and 12 days post-priming (panels A and D, B and E, C and F, respectively). Values related to priming with B. thuringiensis and S. entomophila are represented by green and red bars, respectively. Data are represented as mean expression in primed condition relative to PBS-injected control. A ‘*’ above the bar indicates a significant over- or under-expression in primed condition compared to control (Mann-Whitney test, p<0.05).
Fig 5.
Mothers protect their eggs at 5 days–but not 1 day–post bacterial priming.
Proportion of females with protected eggs in function of the maternal treatment. Data on 7-day old eggs laid by females after 1 day and 5 days post-priming are represented in blue and red, respectively. Eggs (3 per female) collected at day 1 were from 22 naïve females, 17 PBS, 18 Bt and 17 Se. Eggs collected at day 5 were from 17 naïve females, 19 PBS, 19 Bt and 14 Se. A total of 321 eggs were included in the analysis. Error = confidence interval 95% (logistic regression).
Fig 6.
Four complementary protocols for female adult priming and egg sampling were conducted to elucidate the different scenarios of TGIP in T. molitor.
Egg sampling strategies for proteomic approach (panel A), antimicrobial activity following RNAi experiment (panel B), kinetics of gene expression by RT-qPCR (panel C) and antibacterial activity following mothers priming (panel D) are indicating. A full description of the procedure for each experiment is described in details in the corresponding part of the Material and Methods section.