Table 1.
Susceptibility of Tc to Bt spore-crystal mixtures.
Figure 1.
Dose-mortality assays with Cry3Ba, Cry23Aa/Cry37Aa and Cry1Ac spore-crystal mixtures in Tc larvae.
Bioassays were performed on eight to ten day-old larvae fed on flour discs containing spore-crystal mixtures of Cry3Ba, Cry23Aa/Cry37Aa and Cry1Ac toxin producing Bt strains. Arrow points to images of 10 µL flour discs on which Tc larvae were fed for two days containing water (control), an approximately LC50 of Cry3Ba and Cry23Aa/37Aa spore-crystal preparations (12.5 µg/µL flour disc and 5.0 µg/µL flour disc, respectively), and 25 µg Cry1Ac spore-crystal mixture/µL flour disc (negative control).
Figure 2.
Quantitative iTRAQ proteomic analysis.
A work-flow indicating the steps followed in the iTRAQ analysis performed on two biological replicates of each toxin treatment and the classification of the iTRAQ identified proteins according to GO molecular function categories are shown. Proteins showing an i-TRAQ ratio (treatment vs control) with a Protein Pilot p-value≤0.05 at least in one replicate sample were 21 in Cry3Ba spore-crystal treated larvae and 24 in Cry23Aa/Cry37Aa spore-crystal treated larvae. Seven differentially regulated proteins are listed for Cry3Ba and Cry23Aa/Cry37Aa treatments and were selected according to the following cut-off criteria: (i) i-TRAQ ratio p-value≤0.05 in both biological replicates in each treatment, (ii) error factor <2 (generated by ProteinPilotTM), (iii) an average of ±1.5-fold in abundance in response to Bt intoxication in both biological replicates at least in one of the treatments. Using Student's t-test, statistically significant differences between toxin treatments were detected for pyruvate dehydrogenase Eα subunit, cuticular protein and ribosomal protein L13a (p<0.05).
Figure 3.
Tc OBP transcriptional analysis.
qRT-PCR analysis of mRNA expression levels of Tc OBP in larvae exposed to Cry3Ba, Cry23Aa/Cry37Aa and Cry1Ac spore-crystal mixtures relative control larvae, normalized to the RPS18 mRNA. Error bars indicate standard errors of the means from two biological replicates of twenty-four individuals per replicate.
Figure 4.
Tc OBP protein structural features.
(A) Sequence alignment of the OBP amino acid sequences of Tc and Tm using Clustal omega [56]. (B) Predicted three-dimensional homology model of the Tc OBP protein using SWISS-MODEL Workspace server [32] based on Tm THP12 (1C3Y.pdb). The Tm THP12 fold (1C3Y.pdb) is shown for comparison.
Figure 5.
Unrooted phylogenetic tree of insect OBP proteins.
The phylogenetic reconstruction was generated with MEGA version 4.0.2 software [35]. In red is shown the Tc OBP up-regulated upon Cry3Ba and Cry23Aa/Cry37Aa treatment challenge and in green, homologous Tc OBPs identified searching the BeetleBase protein database. Depicted in blue are other coleopteran OBPs, in grey lepidopteran OBPs, in yellow dipteran OBPs and in pink hymenopteran OBPs, which were identified to show homology to Tc OBP by searching the NCBI protein database.
Figure 6.
Tc OBP functional characterization.
(A) qRT-PCR analysis of the OBP mRNA expression levels in Tc larvae of different weight, using RPS18 mRNA as reference. (B) OBP silencing in Tc larvae in response to injection of dsRNA against Tc OBP. The relative amount of Tc OBP transcript estimated by qRT-PCR in control and silenced larvae was compared normalized to the expression of RPS18 gene. The statistical significance of the gene expression between the two samples was evaluated using Student's t-test and significant knockdown was observed (p<0.05). Error bars represent standard error of the mean of two biological replicates. (C) Mortality percentage following Cry3Ba, Cry23Aa/Cry37Aa or Cry1Ac spore-crystal treatments on either buffer-injected (control) or OBP dsRNA-injected larvae. Mortality experiments were performed using fifty Tc larvae in each spore-crystal treatment and the corresponding controls. Error bars represent standard error of the mean of at least two biological replicates. Mortality increase observed upon Bt treatments in OBP silenced larvae respect to buffer-injected larvae was statistically significant using Student's t-test (p<0.05).
Figure 7.
Tc differential response to Cry3Ba and Cry23Aa/Cry37Aa spore-crystal treatments.
(A) Relative levels of Tc pyruvate dehydrogenase (Pdh), cuticular protein and ribosomal protein L13a (RPL13a) mRNAs normalized to the corresponding untreated control, determined by qRT-PCR analysis in control larvae and larvae exposed to Cry3Ba and Cry23Aa/Cry37Aa spore-crystal mixtures, using RPS18 mRNA as reference. Error bars indicate standard errors of the means from two biological replicates of twenty-four individuals per replicate. (B) Monitoring of Tc larvae development following Cry3Ba and Cry23Aa/Cry37Aa spore-crystal treatments that produced 15–20% mortality. Percentage of pupae was recorded after seven days of spore-crystal treatment and up to day 35, once surviving treated larvae were transferred to spore-crystal-free diet.