Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin

Abstract

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.

Figure 1. Gene Ontology (GO) terms and numerical identifications (ID) from annotations enriched significantly (Fisher’s Exact Test, p<0.05) in a Blast2GO analysis of transcripts from the gut of Tenebrio molitor larvae fed diet containing 0.1% Cry3Aa for 24 h compared to larvae fed control diet, in the categories Cellular Component (above black line) and Biological Process (below black line).

Figure 2. Venn diagrams of the number of genes that were significantly (p<0.05) induced or repressed, as determined by a pairwise analysis of microarray data from Tenebrio molitor larvae fed 0.1% Cry3Aa for 6, 12, or 24 h.

Figure 3. Principle component analysis (Manhattan distance, Average linkage) of datasets from control, 6 h, 12 h, or 24 h Cry3Aa-intoxicated (Bt) Tenebrio molitor larvae.

Figure 4. ANOVA of significant (p<0.05) differences in gene expression patterns of transcripts from Tenebrio molitor larvae fed 0.1% Cry3Aa for 6, 12, or 24 h compared to those fed control diet, hybridized to a custom microarray and expressed as relative fold difference.

Microarray contig# and putative protein are on the left, based on tBLASTx of NCBI nr; na, not available due to lack of homology or annotation.

Figure 5. RNA-Seq expression of transcripts encoding putative Bt toxin receptors in control and Cry3Aa-treated Tenebrio molitor larvae, comparing the number of reads of transcripts encoding proteins related to cadherin, aminopeptidase N, and alkaline phosphatase in each treatment group.

Figure 6. Relative expression of Tenebrio molitor gut transcripts encoding proteins containing chitin-binding domain 3 in Cry3Aa-intoxicated larvae compared to control.