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.

Infection routes matter in population-specific responses of the red flour beetle to the entomopathogen Bacillus thuringiensis

“…Generally, CSPs and OBPs are involved in olfaction and chemical communication in insects, but the functions of members of these large families of genes might be diverse [8, 45]. Elevated levels of CSP gene transcripts were previously reported after oral exposure to B. thuringiensis or its toxins in Tenebrio molitor [46] and T. castaneum [16], and a role of OBP-C12 in T. castaneum defense against B. thuringiensis was also demonstrated [16]. The beetle populations differed in the specific sets and the expression dynamics of these genes, some of which were also differentially expressed upon pricking infection.…”

Biotoxicity of Cry1Ab protein on wolf spider Pardosa pseudoannulata

“…These results are similar to that in Tenebrio molitor. The genes expression level involved in mitochondrial respiration was significantly increased in Tenebrio molitor larvae when they intake Cry3Aa protein (Oppert et al 2012). The mitochondria is a powerhouse in cell, providing energy for various intracellular metabolic reaction (Conley 2016).…”

Identification and Characterization of Receptors for Insecticidal Toxins from Bacillus thuringiensis

“…Some of the studies of differential gene expression in insects using massive sequencing technologies have been made using the 454 platform of Roche (Park et al 2009;Pauchet et al 2010;Oppert et al 2012); however, even when the reads are large in average size which facilitates the assembly and subsequent analysis, they are relatively few compared to those obtained from massive sequencing experiments using other platforms such as Illumina platforms Genome Analyzer GAIIx and HiSeq (He et al 2012;Ma et al 2012;Lei et al 2014). The sequence data obtained should be analyzed using a limited number of programs currently available on the Internet.…”

“…In an analysis of RNA-Seq complemented with microarray experiments (using the 454 platform of Roche) performed on Tenebrio molitor, a stored grain pest, intoxicated with toxin Cry3Aa of Bt, Oppert et al (2012) found that the expression of the three best characterized receptors in insects to Bt toxin, cadherin, aminopeptidase N, and ALP, occurs in a very particular way. No differences were observed in the expression pattern of the cadherin and aminopeptidase N receptors amongst the control larvae and larvae intoxicated with the Cry3Aa after 24 h of intoxication.…”

“…Interestingly, increased levels of ALP were observed in Cry1Ab-resistant European corn borer, O. nubilalis (Khajuria et al 2009). In a subsequent analysis using a custom script for function assignment, enrichment for biological processes was obtained, specifically an enrichment of genes associated with transport, suggesting that once the larva is intoxicated it tries to maintain ionic homeostasis (Oppert et al 2012).…”