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

Author
item Oppert, Brenda
item DOWD, SCOT - Research & Testing Laboratory
item BOUFFARD, PASCAL - Life Science Consortium
item LI, LEWYN - Life Science Consortium
item CONESA, ANA - Centro De Investigacion
item LORENZEN, MARCE - North Carolina State University
item TOUTGES, MICHELLE - Non ARS Employee
item MARSHALL, JEREMY - Kansas State University
item HUESTIS, DIANA - Kansas State University
item Fabrick, Jeffrey
item OPPERT, CRIS - University Of Tennessee
item JURAT-FUENTES, JUAN LUIS - University Of Tennessee

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/2/2012
Publication Date: 4/25/2012
Citation: Oppert, B.S., Dowd, S.E., Bouffard, P., Li, L., Conesa, A., Lorenzen, M.D., Toutges, M., Marshall, J., Huestis, D., Fabrick, J.A., Oppert, C., Jurat-Fuentes, J. 2012. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin. PLoS One. 7(4): e34624. 12pp. Available: http://dx.plos.org/10.1371/journal.pone.0034624.

Interpretive Summary: Some insecticides are based on toxins produced by bacteria or other microbes, but these insecticides often don’t work well for pest beetles. In order to use microbial toxins to control beetle pests, we need to make the toxins more effective. To better understand how the toxins work, we studied the gut response of mealworm larvae to microbial toxins. We used a new sequencing technology that gives us lots of different genetic (DNA) sequences, and we used these sequences to explore gene expression in a technology called “microarrays”. Using this approach, we found that toxin-fed mealworm larvae turn off expression of genes associated with the digestion of food, and they increase expression of other genes, such as those that are related to respiration and immunity. Knowledge of the mealworm response to toxin provides information critical to the improvement of microbial toxins for beetle pest control.

Technical Abstract: Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against some coleopteran pests, but improvements are needed in both efficacy and “time to kill” for Cry toxins to become valuable tools for use in integrated pest management. To gain insight into Bt intoxication of Coleoptera, we performed RNA-Seq on cDNA from the guts of Tenebrio molitor larvae that had been fed either a control (untreated) diet or a diet containing 0.1% Cry3Aa for 24 hours. 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 in Blast2Go indicated that functions associated with mitochondrial respiration, membrane restructuring, protein recycling/synthesis, signal transduction, and stress responses were increased significantly (p<0.05) in Cry3Aa-treated larvae, whereas many metabolic processes, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis, were decreased. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 hours of Cry3Aa exposure. At 6 h, Cry3Aa-intoxicated larvae had significant differential expression of genes encoding an allergen-like protein, a chitin deacetylase, and one nonannotated protein; at 12 h, differential expression was found in transcripts encoding proteins that interact with chitin or lipid, hydrolases, immunity, nuclear regulatory and metabolic proteins, and signaling; and at 24 h, Cry3Aa-intoxicated larvae continued to display differential expression of transcripts associated with chitin, hydrolases, and immunity. Statistical analysis of transcript expression in control and Cry3Aa-treated larvae from all time-points indicated that an ortholog to Drosophila CG4367 previously reported to be induced in response to gram-negative bacterial infection [N Buchon et al., Cell. Host Microbe, 2009, 5, 200–211] was most dramatically induced in Cry3Aa-intoxicated larvae. Overall, the data suggest that Cry3Aa-intoxicated T. molitor larvae induce the transcription of genes associated with mitochondrial respiration, membrane restructuring, immunity, and signaling, and concomitantly down-regulate selected transcripts associated with metabolism of food. This study demonstrates a novel approach to study Cry intoxication in insects lacking a sequenced genome, and at the same time provides sequence data to aid in future studies.