Gene expression in Tribolium castaneum life stages: Identifying a species-specific target for pest control applications

Authors: Perkin, Lindsey; Oppert, Brenda

Submitted to: PeerJ
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2019
Publication Date: 5/23/2019
Citation: Perkin, L.C., Oppert, B.S. 2019. Gene expression in Tribolium castaneum life stages: Identifying a species-specific target for pest control applications. PeerJ. 7:e6946. https://doi.org/10.7717/peerj.6946.
DOI: https://doi.org/10.7717/peerj.6946

Interpretive Summary: Many beetle species are important pests of stored products such as grains and cereals and new molecular based pesticides may provide safer and more targeted tools for managing these pests. An important step in developing molecular based pesticides is the identification of targets that are specific to the pest and that are effective at low doses. Using the genetic model for stored product beetles, the red flour beetle Tribolium castaneum, with RNA sequencing and statistical analyses we identified genes that were differentially expressed in the different developmental stages of this pest insect. Categorized genes were further sorted into those with high or low expression levels and for genes with low expression levels there were 273 genes unique to adults, 167 to unique to eggs, 139 to unique to larvae, and 14 unique to pupae. Larvae are an important pest management target because this stage consumes large amounts of grain. Six cuticle genes were identified as unique to larvae and expressed at low levels. We chose one gene, cuticle protein, to evaluate as a potential control target, since it met our criteria of being potentially lethal or disruptive to development, had regions where molecular disruption using RNAi would minimize off target effects, and was expressed at low levels so that lower amounts of injected dsRNA as part of RNAi treatment could be used. Our functional test with RNAi showed knockdown in larvae caused mortality compared to non-injected controls. Interestingly, knockdown also occurred in injected with only water and there was also some mortality, although not to the same degree as with the dsRNA. Therefore, puncturing the cuticle likely causes a decrease in expression in this gene, and injecting dsRNA reduces transcript expression further. This study provided useful information on stage specific gene expression in the red flour beetle and identified a potential insect control product based on using dsRNA to disrupt a cuticle gene target. Because the gene is expressed in the cuticle of the insect, either oral or topical application of dsRNA can be investigated as a strategy to control larval stages of the red flour beetle.

Technical Abstract: Tribolium castaneum, the red flour beetle, is a genetic model for the largest group of insects, coleopterans, and a major stored product agricultural pest that damages post-harvest products. Due to limited pesticides and development of pesticide resistance, new control strategies for T. castaneum and other coleopterans are needed, such as molecular-based pesticides. An important step in designing molecular targets for new control products is to identify genes that will be specific to a pest or small range of pest insects, yet effective at low doses of treatment (i.e. targeting genes expressed at low levels). To this end, we used RNA-seq to identify genes expressed in four major life stages of T. castaneum, categorized genes via sorting data into groups based on high or low expression levels, and compared expression among all life stages. We searched low-expression genes with potential for pest control. We identified a target encoding a cuticle protein unique to T. castaneum and expressed in the larval stage. RNA interference of cuticle protein transcripts caused significant mortality, and RNA-seq discovered changes in gene expression of other cuticle proteins. RNA-seq combined with bioinformatics can identify potential targets for the development of new biologically-based control products for insect pests.