RNA-Seq Validation of RNAi Identifies Additional Gene Connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

RNA interference (RNAi) is a functional genomics tool to correlate genotype and phenotype by delivering targeted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to reduce gene expression. In the red flour beetle, Tribolium castaneum, RNAi has been successful via injected dsRNA at all life stages. Traditionally, successful transcript knockdown has been quantified by qPCR on a gene-by-gene basis, where only expression of the target gene and normalization genes are evaluated. In this study, RNA-Seq was used to quantify transcript expression in larvae injected with dsRNA for aspartate 1-decarboxylase (ADC), which gives a reliable phenotype of an adult with a black cuticle instead of the wild-type red-brown. ANOVA of control, mock-injected, and ADC-dsRNA injected larvae indicated that target gene expression was significantly (P = 0.002) reduced 4-fold, and the black phenotype was achieved in all adults injected with ADC-dsRNA as larvae. In a pairwise analysis, significant (P < 0.05) differential expression of other genes in ADC-injected larvae suggested connections between gene pathways. One gene, dopamine receptor 2, was increased in expression 227-fold (P = 0.025), presumably connected to previous data that showed a reduction in expression of ADC results in increased levels of dopamine. To evaluate the hypothesis that increased dopamine levels can affect mobility, T. castaneum adults injected with ADC-dsRNA as larvae were significantly impaired in movement tests compared to controls, similar to black mutants in Drosophila melanogaster. The data demonstrate that RNA-Seq can reveal gene connectivity and provide more complete data validation and analysis compared to qPCR.