Quantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔC_t method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

Odorant-binding proteins (OBPs) play a fundamental role in insect olfaction. In recent years,Galeruca daurica(Joannis) (Coleoptera: Chrysomelidae) has become one of the most important insect pests in the Inner Mongolian grasslands of China. This pest only feeds on the species ofAlliumplants, implying the central role of olfaction in its search for specific host plants. However, the olfaction-related proteins have not been investigated in this beetle. In this study, we identified 29 putative OBP genes, namely GdauOBP1–29, from the transcriptome database ofG. dauricaassembled in our laboratory by using RNA-Seq. All 29 genes had the full-length open reading frames except GdauOBP29, encoding proteins in length from 119 to 202 amino acids with their predicted molecular weights from 12 to 22 kDa with isoelectric points from 3.88 to 8.84. Predicted signal peptides consisting of 15–22 amino acid residues were found in all except GdauOBP6, GdauOBP13 and GdauOBP29. The amino acid sequence identity between the 29 OBPs ranged 8.33–71.83%. GdauOBP1–12 belongs to the Classic OBPs, while the others belong with the Minus-C OBPs. Phylogenetic analysis indicated that GdauOBPs are the closest to CbowOBPs fromColaphellus bowringi. RT-PCR and qRT-PCR analyses showed that all GdauOBPs were expressed in adult antennae, 11 of which with significant differences in their expression levels between males and females. Most GdauOBPs were also expressed in adult heads (without antennae), thoraxes, abdomens, legs and wings. Moreover, the expression levels of the GdauOBPs varied during the different development stages ofG. dauricawith most GdauOBPs expressed highly in the adult antennae but scarcely in eggs and pupae. These results provide insights for further research on the molecular mechanisms of chemical communications inG. daurica.

Juvenile hormone (JH) signaling plays an important role in regulation of reproductive diapause in insects. However, we have little understanding of the effect of JH on gene expression at the transcriptome level in diapause. Galeruca daurica is a new pest in the Inner Mongolia grasslands with obligatory summer diapause in the adult stage. Topical application of a JH analog methoprene at the pre-diapause stage delayed the adults entering diapause and inhibited lipid accumulation whereas it did not during diapause. Using Illumina sequencing technology and bioinformatics tools, 54 and 138 differentially expressed genes (DEGs) were detected at 1 and 2 d after treatment, respectively. The KEGG analysis showed that the DEGs were mainly enriched in the metabolism pathways. qRT-PCR analysis indicated that methoprene promoted the expression of genes encoding vitellogenin, fork head transcription factor and Krüppel homolog 1, whereas suppressed the expression of genes encoding juvenile hormone-binding protein, juvenile hormone esterase, juvenile hormone acid methyltransferase, juvenile hormone epoxide hydrolase and fatty acid synthase 2. These results indicate that JH signaling plays an important role in regulating reproductive diapause of G. daurica.