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.

Galeruca daurica (Joannis) is a pest species with serious outbreaks in the Inner Mongolian grasslands in recent years, and its larvae and eggs are extremely cold-tolerant. To gain a deeper understanding of the molecular mechanism of its cold-tolerant stress response, we performed de novo transcriptome assembly of G. daurica via RNA-Seq and compared the differentially expressed genes (DEGs) of first- and second-instar larvae grown and developed indoors and outdoors, respectively. The results show that cold tolerance in G. daurica is associated with changes in gene expression mainly involved in the glycolysis/gluconeogenesis pathway, the fatty acid biosynthesis pathway and the production of heat shock proteins (HSPs). Compared with the control group (indoor), the genes associated with gluconeogenesis, fatty acid biosynthesis and HSP production were up-regulated in the larvae grown and developed outdoors. While the changes in these genes were related to the physiological metabolism and growth of insects, it was hypothesized that the proteins encoded by these genes play an important role in cold tolerance in insects. In addition, we also investigated the expression of genes related to the metabolic pathway of HSPs, and the results show that the HSP-related genes were significantly up-regulated in the larvae of G. daurica grown and developed outdoors compared with the indoor control group. Finally, we chose to induce significant expression differences in the Hsp70 gene (Hsp70A1, Hsp70-2 and Hsp70-3) via RNAi to further illustrate the role of heat stress proteins in cold tolerance on G. daurica larvae. The results show that separate and mixed injections of dsHSP70A1, dsHsp70-2 and dsHsp70-3 significantly reduced expression levels of the target genes in G. daurica larvae. The super-cooling point (SCP) and the body fluid freezing point (FP) of the test larvae were determined after RNAi using the thermocouple method, and it was found that silencing the Hsp70 genes significantly increased the SCP and FP of G. daurica larvae, which validated the role of heat shock proteins in the cold resistance of G. daurica larvae. Our findings provide an important theoretical basis for further excavating the key genes and proteins in response to extremely cold environments and analyzing the molecular mechanism of cold adaptation in insects in harsh environments.

20-hydroxyecdysone (20E) signaling plays an important role in regulating insect growth, development, and reproduction. However, the effect of 20E on reproductive diapause and its regulatory mechanisms have not been fully understood. Galeruca daurica is a new pest in the Inner Mongolia grasslands, and it aestivates in an obligatory reproductive diapause form. In this study, the complete open reading frame (ORF) sequence of the ecdysone receptor (EcR) was cloned from G. daurica. Application of 20E promoted the expression of EcR, nuclear hormone receptor (HR3), and vitellogenin (Vg), whereas it reduced the expression of fatty acid synthase (FAS) and lipid content, leading to delayed diapause entry in female adults. Silencing EcR or HR3 by RNAi increased the expression of FAS and lipid content, whereas it reduced the expression of Vg, inducing reproductive diapause. These results indicate that 20E may mediate reproductive diapause via a conserved EcR/HR3 cascade in G. daurica.

Galeruca daurica (Joannis) is an oligophagous pest in the grasslands of Inner Mongolia, China, which feed mainly on Allium spp. Odorant receptors (ORs) play an important role in the olfactory system in insects, and function together with olfactory co-receptor (ORco). In this study, 21 OR genes were identified from the transcriptome database of G. daurica adults, and named GdauOR1-20 and GdauORco. The expression profiles were examined by RT-qPCR and RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted to further identify the olfactory functions of GdauOR4, GdauOR11, GdauOR15, and GdauORco. It was found that 15 GdauORs (OR1, OR3-6, OR8, OR11-13, OR15, OR17-20, and ORco) were mainly expressed in antennae, and the expression levels of GdauORs in adults were affected by age. When GdauOR4, GdauOR15, and GdauORco were silenced by RNAi, the electrophysiological responses to host plant volatiles were significantly decreased in G. daurica. This study lays a necessary foundation for clarifying the mechanism on finding host plants in G. daurica.

Plants are important ecological factors and food resources, which can significantly affect the occurrence and distribution of insects. The metabolites in host plants can affect the feeding, spawning, and avoidance behaviors of herbivorous insects. Galeruca daurica (Joannis) is a phytophagous pest that has seriously occurred in the desert steppe of Inner Mongolia in recent years, only infesting the leaves of Allium plants. In order to clarify the effects of plant metabolites on the gene expression in G. daurica larvae at the transcriptome level, we fed the larvae of G. daurica with Allium tuberosum leaves soaked in 10% DMSO solutions containing d-galactose, β-d-glucopyranose, l-rhamnose, isoquercitrin, isoflavone, and rutin, respectively, used the larvae fed on A. tuberosum leaves soaked in a 10% DMSO solution as the control, and screened out the differentially expressed genes (DEGs) by performing high-throughput transcriptome sequencing. The results showed that a total of 291 DEGs were identified compared to the solvent control (DMSO), including 130, 34, 29, 21, 72, and 97 in the isoquercitrin, isoflavone, rutin, d-galactose, β-d-glucopyranose, and l-rhamnose treatment groups, respectively. GO and KEGG enrichment analysis showed that most DEGs were enriched in various metabolic pathways, implying that these six main primary and secondary metabolites in Allium plants may affect various metabolic processes in the larvae of G. daurica.

Metamorphosis in insects is regulated by hormonal signals and metabolic pathways. In this study, we characterized a 20E-mediated miR-2788/Treh1 regulatory axis involved in the larval-to-pupal transition in the leaf beetle Galeruca daurica. Dual-luciferase assays and expression profiling suggest that miR-2788 directly targets and suppresses trehalase 1 (Treh1), a key enzyme for trehalose hydrolysis. We found that exogenous 20E treatment significantly downregulated miR-2788, leading to the derepression of Treh1 and increased expression of chitin biosynthetic genes (CHI, CHS, and GPI). Conversely, miR-2788 overexpression or Treh1 silencing via RNAi resulted in massive trehalose accumulation, reduced chitin synthesis, and lethal developmental arrest during the metamorphic transition. Furthermore, pharmacological inhibition of Treh1 with Validamycin not only recapitulated these chitin-deficiency phenotypes but was also associated with an upregulation of miR-2788. These results describe a signaling cascade where 20E modulates the conversion of carbohydrate to chitin through the miR-2788/Treh1 axis. This study advances our understanding of hormonal–miRNA–metabolic crosstalk and identifies miR-2788 as a potential target for RNAi-based biopesticide development.