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.

Lepidopteran insects mainly rely on sex pheromones to complete sexual communications. Pheromone receptors (PRs) are expressed on the olfactory receptor neurons (ORNs) of the sensilla trichodea and play an essential role in sexual communication. Despite extensive investigations into the mechanisms of peripheral recognition of sex pheromones in Lepidoptera, knowledge about these mechanisms in L. sticticalis remains limited. In this study, five candidate LstiPRs were analyzed in a phylogenetic tree with those of other Lepidopteran insects. Electroantennography (EAG) assays showed that the major sex pheromone component E11-14:OAc elicited a stronger antennal response than other compounds in male moths. Moreover, two types of neurons in sensilla trichodea were classified by single sensillum recordings, of which the “a” neuron specifically responded to E11-14:OAc. Five candidate PRs were functionally assayed by the heterologous expression system of Xenopus oocytes, and LstiPR2 responded to the major sex pheromone E11-14:OAc. Our findings suggest that LstiPR2 is a PR sensitive to L. sticticalis’s major sex pheromone compound, E11-14:OAc. Furthermore, this study offers valuable insights into the sexual communication behavior of L. sticticalis, forming a foundation for further analysis of the species’ central nervous system.

Grassland locusts and grasshoppers play a vital role in driving the dynamic changes of grassland ecosystems. In this study, we propose a lightweight deep learning-based network model for accurate identification of locust and grasshopper genera. Two image datasets were constructed, each containing 60 genera of locusts and grasshoppers. To improve recognition accuracy and computational efficiency while reducing floating-point operations (FLOPs) and the number of parameters, we introduced the channel-wise principal-component attention (CPCA) attention mechanism module and replaced part of the EfficientNet convolution modules with GhostConv, which incorporates the efficient channel attention (ECA) attention mechanism, thereby developing the CGENet model. During training, transfer learning and the Adam optimization algorithm were employed, significantly enhancing accuracy. This study makes precise control of locusts and grasshoppers feasible, thereby helping to reduce the damage they cause to agricultural production.

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.