The basic helix–loop–helix (bHLH) transcription factors possess DNA-binding and dimerization domains and are involved in various biological and physiological processes, such as growth and development, the regulation of secondary metabolites, and stress response. However, the bHLH gene family in C. tinctorius has not been investigated. In this study, we performed a genome-wide identification and analysis of bHLH transcription factors in C. tinctorius. A total of 120 CtbHLH genes were identified, distributed across all 12 chromosomes, and classified into 24 subfamilies based on their phylogenetic relationships. Moreover, the 120 CtbHLH genes were subjected to comprehensive analyses, including protein sequence alignment, evolutionary assessment, motif prediction, and the analysis of promoter cis-acting elements. The promoter region analysis revealed that CtbHLH genes encompass cis-acting elements and were associated with various aspects of plant growth and development, responses to phytohormones, as well as responses to both abiotic and biotic stresses. Expression profiles, sourced from transcriptome databases, indicated distinct expression patterns among these CtbHLH genes, which appeared to be either tissue-specific or specific to certain cultivars. To further explore their functionality, we determined the expression levels of fifteen CtbHLH genes known to harbor motifs related to abiotic and hormone responses. This investigation encompassed treatments with ABA, salt, drought, and MeJA. The results demonstrated substantial variations in the expression patterns of CtbHLH genes in response to these abiotic and hormonal treatments. In summary, our study establishes a solid foundation for future inquiries into the roles and regulatory mechanisms of the CtbHLH gene family.

Reliable identification of pest developmental stages provides a foundation for investigating aphid development and adaptive mechanisms and informs the selection of timely interventions in integrated pest management. In this study, eight morphological indicators of Uroleucon gobonis were distinguishing under a stereomicroscope. Additionally, six candidate genes identified from transcriptomic data were selected to characterize their expression profiles across five developmental instars. These results showed that the body length enabled distinguishing of the aphids from the first instar to the fourth instar, while antennal length and cauda length facilitated distinction between the second–fourth instar nymphs; cornicle length allowed separation of the third instar and subsequent instars. In contrast, body width permitted discrimination primarily between the first and second instars; head width, foreleg length, and hindleg length exhibited substantial overlap across instars, limiting their utility for instar discrimination. Elevated expression of DN1031, DN1019, and DN1093 was a prominent feature of first instar nymphs. DN1098 was persistently increased from the first to the third instar, which facilitated discrimination among these three developmental stages. The fourth instar was characterized by the concurrent down-regulation of DN1098 and DN1031 relative to the third instar, whereas adults exhibited a distinct expression peak of DN136 compared to other developmental stages. This study establishes an integrated instar-identification system for U. gobonis, combining rapid morphological screening with molecular characterization. This framework provides a valuable methodological basis for investigating developmental plasticity and supports the development of targeted pest management strategies.

WRKY is a crucial transcription factor involved in plant growth, development, and responses to abiotic stress. In the present study, a total of 182 AaWRKY transcription factor members were identified across the Artemisia argyi genome and found to be distributed across 17 chromosomes. Evolutionary analysis revealed that segmental duplication served as the primary driver for family expansion, with the evolutionary trajectory shaped by strong purifying selection (Ka/Ks < 1.0). Phylogenetic classification categorized these members into seven highly conserved subgroups, while physicochemical analysis indicated that most AaWRKYs are unstable, hydrophilic proteins, consistent with the rapid turnover required for transcriptional switches. Transcriptomic profiling unveiled significant tissue-specific expression patterns, with over 50% of the members predominantly enriched in roots and specific genes, such as AaWRKY11, implicated in the regulation of leaf senescence. Protein–protein interaction (PPI) network analysis identified AaWRKY110 as a central regulatory hub linking the MAPK signaling pathway with the isoflavonoid biosynthetic machinery. Furthermore, comparative transcriptomic analysis between aphid-resistant (Ai20K) and susceptible (Ai72G) cultivars demonstrated that resistance is conferred by a priming mechanism involving high basal expression of key candidates, including AaWRKY82, 108, 128, and 71. In contrast, the susceptible genotype exhibited a delayed and ineffective hypersensitive-like response. Collectively, these findings elucidate the evolutionary dynamics of the AaWRKY family and provide critical genetic targets for the concurrent improvement of medicinal metabolite accumulation and biotic stress resilience in Artemisia argyi via molecular breeding.