The biological insecticide, Ectropis obliqua nucleopolyhedrovirus (EcobNPV), has been applied to control the major tea-pest Ectropis grisescens. Previously, the virus strain EcobNPV-QF4 showed higher a mortality rate (58.2% vs. 88.2%) and shorter median lethal-time (13.9 d vs. 15.4 d) on E. grisescens than the strain EcobNPV-QV. However, the mechanism of the difference in virulence between the two strains remains unclear. Using the leaf-disc method, we detected the virulence of the two strains on 3^rd-instar larvae, and found that median lethal-dose (LD₅₀) of EcobNPV-QF4 is 55-fold higher than that of EcobNPV-QV (4.35 × 10⁸ vs. 7.89 × 10⁶). Furthermore, fourteen larva transcriptomes of E. grisescens were subsequently sequenced at seven time-points after ingestion of the two virus strains, yielding 410.72 Gb of raw reads. Differential gene-expression analysis shows that 595, 87, 27, 108, 0, 12, and 290 genes were up-regulated in EcobNPV-QF4 at 0, 2, 6, 12, 24, 36 h and 48 h post ingestion (hpi), while 744, 68, 152, 8, 1, 0, 225 were down-regulated. KEGG enrichment showed that when the virus first invades (eats the leaf-discs), EcobNPV-QF4 mainly affects pathways such as ribosome (p-value = 2.47 × 10⁻²⁹), and at 48 hpi EcobNPV-QF4, causes dramatic changes in the amino-acid-synthesis pathway and ribosome pathway (p-value = 6.94 × 10⁻¹³) in E. grisescens. Among these, thirteen key genes related to immunity were screened. The present study provides the first ever comprehensive analysis of transcriptional changes in E. grisescens after ingestion of the two strains of EcobNPV.

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, P_hp, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter P_hp was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40–80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains.

Ectropis obliqua nucleopolyhedrovirus (EcobNPV) is an important biocontrol agent against Ectropis obliqua and E. grisescens. A previously isolated strain, EcobNPV-QF4, exhibits significantly higher virulence than the original strain EcobNPV-QV, yet the molecular basis for this difference remains unclear. Leaf-dipping bioassays demonstrated that EcobNPV-QF4 caused significantly higher larval mortality than EcobNPV-QV from 10 dpi onward, reaching 97.9% versus 33.8% at 16 dpi, and higher pupal mortality (100% versus 47.3%), confirming its superior virulence across both larval and pupal stages. To investigate the transcriptional dynamics underlying virulence variation, we performed a comparative time-course transcriptomic analysis of E. grisescens infected with either strain at 0, 2, 6, 12, 24, 36, and 48 h post-infection. The reliability of the RNA-seq data was validated by qRT-PCR for selected expressed genes. The results showed that, relative to EcobNPV-QV, the EcobNPV-QF4 strain exhibits a transcriptional strategy characterized by comprehensive and accelerated activation: the transcriptional initiation of its functional modules is globally advanced by approximately 6 h compared with EcobNPV-QV, and genes associated with midgut escape, virion assembly, and viral DNA replication are preferentially and coordinately expressed, resulting in a more synchronized transcriptional profile. Based on these findings, we hypothesize that the enhanced virulence of EcobNPV-QF4 is not attributable to a single factor but rather reflects a synergistic, multitiered acceleration of transcriptional progression that may compress the infection cycle and enhance viral dissemination efficiency. These findings provide critical insights into the molecular mechanisms that may underlie baculovirus virulence and provide a basis for future mechanistic studies.

Ectropis grisescens (Lepidoptera: Geometridae) is a destructive pest in tea plantations, leading to significant economic losses through defoliation. Existing control strategies, including chemical insecticides and biological agents, are often limited by environmental concerns, resistance, and variable efficacy. Recent evidence suggests that bacteria influence insect physiology and could be leveraged for pest management, but the postmortem microbial ecology of E. grisescens remains uncharacterized. In this study, we employed 16S rRNA sequencing to investigate temporal changes in the bacterial communities of E. grisescens cadavers at 0, 7, and 21 days following cryogenic mortality. Our results indicate a time-dependent decline in microbial diversity, while species richness initially increased before subsequent reduction. The dominant endosymbiont Wolbachia gradually diminished after host death, whereas Enterobacter remained abundant. Notably, non-dominant genera including Lysinibacillus and Sporosarcina exhibited a transient increase in abundance at day 7 before reverting to control levels by day 21. This study presents the first comprehensive analysis of postmortem microbial succession in a lepidopteran system, highlighting dynamic shifts in bacterial composition and offering potential avenues for microbiome-based pest management strategies.