# Identification and Stability Assessment of Reference Genes in Helicoverpa armigera Under Plant Secondary Substance and Insecticide Stresses

## TL;DR

This study identifies stable reference genes in cotton bollworms under plant and insecticide stress, helping improve gene expression research and pest control strategies.

## Contribution

The study provides condition-specific reference gene combinations and highlights GADD45's role in detoxification under stress.

## Key findings

- RPL32 and RPL27 showed consistent expression under plant and insecticide stress, respectively.
- GADD45 was significantly upregulated in response to various plant compounds and insecticides.
- Optimal reference gene combinations vary depending on the stress type and experimental condition.

## Abstract

The cotton bollworm (Helicoverpa armigera), a globally significant agricultural pest, displays notable host adaptability and insecticide resistance. This study utilized five algorithms to assess the expression stability of eleven candidate reference genes in H. armigera under stress from four plant secondary substances (tannic acid, quercetin, 2-tridecanone, and ZQ-8) and two insecticides (chlorantraniliprole and indoxacarb). The findings indicated that the optimal combination of reference genes varied across different stress conditions. Additionally, ribosomal protein RPL32 and RPL27 exhibited relatively consistent expression levels under plant secondary substance and insecticide treatments, respectively. Additionally, larvae exposed to sublethal doses of plant secondary substances and insecticides showed a significant upregulation of growth arrest and DNA-damage-inducible gene 45 (GADD45) expression, indicating its involvement in the detoxification metabolism of H. armigera. This research establishes a standardized foundation for using real-time quantitative polymerase chain reaction (qPCR) to explore the molecular regulatory pathways of H. armigera in response to plant secondary substance and insecticide stress. It also identifies potential novel target sites for the management of insecticide resistance in this pest.

The cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae) is a globally distributed agricultural pest. When conducting expression analysis of its functional genes, appropriate reference genes should be selected to ensure the reliability of the results. In this study, five algorithms including Delta Ct, GeNorm, Normfinder, BestKeeper, and RefFinder were used to evaluate the expression stability of eleven candidate reference genes under different developmental stages, larval tissues, adult sexes, plant secondary substance stresses, and insecticide treatments in H. armigera. The candidate genes included Actin, Tubulin, EF-1α, RPS3, RPS15, RPL27, RPL32, 28S, GAPDH, SOD, and TRX. The reliability of the recommended reference gene combinations was validated using the growth arrest and DNA-damage-inducible gene 45 (GADD45). The results showed that normalizing relative expression of the target gene with the combination of the two most stable reference genes is recommended. Specifically, the combination of RPS3 + RPL27 is recommended for developmental stage comparisons; RPL32 + RPL27 for larval tissues; RPS3 + RPL27 for adult sex comparisons; GAPDH + RPL32 under tannic acid stress; RPL32 + RPS3 under quercetin stress; RPS15 + RPL32 under 2-tridecanone stress; RPS3 + RPL32 under ZQ-8 stress; RPL27 + TRX following chlorantraniliprole treatment; and RPL27 + RPL32 following indoxacarb treatment. Moreover, larvae exposed to three concentrations of plant secondary substances and to sublethal doses of insecticides exhibited significant upregulation of GADD45: after 4 h of exposure to 1% tannic acid, 0.1% and 1% quercetin, 1% 2-tridecanone, and 0.05% ZQ-8; after 15 h of chlorantraniliprole treatment; and after 24 h of indoxacarb treatment. Thus, GADD45 was overexpressed in response to various plant secondary substances and insecticide treatments, indicating its involvement in the detoxification and metabolism of H. armigera. This study proves to be helpful for selecting reference genes in H. armigera under plant secondary substance and insecticide stress and lays the foundation for further research utilizing GADD45 as a molecular target for pest control.