Selection of reference genes for RT-qPCR analysis of wing dimorphism in English grain aphid, Sitobion avenae (Hemiptera: Aphididae)

Authors: LI, XIANGRUI - Chinese Academy Of Agricultural Sciences; LI, XINAN - Chinese Academy Of Agricultural Sciences; YAN, WEIWEI - Chinese Academy Of Agricultural Sciences; Coates, Brad; ZHOU, XUGUO - University Of Kentucky; WANG, CHAO - Chinese Academy Of Agricultural Sciences; HAIFENG, GAO - Xinjiang Academy Of Agricultural And Reclamation Science; ZHANG, YUNHUI - Chinese Academy Of Agricultural Sciences; ZHU, XUN - Chinese Academy Of Agricultural Sciences

Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/2021
Publication Date: 11/25/2021
Citation: Li, X., Li, X., Yan, W., Coates, B.S., Zhou, X., Wang, C., Haifeng, G., Zhang, Y., Zhu, X. 2021. Selection of reference genes for RT-qPCR analysis of wing dimorphism in English grain aphid, Sitobion avenae (Hemiptera: Aphididae). Journal of Economic Entomology. 115(1):313-324. https://doi.org/10.1093/jee/toab214.
DOI: https://doi.org/10.1093/jee/toab214

Interpretive Summary: Aphids are insects that feed on crop plants, and impact corn, soybean and wheat crop health by spreading disease. Aphids have a unique biology where environmental conditions determine whether genetically identical clones from a single female either develop wings or remain wingless. For instance, winged aphids are more apt to develop when nutritional quality of host plants are poor. Thus, aphids quickly adapt to changing environments within a year by developing wings and flying to and infesting different host plants. The change in proportion of winged aphids is accompanied by changes in the expression of many genes, including genes involved in wing development. In order to understand this complex change, a USDA researcher worked within a collaborative team of domestic university and international researchers to validate laboratory assays that more accurately estimate the variation in gene expression between wingless to winged aphids. This work establishes appropriate protocols for accurately measuring gene expression changes under a broad range of laboratory conditions. These results are important for future investigations into mechanisms controlling wing development and migration of these insect crop pests, and may lead to novel strategies for the reduce levels of crop damage. This research will be of interest to university, government, and industry stakeholders, as well as regulatory agencies interested in understanding the factors affecting arthropod pest species movement within the agroecosystem.

Technical Abstract: The grain aphid, Sitobion avenae (Fabricius), is one of the most destructive pests of wheat crops worldwide, and exhibits plasticity in winged phenotype that facilitates adaptation to changes in seasonal conditions. Although the molecular basis is not fully understood, developmental signals in early nymphal stages are manifested in wing polyphenism traits among adults. Validated reference genes are needed to accurately normalize temporal and spatial variation in gene expression estimates by RT-qPCR. In this study, the stability of RT-qPCR for 11 S. avenae candidate reference gene transcripts were assessed across developmental stages, environmental stresses, and under conditions that affect wing dimorphism using a comprehensive ranking of delta Ct, BestKeeper, NormFinder and geNorm algorithm results with the RefFinder tool. Our results indicate helicase, HEL, is suitable across most of the biotic conditions. RpS27 is the second suitable reference gene in most of the treatment, such as the different developmental stages, thorax and abdomen tissues, different temperature, and alarm pheromone treatment. Ap-NADH and 28S is the most appropriate sets across insecticide and antibiotic treatment, which is also suitable for head tissue and winged tissue, respectively. This study is the first to validate a set of reference genes for wing dimorphism in S. avenae and establishes a standardized qRT-PCR normalization procedure for study of phenotypic plasticity.