# Selection of Sclerodermus pupariae Reference Genes for Quantitative Real-Time PCR

## TL;DR

This study identifies RPS18 as the most stable reference gene for qRT-PCR in Sclerodermus pupariae, a parasitoid wasp used for pest control.

## Contribution

The study provides the first evaluation of reference gene stability in Sclerodermus pupariae across developmental stages and temperature conditions.

## Key findings

- RPS18 was identified as the most stable reference gene for qRT-PCR in Sclerodermus pupariae.
- Gene expression stability was evaluated across developmental stages and temperature conditions.
- Three software tools and RefFinder were used to validate reference gene stability.

## Abstract

The parasitoid wasp Sclerodermus pupariae exhibits strong active attack capabilities and is widely used in the control of wood-boring pests such as longhorn beetles and jewel beetles. Using three software tools and the online platform RefFinder, the expression levels of eight candidate reference genes were analyzed across different developmental stages to identify the most stable reference gene. The stability of the candidate reference genes was further validated under different temperature conditions. The RPS18 gene was identified as the most suitable reference gene for S. pupariae, providing a reliable molecular foundation for future molecular studies of this species. This can be used for the subsequent validation of standardized target gene expression in various tissue preparations and samples at each developmental stage, while also providing an important resource for studying the relevant biological mechanisms of S. pupariae.

S. pupariae is a newly discovered species of parasitoid wasps. Research into its development, behavioral genetics, and molecular mechanisms provides valuable insights for improving integrated pest management strategies. Quantitative real-time PCR (qRT-PCR) is the most commonly used method for analyzing gene expression. This method requires the identification of stable reference genes to accurately evaluate transcriptional level variations. In this study, eight candidate reference genes (TUB, TBP, RPS18, GAPDH, 18S rRNA, RPL32, Actin, and EF1-α) were identified and evaluated for their suitability as reference genes. Gene expression levels across different developmental stages were analyzed using three software tools, GeNorm, NormFinder, and BestKeeper, and the online tool RefFinder. The overall ranking of reference gene stability was as follows: RPS18 > 18S rRNA > RPL32 > GAPDH > Actin > TUB > TPB > EF1-α. Ultimately, RPS18 was determined to be the most stable reference gene.