# Screening and Identification of Reference Genes for Paracarophenax alternatus

## TL;DR

This study identifies GAPDH and RPS18 as the most stable reference genes for Paracarophenax alternatus, a mite used in controlling pine wilt disease.

## Contribution

The study provides validated reference genes for gene expression analysis in Paracarophenax alternatus across different life stages.

## Key findings

- GAPDH and RPS18 were identified as the most stable reference genes using multiple analytical methods.
- qRT-PCR showed amplification efficiency between 90% and 102% for the candidate genes.
- EF1A was not suitable as a reference gene due to a p-value exceeding 0.05.

## Abstract

In this study, we focused on the screening and identification of reference genes for Paracarophenax alternatus Xu and Zhang, which is the primary natural enemy mite targeting the eggs of Monochamus alternatus Hope and has significant potential in the control of pine wilt disease. Samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy, and the expression levels of seven candidate reference genes were detected through qRT-PCR. According to the ΔCt method, as well as the GeNorm, NormFinder, and BestKeeper software, GAPDH and RPS18 were the most stable and represented the most suitable combination of reference genes among the four life stages of P. alternatus. This result lays the foundation for subsequent gene expression studies of this mite, which is believed to play a key role in host-seeking abilities, contributing to research on the biological control of pine wilt disease.

In this study, we focused on the screening and identification of reference genes for Paracarophenax alternatus Xu and Zhang. The laboratory population was used as the laboratory population, and samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy. Then, the expression levels of seven candidate reference genes (α-tubulin, β-tubulin, RPS18, RPL13, GAPDH, EF1A, SDHA) were detected through qRT-PCR. Melting curves showed good gene specificity, and the amplification efficiency ranged from 90% to 102%. ΔCt analysis indicated that GAPDH was the most stable reference gene. The GeNorm software determined that the optimal number of reference genes was two, with GAPDH and RPS18 forming the most stable combination, and NormFinder identified RPS18 as the most stable reference gene. Although the BestKeeper software suggested that EF1A was the most stable, its p-value exceeded 0.05, rendering it unsuitable for use as a reference gene. Finally, through the RefFinder network tool, the most stable reference genes were identified as GAPDH and RPS18.