Screening and Identification of Reference Genes for Paracarophenax alternatus
by
Yangming Zhang
1,2,†, 
Xu Chu
1,2,†,
Ruiheng Lin
1,2,
Yunfeng Zheng
1,2,
Sikai Ke
1,2,
Feiping Zhang
1,2,* and
Songqing Wu
1,2,* 1
Forestry College, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Key Laboratory of Integrated Pest Management in Ecological Forests, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Insects 2026, 17(1), 7; https://doi.org/10.3390/insects17010007
Submission received: 20 October 2025
/
Revised: 15 December 2025
/
Accepted: 17 December 2025
/
Published: 20 December 2025
(This article belongs to the Section Insect Molecular Biology and Genomics)
Simple Summary
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.
Abstract
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.
