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. 2023 May 12;14(5):456.
doi: 10.3390/insects14050456.

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

Ya Liu  1 , Jing Zhou  1 , Zhisong Qiu  1 , Ping Hu  1   2 , Xiao Chen  1 , Zhende Yang  1   2
Affiliations

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

Ya Liu et al. Insects. .

Abstract

Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa's target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes' stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

Keywords: Leptocybe invasa; RT-qPCR; gene stability; reference genes; target genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Reference gene expression levels under various experimental conditions: (A) sex, (B) somite, (C) temperature, (D) diet, (E) pesticide, and (F) all samples. The violin diagram’s white dot depicts the median Ct value, while the black bar indicates the interquartile range. The width of the violin is the richness of this set of data at this value of the vertical coordinate (frequency of each y-axis data). The different colors in the six violin diagrams represent different genes in the same order, from left to right, RPS30, ACTR, ACT, RPL18, GAPDH, 18S rRNA, 28S rRNA, and TUB.
Figure 2
Figure 2
Evaluation of the optimum amount of housekeeping genes under different experimental conditions of L. invasa. When the V value is less than 0.15, there is no need to add additional internal reference genes for normalization.
Figure 3
Figure 3
The expression stability values (M) of the eight housekeeping genes were verified by the geNorm program. The least stable genes with higher M values are on the left side, and the steadiest genes with lower M values are on the right.
Figure 4
Figure 4
The eight housekeeping genes were used as the subject of stability assessments by NormFinder, BestKeeper, comparative ∆Ct, and RefFinder. The pane’s smaller value and lighter hues show the reference gene’s stability.
Figure 5
Figure 5
Eight housekeeping genes for L. invasa were ranked for stability under various treatment conditions using RefFinder.
Figure 6
Figure 6
Relative expression levels of HSP90 under diverse temperatures in adult L. invasa using different housekeeping genes.
See this image and copyright information in PMC

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