Selection and validation of reference genes for RT-qPCR-based analyses of Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae)

Abstract

RT-qPCR remains a vital approach for molecular biology studies aimed at quantifying gene expression in a range of physiological or pathological settings. However, the use of appropriate reference genes is essential to attain meaningful RT-qPCR results. Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae) is an important egg parasitoid wasp and natural enemy of fruit bugs and forest caterpillars. While recent transcriptomic studies have analyzed gene expression profiles in A. japonicus specimens, offering a robust foundation for functional research focused on this parasitoid, no validated A. japonicus reference genes have yet been established, hampering further research efforts. Accordingly, this study sought to address this issue by screening for the most stable internal reference genes in A. japonicus samples to permit reliable RT-qPCR analyses. The utility of eight candidate reference genes (ACTIN, TATA, GAPDH, TUB, RPL13, RPS6, EF1α, RPS3a) was assessed under four different conditions by comparing developmental stages (larvae, pupae, adults), tissues (abdomen, chest, head), sex (male or female adults), or diapause states (diapause induction for 25, 35, 45, or 55 days, or diapause termination). RefFinder was used to calculate gene stability based on the integration of four algorithms (BestKeeper, Normfinder, geNorm, and ΔCt method) to determine the optimal RT-qPCR reference gene. Based on this approach, RPS6 and RPL13 were found to be the most reliable reference genes when assessing different stages of development, while ACTIN and EF1α were optimal when comparing adults of different sexes, RPL13 and EF1α were optimal when analyzing different tissues, and TATA and ACTIN were optimal for different diapause states. These results provide a valuable foundation for future RT-qPCR analyses of A. japonicus gene expression and function under a range of experimental conditions.

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

“…Actin genes include ACT8 and ACT11, which we have long used as housekeeping genes [39]. In fact, under various diapause states and in different tissues, Anastatus japonicus [17] displayed that ACTIN was the most reliable, consistent with earlier findings on Locusta migratoria [40] and Spodoptera litura [41]. In this study, ACT expresses steadily in most cases.…”

“…RefFinder is a comprehensive evaluation tool that generates stability scores by estimating the geometric mean of internal control genes to lessen the effects of a single algorithm’s limitations [ 36 ]. Many species, including Neoceratitis asiatica [ 18 ] and Anastatus japonicus [ 17 ], were studied using a similar strategy. The expression of HSP90 under different temperature treatments was evaluated to authenticate our findings.…”

“…Yet, many researchers have used RT-qPCR to analyze the internal reference genes, and the results demonstrate that no gene can exhibit steady expression in various experimental conditions. Benchmark genes for RT-qPCR analysis depend on the specific experimental context [ 15 , 17 ]. In the case of Anastatus japonicus development, RPS6 and RPL13 were determined to be perfect.…”

“…When analyzing diverse tissues, RPL13 and EF1α performed better than other genes. Finally, TATA and ACTIN were recognized as excellent for evaluating distinct diapause conditions [ 17 ]. RPS18 and EF1α were the two trustiest genes in Neoceratitis asiatica , whereas RPS15 and EF1β were the most untrustworthy [ 18 ].…”

Identification and Comparative Expression Profiles of Candidate Olfactory Receptors in the Transcriptomes of the Important Egg Parasitoid Wasp Anastatus japonicus Ashmead (Hymenoptera: Eupelmidae)

“…Studies concerning the ORs and IRs of parasitoid wasps are essential for understanding the mechanisms of the olfactory system that finds the host pests and for developing an olfactory-based integrated pest management (IPM) strategy. Previous research on A. japonicus focused primarily on its morphology, ecology, and raising [ 1 , 5 , 6 , 7 , 9 , 35 , 36 , 37 ]; little information is available on its chemosensory mechanisms. Two previous studies have reported transcriptome datasets from female A. japonicus antennae [ 38 , 39 ], but neither of these studies involved the female abdominal and male antennal transcriptomes.…”