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

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.


Introduction
Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae) is an important egg parasitoid wasp that preys on a range of fruit bugs and forest caterpillars including Halyomorpha halys Stal, Tessaratoma papillosa Drury, and Riptortus pedestris Fabricius (Zhao et al., 2019a;Chen et al., 2019;Mi et al., 2020;Chen et al., 2021;Zhao et al., 2021;Wei et al., 2022). In commercial settings, A. japonicus is used as a form of biological control agent that is reared within Antheraea pernyi Guerin-Meneville eggs (Lepidoptera: Saturniidae) (Zhao et al., 2021). Moderate springtime temperatures induce diapause in A. japonicus, resulting in delayed adult emergence and interfering with the optimal timing for pest control, thus limiting the biocontrol value of this wasp species (Zhao et al., 2019b;Zhao et al., 2021). Relative to A. japonicus individuals not in diapause, those in diapause exhibit higher survival rates following storage at 10°C for 180 days (Zhao et al., 2021). Our group recently conducted transcriptomic sequencing for non-diapause and diapause A. japonicus individuals (unpublished data), revealing the presence of many genes associated with energy metabolism, development, and oxidation-reduction reactions. Further studies of these genes, however, will be vital to clarify the molecular basis for diapause in A. japonicus. No published studies to date have sought to analyze A. japonicus gene expression profiles, highlighting the need for additional foundational research to support further study of this potentially economically valuable biocontrol species.
Quantitative real-time PCR (RT-qPCR) analyses offer a rapid, sensitive, efficient, accurate, and reproducible means of measuring gene expression under a range of analytical conditions while also permitting single nucleotide polymorphism and restriction fragment length polymorphism analyses (Wang et al., 2019). However, the reliability of RT-qPCR analyses is strongly dependent on factors including the quality and yield of utilized templates, the amplification efficiency of the selected primers, and underlying biological signal (Jiang et al., 2015;Shu et al., 2018;Hu et al., 2019). In the process of RT-qPCR, in order to ensure the accuracy of quantitative results and achieve normalization and correction of different samples, the introduction of internal reference genes is indispensable (Lu et al., 2013;Taylor et al., 2016). Housekeeping genes often used for this purpose across species and treatment conditions include 18S rRNA and the genes encoding tubulin, ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, a growing number of studies have revealed that these commonly utilized housekeeping genes do not exhibit stable expression patterns across all experimental conditions, potentially leading to skewed, inaccurate data. It is thus vital that the most appropriate reference genes for particular RT-qPCR applications be selected in a systematic manner to maximize the reliability and accuracy of associated experimental results.
Here, eight commonly utilized reference genes were evaluated to establish their value when normalizing RT-qPCR data for A. japonicus specimens. These genes included ribosomal protein S3a (RPS3a), GAPDH, ACTIN, elongation factor 1α (EF1α), TATA-box binding protein (TATA), ribosomal protein S6 (RPS6), β -tubulin (TUB), and ribosomal protein L13 (RPL13). All of which have been widely used as reference genes in different organisms because they are considered to have a uniform expression.The EF1α and TUB were thought to be good reference genes for most animals (Zhao et al., 2019a;Wang et al., 2021). However, the absence of poly-A in rRNA makes it impossible to isolate it using oligo (dT)-based techniques, which is a significant disadvantage when employing rRNA as a normalizer in RT-qPCR experiments (Xue et al., 2010). Thus, we focused on genes transcribed by RNA polymerase II whose transcripts can be isolated using oligo (dT), like the genes linked to GAPDH, ACTIN and ribosomal proteins (e.g., RPS3a, RPS6, and RPL13) (Galetto et al., 2013;Xu et al., 2021;Li et al., 2021;Shen et al., 2022). And the TATA gene is required for initiating the RNA polymerase (I, II, and III)-mediated transcription of genes with promoters with or without a TATA box (Pugh, 2000). So, Those eight reference genes were chosen. Expression dynamics for these candidate reference genes were compared with RefFinder, which integrates results from four different algorithms (BestKeeper, NormFinder, geNorm, and the ΔCt method). The stability of these genes was compared across three biotic conditions (sex, tissue type, or developmental stage) and one abiotic condition (diapause state). The goal of this study was to define the A. japonicus reference genes that are most appropriate for use across a range of experimental conditions. These data may offer value as a foundation for future more reliably RT-qPCR-based studies of A. japonicus.

Insect rearing
A. japonicus Ashmead specimens were obtained from the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences, Ministry of Agriculture and Rural Affairs-CABI Biosafety Joint Laboratory. The initial A. japonicus colony was established from brown marmorated stink bug (Halyomorpha halys Stal) eggs that had been parasitized under field conditions and were collected from the suburbs of Beijing. A. japonicus colonies were maintained for 1 year under laboratory conditions (24°C, 16 h light/8 h dark cycle) using Antheraea pernyi eggs. After newly emerging, a solution of 10% honey and water was used to feed A. japonicus individuals in a 32 cm × 25 cm × 9 cm plastic box under these same laboratory conditions.

Sample preparation Biotic factors
All A. japonicus developmental stages were analyzed for this study (including mature larvae (n = 8), pupae (n = 10), and male (n = 20) and female (n = 10) adult individuals sampled on day 1 of the corresponding stage. In addition, samples from the head, chest, and abdomen of~40 adult female individuals were collected for analysis.
Each treatment was repeated in triplicate (n = 15 individuals/ treatment). Samples were stored in 1.5 ml RNA-free tubes, snapfrozen with liquid nitrogen, and stored at −80°C.
All samples exhibited OD260/280 values from 1.9 to 2.1. First-strand cDNA synthesis was performed using a PrimeScript RT kit containing gDNA Eraser (Perfect Real Time, TaKaRa, China). All cDNA samples were diluted 10-fold for RT-qPCR analyses.

Primer design
For this study, RT-qPCR was used to examine the expression of eight commonly utilized reference gene candidates (Table 1), using primers designed with the PrimerQuest Tool (https://sg. idtdna.com/PrimerQuest/Home/Index) based on sequences derived from our recent transcriptomic analysis of A. japonicus (unpublished data).

Quantitative real-time PCR analysis
TB Green ® Premix Ex Taq ™ II (Tli RNaseH Plus) (Takara) and a CFX Connect Real-Time Instrument (Bio-Rad, CA, United States) were used for all RT-qPCR analyses, which were conducted in a total volume of 50 µl containing 25 µl of TB green, 2.5 µl of each primer (F + R; 100 µM), 2.5 µl of diluted cDNA, and 17.5 µl of RNase-free ddH 2 O. Amplification settings were as follows: 95°C for 30 s; 39 cycles of 95°C for 5 s and 55°C for 30 s; Melting curves for the product were determined by raising the temperature from 55°C to 90°C in sequential steps of 0.5°C for 1 s. For each primer, standard curves were produced using a two-fold dilution series of cDNA as the template according to the linear regression model. All analyses were Frontiers in Physiology frontiersin.org 03 conducted using three biological and three technical replicates. RT-qPCR amplification efficiency (E) were measured as follows: E = [10 (−1/slope)−1] × 100 (Pfaffl, 2001).

Data analysis
The number of cycle (Ct) values required for amplification levels to reach a fixed exponential phase threshold (set to 500 for all genes) during PCR analyses was used to compute gene expression. RefFinder (http://blooge.cn/RefFinder/), which is a tool that integrates results from the NormFinder (Andersen et al., 2004), geNorm (Vandesompele et al., 2002), ΔCt method (Silver, et al., 2006) and BestKeeper (Pfaffl et al., 2004)analytical tools, was utilized to assess the stability of these different A. japonicus reference gene candidates. In addition, the geNorm tool was utilized to establish the optimal number of reference genes based on V n /V n+1 values, with a V n /V n+1 > 0.15 necessitating the Frontiers in Physiology frontiersin.org 04 addition of an additional reference gene whereas no further reference genes are required when V n /V n+1 < 0.15, with 0. 15 thus serving as the threshold for additional reference genes gene incorporation (Vandesompele et al., 2002;Liang et al., 2014).

Amplification efficiencies
All eight of the tested reference gene candidates were expressed at detectable levels in the analyzed A. japonicus samples, with 1% agarose gel electrophoresis results for each transcript exhibiting a single amplicon of the expected size. Amplification efficiency for each gene was estimated using two-point standard curves with known RNA concentrations, while melt curves were used to confirm the specificity of these amplification results based on the presence of a single peak without any evidence of primer-dimer peaks (Figure 1). Amplification efficiency (E) values for these genes ranged from 99% to 107.67%, with an R 2 > 0.9909 (Table 1).

Expression profiles of candidate reference genes
Expression levels for these candidate reference genes varied widely across the tested experimental conditions with respect to the measured Ct values (Figure 2), ranging from a Ct value of 17.80 for GAPDH to 28.69 for TATA. Mean respective Ct values for TATA and TUB were 27.65 and 24.28, with these values being notably higher than those for other tested genes. Moderate expression was observed for the other candidate reference genes included in this study, with respective mean Ct values for EF1α, GAPDH, RPS6, RPS3a, RPL13, and ACTIN of 19.53,20.07,20.40,19.88,19.90,and 20.56. Analyses of the stability of A. japonicus reference genes under different experimental conditions and optimum number of genes for normalization Developmental stage analysis When analyzing A. japonicus samples collected during different stages of development, the geNorm, NormFinder, and ΔCt analytical approaches revealed RPS6 and RPL13 as the most stable reference genes (Table 2). In contrast, BestKeeper analyses suggested that Ef1α and RPS3a were instead the most stable, with ACTIN and GAPDH as the least stable genes. Per RefFinder, the overall rank order for the stability of these reference genes was: RPL13, RPS6, RPS3a, EF1α, TATA, TUB, ACTIN, GAPDH ( Figure 3A). In geNorm analyses, the pairwise V2/3 value was below the established 0.15 cut-off threshold ( Figure 4). Overall, RefFinder thus suggested that RPL13 and RPS6 are the most appropriate target genes when analyzing A.

Tissue-based analyses
Different reference genes were found to be the most stable when performing tissue-based analyses of A. japonicus samples using these different software programs (Table 2). Specifically, RPS3a and RPL13 were identified as the most stable reference genes in these different tissue types according to geNorm, while EF1α and ACTIN were the most stable according to NormFinder, EF1α and TUB were the most stable according to BestKeeper, and the ΔCt identified ACTIN and RPS6 as the Frontiers in Physiology frontiersin.org 07 most stable reference gene candidates. GAPDH was identified as being the least stable reference gene in the geNorm, NormFinder, and BestKeeper analyses. Per RefFinder, the overall rank order for the stability of these reference genes was: ACTIN, RPS6, RPL13, EF1α, RPS3a, TUB, TATA, GAPDH ( Figure 3B). The pairwise V2/V3 value identified based on geNorm data was below the established 0.15 cut-off threshold, although the V7/8 value was above this threshold ( Figure 4). Overall, the RefFinder analyses indicated that ACTIN and RPS6 are the most appropriate reference genes for use when normalizing A. japonicus gene expression across different tissue types (Table 2).

Sex-based analyses
When comparing gene expression results for male and female A. japonicus specimens, the geNorm, NormFinder, and ΔCt methods identified EF1α and RPL13 as the most stable candidate reference genes (Table 2). While BestKeeper found ACTIN and GAPDH to be the most stable genes, the other three analytical tools suggested that GAPDH was instead the least stable reference gene. Per RefFinder, the overall rank order for the stability of these reference genes was: RPL13, EF1α, TUB, RPS6, ACTIN, RPS3a, GAPDH, TATA ( Figure 3C). In the geNorm analysis, the V2/V3 pairwise value was below the established 0.15 cut-off threshold (Figure 4). Overall, these RefFinder results suggest that RPL13 and EF1α are the most appropriate reference genes for use when normalizing gene expression levels in A. japonicus specimens of different sexes (Table 2).

Diapause treatment analyses
When evaluating the different tested diapause treatments, ACTIN and TATA were identified by geNorm analyses as the most stable reference genes, whereas RPS6 was the least stable. The ΔCt and NormFinder analyses also yielded similar results (Table 2). BestKeeper, in contrast, identified RPL13 and EF1α as being the most stable. Per RefFinder, the overall rank order for the stability of these reference genes was: TATA, ACTIN, RPL13, RPS3a, EF1α, TUB, GAPDH, and RPS6 ( Figure 3D). All pairwise variation values were under the 0.15 cut-off threshold in geNorm analyses (Figure 4). According to the RefFinder results, ACTIN and TATA were thus the most reliable reference genes when normalizing target gene expression over a range of diapause treatment conditions.

All conditions
When evaluating all treatment conditions, the ΔCt and geNorm analyses revealed that RPL13 and RPS3a were the most stable reference genes (Table 2). From most to least stable, the RefFinder analysis ranked the stability of these candidate A. japonicus reference genes as follows: RPS3a, RPL13, ACTIN, TUB, EF1α, RPS6, TATA, GADPH ( Figure 3E). In geNorm analyses, all pairwise variation values were under the selected cut-off value of 0.15 (Figure 4). Overall, this RefFinder analysis indicated that RPS3a and RPL13 are the most appropriate genes for internal reference use when normalizing A. japonicus gene expression in RT-qPCR analyses (Table 2).

Reference gene validation
FD (Farnesol dehydrogenase) is an important enzyme in the diapause process of insects and can oxidize farnesol to farnesol (the precursor of JH) (Mayoral et al., 2009). Relative FD expression levels were next used for the validation of these reference genes among the tested diapause treatment conditions. In general, expression patterns were similar across conditions, although these expression levels were 54-and 85-fold higher on diapause day 25 relative to non-diapause treatment when respectively normalized to the most stable and least stable reference genes ( Figure 5). When using ACTIN and TATA, which were the most stable reference genes, significant increases in FD expression were observed in D55 relative to D25 samples, whereas no significant differences were evident between these two time points when using unstable reference genes such as GAPDH and RPS6.

Discussion
Appropriate reference gene selection is dependent on species-and development stage-specific variability. For example, EF1α and Arm have been identified as reliable Frontiers in Physiology frontiersin.org targets for normalizing RT-qPCR data derived from Australian plague locusts under varying rearing density conditions , while GAPDH and UCCR have served as Spodoptera litura reference genes (Lu et al., 2013). Indeed, no one reference gene or set of reference genes is universally appropriate across insect species. Zhang et al. (2015) demonstrated that while RPS15 and RPL13 were the most stable reference genes when comparing a range of Helicoverpa armigera larval tissue types, RPL27 and EF were more appropriate when evaluating adult samples. Five studies have explored reference gene selection in Bemisia tabaci, yet these studies have yielded conflicting results Su et al., 2013;Collins et al., 2014;Liang et al., 2014;Dai et al., 2017).
To date, reliable reference genes have been identified for many Hymenoptera species (Table 3), including Cotesia chilonis (Li et al., 2019), Apis mellifera (Lourenço et al., 2008), Lysiphlebia japonica (Gao et al., 2017a), Aphidius gifuensis (Gao et al., 2017b), Tamarixia radiate (Guo et al., 2020), and Solenopsis invicta (Cheng et al., 2013). A. japonicus is an important natural predator of a range of agricultural pests, and further molecular analyses of this species may thus support further biocontrol efforts. However, no prior studies have selected or validated reference genes in this parasitic wasp species. As such, eight commonly utilized reference genes were herein analyzed for their stability under four different sets of experimental conditions using a panel of software programs.
Ribosomal proteins are highly conserved across different species (Petibon et al., 2021), and many ribosomal genes have successfully been leveraged as stable reference genes when conducting transcriptomic analyses (Liu et al., 2016;Gao et al., 2017a). Consistently, RPS3a and RPL13 were herein identified as the most stable A. japonicus reference genes across all conditions. In a prior study of Agasicles hygrophilao, these two genes were similarly found to exhibit stable expression in most tested experimental samples (Wang et al., 2019).In the present study, RPS6 was the second most stable reference gene across different tissue types and developmental stages in A. japonicus, while RPL13 was the most effective reference target when analyzing samples from different developmental stages or adults of different sexes. In Rhopalosiphum padi, RPS18 and RPL13 have been reported as the most stable reference genes across tissue types, while RPS6 was the second most stable such reference gene in response to temperature or antibiotic treatment conditions .
ACTIN is an integral component of the cytoskeleton, controlling the structural integrity of cells such that it can be readily leveraged as a reference gene . Indeed, ACTIN was found to be the most stable tested reference gene in A. japonicus under different tested diapause treatment conditions and in various tissues, in line with prior evidence regarding insecticide stress conditions in Locusta migratoria (Yang et al., 2014) and Spodoptera litura (Lu et al., 2013). Similarly, ACTIN showed high stability in other insects under different experimental sets, for the Orchesella cincta, ACTIN was the dominant most stable gene under temperature, desiccation and starvation treatments (de Boer et al., 2009), while high levels of ACTIN stability were evident in Bradysia odoriphaga specimens exposed to insecticides including chlorfluazuron or chlorpyrifos, in addition to remaining stable across developmental stages and tissue types (Fu et al., 2022). However, these results are not universal, with ACTIN having been established as the least stable gene in Henosepilachna vigintioctopunctata across different tissues, host plants, and stages of development (Lü et al., 2018). These findings emphasize the importance of individually evaluating reference gene stability under experimental conditions of interest. Here reference gene stability was tested with the geNorm, NormFinder, BestKeeper, and ΔCt programs, and these different software applications each yielded differing results with respect to target gene stability. Under the developmental stages, the analysis results of NormFinder, geNorm and ΔCt are relatively similar, and the analysis results of BestKeeper are quite different from the other three analysis results. For example, RPS6/ RPL13 ranks as the most stable reference gene in NormFinder, geNorm and ΔCt, but ranks 4th and 3rd in BestKeeper, respectively. Inconsistent results were obtained for these four software programs across tissue types, with RPS3a/ RPL13, EF1α/Actin, TUB, and ACTIN ultimately being the most stable reference genes. When comparing specimens of different sexes, NormFinder and geNorm yielded similar results with EF1α and RPL13 as the two top-ranked genes, whereas TATA, Actin, and GAPDH were the bottom-ranked genes. In contrast, BestKeeper identified Actin and GAPDH as the two top-ranked genes. Under the tested diapause conditions, TATA and ACTIN were the top-ranked reference genes according to geNorm, NormFinder, and ΔCt, with Actin and TATA respectively being ranked first in the NormFinder and were TATA and ΔCt analyses. However, BestKeeper ranked RPL13 and EF1α as the two top genes. These inconsistent results across programs are attributable to the differences in the calculation strategies that they employ, with NormFinder and geNorm yielding more stable results for each reference gene. geNorm calculates M values for each reference gene to determine the optimal reference gene, with NormFinder using a similar approach while also analyzing each independent internal reference gene (Fu et al., 2013). BestKeeper produces correlation coefficients, standard deviations, and coefficients of variation for each gene pairing, enabling the simultaneous analysis of up to 10 genes (Chen and Lu, 2014). The inconsistent ranked lists produced by these programs can Frontiers in Physiology frontiersin.org make the selection of optimal reference genes challenging. As such, RefFinder was used in this study owing to its ability to integrate these algorithms and to thereby better clarify overall candidate gene stability. These results revealed marked differences in the optimal internal reference genes for use when comparing different sample types. This aligns well with growing interest in recent years to utilize more than one reference gene when normalizing gene expression data . The geNorm software offers the ability to establish the optimal number of reference genes for use through the calculation of pairwise variation (V n/n + 1 ) based on normalization factors NF n and NF n + 1 , where n is greater than or equal to 2. Using this algorithm, n is defined as the optimal number of reference genes when V n/n + 1 is under 0.15. Accordingly, the optimal number of reference gene combinations identified herein when comparing A. japonicus samples corresponding to different development stages, tissue types, sexes, and diapause treatments were RPL13 and RPS6, ACTIN and RPS6, RPL13 and EF1α, and ACTIN and TATA, respectively.

Conclusion
In conclusion, these results underscore the importance of selecting application-specific reference genes when conducting RT-qPCR analyses of A. japonicus, as no one reference gene is optimal under all conditions. In this study, the most stable sets of reference genes were compared, leading to the identification of a combination of RPS3a and RPL13 as the most appropriate reference genes on average across the tested experimental conditions. However, the ideal reference genes ultimately varied among experiments, with RPL13 and RPS6 being the most reliable for developmental stagebased analyses, ACTIN and RPS6 as the most reliable when comparing different tissue types, ACTIN and TATA as the most stable when comparing different diapause treatments, and RPL13 and EF1α as the most stable when analyzing female and male A. japonicus specimens (de Boer et al., 2009;Chen and Lu, 2014;Lü et al., 2018;Wang et al., 2020).

Data availability statement
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Author contributions
CZ and ZL conceived and designed research. ZL and JX conducted the experiments. ZL analyzed the data and wrote the manuscript. YX and QW contributed material. CZ and DL revised the manuscript. All authors have read and approved the manuscript.