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Article

Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses

1
Henan Key Laboratory of Crop Pest Control, Key Laboratory of Integrated Pest Management on Crops in Southern Region of North China, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
2
Luohe Academy of Agricultural Sciences, Luohe 462300, China
3
Institute of Plant Protection, Luoyang Academy of Agricultural and Forestry Sciences, Luoyang 471027, China
*
Author to whom correspondence should be addressed.
Insects 2024, 15(9), 661; https://doi.org/10.3390/insects15090661
Submission received: 29 July 2024 / Revised: 25 August 2024 / Accepted: 29 August 2024 / Published: 30 August 2024
(This article belongs to the Section Insect Molecular Biology and Genomics)

Abstract

:

Simple Summary

The dark black chafer, Holotrichia parallela Motschulsky (Coleoptera: Scarabaeidae), is an important subterranean insect in China due to its wide distribution and the high degree of damage it causes. Selecting stable reference genes is crucial for accurate quantitative polymerase chain reaction (qPCR) and gene expression analysis. This study evaluated the expression stability of 11 candidate reference genes in H. parallela under various biological conditions and environmental stresses. Our findings suggest that the optimum reference genes were as follows: RPL18 and RPL13a for developmental stages and RNAi conditions, RPL13a, RPL18, and RPL32 for female and male adults, RPL13a and RPS3 for different tissues, RPL32, RPL13a, and RPS3 for varying photoperiod conditions, and Actin and RPL13a for different temperatures. These discoveries will serve as a foundation for subsequent precise qPCR and gene expression studies in H. parallela and other closely related insect species.

Abstract

Holotrichia parallela is among the world’s most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in H. parallela research is lacking. In this study, 11 reference genes (GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB,and EF1A) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, RPL18 and RPL13a exhibited the greatest stability. RPL13a, RPL18, and RPL32 were the most stable genes in both male and female adults. Under differing tissue conditions, RPL13a and RPS3 stood out as the most reliable. Moreover, under varying photoperiod conditions, RPL13a, RPS3 and RPL32 were the most stable genes. Lastly, Actin and RPL13a were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in H. parallela using RT-qPCR.

Graphical Abstract

1. Introduction

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an efficient, dependable, and reproducible technique that facilitates the accurate measurement of gene expression during various biological processes [1]. A widely adopted strategy in RT-qPCR for normalizing gene expression data involves the concurrent measurement of the expression of an reference gene within the same sample, ensuring the accuracy and reliability of the results [1]. However, it is important to note that the levels of expression for frequently used reference genes may exhibit considerable variation across diverse experimental settings [2,3,4,5,6]. Therefore, it is advisable to conduct a thorough and species-specific investigation to ascertain appropriate reference genes [3,7].
Holotrichia parallela is an important subterranean pest in China because of its wide distribution and the significant damage it causes, which is usually between 20 and 30%. In severe cases, losses may exceed 50% [8,9,10]. The larvae, referred to as white grubs, dwell underground and consume the roots of crops [11]. This makes the larvae susceptible to infection by pathogens present in the soil [12].
In recent times, the adoption of agricultural practices, such as no-till and reduced tillage methods, along with straw return, has fostered favorable conditions for the survival and proliferation of white grubs, resulting in a significant increase in the H. parallela population [13]. Although chemical insecticides have been widely utilized to effectively control white grub infestations, this approach has unfortunately resulted in soil and groundwater contamination, leading to significant environmental issues [14]. Furthermore, the indiscriminate and improper utilization of chemical pesticides has fostered the emergence of resistance in grubs, thereby contributing to the progressive intensification of pest infestations in certain regions on an annual basis [12].
The advent of RNA interference (RNAi) technology has revolutionized the field of functional genomics, emerging as a potent tool for deciphering gene function and validating genetic targets [15,16]. RNAi serves as a pivotal strategy in agricultural plant protection [17]. Gene expression analyses are crucial for understanding the molecular mechanisms of physiological, developmental and reproductive processesin coleopteran insects, particularly given the high effectiveness and systemic penetration of RNAi in these species [15]. To date, these research efforts have successfully identified several genes in H. parallela [18,19,20,21]. However, to further our understanding and ultimately develop effective control strategies for this pest, it is imperative to precisely ascertain the gene expression levels of this pest under diverse biological conditions and environmental stresses.
The aim of this study was to establish a set of stably expressed reference genes in H. parallela for RT-qPCR analysis under various conditions, including different developmental stages, tissues, sexes, temperature treatments, light treatments, and RNAi treatments. To accomplish this, 11 potential reference genes were selected from the transcriptomes of H. parallela. These genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Vacuolar-type ATPase B (VATB), actin (Actin), elongation factor 1-alpha (EF1A), and ribosomal proteins (L7a, L13a, L18, L32, S3, S7, and S18). Many of these genes encode for proteins involved in essential cellular processes, such as glycolysis (GAPDH), energy transduction (VATB), cytoskeleton organization (Actin), protein synthesis (EF1A), and ribosome assembly (ribosomal proteins). All these genes are commonly used reference genes and were previously examined across other insect species [22,23,24]. The stability of these genes was then evaluated using four commonly used algorithms, namely BestKeeper, NormFinder, geNorm, and comparative ΔCt, as well as a comprehensive RefFinder program. These findings will enhance gene expression studies and facilitate gene function research in H. parallela, benefiting future investigations.

2. Materials and Methods

2.1. Experimental Materials and Treatment Methods

The H. parallela specimens utilized in the study were captured under a black light or collected from elm trees near the Henan Research and Development Center for Modern Agriculture, located in Yuanyang County, Henan, China (35°00′ N, 113°40′ E). Laboratory-reared H. parallela beetles were maintained under controlled conditions of 25 °C ± 1 °C temperature, 70% humidity, and a 16-hour light/8-hour dark photoperiod [25]. Samples were collected and dissected in accordance with rigorous experimental guidelines. The study examined the developmental stages of H. parallela, ranging from eggs to third-instar larvae. Adult tissues, including the head, prothorax, legs, and fat body, were dissected. Sex-specific samples, including the head and ovary or testis, were dissected for female and male adults, respectively.
The female beetles, comprising a total of 5 groups with 20 individuals in each, were placed in culture boxes with dimensions of 16 × 24 cm. Each culture box contained a layer of 30 cm of sterile soil. For the photoperiod experiments, adults were treated under five different light conditions (24L:0D, 16L:8D, 12L:12D, 8L:16D, 0L:24D) for four days [26]. Randomly, three beetles were chosen from within each group and dissected to isolate their heads.
Third-instar larvae were selected for temperature effect assessment [27] and cleaned before being subjected to treatments at 4 and 30 °C for 2 h each, with consistent humidity and light conditions between treatments.
Female beetles were fed elm leaves soaked in RNAi solutions (ddH2O, dsGFP, and dsFcp3C) for 11 days, as documented in prior research [20]. The ovaries of female beetles from each treatment were randomly selected, dissected, and transferred into sterile 1.5 mL centrifuge tubes.
The tissue dissection method for H. parallela was as follows. The samples to be dissected were fixed on paraffin dishes with clean insect needles and observed under a stereomicroscope. Chilled 1×PBS buffer solution was poured into the dish, and the tissues required for the experiment were dissected using dissecting forceps according to experimental requirements. Three biological replicates per sample group were frozen in liquid nitrogen and stored in 1.5 mL centrifuge tubes at −70 °C for future use.

2.2. RNA Isolation and Reverse Transcription

Total RNA was isolated from each sample using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA purity and concentration were analyzed spectrophotometrically using a Thermo NanoDrop. Subsequently, cDNA synthesis was performed using the PrimeScript™ RT reagent kit (Takara, Dalian, China).

2.3. Selection of Reference Genes and Primer Formulation

The sequences of 11 candidate reference genes commonly used in insect studies, including GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB, and EF1A, were obtained from the transcriptome data for H. parallela [20]. Primer design and evaluation were conducted using DNAMAN 8.0 software and Primer3 (v. 0.4.0) software (accessible at http://bioinfo.ut.ee/primer3/, accessed on 10 June 2022) following the principles for designing qRT-PCR primers.

2.4. Fluorescent Quantitative PCR Reaction Conditions

RT-qPCR was performed on the CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using TB Green® Premix Ex Taq™ II kit (Takara, Dalian, China). Primer sequences are detailed in Table 1. Each 10 μL reaction contained 5 μL of TB Green Premix, 0.4 μL of each primer, 0.8 μL of cDNA, and 3.4 μL of sterile water. The thermal cycling conditions included an initial denaturation step followed by 40 cycles of denaturation, annealing, and extension, and a final melting curve analysis. Melting curve and standard curve were conducted to ensure specificity and assess amplification efficiency. A standard curve was constructed using serial 10-fold dilutions of cDNA, which yielded the following five concentration gradients of cDNA templates: 100 (500 ng/μL), 10−1, 10−2, 10−3, and 10−4. The amplification efficiency (E) value was then calculated for all primers as a percentage, as follows: E = (10[−1/slope]−1) × 100%.

2.5. Assessment of Reference Gene Stability

To assess the stability of candidate reference genes, this study employed a range of algorithms, including geNorm [1], NormFinder [28], BestKeeper [29], and the ΔCt method [30], providing a comprehensive evaluation. After this thorough assessment, the RefFinder [31] was employed to compare and rank the candidate genes, facilitating their prioritization.

2.6. Validation of Selected Reference Genes

The forkhead transcription factor FoxL2 gene and the vitellogenin receptor (VgR) gene are associated with ovarian differentiation and egg development in insects. In this study, FoxL2 and VgR were chosen as the target genes. To standardize the data, diverse internal reference genes were utilized and their stability was verified. The expression profiles of the target genes, VgR and FoxL2, were determined utilizing the 2−ΔΔCt method. The SPSS Statistics 22 software package was then applied to evaluate the variance in expression levels of the genes of interest through an independent-samples t-test.

3. Results

3.1. Assessment of Primer Efficiency and Specificity in Amplification

Prior to assessing the suitability of reference genes, rigorous validation of the PCR amplification’s specificity and efficiency is essential. Each PCR amplicon was examined using a 1.5% agarose gel and displayed a single band that matched the anticipated length. Furthermore, the melting curves of PCR amplifications with each primer set exhibited a unique peak, demonstrating their specificity (Figure S1). All primer pairs, excluding the VATB primer, demonstrated high amplification efficiency (ranging from 105.82% to 114.35%), with regression coefficients exceeding the threshold of 0.990, indicating reliable qPCR results (Table 1, Figure S2).

3.2. Expression Profile of Reference Genes

The threshold-cycle (Ct) represents the transcript level of the mRNA. Under varying treatment conditions, the Ct values of the 11 candidate reference genes for H. parallela ranged from 13.48 to 30.01. The mean Ct values spanned a range, with RPL32 exhibiting the lowest value at 19.05 and VATB having the highest value among the samples at 24.36 (Figure 1). Based on the coefficient of variation (CV), RPL18 exhibited the highest stability (CV = 8.92) while RPL32 displayed the greatest variability (CV = 15.94). (Table S2).

3.3. Identifying Reference Genes with Consistent Expression Stability

The expression stability of 11 candidate internal reference genes in H. parallela under varying treatment conditions was assessed using the following four analysis methods: the ΔCt method, geNorm, NormFinder, and BestKeeper. RefFinder was used to comprehensively evaluate the expression stability of 11 candidate reference genes in H. parallela under different treatment conditions (Figure 2, Table S3).
For different developmental stages of H. parallela, the expression analysis of candidate reference genes was performed using the ΔCt method and NormFinder (Version dated 05/01-2015) software . It was found that RPL18, RPS3, and RPL13a showed the highest expression stability. The geNorm analysis revealed that RPL18 and RPL13a were the most stable reference genes, while BestKeeper analysis indicated that the reference gene VATB was the most stable. All four analyses revealed that RPS7 and EF1A were relatively unstable internal reference genes (Figure 2, Table S3). RefFinder analysis ranked the expression stability of candidate reference genes in H. parallela during development from highest to lowest, as follows: RPL18>RPL13a>RPS3>GAPDH>RPL32>VATB>Actin>RPL7A>RPS18>RPS7>EF1A (Figure 3, Table S3).
For female and male H. parallela tissues, analysis using the ΔCt method showed that RPL13a and RPS18 exhibited the most stable expression. The geNorm software (Version 3.5) evaluation recognized RPL32 and RPL13a as the top stable reference genes, whereas NormFinder pointed to RPL18 as exhibiting the highest expression stability. All three analyses revealed that the expression of GAPDH and EF1A exhibited relatively poor stability. However, the BestKeeper software (Version 1) analysis suggested that GAPDH and EF1A showed the most stable expression while RPL7A and Actin exhibited poorer stability (Figure 2, Table S3). The RefFinder analysis ranked the expression stability of candidate reference genes in male and female adult H. parallela tissues from highest to lowest, as follows: RPL13a>RPL18>RPL32>RPS18>RPS3>GAPDH>VATB>EF1A>RPS7>RPL7A>Actin (Figure 3, Table S3).
For different tissues of H. parallela, the ΔCt analysis and NormFinder revealed that RPL13a exhibited the most stable expression among the candidate internal reference genes. The geNorm analysis identified RPL32 and RPS3 as the most stable reference genes. All three analyses indicated relatively poor stability in the expression of VATB and EF1A. According to the BestKeeper analysis,RPL18 and RPL13a had the most stable expression, while VATB and RPL7A exhibited poorer stability (Figure 2, Table S3). The RefFinder analysis ranked the expression stability of candidate reference genes in different adult H.parallela tissues from highest to lowest, as follows: RPL13a>RPS3>RPL32>RPL18>GAPDH>Actin>RPS7>RPS18>RPL7A>VATB>EF1A (Figure 3, Table S3).
For different light exposure treatments, RPL32 and RPL13a were identified as the most stable reference genes in adult H. parallela based on the ΔCt and geNorm analyses. Specifically, NormFinder favored RPL32, while BestKeeper suggested GAPDH as the preferred option. All four analyses demonstrated that EF1A and Actin had relatively poor expression stability (Figure 2, Table S3). RefFinder analysis ranked the expression stability of candidate reference genes in H. parallela under varying light exposure conditions from highest to lowest, as follows: RPL32>RPL13a>RPS3>GAPDH>RPL18>RPS7>VATB >RPS18>RPL7A>EF1A>Actin (Figure 3, Table S3).
For samples of third-instar H. parallela larvae under different temperature treatments, Actin and RPL32 were identified as the most stable reference genes in adult H. parallela, based on the ΔCt and geNorm analyses. The geNorm analysis showed that RPL13a and RPL18 were the most stable, while all three analyses demonstrated that RPS3 and GAPDH were relatively unstable internal reference genes. The BestKeeper analysis identified EF1A and RPL18 as the most stable, while VATB and RPS3 exhibited poor stability (Figure 2, Table S3). The RefFinder analysis ranked the expression stability of candidate reference genes in third-instar H. parallela larvae samples under different temperature treatments from highest to lowest, as follows: Actin>RPL13a>RPL18>EF1A>RPL32>RPL7A>RPS7>VATB >RPS18>GAPDH>RPS3 (Figure 3, Table S3).
For samples of H. parallela under RNAi feeding treatment, RPL18 and RPL13a were identified as the most stable reference genes in adult H. parallela based on the ΔCt and geNorm analyses. While the geNorm analysis favored RPL7A and RPS7, the BestKeeper analysis identified EF1A as the most stable. All four analyses revealed that RPS18 had relatively poor expression stability (Figure 2, Table S3). The RefFinder analysis ranked the expression stability of candidate reference genes in third-instar H. parallela larvae samples under RNAi feeding treatment from highest to lowest, as follows: RPL18>RPL13a >RPS3>RPL7A>RPS7>EF1A>GAPDH>VATB>RPL32>Actin>RPS18 (Figure 3, Table S3).

3.4. Optimal Number of Reference Genes

Pairwise variation (V) values, which indicate the stability of reference genes, were computed using thegeNorm software to ascertain the optimal number of reference genes required for data normalization under different experimental conditions. The results of the analysis showed that for different developmental stages and adult tissues, V2/3 to V9/10 were all less than 0.15. For third-instar larvae under varying temperature conditions, V2/3 was less than 0.15 and V3/4 to V10/11 were all greater than 0.15. Additionally, for adult H. parallela under RNAi feeding treatment, V2/3 to V10/11 were all less than 0.15. This suggests that two candidate reference genes are sufficient for data normalization in different developmental stages of H. parallela, adult H. parallela tissues, third-instar H. parallela larvae under varying temperature conditions, and adult H. parallela under RNAi feeding treatment. For different tissues of adult male and female H. parallela, V4/5 to V8/9 were all less than 0.15 while V2/3 to V3/4 and V9/10 to V10/11 were all greater than 0.15. Under different light conditions, V3/4 to V8/9 were all less than 0.15 while V2/3 and V9/10 to V10/11 were all greater than 0.15. This indicates that three candidate reference genes should be used for data standardization in the different tissues of male and female adult H. parallela as well as in adult H. parallela under varying light conditions (Figure 4).

3.5. The Influence of Housekeeping Genes on Quantitative PCR Data Interpretation

The stability analysis and pairwise variation (V) calculations of comprehensive candidate reference genes performed under the H. parallela RNAi feeding treatment indicate that utilizing two highly stable reference genes is optimal for normalization. Consequently, in this research, the pairing of the most stable genes RPL18 and RPL13a, in addition to the least stable gene, RPS18, were chosen as reference genes. These genes were employed to analyze the expression levels of the vitellogenin receptor (VgR) gene and the forkhead box L2 (FoxL2) gene, ultimately validating the stability of the selected reference genes.
The follicular cell protein (Fcp3C) gene is related to insect reproduction and oviposition. After RNAi, Fcp3C affects the expression of the VgR and FoxL2 genes. When using the highly stable gene combination of RPL18 and RPL13a as reference genes, the expression level of the target gene VgR was relatively lower in dsFcp3C samples. Utilizing the least stable gene, RPS18, as a reference gene, the expression level of the target gene VgR was relatively lower in both the dsGFP and dsFcp3C samples, with no significant difference between them (Figure 5A). Regarding the expression level of the target gene FoxL2, when using the highly stable gene combination of RPL18 and RPL13a as reference genes, the expression level in dsFcp3C samples was higher than in the control (ddH2O) and dsGFP samples. There was no significant difference in expression level between the control (ddH2O) and dsGFP samples. However, when using RPS18 (the least stable gene) as a reference gene, the expression level of the target gene FoxL2 was higher in the control (ddH2O) sample, whereas it was lower in both the dsGFP and dsFcp3C samples, with the lowest expression level being observed in the dsGFP sample (Figure 5B). Based on the validation results discussed above, using different reference genes for normalizing target genes will indeed yield different experimental results.

4. Discussion

qPCR, which is usually used to determine gene expression levels, is a widely recognized, established method that serves as a gold standard in molecular biology research [32,33,34]. Normalization is essential in qRT-PCR analysis for addressing variations that occur within and between reaction cycles. These variations can lead to inconsistencies, including variations in the quantity of the initial sample material, differences in RNA integrity, discrepancies in the loading amounts of cDNA among samples, and deviations in reverse transcription efficiency [35,36,37]. Thus, the observed reference gene variability encapsulates both the intrinsic variability of the gene itself and the technical errors incurred during the process [36]. Controlling all steps leading up to the PCR measurement is crucial, and selecting an appropriate reference gene is vital for achieving precision across varying experimental setups.
Transcriptomic datasets for H. parallela have been made available to facilitate research into its developmental processes, chemosensory genes, odorant-degrading enzymes, and immune responses [19,20,38,39]. Previous qRT-PCR studies on H. parallela generally utilized conventional insect reference genes, notably Actin and GAPDH [21,38,40,41]. In addition to the highest stability of Actin under different temperature conditions, the present study found that Actin and GAPDH exhibited particularly high variable instability values. Therefore, neither Actin nor GAPDH was the most stable reference gene in most cases. The expression of Actin was less stable in several insects, including Tribolium castaneum, H. oblita, Coleomegilla maculata, Hippodamia convergen and Hippodamia variegata, all belonging to the order Coleoptera [27,42,43,44,45].
The present study identified unique gene stability profiles in H. parallela, aligning with previous research findings [27] that highlighted the variability of gene stability under various experimental conditions in Holotrichia oblita. In the present study on H. parallela, RPL18 and RPL13a were the most stable across the developmental stages and RNAi conditions. This suggests that these two genes can serve as reliable reference gene selections under these experimental conditions. Furthermore, RPL13a, RPL18, and RPL32 displayed high stability in both sexes, indicating their appropriateness as reference genes for sex-specific gene expression analyses in H. parallela. Under varying photoperiods, RPL32, RPL13a, and RPS3 exhibited the greatest stability, while Actin and RPL13a were the most stable across different temperatures.
In contrast, previous studies on H. oblita have reported that RPL13a and RPL18 are the optimal reference genes for investigating different tissues, developmental stages, and temperature. This is in line with the present findings in H. parallela. The consistency in the stability of RPL13a and RPL18 across species suggests that these genes may serve as general reference markers for gene expression analysis in closely related insects, particularly within the Holotrichia genus. However, for photoperiod treatments in H. oblita, RPL13a and RPS3 were found to be the most stable genes, while RPS18 and RPL18 were most suitable for sex-specific gene expression studies. Among the selected genes, including GAPDH, Actin, and 18s rRNAs, GAPDH was selected as a standardized reference gene in various tissues of H. parallela [38]. However, when compared to the other genes analyzed in the current study, RPL18 and RPL13a exhibited the greatest stability, while GAPDH ranked sixth in stability among the 11 genes tested. Similarly, Actin consistently ranked below the median in stability assessments, except when it exhibited the highest stability under different temperature treatments, which supports findings observed in other species [43,45,46,47]. In a previous work, GAPDH and RPL13a were considered the best reference genes for various tissues and the developmental stages of Anomala corpulenta, respectively [48].
Extensive studies across insect species have confirmed that ribosomal proteins are the most reliable reference genes, excelling in expression consistency and robustness compared to other candidates [22]. Consistently, the results of the current analysis ranked ribosomal protein genes among the three most stable reference gene options across diverse experimental settings. In the field of gene expression studies in insects, the ribosomal protein gene, including RPL18, RPS23, PRL13, and RPL32, has emerged as the preferred choice for reference genes, demonstrating superior suitability based on recent comprehensive evaluations [44,49,50,51,52]. Furthermore, RPL18, RPS3, and RPL13a can be considered as proper reference genes for normalizing RT-qPCR data, especially when choosing reference genes from other Coleoptera insects that have not been extensively studied. Nevertheless, the differences observed in the stability of other genes under specific conditions highlight the crucial role of choosing suitable reference genes based on the experimental plan and species under investigation.
In summary, this study offers significant insights into the identification of appropriate reference genes for quantitative gene expression analysis in H. parallela, especially within the context of RNAi treatments. This will guarantee precise and dependable analysis of gene expression for similar gene types in this and other closely related insect species and facilitate the more effective identification of target genes for pest control.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects15090661/s1, Figure S1: Specificity of primer pairs; Figure S2: Amplification efficiency curves; Table S1: Standard curve equations, slopes, and amplification efficiencies of the 11 candidate internal reference genes; Table S2: Ct values and coefficients of variation (CV) for the 11 candidate internal reference genes Table S3: The stability of the 11 candidate reference genes evaluated by ΔCt method, BestKeeper, NormFinder, and geNorm.

Author Contributions

Conceptualization, Z.G. and Y.W.; methodology, J.Z. and Q.C.; software, Z.G., J.Z. and Y.D.; validation, J.Z., Q.C., Y.D., Y.J., T.L., H.L. and Z.Z.; formal analysis, Z.G., J.Z., Y.D., Y.J., T.L. and J.M.; investigation, Z.G., J.Z., H.L. and Z.Z.; resources, Z.G.; data curation, Z.G., J.Z., H.L. and Z.Z.; writing—original draft preparation, Z.G. and J.Z.; writing—review and editing, Y.W.; visualization, Z.G. and H.L.; supervision, Z.Z., J.M. and Y.W.; project administration, Z.G. and J.M.; funding acquisition, Z.G. and Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Advantageous Discipline Cultivation Project of Henan Provincial Science and the Technology Research and Development Plan Joint Fund (232301420114), the China Agriculture Research System of MOF and MARA (CARS-03), Innovative Research Team for Science and Technology of the Henan Academy of Agricultural Sciences (No. 210120005).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Reference Gene Expression under Different Treatments in Holotrichia parallela. In each box, the lower quartile (25th percentile)and upper quartile (75th percentile) are depicted. The whiskers mark the minimum and maximum values of the dataset. The horizontal line within each box marks the median value. Dots represent the reference gene expression for each sample.
Figure 1. Reference Gene Expression under Different Treatments in Holotrichia parallela. In each box, the lower quartile (25th percentile)and upper quartile (75th percentile) are depicted. The whiskers mark the minimum and maximum values of the dataset. The horizontal line within each box marks the median value. Dots represent the reference gene expression for each sample.
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Figure 2. Stability of 11 candidate reference genes assessed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The size of each bubble represents the reference gene stability value, with smaller bubbles indicating more stable genes. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
Figure 2. Stability of 11 candidate reference genes assessed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The size of each bubble represents the reference gene stability value, with smaller bubbles indicating more stable genes. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
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Figure 3. Expression stability of 11 reference genes of Holotrichia parallela under various treatment conditions analyzed using RefFinder. Bubble size represents reference gene stability, with smaller bubbles indicating greater stability. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
Figure 3. Expression stability of 11 reference genes of Holotrichia parallela under various treatment conditions analyzed using RefFinder. Bubble size represents reference gene stability, with smaller bubbles indicating greater stability. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
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Figure 4. geNorm analysis of 11 reference genes in Holotrichia parallela determined the optimal number for accurate normalization. Pairwise variation (Vn/Vn+1) values indicate the stability of reference gene combinations. The dotted line represents the recommended threshold (0.15), below which additional reference genesare not necessary. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
Figure 4. geNorm analysis of 11 reference genes in Holotrichia parallela determined the optimal number for accurate normalization. Pairwise variation (Vn/Vn+1) values indicate the stability of reference gene combinations. The dotted line represents the recommended threshold (0.15), below which additional reference genesare not necessary. De: developmental stage, Ds: dsRNA treatment, Ph: photoperiod treatment, Se: sexes, Te: temperature, Ti: tissues.
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Figure 5. Expression of the target genes VgR (A) and FoxL2 (B) in Holotrichia parallela treated with RNA interference (RNAi) using different reference genes. *: a statistically significant difference with p < 0.05; ns: not significant.
Figure 5. Expression of the target genes VgR (A) and FoxL2 (B) in Holotrichia parallela treated with RNA interference (RNAi) using different reference genes. *: a statistically significant difference with p < 0.05; ns: not significant.
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Table 1. The primer sequences for the candidate reference genes and the two target genes utilized in this study.
Table 1. The primer sequences for the candidate reference genes and the two target genes utilized in this study.
GenePrimer Sequences (5′-3′, F/R)Efficieny (E) (%)Regression Coefficient (R2)
GAPDHAGTCGCCGTAAATGATCCCT110.680.9971
CGTCGACTGTGCCATTGAAT
RPL32GCAAAAACCCGTCATATGCT110.170.998
TGATACGCCATGTGCAATTT
RPL7ATGCAAATACAATCCGAGCTG107.710.9916
GGCAGGAAGAGCACAAGTTC
RPS18CGTGCTGGAGAATGTTCTGA105.820.9972
GCTGGCTATATTTGCCATCAA
RPL13aGGCGTACCTCCACCTTATGA106.760.9978
CAGAATTTGCGACCAGGTTT
RPL18CATTACGCTCACCAACAGGA111.190.998
CTGGAGCAGGACCAAAGTGT
ActinTGTCACTGTATGCCTCTGGT109.670.9978
TACCAGCCAAATCCAAACGC
RPS7CGCGAGCTTGAGAAGAAGTT109.180.9994
AGAACGTGGACGCTTCTGTT
RPS3ATCCACTCAGGTGACCCTTG114.350.9956
AACGGCCTCTTAGGTCCAAT
VATBGGTCTACCGCACAACGAAAT126.920.9933
ACCTAGCGGTTTCCATGTTG
EF1AGCCAGAAGCTGTACCTGGAG106.760.9994
TGTCACCGGCTACATAACCA
Target gene
VgRTGGCGAAGACGAGAAAAACT--
TCGTCCGACAAATCGTAACA
FoxL 2CAGCAGCCTATACGCAACAA--
AGGAGGCCAATAAGCTGGAT
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MDPI and ACS Style

Gong, Z.; Zhang, J.; Chen, Q.; Li, H.; Zhang, Z.; Duan, Y.; Jiang, Y.; Li, T.; Miao, J.; Wu, Y. Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects 2024, 15, 661. https://doi.org/10.3390/insects15090661

AMA Style

Gong Z, Zhang J, Chen Q, Li H, Zhang Z, Duan Y, Jiang Y, Li T, Miao J, Wu Y. Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects. 2024; 15(9):661. https://doi.org/10.3390/insects15090661

Chicago/Turabian Style

Gong, Zhongjun, Jing Zhang, Qi Chen, Huiling Li, Ziqi Zhang, Yun Duan, Yueli Jiang, Tong Li, Jin Miao, and Yuqing Wu. 2024. "Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses" Insects 15, no. 9: 661. https://doi.org/10.3390/insects15090661

APA Style

Gong, Z., Zhang, J., Chen, Q., Li, H., Zhang, Z., Duan, Y., Jiang, Y., Li, T., Miao, J., & Wu, Y. (2024). Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects, 15(9), 661. https://doi.org/10.3390/insects15090661

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