Agrilus zanthoxylumi Hou (Coleoptera: Buprestidae) is the main stem-boring pest of Chinese prickly ash, Zanthoxylum bungeanum Maxim, an important economic crop in northern China. Agrilus zanthoxylumi is monophagous, attacking only Z. bungeanum (Wu 2006), and exhibits one generation per year. Adults feed on foliage causing notches on leaf edges (Cui 1990). Larvae overwinter in the bark but actively feed in the phloem and gradually consume the cambium creating decaying pupal channels in the xylem. Characteristic symptoms of larval damage are large volumes of exudates, softening, decay, etc., causing yellowing of foliage, death of entire branches or the crown, and even death of the tree (Kai 1991).

Pheromones have been proposed for the management of A. zanthoxylumi for monitoring populations, trap-and-kill methods, and mating or host detection confusion (Hamadttu et al. 2020). Knowledge of gene expression involved in chemical detection and sensing is an important step in development and efficacious use of these chemistries for A. zanthoxylumi management.

We report herein our assessment of selected reference genes that might be reliably and accurately used in reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) for detecting gene expression levels in various tissues of A. zanthoxylumi. RT-qPCR has been used extensively in insect transcriptome verification and gene expression analysis because of its relative speed, high sensitivity, replicability, and accuracy (Lü et al. 2018, Shakeel et al. 2015, Shi et al. 2017, VanGuilder et al. 2008, Wang and Xue. 2016). RT-qPCR results are affected by many factors (e.g., quality of initial RNA, transcription and amplification variations, complementary DNA [cDNA] synthesis efficiency, etc.) (Heid et al. 1996); therefore, suitable reference genes are used as quantitative standard references in order to ensure the accuracy and reliability of data analysis.

In this study, we assessed six candidate reference genes. These were histone, β-tubulin, actin, 18S rRNA, 28S-1 rRNA and 28S-2 rRNA. The expression stability of these six genes was established using fluorescent qPCR. Data were also evaluated using the GeNorm (Biogazelle, Zwijnaarde, Belgium; Vandesompele et al. 2002), NormFinder (Aarhus University Hospital, Denmark; Claus et al. 2004), and BestKeeper (Pfaffl et al. 2004) algorithms.

Materials and Methods

Sample preparation and total RNA extraction. The samples used in this study were collected from the base of Chinese prickly ash in the mountainous area of Zhoudong Village, Puhua Town, Lantian County, Shaanxi Province (May to July 2019). It is a semiclosed basin with a mountainous terrain. The climate is warm temperate and semihumid continental climate with distinct seasons. The average annual temperature is 12°C, and the central location is N 34°13′ and E 109°31′. Active A. zanthoxylumi adults were collected from branches and foliage of Z. bungeanum, placed in tubes (1.3-cm diameter, 4-cm height) with air holes, transported to the laboratory, and held at 25°C (Gong et al. 2019). Periodically, the Z. bungeanum foliage was replaced with fresh leaves, and frass and leaf residues were removed. Tissues from the head, thorax, abdomen, legs, and wings of adult insects were dissected and collected from male and female pairs, inserted into a centrifuge tube without RNA enzyme, and held at –80°C until assayed.

Total RNA was extracted using the column-type insect RNA extraction kit (BTN81220, Biolab Company, Beijing). As per the kit instructions, total RNA was extracted from five parts of female and male adults: heads, thorax, abdomen, legs, and wings. Each part was subjected to three repetitions and marked with designations for body region, sex, and replicate number (e.g., ANF1∼3, HF1∼3, TF1∼3, ABF1∼3, LF1∼3, and WIF 1∼3 for females, and ANM1∼3, HM1∼3, TM1∼3, ABM1∼3, LM1∼3, and WIM1∼3 for males). The integrity was detected by agarose gel electrophoresis, and the reverse transcription was performed according to the instructions of the Vazyme reverse transcription kit (R133-01). The first strand of cDNA was stored at –20°C for subsequent real-time fluorescence quantitative detection.

Primer design and qRT-PCR. Six candidate genes for reference (e.g., histone, β-tubulin, actin, 18S, and 28S-1 and 28S-2) were screened from the transcription group data of A. zanthoxylumi. According to the obtained gene sequence, primers for quantitative analysis were designed by using Primer-BLAST method with the information for each primer (Table 1). The synthesized first-strand cDNA was used as a template to detect the expression amount of the reference gene by RT-qPCR, and the reactions were performed in duplicate using the following protocol: 95°C for 10 min, followed by 35 cycles of 95°C for 1 min, 58°C for 30 s, and 72°C for 30 s.

Table 1 Candidate reference genes of Agrilus zanthoxylumi.

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The cDNA was prepared into templates with seven gradient concentrations by a two-time dilution method. The cycle threshold (Ct) values of histone, β-tubulin, actin, 18S, 28S-1, and 28S-2 at different concentrations were measured by the CFX96 real-time PCR detection system, using the following protocol: 95°C for 10 s, and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. According to the results, the linear relationship between the concentration of cDNA template and Ct value was analyzed by the linear correlation method, yielding a correlation coefficient (R²) and slope for each. The amplification efficiency of the primers was calculated by the formula E = (10^[–1/slope] – 1) × 100 (Radonic et al. 2004).

Data analysis. With the quantitative Ct value of candidate reference genes in each sample as a reference, the data were organized according to the requirements of the three software programs (GeNorm, NormFinder, and BestKeeper). The GeNorm automatically calculates an M value for each reference gene, with the more stable genes indicated by lower M values. The calculation principle of the NormFinder is similar to that of GeNorm. The stable value of reference gene expression is obtained first, and then the most appropriate reference gene is selected according to that value. The judgment criterion is that the reference gene with the minimum expression stability value is the most appropriate reference gene. The correlation coefficient (R), standard deviation (SD), and coefficient of variation (CV) of pairing between each gene can be obtained through the calculation of the BestKeeper, with lower SD values signifying more stable genes. Finally, the geometric mean of the six reference genes ranking in three different evaluation procedures was calculated, and then the comprehensive ranking was conducted to determine the stability of the six reference genes.

Results

The transcriptome sequencing identified five types of candidate reference genes (Table 1). These were histone, β-tubulin, actin, 18S, and 28S. Among those, two genes in 28S can be successfully cloned and were thus named 28S-1 and 28S-2. The six candidate reference genes of A. zanthoxylumi and the corresponding genes of other coleopteran insects have high nucleotide sequence identity, withthe highest consistency with Agrilus planipennis Fairmaire, both >87% and some approaching 100%.

In analyzing amplification efficiency and specificity of the expression levels of the six candidate reference genes of A. zanthoxylumi male and female adults, we found that the slopes of the lines ranged from –2.9 to –3.6 and linear correlation coefficients were >0.9800. This indicated that there was a clear linear relationship between the initial template amount of cDNA and the corresponding Ct value, that the obtained linear correlation equation was reliable, and that the amplification efficiency was between 90% and 120%, indicating the effectiveness of the RT-qPCR reaction (Table 2). The dissolution curves of the six reference genes were basically single peaks, indicating that the primers for the reference genes amplified well under the real-time fluorescence quantification condition, thus meeting the basic requirements of real-time fluorescence quantification. The changes of Ct values of each candidate reference genes in this study showed that in both sexes (Figs. 1, 2), the expressions of the Class 28S gene and the histone gene were relatively low, and the expression of the actin gene was highest of the six reference genes.

Table 2 Amplification efficiency, correlation coefficient, and slope of primers in various tissues of male and female adult Agrilus zanthoxylumi.

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Table 2 Continued.

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Expression stability analysis by GeNorm. In the GeNorm software, and the average expression stability (M) values are an indication of gene stability and are inversely proportional to reference gene stability. Based on our analysis (Table 3), gene stability of the six candidate reference genes in female A. zanthoxylumi was, from highest to lowest, in the head tissues was actin > histone > β-tubulin > 28S-1 > 18 S > 28S-2; in the thorax tissues, 28S-2 > 18S > β-tubulin > 28S-1 > histone > actin; in the abdominal tissues, 28S-2 > actin > histone > β-tubulin > 18S > 28S-1; in the legs, 28S-1 > 18S > actin > histone > β-tubulin > 28S-2; and in the wings, 28S-2 > 18S > 28S-1 > actin > β-tubulin > histone. In males, gene stability from highest to lowest was 18S > β-tubulin > 28S-2 > actin > 28S-1 > histone in head tissues; 28S-1 > β-tubulin > 28S-2 > 18S > actin > histone in thoracic tissues; 28S-2 > actin > 28S-1 > 18S > histone > β-tubulin in abdominal tissues; 28S-2 > β-tubulin > 18S > histone > actin > 28S-1 in the legs; and actin > histone > β-tubulin > 28S-1 > 18S > 28S-2 in the wings. Overall, regardless of sex, gene stability among these six reference genes was 28S-2 > β-tubulin > 28S-1 >18S > actin > histone based on the GeNorm analysis.

Table 3 GeNorm reference gene stability values (M) of Agrilus zanthoxylumi.

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Expression stability analysis by NormFinder. NormFinder software analysis also depends on a stability value to judge the stability of the reference gene. Those values of the six candidate reference genes in female and male tissues are shown in Table 4. The two 28S reference genes exhibited the highest stability in the thorax, leg, and wing tissues of female A. zanthoxylumi, while β-tubulin showed the highest stability in the head and abdominal tissues. Among males, actin exhibited the highest stability in the head and abdominal tissues, while β-tubulin showed highest stability in the thoracic tissues with 18S and histone having the highest stability in legs and wings.

Table 4 NormFinder reference gene stability values of Agrilus zanthoxylumi.

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Expression stability analysis by BestKeeper. BestKeeper analysis requires calculation of correlation coefficient, SD, and CV of each candidate reference gene, and the relative stability of each gene is judged according to the SD of each gene. Smaller standard coefficient of deviation (SD) and the CV correlationvalues, along with larger Pearson correlation coefficient values are indicators of higher expression stability of the gene. Our analysis of the six candidate reference genes with BestKeeper (Table 5) showed that the genes with the highest expression stability in the respective tissues of female A. zanthoxylumi head, thorax, abdomen, legs, and wings of adults were 28S-1 in the head, 28S-2 in the thorax, histone in the abdomen, 28S-2 in the legs, and 18S in the wings. In males, the highest stability expressions were 28S-1 in the head, β-tubulin in the thorax, actin in the abdomen, and histone in the legs and wings.

Table 5 BestKeeper reference gene stability values of Agrilus zanthoxylumi.

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Overall ranking of stability. We calculated the arithmetic mean of the relative ranking (1–6) of each of the six candidate reference genes from the three calculations in order to determine the reference gene that was most suitable for A. zanthoxylumi, thus providing a basis for future research. These calculations for females and males are shown in Tables 6 and 7, respectively. The 28S-2 gene was the most stable of the six genes in all tissues of female A. zanthoxylumi; 28S-1 also was relatively stable in female tissues from those body regions. In males, the expression of actin gene was the most stable in all tissues followed by 28S-2 gene.

Table 6 Comprehensive ranking of reference genes in female tissues of Agrilus zanthoxylumi.

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Table 7 Comprehensive ranking of reference genes in male tissues of Agrilus zanthoxylumi.

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Discussion

Real-time fluorescent qPCR has been widely used in the quantitative study of gene expression (Heid et al. 1996). Yet, RT-qPCR also has its limitations in that expression results are often greatly influenced by biological and abiotic factors. Therefore, reference genes are often introduced in the quantitative gene expression by RT-qPCR to correct and standardize the test data, so as to offset the errors between the test data measured by the instrument (Chen et al. 2015, T.F. Li 2018). Selecting appropriate reference genes can effectively improve the reliability and accuracy of the test results (Bustin et al. 2009).

Ideal reference genes refer to a class of genes that can be stably expressed in different types of tissues and different treatments of the same tissue, and whose expression levels are not affected by any endogenous or exogenous factors (Janská et al. 2013). However, various studies have screened and identified reference genes for a variety of insects and found that the optimal reference genes were not the same under different test conditions, that is, different species, different tissues, and different development stages (Li et al. 2018, Morales et al. 2016, Tan et al. 2017, Yang et al. 2013).

In studies with coleopterans, reference genes screened differed among the reported studies. For example, Liu et al. (2014) screened eight reference genes, including β-actin, GAPDH, α-tubulin, SYN1, SYN6, RPS3, RPS8, and RPS13a in Tribolium castneum (Herbst) and determined that suitable reference genes for gene expression analysis of different strains of T. castneum induced by phosphine were RPS8 and RPL13a. Li et al. (2018) found that the expression levels of four reference genes (TUB, TUA, RPS20, and RPL 12) in different tissues of the adult weevil Sympiezomias velatus Chevrolat were relatively stable. Feng et al. (2016) found that GAPDH was the preferred reference gene for expression analysis of the chemosensory gene of Monochamus alternatus White, and the combination of the reference genes GAPDH and TUB was sufficient to effectively correct the expression of the chemosensory gene of M. alternatus. In other coleopteran insects, such as Anoplophora glabripennis (Motschulsky) and Galeruca daurica Joannis, RPL32(RP49) is reportedly one of the most suitable reference genes for gene expression in different tissues (Rodrigues et al. 2017, Tan et al. 2017).

All of these studies have shown that there are differences in the expression stability of the reference gene in different species, development stages, and tissues (De et al. 2007, Huis et al. 2010, Sun et al. 2009). Furthermore, no housekeeping gene has been found to be stably expressed under all conditions or suitable for all cell and tissue types, and it is difficult to meet the requirements of universality even though it may be highly conserved during evolution (Yang et al. 2015).

In this study, six candidate reference genes were identified from A. zanthoxylumi, and the reference genes were screened in different tissues of male and female adults using RT-qPCR technology and three analysis software programs. We determined that the expression of the 28S-2 gene, followed by 28S-1, was the most stable in all tissues of female A. zanthoxylumi. The actin gene was the most stable expression in each tissue of males, followed by the 28S-2 gene.

The screening of the optimal reference gene will provide basic information for analyzing the gene expression of A. zanthoxylumi and also provide a certain basis for the selection of reference genes for other insects. Since the stability of the reference gene was highly correlated with the experimental conditions, species type, and tissue location (Sun et al. 2009), it is necessary to further study the applicability of the RT-qPCR derived from the screening of the reference gene from adult A. zanthoxylumi to other insects.