Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Abstract

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ∆Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18S were selected as the most stable genes by both the ∆Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase (JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.

Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

“…For example, TATA was identified as a stable reference gene for normalizing target gene expression in D. melanogaster treated with acetic acid (Kim et al, 2020). Additionally, TATA expression was revealed to be moderately stable in female and male P. solenopsis at different developmental stages (Zheng et al, 2019). Regarding P. tsushimanus, TATA is the most stable reference gene for analyzing gene expression among plant samples, but it is the least stable reference gene for gene expression analyses of developmental stages (Chen C. et al, 2020).…”

“…Considering the importance of reference genes for qRT-PCR, previous studies validated reference gene sets in various insect species, including Lymantria dispar (Yin et al, 2020), Drosophila melanogaster (Kim et al, 2020), Pagiophloeus tsushimanus (Chen C. et al, 2020), Phenacoccus solenopsis (Zheng et al, 2019), Chilo partellus (Adeyinka et al, 2019), Harmonia axyridis (Yang et al, 2018), Henosepilachna vigintioctomaculata (Lu et al, 2018), Liriomyza trifolii (Chang et al, 2017), Myzus persicae (Kang et al, 2017), Bradysia odoriphaga (Shi et al, 2016), Lipaphis erysimi (Koramutla et al, 2016), Helicoverpa armigera (Shakeel et al, 2015), Sesamia inferens (Lu et al, 2015), and Spodoptera exigua (Zhu et al, 2014). In a previous study, the gene expression stability of four potential housekeeping genes was evaluated for viruliferous winged and wingless R. padi adults (Wu et al, 2014), which is an insufficient number of genes.…”

Reference Gene Selection for Analyzing the Transcription Patterns of Two Fatty Acyl-CoA Reductase Genes From Paracoccus marginatus (Hemiptera: Pseudococcidae)

“…Additionally, the geNorm program can calculate the optimal number of reference genes for valid normalization of RT-qPCR, and the method is as follows: first select the two reference genes with the best stability to calculate the normalization factor (NF), and then add the next gene one by one for calculation. If NF value is less than 0.15, it corresponds to the number of used reference genes, and if more than one combination has NF values lower than 0.15, then the minimum number of reference genes are usually chosen ( Zheng et al 2019 ), as an additional reference gene will not significantly improve the normalization ( Yang et al 2014 ). To input the transcription data for NormFinder analysis, the Cq values were also transformed to quantities using the formula 2 ΔCq .…”

“…To date, studies about reference gene selection in mealybug species were all concentrated in Ph. solenopsis ( Arya et al 2017 , Singh et al 2019 , Zheng et al 2019 ) but none concentrated in P. marginatus . We carried out the experiments to clone, identified the FAR genes from P. marginatus , and then found a set of suitable reference genes across all developmental stages.…”

“…However, increasing evidence has suggested that there are no universal reference genes that are suitable for all experimental conditions in all species ( Morales et al 2016 ). With the development of large-scale high-throughput sequence technologies to identify genes that are differentially expressed between conditions, finding a reference gene that has stable expression under specific experimental conditions become much more convenient ( Arya et al 2017 , Zheng et al 2019 ).…”