Reference Gene Selection in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) and Their Normalization Impact on Gene Expression in RNAi Studies

Phenacoccus solenopsis, the cotton mealybug (Hemiptera: Pseudococcidae), is one of the major cotton pests in India. Scanty information is available on molecular studies in this insect due to limited genomic or transcriptomic sequence data. With advancement in sequencing technology, enormous genomic and sequencing data are being generated, and RNAi studies are being undertaken in insects, which require reverse transcription quantitative polymerase chain reaction evaluation. These gene expression studies require normalization of mRNA levels with reference genes to account for sample variability. To supplement the molecular studies in this insect, candidate reference genes were identified and evaluated for their expression stability across various developmental stages and starvation stress. Fourteen candidate reference genes including several commonly used ones were investigated across five different stages and under starvation stress using four different statistical algorithms (NormFinder, genNorm, BestKeeper, and RefFinder). Based on this analysis, GST (third, fourth, and adult stage), Actin (Crawler, second instar), TFIID (starvation stress), SDHA, and 28s were identified as best reference genes for expression studies in mealybug.