Stability analysis of reference genes in Maconellicoccus hirsutus across various conditions including dsRNA treatment

Maconellicoccus hirsutus is a polyphagous pest globally distributed in tropical and subtropical regions. In India, it is reported to adversely affect commercially significant crops, including grapes, guava, custard apple, and mulberry. Gene expression studies have become crucial for comprehending gene function and understanding molecular pathways, which are essential for developing effective pest management strategies. The RT-qPCR is a highly sensitive technique used to study the expression patterns of target genes. However, it's crucial to use a suitable internal reference gene, which is often overlooked before delving into the analysis of gene expression via RT-qPCR. The present study evaluates the expression stability of six candidate reference genes (28S, ATP51a, β-tubulin, GAPDH, α-tubulin, and Actin) under different experimental conditions (dsRNA-treated condition, starved conditions, different food sources: pumpkin, hibiscus, and sucrose; and sex-specific) using four statistical algorithms like geNorm, NormFinder, BestKeeper, and RefFinder. The results showed that GAPDH and β-tubulin expression were stable in dsRNA treatment. Reference genes GAPDH and ATP51a exhibited stability in both sex-specific and starvation conditions, while GAPDH and α-tubulin exhibited stability in different food sources. Our findings will aid in enhancing the precision of RT-qPCR analysis for future functional studies of the target gene in M. hirsutus.