Reference Genes for Expression Analyses by RT-qPCR in Hyblaea puera (Lepidoptera: Hyblaeidae)

Area of Science:

• Molecular Biology
• Entomology
• Gene Expression Analysis

Background:

• Hyblaea puera is a significant pest affecting teak and mangrove ecosystems.
• Accurate gene expression studies using RT-qPCR depend on validated reference genes for normalization.
• No reference genes have been previously validated for Hyblaea puera.

Purpose of the Study:

• To evaluate the expression stability of ten candidate reference genes in Hyblaea puera.
• To identify the most stable reference genes for RT-qPCR normalization under diverse experimental conditions.
• To provide reliable reference gene sets for future molecular research on Hyblaea puera.

Main Methods:

• Candidate reference gene expression was assessed using RT-qPCR.
• Expression stability was analyzed using the delta Ct method, BestKeeper, NormFinder, and geNorm.
• RefFinder tool was employed for comprehensive stability ranking and selection of optimal gene combinations.

Main Results:

• All ten candidate genes exhibited high amplification efficiencies (91.67–100.82%) and R² values (>0.9901).
• Optimal reference gene pairs varied depending on experimental conditions, including temperature, larval instars, adult sexes, developmental stages, and tissues.
• Specific recommended pairs include RPL27/RPL10 (temperature), Actin/RPL10 (larval instars), RPS5/EF-1a (adult sexes), RPL10/EF-1a (developmental stages), RPL10/RPS5 (tissues), and EF-1a/Actin (all conditions).

Conclusions:

• The study successfully identified and validated a set of reference genes for Hyblaea puera.
• The selection of appropriate reference genes is crucial for accurate RT-qPCR data normalization in this pest.
• These findings will enhance the reliability of future molecular studies on Hyblaea puera, including host plant adaptation, temperature tolerance, and larval development.