Reference Genes for Expression Analyses by qRT-PCR in Propsilocerus akamusi (Diptera: Chironomidae)

Area of Science:

• Environmental Science
• Molecular Biology
• Ecotoxicology

Background:

• Propsilocerus akamusi larvae are vital bioindicators for assessing water quality.
• Accurate gene expression analysis using RT-qPCR requires reliable reference genes.
• Identifying stable reference genes is essential for robust environmental monitoring studies.

Purpose of the Study:

• To identify optimal reference genes for RT-qPCR in Propsilocerus akamusi under various experimental conditions.
• To evaluate the expression stability of candidate reference genes across different developmental stages, body parts, temperatures, and chemical exposures.
• To provide a validated set of reference genes for future ecotoxicological and water quality studies using P. akamusi.

Main Methods:

• Selected fifteen candidate reference genes based on common usage in insect species.
• Identified homologous genes in P. akamusi via sequence alignment with Drosophila melanogaster.
• Assessed gene expression stability using geNorm, NormFinder, BestKeeper, and ΔCt methods across diverse conditions.

Main Results:

• RPL32 and RPL4 showed high stability in adult body parts and under varying temperatures.
• RPS11 and RPL8 demonstrated the greatest stability across developmental stages, larval temperatures, and deltamethrin/nickel chloride exposures.
• Reference gene stability varied significantly depending on the experimental condition.

Conclusions:

• RPS11 and RPL8 are recommended as the most reliable reference genes for RT-qPCR in Propsilocerus akamusi larvae under environmental stress and developmental changes.
• The findings support enhanced ecosystem-impact insights and improved water quality monitoring strategies.
• Validated reference genes will improve the accuracy and reliability of RT-qPCR-based ecotoxicological assessments.