Abstract
Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from Thermobia domestica, a representative species of Zygentoma, including Actin 5C (Actin5C), Elongation factor-1 alpha (EF1A), Ribosome protein S26 (RPS26), Ribosome protein L32 (RPL32), and Superoxide dismutase 2 (SOD2), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of RPS26 and RPL32 was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of Myoglianin (Myo), a crucial gene-regulating insect metamorphosis, in ametabolous T. domestica, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.
Highlights
The basal insect order Zygentoma includes the so-called silverfish and firebrats
Discussion quantitative RT-PCR (qPCR) is an efficient and convenient method for gene expression analysis, and using an appropriate reference gene or genes is the basic requirement to achieve qPCR reliability
Under RNA interference (RNAi) conditions at the 8th instar of T. domestica, the T. domestica Myoglianin (Tdmyo) mRNA level was significantly reduced in dsTdmyo-treated firebrats compared with dsMuslta-treated firebrats (Figure 6C), which suggested a high efficiency of RNAi Myo in firebrat nymphs
Summary
The basal insect order Zygentoma includes the so-called silverfish and firebrats. In contrast to winged insects, primitively wingless members of Zygentoma molt throughout their entire lives and do not undergo distinct morphological changes except for increases in size. As the extant ametabolous group closest to pterygote insects [1], Zygentoma occupies a key phylogenetic position for studying the evolution of insect metamorphosis. To date, Zygentoma has received little attention regarding genetic analysis. As a potential model organism of ametabolous insects, the firebrat Thermobia domestica is widely distributed worldwide. Firebrats are easy to maintain, breed, and synchronize in a laboratory. Rough genomic data from T. domestica have been reported recently [2], which facilitates genetic studies of T. domestica
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