Abstract
Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.
Highlights
Differential gene expression (DGE) studies are fundamental to evaluate the effects of physiological responses on biological variation in insect populations at the molecular level
Development Stages and Different Tissue Types Samples were collected from different life stages of Ips sexdentatus (ISx): three larval stages (L1, L2, and L3), pupae, (P), callow male (CM) and female (CF), and fed adult male (AMF) and female (AFF)
real-time quantitative reverse transcription PCR (RT-qPCR) products generated with each primer set against target genes were evaluated by the occurrence of a single peak in melting curve analyses (Supplementary Figure 2) and specific bands of the expected size in agarose gel electrophoresis (Supplementary Figure 3)
Summary
Differential gene expression (DGE) studies are fundamental to evaluate the effects of physiological responses on biological variation in insect populations at the molecular level. There are several studies on gene function analysis in insects using reference genes such as β-actin (Actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1A), β-Tubulin (β-Tubulin), ribosomal proteins (RPs), ubiquitin (UbiQ), superoxide dismutase (SOD), heat shock protein 90 (HSP90), and vacuolar-type H+-ATPase subunit B (V-ATPase-B) for gene expression normalization (Lu et al, 2018; Qu et al, 2018; Gao et al, 2020; Wang et al, 2020) This suggests that reference genes can be differentially expressed in insects under varied experimental conditions. Our study delivers a catalog of genes that should be used for DGE studies on ISx under varied experimental conditions and commences the possibility for aggressive management of bark beetles using molecular approaches such as RNA interference (RNAi) (Joga et al, 2021)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
