Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying expression levels of targeted genes during various biological processes in numerous areas of clinical and biological research. Selection of appropriate reference genes for RT-qPCR normalization is an elementary prerequisite for reliable measurements of gene expression levels. Here, by analyzing datasets published between 2008 and 2017, we summarized the current trends in reference gene selection for insect gene expression studies that employed the most widely used SYBR Green method for RT-qPCR normalization. We curated 90 representative papers, mainly published in 2013–2017, in which a total of 78 insect species were investigated in 100 experiments. Furthermore, top five journals, top 10 frequently used reference genes, and top 10 experimental factors have been determined. The relationships between the numbers of the reference genes, experimental factors, analysis tools on the one hand and publication date (year) on the other hand was investigated by linear regression. We found that the more recently the paper was published, the more experimental factors it tended to explore, and more analysis tools it used. However, linear regression analysis did not reveal a significant correlation between the number of reference genes and the study publication date. Taken together, this meta-analysis will be of great help to researchers that plan gene expression studies in insects, especially the non-model ones, as it provides a summary of appropriate reference genes for expression studies, considers the optimal number of reference genes, and reviews the average number of experimental factors and analysis tools per study.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the ΔCt method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

RNA interference (RNAi) has emerged as a powerful tool for developing novel management strategies for controlling insect pests. The 28‐spotted ladybeetle, Henosepilachna vigintioctopunctata is one of the most important pests attacking solanaceous plants in Asia. In this study, the potential of dietary RNAi to manage H. vigintioctopunctata was investigated using both in vitro synthesized and bacterially expressed double‐stranded RNAs (dsRNAs) of HvvATPase A and HvvATPase E. The expression levels of HvvATPase A and HvvATPase E were higher in Malpighian tubules than in other tissue types. The silencing of HvvATPase A and HvvATPase E led to significant mortality in H. vigintioctopunctata larvae. In addition, the ingestion of HvvATPase A and HvvATPase E significantly deterred feeding behavior and subsequently arrested the development of H. vigintioctopunctata. Notably, the bacterially expressed dsRNAs consistently caused higher mortality in larvae and adults. Finally, the nontarget effects of the dsRNAs of H. vigintioctopunctata on the predatory ladybeetle Propylaea japonica were evaluated. P. japonica 1st instar larvae were administered vATPase A and vATPase E dsRNAs from H. vigintioctopunctata and P. japonica under the worst‐case scenario, in which dsGFP served as negative control. There were significant effects of dsHvvATPase A on P. japonica at the transcriptional level but not at the organismal level, whereas dsHvvATPase E did not effect P. japonica at either the transcriptional or the organismal level. Collectively, the results of the study suggest that HvvATPase A and HvvATPase E can act as novel molecular targets for the control of H. vigintioctopunctata.

The gut bacteria of insects positively influence the physiology of their host, however, the dynamics of this complicated ecosystem are not fully clear. To improve our understanding, we characterized the gut prokaryotic of Henosepilachna vigintioctopunctata that fed on two host plants, Solanum melongena (referred to as QZ hereafter) and Solanum nigrum (referred to as LK hereafter), by sequencing the V3-V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq system. The results revealed that the gut bacterial composition varied between specimens that fed on different host plants. The unweighted pair group method with arithmetic mean analyses and principal coordinate analysis showed that the bacterial communities of the LK and QZ groups were distinct. Four phyla (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were present in all H. vigintioctopunctata gut samples. It is noteworthy that bacteria of the phylum Cyanobacteria were only found in the LK group, with a low relative abundance. Proteobacteria and Enterobacteriaceae were the predominant phylum and family, respectively, in both the LK and QZ groups. Linear discriminant analysis effect size (LEfSe) analyses showed that the QZ group enriched the Bacilli class and Lactococcus genus; while the LK group enriched the Alphaproteobacteria class and Ochrobactrum genus. PICRUSt analysis showed that genes predicted to be involved in xenobiotic biodegradation and metabolism, metabolism of other amino acids, signaling molecules, and interaction were significantly higher in the QZ group. Genes predicted to be involved in the metabolism of cofactors and vitamins were significantly higher in the LK group. Furthermore, the complexity of the network structure and the modularity were higher in the LK group than in the QZ group. This is the first study to characterize the gut bacteria of H. vigintioctopunctat, our results demonstrate that the two host plants tested had a considerable impact on bacterial composition in the gut of H. vigintioctopunctata and that the bacterial communities were dominated by relatively few taxa.