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.

BACKGROUNDRNA interference (RNAi) is a potential tool for plant protection against insect pests. The great challenge for effective pest control using RNAi in the field is the development of efficient and reliable methods for the production and delivery of double‐stranded RNA (dsRNA).RESULTSIn the present study, we investigated the potential of feeding in vitro synthesized or bacterially expressed dsRNA to populations of the 28‐spotted ladybeetle Henosepilachna vigintioctopunctata as a method of biological pest control. Ingestion of in vitro synthesized dsHvRPS18 or dsHvRPL13 led to significant down‐regulation of the ribosomal protein‐encoding genes HvRPS18 and HvRPL13, respectively, and significantly decreased the survival of H. vigintioctopunctata. Such silencing of HvRPS18 or HvRPL13 expression appeared to be partially dose‐dependent and also inhibited the growth of H. vigintioctopunctata and significantly suppressed the expression of digestive enzyme‐related genes. Finally, ingestion of bacterially expressed dsHvRPS18 or dsHvRPL13 induced significant mortality in the first and third instars, and in adults.CONCLUSIONThe effectiveness of RNAi‐based gene silencing in H. vigintioctopunctata provides a powerful reverse genetic tool for the functional annotation of its genes. This study demonstrates that HvRPS18 and HvRPL13 represent candidate genes for RNAi‐based biological control of H. vigintioctopunctata. © 2020 Society of Chemical Industry

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.

BACKGROUND Use of RNA interference (RNAi) technology in effective pest management has been explored for decades. Henosepilachna vigintioctopunctata is a major solanaceous crop pest in Asia. In this study, the effects of the RNAi‐mediated silencing of clathrin heavy chain in H. vigintioctopunctata were investigated. RESULTS Feeding either the in vitro‐synthesized or the bacterially expressed double‐stranded RNAs (dsRNAs) significantly impaired the normal physiology of H. vigintioctopunctata instars and adults. However, the bacterially expressed dsHvChc caused higher mortality than the in vitro‐synthesized ones in the larvae and adults. Moreover, on evaluating the potential risk of dsHvChc on Propylea japonica, significant transcriptional effects of dsHvChc1 were observed, while the organismal level effects were not significant. On the contrary, dsHvChc2 did not affect P. japonica at either level. A similar test revealed significant transcriptional effects of dsPjChc1 on H. vigintioctopunctata, while staying ineffective at the organismal levels. Conversely, dsPjChc2 did not affect H. vigintioctopunctata at either level. Importantly, no effect of dsPjChc1 exposure on H. vigintioctopunctata suggested that other factors besides the 21‐nucleotide (nt) matches between sequences were responsible. Finally, ingestion of dsHvmChc1 derived from H. vigintioctomaculata, containing 265‐nt matches with dsHvChc1, caused 100% mortality in H. vigintioctopunctata. CONCLUSIONS We conclude that (i) species with numerous 21‐nt matches in homologous genes are more likely to be susceptible to dsRNA; (ii) dsRNA can be safely designed to avoid negative effects on non‐target organisms at both transcriptional and organismal levels; (iii) HvChc can be used as an efficient RNAi target gene to effectively manage H. vigintioctopunctata. © 2021 Society of Chemical Industry.

RNA interference (RNAi) techniques have emerged as powerful tools in the development of novel management strategies for the control of insect pests, such as Henosepilachna vigintioctopunctata, which is a major solanaceous pest in Asia. Our results showed that levels of HvSnf7 expression were greater in larval midguts than in other tissues. Silencing of HvSnf7 led to greater H. vigintioctopunctata mortality rates and appeared to be time- and partially dose-dependent. Bacterially expressed dsHvSnf7 that was applied to detached plant leaves caused 98, 88, and 60% mortality in 1st and 3rd instars, and adults after 10, 12, and 14 d, respectively; when applied to living plants, bacterially expressed dsHvSnf7 led to mortality in 1st and 3rd instars, with no effect on adults. Bacterially expressed dsHvSnf7 led to improved plant protection against H. vigintioctopunctata. Ultrastructural changes caused by HvSnf7-RNAi in larval midguts showed extensive loss of cellular contents that indicate loss of membrane integrity. This study indicate that HvSnf7 potentially can be used as RNAi target gene for controlling 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.

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