Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), α-tubulin (α-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1α and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1α and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1α and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1α and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella.

Fushi Tarazu Factor 1 (FTZ-F1), a member of the nuclear receptor superfamily, is the downstream factor of 20-hydroxyecdysone signaling. In Drosophila melanogaster, alternative transcription start and splicing in the FTZ-F1 gene generate αFTZ-F1 and βFTZ-F1 isoforms, which are vital for pair-rule segmentation in early embryogenesis and post-embryonic development, respectively. However, whether the same mRNA isoforms are present and exert the conservative roles remains to be clarified in other insects. In the present paper, we first mined the genomic data of representative insect species and unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. Our expression data in Henosepilachna vigintioctopunctata, a serious polyphagous defoliator damaging a wide range of crops in Solanaceae and Cucurbitaceae, showed that both αFTZ-F1 and βFTZ-F1 were actively transcribed throughout the development, from embryo to adult. The RNA interference-aided knockdown of both isoforms completely arrested larval ecdysis from the third to the fourth instar, in contrast to the depletion of either isoform. In contrast, silencing βFTZ-F1, rather than αFTZ-F1, severely impaired the larval–pupal transformation. We accordingly propose that both FTZ-F1 isoforms are essential but mutually interchangeable for larval–larval molting, while βFTZ-F1 is necessary for the larval–pupal transition and sufficient to exert the role of both FTZ-F1s during larval–pupal metamorphosis in H. vigintioctopunctata.

Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, we determined the functions of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. We uncovered that E93 was abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, our results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval–pupal–adult transition.

The diamondback moth, Plutella xylostella, is a major pest of brassica vegetables and oilseed crops, posing a serious threat to China’s grain and oil production. RNA interference (RNAi) has been developed as an efficient strategy to control pests. In this study, the effects of RNAi on P. xylostella were evaluated by injecting two doses of synthesized dsPxvATPasea. The transcripts of PxvATPasea were widely transcribed during different developmental stages from egg to adult. They were abundantly expressed in the hindgut and Malpighian tubules, compared with other tissue types. Introduction of 800 ng dsPxvATPasea in the fourth-instar larvae greatly reduced corresponding mRNA levels by 3.1 and 1.4 times on day 2 and 3, respectively, causing 66.6% mortality and 33.4% treated larvae pupated. Silencing PxvATPasea by injecting 1200 ng dsRNA significantly decreased the expression level by 5.0 and 2.0 times on the second and third day, leading to 79.2% larval lethality and 20.8% depleted larvae pupated. Moreover, introducing 800 ng or 1200 ng dsPxvATPasea finally reduced larval fresh weight by 22.1% and 28.8%, respectively. The results indicated that the silencing efficiency of PxvATPasea worked in a dose-dependent way. Consequently, PxvATPasea is a potential molecular target gene. Our findings will facilitate the application of RNAi technology to manage P. xylostella.