Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.     

The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20‐hydroxyecdysone in the Colorado potato beetle,Leptinotarsa decemlineata

Ecdysone 20‐monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20‐hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix‐C, Helix‐I, Helix‐K, PxxFxPE/DRF (PERF) and heme‐binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double‐stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd‐dsRNA by the fourth instars also down‐regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd‐dsRNA‐ingested second instars, and of halofenozide into the LdShd‐dsRNA‐ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.     

The nuclear hormone receptor E75A regulates vitellogenin gene (Al‐Vg) expression in the mirid bug Apolygus lucorum

Apolygus lucorum is the predominant pest of Bacillus thuringiensis (Bt) cotton in China. 20‐hydroxyecdysone (20E) plays a key role in the reproduction of this insect. To better understand the mechanism underlying 20E‐regulated reproduction, the nuclear hormone receptor E75 isoform‐A of Ap. lucorum (Al‐E75A) was cloned and its expression analysed. A 2241‐bp sequence of Al‐E75A cDNA encoded an open reading frame of a polypeptide with a predicted molecular mass of 69.04 kDa. Al‐E75A mRNA was detected in female adult stages of Ap. lucorum with peak expression in 7‐day‐old animals. Al‐E75A was also expressed in several tissues, particularly in the fat body and ovary. A 3.2 kb Al‐E75A mRNA was detected in all tissues by Northern blot. The fecundity and longevity were significantly decreased in female adults treated with Al‐E75A small interfering RNA. The rates of egg incubation rates were considerably lower in the RNA interference‐treated animals compared to the untreated controls. In order to investigate the molecular mechanism underlying the effects described above, vitellogenin (Al‐Vg) was selected for further investigation. The expression pattern of Al‐Vg was similar to that of Al‐E75A and was up‐regulated by 20E. After knockdown of Al‐E75A, the expression profile of Al‐Vg and the protein levels were down‐regulated. These findings suggest that Al‐E75A plays a crucial role in the regulation of Al‐Vg expression in Ap. lucorum.     

Molecular characterization and enzymatic analysis of juvenile hormone epoxide hydrolase genes in the red flour beetle Tribolium castaneum

Juvenile hormone epoxide hydrolases (JHEHs) degrade juvenile hormones (JHs) and are important for JH titre regulation. Here, we report the cloning and analysis of five jheh‐related (jheh‐r1–r5) genes in the red flour beetle, Tribolium castaneum, a model species for the coleopteran insects. T. castaneum JHEH‐r (TcJHEH‐r) proteins show high homology to lepidopteran JHEHs and also to human microsomal epoxide hydrolase. In the phylogenetic tree, Tcjheh‐rs were clustered, and interestingly, they were also clustered in the genome. Examination of enzymatic activities using recombinant TcJHEH‐r proteins showed that TcJHEH‐r3 had strong degradation activity for JH III, whereas TcJHEH‐r4 had weak activity. The study has yielded significant information that will facilitate further analysis of JHEHs and epoxide hydrolases.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.