Dynamics and regulation of glycolysis‐tricarboxylic acid metabolism in the midgut of Spodoptera litura during metamorphosis

Significant changes usually take place in the internal metabolism of insects during metamorphosis. The glycolysis‐tricarboxylic acid (glycolysis‐TCA) pathway is important for energy metabolism. To elucidate its dynamics, the mRNA levels of genes involved in this pathway were examined in the midgut of Spodoptera litura during metamorphosis, and the pyruvate content was quantified. The expression patterns of these genes in response to starvation were examined, and the interaction between protein phosphatase 1 (PP1) and phosphofructokinase (PFK) was studied. The results revealed that the expression or activities of most glycolytic enzymes was down‐regulated in prepupae and then recovered in some degree in pupae, and all TCA‐related genes were remarkably suppressed in both the prepupae and pupae. Pyruvate was enriched in the pupal midgut. Taken together, these results suggest that insects decrease both glycolysis and TCA in prepupae to save energy and then up‐regulate glycolysis but down‐regulate TCA in pupae to increase the supply of intermediates for construction of new organs. The expression of all these genes were down‐regulated by starvation, indicating that non‐feeding during metamorphosis may be a regulator of glycolysis‐TCA pathway in the midgut. Importantly, interaction between PP1 and PFK was identified and is suggested to be involved in the regulation of glycolysis.     

Molecular characterization of a gene encoding juvenile hormone esterase in the red flour beetle,Tribolium castaneum

Juvenile hormone esterases (JHEs) are required for the degradation of juvenile hormones (JHs) in insects. Here, we report the cloning and analysis of the jhe gene in the red flour beetle, Tribolium castaneum, a model insect of Coleoptera. The Tcjhe gene was strongly expressed at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. A recombinant TcJHE protein efficiently degraded JH III, suggesting that the enzyme functions in vivo as a JH‐specific degradation enzyme. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was shown in Coleoptera, and the new data reported here will aid elucidation of the mechanism of JH titer regulation in insects.     

Identification of an E‐box DNA binding protein,activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis‐activating neuropeptide in Helicoverpa armigera

Activated protein 4 (AP‐4), an E‐box DNA‐binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har‐AP‐4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro‐translated Har‐AP‐4 can bind specifically to the E‐box motif on the promoter of the diapause hormone and pheromone biosynthesis‐activating neuropeptide (DH‐PBAN). Har‐AP‐4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har‐AP‐4 can significantly activate the promoter of the DH‐PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har‐AP‐4, which binds to the promoter of DH‐PBAN, may play a role in regulating pupal development in H. armigera.     

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.     

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.     

Isolation,purification and biochemical characterization of Conopomorpha cramerella farnesol dehydrogenase

In Southeast Asia, Conopomorpha cramerella (Snellen) which is commonly known as the cocoa pod borer (CPB) moth has been identified as the most detrimental pest of Theobroma cacao L. Apart from the various side effects on human health and non-target organisms, heavily relying on synthetic pyrethroid insecticides to control CPB infestations also increases the environmental contamination risks. Thus, developing biorational insecticides that minimally affect the non-target organism and environment by targeting the insect growth regulation process is needed to manage the pest population. In insects, juvenile hormones (JH) regulate critical biological events, especially metamorphosis, development and reproduction. Since the physiological roles of JH III vary among different organisms, the biochemical properties, especially substrate specificity and analogue inhibition, may also be different. Therefore, studies on the JH III biosynthetic pathway enzymes in both plants and insects are beneficial to discover more effective analogues. Bioinformatic analysis and biochemical characterization of a NADP⁺-dependent farnesol dehydrogenase, an intermediate enzyme of the JH III pathway, from C. cramerella (CcFolDH), were described in this study. In addition, the farnesol analogues that may act as a potent analogue inhibitor for CcFolDH ware determined using in vitro enzymatic study. The phylogenetic analysis indicated that CcFolDH shared a close phylogenetic relationship to the honeybee's short-chain dehydrogenase/reductase. The 27 kDa CcFolDH has an NADP(H) binding domain with a typical Rossmann fold and is likely a homotetrameric protein in the solution. The enzyme had a greater preference for substrate trans, trans-farnesol and coenzyme NADP⁺. In terms of analogue inhibitor inhibition, hexahydroxyfarnesyl acetone showed the highest inhibition (the lowest K_i) compared to other farnesol analogues. Thus, hexahydroxyfarnesyl acetone would serve as the most potent active ingredient for future biorational pesticide management for C. cramerella infestation. Based on the bioinformatic analyses and biochemical characterizations conducted in this research, we proposed that rCcFolDH differs slightly from other reported farnesol dehydrogenases in terms of molecular weight, substrate preference, coenzymes utilization and analogue inhibitors selection.