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.     

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT‐PCR using degenerate primers based on the N‐terminal amino acid sequence of purified hJHBP and a conserved region near the C‐terminus of other lepidopteran hJHBPs. 5′‐ and 3′‐ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP‐mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS‐PAGE with a M_r of 32 kDa and showed a K_d of 4.5 × 10^?7 M with JH III, both similar to those of native hJHBP.     

Coordinate induction of peroxisomal beta-oxidation activity and cytosolic epoxide hydrolase activity

The effect of four hypolipidemic compounds (tiadenol, clofibrate, acetylsalicylic acid, 1-benzylimidazole) on the specific activities of peroxisomal beta-oxidation and cytosolic and microsomal epoxide hydrolase of rat liver was investigated. Since specific activity of cytosolic epoxide hydrolase from outbred Sprague-Dawley rats showed large interindividual variations (approximately 38-fold), induction studies were performed with inbred Fischer F-344 rats, which showed only low interindividual variations (approximately 2-fold). Clofibrate, tiadenol and acetylsalicylic acid caused a 8-, 13- and 4.5-fold induction of cEH and a 13-, 19- and 5-fold induction of peroxisomal beta-oxidation activity, respectively. Microsomal epoxide hydrolase activity was only slightly increased (less than 1.5-fold). 1-Benzylimidazole induced both cytosolic epoxide hydrolase and peroxisomal beta-oxidation activity about 2-fold, whereas microsomal epoxide hydrolase activity was increased about 4-fold. Increase in cytosolic epoxide hydrolase activity was not due to enzyme activation as demonstrated by in vitro studies. On the other hand, these in vitro studies showed that the increase in microsomal epoxide hydrolase activity by 1-benzylimidazole may partially be due to activation of the enzyme.     

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.     

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.     

Inhibition of human liver microsomal epoxide hydrolase by valproate and valpromide: in vitro/in vivo correlation

On the basis of drug interactions with carbamazepine epoxide, it has been hypothesized that valproic acid and valpromide are inhibitors of epoxide hydrolase, but the role of epoxide hydrolase in these interactions has not been clearly established. In this study, therapeutic concentrations of valproic acid (less than 1 mmol/L) and valpromide (less than 10 mumol/L) inhibited hydrolysis of carbamazepine epoxide and styrene oxide in human liver microsomes and in preparations of purified human liver microsomal epoxide hydrolase. Valpromide (KI = 5 mumol/L) was 100 times more potent than valproic acid (KI = 550 mumol/L) as an inhibitor of carbamazepine epoxide hydrolysis in microsomes. After administration of carbamazepine epoxide to volunteers, the transdihydrodiol formation clearance was decreased 20% by valproic acid (blood concentration approximately 113 mumol/L) and 67% by valpromide (blood concentration less than 10 mumol/L). For both valproic acid and valpromide, a striking similarity exists between in vitro and in vivo inhibitory potencies. Valproic acid and valpromide are the first drugs known to inhibit microsomal epoxide hydrolase, an important detoxification enzyme, at therapeutic concentrations.     

Isolation, endocrine regulation and mRNA distribution of the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae)

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.     

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.     

High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics

Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene‐tolerant [Met], Krüppel‐homolog1, Broad‐Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body‐part‐specific activation of JH signalling pathways.     

Multidrug resistance protein gene expression in Trichoplusia ni caterpillars

Many insect species exhibit pesticide‐resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real‐time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.