Chieka Minakuchi

Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum.

Juvenile hormone (JH) prevents ecdysone-induced metamorphosis in insects. However, our knowledge of the molecular mechanisms of JH action is still fragmented. Krüppel homolog 1 (Kr-h1) is a JH-inducible transcription factor in Drosophila melanogaster (Minakuchi, C., Zhou, X., Riddiford, L.M., 2008b. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech. Dev. 125, 91-105). Analysis of expression of the homologous gene (TcKr-h1) in the beetle Tribolium castaneum showed that its transcript was continuously present in the larval stage but absent in the pupal stage. Artificial suppression of JH biosynthesis in the larval stage caused a precocious larval-pupal transition and a down-regulation of TcKr-h1 mRNA. RNAi-mediated knockdown of TcKr-h1 in the larval stage induced a precocious larval-pupal transition. In the early pupal stage, treatment with an exogenous JH mimic (JHM) caused formation of a second pupa, and a rapid and large induction of TcKr-h1 transcription. JHM-induced formation of a second pupa was counteracted by the knockdown of TcKr-h1. RNAi experiments in combination with JHM treatment demonstrated that in the larval stage TcKr-h1 works downstream of the putative JH receptor Methoprene-tolerant (TcMet), and in the pupal stage it works downstream of TcMet and upstream of the pupal specifier broad (Tcbr). Therefore, TcKr-h1 is an early JH-response gene that mediates JH action linking TcMet and Tcbr.

Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster

Juvenile hormone (JH) given at pupariation inhibits bristle formation and causes pupal cuticle formation in the abdomen of Drosophila melanogaster due to its prolongation of expression of the transcription factor Broad (BR). In a microarray analysis of JH-induced gene expression in abdominal integument, we found that Krüppel homolog 1 (Kr-h1) was up-regulated during most of adult development. Quantitative real-time PCR analyses showed that Kr-h1 up-regulation began at 10h after puparium formation (APF), and Kr-h1 up-regulation occurred in imaginal epidermal cells, persisting larval muscles, and larval oenocytes. Ectopic expression of Kr-h1 in abdominal epidermis using T155-Gal4 to drive UAS-Kr-h1 resulted in missing or short bristles in the dorsal midline. This phenotype was similar to that seen after a low dose of JH or after misexpression of br between 21 and 30 h APF. Ectopic expression of Kr-h1 prolonged the expression of BR protein in the pleura and the dorsal tergite. No Kr-h1 was seen after misexpression of br. Thus, Kr-h1 mediates some of the JH signaling in the adult abdominal epidermis and is upstream of br in this pathway. We also show for the first time that the JH-mediated maintenance of br expression in this epidermis is patterned and that JH delays the fusion of the imaginal cells and the disappearance of Dpp in the dorsal midline.

Transcriptional regulation of juvenile hormone-mediated induction of Krüppel homolog 1, a repressor of insect metamorphosis

The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix–loop–helix Per-ARNT-Sim (bHLH–PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH–PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.

RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum.

Juvenile hormone controls the timing of insect metamorphosis. As a final step of juvenile hormone biosynthesis, juvenile hormone acid O‐methyltransferase (JHAMT) transfers the methyl group from S‐adenosyl‐l‐methionine to the carboxyl group of farnesoic acid and juvenile hormone acid. The developmental expression profiles of JHAMT mRNA in the silkworm Bombyx mori and the fruitfly Drosophila melanogaster suggest that the suppression of JHAMT transcription is critical for the induction of larval–pupal metamorphosis, but genetic evidence for JHAMT function in vivo is missing. In this study, we identified three methyltransferase genes in the red flour beetle Tribolium castaneum (TcMT1, TcMT2 and TcMT3) that are homologous to JHAMT of Bombyx and Drosophila. Of these three methyltransferase genes, TcMT3 mRNA was present continuously from the embryonic stage to the final larval instar, became undetectable before pupation, and increased again in the adult stage. TcMT3 mRNA was localized in the larval corpora allata. Recombinant TcMT3 protein methylated farnesoic acid and juvenile hormone III acid, but TcMT1 and TcMT2 proteins did not. Furthermore, RNA interference‐mediated knockdown of TcMT3 in the larval stage resulted in precocious larval–pupal metamorphosis, whereas knockdown of either TcMT1 or TcMT2 showed no visible effects on metamorphosis. Importantly, precocious metamorphosis caused by TcMT3 RNA interference was rescued by an application of a juvenile hormone mimic, methoprene. Together, these results demonstrate that TcMT3 encodes a functional JHAMT gene that is essential for juvenile hormone biosynthesis and for the maintenance of larval status.

Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata

cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted. Amino‐acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a 12.4‐kb message for the LdEcR A‐isoform, a 10.5‐kb message for the LdEcR B1‐isoform and a 5.7‐kb message for the LdUSP, in fat body, gut, integument, testis and ovaries. In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP. LdEcR/LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (KD = 2.8 nm), while LdEcR alone showed only weak binding (KD = 73.4 nm), and LdUSP alone did not show any binding. The receptor‐binding affinity of various ecdysone agonists to LdEcR/LdUSP was not correlated to their larvicidal activity to L. decemlineata. From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agonists in L. decemlineata.

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Sf-9 cell line.

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [(3)H]ponasterone A ([(3)H]PoA) in intact Sf-9 cells (Spodoptera frugiperda). The amount of [(3)H]PoA binding varied in a concentration-dependent manner. According to the IC(50), concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A > or = inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [(3)H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [(3)H]PoA at 25 microM. However, their binding activity in terms of pIC(50) was either very low or not evaluated.

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.

Relationships between structure and molting hormonal activity of tebufenozide, methoxyfenozide, and their analogs in cultured integument system of Chilo suppressalis Walker.

The molting hormonal activity of methoxyfenozide (RH-2485), tebufenozide (RH-5992), five analogs with various alkyl groups, and 18 acyl analogs was measured by using cultured integument of rice stem borers, Chilo suppressalis Walker. The hormonal activity of methoxyfenozide was remarkably high (EC(50) = 1.1 x 10(-9) M), being equivalent to that of tebufenozide (RH-5992). The hormonal activity of several tebufenozide analogs with varying alkyl groups such as CH(3), n-C(3)H(7), i-C(3)H(7), n-C(4)H(9) and n-C(5)H(11) at the para-position of the benzene ring furthest from the tert-butyl group was lower than that of tebufenozide (alkyl group is C(2)H(5)). The activity decreased to varying degrees as a result of replacement of the 3,5-dimethylphenyl moiety of tebufenozide with either a phenyl, naphthyl, or cyclohexyl group. Both 1- and 2-naphthyl derivatives were very active (EC(50) = 4.3 x 10(-8) M and 3.2 x 10(-8) M, respectively) without any significant difference between them. The activity of the 1-cyclohexenyl analog (EC(50) = 1.0 x 10(-7) M) was about 40x that of the corresponding 3-cyclohexenyl analog (EC(50) = 4.4 x 10(-6) M), but 1/100 that of tebufenozide. The activity varied parabolically with respect to the molecular hydrophobicity, and decreased with longer acyl moieties.

Antimicrobial peptide gene induction, involvement of Toll and IMD pathways and defense against bacteria in the red flour beetle, Tribolium castaneum

Using Tribolium castaneum, we quantitatively investigated the induction of nine antimicrobial peptide (AMP) genes by live gram-negative bacteria (Escherichia coli and Enterobacter cloacae), gram-positive bacteria (Micrococcus luteus and Bacillus subtilis) and the budding yeast (Saccharomyces cerevisiae). Then, five representative AMP genes were selected, and the involvement of the Toll and IMD pathways in their induction by E. coli, M. luteus and S. cerevisiae was examined by utilizing RNA interference of either MyD88 or IMD. Results indicated: Robust and acute induction of three genes by the two bacterial species was mediated mainly by the IMD pathway; slow and sustained induction of one gene by the two bacteria was mediated mainly by the Toll pathway; induction of the remaining one gene by the two bacteria was mediated by both pathways; induction of the five genes by the yeast was mediated by the Toll and/or IMD pathways depending on respective genes. These results suggest that more promiscuous activation and usage of the two pathways may occur in T. castaneum than in Drosophila melanogaster. In addition, the IMD pathway was revealed to dominantly contribute to defense against two bacterial species, gram-negative E. cloacae and gram-positive B. subtilis that possesses DAP-type peptidoglycan.

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.