Manabu Kamimura

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.

cDNA cloning and characterization of Bombyx mori juvenile hormone esterase: an inducible gene by the imidazole insect growth regulator KK-42

The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3H-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.

Activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggered mechanism

The insect cytokine paralytic peptide (PP) induces muscle contraction in silkworm larvae. Here we demonstrate that bacterial and fungal cell wall components peptidoglycan and glucan stimulate muscle contraction via activation of PP in the hemolymph. Anti-PP antibody suppressed the muscle contraction induced by PP, peptidoglycan, or glucan. The contraction was also inhibited by free radical scavengers and serine protease inhibitors. Moreover, injecting live silkworms with peptidoglycan or glucan generated the active form of PP. The active form of PP was also produced in vitro when peptidoglycan or glucan was incubated with hemolymph containing the PP precursor. Generation of the active form of PP was suppressed by free radical scavengers and serine protease inhibitors. Furthermore, PP activation in isolated hemolymph was inhibited by potassium cyanide, suggesting that cellular activity is involved. Stimulation by peptidoglycan promoted the generation of reactive oxygen species by silkworm hemocytes. The addition of either the active form of PP or anti-PP antibody to Staphylococcus aureus injected into silkworm larvae delayed or enhanced, respectively, the killing effect of S. aureus, suggesting that activated PP contributes to host resistance to infectious pathogens. These findings suggest that immunologic stimulants such as peptidoglycan or glucan induce reactive oxygen species production from larval hemocytes, followed by the activation of serine protease, which mediates the PP processing reaction and leads to defensive responses.

Insect cytokine paralytic peptide (PP) induces cellular and humoral immune responses in the silkworm Bombyx mori.

In the blood (hemolymph) of the silkworm Bombyx mori, the insect cytokine paralytic peptide (PP) is converted from an inactive precursor to an active form in response to the cell wall components of microorganisms and contributes to silkworm resistance to infection. To investigate the molecular mechanism underlying the up-regulation of host resistance induced by PP, we performed an oligonucleotide microarray analysis on RNA of blood cells (hemocytes) and fat body tissues of silkworm larvae injected with active PP. Expression levels of a large number of immune-related genes increased rapidly within 3 h after injecting active PP, including phagocytosis-related genes such as tetraspanin E, actin A1, and ced-6 in hemocytes, and antimicrobial peptide genes cecropin A and moricin in the fat body. Active PP promoted in vitro and in vivo phagocytosis of Staphyloccocus aureus by the hemocytes. Moreover, active PP induced in vivo phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in the fat body. Pretreatment of silkworm larvae with ML3403, a pharmacologic p38 MAPK inhibitor, suppressed the PP-dependent induction of cecropin A and moricin genes in the fat body. Injection of active PP delayed the killing of silkworm larvae by S. aureus, whereas its effect was abolished by preinjection of the p38 MAPK inhibitor, suggesting that p38 MAPK activation is required for PP-dependent defensive responses. These findings suggest that PP acts on multiple tissues in silkworm larvae and acutely activates cellular and humoral immune responses, leading to host protection against infection.

In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ.

The lepidopteran hematopoietic process is poorly understood. We therefore examined the fundamental properties of hematopoiesis in the silkworm Bombyx mori using hematopoietic organ culture. In a medium containing larval plasma taken from the fourth day of the final larval stadium, over 50,000 hemocytes per hematopoietic organ were discharged within 48 h, with the number of cells comprising the hematopoietic organ simultaneously increasing from approximately 20,000 to 40,000. However, in the absence of plasma, cell numbers comprising the hematopoietic organ were unchanged and the number of discharged cells was much less. Hematopoietic organs cultured with plasma showed strong mitotic indices in a BrdU incorporation assay, but did not when cultured without plasma, indicating that plasma contains hematopoietic factor(s). The hematopoietic stimulation ability of larval plasma was observed from the last day of the penultimate larval stadium to the prepupal stage. The response of the hematopoietic organs to larval plasma was highest at the beginning of the final larval stadium and decreased with aging. Most cells discharged from the hematopoietic organ were plasmatocytes and prohemocytes, irrespective of location and developmental stage. Using this in vitro culture method, we tested the effects of 20-hydroxyecdysone (20E) and juvenile hormone-I (JH-I) on B. mori hematopoiesis. 20E showed a weak, but significant, hematopoietic activity, whereas JH-I did not, suggesting that a part of larval hematopoiesis is endocrinally regulated.

Expression of ecdysone receptor isoforms and trehalase in the anterior silk gland of Bombyx mori during an extra larval molt and precocious pupation induced by 20-hydroxyecdysone administration

When newly molted 5th (last) instar larvae of Bombyx mori were fed an artificial diet containing 20-hydroxyecdysone (20E), an extra larval molt was induced and the cuticle layer of the anterior silk gland was renewed. In contrast, feeding 20E to 5th instar larvae allatectomized in the mid-4th instar induced pupational responses and anterior silk gland degeneration. Topical application of a juvenile hormone analogue, fenoxycarb, to allatectomized larvae before 20E treatment nullified the effects of allatectomy and induced an extra molt. In the two cases where an extra larval molt was induced, mRNA expression of the ecdysone receptor A isoform (EcR-A) preceded that of the B1 isoform (EcR-B1) in the anterior silk gland, while EcR-A and EcR-B1 were expressed synchronously during precocious pupation. Precedence of EcR-A over EcR-B1 and the synchronization of both are expression patterns observed during the normal 4th and 5th instars. These results suggest that temporal expression profiles of EcR isoforms are important for regulating stage-specific responses of this tissue to ecdysteroids. Trehalase was also identified as a metamorphosis-specific gene in the anterior silk gland by mRNA differential display between the extra larval molt and precocious pupation. During normal 4th and 5th instar, trehalase mRNA was expressed at the end of the 5th instar only. Thus, extra larval molt and precocious pupation induced by dietary application of 20E mimic normal larval molt and pupation well and can be a useful tool in isolating stage-specific ecdysteroid-regulated genes. Arch. Insect Biochem. Physiol. 41:79–88, 1999.

Purification and characterization of silkworm hemocytes by flow cytometry.

Hemocyte functions are well-investigated in the silkworm, Bombyx mori, however, detailed analysis of each hemocyte subset has been hampered by the lack of appropriate separation method. Here we use an array of flow cytometric analyses to characterize silkworm hemocytes with various molecular probes, such as propidium iodide, green fluorescence protein, monoclonal antibodies, and fluorescent lectins. Of these, separation using propidium iodide was the simplest and provided most reliable results for the isolation of the hemocyte subsets. cDNAs were then synthesized from these sorted populations and subset-specific gene expression was examined by RT-PCR. Granulocytes, plasmatocytes, and oenocytoids expressed different classes of immune genes, suggesting that they have multiple roles in silkworm immunity. In contrast, a contribution of spherulocytes to immunity was not documented in that they failed to express most of the genes. The functions of spherulocytes are thus likely to be distinct from those of the other three hemocyte subsets.

Bombyx mori orphan receptor, BmHR78: cDNA cloning, testis abundant expression and putative dimerization partner for Bombyx ultraspiracle

We have identified a novel member of the nuclear receptor superfamily from the silkworm Bombyx mori, and named it as BmHR78, the B. mori hormone receptor. The DNA binding domain of BmHR78 shows high similarities to those of Tenebrio molitor hormone receptor 78, Drosophila hormone receptor 78, and mammalian testicular receptor 2, whereas the ligand binding domain is not well conserved. Northern blot analysis showed that BmHR78 gene was most abundantly expressed in the testis. From the fourth to fifth instar, BmHR78 gene was constantly expressed in the testis. In the anterior silk gland, the level of BmHR78 gene expression was developmentally changed. From day 10.0 to 11.0 in the fifth instar, another BmHR78 transcript with the smaller size appeared. Ultraspiracle (USP) isoform also appeared at the same stages in this tissue. BmHR78 forms not only a homodimer, but also a heterodimer with USP in a yeast two hybrid assay. The direct interaction between BmHR78 and USP was confirmed by pull down assay. Deletion mutant analysis showed that BmHR78 interacts with USP via the ninth heptad repeat in helix ten of the E region. This repeat is well conserved in RXR and its heterodimer partners, and shown to be an interface for their dimerization. In insect, only the ecdysone receptor and hormone receptor 38 are known thus far to dimerize with USP. Thus, BmHR78 is a third dimerization partner for USP and may modulate the molecular action of USP, including the ecdysone signal cascades.

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 Mr of 32 kDa and showed a Kd of 4.5 × 10−7 M with JH III, both similar to those of native hJHBP.

A new chitinase-related gene, BmChiR1, is induced in the Bombyx mori anterior silk gland at molt and metamorphosis by ecdysteroid.

A novel ecdysteroid-inducible gene was isolated from the anterior silk gland of the silkworm by mRNA differential display and named Bombyx mori chitinase-related gene 1 (BmChiR1). cDNA for BmChiR1 is 3.7 kbp encoding 1080 amino acids. Its predicted protein sequence consists of two tandem-repeated sequences, both showing high similarities to arthropod chitinases but lacking the active site glutamate essential for catalytic activity, suggesting that BmChiR1 protein has no chitinolytic activity. BmChiR1 mRNA was expressed simultaneously with chitinase mRNA in the anterior silk gland at the ends of the penultimate and last larval instar. Injection of 20-hydroxyecdysone (20E) into feeding last instar larvae induced accumulation of BmChiR1 mRNA. Topical application of a juvenile hormone analog, fenoxycarb, just after the 20E injection, suppressed this induction. BmChiR1 expression is therefore upregulated by ecdysteroid and downregulated by juvenile hormone.