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.

Fungal ecdysteroid-22-oxidase, a new tool for manipulating ecdysteroid signaling and insect development.

Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 µM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.

EST sequencing and fosmid library construction in a non-model moth, Mamestra brassicae, for comparative mapping.

Genome data are useful for both basic and applied research; however, it is difficult to carry out large-scale genome analyses using species with limited genetic or genomic resources. Here, we describe a cost-effective method to analyze the genome of a non-model species, using the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae). First, we conducted expression sequence tag (EST) analysis. In this analysis, we performed PCR-based prescreening of a non-normalized embryonic cDNA library to eliminate already sequenced cDNAs from further sequencing, which significantly increased the percentage of unique genes. Next, we constructed a fosmid library of M. brassicae and isolated 120 clones containing 119 putative single copy genes by PCR-based screening with primer sets designed from the ESTs. Finally, we showed that the isolated fosmid clones could be used as probes for multicolor fluorescence in situ hybridization (FISH) analysis against an M. brassicae chromosome and confirmed conserved gene order between M. brassicae and the silkworm, Bombyx mori. Thus, we developed new genomic resources for comparative genome analysis in M. brassicae using robust and relatively low cost methods that can be applied to any non-model organism.

A eukaryotic (insect) tricistronic mRNA encodes three proteins selected by context-dependent scanning.

Eukaryotic mRNAs are generally considered monocistronic and encode only one protein. Although dicistronic mRNAs encoding two proteins were found in fungi, plants, and animals, polycistronic mRNAs encoding more than two proteins have remained elusive so far in any eukaryote. Here we demonstrate that a single mRNA from silkworm encodes the precursor of an insect cytokine paralytic peptide (PP) and two new cytokine precursor-like proteins, uENF1 and uENF2. RT-PCR analysis showed that this mRNA is widely conserved in moths. Western blot analyses and reporter assays using its modified mRNAs, created by replacing each one of the three ORFs with the firefly luciferase ORF, showed that all three proteins were translated from this mRNA in cell lines, larval tissues, and cell-free systems. Insertion experiments using the Renilla luciferase ORF or a stem loop ruled out the possible involvement of internal ribosome entry site in the three protein translation. On the other hand, systematic mutation analysis of the translation initiation sequence of the 5'-proximal uENF1 ORF suggested that the context-dependent leaky-scanning mechanism is involved in translation of the downstream uENF2 and PP ORFs. In vitro, a synthetic peptide corresponding to the putative mature form of uENF1 stimulated spreading of hemocytes as did the synthetic PP, whereas that of uENF2 antagonized the stimulating activities of PP and the uENF1 peptide, suggesting that the three proteins control cellular immunity interactively. Thus, eukaryotes have a cellular tricistronic mRNA that encodes three functionally related proteins as in an operon.

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.

Isolation of BAC clones containing conserved genes from libraries of three distantly related moths: a useful resource for comparative genomics of Lepidoptera.

Lepidoptera, butterflies and moths, is the second largest animal order and includes numerous agricultural pests. To facilitate comparative genomics in Lepidoptera, we isolated BAC clones containing conserved and putative single-copy genes from libraries of three pests, Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella, harboring the haploid chromosome number, n = 31, which are not closely related with each other or with the silkworm, Bombyx mori, (n = 28), the sequenced model lepidopteran. A total of 108-184 clones representing 101-182 conserved genes were isolated for each species. For 79 genes, clones were isolated from more than two species, which will be useful as common markers for analysis using fluorescence in situ hybridization (FISH), as well as for comparison of genome sequence among multiple species. The PCR-based clone isolation method presented here is applicable to species which lack a sequenced genome but have a significant collection of cDNA or EST sequences.

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head.

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.

Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ.

Only a few extracellular hematopoietic factors have been identified in insects. We previously developed an in vitro culture system for the larval hematopoietic organ (HPO) of the silkworm Bombyx mori, and found that cell proliferation is linked to hemocyte discharge from the HPO. In this study, we tested hematopoietic activity of bombyxin, a peptide in the insulin family. When silkworm HPO was cultured with synthetic bombyxin-II, the number of discharged hemocytes increased in a dose-dependent manner, indicating that bombyxin promoted cell proliferation in the HPO. However, a neutralization experiment using anti-bombyxin-II antibody revealed that bombyxin is not the primary effector in larval plasma. Similarly, bovine insulin showed hematopoietic activity. Addition of molting hormone, 20-hydroxyecdysone, circumstantially enhanced the hematopoietic activity of bombyxin and insulin. Bombyxin and insulin induced phosphorylation of different sets of proteins in the HPO, suggesting that their signaling pathways are different.

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid--a mechanism that determines the timing of metamorphosis.

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.

Identification of two juvenile hormone inducible transcription factors from the silkworm, Bombyx mori.

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mß (E(spl)mß), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mß was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mß was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mß are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.

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.

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.

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.

Solution structure of paralytic peptide of silkworm, Bombyx mori.

Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of "ENF" is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second beta-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.

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.

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland.

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.

Insect cytokine paralytic peptide activates innate immunity via nitric oxide production in the silkworm Bombyx mori.

Insect cytokine paralytic peptide (PP) upregulates the expression of immune-related genes and contributes to host defense in the silkworm Bombyx mori. The present findings demonstrated that PP promotes nitric oxide (NO) production and induces the expression of NO synthase. A pharmacologic NO synthase inhibitor suppressed the PP-dependent (i) induction of immune-related genes, (ii) activation of p38 mitogen-activated protein kinase, and (iii) killing delay of silkworm larvae by Staphylococcus aureus. The upstream mechanism of NO synthesis in insect immunity has been unknown, and the present results suggest for the first time that an insect cytokine induces NO and contributes to self-defense.

Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at -2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

Two hemocyte lineages exist in silkworm larval hematopoietic organ.

BACKGROUND: Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. CONCLUSIONS/SIGNIFICANCE: From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.