Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

Janelia Farm Research Campus, Howard Hughes Medical Institute, United States of America
PLoS Genetics (Impact Factor: 7.53). 03/2012; 8(3):e1002486. DOI: 10.1371/journal.pgen.1002486
Source: PubMed


Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

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Available from: Toru Shimada, Oct 01, 2015
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    • "The detection of highly variable expression profiles strongly indicates that the silkworm enhancer trap system can contribute to the identification of a wide variety of novel genes and/or their promoter/enhancer elements (Uchino et al. 2008). These strains are also valuable resources for tissue-specific driver strains since the transposon contains a GAL4 gene (Ito et al. 2008; Daimon et al. 2012). "
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    ABSTRACT: Transgenic techniques offer a valuable tool for determining gene functions. Although various promoters are available for use in gene overexpression, gene knockdown and identification of transgenic individuals, there is nevertheless a lack of versatile promoters for such studies and this dearth acts as a bottleneck, especially with regard to non-model organisms. Here, we succeeded in identifying a novel strong and ubiquitous promoter/enhancer in the silkworm. We identified a unique silkworm strain whose reporter gene showed strong and ubiquitous expression during the establishment of enhancer trap strains. In this strain the transposon was inserted into the 5'UTR of hsp90, a housekeeping gene that is abundantly expressed in a range of tissues. To determine whether the promoter/enhancer of hsp90 could be used to induce strong gene expression, a 2.9kb upstream genomic fragment of hsp90 was isolated (hsp90(P2.9k)) and its transcriptional activation activity was examined. Strikingly, hsp90(P2.9k) induced strong gene expression in silkworm cell cultures and also strongly induced gene expression in various tissues and developmental stages of the silkworm. hsp90(P2.9k) also exhibited significant promoter/enhancer activity in Sf9, a cell culture from the armyworm, suggesting that this fragment might possibly be used as a gene expression tool in other Lepidoptera. We further found that 2.0 kb of hsp90(P2.9k) is sufficient for the induction of strong gene expression. We believe that this element will be of value for a range of studies such as targeted gene overexpression, gene knockdown and marker gene expression, not only in the silkworm but also in other insect species.
    G3-Genes Genomes Genetics 05/2014; 4(7). DOI:10.1534/g3.114.011643 · 3.20 Impact Factor
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    • "If so, an expression pattern similar to that of wnt-1 may be found at the early developmental stages in both the O. excavata leaf pattern and the T. juno nonmimetic pattern, and subsequent expression patterns may become different to establish different modules. Testing this hypothesis will require a detailed analysis of the pattern formation processes using well-developed molecular techniques [63-70]. "
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    BMC Evolutionary Biology 07/2013; 13(1):158. DOI:10.1186/1471-2148-13-158 · 3.37 Impact Factor
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    • "The development of the germline transformation technique and the completion of genome sequencing have also prompted functional analyses of various genes in the silkworm (Tamura et al., 2000; The International Silkworm Genome Consortium, 2008). When identifying a mutant gene, one can execute homology searches on the genome data or positional cloning to narrow down candidate genes, which can then be examined functionally by expressing them in the mutant (Yamamoto, 2009; Fujii et al., 2010; Atsumi et al., 2012; Daimon et al., 2012). However, phenotypic changes cannot be studied if the introduced gene has deteriorating effects when it is expressed systemically or uniformly in a tissue. "
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    ABSTRACT: A clonal analysis system in the silkworm, Bombyx mori, was developed to achieve expression of genes of interest in portions of tissues. We established two types of transgenic silkworm strains: one expresses yeast FLP recombinase under the control of a heat shock promoter, and the other possesses the DsRed gene interrupted with the GFP gene flanked at both ends with two FLP recombinase target (FRT) sites. The F1 eggs between the two strains were heat-shocked to induce FLP recombinase, which catalyzed intrachromosomal recombination to excise the GFP gene, resulting in the expression of DsRed in randomly formed cell clones.
    Journal of Insect Biotechnology and Sericology 06/2012; 81(2):63-67. DOI:10.11416/jibs.81.2_3_063
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