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Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wildtype neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30[degrees]C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (schI) do not hatch even at room temperature (25[degrees]C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BroTh coding sequences were identical among sch, schl, and wild-type. However, in sch the ~70-kb sequence was replaced with ~4.6 kb of a Tc1-mariner type transposon located ~6 kb upstream of BroTh, and in schl, a large fragment of an LIBm retrotransposon was inserted just in front of the transcription start site of BroTh. In both cases, we observed a drastic reduction of BroTh expression. Use of RNAi with BroTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, [pnd.sup.+] and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BrnTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color. doi/ 10.1073/pnas.1001725107

Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2, which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects. virus resistance | positional cloning | transgenesis

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an [F.sub.1] male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an [F.sub.1] female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of [approximately equal to]3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated. silkworm | microsatellite

Enhanced locomotory activity (ELA), such as wandering, is a normal behavior that occurs at the end of the larval stage in lepidopteran (butterflies and moths) insects. Baculovirus infection can also induce ELA in lepidopteran larvae. The belief is that the virus induces this behavior to increase its transmission [Goulson, D. (1997) Oecologia 109, 219-228]. Here we show that a baculovirus-encoded protein tyrosine phosphatase (PTP) gene (ptp) induces ELA that is activated by light. ELA was induced in silkworm Bombyx mori infected with the baculovirus B. mori nucleopolyhedrovirus (BmNPV) beginning at [approximately equal to] 3.75 days postinfection (p.i.) and continued until 4.75 days p.i. The intensity of the ELA was dramatically reduced immediately before death at 5.25 days p.i. Light activated the intensity of the ELA by [approximately equal to] 3-fold, and larvae with ELA showed positive phototropism. ELA was not induced in larvae of B. mori infected with a BmNPV ptp knockout mutant (BmPTPD). However, when a silkworm-derived ptp gene (Bmptp-h) was inserted into BmPTPD, ELA was partially recovered. Bmptp-h was identified from silkworms at 2 days after the start of the natural wandering stage. The deduced amino acid sequence of Bmptp-h showed 48.2% identify (80.7% similarity) to the deduced amino acid sequence of BmNPV ptp. On the basis of the high homology and larval stage at which Bmptp-h was isolated, we postulate that the modern baculovirus may have acquired its ptp gene from an ancestral host and that this gene was selectively maintained because it increases virus transmission. Bombyx mori | enhanced locomotory response | protein tyrosine phosphatase gene | nucleopolyhedrovirus

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 morí. In a B. morí cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (/cJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of fcJHRE (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 WHRE reporter was activated JH-dependently in HEK2g3 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with /cJHRE. 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. [ABSTRACT FROM AUTHOR]

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an Female mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F1 female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR- based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of ≈3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated. [ABSTRACT FROM AUTHOR]

The article discusses the Bacillus thuringiensis (Bt) toxin resistance in silkworm, Bombyx mori by the single amino acid mutation in an Adenosine-5'-triphosphate (ATP) binding cassette transporter gene. The resistance is said to involve proteins with active functions in the gut and amino acid insertion and deletion can be used to distinguish resistant from susceptible strains of silkworm. It also informs that Bt resistance is a recessive trait that requires two nonfunctioning gene copies.

To build a foundation for the complete genome analysis of Bombyx mori, we have constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains 35,000 ESTs from 36 cDNA libraries, which are grouped into approximately 11,000 nonredundant ESTs with the average length of 1.25 kb. The comparison with FlyBase suggests that the present EST database, SilkBase, covers >55% of all genes of Bombyx. The fraction of library-specific ESTs in each cDNA library indicates that we have not yet reached saturation, showing the validity of our strategy for constructing an EST database to cover all genes. To tackle the coming saturation problem, we have checked two methods, subtraction and normalization, to increase coverage and decrease the number of housekeeping genes, resulting in a 5-11% increase of library-specific ESTs. The identification of a number of genes and comprehensive cloning of gene families have already emerged from the SilkBase search. Direct links of SilkBase with FlyBase and WormBase provide ready identification of candidate Lepidoptera-specific genes.