Male-killing virus in a noctuid moth Spodoptera litura.

A female-biased sex ratio is considered advantageous for the cytoplasmic elements that inhabit sexually reproducing organisms. There are numerous examples of bacterial symbionts in the arthropod cytoplasm that bias the host sex ratio toward females through various means, including feminization and male killing. Recently, maternally inherited RNA viruses belonging to the family Partitiviridae were found to cause male killing in moths and flies, but it was unknown whether male-killing viruses were restricted to Partitiviridae or could be found in other taxa. Here, we provide compelling evidence that a maternally inherited RNA virus, Spodoptera litura male-killing virus (SlMKV), selectively kills male embryos of the tobacco caterpillar Spodoptera litura, resulting in all-female broods. SlMKV injected into uninfected S. litura can also be inherited maternally and causes male killing. SlMKV has five genomic segments encoding seven open reading frames, has no homolog of known male-killing genes, and belongs to an unclassified group of arthropod-specific viruses closely related to Tolivirales. When transinfected into larvae, both male and female recipients allow SlMKV to proliferate, but only males die at the pupal stage. The viral RNA levels in embryonic and pupal male killing suggest that the mechanism of male killing involves the constitutive expression of viral products that are specifically lethal to males, rather than the male-specific expression of viral products. Our results, together with recent findings on male-killing partiti-like viruses, suggest that diverse viruses in arthropods tend to acquire male killing independently and that such viruses may be important components of intragenomic conflict in arthropods.

W chromosome sequences of two bombycid moths provide an insight into the origin of Fem.

Fem is a W-linked gene that encodes a piRNA precursor, and its product, Fem piRNA, is a master factor of female determination in Bombyx mori. Fem has low similarity to any known sequences, and the origin of Fem remains unclear. So far, two hypotheses have been proposed for the origin of Fem: The first hypothesis is that Fem is an allele of Masc, which assumes that the W chromosome was originally a homologous chromosome of the Z chromosome. The second hypothesis is that Fem arose by the transposition of Masc to the W chromosome. To explore the origin of Fem, we determined the W chromosome sequences of B. mori and, as a comparison, a closely relative bombycid species of Trilocha varians with a Fem-independent sex determination system. To our surprise, although the sequences of W and Z chromosomes show no homology to each other, a few pairs of homologues are shared by W and Z chromosomes, indicating the W chromosome of both species originated from Z chromosome. In addition, the W chromosome of T. varians lacks Fem, while the W chromosome of B. mori has over 100 copies of Fem. The high-quality assembly of the W chromosome of B. mori arose the third hypothesis about the origin of Fem: Fem is a chimeric sequence of multiple transposons. More than half of one transcriptional unit of Fem shows a significant homology to RTE-BovB. Moreover, the Fem piRNA-producing region could correspond to the boundary of the two transposons, gypsy and satellite DNA.

Dimorphic sperm formation by Sex-lethal.

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of Sxl in the lepidopteran insect Bombyx mori In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori Therefore, the ancestral function of Sxl might be related to germline development.

Unique sex determination system in the silkworm, Bombyx mori: current status and beyond.

The silkworm Bombyx mori has been used for silk production for over 5,000 years. In addition to its contribution to sericulture, B. mori has played an important role in the field of genetics. Classical genetic studies revealed that a gene(s) with a strong feminizing activity is located on the W chromosome, but this W-linked feminizing gene, called Feminizer (Fem), had not been cloned despite more than 80 years of study. In 2014, we discovered that Fem is a precursor of a single W chromosome-derived PIWI-interacting RNA (piRNA). Fem-derived piRNA binds to PIWI protein, and this complex then cleaves the mRNA of the Z-linked Masculinizer (Masc) gene, which encodes a protein required for both masculinization and dosage compensation. These findings showed that the piRNA-mediated interaction between the two sex chromosomes is the primary signal for the sex determination cascade in B. mori. In this review, we summarize the history, current status, and perspective of studies on sex determination and related topics in B. mori.

Variation in nesting behavior of eight species of spider mites, Stigmaeopsis having sociality.

Nesting behavior is considered to be an important element of social living in animals. The spider mites belonging to the genus Stigmaeopsis spend their lives within nests produced from silk threads. Several of these species show cooperative sociality, while the others are subsocial. In order to identify the origins of this social behavior, comparisons of nest sizes, nesting behaviors (making nests continuously or separately), and their associated traits (fecal deposition patterns) were made for eight cogeneric Stigmaeopsis species showing various levels of social development. All of these species inhabit bamboo plants (Poaceae). We initially addressed the proximate factor of nest size variation. The variation in nest size of the eight species corresponded well with the variation in dorsal seta sc1 length, suggesting that nest size variation among species may have a genetic basis. The time spent within a nest (nest duration) increased with nest size on the respective host plants. Nest arrangement patterns varied among species showing different sized nests: Large nest builders continuously extended their nests, while middle and small nest-building species built new separate nests, which resulted in different social interaction times among species, and is thought to be closely related to social development. Fecal deposition behaviors also varied among Stigmaeopsis species, suggesting diversity in anti-predatory adaptations. Finally, we discuss how the variation in sociality observed within this genus is likely the result of nest size variation that initially evolved as anti-predator strategies.

Neo-sex chromosomes and adaptive potential in tortricid pests.

Changes in genome architecture often have a significant effect on ecological specialization and speciation. This effect may be further enhanced by involvement of sex chromosomes playing a disproportionate role in reproductive isolation. We have physically mapped the Z chromosome of the major pome fruit pest, the codling moth, Cydia pomonella (Tortricidae), and show that it arose by fusion between an ancestral Z chromosome and an autosome corresponding to chromosome 15 in the Bombyx mori reference genome. We further show that the fusion originated in a common ancestor of the main tortricid subfamilies, Olethreutinae and Tortricinae, comprising almost 700 pest species worldwide. The Z-autosome fusion brought two major genes conferring insecticide resistance and clusters of genes involved in detoxification of plant secondary metabolites under sex-linked inheritance. We suggest that this fusion significantly increased the adaptive potential of tortricid moths and thus contributed to their radiation and subsequent speciation.

Dynamic karyotype evolution and unique sex determination systems in Leptidea wood white butterflies.

BACKGROUND: Chromosomal rearrangements have the potential to limit the rate and pattern of gene flow within and between species and thus play a direct role in promoting and maintaining speciation. Wood white butterflies of the genus Leptidea are excellent models to study the role of chromosome rearrangements in speciation because they show karyotype variability not only among but also within species. In this work, we investigated genome architecture of three cryptic Leptidea species (L. juvernica, L. sinapis and L. reali) by standard and molecular cytogenetic techniques in order to reveal causes of the karyotype variability. RESULTS: Chromosome numbers ranged from 2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from Czech populations) to 2n = 51 to 55 in L. reali (Spanish population). We observed significant differences in chromosome numbers and localization of cytogenetic markers (rDNA and H3 histone genes) within the offspring of individual females. Using FISH with the (TTAGG) n telomeric probe we also documented the presence of multiple chromosome fusions and/or fissions and other complex rearrangements. Thus, the intraspecific karyotype variability is likely due to irregular chromosome segregation of multivalent meiotic configurations. The analysis of female meiotic chromosomes by GISH and CGH revealed multiple sex chromosomes: W1W2W3Z1Z2Z3Z4 in L. juvernica, W1W2W3Z1Z2Z3 in L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali. CONCLUSIONS: Our results suggest a dynamic karyotype evolution and point to the role of chromosomal rearrangements in the speciation of Leptidea butterflies. Moreover, our study revealed a curious sex determination system with 3-4 W and 3-4 Z chromosomes, which is unique in the Lepidoptera and which could also have played a role in the speciation process of the three Leptidea species.

Rapid turnover of the W chromosome in geographical populations of wild silkmoths, Samia cynthia ssp.

Our previous studies revealed a considerably high level of chromosomal polymorphism in wild silkmoths, Samia cynthia ssp. (Lepidoptera: Saturniidae). Geographical populations of this species complex differ in chromosome numbers and show derived sex chromosome systems including Z0/ZZ in S. cynthia ricini (2n = 27/28; Vietnam), neo-Wneo-Z/neo-Zneo-Z in S. cynthia walkeri (2n = 26/26; Sapporo, Hokkaido) and neo-WZ1Z2/Z1Z1Z2Z2 in S. cynthia subsp. indet. (2n = 25/26; Nagano, Honshu). In this study, we collected specimens of S. cynthia pryeri in Japanese islands Kyushu, Shikoku and Honshu, with an ancestral-like karyotype of 2n = 28 in both sexes and a WZ/ZZ sex chromosome system, except for one population, in which females have lost the W chromosome. However, the S. cynthia pryeri W chromosome showed a very unusual morphology: It was composed of a highly heterochromatic body, which remained condensed throughout the whole cell cycle and of a euchromatin-like "tail." We examined molecular composition of the W and neo-W chromosomes in S. cynthia subspecies by comparative genomic hybridisation and fluorescence in situ hybridisation with W chromosome painting probes prepared from laser-microdissected W chromatin of S. cynthia pryeri. These methods revealed that the molecular composition of highly heterochromatic part of the S. cynthia pryeri W chromosome is very different and lacks homology in the genomes of other subspecies, whereas the euchromatin-like part of the W chromosome corresponds to a heterochromatic part of the neo-W chromosomes in S. cynthia walkeri and S. cynthia subsp. indet. Our findings suggest that the curious WZ system of S. cynthia pryeri may represent an ancestral state of the Samia species complex but do not exclude an alternative hypothesis of its derived origin.

Sex chromosome evolution in moths and butterflies.

Lepidoptera, i.e. moths and butterflies, have a female heterogametic sex chromosome system, with most females having a WZ constitution while males are ZZ. Besides this predominant WZ/ZZ system, Z/ZZ, WZ(1)Z(2)/Z(1)Z(1)Z(2)Z(2) and W(1)W(2)Z/ZZ systems also occur. Sex is determined by an unknown W-linked gene or genes in Bombyx mori, but by dosage-dependent and equally unknown Z-linked genes in all Z/ZZ species. The female heterogametic sex chromosome system has been conserved for at least 180 MY in the phylogenetic branch that combines Lepidoptera and Trichoptera. The W chromosome, which is present in most lepidopteran species, was incorporated in the sex chromosome system much later, about 90-100 MY ago. The Z chromosomes are highly conserved among Lepidoptera, much like the Z in birds or the X in mammals. The W, on the other hand, is evolving rapidly. It is crammed with repetitive elements which appear to have a high turnover rate but poor in or even devoid of protein-coding genes. It has frequently undergone fusion with autosomes or sporadically lost altogether.

Lepidopteran Synteny Units reveal deep chromosomal conservation in butterflies and moths.

DNA is compacted into individual particles or chromosomes that form the basic units of inheritance. However, different animals and plants have widely different numbers of chromosomes. This means that we cannot readily tell which chromosomes are related to which. Here, we describe a simple technique that looks at the similarity of genes on each chromosome and thus gives us a true picture of their homology or similarity through evolutionary time. We use this new system to look at the chromosomes of butterflies and moths or Lepidoptera. We term the associated synteny units, Lepidopteran Synteny Units (LSUs). Using a sample of butterfly and moth genomes from across evolutionary time, we show that LSUs form a simple and reliable method of tracing chromosomal homology back through time. Surprisingly, this technique reveals that butterfly and moth chromosomes show conserved blocks dating back to their sister group the Trichoptera. As Lepidoptera have holocentric chromosomes, it will be interesting to see if similar levels of synteny are shown in groups of animals with monocentric chromosomes. The ability to define homology via LSU analysis makes it considerably easier to approach many questions in chromosomal evolution.

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.

Silk threads function as an 'adhesive cleaner' for nest space in a social spider mite.

Individuals of the social spider mite Stigmaeopsis longus live communally in narrow, humid nests made from silk threads and display nest sanitation behaviour through the coordinated deposition of faeces. We used artificial dust to experimentally determine that females of this species use silk threads to perform regular cleaning of the nest space and eggs. We first learned that silk-weaving behaviour is not a by-product of nest building (nest reinforcement); rather, it is actively performed as a function of cleaning the living space and eggs. Furthermore, we determined the effectiveness of the attending females by artificially manipulating their natural habitat, which is clearly connected to the cleaning behaviour by parental females. As such, we have uncovered an extraordinary new role of silk threads as devices for cleaning the nest space and/or eggs. These results strongly indicate that special adaptations for maintaining clean habitats are essential for animals to evolve aggregative social lives.

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.

Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera).

We examined chromosomal distribution of major ribosomal DNAs (rDNAs), clustered in the nucleolar organizer regions (NORs), in 18 species of moths and butterflies using fluorescence in situ hybridization with a codling moth (Cydia pomonella) 18S rDNA probe. Most species showed one or two rDNA clusters in their haploid karyotype but exceptions with 4-11 clusters also occurred. Our results in a compilation with previous data revealed dynamic evolution of rDNA distribution in Lepidoptera except Noctuoidea, which showed a highly uniform rDNA pattern. In karyotypes with one NOR, interstitial location of rDNA prevailed, whereas two-NOR karyotypes showed mostly terminally located rDNA clusters. A possible origin of the single interstitial NOR by fusion between two NOR-chromosomes with terminal rDNA clusters lacks support in available data. In some species, spreading of rDNA to new, mostly terminal chromosome regions was found. The multiplication of rDNA clusters without alteration of chromosome numbers rules out chromosome fissions as a major mechanism of rDNA expansion. Based on rDNA dynamics in Lepidoptera and considering the role of ordered nuclear architecture in karyotype evolution, we propose ectopic recombination, i.e., homologous recombination between repetitive sequences of non-homologous chromosomes, as a primary motive force in rDNA repatterning.

An HDAC inhibitor increases AcMNPV gene expression in mammalian cells.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is used as a safer viral vector in mammalian cells with potential applications in gene therapy. However, the mechanism for the insusceptibility of mammalian cells to proliferative infection by entomopathogenic viruses is not well understood. Here, we studied the significance of epigenetic modifications such as histone acetylation, histone methylation and HP1 accumulation for AcMNPV gene expression in mammalian BHK cells. Real-time PCR and chromatin immunoprecipitation with sodium butyrate revealed an important relationship between viral gene expression and histone acetylation, with implications for a mechanism of suppression of AcMNPV gene expression in BHK cells.

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle.

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130kDa Cry8Da protein to produce a 64kDa protein. This 64kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M(1) to F(63) was removed. As in the case of Cry3Aa, the proteases further digested the 64kDa protein to two 8kDa and 54kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8kDa fragment consists of Alpha 1-3 of Domain I and that the 54kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8kDa proteins suggesting that the 54kDa and 8kDa fragments are still forming the toxin complex equivalent to the 64kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54kDa fragment bound to the BBMV preparations but not the 64kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.

Reprobing multicolor FISH preparations in lepidopteran chromosome.

Multicolor fluorescence in-situ hybridization (FISH) and subsequent reprobing of chromosome preparations increase the number of chromosomes and/or anchor loci on the chromosomes simultaneously identified. Reprobing techniques have been widely applied to chromosomes of vertebrates and plants. We have developed a novel reprobing protocol that utilizes multicolor FISH and bacterial artificial chromosome (BAC) probes to examine chromosome preparations in a model lepidopteran species, the silkworm, Bombyx mori. With standard two-color BAC-FISH, routinely used to map genes on B. mori chromosomes, we could localize only two probes on one preparation, whereas our new protocol combining five-color BAC-FISH and preparation reprobing enabled us to simultaneously map 10 probes, as demonstrated with the Bombyx Z chromosome. The improved BAC-FISH technique will facilitate karyotyping and synteny analysis in Lepidoptera.

Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles.

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 microg per 1 cm(2) of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D's and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.

Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera.

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1-2, PBP-A-D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69-149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects.

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.