Toru Shimada

In this study, we found two embryonic lethal mutations, t04 lethal (l-t04) and m04 lethal (l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkworm Bombyx mandarina, is substituted with chromosomes of the domesticated silkworm B. mori, and l-t04 and l-m04 mutations are located on B. mandarina-derived chromosome 4. To clarify the cause of the lethalities and the genes responsible for these mutations, positional cloning and CRISPR/Cas9 mediated knockout screening were performed. Finally, genetic complementation tests l-t04l-m04 identified the mutations responsible for the l-t04 and l-m04 as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA box binding protein-associated factor 5 (BmTaf5), respectively. Lethal stages of each knockout mutant indicated that the importance of these genes in B. mori late embryogenesis. The lethal mutations responsible for l-t04 and l-m04 were not found in parental strains or wild B. mandarina collected from 39 distinct locations in Japan, indicating that both mutations were independently introduced during or after the development of the semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4.

In this study, we found two embryonic lethal mutations,t04 lethal(l-t04) andm04 lethal(l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkwormBombyx mandarina, is substituted with chromosomes of the domesticated silkwormB. mori, andl-t04andl-m04mutations are located onB. mandarina-derived chromosome 4. The mutations responsible for thel-t04andl-m04were identified as theBombyxhomolog ofimaginal discs arrested(Bmida) andTATA Box Binding Protein-associated factor 5(BmTaf5), respectively. These findings indicate that both mutations were independently introduced during or after the development of semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4.

<title>Abstract</title> Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Samia ricini, a gigantic saturniid moth, has the potential to be a novel lepidopteran model species. Samia ricini is far more resistant to diseases than the current model species Bombyx mori, and therefore can be more easily reared. In addition, genetic resources available for S. ricini rival those for B. mori: at least 26 ecoraces of S. ricini are reported and S. ricini can hybridize with wild Samia species, which are distributed throughout Asian countries, and produce fertile progenies. Physiological traits such as food preference, integument colour and larval spot pattern differ among S. ricini strains and wild Samia species so that those traits can be targeted in forward genetic analyses. To facilitate genetic research in S. ricini, we determined its whole genome sequence. The assembled genome of S. ricini was 458 Mb with 155 scaffolds, and the scaffold N50 length of the assembly was ~ 21 Mb. In total, 16,702 protein coding genes were predicted. While the S. ricini genome was mostly collinear with that of B. mori with some rearrangements and few S. ricini-specific genes were discovered, chorion genes and fibroin genes seemed to have expanded in the S. ricini lineage. As the first step of genetic analyses, causal genes for "Blue," "Yellow," "Spot," and "Red cocoon" phenotypes were mapped to chromosomes.

Gene duplication provides a major source of new genes for evolutionary novelty and ecological adaptation. However, the maintenance of duplicated genes and their relevance to adaptive evolution has long been debated. Insect trehalase (Treh) plays key roles in energy metabolism, growth, and stress recovery. Here, we show that the duplication of Treh in Lepidoptera (butterflies and moths) is linked with their adaptation to various environmental stresses. Generally, two Treh genes are present in insects: Treh1 and Treh2. We report three distinct forms of Treh in lepidopteran insects, where Treh1 was duplicated into two gene clusters (Treh1a and Treh1b). These gene clusters differ in gene expression patterns, enzymatic properties, and subcellular localizations, suggesting that the enzymes probably underwent sub- and/or neofunctionalization in the lepidopteran insects. Interestingly, selective pressure analysis provided significant evidence of positive selection on duplicate Treh1b gene in lepidopteran insect lineages. Most positively selected sites were located in the alpha-helical region, and several sites were close to the trehalose binding and catalytic sites. Subcellular adaptation of duplicate Treh1b driven by positive selection appears to have occurred as a result of selected changes in specific sequences, allowing for rapid reprogramming of duplicated Treh during evolution. Our results suggest that gene duplication of Treh and subsequent functional diversification could increase the survival rate of lepidopteran insects through various regulations of intracellular trehalose levels, facilitating their adaptation to diverse habitats. This study provides evidence regarding the mechanism by which gene family expansion can contribute to species adaptation through gene duplication and subsequent functional diversification.

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).

© 2018, The Author(s). In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

The Masculinizer gene (Masc) encodes a CCCH tandem zinc finger protein essential for masculinization and dosage compensation in the silkworm Bombyx mori. Previously we identified a Masc orthologue from the crambid Ostrinia furnacalis (OfMasc) and observed its masculinizing activity in the B. mori cultured cell line BmN-4. However, the role of OfMasc in masculinization of O. furnacalis has not been assessed. In this study, we unexpectedly discovered that all of the male larvae that escaped from Wolbachia-induced embryonic male-killing by OfMasc cRNA injection expressed the female-type splicing variants of O. furnacalis doublesex (Ofdsx). To clarify the role of OfMasc in the masculinization process in vivo, we established a system to monitor both sex chromosome- and dsx splicing-based sexes from a single O. furnacalis embryo. Using this system, we investigated the effects of OfMasc knockdown in early embryos on Ofdsx splicing and found that depletion of OfMasc mRNA in male embryos induced the production of the female-type splicing variants of Ofdsx. This result indicates that OfMasc is required for masculinization in O. furnacalis, and that the Masc protein possesses masculinizing activity in an insect species that is phylogenetically distant from Bombycidae.

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the + nsd-1 gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6–6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.

Lepidopteran nucleopolyhedroviruses have distinct viral tissue tropisms in host larvae. We previously identified the Bm8 gene of Bombyx mori nucleopolyhedrovirus (BmNPV), the product of which inhibits viral propagation in the middle silk gland (MSG). However, it is unknown whether this inhibitory function of the Bm8 protein is specific to MSGs. Here we generated a Bm8-disrupted recombinant BmNPV expressing green fluorescent protein (GFP) and examined viral propagation in B. mori cultured cells and larvae. We found that Bm8-disrupted BmNPV produced fewer budded viruses and more occlusion bodies (OBs) than the wild-type virus in both cultured cells and larvae. Microscopic observation of OB production and GFP expression revealed that Bm8 disruption accelerated the progression of viral infection in various larval tissues. Furthermore, quantitative reverse transcription-polymerase chain reaction experiments showed that the loss of Bm8 enhanced viral gene expression in BmNPV-infected larval tissues. These results indicate that the Bm8 protein suppresses viral propagation to varying degrees in each larval tissue, which may establish BmNPV tissue tropisms in B. mori larvae.

The Drosophila eye color gene brown is known to control the transport of pteridine precursors in adult eyes. The Brown protein belongs to the ATP-binding cassette (ABC) transporter G family, which includes proteins encoded by the genes brown, scarlet, and white. These genes are responsible for pigmentation in Drosophila and the domestic silkworm Bombyx mori. Although orthologs of brown are conserved among insects, the function of this gene is only known in Drosophila. Here, we elucidated the function of the B. mori ortholog Bm-brown. We examined the spatial and temporal expression profiles of Bm-brown and found that this gene was specifically and continuously expressed in larval Malpighian tubules (MTs), indicating this gene has a special function in MTs. We then successfully obtained a Bm-brown knockout (KO) strain based on a wild-type (WT) strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system. We found that larval MTs of the KO strain were white, whereas those of WT were yellow. It is known that larval yellow MTs of WT are due to the accumulation of riboflavin. Therefore, we compared the riboflavin contents of MTs of KO and WT strains, and found that the riboflavin level in the KO strain was 20 fold less than that in WT during the 5th instar period. MTs are known to exhibit a similar milky color in w-3 mutant larvae due to a deficiency of riboflavin accumulation. The responsible gene for w-3 mutant is the Bmwh3 gene, which is orthologous to Drosophila white. Thus, we speculate that Bm-brown is heterodimerized with Bmwh3, similar to Brown/White in Drosophila, and acts as a riboflavin transporter in silkworm MTs.

How to wire a neural circuit is crucial for the functioning of the nervous system. Here, we describe the neuroanatomy of the olfactory neurons in the spli mutant strain of silkmoth (Bombyx mori) to investigate the function of a transcription factor involved in neuronal wiring in the central olfactory circuit. The genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted in the spli mutant. We report the neuroanatomical abnormality in the morphology of the antennal lobe projection neurons (PNs) that process the sex pheromone. In addition to the mistargeting of dendrites and axons, we found axonal bifurcation within the PNs. These results indicate that the morphology of neurons in the pheromone processing pathway is modified by Bmacj6.

"Tanaka's mottled translucent" (otm) is a mutation of the silkworm Bombyx mori that exhibits translucent skin during larval stages. We performed positional cloning of the gene responsible for otm and mapped it to a 364-kb region on chromosome 5 that contains 22 hypothetical protein-coding genes. We performed RNA-seq analysis of the epidermis and fat body of otm larvae and determined that the gene BGIBMGA002619 may be responsible for the otm mutation. BGIBMGA002619 encodes the biosynthesis of lysosome-related organelles complex 1 (BLOC-1) subunit 5, whose ortholog is responsible for the Muted mutant in mouse. Accordingly, we named this gene Bm-muted. We discovered that the expression of Bm-muted in the epidermis and fat body of otm mutants was dramatically suppressed compared with the wild type. We determined the nucleotide sequences of the full-length cDNA and genomic region corresponding to Bm-muted and found that a 538-bp long DNA sequence similar to B. mori transposon Organdy was inserted into the 3' end of the first intron of Bm-muted in two otm strains. The Bm-muted cDNA of otm mutants lacked exon 2, and accordingly generated a premature stop codon in exon 3. In addition, short interfering RNA (siRNA)-mediated knockdown of this gene caused localized partial translucency of larval skin. These data indicate that the mutation in Bm-muted caused the oats-mutant phenotype. We propose that the insertion of Organdy caused a splicing disorder in Bm-muted in the otm mutant, resulting in a null mutation of Bm-muted. This mutation is likely to cause deficiencies in urate granule formation in epidermal cells that result in translucent larval skin.

PIWI-interacting RNAs (piRNAs) play essential roles in the defense system against selfish elements in animal germline cells by cooperating with PIWI proteins. A subset of piRNAs is predicted to be generated via the "ping-pong" cascade, which is mainly controlled by two different PIWI proteins. Here we established a cell-based artificial piRNA production system using a silkworm ovarian cultured cell line that is believed to possess a complete piRNA pathway. In addition, we took advantage of a unique silkworm sex-determining one-to-one ping-pong piRNA pair, which enabled us to precisely monitor the behavior of individual artificial piRNAs. With this novel strategy, we successfully generated artificial piRNAs against endogenous protein-coding genes via the expected back-and-forth traveling mechanism. Furthermore, we detected "primary" piRNAs from the upstream region of the artificial "ping-pong" site in the endogenous gene. This artificial piRNA production system experimentally confirms the existence of the "ping-pong" cascade of piRNAs. Also, this system will enable us to identify the factors involved in both, or each, of the "ping" and "pong" cascades and the sequence features that are required for efficient piRNA production.

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. (C) 2016 Elsevier Inc. All rights reserved.

The silkworm Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation. Masc protein is a nuclear protein, but the mechanism underlying the transport of this protein into the nucleus has not yet been elucidated. Here, we identified a functional bipartite nuclear localization signal (NLS) located between residues 274 and 290 of the Masc protein. Sequence comparison revealed that this bipartite NLS is evolutionarily conserved in Masc proteins from other lepidopteran insects. Furthermore, we showed that the degree of nuclear localization is not associated with the masculinizing activity of the Masc protein.

Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd-2. We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), +(nsd-2) (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and +(nsd-2) were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of +(nsd-2) expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of +(nsd-2) in the midgut and that viral infection is supported even by very faint expression of +(nsd-2). By contrast, the expression levels of +(nsd-2) were exceedingly low or undetectable in the middle part of the mid gut, indicating that BmBDV infection might occur via another mechanism, independent of +(nsd-2), in the middle part of the midgut. (C) 2016 Elsevier Inc. All rights reserved.

Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing-and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to similar to 2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing-and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera.

Diverse color patterns on the integument of lepidopteran larvae play important roles in their survival through camouflage, mimicry, sexual signaling, and aposematism. In the silkworm Bombyx mari, many color pattern variations have been preserved in inbred strains making them a good model for elucidating the molecular mechanisms that underlie color pattern formation. In this study, we focused on the silkworm quail (q) mutant, which exhibits abnormalities in multiple pigment biosynthesis pathways. Positional cloning of the q gene revealed that disruption of a guanylyl cyclase gene, BmGC-I, is responsible for its abnormal pigmentation. In q mutants, we identified a 16-bp deletion in the BmGC-I transcript, resulting in the production of a premature stop codon. Knockout of the BmGC-I gene resulted in the q-like abnormal pigmentation, thereby demonstrating that the BmGC-I gene is involved in the pigment biosynthesis pathway in the integument. Moreover, quantitative reverse transcription polymerase chain reaction showed that BmGC-I was strongly expressed in the fourth instar on day 2. Our results suggest that BmGC-I deficiency affects the pigment biosynthesis pathway, which supports the involvement of guanylyl cyclase in larval coloration. (C) 2015 Elsevier Ltd. All rights reserved.

We recently showed that the Masculinizer gene (Masc) plays a primary role in sex determination in the lepidopteran model insect Bombyx mori. However, it remains unknown whether this Masc protein-dependent sex determination system is conserved amongst lepidopteran insects or within the family Bombycidae. Here we cloned and characterized a Masc homologue (TvMasc) in Trilocha varians (Lepidoptera: Bombycidae), a species closely related to B. mori. To elucidate the role of TvMasc in the sex determination cascade of T. varians, TvMasc expression was knocked down in early embryos by the injection of small interfering RNAs (siRNAs) that targeted TvMasc mRNAs. Both female- and male-type splice variants of Tvdsx, a doublesex (dsx) homologue in T. varians were observed in control siRNA-injected embryos. By contrast, only female-type splice variants were observed in TvMasc siRNA-injected embryos. These results indicate that the TvMasc protein directly or indirectly regulates the splicing patterns of Tvdsx. Furthermore, we found that male-type splice variants of B. mori dsx (Bmdsx) were produced in TvMasc-overexpressing BmN4 cells. The mRNA level of B. mori Imp, a gene whose product induces male-specific Bmdsx splicing also increased. These results suggest that Masc genes play similar roles in the sex-determination cascade in Bombycidae.

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.

Pathogens are known to manipulate the reproduction and development of their hosts for their own benefit. Wolbachia is an endosymbiotic bacterium that infects a wide range of insect species. Wolbachia is known as an example of a parasite that manipulates the sex of its host's progeny. Infection of Ostrinia moths by Wolbachia causes the production of all-female progeny, however, the mechanism of how Wolbachia accomplishes this male-specific killing is unknown. Here we show for the first time that Wolbachia targets the host masculinizing gene of Ostrinia to accomplish male-killing. We found that Wolbachia-infected O. furnacalis embryos do not express the male-specific splice variant of doublesex, a gene which acts at the downstream end of the sex differentiation cascade, throughout embryonic development. Transcriptome analysis revealed that Wolbachia infection markedly reduces the mRNA level of Masc, a gene that encodes a protein required for both masculinization and dosage compensation in the silkworm Bombyx mori. Detailed bioinformatic analysis also elucidated that dosage compensation of Z-linked genes fails in Wolbachia-infected O. furnacalis embryos, a phenomenon that is extremely similar to that observed in Masc mRNA-depleted male embryos of B. mori. Finally, injection of in vitro transcribed Masc cRNA into Wolbachia-infected embryos rescued male progeny. Our results show that Wolbachia-induced male-killing is caused by a failure of dosage compensation via repression of the host masculinizing gene. Our study also shows a novel strategy by which a pathogen hijacks the host sex determination cascade.

The actin rearrangement-inducing factor 1 (arif-1) gene is a baculoviral early gene conserved in most alphabaculoviruses. Previous studies reported that Autographa californica nucleopolyhedrovirus ARIF-1 protein induces filamentous actin concentration on the plasma membrane during the early stage of infection in Trichoplusia ni TN-368 cells, but its role in larval infection remains unknown. In this study, we performed behavioural screening using Bombyx mori larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) mutants and found that larvae infected with arif-1-mutated BmNPVs did not show locomotor hyperactivity that was normally observed in BmNPV-infected larvae. arif-1-deficient BmNPVs also showed reduced pathogenicity and total viral propagation in B. mori larvae, whereas viral propagation of arif-1-deficient viruses was comparable with that of control viruses in B. mori cultured cells. An arif-1-defective BmNPV expressing the GFP gene (gfp) was used to monitor the progression of infection in B. mori larvae. GFP expression and quantitative reverse transcription-PCR analyses revealed that infection by the arif-1-disrupted virus was significantly delayed in trachea, fat body, suboesophageal ganglion and brain. These results indicated that BmNPV ARIF-1 enhanced systemic infection in B. mori larvae.

Several lines of evidence have shown that the deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the nucleopolyhedrovirus (NPV) genome increases the killing speed of host lepidopteran larvae. However, it has not been investigated in detail whether the effects of egt deletion depend on the larval stages of the host insect. In this study, we performed bioassays using 10 continuous larval stages of the 4th- or 5th-instar Bombyx mori larvae and B. mori NPV egt mutants. The fast-killing phenotype was observed in the egt mutants only when the infection process progressed through larval-larval transition. All day-2 4th-instar larvae infected with the egt mutants entered the molting stage and died much earlier than wild-type-infected larvae. Bodies of egt mutant-infected larvae were filled with excessive fluid immediately after head capsule slippage, owing presumably to the degeneration of Malpighian tubules. Fourth- or 5th-instar larvae infected with the egt mutants at early stages of each instar died similarly to those infected with the wild-type virus. Under infection in the middle stages of the 5th-instar, the survival time of egt mutant-infected larvae was significantly longer than that of the wild-type virus-infected larvae. These results clearly show that the effects of egt deletion on killing speed of NPV are largely dependent on the developmental stage of the host larvae infected by the virus. (C) 2015 Elsevier Inc. All rights reserved.

Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry. (C) 2015 Elsevier Ltd. All rights reserved.

In the silkworm, Bombyx mori, females are heterogametic (WZ) whereas males have two Z chromosomes. Femaleness of B. mori is determined by the presence of the W chromosome, suggesting that there is a dominant feminizing gene on this chromosome. Recently, by transcriptome analysis of B. mori embryos, we discovered that a single W-chromosome-derived PIWI-interacting RNA (piRNA) is the long-sought primary determinant of femaleness in B. mori. However, sexual bias in the transcriptome of B. mori early embryos has not yet been well characterized. Using deep sequencing data from molecularly sexed RNA of B. mori embryos, we identified and characterized 157 transcripts that are statistically differentially expressed between male and female early embryos. Most of the female-biased transcripts were transposons or repeat sequences that are produced presumably from the W chromosome. Bioinformatic analysis revealed that these repetitive sequences are piRNA precursors. In contrast, male-biased genes were frequently transcribed from the Z chromosome, suggesting that dosage compensation in Z-linked genes does not occur or is incomplete at early embryonic stages. Our analysis has drawn a picture of a global landscape of sexually biased transcriptome during early B. mori embyogenesis and has suggested for the first time that most sexually biased embryonic transcripts depend on sex chromosomes.

Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of the superoxide anion. Homologs of a Cu/Zn-SOD gene were found in the genomes of most lepidopteran baculoviruses; however, its function is poorly understood. In this study, we characterized the sod gene of Bombyx mori (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV). Western blot analysis revealed that His-tagged BmNPV SOD was expressed as an approximately 17-kDa protein. Reverse transcription-quantitative polymerase chain reaction demonstrated that BmNPV sod mRNA was detected at 8 h postinfection (hpi), peaked at 36 hpi, and declined gradually. In contrast, B. mori sod declined rapidly and was almost completely diminished from 36 hpi. To comprehend the role of BmNPV sod during virus infection, we used an sod-disrupted BmNPV bacmid. We found that deletion of sod from the BmNPV genome had little effect on virus propagation in cultured cells, whereas production of budded viruses and occlusion bodies were significantly delayed in B. mori larvae. These results indicate that BmNPV SOD plays a role in efficient virus propagation in B. mori larvae.

Although some viruses have been shown to encode long non-coding RNAs (lncRNAs), how they function during their infection cycles remains elusive. We previously found an unexpectedly large number of novel transcripts, including putative lncRNAs, which were expressed from the genome of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV). To investigate the function of baculoviral antisense lncRNAs, we selected 15 BmNPV lncRNAs expressed from the baculovirus early or late promoter motif, and constructed the corresponding promoter knockout (PKO) viruses in which nucleotide substitutions were introduced at the transcription start sites of lncRNAs. We investigated the production of budded viruses (BVs) and occlusion bodies (OBs) in PKO virus-infected cultured cells and silkworm larvae. No defects in BV and OB production were observed for most of the PKO viruses, whereas KO122AS, in which an antisense lncRNA in the Bm122 locus was disrupted, produced fewer OBs than the wild-type or repair virus both in cultured cells and in silkworm larvae. Northern blot analysis revealed that loss of this antisense lncRNAs reduced the amounts of the corresponding sense mRNA. These results suggest that the lncRNA transcribed from the Bm122 locus might contribute to virus propagation by regulating viral gene expression. This is the first study to characterize the properties and functions of baculoviral lncRNAs.

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5 splice site. Segmental duplication in Ge-2, an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species and is crucial for heterochromatin establishment and maintenance. The silkworm, Bombyx mori, encodes two HP1 proteins, BmHP1a and BmHP1b. In order to investigate the role of BmHP1a in transcriptional regulation, we performed genome-wide analyses of the transcriptome, transcription start sites (TSSs), chromatin modification states and BmHP1a-binding sites of the silkworm ovary-derived BmN4 cell line. We identified a number of BmHP1a-binding loci throughout the silkworm genome and found that these loci included TSSs and frequently co-occurred with neighboring euchromatic histone modifications. In addition, we observed that genes with BmHP1a-associated TSSs were relatively highly expressed in BmN4 cells. RNA interference-mediated BmHP1a depletion resulted in the transcriptional repression of highly expressed genes with BmHP1a-associated TSSs, whereas genes not coupled with BmHP1a-binding regions were less affected by the treatment. These results demonstrate that BmHP1a binds near TSSs of highly expressed euchromatic genes and positively regulates their expression. Our study revealed a novel mode of transcriptional regulation mediated by HP1 proteins.

The baculovirus Bombyx mod nucleopolyhedrovirus (BmNPV) possesses two genes, iap1 and iap2, which encode inhibitor of apoptosis (IAP) proteins. We previously showed that although both genes are dispensable for viral propagation, iap2 is required for efficient viral propagation in cultured cells. BmNPV IAP2 contains three putative functional domains: a baculovirus IAP repeat (BIR), a BIR-like (BIRL) domain, and a RING finger domain. To identify the domain affecting viral growth, we generated a series of BmNPV bacmids expressing iap2 derivatives lacking one or two domains, or possessing a single amino acid substitution to abolish IAP2 ubiquitin ligase activity. We examined their properties in both cultured cells and B. mod larvae. We found that either the BIR or BIRL domain of IAP2 plays an important role in BmNPV infection, and that the RING finger domain, which is required for ubiquitin ligase activity, does not greatly contribute to BmNPV propagation. This is the first study to identify functional domains of the baculovirus IAP2 protein. (C) 2014 Elsevier Inc. All rights reserved.

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Newly ecdysed fifth instar larvae of the Bombyx mori strain N4 were divided into three groups and fed one of three different diets until they began spinning a cocoon. One group was fed the basal diet, in which carotenoids consisted mainly of lutein (53.3μg/g wet) and β-carotene (14.4μg/g wet), while the two other groups were fed the basal diet supplemented with astaxanthin from AstaREAL® or capsanthin from paprika-red®. High performance liquid chromatography was performed to identify the carotenoids in lipophorin, the middle silk glands, and the cocoons. Lutein, as well as astaxanthin and capsanthin, was accumulated in the cocoons after feeding with the astaxanthin- or capsanthin-supplemented diet. Quantitative analysis of the strain N4 and c10 demonstrated competitive accumulation in the cocoon between lutein and astaxanthin. In fifth instar larvae of the white cocoon producer, the strain c05 fed a diet containing lutein and astaxanthin, however, they produced only white cocoon. These results indicated that lutein and astaxanthin transport share common pathways, and that the delivery system in the silkworm can mediate the transfer and accumulation of carotenoids which are not derived from mulberry leaves, such as astaxanthin and capsanthin.<br>

We investigated the neuroanatomy of the macroglomerular complex (MGC), which is involved in sex pheromone processing, in five species in the subfamily Bombycinae, including Ernolatia moorei, Trilocha varians, Rondotia menciana, Bombyx mandarina and Bombyx mori. The glomerulus located at the dorsal-most part of the olfactory centre shows the largest volume in moth species examined to date. Such normal glomerular organization has been observed in E. moorei and T. varians, which use a two-component mixture and includes the compound bombykal as a mating signal. By contrast, the other three species, which use another component as a single attractant, exhibited a modified arrangement of the MGC. This correlation between pheromone usage and neural organization may be useful for understanding the process of speciation.

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth's olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.