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).

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.

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.

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.

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.

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.

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.