Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Expression of the Wolbachia male-killing factor Oscar impairs dosage compensation in lepidopteran embryos.

Wolbachia are intracellular bacteria in insects that can manipulate the sexual development and reproduction by male killing or other methods. We have recently identified a Wolbachia protein named Oscar that acts as a male-killing factor for lepidopteran insects. Oscar interacts with the Masculinizer (Masc) protein, which is required for both masculinization and dosage compensation (DC) in lepidopteran insects. Embryonic expression of Oscar inhibits masculinization and causes male killing in two lepidopteran species, Ostrinia furnacalis and Bombyx mori. However, it remains unknown whether Oscar-induced male killing is caused by a failure of DC. Here, we performed a transcriptome analysis of Oscar complementary RNA-injected O. furnacalis and B. mori embryos, and found that Oscar primarily targets the Masc protein, resulting in male killing by interfering with DC in lepidopteran insects.

A method for screening the suppressor genes of siRNA and piRNA pathways using cultured silkworm cells.

The BmN-4 cell line originates from the ovaries of silkworm, Bombyx mori , and possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. BmN-4 cells are latently infected with Bombyx mori latent virus (BmLV), an RNA virus whose replication is strictly controlled by both siRNA and piRNA pathways. Knockdown or knockout of the core factors of these two small RNA pathways increases BmLV RNA amount, which in turn inhibits cell growth. Here, we used the known RNAi suppressor CrPV-1A to assess whether the BmN-4 cell line can be used for screening the suppressors of siRNA and piRNA pathways.

Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.

A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses.

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.

Masculinizer is not post-transcriptionally regulated by female-specific piRNAs during sex determination in the Asian corn borer, Ostrinia furnacalis.

Lepidopteran insects are heterogametic in females, although most insect species are heterogametic in males. In a lepidopteran model species, the silkworm Bombyx mori (Bombycoidea), the uppermost sex determinant Feminizer (Fem) has been identified on the female-specific W chromosome. Fem is a precursor of PIWI-interacting small RNA (piRNA). Fem piRNA forms a complex with Siwi, one of the two B. mori PIWI-clade Argonaute proteins. In female embryos, Fem piRNA-Siwi complex cleaves the mRNA of the male-determining gene Masculinizer (Masc), directing the female-determining pathway. In male embryos, Masc activates the male-determining pathway in the absence of Fem piRNA. Recently, W chromosome-derived piRNAs complementary to Masc mRNA have also been identified in the diamondback moth Plutella xylostella (Yponomeutoidea), indicating the convergent evolution of piRNA-dependent sex determination in Lepidoptera. Here, we show that this is not the case in the Asian corn borer, Ostrinia furnacalis (Pyraloidea). Although our previous studies demonstrated that O. furnacalis Masc (OfMasc) has a masculinizing function in the embryonic stage, the expression level of OfMasc was indistinguishable between the sexes at the timing of sex determination. Deep sequencing analysis identified no female-specific small RNAs mapped onto OfMasc mRNA. Embryonic knockdown of two PIWI genes did not affect the expression level of OfMasc in either sex. These results demonstrated that piRNA-dependent reduction of Masc mRNA in female embryos is not a common strategy of sex determination, which suggests the possibility of divergent evolution of sex determinants across the order Lepidoptera.

Recessive embryonic lethal mutations uncovered in heterozygous condition in silkworm semiconsomic strains.

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 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 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.

Expression and localization of Bombyx mori nucleopolyhedrovirus GP37.

Mitochondria play an essential role in intracellular energy metabolism. This study described the involvement of Bombyx mori nucleopolyhedrovirus (BmNPV) GP37 (BmGP37) in host mitochondria. Herein, the proteins associated with host mitochondria isolated from BmNPV-infected or mock-infected cells by two-dimensional gel electrophoresis were compared. One mitochondria-associated protein in virus-infected cells was identified as BmGP37 by liquid chromatography-mass spectrometry analysis. Furthermore, the BmGP37 antibodies were generated, which could react specifically with BmGP37 in the BmNPV-infected BmN cells. Western blot experiments showed that BmGP37 was expressed at 18 h post-infection and was verified as a mitochondria-associated protein. Immunofluorescence analysis demonstrated that BmGP37 localized to the host mitochondria during BmNPV infection. Furthermore, western blot analysis revealed that BmGP37 is a novel component protein of the occlusion-derived virus (ODV) of BmNPV. The present results indicated that BmGP37 is one of the ODV-associated proteins and may have important roles in host mitochondria during BmNPV infection.

Two Complete Genomes of Male-Killing Wolbachia Infecting Ostrinia Moth Species Illuminate Their Evolutionary Dynamics and Association with Hosts.

Wolbachia is an extremely widespread intracellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of Ostrinia furnacalis and Ostrinia scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species' infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, two possible scenarios were proposed: (1) Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis, or (2) Wolbachia infection in these species was introgressively transferred from a currently unidentified relative. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint.

The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells.

PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still "alive" and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs.

A Wolbachia factor for male killing in lepidopteran insects.

Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression of Oscar inhibits Masc-induced masculinization and leads to male killing in two lepidopteran insects, O. furnacalis and the silkworm Bombyx mori. Our study identifies a mechanism by which Wolbachia induce male killing of host progeny.

The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage.

The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.

Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm, Bombyx mori.

The evolution of dosage compensation produces similar expression of sex-linked and autosomal genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran insect, has a female heterogametic WZ sex determination system. A Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation in B. mori. However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analysed allele-specific expression to distinguish these alternatives. Our analysis revealed that genes on both the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. We therefore conclude that both Z chromosomes are transcriptionally downregulated in B. mori males, similar to the system in Caenorhabditis elegans.

Mutations in the polyhedrin NLS affect the assembly and polyhedral shape of alphabaculovirus occlusion bodies.

Alphabaculoviruses produce occlusion bodies (OBs) in the nucleus of the infected cells at the late stage of infection. OBs are mainly composed of a single viral protein called polyhedrin (POLH). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) POLH possesses a monopartite nuclear localization signal sequence (NLS), KRKK, from 32nd to 35th residues. However, the functions of POLH NLS of other alphabaculoviruses remain unknown. Here, POLH NLS mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated and NLS function as well as the relationship between NLS and OB localization or morphology was investigated. Deletion or mutation of BmNPV POLH NLS severely affected POLH and OB intracellular localization. Additionally, viruses in which the arginine residue at the 33rd position of POLH was mutated produced a lower number of OBs, which was presumably due to decreased POLH accumulation in the infected cells. Furthermore, cytoplasmic OBs were morphologically aberrant, even though nuclear OB morphology was normal in the same cell. These results indicate that NLS is required for nuclear localization and efficient accumulation of BmNPV POLH, which heavily affect the number and morphology of OBs.

Resistance mechanism of Nid-1, a dominant non-susceptibility gene, against Bombyx mori densovirus 1 infection.

Bombyx mori densovirus 1 (BmDV1) is a pathogen that causes flacherie disease in mulberry silkworms (B. mori). The absolute resistance (non-susceptibility) to BmDV1 of certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. Previously, we investigated the expression of viral transcript in virus-inoculated silkworms carrying different nsd-1 and Nid-1 genotypes, and observed that nsd-1 and Nid-1 expression blocked the early and late steps of BmDV1 infection, respectively. In addition, we found that nsd-1 encoded a Bombyx-specific mucin-like membrane protein only present on the surface of the midgut, where BmDV1 could infect. In this study, we dissected the resistance mechanism by Nid-1 against BmDV1 infection by investigating the sequential changes in the accumulation of viral DNA, transcripts, and proteins derived from BmDV1 in susceptible strain (pxj) and Nid-1-carrying resistant strain (No. 908) after inoculation with BmDV1. Genomic PCR results showed that the BmDV1 DNA was detected immediately after the infection in both strains but rapidly decreased in the Nid-1-carrying strain No. 908 compared with the susceptible strain pxj. RT-PCR results also showed that the BmDV1 transcripts of Nid-1-carrying strain No. 908 were rapidly decreased after the infection. Moreover, BmDV1-derived proteins were not detected in No. 908 throughout the infection. These results suggest that Nid-1 expression might inhibit the accumulation of viral DNA and transcripts. As Nid-1 has not been molecularly characterized, its identification will contribute to the elucidation of the interactions between the silkworm and BmDV1.

SilkBase: an integrated transcriptomic and genomic database for Bombyx mori and related species.

We introduce SilkBase as an integrated database for transcriptomic and genomic resources of the domesticated silkworm Bombyx mori and related species. SilkBase is the oldest B. mori database that was originally established as the expressed sequence tag database since 1999. Here, we upgraded the database by including the datasets of the newly assembled B. mori complete genome sequence, predicted gene models, bacterial artificial chromosome (BAC)-end and fosmid-end sequences, complementary DNA (cDNA) reads from 69 libraries, RNA-seq data from 10 libraries, PIWI-interacting RNAs (piRNAs) from 13 libraries, ChIP-seq data of 9 histone modifications and HP1 proteins and transcriptome and/or genome data of four B. mori-related species, i.e. Bombyx mandarina, Trilocha varians, Ernolatia moorei and Samia ricini. Our new integrated genome browser easily provides a snapshot of tissue- and stage-specific gene expression, alternative splicing, production of piRNAs and histone modifications at the gene locus of interest. Moreover, SilkBase is useful for performing comparative studies among five closely related lepidopteran insects. Database URL: https://silkbase.ab.a.u-tokyo.ac.jp.

Transcriptome analysis in the silkworm Bombyx mori overexpressing piRNA-resistant Masculinizer gene.

Dosage compensation is a process that produces a similar expression of sex-linked and autosomal genes. In the silkworm Bombyx mori with a WZ sex-determination system, the expression from the single Z in WZ females matches that of ZZ males due to the suppression of Z-linked genes in males. A primary maleness determinant gene, Masculinizer (Masc), is also required for dosage compensation. In females, silkworm Piwi is complexed with the W chromosome-derived female-specific Feminizer (Fem) PIWI-interacting RNA (piRNA) and cleaves Masc mRNA. When Fem piRNA-resistant Masc cDNA (Masc-R) is overexpressed in both sexes, only female larvae are dead during the larval stage. In this study, transcriptome analysis was performed in neonate larvae to examine the effects of Masc-R overexpression on a global gene expression profile. Z-linked genes were globally repressed in Masc-R-overexpressing females due to force-driven dosage compensation. In contrast, Masc-R overexpression had little effect on the expression of Z-linked genes and the male-specific isoform of B. mori insulin-like growth factor II mRNA-binding protein in males, indicating that excessive Masc expression strengthens neither dosage compensation nor maleness in males. Fourteen genes were differentially expressed between Masc-R-overexpressing and control neonate larvae in both sexes, suggesting Masc functions other than dosage compensation and masculinization.

Functional Characterization of Silkworm PIWI Proteins by Embryonic RNAi.

The molecular mechanisms of sex-determination systems among insect orders and species are diverse. Therefore, genes involved in sex determination are strong candidates for insect pest management. Even though lepidopterans are major agricultural insect pests that cause widespread economic damage to various crops, their sex-determination systems have not been fully elucidated, even in the silkworm (Bombyx mori), a model lepidopteran insect. In 2014, we found that a female-specific W chromosome-derived PIWI-interacting RNA (piRNA) determines femaleness in silkworms. To analyze the function of two core silkworm piRNA biogenesis pathway genes, Siwi and BmAgo3, in the sex-determination system, we developed a genomic DNA and total RNA extraction strategy for a siRNA-injected single embryo. The siRNA-injected embryo can be molecularly sexed by W chromosome-specific DNA markers. Using complementary DNA (cDNA) reverse transcribed from the sexed RNA, we evaluated the knockdown effect of the PIWI protein-coding genes on a sexual development-related gene, Bombyx mori doublesex.

Mutations in a ß-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori.

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the ß-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes.

RNase κ promotes robust piRNA production by generating 2',3'-cyclic phosphate-containing precursors.

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.