Biology subjects

Katsuma, S.

Publications and source records attributed to Katsuma, S..

14 recordsLinked to original sources

bioRxiv2025

The Masc-PSI complex directly induces male-type doublesex splicing in silkworms

The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori, a lepidopteran model insect, females carry the W chromosome with the feminizing gene Feminizer (Fem), which is the source of a female-specific PIWI-interacting RNA (piRNA). The Fem piRNA-PIWI complex cleaves an mRNA encoding the masculinizing protein Masculinizer (BmMasc), resulting in the production of the female-type splice variant of Bombyx mori doublesex (Bmdsx), which is the master genetic switch of B. mori sex differentiation. In contrast, in males, BmMasc induces the production of the male-type Bmdsx splice variant (BmdsxM). However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the male-specific Bmdsx splicing event remains unknown. In this study, we showed that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), that is a RNA binding protein required for BmdsxM expression. BmMasc overexpression resulted in the production of BmdsxM in B. mori ovary-derived BmN-4 cells, but this induction was severely inhibited in BmPSI-knocked down cells, indicating that BmPSI is essential for the masculinizing activity of BmMasc. We also identified that BmMasc-containing protein complex was associated with a region of Bmdsx pre-mRNA spanning introns 2 to 4, particularly around the junctions of intron 2 with exon 3 and intron 3 with exon 4. Exons 3 and 4 of Bmdsx are female-specific units and are skipped in BmdsxM. Together with a previous report of the binding of BmPSI to the CE1 sequence located in exon 4 of Bmdsx, the current results strongly suggest that the BmMasc-BmPSI complex is required for male-specific exon skipping in Bmdsx pre-mRNA through its binding to the female-specific Bmdsx introns and exons.

molecular biology↗

bioRxiv2025

Evolution of Wolbachia male-killing mechanism within a host species

Male-killing bacterial symbionts, prevalent in arthropods, skew population sex ratios by selectively killing male progeny, profoundly impacting ecology and evolution of their hosts. Male-killing is a convergently evolved trait, with microbes evolving diverse male-killing mechanisms across host species with widely divergent sex determination pathways. A common evolutionary response to MK presence is the spread of suppressor mutations that restore male survival. In this study, we demonstrate evolution of a novel male-killing mechanism that is insensitive to an existing male-killing suppressor. Hypolimnas bolina butterflies from Yogyakarta, Indonesia, showed extreme female biased population sex ratio associated with high prevalence of a male-killing Wolbachia. This strain, wBol1Y, shared a very recent common ancestor with the previously characterized suppressed male-killing strain in the species, wBol1, but retained its male-killing ability in the presence of the male-killing suppressor. The genome of wBol1Y differed from the suppressed wBol1 in carrying an additional prophage that included strong candidate genes for male-killing. In vitro and in vivo data demonstrated that wBol1Y feminized splicing and expression of lepidopteran sex determination pathway genes, and that the gene Hb-oscar - present on wBol1Ys unique prophage insert - was sufficient to disrupt the male sex determination pathway. Our study demonstrates the diversity of male-killing mechanisms is a product both of interaction with varying insect sex determination systems and evolution of male-killing within a host species. Our data indicate male-killer and host may be involved in escalating arms races, where spread male-killing suppression drives evolution of additional systems that reestablish male killing by the symbiont.

evolutionary biology↗

bioRxiv2024

Prophage-encoded Hm-oscar gene recapitulates Wolbachia-induced male killing in the tea tortrix moth Homona magnanima

Wolbachia are maternally transmitted bacterial symbionts that are ubiquitous among arthropods. They can hijack host reproduction in various ways, including male killing (MK), where the sons of infected mothers are killed during development. The recent discovery of MK-associated Wolbachia genes, i.e., oscar in Ostrinia moths and wmk in Drosophila flies, stimulates our interest in the diversity and commonality of MK mechanisms, which remain largely unclear. We recently discovered that a Wolbachia symbiont of the moth Homona magnanima carries an MK-associated prophage region encoding homologs of oscar (Hm-oscar) and wmk (wmk-1-4). Here, we investigated the effects of these genes in the native host. Upon transient overexpression, Hm-oscar, but not wmk, induced male lethality in H. magnanima, in contrast to our observations in Drosophila, where the wmk homologs, but not Hm-oscar, killed the males. Hm-oscar disrupted sex determination in male embryos by inducing a female-type doublesex splicing and impaired dosage compensation, recapitulating the Wolbachia phenotype. Cell-based transfection assays confirmed that Hm-oscar suppressed the function of masculinizer, the primary male sex determinant involved in lepidopteran dosage compensation. Our study highlights the conserved roles of oscar homologs in Wolbachia-induced lepidopteran MK and argues that Wolbachia have evolved multiple MK mechanisms in insects.

evolutionary biology↗

bioRxiv2024

Parasitoid wasp venoms degrade Drosophila imaginal discs for successful parasitism

Parasitoid wasps, one of the most diverse and species-rich animal taxa on Earth, produce venoms that manipulate host development and physiology to exploit host resources. However, mechanisms of venom action remain poorly understood. Here, we show that infection of host Drosophila by the endoparasitoid wasp, Asobara japonica, triggers imaginal disc degradation (IDD) by inducing apoptosis, autophagy, and mitotic arrest, leading to impaired host metamorphosis. A multi-omics approach identified two venom proteins of A. japonica necessary for IDD. Knockdown experiments targeting the venom genes revealed that in concert with host immune suppression, IDD is essential for successful parasitism. Our study highlights a venom-mediated hijacking strategy of the parasitoid wasp that allows host larvae to grow, but ultimately kills the hosts.

developmental biology↗

bioRxiv2022

Deleterious mutations in a heterozygous chromosome result in recessive lethality 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. The mutations responsible for the l-t04 and l-m04 were identified as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/521575v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1c59ea3org.highwire.dtl.DTLVardef@131fbf0org.highwire.dtl.DTLVardef@93515borg.highwire.dtl.DTLVardef@1c23afe_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

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.

biochemistry↗

bioRxiv2022

Baculovirus bv/odv-e26 is required for host behavioral manipulation by optimizing viral virulence to lepidopteran hosts

Host behavioral manipulation is a widely used strategy for parasites to enhance their transmission. Lepidopteran nucleopolyhedroviruses (NPVs) induce hyperactivity and alteration of behavioral patterns in host insect larvae. Previous studies revealed that Bombyx mori nucleopolyhedrovirus (BmNPV) does not induce hyperactivity in larval Bombyx mori, when Bm8, a homologue of the baculovirus bv/odv-e26 gene, is knocked out. Bm8 knockout also enhances pathogenicity, but the relationship between Bm8 functions and host behavioral manipulation is still unknown. This study revealed that Bm8 is dispensable for altering behavioral patterns but crucial for inducing hyperactivity. Bm8 overexpression decreased pathogenicity in B. mori larvae and delayed viral infection in cultured cells. These results suggest that Bm8 maintains larval locomotory activity and synchronizes hyperactivity induction with behavioral pattern alteration by suppressing viral virulence. Conserved amino acid residues were also identified in the coiled-coil domain of the Bm8 protein, which is required for suppressing viral virulence in cultured cells and larval hosts. Although bv/odv-e26 is previously thought to be conserved in a limited group of lepidopteran NPVs, our synteny-based bioinformatics approach discovered putative homologues in the genomes of most lepidopteran NPVs. Functional analyses revealed that phylogenetically close and distant bv/odv-e26 homologues possess suppressive activity with functionally essential common residues in their coiled-coil domains. Collectively, these findings indicate that baculovirus bv/odv-e26 is a conserved gene optimizing viral virulence for establishing behavioral manipulation in NPV-infected larval hosts. Author SummaryBaculovirus-induced host abnormal behavior has been described in various lepidopteran insects for a long time, although it is still largely unknown how the virus establishes this complex trait. We showed that baculovirus bv/odv-e26 is a factor that suppresses viral virulence and synchronizes hyperactivity and behavioral pattern alteration. We also revealed that putative bv/odv-e26 homologues are encoded by a wider range of baculoviruses than previously thought, with functionally conserved residues. We confirmed the suppressive function in both phylogenetically close and distant bv/odv-e26 homologues. These results indicate that acquiring a suppressive factor for viral virulence is a crucial event for the evolution of a host manipulation strategy by baculoviruses.

microbiology↗

bioRxiv2022

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, P-element induced wimpy testis (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. HighlightsO_LITranscriptome analysis was performed in Masc-R-overexpressing neonate Bombyx mori larvae. C_LIO_LIZ-linked genes were globally suppressed in Masc-R-overexpressing females. C_LIO_LIMasc-R overexpression had little effect on BmImpM expression in males. C_LIO_LISeveral genes may be controlled by Masc-R regardless of sex. C_LI

genetics↗

bioRxiv2022

KaicoTracker: a robust and automated locomotory analysis for baculovirus-infected silkworm larvae

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen of the domestic silkworm, Bombyx mori, whose infection drastically alters larval locomotory patterns and activity. As B. mori is the only insect that has been completely domesticated, its larvae are typically inactive. In contrast, BmNPV-infected larvae exhibit vigorous movement. Recently, we developed a method for locomotory analysis of BmNPV-infected B. mori larvae, which yielded high-resolution data on BmNPV-induced host locomotory alteration. However, this method had a disadvantage when analyzing the behavior of uninfected larvae, as they were typically inactive and difficult to distinguish from the background. In addition, the analysis processes involve many manual steps, which reduces analysis throughput. We have developed KaicoTracker, a new tool for automated locomotory analysis of BmNPV-infected B. mori larvae, using Python and OpenCV. This tool is an all-inclusive package that tracks and quantifies larval locomotion and verifies the accuracy of the tracking. Larval locomotion is tracked by motion tracking function based on k-Nearest Neighbor implemented in OpenCV. Based on the tracking results, the positions of the larvae are determined, and locomotory distance, speed, duration, and frequency are calculated. The larval positions are marked on the original video, allowing tracking precision to be examined. By analyzing previously obtained data, we confirmed that this tool provides a robust analysis of inactive larvae with fewer manual steps.

microbiology↗

bioRxiv2022

Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts

Wolbachia is an extremely widespread endocellular 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 O. furnacalis and O. 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, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis. 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. SignificanceDespite the growing number of publicly available Wolbachia genome sequences, only a few high-quality male-killer genomes exist, particularly those found in lepidopteran hosts. The complete genomes of two male-killing Wolbachia of Ostrinia moth hosts were determined in this study. The genomic data obtained here will be used to elucidate the mechanism of reproductive manipulation and the origins of this endosymbionts extraordinary diversity. Additionally, phylogenetic analysis of mitochondria and Wolbachia revealed the evolutionary history of Ostrinia hosts and Wolbachia. The inferred dynamic pattern of infection adds to our understanding of evolution and ecology of Wolbachia endosymbiont, a promising agent for biological pest control.

microbiology↗

bioRxiv2022

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

Dosage compensation balances the expression of sex-chromosome-linked genes with autosome-linked genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran model insect, uses the female heterogametic WZ sex determination system. In B. mori, a Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation. 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 analyzed the allele-specific expression (ASE) to solve the problem. The ASE analysis revealed that the genes located on the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. Also, our results revealed that two Z chromosomes are transcriptionally downregulated in B. mori males as observed in Caenorhabditis elegans.

genetics↗

bioRxiv2021

Mutations in a β-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 {beta}-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. HighlightO_LIResponsible region for the brown egg 4 (b-4) mutation was narrowed down by double digest restriction-site associated DNA sequencing (ddRAD-seq). C_LIO_LIThe gene structure was disrupted in one of the candidate genes, Bombyx mori mahogany (Bmmah), in the b-4 mutant strains. C_LIO_LICRISPR/Cas9-mediated gene knockout and complementation test confirmed that the Bmmah is responsible for the b-4 phenotypes. C_LIO_LIThe Bmmah encoded a putative amino acid transporter that belongs to the {beta}-group of solute carrier family. C_LIO_LIThe Bmmah gene is essential for normal colorization of eggs, compound eyes, and ganglions. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/436376v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10d1d56org.highwire.dtl.DTLVardef@12df5org.highwire.dtl.DTLVardef@1703701org.highwire.dtl.DTLVardef@2b5bcf_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2020

Reprogramming of larval locomotory pattern during baculovirus-induced host manipulation

Many parasites manipulate host behaviour to enhance their transmission. Baculovirus induces enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points. However, as ELA continues for several hours, these methods are unlikely to evaluate larval locomotion precisely during ELA. We developed a novel method to continuously trace the locomotion of Bombyx mori larvae using time-lapse imaging. This method successfully quantified the locomotory activities of larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) for 24 h. We found that both mock- and BmNPV-infected larvae periodically repeated moving and pausing with a similar frequency. In contrast, BmNPV-infected larvae showed fast and long-lasting locomotion compared with mock-infected larvae, which resulted in longer locomotory distances in infected larvae. Moreover, BmNPV-infected larvae exhibited biphasic behaviour. Initially, BmNPV-infected larvae showed longer locomotory distances, but the locomotory pattern was similar to mock-infected larvae. However, during the second phase, the locomotory pattern was drastically altered, with an extremely larger locomotory area. These results indicate that BmNPV reprograms host locomotory pattern, which is a turning point for the process of BmNPV-induced host manipulation.

microbiology↗

bioRxiv2019

CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

molecular biology↗