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1.
Methods Mol Biol ; 2360: 19-31, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34495504

RESUMO

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.

2.
Insect Biochem Mol Biol ; 137: 103624, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34333110

RESUMO

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.

3.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

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.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
4.
Mol Biol Evol ; 38(7): 2897-2914, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33739418

RESUMO

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 ß-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of ß-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of ß-fructofuranosidase were found in Coleoptera and Hymenoptera. ß-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran ß-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 ß-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of ß-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.


Assuntos
Evolução Biológica , Duplicação Gênica , Transferência Genética Horizontal , Lepidópteros/genética , beta-Frutofuranosidase/genética , Animais , Feminino , Homeostase , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/enzimologia , Masculino , Sacarase/metabolismo
5.
Arch Insect Biochem Physiol ; 106(3): e21768, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33644912

RESUMO

Bombyx mori Masculinizer protein (BmMasc) is essential for both masculinization and dosage compensation in B. mori. We previously identified a bipartite nuclear localization signal (NLS) of BmMasc and two essential residues (lysine at 274 [K274] and arginine at 275 [R275]) implicated in its function. Sequence comparison showed the presence of putative NLSs in lepidopteran Masc proteins, but their functional properties and critical residues are unknown. Here we characterized a putative NLS of Ostrinia furnacalis Masc (OfMasc) using B. mori ovary-derived BmN-4 cell line. Deletion and alanine scanning mutagenesis revealed that a putative NLS is required for nuclear localization of OfMasc. However, mutations at both K227 and R228, which correspond to K274 and R275 of BmMasc, respectively, do not greatly abolish the NLS activity. Additional mutagenesis analysis revealed that triple mutations at K227, R228, and K240 almost completely inhibited OfMasc nuclear localization. These results suggest that lepidopteran Masc proteins possess a common functional NLS, but the critical residues for its activity are different. Moreover, we examined the masculinizing activity of OfMasc derivatives and found that nuclear localization is not required for the masculinizing activity of OfMasc. The results from our studies indicate that lepidopteran Masc proteins function in the cytoplasm to drive masculinizing cascade.


Assuntos
Mariposas/genética , Sinais de Localização Nuclear , Animais , Bombyx/genética , Linhagem Celular , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo
6.
Mol Ecol Resour ; 21(1): 327-339, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32985129

RESUMO

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.

7.
Gene ; 768: 145314, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33220342

RESUMO

The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.


Assuntos
Proteínas Argonauta/genética , Bombyx/citologia , Mutagênese Sítio-Dirigida/métodos , Tymoviridae/genética , Animais , Bombyx/genética , Sistemas CRISPR-Cas , Proliferação de Células , Células Cultivadas , Edição de Genes , Proteínas de Insetos/genética , Mutação com Perda de Função , RNA Interferente Pequeno/genética , RNA Viral/genética , Transdução de Sinais
8.
Curr Biol ; 30(4): 561-572.e10, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032508

RESUMO

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 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding 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, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN 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, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.


Assuntos
Cromossomos de Insetos/genética , Proteínas de Insetos/genética , Cinetocoros , Lepidópteros/genética , Sequência de Aminoácidos , Animais , Bombyx/química , Bombyx/genética , Bombyx/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Lepidópteros/metabolismo , Alinhamento de Sequência
9.
FEBS Open Bio ; 9(9): 1573-1579, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31294930

RESUMO

The Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. We previously measured the masculinizing activity of the lepidopteran Masc proteins using B. mori ovary-derived cell line BmN-4. Here, we established an RNA-seq data-based assay system in which the level of B. mori Masc (BmMasc)-induced dosage compensation can be estimated in BmN-4 cells. Using this system, we found that a cysteine residue at position 301, which was shown to be essential for the masculinizing activity of BmMasc, is also required for dosage compensation. We further investigated the relationships between Masc-induced cell growth inhibition, masculinizing activity, and the level of dosage compensation, using Masc genes from three lepidopteran insects. In summary, we have established a cell-based system to monitor levels of Masc-induced dosage compensation.


Assuntos
Compensação de Dosagem (Genética)/genética , Proteínas de Insetos/genética , Animais , Bombyx , Proliferação de Células , Células Cultivadas , Cisteína/genética , Cisteína/metabolismo , Proteínas de Insetos/metabolismo
10.
Insect Biochem Mol Biol ; 107: 53-62, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30802494

RESUMO

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).


Assuntos
Bombyx/genética , Animais , Genoma de Inseto , Sequenciamento de Nucleotídeos em Larga Escala
11.
Insect Biochem Mol Biol ; 104: 30-38, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529581

RESUMO

The Masculinizer (Masc) gene encodes a novel lepidopteran-specific protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. The Masc protein possesses two CCCH-type zinc finger domains (ZFs), a nuclear localization signal, and an 11-amino-acid region that is highly conserved among lepidopteran insects. Using a cell-based assay system, we revealed that two cysteine residues localized in the conserved region, but not ZFs, are required for masculinization. In addition, nuclear localization of the Masc protein is not associated with masculinizing activity. Because dosage compensation is considered to occur in the nucleus, we inferred that the two ZFs play a role in the establishment of dosage compensation. To investigate this hypothesis at the organism level, we utilized the CRISPR/Cas9 system and established three B. mori strains whose Masc is partially deleted at different regions. The strain lacking the 210 C-terminal amino acids of the Masc protein showed male-specific embryonic lethality due to its low abundance and/or instability. The male embryos of this strain expressed the female-type splice variants of B. mori doublesex and did not express the male-type mRNA of B. mori IGF-II mRNA-binding protein. Furthermore, mRNA levels of Z-linked genes were abnormally enhanced only in male embryos. In contrast, the strain lacking both ZFs grew normally and did not show any defective phenotypes including sexual differentiation and the expression of Z-linked genes, demonstrating that the two CCCH-type ZFs, which are conserved in lepidopteran Masc homologs, are dispensable for masculinization and dosage compensation.


Assuntos
Compensação de Dosagem (Genética) , Processos de Determinação Sexual/fisiologia , Diferenciação Sexual/fisiologia , Animais , Bombyx/genética , Bombyx/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Dedos de Zinco
12.
PLoS One ; 13(10): e0205758, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30321229

RESUMO

The white color in the larval integument of the silkworm Bombyx mori is considered the result of uric acid accumulation in its epidermal cells. Larvae of the eri silkworm Samia ricini (Lepidoptera; Saturniidae) also have a white and opaque integument, but little is known about its coloration mechanism. In this study, we first performed a feeding assay of S. ricini larvae using allopurinol, an inhibitor of xanthine oxidase, which catalyzes the degradation of xanthine to uric acid. This treatment induced a clear translucent integument phenotype, indicating that the larval color of S. ricini is also determined by uric acid accumulation. Next, to investigate the genetic basis that controls uric acid accumulation in S. ricini larvae, we isolated and characterized the S. ricini homolog of mammalian biogenesis of lysosome-related organelles complex 1, subunit 2 (BLOS2), which is known to play a crucial role in urate granule biosynthesis. We created a transcription activator-like effector nuclease (TALEN)-mediated gene knockout of S. ricini BLOS2 (SrBLOS2) and succeeded in establishing SrBLOS2 knockout strains (SrBLOS2KO). SrBLOS2KO mutants exhibited a translucent larval integument phenotype and lacked uric acid in the epidermis, as also observed in allopurinol-fed larvae. In addition, electron microscopy revealed that urate granules were rarely observed in the epidermis of SrBLOS2KO larvae, whereas abundant granules were found in the epidermis of wild-type larvae. These results clearly demonstrated that larval S. ricini accumulates uric acid as urate granules in the epidermis and that the genetic basis that controls uric acid accumulation is evolutionarily conserved in S. ricini and B. mori.


Assuntos
Mariposas/metabolismo , Ácido Úrico/metabolismo , Alopurinol/farmacologia , Animais , Cor , DNA/genética , Epiderme/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/ultraestrutura , Filogenia , Análise de Sequência de DNA , Xantina/metabolismo , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/metabolismo
13.
Genetica ; 146(4-5): 425-431, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30094710

RESUMO

A variety of insects accumulate high contents of riboflavin (vitamin B2) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.


Assuntos
Bombyx/metabolismo , Túbulos de Malpighi/metabolismo , Deficiência de Riboflavina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Bombyx/genética , Proteínas de Insetos/genética , Insetos/genética , Larva/genética , Mutação , Filogenia , Pigmentação/genética , Riboflavina/metabolismo , Análise de Sequência de DNA/métodos
14.
Sci Rep ; 8(1): 11942, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093702

RESUMO

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.


Assuntos
Bombyx/fisiologia , Herbivoria/fisiologia , Morus/parasitologia , Folhas de Planta/parasitologia , Terpenos/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Animais , Bombyx/classificação , Bombyx/genética , Resistência à Doença/fisiologia , Morus/metabolismo , Filogenia , Doenças das Plantas/parasitologia , Folhas de Planta/metabolismo , Terpenos/química , Compostos Orgânicos Voláteis/química , Volatilização
15.
Biochem Biophys Res Commun ; 503(3): 1768-1772, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30064911

RESUMO

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.


Assuntos
Proteínas de Insetos/genética , Mariposas/genética , Cromossomos Sexuais/genética , Processos de Determinação Sexual/genética , Animais , Feminino , Proteínas de Insetos/metabolismo , Masculino , Mariposas/embriologia , Cromossomos Sexuais/metabolismo
16.
Proc Jpn Acad Ser B Phys Biol Sci ; 94(5): 205-216, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760316

RESUMO

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


Assuntos
Bombyx/genética , Processos de Determinação Sexual , Animais , Sequência de Bases , Bombyx/microbiologia , Bombyx/fisiologia , Cromossomos de Insetos/genética , Perfilação da Expressão Gênica , Caracteres Sexuais , Wolbachia/fisiologia
17.
Arthropod Struct Dev ; 47(3): 238-247, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29518525

RESUMO

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.


Assuntos
Bombyx/fisiologia , Células Quimiorreceptoras/fisiologia , Sensilas/fisiologia , Animais , Bombyx/anatomia & histologia , Bombyx/ultraestrutura , Células Quimiorreceptoras/citologia , Células Quimiorreceptoras/ultraestrutura , Extremidades , Feminino , Masculino , Microscopia Eletrônica de Varredura , Sensilas/citologia , Sensilas/ultraestrutura , Especificidade da Espécie
18.
DNA Res ; 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29360973

RESUMO

Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.

19.
Insect Biochem Mol Biol ; 92: 65-72, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29191464

RESUMO

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.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Bombyx/genética , Proteínas de Insetos/genética , Túbulos de Malpighi/metabolismo , Riboflavina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Pigmentação
20.
Gene ; 629: 92-100, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28768207

RESUMO

"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 otm-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.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Proteínas de Insetos/genética , Proteínas de Transporte Vesicular/genética , Animais , Proteínas de Transporte/metabolismo , Clonagem Molecular , Proteínas de Insetos/metabolismo , Complexos Multiproteicos/metabolismo , Mutação , Interferência de RNA , Ácido Úrico/metabolismo , Proteínas de Transporte Vesicular/metabolismo
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