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Genome sequences contain the fundamental genetic information that largely determines the biology of a species. Over the past 20 years, advancements in high-throughput sequencing technologies and bioinformatics tools have matured, facilitating genome assembly and ushering in the telomere-to-telomere (T2T) era. Bombyx mori is renowned as a silk-producing insect and serves as an important model organism extensively studied across various fields of biology. In this study, we present the first assembled T2T genome by integrating HiFi, ultra-long ONT, NGS, and Hi-C data. This assembly comprises 450,267,439 base pairs from 28 chromosomes and includes annotations for a total of 18,253 protein-coding genes. A completeness evaluation revealed that 99.1% of conserved single-copy genes were included, as determined by a BUSCO analysis. Furthermore, the consensus quality (QV) assessed through Merqury was recorded at 59.88. The proportion of repeat sequence achieved 60.77%, marking it as the highest reported value for B. mori to date. In comparison to previously published genomes, our assembly offers a more complete and higher quality representation, particularly concerning highly homologous tandem regions such as telomeres, rDNA clusters, and Gr family regions. Furthermore, our extensive experience in genome assembly, including sample preparation experience and assembly strategies to reduce complexity, will provide valuable references for other species aiming to achieve their own T2T genome assemblies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cry1Ab has emerged as a bio‐insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag‐based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up‐regulated and significantly enriched in the serine protease activity pathway, and up‐regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down‐regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein–protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt‐resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies. Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical features of their function and evolution. To this end, we constructed an expression library from the silk glands of the ghost moth, Hepialus californicus, and characterized light chain fibroin and heavy chain fibroin gene transcripts. The predicted H. californicus silk fibroins share many elements with other lepidopteran and trichopteran fibroins, such as conserved placements of cysteine, aromatic, and polar amino acid residues. Further comparative analyses were performed to determine site-specific signatures of selection and to assess whether fibroin genes are informative as phylogenetic markers. We found that purifying selection has constrained mutation within the fibroins and that light chain fibroin is a promising molecular marker. Thus, by characterizing the H. californicus fibroins, we identified key functional amino acids and gained insight into the evolutionary processes that have shaped these adaptive molecules.

The larval head cuticle and anal plates of the silkworm mutant cheek and tail spot (cts) have chocolate-colored spots, unlike the entirely white appearance of the wild-type (WT) strain. We report the identification and characterization of the gene responsible for the cts mutation. Positional cloning revealed a cts candidate on chromosome 16, designated BmMFS, based on the high similarity of the deduced amino acid sequence between the candidate gene from the WT strain and the major facilitator superfamily (MFS) protein. BmMFS likely encodes a membrane protein with 11 putative transmembrane domains, while the putative structure deduced from the cts-type allele possesses only 10-pass transmembrane domains owing to a deletion in its coding region. Quantitative RT-PCR analysis showed that BmMFS mRNA was strongly expressed in the integument of the head and tail, where the cts phenotype is observed; expression markedly increased at the molting and newly ecdysed stages. These results indicate that the novel BmMFS gene is cts and the membrane structure of its protein accounts for the cts phenotype. These expression profiles and the cts phenotype are quite similar to those of melanin-related genes, such as Bmyellow-e and Bm-iAANAT, suggesting that BmMFS is involved in the melanin synthesis pathway. Key words: Bombyx mori, cts mutant, positional cloning, melanin, major facilitator superfamily (MFS). La cuticule de la tete et les plaques anales chez les larves du mutant cheek and tail spot (cts) du vers a soie presentent des taches de couleur chocolat contrairement au type sauvage (WT) qui est entierement blanc. Les auteurs rapportent l'identification et la caracterisation du gene responsable de la mutation cts. Le clonage positionnel a permis d'identifier un gene candidat sur le chromosome 16; celui-ci a ete nomme BmMFS en raison de la grande similarite entre la sequence peptidique predite chez le WT et les proteines de la superfamille MFS (' major facilitator superfamily '). Le gene BmMFS code vraisemblablement pour une proteine membranaire avec 11 domaines transmembranaires, tandis que la structure predite pour l'allele cts n'en presenterait que 10 en raison d'une deletion au sein de la region codante. Des analyses RT-PCR quantitatives ont revele que l'ARNm de BmMFS est exprime fortement au sein du tegument de la tete et de la queue, la ou le phenotype s'observe. L'expression augmente de maniere importante au cours de la mue et des stades qui en resultent. Ces resultats indiquent que le gene BmMFS correspondrait a cts et que la structure membranaire de sa proteine expliquerait le phenotype mutant cts. Ces profils d' expression et le phenotype cts sont tres semblables a ceux des genes associes a la melanine comme Bmyellow-e et Bm-iAANAT, ce qui suggere que BmMFS serait implique dans le sentier de synthese de la melanine. Mots-cles : Bombyx mori, mutant cts, clonage positionnel, melanine, superfamille MFS. [Traduit par la Redaction], Introduction Many lepidopteran insects show body color variations. The high phenotypic diversity observed in butterfly wings and larval body patterns offers an opportunity to study adaptive morphological evolution (Ferguson et [...]

On the W chromosome of the silkworm, Bombyx mori, we found a novel piggyBac-like DNA transposon that potentially encodes an intact transposase (610 amino acid residues), which is flanked by 16-bp perfect inverted terminal repeats and a duplicated TTAA target site. Interestingly, we also identified another intact copy of this transposon on an autosome (chromosome 21), which showed 99.6% identity in the DNA sequence of the transposase (99.3% amino acid identity). These features raised the possibility that this novel piggyBac-like DNA transposon, designated as yabusame, may retain transposition activity. Here we report the identification and characterization of yabusame transposons from the silkworm. We cloned the full length of the yabusame transposon on the W chromosome (yabusame-W) and its autosomal copy (yabusame-1). Southern blot analysis showed that there are interstrain polymorphisms in yabusame elements for their insertion sites and copy number. We also found strong evidence for the recent transposition of yabusame elements in the silkworm genome. Although our in vitro excision assays suggested that the transposition activity of yabusame-1 and yabusame-W has been lost almost entirely, our data will lead to a greater understanding of the characteristics of piggyBac superfamily elements. Key words: insect, Bombyx, piggyBac, transposable element, transgenesis. Sur le chromosome W du vers a soie, Bombyx mori, les auteurs ont trouve un nouveau transposon a ADN de type piggyBac qui code potentiellement pour une transposase intacte (610 acides amines), laquelle serait bordeedesequences terminales repetees inversees de 16 pb et produirait une duplication TTAA au site d'integration. Les auteurs ont egalement identifie une autre copie complete de ce transposon sur un autosome (chromosome 21), laquelle est identique a 99,6 % au sein de la region d'ADN qui code pour la transposase (99,3 % d'identite des acides amines). Ces caracteristiques ont suggere la possibilite que ce nouveau transposon de type piggyBac, nomme yabusame, soit encore actif en matiere de transposition. Les auteurs rapportent ici l'identification et la caracterisation des transposons yabusame chez le vers a soie. Les auteurs ont clone les copies completes des transposons yabusame situes sur le chromosome W (yabusame-W) et l'autosome (yabusame-1). Une hybridation Southern a revele qu'il y a du polymorphisme entre les souches pour ce qui est des sites d'insertion et du nombre de copies des elements yabusame. Les auteurs ont egalement trouve des evidences fortes de transpositions recentes des elements yabusame au sein du genome du vers a soie. Bien qu'un systeme in vitro de mesure de l'excision suggere que l'activite de transposition des elements yabusame-1 et yabusame-W ait ete presque completement perdue, ces donneees vont contribuer a une meilleure comprehension des caracteristiques des elements de la superfamille piggyBac. Mots-cles: insecte, Bombyx, piggyBac,eleement transposable, transgnese. [Traduit par la Redaction], Introduction Germline transformation of insects is a field of intensive research because there is considerable interest in genetic modification of insects for controlling insect pests or utilizing insects as bioreactors [...]

We carried out genetic and cytogenetic analyses of X-ray-induced deleterious Z chromosomes that result in a soft and pliable (spli) phenotype in the silkworm, Bombyx mori. In a B. mori strain with a spli phenotype, we found the Z chromosome broken between the sch (1-21.5) and od (1-19.6) loci. We also found a chromosomal fragment bearing a fifth-chromosome locus for egg and eye pigmentation fused to a Z chromosome fragment. By means of fluorescence in situ hybridization using bacterial artificial chromosome clones as probes, we confirmed that the fused chromosome is composed of a fragment of chromosome 5 and a fragment of the Z chromosome. Moreover, a predicted gene, GA002017, the Bombyx ortholog of the Drosophila gene acj6 (Bmacj6), was completely deleted by the Z chromosome breakage event. The relationship between Bmacj6 and the spli phenotype is discussed. Key words: chromosome breakage, X-ray-induced mutant, telomere, Z chromosome, acj6. Les auteurs ont realise des analyses genetiques et cytogenetiques sur des chromosomes Z deleteres induits par des rayons X, lesquelles produisent un phenotype spli (<< soft and pliable >>) chez le ver a soie Bombyx mori. Chez une souche du B. mori a phenotype spli, les auteurs ont observe un chromosome Z casse entre les locus sch (1-21.5) et od (1-49.6). Ils ont egalement trouve un fragment chromosomique portant un locus pour la pigmentation des oeufs et des yeux, lequel est normalement situe sur le chromosome 5 mais qui est ici fusionne a un fragment de chromosome Z. Par hybridation in situ en fluorescence avec des clones de chromosomes bacteriens artificiels, les auteurs ont confirme que le chromosome fusionne etait compose; du chromosome 5 et du chromosome Z. De plus, un gene predit, GA002017, l'orthologue chez le Bombyx du gene acj6 de la drosophile (Bmacj6), a ete elimine completement suite a la cassure chromosomique. La relation entre Bmacj6 et le phenotype spli est discutee. Mots-cles: cassure chromosomique, mutant induit par rayons X, telomere, chromosome Z, acj6. [Traduit par la Redaction], Introduction In the silkworm, Bombyx mori, more than 400 Mendelian mutations have been reported (Fujii et al. 1998). These mutations are useful resources for understanding the function of the genes [...]

The role of sex chromosomes in sex determination has been well studied in diverse groups of organisms. However, the role of the genes on the sex chromosomes in conferring sexual dimorphism is still being experimentally evaluated. An unequal complement of sex chromosomes between two sexes makes them amenable to sex-specific evolutionary forces. Sex-linked genes preferentially expressed in one sex over the other offer a potential means of addressing the role of sex chromosomes in sexual dimorphism. We examined the testis transcriptome of the silkworm, Bombyx mori, which has a ZW chromosome constitution in the female and ZZ in the male, and show that the Z chromosome harbors a significantly higher number of genes expressed preferentially in testis compared to the autosomes. We hypothesize that sexual antagonism and absence of dosage compensation have possibly led to the accumulation of many male-specific genes on the Z chromosome. Further, our analysis of testis-specific paralogous genes suggests that the accumulation on the Z chromosome of genes advantageous to males has occurred primarily by translocation or tandem duplication. DOI: 10.1534/ge netics. 108.099994

Many larval color mutants have been obtained in the silkworm Bombyx mori. Mapping of melaninsynthesis genes on the Bombyx linkage map revealed that yellow and ebony genes were located near the chocolate (ch) and sooty (so) loci, respectively. In the ch mutants, body color of neonate larvae and the body markings of elder instar larvae are reddish brown instead of normal black. Mutations at the so locus produce smoky larvae and black pupae. [F.sub.2] linkage analyses showed that sequence polymorphisms of yellow and ebony genes perfectly cosegregated with the ch and so mutant phenotypes, respectively. Both yellow and ebony were expressed in the epidermis during the molting period when cuticular pigmentation occurred. The spatial expression pattern of yellow transcripts coincided with the larval black markings. In the ch mutants, nonsense mutations of the yellow gene were detected, whereas large deletions of the ebony ORF were detected in the so mutants. These results indicate that yellow and ebony are the responsible genes for the ch and so loci, respectively. Our findings suggest that Yellow promotes melanization, whereas Ebony inhibits melanization in Lepidoptera and that melanin-synthesis enzymes play a critical role in the lepidopteran larval color pattern.

Mutations at the flugellos (fl) locus in Bombyx mori produce wingless pupae and moths because of the repressed response of wing discs to ecdysteroid. Four recessive fl alleles occurred spontaneously and were mapped at 13.0 of the silkworm genetic linkage group 10. By positional cloning, we confirmed that the gene responsible for fl is fringe (fng) encoding Fng glycosyltransferase, which is involved in regulating the Notch signaling pathway. In four different fl alleles, we detected a large deletion of the fng gene in [fl.sup.k] and nonsense mutations in fl, [fl.sup.0], and [fl.sup.n]. In the wild-type (WT) silkworm, fng is expressed actively in the wing discs, brain, and reproductive organs from the fourth to final instars but barely in the other tissues tested. In. situ hybridization showed that fng mRNA is expressed in the dorsal layer of the WT wing discs. The wingless (wg) mRNA, a downstream marker of Fng-mediated Notch signaling, is localized at the dorsoventral boundary in the WT wing discs but repressed markedly in the fl wing discs. Although null mutants of Drosophila fng result in postembryonic lethality, loss of fng function in Bombyx affects only wing morphogenesis, suggesting different essential roles for fng in tissue differentiation among insects.

Abstract Doubletime (DBT), a homolog of casein kinase Iζ(CKIζ), is an essential circadian clock component and developmental regulator in Drosophila melanogaster. The authors cloned a dbt homolog from the silkworm, [...]

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an [F.sub.1] (p50T X C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an [F.sub.1] male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an [F.sub.1] female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of [approximately equal to]3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated. silkworm | microsatellite

The polyubiquitin gene is an evolutionarily conserved eukaryotic gene, encoding tandemly repeated multiple ubiquitins, and is considered to be subject to concerted evolution. Here, we present the nucleotide sequences of new alleles of the polyubiquitin gene UbC in humans and CHUB2 in Chinese hamster, which encode a different number of ubiquitin units from those of previously reported genes. And we analyze the concerted evolution of these genes on the basis of their orthologous relationship. That the mean of the synonymous sequence difference [K.sub.s], which is defined as the number of synonymous substitution relative to the total number of synonymous sites, within the UbC and CHUB2 genes (0.192 [+ or -] 0.096) is significantly less than [K.sub.s] between these genes (0.602 [+ or -] 0.057) provides direct evidence for concerted evolution. Moreover, it also appears that concerted evolutionary events have been much more frequent in CHUB2 than in UbC, because [K.sub.s] within CHUB2 (0.022 [+ or -] 0.018) is much less than that within UbC (0.362 [+ or -] 0.192). By a numerical simulation, postulating that the major mechanism of concerted evolution in polyubiquitin genes is unequal crossing over, we estimated the frequency of concerted evolutionary events of CHUB2 at 3.3 X [10.sup.-5] per year and that of UbC at no more than 5.0 x [10.sup.-7] per year.

Abstract Background Body coloration is an ecologically important trait that is often involved in prey-predator interactions through mimicry and crypsis. Although this subject has attracted the interest of biologists and the general public, our scientific knowledge on the subject remains fragmentary. In the caterpillar of the swallowtail butterfly Papilio xuthus, spectacular changes in the color pattern are observed; the insect mimics bird droppings (mimetic pattern) as a young larva, and switches to a green camouflage coloration (cryptic pattern) in the final instar. Despite the wide variety and significance of larval color patterns, few studies have been conducted at a molecular level compared with the number of studies on adult butterfly wing patterns. Results To obtain a catalog of genes involved in larval mimetic and cryptic pattern formation, we constructed expressed sequence tag (EST) libraries of larval epidermis for P. xuthus, and P. polytes that contained 20,736 and 5,376 clones, respectively, representing one of the largest collections available in butterflies. A comparison with silkworm epidermal EST information revealed the high expression of putative blue and yellow pigment-binding proteins in Papilio species. We also designed a microarray from the EST dataset information, analyzed more than five stages each for six markings, and confirmed spatial expression patterns by whole-mount in situ hybridization. Hence, we succeeded in elucidating many novel marking-specific genes for mimetic and cryptic pattern formation, including pigment-binding protein genes, the melanin-associated gene yellow-h3, the ecdysteroid synthesis enzyme gene 3-dehydroecdysone 3b-reductase, and Papilio-specific genes. We also found many cuticular protein genes with marking specificity that may be associated with the unique surface nanostructure of the markings. Furthermore, we identified two transcription factors, spalt and ecdysteroid signal-related E75, as genes expressed in larval eyespot markings. This finding suggests that E75 is a strong candidate mediator of the hormone-dependent coordination of larval pattern formation. Conclusions This study is one of the most comprehensive molecular analyses of complicated morphological features, and it will serve as a new resource for studying insect mimetic and cryptic pattern formation in general. The wide variety of marking-associated genes (both regulatory and structural genes) identified by our screening indicates that a similar strategy will be effective for understanding other complex traits.