Toru Shimada

In this study, we found two embryonic lethal mutations, t04 lethal (l-t04) and m04 lethal (l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkworm Bombyx mandarina, is substituted with chromosomes of the domesticated silkworm B. mori, and l-t04 and l-m04 mutations are located on B. mandarina-derived chromosome 4. To clarify the cause of the lethalities and the genes responsible for these mutations, positional cloning and CRISPR/Cas9 mediated knockout screening were performed. Finally, genetic complementation tests l-t04l-m04 identified the mutations responsible for the l-t04 and l-m04 as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA box binding protein-associated factor 5 (BmTaf5), respectively. Lethal stages of each knockout mutant indicated that the importance of these genes in B. mori late embryogenesis. The lethal mutations responsible for l-t04 and l-m04 were not found in parental strains or wild B. mandarina collected from 39 distinct locations in Japan, indicating that both mutations were independently introduced during or after the development of the semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4.

<title>Abstract</title> Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Samia ricini, a gigantic saturniid moth, has the potential to be a novel lepidopteran model species. Samia ricini is far more resistant to diseases than the current model species Bombyx mori, and therefore can be more easily reared. In addition, genetic resources available for S. ricini rival those for B. mori: at least 26 ecoraces of S. ricini are reported and S. ricini can hybridize with wild Samia species, which are distributed throughout Asian countries, and produce fertile progenies. Physiological traits such as food preference, integument colour and larval spot pattern differ among S. ricini strains and wild Samia species so that those traits can be targeted in forward genetic analyses. To facilitate genetic research in S. ricini, we determined its whole genome sequence. The assembled genome of S. ricini was 458 Mb with 155 scaffolds, and the scaffold N50 length of the assembly was ~ 21 Mb. In total, 16,702 protein coding genes were predicted. While the S. ricini genome was mostly collinear with that of B. mori with some rearrangements and few S. ricini-specific genes were discovered, chorion genes and fibroin genes seemed to have expanded in the S. ricini lineage. As the first step of genetic analyses, causal genes for "Blue," "Yellow," "Spot," and "Red cocoon" phenotypes were mapped to chromosomes.

Gene duplication provides a major source of new genes for evolutionary novelty and ecological adaptation. However, the maintenance of duplicated genes and their relevance to adaptive evolution has long been debated. Insect trehalase (Treh) plays key roles in energy metabolism, growth, and stress recovery. Here, we show that the duplication of Treh in Lepidoptera (butterflies and moths) is linked with their adaptation to various environmental stresses. Generally, two Treh genes are present in insects: Treh1 and Treh2. We report three distinct forms of Treh in lepidopteran insects, where Treh1 was duplicated into two gene clusters (Treh1a and Treh1b). These gene clusters differ in gene expression patterns, enzymatic properties, and subcellular localizations, suggesting that the enzymes probably underwent sub- and/or neofunctionalization in the lepidopteran insects. Interestingly, selective pressure analysis provided significant evidence of positive selection on duplicate Treh1b gene in lepidopteran insect lineages. Most positively selected sites were located in the alpha-helical region, and several sites were close to the trehalose binding and catalytic sites. Subcellular adaptation of duplicate Treh1b driven by positive selection appears to have occurred as a result of selected changes in specific sequences, allowing for rapid reprogramming of duplicated Treh during evolution. Our results suggest that gene duplication of Treh and subsequent functional diversification could increase the survival rate of lepidopteran insects through various regulations of intracellular trehalose levels, facilitating their adaptation to diverse habitats. This study provides evidence regarding the mechanism by which gene family expansion can contribute to species adaptation through gene duplication and subsequent functional diversification.

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).