Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.T HE cuticle covering the entire body surface of insects not only participates in defense against pathogens and adverse environmental factors, but is also indispensable for constructing and maintaining external morphological characteristics and locomotion during the entire developmental process (Wigglesworth 1957;Delon and Payre 2004;Moussian et al. 2005). Therefore, the cuticle greatly enhances survival ability and adaptability of insects, ensuring its continued existence as one of the most successful life forms in the animal kingdom.The cuticle is a complex composite material mainly comprising chitin fibers and proteins (Andersen et al. 1995;Moussian 2010). Chitin is the polymer of b-1,4-linked N-acetyl-D-glucosamine (Gilbert 2011, Chap. 7). In procuticles, chitin fibers are arranged in laminae in an antiparallel manner and superimpose each other, forming sheets of fibrils that are stacked in a helicoidal fashion, maintaining cuticle structure, elasticity, and stability (Bouligand 1965;Neville and Luke 1969;Moussian 2010). In terrestrial insects, the chitin content is positively correlated with body size, suggesting a close relationship with cuticle extension and expansion (Merzendorfer and Zimoch 2003;Lease and Wolf 2010).Cuticular proteins (CPs), the principal structural constituents of cuticle, are encoded by more than 100 genes in known insect genomes (Andersen et al. 1995 The soft, flexible cuticle of Lepidoptera larvae not only bears pressure from ...

The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in , a gene encoding a cuticular protein with low complexity sequence. Spatiotemporal expression analyses showed that is expressed in the larval epidermis postecdysis. RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of produced the abnormal body shape and the inhibited pigment typical of the mutant phenotype. In addition, our results showed that may be involved in the synthesis of endocuticle and its disruption-induced apoptosis of epidermal cells that accompanied the reduced expression of R&R-type larval cuticle protein genes and pigmentation gene Strikingly,, a fast-evolving gene, has evolved a new function responsible for the assembly of silkworm larval cuticle and has evolved to be an indispensable factor maintaining the larval body shape and its coloration pattern. This is the first study to identify a molecule whose pleiotropic function affects the development of body shape and color patterns in insect larvae.

The silkworm Bombyx mori is an important economic insect for producing silk, the “queen of fabrics”. The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knockout mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.