The tobacco cutworm, Spodoptera litura, is among the most widespread and destructive agricultural pests, feeding on over 100 crops throughout tropical and subtropical Asia. By genome sequencing, physical mapping and transcriptome analysis, we found that the gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione-S-transferase, were massively expanded in this polyphagous species, enabling its extraordinary ability to detect and detoxify many plant secondary compounds. Larval exposure to insecticidal toxins induced expression of detoxification genes, and knockdown of representative genes using short interfering RNA (siRNA) reduced larval survival, consistent with their contribution to the insect’s natural pesticide tolerance. A population genetics study indicated that this species expanded throughout southeast Asia by migrating along a South India–South China–Japan axis, adapting to wide-ranging ecological conditions with diverse host plants and insecticides, surviving and adapting with the aid of its expanded detoxification systems. The findings of this study will enable the development of new pest management strategies for the control of major agricultural pests such as S. litura.

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.

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 butterflyPapilio 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 forP. xuthus, andP. polytesthat 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 inPapiliospecies. 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-mountin situhybridization. Hence, we succeeded in elucidating many novel marking-specific genes for mimetic and cryptic pattern formation, including pigment-binding protein genes, the melanin-associated geneyellow-h3, the ecdysteroid synthesis enzyme gene3-dehydroecdysone 3b-reductase, andPapilio-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,spaltand ecdysteroid signal-relatedE75, as genes expressed in larval eyespot markings. This finding suggests thatE75is 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.

Insect cuticle is considered an adaptable and versatile building material with roles in the construction and function of exoskeleton. Its physical properties are varied, as the biological requirements differ among diverse structures and change during the life cycle of the insect. Although the bulk of cuticle consists basically of cuticular proteins (CPs) associated with chitin, the degree of cuticular sclerotization is an important factor in determining its physical properties. Spodoptera litura, the tobacco cutworm, is an important agricultural pest in Asia. Compared to the domestic silkworm, Bombyx mori, another lepidopteran whose CP genes have been well annotated, S. litura has a shorter life cycle, hides in soil during daytime beginning in the 5th instar and is exposed to soil in the pupal stage without the protection of a cocoon. In order to understand how the CP genes may have been adapted to support the characteristic life style of S. litura, we searched its genome and found 287 putative cuticular proteins that can be classified into 9 CP families (CPR with three groups (RR-1, RR-2, RR-3), CPAP1, CPAP3, CPF, CPFL, CPT, CPG, CPCFC and CPLCA), and a collection of unclassified CPs named CPH. There were also 112 cuticular proteins enriched in Histidine residues with content varying from 6% to 30%, comprising many more His-rich cuticular proteins than B. mori. A phylogenetic analysis between S. litura, M. sexta and B. mori uncovered large expansions of RR-1 and RR-2 CPs, forming large gene clusters in different regions of S. litura chromosome 9. We used RNA-seq analysis to document the expression profiles of CPs in different developmental stages and tissues of S. litura. The comparative genomic analysis of CPs between S. litura and B. mori integrated with the unique behavior and life cycle of the two species offers new insights into their contrasting ecological adaptations.