Micropterigidae is regarded as the sister group of all the other Lepidoptera, providing important insights into the evolution of Lepidoptera. However, the gene and protein profiles of silk from Micropterigidae remain unidentified. In this study, we investigated the components of silk cocoons of the micropterigid species Neomicropteryx cornuta. We characterized the silk glands and cocoons using light and electron microscopy, X-ray microtomography, and molecular techniques. Our results showed that the protein fibroin heavy chain (FibH) is absent in the silk of N. cornuta and that the putative homolog of fibroin light chain (FibL) is also absent or severely altered. This was confirmed by transcriptome and genome analyses of the conserved regions in this species. The examination of the synteny around the fibH genes in several Lepidoptera and Trichoptera species showed that the genomic region containing this gene was absent in another micropterigid species, Micropterix aruncella. In contrast, we found putative orthologs of fibH and fibL in the representative transcripts of another distinct clade, Eriocraniidae. This study showed that the loss of FibH and the loss or severe divergence of FibL occurred specifically in the family Micropterigidae and revealed dynamic evolutionary changes in silk composition during the early evolution of Lepidoptera.

Total RNA samples from dissected larval (last-instar) SG and whole T. bisselliella last-instar larvae were extracted using TRIzol reagent (Life Technologies, Carlsbad, USA) and used to prepare cDNA libraries for the Illumina sequencing platform. A RiboMinus Eukaryote Kit for RNA-Seq (Ambion, Austin, USA) was used to remove rRNA, after which the poly-A mRNA was enriched with the aid of a Dynabeads Oligo (dT)25 mRNA Purification Kit (Thermo Fisher Scientific, Waltham, USA). The cDNA library was created with a NEXTflex Rapid RNA-Seq Kit (Bioo Scientific, Austin, USA). Sequencing was performed using a MiSeq (Illumina, San Diego, USA), producing sequences in the 2 × 150 nt pair-end format. A total of 1.6 × 107 reads were assembled and mapped to the N. cornuta publicly available genome ASM2038319v1 using RNA Star, and the transcriptome was constructed using BRAKER3 (Galaxy Version 3.0.6+ galaxy2).