Fibroin and sericin
Fibroin is the major component of silk protein. Although fibroin ofB. mori consists three polypeptides, namely heavy-chain (Fib-H), light-chain (Fib-L) and fibrohexamerin (p25) (Inoue et al., 2000), it was biochemically confirmed that fibroin of S. ricini lacks Fib-L and p25 and it consists of Fib-H/Fib-H homodimer (Tamura, & Kubota, 1988).The complete amino acid sequence of Fib-H (SrFib-H) was already determined by Sezutsu and Yukuhiro (2014), but our gene prediction was unable to properly construct the gene model for SrFib-H , mainly because of its repetitive sequences. However, TBLASTN search using SrFib-H as query detected the near-complete coding sequence ofSrFib-H (Fig. S4A), supporting the accuracy of the assembly. The genome information also revealed that S. ricini genome has three copies of p25 , in addition to Fib-H , but lacksFib-L (Table 3). In addition, we confirmed that Fib-L is absent in the genome of A. yamamai (Kim et al., 2018), another saturniid moth, through TBLASTN search (Fig. S4B). Because other lepidopteran species, including B. mori , P. xylostella ,P. xuthus and Corcyra cephalonica (Chaitanya, & Dutta-Gupta, 2010), possess Fib-L gene, absence of Fib-Lin saturniid moths can be ascribed to the loss of Fib-L in the common ancestor of saturniid moths.
As described above, silk fibroin of B. mori consists H-chain, L-chain and P25. 3 fibroin polypeptides assemble with a 6:6:1 molecular ratio, which is considered to be indispensable for proper secretion of fibroin: mutations in Fib-H or Fib-L cause fibroin secretion deficiency (Inoue et al., 2000; Ma et al., 2014). B. mori strains with deletions in Fib-H or Fib-L cannot properly secrete fibroin protein to lumen in silk gland, and their cocoons are mainly composed of sericin. Therefore, it has been hypothesized that B. mori has a mechanism which recognizes three‐dimensional structure of fibroin assembled by the three polypeptides with 6:6:1 molecular ratio and selectively transport the fibroin polypeptide complex to lumen in silk gland. Since saturniid species lack Fib-L gene, fibroin transportation and secretion system in saturniid species must be different from that in B. mori .
So far, biological function of p25 is still unclear. Whether knockout ofp25 affects the secretion of fibroin or not remains to be answered. Since p25 protein is undetectable in S. ricini silk,p25 could take on different function other than being the part of complex structure of fibroin. The presence of multi-copies of p25in S. ricini genome are posing the possibility of functional differentiation among paralogous p25 s (Table 3).
Sericin occupies the second largest proportion of silk protein, following fibroin. Unlike fibroin, sericin is soluble to water and consisting the most outer layer of silk. B. mori has threesericin genes, Ser1 , Ser2 , and Ser3 (Tsubota et al., 2015). While Ser1 and Ser3 were the components of cocoon protein, Ser2 is not present in cocoon (Takasu, Hata, Uchino, & Zhang, 2010). Two proteins derived from alternative splicing of Ser2 can be found in larval silk produced during the growing stages (Takasu et al., 2010). So far, nine transcripts are registered to NCBI genbank asSericin -encoding genes or Sericin -like genes in S. ricini (Table S8) (Dong et al., 2015; Tsubota et al., 2015). BLAST analysis successfully confirmed that all of them are present in S. ricini genome and transcribed from seven locus, meaning that S. ricini has 7 putative Sericin genes. Phylogenetic analysis showed that four out of seven genes are categorized into Ser1/3class and the other three genes were included in Ser2 class (Fig. S5). Despite belonging to the same family (Saturniidae), sericin gene repertoires of A. yamamai and S. ricini were quite different: Ser1/3 class genes seemed to multiplicated in A. yamamai . Phylogenetic analysis revealed that all sericin genes inA. yamamai belong to Ser1/3 class and Ser2 class genes were not identified while S. ricini possess three Ser2 class genes (Fig. S5). The diversity of sericin genes among these saturniids may reflect the differences of their indigenous environments. However, whether proteins encoded by seven putative Ser genes in S. riciniare present in cocoons remains to be elucidated. Proteomic analysis onS. ricini cocoons should be carried out to reveal the protein composition.