Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.

Biology Centre of the Czech Academy of Sciences and Faculty of Sciences University of South Bohemia 370 05 České Budějovice Czech Republic

Biology Centre of the Czech Academy of Sciences and Faculty of Sciences University of South Bohemia 370 05 České Budějovice Czech Republic; Faculty of Science University of South Bohemia Branisovska 31 370 05 Ceske Budejovice Czech Republic

Institute of Sericulture Iikura 1053 300 0324 Ami machi Ibaraki Japan

National Agriculture and Food Research Organization 1 2 Owashi Tsukuba Ibaraki 305 8634 Japan

National Agriculture and Food Research Organization 1 2 Owashi Tsukuba Ibaraki 305 8634 Japan; Biology Centre of the Czech Academy of Sciences and Faculty of Sciences University of South Bohemia 370 05 České Budějovice Czech Republic

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