Bombyx mori cocoon has a multi-layer structure that provides optimal protection for silkworm pupa. Research on the mechanical properties of the multi-layer structure revealed structure-property relationships of the cocoon. Here, we investigated the protein components of the B. mori cocoon in terms of its multi-layer structure. Liquid chromatography-tandem mass spectrometry identified 286 proteins from the multiple cocoon layers. In addition to fibroins and sericins, we identified abundant protease inhibitors, seroins and proteins of unknown function. By comparing protein abundance across layers, we found that the outermost layer contained more sericin1 and protease inhibitors and the innermost layer had more seroin1. As many as 36 protease inhibitors were identified in cocoons, showing efficient inhibitory activities against a fungal protease. Thus, we propose that more abundant protease inhibitors in the outer cocoon layers may provide better protection for the cocoon. This study increases our understanding of the multi-layer mechanism of cocoons, and helps clarify the biological characteristics of cocoons. The data have been deposited to the ProteomeXchange with identifier PXD001469.

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography-tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49%) were predicted to function as binding proteins and 46% were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.

The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography–tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study.

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