A clip domain serine protease involved in moulting in the silkworm, Bombyx mori: cloning, characterization, expression patterns and functional analysis

Abstract

Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signalling cascades in various biological processes, especially in innate immunity and the embryonic development of insects. Additionally, CLIPs may have additional non-immune functions in insect development. In the present study, the clip domain serine protease gene Bombyx mori serine protease 95 (BmSP95), which encodes a 527-residue protein, was cloned from the integument of B. mori. Bioinformatics analysis indicated that BmSP95 is a typical CLIP of the subfamily D and possesses a clip domain at the N terminus, a trypsin-like serine protease (tryp_spc) domain at the C terminus and a conserved proline-rich motif between these two domains. At the transcriptional level, BmSP95 is expressed in the integument during moulting and metamorphosis, and the expression pattern is consistent with the fluctuating 20-hydroxyecdysone (20E) titre in B. mori. At the translational level, BmSP95 protein is synthesized in the epidermal cells, secreted as a zymogen and activated in the moulting fluid. Immunofluorescence revealed that BmSP95 is distributed into the old endocuticle in the moulting stage. The expression of BmSP95 was upregulated by 20E. Moreover, expression of BmSP95 was downregulated by pathogen infection. RNA interference-mediated silencing of BmSP95 led to delayed moulting from pupa to moth. These results suggest that BmSP95 is involved in integument remodelling during moulting and metamorphosis.

Clip-Domain Serine Protease Gene (LsCLIP3) Is Essential for Larval–Pupal Molting and Immunity in Lasioderma serricorne

“…Similar expression was also observed in P. puparum, in which the Clip-SP16 was highly expressed in larvae and pupae but expressed at low levels in older mated adults (Yang et al, 2017). In B. mori, CLIP-SP95 was highly expressed in larvae, but specifically in the molting process and wandering stage of pupation (Liu et al, 2017). In most insects, newly emerged adults will darken in association with the melanization process.…”

“…Clip-SP10 in P. puparum showed a higher expression level in the venom gland than other tissues analyzed, suggesting that it plays crucial roles in parasitism (Yang et al, 2017). In B. mori, CLIP-SP95 was mainly expressed in cuticle, but with low expression in hemocytes (Liu et al, 2017). Similarly, we characterized high expression of two L. serricorne CLIP-Cs in cuticle (Chen et al, 2019).…”

“…Larval-specific expression of CLIP occurs in P. puparum and N. lugens (Bao et al, 2014;Yang et al, 2017). Tissue-specific expression of CLIP-SP95 has been demonstrated in the integument of molting stages in B. mori and was functionally validated in the molting process (Liu et al, 2017). The timing of gene expression in B. mori integument was correlated with ecdysteroid titer (Liu et al, 2017).…”

“…Two CLIPs, CLIP8 and CLIP11, had an evolutionary relationship with homologs that were involved in the immune response (Kanost et al, 2001). The BmSP95 CLIP is involved in the pupa to adult molt (Liu et al, 2017). While this is a large family of enzymes, limited functions have been documented, and the roles of most CLIPs remain unknown.…”

Genome-wide gene expression profiling of the melon fly, Zeugodacus cucurbitae, during thirteen life stages

“…Complex biochemical processes also take place during the pupa stage in which the tissues were remodeled, e.g. SP95 involved in integument remodeling 28 . Gene expression during the specific time strictly in the process of the molting from old larva to the early pupa was also critical to successful molting, e.g.…”