Characterization and comparative analysis of sericin protein 150 in Bombyx mori.

Lepidopteran silk is a complex mixture of proteins, consisting mainly of fibroins and sericins. Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers. So far, five genes encoding sericin proteins have been identified in Bombyx mori. Having previously identified sericin protein 150 (SP150) as a major sericin-like protein in the cocoons of the pyralid moths Galleria mellonella and Ephestia kuehniella, we describe the identification of its homolog in B. mori. Our refined gene model shows that it consists of four exons and a long open reading frame with a conserved motif, CXCXCX, at the C-terminus, reminiscent of the structure observed in a class of mucin proteins. Notably, despite a similar expression pattern, both mRNA and protein levels of B. mori SP150 were significantly lower than those of its pyralid counterpart. We also discuss the synteny of homologous genes on corresponding chromosomes in different moth species and the possible phylogenetic relationships between SP150 and certain mucin-like proteins. Our results improve our understanding of silk structure and the evolutionary relationships between adhesion proteins in the silk of different lepidopteran species.

Molecular Characterization of the Functional Genes Associated with Silk Assembly, Transport, and Protection in the Silk Glands of Popular Multivoltine Breeds of Silkworm Bombyx mori. L.

Bombyx mori is an agriculturally important insect used extensively for silk production. India, especially the eastern regions, is mostly dependent on the multivoltine breeds of silkworm Bombyx mori and their hybrids/crossbreeds. The multivoltine breeds are indigenous and superior in survival and hardiness but are relatively inferior in terms of qualitative traits, typically the silk quality. Therefore, it is highly relevant to understand the mechanism of silk production in the multivoltine breeds to decipher the reasons for the inferior quality of silk produced by the multivoltine breeds and thus gain leads to improve the quality of silk production in multivoltine breeds. With this background, study was carried to identify differential expression of the major genes associated with silk proteins in the silk gland region of the popular multivoltine breeds. Our results indicated that although fib-L, fib-H, Sericins, and P25 are the major genes associated with silk filament, a few other genes associated with silk assembly, transport, and protection in the silk glands are the ones that largely contribute towards efficient silk production. The differential expression of these genes had a major effect on the movement of silk proteins within the silk gland and the efficiency of silk production as well. The Pearson correlation revealed a positive correlation amongst the genes dealt with in this study, indicating that the concurrent increase in expression of both the types of genes in the silk glands, significantly improves the silk production.

Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm.

Bombyx mori silk is a super-long natural protein fiber with a unique structure and excellent performance. Innovative silk structures with high performance are in great demand, thus resulting in an industrial bottleneck. Herein, the outer layer sericin SER3 is ectopically expressed in the posterior silk gland (PSG) in silkworms via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a fiber with sericin microsomes dispersed in fibroin fibrils. The water-soluble SER3 protein secreted by PSG causes P25's detachment from the fibroin unit of the Fib-H/Fib-L/P25 polymer, and accumulation between the fibroin layer and the sericin layer. Consequently, the water solubility and stability of the fibroin-colloid in the silk glandular cavity, and the crystallinity increase, and the mechanical properties of cocoon fibers, moisture absorption and moisture liberation of the silk also improve. Meanwhile, the mutant overcomes the problems of low survival and abnormal silk gland development, thus enabling higher production efficiency of cocoon silk. In summary, we describe a silk gland transgenic target protein selection strategy to alter the silk fiber structure and to innovate its properties. This work provides an efficient and green method to produce silk fibers with new functions.

Identification and location of sericin in silkworm with anti-sericin antibodies.

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Insights into regulatory characteristics of the promoters of Sericin 1 and Sericin 3 in transgenic silkworms.

Sericin, produced in the middle silk gland (MSG) of silkworms, is a group of glue proteins that coat and cement silk fibers. Several genes are known to encode sericin, but their spatiotemporal regulation has yet to be fully elucidated. Here, we report in detail the expression profiles of the promoters of two major sericin-coding genes, Sericin 1 (Ser1)and Sericin 3 (Ser3), by analyzing Gal4/UAS transgenic silkworms. We found that UAS-linked EGFP fluorescence in transgenic silkworms driven by Ser1-Gal4was detected in only the R3, R4 and R5 regions of MSG starting inday-3 fifth-instar larvae and was continuously expressed until silk gland degradation. In transgenic silkworms driven by Ser3-Gal4, EGFP fluorescence was detected at a low level in the R2 region of MSG since the last day of fifth-instar larvae, and the expression increased during the wandering stages and was continuously detected until silk gland degradation. The molecular detection of EGFP expression in each of the Gal4/UAS transgenic silkworms was consistent with fluorescence observations. These findings reveal clear differences in the regulatory characteristics of the promoters of Ser1and Ser3 and provide new insights into the regulatory mechanism of the expression of sericin-coding genes.

Bmo-miR-2780a regulates the expression of the sericin-1 gene of Bombyx mori.

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.

Identification of Bombyx mori sericin 4 protein as a new biological adhesive.

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.

The expansion of genes encoding soluble silk components in the greater wax moth, Galleria mellonella.

Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera.

bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro.

We hypothesized that bmo-miR-275 has a potential regulatory function regarding the expression of sericin gene 2 (BmSer-2). First, we examined the expression of bmo-miR-275 and its target gene BmSer-2 in seven different tissues from 5th instar day-3 silkworm larvae. The results showed that they were both specifically expressed in the middle silk gland, implying that spatio-temporal conditions are required for bmo-miR-275 to regulate the expression of BmSer-2. To test this hypothesis, we constructed a pri-bmo-miR-275 expressing plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and BmSer-2-3´UTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-3' UTR-SV40]. Finally, BmN cells were harvested and luciferase activity was detected. Results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected with pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and pGL3.0 [A3-luc-Ser-2-3'UTR-SV40], suggesting that bmo-miR-275 can down-regulate the expression of BmSer-2 in vitro. Our results improve the understanding of the regulatory function of Bombyx mori miRNA on the expression of genes regulating silk formation.

Bone regeneration is associated with the concentration of tumour necrosis factor-α induced by sericin released from a silk mat.

To understand the osteogenic effect of the middle layer of the silk cocoon, sericin was examined for its cellular effects associated with tumor necrosis factor-α (TNF-α) signaling in this study. The fragmented sericin proteins in the silk mat were evaluated for the TNF-α expression level in murine macrophages. The concentration of protein released from silk mats was higher in the outermost and the innermost layers than in the middle layers, and the protein released from the silk mat was identified as sericin. The level of TNF-α in murine macrophages was dependent on the applied concentration of sericin, and the expression of genes associated with osteogenesis in osteoblast-like cells was dependent on the applied concentration of TNF-α. In animal experiments, silk mats from the middle layers led to a higher regenerated bone volume than silk mats from the innermost layer or the outermost layer. If TNF-α protein was incorporated into the silk mats from the middle layers, bone regeneration was suppressed compared with unloaded silk mats from the middle layers. Accordingly, silk mats from the silk cocoon can be considered to be a fragmented sericin-secreting carrier, and the level of sericin secretion is associated with TNF-α induction and bone regeneration.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1.

Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana.

Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon.

Sericin Composition in the Silk of Antheraea yamamai.

The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes.

Comparative transcriptome analyses on silk glands of six silkmoths imply the genetic basis of silk structure and coloration.

BACKGROUND: Silk has numerous unique properties that make it a staple of textile manufacturing for several thousand years. However, wider applications of silk in modern have been stalled due to limitations of traditional silk produced by Bombyx mori. While silk is commonly produced by B. mori, several wild non-mulberry silkmoths--especially members of family Saturniidae--produce silk with superior properties that may be useful for wider applications. Further utilization of such silks is hampered by the non-domestication status or limited culturing population of wild silkworms. To date there is insufficient basic genomic or transcriptomic data on these organisms or their silk production. RESULTS: We sequenced and compared the transcriptomes of silk glands of six Saturniidae wild silkmoth species through next-generation sequencing technology, identifying 37758 ~ 51734 silkmoth unigenes, at least 36.3% of which are annotated with an e-value less than 10(-5). Sequence analyses of these unigenes identified a batch of genes specific to Saturniidae that are enriched in growth and development. Analyses of silk proteins including fibroin and sericin indicate intra-genus conservation and inter-genus diversification of silk protein features among the wild silkmoths, e.g., isoelectric points, hydrophilicity profile and amino acid composition in motifs of silk H-fibroin. Interestingly, we identified p25 in two of the silkmoths, which were previously predicted to be absent in Saturniidae. There are rapid evolutionary changes in sericin proteins, which might account for the highly heterogeneity of sericin in Saturniidae silkmoths. Within the six sikmoths, both colored-cocoon silkmoth specific transcripts and differentially expressed genes between the colored-cocoon and non-colored-cocoon silkmoths are significantly enriched in catalytic activity, especially transferase activity, suggesting potentially viable targets for future gene mining or genetic manipulation. CONCLUSIONS: Our results characterize novel and potentially valuable gene resources of saturniid silkmoths that may facilitate future genetic improvement and modification of mulberry silkworms. Our results suggest that the disparate features of silk--coloration, retention, strength, etc. --are likely not only due to silk proteins, but also to the environment of silk assembly, and more specifically, that stable silk coloration exhibited by some Saturniidae silkmoths may be attributable to active catalytic progress in pigmentation.

A juvenile hormone transcription factor Bmdimm-fibroin H chain pathway is involved in the synthesis of silk protein in silkworm, Bombyx mori.

The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori.

Functional conservation and structural diversification of silk sericins in two moth species.

Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning.