High mechanical property silk produced by transgenic silkworms expressing the Drosophila Dumpy.

Extensive research has been conducted on utilizing transgenic silkworms and their natural spinning apparatus to produce high-performance spider silk fibers. However, research on using non-spider biological proteins to optimize the molecular structure of silk protein and improve the mechanical performance of silk fibers is still relatively scarce. Dumpy, a massive extracellular matrix polypeptide, is essential for preserving the shape and structural integrity of the insect cuticle due to its remarkable tension and elasticity. Here, we constructed two transgenic donor plasmids containing the fusion genes of FibH-Dumpy and FibL-Dumpy. The results indicated the successful integration of two exogenous gene expression cassettes, driven by endogenous promoters, into the silkworm genome using piggyBac-mediated transgenic technology. Secondary structure analysis revealed a 16.7% and 13.6% increase in the ß-sheet content of transgenic silks compared to wild-type (WT) silk fibers. Mechanical testing demonstrated that, compared to the WT, HDUY and LDUY transgenic silk fibers exhibited respective increases of 39.54% and 21.45% in maximum stress, 44.43% and 45.02% in toughness, and 24.91% and 28.51% in elastic recovery rate. These findings suggest that Drosophila Dumpy significantly enhanced the mechanical properties of silk, positioning it as an excellent candidate for the development of extraordinary-performance fibers. This study provides rich inspiration for using other biological proteins to construct high-performance silk fibers and expands the possibilities for designing and applying novel biomaterials.

Modeling the 3-dimensional structure of the silkworm's spinning apparatus in silk production.

The silk-spinning process of the silkworms transforms the liquid silk solution to a solid state under mild conditions, making it an attractive model for bioinspiration However, the precise mechanism behind silk expulsion remains largely unknown. Here we selected the silkworms as representative models to investigate the silk-spinning mechanism. We used serial block-face scanning electron microscopy (SBF-SEM) to reconstruct the three-dimensional structures of the spinnerets in silkworms at various stages and with different gene backgrounds. By comparing the musculature and duct deformation of these spinneret models during the spinning process, we were able to simulate the morphological changes of the spinneret. Based on the results, we proposed three essential factors for silkworm spinning: the pressure generated by the silk gland, the opening duct, and the pulling force generated by head movement. Understanding the silkworm spinning process provides insights into clarify the fluid-ejecting mechanism of a group of animals. Moreover, these findings are helpful to the development of biomimetic spinning device that mimics the push-and-pull dual-force system in silkworms. STATEMENT OF SIGNIFICANCE: The silkworms' spinning system produces fibers under mild conditions, making it an ideal candidate for bioinspiration. However, the mechanism of silk expulsion is unknown, and the three-dimensional structure of the spinneret is still uncertain. In this study, we reconstructed a detailed 3-dimensional model of the spinneret at near-nanometer resolution, and for the first time, we observed the changes that occur before and during the silk-spinning process. Our reconstructed models suggested that silkworms have the ability to control the spinning process by opening or closing the spinning duct. During the continuously spinning period, both the pressure generated by the silk gland and the pulling force resulting from head movement work in tandem to expel the silk solution. We believe that gaining a full understanding of the spinning process steps can advance our ability to spin synthetic fibers with properties comparable to those of native fibers by mimicking the natural spinning process.

Drosophila melanogaster resilin improves the mechanical properties of transgenic silk.

Resilin is a natural protein with high extensibility and resilience that plays a key role in the biological processes of insects, such as flight, bouncing, and vocalization. This study used piggyBac-mediated transgenic technology to stably insert the Drosophila melanogaster resilin gene into the silkworm genome to investigate whether exogenous protein structures improve the mechanical properties of silkworm silk. Molecular detection showed that recombinant resilin was expressed and secreted into silk. Secondary structure and mechanical property analysis showed that the ß-sheet content in silk from transgenic silkworms was higher than in wild-type silk. The fracture strength of silk fused with resilin protein was 7.2% higher than wild-type silk. The resilience of recombinant silk after one-time stretching and cyclic stretching was 20.5% and 18.7% higher than wild-type silk, respectively. In summary, Drosophila resilin can enhance the mechanical properties of silk, and this study is the first to improve the mechanical properties of silk using proteins other than spider silk, which broadens the possibilities for the design and application of biomimetic silk materials.

Sex separation by body color via a W-chromosome-linked transgene.

Sex separation processes are important for commercial insect production and sterile insect techniques. Here, we describe the transgenic insertion of a DsRed expression cassette driven by the enhancer HR3 and strong promoter IE1 into the silkworm W chromosome as a dominant visible marker of sex separation. The obtained transgenic lines showed female-specific body color visible to the naked eye at the second- to fifth-instar larval, pupal and adult stages, and their performance traits were comparable to those of a nontransgenic practical silkworm variety. This strategy can greatly facilitate the sex separation of silkworms for male-only rearing and to obtain hybrids while avoiding sibling mating, and it can also be applied to the sex separation of other light-colored insects.

Mechanism of silk secretion revealed by proteomic analysis of silkworm cocoons with fibroin light chain mutations.

Silkworm is an economically important insect due to its efficient production of silk proteins. Silk itself and the silk trade have enriched human civilization through art and culture and contributed to early globalization in the Silk Road era for nearly two thousand years. Although a large number of studies on silk have been carried out, the mechanism of silk secretion in silkworms has not been thoroughly studied thus far. As the main component of fibroin, fibroin light chain (Fib-L) plays a key role in the secretion of silk. In this study, we constructed a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. Strikingly, fibrohexamerin (P25) was secreted almost normally in the homozygous mutant. Further analysis of cocoon proteins revealed that the mutant responded to greater environmental stress caused by a dramatic decrease in fibroin by significantly increasing the secretion of protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm. SIGNIFICANCE: This study generated a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. The analysis of the abundance of silk proteins in the cocoons revealed that P25 could be secreted almost normally. The analysis of the abundance of cocoon proteins other than silk proteins showed that the homozygous mutants responded to greater environmental stress by increasing the secretion of defense-related proteins, such as protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm.

Horizontal Gene Transfer and Gene Duplication of ß-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits.

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect ß-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of ß-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of ß-fructofuranosidase were found in Coleoptera and Hymenoptera. ß-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran ß-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of ß-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of ß-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Physiological adaptations to sugar-mimic alkaloids: Insights from Bombyx mori for long-term adaption and short-term response.

Insects evolved adaptive plasticity to minimize the effects of the chemical defenses of their host plants. Nevertheless, the expressional response and adaptation of phytophagous specialists for long-term adaption and short-term response to host phytochemicals remains largely unexplored. The mulberry (Morus alba)-silkworm (Bombyx mori) interaction is an old and well-known model of plant-insect interaction. In this study, we examined the long-term adaption and short-term response of the mulberry-specialist silkworm to two sugar-mimic alkaloids in mulberry: the commonly encountered 1-deoxynojirimycin (1-DNJ) and occasionally encountered 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1), respectively. Global transcriptional patterns revealed that the physiological responses induced by the selective expression of genes involved in manifold cellular processes, including detoxification networks, canonical digestion processes, target enzymes, and other fundamental physiological processes, were crucial for regulating metabolic homeostasis. Comparative network analysis of the effects of exposure to D-AB1 and 1-DNJ supported the contention that B. mori produced similar and specific trajectories of changed gene expression in response to different sugar-mimic alkaloids. D-AB1 elicited a substantial proportion of downregulated genes relating to carbohydrate metabolism, catabolic process, lipid metabolism, and glycan biosynthesis and metabolism. This study dramatically expands our knowledge of the physiological adaptations to dietary sugar-mimic alkaloid intake and uncovered both metabolic evolutionarily responses and unique adaptive mechanisms previously unknown in insects.

Expression plasticity and evolutionary changes extensively shape the sugar-mimic alkaloid adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects.

During the co-evolutionary arms race between plants and herbivores, insects evolved systematic adaptive plasticity to minimize the chemical defence effects of their host plants. Previous studies mainly focused on the expressional plasticity of enzymes in detoxification and digestion. However, the expressional response and adaptive evolution of other fundamental regulators against host phytochemicals are largely unknown. Glucosidase II (GII), which is composed of a catalytic GIIα subunit and a regulatory GIIß subunit, is an evolutionarily conserved enzyme that regulates glycoprotein folding. In this study, we found that GIIα expression of the mulberry-specialist insect was significantly induced by mulberry leaf extract, 1-deoxynojirimycin (1-DNJ), whereas GIIß transcripts were not significantly changed. Moreover, positive selection was detected in GIIα when the mulberry-specialist insects diverged from the lepidopteran order, whereas GIIß was mainly subjected to purifying selection, thus indicating an asymmetrically selective pressure of GII subunits. In addition, positively selected sites were enriched in the GIIα of mulberry-specialist insects and located around the 1-DNJ-binding sites and in the C-terminal region, which could result in conformational changes that affect catalytic activity and substrate-binding efficiency. These results show that expression plasticity and evolutionary changes extensively shape sugar-mimic alkaloids adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects. Our study provides novel insights into a deep understanding of the sequestration and adaptation of phytophagous specialists to host defensive compounds.

Comparative transcriptome analysis of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) reveals novel insights into heat stress tolerance in insects.

BACKGROUND: Heat tolerance is a key parameter that affects insect distribution and abundance. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a devastating pest of mulberry in the main mulberry-growing regions and can cause tremendous losses to sericulture by directly feeding on mulberry leaves and transmitting viruses to Bombyx mori. Moreover, G. pyloalis shows a prominent capacity for adaptation to daily and seasonal temperature fluctuations and can survive several hours under high temperature. To date, the molecular mechanism underlying the outstanding adaptability of this pest to high temperature remains unclear. RESULTS: In this study, we performed comparative transcriptome analyses on G. pyloalis exposed to 25 and 40 °C for 4 h. We obtained 34,034 unigenes and identified 1275 and 1222 genes significantly upregulated or downregulated, respectively, by heat stress. Data from the transcriptome analyses indicated that some processes involved in heat tolerance are conserved, such as high expression of heat shock protein (HSP) genes and partial repression of metabolism progress. In addition, vitamin digestion and absorption pathways and detoxification pathways identified here provided new insights for the investigation of the molecular mechanisms of heat stress tolerance. Furthermore, transcriptome analysis indicated that immune and phosphatidylinositol signaling system have a close relationship with heat tolerance. In addition, the expression patterns of ten randomly selected genes, such as HSP and cytochrome P450, were consistent with the transcriptome results obtained through quantitative real-time PCR. CONCLUSIONS: Comparisons among transcriptome results revealed the upregulation of HSPs and genes involved in redox homeostasis, detoxication, and immune progress. However, many metabolism progresses, such as glycolysis/gluconeogenesis and fatty acid biosynthesis, were partially repressed. The results reflected that the heat tolerance of G. pyloalis is a fairly complicated process and related to a broad range of physiological regulations. Our study can improve understanding on the mechanisms of insect thermal tolerance.

Molecular evolutionary mechanisms driving functional diversification of α-glucosidase in Lepidoptera.

The digestive tract of lepidopteran insects is unique given its highly alkaline pH. The adaptive plasticity of digestive enzymes in this environment is crucial to the highly-efficient nutritional absorption in Lepidoptera. However, little is known about the molecular adaptation of digestive enzymes to this environment. Here, we show that lepidopteran α-glucosidase, a pivotal digestive enzyme, diverged into sucrose hydrolase (SUH) and other maltase subfamilies. SUH, which is specific for sucrose, was only detected in Lepidoptera. It suggests that lepidopteran insects have evolved an enhanced ability to hydrolyse sucrose, their major energy source. Gene duplications and exon-shuffling produced multiple copies of α-glucosidase in different microsyntenic regions. Furthermore, SUH showed significant functional divergence (FD) compared with maltase, which was affected by positive selection at specific lineages and codons. Nine sites, which were involved in both FD and positive selection, were located around the ligand-binding groove of SUH. These sites could be responsible for the ligand-binding preference and hydrolytic specificity of SUH for sucrose, and contribute to its conformational stability. Overall, our study demonstrated that positive selection is an important evolutionary force for the adaptive diversification of α-glucosidase, and for the exclusive presence of membrane-associated SUHs in the unique lepidopteran digestive tract.

Negative regulation of DAB2IP by Akt and SCFFbw7 pathways.

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCF(Fbw7). DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1δ. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development.