Time-course transcriptome data of silk glands in day 0-7 last-instar larvae of Bombyx mori (w1 pnd strain).

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

A major endogenous glycoside hydrolase mediating quercetin uptake in Bombyx mori.

Quercetin is a common plant flavonoid which is involved in herbivore-plant interactions. Mulberry silkworms (domestic silkworm, Bombyx mori, and wild silkworm, Bombyx mandarina) take up quercetin from mulberry leaves and accumulate the metabolites in the cocoon, thereby improving its protective properties. Here we identified a glycoside hydrolase, named glycoside hydrolase family 1 group G 5 (GH1G5), which is expressed in the midgut and is involved in quercetin metabolism in the domestic silkworm. Our results suggest that this enzyme mediates quercetin uptake by deglycosylating the three primary quercetin glycosides present in mulberry leaf: rutin, quercetin-3-O-malonylglucoside, and quercetin-3-O-glucoside. Despite being located in an unstable genomic region that has undergone frequent structural changes in the evolution of Lepidoptera, GH1G5 has retained its hydrolytic activity, suggesting quercetin uptake has adaptive significance for mulberry silkworms. GH1G5 is also important in breeding: defective mutations which result in discoloration of the cocoon and increased silk yield are homozygously conserved in 27 of the 32 Japanese white-cocoon domestic silkworm strains and 12 of the 30 Chinese ones we investigated.

An Effective Chemical Permeabilization of Silkworm Embryos.

The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid layer also prevents the flux of chemicals into the embryos, such as cryoprotectants, which are required for successful cryopreservation. The permeabilization studies of silkworm embryos remain insufficient. Therefore, in this study, we developed a permeabilization method to remove the lipid layer in the silkworm, Bombyx mori, and examined factors affecting the viability of dechorionated embryos, including the types and exposure times of chemicals and embryonic stages. Among the chemicals used, hexane and heptane were effective for permeabilization, whereas Triton X-100 and Tween-80 were less effective. Regarding the embryonic stages, there were significant differences between 160 and 166 h after egg laying (AEL) at 25 °C. Consequently, we found that the treatment of 160 AEL embryos with hexane for 30 s was the best condition for the permeability and viability of embryos, in which over 62% of the permeabilized embryos grew up to the second larval instar and their moths could lay fertilized eggs. Our method can be used for various purposes, including permeability investigations using other chemicals and embryonic cryopreservation.

Role of a single odorant receptor in the chemotaxis behavior of Bombyx mori.

Silkworm (Bombyx mori), an insect herbivore, is attracted to cis-jasmone released from mulberry leaves. Its olfactory receptor, BmOr56, specifically responds to cis-jasmone. In this study, we constructed a BmOr56 deletion line and found that the attractive behavior of cis-jasmone was completely lost in the mutant, suggesting the involvement of a single receptor in this specific chemoattractive behavior.

Highly accurate genome assembly of an improved high-yielding silkworm strain, Nichi01.

The silkworm (Bombyx mori) is an important lepidopteran model insect and an industrial domestic animal traditionally used for silk production. Here, we report the genome assembly of an improved Japanese strain Nichi01, in which the cocoon yield is comparable to that of commercial silkworm strains. The integration of PacBio Sequel II long-read and ddRAD-seq-based high-density genetic linkage map achieved the highest quality genome assembly of silkworms to date; 22 of the 28 pseudomolecules contained telomeric repeats at both ends, and only four gaps were present in the assembly. A total of 452 Mbp of the assembly with an N50 of 16.614 Mbp covered 99.3% of the complete orthologs of the lepidopteran core genes. Although the genome sequence of Nichi01 and that of the previously reported low-yielding tropical strain p50T assured their accuracy in most regions, we corrected several regions, misassembled in p50T, in our assembly. A total of 18,397 proteins were predicted using over 95 Gb of mRNA-seq derived from 10 different organs, covering 96.9% of the complete orthologs of the lepidopteran core genes. The final assembly and annotation files are available in KAIKObase (https://kaikobase.dna.affrc.go.jp/index.html) along with a genome browser and BLAST searching service, which would facilitate further studies and the breeding of silkworms and other insects.

Genome Editing of Silkworms.

Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available, and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knockout is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the precise integration into target chromosome (PITCh) method. Here we describe protocols for ZFN (zinc finger nuclease)-, TALEN (transcription activator-like effector nuclease)-, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm. We consider that all of these techniques can contribute to the further promotion of various biological studies in the silkworm and other insect species.

Insights into the quality of recombinant proteins produced by two different Bombyx mori expression systems.

The silkworm, Bombyx mori, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been widely used for recombinant protein production in B. mori: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined the quality of the resulting proteins in terms of N-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an N-glycoprotein. Although the production in the baculovirus/silkworm expression system was much more efficient than that in the transgenic silkworm expression system, unexpected variants of IFN-γ were also produced in the former system due to the different N-glycosylation and C-terminal truncations. These results indicate that while high protein production could be achieved in the baculovirus/silkworm expression system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of N-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in B. mori.

Abolition of egg diapause by ablation of suboesophageal ganglion in parental females is compatible with genetic engineering methods.

Microinjection of genetic material into non-diapause eggs is required for genetic engineering of silkworms. Besides diapause could be useful for maintaining transgenic lines, a drawback of this technology is that most standard silkworm strains and experimental lines of interest produce diapausing eggs. Several approaches have been developed to abolish diapause but none are very efficient. Here, we investigated the ablation of the suboesophageal ganglion (SG) in female pupae, which is a source of the hormone required to trigger egg diapause, as a mean to abolish diapause. We showed that SG-ablation is a reliable method to produce nondiapause eggs. Additionally, the challenge associated with lower fecundity of females with SG ablation was resolved by injecting pilocarpine into the mated female. We also investigated the suitability of nondiapause eggs laid by SG-ablated females for transgenesis, targeted mutagenesis, and induction of parthenogenetic development. Our results demonstrated SG-ablation to be a useful and simple method for expanding the possibilities associated with genetic engineering in silkworms.

MicroRNA let-7 is required for hormonal regulation of metamorphosis in the silkworm, Bombyx mori.

The heterochronic microRNA let-7, which was first identified in Caenorhabditis elegans, controls the timing of developmental programs, and let-7 triggers the onset of the juvenile-adult transition in bilaterians. The expression of let-7 is strongly induced during the last larval stage of C. elegans and is highly expressed in the late last instar larvae/nymphs of the fly Drosophila melanogaster and the cockroach Blattella germanica. In the silkworm Bombyx mori, the expression of let-7 remarkably increases in the corpus cardiacum-corpus allatum complex (CC-CA) at the beginning of the last larval instar and is maintained at high levels during this instar. To determine the biological function of let-7 in B. mori, we generated a let-7 knockout line and a transgenic UAS-let-7 line. The let-7 knockout larvae were developmentally arrested in the prepupal stage and became pupal-adult intermediates after apolysis. When let-7 was ubiquitously overexpressed under the transcriptional control of an Actin3-GAL4 driver, developmental timing and growth of larvae were severely impaired in the penultimate (L4) instar, and these larvae underwent precocious metamorphosis from L4. Furthermore, our results showed that reception and signaling of ecdysteroids and juvenile hormones (JHs) normally occurred in the absence of let-7, whereas the biosynthesis of ecdysone and JHs were affected by disruption and overexpression of let-7. Together, the present study demonstrates that let-7 is required for the coordination of the biosynthesis of ecdysone and JH to ensure the developmental transition during the metamorphosis of B. mori.

The Exact Timing of Microinjection of Parthenogenetic Silkworm Embryos Is Crucial for Their Successful Transgenesis.

The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol.

Temporal analysis of N-acetylglucosamine extension of N-glycans in the middle silk gland of silkworm Bombyx mori.

N-glycosylation of proteins is an important post-translational modification in eukaryotic cells. One of the key modifications in protein N-glycosylation is N-acetylglucosamine (GlcNAc) extension mediated by N-acetylglucosaminyltransferase I (GNTI), which triggers N-glycan maturation from high-mannose-type to hybrid- and complex-type structures in Golgi. However, the temporal contributions of GNTI to GlcNAc extension and the resultant N-glycan structures in insects have not been analyzed. Here, focusing on GlcNAc extension of N-glycan in the silkworm Bombyx mori, we analyzed the temporal N-glycan alterations in the middle silk gland (MSG) and characterized the property of key enzyme for complex-type N-glycan biosynthesis, B. mori GNTI (BmGNTI). N-glycan analysis of N-glycoproteins in the MSG demonstrated that BmGNTI identified and characterized in this study consistently contributed to GlcNAc extension of N-glycans, which led to the accumulation of GlcNAc-extended N-glycans as predominant structures throughout the MSG development. The expression profile of GlcNAc extension-related genes revealed that the enzymes contributing to the hydrolysis of GlcNAc showed stage-specific expressions, thereby resulting in accumulations of the end product N-glycans of the enzyme. These results lead to the speculation that not BmGNTI but rather glycosylhydrolases critically influenced the structural formations and the changes in the ratio of N-glycans with GlcNAc residue(s) in MSG.

Identification and characterization of a serine racemase in the silkworm Bombyx mori.

The pupae of lepidopterans contain high concentrations of endogenous d-serine. In the silkworm Bombyx mori, d-serine is negligible during the larval stage but increases markedly during the pupal stage, reaching 50% of the total free serine. However, the physiological function of d-serine and the enzyme responsible for its production is unknown. Herein, we identified a new type of pyridoxal 5'-phosphate (PLP)-dependent serine racemase (SR) that catalyses the racemization of l-serine to d-serine in B. mori. This silkworm SR (BmSR) has an N-terminal PLP-binding domain that is homologous to mammalian SR and a C-terminal putative ligand-binding regulatory-like domain (ACT-like domain) that is absent in mammalian SR. Similar to mammalian SRs, BmSR catalyses the racemization and dehydration of both serine isomers. However, BmSR is different from mammalian SRs as evidenced by its insensitivity to Mg2+/Ca2+ and Mg-ATP-which are required for activation of mammalian SRs-and high d-serine dehydration activity. At the pupal stage, the SR activity was predominantly detected in the fat body, which was consistent with the timing and localization of BmSR expression. The results are an important first step in elucidating the physiological significance of d-serine in lepidopterans.

Co-Expression Network and Time-Course Expression Analyses to Identify Silk Protein Regulatory Factors in Bombyx mori.

Bombyx mori is an important economic insect and an animal model in pharmacomedical research. Although its physiology has been studied for many years, the mechanism via which silk protein genes are regulated remains unclear. In this study, we performed two-step expression screening, namely co-expression network and time-course expression analyses to screen silk protein regulation factors. A co-expression network analysis using RNA-seq data that were obtained from various tissues, including the silk glands of B. mori, was performed to identify novel silk protein regulatory factors. Overall, 91 transcription factors, including some known ones, were found to be co-expressed with silk protein genes. Furthermore, time-course expression analysis during the fifth instar larvae stage revealed that the expression pattern of 13 novel transcription factors was highly relevant to that of silk protein genes and their known regulatory factor genes. In particular, the expression peak of several transcription factors (TFs) was detected before the expression of silk protein genes peak. These results indicated that a larger number of genes than expected may be involved in silk protein regulation in B. mori. Functional analyses of function-unknown transcription factors should enhance our understanding of this system.

The red egg gene as a novel effective egg color marker for silkworm transgenesis.

Ommochromes are major pigments involved in coloration of eggs, eyes, and epidermis of arthropods. The recessive homozygous of egg and eye color mutant of Bombyx mori, red egg (re), exhibits red eggs and dark red eyes instead of normal purplish-brown eggs and black eyes, due to a defect in ommochrome pigment synthesis. The gene responsible for the re mutant is a major facilitator superfamily transporter gene, Bm-re. Here, we demonstrate that the re phenotype can be effectively rescued by an intact Bm-re gene driven by the Bombyx Actin A3 promoter or the baculovirus Immediate Early 1 promoter, indicating that the Bm-re gene can be used as a marker gene for visual screening of transgenic silkworms. The coloration of eggs rescued by the Bm-re transgene could be distinguished from that of host mutant eggs from diapausing period through head pigmentation stage. This allows transgenic screening at earlier embryonic stages and over a longer time period compared to conventional 3xP3 fluorescent markers, without requiring the skill and equipment to detect stemmata fluorescence.

Effect of chemical dechorionation on silkworm embryo viability.

The chorion covering/protecting insect egg, which has some effective functions such as providing mechanical strength, protecting eggs from external environments, and keeping moisture adjustment, is one of the principal barriers to manipulation, cryopreservation, and study of insect embryos. Here we evaluated the silkworm embryo viability after dechorionation using chemical reagents. We have developed an easy and effective method for chemical dechorionation that enables embryos to develop in culture, so that the larvae could normally grow. Eggs attached to a nylon net were treated with potassium hydroxide (KOH) and sodium hypochlorite (NaClO) to remove the chorion, washed with the Grace's insect medium, and then cultured using a dry-moist method which we created. The most effective treatment with regard to embryonic development, hatching, and production of second instar larvae was 30% KOH for 7 min and 2% NaClO for 5 min at 27 °C. Embryos at later embryonic stages were more tolerant to chemical dechorionation and over 75% of embryos treated at 168 h-old (Stage 25, appearance of taenidium) survived to the second larval instar, moreover, the larvae derived from the dechorionated embryos have developed into the moths which can lay the fertilized eggs. Our method would contribute to the establishment of cryopreservation using embryos and analysis of silkworm embryogenesis and might also be applicable to other insect species.

Dynamic alterations of circulating T lymphocytes and the clinical response in patients with head and neck squamous cell carcinoma treated with nivolumab.

Cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). However, only approximately 20-30% of patients with recurrent/metastatic (R/M) HNSCC benefit. Moreover, the mechanisms underlying the response to ICIs remain unclear. We investigated the proportion, activation status, and expression level of immune checkpoint molecules in circulating T cell subsets in R/M HNSCC patients treated with nivolumab using flow cytometry and mass cytometry, and then determined whether treatment response was associated with these values. We also assessed the changes in the frequency of tumor-associated antigens, MAGE-A4 and p53, -specific T cells prior to and after nivolumab treatment using the IFN-γ ELISPOT assay. The proportion of activated CD4+ and CD8+ TEMRA cells significantly increased in the disease-controlled patients but not in disease-progressed patients. As expected, the expression of PD-1 in T cells markedly decreased regardless of the therapeutic response. Meanwhile, T cell immunoglobulin mucin-3 expression on CD8+ T cells was significantly higher in patients with disease progression than in disease-controlled patients after treatment. The frequency of the tumor-associated antigens, MAGE-A4- and p53-specific T cells, was not correlated with clinical responses; however, in the disease-controlled patients, the frequency of MAGE-A4-specific T cells was significantly augmented. We concluded that in R/M HNSCC patients treated with nivolumab, circulating T cells show dynamic alterations depending on treatment efficacy. An analysis of the immunokinetics of circulating T cells could thus provide new insights into rational therapeutic strategies in cancer immunotherapy for HNSCC.

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) - an immuno-stimulative agent - into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

Reference Transcriptome Data in Silkworm Bombyx mori.

Herein, we performed RNA-seq analysis of ten major tissues/subparts of silkworm larvae. The sequences were mapped onto the reference genome assembly and the reference transcriptome data were successfully constructed. The reference data provided a nearly complete sequence for sericin-1, a major silk gene with a complex structure. We also markedly improved the gene model for other genes. The transcriptomic expression was investigated in each tissue and a number of transcripts were identified that were exclusively expressed in tissues such as the testis. Transcripts strongly expressed in the midgut formed tight genomic clusters, suggesting that they originated from tandem gene duplication. Transcriptional factor genes expressed in specific tissues or the silk gland subparts were also identified. We successfully constructed reference transcriptome data in the silkworm and found that a number of transcripts showed unique expression profiles. These results will facilitate basic studies on the silkworm and accelerate its applications, which will contribute to further advances in lepidopteran and entomological research as well as the practical use of these insects.

Production of cloned transgenic silkworms by breeding non-diapausing parthenogenetic strains.

Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.

Mutation in Bombyx mori fibrohexamerin (P25) gene causes reorganization of rough endoplasmic reticulum in posterior silk gland cells and alters morphology of fibroin secretory globules in the silk gland lumen.

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.