Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori.

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

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.

Complexities in Bombyx germ cell formation process revealed by Bm-nosO (a Bombyx homolog of nanos) knockout.

Inheritance (sequestration of a localized determinant: germplasm) and zygotic induction are two modes of metazoan primordial germ cell (PGC) specification. vasa and nanos homologs are evolutionarily conserved germline marker genes that have been used to examine the ontogeny of germ cells in various animals. In the lepidopteran insect Bombyx mori, although the lack of vasa homolog (BmVLG) protein localization as well as microscopic observation suggested the lack of germplasm, classical embryo manipulation studies and the localization pattern of Bm-nosO (one of the four nanos genes in Bombyx) maternal mRNA in the egg raised the possibility that an inheritance mode is operating in Bombyx. Here, we generated Bm-nosO knockouts to examine whether the localized mRNA acts as a localized germ cell determinant. Contrary to our expectations, Bm-nosO knockout lines could be established. However, these lines frequently produced abnormal eggs, which failed to hatch, to various extent depending on the individuals. We also found that Bm-nosO positively regulated BmVLG expression at least during embryonic stage, directly or indirectly, indicating that these genes were on the same developmental pathway for germ cell formation in Bombyx. These results suggest that these conserved genes are concerned with stable germ cell production. On the other hand, from the aspect of BmVLG as a PGC marker, we showed that maternal Bm-nosO product(s) as well as early zygotic Bm-nosO activity were redundantly involved in PGC specification; elimination of both maternal and zygotic gene activities (as in knockout lines) resulted in the apparent lack of PGCs, indicating that an inheritance mechanism indeed operates in Bombyx. This, however, together with the fact that germ cells are produced at all in Bm-nosO knockout lines, also suggests the possibility that, in Bombyx, not only this inheritance mechanism but also an inductive mechanism acts in concert to form germ cells or that loss of early PGCs are compensated for by germline regeneration: mechanisms that could enable the evolution of preformation. Thus, Bombyx could serve as an important organism in understanding the evolution of germ cell formation mechanisms; transition between preformation and inductive modes.

Meeting report of the OECD conference on "Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation".

The "OECD Conference on Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation" was held on the 28-29 June 2018 at the OECD headquarter and conference centre in Paris, France. It brought together policy makers, academia, innovators and other stakeholders involved in the topic, in order to take stock of the current technical developments and implementations of genome editing, as well as their applications in various areas of agriculture and the implications they give rise to (More information on the "OECD Conference on Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation" can be found on the OECD Genome Editing hub: http://www.oecd.org/environment/genome-editing-agriculture/ ; the hub also contains the detailed conference programme, the biographies of all conference speakers, the detailed conference abstracts, and the presentations of the two-day conference). The conference aimed to provide a clearer understanding of the regulatory considerations raised by products of genome editing, pointing towards a coherent policy approach to facilitate innovations involving genome editing.

Deficiency of a pyrroline-5-carboxylate reductase produces the yellowish green cocoon 'Ryokuken' of the silkworm, Bombyx mori.

The silkworm cocoon colour has attracted researchers involved in genetics, physiology and ecology for a long time. 'Ryokuken' cocoons are yellowish green in colour due to unusual flavonoids, prolinylflavonols, while 'Sasamayu' cocoons are light green and contain only simple flavonol glucosides. We found a novel gene associated with the cocoon colour change resulting from a change in flavonoid composition and named it Lg (light green cocoon). In the middle silk glands of the + Lg /+ Lg larvae, 1-pyrroline-5-carboxylic acid (P5C) was found to accumulate due to a decrease in the activity of pyrroline-5-carboxylate reductase (P5CR), an enzyme reducing P5C to proline. Sequence analysis of BmP5CR1, the candidate gene for Lg, revealed a 1.9 kb insertion and a 4 bp deletion within the 1st intron, a 97 bp deletion within the 4th intron, and a > 300 bp insertion within the 3'-UTR, in addition to two amino acid changes on exons 3 and 4 in + Lg /+ Lg compared to Lg/Lg. Decreased expression of BmP5CR1 was observed in all of the investigated tissues, including the middle silk glands in + Lg /+ Lg , which was probably caused by structural changes in the intronic regions of BmP5CR1. Furthermore, a BmP5CR1 knockout strain exhibited a yellowish green cocoon with the formation of prolinylflavonols. These results indicate that the yellowish green cocoon is produced by a BmP5CR1 deficiency. To our knowledge, this is the first report showing that the defect of an enzyme associated with intermediate metabolism promotes the conjugation of phytochemicals derived from foods with endogenously accumulating metabolites in animal tissues.

The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly.

Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.

Recent progress in genome engineering techniques in the silkworm, Bombyx mori.

Rapid advances in genome engineering tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas) system, have enabled efficient gene knockout experiments in a wide variety of organisms. Here, we review the recent progress in targeted gene disruption techniques in the silkworm, Bombyx mori. Although efficiency of targeted mutagenesis was very low in an early experiment using ZFNs, recent studies have shown that TALENs can induce highly efficient mutagenesis of desired target genes in Bombyx. Notably, mutation frequencies induced by TALENs can reach more than 50% of G0 gametes. Thus, TALENs can now be used as a standard tool for gene targeting studies, even when mutant phenotypes are unknown. We also propose guidelines for experimental design and strategy for knockout experiments in Bombyx. Genome editing technologies will greatly increase the usefulness of Bombyx as a model for lepidopteran insects, the major agricultural pests, and lead to sophisticated breeding of Bombyx for use in sericulture and biotechnology.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori.

The exceptional quality of silkworm silk is attributed to the amino acid sequence of its fibroin heavy chain (Fib-H) protein. The large central domain of Fib-H, which consists of glycine- and alanine-rich crystalline regions interspersed with amorphous motifs of approximately 30 amino acid residues, is considered crucial for fibrilization and determines the properties of the silk fiber. We established a technical platform to modify the Fib-H core region systematically using transcription activator-like effector nuclease-mediated homologous recombination through a somatic and germline gene knockin assay along with PCR-based screening. This efficient knockin system was used to generate a silkworm strain carrying a mutant Fib-H allele, in which the core region was replaced with a highly ordered synthetic repeat sequence of a length comparable with native Fib-H core. Heterozygous knockin mutants produced seemingly normal cocoons, whereas homozygotes did not and exhibited considerable degradation in their posterior silk glands (PSGs). Cross-sectional examination of the PSG lumen and tensile tests conducted on reeled silk threads indicated that the mutant Fib-H, which exhibited reduced stability in the PSG cells and lumen, affected the mechanical properties of the fiber. Thus, sequence manipulation of the Fib-H core domain was identified as a crucial step in successfully creating artificial silk using knockin technology.

ABC transporter subfamily B1 as a susceptibility determinant of Bombyx mori larvae to Cry1Ba, Cry1Ia and Cry9Da toxins.

ATP binding cassette (ABC) transporters are a diverse family of transmembrane proteins. Specific subfamily members expressed in the lepidopteran midgut can act as susceptibility determinants for several insecticidal Bt Cry proteins. However, the susceptibility determinants to many Cry toxins still remain unclear. Therefore, we knocked out a series of ABC transporters that are highly expressed in the midgut of Bombyx mori larvae by transcription activator-like effector nuclease (TALEN)-mediated gene editing, and the lineages that became resistant to Cry toxins were searched by toxin overlay bioassay. As a result, the B. mori ABC transporter subfamily B1 (BmABCB1) knockout lineage showed 19.17-fold resistance to Cry1Ba, 876.2-fold resistance to Cry1Ia, and 29.1-fold resistance to Cry9Da, suggesting that BmABCB1 is the determinant of susceptibility to these toxins. BmABCC2 and BmABCC3 have been shown to be susceptibility determinants based on their function as receptors. Therefore, we next heterologously expressed these ABC transporters in HEK293T cells and performed a cell swelling assay to examine whether these molecules could exert receptor functions. As a result, BmABCB1-expressing cells showed swelling response to Cry1Ia and Cry9Da, and cells expressing PxABCB1, which is the Plutella xylostella ortholog of BmABCB1, showed swelling for Cry1Ba, suggesting that ABCB1 is a susceptibility determinant by functioning as a receptor to these toxins. Furthermore, in order to clarify how high binding affinity is based on receptor function, we performed surface plasmon resonance analysis and found that each KD of Cry1Ba, Cry1Ia, and Cry9Da to BmABCB1 were 7.69 × 10-8 M, 2.19 × 10-9 M, and 4.17 × 10-6 M respectively.

ATP-binding cassette transporter subfamily C members 2, 3 and cadherin protein are susceptibility-determining factors in Bombyx mori for multiple Bacillus thuringiensis Cry1 toxins.

Field-evolved resistance of insect pests to Bacillus thuringiensis (Bt) toxins (Cry toxins) is a threat to the efficacy of Bt-based bio-insecticides and transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) and cadherin-like receptor play important roles in conferring susceptibility to Cry1 toxins. However, the receptors involved in Bt susceptibility in each insect remain unclear. To determine the receptors that are involved in the susceptibility of Bombyx mori to Cry1 toxins (1Ab, 1Ac and 1Fa), we conducted diet overlay bioassay using B. mori strains disrupted with one or two receptor (s) among BmABCC2, BmABCC3, and cadherin-like receptor (BtR175) generated by transcription activator-like effector nuclease (TALEN)-mediated gene editing. The single-knockout strains for BmABCC2 showed resistance to Cry1Ab and Cry1Ac, whereas only strains with double knockout of BmABCC2 and BmABCC3 exhibited high resistance to Cry1Fa. Progeny populations generated from the crossing of heterozygotes for BtR175 knockout allele included 25% theoretical homozygotes for the BtR175 knockout allele and they showed resistance to Cry1Ab and Cry1Ac. Then, through a cell swelling assay using Sf9 cells ectopically expressing the receptor, we analyzed the mechanisms underlying the different contributions of BmABCC2, BmABCC3, and BtR175 to larval susceptibility. The receptor activity of BmABCC2 for Cry1Ab and Cry1Ac was far higher than that of BmABCC3, and BtR175 synergistically enhanced the receptor activity of BmABCC2. This result well explained the important involvement of BmABCC2 and BtR175 in the larval susceptibility to Cry1A toxins. By contrast, the receptor activities of BmABCC2 and BmABCC3 for Cry1Fa were observed at a similar level and synergistic effect of BtR175 was small. This finding explains the equal importance of BmABCC2 and BmABCC3 and very small contribution of BtR175 on larval susceptibility to Cry1Fa. Thus, we demonstrated the different importance of BmABCC2, BmABCC3, and BtR175 to various Cry1 toxins as susceptibility-determining factors in B. mori larvae and the underlying basis for the observed differences. Furthermore, a weak correlation was indicated between the binding affinity and receptor activities of BmABCC2 and BmABCC3 to Cry1 toxins.

Comparisons in temperature and photoperiodic-dependent diapause induction between domestic and wild mulberry silkworms.

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother's embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.

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.

ATP-Binding Cassette Subfamily A Member 2 is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, but not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins.

: Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins.

Functional Analysis of Adipokinetic Hormone Signaling in Bombyx mori.

Insect adipokinetic hormones (AKHs) are short peptides produced in the corpora cardiaca and are responsible for mobilizing energy stores from the fat body to the hemolymph. Three related peptides, AKH1, AKH2, and AKH/corazonin-related peptide (ACP) as well as three AKH receptors have been reported in Bombyx mori. AKH1 and AKH2 are specific for the AKHR1 receptor, whereas ACP interacts with the other two AKHRs. To assess the effect of the two silkworm AKHs and ACP in the regulation of energy homeostasis we examined the expression pattern of the three peptides and their receptors as well as their effect on the level of carbohydrates and lipids in the hemolymph. Our results support the hypothesis that only AKH1 and AKH2 peptides together with the AKHR1 receptor are involved in the maintenance of energy homeostasis. Because Bombyx AKHR1 (BmAKHR1) seems to be a true AKHR we generated its mutation. The BmAKHR1 mutant larvae display significantly lower carbohydrate and lipid levels in the hemolymph and reduced sensitivity to starvation. Our study clarifies the role of BmAKHR1 in energy homeostasis.

Policy Considerations Regarding Genome Editing.

The international Organisation for Economic and Co-operative Development (OECD) conference on genome editing (June 2018) provided a timely platform for scientists, risk assessors, policy-makers, and regulators to discuss the applications and implications of this technology in various agriculture areas and the related policy considerations; in addition questions related to appropriate safety assessments and the regulation of genome-edited products were debated.

Genome Editing Advances the Structural Study of Silk.

We first applied the genome edited silkworm silk (GE-silk) to interpret X-ray fiber diagram, and implied a great potential for the application of genome editing technology to the structural study of silk. The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the X-ray fiber diagram of Bombyx mori silkworm silk, has been widely believed but not experimentally proven to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. The above hypothesis was experimentally demonstrated from X-ray measurements of GE-silk.

[Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms].

　We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.

Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori.

Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for the protein, we determined the primary structures of its partial peptides, and the gene was searched using the silkworm genomic databases. Three contigs containing the corresponding nucleotide sequences were identified and categorized as one group. The gene structure covering the 5' flanking and the 3' end was determined by PCR fragments from genomic DNA, RT-PCR, and 5' and 3' RACE. The amino acid sequence deduced from the nucleotide sequence mainly consists of two serine-rich regions of 86-amino acid motif and 8-amino acid repeated sequence. The expression of the gene is limited to the anterior and middle parts of the middle silk gland. In addition, because the sericin gene appeared different from the sericin 1 and 2 genes reported earlier, we designated the newly discovered gene as sericin 3.

Targeted Mutagenesis in Bombyx mori Using TALENs.

Bombyx mori is a valuable model organism of high economic importance. Its genome sequence is available, as well as basic genetic and molecular genetic tools and markers. The introduction of genome editing methods based on engineered nucleases enables precise manipulations with genomic DNA, including targeted DNA deletions, insertions, or replacements in the genome allowing gene analysis and various applications. We describe here the use of TALENs which have a simple modular design of their DNA-binding domains, are easy to prepare and proved to be efficient in targeting of a wide range of cleavage sites. Our procedure often allows the production of individuals carrying homozygous mutations as early as in the G1 generation.