Emission of water-window soft x-rays under optically thin conditions using low-density foam targets.

The effect of optical thickness in a bismuth water-window soft x-ray source is considered by comparing the emission from laser-produced plasmas of a 7.5% atomic density foam target and a solid-density target. The number of photons recorded in the 4 nm region was comparable for both targets at a plasma-initiating laser pulse duration of 6 ns. From experiments at different pulse durations of 150 ps and 6 ns, self-absorption (opacity) effects were found to be relatively small for bismuth plasmas as compared to those of tin, based on the same emission mechanism and which are used in 13.5 nm sources for extreme ultraviolet lithography.

Selection of target elements for laser-produced plasma soft x-ray sources.

We demonstrated the upper limitation to the number of shots, i.e., target lifetime, together with the number of photons emitted in the water-window soft x-ray spectral region from a number of targets used as sources in this spectral region, for multi-shot irradiation at the same position on the target surface. The spectra involved result from unresolved transition arrays originating from n=3-n=4 transitions in medium-Z element plasmas and from n=4-n=4 transitions originating in high-Z plasmas. The output flux was maintained for the highest number of shots in the case of the high melting point element molybdenum, and the total output in the water window was 7.7×1013 photons/sr at a laser power density of 1.2×1014 W/cm2.

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons.

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis, in the silkworm Bombyx mori.

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval-larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.

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.

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori.

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists.

Polyclonal activation of naïve T cells by urease deficient-recombinant BCG that produced protein complex composed of heat shock protein 70, CysO and major membrane protein-II.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs. METHODS: Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags). RESULTS: BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB. CONCLUSIONS: The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation.

A single sex pheromone receptor determines chemical response specificity of sexual behavior in the silkmoth Bombyx mori.

In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.

Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway.

Ommochromes are one of the major pigments involved in coloration of eggs, eyes, and body surface of insects. However, the molecular mechanisms of the final steps of ommochrome pigment synthesis have been largely unknown. The eggs of the silkworm Bombyx mori contain a mixture of ommochrome pigments, and exhibit a brownish lilac color. The recessive homozygous of egg and eye color mutant, red egg (re), whose eggs display a pale orange color instead of normal dark coloration, has been long suggested to have a defect in the biosynthesis of the final ommochrome pigments. Here, we identify the gene responsible for the re locus by positional cloning, mutant analysis, and RNAi experiments. In the re mutants, we found that a 541-bp transposable element is inserted into the ORF of BGIBMGA003497-1 (Bm-re) encoding a novel member of a major facilitator superfamily transporter, causing disruption of the splicing of exon 9, resulting in two aberrant transcripts with frameshifts yielding nonfunctional proteins lacking the C-terminal transmembrane domains. Bm-re function in pigmentation was confirmed by embryonic RNAi experiments. Homologs of the Bm-re gene were found in all insect genomes sequenced at present, except for 12 sequenced Drosophila genomes, which seemed to correlate with the previous studies that have demonstrated that eye ommochrome composition is different from other insects in several Dipterans. Knockdown of the Bm-re homolog by RNAi in the red flour beetle Tribolium castaneum caused adult compound eye coloration defects, indicating a conserved role in ommochrome pigment biosynthesis at least among holometabolous insects.

Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori.

Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 degrees C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (sch(l)) do not hatch even at room temperature (25 degrees C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, sch(l), and wild-type. However, in sch the approximately 70-kb sequence was replaced with approximately 4.6 kb of a Tc1-mariner type transposon located approximately 6 kb upstream of BmTh, and in sch(l), a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd(+) and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color.

Soaking RNAi in Bombyx mori BmN4-SID1 cells arrests cell cycle progression.

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes.

[Towards novel tuberculosis and leprosy vaccine development: the role of Th1-inducing peptide in cytotoxic T cell differentiation].

The effectiveness of a vaccine against tuberculosis and leprosy is mainly judged by its capability to induce memory CD8 cytotoxic T cells (CTL). It has been reported that 'help' from CD4+ T cells is required to induce memory CTL. However, how CD4+ T cells instruct or support memory CTL during priming phase has not been resolved in detail. Therefore, we examined the helper function of CD4+ T cells in CTL differentiation. Peptide-25 is the major T cell epitope of Ag85B of Mycobacterium tuberculosis. We found that this peptide induced the expression of T-bet and TATA box binding protein-associated factor that can induce the chromatin remodeling of ifn-gamma gene, and as a result induced Th1 differentiation even in the absence of IFN-gamma and IL-12. Next, we established an in vitro CTL differentiation system using Peptide-25, Peptide-25 specific CD4+ T cells, OVA specific CD8+ T cells and splenic DC. By using this system, we found that CD4+ T cells activated DC even in the absence of IFN-gamma and CD40 ligand association, and the activated DC induced the functional differentiation of CTL. To identify the regulatory factors for DC activation, we analyzed the gene expression profile of helper CD4 T cells and identified 27 genes. Taken together, these results suggest that the inducing factors for Th1 differentiation are not indispensable to induce the functional differentiation of CTL.

Positional cloning of silkworm white egg 2 (w-2) locus shows functional conservation and diversification of ABC transporters for pigmentation in insects.

The white, scarlet and brown genes of Drosophila melanogaster encode three half-type ATP-binding cassette (ABC) transporters. In Drosophila, precursors of ommochromes and pteridines are transported by White/Scarlet and White/Brown heterodimers, respectively. The white egg 2 (w-2) mutant of the silkworm, Bombyx mori, has white eggs and eyes because of lack of ommochrome granules in the serosa and eyes. Here, we report that the silkworm w-2 locus encodes an ortholog of Drosophila scarlet. Our results indicate that Bombyx Scarlet forms a heterodimer with Bombyx White to transport ommochrome precursors, suggesting that formation of a White/Scarlet heterodimer and its involvement in the transport of ommochrome precursors are evolutionarily ancient and widely conserved traits in insects. Contrary to dipteran insects, white and scarlet were juxtaposed in a head-to-tail orientation in the silkworm genome, suggesting that the origin of white and scarlet was a tandem duplication of their ancestral transporter gene. In Bombyx, White is also essential for the transport of uric acid in larval epidermis. However, our results suggest that a Bombyx White/Scarlet heterodimer is not involved in this process. Our results emphasize the functional conservation and diversification of half-type ABC transporter families in insects, which may contribute to their extremely diverse color patterns.

Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1.

RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis.

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

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.

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.

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.