ETS transcription factors regulate precise matrix metalloproteinase expression and follicle rupture in Drosophila.

Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1 kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.

The bHLH-PAS transcriptional complex Sim:Tgo plays active roles in late oogenesis to promote follicle maturation and ovulation.

Across species, ovulation is a process induced by a myriad of signaling cascades that ultimately leads to the release of encapsulated oocytes from follicles. Follicles first need to mature and gain ovulatory competency before ovulation; however, the signaling pathways regulating follicle maturation are incompletely understood in Drosophila and other species. Our previous work has shown that the bHLH-PAS transcription factor Single-minded (Sim) plays important roles in follicle maturation downstream of the nuclear receptor Ftz-f1 in Drosophila. Here, we demonstrate that Tango (Tgo), another bHLH-PAS protein, acts as a co-factor of Sim to promote follicle cell differentiation from stages 10 to 12. In addition, we discover that re-upregulation of Sim in stage-14 follicle cells is also essential to promote ovulatory competency by upregulating octopamine receptor in mushroom body (OAMB), matrix metalloproteinase 2 (Mmp2) and NADPH oxidase (NOX), either independently of or in conjunction with the zinc-finger protein Hindsight (Hnt). All these factors are crucial for successful ovulation. Together, our work indicates that the transcriptional complex Sim:Tgo plays multiple roles in late-stage follicle cells to promote follicle maturation and ovulation.

The small RNA landscape is stable with age and resistant to loss of dFOXO signaling in Drosophila.

Aging can be defined as the progressive loss of physiological homeostasis that leads to a decline in cellular and organismal function. In recent years, it has become clear that small RNA pathways play a role in aging and aging related phenotypes. Small RNA pathways regulate many important processes including development, cellular physiology, and innate immunity. The pathways illicit a form of posttranscriptional gene regulation that relies on small RNAs bound by the protein components of the RNA-induced silencing complexes (RISCs), which inhibit the expression of complementary RNAs. In Drosophila melanogaster, Argonaute 1 (Ago1) is the core RISC component in microRNA (miRNA) silencing, while Argonaute 2 (Ago2) is the core RISC component in small interfering RNA (siRNA) silencing. The expression of Ago1 and Ago2 is regulated by stress response transcription factor Forkhead box O (dFOXO) increasing siRNA silencing efficiency. dFOXO plays a role in multiple stress responses and regulates pathways important for longevity. Here we use a next-generation sequencing approach to determine the effects of aging on small RNA abundance and RISC loading in male and female Drosophila. In addition, we examine the impact of the loss of dFOXO on these processes. We find that the relative abundance of the majority of small RNAs does not change with age. Additionally, under normal growth conditions, the loss of dFOXO has little effect on the small RNA landscape. However, we observed that age affects loading into RISC for a small number of miRNAs.

Draft genome of the cotton aphid Aphis gossypii.

The cotton aphid Aphis gossypii Glover is a worldwide agricultural pest that feeds on cotton, melon, and other landscape plants, causing a high level of economic loss. In addition to the common characteristics shared with other aphids, Ap. gossypii has evolved multiple biotypes that present substantial differences in host adaption. These intriguing biological features are of interest from both a fundamental and applied perspective. However, the molecular studies of Ap. gossypii have been restrained by the lack of a reference genome. Furthermore, in order to establish a platform for the development of novel and sustainable control methods, it is necessary to generate genomic resources for Ap. gossypii. Here, we present a 294 Mb draft genome sequence of Ap. gossypii, which consists of 4,724 scaffolds with an N50 size of 438 kb. Compared to other aphid species with published genomes, Ap. gossypii presents the most compact genome size. A total of 14,694 protein-coding genes were predicted and annotated in the consensus gene set, 98.03% of CEGMA genes and 93.5% of BUSCO genes were captured respectively. Genome-wide selection analyses revealed that significantly evolving pathways in the genus Aphis are related to biological processes of detoxification, steroid biosynthesis, and ethylbenzene degradation. The acquisition of the genome of Ap. gossypii makes it possible to understand the molecular mechanism of intricate biological traits of this species, and will further facilitate the study of aphid evolution.

MicroRNA-14 regulates larval development time in Bombyx mori.

MicroRNAs (miRNA) regulate multiple physiological processes including development and metamorphosis in insects. In the current study, we demonstrate that a conserved invertebrate miRNA-14 (miR-14) plays an important role in ecdysteroid regulated development in the silkworm Bombyx mori, a lepidopteran model insect. Ubiquitous transgenic overexpression of miR-14 using the GAL4/UAS system resulted in delayed silkworm larval development and smaller body size of larva and pupa with decrease in ecdysteriod titers. On the contrary, miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage with increase in ecdysteriod titers. We identified that the hormone receptor E75 (E75) and the ecdysone receptor isoform B (ECR-B), which both serve as essential mediators in the ecdysone signaling pathway, as putative target genes of miR-14 by in silico target prediction. Dual-luciferase reporter assays confirmed the binding of miR-14 to the 3'UTRs of E75 and ECR-B in a mammalian HEK293T cell line. Furthermore, transcription levels of E75 and ECR-B were significantly affected in both miR-14 overexpression and knockout transgenic animals. Taken together, our data suggested that the canonical invertebrate miR-14 is a general regulator in maintaining ecdysone homeostasis for normal development and metamorphosis in B. mori.

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest.

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms.

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control.

The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, Bombyx mori.

Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. However, how FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. Here, using the transgene-based CRISPR/Cas9 system, we generated and characterized mutants of the silkworm Bombyx mori FOXO (BmFOXO) to elucidate its physiological functions during development of this lepidopteran insect. The BmFOXO mutant (FOXO-M) exhibited growth delays from the first larval stage and showed precocious metamorphosis, pupating at the end of the fourth instar (trimolter) rather than at the end of the fifth instar as in the wild-type (WT) animals. However, different from previous reports on precocious metamorphosis caused by juvenile hormone (JH) deficiency in silkworm mutants, the total developmental time of the larval period in the FOXO-M was comparable with that of the WT. Exogenous application of 20-hydroxyecdysone (20E) or of the JH analog rescued the trimolter phenotype. RNA-seq and gene expression analyses indicated that genes involved in JH degradation but not in JH biosynthesis were up-regulated in the FOXO-M compared with the WT animals. Moreover, we identified several FOXO-binding sites in the promoter of genes coding for JH-degradation enzymes. These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in B. mori In conclusion, we have uncovered a pivotal role for FOXO in regulating JH signaling to control insect development.

Bombyx mori P-element Somatic Inhibitor (BmPSI) Is a Key Auxiliary Factor for Silkworm Male Sex Determination.

Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination.

Sexually dimorphic traits in the silkworm, Bombyx mori, are regulated by doublesex.

The DM domain genes, doublesex (dsx) in insects, or their structural homologs, male abnormal 3 (mab-3) in nematodes and Dmrt1 (doublesex and mab-3-related transcription factor 1) in mammals, are downstream regulators of the sex determination pathway that control sexually dimorphic development. Despite the functional importance of dsx and its potential applications in sterile insect technologies (SITs), the mechanisms by which it controls sexually dimorphic traits and the subsequent developmental gene networks in insects are poorly understood. Phylogenetic analyses indicate that insect dsx genes have sex-specific alternative splicing isoforms, whereas other taxa do not. We exploited genome editing and transgenesis technologies to induce mutations in either the male-specific isoform (dsxM) or common region (dsxC) of dsx in the somatic tissues of the lepidopteran model insect Bombyx mori. Disruptions of gene function produced either male-specific sexually-dimorphic defects or intersexual phenotypes; these results differ from those observed in other insects, including Drosophila melanogaster. Our data provide insights into the divergence of the insect sex determination pathways related to the most conserved downstream component dsx.

CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in the global pest, diamondback moth (Plutella xylostella).

The diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to multiple classes of insecticides. Genetics-based approaches show promise as alternative pest management approaches but require functional studies to identify suitable gene targets. Here we use the CRISPR/Cas9 system to target a gene, abdominal-A, which has an important role in determining the identity and functionality of abdominal segments. We report that P. xylostella abdominal-A (Pxabd-A) has two structurally-similar splice isoforms (A and B) that differ only in the length of exon II, with 15 additional nucleotides in isoform A. Pxabd-A transcripts were detected in all developmental stages, and particularly in pupae and adults. CRISPR/Cas9-based mutagenesis of Pxabd-A exon I produced 91% chimeric mutants following injection of 448 eggs. Phenotypes with abnormal prolegs and malformed segments were visible in hatched larvae and unhatched embryos, and various defects were inherited by the next generation (G1). Genotyping of mutants demonstrated several mutations at the Pxabd-A genomic locus. The results indicate that a series of insertions and deletions were induced in the Pxabd-A locus, not only in G0 survivors but also in G1 individuals, and this provides a foundation for genome editing. Our study demonstrates the utility of the CRISPR/Cas9 system for targeting genes in an agricultural pest and therefore provides a foundation the development of novel pest management tools.

Expansion of CRISPR targeting sites in Bombyx mori.

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori.

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval-pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.

MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori.

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.

Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.

Site-specific, TALENs-mediated transformation of Bombyx mori.

Transposon-based genetic transformation has facilitated insect functional genomics and new strategies of pest management. However, there is a need for alternative, site-specific approaches to overcome limitations of random integration (and associated position-effects) and potential instability of inserted transgenes. Here we describe a transposon-free, site-specific genetic transformation system mediated by transcription activator-like effector nucleases (TALENs) in the silkworm, Bombyx mori, a lepidopteran model insect. We successfully established a site-specific transgenic system with comparable transformation efficiency to transposon-based genetic transformation through microinjection of TALENs mRNA targeting the BmBLOS2 locus and a linearizable donor plasmid encoding an expression cassette of the DsRed2 red fluorescent protein. This system provides a valuable approach for insect transgenesis and will enable future functional gene analysis and generate novel applications in agricultural and medical insect pest-management technologies.

CYP18A1 regulates tissue-specific steroid hormone inactivation in Bombyx mori.

Insect development and metamorphosis are regulated by two major hormones, juvenile hormone and ecdysteroids. Despite being the key regulator of insect developmental transitions, the metabolic pathway of the primary steroid hormone, 20-hydroxyecdysone (20E), especially its inactivation pathway, is still not completely elucidated. A cytochrome P450 enzyme, CYP18A1, has been shown to play key roles in insect steroid hormone inactivation through 26-hydroxylation. Here, we identified two CYP18 (BmCYP18A1 and BmCYP18B1) orthologs in the lepidopteran model insect, Bombyx mori. Interestingly, BmCYP18A1 gene is predominantly expressed in the middle silk gland (MSG) while BmCYP18B1 expresses ubiquitously in B. mori. BmCYP18A1 is induced by 20E in vitro, suggesting its role in 20E metabolism. Using the binary Gal4/UAS transgenic system, we ectopically overexpressed BmCYP18A1 in a MSG-specific manner with a Sericin1-Gal4 (Ser-Gal4) driver or in a ubiquitous manner with an Actin3-Gal4 (A3-Gal4) driver. Ectopic overexpression of BmCYP18A1 in MSG or in all tissues resulted in developmental arrestment of transgenic animals during the final instar larval stage. The 20E titers in the transgenic animals expressing BmCYP18A1 were lower compared to the levels in the control animals. Although the biological significance of MSG-specific expression of BmCYP18A1 is unclear, our results provide the first evidence that BmCYP18A1, which is conserved in most arthropods, is involved in a tissue-specific steroid hormone inactivation in B. mori.

Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.

MicroRNA Let-7 regulates molting and metamorphosis in the silkworm, Bombyx mori.

MicroRNAs (miRNAs) are a class of endogenous, non-coding, regulatory RNA molecules that post-transcriptionally regulate gene expression by binding to the 3'UTRs of mRNA targets and thus cause their degradation or translational inhibition. In insects, important roles of miRNAs in various biological processes have been demonstrated in Drosophila melanogaster. However, biological roles of miRNAs are barely unveiled in the majority of insect species due to limited genetic tools. In the present study, we introduce the transgenic miRNA sponge (miR-SP) technology combining with the binary GAL4/UAS system in the domesticated silkworm, Bombyx mori, to exploit the biological function of an evolutionally conserved miRNA, let-7. We successfully established transgenic silkworm lines in which a miRNA sponge construct targeting BmLet-7 seed region was expressed in a ubiquitous manner directed by A3-GAL4 driver. Transgenic animals showed decreased expression of BmLet-7, leading to developmental arrestment during the larval-larval and larval-pupal transition. Simultaneously, expression levels of the predicted BmLet-7 target genes, FTZ-F1 and Eip74EF (E74), key regulatory factors in the ecdysone pathway, were elevated in transgenic animals. The current study is the first report on application of the transgenic miR-SP technology in non-drosophilid insects, which will not only contribute to better understanding of let-7 biological roles, but also greatly facilitate future miRNA functional analysis in insects.