Manipulating the Generation of Photonic Moiré Lattices Using Plasmonic Metasurfaces.

The generation of moiré lattices by superimposing two identical sublattices at a specific twist angle has garnered significant attention owing to its potential applications, ranging from two-dimensional materials to manipulating light propagation. While macroscale moiré lattices have been widely studied, further developments in manipulating moiré lattices at the subwavelength scale would be crucial for miniaturizing and integrating platforms. Here, we propose a plasmonic metasurface design consisting of rotated nanoslits arranged within N + N' round apertures for generating focused moiré lattices. By introducing a spin-dependent geometric phase through the rotated nanoslits, an overall lens and spiral phase can be achieved, allowing each individual set of round apertures to generate a periodic lattice in the focal plane. Superimposing two sets of N and N' apertures at specific twist angles and varying phase differences allows for the superposition of two sublattices with different periods, leading to the formation of diverse moiré patterns. Our simulations and theoretical results demonstrate the feasibility of our proposed metasurface design. Due to their compactness and tunability, the utilization of metasurfaces in creating nanoscale photonic moiré lattices is anticipated to find extensive applications in integrated and on-chip optical systems.

Time-Resolved Four-Channel Jones Matrix Measurement of Birefringent Materials Using an Ultrafast Laser.

A method for ultrafast time-resolved four-channel Jones matrix measurement of birefringent materials using an ultrafast laser is investigated. This facilitated the acquisition of a four-channel angular multiplexing hologram in a single shot. The Jones matrix information of a birefringent sample was retrieved from the spatial spectrum of a hologram. The feasibility of this approach was established by measuring the Jones matrix of starch granules in microfluidic chips and the complex amplitude distribution and phase delay distribution of liquid crystal cell at different voltages. Moreover, when the picosecond laser was switched to a femtosecond laser, ultrafast measurements were possible provided that the time interval between two detection pulses was larger than the pulse width.

Metasurfaces for Amplitude-Tunable Superposition of Plasmonic Orbital Angular Momentum States.

The superposition of orbital angular momentum (OAM) in a surface plasmon polariton (SPP) field has attracted much attention in recent years for its potential applications in classical physics problems and quantum communications. The flexible adjustment of the amplitudes of two OAM states can provide more freedom for the manipulation of superposed states. Here, we propose a type of plasmonic metasurface consisting of segmented spiral-shaped nanoslits that not only can generate the superposition of two OAM states with arbitrary topological charges (TCs), but also can independently modulate their relative amplitudes in a flexible manner. The TCs of two OAM states can be simultaneously modulated by incident light, the rotation rate of the nanoslits, and the geometric parameters of the segmented spiral. The relative amplitudes of the two OAM states are freely controllable by meticulously tuning the width of the nanoslits. Under a circularly polarized beam illumination, two OAM states of opposite TCs can be superposed with various weightings. Furthermore, hybrid superposition with different TCs is also demonstrated. The presented design scheme offers an opportunity to develop practical plasmonic devices and on-chip applications.

Transcriptional Dynamics Induced by Diapause Hormone in the Silkworm, Bombyx mori.

Diapause is a form of dormancy that organisms use to adapt to extreme environments by exhibiting developmental arrest. In the silkworm, Bombyx mori, diapause is thought to be elicited by diapause hormone (DH) signaling, which consists of interactions between DH and the DH receptor (DHR). However, the steps downstream of the DH signaling pathway are largely unknown. In the present study, we directly injected synthesized DH into the female pupae of a multivoltine, non-diapausing strain at 36 h after pupation. We found that the mRNA level of DHR declined at 4 h and recovered at 12 h after the injection of DH. Thus, we sequenced the transcriptome of the ovaries at 4 h and 12 h after the injection of DH. We identified 60 and 221 differentially expressed genes at 4 h and 12 h after the injection, respectively. All DEGs were identified, relating to 20E-related genes, JH-related genes, cellular detoxification, ribosomal proteins, lipid metabolism, and epigenetic modifications. Eleven genes were selected from the above categories to verify the transcriptome data. The qRT-PCR and RNA-Seq expression patterns of the genes were consistent, which indicated the authenticity and reliability of the transcriptome data. This study dramatically expands upon our knowledge of gene expression variation at the early phase of DH release.

Arbitrary superposition of plasmonic orbital angular momentum states with nanostructures.

A kind of plasmonic nanostructure is proposed that can generate the arbitrary superposition of orbital angular momentum (OAM) states in surface plasmons (SPs), which is achieved by combining the segmented spirals with nanoslit pairs. The structures can independently modulate both the phase and amplitude of SP waves, and thus enable the superposition of two OAM states with arbitrary topological charges (TCs) as well as free control of their relative amplitudes. Superposed states distributed over the entire Bloch sphere and hybrid superposed states with different TCs were constructed and experimentally demonstrated. This work will offer more opportunities for multifunctional plasmonic devices.

Presenilin is involved in larval-pupal metamorphosis development of Bombyx mori.

Disruption of the presenilin (ps) genes are the major genetic cause of familial Alzheimer's disease. The silkworm, Bombyx mori (B. mori), is an important model insect. The ps homologue gene in B. mori was identified and characterized. However, the role of ps in B. mori was poorly understood. Here, we found that Bmps was ubiquitously expressed in all the tested tissues during metamorphosis. In the current study, loss-of-function analysis of Bmps was performed by the binary transgenic CRISPR/cas9 system. Compared with the wild type, the developmental time of ∆Bmps animals were significantly delayed. In addition, ∆Bmps showed abnormal appendage including antenna, leg, wing and eye during pupal and adult stages. RNA-seq analysis indicated that apoptosis and proliferation related pathways were affected in ∆Bmps. Moreover, the Hippo pathway was affected by Bmps depletion in brain and wing disc. Our results suggest that PS is essential for maintaining the dynamic balance of apoptosis and proliferation during metamorphosis.

T follicular regulatory cells suppress Tfh-mediated B cell help and synergistically increase IL-10-producing B cells in breast carcinoma.

T follicular regulatory (Tfr) cell is a recently discovered subset of T regulatory (Treg) cells. The main function of Tfr cells is thought to suppress germinal cancer reaction and inhibit B cell proliferation and Ig production. However, recent studies demonstrate that Tfr cells may be required for high-affinity Ig formation during acute virus infections. The role of Tfr cells in breast cancer is not thoroughly investigated. In this study, total circulating CD4 T cells were sorted into CD25+CXCR5- Treg-like, CD25+CXCR5+ Tfr-like, and CD25-CXCR5+ Tfh-like subsets. Data showed that the Tfr-like subset presented intermediate levels of both Foxp3 and Bcl-6, while the Treg-like subset was high in Foxp3 and low in Bcl-6, and the Tfh-like was high in Bcl-6 and low in Foxp3. Of note, the frequencies of Tfr-like and Treg-like cells were significantly elevated in breast cancer (BC) patients than in non-cancer (NC) controls. Tfr-like cells in BC patients also expressed significantly higher levels of Foxp3 than those in NC controls. Neither Treg-like nor Tfr-like cells could support Ig production from naive B cells, while Tfh-like cells potently supported Ig production from naive B cells. Tfr-like cells increased the availability of IL-10, both by directly producing IL-10 and by increasing IL-10 production from B cells. Interestingly, Tfr-like cells increased IL-10 production from B cells synergistically with Tfh cells, but at the same time, significantly reduced Ig production in the Tfh-B cell coculture. These Tfr-mediated effects on Tfh cells were not found in canonical Treg cells. Overall, this study demonstrates several distinctive features in circulating Tfr cells and suggests that Tfr cells may promote the formation of IL-10-producing B cells in BC.

Identification of a germline-expression promoter for genome editing in Bombyx mori.

Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmß4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.

Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori.

Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element-binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl3 ) in both dose- and time-dependent manners. When AlCl3 solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl3 dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl3 , SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p-CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p-CREB. Our data suggest that B. mori can serve as a model animal for studying Al-induced neurotoxicity or neurodegeneration.

Differentiation of lepidoptera scale cells from epidermal stem cells followed by ecdysone-regulated DNA duplication and scale secreting.

Integuments are the first line to protect insects from physical damage and pathogenic infection. In lepidopteran insects, they undergo distinct morphology changes such as scale formation during metamorphosis. However, we know little about integument development and scale formation during this stage. Here, we use the silkworm, Bombyx mori, as a model and show that stem cells in the integument of each segment, but not intersegmental membrane, divide into two scale precursor cells during the spinning stage. In young pupae, the scale precursor cell divides again. One of the daughter cells becomes a mature scale-secreting cell that undergoes several rounds of DNA duplication and the other daughter cell undergoes apoptosis later on. This scale precursor cell division is crucial to the development and differentiation of scale-secreting cells because scale production can be blocked after treatment with the cell division inhibitor paclitaxel. Subsequently, the growth of scale-secreting cells is under the control of 20-hydroxyecdysone but not juvenile hormone since injection of 20-hydroxyecdysone inhibited scale formation. Further work demonstrated that 20-hydroxyecdysone injection inhibits DNA duplication in scale-secreting cells while the expression of scale-forming gene ASH1 was down-regulated by BR-C Z2. Therefore, this research demonstrates that the scale cells of the silkworm develops through stem cell division prior to pupation and then another wave of cell division differentiates these cells into scale secreting cells soon after entrance into the pupal stage. Additionally, DNA duplication and scale production in the scale-secreting cells were found to be under the regulation of 20-hydroxyecdysone.

YorkieCA overexpression in the posterior silk gland improves silk yield in Bombyx mori.

The traditional hybrid breeding techniques can no longer meet the increasing demands for silk production by the silkworm, Bombyx mori, and further improvement of the silk yield will depend on modern molecular breeding techniques. Here, we report improved silk yield in transgenic silkworms overexpressing the oncogene YorkieCA specifically in the posterior silk gland (PSG). The YorkieCA cDNA was ligated downstream of the hr3 enhancer and the fibroin L-chain (Fil) promoter, then inserted into a piggyBac vector for transgene. Overexpression of YorkieCA in the PSG significantly increased the weight of the PSG, and also increased the weight of the cocoon, larval body, and pupal body to decreasing degrees. Overexpression of YorkieCA up-regulated the Yorkie target genes resulting in increased cell size, endomitosis, the number of protein synthesis organelles, the expression of fibroin genes in the PSG, and eventually silk yield. Additionally, as we reported previously using the binary GAL4/UAS system, transgenic silkworms overexpressing Ras1CA with the hr3 enhancer and the Fil promoter also showed improved silk yield. Unfortunately, the hybrid progeny of YorkieCA-overexpressing silkworms and Ras1CA-overexpressing silkworms did not show overlapping improved silk yield due to the failure to increase expression of both Yorkie and Ras1.

Analysis of gene expression in the midgut of Bombyx mori during the larval molting stage.

BACKGROUND: Insects can be models for understanding human intestinal infection and pathology. Molting, a special period during which the old insect cuticle is shed and a new one is produced, is crucial for insect development. Holometabolous insects may experience several larva-to-larva moltings to become larger, a pupal molt and adult eclosion to become adults. During the larval molts, they stop feeding and become quiescent. Although the molting larvae become quiescent, it is not known if changes in microbiome, physiology, development and immunity of midguts occur. RESULTS: Transcriptome analysis indicated that functions such as metabolism, digestion, and transport may become reduced due to the downregulated expression of many associated genes. During the molting stage, midguts harbor less microflora and DNA synthesis decreases. Both ecdysone and juvenile hormone in the larval midgut likely degrade after entering the larva-to-larva molting stage. However, at 12 h after ecdysis, the feeding larvae of 5th instars that were injected with 20-hydroxyecdysone entered a molting-like stage, during which changes in midgut morphology, DNA synthesis, gene expression, and microflora exhibited the same patterns as observed in the actual molting state. CONCLUSION: This study is important for understanding insect midgut physiology, development and immunity during a special development stage when no food is ingested. Although the molting larva becomes immobile and quiescent, we demonstrate that numerous changes occur in midgut morphology, physiology, metabolism and microbiome during this period.

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx.

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated Yorkie(CA) overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm.

Gut immunity in Lepidopteran insects.

Lepidopteran insects constitute one of the largest fractions of animals on earth, but are considered pests in their relationship with man. Key to the success of this order of insects is its ability to digest food and absorb nutrition, which takes place in the midgut. Because environmental microorganisms can easily enter Lepidopteran guts during feeding, the innate immune response guards against pathogenic bacteria, virus and microsporidia that can be devoured with food. Gut immune responses are complicated by both resident gut microbiota and the surrounding peritrophic membrane and are distinct from immune responses in the body cavity, which depend on the function of the fat body and hemocytes. Due to their relevance to agricultural production, studies of Lepidopteran insect midgut and immunity are receiving more attention, and here we summarize gut structures and functions, and discuss how these confer immunity against different microorganisms. It is expected that increased knowledge of Lepidopteran gut immunity may be utilized for pest biological control in the future.

DNA duplication is essential for the repair of gastrointestinal perforation in the insect midgut.

Invertebrate animals have the capacity of repairing wounds in the skin and gut via different mechanisms. Gastrointestinal perforation, a hole in the human gastrointestinal system, is a serious condition, and surgery is necessary to repair the perforation to prevent an abdominal abscess or sepsis. Here we report the repair of gastrointestinal perforation made by a needle-puncture wound in the silkworm larval midgut. Following insect gut perforation, only a weak immune response was observed because the growth of Escherichia coli alone was partially inhibited by plasma collected at 6 h after needle puncture of the larval midgut. However, circulating hemocytes did aggregate over the needle-puncture wound to form a scab. While, cell division and apoptosis were not observed at the wound site, the needle puncture significantly enhanced DNA duplication in cells surrounding the wound, which was essential to repair the midgut perforation. Due to the repair capacity and limited immune response caused by needle puncture to the midgut, this approach was successfully used for the injection of small compounds (ethanol in this study) into the insect midgut. Consequently, this needle-puncture wounding of the insect gut can be developed for screening compounds for use as gut chemotherapeutics in the future.

Molecular cloning and characterization of presenilin gene in Bombyx mori.

Presenilin (PS), the catalytic core of the γ-secretase complex, is considered to be a causative protein of the early­onset familial form of Alzheimer's disease. Aging is a risk factor for Alzheimer's disease and a number of genetic studies have utilized Bombyx mori (B. mori) as a model, making it possible to use B. mori to investigate Alzheimer's disease. However, the homologous gene of human PS in B. mori has remained to be elucidated. In the present study, the PS homologue gene in B. mori was identified and characterized, and six B. mori presenilin (BmPS) mRNA transcripts were generated by selecting multiple transcription start sites and/or alternative splice sites. The longest mRNA of BmPS (termed BmPS1) contains a 153 nt 5' untranslated region (UTR), a 1,440 nt open reading frame and a 1,063 nt 3' UTR. The predicted protein of BmPS1 consists of 479 amino acid residues and has two highly­conserved aspartate residues, which form the catalytic core of aspartic proteases. It exhibits a sequence identity of ~44 and 51% with homologues in Homo sapiens and Drosophila melanogaster, respectively. However, the amino acid sequence of the BmPS loop region does not completely match between the two B. mori strains R13Q and Dazao. Genomic analysis revealed that B. mori had a single copy of the BmPS gene, which was composed of 14 exons. A total of four isoforms of BmPS (BmPS­A, ­B, ­C and ­D) owing to multiple transcriptional start sites and alternative splice sites were identified. The alternative splicing events occurring in the loop region improved the diversity of the BmPS protein and were detectable in all tissues, as determined using reverse transcription quantitative polymerase chain reaction (RT­qPCR). Furthermore, the expression levels of BmPS in the brain at different developmental stages were detected using RT­qPCR, and significantly higher expression levels of BmPS were found in the adult stage compared with those in the larval and pupal stages. The present study on BmPS provided insight into the pathogenesis of Alzheimer's disease and mechanisms of silkworm developmental regulation.

A droplet-based microfluidic electrochemical sensor using platinum-black microelectrode and its application in high sensitive glucose sensing.

We describe a droplet-based microfluidic electrochemical sensor using platinum-black (Pt-black) microelectrode. Pt-black microelectrode was generated by electrodeposition of Pt nanoparticles on bare Pt microelectrode. Scanning electron microscope (SEM) image displays a flower-like microstructure of Pt nanoparticels. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) indicate that the Pt-black efficiently decreased the charge transfer resistance and improved the electrocatalytic activity towards oxidation of hydrogen peroxide (H2O2). Compared with bare Pt microelectrode, the current response on Pt-black microelectrode increased 10.2 folds. The effect of applied potential and electrodeposition time has been investigated in detail. The proposed sensor was validated by performing enzyme activity assay in flowing droplets. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of ß-D-glucose to the product H2O2. The enzyme activity of GOx was evaluated by measuring the electrochemical current responding to various glucose concentrations. And the results indicate that this microfluidic sensor holds great potential in fabricating novel glucose sensors with linear response up to 43.5mM. The analytical applications of the droplet-based microfluidic sensor were tested by using human blood serum samples. Reproducibility, interferences, and long-term stability of the modified electrode were also investigated. The present approach shows the feasibility and great potentials in constructing highly sensitive and low-consumption sensors in the field of droplet microfluidics.

Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5' splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5' splice site at intron 3 of Lepidopteran dsx. We propose that the 5' splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5' splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx.

[Advances on Drosophila presenilin gene].

In Drosophila, presenilin is an aspartyl protease that plays important roles in the development and calcium homeostasis. It has been expressed all through the fly developmental process. The loss of Drosophila presenilin (DPS) function causes significantly decreased Notch signaling and neuron apoptosis and increased cytoplasm calcium. This subsequently led to impaired long term memory and cognitive deficits. Therefore, study of DPS is one of the most popular models for Alzheimer's disease research, and has provided important insights into the pathological mechanisms of AD. This review is to summarize the AD related function of DPS gene.

Molecular cloning and characterization of Bombyx mori CREB gene.

The cAMP response element binding protein (CREB), as one of the best characterized stimulus-induced transcription factors, plays critical roles in activating transcription of target genes in response to a variety of environmental stimuli. To characterize this important molecule in the silkworm, Bombyx mori, we cloned a full-length cDNA of CREB gene from B. mori brains by using RACE-PCR. The sequence of B. mori CREB (named BmCREB1) gene contains a 88 bp 5' UTR, a 783 bp open reading frame (ORF) encoding 261 amino acids and a 348 bp 3' UTR. The deduced BmCREB amino acid sequence has 56.7% and 37.2% homology with CREB from Apis mellifera carnica and Drosophila melanogaster, respectively. The primary structure of the deduced BmCREB1 protein contains a kinase-inducible domain (KID) and a basic region/leucine zipper (bZIP) dimerization domain which exists in all CREB family members. Genomic analysis showed there are 9 exons and 5 introns in B. mori CREB genome sequences. We identified three different isoforms of BmCREB (BmCREB1, BmCREB2 and BmCREB3) through alternative splicing in C terminal. In addition, the expression of BmCREB in different developmental stages was investigated by using quantitative real-time PCR in both diapause and non-diapause type of B. mori bivoltine race (Dazao). BmCREB transcripts showed two peaks in embryonic stage and pupal stage in both types of bivoltine race. However, consistently higher expression of BmCREB was found throughout the developmental stages in the diapause type than in the non-diapause type. These results suggest that BmCREB is involved in the process of diapause induced by environmental factors.