Search

Complement receptor 3 (CD11b/CD18) is an important receptor that mediates adhesion, phagocytosis and chemotaxis in various immunocytes. The conidia of the medically-important pathogenic fungus, Aspergillus fumigatus can be internalized into alveolar epithelial cells to disseminate its infection in immunocompromised host; however, the role of CR3 in this process is poorly understood. In the present study, we investigated the potential role of CR3 on A. fumigatus internalization into type II alveolar epithelial cells and its effect on host intracellular PA content induced by A. fumigatus. We found that CR3 is expressed in alveolar epithelial cells and that human serum and bronchoalveolar lavage fluid (BALF) could improve A. fumigatus conidial internalization into A549 type II alveolar epithelial cell line and mouse primary alveolar epithelial cells, which were significantly inhibited by the complement C3 quencher and CD11b-blocking antibody. Serum-opsonization of swollen conidia, but not resting conidia led to the increase of cellular phosphatidic acid (PA) in A549 cells during infection. Moreover, both conidial internalization and induced PA production were interfered by CD11b-blocking antibody and dependent on FAK activity, but not Syk in alveolar epithelial cells. Overall, our results revealed that CR3 is a critical modulator of Aspergillus fumigatus internalization into alveolar epithelial cells.

RNA interference (RNAi), one of the strategies that organisms use to defend against invading viruses, is an important tool for functional genomic analysis. In insects, the efficacy of RNAi varies amongst taxa. Lepidopteran insects are, in large part, recalcitrant to RNAi. The overall goal of this study is to overcome such insensitivity in lepidopterans to RNAi. We hypothesize that over-expression of core RNAi machinery enzymes can improve RNAi efficacy in traditionally recalcitrant species. A transgenic Bombyx mori strain, Baculovirus Immediate-Early Gene, ie1, promoter driven expression of silkworm Dicer2 coding sequence (IE1-BmDicer2), which over-expresses BmDicer2, was generated by piggyBac transposon-mediated transgenesis. Two indexes, the ratio of animals that showed a silencing phenotype and the duration of silencing, were used to evaluate silencing efficiency. Significant knockdown of target gene expression was observed at 48 h postinjection at both the transcriptional and translational levels. Furthermore, we coexpressed B. mori Argonaute 2 BmAgo2）and BmDicer 2 and found that 22% of the animals (n = 18) showed an obvious silencing effect even at 72 h, suggesting that coexpression of these two RNAi core machinery enzymes further increased the susceptibility of B. mori to injected double-stranded RNAs. This study offers a new strategy for functional genomics research in RNAi-refractory insect taxa in general and for lepidopterans in particular.

The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.

Background The glyoxalase system usually comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII). This system converts cytotoxic methylglyoxal (MG) into non-toxic D-lactate in the presence of reduced glutathione (GSH) in two enzymatic steps. Recently, a novel type of glyoxalase III (GLYIII) activity has observed in Escherichia coli that can detoxify MG into D-lactate directly, in one step, without a cofactor. Investigation of the glyoxalase enzymes of a number of plant species shows the importance of their roles in response both to abiotic and to biotic stresses. Until now, glyoxalase gene families have been identified in the genomes of four plants, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula but no similar study has been done with the grapevine Vitis vinifera L. Results In this study, four GLYI-like, two GLYII-like and three GLYIII-like genes are identified from the genome database of grape. All these genes were analysed in detail, including their chromosomal locations, phylogenetic relationships, exon-intron distributions, protein domain organisations and the presence of conserved binding sites. Using quantitative real-time PCR analysis (qRT-PCR), the expression profiles of these genes were analysed in different tissues of grape, and also when under infection stress from downy mildew (Plasmopara viticola). The study reveals that most VvGLY-like genes had higher expressions in stem, leaf, tendril and ovule but lower expressions in the flower. In addition, most of the VvGLY-like gene members were P. viticola responsive with high expressions 6-12 h and 96-120 h after inoculation. However, VvGLYI-like1 was highly expressed 48 h after inoculation, similar to VvPR1 and VvNPR1 which are involved in the defence response. Conclusions This study identified the GLYI-like, GLYII-like and GLYIII-like full gene families of the grapevine. Based on a phylogenetic analysis and the presence of conserved binding sites, we speculate that these glyoxalase-like genes in grape encode active glyoxalases. Moreover, our study provides a basis for discussing the roles of VvGLYI-like, VvGLYII-like and VvGLYIII-like genes in grape’s response to downy mildew infection. Our results shed light on the selection of candidate genes for downy mildew tolerance in grape and lay the foundation for further functional investigations of these glyoxalase genes. Electronic supplementary material The online version of this article (10.1186/s12864-019-5733-y) contains supplementary material, which is available to authorized users.

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval-pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.

Production of seedless V. vinifera L. cultivars is one of the major goals of table grape breeding. Traditional methods result in only a low percentage of seedless progeny. Modern techniques, such as embryo rescue, are used to improve production efficiency of seedless cultivars. In this study, the effects of various putrescine concentrations (0, 1, 2, 3, 4, and 5 mmol L−1) and different stages of embryo development (globular-, heart-, torpedo-, and cotyledon-shaped embryo) on the success rate of embryo rescue were assessed in three lines of the F1 Vitis vinifera L., cross ‘Delight’ × ‘Ruby Seedless,’ ‘DR2,’ ‘DR3,’ and ‘DR6’ (with small seed traces). The plant development rates of ‘DR2,’ ‘DR3,’ and ‘DR6’, which contain small seed traces, as female parents, were significantly higher than those of seedless varieties. The rates of embryo development on woody plant medium (WPM) of ‘DR2’ × ‘Thompson Seedless,’ ‘DR3’ × ‘Thompson Seedless,’ and ‘DR6’ × ‘Monukka’ were highest with 2, 2, and 4 mmol L−1 putrescine, respectively. The germination rates of torpedo- and cotyledon-shaped embryos were significantly higher than those of globular- and heart-shaped embryos. Characteristics of the embryo germination progress of ‘DR2’ × ‘Thompson Seedless’ at different developmental stages were recorded. The Grape Seedless gene Probe 1 (GSLP1) marker was used to help identify the seedless descendants; 14 plantlets showed a corresponding electrophoresis band at 569 bp and were preliminarily defined as seedless.

The embryo rescue technique has been proved to be highly effective and become a standard tool in seedless grape breeding. The main factors limiting the success of in vitro embryo rescue are the ger...

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.

As aviation technology has developed, there has been more emphasis on exploitation and utilization of the space frontier. The lepidopteran insect Bombyx mori has advantages, including small body size, light weight, short life cycle, and well-characterized genetics, when used as a model for biological investigations in space compared with other animals. In preparation for experiments in space, we carried out a simulation experiment, and the number of embryos and the culture temperature and humidity were optimized. The silkworm incubator was launched with China’s SJ-10 recoverable microgravity experimental satellite and was in orbit for 12 days and 15 h in 2016. The embryos were cultured in space. Images of the silkworm embryos were obtained during flight. The embryos cultured in space hatched properly after returning to the ground, but silkworm larva obtained from cultures grown on the SJ-10 satellite grew more rapidly than the ground control group. Analyses of subsequent generations and genome, transcriptome, and proteome analyses are ongoing.

Table S2. Conserved binding sites analysis of genes previously reported as GLYI proteins of Arabidopsis, rice, soybean and Medicago truncatula that are not likely to be GLYs. (DOC 94 kb)

Table S3. Conserved binding sites analysis of all putative GLYII proteins from Vitis vinifera, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula. (DOCX 14 kb)

Table S8. Primers used in expression analysis of glyoxalase-like gene families in grape. (DOCX 18 kb)

Table S5. The number of previously reported glyoxalase genes and glyoxalase genes with the critical conserved binding sites in grape, rice, Arabidopsis, soybean and Medicago truncatula. (DOCX 17 kb)

Figure S3. Sequence alignment of the N-terminal and C-terminal DJ-1/PfpI domains of GLYIII-like proteins. Both the N-terminal and C-terminal DJ-1/PfpI domains of VvGLYIII-like proteins were aligned with other well characterized DJ-1/PfpI superfamily proteins from human, mouse, Drosophila, Caenorhabditis elegans, Escherichia coli and orthologs from Arabidopsis, rice, soybean and Medicago truncatula. Three residues relate to the activity are marked with black boxes. Proteins without the cysteine in the N-terminal or C-terminal are marked with red boxes respectively. (DOCX 1457 kb)

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination., Author summary Sex determination is an essential and universal process for metazoan reproduction and development. Insect sex determination is highly diverse, especially for the primary signal and transductory genes. Mechanism of sex determination in the model lepidopteran insect, Bombyx mori, is largely unknown, although a piRNA, named Fem, has been identified recently as the initial factor. In the current report, we generate somatic mutants for the two silkworm piRNA-bound proteins, BmSiwi and BmAgo3, and identify that the histone methyltransferase BmAsh2 is involved in silkworm sex determination. Loss of BmAsh2 function produces a phenocopy of BmSiwi mutation and induces partial female-to-male sexual reversal. Importantly, we find the co-localization and protein interaction between BmAsh2 and BmSiwi, further supporting critical roles of BmAsh2 in the piRNA-mediated sex determination in B. mori.

A program for producing new seedless table and raisin grape cultivars was initiated using embryo culture methods. F1 descendants of the cross Delight × Ruby Seedless (DRs) were used as the female parent and crossed with various seedless cultivars. The berries of DRs have excellent table traits but they retain small seed traces at maturity. Blush Seedless and DRs were used as female parents, and 354 ovules from five crosses were tested. The percentage of embryos that developed using DRs as the female parent was higher (26.7–35.8%) than when a seedless cultivar was the female parent (5.9–6.7%). The best ovule excision time [days after flowering (DAF)] for ovule inoculation was tested in another set of crosses and found to be DAF 48, 50, and 50 for DR1 × Monukka, DR6 × Thompson Seedless, and DR7 × Zhengguo Seedless, respectively. A total of 569 hybrid progeny were obtained from 13 hybrid combinations; 311 of these survived and were established in soil after hardening. We used three molecular markers (SCC8, SCF27, and GSLP1) to analyze 15 parents used in the hybrid combinations. SCC8 had a 1018-bp band in the seedless parents and in some of the DR parents, while SCF27 had a 2000-bp band in all seedless and DR parents. GSLP1 had a 569-bp band in all of the seedless parents tested, whereas the seeded and DR parents had no bands of this size. Therefore, GSLP1 was used to screen the progeny for possible seedlessness. Eight strains had the 569-bp band; these were preliminarily identified as being seedless since they are in juvenile phase, but they have not yet been evaluated for fruiting characteristics. If the strains identified by using GSLP1 are confirmed to be seedless, this marker will be a valuable tool in breeding for seedlessness in grape.

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.

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.

Summary As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.

Background The HD-Zip family has a diversity of functions during plant development. In this study, we identify 33 HD-Zip transcription factors in grape and detect their expressions in ovules and somatic embryos, as well as in various vegetative organs. Results A genome-wide survey for HD-Zip transcription factors in Vitis was conducted based on the 12 X grape genome (V. vinifera L.). A total of 33 members were identified and classified into four subfamilies (I-IV) based on phylogeny analysis with Arabidopsis, rice and maize. VvHDZs in the same subfamily have similar protein motifs and intron/exon structures. An evaluation of duplication events suggests several HD-Zip genes arose before the divergence of the grape and Arabidopsis lineages. The 33 members of HD-Zip were differentially expressed in ovules of the stenospermic grape, Thompson Seedless and of the seeded grape, Pinot noir. Most have higher expressions during ovule abortion in Thompson Seedless. In addition, transcripts of the HD-Zip family were also detected in somatic embryogenesis of Thompson Seedless and in different vegetative organs of Thompson Seedless at varying levels. Additionally, VvHDZ28 is located in the nucleus and had transcriptional activity consistent with the typical features of the HD-Zip family. Our results provide a foundation for future grape HD-Zip gene function research. Conclusions The identification and expression profiles of the HD-Zip transcription factors in grape, reveal their diverse roles during ovule abortion and organ development. Our results lay a foundation for functional analysis of grape HDZ genes. Electronic supplementary material The online version of this article (10.1186/s12864-017-4110-y) contains supplementary material, which is available to authorized users.

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.

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.

Engineering sex-specific sterility is critical for developing transgene-based sterile insect technology. Targeted genome engineering achieved by customized zinc-finger nuclease, transcription activator-like effector nuclease (TALEN) or clustered, regularly interspaced, short palindromic repeats/Cas9 systems has been exploited extensively in a variety of model organisms; however, screening mutated individuals without a detectable phenotype is still challenging. In addition, genetically recessive mutations only detectable in homozygotes make the experiments time-consuming. In the present study, we model a novel genetic system in the silkworm, Bombyx mori, that results in female-specific sterility by combining transgenesis with TALEN technologies. This system induces sex-specific sterility at a high efficiency by targeting the female-specific exon of the B. mori doublesex (Bmdsx) gene, which has sex-specific splicing isoforms regulating somatic sexual development. Transgenic animals co-expressing TALEN left and right arms targeting the female-specific Bmdsx exon resulted in somatic mutations and female mutants lost fecundity because of lack of egg storage and abnormal external genitalia. The wild-type sexual dimorphism of abdominal segment was not evident in mutant females. In contrast, there were no deleterious effects in mutant male moths. The current somatic TALEN technologies provide a promising approach for future insect functional genetics, thus providing the basis for the development of attractive genetic alternatives for insect population management.

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5′-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval−pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.

The PAR-domain protein 1 (PDP1) is essential for locomotor activity of insects. However, its functions in insect growth and development have not been studied extensively, which prompted our hypothesis that PDP1 acts in energy metabolism. Here we report identification of TcPDP1 in the red flour beetle, Tribolium castaneum, and its functional analysis by RNAi. Treating larvae with dsTcPDP1 induced pupae developmental arrestment, accompanied by accelerated fat body degradation. dsTcPDP1 treatments in adults resulted in reduced female fecundity. Disruption of TcPDP1 expression affected the transcription of genes involved in insulin signaling transduction and mechanistic target of rapamycin (mTOR) pathway. These results support our hypothesis that TcPDP1 acts in energy metabolism in T. castaneum.

Insect development and metamorphosis are regulated by the coordination of ecdysone and juvenile hormones. Insect microRNAs (miRNAs) also act in insect development and metamorphosis by regulating genes in the ecdysone cascade. Although hundreds of insect miRNAs have been identified, the physiological functions of most remain poorly understood. Here, we report that a conserved insect miRNA, microRNA-281 (miR-281), regulates the ecdysone receptor (EcR), in an isoform-specific manner in the silkworm Bombyx mori. The B. mori EcR (BmEcR) gene encodes three isoforms: BmEcR-A, BmEcR-B1 and BmEcR-B2. The 3'UTR regions of A and B genes, which contain multiple potential microRNA targeting sites, are distinct. Target prediction revealed that miR-281 may specifically target the 3'UTR of BmEcR-B. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-281 suppressed transcription of BmEcR-B but not BmEcR-A. The expression of miR-281 and BmEcR-B are well coordinated in the Malpighian tubules from the fourth larval molt to pupation. In the Malpighian tubules of fifth instar larvae, BmEcR-B protein expression was down-regulated after injection of a miR-281 mimic while up-regulated after injection of a miR-281 inhibitor. miR-281 expression was suppressed by 20-hydroxyecdysone treatments but not affected by juvenile hormone treatments. Based on these findings, we propose that miR-281 participates in B. mori developmental regulation in the Malpighian tubules through suppression of BmEcR-B expression.

Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori . We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.

MucK is a member of the major facilitator super family (MFS) and is within a subgroup involved in the uptake of organic acids. Here, we provide the first evidence that mucK is required for normal body color pattern in insect larvae. In the cts mutant strain of Bombyx mori (Linnaeus), the larval anal plates, prothoracic tergum, and the head are reddish brown, as opposed to the white color found in the wild type. Positional cloning of the cts gene was carried out and it was mapped within 90kb on the 16th chromosome. Two molecular markers, 182A4 and 242GA, were found co-segregated with the cts phenotype. Only one candidate gene in the region, BGIBMGA013242, which codes for a major facilitator super family protein named BmMucK, located between the two loci. Sequence analysis revealed that a ∼3kb deletion in the genome resulting in a 126bp deletion in the open reading frame (ORF) of BmmucK in the cts mutant. Fluorescence in situ hybridization (FISH) and quantitative reverse transcription PCR showed that BmmucK expression coincided with reddish brown pigmented regions in the cts mutant strain. BmmucK expression in the head and anal plates were markedly elevated at the molting stages. We performed RNA interference of the mucK homolog in Tribolium castaneum and observed brown coloration accompanied by lethality in ∼1/3 of the insects. The results suggest that MucK plays an important role in the pigmentation process of insect larvae.

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.

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.

Powdery mildew (PM), which is caused by the pathogen Erysiphe necator (Schw.) Burr., is the single most damaging disease of cultivated grapes (Vitis vinifera) worldwide. However, little is known about the transcriptional response of grapes to infection with PM. RNA-seq analysis was used for deep sequencing of the leaf transcriptome to study PM resistance in Chinese wild grapes (V. pseudoreticulata Baihe 35-1) to better understand the interaction between host and pathogen. Greater than 100 million (M) 90-nt cDNA reads were sequenced from a cDNA library derived from PM-infected leaves. Among the sequences obtained, 6541 genes were differentially expressed (DEG) and were annotated with Gene Ontology terms and by pathway enrichment. The significant categories that were identified included the following: defense, salicylic acid (SA) and jasmonic acid (JA) responses; systemic acquired resistance (SAR); hypersensitive response; plant-pathogen interaction; flavonoid biosynthesis; and plant hormone signal transduction. Various putative secretory proteins were identified, indicating potential defense responses to PM infection. In all, 318 putative R-genes and 183 putative secreted proteins were identified, including the defense-related R-genes BAK1, MRH1 and MLO3 and the defense-related secreted proteins GLP and PR5. The expression patterns of 16 genes were further illuminated by RT-qPCR. The present study identified several candidate genes and pathways that may contribute to PM resistance in grapes and illustrated that RNA-seq is a powerful tool for studying gene expression. The RT-qPCR results reveal that effective resistance responses of grapes to PM include enhancement of JA and SAR responses and accumulation of phytoalexins.

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.