Unpredictable recombination of PB transposon in Silkworm: a potential risk.

The piggyBac (PB) transposon is the most widely used vector for generating transgenic silkworms. The stability of the PB transposon in the receptor is a serious concern that requires attention because of biosafety concerns. In this study, we found that the transgene silkworm developed loss of reporter gene traits. To further investigate the regularity, we traced the genes and traits of this silkworm. After successful alteration of the silkworm genome with the MASP1 gene (named red-eyed silkworm; RES), silkworm individuals with lost reporter genes were found after long-term transgenerational breeding and were designated as the white-eyed silkworm (WES). PCR amplification indicated that exogenous genes had been lost in the WES. Testing was conducted on the PB transposons, and the left arm (L arm) did not exist; however, the right arm (R arm) was preserved. Amino acid analysis showed that the amino acid content of the WES changed versus the common silkworm and RES. These results indicate that the migration of PB transposons in Bombyx mori does occur and is unpredictable. This is because the silkworm genome contains multiple PB-like sequences that might influence the genetic stability of transgenic lines. When using PB transposons as a transgene vector, it is necessary to fully evaluate and take necessary measures to prevent its re-migration in the recipient organism. Further experiments are needed if we want to clarify the regularity of the retransposition phenomenon and the direct and clear association with similar sequences of transposons.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.

Diet choice: The two-factor host acceptance system of silkworm larvae.

Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and ß-sitosterol (ßsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.

Imaginal disc growth factor maintains cuticle structure and controls melanization in the spot pattern formation of Bombyx mori.

The complex stripes and patterns of insects play key roles in behavior and ecology. However, the fine-scale regulation mechanisms underlying pigment formation and morphological divergence remain largely unelucidated. Here we demonstrated that imaginal disc growth factor (IDGF) maintains cuticle structure and controls melanization in spot pattern formation of Bombyx mori. Moreover, our knockout experiments showed that IDGF is suggested to impact the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3, to further control the melanin metabolism. Furthermore, the untargeted metabolomics analyses revealed that BmIDGF significantly affected critical metabolites involved in phenylalanine, beta-alanine, purine, and tyrosine metabolism pathways. Our findings highlighted not only the universal function of IDGF to the maintenance of normal cuticle structure but also an underexplored space in the gene function affecting melanin formation. Therefore, this study furthers our understanding of insect pigment metabolism and melanin pattern polymorphisms.

Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition.

The egg stage is one of the most critical periods in the life history of silkworms, during which physiological processes such as sex determination, tissue organ formation and differentiation, diapause and pigmentation occur. In addition, egg color gradually emerges around 36h after oviposition. The red egg mutant rep-1, which was recently discovered in the C1(H) wild-type, C1(H) exhibits a brown egg color. In this study, the transcriptome of the eggs was analyzed 36h after oviposition. Between the rep-1 mutant and the C1(H) wild-type, 800 differentially expressed genes (DEGs) were identified, including 325 up-regulated genes and 475 down-regulated genes. These DEGs were mainly involved in biological processes (metabolic process, cellular process, biological regulation and regulation of biological process and localization), cellular components (membrane, membrane part, cell, cell part and organelle) and molecular functions (binding, catalytic activity, transporter activity, structural molecule activity and molecular transducer activity). The pathway enrichment of these DEGs was performed based on the KEGG database, and the results indicated that these DEGs were mainly involved in pathways in the following categories: metabolic pathways, longevity-regulating pathway-multiple species, protein processing in endoplasmic reticulum, peroxisome, carbon metabolism and purine metabolism. Further analysis showed that a large number of silkworm growth- and development-related genes and ommochrome synthesis- and metabolism-related genes were differentially expressed, most of which were up-regulated in the mutant. Our research findings provide new experimental evidence for research on ommochrome pigmentation and lay the foundation for further research on the mechanism of the rep-1 mutant.

A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori.

Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. In the current study, we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.

A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated.

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret.

Morphology and physiology of olfactory neurons in the lateral protocerebrum of the silkmoth Bombyx mori.

Insect olfaction is a suitable model to investigate sensory processing in the brain. Olfactory information is first processed in the antennal lobe and is then conveyed to two second-order centres-the mushroom body calyx and the lateral protocerebrum. Projection neurons processing sex pheromones and plant odours supply the delta area of the inferior lateral protocerebrum (∆ILPC) and lateral horn (LH), respectively. Here, we investigated the neurons arising from these regions in the brain of the silkmoth, Bombyx mori, using mass staining and intracellular recording with a sharp glass microelectrode. The output neurons from the ∆ILPC projected to the superior medial protocerebrum, whereas those from the LH projected to the superior lateral protocerebrum. The dendritic innervations of output neurons from the ∆ILPC formed a subdivision in the ∆ILPC. We discuss pathways for odour processing in higher order centres.

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland.

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step "IAAld → IAA" from a silk-gland extract of Bombyx mori. The enzyme, designated "BmIAO1", contains two 2Fe-2S iron-sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp. Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Trp: tryptophan.

Structure analysis of the spinneret from Bombyx mori and its influence on silk qualities.

Silk is an excellent natural fiber, which has been widely used in versatile fields. Silk spinning is a complex process involving the larval spinneret. The spinneret is essential for silk spinning, but the sectional morphology of the spinneret that determines the silk monofilament, the muscular activities around the silk press as well as the relationships between the spinneret and the properties of the resulting silk remain poorly understood. We studied these factors by dissecting the spinneret and analyzing silk from different Bombyx mori strains. The sectional morphology of silk monofilament was found to be largely determined by the spinneret, especially by the silk press. Moreover, contractile activity of the muscles around the silk press is high, and the contraction frequency of the muscles was estimated to range from 11.42 to 50 HZ. A comparison of the fibroin filaments before they entered the common tube indicated that the spinneret determines both silk shape and silk size. This study provides insight into the silk spinning process, which may help develop bionic spinning in further studies and also provides a rationale to study the effect of the spinneret on silk fineness at the molecular level.

Proteomic Analysis of Larval Integument in a Dominant Obese Translucent (Obs) Silkworm Mutant.

The dominant obese translucent (Obs) mutant of the silkworm (Bombyx mori) results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays deficiency in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae were indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which were involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAPs), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially expressed proteins. Moreover, 22 of the 26 cuticular proteins were downregulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant might be related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results could lay a basis for further identification of the gene responsible for the Obs mutant. The data have been deposited to ProteomeXchange with identifier PXD010998.

Comparative Analysis of the Integument Transcriptomes between Stick Mutant and Wild-Type Silkworms.

In insects, the integument provides mechanical support for the whole body and protects them from infections, physical and chemical injuries, and dehydration. Diversity in integument properties is often related to body shape, behavior, and survival rate. The stick (sk) silkworm is a spontaneous mutant with a stick-like larval body that is firm to the touch and, thus, less flexible. Analysis of the mechanical properties of the cuticles at day 3 of the fifth instar (L5D3) of sk larvae revealed higher storage modulus and lower loss tangent. Transcriptome sequencing identified a total of 19,969 transcripts that were expressed between wild-type Dazao and the sk mutant at L5D2, of which 11,596 transcripts were novel and detected in the integument. Differential expression analyses identified 710 upregulated genes and 1009 downregulated genes in the sk mutant. Gene Ontology (GO) enrichment analysis indicated that four chitin-binding peritrophin A domain genes and a chitinase gene were upregulated, whereas another four chitin-binding peritrophin A domain genes, a trehalase, and nine antimicrobial peptides were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that two functional pathways, namely, fructose and mannose metabolism and tyrosine metabolism, were significantly enriched with differentially-expressed transcripts. This study provides a foundation for understanding the molecular mechanisms underlying the development of the stiff exoskeleton in the sk mutant.

Morphology of visual projection neurons supplying premotor area in the brain of the silkmoth Bombyx mori.

Sex pheromones orient male moths toward conspecific female moths; the presence of visual information modulates this behavior. In the current study, we explore candidate neuronal pathways for the interaction between vision and the locomotor signal for pheromone orientation. We describe the connectivity between visual neuropils and brain premotor centers, the posterior slope (PS) and the lateral accessory lobe (LAL), in the silkmoth Bombyx mori. Using a single-cell labeling technique, we analyze visual projection neurons supplying these areas. Neurons from both the medulla and lobula complex projected to the PS but only the neurons originating in the lobula complex had additional processes to the LAL. Further, we identified populations of putative feedback neurons from the premotor centers to the optic lobe. Neurons originating in the PS were likely to project to the medulla, whereas those originating in the LAL were likely to project to the lobula complex. The anatomical study contributes to further understanding of integration of visual information on the locomotor control in the insect brain.

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori.

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6-6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.

Molecular and biochemical responses in the midgut of the silkworm, Bombyx mori, infected with Nosema bombycis.

BACKGROUND: Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori. METHODS: A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories 'molecular function', 'cellular component' and 'biological process'. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori. RESULTS: A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori midgut tissues decreased significantly, whereas the fatty acid content did not significantly change after four days of N. bombycis infection. Microsporidia N. bombycis infection upregulated the expression level of genes involved in host ATP synthesis, protein and fat degradation, which eventually causes the obvious decline of protein content and ATP synthesis in the host midgut, whereas the fatty acids content did not change significantly. CONCLUSIONS: This study suggested to some extent that N. bombycis invasion can activate the host protein degradation and accelerate the production of host ATP. Microsporidia of N. bombycis show preference for proteins rather than fatty acids from the host to ensure the material preparation required by their parasitic life-cycle. Requirements of N. bombycis for energy were also mainly dependent on the host ATP production. This study provides a new data that may help our understanding of the molecular mechanisms of obtaining energy and nutrients from the host by the microsporidium N. bombycis.

The pivotal role of aristaless in development and evolution of diverse antennal morphologies in moths and butterflies.

BACKGROUND: Antennae are multi-segmented appendages and main odor-sensing organs in insects. In Lepidoptera (moths and butterflies), antennal morphologies have diversified according to their ecological requirements. While diurnal butterflies have simple, rod-shaped antennae, nocturnal moths have antennae with protrusions or lateral branches on each antennal segment for high-sensitive pheromone detection. A previous study on the Bombyx mori (silk moth) antenna, forming two lateral branches per segment, during metamorphosis has revealed the dramatic change in expression of antennal patterning genes to segmentally reiterated, branch-associated pattern and abundant proliferation of cells contributing almost all the dorsal half of the lateral branch. Thus, localized cell proliferation possibly controlled by the branch-associated expression of antennal patterning genes is implicated in lateral branch formation. Yet, actual gene function in lateral branch formation in Bombyx mori and evolutionary mechanism of various antennal morphologies in Lepidoptera remain elusive. RESULTS: We investigated the function of several genes and signaling specifically in lateral branch formation in Bombyx mori by the electroporation-mediated incorporation of siRNAs or morpholino oligomers. Knock down of aristaless, a homeobox gene expressed specifically in the region of abundant cell proliferation within each antennal segment, during metamorphosis resulted in missing or substantial shortening of lateral branches, indicating its importance for lateral branch formation. aristaless expression during metamorphosis was lost by knock down of Distal-less and WNT signaling but derepressed by knock down of Notch signaling, suggesting the strict determination of the aristaless expression domain within each antennal segment by the combinatorial action of them. In addition, analyses of pupal aristaless expression in antennae with various morphologies of several lepidopteran species revealed that the aristaless expression pattern has a striking correlation with antennal shapes, whereas the segmentally reiterated expression pattern was observed irrespective of antennal morphologies. CONCLUSIONS: Our results presented here indicate the significance of aristaless function in lateral branch formation in B. mori and imply that the diversification in the aristaless expression pattern within each antennal segment during metamorphosis is one of the significant determinants of antennal morphologies. According to these findings, we propose a mechanism underlying development and evolution of lepidopteran antennae with various morphologies.

Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca2+ release effect in Sf9 cells.

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro, in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca2+-Mg2+-ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms.

Body Shape and Coloration of Silkworm Larvae Are Influenced by a Novel Cuticular Protein.

The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in BmorCPH24, a gene encoding a cuticular protein with low complexity sequence. Spatiotemporal expression analyses showed that BmorCPH24 is expressed in the larval epidermis postecdysis. RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of BmorCPH24 produced the abnormal body shape and the inhibited pigment typical of the mutant phenotype. In addition, our results showed that BmorCPH24 may be involved in the synthesis of endocuticle and its disruption-induced apoptosis of epidermal cells that accompanied the reduced expression of R&R-type larval cuticle protein genes and pigmentation gene Wnt1 Strikingly, BmorCPH24, a fast-evolving gene, has evolved a new function responsible for the assembly of silkworm larval cuticle and has evolved to be an indispensable factor maintaining the larval body shape and its coloration pattern. This is the first study to identify a molecule whose pleiotropic function affects the development of body shape and color patterns in insect larvae.

Timing of autophagy and apoptosis during posterior silk gland degeneration in Bombyx mori.

Over the years, the silkworm, Bombyx mori, has been manipulated by means of chemical and genetic approaches to improve silk production both quantitatively and qualitatively. The silk is produced by the silk gland, which degenerates quickly once the larva has finished spinning the cocoon. Thus, interfering with this degeneration process could help develop new technologies aimed at ameliorating silk yield. To this end, in this work we studied the cell death processes that lead to the demise of the posterior silk gland of B. mori, directing in particular our attention to autophagy and apoptosis. We focused on this portion of the gland because it produces fibroin, the main component of the silk thread. By using multiple markers, we provide a morphological, biochemical and molecular characterization of the apoptotic and autophagic processes and define their timing in this biological setting. Our data demonstrate that the activation of both autophagy and apoptosis is preceded by a transcriptional rise in key regulatory genes. Moreover, while autophagy is maintained active for several days and progressively digests silk gland cells, apoptosis is only switched on at a very late stage of silk gland demise.

iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. SIGNIFICANCE: The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes the silkworm to death, which negatively affects the sericulture industry. Studies on insect antiviral immunity and on interactive mechanisms between host cells and BmCPV are in their infancy and remain insufficient. In order to obtain an overall view of silkworm response to BmCPV infection, we performed a proteomic analysis of the midgut of silkworm responses to BmCPV infection by iTRAQ. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection.