The BTB-ZF gene Bm-mamo regulates pigmentation in silkworm caterpillars.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects.

High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation.

The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

Effect of Wear-Induced Surface Deformation on Stick-Slip Friction of Galvanized Automotive Steels.

This study examined the influence of worn surfaces on the stick-slip friction of galvanized automotive steel and revealed the intrinsic role of surface topography parameters in the stick-slip friction with wear. The results show that the surface deformation induced by wear significantly affects the stick-slip friction. The stick-slip friction can be suppressed by increasing the vertical area of the surface deformation because of reductions in the difference between the static and kinetic friction coefficients. The friction behavior changes from stick-slip friction to smooth sliding when the skewness exceeds a critical value, thus suggesting that this parameter can be used as an effective surface topography parameter to describe the stick-slip friction of galvanized automotive steel with worn surfaces. The findings can help improve the understanding of the mechanism of the stick-slip friction of materials with worn surfaces and provide an approach to suppress the noise and vibration caused by the stick-slip friction of galvanized automotive steel.

The evolution and genetics of lepidopteran egg and caterpillar coloration.

Insect colors and color patterns have fascinated biologists for centuries. While extensive research has focused on the adult colors of Drosophila and butterflies, our understanding of how colors are generated and diversified in embryonic and larval stages remains limited, especially, the genetics behind the protective coloration of the immobile embryonic and larval stages. Lepidoptera, one of the most widespread and species-rich insect orders, are extremely helpful uncovering those mechanisms due to their remarkable diverse colors in eggs and caterpillars within or among species, and these colors usually are variable in different developmental stages or in response to different environments. Here we review the recent progress on coloration of lepidopteran eggs and caterpillars, focusing on the genetic basis, developmental mechanisms, ecology, and evolution underlying the remarkable color diversity.

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death.

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant rel shows both red egg color and embryo mortality. We studied the molecular basis of the rel phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant rel . BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm rel mutant formation and indicated that the Sema1a gene is essential for insect embryo development.

In situ synthesis of superorganism-like Au NPs within microgels with ultra-wide absorption in visible and near-infrared regions for combined cancer therapy.

The whole is a collection of parts and fulfills specific functions that the parts do not have. In this work, 50 nm Au NPs were in situ synthesized and close packed into a superorganism-like superstructure by means of microgel 3D networks. The combined microgel is endowed with ultra-wide absorption in visible and near-infrared regions between 500 and 1100 nm in spite of Au NPs not having this property. The strong collective plasmon coupling between neighboring Au NPs induces high photothermal conversion efficiency of the microgel system under irradiation at various laser wavelengths. Due to the good loading capability, microgels with nanocomposites can also load photosensitive drugs simultaneously and be used for combined cancer treatments of photothermal therapy and photodynamic therapy.

Flight Muscle and Wing Mechanical Properties are Involved in Flightlessness of the Domestic Silkmoth, Bombyx mori.

Flight loss has occurred in many winged insect taxa. The flightless silkmoth Bombyx mori, is domesticated from the wild silkmoth, Bombyx mandarina, which can fly. In this paper, we studied morphological characteristics attributed to flightlessness in silkmoths. Three domestic flightless B. mori strains and one B. mandarina population were used to compare morphological components of the flight apparatus, including wing characteristics (shape, forewing area, loading, and stiffness), flight muscle (weight, ratio, and microscopic detail) and body mass. Compared with B. mandarina, B. mori strains have a larger body, greater wing loading, more flexible wings and a lower flight muscle ratio. The arrangement in microscopy of dorsal longitudinal flight muscles (DLFMs) of B. mori was irregular. Comparative analysis of the sexes suggests that degeneration of flight muscles and reduction of wing mechanical properties (stiffness) are associated with silkmoth flightlessness. The findings provide important clues for further research of the molecular mechanisms of B. mori flight loss.

Comparative analysis of integument transcriptomes identifies genes that participate in marking pattern formation in three allelic mutants of silkworm, Bombyx mori.

The diversity markings and pigment patterns in insects are outcomes of adaptive evolution. The elucidation of the molecular mechanism underlying variations in pigment patterns may improve our understanding of the origin and evolution of these spectacular diverse phenotypes. Melanin, ommochrome, and pteridine are the three main types of insect pigments, and the genes that directly participate in pigment biosynthesis have been extensively studied. However, available information on gene interactions and the whole pigment regulatory network is limited. In this study, we performed integument transcriptome sequencing to analyze three larval marking allelic mutants, namely, multi lunar (L), LC, and LCa, which have similar twin-spot markings on the dorsal side of multiple segments. Further analysis identified 336 differentially expressed genes (DEGs) between L and Dazao (wild type which exhibits normal markings), 68 DEGs between LC/+ and +LC/+LC, and 188 DEGs between LCa/+ and +LCa/+LCa. Gene Ontology (GO) analysis indicated a significant DEG enrichment of the functional terms catalytic activity, binding, metabolic process, and cellular process. Furthermore, three mutants share six common enriched KEGG pathways. We finally identified eight common DEGs among three pairwise comparisons, including Krueppel-like factor, TATA-binding protein, protein patched, UDP-glycosyltransferase, an unknown secreted protein, and three cuticular proteins. Microarray-based gene expression analysis revealed that the eight genes are upregulated during molting, which coincides with marking formation, and are significantly differentially expressed between marking and non-marking regions. The results suggest that the eight common genes are involved in the construction of the multiple twin-spot marking patterns in the three mutants.

BmBlimp-1 gene encoding a C2H2 zinc finger protein is required for wing development in the silkworm Bombyx mori.

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the most common class of transcription factors. These factors have great potential for the management of developmental progression by regulating the specific spatiotemporal expression of genes. In this study, we cloned one C2H2-ZF protein gene of Bombyx mori, BGIBMGA000319, that is orthologous to B-lymphocyte-induced maturation protein-1 (Blimp-1); we thus named it as Bombyx mori Blimp-1 (BmBlimp-1). In the silkworm, the BmBlimp-1 gene is specifically upregulated during day 2 of the pupal to adult stage and is highly expressed in wing discs on day 3 of the pupa. Knockdown of its expression level in the pupal stage results in a crumpled-winged silkworm moth. Using the predicted DNA-binding sequences of BmBlimp-1 to search the silkworm genome to screen target genes of BmBlimp-1, 7049 genes were identified to have at least one binding site of BmBlimp-1 on their 1 kb upstream and downstream genome regions. Comparisons of those genes with a reported pupal wing disc transcriptome data resulted in 4065 overlapping genes being retrieved. GO enrichment analysis of the overlapping genes showed that most of the genes were enriched in the binding term. Combining functional annotation and real-time quantitative PCR, 15 genes were identified as the candidate target genes of BmBlimp-1, including several wing cuticular protein genes, chitin synthase A, and wing disc development genes, such as Wnt1, cubitus interruptus (ci) and engrailed (en). Moreover, the amino acid sequence of the zinc finger motif of Blimp-1 gene was highly conserved among the 15 insect species. We propose that BmBlimp-1 is an important regulatory factor in silkworm wing development.

Genome-wide identification and expression profiling of the C2H2-type zinc finger protein genes in the silkworm Bombyx mori.

Cys2-His2 zinc finger (C2H2-ZF) proteins comprise the largest class of putative eukaryotic transcription factors. The zinc finger motif array is highly divergent, indicating that most proteins will have distinctive binding sites and perform different functions. However, the binding sites and functions of the majority of C2H2-ZF proteins remain unknown. In this study, we identified 327 C2H2-ZF protein genes in the silkworm, 290 in the monarch butterfly, 243 in the fruit fly, 107 in elegans, 673 in mouse, and 1,082 in human. The C2H2-ZF protein genes of the silkworm were classified into three main grouping clades according to a phylogenetic classification, and 312 of these genes could be mapped onto 27 chromosomes. Most silkworm C2H2-ZF protein genes exhibited specific expression in larval tissues. Furthermore, several C2H2-ZF protein genes had sex-specific expression during metamorphosis. In addition, we found that some C2H2-ZF protein genes are involved in metamorphosis and female reproduction by using expression clustering and gene annotation analysis. Among them, five genes were selected, BGIBMGA002091 (CTCF), BGIBMGA006492 (fru), BGIBMGA006230 (wor), BGIBMGA004640 (lola), and BIGBMGA004569, for quantitative real-time PCR analysis from larvae to adult ovaries. The results showed that the five genes had different expression patterns in ovaries, among which BGIBMGA002091 (CTCF) gene expression level was the highest, and its expression level increased rapidly in late pupae and adult stages. These findings provide a basis for further investigation of the functions of C2H2-ZF protein genes in the silkworm, and the results offer clues for further research into the development of metamorphosis and female reproduction in the silkworm.

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.

Disruption of PTPS Gene Causing Pale Body Color and Lethal Phenotype in the Silkworm, Bombyx mori.

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene or by defects in the tetrahydrobiopterin (BH4) synthesis pathway. Here, by positional cloning, we report that the 6-pyruvoyl-tetrahydropterin synthase (PTPS) gene, encoding a key enzyme of BH4 biosynthesis, is responsible for the alc (albino C) mutation that displays pale body color, head shaking, and eventually lethality after the first molting in silkworm. Compared to wild type, the alc mutant produced more substrates (phenylalanine (Phe) and tyrosine (Tyr)) and generated less DOPA and dopamine. Application of 2,4-diamino-6-hydroxypyrimidine (DAHP) to block BH4 synthesis in the wild type effectively produced the alc-like phenotype, while BH4 supplementation rescued the defective body color and lethal phenotype in both alc and DAHP-treated individuals. The detection of gene expressions and metabolic substances after drugs treatments in alc and normal individuals imply that silkworms and humans have a high similarity in the drugs metabolic features and the gene pathway related to BH4 and the dopamine biosynthesis. We propose that the alc mutant could be used as an animal model for drug evaluation for BH4-deficient PKU.

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.

Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori.

The insect cuticle is a critical protective shell that is composed predominantly of chitin and various cuticular proteins and pigments. Indeed, insects often change their surface pigment patterns in response to selective pressures, such as threats from predators, sexual selection and environmental changes. However, the molecular mechanisms underlying the construction of the epidermis and its pigmentation patterns are not fully understood. Among Lepidoptera, the silkworm is a favorable model for color pattern research. The black dilute (bd) mutant of silkworm is the result of a spontaneous mutation; the larval body color is notably melanized. We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd. In these experiments, during an early stage of the fourth molt, a stage at which approximately 51% of genes were expressed genome wide (RPKM ≥1) in each strain. A total of 254 novel transcripts were characterized using Cuffcompare and BLAST analyses. Comparison of the transcriptome data revealed 28 differentially expressed genes (DEGs) that may contribute to bd larval melanism, including 15 cuticular protein genes that were remarkably highly expressed in the bd/bd mutant. We suggest that these significantly up-regulated cuticular proteins may promote melanism in silkworm larvae.

Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of ß-alanine and N-ß-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of ß-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant.

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of ß-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.

[Effects and significance of gas explosion on expression of NF-kappaB and ICAM1 in lung tissue of rats].

OBJECTIVE: To explore the expression of NF-kappaB and ICAM-1 in the gas explosion wounded lung of rats and the relationship. METHODS: Digoxin labeled NF-kappaB was used as probe. In situ hybridization was performed to detect the NF-kappaB mRNA. Immunohistochemistry was used to detect the expression of NF-kappaB and ICAM-1. RESULTS: The levels of NF-kappaB mRNA, the expression of NF-kappaB and ICAM-1 in the wounded rats were significantly increased and reached their peak two hours after injury. Pathology of lung tissue showed that some crachea epithelium mucosae were desquamated; congestion, edema of trachea wall and infiltration of neutrophilic granulocytes were found; hemorrhage, edema and infiltration of lots of inflammatory cells were present in alveolus cells. Electron microscope showed that type I, especially type II alveolus epithelia had degeneration and desquamation. CONCLUSION: The injury of gas explosion can activate NF-kappaB, which has close correlation with the acute injury to lung.

[A study on relationship between rat brain tissues hurts of the gas explosion and the expression of protein kinase CamRNA and c-fos gene.].

OBJECTIVES: To explore relationship between rat brain tissues hurts of gas explosion and the expression of Protein Kinase C alpha mRNA. METHODS: Build up rat hurt model of gas explosion. In Situ Hybridization (IDH) technique was used to test Protein Kinase C alpha mRNA. Immunohistochemical Assays (IHA) was used to determine c-fos gene protein. RESULTS: Only a little a mount expression of PKC alpha mRNA and c-fos of the control group was detected. The expression of the cerebral cortex and hippocampus of PKC alpha mRNA 24 h, 48 h and the 48 h increased obviously, and the 48 h reached the peak of expression; (t = 4.12 P < 0.01). The expression of c-fos protein of the cerebral cortex and hippocampus started to increase obviously at 0.5 h and the 4 h reached the peak, then the strength lowered gradually and the expression level came back normal level on fifth day. CONCLUSION: The anoxia of brain tissues due to the gas explosion may promote the expression of PKCamRNA, and PKCamRNA could regulate the expression of the gene of c-fos. Both PKCamRNA and the gene of c-fos are involved in harmful processes to the nerve cells.