SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

The Hox gene Antennapedia is essential for wing development in insects.

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. This article has an associated 'The people behind the papers' interview.

Deciphering the Genetic Basis of Silkworm Cocoon Colors Provides New Insights into Biological Coloration and Phenotypic Diversification.

The genetic basis of phenotypic variation is a long-standing concern of evolutionary biology. Coloration has proven to be a visual, easily quantifiable, and highly tractable system for genetic analysis and is an ever-evolving focus of biological research. Compared with the homogenized brown-yellow cocoons of wild silkworms, the cocoons of domestic silkworms are spectacularly diverse in color, such as white, green, and yellow-red; this provides an outstanding model for exploring the phenotypic diversification and biological coloration. Herein, the molecular mechanism underlying silkworm green cocoon formation was investigated, which was not fully understood. We demonstrated that five of the seven members of a sugar transporter gene cluster were specifically duplicated in the Bombycidae and evolved new spatial expression patterns predominantly expressed in silk glands, accompanying complementary temporal expression; they synergistically facilitate the uptake of flavonoids, thus determining the green cocoon. Subsequently, polymorphic cocoon coloring landscape involving multiple loci and the evolution of cocoon color from wild to domestic silkworms were analyzed based on the pan-genome sequencing data. It was found that cocoon coloration involved epistatic interaction between loci; all the identified cocoon color-related loci existed in wild silkworms; the genetic segregation, recombination, and variation of these loci shaped the multicolored cocoons of domestic silkworms. This study revealed a new mechanism for flavonoids-based biological coloration that highlights the crucial role of gene duplication followed by functional diversification in acquiring new genetic functions; furthermore, the results in this work provide insight into phenotypic innovation during domestication.

Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

The beta-1, 4-N-acetylglucosaminidase 1 gene, selected by domestication and breeding, is involved in cocoon construction of Bombyx mori.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that ß-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.

Juvenile hormone suppresses the FoxO-takeout axis to shorten longevity in male silkworm.

Juvenile hormone (JH) plays a crucial endocrine regulatory role in insect metamorphosis, reproduction, and longevity in multiple organisms, such as flies, honeybees, and migratory monarch butterflies. However, the molecular mechanism of JH affecting longevity remains largely unknown. In this study, we showed that JH III and its analog methoprene shortened the survival days significantly in the adulthood of male silkworm. At the same time, the allatostatin, a neuropeptide that inhibits the secretion of JH by the corpora allata, could extend the survival days dramatically after adult eclosion in male silkmoth. Interestingly, a central pro-longevity FoxO transcription factor was reduced upon JH stimulation in silkworm individuals and BmN-SWU1 cells. Furthermore, the analysis of the upstream sequence of the FoxO gene identified a JH response element which suggested that FoxO might be regulated as a target of JH. Surprisingly, we identified a Bmtakeout (BmTO) gene that encodes a JH-binding protein and contains a FoxO response element. As expected, FoxO overexpression and knockdown up- and down-regulated the expression of BmTO respectively, indicating that BmTO functions as a FoxO target. BmTO overexpression could release the inhibitory effect of JH on the BmFoxO gene by reducing JH bioavailability to block its signal transduction. Collectively, these results may provide insights into the mechanism of the JH-FoxO-TO axis in aging research and pest control.

How the Material Characteristics of Optical Fibers and Soil Influence the Measurement Results of Distributed Acoustic Sensing.

Fiber optic distributed acoustic sensing (DAS) technology is widely used in security surveillance and geophysical survey applications. The response of the DAS system to external vibrations varies with different types of fiber optic cable connections. The mechanism of mutual influence between the cable's characteristics and DAS measurement results remains unclear. This study proposed a dynamic model of the interaction between the optical cable and the soil, analyzed the impact of the dynamic parameters of the optical cable and soil on the sensitivity of the DAS system, and validated the theoretical analysis through experiments. The findings suggest that augmenting the cable's bending stiffness 5.5-fold and increasing its unit mass 4.2-fold result in a discernible reduction of the system's response to roughly 0.15 times of its initial magnitude. Cables with lower unit mass and bending stiffness are more sensitive to vibration signals. This research provides a foundation for optimizing vibration-enhanced fiber optic cables and broadening the potential usage scenarios for DAS systems.

Lycium barbarum polysaccharide improves dopamine metabolism and symptoms in an MPTP-induced model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease in middle-aged and elderly populations, whereas there is no cure for PD so far. Novel animal models and medications await development to elucidate the aetiology of PD and attenuate the symptoms, respectively. METHODS: A neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was used in the current study to establish a PD pathologic model in silkworms. The time required to complete specific behaviours was recorded. Dopamine content was detected by ultra-performance liquid chromatography (UPLC). The activity of insect tyrosine hydroxylase (TH) was determined using a double-antibody sandwich method. Oxidative stress was assessed by changes in antioxidant enzyme activity and the content of oxidative products. RESULTS: MPTP-treated silkworms were characterized by impaired motor ability, reduced dopamine content, and elevated oxidative stress level. The expression of TH, a dopamine biosynthetic enzyme within dopaminergic neurons in the brain, was significantly reduced, indicating that dopaminergic neurons were damaged. Moreover, MPTP-induced motility impairment and reduced dopamine level in the silkworm PD model could be rescued after feeding a combination of levodopa (L-dopa [LD]) and carbidopa (CD). MPTP-induced oxidative damage was also alleviated, in ways consistent with other PD animal models. Interestingly, administration of Lycium barbarum polysaccharide (LBP) improved the motor ability, dopamine level, and TH activity, and the oxidative damage was concomitantly reduced in the silkworm PD model. CONCLUSIONS: This study provides a promising animal model for elucidating the pathogenesis of PD, as well as a relevant preliminary drug screening (e.g., LBP) and evaluation.

Unusual tertiary pairs in eukaryotic tRNAAla.

tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb database (the Lowe Laboratory, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for alanine compared to other tRNA species, including tRNAs specific for glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair generally observed as a Watson-Crick pair throughout phylogeny. Second, the tertiary pair R15/Y48 occurs as a purine-purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine-purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to a variable extent depending on phylogeny. Among eukaryotes, insects display all variations simultaneously, whereas mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, nor archaeal, tRNAs, probably because of the avoidance of A34-containing anticodons in four-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect, besides aminoacylation, transcription rates (e.g., 54/58) or ribosomal translocation (30/40).

Precursor Customized Assembly of Wafer-Scale Polymerized Aniline Thin Films for Ultrasensitive NH3 Detection.

2D conducting polymer thin film recently has garnered numerous interests as a means of combining the molecular aggregate ordering and promoting in-plane charge transport for large-scale/flexible organic electronics. However, it remains far from satisfactory for conducting polymer chains to achieve desirable surface topography and crystallinity due to lack of control over the precursor-involved interfacial assembly. Herein, wafer-size polyaniline (PANI) and tetra-aniline thin films are developed via a controlled interfacial synthesis with customized surface morphology and crystallinity through two typical aniline precursors selective polymerization. Two crucial competing assembly mechanisms, a) direct interfacial polymerization, b) solution polymerization and subsequent interfacial assembly, are investigated to play a vital role in determining elemental chain length and aggregate architecture. The optimal PANI thin film manifests ultraflat surface topography and unambiguous crystalline domains, which also enabling fascinating ammonia sensing capability with 31.4% ppm-1 sensitivity, fast response time (88 s) with astonishing selectivity, repeatability, and recovery capability. The thus-demonstrated strategy with wafer-scale processing potential and flexible microdevice offers a promising route for large-scale manufacturing thin-film organic electronics.

A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

The Role of Chitooligosaccharidolytic ß-N-Acetylglucosamindase in the Molting and Wing Development of the Silkworm Bombyx mori.

The insect glycoside hydrolase family 20 ß-N-acetylhexosaminidases (HEXs) are key enzymes involved in chitin degradation. In this study, nine HEX genes in Bombyx mori were identified by genome-wide analysis. Bioinformatic analysis based on the transcriptome database indicated that each gene had a distinct expression pattern. qRT-PCR was performed to detect the expression pattern of the chitooligosaccharidolytic ß-N-acetylglucosaminidase (BmChiNAG). BmChiNAG was highly expressed in chitin-rich tissues, such as the epidermis. In the wing disc and epidermis, BmChiNAG has the highest expression level during the wandering stage. CRISPR/Cas9-mediated BmChiNAG deletion was used to study the function. In the BmChiNAG-knockout line, 39.2% of female heterozygotes had small and curly wings. The ultrastructure of a cross-section showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the correct wing vein structure. The molting process of the homozygotes was severely hindered during the larva to pupa transition. Epidermal sections showed that the endocuticle of the pupa was not degraded in the mutant. These results indicate that BmChiNAG is involved in chitin catabolism and plays an important role in the molting and wing development of the silkworm, which highlights the potential of BmChiNAG as a pest control target.

The genomic landscape of young and old lung cancer patients highlights age-dependent mutation frequencies and clinical actionability in young patients.

The aim of the study was to investigate age-dependent tendency of genomic alterations in lung cancer, and also to examine mutational profiles and its association with clinical treatment outcomes in young adenocarcinoma patients. By studying 7858 lung cancer samples using targeted-gene sequencing, we investigated genomic differences and clinical on-treatment time (OTT) to different therapies between young (≤ 45 years) and old (> 45 years) patients. The age-dependent trend test for genomic alterations in all patients revealed steady increases in tumor mutation burden and alterations in a number of genes with age, including KRAS, MET, CDKN2A, PIK3CA and MDM2, while the frequencies of ALK, ROS1 and RET fusions and ERBB2 mutations were decreasing. The highest rate of EGFR alterations was observed in the 45 ~ 50 years age group. Comparisons of young and old adenocarcinoma patients found that young patients were characterized by a higher prevalence of ALK, ROS1 and RET fusions, and ERBB2 exon-20 insertions and EGFR exon-19 deletions. Actionable mutations were highly prevalent in young adenocarcinoma patients, with 88% of patients harboring at least one actionable genetic alteration. First-line therapies in EGFR-positive patients (n = 979) by EGFR tyrosine kinase inhibitors or chemotherapy resulted in similar OTT between young and old patients. Somatic interaction analyses implied that young EGFR-positive patients were more likely to also have PIK3CA, MET, TP53 and RB1 mutations than old patients. Lung cancer in young patients, and especially those with adenocarcinoma, exhibited different clinical features and genomic attributes compared to old patients, which should be considered for therapeutic decision-making purposes.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome. RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

Whole-genome resequencing reveals loci under selection during silkworm improvement.

Breeding or genetic improvement refers to the process of artificial selection following domestication; as such, it has had a major influence on modern agriculture and animal production. Improvement generally focuses on traits that greatly affect the economic performance. Therefore, understanding the genetic basis underlying improvement will contribute to the identification of genes controlling economic traits and will facilitate future crop and animal breeding. However, genome-wide study of the molecular basis underlying improvement remains rare. The silkworm is a unique, entirely domesticated economically important invertebrate; genetic improvement has had a huge effect on the silkworm regarding silk-related traits. Herein, we performed whole-genomic sequencing on local and genetically improved silkworm lines to identify the genomic regions under strong selection in silkworm breeding/improvement. By genomic-wide selective sweeping analysis, we identified 24 genomic regions with strong selection signals, eight of which contained 13 candidate genes underlying silkworm breeding. Interestingly, six of these genes were annotated with functions related to neural signal response. Among the six genes, BGIBMGA004050 encodes silkworm CREB-regulated_transcription_coactivator_1 (BmCRTC1), which was reported to be involved in energy-sensing pathways. These results suggested that improvement may have affected the nervous system of the silkworm. This research will provide new insights into the genetic basis underlying the genetic improvement of silkworms and possibly of other species.

Identification, expression, and artificial selection of silkworm epigenetic modification enzymes.

BACKGROUND: Understanding the genetic basis of phenotype variations during domestication and breeding is of great interest. Epigenetics and epigenetic modification enzymes (EMEs) may play a role in phenotypic variations; however, no comprehensive study has been performed to date. Domesticated silkworm (Bombyx mori) may be utilized as a model in determining how EMEs influence domestication traits. RESULTS: We identified 44 EMEs in the genome of silkworm (Bombyx mori) using homology searching. Phylogenetic analysis showed that genes in a subfamily among different animals were well clustered, and the expression pattern of EMEs is constant among Bombyx mori, Drosophila melanogaster, and Mus musculus. These are most highly expressed in brain, early embryo, and internal genitalia. By gene-related selective sweeping, we identified five BmEMEs under artificial selection during the domestication and breeding of silkworm. Among these selected genes, BmSuv4-20 and BmDNMT2 harbor selective mutations in their upstream regions that alter transcription factor-binding sites. Furthermore, these two genes are expressed higher in the testis and ovary of domesticated silkworm compared to wild silkworms, and correlations between their expression pattern and meiosis of the sperm and ova were observed. CONCLUSIONS: The domestication of silkworm has induced artificial selection on epigenetic modification markers that may have led to phenotypic changes during domestication. We present a novel perspective to understand the genetic basis underlying animal domestication and breeding.

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.

Identification and effect of Zf-AD-containing C2H2 zinc finger genes on BmNPV replication in the silkworm (Bombyx mori).

Zf-AD-containing C2H2 zinc -finger genes (ZAD) are uniquely present and have lineage-specific expansion in arthropods. Arthropods are also the hosts of Baculoviruses. We studied the possible relationship between the lineage-specific expansion of ZAD genes and arthropod-Baculovirus co-evolution. We used the silkworm (Bombyx mori) as a model. We identified 73 ZAD genes (BmZAD) in the silkworm. Sequence-based similarity analysis showed that nine clusters involving 28 BmZADs may have undergone species-specific expansion in the silkworm. Expression pattern analysis showed that the BmZADs were divided into five groups. Group I comprised 10 genes with high expression in multiple tissues, suggesting that BmZADs may play roles in the development of various tissues. We identified six BmZADs that could be induced by the Nucleopolyhedrovirus (BmNPV). Among them, BmZAD69 expression is capable of responding to BmNPV infection, and the ZAD domain is indispensable for the function of BmZAD69 in BmNPV replication. We also detected a 3 bp deletion at 1.7 kb upstream of BmZAD69, which may make it more sensitive to BmNPV infection, and thus elevate the BmNPV resistance in Qiufeng_N, a strain with strong virus resistance. These data suggest that BmZADs may be involved in BmNPV infection and that ZAD genes may play a role in arthropod-Baculovirus co-evolution.

Hippo pathway regulates somatic development and cell proliferation of silkworm.

Hippo signaling pathway (signaling pathway Hippo, hereinafter referred to as the Hippo pathway) was the earliest found in Drosophila (Schneck [1]), which can regulate the development of tissues and organs, even some components of the pathway were identified as tumor suppressor [2]. The pathway was more concerned in fruit flies and mice (Schneck [1]), but little attention has been given in silkworm, an important economic and lepidopteran model insect. In this manuscript, we identified major Hippo pathway related genes (Hippo, Salvador, Warts, Mats, Yorkie) in silkworm and named BmHpo, BmSav, BmWts, BmMats, BmYki. The domain organization of these genes was highly conserved in silkworm and other organisms suggesting that they could use similar protein-protein interactions to construct the Hippo kinase cascades. The expression profiles of these genes in silkworm during embryonic, larval, wandering, pupal and adult stages were analyzed, 14 tissues/organs of the day 3, 5th instar larvae (L5D3) as well. Experimental results showed that the expression of Hippo pathway had some influence on the development of silkworm. In order to find out the mechanism of Hippo pathway affecting silkworm development, BmHpo and BmYki were up-regulated and de-regulated in the cell line of Bombyx mori-BmN-SWU1(NS), and the changes of cell proliferation activity and cell cycle were detected. The distribution of BmYki was detected by immunofluorescence technique. This study provides insights into the genes of Hippo pathway which have a certain effect on somatic development and cell proliferation in silkworm.

Identification and characterization of a new long noncoding RNA iab-1 in the Hox cluster of silkworm, Bombyx mori identification of iab-1.

Long noncoding RNAs (lncRNAs) areinvolvedin a variety of biological processes. In silkworm, numerous lncRNAs have been predicted through deep transcriptome sequencing, but no functional role has been experimentally validated yet. Here, we characterized a new lncRNA iab-1 that was mainly encoded by the intergenic region between Bmabd-A and Bmabd-B in the Homeobox (Hox) cluster of the silkworm, Bombyx mori. More than seven alternative splicing isoforms of lncRNA iab-1 were cloned, which were subgrouped into types 1 and 2 based on the location of the 3'-ends. The iab-1 was expressed at a low level, but the expression of iab-1 peaked at several specific development stages, including 3 to 4 days during the embryonic stage, stages before fourth molting, and the sixth hour after the fourth molting, and early stages during metamorphosis. It was highly expressed in the nervus and epidermis, especially the epidermis of the posterior abdomen at the fourth instar premolting stage. The relationship between iab-1 and nearby Hox genes was analyzed at different developmental stages. Iab-1 expression was highly associated with Bmabd-A as well as Bmabd-B in the embryonic and larval stages, while this association was decreased at the metamorphic stage; iab-1 expression was highly associated with BmUbx only in the embryonic stage. Downregulation of iab-1 expression by small interfering RNA led to the death of most of the treated individuals at the larval stage, suggesting that iab-1 transcript expression might be involved in certain relevant physiological processes. The expression of Bmabd-A and Bmabd-B did not change in iab-1 downregulated individuals, indicating that the relevance between the two genes and iab-1 was not induced by iab-1 transcript. Collectively, the results showed that the newly identified iab-1 may be involved in some physiological processes, and the interaction between iab-1 and Hox genes was also preliminarily analyzed.