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Journal of Forestry Research 2018年第3期29卷 822-829页

作者： Ik Je Choi Tae Sung Kwon Jongok Lim silkworm and insect management center Gyeongsangbuk-do Agricultural Resource Management Institute Forest insect Pests and Disease Division Korea Forest Research Institute Division of Forest Biodiversity Korea National Arboretum

The cerambycid genus Miaenia Pascoe(Coleoptera: Cerambycoidea) of South Korea is taxonomically reviewed. Two species, Miaenia fasciata fasciata(Matsushita) and Miaenia maritima Tsherepanov have been recorded. In this paper, M. tonsa(Bates) is added to the Korean cerambycid fauna for the first time. A key to Korean Miaenia species, re-description and diagnostic illustrations, including male genitalia for each species, are provided.

关键词： Acanthocinini Cerambycidae Miaenia New species South Korea Taxonomy

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

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insect Science 2009年第6期16卷 465-473页

作者： Isao Kobayashi Katsura Kojima Hideki Sezutsu Keiro Uchino Toshiki Tamura Transgenic silkworm Research center Silk Materials Research Unit National Institute of Agrobiological Sciences Ibaraki Japan

To understand the evolutionary conservation ofthe gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamarnai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori-type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non-transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.

关键词： Antheraea yamamai Bombyx mori evolution fibroin silk transgenic

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori

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Journal of Genetics and Genomics 2017年第9期44卷 451-459页

作者： Yuanyuan Liu Sanyuan Ma Jiasong Chang Tong Zhang Xiaogang Wang Run Shi Jianduo Zhang Wei Lu Yue Liu Qingyou Xia State Key Laboratory of silkworm Genome Biology Southwest University Chongqing Engineering and Technology Research center for Novel Silk Materials Southwest University DBNBiotechnology center

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm,Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA（gRNA）, they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing *** perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines,one expressing Cas9 protein in the posterior silk glands（PSGs） and the other constitutively expressing BmlaminA/C（BmLMN） gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%,and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.

关键词： CRISPR/Cas9 system Genome editing PiggyBac transposon Posterior silk gland Endoreplication

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

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insect Science 2021年第5期28卷 1277-1289页

作者： Feng Wang Yan-Ting Ji Chi Tian Yuan-Cheng Wang Shen Xu Ri-Yuan Wang Qian-Qian Yang Ping Zhao Qing-You Xia State Key Laboratory of silkworm Genome Biology Chongqing Engineering and Technology Research Center for Novel Silk MaterialsBiological Science Research CenterSouthwest UniversityChongqingChina

Inducible gene-expression systems play important roles in gene functional assays in the post-genome *** phage-derived phiC31 integrase,which mediates an irreversible site-specific cassette exchange between the phage attachment site(attP)and the bacterial attachment site(attB),provides a promising option for the construction of a controllable gene-expression ***,we report a phiC3I integrase-mediated promoter flip system(FLIP)for the inducible expression of target genes in silk-worm(Bombyx mori).First,we constructed a FLIP reporter system,in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter *** coexpression of a C-terminal modified phiC3 I-NLS integrase carrying a simian virus 40(SV40)nuclear localization signal(NLS)effectively flipped the BmAct4 promoter through an attBlattP exchange,thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line,***,the FLIP system,together with a system continuously expressing the phiC3 I-NLS integrase,was used to construct binary transgenic silkworm *** between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter,with an approximately 39%heritable transformation efficiency in silkworm offispring,leading to the constitutive and high-level expression of DsRed in silkworms,which accounted for approximately 0.81%of the silkworm pupal *** successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

关键词： inducible gene expression PhiC31 integrase promoter flipping silkworm

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

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insect Science 2024年第1期31卷 47-58页

作者： Hao Sun Jingya Chen Ruolin Wang Dan Liu Na Zhang Tong Zhang Ling Jia Sanyuan Ma Qingyou Xia State Key Laboratory of silkworm Genome Biology Southwest UniversityChongqingChina Biological Science Research center Southwest UniversityChongqingChina

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly ***-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host ***,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor *** this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of *** annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and *** enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell *** screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to *** findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

关键词： CRISPR/Cas9 CRISPR library screening ecdysone 20E signal transduction silkworm whole genome editing

BmCaspase-8-like regulates autophagy by suppressing BmDREDD-mediated cleavage of BmATG6

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insect Science 2023年第2期30卷 365-374页

作者： Chang Sun Dongmei Wei Yumeng Pan Xiaoyi Xiao Fei Wang State Key Laboratory of silkworm Genome Biology Southwest UniversityChongqingChina Biological Science Research center Southwest UniversityChongqingChina

Autophagy plays an important role in tissue remodeling during insect *** interplay between autophagy-related(ATG)proteins and caspases regulates the autophagic activity of ATGs,thereby modulating the process of *** previous study characterized BmCaspase-8-like(BmCasp8L)as a caspase suppressor that inhibits apoptosis and immune signaling by suppressing the activation of death-related ced-3/Nedd2-like caspase(DREDD),a caspase-8 homolog in *** this study,we explored the regulatory role of BmCasp8L in *** found that the expression of Bmcasp8l increased from the late spinning stage to the pupa stage in the posterior silk gland(PSG),correlating with the expression patterns of Bmatg8 and *** interference-mediated downregulation of BmCasp8L expression significantly decreased starvation-induced autophagic influx as determined by the levels of BmATG8–phosphatidylethanolamine and the percentage of cells displaying punctate enhanced green fluorescent ***,the overexpression of BmCasp8L significantly increased autophagic *** also found that BmCasp8L underwent autophagic degradation induced by starvation and that it was colocalized with ***,we demonstrated that BmDREDD attenuated autophagy and BmCasp8L suppressed BmDREDD-mediated cleavage of *** together,our results demonstrated that BmCasp8L is a novel proautophagic molecule which suppresses BmDREDD-mediated cleavage of BmATG6 and is a target for autophagy.

关键词： ATG6 ATG8 autophagy Bombyx mori Caspase-8-like DREDD

Molecular cloning and comparative analysis of transcripts encoding chemosensory proteins from two plant bugs, Lygus lineolaris and Lygus hesperus

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insect Science 2020年第3期27卷 404-424页

作者： J.Joe Hull Omaththage P.Perera Mei-Xian Wang USDA-ARS Arid Land Agricultural Research center MaricopaArizonaUSA USDA-ARS Southern Insect Management Research UnitStonevilleMississippiUSA College of Animal Sciences Zhejiang UniversityHangzhouChina

Chemosensory proteins(CSPs)are soluble carrier proteins typically characterized by a six‐helix bundle structure joined by two disulfide bridges and a conserved Cys spacing pattern(C1‐X6‐8‐C2‐X16‐21‐C3‐X2‐C4).CSPs are functionally diverse with reported roles in chemosensation,immunity,development,and *** expand our molecular understanding of CSP function in plant bugs,we used recently developed transcriptomic resources for Lygus lineolaris and Lygus hesperus to identify 17 and 14 CSP‐like sequences,*** Lygus CSPs are orthologous and share significant sequence identity with previously annotated *** of the CSPs are predicted to deviate from the typical CSP structure with either five or seven helical segments rather than *** seven helix CSP is further differentiated by an atypical C3‐X3‐C4 Cys spacing *** transcriptase PCR‐based profiling of CSP transcript abundance in adult *** tissues revealed broad expression for most of the CSPs with antenna specific expression limited to a subset of the *** sequence analyses and homology modeling suggest that variations in the amino acids that comprise the Lygus CSP binding pockets affect the size and nature of the ligands accommodated.

关键词： chemosensation chemosensory protein homology modeling Lygus plant bug mired transcriptome

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

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Journal of Integrative Agriculture 2019年第5期18卷 1042-1049页

作者： WANG Yu-sheng TIAN Hu WAN Fang-hao ZHANG Gui-fen State Key Laboratory for Biology of Plant Diseases and insect Pests/Key Laboratory of Integrated Pest management of Crop Ministry of Agriculture/Institute of Plant Protection Chinese Academy of Agricultural Sciences Caofeidian Sub-center of Hebei Entry-Exit Inspection and Quarantine Technical center

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable *** slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for *** is *** the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of *** pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among *** and related mealybug *** of the primer pair was validated on 21 closely related *** tests were performed on four immature developmental stages and female *** tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all ***,the SS-COI primer pair was assayed on three populations of *** from major exporting countries of *** PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of *** tool will be useful for quarantine,monitoring,and management of this invasive pest.

关键词： Phenacoccus manihoti cassava mealybug invasive pest molecular identification species-specific COI primers

Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid,Diaphorina citri

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insect Science 2024年第4期31卷 1211-1221页

作者： Xue Dong Chen Justin George Lauren M.Diepenbrock Hunter Gossett Guoping Liu Jawwad A.Qureshi Lukasz L.Stelinski Entomology and Nematology Department University of FloridaCitrus Research and Education CenterLake AlfredFloridaUSA Southern insect management Research Unit USDA-ARSStonevilleMississippiUSA Entomology and Nematology Department University of FloridaSouthwest Florida Research and Education CenterImmokaleeFloridaUSA

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid,Diaphorina citri Kuwayama,which serves as vector of phytopathogens causing citrus ***,development of resistance to neonicotinoids among populations of *** has coincided with occasional control failures in the *** objectives of this research were to(1)survey current levels of imidacloprid resistance in Florida citrus;(2)compare feeding behavior between imidacloprid-resistant and susceptible *** using electrical penetration graph recordings,and(3)investigate the possible amplification of insecticide hormoligosis associated with *** surveys confirmed that the susceptibility of *** populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible *** 12 generations of selection,resistance to imidacloprid increased by 438 fold compared with the susceptible ***-susceptible *** feeding on citrus exhibited significantly more bouts associated with intercellular pathway(C),phloem penetration(D),phloem salivation(E1),and nonprobing(Np)activities than imidacloprid-resistant ***,there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant *** was no statistical difference in fecundity between resistant and susceptible ***,the fecundity of imidacloprid-susceptible female *** treated with a sublethal concentration of imidacloprid(LC_(25))increased significantly compared with controls,while such hormoligosis was less pronounced among imidacloprid-resistant *** results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide,indicative of a behavioral resistance mechanism.

关键词： artificial selection electrical penetration graph feeding behavior hormoligosis insecticide resistance sublethal exposure

Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems

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insect Science 2019年第6期26卷 983-990页

作者： Xiao-Gang Wang San-Yuan Ma Jia-Song Chang Run Shi Ruo-Lin Wang Ping Zhao Qing-You Xia State Key Laboratory of silkworm Genome Biology Southwest University ChongqingChina Chongqing Engineering and Technology Research center for Novel Silk Materials Southwest University Chongqing China

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpfl) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.

关键词： Bombyx mori CRISPR/dCas9 system transcriptional activation VP64 VPR