查看更多>>摘要：RNA interference(RNAi)is a form of gene silencing triggered by double-stranded RNA(dsRNA)that operates in all eukaryotic cells.RNAi has been widely inves-tigated in insects to determine the underlying molecular mechanism,to investigate its role in systemic antiviral defense,and to develop strategies for pest control.When insect cells are infected by viruses,viral dsRNA signatures trigger a local RNAi response to block viral replication and generate virus-derived DNA that confers systemic immunity.RNAi-based insect pest control involves the application of exogenous dsRNA targeting genes essential for insect development or survival,but the efficacy of this approach has limited potency in many pests through a combination of rapid dsRNA degradation,inefficient dsRNA uptake/processing,and ineffective RNAi machinery.This could be addressed by dsRNA screening and evaluation,focusing on dsRNA design and off-target management,as well as dsRNA production and delivery.This review summarizes recent progress to determine the role of RNAi in antiviral defense and as a pest control strategy in insects,addressing gaps between our fundamental understanding of the RNAi mechanism and the exploitation of RNAi-based pest control strategies.
查看更多>>摘要：Diaphorina citri is a global citrus pest.As a vector insect,it can transmit the causative agents of citrus huanglongbing,causing irreversible losses to the citrus industry.The acquisition of genomic information can provide a molecular genetic basis for effective control of D.citri.Here,the DNBSEQTM,Oxford Nanopore Technologies,and Hi-C tech-nologies are applied to generate a high-quality chromosome-level genome of D.citri.The genome size of D.citri was 523.78 Mb with a scaffold N50 of 47.05 Mb distributed on 13 chromosomes.A total of250.64 Mb(47.85％)repeat sequences and 24 048 protein-coding genes were predicted.Genome resequencing of female and male individuals indicated that the sex chromosome system of D.citri is XO.Phylogenetic analysis demonstrated that D.citri and Pachypsylla venusta,which separated from their most recent common ancestor about 336.62 million years ago,were the most closely related.Additionally,we identified genes potentially involved in detoxification metabolism,pathogen transmission,and hon-eydew secretion for further investigation.The high-quality genome provides an important reference for developing effective management strategies of D.citri.
查看更多>>摘要：The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products.In sericulture,only the first filial generation(F1)hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms,but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hy-brid transgenic eggs.To address this issue,we developed a safe and efficient strategy using the GAL4/Upstream activating sequence(UAS)system,the FLP/flippase recogni-tion target(FRT)system,and the gonad-specific expression gene promoters(RSHP1p and Nanosp)for the germ cell-specific automatic excision of foreign DNA in the F 1 hy-brid transgenic silkworms.We established 2 types of activator strains,R1p::GAL4-Gr and Nsp::GAL4-Gr,containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp,respectively,and 1 type of effector strain,UAS::FLP-Rg,containing the UAS-linked FLP gene expression cassette.The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silk-worms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100％,whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73％to 80.3％.Additionally,we identified a gene,sw11114,that is expressed in both testis and ovary of Bombyx mori,and can be used to establish novel gonad-specific expression systems in transgenic silkworms.This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.
查看更多>>摘要：Metamorphosis is a complex developmental process involving multiple path-ways 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 under-stood.Genome-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 interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and success-fully revealed the genes involved in biotic or abiotic stress factor responses.In 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 action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nu-trition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our 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.
查看更多>>摘要：The Indian meal moth,Plodia interpunctella(Lepidoptera:Pyralidae),a globally distributed storage pest,relies on odors that are emitted from stored foods to select a suitable substrate for oviposition.However,the molecular mechanism underly-ing the chemical communication between P interpunctella and its host remains elusive.In this study,130 chemosensory genes were identified from the transcriptomes of 7 P.interpunctella tissues,and the quantitative expression levels of all 56 P interpunctella odorant receptor genes(PintORs)were validated using real-time quantitative polymerase chain reaction.The functional characteristics of 5 PintORs with female antennae-biased expression were investigated using 2-electrode voltage clamp recordings in Xenopus lae-vis oocytes.PintOR23 was found to be specifically tuned to acetophenone.Acetophenone could elicit a significant electrophysiological response and only attracted mated females when compared with males and virgin females.In addition,molecular docking predicted that the hydrogen bonding sites,TRP-335 and ALA-167,might play key roles in the bind-ing of PintOR23 to acetophenone.Our study provides valuable insights into the olfac-tory mechanism of oviposition substrate detection and localization in P interpunctella and points toward the possibility of developing eco-friendly odorant agents to control pests of stored products.
查看更多>>摘要：Spermatogenesis is a critical part of reproduction in insects;however,its molecular mechanism is still largely unknown.In this study,we identified a testis-specific gene CG3526 in Drosophila melanogaster.Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2H2 type zinc fingers,and it is clustered to the vertebrate really interesting new gene(RING)family E3 ubiquitin-protein ligases.When CG3526 was knocked down by RNA interference(RNAi),the testis became much smaller in size,and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis.More importantly,compared to the control flies,only a few mature sperm were present in the seminal vesicle of C587-Gal4＞CG3526 RNAi flies.Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the home-ostasis of testis stem cell niche was disrupted,cell distribution in the apical tip was scat-tered,and the process of spermatogenesis was not completed.Furthermore,we found that the phenotype of CG3526 RNAi flies'testis was similar to that of testis of Stat92E RNAi flies,the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies,and the promoter activity of CG3526 was upregulated by STAT92E.Taken to-gether,our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.
查看更多>>摘要：Apolipoprotein D(ApoD),a member of the lipocalin superfamily of proteins,is involved in lipid transport and stress resistance.Whereas only a single copy of the ApoD gene is found in humans and some other vertebrates,there are typically several ApoD-like genes in insects.To date,there have been relatively few studies that have exam-ined the evolution and functional differentiation of ApoD-like genes in insects,particularly hemi-metabolous insects.In this study,we identified 10 ApoD-like genes(NlApoD1-10)with distinct spatiotemporal expression patterns in Nilaparvata lugens(BPH),which is an important pest of rice.NlApoD1-10 were found to be distributed on 3 chromosomes in a tandem array of NlApoD1/2,NlApoD3-5,and NlApoD7/8,and show sequence and gene structural divergence in the coding regions,indicating that multiple gene duplica-tion events occurred during evolution.Phylogenetic analysis revealed that NlApoD1-10 can be clustered into 5 clades,with NlApoD3-5 and NlApoD7/8 potentially evolving exclusively in the Delphacidae family.Functional screening using an RNA interference approach revealed that only NlApoD2 was essential for BPH development and survival,whereas NlApoD4/5 are highly expressed in testes,and might play roles in reproduction.Moreover,stress response analysis revealed that NlApoD3-5/9,NlApoD3-5,and NlA-poD9 were up-regulated after treatment with lipopolysaccharide,H2O2,and ultraviolet-C,respectively,indicating their potential roles in stress resistance.
查看更多>>摘要：High fecundity is a common characteristic of insect pests which increases the difficulty of population control.Serine/threonine kinase Akt is an indispensable compo-nent of the insulin signaling pathway.Silencing of LsAkt severely hinders reproduction in Lasioderma serricorne,a stored product insect pest.However,the post-transcriptional pathway of LsAkt in L.serricorne remains unknown.This study identified 2 binding sites of miR-9c-5p and novel-mir50 in the coding sequences ofLsAkt.The expression profiles of 2 microRNAs(miRNAs)and LsAkt displayed an opposite pattern during the adult stages.Luciferase reporter assay showed that novel-mir50 and miR-9c-5p could downregulate the expression of LsAkt.Overexpression of miR-9c-5p and novel-mir50 by injection of mimics inhibited the expression of LsAkt and reduced oviposition,decreased egg hatchability,and blocked ovarian development.It also decreased the expression of genes involved in ovar-ian development(LsVg and LsVgR)and the nutritional signaling pathway(LsTOR,LsS6K,and Ls4EBP),and reduced the phosphorylation of Akt.Conversely,injection of miR-9c-5p and novel-mir50 inhibitors induced the expressions of LsAkt,LsVg,LsVgR,LsTOR,LsS6K,and Ls4EBP,enhanced Akt phosphorylation level,and accelerated ovarian devel-opment.Injection of bovine insulin downregulated the expression of miR-9c-5p and novel-mir50 and upregulated the LsAkt expression.It also rescued the reproductive development defects associated with miR-9c-5p/novel-mir50 overexpression,forming a positive regula-tory loop of insulin signaling.These results indicate that miR-9c-5p/novel-mir50 regulates the female reproduction of L.serricorne by targeting Akt in response to insulin signaling.The data also demonstrate the effects of the insulin/miRNA/Akt regulatory axis in insect reproduction.
查看更多>>摘要：RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsR-Nase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects'dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When compar-ing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Sub-sequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.
查看更多>>摘要：Monochamus alternatus is the primary carrier of pine wood nematodes,which pose a serious threat to Pinus spp.in many countries.Newly emerging M.alternatus adults feed on heathy host pines,while matured adults transfer to stressed host pines for mat-ing and oviposition.Several odorant-binding proteins(OBPs)of M.alternatus have been proved to aid in the complex process of host location.To clarify the corresponding rela-tions between OBPs and pine volatiles,more OBPs need to be studied.In this research,MaltOBP19 showed a specific expression in the antennae and mouthparts of M.alterna-tus,and it was marked in 4 types of antenna sensilla by immunolocalization.Fluorescence binding assays demonstrated the high binding affinity of MaltOBP19 with camphene and myrcene in vitro.In Y-tube olfactory experiments,M.alternatus adults were attracted by camphene and RNAi of OBP19 via microinjection significantly decreased their attraction index.Myrcene induced phobotaxis,but RNAi had no significant effect on this behavior.Further,we found that ingesting dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of MaltOBP 19.These results suggest that MaltOBP 19 may play a role in the process of host conversion via the recog-nition of camphene,which has been identified to be strongly released in stressed host pines.In addition,it is proved that knockdown of OBP can be achieved by oral admin-istration of bacteria-expressed double-stranded RNA in M.alternatus adults,providing a new perspective in the control of M.alternatus.
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