The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis...The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis,mitigating aminoacidemia by assimilating free amino acids,and reallocating nitrogen during metamorphosis through programmed cell death.However,the specific functions of nitrogen metabolism-related genes in this process remain unclear.Using CRISPR/Cas9-based gene editing,mutations were generated in glutamine synthetase(GS),glutamate synthetase(GOGAT),asparagine synthetase(AS),glutamate dehydrogenase(GDH)and glutamate oxaloacetate transaminase 1(GOT1).Disruption of GS,GOGAT,and AS consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription,whereas GOT1 mutation had no such effect.GOGAT mutants exhibited abnormally enlarged silk glands,whereas GS and AS mutants showed delayed programmed cell death in the silk glands.In contrast,GOT1 mutants displayed normal silk gland morphology but were consistently smaller.Disruption of GS,GOGAT,and AS led to more extensive transcriptional changes,including altered expression of transcription factors in the silk glands,compared with GOT1 mutants.Both GS and GOGAT mutants exhibited up-regulation of AS and GDH,while only GOGAT mutants displayed elevated AS enzymatic activity,suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis.AS mutants showed significantly elevated GOT activity and up-regulation of several metabolic pathways,indicating that AS may functionally interact with GOT in regulating both silk gland development and programmed cell death during metamorphosis.展开更多
The insect brain is the central part of the neurosecretory system,which controls morphology,physiology,and behavior during the insect’s lifecycle.Lepidoptera are holometabolous insects,and their brains develop during...The insect brain is the central part of the neurosecretory system,which controls morphology,physiology,and behavior during the insect’s lifecycle.Lepidoptera are holometabolous insects,and their brains develop during the larval period and metamorphosis into the adult form.As the only fully domesticated insect,the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process.Hormonal regulation in insects is a key factor in multiple processes.However,how juvenile hormone(JH)signals regulate brain development in Lepidoptera species,especially in the larval stage,remains elusive.We recently identified the JH receptor Methoprene tolerant 1(Met1)as a putative domestication gene.How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin’s theory on domestication.Here,CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain,unlike precocious pupation of the cuticle.At the whole transcriptomelevel,theecdysteroid(20-hydroxyecdysone,20 E)signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain.Pathways related to cell proliferation and specialization processes,such as extracellular matrix(ECM)-receptor interaction and tyrosine metabolism pathways,were suppressed in the brain.Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B.mori,which decreased transcriptional binding activity.The B.mori MET1 protein showed a changed structure and dynamic features,as well as a weakened co-expression gene network,compared with B.mandarina.Based on comparative transcriptomic analyses,we proposed a pathway downstream of JH signaling(i.e.,tyrosine metabolism pathway)that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.展开更多
Fatty acid-bindi ng proteins(FABPs)are a family of lipid chaper on es,which con tribute to systemic metabolic regulati on through diverse lipid signalings.In this study,a midgut-specific FABP gene(Slfabp2)was cloned f...Fatty acid-bindi ng proteins(FABPs)are a family of lipid chaper on es,which con tribute to systemic metabolic regulati on through diverse lipid signalings.In this study,a midgut-specific FABP gene(Slfabp2)was cloned from Spodoptera litura.RT-PCR and Western blot analysis indicated that RNA and protein levels of S/FABP2 gradually increased and reached a peak at the prepupal stage and maintained a high level during the pupal stage.The expression of S/FABP2 protein was induced by starvation treatment.In vitro binding assay revealed that the recombinant S/FABP2 had high mffinities of binding Iong-chain fatty acids,such as palmitic acid,arachidonate and oleic acid.The results suggest that S/FABP2 may have a unique function that transports intracellular fatty acids and can regulate the metabolism of lipids in metamorphosis.This work provides experimental clues for understanding the potential function of S/FABP2 in fatty acid metabolism in S.litura.展开更多
Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our publ...Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.展开更多
Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene ex...Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression.However,the mechanisms by which DNA methylation regulates these processes in insects remain unclear.Here,we studied the impacts of DNA methylation on early embryonic development in Bombyx mori.Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5'region of protein metabolism-related genes.The transcription factor gene zinc finger protein 615(ZnF615)was methylated by DNA methyltransferase 1(Dnmt1)to be up-regulated and bind to protein metabolism-related genes.Dnmt1 RNA interference(RNAi)revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615.The same sites in the ZnF615 gene were methylated in ovaries and embryos.Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout.Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo.Thus,Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.展开更多
The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we...The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.展开更多
Two new species of Protura are described:Baculentulus wenyingae sp.nov.and Pseudanisentomon lishuiensis sp.nov.Baculentulus wenyingae sp.nov.is characterized by the combined characters of the presence of foretarsal se...Two new species of Protura are described:Baculentulus wenyingae sp.nov.and Pseudanisentomon lishuiensis sp.nov.Baculentulus wenyingae sp.nov.is characterized by the combined characters of the presence of foretarsal sensillum b',differentiated sensilla on the maxillary palp,leaf-shaped sensillum on the labial palp,absence of P1a seta on tergites I–VI,eight anterior setae on tergite VI and 4/18 chaetotaxy on tergite VII.An updated key to the 13 Baculentulus species from China is also provided.Pseudanisentomon lishuiensis sp.nov.is characterized by the absence of foretarsal sensillum b'-2,b longer than c,the empodial appendage of II and III leg very short,the tergite I with 12 posterior setae,tergites V–VII lack seta A3,and sternites VIII–X with 2/7-4-4 chaetotaxy respectively.It was compared with the affiliated species P.guangxiensis(Yin&Zhang,1982)in detail.In addition,a checklist and the distribution of 39 Protura species from Zhejiang Province are provided,and the Protura fauna in Zhejiang is also discussed.展开更多
Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic assoc...Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis.We found significant differences in the silk gland transcriptomic landscape between the two systems,including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents.In the quaternary hybrid system,hybrid vigor was primarily due to upregulated genes and the parent-dominant upregulated expression pattern,involving multiple transport processes,cellular nitrogen compound catabolism,glucose metabolism,and tricarboxylic acid cycle.In the binary system,hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern,mainly involving basic nitrogen synthesis metabolism and body function.We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes.Thus,this study revealed two alternative mechanisms that may contribute to silkworm heterosis,both of which facilitate the efficient utilization of energy and nitrogen for silk production.展开更多
Insects represent one of the most evolutionarily successful groups,with their diversity hypothesized to be related to the regulatory roles of Hox genes,a set of related genes encoding homeodomain transcription factors...Insects represent one of the most evolutionarily successful groups,with their diversity hypothesized to be related to the regulatory roles of Hox genes,a set of related genes encoding homeodomain transcription factors determining the identity of segments along the anterior-posterior axis of the embryo.However,functional insights into the roles of Hox genes in primitive ametabolous insects,which represent the critical transition from aquatic crustaceans to winged insects,have been limited.In this study,we identified complete protein-coding sequences of 10 Hox genes in the Zygentoma Thermobia domestica,and applied clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas 9)mediated gene knockout(KO)to decipher their functions.We found that the roles of pb,Dfd,and Scr are vital in specifying the appendages of the head in T.domestica,and these roles are relatively conserved in crustaceans and winged insects.Antp is essential for the development of the prothorax segment and the first pair of legs in T.domestica.Ubx and abd-A fully repress appendage development in the abdomen of T.domestica,which implies a functional switch from crustaceans to insects.Additionally,the role of ftz in segmenting the abdomen of T.domestica suggests it has acquired new functions in primitive insects,beyond its traditional Hox-like roles.Although KOs of lab,Hox3,and Abd-B did not result in obvious external phenotypic changes,they led to a significant decrease in hatching rates and substantial deviations in daily survival numbers compared to the negative control.These findings underscore the indispensable roles of all Hox genes during the embryonic development of T.domestica.Our study sheds new light on the functional evolution of Hox genes in ametabolous insects and enhances our understanding of the genetic underpinnings of insect development and diversification.展开更多
Cadherin,aminopeptidase N(APN)and alkaline phosphatase(ALP)have been characterized as Cry receptors.In this study,comparative genomic analysis of the 3 receptor families was performed in 7 insects.ALPs and APNs are di...Cadherin,aminopeptidase N(APN)and alkaline phosphatase(ALP)have been characterized as Cry receptors.In this study,comparative genomic analysis of the 3 receptor families was performed in 7 insects.ALPs and APNs are divided into three and eight clades in phylogenetic trees,respectively.ALPs in clade 3 and APNs in clade 1 contain multiple paralogs within each species and most paralogs are located closely in chromosomes.Drosophila melanogaster has expanded APNs in clade 5 and were lowly expressed in midgut.Cadherins are divided into 16 clades;they may diverge before holometabolous insect speciation except for BtR and Cad89D-like clades.Eight insects from different orders containing BtR orthologs are sensitive to Cryl A or Cry3A,while five species without BtR are insensitive to both toxins.Most APNs in clade 1,several ALPs in clade 3,BtR and Cad89D-like genes were highly or moderately expressed in larval midgut of Spodoptera litura and the other six species,and several members in these clades have been identi-•fied as Cry receptors.Expressions of putative 5.litura Cry receptors in the midgut after exposing to Bt toxins were also analyzed.展开更多
Regeneration,as a fascinating scientific field,refers to the ability of animals replacing lost tissue or body parts.Many metazoan organisms have been reported with the regeneration phenomena,but showing evolutionarily...Regeneration,as a fascinating scientific field,refers to the ability of animals replacing lost tissue or body parts.Many metazoan organisms have been reported with the regeneration phenomena,but showing evolutionarily variable abilities.As the most diverse metazoan taxon,hundreds of insects show strong appendage regeneration ability.The regeneration process and ability are dependent on many factors,including macroscopic physiological conditions and microscopic molecular mechanisms.This article reviews research progress on the physiological conditions and internal underlying mechanisms controlling appendage regeneration in insects.展开更多
Continuous and long-term use of traditional and new pesticides can result in cross-resistance among pest populations in different fields.Study on the mechanism of cross-resistance and related genes will help resistanc...Continuous and long-term use of traditional and new pesticides can result in cross-resistance among pest populations in different fields.Study on the mechanism of cross-resistance and related genes will help resistance management and field pest control.In this study,the pesticide-resistance mechanism in Spodoptera frugiperda(FAW)was studied with field populations in 3 locations of South China.Field FAW populations were highly resistant to traditional insecticides,chlorpyrifos(organophosphate)and deltamethrin(pyrethroid),and had higher levels of cytochrome P450 activity than a non-resistant laboratory strain.Inhibition of P450 activity by piperonyl butoxide significantly increased the sensitivity of resistant FAW in 3 locations to chlorpyrifos,deltamethrin and chlorantraniliprole(amide),a new type of insecticide,suggesting that P450 detoxification is a critical factor for insecticide resistance in field FAW populations.Transcriptomic analysis indicated that 18 P450 genes were upregulated in the field FAW populations collected in 3 regions and in 2 consecutive years,with CYP321A8,the most significantly upregulated one.Knockdown of CYP321A8 messenger RNA by RNA interference resulted in an increased sensitivity to the 3 tested insecticides in the field FAW.Enzyme activity and molecular docking analyses indicated that CYP321A8 enzyme was able to metabolize the 3 tested insecticides and interact with 8 other types of insecticides,confirming that CYP321A8 is a key cross-resistance gene with a wide range of substrates in the field FAW populations across the different regions and can be used as a biomarker and target for management of FAW insecticide resistance in fields.展开更多
Antimicrobial peptides(AMPs)are critical components of innate immunity in diverse organisms,including plants,vertebrates,and insects.This study identified and characterized a novel Lepidoptera-specific AMP,named lepid...Antimicrobial peptides(AMPs)are critical components of innate immunity in diverse organisms,including plants,vertebrates,and insects.This study identified and characterized a novel Lepidoptera-specific AMP,named lepidoptin,from the invasive pest Spodoptera frugiperda(Lepidoptera:Noctuidae).Lepidoptin is a 116-amino acid protein containing a signal peptide and a novelβ-sandwich domain that is distinct from previously reported AMPs.Temporal and spatial expression analyses revealed a significant upregulation of the lepidoptin gene in vivo and in cultured SF9 cells in response to pathogens.Molecular docking analysis identified a specific binding cavity.Enzymelinked immunosorbent assay and binding assays confirmed that lepidoptin can bind to pathogen-associated molecular patterns,bacteria,and fungi.Recombinant lepidoptin exhibited potent antibacterial activity by inducing bacterial agglutination,inhibiting bacterial growth,increasing bacterial membrane permeability,and preventing biofilm formation.Lepidoptin also showed antifungal activity against the entomopathogenic fungus Beauveria bassiana by inhibiting spore germination,increasing fungal cell permeability,and increasing reactive oxygen species.Injection of recombinant lepidoptin into S.frugiperda larvae increased survival after B.bassiana infection,whereas knockdown of lepidoptin by RNA interference decreased larval survival.In addition,lepidoptin showed antimicrobial activity against the plant pathogen Fusarium graminearum by inhibiting spore germination and alleviating disease symptoms in wheat seedlings and cherry tomatoes.This study demonstrates the remarkable dual functionality of lepidoptin in enhancing S.frugiperda immunity and controlling plant pathogens,making it a promising candidate for biocontrol strategies in both pest management and plant disease prevention.展开更多
Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of...Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.展开更多
Basic helix-loop-helix(bHLH)transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes,and bHLH proteins also participate in immune responses,especially in plan...Basic helix-loop-helix(bHLH)transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes,and bHLH proteins also participate in immune responses,especially in plants.However,their roles in insects upon entomopathogen infection are unknown.In this study,54 bHLH genes in 41 families were identified in a polyphagous pest,Spodoptera litura,including a new bHLH gene in group B,which is specifically present in Lepidoptera and was thus named Lep.The conserved amino acids in the bHLH domain,structural architecture,and chromosomal distribution of bHLH genes in S.litura were analyzed.The bHLH genes in Plutella xylostella and Apis mellifera were also updated,and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed.The expression profiles of S.litura bHLH(SlbHLH)genes in 3 tissues at different developmental stages and their responses to S.litura nucleopolyhedrovirus(SpltNPV),Nomuraea rileyi(Nr),and Bacillus thuringiensis(Bt)infection were investigated.More SlbHLHs in group B were expressed and differentially expressed during pathogen infections,and SlbHLHs tended to be downregulated in the midgut of S.litura larvae after B.thuringiensis treatment.Our study provides an overview of bHLH family members in S.litura and their responses to different pathogens used for pest biocontrol.These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.展开更多
It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds t...It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity.However,the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear.In this study,binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied.The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs(RRM)of BmLARK are simultaneously required for the binding with the G4 structure.Either RRM1 or RRM2 alone could not bind with the G4 structure.The zinc-finger motif was not involved in the binding.A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction.The results indicated thatβsheets,especially theβ1 andβ3 sheets,in the RRM domains of BmLARK played critical roles in the binding with the G4 structure.Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1(RNP1)(motif inβ3 strand)and RNP2(motif inβ1 strand)caused loss of binding ability,indicating that these amino acids are the key sites for the binding.All the results suggest that RRM domains,particularly their the RNP1 and RNP2 motifs,play important roles not only in RNA recognition,but also in the G4 structure binding.展开更多
Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investiga...Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica;however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.展开更多
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.Bioinformatic...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 homeostasis of testis stem cell niche was disrupted,cell distribution in the apical tip was scattered,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 together,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.展开更多
20-hydroxyecdysone(20E)induced transcription factor E93 is important for larval–adult transition,which functions in programmed cell death of larval obsolete tissues,and the formation of adult new tissues.However,the ...20-hydroxyecdysone(20E)induced transcription factor E93 is important for larval–adult transition,which functions in programmed cell death of larval obsolete tissues,and the formation of adult new tissues.However,the apoptosis-related genes directly regulated by E93 are still ambiguous.In this study,an E93 mutation fly strain was obtained by clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-mediated long exon deletion to investigate whether and how E93 induces apoptosis during larval tissues metamorphosis.The transcriptional profile of E93 was consistent with 3 RHG(rpr,hid,and grim)genes and the effector caspase gene drice,and all their expressions peaked at the initiation of apoptosis during the degradation of salivary glands.The transcription expression of 3 RHG genes decreased and apoptosis was blocked in E93 mutation salivary gland during metamorphosis.In contrast,E93 overexpression promoted the transcription of 3 RHG genes,and induced advanced apoptosis in the salivary gland.Moreover,E93 not only enhance the promoter activities of the 3 RHG genes in Drosophila Kc cells in vitro,but also in the salivary gland in vivo.Our results demonstrated that 20E induced E93 promotes the transcription of RHG genes to trigger apoptosis during obsolete tissues degradation at metamorphosis in Drosophila.展开更多
Apoptosis is an important process for organism development that functions to eliminate cell damage,maintain homeostasis,and remove obsolete tissues during morphogenesis.In mammals,apoptosis is accompanied by the relea...Apoptosis is an important process for organism development that functions to eliminate cell damage,maintain homeostasis,and remove obsolete tissues during morphogenesis.In mammals,apoptosis is accompanied by the release of cytochrome C(Cyt-c)from mitochondria to the cytoplasm.However,whether this process is conserved in the fruit fly,Drosophila melanogaster,remains controversial.In this study,we discovered that during the degradation of Drosophila salivary gland,the transcription of mitochondria apoptosis factors(MAPFs),Cyt-c,and death-associated APAF1-related killer(Dark)encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes.The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation.Meanwhile,the Cyt-c protein colocates with mito-GFP,the marker indicating cytoplasmic mitochondria,and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm.Moreover,impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland,and accordingly decreases or increases the mitochondrial membrane potential,respectively.Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation,shedding light on partial conserved mechanism in apoptosis between insects and mammals.展开更多
基金supported by the National Natural Science Foundation of China(32270458,32070411,2023A1515010657)State Key Laboratory of Resource Insects(SKLSGB-ORP202209)。
文摘The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis,mitigating aminoacidemia by assimilating free amino acids,and reallocating nitrogen during metamorphosis through programmed cell death.However,the specific functions of nitrogen metabolism-related genes in this process remain unclear.Using CRISPR/Cas9-based gene editing,mutations were generated in glutamine synthetase(GS),glutamate synthetase(GOGAT),asparagine synthetase(AS),glutamate dehydrogenase(GDH)and glutamate oxaloacetate transaminase 1(GOT1).Disruption of GS,GOGAT,and AS consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription,whereas GOT1 mutation had no such effect.GOGAT mutants exhibited abnormally enlarged silk glands,whereas GS and AS mutants showed delayed programmed cell death in the silk glands.In contrast,GOT1 mutants displayed normal silk gland morphology but were consistently smaller.Disruption of GS,GOGAT,and AS led to more extensive transcriptional changes,including altered expression of transcription factors in the silk glands,compared with GOT1 mutants.Both GS and GOGAT mutants exhibited up-regulation of AS and GDH,while only GOGAT mutants displayed elevated AS enzymatic activity,suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis.AS mutants showed significantly elevated GOT activity and up-regulation of several metabolic pathways,indicating that AS may functionally interact with GOT in regulating both silk gland development and programmed cell death during metamorphosis.
基金supported by the National Natural Science Foundation of China(32070411,31720103916,31330071,31672494)Natural Science Foundation of Guangdong Province(2019A1515011012)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11010600)。
文摘The insect brain is the central part of the neurosecretory system,which controls morphology,physiology,and behavior during the insect’s lifecycle.Lepidoptera are holometabolous insects,and their brains develop during the larval period and metamorphosis into the adult form.As the only fully domesticated insect,the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process.Hormonal regulation in insects is a key factor in multiple processes.However,how juvenile hormone(JH)signals regulate brain development in Lepidoptera species,especially in the larval stage,remains elusive.We recently identified the JH receptor Methoprene tolerant 1(Met1)as a putative domestication gene.How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin’s theory on domestication.Here,CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain,unlike precocious pupation of the cuticle.At the whole transcriptomelevel,theecdysteroid(20-hydroxyecdysone,20 E)signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain.Pathways related to cell proliferation and specialization processes,such as extracellular matrix(ECM)-receptor interaction and tyrosine metabolism pathways,were suppressed in the brain.Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B.mori,which decreased transcriptional binding activity.The B.mori MET1 protein showed a changed structure and dynamic features,as well as a weakened co-expression gene network,compared with B.mandarina.Based on comparative transcriptomic analyses,we proposed a pathway downstream of JH signaling(i.e.,tyrosine metabolism pathway)that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.
基金The study was supported by the National Natural Science Foundation of China(31330071 and 31772519)and the Natural Science Foundation of Guangdong Province,China(2017A030313210).
文摘Fatty acid-bindi ng proteins(FABPs)are a family of lipid chaper on es,which con tribute to systemic metabolic regulati on through diverse lipid signalings.In this study,a midgut-specific FABP gene(Slfabp2)was cloned from Spodoptera litura.RT-PCR and Western blot analysis indicated that RNA and protein levels of S/FABP2 gradually increased and reached a peak at the prepupal stage and maintained a high level during the pupal stage.The expression of S/FABP2 protein was induced by starvation treatment.In vitro binding assay revealed that the recombinant S/FABP2 had high mffinities of binding Iong-chain fatty acids,such as palmitic acid,arachidonate and oleic acid.The results suggest that S/FABP2 may have a unique function that transports intracellular fatty acids and can regulate the metabolism of lipids in metamorphosis.This work provides experimental clues for understanding the potential function of S/FABP2 in fatty acid metabolism in S.litura.
文摘Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.
基金supported by the National Natural Science Foundation of China(31872286,32100374)。
文摘Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression.However,the mechanisms by which DNA methylation regulates these processes in insects remain unclear.Here,we studied the impacts of DNA methylation on early embryonic development in Bombyx mori.Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5'region of protein metabolism-related genes.The transcription factor gene zinc finger protein 615(ZnF615)was methylated by DNA methyltransferase 1(Dnmt1)to be up-regulated and bind to protein metabolism-related genes.Dnmt1 RNA interference(RNAi)revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615.The same sites in the ZnF615 gene were methylated in ovaries and embryos.Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout.Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo.Thus,Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.
基金This work was supported by the National Natural Science Foundation of China(32070502,31730074,32072419 and 31501905).
文摘The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.
基金supported by the National Natural Science Foundation of China (31772509, 31970438, 31471958, 31772510)the Basic Research Foundation of Shanghai Science & Technology Museum
文摘Two new species of Protura are described:Baculentulus wenyingae sp.nov.and Pseudanisentomon lishuiensis sp.nov.Baculentulus wenyingae sp.nov.is characterized by the combined characters of the presence of foretarsal sensillum b',differentiated sensilla on the maxillary palp,leaf-shaped sensillum on the labial palp,absence of P1a seta on tergites I–VI,eight anterior setae on tergite VI and 4/18 chaetotaxy on tergite VII.An updated key to the 13 Baculentulus species from China is also provided.Pseudanisentomon lishuiensis sp.nov.is characterized by the absence of foretarsal sensillum b'-2,b longer than c,the empodial appendage of II and III leg very short,the tergite I with 12 posterior setae,tergites V–VII lack seta A3,and sternites VIII–X with 2/7-4-4 chaetotaxy respectively.It was compared with the affiliated species P.guangxiensis(Yin&Zhang,1982)in detail.In addition,a checklist and the distribution of 39 Protura species from Zhejiang Province are provided,and the Protura fauna in Zhejiang is also discussed.
基金supported by the National Natural Science Foundation of China(31371286,32070411,81872299,31830094,U20A2058)Guangzhou Science Technology Project(201904010007)+1 种基金Shenzhen Science and Technology Program(JCYJ20190807160011600,JCYJ20210324124808023)Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology(GDKCFY2101)。
文摘Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis.We found significant differences in the silk gland transcriptomic landscape between the two systems,including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents.In the quaternary hybrid system,hybrid vigor was primarily due to upregulated genes and the parent-dominant upregulated expression pattern,involving multiple transport processes,cellular nitrogen compound catabolism,glucose metabolism,and tricarboxylic acid cycle.In the binary system,hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern,mainly involving basic nitrogen synthesis metabolism and body function.We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes.Thus,this study revealed two alternative mechanisms that may contribute to silkworm heterosis,both of which facilitate the efficient utilization of energy and nitrogen for silk production.
基金National Natural Science Foundation of China(Nos.32170425,32470443,32300388).
文摘Insects represent one of the most evolutionarily successful groups,with their diversity hypothesized to be related to the regulatory roles of Hox genes,a set of related genes encoding homeodomain transcription factors determining the identity of segments along the anterior-posterior axis of the embryo.However,functional insights into the roles of Hox genes in primitive ametabolous insects,which represent the critical transition from aquatic crustaceans to winged insects,have been limited.In this study,we identified complete protein-coding sequences of 10 Hox genes in the Zygentoma Thermobia domestica,and applied clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas 9)mediated gene knockout(KO)to decipher their functions.We found that the roles of pb,Dfd,and Scr are vital in specifying the appendages of the head in T.domestica,and these roles are relatively conserved in crustaceans and winged insects.Antp is essential for the development of the prothorax segment and the first pair of legs in T.domestica.Ubx and abd-A fully repress appendage development in the abdomen of T.domestica,which implies a functional switch from crustaceans to insects.Additionally,the role of ftz in segmenting the abdomen of T.domestica suggests it has acquired new functions in primitive insects,beyond its traditional Hox-like roles.Although KOs of lab,Hox3,and Abd-B did not result in obvious external phenotypic changes,they led to a significant decrease in hatching rates and substantial deviations in daily survival numbers compared to the negative control.These findings underscore the indispensable roles of all Hox genes during the embryonic development of T.domestica.Our study sheds new light on the functional evolution of Hox genes in ametabolous insects and enhances our understanding of the genetic underpinnings of insect development and diversification.
基金This research is supported by the National Key R&D Program of China(No.2019YFD1002100)National Natural Science Foundation of China(No.31901941 and No.31970474)Hunan Provincial Natural Science Foundation of China(2020JJ4178).
文摘Cadherin,aminopeptidase N(APN)and alkaline phosphatase(ALP)have been characterized as Cry receptors.In this study,comparative genomic analysis of the 3 receptor families was performed in 7 insects.ALPs and APNs are divided into three and eight clades in phylogenetic trees,respectively.ALPs in clade 3 and APNs in clade 1 contain multiple paralogs within each species and most paralogs are located closely in chromosomes.Drosophila melanogaster has expanded APNs in clade 5 and were lowly expressed in midgut.Cadherins are divided into 16 clades;they may diverge before holometabolous insect speciation except for BtR and Cad89D-like clades.Eight insects from different orders containing BtR orthologs are sensitive to Cryl A or Cry3A,while five species without BtR are insensitive to both toxins.Most APNs in clade 1,several ALPs in clade 3,BtR and Cad89D-like genes were highly or moderately expressed in larval midgut of Spodoptera litura and the other six species,and several members in these clades have been identi-•fied as Cry receptors.Expressions of putative 5.litura Cry receptors in the midgut after exposing to Bt toxins were also analyzed.
基金supported by the Natural Science Foundation of Guangdong Province(Grant Nos.2021B1515020044 and 2020A1515011267)the National Natural Science Foundation of China(Grant Nos.32070500,31930014,32100384 and 31702055)+3 种基金the Laboratory of Lingnan Modern Agriculture Project(Grant No.NT2021003)the Department of Science and Technology in Guangdong Province(Grant Nos.2019B090905003 and 2019A0102006)the Department of Science and Technology in Guangzhou(Grant No.202102020110)the Shenzhen Science and Technology Program(Grant No.KQTD20180411143628272).
文摘Regeneration,as a fascinating scientific field,refers to the ability of animals replacing lost tissue or body parts.Many metazoan organisms have been reported with the regeneration phenomena,but showing evolutionarily variable abilities.As the most diverse metazoan taxon,hundreds of insects show strong appendage regeneration ability.The regeneration process and ability are dependent on many factors,including macroscopic physiological conditions and microscopic molecular mechanisms.This article reviews research progress on the physiological conditions and internal underlying mechanisms controlling appendage regeneration in insects.
基金supported by the National Key R&D Program of China(No.2021YFD1400704)Guangdong Province Basic and Applied Basic Research Foundation of China(No.2022A1515012601).
文摘Continuous and long-term use of traditional and new pesticides can result in cross-resistance among pest populations in different fields.Study on the mechanism of cross-resistance and related genes will help resistance management and field pest control.In this study,the pesticide-resistance mechanism in Spodoptera frugiperda(FAW)was studied with field populations in 3 locations of South China.Field FAW populations were highly resistant to traditional insecticides,chlorpyrifos(organophosphate)and deltamethrin(pyrethroid),and had higher levels of cytochrome P450 activity than a non-resistant laboratory strain.Inhibition of P450 activity by piperonyl butoxide significantly increased the sensitivity of resistant FAW in 3 locations to chlorpyrifos,deltamethrin and chlorantraniliprole(amide),a new type of insecticide,suggesting that P450 detoxification is a critical factor for insecticide resistance in field FAW populations.Transcriptomic analysis indicated that 18 P450 genes were upregulated in the field FAW populations collected in 3 regions and in 2 consecutive years,with CYP321A8,the most significantly upregulated one.Knockdown of CYP321A8 messenger RNA by RNA interference resulted in an increased sensitivity to the 3 tested insecticides in the field FAW.Enzyme activity and molecular docking analyses indicated that CYP321A8 enzyme was able to metabolize the 3 tested insecticides and interact with 8 other types of insecticides,confirming that CYP321A8 is a key cross-resistance gene with a wide range of substrates in the field FAW populations across the different regions and can be used as a biomarker and target for management of FAW insecticide resistance in fields.
基金supported by the Anhui Province Natural Science Foundation(Grant No:2008085J17)the National Natural Science Foundation of China(Grant No:31872284)the Anhui Graduate Education Quality Project(Grant No:2023xscx047).
文摘Antimicrobial peptides(AMPs)are critical components of innate immunity in diverse organisms,including plants,vertebrates,and insects.This study identified and characterized a novel Lepidoptera-specific AMP,named lepidoptin,from the invasive pest Spodoptera frugiperda(Lepidoptera:Noctuidae).Lepidoptin is a 116-amino acid protein containing a signal peptide and a novelβ-sandwich domain that is distinct from previously reported AMPs.Temporal and spatial expression analyses revealed a significant upregulation of the lepidoptin gene in vivo and in cultured SF9 cells in response to pathogens.Molecular docking analysis identified a specific binding cavity.Enzymelinked immunosorbent assay and binding assays confirmed that lepidoptin can bind to pathogen-associated molecular patterns,bacteria,and fungi.Recombinant lepidoptin exhibited potent antibacterial activity by inducing bacterial agglutination,inhibiting bacterial growth,increasing bacterial membrane permeability,and preventing biofilm formation.Lepidoptin also showed antifungal activity against the entomopathogenic fungus Beauveria bassiana by inhibiting spore germination,increasing fungal cell permeability,and increasing reactive oxygen species.Injection of recombinant lepidoptin into S.frugiperda larvae increased survival after B.bassiana infection,whereas knockdown of lepidoptin by RNA interference decreased larval survival.In addition,lepidoptin showed antimicrobial activity against the plant pathogen Fusarium graminearum by inhibiting spore germination and alleviating disease symptoms in wheat seedlings and cherry tomatoes.This study demonstrates the remarkable dual functionality of lepidoptin in enhancing S.frugiperda immunity and controlling plant pathogens,making it a promising candidate for biocontrol strategies in both pest management and plant disease prevention.
基金the National Natural Science Foundation of China (Grant No. 31330071,31772519)Natural Science Foundation of Guangdong Province (Grant No. S2012010010185,2017A030313210).
文摘Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
基金This research was funded by grants from the National Key R&D Program of China(No,2019YFD1002100)National Natural Science Foundation of China(No.31901941 and No.31970474)+1 种基金Natural Science Foun-dation of Guangdong Province(No.202102020966)China Postdoctoral Science Foundation(No.2020M682752).The authors are grateful for the generous gift of S.litura nucleopolyhedrovirus(SpItNPV)from Professor Kai Yang at Sun Yat-Sen University,Nomuruea rileyi(Nr06)from Professor Xusheng Liu at Central China Normal University,and Bacillus thuringiensis(WB9)from Professor Xiong Guan at Fujian Agriculture and Forestry University.
文摘Basic helix-loop-helix(bHLH)transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes,and bHLH proteins also participate in immune responses,especially in plants.However,their roles in insects upon entomopathogen infection are unknown.In this study,54 bHLH genes in 41 families were identified in a polyphagous pest,Spodoptera litura,including a new bHLH gene in group B,which is specifically present in Lepidoptera and was thus named Lep.The conserved amino acids in the bHLH domain,structural architecture,and chromosomal distribution of bHLH genes in S.litura were analyzed.The bHLH genes in Plutella xylostella and Apis mellifera were also updated,and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed.The expression profiles of S.litura bHLH(SlbHLH)genes in 3 tissues at different developmental stages and their responses to S.litura nucleopolyhedrovirus(SpltNPV),Nomuraea rileyi(Nr),and Bacillus thuringiensis(Bt)infection were investigated.More SlbHLHs in group B were expressed and differentially expressed during pathogen infections,and SlbHLHs tended to be downregulated in the midgut of S.litura larvae after B.thuringiensis treatment.Our study provides an overview of bHLH family members in S.litura and their responses to different pathogens used for pest biocontrol.These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.
基金This work was supported by the grants of the Chinese National Natural Science Foundation(Grant no.:31672494,31720103916,31930102)。
文摘It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity.However,the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear.In this study,binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied.The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs(RRM)of BmLARK are simultaneously required for the binding with the G4 structure.Either RRM1 or RRM2 alone could not bind with the G4 structure.The zinc-finger motif was not involved in the binding.A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction.The results indicated thatβsheets,especially theβ1 andβ3 sheets,in the RRM domains of BmLARK played critical roles in the binding with the G4 structure.Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1(RNP1)(motif inβ3 strand)and RNP2(motif inβ1 strand)caused loss of binding ability,indicating that these amino acids are the key sites for the binding.All the results suggest that RRM domains,particularly their the RNP1 and RNP2 motifs,play important roles not only in RNA recognition,but also in the G4 structure binding.
基金This work was supported by the National Natural Science Foundation of China(Grant No.31772533).
文摘Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica;however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.
基金This work was supported by the National Natural Science Foundation of China(No.31970474 and No.32272606)China Postdoctoral Science Foundation(No.2022M721219).
文摘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 homeostasis of testis stem cell niche was disrupted,cell distribution in the apical tip was scattered,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 together,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.
基金the National Science Foundation of China(Grants No.32070491 to Kang Li)Laboratory of Lingnan Modern Agriculture Project(NT2021003 to Sheng Li)the National Science Foundation of Guangdong Province(Grants No.2022A1515010457 to Kang Li),English was polished by the Nature Publishing Group.
文摘20-hydroxyecdysone(20E)induced transcription factor E93 is important for larval–adult transition,which functions in programmed cell death of larval obsolete tissues,and the formation of adult new tissues.However,the apoptosis-related genes directly regulated by E93 are still ambiguous.In this study,an E93 mutation fly strain was obtained by clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-mediated long exon deletion to investigate whether and how E93 induces apoptosis during larval tissues metamorphosis.The transcriptional profile of E93 was consistent with 3 RHG(rpr,hid,and grim)genes and the effector caspase gene drice,and all their expressions peaked at the initiation of apoptosis during the degradation of salivary glands.The transcription expression of 3 RHG genes decreased and apoptosis was blocked in E93 mutation salivary gland during metamorphosis.In contrast,E93 overexpression promoted the transcription of 3 RHG genes,and induced advanced apoptosis in the salivary gland.Moreover,E93 not only enhance the promoter activities of the 3 RHG genes in Drosophila Kc cells in vitro,but also in the salivary gland in vivo.Our results demonstrated that 20E induced E93 promotes the transcription of RHG genes to trigger apoptosis during obsolete tissues degradation at metamorphosis in Drosophila.
基金This study was supported by Laboratory of Lingnan Modern Agriculture Project(NT2021003 to S.Li)the National Natural Science Foundation of China(Grants No.32070491 to K.Li and 31900355 to N.Li)the National Science Foundation of Guangdong Province(Grants No.2022A1515010457 to K.Li and 2022A1515011759 to N.Li).English was edited by the Nature Publishing Group.
文摘Apoptosis is an important process for organism development that functions to eliminate cell damage,maintain homeostasis,and remove obsolete tissues during morphogenesis.In mammals,apoptosis is accompanied by the release of cytochrome C(Cyt-c)from mitochondria to the cytoplasm.However,whether this process is conserved in the fruit fly,Drosophila melanogaster,remains controversial.In this study,we discovered that during the degradation of Drosophila salivary gland,the transcription of mitochondria apoptosis factors(MAPFs),Cyt-c,and death-associated APAF1-related killer(Dark)encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes.The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation.Meanwhile,the Cyt-c protein colocates with mito-GFP,the marker indicating cytoplasmic mitochondria,and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm.Moreover,impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland,and accordingly decreases or increases the mitochondrial membrane potential,respectively.Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation,shedding light on partial conserved mechanism in apoptosis between insects and mammals.
