C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity ...C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity in insects.In this study,we report a novel dual carbohydrate-recognition domain(CRD)CTL from Plutella xylostella which we designate PxIML.PxIML is a protein with a 969 bp open reading frame(ORF)encoding 322 amino acids,containing a signal peptide and a dual-CRD with EPN(Glu_(124)-Pro_(125)-Asn_(126))and QPD(Gln_(274)-Pro_(275)-Asp_(276))motifs.The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut.The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010(Bt8010)at 18 h,while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris.The recombinant PxIML(rPxIML)protein could bind to the tested pathogen-associated molecular patterns(PAMPs),and the bacteria of Enterobacter sp.IAE5,S.marcescens IAE6,Staphylococcus aureus,Escherichia coli BL21,and Bt8010 in a Ca^(2+)-dependent manner,however,it showed limited binding to the fungus,P.pastoris.The rPxIML exhibited strong activity in the presence of Ca^(2+) to agglutinate Bt8010,Enterobacter sp.IAE5 and S.aureus,but it only weakly agglutinated with E.coli BL21,and could not agglutinate with S.marcescens IAE6 or P.pastoris.Furthermore,the rPxIML could bind to hemocytes,promote the adsorption of hemocytes to beads,and enhance the phenoloxidase(PO)activity and melanization of P.xylostella.Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P.xylostella.展开更多
Orphan genes are genes with no sequence homologues in other species. Here, we identified two orphan genes, namely, Tssor-3 and Tssor-4, in Plutella xylostella. Both genes contained a signal peptide sequence, suggestin...Orphan genes are genes with no sequence homologues in other species. Here, we identified two orphan genes, namely, Tssor-3 and Tssor-4, in Plutella xylostella. Both genes contained a signal peptide sequence, suggesting their functions as secreted proteins. Expression pattern analysis based on real-time quantitative PCR(qPCR) showed that both orphan genes were specifically expressed in all male gonads except the testes. The expression of both the orphan genes peaked at the male adult stage. Immunofluorescence assays suggested that the two proteins were seminal proteins, indicating their potential roles in male reproductive regulation. To further explain their functions, we knocked down the expression of these two genes by RNA interference(RNAi). The results showed that the expression of Tssor-3 and Tssor-4 was significantly downregulated at 24 h after injection compared to that of the controls. Biological assays showed that the number of laid eggs and the hatching rate of offspring eggs were significantly reduced when the expression of Tssor-3 and Tssor-4 was reduced, suggesting that the two orphan genes played a role in male fertility in P. xylostella. Our results provide evidence that orphan genes are involved in male reproductive regulation, which is important for male fitness during evolution.展开更多
Earth's rotation shapes a 24-h cycle,governing circadian rhythms in organisms.In mammals,the core clock genes,CLOCK and BMAL1,are regulated by PERIODs(PERs)and CRYPTOCHROMEs(CRYs),but their roles remain unclear in...Earth's rotation shapes a 24-h cycle,governing circadian rhythms in organisms.In mammals,the core clock genes,CLOCK and BMAL1,are regulated by PERIODs(PERs)and CRYPTOCHROMEs(CRYs),but their roles remain unclear in the diamondback moth,Plutella xylostella.To explore this,we studied P.xylostella,which possesses a simplified circadian system compared to mammals.In P.xylostella,we observed rhythmic expressions of the Pxper and Pxcry2 genes in their heads,with differing phases.In vitro experiments revealed that PxCRY2 repressed monarch butterfly CLK:BMAL1 transcriptional activation,while PxPER and other CRY-like proteins did not.However,PxPER showed an inhibitory effect on PxCLK/PxCYCLE.Using CRISPR/Cas9,we individually and in combination knocked out Pxper and Pxcry2,then conducted gene function studies and circadian transcriptome sequencing.Loss of either Pxper or Pxcry2 eliminated the activity peak after lights-off in light–dark cycles,and Pxcry2 loss reduced overall activity.Pxcry2 was crucial for maintaining endogenous rhythms in constant darkness.Under light–dark conditions,1098 genes exhibited rhythmic expression in wild-type P.xylostella heads,with 749 relying on Pxper and Pxcry2 for their rhythms.Most core clock genes lost their rhythmicity in Pxper and Pxcry2 mutants,while Pxcry2 sustained rhythmic expression,albeit with reduced amplitude and altered phase.Additionally,rhythmic genes were linked to biological processes like the spliceosome and Toll signaling pathway,with these rhythms depending on Pxper or Pxcry2 function.In summary,our study unveils differences in circadian rhythm regulation by Pxper and Pxcry2 in P.xylostella.This provides a valuable model for understanding circadian clock regulation in nocturnal animals.展开更多
The diamondback moth,Plutella xylostella(L.),is an economically important pest of cruciferous crops worldwide.This pest is notorious for rapid evolution of the resistance to diferent classes of insecticides,making it ...The diamondback moth,Plutella xylostella(L.),is an economically important pest of cruciferous crops worldwide.This pest is notorious for rapid evolution of the resistance to diferent classes of insecticides,making it increasingly dificult to control.Genetics-based control approaches,through manipulation of target genes,have been reported as promising supplements or alternatives to traditional methods of pest management.Here we identified a gene of pigmentation(yellow)in P.xylostella,Pxyellow,which encodes 1674 bp complementary DNA sequence with four exons and three introns.Using the clustered regularly interspersed palindromic repeats(CRISPR)CRISPR-associated protein 9 system,we knocked out Pxyellow,targeting two sites in Exon III,to generate 272 chimeric mutants(57%of the CRISPR-treated individuals)with color-changed phenotypes of the Ist to 3rd instar larvae,pupae,and adults,indicating that Pxyellow plays an essential role in the body pigmentation of P xlostella.Fitness analysis revealed no significant difference in the oviposition of adults,the hatchability of eggs,and the weight of pupac between homozygous mutants and wildtypes,suggesting that Pxyellow is not directly involved in regulation of growth,development,or reproduction.This work advances our understanding of the genetic and insect science molecular basis for body pigmentation of P xylostella,and opens a wide avenue for development of the genctcally based pest control techniques using Pxyellow as a screening marker.展开更多
Odorant binding proteins(OBPs)are a group of soluble proteins functioning as odorant carriers in insect antennae,mouth parts and other chemosensory organs.However,multiple insect OBPs have been detected in other tissu...Odorant binding proteins(OBPs)are a group of soluble proteins functioning as odorant carriers in insect antennae,mouth parts and other chemosensory organs.However,multiple insect OBPs have been detected in other tissues and various functions have been proposed.Therefore,a detailed expression profile including stages,tissues and sexes where OBPs are expressed will assist in building the links to their potential functions,enhancing the functional studies of insect OBPs.Here,we identified 39 putative OBP genes from its genome and transcriptome sequences of diamondback moth(DBM),Plutella xylostella.The expression patterns of identified PxylOBPs were further investigated from eggs,larvae,pupae,virgin adults,mated adults,larval midgut,larval heads,adult antennae,adult heads and adult tarsi.Moreover,P.xylostella larvae and adults with and without host plants for 5 h were utilized to study the interactions between OBP expression and host plants.The results showed that most PxylOBPs were highly expressed in male and female adult antennae.The expression levels of certain PxyOBPs could be regulated by mating activities and feeding host plants.This study advances our knowledge of P.xylostella OBPs,which may help develop new strategies for more environmentally sustainable management of P.xylostella.展开更多
Circadian clocks control the rhythmicity of many behaviors and physiological features of insects.To study the circadian clock of the moth Plutella xylostella,we employed CRISPR/Cas9-mediated genome editing to investig...Circadian clocks control the rhythmicity of many behaviors and physiological features of insects.To study the circadian clock of the moth Plutella xylostella,we employed CRISPR/Cas9-mediated genome editing to investigate the effect of loss of the clock gene period on the circadian rhythms.P.xylostella harbors a single copy of period.Phylogenetic analysis showed that P.xylostella PERIOD is more homologous to mouse PERIOD than the PERIOD proteins from bees,flies,mosquitos,and many other Lepidoptera,such as Danaus plexippus and Bombyx mori.The circadian rhythms in adult locomotor activity were altered in the period knockout strain of P.xylostella under light–dark(LD)and continuous dark(DD)conditions.Under the LD cycle,the wild-type moths displayed nocturnal activity with activity peaking very early after lights off and quickly declining after lights on.In contrast,the period knockout strain had no peak in activity when the lights were turned off and exhibited steady activity throughout the hours of darkness.Interestingly,under DD conditions,our results showed that the locomotor rhythm can be maintained without period gene,but at a lower rhythmicity ratio than wild-type.In addition,knockout of period in P.xylostella changed circadian rhythms patterns related to pupal eclosion,mating,egg-laying,and egg hatching.Mechanistically,loss of PERIOD disrupted the molecular rhythm of period and changed the clock transcription rhythm in the heads of the moths under LD and DD conditions.Together,our study indicates that the PERIOD is required for normal expression of many behavioral rhythms in P.xylostella.展开更多
Cryptochrome 1(CRY1)functions as a light-responsive photoreceptor,which is crucial for circadian rhythms.The identity and function of CRY1 in Plutella xylostella remain unknown.In this study,cryl was cloned and identi...Cryptochrome 1(CRY1)functions as a light-responsive photoreceptor,which is crucial for circadian rhythms.The identity and function of CRY1 in Plutella xylostella remain unknown.In this study,cryl was cloned and identified in P xylostella.Then,a cry1-knockout strain(Cry1-KO)of P xylostella with a 2-bp deletion was established from the strain Geneva 88(G88)using the CRISPR/Cas9 technology.No daily temporal os-cillation of cryl was observed in G88 and Cry1-KO,and cryl mean daily transcription of Cry1-KO was lower than that of G88.Both G88 and Cry1-KO demonstrated rhythmic locomotion under the light/dark condition with Cryl-KO being more active than G88 in the daytime,whereas Cry1-KO completely lost rhythmicity under constant darkness.The developmental period of pre-adult of Cry1-KO was longer than that of G88;the lifespan of the Cry1-KO male adult was shorter than that of G88;the fecundity of Cry1-KO was lower than that of G88;and Cry1-KO showed lower intrinsic rate of increase(r),net repro-duction rate(Ro),finite increase rate(a),and longer mean generation time(T)than G88.Our results indicate that cryl is involved in the regulation of locomotor circadian rhythm and development in P xylostella,providing a potential target gene for controlling the pest and a basis for further investigation on circadian rhythms in lepidopterans.展开更多
基金the project of the National Key R&D Program of China(2017YFE0122000)the National Natural Science Foundation of China(31871968)the Natural Science Foundation of Fujian Province,China(2018J01614).
文摘C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity in insects.In this study,we report a novel dual carbohydrate-recognition domain(CRD)CTL from Plutella xylostella which we designate PxIML.PxIML is a protein with a 969 bp open reading frame(ORF)encoding 322 amino acids,containing a signal peptide and a dual-CRD with EPN(Glu_(124)-Pro_(125)-Asn_(126))and QPD(Gln_(274)-Pro_(275)-Asp_(276))motifs.The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut.The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010(Bt8010)at 18 h,while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris.The recombinant PxIML(rPxIML)protein could bind to the tested pathogen-associated molecular patterns(PAMPs),and the bacteria of Enterobacter sp.IAE5,S.marcescens IAE6,Staphylococcus aureus,Escherichia coli BL21,and Bt8010 in a Ca^(2+)-dependent manner,however,it showed limited binding to the fungus,P.pastoris.The rPxIML exhibited strong activity in the presence of Ca^(2+) to agglutinate Bt8010,Enterobacter sp.IAE5 and S.aureus,but it only weakly agglutinated with E.coli BL21,and could not agglutinate with S.marcescens IAE6 or P.pastoris.Furthermore,the rPxIML could bind to hemocytes,promote the adsorption of hemocytes to beads,and enhance the phenoloxidase(PO)activity and melanization of P.xylostella.Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P.xylostella.
基金supported by the Natural Science Foundation of Fujian Province, China (2020J01525)the National Natural Science Foundation of China (31320103922 and 31230061)the Major Science and Technology Projects in Fujian Province, China (2018NZ010100130)。
文摘Orphan genes are genes with no sequence homologues in other species. Here, we identified two orphan genes, namely, Tssor-3 and Tssor-4, in Plutella xylostella. Both genes contained a signal peptide sequence, suggesting their functions as secreted proteins. Expression pattern analysis based on real-time quantitative PCR(qPCR) showed that both orphan genes were specifically expressed in all male gonads except the testes. The expression of both the orphan genes peaked at the male adult stage. Immunofluorescence assays suggested that the two proteins were seminal proteins, indicating their potential roles in male reproductive regulation. To further explain their functions, we knocked down the expression of these two genes by RNA interference(RNAi). The results showed that the expression of Tssor-3 and Tssor-4 was significantly downregulated at 24 h after injection compared to that of the controls. Biological assays showed that the number of laid eggs and the hatching rate of offspring eggs were significantly reduced when the expression of Tssor-3 and Tssor-4 was reduced, suggesting that the two orphan genes played a role in male fertility in P. xylostella. Our results provide evidence that orphan genes are involved in male reproductive regulation, which is important for male fitness during evolution.
基金supported by the key project of the Natural Science Foundation of Fujian Province(2023J02009)supported by the National Natural Science Foundation of China(31970461)+1 种基金the Natural Science Foundation of Fujian Province(2020J02027)the Qi-Shan scholar grant of Fuzhou University(GXRC-20070).
文摘Earth's rotation shapes a 24-h cycle,governing circadian rhythms in organisms.In mammals,the core clock genes,CLOCK and BMAL1,are regulated by PERIODs(PERs)and CRYPTOCHROMEs(CRYs),but their roles remain unclear in the diamondback moth,Plutella xylostella.To explore this,we studied P.xylostella,which possesses a simplified circadian system compared to mammals.In P.xylostella,we observed rhythmic expressions of the Pxper and Pxcry2 genes in their heads,with differing phases.In vitro experiments revealed that PxCRY2 repressed monarch butterfly CLK:BMAL1 transcriptional activation,while PxPER and other CRY-like proteins did not.However,PxPER showed an inhibitory effect on PxCLK/PxCYCLE.Using CRISPR/Cas9,we individually and in combination knocked out Pxper and Pxcry2,then conducted gene function studies and circadian transcriptome sequencing.Loss of either Pxper or Pxcry2 eliminated the activity peak after lights-off in light–dark cycles,and Pxcry2 loss reduced overall activity.Pxcry2 was crucial for maintaining endogenous rhythms in constant darkness.Under light–dark conditions,1098 genes exhibited rhythmic expression in wild-type P.xylostella heads,with 749 relying on Pxper and Pxcry2 for their rhythms.Most core clock genes lost their rhythmicity in Pxper and Pxcry2 mutants,while Pxcry2 sustained rhythmic expression,albeit with reduced amplitude and altered phase.Additionally,rhythmic genes were linked to biological processes like the spliceosome and Toll signaling pathway,with these rhythms depending on Pxper or Pxcry2 function.In summary,our study unveils differences in circadian rhythm regulation by Pxper and Pxcry2 in P.xylostella.This provides a valuable model for understanding circadian clock regulation in nocturnal animals.
基金the National Natural Science Foundation of China(31972271)the Strait Postdoctoral Exchange Program of Fujian(2018B002)Fujian Science and Technology Major Program(2018NZ01010013).
文摘The diamondback moth,Plutella xylostella(L.),is an economically important pest of cruciferous crops worldwide.This pest is notorious for rapid evolution of the resistance to diferent classes of insecticides,making it increasingly dificult to control.Genetics-based control approaches,through manipulation of target genes,have been reported as promising supplements or alternatives to traditional methods of pest management.Here we identified a gene of pigmentation(yellow)in P.xylostella,Pxyellow,which encodes 1674 bp complementary DNA sequence with four exons and three introns.Using the clustered regularly interspersed palindromic repeats(CRISPR)CRISPR-associated protein 9 system,we knocked out Pxyellow,targeting two sites in Exon III,to generate 272 chimeric mutants(57%of the CRISPR-treated individuals)with color-changed phenotypes of the Ist to 3rd instar larvae,pupae,and adults,indicating that Pxyellow plays an essential role in the body pigmentation of P xlostella.Fitness analysis revealed no significant difference in the oviposition of adults,the hatchability of eggs,and the weight of pupac between homozygous mutants and wildtypes,suggesting that Pxyellow is not directly involved in regulation of growth,development,or reproduction.This work advances our understanding of the genetic and insect science molecular basis for body pigmentation of P xylostella,and opens a wide avenue for development of the genctcally based pest control techniques using Pxyellow as a screening marker.
基金We are very grateful for the financial support from the National Natural Science Foundation of China(31320103922,31301666)Dr Wei Xu is the recipient of an Australian Research Council Discovery Early Career Researcher Award(DECRA)(DE160100382).
文摘Odorant binding proteins(OBPs)are a group of soluble proteins functioning as odorant carriers in insect antennae,mouth parts and other chemosensory organs.However,multiple insect OBPs have been detected in other tissues and various functions have been proposed.Therefore,a detailed expression profile including stages,tissues and sexes where OBPs are expressed will assist in building the links to their potential functions,enhancing the functional studies of insect OBPs.Here,we identified 39 putative OBP genes from its genome and transcriptome sequences of diamondback moth(DBM),Plutella xylostella.The expression patterns of identified PxylOBPs were further investigated from eggs,larvae,pupae,virgin adults,mated adults,larval midgut,larval heads,adult antennae,adult heads and adult tarsi.Moreover,P.xylostella larvae and adults with and without host plants for 5 h were utilized to study the interactions between OBP expression and host plants.The results showed that most PxylOBPs were highly expressed in male and female adult antennae.The expression levels of certain PxyOBPs could be regulated by mating activities and feeding host plants.This study advances our knowledge of P.xylostella OBPs,which may help develop new strategies for more environmentally sustainable management of P.xylostella.
基金Wenfeng Chen's lab is supported by the Qi-Shan scholar grant of Fuzhou University(GXRC-20070)the National Natural Science Foundation of China(31970461)+1 种基金the Natural Science Foundation of Fujian Province(2020J02027)Guang Yang's work is supported by the National Natural Science Foundation of China(grant number 31772237).
文摘Circadian clocks control the rhythmicity of many behaviors and physiological features of insects.To study the circadian clock of the moth Plutella xylostella,we employed CRISPR/Cas9-mediated genome editing to investigate the effect of loss of the clock gene period on the circadian rhythms.P.xylostella harbors a single copy of period.Phylogenetic analysis showed that P.xylostella PERIOD is more homologous to mouse PERIOD than the PERIOD proteins from bees,flies,mosquitos,and many other Lepidoptera,such as Danaus plexippus and Bombyx mori.The circadian rhythms in adult locomotor activity were altered in the period knockout strain of P.xylostella under light–dark(LD)and continuous dark(DD)conditions.Under the LD cycle,the wild-type moths displayed nocturnal activity with activity peaking very early after lights off and quickly declining after lights on.In contrast,the period knockout strain had no peak in activity when the lights were turned off and exhibited steady activity throughout the hours of darkness.Interestingly,under DD conditions,our results showed that the locomotor rhythm can be maintained without period gene,but at a lower rhythmicity ratio than wild-type.In addition,knockout of period in P.xylostella changed circadian rhythms patterns related to pupal eclosion,mating,egg-laying,and egg hatching.Mechanistically,loss of PERIOD disrupted the molecular rhythm of period and changed the clock transcription rhythm in the heads of the moths under LD and DD conditions.Together,our study indicates that the PERIOD is required for normal expression of many behavioral rhythms in P.xylostella.
基金funded by the National Key R&D Program of China,grant number 2017YFD0200400the Special Key Project of Fujian Province,grant number 2018NZ01010013,and a fund of"111"program,grant numberKRA16001A.
文摘Cryptochrome 1(CRY1)functions as a light-responsive photoreceptor,which is crucial for circadian rhythms.The identity and function of CRY1 in Plutella xylostella remain unknown.In this study,cryl was cloned and identified in P xylostella.Then,a cry1-knockout strain(Cry1-KO)of P xylostella with a 2-bp deletion was established from the strain Geneva 88(G88)using the CRISPR/Cas9 technology.No daily temporal os-cillation of cryl was observed in G88 and Cry1-KO,and cryl mean daily transcription of Cry1-KO was lower than that of G88.Both G88 and Cry1-KO demonstrated rhythmic locomotion under the light/dark condition with Cryl-KO being more active than G88 in the daytime,whereas Cry1-KO completely lost rhythmicity under constant darkness.The developmental period of pre-adult of Cry1-KO was longer than that of G88;the lifespan of the Cry1-KO male adult was shorter than that of G88;the fecundity of Cry1-KO was lower than that of G88;and Cry1-KO showed lower intrinsic rate of increase(r),net repro-duction rate(Ro),finite increase rate(a),and longer mean generation time(T)than G88.Our results indicate that cryl is involved in the regulation of locomotor circadian rhythm and development in P xylostella,providing a potential target gene for controlling the pest and a basis for further investigation on circadian rhythms in lepidopterans.
