Functional identification of C-type lectin in the diamondback moth,Plutella xylostella(L.)innate 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.

Functional analysis of the orphan genes Tssor-3 and Tssor-4 in male Plutella xylostella

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.

Identification of chorion genes and RNA interference-mediated functional characterization of chorion-1 in Plutella xylostella

Choriogenesis is the last step of insect oogenesis,a process by which the chorion polypeptides are produced by the follicular cells and deposited on the surface of oocytes in order to provide a highly specialized protective barrier to the embryo.The essential features of chorion genes have yet to be clearly understood in the diamondback moth,Plutella xylostella,a worldwide Lepidoptera pest attacking cruciferous crops and wild plants.In this study,complete sequences for 15 putative chorion genes were identified,and grouped into A and B classes.Phylogenetic analysis revealed that both classes were highly conserved and within each,branches are also species-specific.Chorion genes from each class were located in pairs on scaffolds of the P.xylostella genome,some of which shared the common promoter regulatory region.All chorion genes were highly specifically expressed in the P.xylostella adult females,mostly in the ovary with full yolk,which is a crucial period to build the shells of the eggs.RNAi-based knockdown of chorion-1,which is located on the Px_scaffold 6 alone,although had no effect on yolk deposition,resulted in smaller eggs and sharply reduced hatchability.Additionally,inhibition of PxCho-1 expression caused a less dense arrangement of the columnar layers,reduced exochorion roughness and shorter microvilli.Our study provides the foundation for exploring molecular mechanisms of female reproduction in P.xylostella,and for making use of chorion genes as the potential genetic-based molecular target to better control this economically important pest.

Knockout of cryptochrome 1 disturbs the locomotor circadian rhythm and development of Plutella xylostella

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.

CRISPR/Cas9-mediated knockout of period reveals its function in the circadian rhythms of the diamondback moth Plutella xylostella

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.

Cell Iines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in R xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from IS to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhe. drovirus from Autographa californica, AcMNPV four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the R xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1、was detected. The R xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.

Structure and expression of sulfatase and sulfatase modifying factor genes in the diamondback moth,Plutella xylostella

The diamondback moth,Plutella xylostella (L.),uses sulfatases (SULF)to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding.Sulfatase activity is regulated by post-translational modi- fication of a cysteine residue by sulfatase modifying factor 1(SUMF1).We identified 12 SULF genes (PxylSulfs)and two SUMF1 genes (PxylSumfls)in the P.xylostella genome. Phylogenetic analysis of SULFs and SUMFs from P.xylostella,Bombyx mori,Manduca sexta,Heliconius melpomene,Danaus plexippus,Drosophila melanogaster,Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups,and the SUMFs could be divided into two groups.Profiling of the expression of PxylSulfs and Pxyl Sumfs by RNA-seq and by quantitative real-time polymerase chain reaction showed that two glucosinolate sulfatase genes (GSS),PxylSulf2and PxylSulf3,were primarily expressed in the midgut of 3rd-and 4th-instar larvae.Moreover,expression of sulfatases PxylSulf2, PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying fac tor PxylSumfla.The findings from this study provide new insights into the structure and expression of SUMF1and PxylSulf genes that are considered to be key factors for the evolutionary success ofP.xylostella as a specialist herbivore of cruciferous plants.

Chinese species of the genus Prochiloneurus Silvestri with description of a new species(Hymenoptera: Encyrtidae)

Four species of the genus Prochiloneurus Silvestri, belonging to the family Encyrtidae of Hymenoptera, are reported from China. Among them, P. stenopterus sp. nov., which is reared as the hyperparasitoid of Phenacoccus solenopsis Tinsley （Hemiptera： Pseudococcidae）, an invasive mealybug to China, is reported as new to science. A key of the genus is provided for the recognition of the females of Chinese species. Notes on the parasitoid and hyperparasitoid of the mealybug Phenacoccus solenopsis are provided.

Identification and characterization of odorant binding proteins in the diamondback moth,Plutella xylostella

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.

Population characteristics of Macrocheles glaber (Acari: Macrochelidae) and Stratiolaelaps scimitus (Acari: Laelapidae) reared on a mushroom fly Coboldia fuscipes (Diptera: Scatopsidae)

Subterranean predatory mites are important biological control agents of pests in soil. In order to understand the population characteristics of two predatory mites, Macrocheles glaber Miiller and Stratiolaelaps scimitus Womersley, we studied their development, survival and fecundity data under laboratory conditions using Coboldia fuscipes Meigen as a food source and analyzed them with the age-stage, two-sex life table. Macrocheles glaber had a significantly shorter developmental time, oviposition period, longevity and lower fecundity than those of S. scimitus. The intrinsic rate of increase (λ), finite rate of increase (r), net reproductive rate (C0),net predation rate (C0), and finite predation rate (ω) of M. glaber were significantly lower than those of S. scimitus. Both population parameters and computer simulation implied that S. scimitus is a potential powerful biocontrol agent compared to M. glaber.

CRISPR/Cas9-based functional analysis of yellow gene in the diamondback moth,Plutella xylostella

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.

FgRab5 and FgRab7 are essential for endosomes biogenesis and nonredundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex,composed of the cargo-selective complex(CSC)Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17,mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells.Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation,budding,transport,tethering,docking and fusion with target membranes.Herein,we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches.We found that only FgRab5(FgRab5A and FgRab5B)and FgRab7 associate with the retromer complex.Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm ofΔFgrab5A,ΔFgrab5B andΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type.FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes.Most strikingly,we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells,while they were very clearly seen in the wild-type PH-1.Furthermore,FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes,whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes.Thus,we conclude that the Rab proteins FgRab5A,FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F.graminearum.