A viral movement protein targets host catalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat

The infection of host plants by many different viruses causes reactive oxygen species(Ros)accumulation and yellowing symptoms,but the mechanisms through which plant viruses counteract RoS-mediated immunity to facilitate infection and symptom development have not been fully elucidated.Most plant viruses are transmitted by insect vectors in the field,but the molecular mechanisms underlying virus-host-insect interactions are unclear.In this study,we investigated the interactions among wheat,barley yellow dwarf virus(BYDV),and its aphid vector and found that the BYDV movement protein(MP)interacts with both wheat catalases(CATs)and the 26S proteasomeubiquitin receptor non-ATPase regulatorysubunit2homolog(PSMD2)to facilitate the 26S proteasome-mediateddegradation of CATs,promotingviral infection,disease symptom development,and aphid transmission.Overexpression of the BYDV MP gene in wheat enhanced the degradation of CATs,which leading to increased accumulation of ROS and thereby enhanced viral infection.Interestingly,transgenic wheat lines overexpressing BYDV MP showed significantly reduced proliferation of wingless aphids and an increased number of winged aphids.Consistent with this observation,silencing of CAT genes also enhanced viral accumulation and reduced the proliferation of wingless aphids but increased the occurrence of winged aphids.In contrast,transgenic wheat plants overexpressing TaCAT1 exhibited the opposite changes and showed increases in grain size and weight upon infection with BYDV.Biochemical assays demonstrated that BYDV MP interacts with PSMD2 and promotes 26S proteasome-mediated degradation of TaCAT1 likely in a ubiquitination-independent manner.Collectively,our study reveals a molecular mechanism by which a plant virus manipulates the Ros production system of host plants to facilitate viral infection and transmission,shedding new light on the sophisticated interactions among viruses,host plants,and insect vectors.

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation role of DSC1 under insecticide stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and we found that HA has excellent insecticidal activity and is worthy of further study as a botanical insecticide. Herein, we performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1/DSC1−/−, electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w1118, parats1, and DSC1−/− strains. Moreover, the neuromuscular synapse excitatory activities of parats1/DSC1−/− larvae were more sensitive to HA than DSC1−/− larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, our study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle.Lipophorin loads and transports lipids,including hydrocarbons,from one tissue to another.In some insects,the lipophorin receptor(LpR),which binds to lipophorin and accepts its lipid cargo,is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes.However,it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown.We herein present the results of our molecular characterization,phylogenetic analysis,and functional annotation of the pea aphid(Acyrthosiphon pisum)LpR gene(ApLpR).This gene was transcribed throughout the A.pisum life cycle,but especially during the embryonic stage and in the abdominal cuticle.Furthermore,we optimized the RHA interference(RNAi)parameters by determining the ideal dose and duration for gene silencing in the pea aphid.We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity.Additionally,a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress,with decreased survival rates under simulated drought conditions.Moreover,ApLpR expression levels significantly increased in response to the desiccation treatment.These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress.Furthermore,this gene is vital for aphid reproduction.Therefore,the ApLpR gene of A.pisum may be a novel RNAi target relevant for insect pest management.

Two putative fatty acid synthetic genes of BgFas3 and BgElo1 are responsible for respiratory waterproofing in Blattella germanica

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.

Aspartate-β-alanine-NBAD pathway regulates pupal melanin pigmentation plasticity of ladybird Harmonia axyridis

Phenotypic plasticity is observed in many animal species and it is effective for them to cope with many types of environmental threats.The multicolored Asian ladybird Harmonia axyridis shows a cuticular pigmentation plasticity that can be rapidly induced by temperature changes,and in the form of changeable melanin spot patterns to adjust heat-absorbing.Here,H.axyridis with thermal stimulation were selected for determining the molecular regulations behind it.First,we confirmed the melanin level changes of H.axyridis pupa could be induced by temperature,and then screened the efficient time window for thermal sensing of H.axyridis pre-pupa;it is suggested that the late stage of pre-pupa(late stage of 4th instar larva)is the critical period to sense thermal signals and adjust its pupal melanin spot area size to adapt to upcoming thermal conditions.The Ha-ADC(aspartate decarboxylase)and Ha-ebony(NBAD synthase)of aspartate-β-alanine-NBAD pathway were then proved in regulation of cuticular melanization for pupa through RNA interference experiments;knockdown of these two genes enlarged the melanin spot size.Finally,we designed a random injection of Ha-ADC at different pre-pupal stages,to further study the regulation window during this process.Combined with all evidence observed,we suggested the spot size determination can be regulated very close to the time point of pupation,and genes of the aspartate-β-alanine-NBAD pathway play an important role at the molecular level.In brief,H.axyridis exhibits a flexible active physiological regulation through transcriptional modification to thermal changes.

Acetyl coenzyme A carboxylase modulates lipogenesis and sugar homeostasis in Blattella germanica

Lipid and sugar homeostasis is critical for insect development and survival.In this study,we characterized an acetyl coenzyme A carboxylase gene in Blattella germanica(BgACC)that is involved in both lipogenesis and sugar homeostasis.We found that BgACC was dominantly expressed in the fat body and integument,and was significantly upregulated after molting.Knockdown of BgACC in 5th-instar nymphs did not affect their normal molting to the next nymphal stage,but it caused a lethal phenotype during adult emergence.BgACC-RNA interference(RNAi)significantly downregulated total free fatty acid(FFA)and triacylglycerol(TAG)levels,and also caused a significant decrease of cuticular hydrocarbons(CHCs).Repression of BgACC in adult females affected the development of oocytes and resulted in sterile females,but BgACC-RNAi did not affect the reproductive ability of males.Interestingly,knockdown of BgACC also changed the expression of insulin-like peptide genes(BglLPs),which mimicked a physiological state of high sugar uptake.In addition,BgACC was upregulated when B.germanica were fed on a high sucrose diet,and repression of BgACC upregulated the expression of the glycogen synthase gene(BgGlyS).Moreover,BgACC-RNAi increased the circulating sugar levels and glycogen storage,and a longevity assay suggested that BgACC was important for the survival of B.germanica under conditions of high sucrose uptake.Our results confirm that BgACC is involved in multiple lipid biogenesis and sugar homeostasis processes,which further modulates insect reproduction and sugar tolerance.This study benefits our understanding of the crosstalk between lipid and sugar metabolism.

Abdominal-B regulates structure and development of the Harmonia axyridis cremaster

The ladybird Harmonia axyridis is an insect that exhibits pupal attachment to plants,which facilitates development and environmental adaptation.The cremaster is highly specialized for this behavior.However,the underlying molecular regulation of the cremaster remains unclear;therefore,we performed experiments to investigate the transcriptional regulation of cremaster development.First,we examined the morphological structure of the cremaster to reveal its function in pupal attachment of H.axyridis.Next,we analyzed the Hox gene Ha-Abd-B using RNA interference(RNAi)to determine its function in regulating cremaster formation;Ha-Abd-B up-regulation promoted effective pupal attachment,whereas successful RNAi caused severe down-regulation of this gene,and pupae were unable to attach.Furthermore,successful RNAi and subsequent Ha-Abd-B down-regulation caused phenotypic changes in cremaster structure,including its complete disappearance from some individuals.Finally,we observed unique development of the cremaster and dynamic expression of Ha-Abd-B during pre-pupal development;consequently,we hypothesized that there was specific pre-pupal development of the cremaster.Overall,based on these results,the specialized cremasteric structure located on the posterior side of H.axyridis was determined to be a key organ for pupal attachment.Cremaster identification in H.axyridis is regulated by Ha-Abd-B and exhibits preferential development.Pupal attachment of H.axyridis reveals an environmental adaptation of this species;thus,this study and future molecular studies will help determine the role of Hox genes in regulation of insect attachment and further our understanding of the multiple functions of Hox genes.

Molecular and functional characterization of three odorant-binding proteins from the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana,a periodic but devastating wheat pest,relies on wheat spike volatiles as a cue in sclecing hosts for oviposition.Insect odorant-binding proteins(OBPs)are thought to play essential roles in filtering,binding and transporting hydropho-bic odorant molecules to specific receptors.To date,the molecular mechanisms underlying S.mosellana olfaction are poorly understood.Here,three S.mosellana antenna-specific OBP genes,SmosOBPII,16 and 21,were cloned and bacterially expressed.Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were in-vestigated using fluorescence competitive binding assays.Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily.Ligand-binding analysis showed that all three SmosOBPs preferentially bound alcohol,ester and ketone com-pounds,and SmosOBP11 and 16 also selectively bound terpenoid compounds.In par-ticular,the three SmosOBPs had high binding affinities(Ki<20μmol/L)to 3-hexanol and cis-3-hexenylacetate that elicited strong electroantennogram(EAG)response fromfemale antennae.In addition,SmosOBP11 displayed significantly higher binding(Ki<8μmo/L)than SmosOBP16 and 21 to l-octen-3-ol,D-panthenol,a-pinene and heptyl acetate which elicited significant EAG response,suggesting that SmosOBP11 plays a ma-jor role in recognition and transportation of these volatiles.These findings have provided important insight into the molecular mechanism by which S.mosellana specifically rec-ognizes plant volatiles for host selection,and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides

Nicotinamide adenine dinucleotide phosphate(NADPH)-cytochrome P450 reductase(CPR)is involved in the metabolism of endogenous and exogenous substances,and detoxification of insecticides.RNA interference(RNAi)of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides.However,the CPR of Acyrthosiphon pisum has not been characterized,and its function is still not understood.In this study,we investigated the biochemical functions of A.pisum CPR(ApCPR).ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage,and in the gut,head,and abdominal cuticle.After optimizing the dose and silencing duration of RNAi for downregulating ApCPR,we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents,and of cuticular waxy coatings.Deficiency in cuticular hydrocarbons(CHCs)decreased the survival rate of A.pisum under desiccation stress and increased its susceptibility to contact insecticides.Moreover,desiccation stress induced a significant increase in ApCPR mRNA levels.We further confirmed that ApCPR participates in CHC production.These results indicate that ApCPR modulates CHC production,desiccation tolerance,and insecticide susceptibility in A.pisum,and presents a novel target for pest control.

Fitness traits and underlying genetic variation related to host plant specialization in the aphid Sitobion avenae

Sitobion avenae （E） is an important cereal pest worldwide that can survive on various plants in the Poaceae, but divergent selection on different host plants shouldpromote the evolution of specialized genotypes or host races. In order to evaluate their resource use strategies, clones of S. avenae were collected from oat and barley. Host-transfer experiments for these clones were conducted in the laboratory to compare their fitness traits. Our results demonstrated that barley clones had significantly lower fecundityand tended to have longer developmental times when transferred from barley to oat. However, oat clones developed faster after they were transferred to barley. Clones fromoat and barley had diverged to a certain extent in terms of fecundity and developmental time of the nymphs. The separation of barley clones and oat clones of S. avenae was alsoevident in a principal component analysis. Barley clones tended to have higher broad-sense heritabilities for fitness traits than oat clones, indicating the genetic basis of differentiationbetween them. Barley clones showed significantly higher extent of specialization compared to oat clones from two measures of specialization （i.e., Xsp and Ysp）. Therefore, barleyclones were specialized to a certain extent, but oat clones appeared to be generalized. The fitness of S. avenae clones tended to increase with higher extent of specialization. Theevolution toward ecological specialization in S. avenae clones, as well as the underlying genetic basis, was discussed.

Effects of mummy consumption on fitness and oviposition site selection on Harmonia axyridis

Intraguild predation(IGP)has been commonly reported between predators and parasitoids used as biological control agents as predators consuming parasitoids within their hosts.However,the effect of parasitoid-mummy consumption on the fitness of the predator and subsequent oviposition site selection have not been well studied.In our study,we conducted two laboratory experiments to examine the influence of Aphidius gifuen-sis Ashmead(Hymenoptera:Braconidae)mummies as prey on fitness and subsequently oviposition site selection of Harmonia axyridis(Pallas)(Coleoptera:Coccnellidae).Re-sults indicate that when H.aryridis was reared on A.gifiuensis mummies only,its larval development was prolonged,and body weight of the 4th instar larvae and newly emerged adults,and fecundity decreased.Moreover,H.axyridis did not exhibit oviposition pref-erence on plants infested with unparasitized aphids or aphids parasitized for shorter than 9 days.However,compared with plants with mummies(parasitized≥9 days),H.axryridis laid more eggs on plants with unparasitized aphids.In contrast,H.axryridis previously fed with A.gifuensis mummies did not show a significant oviposition preference between plants with unparasitized aphids and those with mummies(parasitized≥9 days).Overall,our results suggest that mummy consumption reduced the fitness of H.axyridis.Although H.axyridis avoided laying eggs on plants with A.gifuensis mummies,prior feeding ex-perience on A.gifuensis mummies could alter the oviposition site preference.Thus,in biological control practice,prior feeding experience of H.axyridis should be carflly considered for reduction of IGP and increase of fitness of H.axyridis on A.gifuensis.

High basal defense gene expression determines sorghum resistance to the whorl-feeding insect southwestern corn borer

Southwestern corn borer （SWCB, Diatraea grandiosella） and fall armyworm （FAW, Spodopterafrugiperda） are major pests of sorghum in the southern United States. Host plant resistance is a desirable means for reducing plant damage and yield losses from both insects. In this study, we evaluated 12 sorghum lines for whorl-stage resistance to leaf-feeding SWCB and FAW in greenhouse and laboratory bioassays. Differential plant responses were detected against the two insects. Among 12 lines tested, CM1821, Della and PI196583 were resistant to both insects, while BTx2752 was largely susceptible. Line R.09110 was resistant to SWCB, but susceptible to FAW, whereas Redbine-60 was suscep- tible to SWCB, but not to FAW. In addition, we quantified various chemical components in the plants and determined their association with insect resistance. Tannin and chloro- phyll in leaves did not show any significant correlation with resistance to either insects, but contents of soluble protein in general were negatively correlated with resistance to both insects. Endogenous soluble sugar and dhurrin were only positively correlated with resistance to SWCB, but not with FAW resistance. To gain some molecular insight into resistance mechanism of sorghum to SWCB, we performed qPCR reactions for key genes encoding enzymes involved in dhurrin and jasmonic acid （JA） biosynthesis on selected resistant or susceptible lines. Although these genes were rapidly and strongly induced by insect feeding in all lines, the observed resistance is likely explained by higher constitutive dhurrin contents in some resistant lines and higher basal JA biosynthesis in others. Our results suggest that sorghum utilizes multiple strategies to defend itself against SWCB.

Deleterious effects of electron beam irradiation on development and reproduction of tomato/potato psyllids, Bactericera cockerelli

The potato/tomato psyllid Bactericera cockerelli causes serious damage to several solanaceous crops by direct feeding and vectoring Candidatus Liberibacter solanacearum,a bacterial pathogen.Electron beam(eBeam)irradiation is an environmentally friendly,chemical-free alternative method that is increasing in use for disinfestation of insect pests.We hypothesize that this irradiation technology will have detrimental effects on potato psyllid and thus impede its disease vectoring.To this end,we explored the effects of eBeam treatment ranging from 50 to 500 Gy on survival,development and reproduction of this pest.Impact on psyllids was apparently dose-dependent.When irradiated at 350 Gy,eggs could not hatch,1st instar nymphs failed to emerge,and although a small portion of irradiated 5th instar nymphs survived,the emerged adults were mostly deformed.Abnormality in eclosed adults suggests harmful effects of eBeam on metamorphosis.Reproduction was seriously impaired when female psyllids were exposed to eBeam at the 5th instar nymphal or young adult stage,presumably due to inability to form oocytes.In addition,reciprocal crosses between irradiated and untreated psyllids indicated that female psyllids were more radiosensitive than males to eBeam.Taken together,these findings indicate that eBeam negatively impacted potato psyllid development and reproduction,which would inevitably compromise its disease transmission capacity.A dose of 350 Gy can be considered as a reference dose for effective control of potato psyllids.