Regional selection of insecticides and fungal biopesticides to control aphids and thrips and improve the forage quality of alfalfa crops

The efficacies of biological and conventional chemical insecticides against two major insect pests of alfalfa(aphids and thrips)were compared in three sites across China’s alfalfa belt.In addition,the persistence of the residues of chemical insecticides in alfalfa and their influence on the quality of alfalfa hay were examined.Efficacy varied among the different biological and chemical insecticides.The chemical insecticides were significantly more effective than biopesticides in a short time-frame.The efficacy period of biopesticides was significantly longer than that of chemical insecticides,and the corrected mortality rate of aphids in all regions was above 50%at 14 days after application.The analysis of pesticide residues showed that the residual doses of all the pesticides were within the allowed ranges after the safe period.The acid detergent fiber and neutral detergent fiber contents in alfalfa hay were higher and the protein content was lower in chemical insecticide treatments than in biopesticide treatments in Hebei.The relative feeding value of alfalfa hay treated with Metarhizium anisopliae IPP330189 was the highest among the treatments.Compared with chemical insecticides,the yield of alfalfa hay was higher in the biopesticides treatments.Biopesticides show a stronger control effect on insect populations and also a better improvement in the quality of alfalfa hay than chemical insecticides.This study provides a basis for exploring and developing a comprehensive control regime for alfalfa insect pests in the different alfalfa-growing regions in China,and for reducing chemical insecticide usage and improving forage quality.

Model fitting of the seasonal population dynamics of the soybean aphid, Aphis glycines Matsumura, in the field

The soybean aphid, Aphis glycines Matsumura(Hemiptera: Aphididae), is one of the greatest threats to soybean production, and both trend analysis and periodic analysis of its population dynamics are important for integrated pest management(IPM). Based on systematically investigating soybean aphid populations in the field from 2018 to 2020, this study adopted the inverse logistic model for the first time, and combined it with the classical logistic model to describe the changes in seasonal population abundance from colonization to extinction in the field. Then, the increasing and decreasing phases of the population fluctuation were divided by calculating the inflection points of the models, which exhibited distinct seasonal trends of the soybean aphid populations in each year. In addition, multifactor logistic models were then established for the first time, in which the abundance of soybean aphids in the field changed with time and relevant environmental conditions. This model enabled the prediction of instantaneous aphid abundance at a given time based on relevant meteorological data. Taken as a whole, the successful approaches implemented in this study could be used to build a theoretical framework for practical IPM strategies for controlling soybean aphids.

Identification of candidate genes for aphid resistance in upland cotton by QTL mapping and expression analysis

Lignin is one of the main components of cell walls and is essential for resistance to insect pests in plants.Cotton plants are damaged by aphid(Aphis gossypii) worldwide but resistant breeding is undeveloped due to scarce knowledge on resistance genes and the mechanism. This study reported a lignin biosynthesisrelated gene identified in the F_(2) population derived from the cross between cotton cultivars Xinluzao 61(resistant to aphid) and Xinluzao 50(susceptible to aphid). A quantitative trait locus was mapped on chromosome D04 with a logarithm of odds(LOD) score of 5.99 and phenotypic effect of 27%. RNA-seq analysis of candidate intervals showed that the expression level of GH_D04G1418 was higher in the resistant cultivar than in the susceptible cultivar. This locus is close to AtLAC4 in the phylogenetic tree and contains a conserved laccase domain. Hence, it was designated GhLAC4-3. Silencing of GhLAC4-3 in Xinluzao 61 via virus-induced gene silencing(VIGS) resulted in decreased lignin content and increased susceptibility to aphids. These results suggest that GhLAC4-3 might enhance aphid resistance by regulating lignin biosynthesis in cotton.

Identification of Early Peach Aphid Infestation Based on Hyperspectral Imaging Technology

Peach aphid is a common pest and hard to detect.This study employs hyperspectral imaging technology to identify early damage in green cabbage caused by peach aphid.Through principal component transformation and multiple linear regression analysis,the correlation relation between spectral characteristics and infestation stage is analyzed.Then,four characteristic wavelength selection methods are compared and optimal characteristic wavelengths subset is determined to be input for modelling.One linear algorithm and two nonlinear modelling algorithms are compared.Finally,support vector machine(SVM)model based on the characteristic wavelengths selected by multi-cluster feature selection(MCFS)acquires the highest identification accuracy,which is 98.97%.These results indicate that hyperspectral imaging technology have the ability to identify early peach aphid infestation stages on green cabbages.

Resistance in Barley (<i>Hordeum vulgare</i>L.) to New Invasive Aphid, Hedgehog Grain Aphid (<i>Sipha maydis</i>, Passerini) (Hemiptera: Aphididae)

Sipha maydis Passerini (Hemiptera: Aphididae) is a pest of cereals in many regions of the world and was identified as an invasive pest of the US in 2007. Regional surveys from 2015-2017 revealed this pest was broadly distributed throughout many of the western Great Plains states where it is a potential threat to cereal production. The common name hedgehog grain aphid, HGA, has been associated with Sipha maydis in the US. Cross-resistance where a plant is resistant to one aphid species and is also resistant to another species that is known to occur. Six barleys were evaluated for cross-resistance to HGA: Russian wheat aphid, RWA, resistant germplasms STARS 9301B and STARS 9577B and cultivar “Mesa”;greenbug, GB, resistant germplasm STARS 1501B and cultivar “Post 90”;and RWA and GB resistant experimental line 00BX 11-115. Cultivars “Morex” and “Schuyler” were susceptible controls. Antixenosis was measured 5 days after infestation by HGA. Seedling damage ratings and reductions in seedling growth were recorded after 17 days of infestation. Intrinsic rate of increase, rm, of HGA was determined by following the development of newborn aphids to adulthood and reproduction. 00BX 11-115 and Post 90 had significantly greater antixenosis (fewer aphids/seedling), significantly lower plant damage ratings, and significantly lower intrinsic rates of increase than other entries. Differences in seedling growth were not significant. 00BX 11-115 and Post 90 were the only entries with the Rsg1 greenbug resistance gene. Rsg1 greenbug resistance confers cross-resistance to HGA in the seedling stage.

The biotypes and host shifts of cotton-melon aphids Aphis gossypii in northern China

Aphis gossypfi is a globally distributed species and therefore has a highly variable life cycle. Populations of A. gossypii in northern China exhibit greater genotypic diversity and a broader host range, yet the details of life cycles of different biotypes is still unclear. In this study, the Cytb and 16S gene regions of A. gossypfi collected from 5 common summer hosts and 4 primary hosts were analyzed. A total of 57 haplotypes were obtained from 1 046 individual A. gossyp# sequences. The sequence included 44 variable sites, 27 of which were parsimony informative sites and 17 of which were singleton variable sites. The most frequent 3 haplotypes were found in 896 individuals, representing a total of 85.7% of all individuals and 36 haplotypes were found in 1 individual. A neighbor-joining tree was constructed using 21 haplotypes that were found in more than 2 individuals. Considering the individual host plant, 5 biotypes were identified. Type 1 corresponded exactly to the cucurbit host-race and the other 4 biotypes were found as cotton host-races. Type 3 was the most abundant biotype in cotton fields in northern China.

Aphids and their transmitted potato viruses: A continuous challenges in potato crops

Aphid is one of the most destructive insect pests on cultivated plants in temperate regions.Their piercing-sucking mouthparts and phloem feeding behavior directly damage crops and deplete plant nutrients.Potato(Solanum tuberosum L.)is one of the most important food sources on the planet,and several aphid species,e.g.,Myzus persicae(Sulzer)(green peach aphid)and Macrosiphum euphorbiae(Thomas)(potato aphid)(Hemiptera:Aphididae)colonize potato and transmit several economically important viruses.Aphid-transmitted potato viruses have been emerging all over the world as a very serious problem in potato production,inducing a wide variety of foliar and tuber symptoms,leading to severe yield reduction and loss of tuber quality.In this review,recent advances in understanding the interactions of potato viruses with their hosts,aphid vectors and the environment are described.

Identification of Soybean Resources of Resistance to Aphids

Four soybean [Glycine max （L.） Merr.] cultivars with soybean aphid resistance （Aphis glycines Matsmura）, p189, P203, P574, and P746, were identified in field test, choice test, and non-choice test, The grade of resistance to aphids and the damage index of P189, P203, and P746 were significantly different from the susceptible cultivars （P=0.05）. P574 and P746 showed antibiosis resistance, preventing aphids from reproducing on the plants. P203 showed antixenosis resistance, preventing aphids from reproducing in field test and choice test, but susceptible in non-choice test. Population development on plants was significantly different in field test, choice test, and non-choice test, which was caused by different selective pressures.

Identification of Molecular Markers for a Aphid Resistance Gene in Sorghum and Selective Efficiency Using These Markers

In this study, an F2 segregated population obtained by hybridization between the aphid-sensitive sorghum strain Qiansan and aphid-resistant cultivar Henong 16 was used to establish an aphid-resistant pool and an aphid-sensitive pool. 192 pairs of AFLP （amplified fragment length polymorphism） marker primers were screened in these pools using BSA （bulked segregant analysis）. Three pairs of EcoR I-CTG/Mse I-CCT, EcoR I-CTG/Mse I-CAT, and EcoR I-AGT/Mse I-CCC showed linkage with aphis resistance. EcoR I-CTG/Mse I-CCT-475, EcoR I-CTG/Mse I-CAT-390, and EcoR I-AGT/Mse I-CCC- 350 （E42/M52-350） were mapped within 6, 10, and 13 cM distances with the aphid-resistant gene by using Mapmaker 3.0 software. The bands amplified by EcoR I-CTG/Mse I-CCT-475 and EcoR I-CTG/Mse I-CAT-390 were extracted, cloned, and sequenced. Specific primers of SCAR （sequence characterized amplified regions） were then designed from these bands. A specific band of 300 bp was amplified by a pair of SCAR primers designed based on the sequence obtained from the EcoR I-CTG/Mse I-CAT-390 marker. The SCAR marker was named SCAS0. The marker was used to detect the F2, BC1, and F2：3 populations. The selective efficiency was 86.8, 91.1, and 86.3% in the BC1, F2, and F2：3 populations, respectively. The average selective efficiency was 88.2%.

Transcriptome approach to understand the potential mechanisms of resistant and susceptible alfalfa(Medicago sativa L.) cultivars in response to aphid feeding

Plant breeding for resistance to agricultural pests is an essential element in the development of integrated crop management systems, however, the molecular and genetic mechanisms underlying resistance are poorly understood. In this pilot study, we conducted a transcriptomic analysis of a resistant （R） and susceptible （S） variety of alfalfa, with （＋A） or without （-A） aphids （totally four treatments）. We used the resistant cultivar Zhongmu t and the susceptible cultivar Soca. A total of 3 549 mRNAs were differentially expressed, of which 1 738 up-regulated and 1 307 down-regulated genes were identified in S＋A/S-A plants, while 543 up-regulated and 331 down-regulated genes were identified in the R＋A/R-A plants. KEGG analysis mapped 112 and 546 differentially expressed genes to 8 and 17 substantially enriched pathways for Zhongmu 1 and Soca, respectively. Six shared pathways were linked to plant resistance traits, including phenylpropanoid biosynthesis associated with salicylic acid synthesis, and linoleic acid metabolism associated with both jasmonic acid and flavonoid biosynthesis. Ultimately, we proposed a preliminary regulatory mechanism of alfalfa cultivar resistance response to aphids feeding based on transcriptome analyses and published documents.

Physiology and defense responses of wheat to the infestation of different cereal aphids

Cereal aphids are major insect pests of wheat,which cause significant damages to wheat production.Previous studies mainly focused on the resista nee of differe nt wheat varieties to one specific aphid species.However,reports on the physiology and defense responses of wheat to different cereal aphids are basically lacking.In this work,we studied the feeding behavior of three cereal aphids:the grain aphid,Sitobion avenae(Fabricius),the greenbugs,Schizaphis graminum(Ron dani),and the bird cherry-oat aphid,Rhopalosiphum padi(Linn aeus)on win ter wheat,and the physiology and defense responses of wheat to the infestation of these cereal aphids with focus on how these cereal aphids utilize divergent strategies to optimize their nutrition requirement from wheat leaves.Our results indicated that S.graminum and R.padi were better adapted to penetrating phloem tissue and to collect more nutrition than S.avenae.The harm on wheat physiology committed by S.graminum and R.padi was severer than that by S.avenae,through reducing chlorophyll concentration and interfering metabolism genes.Furthermore,cereal aphids manipulated the plant nutrition metabolism by increasing the relative concentration of major amino acids and percentage of essential amino acids.In addition,different cereal aphids triggered specific defense response in wheat.All of these results suggested that different cereal aphids utilize diverge nt strategies to cha nge the physiological and defe nse resp on ses of their host plants in order to optimize their nutriti on absorption and requireme nt.These fin dings not only exte nd our current knowledge on the insect-pla nt in teractions but also provide useful clues to develop no vel biotech no logical strategies for enhancing the resistance and toleranee of crop plants against phloem-feeding insects.

Reproductive polyphenism and its advantages in aphids:Switching between sexual and asexual reproduction

Reproductive polyphenism,which allows one genotype to produce sexual and asexual morphs,is an extreme case of phenotypic plasticity and is commonly observed in aphids.Aphids are typical species that switch these reproductive modes,and the pathway orientation is triggered by the environmental conditions(mainly photoperiod and temperature).The typical arm ual life of aphids in eludes a successi on of parthenogenetic gen erations duri ng the spri ng and summer and a single sexual generation in autumn.In this review,we describe how the environmental cues orientate the reproductive mode of aphids from photoperiodic perception to endocrine regulation,and how juvenile hormones may act on the target cells(oocytes)to initiate the gametogenesis and embryogenesis in sexual and asexual reproduction.We also discuss the paradox of sex,especially the advantages of sexual reproduction in aphids.With the recent development of genomic resources in aphids,many potential genes involved in the reproductive polyphenism will enter the public's awareness.In particular,we describe a novel RNAi method in aphids,which may provide a molecular technique for determining the developmental fate and multiple reproductive strategies.

Effects of imidacloprid and thiamethoxam as seed treatments on the early seedling characteristics and aphid-resistance of oilseed rape

Seed treatments with the neonicotinoid insecticides imidacloprid and thiamethoxam were evaluated to determine whether the chemicals at effective concentrations for aphid control would influence the germination and early growth of oilseed rape,Brassica napus.Treatment with imidacloprid or thiamethoxam did not affect the cumulative germination rate,but significantly inhibited establishment potential by suppressing root system development in the cotyledon stage.However,these alterations in seedling development in the thiamethoxam-treated seeds appeared not to be detrimental as leaves developed;in contrast,for the seedlings with imidacloprid as seed treatment agent,a significantly decreased shoot/root ratio was still evident at the late two-leaf stage.After two leaves developed,chlorophyll content per leaf in the thiamethoxam treatment was significantly higher than that of the control,while chlorophyll content per leaf in the imidacloprid treatment remained close to that in the control.Most other parameters,i.e.,height,leaf area,weight of stem,leaf or root,and other growth indexes,between the treatments and the control showed no significant difference.Additionally,it was found that storage time of the treated seeds had a significant effect on cumulative germination rate.Treatment 30 d before planting significantly reduced germination relative to that of the control.All of the plants treated with neonicotinoids were shown to have significant anti-aphid characteristics that persisted until the end of the trial.

Performance and transcriptomic response of the English grain aphid, Sitobion avenae, feeding on resistant and susceptible wheat cultivars

Plant resistance against insects mainly depends on nutrient restriction and toxic metabolites, but the relative importance of nutrition and toxins remains elusive. We examined performance, nutrition ingestion, and transcriptome response of the English grain aphid, Sitobion avenae, feeding on resistant Xiaoyan 22(XY22) and susceptible Xinong 979(XN979) wheat cultivars. Aphids had lower body weight and fecundity when feeding on XY22 than on XN979, although the phloem sap of XY22 had a higher nutritive quality(in terms of amino acid:sucrose ratio). Aphids feeding on XY22 also had a lower honeydew excretion rate than those on XN979, suggesting that aphids ingested less phloem sap from XY22. The transcriptome data showed 600 differentially expressed genes(DEGs), and 11 of the top 20 KEGG pathways significantly enriched in DEGs were involved in nutrient metabolism. We found 81 DEGs associated with the metabolism of sugars, lipids, and amino acids, 59 of which were significantly downregulated in aphids feeding on XY22. In contrast, there were 18 DEGs related to detoxifying metabolism, namely eight UDP-glucuronosyltransferases, six cytochromes P450 monooxygenases, one glutathione S-transferase, two ATP-binding cassette transporters, and one major facilitator superfamily transporter;12 of these were upregulated in the aphids feeding on XY22. Our results indicated that both the quantity and quality of phloem nutrition available to aphids are critical for the growth and development of aphids, and the higher resistance of XY22 is mainly due to the reduction in phloem sap ingested by aphids, rather than toxic metabolites.

Including predator presence in a refined model for assessing resistance of alfalfa cultivar to aphids

The aphid quantity ratio（AQR） is defined as the number of aphids on each cultivar divided by the number of aphids on all cultivars. AQR is based on the correlation between aphid populations and their host plants and is an important tool that has been utilized in evaluating Medicago sativa（alfalfa） cultivar resistance to aphids. However, assessment of alfalfa resistance to aphids can be confused by the presence of aphid predators, causing the assessment of plant resistance to aphids to be based on incorrect aphid population data. To refine the AQR and account for the effect of predators on aphid population assessments, we introduced a parameter ‘α＇, corresponding to the predator quantity ratio, and used αAQR as the ratio to quantify aphid populations. Populations of both aphids（4 species） and their predators（12 species） occurring in 28 M. sativa cultivars were sampled over two years at a research station near Cangzhou, Hebei Province, China. Results showed that the most suitable evaluation period was from May to June, as the aphid population was stable during this period. Compared with the AQR method, the predator population numbers based on the αAQR had a significant inverse relationship with aphid population numbers and the 28 cultivars were clustered into three classes： the resistant class, tolerant class, and susceptible class. In addition, 17 cultivars were reassigned when evaluated using αAQR. All numerical values calculated by αAQR were displayed as a Gaussian distribution, which showed that the 28 cultivars could be clustered into nine groups using a median value（±SE） of 1±0.1. Hence, ongoing alfalfa breeding trials will be assessed using the αAQR to establish a robust system that includes agronomic performance parameters in order to generalize the new method for further studies.

Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

Inter-species mRNA transfer among green peach aphids,dodder parasites,and cucumber host plants

mRNAs are transported within a plant through phloem.Aphids are phloem feeders and dodders(Cuscuta spp.)are parasites which establish phloem connections with host plants.When aphids feed on dodders,whether there is trafficking of mRNAs among aphids,dodders,and host plants and if aphid feeding a匚fects the mRNA transfer between dodders and hosts are unclear.We constructed a green peach aphid(GPA,Myzus persicae)-dodder(Cuscuta austra/is)-cucumber(Cucumis sativus)tritrophic system by infesting GPAs on C.australis,which parasitized cucumber hosts.We found that GPA feeding activated defense-related phytohormonal and transcriptomic responses in both C.australis and cucumbers and large numbers of mRNAs were found to be transferred between C.australis and cucumbers and between C.australis and GPAs;importantly,GPA feeding on C.australis greatly altered inter-species mobile mRNA profiles.Furthermore,three cucumber mRNAs and three GPA mRNAs could be respectively detected in GPAs and cucumbers.Moreover,our statistical analysis indicated that mRNAs with high abundances and long transcript lengths are likely to be mobile.This study reveals the existence of inter-species and even inter-kingdom mRNA movement among insects,parasitic plants,and parasite hosts,and suggests complex regulation of mRNA trafficking.

Analysis of Leaf Volatiles of Crabapple (<i>Malus</i>sp.) Individuals in Different Aphids’ Resistance

The aim of this experiment was to analyze the leaf volatiles of crabapple (Malus sp.) individuals at different aphid’s resistance, to ascertain the particular ingredients which has lure or aversion effects on aphid, and to provide reference for finding out a simple method to control effectively aphids. Volatiles of leaves from twenty-one different crabapple individuals were evaluated with the method of head space-solid phase micro-extraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS). Volatiles profiles of them were then compared. There are one hundred eighty-six kinds of volatiles were detected with varied contents found in different individuals. And all plants contain eight kinds of common components: 3-Hexen-1-ol, acetate, (Z)-, 4-Hexen-1-ol, (Z)-, n-Decanal, n-Tetradecane, .alpha.-Farnesene, Diethyl Phthalate, Oxime-, methoxy-phenyl- and Dibenzofuran-, wherein the relative content of higher have 3-Hexen-1-ol, acetate, (Z)- and 4-Hexen-1-ol, (Z)-. Specific volatile substances in high resistance plants contain 3-Hexenal, (Z)- and 2-Hexenal, (E)-. Leaf volatiles differ in twenty-one crabapple individuals. High resistance plants specific volatile substances contains 3-Hexenal, (Z)-, 2-Hexenal, (E)- and other small molecular volatile substances, and no-resistance plants all have .beta.-Farnesene.

Control Effect of Slow-release, Long-acting and Multi-functional New Pesticides on Sugarcane Borders and Woolly Aphids

In order to select the long-acting,low toxic,low-risk and multi-functional new pesticides for the control of sugarcane borders and woolly aphids and precise and efficient application technology,the control effect of 10% monosultap · thiamethoxam granular formulation and 1% Bt · clothianidin granular formulation on sugarcane borders and woolly aphids were studied.The results showed that 10% monosultap·thiamethoxam GR and 1% Bt·clothianidin GR had good control effects on sugarcane borders and woolly aphids.They were ideal slow-release,long-acting,low toxic and multi-functional new pesticides used to control sugarcane borders and woolly aphids.They could be used alternately with other pesticides to delay the emergence and development of pest resistance to pesticides.The best dosage of the two pesticides in the field was 45 kg/hm^2.They could be mixed with fertilizer( 1200-1800 kg/hm^2),scattered in sugarcane ditches or at the base of sugarcane plants,and covered with soil or film from January to July.The control effect on dead heart seedlings damaged by borers could be up to above 79.2%,and the control effect on sugarcane woolly aphids could reach more than 98.8%.In comparison with the control group,the actual yield and sugar content of sugarcane increased by above 41 555 kg/hm^2 and 6.5% respectively.The application of slow-release,long-acting,strong systemic and multi-functional new agents with fertilizer around roots is convenient,precise and efficient,labor-saving,time-saving and environmentally friendly,and is worthy of being widely applied in sugarcane areas.

Insights into wing dimorphism in worldwide agricultural pest and hostalternating aphid Aphis gossypii

Background:The worldwide pest Aphis gossypii has three-winged morphs in its life cycle,namely,winged parthenogenetic female(WPF),winged gynopara(GP),and winged male,which are all produced by a wingless parthenogenetic female(WLPF).Most studies on A.gossypii have focused on WPF,while few have investigated GP and male.The shared molecular mechanism underlying the wing differentiation in the three wing morphs of A.gossypii remains unknown.The wing differentiation of WPF was explored in a previous study.Herein,GP and male were induced indoors.The characters of the body,internal genitals,wing veins,and fecundity of GP and male were compared with those of WPF or WLPF.Compared with WLPF,the shared and separate differentially expressed genes(DEGs)were identified in these three-wing morphs.Results:Newly-born nymphs reared in short photoperiod condition(8 L:16D,18°C)exclusively produced gynoparae(GPe)and males in adulthood successively,in which the sex ratio was GP biased.A total of 14 GPe and 9 males were produced by one mother aphid.Compared with WLPF,the three-wing morphs exhibited similar morphology and wing vein patterns but were obviously discriminated in the length of fore-and underwings,reproductive system,and fecundity.A total of 37090 annotated unigenes were obtained from libraries constructed using the four morphs via RNA sequencing(RNA-Seq).In addition,10867 and 19334 DEGs were identified in the pairwise comparison of GP versus WLPF and male versus WLPF,respectively.Compared with WLPF,the winged morphs demonstrated 2335 shared DEGs(1658 upregulated and 677 downregulated).The 1658 shared upregulated DEGs were enriched in multiple signaling pathways,including insulin,FoxO,MAPK,starch and sucrose metabolism,fatty acid biosynthesis,and degradation,suggesting their key roles in the regulation of wing plasticity in the cotton aphid.Forty-four genes that spanned the range of differential expression were chosen to validate statistical analysis based on RNA-Seq through the reverse transcription quantitative real time polymerase chain reaction(RT-qPCR).The comparison concurred with the expression pattern(either up-or downregulated)and supported the accuracy and reliability of RNA-Seq.Finally,the potential roles of DEGs related to the insulin signaling pathway in wing dimorphism were discussed in the cotton aphid.Conclusions:The present study established an efficiently standardized protocol for GP and male induction in cotton aphid by transferring newly-born nymphs to short photoperiod conditions(8 L:16D,18°C).The external morphological characters,especially wing vein patterns,were similar among WPFs,GPe,and males.However,their reproductive organs were strikingly different.Compared with WLPF,shared(2335)and exclusively(1470 in WLPF,2419 in GP,10774 male)expressed genes were identified in the three-wing morphs through RNA-Seq,and several signaling pathways that are potentially involved in their wing differentiation were obtained,including insulin signaling,starch and sucrose metabolism.