The miR-9b microRNA mediates dimorphismand development of wing in aphidsFeng Shanga,b,1

, Jinzhi Niua,b,1, Bi-Yue Dinga,b,1, Wei Zhanga,b, Dan-Dan Weia,b, Dong Weia,b, Hong-Bo Jianga,b,and Jin-Jun Wanga,b,2

aKey Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, 400716 Chongqing, China; and bStateCultivation Base of Crop Stress Biology for Southern Mountainous Land, Academy of Agricultural Sciences, Southwest University, 400716 Chongqing, China

Edited by Alexander S. Raikhel, University of California, Riverside, California, and approved March 5, 2020 (received for review November 1, 2019)

Wing dimorphism is a phenomenon of phenotypic plasticity inaphid dispersal. However, the signal transduction for perceivingenvironmental cues (e.g., crowding) and the regulation mecha-nism remain elusive. Here, we found that aci-miR-9b was the onlydown-regulated microRNA (miRNA) in both crowding-induced wingdimorphism and during wing development in the brown citrusaphid Aphis citricidus. We determined a targeted regulatory rela-tionship between aci-miR-9b and an ABC transporter (AcABCG4).Inhibition of aci-miR-9b increased the proportion of winged off-spring under normal conditions. Overexpression of aci-miR-9bresulted in decline of the proportion of winged offspring undercrowding conditions. In addition, overexpression of aci-miR-9balso resulted in malformed wings during wing development. Thisrole of aci-miR-9b mediating wing dimorphism and developmentwas also confirmed in the pea aphid Acyrthosiphon pisum. Thedownstream action of aci-miR-9b-AcABCG4 was based on the in-teraction with the insulin and insulin-like signaling pathway. Amodel for aphid wing dimorphism and development was demon-strated as the following: maternal aphids experience crowding,which results in the decrease of aci-miR-9b. This is followed bythe increase of ABCG4, which then activates the insulin andinsulin-like signaling pathway, thereby causing a high proportionof winged offspring. Later, the same cascade, “miR-9b-ABCG4-insulin signaling,” is again involved in wing development. Takentogether, our results reveal that a signal transduction cascademediates both wing dimorphism and development in aphids viamiRNA. These findings would be useful in developing potentialstrategies for blocking the aphid dispersal and reducing viraltransmission.

microRNA | ABC transporter | trehalose | insulin | population density

Evolution of wing-facilitated flying ability shapes the environ-mental adaptation behavior of insects, and wing diversity is

one of the most fascinating of phenomena. Among them, wingdimorphism functions as an energy trade-off between migrationand reproduction as an adaptive switch to environmental changes.Flightless morphs (short-winged or wingless [apterous] morphs)allocate energy into offspring production while winged morphstypically produce fewer offspring but can migrate long distances insearch of suitable habitats (1, 2). Factors contributing to insectwing dimorphism include population density, microorganisms,temperature, photoperiod, and host quality (1–3). Ecdysteroids,juvenile hormone, c-Jun NH2-terminal kinases, and insulin/insulin-like growth factor signaling (IIS) pathways contribute tothe regulation of wing dimorphism. These pathways have beenstudied in aphids and planthoppers using RNA interference(RNAi)-based approaches (4–8). Two laterally transferred viralgenes (Apns-1 and Apns-2) have been associated with crowding-induced wing dimorphism in the pea aphid Acyrthosiphon pisum(9). In aphids, wing dimorphism is the process that determineswing morphs (proportion), and then wing development is theprocess that develops wing buds (wing growth). Thus, wingdimorphism and wing development are indispensable continu-ous processes for aphid wing formation in order to successfully

escape unfavorable conditions and to locate new habitats.Therefore, it is important to understand how aphids perceiveenvironmental cues in triggering wing dimorphism and exploitspecific approaches to target this process for aphid pest control.However, the signal transduction of this aspect still remainsunclear.microRNAs (miRNAs) are critical components of post-

transcriptional gene expression regulation (10, 11). The abundanceof miRNAs can be altered by environmental stressors such as in-secticides, plant defenses, parasites, and extreme temperatures (10).Thus, miRNAs may facilitate the adaptation of insects to changingenvironmental conditions. We used the brown citrus aphid Aphiscitricidus (SI Appendix, Fig. S1), the main vector of Citrus tristezavirus (one of the most widely distributed pathogens that causesdestructive losses in the citrus industry worldwide), as a case study.Here, we report that the miR-9b miRNA plays a key role in theregulation of wing dimorphism and wing development in aphids.Our work promotes the understanding of molecular mechanismsfor phenotypic plasticity in aphids as well as the regulation of in-sect adaptation to the changing environment. The finding of thisstudy might also be helpful in identifying potential targets inpest control through blocking dispersal of flying insect pests.

Significance

Evolution of various wings plays an important role in the suc-cess of insects in environmental adaptation. During evolution,aphids deploy a wing dimorphism strategy to escape unfavor-able conditions. This strategy also poses high risks to crops bydistant transmission of viral diseases vectored by aphids. Thus, itis important to understand how aphids perceive environmentalcues in triggering wing dimorphism and exploit specific ap-proaches to target this process. Here, we report a microRNA(miR-9b)-mediated signal cascade that controls high-population-density–induced wing dimorphism in aphids. This findinghighlights that small RNAs are important in regulating signaltransduction of insect phenotypic plasticity when insects faceenvironmental challenges, which may provide an alternative forthe development of dispersal restriction-based pest control.

Author contributions: F.S., J.N., H.-B.J., and J.-J.W. designed research; F.S. and B.-Y.D.performed research; F.S., B.-Y.D., W.Z., D.-D.W., and D.W. analyzed data; and F.S., J.N.,B.-Y.D., and J.-J.W. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Sequence data have been deposited in the National Center for Biotech-nology Information’s Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra (accessionno. PRJNA578247 and PRJNA576278).1F.S., J.N., and B.-Y.D. contributed equally to this work.2To whom correspondence may be addressed. Email: wangjinjun@swu.edu.cn.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1919204117/-/DCSupplemental.

First published March 26, 2020.

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ResultsThe aci-miR-9b Emerged as the Only Candidate miRNA Associatedwith Wing Dimorphism and Wing Development in Aphids. Trans-generational wing dimorphism was observed in A. citricidus inwhich crowding of the parent (wingless adult) had increased thewinged offspring proportion to ∼50% (Fig. 1A). In addition, itwas observed that the wing buds developed slowly in nymphalinstars until the fully formed wings unfolded after adult emer-gence in A. citricidus (Fig. 1B). Thus, to identify the potentialmiRNAs mediating wing dimorphism and wing development, weperformed small RNA sequencing for three comparisons: normalvs. crowding (for exploration of the potential miRNAs involved inwing dimorphism), fourth instar winged nymphs (N4-WD) vs.winged adults (AD-WD) (for exploration of the potential miR-NAs involved in wing development), and fourth instar winglessnymphs (N4-WL) vs. wingless adults (AD-WL) (as a control forexcluding the miRNAs involved in aphid development but notspecifically for wing development) (Fig. 1B). Among these threecomparisons, only one miRNA (aci-miR-9b) was down-regulatedin both wing dimorphism (normal vs. crowding) and wing devel-opment (N4-WD vs. AD-WD) but had no change in the control(N4-WL vs. AD-WL) (Fig. 1C, SI Appendix, Fig. S2A, and DatasetsS1 and S2). Additionally, the expression level of aci-miR-9b waslower in the winged morphs than in the wingless morphs (N4-WDvs. N4-WL and AD-WD vs. AD-WL) (SI Appendix, Fig. S3). Takentogether, the RNA sequencing (RNA-seq) results suggested thataci-miR-9b might act as a key regulator in wing dimorphism anddevelopment in aphids.

The aci-miR-9b Targets AcABCG4. To explore the targets of aci-miR-9b, four miRNA-messenger RNA (mRNA) target prediction pro-grams were performed (SI Appendix, Fig. S4A), and three targets

(EVM0006751 [uncharacterized protein LOC111032307], EVM0005413[phosphoenolpyruvate carboxykinase (GTP)-like], and EVM00015677[ATP-binding cassette subfamily G member 4, AcABCG4]) werepredicted to fit the “down-up” miRNA-target regulation pattern(SI Appendix, Figs. S2B and S4B and Datasets S3–S5). Of thesethree candidate targets, only the expression levels of AcABCG4altered accordingly in the aphids treated with aci-miR-9b mimic orinhibitor (Fig. 2 and SI Appendix, Fig. S4 C and D), indicating thatAcABCG4 is the target of aci-miR-9b.To determine if aci-miR-9b specifically targets AcABCG4, we

used several experimental approaches as follows: 1) aci-miR-9bwas observed to have an opposite expression trend with AcABCG4under crowding treatment (Fig. 2A) and during wing developmentby quantitative reverse transcription PCR (RT-qPCR) (Fig. 2B).The coding sequencing (CDS) of AcABCG4 exhibited the po-tential target site of aci-miR-9b (Fig. 2C). 2) The protein level ofAcABCG4 decreased or increased (based on Western blot anal-ysis) after the aphids fed on the aci-miR-9b mimic or aci-miR-9binhibitor, respectively (Fig. 2 D and E and SI Appendix, Fig. S5). 3)To validate the binding activity of aci-miR-9b to AcABCG4, adual-luciferase reporter assay was performed by cloning an ∼400-bp CDS fragment of AcABCG4 containing the target sequences ofthe miRNA. The luciferase activities declined when the aci-miR-9b mimic and AcABCG4 target were cotransfected, while the re-porter activity recovered when the construct containing themutated AcABCG4 sequences (Fig. 2F). 4) An RNA immuno-precipitation assay showed that the abundance of AcABCG4mRNA was 5.77-fold up-regulated when the aci-miR-9b mimicwas supplied in the Ago-1 antibody-mediated RNA complexcompared to an IgG control (Fig. 2G and SI Appendix, Fig. S6).These data showed that AcABCG4 was targeted by aci-miR-9b.

Fig. 1. RNA-seq reveals that aci-miR-9b is involved in wing dimorphism and wing development in A. citricidus. (A) The proportion of winged offspring undercrowding. normal, 10 adults in a stem-leaf device; crowding, 80 adults in a stem-leaf device. Mean (±SE) is based on four biological replicates. The significantdifference between crowding and normal is indicated by asterisks (***P < 0.001). (B) Schematic diagram of wing dimorphism and wing development andstrategy for RNA-seq in A. citricidus. The red triangles represent the location of the wing (wing bud) in winged morphs. The comparisons inside the dashedbox indicate the strategy of RNA-seq. Red-dashed boxes indicate the potential miRNAs mediating wing dimorphism (normal vs. crowding) and wing de-velopment (N4-WD vs. AD-WD) while the blue-dashed box is used to exclude the miRNAs in aphid development but not specifically for wing development(N4-WL vs. AD-WL). (C) The differentially expressed miRNAs among crowding vs. normal, AD-WD vs. N4-WD, and AD-WL vs. N4-WL. miRNAs with an adjustedP value < 0.01 and the absolute value of a fold change > 1.25 found by DESeq were assigned as differentially expressed. In each comparison, the relativeexpression of miRNAs was clustered based on z-scores from low to high value (with a scale from −1 to 1) among all of the biological replicates. Up-regulationis represented by red shading and down-regulation is represented by blue shading. R1 to R4 represent biological replicates; normal, wingless adult undernormal condition; crowding, wingless adult under crowding; N1, first instar nymph; N2, second instar nymph; N3-WD, third instar winged nymph; N3-WL,third instar wingless nymph; N4-WD, fourth instar winged nymph; N4-WL, fourth instar wingless nymph; AD-WD, winged adult; AD-WL, wingless adult.

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The aci-miR-9b Is Involved in Wing Dimorphism and Wing Development.To determine the role of aci-miR-9b and its target in wing di-morphism and wing development, we combined different treat-ments by feeding aphids with the aci-miR-9b inhibitor dsAcABCG4and the aci-miR-9b mimic (SI Appendix, Fig. S7).In the wing dimorphism experiment, two population-density—

normal (10 adults per stem-leaf device) and crowding (80 adultsper stem-leaf device)—were used (SI Appendix, Fig. S7A). Undernormal conditions, 53% of offspring were winged morphs whenaci-miR-9b was inhibited, while the proportion of winged morphsdid not change (<7%) in the dsAcABCG4-fed group, the aci-miR-9b mimic-fed group, and the controls (Fig. 3A and SI Appendix,Figs. S8 and S9). However, under crowding, the proportions ofwinged offspring in the dsAcABCG4-fed group (29%) and the aci-miR-9b mimic-fed group (23%) were significantly lower than thosein the controls (50% in average) and the aci-miR-9b inhibitor-fedgroup (57%) (Fig. 3B, SI Appendix, Fig. S8, and Dataset S6). Theresults indicated that aci-miR-9b and its target gene AcABCG4 areinvolved in crowding-induced wing dimorphism in A. citricidus.In the wing development experiment, 51% of adults had

malformed wings after depletion of AcABCG4 (Fig. 3 C andD andSI Appendix, Fig. S8). A high percentage of aphids with malformedwings (41%) occurred after aci-miR-9b mimic feeding, while themalformed wing phenotype did not occur with inhibition of aci-miR-9b (Fig. 3 C and D and SI Appendix, Fig. S8). The aphidforewing size of dsAcABCG4 and aci-miR-9b mimic-fed groupsdeclined compared to controls (declined 48 and 39%, respectively)(SI Appendix, Fig. S10). However, no other phenotypic differences(e.g., adult emergence and body size) were observed in A. citricidusupon miRNA inhibitor/double-stranded RNA (dsRNA)/miRNA

mimic treatments (SI Appendix, Figs. S10 and S11). The resultssuggest that aci-miR-9b and its target gene AcABCG4 play a specificrole in wing development.Taken together, these results demonstrate that aci-miR-9b neg-

atively regulates the expression of AcABCG4 to modulate wingplasticity in both wing dimorphism and wing development. Thismechanism was also tested in A. pisum: inhibition of api-miR-9bincreased the proportion of winged A. pisum morphs under nor-mal conditions, whereas the silencing of ApABCG4 or the over-supply of api-miR-9b reduced the winged offspring proportionunder crowding conditions. During wing development, ∼50% ofthe aphids showed malformed wings due to silencing of ApABCG4or an oversupply of api-miR-9b (SI Appendix, Figs. S12 and S13).Furthermore, silencing of ABCG4 decreased the expression of 20wing-patterning network genes, including decapentaplegic, notch,apterous, wingless, nubbin, and ultrablthorax (SI Appendix, Fig.S14), indicating that the canonical insect wing development path-way was also conserved in aphids (12–14).

Downstream Action of aci-miR-9b. To clarify the downstreampathway of aci-miR-9b in aphids, we analyzed the tissue-specificexpression pattern of aci-miR-9b and its target gene. The resultsshowed that both aci-miR-9b and AcABCG4 are highly expressedin the fat body (SI Appendix, Fig. S15). It is possible that the fatbody could regulate insulin-like peptide (ILP) secretion and in-fluence IIS pathway activity in response to the nutritional environ-ment (15). The IIS pathway is a conserved nutrient-sensing pathwaythat is influenced by carbohydrate content (16). Therefore, we hy-pothesized that the perception of environmental stressors (e.g.,crowding) by aphids triggers the regulation of aci-miR-9b, which

Fig. 2. aci-miR-9b targets AcABCG4. (A) The expression profiles of aci-miR-9b and AcABCG4 for the wingless adult upon crowding after 24 h by RT-qPCR.normal, 10 adults in a stem-leaf device; crowding, 80 adults in a stem-leaf device. (B) aci-miR-9b and AcABCG4 present the opposite trend in different de-velopmental stages during wing development. N1, first instar nymph; N2, second instar nymph; N3-WD, third instar winged nymph; N4-WD, fourth instarwinged nymph; AD-WD, winged adult. (C) The putative aci-miR-9b–binding sites in AcABCG4 were predicted by miRanda, PITA, RIsearch, and RNAhybrid. (D)The mRNA level (RT-qPCR) of ABCG4 in N4-WD treated with miR-9b mimic after 24 h, and the protein level (Western blot) of ABCG4 after 48 h. (E) The mRNAlevel (RT-qPCR) of ABCG4 in a wingless adult under normal conditions treated with miR-9b mimic after 24 h and the protein level (Western blot) of ABCG4after 48 h. (Inset) Protein level for ABCG4 after aphids treated with miR-9b mimic (D) or inhibitor (E) through Western blot. U6, small nuclear RNA U6; EF1α,elongation factor-1; β-ACT, beta-actin. (F) aci-miR-9b directly targets AcABCG4 in vitro by using a luciferase reporter assay. (G) aci-miR-9b targets AcABCG4in vivo demonstrated by RNA immunoprecipitation assay. All of the mean (±SE) is based on four biological replicates. The significant differences amongdifferent treatments are indicated by lowercase letters above each bar (one-way ANOVA followed by the least significant difference (LSD) test, P < 0.05) for D,E, and F. The significant differences between treatment and control are indicated by asterisks in A and G (Student’s t test, **P < 0.01; ***P < 0.001).

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negatively regulates the expression of AcABCG4 and might conse-quently act on the IIS pathway in aphids, ultimately determiningwing dimorphism and wing development.To test this hypothesis, we first measured the level of the main

carbohydrate, trehalose. During wing dimorphism, inhibition ofaci-miR-9b increased the level of trehalose under normal conditions

(Fig. 4A), while during wing development, the silencing of AcABCG4or feeding with the aci-miR-9b mimic decreased the level of trehalose(Fig. 4A). These data revealed the association of aci-miR-9b withtrehalose level. We then tested whether aci-miR-9b/AcABCG4interacts with the IIS pathway activity. The expression levels of IISpathway genes were consistent with the trends of AcABCG4

Fig. 3. aci-miR-9b mediates wing dimorphism and wing development in A. citricidus. (A) The proportion of winged offspring of A. citricidus after a wingless adultwas treated by aci-miR-9b inhibitor, dsAcABCG4, and aci-miR-9b mimic under normal. (B) The proportion of winged offspring of A. citricidus after wingless adulttreated by aci-miR-9b inhibitor, dsAcABCG4, and aci-miR-9b mimic under crowding. normal, 10 adults in a stem-leaf device; crowding, 80 adults in a stem-leafdevice. (C) The proportion of A. citricidus adults with malformed wings after N4-WD treated by aci-miR-9b inhibitor, dsAcABCG4, and aci-miR-9b mimic. (D) Wing-defect phenotypes of A. citricidus under dsAcABCG4 and aci-miR-9b mimic treatment. All of the mean (±SE) is based on four biological replicates. The significantdifferences among different treatments are indicated by lowercase letters above each bar (one-way ANOVA followed by the LSD test, P < 0.05) for A, B, and C.

Fig. 4. Mechanism of aci-miR-9b in the regulation of wing dimorphism and wing development in A. citricidus. (A) Trehalose content of AD-WL after aci-miR-9b inhibitor feeding under normal and N4-WD after dsAcABCG4 and aci-miR-9b mimic feeding. (B) The expression level of AcILP3 of AD-WL after aci-miR-9binhibitor feeding and N4-WD after dsAcABCG4 and aci-miR-9b mimic feeding. (C) The proportion of A. citricidus adults with malformed wings under dsAcILP3treatment. (D) Cofeeding bovine insulin rescued the wing defects caused by dsAcABCG4. A “+” indicates with the treatments, and a “−” indicates without thetreatments. All of the mean (±SE) is based on four biological replicates. normal, wingless adult under normal N4-WD, fourth instar winged nymph. (E)Proposed model of miR-9b/ABCG4 in the regulation of wing dimorphism and wing development in aphids. Red color indicates increase and blue color in-dicates decrease. All of the mean (±SE) is based on four biological replicates. The significant differences among different treatments are indicated bylowercase letters above each bar (one-way ANOVA followed by the LSD test, P < 0.05) in A and B (data from normal and N4-WD were compared separately)and in D. The significant differences between treatment and control are indicated by asterisks (Student’s t test, **P < 0.01; ***P < 0.001) in C.

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expression dynamics (Fig. 4B and SI Appendix, Fig. S16). Theupstream component of the IIS pathway is ILPs and three ILPs(AcILP1, AcILP3, and AcILP5) were identified based on the ge-nome sequence of A. citricidus (SI Appendix, Fig. S17A). The tissue-specific expression patterns showed that AcILP3 and AcILP5 werehighly expressed in the fat body, consistent with the expressionof aci-miR-9b and AcABCG4, while AcILP1 was specificallyexpressed in the brain (SI Appendix, Fig. S17B). Silencing ofAcILP3 increased the expression of AcABCG4, indicating thatAcABCG4 and AcILP3 may be associated (SI Appendix, Fig.S17C). Silencing of AcILP3 resulted in 53% of aphids with mal-formed wings while silencing of AcILP1 resulted in 48% of theaphids with inhibited adult emergence (Fig. 4C and SI Appendix,Fig. S17 D and E). A rescue experiment was conducted usingfeeding with bovine insulin. The proportion of malformed wingswas 44% under dsAcABCG4 combined with HEPES buffertreatment, while no adults had malformed wings when feddsAcBCG4 combined with bovine insulin (Fig. 4D). These resultsindicate that insulin rescued the wing defects caused by thesilencing of AcABCG4 and confirmed that the downstream actionof aci-miR-9b/AcABCG4 is through the IIS pathway.

DiscussionPopulation density is a key environmental factor inducing wingedmorphs in aphid species (3, 17, 18). We found a key regulator,miR-9b, that controls crowding-induced wing dimorphism inaphids. miRNAs jointly regulate wing development in nonwingdimorphic insects (19–21). Based on a target-miRNA reverse pre-diction strategy, the insulin receptor gene of the IIS pathway wastargeted by miR-34 in a planthopper (22). Here, based on an RNA-seq approach, we found that only the expression of aci-miR-9b wasaltered in both the wing dimorphism and the wing developmentperiod but not in wingless development from fourth instar nymphsto adults. The differential expression of miR-9b between wingedand wingless morphs might reflect a maternal effect of thecrowding. Indeed, via experimental manipulation of miR-9b,the wing dimorphism and development were altered. Inhibitionof miR-9b increased the winged offspring proportion whileoverexpression of miR-9b could decline the high proportion ofwinged offspring induced by crowding. During wing development,the overexpression of miR-9b also led to adults with malformedwings. Upon miR-9b inhibitor exposure, the treated aphids couldnot produce more winged offspring than other treatments undercrowding for two possible reasons: 1) The expression of miR-9bcould not be completely depleted through feeding on the miR-9binhibitor (the expression of aci-miR-9b declined 44 to 87%, SIAppendix, Fig. S8). A genome-editing approach may clarify this infuture studies (23). 2) Alternatively, different genetic backgroundsof aphids may exhibit variation of winged offspring proportion in arange from “weak-inducible” to “high-inducible” genotypes afteraphids perceiving environmental cues (e.g., crowding) (9). In thecurrent study, the additional miR-9b inhibitor in crowding did notincrease the proportion of winged offspring in A. citricidus. Since theproportions of winged offspring under the miR-9b inhibitor treat-ment alone or under crowding conditions were both ∼50%, thissuggested that an ∼50% winged proportion might be the thresholdfor this genetic background, which confirms that miR-9b is the keyregulator in crowding-induced wing dimorphism in A. citricidus.We found that miR-9b down-regulates ABCG4 and also reg-

ulates the activity of the carbohydrate-stimulated IIS pathway.Phosphorylation by the corresponding ligands is an importantindicator to reflect the activity of the IIS pathway. In addition tophosphorylation, the expression levels of downstream intracellularsignaling can also reflect the diminished or enhanced IIS activities(24). The link between miR-9b and ABCG4 to IIS throughILP3 was based on the following: 1) The expression patterns ofILP3 were consistent with ABCG4 upon miR-9b inhibitor/dsABCG4/miR-9b mimic treatments. 2) Silencing of ILP3 resulted in

malformed wings, which was the same phenotype caused by theoverexpression of miR-9b or silencing of ABCG4. 3) Feeding bovineinsulin to A. citricidus rescued the malformed wing phenotypescaused by dsABCG4 treatment. 4) The expression patterns ofdownstream components of IIS exhibited similar dynamics asABCG4 upon miR-9b inhibitor/dsABCG4/miR-9b mimic. It is knownthat there is a transgenerational effect of wing dimorphism in severalaphid species, which implies that the parent senses the environmentalcues (e.g., crowding) and the proportion of winged offspring is al-tered (7, 18, 25), which was confirmed in A. citricidus and A. pisumin this study. In addition, we also observed that the expression ofaci-miR-9b was reduced in nymphs (N3-WD and N3-WL) uponcrowding (SI Appendix, Fig. S18), which indicated that the wingdimorphism could also occur throughout nymphal stages. A pre-vious study showed that the interactions among the insulin signalingpathway, ecdysone signaling, and juvenile hormone signaling are in-volved in wing dimorphism (22). In this study, we found that thisphysiological process was regulated by the cascade “miR-9b-ABCG4-insulin signaling.”However, how this signal transduces to the offspring(embryo) in aphids, and the sensitive period of wing dimorphism inaphids still requires further study.As primary-active proteins that bind and hydrolyze ATP, ABC

transporters facilitate the transportation of many substrates suchas amino acids, sugars, lipids, and toxic metabolites across mem-branes (26). However, beyond miR-9b in regulating ABCG4, it is notyet clear how ABCG4 participates in the carbohydrate-stimulatedIIS pathway in aphids. In mouse pancreatic β cells (MIN6 cells),miR-463 targets ABCG4 and controls insulin secretion, which isbased on the concentration of glucose (27). High carbohydratecontent increased wing growth (4, 28) while the triglyceride con-tent showed an opposite trend with trehalose level during wingdimorphism and development (SI Appendix, Fig. S19). This is alsosupported by transcriptome and proteome-based analysis, whichrevealed a high level of glycolysis/gluconeogenesis as well as pyruvatemetabolism in winged morphs of A. citricidus and A. pisum (29, 30).The insect fat body is necessary for energy storage and metab-

olism, and it may function as a nutrient sensor through regulationof ILP secretion (31). In the fruit fly Drosophila melanogaster andthe yellow fever mosquito Aedes aegypti, only ILP6 was secreted bythe fat body (32, 33). In the present study, we found that two ILPs(ILP3 and ILP5) were highly expressed in the fat body of A. citricidus,and ILP3 appeared to be specifically related to wing dimorphism.In conclusion, we found a miRNA, aci-miR-9b, that is a critical

regulator of wing dimorphism and wing development in aphids.For wing dimorphism, aphids perceive crowding which decreasesthe expression of miR-9b, thereby increasing the expression of itstarget gene ABCG4 and carbohydrate content, which subsequentlyactivate the IIS pathway leading to an increase in the proportionof winged morphs. This cascade was termed as miR-9b-ABCG4-insulin signaling. For wing development, the same cascade, miR-9b-ABCG4-insulin signaling, was again involved in the transitionfrom fourth instar winged nymph to winged adult. (Fig. 4E). Theresults increase our knowledge of miRNA as a sensory factor thatcan help insects adapt to a changing environment. The informa-tion may stimulate the development of methods for controlling thedispersal of aphid pests and virus transmission.

Materials and MethodsInsects, sample preparation for RNA-seq, transcriptome and small RNA se-quencing, luciferase activity assay, RNA immunoprecipitation assay, dsRNAsynthesis and RNAi assay, RT-qPCR, determination of trehalose content,Western blot, and statistics used in this study are included in SI Appendix.

See SI Appendix, Materials and Methods for details.

Data Availability. Sequence data have been deposited in the National Centerfor Biotechnology Information’s Sequence Read Archive (accession nos.PRJNA578247 and PRJNA576278).

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ACKNOWLEDGMENTS.We thank Dr. Guy Smagghe (Ghent University) for hiscritical comments on experimental design. This research was supported bythe National Key R&D Program of China (Grant 2017YFD0200900); the China

Postdoctoral Science Foundation (Grant 2018M640894); the 111 Project(Grant B18044); and the earmarked fund for the Modern Agro-industry (Cit-rus) Technology Research System of China (Grant CARS-26).

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