Transcriptome Analysis of the Regulatory Mechanism of Foxo on Wing Dimorphism in the Brown Planthopper, Nilaparvata Lugens (Hemiptera: Delphacidae)

insects

Article Transcriptome Analysis of the Regulatory Mechanism of FoxO on Wing Dimorphism in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

Nan Xu 1, Sheng-Fei Wei 1 and Hai-Jun Xu 1,2,3,*

1 State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou 310058, China; [email protected] (N.X.); [email protected] (S.-F.W.) 2 Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, China 3 Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China * Correspondence: [email protected]; Tel.: +86-571-88982996

Simple Summary: The brown planthopper (BPH) Nilaparvata lugens can develop into either long- winged or short-winged adults depending on environmental stimuli received during larval stages. The transcription factor NlFoxO serves as a key regulator determining alternative wing morphs in BPH, but the underlying molecular mechanism is largely unknown. Here, we investigated the transcriptomic proﬁle of forewing and hindwing buds across the 5th-instar stage, the wing-morph decision stage. Our results indicated that NlFoxO modulated the developmental plasticity of wing buds mainly by regulating the expression of cell proliferation-associated genes.

Abstract: The brown planthopper (BPH), Nilaparvata lugens, can develop into either short-winged (SW) or long-winged (LW) adults according to environmental conditions, and has long served as a model organism for exploring the mechanisms of wing polyphenism in insects. The transcription Citation: Xu, N.; Wei, S.-F.; Xu, H.-J. factor NlFoxO acts as a master regulator that directs the development of either SW or LW morphs, Transcriptome Analysis of the but the underlying molecular mechanism is largely unknown. Here, we microinjected SW-destined Regulatory Mechanism of FoxO on morphs with double stranded-RNA (dsRNA) targeting NlFoxO (dsNlFoxO) to change them into Wing Dimorphism in the Brown LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the Planthopper, Nilaparvata lugens gene encoding green fluorescence protein (dsGfp) served as a negative control. The forewing and (Hemiptera: Delphacidae). Insects 2021, 12, 413. https://doi.org/ hindwing buds of 5th-instar nymphs collected at 24, 36, and 48 h after eclosion (hAE) were used for 10.3390/insects12050413 RNA sequencing. We obtained a minimum of 43.4 million clean reads from forewing and hindwing buds at a single developmental time. Differentially expressed genes (DEGs) were significantly Received: 26 March 2021 enriched in various Gene Ontology (GO) terms, including cellular process, binding, and cell part. Accepted: 30 April 2021 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that up- Published: 4 May 2021 regulated genes in dsNlFoxO-treated forewing and hindwing buds were largely associated with the cell cycle and DNA replication. Furthermore, most up-regulated genes displayed higher expression Publisher’s Note: MDPI stays neutral at 24-, and 36-hAE relative to 48 hAE, indicating that wing cells in LW-destined wings might actively with regard to jurisdictional claims in proliferate during the first 36 h in 5th-instar nymphs. Our findings indicated that LW development published maps and institutional affil- in BPH was likely dependent on the duration of cell proliferation in the 5th-instar stage, which sheds iations. light on the molecular basis of wing polymorphism in insects.

Keywords: planthopper; wing polyphenism; RNA-seq; FoxO; cell proliferation

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons The brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is the Attribution (CC BY) license (https:// most destructive rice pest in Asia [1]. BPH feeds exclusively on the phloem sap of rice creativecommons.org/licenses/by/ plants and transmits plant viruses such as rice ragged stunt viruses and rice grassy stunt 4.0/). viruses, leading to huge losses in rice yields [2,3]. BPH is one of the most extensively

Insects 2021, 12, 413. https://doi.org/10.3390/insects12050413 https://www.mdpi.com/journal/insects Insects 2021, 12, 413 2 of 15

studied wing-dimorphic insects due to the natural occurrence of short- and long-winged (SW and LW) adults [4]. As a hemimetabolous insect, newly hatched first-instar nymphs look like miniature adults, and wing buds grow gradually as the developmental stages advance. Our recent studies showed that the N. lugens FoxO homolog NlFoxO relied on the insulin/insulin-like signaling (IIS) activity to direct wing buds developing into SW or LW morphs during the wing-morph decisive stage (the 5th-instar stage). High IIS activity inhibited NlFoxO activity, leading to LW morphs, and vice versa [5–7]. However, the molecular basis by which NlFoxO influences alternative wing morphs remains largely unknown. FoxO transcription factors belong to the large Forkhead family of proteins, which are characterized by a conserved DNA-binding domain termed the “forkhead box” [8]. FoxO proteins are tightly involved in various cellular processes including regulating the expression of genes associated with cell cycle, DNA repair, apoptosis, and energy balance. Loss of FoxO is associated with increased cancer in mammals [9,10]. In the nematode worm Caenorhabditis elegans, the FoxO homolog daf-16 was initially isolated as a dauer defective mutant [11], and overexpression of daf-16 can extend lifespan via dauer formation [12]. In addition, FoxO mediates the effects of the IIS pathway in stress resistance, fat storage, immunity, and reproduction [13–18]. In the absence of IIS activity, FoxO induces cell cycle arrest and entrance into cellular quiescence [19]. FoxO transcription factors play a major role in G1 arrest by both up-regulating cell cycle inhibitors (p21 and p27) and by repressing cell cycle activators (cyclin D1/D2) [20,21]. In the model insect Drosophila melanogaster, fly FoxO homolog dFoxO mutants were viable and of normal size, but lost protection against oxidative stress [22]. In contrast, ectopic expression of dFoxO caused a decrease in cell number and cell size [22–24], leading to a reduction in body size. The emergence of next-generation sequencing technology has profoundly improved our understanding of the molecular basis of wing polymorphism in insects. Our early RNA sequencing (RNA-seq) study identified hundreds of genes including those correlated to respiration and energy metabolism were up-regulated in LW versus SW BPH adults, indicating LW BPH adults might require more energy than SW for flight [25]. In the cotton aphids (Aphis gossypii), genes associated with flight-reproduction trade-offs were differentially expressed in winged versus wingless morphs through RNA-seq analysis [26]. In the brown citrus aphid (Toxoptera citricida), RNA-seq identified both lipid and glyco- gen metabolism-associated genes that were differentially expressed between winged and wingless adults, indicating that these genes might contribute to energy metabolism during aphid wing development [27]. In the present study, we microinjected SW-destined morphs with double stranded- RNA (dsRNA) targeting NlFoxO (dsNlFoxO) to change them into LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the gene encoding green fluorescence protein (dsGfp) served as a negative control. Forwing and hindwing buds were dissected from dsNlFoxO- and dsGfp-treated nymphs at the wing-morph decisive stage (the 5th-instar stage), and were then used for RNA-sequencing. Comparative transcriptomic analysis indicated that NlFoxO-regulated alternative wing morphs were likely through modulating genes involved in cell proliferation. These results advanced our understanding of the molecular basis of wing dimorphism in insects.

2. Materials and Methods 2.1. Insect Colony The SW N. lugens strain (SW > 95%) was initially collected from a rice ﬁeld in Hangzhou, China, in 2009 [25]. Insects were maintained on rice seedings (rice variety: Xiushui134) in a walk-in chamber at 26 ± 0.5 ◦C with a relative humidity of 50 ± 10% and a photoperiod of 16:8 h (light: dark). Insects 2021, 12, 413 3 of 15

2.2. RNA Extraction and cDNA Library Construction 4th-instar nymphs were collected for microinjection with dsNlFoxO to generate LW- destined morphs. For a negative control, 4th-instar nymphs were microinjected with dsGfp, which produced SW-destined adults as the SW N. lugens strain. For transcriptome sequencing, forewing and hindwing buds were dissected from 5th-instar nymphs (n = 200 for each biological replicate) at 24, 36, and 48 h after ecdysis (hAE). Three independent biological replicates were performed at each designed time. Total RNA was isolated from wing buds using TRIzol (Takara, Dalian, China), and the RNA concentration and quality were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). RNA samples (>2 µg) were subsequently used for cDNA library construction using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to manufacturer’s recommendations, and index codes were added to each sequence. Library fragments of 250–300 bp in length were preferentially puriﬁed using an AMPure XP system (Beckman Coulter, Woerden, The Netherlands), and library quality was assessed using the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a HiSeq PE Cluster Kit v4-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer’s instructions. The cDNA libraries were sequenced on an Illumina Hiseq platform and 150-bp paired-end reads were generated.

2.3. Gene Annotation and Mapping Clean reads were derived from raw reads by removing adapter reads, low-quality reads, and reads containing poly-N and ambiguous bases using the trimmomatic [28]. Clean reads were aligned to the reference genome [29] using Hisat2 [30] for gene expression analysis. Differential genes expression (DGE) in dsNlFoxO versus dsGfp was analyzed using DEseq2 [31]. False discovery rate (FDR) was used to measure the threshold p-value in multiple tests, and transcripts with fold change ≥2 and FDR < 0.05 were subjected to differentially expressed gene (DEG) analysis [32,33].

2.4. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Pathway Analyses Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed using an online platform (https://www.omicshare.com/tools/) on 15 February 2021. GO terms with corrected p-value <0.05 were deﬁned as signiﬁcantly enriched GO terms [29]. DEGs were used for KEGG analysis to identify markedly enriched metabolic pathways or signal transduction pathways with corrected p-value < 0.05 [34].

3. Results 3.1. Overview of Transcriptomes A total of 36 cDNA libraries were prepared from 12 samples with three biological replicates each, then used for Illumina sequencing. More than 44.6 million raw reads were generated from each cDNA library with Q30 values in all libraries exceeding 93.85%. Following the removal of adaptors, poly-N and low-quality reads, more than 43.4 million clean reads were retained for each sample (Table S1). The mapping rate of clean reads against the N. lugens reference genome ranged from 66.25% to 74.78%.

3.2. DEGs in Wing Buds of 5th-Instar Nymphs Treated with dsNlFoxO and dsGfp Using the fold change ≥2 and FDR < 0.05 as criteria, we identiﬁed 2128 (784 up- regulated and 1344 down-regulated), 2902 (1473 up-regulated and 1429 down-regulated), and 1813 (732 up-regulated and 1081 down-regulated) DEGs in forewing buds of dsNlFoxO- treated 5th-instar nymphs at 24, 36, and 48 hAE, respectively, relative to dsGfp treatment. For hindwing buds, 3466 (1294 up-regulated and 2172 down-regulated), 2533 (1496 up- regulated and 1037 down-regulated), and 2279 (1381 up-regulated and 898 down-regulated) DEGs were identiﬁed in dsNlFoxO-treated 5th-instar nymphs at 24, 36, and 48 hAE, respectively (Figure1B). Insects 2021, 12, x FOR PEER REVIEW 4 of 15

Insects 2021, 12, 413 4 of 15 regulated) DEGs were identified in dsNlFoxO-treated 5th-instar nymphs at 24, 36, and 48 hAE, respectively (Figure 1B).

FigureFigure 1.1.Schematic Schematic depictiondepiction of long-wingedlong-winged (LW)-(LW)- and short short-winged-winged (SW) (SW)-- destined destined BPHs, BPHs, and and differentiallydifferentially expressed expressed genes genes (DEGs) (DEGs) in in wing wing buds. buds. ( A(A) 4th-instar) 4th-instar nymphs nymphs were were microinjected microinjected with dswithNlFoxO dsNlFoxOand ds andGfp dstoGfp generate to generate LW and LW SW and destined SW destined BPHs, BPHs, respectively. respectively. Forewing Forewing and hindwing and budshindwing were dissectedbuds were from dissected 5th-instar from nymphs 5th-instar at 24, nymphs 36, and at 48 24, h after36, and ecdysis 48 h after (hAE), ecdysis then collected (hAE), then for RNA-sequencing.collected for RNA (-Bsequencing.) DEGs in LW-destined (B) DEGs in (dsLWNlFoxO-destined) versus (dsNlFoxO SW-destined) versus (ds SWGfp-destined) wing buds.(dsGfp) wing buds. 3.3. Functional Enrichment Analysis of DEGs in Forewing and Hindwing Buds at 24 hAE 3.3. Functional Enrichment Analysis of DEGs in Forewing and Hindwing Buds at 24 hAE To better understand the regulatory mechanisms of long wing development regulated To better understand the regulatory mechanisms of long wing development regu- by dsNlFoxO, DEGs were used for GO term and KEGG pathway enrichment analyses. lated by dsNlFoxO, DEGs were used for GO term and KEGG pathway enrichment anal- For forewing buds at 24 hAE, 2128 DEGs were enriched in 54 GO terms associated with yses. For forewing buds at 24 hAE, 2128 DEGs were enriched in 54 GO terms associated 24 biological process (BP) categories (GO:0008150), 16 cellular component (CC) categories with 24 biological process (BP) categories (GO:0008150), 16 cellular component (CC) cate- (GO:0005575), and 14 molecular function (MF) categories (GO:0003674) with FDR < 0.05 gories (GO:0005575), and 14 molecular function (MF) categories (GO:0003674) with FDR as the criterion (Table S2; Figure2A). The top-ranked terms included cellular process (259< 0.05 up-regulated as the criterion and 534 (Table down-regulated S2; Figure 2A). genes), The bindingtop-ranked (239 terms up-regulated included and cellular 462 down- pro- regulatedcess (259 up genes),-regulated and cell and part 534 (211 down up-regulated-regulated and genes 438), down-regulatedbinding (239 up- genes;regulated Figure and2A). 462 KEGGdown- enrichmentregulated genes pathway), and analysis cell part showed (211 thatup-regulated the 2128 DEGs and 438 of forewing down-regulated buds at 24 genes hAE ; wereFigure annotated 2A). KEGG into enrichment six KEGG classespathway (Figure analysis3A); showed genetic that information the 2128 DEGs processing of forewing (four pathways),buds at 24 metabolismhAE were annotated (12 pathways), into six human KEGG diseases classes (eight(Figure pathways), 3A); genetic environmental information informationprocessing (four processing pathways), (three metabolism pathways), (12 organismal pathways), systems human (10 diseases pathways), (eight and pathways), cellular processesenvironmental (four pathways) information (Figure processing3A). The (three “global pathways), and overview organismal maps” pathwaysystems (10 was path- the mostways), enriched, and cellular followed processes by “signal (four transductions” pathways) (Figure and “infectious 3A). The diseases” “global and pathways. overview maps”Subsequently, pathway was up-regulated the most enriched, and down-regulated followed by DEGs“signal were transductions” further separately and “infec- col- latedtious fordiseases” KEGG pathways. pathway analysis. Out of 784 up-regulated DEGs, only 54 genes were assignedSubsequently, to KEGG pathwaysup-regulated (p-values and down < 0.05;-regulated Table S3; DEGs Figure were4A), further and theseparately ribosome col- pathwaylated for (ko03010)KEGG pathway was at analysis. the top Out of the of 784 list up (q-values-regulated < 0.05; DEGs, Figure only4 A).54 genes Out ofwere 1344 as- down-regulatedsigned to KEGG DEGs,pathways 124 out(p-values of 1344 < genes0.05; Table were S3; the Figure most significantly4A), and theenriched ribosome in path- the metabolicway (ko03010) pathway was (ko01100; at the top Table of the S3; list Figure (q-values4B). < 0.05; Figure 4A). Out of 1344 down- regulatedFor hindwing DEGs, 124 buds out atof 241344 hAE, genes 3466 were DEGs the includingmost significantly 1294 up-regulated enriched in andthe meta- 2172 down-regulatedbolic pathway (ko01100; genes were Table assigned S3; Figure to 54 4B) GO. terms (Table S4; Figure2B), among which the terms cellular process (531 up-regulated and 1027 down-regulated genes), cell part (468 up-regulated and 828 down-regulated genes), and binding (443 up-regulated and 841 down-regulated genes) contained the most enriched DEGs (Figure2B). This observation is in line with GO analysis of forewing buds at 24 hAE. The 3466 DEGs were classified into six KEGG classes, and the top-ranked pathways included “global and overview maps” and “signal transduction” (Figure3B). For up-regulated DEGs in hindwings, 218 out of 1294 genes could be matched to KEGG pathways. Analogous to forewings, “ribosome” was the most enriched term, followed by spliceosome, and DNA replication (ko03030; Table S3; Figure4C). For down-regulated DEGs, 217 out of 1081 genes were assigned to KEGG pathways, and “metabolic” was the most significantly enriched pathway (Table S3; Figure4D), consistent with forewing buds at 24 hAE.

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FigureFigure 2. 2. GeneGene O Ontologyntology (GO) (GO) classification classiﬁcation of ofdifferentially differentially expressed expressed genes genes in inforewing forewing (A ()A and) and hindwinghindwing (B (B) buds) buds of of 24 24 hAE hAE-5th-instar-5th-instar nymphs nymphs with with NlFoxONlFoxO andand GfpGfp knockdown.knockdown. GO GOanalysis analysis is summarizedis summarized as three as three main main categories: categories: cellular cellular component component (CC), (CC),molecular molecular function function (MF), (MF),and bio- and logicalbiological process process (BP). (BP).

3.4.For Functional hindwing Enrichment buds at Analysis 24 hAE, of 3466 DEGs DEGs in Forewing including and Hindwing 1294 up-regulated Buds at 36 andhAE 2172 down-Forregulated forewing genes buds were at 36 assigned hAE, 2902 to DEGs 54 GO (1473 terms up-regulated (Table S4; Figure and 1429 2B) down-regulated), among which thewere terms categorized cellular process into 24 BP,(531 14 up MF-regulated and 16 CC and GO 1027 terms down based-regulated on FDR genes < 0.05), (Tablecell part S5). (468As shownup-regulated in Figure and5, 828 cellular down process-regulated (732 genes up-regulated), and binding and 524 (443 down-regulated up-regulated and genes), 841 downbinding-regulated (710 up-regulated genes) contained and 424 the down-regulated most enriched genes), DEGs and(Figure cell part2B). This (613 up-regulatedobservation isand in line 403 down-regulatedwith GO analysis genes) of forewing were the buds top-ranked at 24 hAE. GO terms,The 3466 respectively. DEGs were KEGG classified analy- intosis showedsix KEGG that classes, DEGs in and forewing the top buds-ranked at 36 hAE pathways were signiﬁcantly included “global enriched and in overview six KEGG maps”classes and including “signal genetic transduction” information (Figure processing 3B). For (4 up pathways),-regulated human DEGs diseasesin hindwings, (eight path-218 outways), of 1294 organismal genes could systems be matched (10 pathways), to KEGG cellular pathways. processes An (fouralogous pathways), to forewings, environmen- “ribosome”tal information was the processingmost enriched (three term, pathways), followed and by metabolism spliceosome, (12 and pathways) DNA replication(Figure6A) . (ko03030;The “global Table and S3; overview Figure maps”4C). For and down “signal-regulated transductions” DEGs, 217 were out the of two1081 most genes enriched were assignedKEGG pathways to KEGG (Figurepathways,6A), and including “metabolic” 227 and was 212 the genes, most respectively.significantly KEGGenriched analysis path- wayof the (Table 1473 S3; up-regulated Figure 4D), genes consistent showed with that forewing the top buds 20 enriched at 24 hAE. pathways included DNA

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replication (ko03030), cell-cycle yeast (ko04111), and cell-cycle (ko04110), suggesting that Insects 2021, 12, x FOR PEER REVIEWthese pathways played important roles in long wing development (Figure7A) . KEGG6 anal-of 15 Insects 2021, 12, x FOR PEER REVIEW 6 of 15 ysis of the 1429 down-regulated genes showed that the metabolic pathway was apparently the most enriched pathway (Figure7B).

FigureFigure 3.3. KyotoKyoto EncyclopediaEncyclopedia ofof GenesGenes andand GenomesGenomes (KEGG) (KEGG) pathway pathway classiﬁcation classification of of DEGs DEGs in in the Figure 3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification of DEGs in forewingthe forewing (A) and(A) and hindwing hindwing (B) buds(B) buds of 24 of hAE-5th-star 24 hAE-5th- nymphsstar nymphs with withNlFoxO NlFoxOand Gfpandknockdown. Gfp knock- thedown. forewing (A) and hindwing (B) buds of 24 hAE-5th-star nymphs with NlFoxO and Gfp knockdown.

FigureFigure 4.4. TopTop 2020 enrichedenriched KEGGKEGG pathwayspathways ofof DEGsDEGs inin 2424 hAE-5th-instarhAE-5th-instar nymphs nymphs with withNlFoxO NlFoxOand GfpFigureandknockdown. Gfp 4. knockdown. Top 20 (enrichedA,B )(A Top,B KEGG) 20Top enriched 20 pathways enriched KEGG of KEGG pathwaysDEGs pathways in 24 of hAE up-regulated of-5 upth--instarregulated and nymphs down-regulated and withdown NlFoxO-regulated genes inandgenes forewing Gfp in knockdown. forewing buds, respectively.buds, (A, Brespectively.) Top (20C, Denriched) Top(C,D 20) KEGGTop enriched 20 pathwaysenriched KEGG KEGG pathwaysof up- regulatedpathways of up-regulated and of up down-regulated- andregulated down- and regulatedgenesdown -inregulated forewing genes ingenes buds, hindwing in respectively. hindwing buds, respectively.buds, (C,D respectively.) Top 20 The enrichedq-values The q KEGG-values in different pathways in different colors of colors denoteup-regulated denote signiﬁcant andsig- down-regulated genes in hindwing buds, respectively. The q-values in different colors denote sig- enrichment.nificant enrichment. Different Different sizes of dotssizes represent of dots represent the number the ofnumber genes of enriched genes enriched in each pathway. in each path- nificantway. enrichment. Different sizes of dots represent the number of genes enriched in each pathway. 3.4. Functional Enrichment Analysis of DEGs in Forewing and Hindwing Buds at 36 hAE 3.4. Functional Enrichment Analysis of DEGs in Forewing and Hindwing Buds at 36 hAE For forewing buds at 36 hAE, 2902 DEGs (1473 up-regulated and 1429 down-regulated)For were forewing categorized buds intoat 36 24 hAE, BP, 142902 MF DEGs and 16 (1473 CC GO up -termsregulated based and on 1429FDR

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S5). As shown in Figure 5, cellular process (732 up-regulated and 524 down-regulated genes), binding (710 up-regulated and 424 down-regulated genes), and cell part (613 up- regulated and 403 down-regulated genes) were the top-ranked GO terms, respectively. KEGG analysis showed that DEGs in forewing buds at 36 hAE were significantly enriched in six KEGG classes including genetic information processing (4 pathways), human diseases (eight pathways), organismal systems (10 pathways), cellular processes (four pathways), environmental information processing (three pathways), and metabolism (12 pathways) (Figure 6A). The “global and overview maps” and “signal transductions” were the two most enriched KEGG pathways (Figure 6A), including 227 and 212 genes, respectively. KEGG analysis of the 1473 up-regulated genes showed that the top 20 enriched pathways included DNA replication (ko03030), cell-cycle yeast (ko04111), and cell-cycle (ko04110), suggesting that these pathways played important roles in long wing develop- Insects 2021, 12, 413 7 of 15 ment (Figure 7A). KEGG analysis of the 1429 down-regulated genes showed that the metabolic pathway was apparently the most enriched pathway (Figure 7B).

FigureFigure 5. 5.GOGO classification classiﬁcation of DEGs of DEGs in forewing in forewing (A)( andA) and hindwing hindwing (B) buds (B) buds of 36 of hAE 36 hAE-5th-instar-5th-instar nymphsnymphs with with NlFoxONlFoxO andand GfpGfp knockdown.knockdown. GO GO is summarized is summarized as three as three main main categories categories (CC, (CC, MF MF andand BP). BP).

For hindwing buds at 36 hAE, 2533 (1496 up-regulated and 1037 down-regulated) DEGs were annotated using GO classifications, including 25 BP, 13 MF, and 16 CC terms,

among which cellular process (807 up-regulated and 412 down-regulated), binding (753 up- regulated and 327 down-regulated), and cell part (691 up-regulated and 300 down-regulated) were the top-ranked terms (Table S6; Figure5B). In line with forewing buds at 36 hAE, the 2533 DEGs in hindwing buds at 36 hAE were distributed in 41 KEGG pathways, and the “global and overview maps” and “signal transductions” pathways contained the most DEGs (Figure6B). KEGG analysis of the 1496 up-regulated genes showed that DNA replication and cell cycle were signiﬁcantly enriched (Figure7C), consistent with the results for forewing buds at 36 hAE. For the 1037 down-regulated DEGs, a metabolic pathway contained the most DEGs in hindwing buds at 36 hAE (Figure7D). InsectsInsectsInsects 20212021 2021,, 1212, 12,, x 413, xFOR FOR PEER PEER REVIEW REVIEW 8 8of ofof 15 15 15

FigureFigureFigure 6. 6. 6. KEGG KEGG pathway pathway classification classification classiﬁcation of of ofDEGs DEGs DEGs in in the in the theforewing forewing forewing (A (A) (and)A and) andhindwing hindwing hindwing (B (B) buds) (budsB)buds of of 36 36 of hAE36hAE hAE-5th-star-5-th5th-star-star nymphs nymphs nymphs with with with NlFoxO NlFoxONlFoxO and andand Gfp GfpGfp knockdown. knockdown.knockdown.

FigureFigureFigure 7. 7. 7. TopTop Top 20 20 20 enriched enriched enriched KEGG KEGG pathways pathways pathways of of DEGs DEGs in in 36 36 hAE hAE-5th-instar hAE-5-th5th-instar-instar nymphs nymphs with with NlFoxO NlFoxO and andGfpand knockdown.Gfp Gfp knockdown knockdown (A,.B (.)A ( TopA,B,B) Top) 20 Top enriched 20 20 enriched enriched KEGG KEGG KEGG pathways pathways pathways of up-regulated of of up up-regulated-regulated and down-regulatedand and down down-regulated-regulated genes genesingenes forewing in in forewing forewing buds, buds, respectively. buds, respectively. respectively. (C,D) ( TopC (C,D,D 20) Top) enrichedTop 20 20 enriched enriched KEGG KEGG pathwaysKEGG pathways pathways of up-regulated of of up up-regulated-regulated and down- and and downregulateddown-regulated-regulated genes genes in genes hindwing in in hindwing hindwing buds, buds, respectively. buds, respec respectively Thetivelyq. -valuesThe. The q -qvalues- invalues different in in different different colors color denotecolors sdenote denote signiﬁcant sig- sig- nificantenrichment.nificant enrichment. enrichment. Different Different Different sizes of sizes dots sizes representof of dots dots represent represent the number the the number of number genes of enrichedof genes genes enriched inenriched each pathway.in in each each path- path- way.way.

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3.5. Functional Enrichment Analysis of DEGs in Forewing Buds and Hindwing Buds at 48 hAE For forewing buds at 48 hAE, the 1813 (732 up-regulated and 1081 down-regulated) DEGs were mapped to 52 GO terms (Table S7; Figure8A). Analogous to forewing buds at 24- and 36-hAE, most genes were assigned to single-organism processes, binding, and cell part terms (Figure8A). KEGG analysis showed that “global and overview maps” and “signal transductions” were the two most enriched pathways (Figure9A), including 156 and 115 genes, respectively. In addition, KEGG analysis of the up-regulated genes showed that the top enriched pathways included DNA replication and cell cycle (Table S3; Insects 2021, 12, x FOR PEER REVIEW 10 of 15 Figure 10A), while metabolic pathways was the most signiﬁcantly enriched pathway in the down-regulated genes (Table S3; Figure 10B).

FigureFigure 8. 8. GOGO classification classiﬁcation of ofDEGs DEGs in inforewing forewing (A ()A and) and hindwing hindwing (B) (budsB) buds of 48 of hAE 48 hAE-5th-instar-5th-instar nymphsnymphs with with NlFoxONlFoxO andand GfpGfp knockdown.knockdown. GO GO was was summarized summarized as as three three main main categories categories (CC, (CC, MF MF andand BP). BP).

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FigureFigure 9.9. KEGGKEGGKEGG pathwaypathway pathway classificationclassification classiﬁcation ofof ofDEGsDEGs DEGs inin the inthe the forewingforewing forewing ((AA)) (andandA) and hindwinghindwing hindwing ((BB)) budsbuds (B) buds ofof 4848 of hAE48hAE hAE-5th-star--55thth--starstar nymphsnymphs nymphs withwith with NlFoxONlFoxONlFoxO andandand GfpGfpGfp knockdown.knockdown.knockdown.

Figure 10. Top 20 enriched KEGG pathways of DEGs in 48 hAE-5th-instar nymphs with NlFoxO and FigureFigure 10.10. TopTop 2020 enrichedenriched KEGGKEGG pathwayspathways ofof DEGsDEGs inin 4848 hAEhAE--55thth--instarinstar nymphsnymphs withwith NlFoxONlFoxO andGfpand knockdown. GfpGfp knockdown.knockdown. (A,B ()(AA Top,,BB)) Top 20Top enriched 2020 enrichedenriched KEGG KEGGKEGG pathways pathwayspathways of up-regulated ofof upup--regulatedregulated and down-regulated andand downdown--regulatedregulated genes genesingenes forewing inin forewingforewing buds, buds, respectively.buds, respectively.respectively. (C,D) ( Top(CC,,DD 20)) TopTop enriched 2020 enrichedenriched KEGG KEGGKEGG pathways pathwayspathways of up-regulated ofof upup--regulatedregulated and down- andand downregulateddown--regulatedregulated genes genes ingenes hindwing inin hindwinghindwing buds, buds, respectively.buds, respectively.respectively. The q -values TheThe qq--valuesvalues in different inin differentdifferent colors colorscolors denote denotedenote signiﬁcant sig-sig- nificantenrichment.nificant enrichment.enrichment. Different DifferentDifferent sizes of sizes dotssizes represent ofof dotsdots representrepresent the number thethe numbernumber of genes ofof enriched genegeness enrichedenriched in each pathway. inin eaceachh path-pathway.way.

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For hindwing buds at 48 hAE, greater percentages of the 2279 DEGs (1381 up-regulated and 898 down-regulated) were mapped to the single-organism process, binding, and cell part terms (Table S7; Figure8B). KEGG analysis showed that “global and overview maps” and “signal transductions” were the two most enriched pathways (Figure9B), including 192 and 165 genes, respectively. The top enriched pathways in the up-regulated genes included endocrine resistance, human papillomavirus infection, and cell cycle (Table S3; Figure 10C), while “metabolic pathways” was the most signiﬁcantly enriched pathway in the down-regulated genes (Table S3; Figure 10B).

3.6. Expression Pattern of Cell Proliferation-Associated Genes in LW- and SW-Destined Wing Buds Because the cell cycle and DNA replication pathways were repeatedly detected in up- regulated genes of LW-destined buds (dsNlFoxO treatment; Figures4A,C,7A,C and 10A,C) , we investigated their expressional profile across 5th-instar stage using heat-maps with p- value <0.05 as a cutoff criterion. A total of 39 cell-cycle- and 25 DNA-replication-associated genes were significantly regulated by dsNlFoxO versus dsGfp in forewing and hindwing buds (Table S9; Figure 11). For both forewing and hindwing buds treated with dsNlFoxO, most genes exhibited a higher expression at 24-, and 36-hAE relative to 48 hAE (Figure 11), indicating that cells of LW-destined wings might actively proliferate within the first 36 h of 5th-instar nymphs. In contrast, for both forewing and hindwing buds treated with dsGfp, a higher expression level was detected at 24 hAE than 36- and 48-hAE, indicating that wing Insects 2021, 12, x FOR PEER REVIEW cells in SW-destined wings might proliferate within the first 24 h of 5th-instar nymphs.12 of 15

Thus, these observations indicated that the duration of cell proliferation might contribute signiﬁcantly to long wing development in dsNlFoxO-treated BPHs.

Figure 11. Cont.

Figure 11. Heatmap of DEGs in cell cycle and DNA replication pathways. Transcripts with fold change ≥ 2 and FDR < 0.05 were subjected to heatmap analysis.

4. Discussion Previous studies showed that inactivation of NlFoxO resulted in a switch from SW to LW morphs in BPH [4,5,35]. To further explore the molecular basis regulated by NlFoxO, transcriptomic profiles were conducted on LW- and SW-destined wing buds of 5th-instar nymphs, the wing-morph decisive stage of BPH [7]. We showed that knockdown of NlFoxO significantly altered the expression of 2128, 2902, and 1813 genes in forewing buds

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Insects 2021, 12, 413 12 of 15

Figure 11. Heatmap of DEGs in cell cycle and DNA replication pathways. Transcripts with fold Figurechange 11.≥ Heatmap2 and FDR of DEGs < 0.05 in were cell subjectedcycle and toDNA heatmap replication analysis. pathways. Transcripts with fold change ≥ 2 and FDR < 0.05 were subjected to heatmap analysis. 4. Discussion 4. DiscussionPrevious studies showed that inactivation of NlFoxO resulted in a switch from SW to LWPrevious morphs studies in BPH showed [4,5,35]. that To further inactivation explore of theNlFoxO molecular resulted basis in a regulated switch from by Nl SWFoxO, to LWtranscriptomic morphs in BPH proﬁles [4,5,35] were. To conducted further explore on LW- the and molecular SW-destined basis wingregulated buds by of Nl 5th-instarFoxO, transcriptomicnymphs, the profiles wing-morph were conducted decisive stage on LW of- BPHand SW [7].-destined We showed wing thatbuds knockdown of 5th-instar of nymphs,NlFoxO thesigniﬁcantly wing-morph altered decisive the expression stage of BPH of 2128, [7]. We2902, showed and 1813 that genes knockdown in forewing of NlFoxObudsof significantly 5th-instar nymphsaltered the at 24,expression 36, 48 hAE, of 2128, respectively, 2902, and and 1813 simultaneously genes in forewing altered buds the expression of 3466, 2533, and 2279 genes in hindwing buds. This observation indicated that differentiation of wing morph caused a remarkable change in gene expression in wing cells. Based on the comparative transcriptomic data, we noticed that dsNlFoxO-treated forewing had a similar gene expression pattern to that of hindwing buds at 24, 36, and 48 hAE. GO analysis showed that DEGs were mostly enriched in cellular process, binding, and cell part terms in forewing and hindwing buds at each time-point (Figures2,5 and8) . KEGG pathway analysis showed that the up-regulated DEGs were largely assigned to the ribosome pathway in both forewing and hindwing buds at 24 hAE. However, the top enriched KEGG pathway was switched from ribosome to DNA replication and cell cycle as developmental time proceeded into 36 hAE. This observation suggested that knockdown of NlFoxO might increase the expression levels and duration of cell proliferation-associated genes in wing buds, which might be the main driver of long wing development. This assumption was also supported by heatmap analysis on cell-cycle- and DNA-replication- associated genes across the 5th-instar developmental stage (Figure 11). We noticed that genes were highly expressed in dsNlFoxO-treated forewing and hindwing at both 24 and 36 hAE. In contrast, cell proliferation-associated genes were only active at 24 hAE in dsGfp-treated forewing and hindwing buds (Figure 11). Previous studies in mammals showed that FoxO transcription factors play a major role in cell cycle arrest at the G1/S and G2/M transitions, two checkpoints that are critical in the cellular process [13]. FoxOs promote cell cycle arrest at the G1/S boundary by both up-regulating cell cycle inhibitors (p21 and p27) [36,37] and by repressing cell cycle activators (cyclin D1 and D2) [21,38]. FoxOs arrest cell progression at the G2/M boundary by regulating the expression of cyclin G2 [39], and trigger DNA repair by modulating the expression of growth arrest and DNA damage-inducible protein 45 (Gadd45)[40,41]. This regulatory mechanism may explain the decrease in cell number [22–24] and cell size [22,23] Insects 2021, 12, 413 13 of 15

in Drosophila caused by ectopic expression of dFoxO. In the present, our results show that knockdown of NlFoxO signiﬁcantly enhanced the expression of cyclin D2 (Nl.scaffold.0432; Table S9; Figure 11) in wing cells of BPH, which may partially explain the long wing development in BPH upon NlFoxO knockdown.

5. Conclusions This study investigated the transcriptional profile of wing-morph transition from SW (dsGfp) to LW (dsNlFoxO) in 5th-instar nymphs, the wing-morph decisive stage. Com- parative transcriptome analysis revealed that a large percentage of genes (10–19%) were significantly differentially expressed in both forewing and hindwing buds. GO enrichment analysis showed that DEGs were mainly related to cellular process, binding, and cell part categories. The up-regulated genes were significantly enriched in DNA replication (ko03030), and cell cycle (ko04110 and ko04111) pathways in KEGG analysis. Thus, the duration of cell proliferation might contribute significantly to long wing development modulated by NlFoxO.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/insects12050413/s1. Supplementary Table S1: Data quality of RNA-seq. Supplementary Table S2: Differentially expressed genes of GO classification in forewing at 24 hAE. Supplementary Table S3: KEGG enrichment of differentially expressed genes in forewing and hindwing buds at 24, 36, and 48 hAE. Supplementary Table S4: Differentially expressed genes of GO classification in hindwing at 24 hAE. Supplementary Table S5: Differentially expressed genes of GO classification in forewing at 36 hAE. Supplementary Table S6: Differentially expressed genes of GO classification in hindwing at 36 hAE. Supplementary Table S7: Differentially expressed genes of GO classification in forewing at 48 hAE. Supplementary Table S8: Differentially expressed genes of GO classification in hindwing at 48 hAE. Supplementary Table S9: Differentially expressed genes associated with cell cycle and DNA replication. Author Contributions: Conceptualization, H.-J.X.; methodology, N.X.; software, N.X.; validation, N.X.; formal analysis, N.X.; investigation, N.X.; resources, S.-F.W.; data curation, N.X., S.-F.W.; writing—original draft preparation, N.X.; writing—review and editing, H.-J.X.; visualization, H.-J.X.; supervision, H.-J.X.; project administration, H.-J.X.; funding acquisition, H.-J.X. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants from National Natural Science Foundation of China (Grants 31772158 and 31972261 to H.-J.X.), Natural Science Foundation of Zhejiang province (LZ21C14 0002) and China postdoctoral science foundation (Grant 2020M671739 to JLZ). Institutional Review Board Statement: Not applicable. Data Availability Statement: The clean data from the RNA-seq were submitted to GenBank (BioPro- ject accession number: PRJNA715841). Conflicts of Interest: The authors declare no conflict of interest.

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