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Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens

['Wen-Hua Xue', 'Institute Of Insect Sciences', 'Zhejiang University', 'Hangzhou', 'Nan Xu', 'Sun-Jie Chen', 'Xin-Yang Liu', 'Jin-Li Zhang', 'Hai-Jun Xu', 'State Key Laboratory Of Rice Biology']

Date: 2021-08

Abstract A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged–destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates.

Author summary The highly conserved insulin/insulin-like growth factor signaling pathway plays a pivotal role in growth, development, and various physiological processes across a wide phylogeny of organisms. Unlike a single InR in the model species such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, most insect lineages have two or even three InR copies. However, the function of the alternative InRs remains elusive. Here, we created a homozygous mutation for a second insulin receptor (InR2) in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens, using the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas9) system. Our findings revealed that InR2 possesses functions distinct from the BPH InR homologue (NlInR1), indicating that multiple InR paralogues may have evolved independently and may have functionally diversified in ways more complex than previously expected in invertebrates.

Citation: Xue W-H, Xu N, Chen S-J, Liu X-Y, Zhang J-L, Xu H-J (2021) Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens. PLoS Genet 17(6): e1009653. https://doi.org/10.1371/journal.pgen.1009653 Editor: Subba Reddy Palli, University of Kentucky, UNITED STATES Received: December 21, 2020; Accepted: June 9, 2021; Published: June 28, 2021 Copyright: © 2021 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The raw data from the RNA-seq on wingbuds and female adults were submitted to GenBank with SRA accession numbers PRJNA675314 and PRJNA724037. Sequences of NlInR1 and NlInR2 are available with GenBank accession numbers KF974333 and KF974334. Funding: This work was supported by grants from National Natural Science Foundation of China (Grants 31522047, 31772158, and 31972261 to HJX) and China postdoctoral science foundation (Grant 2020M671739 to JLZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that they have no competing interests exist.

Introduction The highly conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is well established as a critical regulator of growth, development, and various physiological processes, including metabolic homeostasis, lifespan, reproduction, and stress responses, across a wide phylogeny of organisms, ranging from nematodes to humans [1–7]. The IIS cascade is activated upon ligand binding, and the actions of insulin and insulin-like growth factors (IGFs) in mice and humans are mediated by insulin receptor (InR) and IGF-1 receptor (IGF-1R), respectively, two distinct transmembrane tyrosine kinases [8,9]. Mice lacking the InR gene were born at term with slight growth retardation and died of ketoacidosis soon after birth [10], while mice lacking Igf-1r died at birth because of respiratory failure [11,12]. However, a single InR but not Igf-1r was identified in the fly Drosophila melanogaster and in the nematode Caenorhabditis elegans [9,13–16]. Genetic evidence derived from targeted Drosophila mutants indicated that the allele Drosophila InR (dInR) mutants were recessive embryonic or early larval lethal, although some heteroallelic complementations of dInR alleles were viable and yielded adults with severe developmental delay, reduced body and organ sizes, extended adult longevity, and female sterility [13,17–19]. The effect of dInR on the reduction of body and organ size in Drosophila was primarily mediated through reduced cell size and number [17,18,20]. In C. elegans, InR homologue (daf2) mutants showed arrested development at the dauer larval stage and increased longevity [15,21]. In addition, InRs have been implicated in the regulation of phenotypic plasticity in some insects. Male rhinoceros beetles (Trypoxylus dichotomus) wield a forked horn on their heads with hyper-variability in its size ranging from tiny bumps to exaggerated structures two-thirds the length of a male’s body. Knockdown of T. dichotomus InR homologue induced a major decrease in the size of the horns [22]. The damp-wood termite (Hodotermopsis sjostedti) exhibits various morphological castes associated with the division of labor within a colony. Knockdown of H. sjostedti InR homologue in pre-soldier termites disrupted soldier-specific morphogenesis including mandibular elongation [23,24]. These lines of findings provide insights into our understanding of the functional diversity of the conserved InR in invertebrates. In contrast to the single InR gene in Drosophila, two or even three InR copies are conserved in most insect lineages [25–28]. Analysis of the InR sequences in 118 insect species from 23 orders indicated that this InR multiplicity might result from duplication of the InR gene prior to the evolution of flight, followed by multiple secondary losses of one InR paralogue in individual lineages, such as in most Diptera [28]. Multiple InR paralogues might have distinctly functional implications throughout the life cycle in some insects. The linden bug Pyrrhocoris apterus (Hemiptera: Pyrrhocoridae) encoded InR1a, InR1b and InR2 paralogues, of which InR1a was probably originated through reverse transcription of InR1b [28]. Knockdown of InR1a or InR2 in P. apterus nymphs led to long-winged (LW) adults; whereas knockdown of InR1b led to short-winged (SW) adults [28]. In addition, accumulated evidences indicated that multiple InRs were caste-specifically expressed in the honey bee Apis mellifera [29], bumblebee Bombus terrestris [30], and fire ant Solenopsis invicta [31], suggesting that InRs might be involved into caste polyphenism of social insects. Furthermore, in addition to functional discrepancy, multiple InR paralogues may have overlapping functions on some aspects of life-history traits in some insects. Knockdown of either two InR paralogues impaired fecundity in the green lacewing Chrysopa pallens [32] and red flour beetle Tribolium castaneum [33], and disrupted nymph-adult transition in the brown citrus aphid Aphis (Toxoptera) citricidus [34]. These evidences raise an intriguing question regarding to the extent of functional conservation between multiple InR paralogues in insects. The wing-dimorphic brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is a classic and representative example of wing polyphenism in insects [35]. BPH nymphs can develop into SW or LW adults in response to environmental cues, the former have fully developed wings and functional indirect flight muscles (IFM), while the latter exhibit reduced wings and underdeveloped IFM. Although persuasive direct evidence is lacking, juvenile hormone (JH) has long been considered to be the main subject of endocrine regulation of wing polyphenism in BPH as well as in several other wing-polyphenic insects [35–37]. Topical application of JH and its agonists at certain juvenile stages could significantly decrease the percentage of LW morphs in BPH [38–40], the aphid Aphis fabae [41], and the cricket Gryllus rubens [42,43]. In contrast, treatment with a JH antagonist (precocene II) induced LW morphs in SW BPH population [44,45]. Recently, genetic analysis showed that the activity of IIS cascade determines alternative wing morphs in BPH. BPH has four insulin-like peptides and two InR paralogues, NlInR1 and NlInR2, and they share a high sequence similarity and resemble domain structures. However, only NlInR1 was functionally analogous to dInR with respect to development, metabolic homeostasis, stress responses, lifespan, reproduction, and starvation tolerance [25,46,47]. Activation of NlInR1 activates of the phosphatidylinositol-3-OH kinase [NlPI(3)K]-protein kinase B (NlAkt) signaling cascade, which in turn inactivates the forkhead transcription factor subgroup O (NlFoxO), thus leading to the LW morph. However, NlInR2 can antagonize the NlInR1 activity, and as a result activates the NlFoxO activity, leading to the SW morph [46]. Hence, RNA interference (RNAi)-mediated silencing of NlInR1 (NlInR1RNAi) and NlInR2 (NlInR2RNAi) led to SW and LW, respectively, through common signaling elements of the NlPI(3)K-NlAkt-NlFoxO cascade [46]. This finding provides an additional layer of regulatory mechanism underlying wing dimorphism in BPHs. Here, we aimed to elucidate the function of a second InR copy in insects by creating loss-of-function NlInR2 mutations in N. lugens using the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas9) system. We demonstrated that NlInR2 shared analogous functions with NlInR1 in terms of organism development and fertility, but differed in the effects on fuel metabolism, adult lifespan, starvation tolerance, and tissue growth. Furthermore, NlInR2 might play an important role in symmetrical patterning of wing veins. These findings provide the first direct evidence of distinct functions for the two InR paralogues in insects, and thus further our understanding of the evolution of InRs in invertebrates.

Discussion Owing to the extensive molecular genetic toolbox, studies of Drosophila dInR mutants have contributed greatly to our understanding of the complex regulation of InR in the life cycle of a wide variety of insects, and its functional conservation across the animal kingdom. However, in contrast to the single InR gene in Drosophila, most insect lineages have two or even three InR copies, although our understanding of their functions remains limited. Here, we used BPH as a model system to query the role of a second InR copy in the life-history traits of insects. Our findings revealed distinct and overlapping functions of NlInR1 and NlInR2 in BPH, indicating that multiple InR paralogues may have evolved independently and may have biological functions more complex than previously expected. Although NlInR1 and NlInR2 share a high sequence identity [46]; (S1 Fig), only NlInR1 resembles Drosophila dInR in terms of the regulation of growth, development, and a wide spectrum of physiological process. RNAi-mediated silencing of NlInR1, but not NlInR2, led to dwarf SW BPHs, which exhibited growth retardation, reduced body weight, an extended adult lifespan, an enhanced starvation tolerance, a decreased fertility, and impaired carbohydrate and lipid metabolism [46,47]. In addition, like Drosophila dInR, homozygous NlInR1 mutants were early embryonic lethal, whereas heterozygous mutants resembled NlInR1RNAi [55]. In the present study, we created viable homozygous NlInR2-null mutants by disrupting exons 4 and 5 of NlInR2 using CRISPR/Cas9. Like NlInR1RNAi, NlInR2-null mutants resulted in developmental delay and decreased fertility. However, unlike NlInR1RNAi, NlInR2-null mutants had marginal effects on fuel metabolism, lifespan, and decreased starvation tolerance. This observation stands in sharp contrast to the extended longevity of InR mutants in major model organisms, including worms [15], flies [19], and mice [56]. Although the exact mechanisms are unknown, this phenotypic feature in NlInR2 mutants may challenge the evolutionarily conserved roles of InR in fuel metabolism and longevity. One important and seemingly paradoxical difference between NlInR1 and NlInR2 was that knockdown of NlInR1 led to SW BPHs, while NlInR2-null mutation induced wing development, leading to LW BPHs. Previous studies in Drosophila showed that dInR-deficient flies had smaller bodies and organs because of a reduction in both cell size and cell number [17,18,20]. This mechanism may also explain the small body and wing sizes in NlInR1RNAi BPHs [46]. However, NlInR2-null mutation likely increased wing-cell number by accelerating cell proliferation during the first 48 h after 5th-instar eclosion. The effect of NlInR2 on cell proliferation was further evidenced by RNA-seq analysis on wing buds of 5th-instar NlInR2E4 nymphs at 24 hAE, which showed that the most up-regulated genes were associated with DNA replication and cell cycle process. One possible explanation for the opposite effects of NlInR1 and NlInR2 is that NlInR2 may serve as a negative regulator of NlInR1, as proposed previously [46], and depletion of NlInR2 thus activates the canonical IIS pathway to stimulate cell proliferation. Intriguingly, RNA-seq on female adults showed that only 884 and 417 genes were differentially expressed by NlInR1RNAi and NlInR2E4 (S2 Data and S16–S19 Tables), which accounted for 4.8% and 2.2% of BPH encoding genes (18,534), respectively. Moreover, only 101 genes were commonly regulated by NlInR1RNAi and NlInR2E4. Thus, this observation is in contrast to the notion that NlInR2 is a negative regulator of NlInR1. Previous studies indicated that NlFoxO is a main IIS downstream effector relaying the NlInR1 and NlInR2 activities. Lin et al. recently found that wing cells in SW-destined BPHs were largely in the G2/M phase of the cell cycle, whereas those in LW individuals (NlFoxO RNAi) were largely in G1 [57]. This is consistent with the higher cell proliferation rate of NlInR2E4 wing buds in the current study. However, how the single transcription factor NlFoxO exerts different functions in respond to NlInR1 and NlInR2 activities is still an open question. Previous studies indicated that NlInR2 was mainly expressed in wing buds [46], which may explain why NlInR2 null had a marginal effect on body size. Intriguingly, depletion of NlInR2 decreased the phallus length, indicating that male genitalia could respond to the NlInR2 activity. A similar phenomenon was observed in the dung beetle Onthophagus taurus when the activity of O. taurus InR1 homologue was compromised by RNAi knockdown [58]. In addition, NlInR2-null mutation tempo-spatially elevated expression levels of wing-patterning genes that were previously well-established in Drosophila. If the regulatory mechanism of wing patterning in Drosophila (a holometabolous insect) applies to BPH (a hemimetabolous insect), it will be interesting to determine how NlInR2 regulates LW development by exquisitely orchestrating the expression of wing-patterning genes. Another interestingly finding in this study was that depletion of NlInR2 led to asymmetric vein patterning, but this phenotype was not observed in NlInR2RNAi BPHs [46]. Therefore, one speculative explanation for this different phenotypes could be that a basal level of NlInR2 might be necessary for vein patterning in BPHs. Notably, several case studies on wing polyphenism in Hemiptera insects and polyphonic horns in beetles indicated that the regulatory roles of multiple InRs on phenotypic plasticity might be insect lineage specific. In BPH and the linden bug P. apterus, two hemiptera insects, two InR paralogues had opposite roles in determining alternative wing morphs [28,46]. However, knockdown of InR1 or InR2 orthologues in the red-shouldered soapberry bug, Jadera haematoloma (Hemiptera: Rhopalidae), had a marginal role on wing-morph switching [59]. In the dung beetle O. taurus, knockdown of O. taurus InR1 or InR2 homologue had no significant effect on horn size [58], which is in marked contrast to shorted horns derived from knockdown of InR1 in the rhinoceros beetle T. dichotomus [22]. The two BPH InR paralogues have evolved different functions for controlling alternative wing morphs, enabling BPHs to migrate or reside according to changes of heterogeneous environments. However, whether InR paralogues in other insects are involved in additional polyphenisms is still unknown. Although there remains much to be done, the current study on NlInR2-null mutants may provide a better understanding of the co-option of multiple InR paralogues in regulating multiple facets of life-history traits in insects. We believe that future experiments including comparative genomic analysis and functional genetic studies in more non-model insects will improve our understanding of the functional plasticity of multiple InR paralogues in insects.

Materials and methods Insects The BPH strain was originally collected from rice fields in Hangzhou (30°16ˊN, 120°11ˊE), China, in 2008. The WtSW colony was purified by inbreeding for more than 13 generations, and used for genomic DNA sequencing and assembly [60]. All insects were reared at 26 ± 0.5°C under a photoperiod of 16 h light/8 h dark at a relative humidity of 50 ± 5% on rice seedlings (strain: Xiushui 134). In vitro synthesis of Cas9 mRNA and sgRNA The sgRNAcas9 algorithm [61] was used to search sgRNAs in the BPH genome using NlInR2 sequence. The sgRNA was prepared as described previously [62], and then in vitro transcribed using the MEGAscript T7 high yield transcription kit (Thermor Scientific) according to the manufacturer’s instructions. Cas9 mRNA was in vitro transcribed from plasmid pSP6-2sNLS-SpCas9 vector using the mMESSAGE mMACHINE SP6 transcription kit and Poly(A) tailing kit (Thermo Scientific). DNA typing for heterozygosity and homozygosity Genomic DNA (gDNA) was isolated from whole BPH body or forewings to determine the heterozygous or homozygous genotypes of the BPHs. gDNA was extracted from one individual adult, as reported previously [63]. Briefly, one adult was homogenized in a 0.2-ml Eppendorf tube, followed by the addition of 50 μl of extraction buffer (10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl, 0.2 mg/ml proteinase K). The tubes were then incubated for 30 min at 37°C, followed by 2 min at 95°C to inactivate the proteinase K, and the supernatant solution was used directly as a template for PCR. gDNA was isolated from forewings as reported previously [64], with slight modifications. Briefly, forewings were digested in 0.5 ml extraction buffer (0.01 M Tris-HCl, 0.01 M EDTA, 0.1 M NaCl, 0.039 M dithiothreitol, 2% sodium dodecyl sulfate, 20 μg/ml, pH 8.0) for 12h at 37°C, followed by 2 min at 95°C to inactivate proteinase K. The supernatant solution was then used directly as a template for PCR. PCR products spanning NlInR2E4 and NlInR2E5 sgRNA target sites were amplified from the extracted gDNA using primer pairs for E4-iF/E4-iR and E5-iF/E5-iR (S20 Table), respectively. The PCR products were then used for Sanger sequencing or subcloned into pEasy-T3 cloning vector (TransGen Biotech), and then single clones were picked for Sanger sequencing. Embryonic injection and crossing scheme Embryonic injection was performed as described previously [65]. Pre-blastoderm eggs were dissected from rice sheaths within 1 h of oviposition, and microinjection manipulation was accomplished in the following 1 h. To perform cross-mating, a single male or female CRISPR/Cas9-injected G 0 adult was picked to mate with one WtSW female or male to lay eggs for 10 days. Each G 0 adult was then homogenized to determine its genotype. Eggs (G 1 progeny) were reared to adulthood, and gDNA was then isolated from G 1 forewings for genotype determination. A single G 1 adult was picked to mate with one WtSW adult to produce G 2 progeny, and a single G 2 adult was then allowed to mate with one WtSW adult to produce homozygous mutants (G 3 ). The genotype for G2 and G3 generations were determined by dissecting forewings for gDNA isolation. A homozygous mutant population was derived by G 3 self-crossing. Developmental duration, glucose and triglyceride contents, fecundity, and adult longevity Females were allowed to lay eggs for 2 h, and the hatched nymphs were then monitored every 12 h. Newly hatched 1st-instar nymphs (n = 20, 0–12 hAE) were collected, and each individual was raised separately in a glass tube. The developmental times of 1st-, 2nd-, 3rd-, 4th- and 5th-instar stages were monitored every 12 h. Glucose and triglyceride levels were measured in pooled 24h-adult females (n = 15) as reported previously [46]. Glucose levels were measured using glucose oxidase reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Triglyceride contents were quantified by enzymatic hydrolysis using the GPO Trinder method with a tissue triglyceride assay kit (Applygen Technologies), according to the manufacturer’s instructions. The glucose and triglyceride contents were calculated based on three biological replicates. Adult longevity was determined by recording the mortality of newly emerged adult females (0–3 hAE, n = 43 for WTSW and n = 42 for NlInR2E4) and males (0–3 hAE, n = 52 for WTSW and n = 42 for NlInR2E4) every 12 h. To determine fecundity, newly emerged adult females (0–12 hAE) were collected for paired mating assays. Each female (n = 20) was allowed to match with two males in a glass tube. The insects were removed 10 days later and the laid eggs were counted under a Leica S8AP0 stereomicroscope. Starvation tolerance assay Starvation tolerance assay was conducted as reported previously [47]. Briefly, newly emerged WtSW and NlInR2E4 adults (0–6 hAE, n = 30) were collected, and provided with normal food for 24 h. Then, the BPHs were deprived of food and only provided water. Mortality was monitored every 8 h. Western blot analysis Western blot analysis against Vg was performed as previously reported [66]. Briefly, ovaries were dissected from WtSW and NlInR2E4 females at 3 and 5 days after eclosion, and then homogenized in Pierce radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). For immunoblot staining, equal amounts of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with anti-Vg polyclonal rabbit antibody (1: 1000) for 1 h at room temperature (RT), followed by incubation with horseradish peroxidase conjugated goat anti-rabbit antibody (MBL life science) for 1 h at RT, The antibody against ß-actin was used as a loading control. The image of immunoreactivity was taken by the Molecular Imager ChemiDoc XRS+ system (Bio-Rad). Quantification of wing-cell number by qRT-PCR Fifth-instar nymphs (n = 150) collected at 3-, 24-, 36-, 48-, and 72-hAE were used for T2W and T3W dissection. gDNA was isolated from T2W and T3W, respectively, using a Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s instructions. A primer pair (gDNA-F/R) targeting the single copy Ultrabithorax gene was used to quantify gDNA copy number in qRT-PCR assay. qRT-PCR was conducted using a CFX96TM real-time PCR detection system (Bio-Rad). Three independent biological replicates with three technical replicates were used for each sample. EdU staining of wing buds Fifth-instar LW-destined NlInR2E4 and SW-destined WtSW nymphs at 24 hAE were microinjected with EdU (0.5 mM), and T2W and T3W were then removed from 48h-nymphs. Wing buds were fixed and dissected from the outer layer of chitin shell, as described previously [67]. Briefly, wing buds were pre-fixed by incubating in a cocktail solution (6 ml chloroform, 3ml ethanol, 1ml acetic acid) for 10 min at room temperature (RT), followed by fixation in FAA solution (5 ml 37% formaldehyde, 5 ml acetic acid, 81 ml ethanol, 9 ml H 2 O) overnight at RT. After washing with methanol, outer layer of chitin shell was removed with forceps and the wing buds were fixed in 10% formaldehyde for 1 h at RT. After incubating with 1% Triton X-100 for 2h at RT, samples were used for EdU detection using a Click-iT EdU assay (Invitrogen) according to the manufacturer’s instructions. Fluorescent images were acquired using a Zeiss LSM 810 confocal microscope (Carl Zeiss). RNAi-mediated gene silencing The dsRNA synthesis and injection were performed as described previously [46]. Briefly, dsRNA primers targeting NlInR1 (dsNlInR1) and Gfp (dsGfp) were synthesized with the T7 RNA polymerase promoter at both ends (S20 Table). dsNlInR1 and dsGfp were synthesized using a MEGAscript T7 high yield transcription kit (Ambion) according to the manufacturer’s instructions. Microinjection was performed using a FemtoJet microinjection system (Eppendorf). For morphological examination of dsNlInR1-treated BPHs, 4th-instar nymphs were microinjected with dsNlInR1 (100 ng each), and the morphology of the adults was photographed. To examine the antagonistic role of NlInR1 in the context of NlInR2-null BPHs, 12h-5th-instar NlInR2E4 nymphs were microinjected with 150 ng dsNlInR1 or dsGfp, and the numbers of BPHs with alternative wing morphs were counted when the adults emerged. Adults (n = 5 for each of three replicates) at 24 h after eclosion were collected for examination of RNAi efficiency using qRT-PCR. The relative expression of NlInR1 was normalized to the expression level of rps15 with primers in S20 Table. Examination of indirect flight muscles by transmission electron microscope Thoraxes were dissected from 24h-adults for transmission electron microscope, as in described previously [68]. Briefly, thoraxes were fixed in 2.5% glutaraldehyde overnight at 4°C, followed by post-fixation in 1% osmium tetroxide for 1.5 h. Samples were sectioned and stained with 5% uranyl acetate followed by Reynolds’ lead citrate solution. Sections were observed under a JEM-1230 transmission electron microscope (JEOL). Expression of wing-patterning genes in NlInR2-null mutants Fifth-instar NlInR2E4 nymphs (n = 150) were collected at 24-, 48-, and 72-hAE. T2W and T3W were dissected and used for total RNA isolation. First-strand cDNA was synthesized from total RNA using HiScript QRTSuperMix (Vazyme). The qRT-PCR primers for Nlen, Nlhh, Nldpp, Nlap, Nlser, Nlwg, and Nlvg (S20 Table) were designed using Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast). The ribosomal protein gene rps15 was used as an internal reference gene [69]. Statistical comparisons between samples were based on three biological replicates. RNA isolation, cDNA library preparation, and Illumina sequencing Total RNAs were isolated using RNAiso Plus (TaKaRa) according to the manufacturer’s protocol. The quality of the RNA was examined by 1% agarose gels electrophoresis and spectrophotometer (NanoPhotometer, Implen). RNA integrity was assessed using an RNA Nano 6000 Assay Kit with the the Agilent Bioanalyzer 2100 system (Agilent Technologies). A total of 1.5 μg RNA per sample was used for cDNA library construction using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB), following the manufacturer’s recommendations. Read mapping and DEGs After Illumina sequencing, clean reads were generated after removing adapter, polly-N, and low-quality reads from the raw data. All clean reads were aligned to the BPH reference genome using Hisat2 (v2.1.0). The aligned clean reads were coordinately sorted and indexed with Samtools (v1.9). Read counts and number of fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM) were calculated for each gene by using StringTie (v1.3.5). DEseq2 was used to screen DEGs with fold change ≥ 2 and adjusted P-value < 0.01. GO enrichment analysis of differentially expressed genes GO enrichment analysis of DEGs was performed using the online OmicShare tool (https://www.omicshare.com/tools/home/report/goenrich.html and https://www.omicshare.com/tools/Home/Soft/pathwaygsea). Statistical analysis Results were analyzed using the two-tailed Student’s t-tests, Pearson’s Chi-Square test, and log-rank (Mantel-Cox) test. Data are presented as the mean ± standard error of the mean (mean ± s.e.m) for independent biological replicates. Significance levels are indicated as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****).

Acknowledgments We thank Dr. Dan-Ting Li for preparing Fig 3A and International Science Editing (http://www.internationalscienceediting.com) for language editing.

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