Characterization and Seasonal Expression Profiling of a Takeout/Juvenile Hormone‐Binding Protein‐Family Gene Associated With Diapause in Eurygaster integriceps

ABSTRACT

Diapause in the Sunn pest, Eurygaster integriceps, is a key seasonal adaptation associated with migration, overwintering survival, and reproductive timing. However, the molecular components associated with this physiological state remain poorly characterized in this hemipteran pest. In this study, we identified and characterized a takeout‐family transcript from E. integriceps (EiTO1; GenBank OP913374.1) and combined evolutionary, structural, and expression analyses to examine its association with seasonal physiological states. EiTO1 contained a 738‐bp open reading frame encoding a 245‐amino acid protein and was assigned to the juvenile hormone‐binding protein (JHBP) superfamily based on conserved‐domain analysis. Phylogenetic reconstruction recovered EiTO1 as sister to the Nilaparvata lugens–Bemisia tabaci clade; however, the low bootstrap support for this placement prevented confident inference of its closest evolutionary relationship. Structural comparison of the predicted EiTO1 model with experimentally resolved takeout/JHBP‐family proteins indicated a conserved barrel‐like fold and a corresponding internal cavity, consistent with structural features of this protein family. Quantitative PCR profiling revealed marked seasonal and tissue‐specific variation in EiTO1 expression. In female whole‐body samples, EiTO1 transcript abundance was low during spring field activity and in newly emerged adults sampled in June, reached its maximum during the refuge‐associated phase in August (160‐fold), and showed a secondary increase during the late‐overwintering/premigration transition in March (21.5‐fold). Tissue‐resolved assays in females showed the highest expression in the head and intermediate expression in the fat body. Ovarian expression remained at or below the April baseline at most sampling points but showed a transient increase in August. Together, these findings identify EiTO1 as a takeout/JHBP‐family gene whose expression is associated with seasonal physiological transitions in E. integriceps.

EiTO1, a takeout/JHBP‐family gene in Eurygaster integriceps, shows tissue‐specific seasonal expression, with higher levels in the whole body, head, and fat body during diapause‐associated phases, but lower ovarian expression. Comparative sequence analyses support its assignment to the takeout/JHBP family.

Summary

• EiTO1 is a conserved takeout/JHBP‐family gene in Eurygaster integriceps.

• EiTO1 expression increases during diapause‐associated stages.

• Head and fat body show higher EiTO1 expression than ovary.

1. Introduction

The Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is a major constraint on wheat production in West and Central Asia, where feeding by adults and nymphs reduces yield and can disproportionately impair end‐use quality, particularly gluten‐related traits (Allahyari et al. 2010; Dizlek and Özer 2016). Its population dynamics are strongly seasonal: an obligate diapause and predictable movement between wheat agroecosystems and non‐crop refugia generate repeatable transitions from overwintering habitats to spring migration and reproduction in wheat fields (Alborzi et al. 2025, 2026). These predictable switches make the diapause–reproduction transition a mechanistically informative leverage point for phenology‐based management. Diapause is increasingly viewed as an actively regulated alternative physiological state that integrates environmental inputs with endocrine, metabolic, and neurogenetic control, producing a coordinated “diapause syndrome” rather than a passive developmental arrest (Denlinger 2023; Easwaran and Montell 2023). Recent perspectives emphasize that progress will depend on extending mechanistic inference beyond a narrow set of model taxa and embedding molecular regulation within ecologically realistic seasonal contexts (Denlinger 2023).

This framing is especially relevant for hemipteran pests such as E. integriceps, where diapause is closely linked to seasonal movement ecology, host phenology, metabolic depression, and reproductive arrest and/or maturation, yet the specific molecular effectors underlying these links remain relatively poorly resolved (Nielsen et al. 2017; Reynolds et al. 2022; Durak et al. 2023).

In E. integriceps, emerging multitissue transcriptomic work has begun to map endocrine‐associated expression patterns across diapause phases, supporting the feasibility of linking seasonal physiology to gene regulatory outputs in this system (Alborzi et al. 2025). The insect circadian clock ensures that the insect remains in a state of developmental arrest during the scorching summer (estivation) and the freezing winter (hibernation), only resuming reproductive activity when host plants are phenologically optimal in spring. While the ecological aspects of this migration and dormancy have been studied for decades, the molecular “interplay” that synchronizes the internal clock with the endocrine system remains a significant gap in insect physiology.

The initiation and maintenance of insect diapause are primarily governed by the plastic interaction between the circadian clock and the endocrine system. In E. integriceps, the core circadian machinery, comprising genes such as period (per), timeless (tim), clock (clk), and cycle (cyc), functions as the master chronometer (Alborzi et al. 2026). Recent characterizations of these genes in the brain–corpora cardiaca–corpora allata complex have revealed distinct seasonal oscillations, suggesting that the insect perceives seasonal shifts through these molecular oscillators even during dormancy (Alborzi et al. 2026; Meuti et al. 2024). These clock genes do not function in isolation; they must relay temporal information to the endocrine axes that control growth and reproduction, namely the insulin/insulin‐like signaling (IIS) and the juvenile hormone (JH) pathways (Alborzi et al. 2025, 2026).

A key remaining gap is to identify candidate effectors that are simultaneously (i) mechanistically interpretable, belonging to conserved gene families with plausible biochemical functions, and (ii) ecologically interpretable, showing stage‐ and tissue‐resolved regulation across discrete seasonal states (e.g., post‐overwintering, feeding/reproduction, and diapause maintenance).

The takeout/juvenile hormone‐binding protein (JHBP)‐like family is a strong candidate for this integrative agenda. Across insects, takeout/JHBP‐like proteins have repeatedly been implicated at the intersection of circadian state, feeding‐related homeostasis, and hormone‐associated signaling, and recent work in a hemipteran pest suggests roles extending to ecologically consequential behaviors, such as host‐oriented olfactory responses (He et al. 2025). Comparative studies further indicate that takeout/JHBP‐like genes often diversify into lineage‐ and tissue‐biased repertoires with distinct physiological associations (Fujiwara et al. 2023). Structurally, takeout/JHBP proteins share a compact cavity‐containing fold, consistent with ligand binding, supporting their proposed function as carriers of lipophilic molecules relevant to endocrine–metabolic coordination (Meuti et al. 2024; Kim et al. 2020).

The takeout (TO) gene family, originally identified in Drosophila melanogaster as a circadian‐controlled gene regulated by per and tim, is unique in its structural architecture. TO proteins belong to the larger superfamily of JHBP and possess a conserved hydrophobic carrier domain (Sarov‐Blat et al. 2000). Functionally, takeout has been linked to various physiological processes, including feeding behavior, longevity, and, importantly, the regulation of dormancy (Huang et al. 2025; Li et al. 2023; Liu et al. 2024). However, in seasonal pest systems, these candidates remain under‐validated without joint evidence from sequence/structure inference and seasonally grounded, tissue‐resolved expression (Qian et al. 2024).

Here, we characterize a takeout homolog from E. integriceps (EiTO1) and examine its evolutionary placement, conserved features, and seasonal and tissue‐specific expression across biologically defined stages. Leveraging emerging genomic resources for E. integriceps (Alborzi et al. 2025; Kazemi Alamouti et al. 2024) and established structural insights for takeout/JHBP‐like proteins (He et al. 2025; Kim et al. 2020; Dupas et al. 2020), we define the EiTO1 coding sequence, evaluate conservation using phylogenetic and motif‐based analyses, and quantify transcript abundance across seasons (April, June, August, December, and March) and key female tissues (whole body, head, fat body, and ovary). We test the hypothesis that EiTO1 is upregulated during diapause‐associated months and preferentially expressed in neuroendocrine and/or metabolic tissues relative to the ovary, providing an ecologically grounded framework for linking a conserved ligand‐binding protein family to seasonal diapause physiology in a hemipteran pest.

2. Materials and Methods

2.1. Study Species and Seasonal Sampling Design

Adult E. integriceps were sampled across biologically distinct phases of their univoltine annual cycle. Adults collected from wheat fields in Nazarabad, Alborz Province, Iran (35.8920° N, 50.6147° E), in April 2022 represented overwintered adults that had migrated from refuge sites and resumed feeding and reproductive activity. June samples represented newly emerged adults of the next generation that were feeding in wheat fields before migrating to summer and overwintering refuges. Adults were also collected from refuge sites in Khur village, Hashtgerd, Alborz Province (36.0139° N, 50.7081° E), in August, December, and March. August and December represented refuge‐associated summer and overwintering phases, respectively. March adults were collected from beneath Artemisia spp. vegetation at the overwintering site before spring migration and were therefore classified as representing a late‐overwintering/premigration transition rather than confirmed diapause maintenance.

This distinction is supported by a recent study in which March adults collected after migrating to wheat fields showed ovarian development and were classified as post‐diapause and reproductively active (Alborzi et al. 2026). However, ovarian developmental stage, feeding status, and juvenile hormone titers were not assessed in the present study. Therefore, the precise physiological status of the March adults, including whether they were undergoing diapause termination or had entered post‐diapause quiescence, could not be determined. Adult females were hand‐collected and transported to the laboratory for immediate processing. Specimens were either processed as whole‐body samples or dissected for tissue‐specific analyses and were stored at −80°C until molecular assays were performed.

2.2. Tissue Dissection and Sample Pooling

For tissue‐resolved expression analyses, adult females were dissected on dry ice under a stereomicroscope. The head, fat body, and ovary were isolated and processed separately. For each biological replicate, tissues from 4 to 6 individuals were pooled to achieve sufficient RNA yield and reduce individual‐level stochastic variation. Whole‐body samples were prepared from individual adults and processed as pooled biological replicates consistent with the experimental design. All dissected tissues and whole‐body samples were snap‐frozen and maintained at −80°C until RNA extraction.

2.3. RNA Extraction, DNase Treatment, and cDNA Synthesis

Total RNA was extracted from whole‐body or pooled tissue samples using TRIzol Reagent following the manufacturer's instructions. RNA integrity was assessed by agarose gel electrophoresis, and RNA purity was evaluated spectrophotometrically using the A260/A280 ratio. To remove residual genomic DNA, total RNA was treated with DNase I and EDTA, following the Invitrogen Deoxyribonuclease (Thermo Fisher Scientific) protocol. First‐strand cDNA was synthesized from 1 µg of DNase‐treated total RNA using the AddScript cDNA Synthesis Kit (Addbio) with Oligo(dT20) as anchor primers, in accordance with the manufacturer's protocol. Synthesized cDNA was stored at −20°C until qPCR analysis.

2.4. Identification and Primary Characterization of the takeout Gene

EiTO1 was identified from the previously published de novo transcriptomic resource generated from the brain–corpora cardiaca–corpora allata complex and fat body of nondiapausing adult female E. integriceps, as described by Alborzi et al. (2025). Briefly, quality‐filtered paired‐end RNA‐seq reads were assembled de novo using Trinity v2.11.0, coding regions were predicted with TransDecoder, and the predicted proteins were annotated by BLASTP searches against the NCBI nonredundant insect protein database using an E‐value threshold of 1 × 10⁻⁵. The EiTO1 candidate was retrieved from the annotated data set based on its similarity to insect takeout/JHBP‐family proteins.

The nucleotide sequence was analyzed using NCBI ORF Finder, and family assignment was evaluated using the NCBI Conserved Domain Database. The predicted protein was further analyzed using the ExPASy Proteomics Server to estimate its isoelectric point. Subcellular localization was predicted using CELLO v2.5, and the presence of an N‐terminal signal peptide was assessed using SignalP 6.0 with default settings for eukaryotic proteins. Because the available GenBank records are partial and the 5′ and 3′ transcript boundaries were not experimentally validated, the sequence was treated as a transcriptome‐derived predicted ORF rather than a confirmed full‐length mRNA.

2.5. Sequence Alignment, Phylogenetic Reconstruction, and Motif Visualization

To investigate evolutionary relationships and conservation of EiTO1 among insects, deduced amino acid sequences from E. integriceps and representative insect taxa were aligned in Clustal X 2.0.1227. Regions with excessive gaps or missing data were manually trimmed prior to phylogenetic inference. Phylogenetic analyses were conducted in MEGA 12 using Maximum Likelihood, with additional validation; node support was evaluated using 1000 bootstrap replicates. Conserved sequence features were summarized using WebLogo 3 (http://weblogo.berkeley.edu/logo.cgi), with default parameters, to generate motif visualizations from the aligned sequences. Pairwise amino‐acid sequence identity percentages among insect takeout proteins were calculated from aligned sequences using the MBOSS Needle web interface (https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle).

2.6. Motif Discovery and Conserved Domain Assignment

To provide both taxonomically relevant and structurally characterized reference comparisons, the predicted 245‐amino‐acid EiTO1 protein was analyzed together with NlTO1 from Nilaparvata lugens, HhTO1 from Halyomorpha halys, EpTO1 from Epiphyas postvittana (PDB ID: 3E8T, chain A), and BmJHBP from Bombyx mori (PDB ID: 3AOT). The hemipteran sequences were included to provide taxonomically relevant homologs, whereas EpTO1 and BmJHBP were selected because experimentally determined structures are available for these proteins.

Amino‐acid sequences were aligned using Clustal Omega 1.2.4. The resulting alignment was visualized using MView, with EiTO1 designated as the reference sequence and residues colored according to identity with EiTO1. Pairwise coverage and percentage identity values were calculated by MView relative to the EiTO1 reference sequence. Consensus sequences were displayed at conservation thresholds of 100%, 90%, 80%, and 70% using the default MView protein physicochemical‐class scheme.

Recurrent motifs were identified using MEME Suite v5.5.4. MEME was run in the zero‐or‐one‐occurrence‐per‐sequence mode, allowing identification of up to five motifs with widths ranging from 6 to 30 amino acids. Three recurrent motifs were retained in the final output, and their positions were visualized relative to the length of each protein. Motif numbering was retained according to the MEME output ranking rather than their physical order along the protein sequence.

Conserved‐domain assignment of EiTO1 was evaluated separately using the NCBI Conserved Domain Database with default parameters. Domain matches with an E‐value below 1 × 10⁻³ were considered significant.

2.7. Structural Comparison With Experimentally Resolved Takeout/JHBP Proteins

To assess structural conservation within the takeout/JHBP family, experimentally resolved structures of B. mori JHBP in the apo form (PDB ID: 3AOT) and E. postvittana Takeout 1 in the ubiquinone‐bound form (PDB ID: 3E8T) were retrieved from the Protein Data Bank. The EiTO1 structure was generated from the deduced amino acid sequence by comparative homology modeling using the SWISS‐MODEL server (Waterhouse et al. 2018). EpTO1 (PDB ID: 3E8T, chain A) was selected as the highest‐ranked template, with 29.72% sequence identity and 87% target coverage. The resulting model had a GMQE score of 0.66 and a QMEANDisCo score of 0.69 ± 0.06.

The predicted EiTO1 model was visualized and structurally aligned with EpTO1 and BmJHBP using UCSF ChimeraX 1.10. Comparisons focused on the overall fold, secondary‐structure organization, and spatial correspondence of the internal cavity. Because the EiTO1 structure was computationally modeled and no ligand‐binding assay was performed, the observed cavity correspondence was interpreted as predictive evidence of conserved family‐level architecture rather than direct evidence of ligand‐binding activity or functional equivalence.

2.8. Quantitative Real‐Time PCR (qPCR) and Expression Profiling

Relative expression of takeout was quantified across seasons and tissues using SYBR‐based qPCR. qPCR reactions were performed using ExcelTaq 2× Q‐PCR Master Mix (SYBR, ROX; TQ1110‐100, SMOBIO) according to the manufacturer's protocol on a Rotor‐Gene Q system (QIAGEN). Each condition was assessed with three biological replicates and three technical replicates per biological replicate.

Gene‐specific primers for takeout and β‐actin (reference gene) were designed using NCBI Primer‐BLAST. β‐actin was selected as the reference gene because of its widespread use in insect RT–qPCR studies and its previously reported stability under some experimental conditions, including across developmental stages in Diabrotica virgifera virgifera (Barros Rodrigues et al. 2014 ). However, its expression stability across the seasonal stages and tissue types examined in the present study was not independently validated. Because neither gene sequence was available in GenBank at the time of primer design, E. integriceps transcriptome data were used to identify coding regions suitable for primer targeting. Conserved regions were aligned using Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo?stype=dna), and primers were selected to amplify ~180 bp fragments within coding regions. Primer sequences are provided in Table S1. Candidate primers were evaluated in silico using OligoAnalyzer to assess oligonucleotide quality (e.g., potential self‐dimer/hairpin formation) and BLAST to examine sequence specificity against available databases. After assay implementation, amplification specificity was confirmed by melt‐curve analysis; only assays producing a single, well‐defined melting peak were accepted for subsequent quantification and statistical analysis. Amplification efficiencies were determined from standard curves generated using five‐fold serial dilutions of pooled cDNA. Cq values were plotted against the logarithm of the cDNA dilution factor, and amplification efficiency was calculated as (E = (10^{−1/slope} − 1)\times100). The amplification efficiencies were 94.2% for EiTO1 and 88.9% for β‐actin, with R ² values of 0.998 and 0.996, respectively (Table S1). Expression profiling was conducted on whole‐body adult females and dissected female tissues collected across field‐active, refuge‐associated, and late‐overwintering/premigration phases of the annual life cycle.

2.9. Quantification and Statistical Analysis

For RT–qPCR quantification, technical triplicates were averaged within each biological replicate, and data are presented as the mean ± SEM of three independent biological replicates. Relative transcript abundance was calculated using the $2^{-\mathrm{\Delta }\mathrm{\Delta }\mathit{Ct}}$method, with April used as the calibrator and assigned a relative expression value of 1 separately within each sample type. Statistical analyses were performed on ΔCt values rather than on back‐transformed fold‐change values. For each sample type—female whole body, head, fat body, and ovary—differences among the five sampling months were evaluated using one‐way ANOVA. Because the prespecified comparisons were restricted to each month versus the April reference group, Dunnett's multiple‐comparisons test was applied. Statistical significance was defined as a Dunnett‐adjusted p < 0.05.

3. Results

3.1. Phylogenetic Placement of the Eurygaster integriceps EiTO1 Protein

Maximum‐likelihood analysis of 27 takeout/JHBP‐family amino‐acid sequences representing 27 genera and 10 insect orders produced a topology with variable bootstrap support (Figure 1). EiTO1 was recovered as sister to the N. lugens–Bemisia tabaci clade, but this relationship received weak bootstrap support (31%). The internal pairing of N. lugens and B. tabaci was strongly supported (91%). Thus, although the immediate neighboring sequences were hemipteran, the low support for the EiTO1 placement does not permit a confident inference regarding its closest evolutionary relationship.

Figure 1.

Maximum‐likelihood gene tree of the Eurygaster integriceps EiTO1 protein and representative insect takeout/JHBP‐family homologs. A phylogenetic tree was constructed in MEGA 12 from amino acid sequences of takeout proteins from representative insect taxa. Bootstrap values (1000 replicates) are shown above branches, and the scale bar indicates the number of amino acid substitutions per site. The sequences included in the analysis were Eurygaster integriceps (translated predicted ORF from OP913374.1), Nilaparvata lugens (XP_039295782.1), Bemisia tabaci (XP_018897292.1), Tribolium castaneum (XP_966810.1), Bombyx mori (XP_062528695.1), Plutella xylostella (XP_048481921.1), Neodiprion fabricii (XP_046424815.1), Chrysoperla carnea (XP_044741689.1), Trachymyrmex cornetzi (XP_018358135.1), Schistocerca gregaria (XP_049857344.1), Lygus hesperus (JAQ15888.1), Prenolepis imparis (XP_082897998.1), Halyomorpha halys (XP_014290980.1), Apis mellifera (AWC67397.1), Leptidea sinapis (XP_050665597.1), Hetaerina americana (XP_071455445.1), Anabrus simplex (XP_067004639.2), Papilio xuthus (XP_013165952.1), Vanessa cardui (XP_046962309.1), Reticulitermes speratus (BDU67900.1), Cryptotermes secundus (XP_023725619.1), Cimex lectularius (XP_014239931.1), Rhodnius prolixus (JAA76272.1), Gryllus bimaculatus (GLG92888.1), Thrips palmi (XP_034232743.1), Drosophila melanogaster (NP_001287525.1), and Aedes aegypti (AAL60239.1).

Several local relationships received moderate or strong bootstrap support, including B. mori–Plutella xylostella (98%), Reticulitermes speratus–Cryptotermes secundus (88%), and D. melanogaster–Aedes aegypti (68%). Strongly supported associations between sequences from different insect orders were also recovered, including Trachymyrmex cornetzi–Schistocerca gregaria (91%), H. halys–Apis mellifera (99%), and Leptidea sinapis–Hetaerina americana (100%).

The sampled hemipteran sequences were distributed across several regions of the tree rather than forming a single well‐supported clade, and several deeper nodes showed low bootstrap support. Accordingly, the reconstruction should be interpreted as a comparative takeout/JHBP‐family gene tree rather than as a resolved representation of insect order‐level relationships. The weakly resolved position of EiTO1 should therefore be considered together with the conserved‐domain, motif, and structural evidence supporting its family assignment.

3.2. Sequence Conservation and Motif Distribution of EiTO1

Multiple sequence alignment of EiTO1 with two hemipteran homologs and two experimentally characterized takeout/JHBP‐family proteins revealed limited overall amino‐acid identity but recurrent conservation at several positions within the aligned region (Figure 2A). Among the selected comparison proteins, NlTO1 showed the highest percentage identity to EiTO1 (31.2%) and covered 100% of the EiTO1 reference sequence. EpTO1, BmJHBP, and HhTO1 showed identities of 18.2%, 16.4%, and 14.3%, with corresponding coverage values of 87.3%, 83.3%, and 96.3%, respectively.

Figure 2.

Multiple sequence alignment and MEME‐derived motif distribution of EiTO1 and selected insect takeout/JHBP‐family proteins. (A) Multiple sequence alignment of the predicted 245‐amino‐acid EiTO1 protein from Eurygaster integriceps with 2 hemipteran homologs, NlTO1 from Nilaparvata lugens (XP_022201958.2) and HhTO1 from Halyomorpha halys (XP_014287093.3), and 2 experimentally characterized takeout/JHBP‐family proteins, EpTO1 from Epiphyas postvittana (PDB ID: 3E8T, chain A) and BmJHBP from Bombyx mori (PDB ID: 3AOT). EiTO1 was translated from the predicted 738‐nt ORF contained in the transcriptome‐derived partial record OP913374.1. Sequences were aligned using Clustal Omega 1.2.4 and visualized in MView, with EiTO1 designated as the reference sequence. “cov” denotes the percentage of EiTO1 residues aligned with each sequence, whereas “pid” denotes the percentage of identical residues normalized to the ungapped EiTO1 region included in the alignment. The alignment was colored by identity: only residues identical to the corresponding EiTO1 residue are highlighted. Green shades represent hydrophobic residues, red represents positively charged residues, blue represents negatively charged residues, purple represents polar residues, cyan represents serine or threonine, and yellow represents cysteine. Black and white lettering is used only to maintain contrast against the background colors. Consensus lines show exact residue or physicochemical‐class conservation at thresholds of 100%, 90%, 80%, and 70%. Uppercase letters indicate conservation of a specific amino‐acid residue, whereas lowercase symbols indicate shared physicochemical classes: a, aromatic; c, charged; h, hydrophobic; l, aliphatic; o, alcohol; p, polar; s, small; t, turn‐like; and u, tiny. The symbols “+” and “−” denote positively and negatively charged residue classes, respectively, and dots indicate the absence of consensus at the specified threshold. (B) Distribution of three recurrent motifs identified using MEME Suite v5.5.4. Motif 1 is shown in red, Motif 2 in cyan, and Motif 3 in green. Motif locations are drawn to scale relative to protein length. The p values shown beside the sequences are the sequence‐level combined match p values reported in the MEME motif‐location output. Motif numbering follows the MEME output ranking and does not indicate the physical N‐to‐C‐terminal order of the motifs. Motif 2 was detected in all five proteins, whereas Motif 1 was absent from HhTO1 and Motif 3 was absent from BmJHBP. The consensus sequence of each motif is shown below the motif map.

MEME analysis identified three recurrent sequence motifs with variable distributions among the five proteins (Figure 2B). Motif 2 was detected in all five sequences. Motif 1 was present in EiTO1, EpTO1, NlTO1, and BmJHBP but was not detected in HhTO1, whereas Motif 3 was present in EiTO1, EpTO1, NlTO1, and HhTO1 but was not detected in BmJHBP. EiTO1, EpTO1, and NlTO1 displayed the physical motif order 1–3–2; BmJHBP contained Motifs 1 and 2, and HhTO1 contained Motifs 3 and 2.

Conserved‐domain analysis, conducted separately from the alignment and motif visualization, assigned EiTO1 to the JHBP‐related superfamily (pfam06585; E = 2.26 × 10⁻¹¹). Together, the alignment, motif distribution, and conserved‐domain result support assignment of EiTO1 to the broader takeout/JHBP family but do not demonstrate equivalent ligand specificity or physiological function among the compared proteins.

3.3. Structural Comparison of EiTO1 With Experimentally Resolved Takeout/JHBP Proteins

Structural comparison of the predicted EiTO1 model with experimentally determined structures of EpTO1 (E. postvittana) and BmJHBP (B. mori) showed a conserved overall architecture among these proteins (Figure 3). In all three structures, the fold displayed a barrel‐like configuration (Figure 3A). Enlarged views of the binding‐site region indicated that the ligand‐cavity position in EiTO1 corresponded to the analogous cavity region shown for EpTO1 and BmJHBP (Figure 3B). Across the aligned views, the relative placement of the cavity region was retained, supporting structural similarity in the core architecture among the compared proteins (Figure 3).

Figure 3.

Structural comparison of E. integriceps EiTO1 with EpTO1 and BmJHBP proteins. (A) Overall three‐dimensional structures of EpTO1, BmJHBP, and the predicted EiTO1 model. (B) Enlarged views of the ligand‐binding sites (red circles in (A)). Upper panels show top views and lower panels show side views. Structural alignments were performed using ChimeraX 1.10. EiTO1 shares a conserved barrel‐like fold and a similarly positioned ligand‐binding cavity, consistent with functional conservation within the takeout/JHBP family.

3.4. Tissue‐ and Month‐Resolved Expression Dynamics of takeout in Adults

Sampling month significantly affected EiTO1 expression in all four sample types: female whole body, $F\left(\mathrm{4,10}\right)=78.08$; head, $F\left(\mathrm{4,10}\right)=147.1$; fat body, $F\left(\mathrm{4,10}\right)=154.0$; and ovary, $F\left(\mathrm{4,10}\right)=32.01$ (exact omnibus p values are provided in Table S3). Relative expression was normalized separately within each sample type, with April assigned a value of 1; therefore, the values describe temporal variation within individual panels and should not be used to compare absolute expression among sample types.

In female whole‐body samples, EiTO1 expression decreased to 0.269‐fold in June relative to April (Dunnett‐adjusted p = 0.0390), reached a pronounced maximum of approximately 160.02‐fold in August (adjusted p < 0.0001), and decreased to 0.43‐fold in December, which was not significantly different from April (adjusted p = 0.2094). A substantially smaller secondary increase of approximately 21.55‐fold occurred in March (adjusted p = 0.0001) (Figure 4A).

Figure 4.

Relative EiTO1 expression was measured in (A) female whole‐body samples, (B) head, (C) fat body, and (D) ovary. Seasonal and tissue‐specific expression of takeout in adult Eurygaster integriceps. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using one‐way ANOVA followed by Dunnett's multiple comparisons test. Asterisks indicate significant differences compared with the April group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Seasonal sampling points were defined as follows: April, overwintered adults after migration to wheat fields; June, newly emerged adults feeding before migration to refuge sites; August, summer refuge‐associated phase; December, overwintering phase; and March, late‐overwintering/premigration transition. These assignments were based on generation, collection habitat, seasonal timing, and established species phenology rather than direct endocrine or reproductive measurements.

In the head, expression decreased to 0.077‐fold in June (adjusted p < 0.0001), increased to approximately 2.65‐fold in August (adjusted p = 0.0444), and was approximately 1.63‐fold in December, which did not differ significantly from April (adjusted p = 0.5593). Expression reached its maximum of approximately 164.79‐fold in March (adjusted p < 0.0001) (Figure 4B).

In the fat body, expression was approximately 2.56‐fold in June and did not differ significantly from April (adjusted p = 0.0789). Expression increased to approximately 6.69‐fold in August (adjusted p = 0.0017), declined to approximately 0.012‐fold in December (adjusted p < 0.0001), and reached approximately 65.60‐fold in March (adjusted p < 0.0001) (Figure 4C).

In the ovary, expression decreased to approximately 0.205‐fold in June (adjusted p = 0.0009) and increased transiently to 5.62‐fold in August (adjusted p = 0.0011). Expression values of approximately 0.507‐fold in December and 1.32‐fold in March did not differ significantly from April (adjusted p = 0.1586 and 0.8459, respectively) (Figure 4D).

3.5. Supplementary Sequence and In Silico Annotation Outputs

A partial EiTO1 transcript containing a predicted 738‐nt ORF was identified from the published E. integriceps transcriptomic resource. The predicted ORF was bounded by initiation and termination codons and encoded a 245‐amino‐acid protein. The nucleotide and deduced protein sequences were deposited under GenBank accession numbers OP913374.1 and WBH19261.1, respectively (Figure S1). Theoretical protein properties were reported as pI 5.14. A sequence‐logo analysis from an alignment of 70 takeout proteins showed conserved regions concentrated in the central portion of the protein and increased variability at the termini (Figure S2).

In silico localization analysis using CELLO v2.5 yielded the highest score for cytoplasmic localization (1.414), followed by extracellular (1.096) and mitochondrial (0.926), with lower values for nuclear (0.312) and minimal scores for other compartments (Table S2). SignalP output indicated low overall signal‐peptide probability across the sequence, with a modest N‐terminal Sec/SPI peak reaching 0.0175 (Figure S3).

Subcellular localization predictions were partially discordant across tools. While CELLO assigned the highest score to cytoplasmic localization, it also returned a lower score for extracellular localization. By contrast, SignalP did not support a canonical N‐terminal Sec/SPI signal peptide. Taken together, these results indicate mixed in silico localization signals and do not support a definitive conclusion that secretion occurs via the classical secretory pathway. Noncanonical secretion remains a theoretical possibility but cannot be inferred from these predictions alone.

4. Discussion

The expanded taxonomic sampling and removal of redundant congeneric sequences provided a broader comparative context for EiTO1, but the resulting gene tree remained incompletely resolved. EiTO1 was recovered as sister to the N. lugens–B. tabaci clade, although the low bootstrap support for this relationship (31%) prevents its interpretation as a robust phylogenetic placement. Moreover, the sampled hemipteran sequences did not form a single supported clade, and several strongly supported associations involved proteins from different insect orders. These patterns may reflect the limitations of single‐gene reconstruction, lineage‐specific duplication and divergence within the takeout/JHBP family, unequal evolutionary rates, or uncertainty introduced during alignment and trimming. The tree should therefore be treated as complementary comparative evidence, whereas the assignment of EiTO1 to the takeout/JHBP family is more directly supported by conserved‐domain, motif, and structural analyses.

Figure 4 provides the central seasonal expression pattern of this study. EiTO1 transcript abundance varied markedly across distinct phases of the annual life cycle. In female whole‐body samples, expression was low in April, when overwintered adults had returned to wheat fields and resumed feeding and reproduction, and in June, when newly emerged adults of the next generation were feeding before migration to refuge sites. Expression reached its highest level in August, during the early refuge‐associated phase, and showed a secondary increase in March, when adults were still at the overwintering site and approaching spring migration. Accordingly, the March pattern is more appropriately interpreted in relation to the late‐overwintering/premigration transition and possible diapause termination than to the maintenance of confirmed diapause (Alborzi et al. 2026; Denlinger 2023; Hutfilz 2022). These seasonal changes correspond to the major life‐history transition between field activity and reproduction in the overwintered generation and refuge‐associated survival in the new generation. However, they should be interpreted as associations with seasonal physiological state rather than direct evidence that EiTO1 regulates diapause (Alborzi et al. 2026; Denlinger 2023; Hutfilz 2022).

Contemporary syntheses emphasize that diapause is not a passive “shutdown” but a regulated alternative physiological program, typically coordinated through neuroendocrine integration and systemic metabolic remodeling. Within that framework, two features of the female tissue profile are particularly informative. First, the diapause‐elevated signal in the “head” fraction is biologically coherent because this fraction contains key neuroendocrine centers and associated peptidergic signaling that link seasonal timekeeping to endocrine control of diapause (Helfrich‐Förster 2024). Second, the fat body, an established hub for nutrient sensing, lipid storage, and endocrine interorgan communication, also exhibited diapause‐linked elevation, consistent with a systemic seasonal program in which metabolic tissues participate alongside neuroendocrine tissues (Arrese and Soulages 2010; Koyama et al. 2025).

Ovarian EiTO1 expression varied seasonally rather than remaining consistently low. Transcript abundance was at or below the April baseline in June, December, and March, but increased transiently by 5.62‐fold in August. Because each sample type was normalized separately to its April calibrator, this increase reflects temporal variation within the ovary and does not allow direct comparison of absolute expression among tissues. The August elevation coincided with the early refuge‐associated phase and may reflect tissue‐specific transcriptional remodeling rather than reproductive activation. This interpretation is consistent with evidence that diapause‐related responses are tissue‐specific and that ovaries can retain selective molecular activity despite reproductive arrest (Hutfilz 2022; Wei et al. 2024). The observation of undeveloped ovaries in August adults of E. integriceps further supports a cautious interpretation (Alborzi et al. 2026). Because ovarian developmental stage and vitellogenesis markers were not assessed in the present study, the biological significance of this transient increase remains unresolved, and natural variation cannot be excluded. More broadly, such stage‐restricted expression changes are consistent with dynamic transcriptional remodeling during diapause progression and termination (Denlinger 2023; Hutfilz 2022; Koštál 2006; Roncalli et al. 2021; Bao et al. 2022).

Seasonal trajectories are common in insects and can reflect divergent constraints on reproduction and resource allocation, as well as endocrine control of seasonal state transitions, rather than a simple scalar difference in diapause “strength” (Meuti et al. 2024; Hutfilz 2022; Hejníková et al. 2022). Moreover, head‐enriched seasonal signals are biologically coherent within a diapause framework because photoperiodic information is integrated through neuroendocrine pathways centered in the head/brain, and head‐specific transcriptomic shifts have been documented during adult diapause in other insects (Takeda and Suzuki 2022; Dhungana et al. 2025).

Two limitations of this study should be acknowledged. First, the physiological status of seasonal samples was inferred from generation, collection habitat, sampling date, and known species phenology rather than direct ovarian, feeding, or hormonal measurements. Accordingly, March adults were treated as representing a late‐overwintering/premigration transition. Second, β‐actin was used as the sole reference gene without formal validation across seasons and tissues; therefore, the expression patterns should be confirmed in future studies using multiple validated reference genes.

Finally, a practical strength of the present work is that the seasonal transcriptional profiles were derived from field‐relevant time points that align with biologically meaningful phases of migration/feeding/reproduction versus dormancy. Ecologically grounded sampling can complement laboratory diapause paradigms by capturing “real‐world physiology” shaped by fluctuating microclimate, host availability, and movement ecology (Denlinger 2023). In this context, the consistency of diapause‐associated elevation across the integrated data set supports EiTO1 as a robust molecular correlate of diapause‐linked seasonal state in an economically important hemipteran pest.

5. Conclusion

EiTO1 in E. integriceps was identified as a takeout/JHBP‐family gene with conserved motifs and domain architecture, and a predicted fold consistent with a hydrophobic ligand‐binding cavity. Across field‐aligned seasonal stages, EiTO1 transcription varied markedly among reproductive, refuge‐associated, and late‐overwintering transition phases and was enriched in the head and, to a lesser extent, the fat body. Together, these tissue–season profiles indicate that EiTO1 is most consistent with a systemic seasonal physiology marker linked to diapause‐associated state, rather than a primary ovary‐localized transcript. Collectively, the findings provide an evolutionarily and ecologically grounded framework for interpreting the molecular physiology of diapause in the Sunn pest.

Author Contributions

Behnaz Farhadi: writing – original draft, investigation, visualization, formal analysis, data curation, software. Azam Amiri: writing – review and editing, visualization. Houshang Alizadeh: conceptualization, validation. Ali R. Bandani: project administration, conceptualization, funding acquisition, writing – original draft, writing – review and editing, resources, supervision, validation.

Ethics Statement

The authors used Eurygaster integriceps and declared compliance with the ethical standards of the College of Agriculture and Natural Resources, University of Tehran.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

Data Availability Statement

The data sets generated and/or analyzed during the current study are available in the NCBI GenBank repository under Accession Numbers OP913374.1 and WBH19261.1. All other data supporting the findings of this study, including multiple sequence alignments, phylogenetic trees, and qPCR data sets, are included in this published article and its Supporting Information files.