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Article

Unveiling the Regulatory Mechanisms of Irradiation Response in Pseudococcus jackbeardsleyi Under Hypoxic Conditions

by
Li Li
1,†,
Changyao Shan
1,2,3,†,
Qiang Xu
4,
Baishu Li
1,
Haijun Liu
4,* and
Tao Liu
1,3,*
1
Institute of Equipment Technology, Chinese Academy of Quality and Inspection & Testing, No. A3, Gaobeidianbeilu, Chaoyang District, Beijing 100123, China
2
Harry Butler Institute, Murdoch University, Murdoch, WA 6150, Australia
3
College of Environmental and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia
4
Guangzhou Customs District Technology Center, Guangzhou 510623, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(20), 2104; https://doi.org/10.3390/agriculture15202104
Submission received: 19 August 2025 / Revised: 2 October 2025 / Accepted: 6 October 2025 / Published: 10 October 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

Mealybugs are high-priority quarantine pests in fresh-produce trade due to cryptic habits, broad host ranges, and market-access risks. Phytosanitary irradiation (PI) provides a non-residual, process-controlled option that is increasingly integrated with modified-atmosphere (MA/MAP) logistics. Because molecular oxygen enhances indirect radiation damage (oxygen enhancement ratio, OER), oxygen limitation may modulate PI outcomes in mealybugs. The Jack Beardsley mealybug (Pseudococcus jackbeardsleyi) has an IPPC-adopted PI treatment of 166 Gy (ISPM 28, PT 45). We exposed adult females to 166 Gy under air and 1% O2 and generated whole-transcriptome profiles across treatments. Differentially expressed genes and co-differentially expressed genes (co-DEGs) were integrated with protein–protein interaction (PPI) and regulatory networks, and ten hubs were validated by reverse transcription quantitative PCR (RT-qPCR). Hypoxia attenuated irradiation-induced transcriptional disruption. Expression programs shifted toward transport, redox buffering, and immune readiness, while morphogen signaling (Wnt, Hedgehog, BMP) was coherently suppressed; hubs including wg, hh, dpp, and ptc showed stronger down-regulation under hypoxia + irradiation than under irradiation alone. Despite these molecular differences, confirmatory bioassays at 166 Gy under both atmospheres (air and 1% O2) achieved complete control. These results clarify how oxygen limitation modulates PI responses in a quarantine mealybug while confirming the operational efficacy of the prescribed 166 Gy dose. Practically, they support the current international standard and highlight the value of documenting oxygen atmospheres and managing dose margins when PI is applied within MA/MAP supply chains.

1. Introduction

Phytosanitary irradiation (PI) has been established as a mature and widely applied measure that secures market access while maintaining the quality of fresh commodities [1,2]. Within the framework of the International Plant Protection Convention (IPPC), the International Standards for Phytosanitary Measures (ISPM) No. 18 (Guidelines for the Use of Irradiation as a Phytosanitary Measure) and No. 28 (Phytosanitary Treatments for Regulated Pests) prescribe requirements, and adopted phytosanitary treatment (PT) annexes are provided [3]. The absorbed dose is expressed in gray (Gy), an SI-derived unit equal to one joule per kilogram (1 J kg−1). For example, a generic dose of 150 Gy has been specified for Tephritidae (PT 7) [4], whereas 231 Gy has been adopted for several mealybug species, including Dysmicoccus neobrevipes, Planococcus lilacinus, and Planococcus minor (PT 19) [5]. Of particular relevance to this study, PT 45, adopted in 2023, prescribes a treatment dose of 166 Gy for the Jack Beardsley mealybug Pseudococcus jackbeardsleyi, which effectively prevents the development of F1 second-instar nymphs originating from mature adult females at the stipulated level of efficacy [6].
In parallel, modified atmosphere (MA/MAP) technologies, achieved by lowering oxygen concentrations and/or elevating carbon dioxide concentrations, are now widely implemented in postharvest logistics and are frequently combined with PI [7,8,9]. Oxygen availability is a critical covariate for irradiation efficacy because molecular oxygen stabilizes radiation-induced radical lesions on biomolecules. Under the classical oxygen fixation hypothesis, hydroxyl and carbon-centered radicals generated by water radiolysis are converted to relatively stable peroxyl adducts in the presence of O2, increasing the probability that DNA damage becomes non-repairable. When O2 is limited, these reactions are attenuated, apparent radiosensitivity decreases, and the magnitude of the oxygen enhancement ratio (OER) depends on taxa, developmental stage, gas composition, and exposure conditions [10,11,12]. Previous studies on fruit flies and other pest groups have demonstrated a spectrum of outcomes, ranging from pronounced radioprotective effects under extremely low oxygen conditions to negligible impacts under moderate O2/CO2 regimes [13,14], underscoring the necessity of species- and context-specific evidence.
The developmental stage is a major determinant of radiosensitivity in insects. Stages with high mitotic activity, for example, embryos and early larvae, tend to be more sensitive, whereas quiescent or metabolically suppressed stages, for example, late pupae or eggs with low oxygen diffusivity, can be comparatively tolerant. For phytosanitary applications, this necessitates stage-specific dose setting and confirmatory targets and motivates explicit evaluation of oxygen effects in the stage used for operational confirmation [4,5,10,12].
The P. jackbeardsleyi is a highly polyphagous hemipteran pest characterized by its white waxy covering, short lateral filaments, and an ovisac in ovipositing females; adult females are typically 2 to 4 mm. Its cryptic feeding complicates detection and control and feeding injury and honeydew deposition reduce photosynthesis and promote sooty mold, leading to downgrading and rejection of fruit lots and increasing supply chain costs [15]. Major hosts include economically important fruit crops across multiple families, including Citrus spp., Mangifera indica, Carica papaya, Ananas comosus, Musa spp., Psidium guajava, and Selenicereus undatus, as well as numerous ornamentals [16,17,18,19]. The broad host range and quarantine relevance across multiple commodities and regions make it a high-priority target for PI. Although operational treatment doses and confirmatory trials for mealybugs have advanced in recent years [5,6], the molecular basis underlying hypoxia–irradiation interactions in P. jackbeardsleyi remains unresolved.
To address this knowledge gap, we explicitly test whether hypoxia (1% O2) alters the phytosanitary irradiation response of the Jack Beardsley mealybug (Pseudococcus jackbeardsleyi) at the IPPC-adopted dose of 166 Gy (ISPM 28, PT 45). We hypothesize that lowering oxygen diminishes oxygen-dependent radiosensitization and redirects post-irradiation programs toward stress adaptation while retaining efficacy, defined as prevention of development to the F1 second instar. Accordingly, we exposed adult females to 166 Gy under ambient air and 1% O2. To interpret oxygen-dependent differences, we integrated RNA-seq with protein–protein interaction (PPI) and gene regulatory network analyses, and validated ten hub genes by RT-qPCR, delineating the regulatory architecture that links operational performance to mechanism under MA/MAP-relevant conditions.

2. Materials and Methods

2.1. Insect Rearing

P. jackbeardsleyi individuals were originally collected from a mango orchard in Guangxi Zhuang Autonomous Region, China (22.1178° N, 106.7394° E), and a laboratory colony was established at the Chinese Academy of Inspection and Quarantine (CAIQ). Colonies were maintained in ventilated rearing cages (40 × 40 × 50 cm) at 25 ± 1 °C, 70 ± 5% relative humidity (RH), and a 16:8 h light: dark (L:D) photoperiod in ambient air [20]. A solid diet consisting of fresh orange slices together with a supplemental pad of sucrose and enzymatically hydrolyzed yeast (MP Biomedicals, Aurora, OH, USA) mixed at a 3:1 weight ratio was provided throughout rearing to support feeding and oviposition. We selected enzymatic hydrolysate rather than acid-hydrolyzed yeast to better preserve peptides and B-vitamins and to minimize potential formation of heat/acid degradation by-products; such differences in hydrolysis method can alter nutrient availability and, in turn, affect oviposition and nymphal development. Adult females produced ovisacs under these conditions. Eggs were collected from ovisacs produced by females two weeks after female emergence; ovisacs were gently removed with fine forceps and held at 26 ± 1 °C to obtain synchronized cohorts of eggs laid within an 8–12 h interval. To minimize environmental variability and establish a stable genetic background, all colonies were reared on a common solid diet under identical abiotic conditions prior to experimentation. All irradiation assays used adult females of P. jackbeardsleyi from laboratory generations F3 to F6. For confirmatory assays, gravid females were allowed to oviposit on the diet for 1 day, and the resulting progeny cohorts were reared for approximately 30 days under the same conditions until late-stage adult females were obtained (the laboratory lifespan is approximately 35 days) [21].

2.2. Large-Scale Confirmatory Tests

Large-scale confirmatory irradiation tests were conducted to validate the phytosanitary irradiation dose under normoxic and hypoxic conditions. To achieve 99.9968% prevention of reproduction (probit 9 prevention) in P. jackbeardsleyi at the 95% confidence level, a minimum of 93,616 of the most tolerant individuals (late-stage adult females reared for 30 days from oviposition) were required to be treated in accordance with ISPM 18 standards [22,23].

2.2.1. Modified Atmosphere Treatment

Prior to phytosanitary irradiation (PI), late-stage adult females of P. jackbeardsleyi (reared for 30 days from oviposition) were subjected to modified atmosphere (MA) pre-treatment. Approximately 3000 individuals, together with five sprouted potatoes, were placed in perforated plastic containers and sealed inside 3 dm3 gas-tight bags (Dalian Delin Gas Packaging Co., Ltd., Dalian, China). The bags were flushed with a certified gas mixture (99% N2 + 1% O2; provided by Beijing Green Oxygen Tiangang Technology Development Co., Ltd., Beijing, China) for 3 min, and the procedure was repeated three times to ensure full gas replacement.
The sealed bags were incubated under MA conditions. For the 1% O2 + PI (166 Gy) group, bags were removed after 23.5 h and irradiated. All bags were then opened at the same time to ensure consistent sealing duration. In the control and PI-only groups, no gas flushing was applied; insects were sealed in bags and incubated under the same conditions for 24 h.

2.2.2. Phytosanitary Irradiation Treatment

X-ray irradiation was carried out at room temperature using an RS-2000 ProX irradiator (Rad Source Technologies, Buford, GA, USA) at 220 kV and 17.6 mA. Insects sealed in gas-tight bags were placed in a 17 × 15 × 17 inch chamber. A RadCal dosimeter (model 2086) with a 10 × 6−0.6 ion chamber was used to monitor the dose near the samples. Irradiation stopped automatically when the target dose of 166 Gy was reached. The dose rate was approximately 5.0 Gy min−1, with a total exposure time of approximately 33.2 min.

2.2.3. Rearing of Mealybugs After Irradiation

Following irradiation under normoxic and hypoxic conditions, potatoes infested with P. jackbeardsleyi were transferred to plastic rearing containers and maintained under controlled laboratory conditions (25 ± 1 °C, 70 ± 5% RH, 16:8 h L:D photoperiod). Fresh potatoes were periodically supplied as required to support optimal development of the insects to the F1 second-instar stage. To prevent cross-contamination, untreated controls were reared separately in an independent rearing room. For both treated and control groups, the number of adult females and the number of F1 first- and second-instar nymphs that developed were examined and recorded.

2.3. Transcriptional Analysis of P. jackbeardsleyi

Female adults of P. jackbeardsleyi reared under controlled laboratory conditions were used for transcriptomic analysis. Three treatments were designed: Control, Air + 166 Gy, and 1% O2 + 166 Gy. Each treatment included three biological replicates, with 50 female adults per replicate. For hypoxia treatment, oxygen concentration was stabilized at 1% in a sealed chamber and verified prior to irradiation. Surviving insects were collected immediately after treatment, frozen in liquid nitrogen, and stored at −80 °C.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Promega, Madison, WI, USA) following the manufacturer’s protocol. Samples were handled on ice with RNase-free disposables, and homogenization was performed with a bead mill in TRIzol; total RNA for each biological replicate was prepared from pooled whole adult females (pooled 30 individuals per replicate). RNA concentration and purity were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) with prespecified thresholds (A260/280 = 1.9 to 2.1; A260/230 ≥ 2.0), while integrity was assessed by agarose gel electrophoresis and verified with an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) to obtain RIN values; only RNA samples of high quality (RIN ≥ 7.0) proceeded to library preparation. mRNA was purified with poly-T magnetic beads, RNA was fragmented at 94 °C for 15 min, and cDNA was synthesized (random-hexamer priming). After end repair, adapter ligation, and limited-cycle PCR (8 cycles), libraries with an average insert size of approximately 300 bp were purified with AMPure XP beads, validated on the Bioanalyzer, quantified by qPCR, and indexed and multiplexed for sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) in paired-end 150 bp mode at a target depth of ≥20 million read pairs per library, with 3 biological replicates per condition and lane randomization to mitigate batch effects.
Transcript abundance was estimated as TPM (transcripts per million) and raw counts using Salmon or RSEM. Differentially expressed genes (DEGs) between treatments were identified with DESeq2, applying thresholds of |log2FC| ≥ 1 and q < 0.05. Annotated unigenes and transcripts were searched against NR, Swiss-Prot, Pfam, eggNOG, GO, and KEGG databases to assign functional classification. GO enrichment and KEGG pathway analyses of DEGs were performed to explore biological processes and pathways associated with irradiation under normoxic and hypoxic conditions.

2.4. Network Construction and Regulatory Inference

To investigate gene–gene interactions and upstream regulatory mechanisms, a protein–protein interaction (PPI) network was constructed based on the co-DEGs using the STRING database, retaining edges with a combined score > 0.4. The network was visualized in Cytoscape (version 3.9.1), and topological parameters were calculated using the NetworkAnalyzer plugin. Hub genes were identified and ranked by cytoHubba according to multiple centrality measures, with node size and color representing centrality and edge thickness reflecting STRING confidence scores.
To infer upstream regulators of hub genes in P. jackbeardsleyi, transcription factor (TF)–hub interactions were predicted through motif enrichment analysis and TF assignment using insect-focused motif libraries (JASPAR 2024 Insects and CIS-BP). Where available, ChIP-based evidence and validated cis-regulatory elements from public insect datasets were integrated. Candidate miRNA–hub interactions were identified using starBase by intersecting predictions from TargetScan, PicTar, PITA, miRanda/mirSVR, and RNA22, retaining pairs supported by at least three algorithms. All regulatory relationships were integrated and visualized in Cytoscape v3.9.1 to construct comprehensive TF–hub–miRNA regulatory networks.

2.5. Reverse Transcription Quantitative PCR (RT-qPCR) Validation

Key hub genes were selected for RT-qPCR to validate the transcriptome results; primer sequences are listed in Table S1. Total RNA was extracted from P. jackbeardsleyi adult females (irradiation and control groups) using TRIzol (Invitrogen, Carlsbad, CA, USA). Genomic DNA was removed with DNase I (Promega, Madison, WI, USA); first-strand cDNA was synthesized with the FastKing RT Kit (Tiangen, Beijing, China) using oligo(dT) primers according to the manufacturer’s instructions; no-RT controls were included to verify the absence of genomic DNA carryover, and no-template controls (NTC) were negative.
RT-qPCR (qPCR on cDNA) was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in 10 µL reactions containing 0.5 µL cDNA, 0.3 µL of each primer (10 µM), 5 µL GoTaq qPCR Master Mix (Promega), and 3.9 µL nuclease-free water. The cycling program was 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. A melt-curve analysis from 60 to 95 °C confirmed amplicon specificity.
The ribosomal protein gene RPL was used as the internal reference, and relative expression levels were calculated using the 2−ΔΔCt method. All RT-qPCR experiments were performed with three biological replicates. For RT-qPCR, statistics were conducted on ΔCt values. One-way ANOVA followed by Tukey’s HSD was used. Multiple testing was controlled using the Benjamini–Hochberg false discovery rate.

3. Results

3.1. Confirmatory Tests

Large-scale confirmatory tests were conducted to validate the efficacy of the IPPC-prescribed phytosanitary dose (166 Gy) under both normoxic and hypoxic conditions using P. jackbeardsleyi females reared on potato. The target dose was set at 166 Gy. Among approximately 3710 and 3620 treated females, respectively, no F1 generation 2nd instar nymphs were observed, whereas the majority of neonates in the control developed normally (Table 1). The absorbed doses measured by the RadCal dosimeter ranged from 166 to 189 Gy in the irradiation-only test and from 164.8 to 185.4 Gy in the hypoxia + irradiation test, with dose uniformity ratios (DUR) of 1.14 and 1.13, respectively (Table 1). Under control conditions, no adult female mortality was observed from irradiation to oviposition; therefore, treatment efficacy was calculated directly based on the number of treated females without adjustment for control mortality [24]. These results demonstrate that variations in oxygen concentration do not compromise the efficacy of the prescribed phytosanitary irradiation dose.

3.2. General Description of RNA-Seq Data

Sequencing of nine P. jackbeardsleyi transcriptome libraries generated 56.62 Gb of raw data. The experimental design comprised a control group maintained under standard rearing conditions, a normoxia irradiation treatment group (Air + 166 Gy), and a hypoxia irradiation treatment group (1% O2 + 166 Gy). Following quality trimming, 40.32 GB of clean reads were retained. Each library ranged from 5.63 to 7.08 Gb in size, with GC content values between 40.81% and 42.24% and Q30 scores ranging from 96.35% to 97.62% (Table S2). From these datasets, an expression matrix covering 15,414 genes was generated. Correlation analysis among biological replicates yielded coefficients from 0.92 to 0.99, confirming high data consistency (Figure 1A). Principal component analysis (PCA) showed tight clustering of replicates within each treatment, while the control group was clearly separated from both irradiated treatments (166 Gy and 1% O2 + 166 Gy) (Figure 1B).
Further analysis (Figure 1A) showed that the Pearson’s correlation coefficients (r)among biological replicates within the same treatment group were all above 0.92, indicating high reproducibility of the sequencing data. In contrast, correlations between different treatment groups were notably lower, with the most pronounced differences observed between the control and the two irradiation treatments. Principal component analysis (PCA) (Figure 1B) revealed that the first principal component (PC1) accounted for 93.5% of the total variance, effectively separating the control group from the irradiation treatments, while the second principal component (PC2), explaining 3.5% of the variance, primarily distinguished the hypoxia + irradiation treatment from the normoxia irradiation treatment. The three groups formed distinct clusters in the two-dimensional space, indicating that different treatment conditions induced substantial transcriptomic alterations in P. jackbeardsleyi.

3.3. Analysis of Differentially Expressed Genes (DEGs)

To further elucidate the regulatory effects of hypoxia on gene expression in P. jackbeardsleyi under phytosanitary irradiation dosage, we performed differential gene expression analysis across three treatment comparisons, including 1% O2 + 166 Gy vs. Control, Air + 166 Gy vs. Control, and 1% O2 + 166 Gy vs. Air + 166 Gy. Among the 35,422 detected genes, a total of 1197 differentially expressed genes (DEGs) were identified, exhibiting substantial changes (|fold change| ≥ 2 and FDR < 0.05). In the comparison between 1% O2 + 166 Gy and the control, 2077 DEGs were detected (949 upregulated and 1128 downregulated). The Air + 166 Gy vs. Control comparison yielded 1327 DEGs (507 upregulated and 820 downregulated), while 1% O2 + 166 Gy vs. Air + 166 Gy resulted in 1474 DEGs (814 upregulated and 660 downregulated) (Figure 2A–C). The detailed expression profiles of these DEGs are presented in Figure 2E,F. Overall, the results demonstrate substantial transcriptional changes among different treatments, suggesting that the combined effects of hypoxia and irradiation may modulate multiple molecular pathways involved in the physiological responses of P. jackbeardsleyi.

3.4. Functional Annotation and Enrichment Analysis of DEGs

Gene Ontology (GO) enrichment analysis revealed a progressive shift in functional responses from Control to Normoxia + Irradiation (Air + 166 Gy), and further to Hypoxia + Irradiation (1% O2 + 166 Gy).
In the 1% O2 + 166 Gy vs. Control comparison (Figure 3A,B), downregulated genes were mainly linked to lipid metabolism and membrane structure (acyl-CoA hydrolase activity, triglyceride lipase activity, phosphatidylinositol metabolic process, lipid droplet), together with ubiquitin ligase complexes (SCF and cullin–RING) and chitin metabolic processes, indicating suppressed membrane remodeling, lipid catabolism, and protein degradation. Upregulated genes were enriched in mitochondrial and biosynthetic pathways (mitochondrial intermembrane space, L-methionine biosynthesis, aspartate family amino acid biosynthesis, phospholipid/phosphatidylinositol biosynthesis), suggesting a metabolic shift toward energy production and protein synthesis.
In the Air + 166 Gy vs. Control comparison (Figure 3C,D), downregulated DEGs also involved lipid catabolism and membrane turnover (lipase activity, sterol esterase activity, phospholipid metabolism), while upregulated DEGs were associated with RNA processing, protein turnover, and mitochondrial activity (mitochondrial ribonuclease P complex, tRNA processing, protein polyubiquitination), reflecting a stress response aimed at mitochondrial maintenance and nucleic acid metabolism.
In the 1% O2 + 166 Gy vs. Air + 166 Gy comparison (Figure 3E,F), hypoxia under irradiation downregulated ubiquitin-mediated protein degradation (ubiquitin ligase activity, SCF complex) and transcriptional regulation (RNA polymerase II-specific transcription factor activity), while upregulated DEGs enhanced transmembrane transport (sugar/glucose transporters), signal transduction, immune regulation (humoral immune response), and amino acid metabolism (isoleucine metabolism).
Collectively, the progression from the control to normoxia plus irradiation and finally to hypoxia plus irradiation reveals a clear trajectory of metabolic reprogramming in P. jackbeardsleyi under phytosanitary irradiation. While both irradiation treatments suppressed lipid mobilization and membrane renewal, hypoxia further amplified these effects and redirected cellular resources toward energy production, nutrient transport, and immune readiness—at the expense of energy-intensive biosynthetic and nuclear regulatory pathways—representing a survival-oriented metabolic strategy under combined hypoxic and irradiation stress.
KEGG pathway enrichment analysis revealed distinct metabolic and signaling reprogramming patterns in P. jackbeardsleyi under phytosanitary irradiation across oxygen regimes (Figure 4A–C).
In 1% O2 + 166 Gy vs. Control, downregulated DEGs were mainly involved in DNA replication, cell cycle, lipid/membrane biosynthesis, and MAPK signaling, indicating suppressed cell proliferation and membrane renewal. Upregulated DEGs were enriched in lysosome, autophagy, glutathione metabolism, and apoptosis, suggesting activation of catabolic and stress adaptation processes. These patterns are summarized by the top 11 upregulated and 14 downregulated pathways visualized in Figure 4A.
In Air + 166 Gy vs. Control, downregulated DEGs clustered in detoxification (cytochrome P450–mediated xenobiotic metabolism) and lipid mobilization pathways, while upregulated DEGs were linked to hormone biosynthesis, ECM–receptor interaction, and immune-related signaling, reflecting enhanced repair and defense responses. The distribution of terms is captured by the top 7 upregulated and 30 downregulated pathways shown in Figure 4B.
In 1% O2 + 166 Gy vs. Air + 166 Gy, hypoxia under irradiation further suppressed key developmental and survival pathways (MAPK, PI3K–Akt, Hedgehog) and autophagy, while upregulating transmembrane transport, amino acid metabolism, calcium signaling, and JAK–STAT–mediated immune regulation. These shifts are represented by the top 38 upregulated and 10 downregulated pathways in Figure 4C.
Overall, KEGG enrichment patterns indicated that irradiation under both oxygen conditions suppressed lipid mobilization and membrane renewal while activating stress adaptation pathways, whereas hypoxia further repressed growth- and development-associated signaling and redirected resources toward transport, intermediate metabolism, and immune readiness, thereby reflecting a survival-oriented metabolic strategy under combined hypoxic and irradiation stress.
To isolate the net effect of hypoxia on the irradiated background, we defined co-DEGs as genes that are differentially expressed in 1% O2 + 166 Gy vs. Air + 166 Gy and change in the same direction in at least one irradiation–control contrast (Air + 166 Gy vs. Control or 1% O2 + 166 Gy vs. Control). This yielded 376 up-regulated co-DEGs (357 + 19) and 263 down-regulated co-DEGs (255 + 8) (red sectors in Figure 5), and these sets are indicated by the red sectors in Figure 5. By construction, this set filters out irradiation-independent changes and highlights the hypoxia-specific modulation of the post-irradiation transcriptomic response, providing a focused input for the subsequent PPI and regulatory-network analyses.

3.5. PPI Network and Hub-Gene Behavior Under Hypoxic Irradiation

Using DEGs identified across three conditions (1% O2 + 166 Gy, air + 166 Gy, and control), we constructed a STRING-derived PPI network and prioritized central nodes with cytoHubba (Figure 6A). We first collated 639 common differentially expressed genes (co-DEGs) across the pairwise contrasts and queried STRING for interactions among them to assemble the network. The network resolved into three prominent functional clusters: embryonic pattern specification/morphogen signaling, including the Wnt (wg), Hedgehog (hh, ptc), and BMP (dpp) pathways; DNA replication; and pheromone and xenobiotic binding. The most topologically central module comprised ten hubs (wg, ptc, hh, dpp, ftz, odd, hth, sob, hb, and nub) that were densely interconnected (Figure 6B). Nodes were ranked by cytoHubba centrality metrics (MCC, degree, and betweenness). We retained the top ten hubs and required consistent regulation in 1% O2 + 166 Gy vs. air + 166 Gy and in at least one irradiation–control contrast. This yielded the set wg, ptc, hh, dpp, ftz, odd, hth, sob, hb, and nub for RT-qPCR validation (Figure 6B and Figure 8).
In terms of directionality, hypoxic irradiation induced a coherent down-shift in the embryonic-patterning module (Figure 6A), with the corresponding hubs showing lower normalized expression in the heat map (Figure 6C) and in the RT-qPCR validation (Figure 8). Conversely, the DNA-replication cluster was predominantly down-regulated, indicating proliferation restraint/cell-cycle limitation under hypoxic irradiation, while the pheromone/xenobiotic-binding cluster showed an overall attenuation. Together, these findings indicate that hypoxia reshapes the post-irradiation response in P. jackbeardsleyi, dampening replication programs and further suppressing morphogen-driven patterning networks, consistent with reduced oxygen-dependent radiosensitization, yet the cumulative lesion burden remains sufficient to achieve the regulatory endpoint at 166 Gy.

3.6. Layered Regulation Under Hypoxic Irradiation Revealed by TF–Hub and miRNA–Hub Networks

Focusing on the ten hubs identified by the PPI analysis, transcriptional control converged on suppression of morphogen pathways (Wnt, Hedgehog, and BMP), together with segmentation factors and the TALE (three-amino-acid loop extension)/Hox regulatory module. Pioneer and tissue-restricted transcription factors, including Zelda (Zld), Yorkie (Yki)/Scalloped (Sd), Pannier (Pnr), and Apterous (Ap), bridge multiple hubs, indicating system-level integration of repressive signaling and further dampening of the Wnt–Hh–BMP axis under hypoxic irradiation.
The miRNA–hub network exhibits extensive many-to-many connectivity: bantam, miR-8, miR-315, the miR-310–313 cluster, and the let-7 family jointly target morphogen genes and TF hubs, thereby linking developmental and stress-response programs (Figure 7A,B).
Concordant with the transcriptome-wide suppression of embryonic-patterning genes and down-regulation of DNA-replication programs, these networks provide a mechanistic rationale for the attenuated oxygen-dependent radiosensitization and enhanced tolerance observed under hypoxic irradiation. They also nominate a tractable set of regulators—including TFs (Pan, Ci, Mad, Zld, Yki, Sd, Pnr, Ap) and miRNAs (bantam, miR-8, miR-315, the miR-310–313 cluster, and the let-7 family)—as testable candidates for functional perturbation and dose-margin optimization in phytosanitary irradiation of the P. jackbeardsleyi.
Taken together with Figure 8, these layered regulatory changes support a hypoxia-potentiated repression of the Wnt–Hh–BMP axis under phytosanitary irradiation.

3.7. Validation of RNA-Seq Results by RT-qPCR

To verify the RNA-seq–inferred changes within the developmental network, we quantified the mRNA levels of ten hub genes—wg, ptc, hh, dpp, ftz, odd, hth, sob, hb, and nub—by RT-qPCR, using RPL as the internal reference (three biological replicates). Relative expression was calculated by the 2−ΔΔCt method and expressed as log2 fold change relative to the control (Figure 8).
Across all ten genes, irradiation significantly reduced transcript abundance relative to the control. For each gene, expression under 1% O2 + 166 Gy was significantly lower than under air + 166 Gy (p < 0.05). Wnt (wg), Hedgehog (hh, ptc), and BMP (dpp) pathways exhibited the largest reductions, with log2 fold changes between −1.2 and −0.8 under hypoxia + irradiation and between −0.5 and −0.3 under irradiation. Patterning/segmentation factors (ftz, odd, hb, hth, sob, nub) showed moderate down-regulation, ranging from −0.8 to −0.5 under hypoxia + irradiation and from −0.4 to −0.2 under irradiation. Variation among replicates was small (SD, n = 3). Taken together, the RT-qPCR data independently confirm that low oxygen potentiates the irradiation-induced suppression of the Wnt, Hedgehog, and BMP pathways in P. jackbeardsleyi, supporting a hypoxia-dependent attenuation of developmental programs under phytosanitary irradiation.

4. Discussion

Oxygen is recognized as a critical modifier of radiation injury [25,26]. Molecular oxygen stabilizes radical-induced DNA damage and thereby enhances indirect DNA lesions, whereas hypoxia diminishes this stabilization and lowers radiosensitivity [25,26,27]. In this study, transcriptomic disruption in P. jackbeardsleyi at the phytosanitary irradiation dose of 166 Gy was attenuated under hypoxia (1% O2), as evidenced by repression of Wnt, Hedgehog, and BMP signaling pathways and associated hub genes, with validation by RT-qPCR. This expression pattern suggests that under hypoxic irradiation, mealybugs shift toward reduced pressure on development and replication, with enhanced transport, redox, and immune functions, consistent with the classical explanation of the oxygen enhancement effect [26].
Mechanistically, attenuation of oxygen-dependent damage does not equate to radioprotection at the organismal endpoint. At 166 Gy, direct ionization generates clustered and complex DNA lesions, including DSB clusters, that are relatively oxygen-insensitive and difficult to repair [28]. Post-irradiation mitochondrial dysfunction sustains secondary ROS, redox imbalance, and bioenergetic stress. In our data, convergent suppression of morphogenetic signaling pathways (Wnt, Hedgehog, and BMP), together with replication restraint, indicates checkpoint-mediated developmental failure [29]. These processes collectively push the organism below a viability threshold even when radiosensitization is attenuated, explaining why the approved 166 Gy dose remains efficacious under both air and 1% O2 and aligning with phytosanitary standards that emphasize validated dosimetry and process control [24].
Importantly, these molecular effects did not translate into treatment failure at the regulatory endpoint: in mixed life stages, both 166 Gy and 1% O2 + 166 Gy achieved complete control in our assays. This outcome aligns with the broader PI experience that, although severe hypoxia may be mechanistically radioprotective, approved PI doses remain efficacious at their prescribed endpoints across different atmospheres when properly applied [30,31]. For instance, in Tephritidae, a generic 150 Gy schedule reliably prevents adult emergence with very high security (PT-7), and laboratory trials under MAP/low-O2 conditions have not shown increased survival at treatment-relevant doses [32,33,34,35]. Operational assurance at the annexed dose for this quarantine mealybug was thereby supported by the present results.
Within the IPPC framework, P. jackbeardsleyi now has an adopted PI treatment of 166 Gy, which prevents the development of F1 second-instar nymphs from irradiated adult females at the prescribed efficacy. The demonstration of complete control under both air and 1% O2 conditions is therefore fully compatible with PT 45, while also underscoring two practical points already embedded in ISPM 18 and related annexes: first, the presence of live but non-viable stages after irradiation does not imply treatment failure; and second, treatment security depends on validated dosimetry and process control, with documentation of oxygen atmospheres under MA/MAP serving as a logical complement [23,36].
Reports of species- and stage-specific differences in oxygen–irradiation interactions are consistent with our findings. Very low-oxygen atmospheres can induce radioprotective responses in insects, a mechanism that underlies reduced apparent radiosensitivity under anoxia or severe hypoxia [36,37]. Mealybugs possess wax coverings and cryptic habits that may influence micro-oxygenation, and their developmental architecture, dominated by morphogen signaling axes, may be particularly susceptible to hypoxia-induced repression of gene expression programs [38,39]. At the same time, PT 45, adopted as an ISPM 28 annex in 2023, prescribes a species-specific phytosanitary treatment dose of 166 Gy for P. jackbeardsleyi, based on confirmatory trials targeting tolerant female stages [6]. In contrast, for tephritids, factorial O2/CO2 manipulations at approved PI doses generally do not reduce efficacy and may yield additive or even synergistic process outcomes [11,40]. Multiple assessments indicate that modified-atmosphere storage or hypoxic conditioning at tephritid PI doses does not diminish treatment efficacy, including recent controlled studies and international evaluations [41]. These contrasts highlight the importance of species- and stage-resolved data when PI is applied under MA/MAP conditions and align with developmental biology, showing that Wnt, Hedgehog, Notch, and BMP pathways function as oxygen-responsive hubs whose crosstalk can be modulated by hypoxia, providing a mechanistic context for stage-dependent radiosensitivity [42].

5. Conclusions

This study demonstrates that hypoxia (1% O2) modulates, but does not compromise, the phytosanitary irradiation response of P. jackbeardsleyi at the IPPC-adopted dose of 166 Gy (ISPM 28, PT 45). Confirmatory assays under air and 1% O2 prevented development to the F1 second instar, meeting the prescribed endpoint. Integrated analyses show that hypoxia attenuates irradiation-induced transcriptomic disruption, redirects programs toward transport, redox buffering, and immune preparedness, and coherently suppresses morphogenetic signaling (Wnt, Hedgehog, BMP). Collectively, these results mechanistically link the oxygen enhancement effect to developmental control in this quarantine pest and operationally support continued use of PT 45 in MA/MAP supply chains, underscoring the practical value of documenting the oxygen concentration and atmosphere composition during treatment and maintaining prudent dose margins.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15202104/s1, Table S1. Data output quality and mapping rates for the examined samples of Pseudococcus jackbeardsleyi; Table S2. Sequences of primers used for RT-qPCR analysis.

Author Contributions

Conceptualization, C.S., L.L., H.L. and T.L.; methodology, C.S., L.L., H.L. and T.L.; software, C.S.; validation, C.S., L.L. and Q.X.; formal analysis, C.S.; investigation, C.S., L.L., Q.X. and B.L.; resources, C.S. and B.L.; data curation, C.S. and B.L.; writing—original draft preparation, C.S. and L.L.; writing—review and editing, T.L.; visualization, C.S.; supervision, H.L. and T.L.; project administration, H.L. and T.L.; funding acquisition, H.L. and T.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program of China, grant number 2022YFC2601505, and scientific research projects of the general administration of customs of China, grant number 2024HK243.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Chinese Academy of Inspection and Quarantine (Approval Code: 2023S001, and Approval Date: 16 August 2023).

Data Availability Statement

Dataset is available on request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 15 02104 g001
Figure 1. Overall transcriptomic analysis. (A) Heatmap showing replicate samples of different comparison groups of P. jackbeardsleyi. The gradient from light blue to dark blue reflects Pearson correlation coefficients ranging from 0.7 to 1, corresponding to lower to higher correlation levels. (B) Principal component analysis (PCA) of transcriptome profiles for P. jackbeardsleyi, with each color denoting a distinct comparison group.
Figure 1. Overall transcriptomic analysis. (A) Heatmap showing replicate samples of different comparison groups of P. jackbeardsleyi. The gradient from light blue to dark blue reflects Pearson correlation coefficients ranging from 0.7 to 1, corresponding to lower to higher correlation levels. (B) Principal component analysis (PCA) of transcriptome profiles for P. jackbeardsleyi, with each color denoting a distinct comparison group.
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Figure 2. (A–C) Volcano plots illustrating differentially expressed genes (DEGs) in each comparison group, where DEGs were defined as those with |fold change| ≥ 2 and p < 0.05. Up indicates upregulated genes are shown in red; down indicates downregulated genes are shown in blue, and ns indicates non-significant genes are shown in gray. (D–F) Heatmaps displaying the expression profiles of DEGs based on FPKM values, with the X-axis representing sample names and the Y-axis representing DEGs. Color gradients reflect expression levels, where red denotes higher expression and blue denotes lower expression.
Figure 2. (A–C) Volcano plots illustrating differentially expressed genes (DEGs) in each comparison group, where DEGs were defined as those with |fold change| ≥ 2 and p < 0.05. Up indicates upregulated genes are shown in red; down indicates downregulated genes are shown in blue, and ns indicates non-significant genes are shown in gray. (D–F) Heatmaps displaying the expression profiles of DEGs based on FPKM values, with the X-axis representing sample names and the Y-axis representing DEGs. Color gradients reflect expression levels, where red denotes higher expression and blue denotes lower expression.
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Figure 3. GO enrichment analysis of differentially expressed genes (DEGs). The genes were classified into three primary categories: biological process (blue), cellular component (green), and molecular function (pink). The size of each dot represents the number of DEGs. Only the top 10 significantly enriched GO terms within each category are shown. (A) 1% O2 + 166 Gy vs. Control, down-regulated; (B) 1% O2 + 166 Gy vs. Control, up-regulated; (C) Air + 166 Gy vs. Control, down-regulated; (D) Air + 166 Gy vs. Control, up-regulated; (E) 1% O2 + 166 Gy vs. Air + 166 Gy, down-regulated; (F) 1% O2 + 166 Gy vs. Air + 166 Gy, up-regulated.
Figure 3. GO enrichment analysis of differentially expressed genes (DEGs). The genes were classified into three primary categories: biological process (blue), cellular component (green), and molecular function (pink). The size of each dot represents the number of DEGs. Only the top 10 significantly enriched GO terms within each category are shown. (A) 1% O2 + 166 Gy vs. Control, down-regulated; (B) 1% O2 + 166 Gy vs. Control, up-regulated; (C) Air + 166 Gy vs. Control, down-regulated; (D) Air + 166 Gy vs. Control, up-regulated; (E) 1% O2 + 166 Gy vs. Air + 166 Gy, down-regulated; (F) 1% O2 + 166 Gy vs. Air + 166 Gy, up-regulated.
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Figure 4. KEGG pathway enrichment of differentially expressed genes (DEGs) in P. jackbeardsleyi. (A) 1% O2 + 166 Gy vs. Control. (B) Air + 166 Gy vs. Control. (C) 1% O2 + 166 Gy vs. Air + 166 Gy.
Figure 4. KEGG pathway enrichment of differentially expressed genes (DEGs) in P. jackbeardsleyi. (A) 1% O2 + 166 Gy vs. Control. (B) Air + 166 Gy vs. Control. (C) 1% O2 + 166 Gy vs. Air + 166 Gy.
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Figure 5. Effects of hypoxia on irradiation response: Venn diagrams of co-DEGs. Left, down-regulated genes; right, up-regulated genes.
Figure 5. Effects of hypoxia on irradiation response: Venn diagrams of co-DEGs. Left, down-regulated genes; right, up-regulated genes.
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Figure 6. PPI network and hub-gene prioritization. (A) STRING-derived protein–protein interaction (PPI) network of DEGs; red and blue arrows denote up- and down-regulated genes, respectively. Representative functional clusters are annotated: embryonic pattern specification, DNA replication, and pheromone binding/dibutyl phthalate binding. (B) Interaction network of the top 10 hub genes. (C) Heat map of specific transcripts associated with embryonic pattern specification. Colors indicate relative expression levels.
Figure 6. PPI network and hub-gene prioritization. (A) STRING-derived protein–protein interaction (PPI) network of DEGs; red and blue arrows denote up- and down-regulated genes, respectively. Representative functional clusters are annotated: embryonic pattern specification, DNA replication, and pheromone binding/dibutyl phthalate binding. (B) Interaction network of the top 10 hub genes. (C) Heat map of specific transcripts associated with embryonic pattern specification. Colors indicate relative expression levels.
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Figure 7. TF–hub gene interaction network and miRNA–hub gene interaction network. (A) TF–hub gene network; (B) miRNA–hub gene network.
Figure 7. TF–hub gene interaction network and miRNA–hub gene interaction network. (A) TF–hub gene network; (B) miRNA–hub gene network.
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Figure 8. Relative expression levels of hub genes (log2 fold change) under control, irradiation (air + 166 Gy), and hypoxia + irradiation (1% O2 + 166 Gy) conditions. Bars show mean ± SD (n = 3). Different lowercase letters indicate significant differences among conditions for each gene based on one-way ANOVA with Tukey’s HSD at p < 0.05 (tests performed on ΔCt values).
Figure 8. Relative expression levels of hub genes (log2 fold change) under control, irradiation (air + 166 Gy), and hypoxia + irradiation (1% O2 + 166 Gy) conditions. Bars show mean ± SD (n = 3). Different lowercase letters indicate significant differences among conditions for each gene based on one-way ANOVA with Tukey’s HSD at p < 0.05 (tests performed on ΔCt values).
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Table 1. Results of the irradiation tests of late females of P. jackbeardsleyi reared on potato at the phytosanitary treatment dose of 166 Gy for the prevention of F1 generation progeny.
Table 1. Results of the irradiation tests of late females of P. jackbeardsleyi reared on potato at the phytosanitary treatment dose of 166 Gy for the prevention of F1 generation progeny.
TreatmentAbsorbed Dose (Gy)DURNo. PotatoesNo. Late FemalesDeveloped for F1 Generation
No. of NeonatesNo. of 2nd Instars
Air + 166 Gy166–1891.1450396737100
Control0−540038923764
1% O2 + 166 Gy164.8–185.41.1350351436200
Control0−538039443721
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Li, L.; Shan, C.; Xu, Q.; Li, B.; Liu, H.; Liu, T. Unveiling the Regulatory Mechanisms of Irradiation Response in Pseudococcus jackbeardsleyi Under Hypoxic Conditions. Agriculture 2025, 15, 2104. https://doi.org/10.3390/agriculture15202104

AMA Style

Li L, Shan C, Xu Q, Li B, Liu H, Liu T. Unveiling the Regulatory Mechanisms of Irradiation Response in Pseudococcus jackbeardsleyi Under Hypoxic Conditions. Agriculture. 2025; 15(20):2104. https://doi.org/10.3390/agriculture15202104

Chicago/Turabian Style

Li, Li, Changyao Shan, Qiang Xu, Baishu Li, Haijun Liu, and Tao Liu. 2025. "Unveiling the Regulatory Mechanisms of Irradiation Response in Pseudococcus jackbeardsleyi Under Hypoxic Conditions" Agriculture 15, no. 20: 2104. https://doi.org/10.3390/agriculture15202104

APA Style

Li, L., Shan, C., Xu, Q., Li, B., Liu, H., & Liu, T. (2025). Unveiling the Regulatory Mechanisms of Irradiation Response in Pseudococcus jackbeardsleyi Under Hypoxic Conditions. Agriculture, 15(20), 2104. https://doi.org/10.3390/agriculture15202104

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