Changes in Transcriptome and Functional Evaluation of Heat Shock Protein 70 in Predatory Mite Neoseiulus californicus (Hughes) in Response to Extreme High Temperature

TL;DR

This study explores how the predatory mite Neoseiulus californicus adapts to extreme heat by analyzing its transcriptome and the role of a specific heat shock protein, HSP70.

Contribution

The study identifies NcHSP70 as a key gene in heat tolerance and provides insights into molecular mechanisms of heat adaptation in phytoseiid mites.

Findings

NcHSP70 gene showed the highest expression under high temperature exposure.

RNAi suppression of NcHSP70 significantly reduced mite survival under heat stress.

ATPase activity of NcHSP70 increased after heat treatment, indicating functional importance.

Abstract

Neoseiulus californicus, a key biological control agent against phytophagous mites and small insects, is adapted to exposure to relatively high temperatures (HTs) (35–45 °C). Herein, transcriptome sequencing analysis was conducted on N. californicus under 25 °C and 45 °C exposures. Gene functional analysis illustrated that the DEGs were primarily participating in “catalytic activity”, “immune system process”, and the “Calcium signaling pathway”. Furthermore, we identified a heat shock protein 70 gene (NcHSP70) that displayed the highest expression under HT exposure. Functional analysis of RNAi and ATPase activity revealed the important roles of NcHSP70 involved in the heat tolerance of N. californicus. Our outcomes contribute to a deeper comprehension of the HT adaptation of phytoseiid mites and offer a reference for developing sustainable biocontrol strategies for small sap-feeding pests under a global warming environment.

Phytoseiid mites, as effective natural enemies, often experience various environmental stresses, especially extreme HTs under global warming and climate change. However, Neoseiulus californicus from the phytoseiid mite family could endure relatively HT (35–45 °C) exposure. To gain insights into its molecular mechanisms underlying heat adaptation, we conducted a comparative analysis of the transcriptomes exposed at 25 and 45 °C. There were 3117 and 7368 differentially expressed genes (DEGs) identified under the 0.5 and 4 h heat treatments, respectively. The functional enrichment analysis illustrated that DEGs were linked to “catalytic activity”, “metabolic process”, and the “Calcium signaling pathway”. Further DEG annotation and analysis illustrated that the expression of proteins encoding heat shock proteins (HSPs) and protein turnover were significantly induced. We also identified the unigene DN1689_c0 encoding the HSP70 gene (NcHSP70), which exhibited the strongest transcriptional response to heat stress. NcHSP70 inhibition by RNAi suppression had a significant impact on the survival of N. californicus. The ATPase effect of the purified recombinant NcHSP70 protein after HT treatment was significantly elevated. These findings increase our comprehension of the complex molecular mechanisms underlying HT adaptation and determine the important role of NcHSP70 in the heat resistance of N. californicus.

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1. Introduction

Phytoseiid mites, efficient natural enemies, are employed as biological control agents for phytophagous mites and several small insect pests (e.g., spider mites, thrips, and scale insects) [1,2,3]. Environmental alteration or fluctuation provokes several stresses in phytoseiid mites, which negatively impact most aspects of organismal biology and ecology [4]. The populations of phytoseiid mites are often affected by environmental stressors, including extreme temperature [5,6], drought stress [7], ultraviolet radiation [8] and pesticides [9], all of which limit the biocontrol ability of phytoseiid mites. Due to global warming and climate change, heat stress perhaps exerts a predominant abiotic factor regulating the behavior and physiology of phytoseiid mites. Climate sensitivity differs across trophic levels, with a tendency to intensify at elevated trophic levels [10]. Elevated temperatures generally favor the development of phytophagous mites over phytoseiid mites, potentially disrupting biological control [11]. Under extreme high temperature (HT) (>40 °C), the phytoseiid mites Phytoseiulus persimilis and P. macropilis stop movement and enter a heat coma [12]. However, the tolerance to HT varies among different species of phytoseiid mites. Neoseiulus californicus, as a predominant biological regulator from the phytoseiid mite family, could endure relatively extreme constant and fluctuating temperatures (35–45 °C). A 45 °C exposure for 4 h had a slight influence on the adult survival rate, indicating the resistance of N. californicus against HT stress [13]. Therefore, insights into the heat tolerance mechanisms enable more effective integration of this predatory mite into pest management under changing environmental conditions.

Temperature stress is a major driver that underpins critical adaptations in insect development and behavior. The ability of insects to cope with fluctuating and unfavorable temperatures has been a predominant research issue in ecophysiology and evolutionary biology recently. Previous studies have revealed complex biological pathways involved in HT adaptation, encompassing signal transduction, energy metabolism, and protein repair pathways [14]. Ref. [15] illustrated that the calcium signaling pathway has a vital function in heat sensing and the subsequent activation of downstream heat adaptation mechanisms. HT tolerance activates regulatory genes that maintain cellular proteostasis, including those involved in protein transport, degradation, and biosynthesis. High-throughput sequencing has been widely utilized for gene expression quantification and identifying differentially expressed genes (DEGs) associated with heat adaptation. Heat shock proteins (HSPs), functioning as molecular chaperones, are central to refolding denatured or misfolded proteins and preventing aggregation under heat stress. Depending on molecular mass and sequence features, HSPs are separated into four major families: small HSPs, HSP60, HSP70, and HSP90. Among these, the HSP70 family is the most studied and most abundant during stress, and its rapid induction enhances heat tolerance in insects [16,17,18]. However, in spite of the genetic mechanisms of thermotolerance being well-established among many insects, the physiological mechanisms underlying HT adaptation in phytoseiid mites remain poorly understood.

Herein, high-throughput sequencing technology was utilized to explore the mechanistic tolerance of N. californicus to extreme HT. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were utilized to comprehend the DEG biological actions. In addition, we identified the unigene DN1689_c0 encoding the HSP70 gene of N. californicus, which displayed the highest expression level among HSP families under extreme HT. RNAi-mediated suppression of NcHSP70 was performed to investigate its influence on the survival of N. californicus. Finally, we expressed and purified the NcHSP70 protein that was sensitive to heat stress. To determine the in vitro activity function, the NcHSP70 protein was transformed into Escherichia coli to obtain biologically active forms, and then the ATPase activity was assessed.

2. Materials and Methods

2.1. Mites and RNA Extraction

The predatory mite N. californicus was originally introduced from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and kept at 25 ± 1 °C, 70–80% RH, and a 14:10 h (L:D) photoperiod for several generations. The two-spotted spider mite, Tetranychus urticae, reared on cowpea plants, was provided as prey. For heat treatment, about 100 female N. californicus adults were transferred to a plastic transparent sheet (75 mm diameter) kept on a wet sponge (85 mm diameter and 10 mm thickness) in a Petri dish (90 mm diameter), and a piece of paper (20 × 10 mm) folded into a ridge was placed on the plastic transparent sheet to prevent mites from escaping. Then, the female adults were treated with a short-time heat stress (HS-S) of 45 °C for 0.5 h, or a long-time heat stress (HS-L) of 45 °C for 4 h. Three biological replicates were used for each treatment. Treated mites were directly liquid nitrogen-frozen and kept at −80 °C for isolating the RNA.

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to isolate total RNA as per the manufacturer’s protocol, and genomic DNA was eliminated with DNase I (TaKaRa, Dalian, China). The integrity and purity of RNA were estimated via a 2100 Bioanalyzer (Agilent Technologies, version B.02.10, Santa Clara, CA, USA).

2.2. Transcriptome Sequencing and Annotation

Sequencing libraries were conducted as per the manufacturer’s guidelines (Illumina, San Diego, CA, USA), and the Illumina NovaSeq 6000 platform by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) was utilized to sequence the outcomes. The deposition of raw reads was conducted in the NCBI Sequence Read Archive (SRA) (Bioproject no.: PRJNA844499, Accession no.: SRR19514510 to SRR19514518).

Raw reads in fastq format were trimmed and quality controlled. Then, Trinity was utilized to de novo assemble the high-quality clean reads from the samples, and the assembled transcripts were evaluated and adjusted with BUSCO, TransRate, and CD-HIT. The annotation of gene function was conducted by the following databases: the NCBI protein non-redundant (NR), Pfam, Clusters of Orthologous Groups of proteins (COG), GO, Swiss-Prot, and KEGG.

2.3. Differential Gene Expression Analysis

Gene-level quantification was conducted as transcripts per million (TPM) via RSEM. The DESeq2 software (version 1.38.3), which is based on the negative binomial distribution, is directly applied to analyze raw counts. Through specific normalization procedures and filtering criteria, differentially expressed genes between groups were identified. The default parameters were set as follows: adjusted p-value (padjust) < 0.05 and absolute log_2_ fold change (|Log_2_FC|) ≥ 1. Functional enrichment analyses (GO and KEGG) were conducted to detect DEGs in GO terms and metabolic pathways (p-adj ≤ 0.05) relative to the whole-transcriptome background. GO and KEGG were conducted by Goatools (version 1.3.2) and KOBAS (version 3.0).

2.4. qRT-PCR Validation

Eleven unigenes were chosen for qRT-PCR validation in a random manner. α-Tubulin and EF1A served as reference genes, and Table S1 lists the primers. qRT-PCR was conducted as previously mentioned [19], and relative levels were assessed via the 2^−ΔΔCt^ technique [20]. The relationship between qRT-PCR and transcriptome data was assessed via GraphPad Prism 9.0 (https://www.graphpad.com/).

2.5. Functional Analysis of NcHSP70

According to the results of the comparative transcriptome, the expression of DN1689_c0 encoding N. californicus HSP70 under HS-S illustrated the highest overexpression of over 300-fold compared to the control. To further explore the predicted HSP70 gene function, we cloned the full-length HSP70 cDNA, and bioinformatic analysis was conducted. To observe the transcript expression of the HSP70 gene under HT, over 300 female adults (4–5 days old) were subjected to temperatures of 45 °C for 0.5, 1, 2, 3 and 4 h, and the expression levels were detected by qPCR. For RNAi bioassay, NcHSP70 gene-specific double-stranded RNA was synthesized, and mortality affected by RNAi under extreme HT (45 °C) was analyzed. Further, the recombinant plasmid pET28a-NcHSP70 transformation into the E. coli BL21 cell was conducted, and the purified protein was assessed by SDS-PAGE and Western blot. Finally, ATPase activity of recombinant NcHSP70 was tested. The detailed materials and methods used in the functional analysis of NcHSP70 are described in the Supplemental Materials Text S1.

2.6. Statistical Analysis

Statistical analyses were performed using SPSS v.16.0. Differences in gene transcript expression and the mortality rate of adult females among groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. For all post hoc pairwise comparisons conducted after one-way ANOVA (including Tukey’s tests), the built-in alpha-level adjustment inherent to Tukey’s HSD (Honestly Significant Difference) procedure has been employed to control the family-wise error rate. The significance of RNAi efficiency variations and the ATPase activity of NcHSP70 were assessed using Student’s t-test. In all tests, a p value of less than 0.05 was considered statistically significant.

3. Results

3.1. mRNA Sequencing, Assembly, and Annotation

Transcriptome sequencing of nine samples (HS-S 1, HS-S 2, and HS-S 3 at 45 °C for 0.5 h; HS-L 1, HS-L 2, and HS-L 3 at 45 °C for 4 h; and C1, C2, and C3 at 25 °C) was conducted in N. californicus to assess the response to elevated temperature for short and long time periods (Table S2). Approximately 59.13 GB of clean reads were acquired, and a single sample had a GC content of 50.12–51.11%, and the Q30 values were over 93%. Functional annotation revealed that the unigenes were able to annotate 8718 (33.52%), 7716 (29.67%), 9564 (36.77%), 12,755 (49.04%), 8915 (34.28%) and 10,021 (38.53%) unigenes to the GO, eggNOG, NR, KEGG, Swiss-Prot, and Pfam databases, respectively; the annotations covered 13,050 (50.17%) of the total unigenes.

The sequences of N. californicus showed 10,675 (83.69%) matches with Galendromus occidentalis sequences, 453 (3.41%) matches with Varroa destructor sequences, 404 (3.17%) matches with Tropilaelaps mercedesae sequences, and 229 (1.80%) matches with Varroa jacobsoni sequences (Figure S1). Furthermore, after HS-S and HS-L treatments, 3117 and 7368 unigenes were differently expressed. Of these DEGs, 1226 were overexpressed and 1891 were downexpressed by HS-S, and 3620 were overexpressed and 3748 were downexpressed by HS-L (Figure 1). Moreover, 739 DEGs were found in HS-S and HS-L treatments.

3.2. Analysis of Differential Gene Expression Based on Functional Annotation

To elucidate the DEG function, GO and KEGG enrichment analyses were conducted. An annotation of 1333 (HS-S) and 3134 (HS-L) DEGs was conducted across three GO classes—cellular component, molecular function, and biological process—spanning 46 subgroups. Within the cellular component class, “membrane part”, “cell part” and “membrane” were most abundant. In the molecular function class, “binding” and “catalytic activity” predominated, followed by “molecular transducer activity”. In the biological process class, “biological regulation”, “cellular process” and “metabolic process” were most enriched (Figure 2). Table S3 lists the top 20 enriched GO terms for each subgroup.

In total, 1035 (HS-S) and 2502 (HS-L) DEGs were mapped to 238 and 298 KEGG pathways, respectively. “Neuroactive ligand–receptor interaction”, “Complement and coagulation cascades” and the “Calcium signaling pathway” were enriched under both HS-S and HS-L conditions (Figure 3A,B). For the HS-S, the “MAPK signaling pathway” and JAK-STAT were also the dominant pathways. “cAMP signaling pathway” and “Insulin secretion” were also enriched in the HS-L groups (Figure 3A,B). In addition, the DEGs of the calcium signaling pathway all showed downregulated expression under short- and long-term HT stresses (Figure 3C).

3.3. Expression of Unigenes for Heat Tolerance

3.3.1. HSPs

We detected a high transcriptional level of HSPs, categorized into the HSP90, HSP70, HSP60, and HSP20 families, which were either overexpressed or downexpressed in response to heat shock treatment. Among these 13 triggered HSPs, seven genes were in the HSP70 family, two genes were in the HSP90 family, three genes belonged to the HSP60 family, and one gene belonged to the HSP20 family. The HSP genes, except for one HSP70 (DN3247_c0) and one HSP20 (DN18429_c0) gene, were upregulated under HS-S and HS-L. One HSP70 gene (DN2115_c0) was downregulated under HS-L. Especially, HSP70 (DN1689_c0) had a significantly greater than 102- and 151-fold increase in expression under HS-S and HS-L, respectively (Figure 4). Based on the data from Tables S3 and S4, HSP70 (DN1689_c0) also exhibited a highly significant upregulation under both HS-S and HS-L. Under HS-L, DN1689_c0 showed a Log_2_FoldChange of 6.67, ranking it first among the top ten upregulated genes (Table S3). After prolonged heat exposure, its expression increased further, with a Log_2_FoldChange of 7.24, making it the second most upregulated gene under the HS-L condition (Table S4).

3.3.2. Protein Turnover

Apart from genes encoding HSPs, unigenes linked to protein turnover activities are also involved in resisting HT stress. Four proteasome-correlated unigenes (DN8166_c0, DN998_c0, DN7474_c0, and DN1973_c0) were induced both under HS-S and HS-L. Three proteasome-related unigenes (DN17897_c0, DN6936_c0, and DN4182_c0) were upregulated under HS-S, while they were downregulated under HS-L (Figure 5A). Seven ATPase proteins, both under HS-S and HS-L, which participated in protein translation and the control of protein production, were upregulated. However, six ATPase proteins under HS-S and HS-L were downregulated (Figure 5B).

3.4. Validation of DEGs by qRT-PCR

Herein, thousands of DEGs were observed in HS-S and HS-L-treated N. californicus. To validate their levels via qRT-PCR, 11 DEGs with distinct profiles of expression were chosen. The outcomes illustrated that the levels of genes (DN45_c0_g4, DN7453_c0, DN16327_c0, DN10891_c0, DN1689_c0, DN6205_c0, DN1670_c0, DN20861_c0, DN19857_c0, DN18724_c0, and DN11758_c0) linked to heat stress were significantly triggered. The qRT-PCR outcomes aligned with the outcomes of DEG expression profiling (Figure 6).

3.5. Functional Analysis of NcHSP70

3.5.1. Characterization and Phylogenetic Analysis

The sequence analysis illustrated that NcHSP70 (Gene Accession No. OQ995208) contained 1908 bp of ORF length, encoding 635 amino acids with a measured molecular mass of 70.31 kDa and a theoretical isoelectric point of 5.60. The classical HSP70 protein signatures (e.g., IGIDLGTTY, IFDLGGGTFDVSIL, and RIPRIQKLL) were found in the amino acid sequences of NcHSP70. Furthermore, the ATP/GTP-binding site TAEAYLGQP, the deduced bipartite nuclear localization signals (NLS), and the conserved motifs of HSP70, EEVD were all observed in the deduced protein (Figure S2A). The phylogenetic tree of HSP70s was classified into HSP70 in Insecta and HSP70 in Arachnoidea (Figure S2B). The NcHSP70 was grouped into the Arachnoidea group. In the Arachnoidea group, the NcHSP70 homolog was more conserved in Phytoseiidae mites (N. barkeri, G. occidentalis and N. cucumeris).

3.5.2. NcHSP70 Expression Profiles Responding to Heat Stress and Functional Verification by RNA Interference

The mRNA level of the HSP70 gene of N. californicus was significantly affected by 45 °C heat stress (F = 33.172; df = 5, 12; p < 0.001) (Figure 7A). NcHSP70 elevated rapidly once exposed to a short-time HT (0.5 h), and the expression was approximately 150-fold higher than the control. With increasing exposure time, the expression of NcHSP70 showed a gradual elevation from 0.5 to 4 h. Notably, the expression levels of NcHSP70 at 4 h displayed a striking overexpression of over 1100-fold compared to the control.

RNAi was employed to examine the function of NcHSP70 in the heat resistance of N. californicus. Double-stranded RNA oral delivery significantly suppressed NcHSP70 expression, achieving a 72% silencing efficiency in adult females after 48 h of dsRNA ingestion followed by 24 h of feeding on T. urticae (Figure 7B). To assess its function in heat defense, female mortality was measured after dsNcHSP70 treatment and subsequent exposure to 45 °C for 0.5, 1, 2, 3, and 4 h. The dsNcHSP70-treatment females showed higher mortality compared with the dsGFP control under HT exposure (Figure 7C). The mortality of the dsNcHSP70-treatment females increased 3.87-fold, 3.57-fold, 3.02-fold, 2.62-fold, and 3.21-fold compared with the dsGFP control for 0.5, 1, 2, 3, and 4 h, respectively.

3.5.3. Heterologous Expression of Recombinant NcHSP70 and ATPase Activity Assay of NcHSP70

SDS-PAGE analysis of the E. coli BL21 supernatant and purified protein revealed a 70 kDa target band (Figure 8A). Western blotting confirmed that the expressed protein corresponded to the predicted molecular weight of NcHSP70 (70.31 kDa), validating its identity (Figure 8B). The ATPase activity of NcHSP70 was then assessed at 25 and 45 °C. Activity was significantly higher at 45 °C than at the control temperature (Figure 8C).

4. Discussion

Climate change alters both the mean and variability of climatic factors, with temperature being a key abiotic factor regulating the behavior and physiology of poikilotherms [21]. In phytoseiid mites, body temperature closely tracks ambient temperature [22]. Extreme daytime temperatures under climate change can negatively impact phytoseiid mite populations [4,23,24]. N. californicus, as a potential biocontrol agent from the phytoseiid mite family, could endure relatively HT with extreme high constant and fluctuation (35–45 °C). The remarkable heat tolerance of N. californicus sparked significant focus on elucidating its underlying HT response mechanisms.

RNA-seq provides a high-throughput sequencing platform for the genome-wide quantification and comparison of gene expression under varying experimental conditions. Herein, transcriptome analysis revealed substantial differences in gene expression between HS-S and HS-L treatments in N. californicus. These results underscore that the physiological adaptation to heat stress is orchestrated through the differential expression of specific gene sets and the activation of distinct molecular pathways. Here, we detected 3117 (1226 upregulated and 1891 downregulated) and 7368 (3620 upregulated and 3748 downregulated) DEGs in response to HS-S and HS-L treatment, respectively. These DEGs could be related to the excellent thermotolerance actions in N. californicus. GO analysis illustrated that the majority of DEGs under both heat stress conditions were significantly represented in “cell part” and “membrane”, “binding” and “catalytic activity”, and “biological regulation” and “metabolic process”. Consistent with our findings, Tribolium castaneum exhibited comparable responses to heat stress, showing marked enrichment in the membrane, metabolic, and catalytic activities [25]. The enrichment of membrane-associated DEGs may suggest potential heat-induced cellular damage, as membrane integrity is particularly vulnerable to thermal stress and requires active repair mechanisms [26]. Several genes associated with “metabolic process” revealed that extreme temperature could activate some component of the metabolic system, including energy metabolism, immune response, detoxification, and stress signal transduction [27]. Moreover, many highly represented pathways, including “Neuroactive ligand-receptor interaction”, “Complement and coagulation cascades”, and the “Calcium signaling pathway”, were enriched in both HS-S and HS-L treatment, illustrating that these pathways may be crucial in the response to heat stress of N. californicus. The “Calcium signaling pathway” serves as a central hub for transducing various abiotic stresses, including both cold [28,29] and heat shock [15]. Interestingly, the DEGs of the calcium signaling pathway all showed downregulated expression under both HS-S and HS-L treatment. To prevent Ca^2+^ toxicity and conserve energy, a transcriptional negative feedback loop may be engaged. By dampening the expression of channels, pumps, and receptors involved in Ca^2+^ influx, the organism likely reduces pathway sensitivity and establishes a new homeostatic set-point suited to persistent high temperature, thereby conserving cellular energy and minimizing stress-induced apoptosis. Together, these transcriptomic insights reveal a multi-layered adaptive strategy in N. californicus: rapid membrane and metabolic reorganization is accompanied by a calibrated attenuation of specific signaling pathways, such as calcium signaling, to avoid over-activation and promote survival under sustained heat.

HSPs constitute a widespread and highly conserved class of proteins in insects, whose induced expression is a critical component of the physiological response that confers cellular stress tolerance. HSPs have a vital function in keeping proteostasis under thermal stress by acting as molecular chaperones and participating in several cellular activities, such as preventing misfolding, protein refolding and degradation, and stabilizing denatured proteins [30,31]. This investigation illustrated that 11 HSPs from the HSP90, HSP70, HSP60, and HSP20 families in N. californicus were significantly triggered in the heat stress. The expression profiles and count of genes associated with HSP70 were predominant among the upregulated HSPs, indicating that HSP70 is a prominent contributor to heat tolerance in N. californicus. Among these, the unigene DN1689_c0 (later identified as NcHSP70) exhibited extraordinarily high induction, with expression levels rising approximately 102-fold after short-term heat exposure (45 °C for 0.5 h) and 151-fold after prolonged exposure (4 h), relative to unstressed controls. This pattern underscores HSP70 as a central player in the thermal stress response of N. californicus, consistent with its well-documented role as a first-line chaperone in many eukaryotic systems. To further explore the function of this vital unigene, we identified and cloned the HSP70 gene named NcHSP70 from N. californicus. NcHSP70 increased rapidly within just 0.5 h of 45 °C exposure, reaching a level approximately 150-fold higher than the control. NcHSP70 expression exhibited a time-dependent increase from 0.5 to 4 h, culminating in a striking 1100-fold upregulation at the 4 h time point. Earlier investigations have illustrated that a positive relationship between higher HSP70 induction and stronger heat tolerance in arthropods [32,33,34], indicating the remarkable heat adaptation ability of N. californicus. NcHSP70 RNAi suppression in N. californicus significantly decreased survival rates under HT conditions by impairing the induction of heat tolerance, providing direct evidence that NcHSP70 is strongly implicated as critical for this adaptive response. While mortality trends were strongly correlated with significant NcHSP70 transcript knockdown at the group level, future studies employing microinjection could provide more precise individual dosing data. Furthermore, earlier investigations have illustrated that a conserved 45 kDa ATPase domain sac at the N-terminal of the HSP70 protein structure enables HSP70 to switch from ATP- to ADP-binding upon heat stress, resulting in higher affinity for substrates [35,36]. In Aphis aurantii, HT treatment significantly increased the ATPase effect of four purified recombinant AaHSP70 proteins [37]. Similarly, recombinant NcHSP70 in this study exhibited elevated ATPase activity at 45 °C. This biochemical evidence reinforces the notion that HSP70 accumulation is not merely a passive indicator of stress but an active, adaptive response that directly contributes to heat resistance. Collectively, our findings highlight HSP70—and particularly NcHSP70—as a cornerstone of the thermoprotective machinery in N. californicus.

HSPs support trehalose synthesis, the refolding of denatured proteins, and ubiquitin–proteasome-dependent degradation [38]. Mechanistically, the co-chaperone CHIP links HSP90, HSP70, and HSC70 to the ubiquitin–proteasome system, facilitating the clearance of damaged and dysfunctional proteins [39,40]. The ubiquitin–proteasome pathway comprises two steps: (1) the target protein is tagged with a multi-ubiquitin chain through a series of enzymatic reactions, and (2) the 26S proteasome catalyzes the degradation of the tagged protein and releases the ubiquitin moieties for reuse. The process of ubiquitin-conjugated proteins attaching to the 26S proteasome is dependent on ATP [41]. In this study, four proteasome-related unigenes and seven ATPase proteins in this pathway were significantly induced under 45 °C and showed a higher expression under HS-L, suggesting the increased activity of ubiquitin- and proteasome-dependent degradation may be conducive to coping with the acute heat stress.

5. Conclusions

To investigate gene expression in N. californicus under acute heat stress, transcriptome sequencing and differential gene analysis were conducted. GO and KEGG analyses illustrated that DEGs were associated with “catalytic activity”, “metabolic process” and the “Calcium signaling pathway”. Further annotation revealed the significant induction of genes encoding HSPs and proteins involved in protein turnover, suggesting their roles in heat tolerance. Functional assays confirmed the critical role of HSP70: RNAi-mediated knockdown of NcHSP70 increased heat susceptibility, and recombinant NcHSP70 exhibited elevated ATPase activity at HT. These outcomes advance comprehension of the molecular mechanisms of heat tolerance in N. californicus and highlight NcHSP70 as a key factor, providing insights for developing viable regulation approaches for small sap-feeding pests under heat stress.