Selection, Identification, and Transcript Expression Analysis of Antioxidant Enzyme Genes in Neoseiulus barkeri after Short-Term Heat Stress

Phytoseiid mite Neoseiulus barkeri is a crucial biological control agent utilized to control pest mites and many insects in crops all over the world. However, they are vulnerable to multiple environmental pressures, with high-temperature stress being the most significant challenge. Heat stress disrupts the balance of reactive oxygen species (ROS) levels in organisms, resulting in oxidative stress within the body. Antioxidant enzymes play a crucial role in effectively neutralizing and clearing ROS. In this study, comparative transcriptomics and quantitative real-time PCR (qRT-PCR) were employed to assess the impact of short-term heat stress on the transcript expression of antioxidant enzyme genes in N. barkeri. We primarily identified four antioxidant enzyme genes (NbSOD, NbPrx, NbCAT, and NbGPX) in N. barkeri after exposure to short-term heat stress. Then, new data on the expression patterns of these genes were generated. RNA sequencing and bioinformatics analysis revealed that NbSOD belongs to the Fe/Mn family of superoxide dismutase (SOD), which was identified as MnSOD. NbPrx was classified as a 1-Cys peroxiredoxin of the peroxidase family, whereas NbCAT was recognized as a classical catalase, and NbGPX was determined as cytoplasmic glutathione peroxidase-1 (GPX1). Transcriptional expression analysis of these four genes was conducted at different high temperatures: 36 °C, 38 °C, and 40 °C for 2, 4, and 6 h. The results also showed that all four genes exhibited significant up-regulation in response to short-term heat stress. Similarly, the highest expression levels for NbSOD, NbPrx, and NbCAT were observed at 40 °C for 4 h. However, NbGPX displayed its maximum expression value at 38 °C for 4 h. Overall, the obtained data suggest that short-term heat stress increases levels of ROS generated inside living organisms, which disrupts the oxidative balance and leads to alterations in the expression levels of antioxidant enzyme genes.


Introduction
Neoseiulus barkeri (Acari: Phytoseiidae) is a widely distributed and commercially accessible predator of pest mites and many arthropod pests [1].It has been reported in Asia, America, Australia, Africa, and Europe [2].As an ectothermic animal, N. barkeri is very susceptible to temperature stress, and at 16 • C, N. barkeri will undergo diapause, and only after mating can it lay eggs [3].With the increase in temperature, its development period and life span shorten [4,5], its development period cannot be completed at 40 • C [5], and the eggs cannot hatch at 42 • C [6].N. barkeri frequently encounters high-temperature stress in both natural environments and facility agriculture.In spite of numerous attempts that have been made in recent years to study the impact of temperature on the developmental rates of arthropods, there is little information related to mite pests at the molecular level [7].High temperatures can cause a dramatic increase in ROS levels, leading to harmful effects on the population of N. barkeri [6,8,9] and resulting in suppressed potential and low control efficiency on small insects and mite pests [10].
Reactive oxygen species (ROS) are reactive compounds derived from oxygen generated in the cellular metabolism of living cells.Normal levels of ROS are essential for cellular signaling networks and physiological functions such as autophagy, pathogen killing, and resolution of inflammation.In contrast, higher levels of ROS lead to irreversible damage of macromolecules involved in various metabolic processes, which eventually harm cellular components and functions [11][12][13].Several evidences showed that biotic and abiotic stresses may induce a high amount of reactive oxygen species (ROS) concentrations in cells [14].High temperature is one of the abiotic stresses that cause alterations in insect biology, behavior, morphology, and development, as well as cellular and metabolic processes, which can disturb the delicate equilibrium between ROS production and reduction [15][16][17][18][19].The primary way in which ROS is eliminated is through the body's antioxidant defense systems, which include antioxidant enzymes like superoxide dismutases (SOD) and catalase (CAT), as well as direct antioxidants such as vitamin E and glutathione (GSH).These antioxidants are essential for maintaining the balance between oxidation and reduction in the cell and thus play a critical role in reducing ROS levels [20].
The SOD enzyme family is the primary antioxidant defense in organisms.On the one hand, it catalyzes the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide and serves as the first line of enzymatic defense against ROS [21,22].On the other hand, peroxidase (Prx) and CAT decompose H 2 O 2 into nontoxic H 2 O and O 2 , causing a low level of free radicals inside the cells that cannot exert a toxic effect.This protects the intact structure and function of cell membranes [23].Most Glutathione peroxidase (GPX) are selenium-dependent enzymes that catalyze the reduction of H 2 O 2 and organic hydroperoxides to water and the corresponding alcohols, respectively.They typically use glutathione (GSH) as an electron donor [24].Based on our previous research, short-term heat stress resulted in increased expression of SOD, CAT, and Prx activities in N. barkeri, and the enzyme activity reached its maximum at 40 • C-4 h [25].A notable rise in antioxidant enzyme activities suggests the presence of oxidative stress and a positive capability to counteract it by eliminating ROS from cells [26,27].
Although the structure and composition of antioxidant enzyme genes are highly conserved, there is limited understanding of the expression mechanism of these genes in invertebrates, particularly mites.In this study, RNA-seq, qRT-PCR, and comprehensive transcriptome analysis were performed on adult females of the predator mite N. barkeri subjected to short-term high temperatures (25 • C and 40 • C for 4 h).Firstly, we identified four antioxidant genes (NbSOD, NbPrx, NbCAT, and NbGPX), and their differentially expressed genes in response to heat stress were obtained.Second, we conducted bioinformatics analysis on these four genes.Finally, we analyzed the expression patterns of these four antioxidant enzymes after exposure to different temperatures of 36  C for 2, 4, and 6 h.These results will improve our understanding of the molecular response mechanism of N. barkeri to heat stresses and guide their impacts on the efficacy of the biological control of natural enemies against N. barkeri.

Insect Culture
A colony of the predatory mite N. barkeri used in this study was maintained in the laboratory for multiple generations (without pesticide exposure) in the Department of Insect Systematics and Biodiversity at the College of Plant Protection, Gansu Agricultural University, China.The susceptible population of N. barkeri was kept in plastic boxes measuring 17 cm × 12 cm × 10 cm and fed on a bran/mite mixture (a mixture of Aleuroglyphus ovatus Troupeau and bran [6].The colony was continuously reared in an artificial-control chamber, with a 14/10 h light/dark cycle at temperature (25 • C ± 1 • C) and humidity (80 ± 5%).

RNA-Seq Library Construction and Sequencing
Before RNA-Seq library construction and sequencing, the RNA was first extracted.Six hundred newly emerged adult females (1-2 days old) were collected into a 1.5 mL microtube and placed in the artificial-control chamber under two conditions: 25 • C and 40 • C for 4 h.Samples from 25 • C were identified as the control group, while samples from 40 • C-4 h were identified as the treatment group.After treatments, samples were frozen immediately using liquid nitrogen and stored at −80 • C in a refrigerator for subsequent use.RNA was extracted from each sample using Trizol reagent (Thermo Fisher Scientific, New York, NY, USA), and the quality and quantity of RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, California, CA, USA).Only RNA samples with RIN values greater than 8.0 were used for transcriptome sequencing.Each treatment was repeated three times, and 3600 mites were used in the RNA extraction.
After the RNA extraction, 6 RNA-Seq libraries were constructed using 1 µg RNA from each sample, following the protocol described by Chao et al. (2019) [28].The RNA-Seq libraries were collected and sequenced using the DNBSEQ platform.The transcriptome data was uploaded to the NCBI Sequence Read Archive (SRA) with accession number PRJNA993641.

Transcript Assembly, Gene Annotation, and Gene Expression Level Calculation
Before conducting de novo assembly, raw reads from each library were filtered by removing low-quality reads, reads containing adapters, and reads containing >5% unknown bases.The resulting clean reads were assembled using Trinity 2.2.0 software to produce unigenes [29].Unigenes were blasted with non-redundant protein sequences in the nonredundant protein sequence database (NR), gene ontology (GO), Kyoto Encyclopedia of genes and genomes (KEGG), eukaryotic orthologous groups (KOG), Swiss Prot and Pfam databases to obtain annotated information using Blastp software (https://blast.ncbi.nlm.nih.gov/Blast.cgi)[30].Clean reads were aligned to the genome sequence using Bowtie2 (http://bowtie-bio.sourceforge.net/Bowtie2/index.shtml)accessed on 16 April 2023, and gene expression levels were estimated from RNA-Seq data for each sample using the RSEM package.

Selection of Antioxidant Genes
According to the gene expression levels in each sample, the total number of genes identified as differentially expressed (DEG) was detected.Based on gene annotation information obtained from DEG analysis, we screened out the antioxidant enzyme gene regulated by short-term heat stress in N. barkeri.

Cloning the CDS of Antioxidant Genes
Primers were designed based on the analysis of transcriptome information of N. barkeri (Table S1) to amplify CDS fragments of target genes NbSOD, NbPrx, NbCAT, and NbGPX.First-strand cDNA was synthesized using the PrimeScript RT reagent Kit (Takara, Dalian, China), and PCR products were ligated into the pLB-T vector (TIANGEN, China).RNA templates used in cDNA synthesizing were from the control group in Section 2.2.The resulting constructs were transformed into Top10 Escherichia coli cultures (TIANGEN, Beijing, China), and positive clones were selected for sequencing using Tsingke Biotech Co., Ltd.(Beijing, China).

Transcript Expression of Antioxidant Genes
In this study, we investigated the expression of antioxidant enzyme genes at the transcriptional level using RT-qPCR.The housekeeping gene β-actin [31] was used as a reference gene for normalization, and the primers used in the study are listed in Table S2.Six hundred newly emerged adult females (1-2 days old) were collected with a little brush into a 1.5 mL microtube and placed in the artificial-control chamber.Samples were treated at different temperatures (36,38, and 40 • C) for varying durations (2, 4, and 6 h) under controlled humidity (RH 80 ± 5%) and then frozen immediately with liquid nitrogen and stored at −80 • C in a refrigerator after that.Adult females reared at 25 • C were used as negative controls.RNA was extracted from each sample using Trizol reagent (Thermo Fisher Scientific, New York, NY, USA), and the quantity and quality of RNA samples were assessed using a Thermo Scientific NanoDrop TM 2000 UV-VIS Spectrophotometer (Thermo Fisher Scientific, New York, NY, USA).A total of 600 mites were used in RNA extraction in each treatment, and each treatment was replicated three times.Approximately 1 µg RNA from each sample was converted into cDNA using the PrimerScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China).

Statistical Analysis
Quantitative real-time PCR (qRT-PCR) analyses were performed to determine Gene expression using the relative quantification 2 −∆∆CT method [32].

Transcriptional Expression of Antioxidant Enzyme Genes under Different Heat Stress Conditions
The expression patterns of these genes in N. barkeri were found to have differed after short-term high-temperature stress.Interestingly, at 2 h stress time, the highest expression levels of NbSOD, NbPrx, NbCAT, and NbGPX genes were observed at 40 °C followed by 38 °C.On the other hand, at 36 °C, the transcriptional expressions of NbSOD, NbPrx, and NbGPX were not significantly different from those of the control, while NbCAT was significantly lower.At 4 h stress time, the highest expression levels of NbSOD, NbPrx, and NbCAT were observed at 40 °C followed by 38 °C, while the expression of NbGPX was highest at 38 °C and decreased at 40 °C.At 36 °C, the transcriptional expression of NbPrx, NbCAT, and NbGPX was not significantly different from that of the control group, while the expression of NBSOD was significantly higher.At 6 h stress time, the transcription

Transcriptional Expression of Antioxidant Enzyme Genes under Different Heat Stress Conditions
The expression patterns of these genes in N. barkeri were found to have differed after short-term high-temperature stress.Interestingly, at 2 h stress time, the highest expression levels of NbSOD, NbPrx, NbCAT, and NbGPX genes were observed at 40 • C followed by 38 • C. On the other hand, at 36 • C, the transcriptional expressions of NbSOD, NbPrx, and NbGPX were not significantly different from those of the control, while NbCAT was significantly lower.At 4 h stress time, the highest expression levels of NbSOD, NbPrx, and NbCAT were observed at 40 • C followed by 38 • C, while the expression of NbGPX was highest at 38 • C and decreased at 40 • C. At 36 • C, the transcriptional expression of NbPrx, NbCAT, and NbGPX was not significantly different from that of the control group, while the expression of NBSOD was significantly higher.At 6 h stress time, the transcription levels of NbPrx and NbCAT were highest at 36 • C, followed by 38 • C, while the transcription levels of NbSOD and NbGPX were highest at 38 • C, followed by 40 • C (Figure 5).The original data of the results are in Tables S6-S9.levels of NbPrx and NbCAT were highest at 36 °C, followed by 38 °C, while the transcription levels of NbSOD and NbGPX were highest at 38 °C, followed by 40 °C (Figure 5).The original data of the results are in Tables S6-S9.

Discussion
Temperature is the primary abiotic factor that significantly impacts the survival of organisms, particularly those that are poikilothermic and ectothermic [33].N. barkeri frequently encounters high-temperature stress in both natural environments and facility agriculture.Heat stress is a significant factor that disrupts the balance of ROS, resulting in oxidative damage [27].Antioxidant enzymes play a crucial role in efficiently eliminating ROS and mitigating their harmful effects on organisms [26,27].
The exposure of N. barkeri to short-term high temperatures significantly affected the activities of antioxidant enzymes.These effects were further enhanced with increasing temperature and longer exposure duration [27].The transcriptome data revealed differential expression of four antioxidant enzyme genes (NbSOD, NbPrx, NbCAT, and NbGPX) following short-term heat stress, with all four genes showing up-regulation in their expression levels.The up-regulated expression of these four genes indicates that N. barkeri experiences oxidative stress when exposed to short-term high-temperature stress.This up-regulation indirectly enhances the activities of SOD, Prx, CAT, and GPX, thereby providing protection against the harmful effects of ROS on organisms.
Based on the analysis of multiple sequence alignment, NbSOD is categorized as a member of the Fe-Mn SOD family based on the presence of two domains specific to the Fe-Mn SOD family: Mn/Fe-N and Mn/Fe-C (Figure 3A).

Discussion
Temperature is the primary abiotic factor that significantly impacts the survival of organisms, particularly those that are poikilothermic and ectothermic [33].N. barkeri frequently encounters high-temperature stress in both natural environments and facility agriculture.Heat stress is a significant factor that disrupts the balance of ROS, resulting in oxidative damage [27].Antioxidant enzymes play a crucial role in efficiently eliminating ROS and mitigating their harmful effects on organisms [26,27].
The exposure of N. barkeri to short-term high temperatures significantly affected the activities of antioxidant enzymes.These effects were further enhanced with increasing temperature and longer exposure duration [27].The transcriptome data revealed differential expression of four antioxidant enzyme genes (NbSOD, NbPrx, NbCAT, and NbGPX) following short-term heat stress, with all four genes showing up-regulation in their expression levels.The up-regulated expression of these four genes indicates that N. barkeri experiences oxidative stress when exposed to short-term high-temperature stress.This up-regulation indirectly enhances the activities of SOD, Prx, CAT, and GPX, thereby providing protection against the harmful effects of ROS on organisms.
Based on the analysis of multiple sequence alignment, NbSOD is categorized as a member of the Fe-Mn SOD family based on the presence of two domains specific to the Fe-Mn SOD family: Mn/Fe-N and Mn/Fe-C (Figure 3A).Due to the presence of iron sod (FeSOD) in prokaryotes and plants [34,35] and manganese sod (MnSOD) in eukaryotes [36], NbSOD is classified as MnSOD.NbPrx was classified as one of the 1-Cys peroxiredoxins due to the presence of a single conserved Cys residue at position 47 (Figure 3B) [37,38].Peroxiredoxins (Prxs) are a highly conserved family of peroxidases that efficiently reduce peroxides.They contain a conserved cysteine residue known as the "peroxidatic" Cys, which is the site of oxidation by peroxides [39][40][41].NbCAT has been identified as a typical catalase (Figure 3C) and is the most widely present and widely studied monofunctional, heme-containing enzyme in nature [42,43], which also binds NADPH as a second redox-active cofactor.They form tetramers, and in eukaryotic cells, catalases are located in peroxisomes [44,45].Through protein subcellular localization prediction, NbGPX was identified to be situated in the cytoplasm (Tab1), utilizing GSH as its active center (Figure 3D).Consequently, NbGPX is classified as GPX1 within the GPX family [46].GPX1, a tetrameric enzyme, is capable of reacting with H 2 O 2 and soluble low-molecular hydroperoxides, but does not react with more intricate lipid hydroperoxides [47].
The organism's response to oxidative stress begins with an increase in the activity of SOD [48].There are three types of SODs in organisms: copper/zinc SODs (Cu/ZnSOD), manganese SODs (MnSOD), and SODs (FeSOD) [49][50][51].In this study, NbSOD was identified as MnSOD (Figure 3A).MnSOD is predominantly localized within the mitochondria of eukaryotic cells [52,53], which aligns with the findings of protein subcellular localization prediction (Table 1).Mitochondria are responsible for producing energy in the cell and are also known to be a major source of ROS [54].High temperatures accelerate the energy consumption of N. barkeri [55], leading to an increased demand for energy production by the mitochondria.As a result, reactive oxygen species (ROS) are generated as by-products of the mitochondrial electron transport chain [21].Based on this, we hypothesize that the rise in ROS levels in N. barkeri following short-term heat stress is attributed to mitochondrial activity.SOD scavenges free radicals and converts them into H 2 O 2 , while Prx and CAT break down H 2 O 2 [27].Additionally, GPX commonly employs glutathione (GSH) as a reducing agent to catalyze the conversion of H 2 O 2 or organic hydrogen peroxide into H 2 O or corresponding alcohols, respectively [56][57][58].Based on protein subcellular localization prediction (Table 1), NbPrx, NbCAT, and NbGPX are all present in the cytoplasm.Following brief heat stress, N. barkeri generates a significant amount of ROS within the mitochondria, causing an imbalance in ROS levels within the organism.Subsequently, NbSOD converts ROS into H 2 O 2 within the mitochondria, and this H 2 O 2 is subsequently transported to the cytoplasm.In the cytoplasm, NbPrx, NbCAT, and NbGPX each perform distinct mechanisms to break down H 2 O 2 into H 2 O and other harmless substances.This process effectively reduces their harmful effects, safeguarding the integrity and functionality of the cell membrane against oxidative interference and damage.
The transcriptional expression of antioxidant enzyme genes in N. barkeri varies in response to different external stresses.After exposure to UV-B radiation stress, the expression of three SOD genes (Cu/ZnSOD1, Cu/ZnSOD2, and MnSOD) and two phospholipid hydroperoxide GPX genes (PHGPX1 and PHGPX2) in N. barkeri was up-regulated [59].A high-temperature adapted strain (HTAS), the predatory mite N. barkeri was artificially selected via long-term heat acclimation (35 • C) and frequent heat hardenings.Compared to susceptible strains, the HTAS showed increased expression of 2 SOD genes and decreased expression of 3 SOD genes.Additionally, it exhibited increased expression of 6 Prx genes and 1 CAT gene, as well as decreased expression of 3 CAT genes [55].After exposure to Fenpropathrin stress, two GPX genes exhibited up-regulation, whereas only one GPX gene showed up-regulation following pyridazine stress [26].In addition to the variation in the number of genes that regulate the activity of antioxidant enzymes, N. barkeri also exhibited differences in the types of antioxidant enzyme genes in response to external stress.Through a comparison of the four antioxidant genes in Blastp, the NbPrx, NbCAT, and NbGPX were not identified, while the NbSOD was previously reported by Tian et al. [59].
The expression analysis revealed significant differences in the four antioxidant genes in N. barkeri after short-term high-temperature exposure.Similarly, the highest expression levels of all four genes were observed at 40 • C for 4 h, except for NbGPX, which exhibited its maximum expression value at 38 • C for 4 h.These findings are consistent with similar results obtained in other organisms, such as green peach aphid Myzus persicae [60], Drosophila [61], and Mytilus galloprovincialis [62].It suggests that the up-regulation of antioxidant genes may occur within a specific temperature range, as extreme temperatures can disrupt cell redox homeostasis, carbohydrate and energy metabolism [63], and cause damage to cytoskeletal structural elements [64].Furthermore, the negative effects of ex-

Figure 1 .Figure 1 .
Figure 1.The number of differentially expressed genes and their expression levels in adult female individuals of N. barkeri under control (25 °C) vs. treatment (40 °C-4 h) conditions.(A).The number of down-regulated and up-regulated genes in differentially expressed genes.If Log2(Treat FPKM/Control FPKM) > 0, it was identified as an up-regulated gene; if Log2(Treat FPKM/Control FPKM) < 0, it was recognized as a down-regulated gene.(B).Volcano plot displaying the variability pattern, with red points indicating up-regulated genes, blue points indicating down-regulated genes; NbSOD, NbPrx, NbCAT, and NbGPX are shown in black, purple, yellow, and green, respectively.The final result is based on FPKM as the original data, and Log2FC and p-value were calculated using the DEseq2 method.

Figure 1 .Figure 2 .
Figure 1.The number of differentially expressed genes and their expression levels in adult female individuals of N. barkeri under control (25 °C) vs. treatment (40 °C-4 h) conditions.(A).The number of down-regulated and up-regulated genes in differentially expressed genes.If Log2(Treat FPKM/Control FPKM) > 0, it was identified as an up-regulated gene; if Log2(Treat FPKM/Control FPKM) < 0, it was recognized as a down-regulated gene.(B).Volcano plot displaying the variability pattern, with red points indicating up-regulated genes, blue points indicating down-regulated genes; NbSOD, NbPrx, NbCAT, and NbGPX are shown in black, purple, yellow, and green, respectively.The final result is based on FPKM as the original data, and Log2FC and p-value were calculated using the DEseq2 method.

Table 1 .
Detailed information of antioxidant enzyme genes from N. barkeri.