Species of phytoseiid mites are important biological control agents against several mites and small insect pests of fruits, vegetables, and other crop species worldwide in the agriculture industry (Opit et al., 2004; Arthurs et al., 2009; Juan-Blasco et al., 2012; McMurtry et al., 2013). However, their growth and behavior are easily interfered by the ambient temperature (Hart et al., 2002; Palyvos and Emmanouel, 2009; Ghazy et al., 2012). In the context of global warming, the relationship between development rate and temperature has been used to forecast the geographical distribution of mite populations (Perring et al., 1984; Hoffmann and Sgrò, 2011). In the optimum temperature range, phytoseiid mites develop quickly, and have a high survival rate and lay more eggs (Emmert et al., 2008; Gadino and Walton, 2012). In contrary, the sudden rise of the ambient temperature will cause heat stress and exert some negative influence on the population dynamics, and lead to not only the rapid death of insects and mites, but also a decrease, even the loss, of reproduction ability (Michael and Whiting, 1996; Hoffmann et al., 2003; Cui et al., 2008).

Neoseiulus barkeri Hughes (Acari: Phytoseiidae), as the vital commercially produced phytoseiid mite in China, has played a key role in the control of many pest mites in recent years (Li et al., 2021). One of the deleterious consequences when N. barkeri are exposed to heat stress is the production of reactive oxygen species (ROS), i.e., hydroxyl radicals (HO^•−) and superoxide anion radicals (O₂ ^•−), as well as non-radicals, such as hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) (Yang et al., 2010; Ray et al., 2012). In normal situations, a balance exists between the generation of ROS and the antioxidant processes. However, increased levels of ROS generated inside living organisms disrupt the oxidative balance and lead to the oxidative damage. (Gechev et al., 2006; Lopez-Martinez et al., 2008). Surplus ROS, which are highly reactive and deleterious, can result in lipid peroxidation disrupting cell membrane fluidity, nucleic acids, proteins, lipids, and finally leading to apoptosis (Green and Reed, 1998; Lu et al., 2017).

To prevent damage from the ROS, mites have developed antioxidant defense mechanisms and these systems have both enzymatic and non-enzymatic components (Felton and Summers, 1995; Sorensen et al., 2001). Major components of the antioxidant enzymes of insects include superoxide dismutase (SOD) (Yucel and Kayis, 2019), catalase (CAT) (Lu et al., 2014), peroxidase (POD) (Gill and Tuteja, 2010), glutathione-S-transferases (GST) (Dubovskiy et al., 2008), polyphenol oxidase (PPO) (Tamás et al., 2017) as well as ascorbate peroxidase (APX) (Emre et al., 2013). It was reported that SOD, CAT, and POD, which are used as primary antioxidant enzymes to eliminate the ROS so as to maintain free radicals at a low level to prevent the toxic effects to living organisms (Wang et al., 2001). SOD scavenges free radicals to form H₂O₂ and removes the superoxide anion radical (O₂ ^•−) to prevent cells from being damaged. Whereas POD and CAT decompose H₂O₂, resulting in a low level of free radicals which are unable to exert a toxic effect and protecting the intact structure and function of cell membranes (Wang and Li, 2002). The change in the antioxidant enzyme activity is a significant bio indicator because of the role antioxidant enzymes play in maintaining the oxidative balance (Coskun et al., 2020).

The changes in antioxidant enzyme activity are closely associated with external stimulation and xenobiotics (e.g., pesticides, heavy metal, and high temperature) (Li et al., 2021). However, evolutionary adaptation can be rapid and may help species to counter stressful conditions or to realize ecological opportunities arising from the climate change (Bale, 2002). The evolution of insect and mite adaptability to extreme temperatures enables these organisms to withstand the effects of this environmental stress, thus supporting their survival and reproduction (Hoffmann et al., 2003).

We have found that the short-term heat stress does exert a negative effect on the longevity, reproduction characteristics, and functional response of N. barkeri. (Li et al., 2021; Li et al., 2022). In this study, the physiological mechanism of N. barkeri in the response to the short-term heat stress based on relevant antioxidant enzymes was conducted, among which the eggs and the emerged adults were treated at 38, 40, and 42°C for 2, 4, and 6 h, then the changes in the enzyme activity were tested, which aims to clarify the effects of different short-term heat stresses and exposure durations on three protective enzymes in the body of N. barkeri. The results would provide better understanding of revealing the physiological response mechanism of N. barkeri to the short-term heat stress and guidance for releasing as biological agents, and improvement in the practice of integrated control of spider mites as well.

The two-way ANOVA analysis indicated that the protein content was significantly affected by the temperature (F _{(2, 81)} = 218.303, p < 0.001) and the treatment duration (F _{(2, 81)} = 12.320, p = 0.032). The interaction between the temperature and duration was also significant (F _{(4, 81)} = 6.298, p < 0.001).

The protein content of N. barkeri females after high temperatures generally presented the variation tendency of increasing with rise of temperature are shown in Table 1. At 38°C, the protein content first increased, then decreased and then increased again, which made the value reached 0.469 mg/g from 0.165 mg/g. At 40°C, it continued to rise and reached the maximum level of 0.553 mg/g at 40°C-4 h (F _{(1, 18)} = 216.269, p < 0.001), which is more than three times higher than the control of 0.165 mg/g. Then, it descended to 0.354 mg/g but still had a significant difference with that of the control group (F _{(1, 18)} = 151.124, p = 0.002).

Temperature is one of the most important environmental variables that induce physiological change in organisms (Jin et al., 2010; Jia et al., 2011). Arthropods have evolved a series of diverse behavioral and physiological strategies to avoid temperature impairments, such as seeking shelter, changing the fluidity of cell membranes, and accumulating sugars, polyols, anti-freeze proteins and amino acids (Storey, 1997; Wang and Kang, 2005; Zhang et al., 2014). Neoseiulus barkeri, is one of the poikilothermic organisms, whose body temperature is highly affected by the ambient temperature. Moreover, Neoseiulus barkeri has been playing a key role in controlling pest mites in natural filed. However, few studies of thermal stress on the N. barkeri were found. Our previous work and another study all showed that the thermal stress negatively affected the longevity, reproduction characteristics (Li et al., 2021), copulation (Zhang et al., 2016), and functional response (Li et al., 2022). Physiological mechanisms like the change of antioxidant enzymes of Neoseiulus barkeri, when faced thermal stress, were still not clear.

In the present study, the exposure of N. barkeri to high temperatures had significant effects on the activities of SOD, CAT, and POD. The effects were enhanced with the increasing temperature and longer exposure duration. At 38°C, the activity of three enzymes showed a sudden rise after 2 h-exposure, then the CAT continued decreasing while the SOD and POD decreased after 4 h-exposure but slightly increased after 6 h-exposure. The difference in activity between the CAT and other two enzymes could be explained that under normal physiological states, an interacting network of antioxidant enzymes effectively neutralizes the harmful effects of ROS (Davies, 1995; Kayis et al., 2015). This detoxification pathway is the result of multiple enzymes with SOD catalyzing the first step; and then, CAT, POD, and various others removing hydrogen peroxide (Sies, 1997). Specifically, SOD removes O₂-through the process of dismutation to O₂ and H₂O₂; and then, H₂O₂ is sequentially reduced to H₂O and O₂ by CAT and POD (Hoffmann et al., 1997; Kashiwagi et al., 1997).

However, the activity of the three enzymes presented the exact same variation tendency at 40°C. It consistently increased in the first 4 h and reached the maximum value, but it dropped after 6 h-exposure. It means that N. barkeri generated more substances like ROS; thus, enzymes with a higher level activity were needed to protect the body. Comparing with 38°C, 40°C enhanced the negative effect of thermal stress on N. barkeri. A similar pattern was reported by Li Z. M et al., 2010 for the activity of CAT in the adult Tetrastichus brontispae, a parasitoid of Brontispa longissimi. Significant change in POD and CAT activities were also found in the adult Mononychellus mcgregori after exposure to heat stress for 36–42°C (Lu et al., 2014). Thus, those activity changes of antioxidant enzymes might be the reason that N. barkeri could still survive at high temperature even their population characteristics were heavily affected (Coombs and Bale, 2013; Li et al., 2021).

It is known that SOD plays an important role as an antioxidant enzyme by reducing the high level of superoxide radical induced by high surrounding temperature. In this study, the SOD activity significantly increased in early exposure but decreased in the following stressing period, suggesting that SOD was induced by changes in surrounding temperatures and then protect the mites from thermal stress. In addition, N. barkeri might become acclimated to the thermal stress. This result is in consistent with the findings of An and Choi (2010), where early-stage of exposure to acute temperature changes resulted in the oxidative stress regulated by antioxidant enzymes, but continued stress caused by acute temperature exposure resulted in the decreased SOD activity (Yang et al., 2010). It is probably because the well-adapted characteristic that mites owned to their surrounding environment (Hangartner and Hoffmann, 2016; Xu et al., 2001).

In this study, the increased CAT and POD activity was observed together. It is believed that this increase has occurred to eliminate H₂O₂, which is the final product formed during the elimination of superoxide radicals via the increased SOD activity. Previous findings showed that CAT removed H₂O₂ only at high cellular concentrations, whereas it is inefficient for H₂O₂ removal at low concentrations (Ahmad et al., 1991). It is probably because the synergistic work of SOD and POD removed most of the ROS and H₂O₂, which made CAT remain at a low level. It has been reported that the CAT activity was too low to detect in the herbivorous citrus red mite P. citri when exposed to thermal stress (Yang et al., 2010). In the current study, a significant increase of CAT activities was observed in the adults of the carnivorous mite N. barkeri exposed to different thermal stress conditions. Apparently, it enhanced the removal of H₂O₂ and prevented damage by the oxidative stress. The difference in the CAT activity between the predatory mite N. barkeri and herbivorous mite P. citri suggests that the two species differ in the antioxidant defense mechanism though they both belong to the Acari (Zhang et al., 2014).

In this study, correlation analysis showed that the temperature was the primary factor affecting the enzymatic activity, while the exposure duration was the secondary factor. It is probably because the poikilotherms such as N. barkeri are generally well adapted to high temperatures, the longer they are exposed to a high temperature, the more adapted they would be (Hangartner and Hoffmann, 2016). The study by Feng et al. (2008) indicated that non-lethal heat pre-stimulation enhanced the ability of Tetranychus cinnabarinus to resist extremely high temperatures. Other similar studies have presented the same enzymatic change, but none of them pointed out which was more important between the temperature and exposure duration. These physiological changes could to some extent help the organism to resist the adverse environment, however in the meantime, they were also the processes of energy consumption at the expense of potential fertility or survival level, which might be one of the physiological explanations to their shortened longevity in our previous findings (Silbermann and Tatar, 2000; Roux et al., 2010; Zhang et al., 2013).

The present study mainly focused on the three types of primary antioxidant enzymes. Studies have shown that other substances scavenging H₂O₂ also exist when mites encounter heat stress, such as ascorbate peroxidase (APX) (Li et al., 2007), which has a strong affinity and binding force with H₂O₂, and may also be involved in the process of protecting the body. Other non-enzymes may also be used to protect the body from damage, such as ɑ -tocopherol, glutathione, trehalose, polyol, and ascorbic acid. (Ghiselli et al., 2000; Cumming et al., 2014). Antioxidant enzymes also work together with heat shock protein HSPs to reduce the ROS damage to mite bodies (Lopez-Martinez et al., 2008). These related works are expected to be conduct in the following study.