Myzus persicae (Sulzer) (Homoptera: Aphididae), also known as the green peach aphid, is a highly adaptable and polyphagous insect pest that feeds on more than 400 plant species from 40 plant families, including many economically important crops (Kennedy et al., 1962; Stevens and Lacomme, 2017). Aphids are used as a model insect in studies of the effects of climate change on insect biology because they develop over a small temperature range, and their growth rate is highly dependent on temperature (Hulle et al., 2010; Durak et al., 2016).

Temperature fluctuations affect aphid morphology and metabolism, which can lead to cell damage. The defense responses of aphids to high temperatures are of great interest. Aphids can suffer from physiological and ecological stress due to heat stress (Dong et al., 2013; Sentis et al., 2015). The upper temperature threshold for the growth of populations of many aphid species has been estimated to be between 25 and 30°C, while the lower temperature threshold has been estimated to be below 5°C (Barlow, 1962). Optimal temperatures for reproduction were 25°C for M. persicae and 20°C for Macrosiphum euphorbiae (Barlow, 1962). Acclimation temperature affected heat coma; this relationship was linear for Myzus ornatus and Myzus polaris but non-linear for M. persicae (increased tolerance at 10 and 25°C). Upper critical temperatures in M. persicae range from 38.5°C (Broadbent and Hollings, 1951) to 42°C (Hazell et al., 2010). Previous studies indicate that M. persicae has the greatest tolerance to high temperatures (Hazell et al., 2010). Dampc et al. (2020) also focused on three temperatures, i.e., 20, 25, and 28°C, and showed that the adaptive mechanisms were activated by the flexible activity of enzymes, which ran more efficiently at higher temperatures. The defense responses of Aphis pomi varied as a function of temperature at 28°C and survived due to flexible enzyme activity.

There is a close relationship between insect responses to heat and many heat-shock proteins (HSPs), which act as chaperones to aid protein production and refolding after stress and may also enhance immune responses (Wojda, 2017). Numerous studies on the interaction between insects and thermal stress have shown that insects have evolved complex protective mechanisms to protect themselves from high temperatures. HSPs and antioxidant enzymes have been the most prominent effectors in this process (Yang et al., 2010; King and MacRae, 2015). The short forms of heat shock, such as Hsp22, Hsp23, and Hsp27, and mRNAs are approximately 100 nucleotides longer than their respective hormone-induced short forms in Drosophila cells. Expression of the small HSP genes can be induced in cultured Drosophila cells by high-temperature shock and exposure to physiological doses of the insect molting hormone, ecdysterone (Morganelli et al., 1985). The Hsp110 genes function in an ATP-dependent manner as a nucleotide exchange factor that releases peptide substrate from Hsp70. They respond differently to heat stress in different organisms within the same genus, and their expression can be caused by heat stress in animals, according to a recent review (Jones et al., 2018). The enzymatic defense response of aphids is reflected in changes in the activity of enzymatic markers in their tissues (Dampc et al., 2020). As stress proteins, HSPs are involved in the protection of proteins under oxidative and hypertonic stress induced by extreme temperatures, UV radiation, xenobiotic exposure, and parasitoid infestation (Kang et al., 2017). Therefore, knowing the transcription level of HSPs in aphids under different heat stress situations is beneficial for future agricultural management.

In general, the production of reactive oxygen species (ROS) and antioxidant scavenging processes is in equilibrium. Heat stress, in contrast, disturbs this equilibrium and leads to increased production of ROS, resulting in lipid peroxidation (LPO) through the destruction of cell lipids (Dubovskiy et al., 2008; Lalouette et al., 2011; Schieber and Chandel, 2014). The major antioxidant enzymes in insects are superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) (Felton and Summers, 1995; Wang et al., 2001). A significant increase in antioxidant enzyme activities indicates oxidative stress and a sign of a good ability to counteract oxidative stress by removing ROS from cells (Jia et al., 2011; Zhang et al., 2015). Previous work has been done on the antioxidant enzyme activity of M. persicae with different host plants. There have also been changes in antioxidant enzyme function in M. persicae, a highly polyphagous genus, due to its tolerance to different host plants (Abdelsalam et al., 2016), but to the best of our knowledge, no studies have been conducted to calculate the combined effect of different temperatures and time intervals on antioxidant enzyme activity and gene expression of M. persicae. Therefore, this research aimed to observe how temperature changes affect the activities of antioxidant enzymes and HSPs genes in response to short-time heat stress in the green peach aphid. This type of research will lead to new insights into the molecular processes underlying heat resistance in M. persicae, as well as new opportunities to promote the use of heat treatments to control this pest.

This study reports the physiological and molecular response to short-time heat stress in M. persicae. It confirms that the antioxidant enzyme activity of SOD, POD, and CAT and their transcript levels were upregulated by increasing temperatures relative to those at 25°C. This is in agreement with many studies that high temperatures stimulate the defense mechanisms of insects, such as antioxidants and HSPs (Yang et al., 2010; Kang et al., 2017). In this study, our results showed that in all heat shock treatments (27, 30, 33, and 36°C) for four exposure times (1, 3, 6, and 10 h), the expression of SOD, POD, and CAT genes was increased compared with control.

Our research showed that the activity of antioxidant enzymes in M. persicae depends on temperature. We hypothesized that to retain physiological homeostasis, antioxidant enzymes scavenge the ROS generated by thermal stress. The SOD plays an important role in protection against ROS and reducing superoxide radicals produced by high temperatures (Park et al., 2009). An increase in SOD activity is the first stage of aphid response to oxidative stress. The SOD activity of M. persicae can be the adaptive reaction to reduce superoxide anion toxicity caused by high temperatures, as shown in Panonychus citri and Propylaea japonica (Yang et al., 2010; Zhang et al., 2015). CAT is the major H₂O₂ scavenging enzyme and works together with SOD to slowly reduce oxygen (Cui et al., 2011). In this study, the activity of CAT increased in M. persicae under different heat stress (27, 30, 33, and 36°C) compared with control (25°C). The increase in exposure times (1, 3, 6, and 10 h) equally increased CAT activities at 36°C. Previous studies by Dampc et al. (2020) discovered the effects on the activity of enzymatic markers in aphid tissues. A. pomi defense responses varied depending on the temperature at 28°C and survived due to flexible enzyme activity. Similarly, in this study, both the SOD and CAT enzymes in M. persicae tissues increased as the temperature and the stress duration increased, which contributes to their survivability. Studies on Mythimna separata revealed that increasing temperatures beyond the optimal conditions (25°C), increased SOD and CAT activities (Ali et al., 2017). POD oxidizes phenols and other aromatic compounds with the participation of H₂O₂. This group of enzymes plays an important role in the oxidation of secondary metabolites, which greatly facilitates feeding by insects (Urbanska et al., 1998).

In this study, the MDA content increased across all treatments, and a significant increase was observed at 36°C for 3, 6, and 10 h. The increase in H₂O₂ content at 36°C for 10 h was slightly different from the MDA content. This shows that MDA is an oxidative stress biochemical indicator and a major oxidant of peroxidized polyunsaturated fatty acids (Rael et al., 2004; Del Rio et al., 2005). Higher MDA levels were associated with increased electrolyte loss and H₂O₂ accumulation (Nandwal et al., 2007), which has long been used as a marker of stress tolerance due to LPO. The H₂O₂ content at 36°C for 10 h increased, but the MDA concentration significantly increased higher at 36°C for 10 h. These results indicate that the high-temperature stress in M. persicae resulted in increased lipid damage by ROS and may associate with LPO and other oxidative stress responses as reported in P. japonica and Hylyphantes graminicola (Zhang et al., 2015; Zhao et al., 2020).

The oxygen and capacity limitation of thermal tolerance (OCLTT) theory explains the first line of thermal limitation in animals at the whole-organism level and may represent an evolutionary constraint that was modified depending on life stage and climate, as well as during the transition to life in the air (Pörtner et al., 2017). Thermal acclimatization and adaptation change limits via modifying membrane composition, enzyme and mitochondrial capacity, or molecular integrity-protecting mechanisms (Pörtner, 2012). At thermal extremes, oxygen scarcity causes the transition to passive tolerance, associated systemic and cellular stress signals such as hormonal responses or oxidative stress, and the usage of defense mechanisms such as HSPs (Kassahn et al., 2009). In this study, increasing the temperature caused an oxygen shortage, which resulted in oxidative stress or the generation of ROS. As a result, the aphid’s performance capacity was mostly determined by biochemical mechanisms that established the aerobic capacity of cells and tissues in the aphids, specifically the capacity of ventilatory and circulatory organs to supply enough oxygen to pay physiological costs above maintenance. The notion of oxygen- and capacity-limited thermal tolerance (Pörtner and Knust, 2007; Pörtner and Farrell, 2008) describes how insufficient oxygen supply on both sides of the thermal window limits the aerobic scope and determines the performance window in animals (Pörtner and Knust, 2007). In this work, antioxidant activity and gene expression of SOD, CAT, and POD, as well as HSPs, such as Hsp22, Hsp23, and Hsp27, scavenge the ROS as an underlying mechanism of aphid thermal heat tolerance. According to a recent study by Pörtner (2010), a twist in the interpretation of existing knowledge is appropriate for determining the physiological aspects of the environmental niche in which a species or one of its specific life stages exists. The study of performance characters in connection to both biotic and abiotic characters results in such dimensions. As a result of recent insights into the mechanisms of thermal adaptation and limitation, the influence of temperature on terrestrial insects, such as aphids, has emerged as a unifying notion in the cause-and-effect understanding of climate change effects on terrestrial ectotherms.

Previous experiments have shown that HSPs play a significant role in insect thermal resistance (Parsell and Lindquist, 1993) and various environmental stresses, especially heat and cold stress (Kang et al., 2009; Hu et al., 2014). For example, the expressed levels of Hsp23 genes in female silverleaf whiteflies Bemisia tabaci were found to have a crucial role in heat tolerance at 44°C (Lü and Wan, 2011). In insects, forced production of HSP 27 (Hsp27) reverses drug efflux via P-glycoprotein (ABCB1) and MDR1 gene expression (Zhao and Jones, 2012). Furthermore, the expression of the Hsp22 gene was raised in deep diapausing calanoid copepods like Calanus finmarchicus and played a function in both short-term stress responses and shielding proteins from degradation during diapause (Aruda et al., 2011). However, because Hsp22, 23, and 27 have never been shown to play a function in aphid heat tolerance in the short term, we focused on determining their role in M. persicae heat stress tolerance.

In this work, we observed that under short-time heat stress (1, 3, 6, and 10 h), the expression of Hsp22, Hsp23, and Hsp27 was increased. The expression of Hsp22, Hsp23, and Hsp27 genes at 36°C for 10 h was significantly higher than the control at 25°C (without heat) and other temperatures. The gene expression of Hsp27 has been greatly stimulated by thermal stress exposure at 35°C for 2 h in Chironomus riparius (Martínez-Paz et al., 2014). Many experiments have shown that the Hsp23 gene played a vital role in heat tolerance, and insects with higher Hsp gene expression had a higher survival rate under heat shock tolerance (Zhao and Jones, 2012). The expression of Hsp23 was significantly increased at 36°C as the time exposure increased (1, 3, 6, and 10 h). Our results show that Hsp22, Hsp23, and Hsps27 can greatly assist M. persicae in tolerating heat and thus contribute to the evolution of heat tolerance in this pest because Hsp gene expression increased at all temperatures; however, at 36°C for 10 h, Hsp gene expression increased more than other temperatures. These findings indicate that when Hsp22, Hsp23, and Hsp27 levels rise at high temperatures, these genes can support M. persicae in developing heat tolerance. According to our findings, HSPs and antioxidant enzymes work together to defend against thermal damage and are implicated in the antioxidant response to thermal stress in M. persicae adults, allowing them to adequately respond with ROS caused by thermal stress.

In this study, potential mechanisms of antioxidant reaction and HSPs in M. persicae were investigated. According to our knowledge, the physiological and molecular response in M. persicae with different temperatures and exposure times is reported for the first time by this study. According to our findings, short-time heat stress disrupts the function of redox in M. persicae. When the balance of redox processes is disrupted by external influences, oxidative stress develops. The main proposed factor for inducing oxidative stress in M. persicae is heat stress. In the antioxidant reaction to heat stress, antioxidant enzymes, such as SOD, CAT, and POD, as well as HSPs, such as Hsp22, Hsp23, and Hsp27, may be involved in the management of oxidative damage caused by MDA and H₂O₂, thus ROS. Aphids may respond rapidly to environmental stress due to the effective activation of enzymes, which allows them for environmental adaptation. Furthermore, we suggest more studies on M. persicae tolerance to heat stress involving the underlying mechanisms of oxidative stress tolerance.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AK and RI contributed to the study concept and design, and statistical analysis. AK, RI, IU, and SB contributed to the analysis and interpretation of data. CL and AT contributed to the investigation and resources. AK contributed to the drafting of the manuscript. J-JZ contributed to the review, editing, and proofreading of this manuscript. CZL contributed to funding acquisition and study supervision. All authors have read and agreed to this manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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