Exp Appl Acarol DOI 10.1007/s10493-014-9806-y
Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite, Neoseiulus cucumeris (Acari: Phytoseiidae) Guo-Hao Zhang • Huai Liu • Jin-Jun Wang • Zi-Ying Wang
Received: 28 August 2013 / Accepted: 20 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Thermal stress responses are usually associated with the increased generation of reactive oxygen species (ROS), resulting in oxidative damage. In this study, we examined the time-related effect (durations for 1, 2, 3, and 5 h) of thermal stress conditions—i.e., relatively low (0, 5, 10, and 15 °C) or high (35, 38, 41, and 44 °C) temperatures—on the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), and total antioxidant capacity (T-AOC) of the predatory mite Neoseiulus cucumeris. Also the lipid peroxidation (LPO) levels of the predatory mite were measured under thermal stress conditions. The results confirmed that thermal stress results in a condition of so-called oxidative stress and the four antioxidant enzymes play an important role in combating the accumulation of ROS in N. cucumeris. CAT and POX activity changed significantly when the mites were exposed to cold and heat shock, respectively. The elevated levels of SOD and GSTs activity, expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. However, the levels of LPO in N. cucumeris were high, serving as an important signal that these antioxidant enzyme-based defense mechanisms were not always adequate to counteract the surplus ROS. Thus, we hypothesize that thermal stress, especially extreme temperatures, may contribute much to the generation of ROS in N. cucumeris, and eventually to its death. Keywords enzymes
Neoseiulus cucumeris Thermal stress Oxidative stress Antioxidant
Introduction Reactive oxygen species (ROS), such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (HO), are generated endogenously by living organisms
G.-H. Zhang H. Liu (&) J.-J. Wang Z.-Y. Wang Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China e-mail: [email protected]
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during oxidative metabolism (Livingstone 2001). Physiological use of ROS by cells is now being demonstrated in areas such as intracellular signaling and redox regulation (Imai and Nakagawa 2003). In normal situations, a balance exists between the generation of ROS and the antioxidant defenses. However, under various types of environment stress, e.g. relatively low, high or even extreme temperatures, if unbalanced by antioxidant defenses, surplus ROS can trigger severe damage to biological molecules (e.g. lipids, proteins and nucleic acids) (Martindale and Holbrook 2002), with cell membrane fluidity disruption and apoptosis (Green and Reed 1998). To maintain homeostasis and minimize the oxidative damage to cellular components, living organisms have evolved a complex network of antioxidant systems, including various non-enzymatic molecules (e.g., glutathione, vitamins A, C, and E, and flavonoids) as well as antioxidant enzymes (Martindale and Holbrook 2002). Major antioxidant enzymes of insects, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), play an important role in protecting cells and maintaining homeostasis by removing oxidative stress (Rudneva 1999). Both SOD and CAT directly scavenge ROS. SOD removes (O2-) through the process of dismutation to singlet oxygen (O2) and H2O2 (2O2- ? H? ? H2O2 ? O2), and H2O2 produced by SOD is sequentially broken into H2O and O2 (2H2O2 ? 2H2O ? O2) by CAT and POX, respectively (Kashiwagi et al. 1997). GSTs are involved in removing the products of lipid peroxidation or hydroperoxides from cells (Meng et al. 2009). The process of lipid peroxidation (LPO) results in the formation of malondialdehyde (MDA), and its concentration is known to be an important parameter of oxidative damage (Lopez-Martinez et al. 2008). The increased levels of LPO can be determined by measuring MDA concentration (Meng et al. 2009). In addition, total antioxidant capacity (T-AOC) represents the ability of all antioxidants existing in an organism to counteract oxidation (Ghiselli et al. 2000). Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae) is considered a polyphagous predatory mite, feeding on a number of mite and insect species (McMurtry and Croft 1997). Neoseiulus cucumeris has been used successfully in biological control of western flower thrips, Frankliniella occidentalis, in greenhouse vegetable crops worldwide (van Houten et al. 1995; Easterbrook et al. 2001; Messelink et al. 2006; Zilahi-Balogh et al. 2007). Since N. cucumeris was introduced into China from the UK in 1997 by Yanxuan Zhang, it has developed as one of the most effective biological control agents targeting several economical phytophagous mite species, e.g. Schizotetranychus nanjingensis on moso bamboo (Zhang et al. 2000), Panonychus citri on citrus tree (Zhang et al. 2004), and Brevipalpus obovatus on tea bushes (Zhu et al. 2010). Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Recent studies demonstrated that high environmental temperatures promote the occurrence of spider mites, but they demote predatory mite abundance in agroecosystems, and the range of temperatures with positive per capita growth rates was shown to be much wider in prey than in its predators (Roy et al. 2003; Ozawa et al. 2012; Coombs and Bale 2013; Montserrat et al. 2013a,b). In addition, N. cucumeris enter a reproductive diapause which is induced by short-day photoperiods, at low night temperatures (van Houten et al. 1995). Another study showed that cold exposure had great adverse effects on non-diapausing N. cucumeris (Morewood 1993). Harsh environmental conditions, i.e., high as well as low temperatures, are a direct cause of biological control disruption and an increasing challenge for biological control of spider mites, given the intensifying global warming. Li and Fu (2007) found that the optimum temperature zone for development of N. cucumeris feeding on Tetranychus piercei ranges from 24 to 28 °C, and the development, survival and propagation of N. cucumeris are seriously suppressed when the environment
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temperature is higher than 30 °C. However, in south China, summer temperatures may reach 40 °C or even higher, whereas winter temperatures may drop to lower than 0 °C. Therefore, it is likely for N. cucumeris to suffer from thermal stress. Furthermore, a recent study has assessed that antioxidant enzyme in citrus red mite, P. citri, can efficiently deal with ROS induced by thermal stresses (Yang et al. 2010). How does N. cucumeris, an effective natural enemy attacking citrus red mite, deal with thermal stress? Many experimental studies of N. cucumeris have focused on the interactions between the predator and its prey in greenhouse and field crops. Little work has been devoted to investigate the physiological mechanisms of oxidative stress in relation to thermal stress in this predatory mite, or even in the family of Phytoseiidae. Therefore, we first describe the changes of the antioxidant enzyme activities and MDA concentration in response to thermal stress in N. cucumeris. The purpose of this study was to identify the oxidative stress and to evaluate the potential physiological responses of N. cucumeris to thermal stress for various treatment durations.
Materials and methods Test mites In 2010, a non-diapausing laboratory colony of N. cucumeris was established using mites purchased from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Neoseiulus cucumeris was reared on flour mites, Aleuroglyphus ovatus on wheat bran in 4-l translucent plastic rearing boxes closed with a lid where a 16 cm2 fine mesh (45 lm opening) was made to pass air in the middle. The boxes were placed in a 15 m2 temperature chamber (Xutemp Temptech, XT5499-CR20TLH, Hangzhou, China) at 25 ± 1 °C, 70–80 % RH, and a L:D = 14:10 photoperiod. Mites used in this study were maintained in a rearing unit of glass Petri dishes (15 cm in diameter). A round sponge (12 cm in diameter, 1.5 cm thick) soaked in distilled water was firstly placed in the Petri dishes. A piece of black cotton fabric (10 cm in diameter) and transparent plastic film (9 cm in diameter) were then placed on the sponge from the bottom to the top, respectively. The sponge in the Petri dishes was moistened daily with distilled water. To obtain homogeneous individuals for the experiments, approximately 800 female adults from the N. cucumeris stock colony were transferred to the Petri dishes and allowed to lay eggs in the temperature chamber. In order to avoid or reduce the escape of female adult mites during reproduction, a piece of paper (4 cm2) was placed in the centre of transparent plastic film, serving as a shelter. Same-age eggs (\24 h) were transferred into a new dish using a fine camel-hair brush. Thereafter, the offspring were kept till the nymphs emerged, and then A. ovatus including all stages from its stock colony together with wheat bran were supplied as food. After 80 % of the individuals had molted to the adult stage, development times were recorded. Female adult cohorts of 4–5 days old produced via this procedure were used throughout this study. Thermal stress For each treatment, 300 female adults of 4–5 days old from Petri dishes were transferred into 2-ml centrifuge tubes. Three centrifuge tubes were placed into a programmable temperature controller (Ningbo Southeast Instrument, RXZ-260B, China), at each target temperature (0, 5, 10, 15, 35, 38, 41, and 44 °C). The duration of each temperature
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treatment was 1, 2, 3, and 5 h. Mites kept at a temperature of 25 °C served as a control. In order to check mortality, mites were allowed to recover at 25 °C for 10 min. Then, the mites were frozen immediately in liquid nitrogen and stored as a unit sample at -80 °C before our assays. Each treatment was replicated three times on three different days. Sample preparation The samples of treated mites were homogenized rapidly in 200 ll ice-cold 0.05 M pH 7.0 phosphate buffer by using 1-ml glass homogenizers. The resulting crude homogenates were centrifuged at 10,0009g for 15 min at 4 °C and the supernatant was used for further analysis. Protein concentrations were determined according to the Bradford (1976) method using bovine serum albumin as the standard. Enzyme activity, T-AOC, and Lipid peroxidation (LPO) assay The activities of CAT, SOD, POX, T-AOC and LPO levels were determined with assay kits of A007-1, A001-1, A084-1, A015 and A003-1, respectively (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), following the manufacturer’s protocols. Enzyme determination was carried out at 25 ± 1 °C in an air-conditioned room. Absorbance was read at 37 °C in a Microplate Spectrophotometer (XMarkTM, Bio-Rad, Hercules, CA, USA). Lipid peroxidation levels were determined indirectly by measuring MDA concentrations formed by reacting with thiobarbituric acid to give a red product having a maximum absorbance at 532 nm at 37 °C (Meng et al. 2009). MDA concentration was expressed as nmol of MDA produced per mg protein (nmol mg-1 protein). The decomposition reaction of H2O2 by CAT can be terminated immediately by adding ammonium molybdate that reacts with remained H2O2 forming a faint yellow complex compound. CAT activity was detected by measuring the decrease in absorbance resulted from H2O2 decomposition at 405 nm. One unit of CAT activity was defined as the amount that decomposes 1 lmol of H2O2 per s per mg protein (U mg-1 protein). Superoxide dismutase activity was determined at 550 nm by use of xanthine and xanthine oxidase systems. One unit of SOD activity was defined as the amount of enzyme required to cause 50 % inhibition of the xanthine and xanthine oxidase reaction in 1 ml enzyme extraction of 1 mg protein (U mg-1 protein). Peroxidase activity was calculated at 415 nm by catalyzing the oxidation in the presence of H2O2 of a substrate. One unit of POX activity was defined as the amount that catalyses 1 lg substrate per min per mg protein at 37 °C (U mg-1 protein). Glutathione S-transferases activity was determined with 1-chloro-2,4-dinitrobenzene (CDNB; Shanghai Chem.) and reduced GSH (Sigma) as substrates according to the procedure of Habig et al. (1974) with slight modifications. The 96-well microtiter plate with 100 ll CDNB (1 % ethanol (v/v) included) and 100 ll GSH in Tris–HCl buffer in each well were held at 37 °C for 20 min. Then 100 ll enzyme solutions were added per well, giving the final concentrations of 0.2 and 2.0 mM of CDNB and GSH, respectively. The change in absorbance was measured continuously for 5 min at 340 nm and 37 °C in a Microplate Spectrophotometer (XMark). The non-enzymatic reaction of CDNB with GSH measured without enzyme solution served as control. Changes in absorbance per min were converted into nmol CDNB conjugated min-1 mg-1 protein using the extinction coefficient of the resulting 2,4-dinitrophenyl-glutathione: e340 nm = 9.6 mM-1 cm-1 (Habig et al. 1974).
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Total antioxidant capacity was measured based on the generation of the Fe2?-o-phenanthroline complex, as the overall reducing agents in sample supernatant reduced Fe3? to Fe2? in the sample supernatant which reacted with the substrate o-phenanthroline. Stable color of Fe2?-o-phenanthroline complex was measured at 520 nm 37 °C. One unit of T-AOC was defined as the amount necessary to increase the absorbance by 0.01 per min per mg protein at 37 °C (U mg-1 protein). Statistical analysis Treatment effects (temperature and duration) were subjected to one- or two-way analysis of variance (ANOVA) by the general linear model procedure of SPSS 16.0 for Windows (SPSS, Chicago, IL, USA), and when significant effects were obtained, mean differences were separated by Tukey’s least significant difference (LSD) test.
Results Mortality of the predatory mites ranged from 0.3 to 3.6 % in response to cold hardening (0–15 °C), and from 0.4 to 12.7 % under heat shock (35–41 °C), respectively. However, more than 27 % mites were dead being stressed for 5 h at 44 °C. Dead mites were excluded from the assays. Changes in LPO levels Lipid peroxidation levels (expressed as MDA concentration) in response to various thermal stresses are shown in Table 1. Two-way ANOVA revealed that the MDA concentrations in N. cucumeris were significantly affected by treatment temperature (F8,105 = 53.922; P \ 0.001) and duration (F3,105 = 71.153; P \ 0.001), and their interaction (F24,105 = 2.297; P = 0.004). MDA concentration in N. cucumeris, recorded with only 11.1 nmol mg-1 protein, was lowest at 25 °C. However, when the mites were exposed for 1 h it significantly increased among treatments in response to cold hardening or heat shock. For this duration, MDA concentration showed a decline with the rising treatment temperatures range from 0 to 15 °C, whereas it enhanced with temperatures rising from 35 to 44 °C, respectively. When thermal stress lasted for 2 h, it increased continuously among treatment temperatures and peaked at 41 °C (recorded as 36.6 nmol mg-1 protein), 3.3 times higher than the control. For treatment durations longer than 2 h, MDA concentrations decreased with the extension of durations. Changes in activities of antioxidant enzymes Changes in enzyme activities of N. cucumeris after cold hardening or heat shock for different durations are presented in Table 2. ANOVA showed that CAT activities were significantly affected by treatment temperature (F8,105 = 25.786; P \ 0.001), duration (F3,105 = 12.244; P \ 0.001), and their interaction (F24,105 = 3.382; P \ 0.001). Generally, cold stress had more influence on CAT activities than heat stress. For example, exposed to 5 °C for 2, 3 and 5 h, CAT activities significantly increased compared to the controls, whereas it enhanced only slightly among heat treatments (and these differences were not significant).
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123
11.09 11.09 11.09 11.09
± ± ± ±
1.01a 1.01a 1.01a 1.01a
26.13 29.66 21.81 17.76
0
± ± ± ±
1.32cde 0.47bcd 1.86b 1.12bc
23.46 28.90 19.99 18.89
5
± ± ± ±
1.20bcd 1.53bcd 1.36b 1.17bc
21.04 25.64 20.31 18.15
10 ± ± ± ±
0.73b 0.71b 1.21b 1.67bc
21.77 29.77 20.48 16.53
15 ± ± ± ±
2.86bc 0.741bcd 1.32b 1.37b
23.68 26.09 20.53 18.87
35 ± ± ± ±
1.06bcd 1.84bc 1.60b 1.52bc
Means within a row followed by different letters are significantly different (ANOVA followed by LSD: P \ 0.05)
1 2 3 5
25
Duration (h) Temperatures (°C)
25.69 31.01 23.95 21.28
38 ± ± ± ±
0.57cde 1.86d 1.77b 1.39 cd
Table 1 Malondialdehyde (MDA) concentration (mean ± SE) (nmol/mg protein) of Neoseiulus cucumeris exposed to thermal stress
27.73 36.55 28.15 23.03
41 ± ± ± ±
1.01de 1.44e 1.07c 0.73d
28.89 ± 1.33e 30.13 ± 1.25cd 22.61 ± 0.47b –
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Superoxide dismutase activities were influenced by treatment temperature (F8,105 = 21.209; P \ 0.001), duration (F3,105 = 52.163; P \ 0.001) and their interaction (F24,105 = 2.838; P \ 0.001). SOD activities significantly increased in heat treatments (35–44 °C) for 1 h when compared with all cold treatments and the control (25 °C). For 2 h, SOD activity increased at all temperatures, particularly at 41 °C with the peaked value recorded as 242.9 U mg-1 protein. SOD activity decreased at all temperatures when exposure time was extended to 3 h. At 15 °C, SOD activity was significantly declined relative to the control. Glutathione S-transferases activities were affected by temperature (F8,105 = 36.604; P \ 0.001), duration (F3,105 = 28.937; P \ 0.001) and their interaction (F24,105 = 7.510; P \ 0.001). When exposure was extended to 2 h, GSTs activities showed significant increases compared with the control following exposure to 0, 5, 10, 35, 38 and 41 °C, respectively. The maximum value was recorded as 302.3 nmol min-1 mg-1 protein at 5 °C. GSTs activities of the 2-h treatment at 35–41 °C enhanced significantly compared to the control. GSTs activities were very high when exposed for 3 and 5 h to 5 or 10 °C. Peroxidase activities were significantly affected by temperature (F8,105 = 29.635; P \ 0.001), duration (F3,105 = 19.802; P \ 0.001) and their interaction (F24,105 = 4.670; P \ 0.001). POX activities were higher relative to the control after exposure for 1 h to 35, 38 and 41 °C, whereas it showed a notable decrease after cold stress at 0, 5 and 10 °C. Similar effects were found when exposure lasted 2 h, and no notable difference among treatment temperatures was seen when exposed for 3 h. Significant decreases, relative to the 25 °C control, were found after exposure for 5 h to 0, 5, 10, and 35 °C. Total antioxidant capacities were significantly influenced by temperature (F8,105 = 6.952; P \ 0.001), duration (F3,105 = 21.973; P \ 0.001) and their interaction (F24,105 = 3.430; P \ 0.001). When exposed to 0 °C for 1 h, T-AOC significantly increased compared to the control, whereas it reverted to the control level 1 h later. The same situation occurred at 44 °C. No significant difference was observed among treatment temperatures lasting 2 or 3 h. Significant decreases were observed at 5 h exposure to 10, 15, and 35 °C.
Discussion Temperature is one of the most important environmental variables that induce physiological change in organisms (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, antifreeze proteins and amino acids (Storey 1997; Wang and Kang 2005). Previous studies indicated that antioxidant enzymes and heat shock proteins are associated with responses to high temperature and other stresses in nature in a wide range of organisms (Feder and Hofmann 1999; Martindale and Holbrook 2002; Sørensen et al. 2003). The predatory mite N. cucumeris is a poikilothermic organism, and its body temperature is highly affected by the ambient temperature. In this study, we showed that LPO and antioxidant enzymes (CAT, SOD, GSTs, POX, and T-AOC) in N. cucumeris changed significantly under various thermal stresses. The mites used in the current study were mass-reared under optimal conditions for reproduction and development, and they had never been subjected to thermal stress. Commercially available mites are thought to be inherently less cold hardy and to have a lower capacity to acclimate to low temperatures than wild ones (Morewood 1993). Lipid peroxidation is a well-known mechanism of cellular injury in both vertebrates and invertebrates, and acts as a biological marker of oxidative stress in cells and tissues (Del Rio
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10.08 ± 2.11ab 17.958 ± 2.45c
8.06 ± 0.74a
182.93 ± 6.17b
182.93 ± 6.17a
3
5
18.21 ± 0.80a
18.21 ± 0.80c
3
5
26.86 ± 2.34c
15.89 ± 0.56bc
15.93 ± 0.95a
19.67 ± 1.02a
185.70 ± 1.59a
11.26 ± 0.65ab
8.45 ± 0.69a
182.21 ± 6.55a
180.53 ± 6.09b 175.78 ± 5.94a
159.39 ± 2.48a
217.91 ± 3.07cd 197.56 ± 4.87b
181.60 ± 5.50a
13.39 ± 1.04b
12.04 ± 0.49bc
21.70 ± 1.82b
15.88 ± 1.20bc
16.57 ± 0.60a
19.43 ± 1.36a
192.27 ± 4.60a
243.88 ± 6.99b
229.44 ± 8.54ab
13.38 ± 1.10ab
15.31 ± 0.73a
17.96 ± 1.94a
20.67 ± 0.48b
11.01 ± 1.26a
13.62 ± 0.97a
17.13 ± 1.42a
16.41 ± 0.98ab
211.33 ± 3.37ab 227.81 ± 5.75bc
206.28 ± 8.74a
184.21 ± 8.76a
11.39 ± 0.85a
13.44 ± 3.04a
19.11 ± 1.22a
14.87 ± 0.62a
212.37 ± 6.88ab
241.20 ± 19.43b
307.02 ± 26.29c
220.20 ± 10.57ab 268.09 ± 12.46d
201.69 ± 2.2a
208.25 ± 6.85ab 238.16 ± 20.68b
205.80 ± 4.53a
262.65 ± 6.87b
206.39 ± 8.54a
213.73 ± 5.61d
230.67 ± 3.16de
212.05 ± 8.69b
8.73 ± 0.18a
8.38 ± 0.77a
11.61 ± 0.43b
8.47 ± 0.98a
38
9.99 ± 0.99abc
8.53 ± 0.92a
9.52 ± 0.70ab
8.20 ± 0.36a
238.35 ± 2.97b
221.42 ± 5.73c
196.86 ± 8.10a
180.62 ± 5.14b
242.86 ± 7.48e
213.60 ± 2.43b
41
15.35 ± 0.94bc
17.64 ± 1.75a
18.22 ± 1.17a
14.81 ± 2.00a
225.54 ± 7.25bc
248.22 ± 4.60b
292.69 ± 11.05c
313.34 ± 9.19e
187.93 ± 8.80a
15.89 ± 0.37bc
16.02 ± 1.30a
16.53 ± 0.70a
17.02 ± 2.82ab
226.90 ± 5.98bc
240.63 ± 11.69b
290.60 ± 1.75c
302.31 ± 4.71e
189.42 ± 6.34a
189.51 ± 4.60abc 196.31 ± 5.06bc
229.99 ± 10.69b
205.31 ± 9.06abc 199.15 ± 3.49ab
207.99 ± 1.62a
205.82 ± 3.42cd
221.42 ± 1.52cd
202.81 ± 3.82b
8.81 ± 0.59a
8.88 ± 0.41ab
9.03 ± 0.98ab
9.98 ± 0.93abc
35
222.90 ± 10.61d 182.13 ± 12.14ab 172.07 ± 2.59ab
231.77 ± 4.98b
Means within a row followed by different letters are significantly different (ANOVA followed by LSD: P \ 0.05)
18.20 ± 0.80ab
18.21 ± 0.80a
1
2
T-AOC (U mg-1 protein)
204.64 ± 7.45a
233.53 ± 9.78ab 205.57 ± 15.28a 205.88 ± 3.01a
233.53 ± 9.78c
3
5
203.78 ± 6.37a
233.53 ± 9.78bc 202.34 ± 18.81a 204.39 ± 5.08a
233.53 ± 9.78ab 198.21 ± 14.16a 214.67 ± 7.86ab
279.45 ± 8.78b
1
193.39 ± 8.68a
2
POX (U mg-1 protein)
194.79 ± 6.16bc 210.95 ± 6.34cd 255.71 ± 4.36e
194.79 ± 6.16a
3
5
302.31 ± 11.43c
194.80 ± 6.16ab 184.57 ± 6.44a
194.79 ± 6.16a
223.02 ± 5.68b
9.50 ± 1.21ab
15
17.96 ± 1.67cd 15.971 ± 0.76c
12.32 ± 1.32bc
10
208.25 ± 10.15bc 195.50 ± 7.33ab 185.48 ± 4.23a
191.19 ± 3.87a
189.38 ± 3.36bc
222.71 ± 3.08cd
1
192.55 ± 1.79a
188.78 ± 1.79b
215.51 ± 1.30c
197.70 ± 5.77ab 184.15 ± 4.09a
16.66 ± 1.90d
2
GSTs (nmol min-1 mg-1 protein)
182.93 ± 6.17a
182.93 ± 6.17a
1
2
SOD (U mg-1 protein)
5
12.87 ± 1.79c
8.06 ± 0.76a
10.73 ± 0.79abc 19.87 ± 1.05d
3
16.67 ± 1.40c
8.06 ± 0.74a
5
8.06 ± 0.74a
12.97 ± 1.13c
0
2
protein)
25
Temperatures (°C)
1
CAT (U mg
-1
Enzymes Durations (h)
Table 2 Antioxidant enzymes activities (mean ± SE) of Neoseiulus cucumeris exposed to thermal stress
–
15.89 ± 0.86a
17.35 ± 1.91a
27.27 ± 1.26c
–
219.27 ± 4.58ab
243.82 ± 14.82b
251.46 ± 9.73cd
–
165.70 ± 14.62a
194.75 ± 9.86a
202.77 ± 4.60abc
–
190.39 ± 7.75bc
223.76 ± 4.17cd
205.89 ± 4.78b
–
9.23 ± 0.72ab
9.67 ± 0.41ab
10.66 ± 1.03abc
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et al. 2005). A significantly elevated level of MDA concentration in N. cucumeris was observed in response to thermal stress. This clearly demonstrates that thermal stress in N. cucumeris is associated with the process of LPO and other responses to oxidative stress, similar to other arthropods (Yang et al. 2010; Jia et al. 2011; Jena et al. 2013). Initially, MDA concentrations in N. cucumeris induced by thermal exposure increased strongly, but after longer exposure it decreased gradually. The descending MDA with longer exposure was probably a result of the antioxidant system eliminating oxidative stress, and similar phenomenon was found in citrus red mite P. citri (Yang et al. 2010). This suggests that both N. cucumeris and P. citri may become acclimated to thermal stress with the increasing exposure duration. However, the difference between them is that LPO remained at high levels in the predatory mite with extending durations compared to the control, which suggests that this mite may be ineffective in eliminating ROS induced by thermal stress. This matches the general pattern that higher trophic levels are more sensitive to adverse environmental temperatures than lower trophic levels (Voigt et al. 2003; Montserrat et al. 2013b). Catalase, SOD, POX, and GSTs are four crucial antioxidant defense enzymes, which can work synergistically against oxidative stress generated by high concentrations of ROS inside the cell. Among these antioxidant enzymes, CAT is considered to be the principal H2O2 scavenging enzyme (Jena et al. 2013), because arthropods are deficient in seleniumdependent glutathione peroxidase, which is a scavenger in other organisms (Sohal et al. 1990). However, CAT removes H2O2 only at high cellular concentrations, whereas it is inefficient for H2O2 removal at low concentrations (Ahmad et al. 1991). In a previous study, CAT activity was too low to detect in the herbivorous citrus red mite P. citri, 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. cucumeris exposed to the same thermal stress conditions. Apparently, it enhanced the removal of H2O2 and prevented damage by oxidative stress. The difference in CAT activity between the predatory mite N. cucumeris and its prey P. citri suggests that the two species differ in antioxidant defense though they both belong to the Acari. Elevated CAT activities induced by thermal stress were also observed in the oriental fruit fly Bactrocera dorsalis (Jia et al. 2011), and in the fat body of 5th instar silkworm, Bombyx mori (Nabizadeh and Jagadeesh Kumar 2011). It has been well documented that SOD plays an important role in reducing the high levels of superoxide radical induced by low or high surrounding temperatures (Celino et al. 2011). In our study, SOD activities in N. cucumeris under thermal stress significantly increased during early exposures and no significant changes were observed during later compared with control. This suggests that SOD was induced by changes in surrounding temperatures and then protected the mites from thermal stress. In addition, N. cucumeris became acclimated to thermal stress; similar results were found in other studies (An and Choi 2010; Yang et al. 2010; Jena et al. 2013). Both SOD and CAT can directly eliminate surplus ROS in a coordinated way. SOD removes O2- through the process of dismutation to O2 and H2O2, and then H2O2 is sequentially reduced to H2O and O2 by CAT (Kashiwagi et al. 1997). However, when exposed to low temperatures, e.g. 5 and 10 °C, CAT activity was higher than SOD activity. Because CAT removes H2O2 only at high cellular concentrations (Ahmad et al. 1991), it would be attributed to the other possible processes of H2O2 being produced under such cold stresses. Glutathione S-transferases are relevant in the metabolism of LPO products in specific tissues in which they are expressed to protect organisms against oxidative stress (Sawicki et al. 2003; Nair and Choi 2011). Six classes of GSTs were found, including delta, epsilon, omega, sigma, theta, and zeta. The classes of delta and sigma GSTs have been considered
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to play a key role in antioxidant defense in various organisms (Nair and Choi 2011). Sigma GST has a dominant role in protection against oxidative damage in Drosophila melanogaster (Singh et al. 2001). In the present study, GSTs activity of crude enzyme in N. cucumeris was determined by the procedure of Habig et al. (1974), which has been shown to work well in several studies (Yang et al. 2010; Jia et al. 2011; Chen et al. 2013). In this study, the increased activity of the enzyme exposed to 5 °C for 2, 3, and 5 h, suggests that GSTs were acting in removal of toxic LPO products caused by acute thermal stress. However, no significant difference of GSTs activity was observed among treatment temperatures when the mites were exposed for 1 h except for 41 °C. This may indicate that GSTs were involved in the inactivation of accumulated toxic LPO products for 1 h, which agrees with previous findings in the oriental fruit fly B. dorsalis (Jia et al. 2011). In most cases, GSTs activities keep pace with MDA concentrations, mainly because GSTs consume the products of LPO other than H2O2 (Joanisse and Storey 1996). However, in our study, MDA concentrations in each treatment were higher than in control, indicating that the mites could not scavenge LPO products effectively, especially when exposed for a long time. Glutathione S-transferases with POX can metabolize lipid peroxides and POX can break down H2O2 (Jia et al. 2011). In our study, POX activities significantly increased after heat shock for 1–2 h compared to the control, probably as a result of a significant increase of SOD activity in response to increased H2O2 concentration. However, it decreased to control levels or (much) lower with the extension of exposure time. Our result may indicate that POX activity in N. cucumeris was efficiently induced by thermal stress, which congrues with findings in Helicoverpa armigera (Meng et al. 2009). However, in response to low-temperature stress, POX activity in N. cucumeris showed a decrease among all durations. POX activity was also decreased in oriental fruit fly (Jia et al. 2011), whereas in citrus red mite it did not change significantly (Yang et al. 2010). Because the functions of POX in response to thermal stress are not yet fully understood, further studies are needed to explain this pattern. Total antioxidant capacity is a commendable measure of the ability of all antioxidants existing in an organism, and it has been used as a tool to evaluate redox status in recent reports (Ghiselli et al. 2000). In N. cucumeris, T-AOC enhanced significantly when exposed to two extreme temperatures (0 and 44 °C) for 1 h when compared to control. This indicates that T-AOC may be sufficient to cope with oxidative stress and free radical formulation after a short shock. However, when the mites were exposed longer (2–5 h), there appeared to be no obvious change (2 and 3 h) or they were not well equipped (5 h) to defend against ROS, compared with control. Apart from enzymes, some non-enzymatic substances, e.g. trehalose (Mahmud et al. 2010) and vitamin E (a-tocopherol) (Kaur et al. 2009), can play important roles in antioxidant stress. A recent study confirmed that heat shock proteins cooperate with antioxidant enzymes to deal with ROS damage (Rosa et al. 2012). We only determined the activity of (antioxidant) enzymes that play a vital role in antioxidant defense at the physiological level. However, we did not study the synergistic effect of heat-shock proteins and antioxidant enzymes, nor the simple effect of heat shock alone in relation to thermal stress. This deserves further investigation. In conclusion, our results indicated that the redox balance in N. cucumeris was disturbed by thermal stress, which results in oxidative stress. The antioxidant enzymes are intrinsically linked and dependent upon the activity of one another and, therefore, one would expect to see correlative changes in their activity (Rosa et al. 2012). Here, this coordinated effect was confirmed in female adults of N. cucumeris. Increasing levels of SOD activity, expressed in a time-dependent manner, indicated that there was an increase of superoxide
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production. Concomitantly, CAT and POX activity also was elevated when the mites were exposed to cold and heat shock, respectively, possibly to increment the capability to catabolize H2O2 that generated from the dismutation of the O2- radical by SOD. GSTs activity, also expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. Although T-AOC activity in N. cucumeris changed slightly among treatments, it would be of importance as well in mite physiological adaptations. The higher levels of LPO in N. cucumeris serve an important signal demonstrating that antioxidant enzymes are not always adequate to counteract the production of ROS induced by thermal stresses. Further studies of the effects of thermal stress of N. cucumeris in hatch rate of eggs, survival rate and fecundity of female adults should be investigated, based on our results that surplus ROS accumulated after cold hardening or heat shock. Acknowledgments This study was supported by the National Public Benefit Industry (Agriculture) Research Projects, the Ministry of Agriculture, (200903032).
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Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite, Neoseiulus cucumeris (Acari: Phytoseiidae) Guo-Hao Zhang • Huai Liu • Jin-Jun Wang • Zi-Ying Wang
Received: 28 August 2013 / Accepted: 20 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Thermal stress responses are usually associated with the increased generation of reactive oxygen species (ROS), resulting in oxidative damage. In this study, we examined the time-related effect (durations for 1, 2, 3, and 5 h) of thermal stress conditions—i.e., relatively low (0, 5, 10, and 15 °C) or high (35, 38, 41, and 44 °C) temperatures—on the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), and total antioxidant capacity (T-AOC) of the predatory mite Neoseiulus cucumeris. Also the lipid peroxidation (LPO) levels of the predatory mite were measured under thermal stress conditions. The results confirmed that thermal stress results in a condition of so-called oxidative stress and the four antioxidant enzymes play an important role in combating the accumulation of ROS in N. cucumeris. CAT and POX activity changed significantly when the mites were exposed to cold and heat shock, respectively. The elevated levels of SOD and GSTs activity, expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. However, the levels of LPO in N. cucumeris were high, serving as an important signal that these antioxidant enzyme-based defense mechanisms were not always adequate to counteract the surplus ROS. Thus, we hypothesize that thermal stress, especially extreme temperatures, may contribute much to the generation of ROS in N. cucumeris, and eventually to its death. Keywords enzymes
Neoseiulus cucumeris Thermal stress Oxidative stress Antioxidant
Introduction Reactive oxygen species (ROS), such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (HO), are generated endogenously by living organisms
G.-H. Zhang H. Liu (&) J.-J. Wang Z.-Y. Wang Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China e-mail: [email protected]
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during oxidative metabolism (Livingstone 2001). Physiological use of ROS by cells is now being demonstrated in areas such as intracellular signaling and redox regulation (Imai and Nakagawa 2003). In normal situations, a balance exists between the generation of ROS and the antioxidant defenses. However, under various types of environment stress, e.g. relatively low, high or even extreme temperatures, if unbalanced by antioxidant defenses, surplus ROS can trigger severe damage to biological molecules (e.g. lipids, proteins and nucleic acids) (Martindale and Holbrook 2002), with cell membrane fluidity disruption and apoptosis (Green and Reed 1998). To maintain homeostasis and minimize the oxidative damage to cellular components, living organisms have evolved a complex network of antioxidant systems, including various non-enzymatic molecules (e.g., glutathione, vitamins A, C, and E, and flavonoids) as well as antioxidant enzymes (Martindale and Holbrook 2002). Major antioxidant enzymes of insects, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), play an important role in protecting cells and maintaining homeostasis by removing oxidative stress (Rudneva 1999). Both SOD and CAT directly scavenge ROS. SOD removes (O2-) through the process of dismutation to singlet oxygen (O2) and H2O2 (2O2- ? H? ? H2O2 ? O2), and H2O2 produced by SOD is sequentially broken into H2O and O2 (2H2O2 ? 2H2O ? O2) by CAT and POX, respectively (Kashiwagi et al. 1997). GSTs are involved in removing the products of lipid peroxidation or hydroperoxides from cells (Meng et al. 2009). The process of lipid peroxidation (LPO) results in the formation of malondialdehyde (MDA), and its concentration is known to be an important parameter of oxidative damage (Lopez-Martinez et al. 2008). The increased levels of LPO can be determined by measuring MDA concentration (Meng et al. 2009). In addition, total antioxidant capacity (T-AOC) represents the ability of all antioxidants existing in an organism to counteract oxidation (Ghiselli et al. 2000). Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae) is considered a polyphagous predatory mite, feeding on a number of mite and insect species (McMurtry and Croft 1997). Neoseiulus cucumeris has been used successfully in biological control of western flower thrips, Frankliniella occidentalis, in greenhouse vegetable crops worldwide (van Houten et al. 1995; Easterbrook et al. 2001; Messelink et al. 2006; Zilahi-Balogh et al. 2007). Since N. cucumeris was introduced into China from the UK in 1997 by Yanxuan Zhang, it has developed as one of the most effective biological control agents targeting several economical phytophagous mite species, e.g. Schizotetranychus nanjingensis on moso bamboo (Zhang et al. 2000), Panonychus citri on citrus tree (Zhang et al. 2004), and Brevipalpus obovatus on tea bushes (Zhu et al. 2010). Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Recent studies demonstrated that high environmental temperatures promote the occurrence of spider mites, but they demote predatory mite abundance in agroecosystems, and the range of temperatures with positive per capita growth rates was shown to be much wider in prey than in its predators (Roy et al. 2003; Ozawa et al. 2012; Coombs and Bale 2013; Montserrat et al. 2013a,b). In addition, N. cucumeris enter a reproductive diapause which is induced by short-day photoperiods, at low night temperatures (van Houten et al. 1995). Another study showed that cold exposure had great adverse effects on non-diapausing N. cucumeris (Morewood 1993). Harsh environmental conditions, i.e., high as well as low temperatures, are a direct cause of biological control disruption and an increasing challenge for biological control of spider mites, given the intensifying global warming. Li and Fu (2007) found that the optimum temperature zone for development of N. cucumeris feeding on Tetranychus piercei ranges from 24 to 28 °C, and the development, survival and propagation of N. cucumeris are seriously suppressed when the environment
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temperature is higher than 30 °C. However, in south China, summer temperatures may reach 40 °C or even higher, whereas winter temperatures may drop to lower than 0 °C. Therefore, it is likely for N. cucumeris to suffer from thermal stress. Furthermore, a recent study has assessed that antioxidant enzyme in citrus red mite, P. citri, can efficiently deal with ROS induced by thermal stresses (Yang et al. 2010). How does N. cucumeris, an effective natural enemy attacking citrus red mite, deal with thermal stress? Many experimental studies of N. cucumeris have focused on the interactions between the predator and its prey in greenhouse and field crops. Little work has been devoted to investigate the physiological mechanisms of oxidative stress in relation to thermal stress in this predatory mite, or even in the family of Phytoseiidae. Therefore, we first describe the changes of the antioxidant enzyme activities and MDA concentration in response to thermal stress in N. cucumeris. The purpose of this study was to identify the oxidative stress and to evaluate the potential physiological responses of N. cucumeris to thermal stress for various treatment durations.
Materials and methods Test mites In 2010, a non-diapausing laboratory colony of N. cucumeris was established using mites purchased from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Neoseiulus cucumeris was reared on flour mites, Aleuroglyphus ovatus on wheat bran in 4-l translucent plastic rearing boxes closed with a lid where a 16 cm2 fine mesh (45 lm opening) was made to pass air in the middle. The boxes were placed in a 15 m2 temperature chamber (Xutemp Temptech, XT5499-CR20TLH, Hangzhou, China) at 25 ± 1 °C, 70–80 % RH, and a L:D = 14:10 photoperiod. Mites used in this study were maintained in a rearing unit of glass Petri dishes (15 cm in diameter). A round sponge (12 cm in diameter, 1.5 cm thick) soaked in distilled water was firstly placed in the Petri dishes. A piece of black cotton fabric (10 cm in diameter) and transparent plastic film (9 cm in diameter) were then placed on the sponge from the bottom to the top, respectively. The sponge in the Petri dishes was moistened daily with distilled water. To obtain homogeneous individuals for the experiments, approximately 800 female adults from the N. cucumeris stock colony were transferred to the Petri dishes and allowed to lay eggs in the temperature chamber. In order to avoid or reduce the escape of female adult mites during reproduction, a piece of paper (4 cm2) was placed in the centre of transparent plastic film, serving as a shelter. Same-age eggs (\24 h) were transferred into a new dish using a fine camel-hair brush. Thereafter, the offspring were kept till the nymphs emerged, and then A. ovatus including all stages from its stock colony together with wheat bran were supplied as food. After 80 % of the individuals had molted to the adult stage, development times were recorded. Female adult cohorts of 4–5 days old produced via this procedure were used throughout this study. Thermal stress For each treatment, 300 female adults of 4–5 days old from Petri dishes were transferred into 2-ml centrifuge tubes. Three centrifuge tubes were placed into a programmable temperature controller (Ningbo Southeast Instrument, RXZ-260B, China), at each target temperature (0, 5, 10, 15, 35, 38, 41, and 44 °C). The duration of each temperature
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treatment was 1, 2, 3, and 5 h. Mites kept at a temperature of 25 °C served as a control. In order to check mortality, mites were allowed to recover at 25 °C for 10 min. Then, the mites were frozen immediately in liquid nitrogen and stored as a unit sample at -80 °C before our assays. Each treatment was replicated three times on three different days. Sample preparation The samples of treated mites were homogenized rapidly in 200 ll ice-cold 0.05 M pH 7.0 phosphate buffer by using 1-ml glass homogenizers. The resulting crude homogenates were centrifuged at 10,0009g for 15 min at 4 °C and the supernatant was used for further analysis. Protein concentrations were determined according to the Bradford (1976) method using bovine serum albumin as the standard. Enzyme activity, T-AOC, and Lipid peroxidation (LPO) assay The activities of CAT, SOD, POX, T-AOC and LPO levels were determined with assay kits of A007-1, A001-1, A084-1, A015 and A003-1, respectively (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), following the manufacturer’s protocols. Enzyme determination was carried out at 25 ± 1 °C in an air-conditioned room. Absorbance was read at 37 °C in a Microplate Spectrophotometer (XMarkTM, Bio-Rad, Hercules, CA, USA). Lipid peroxidation levels were determined indirectly by measuring MDA concentrations formed by reacting with thiobarbituric acid to give a red product having a maximum absorbance at 532 nm at 37 °C (Meng et al. 2009). MDA concentration was expressed as nmol of MDA produced per mg protein (nmol mg-1 protein). The decomposition reaction of H2O2 by CAT can be terminated immediately by adding ammonium molybdate that reacts with remained H2O2 forming a faint yellow complex compound. CAT activity was detected by measuring the decrease in absorbance resulted from H2O2 decomposition at 405 nm. One unit of CAT activity was defined as the amount that decomposes 1 lmol of H2O2 per s per mg protein (U mg-1 protein). Superoxide dismutase activity was determined at 550 nm by use of xanthine and xanthine oxidase systems. One unit of SOD activity was defined as the amount of enzyme required to cause 50 % inhibition of the xanthine and xanthine oxidase reaction in 1 ml enzyme extraction of 1 mg protein (U mg-1 protein). Peroxidase activity was calculated at 415 nm by catalyzing the oxidation in the presence of H2O2 of a substrate. One unit of POX activity was defined as the amount that catalyses 1 lg substrate per min per mg protein at 37 °C (U mg-1 protein). Glutathione S-transferases activity was determined with 1-chloro-2,4-dinitrobenzene (CDNB; Shanghai Chem.) and reduced GSH (Sigma) as substrates according to the procedure of Habig et al. (1974) with slight modifications. The 96-well microtiter plate with 100 ll CDNB (1 % ethanol (v/v) included) and 100 ll GSH in Tris–HCl buffer in each well were held at 37 °C for 20 min. Then 100 ll enzyme solutions were added per well, giving the final concentrations of 0.2 and 2.0 mM of CDNB and GSH, respectively. The change in absorbance was measured continuously for 5 min at 340 nm and 37 °C in a Microplate Spectrophotometer (XMark). The non-enzymatic reaction of CDNB with GSH measured without enzyme solution served as control. Changes in absorbance per min were converted into nmol CDNB conjugated min-1 mg-1 protein using the extinction coefficient of the resulting 2,4-dinitrophenyl-glutathione: e340 nm = 9.6 mM-1 cm-1 (Habig et al. 1974).
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Total antioxidant capacity was measured based on the generation of the Fe2?-o-phenanthroline complex, as the overall reducing agents in sample supernatant reduced Fe3? to Fe2? in the sample supernatant which reacted with the substrate o-phenanthroline. Stable color of Fe2?-o-phenanthroline complex was measured at 520 nm 37 °C. One unit of T-AOC was defined as the amount necessary to increase the absorbance by 0.01 per min per mg protein at 37 °C (U mg-1 protein). Statistical analysis Treatment effects (temperature and duration) were subjected to one- or two-way analysis of variance (ANOVA) by the general linear model procedure of SPSS 16.0 for Windows (SPSS, Chicago, IL, USA), and when significant effects were obtained, mean differences were separated by Tukey’s least significant difference (LSD) test.
Results Mortality of the predatory mites ranged from 0.3 to 3.6 % in response to cold hardening (0–15 °C), and from 0.4 to 12.7 % under heat shock (35–41 °C), respectively. However, more than 27 % mites were dead being stressed for 5 h at 44 °C. Dead mites were excluded from the assays. Changes in LPO levels Lipid peroxidation levels (expressed as MDA concentration) in response to various thermal stresses are shown in Table 1. Two-way ANOVA revealed that the MDA concentrations in N. cucumeris were significantly affected by treatment temperature (F8,105 = 53.922; P \ 0.001) and duration (F3,105 = 71.153; P \ 0.001), and their interaction (F24,105 = 2.297; P = 0.004). MDA concentration in N. cucumeris, recorded with only 11.1 nmol mg-1 protein, was lowest at 25 °C. However, when the mites were exposed for 1 h it significantly increased among treatments in response to cold hardening or heat shock. For this duration, MDA concentration showed a decline with the rising treatment temperatures range from 0 to 15 °C, whereas it enhanced with temperatures rising from 35 to 44 °C, respectively. When thermal stress lasted for 2 h, it increased continuously among treatment temperatures and peaked at 41 °C (recorded as 36.6 nmol mg-1 protein), 3.3 times higher than the control. For treatment durations longer than 2 h, MDA concentrations decreased with the extension of durations. Changes in activities of antioxidant enzymes Changes in enzyme activities of N. cucumeris after cold hardening or heat shock for different durations are presented in Table 2. ANOVA showed that CAT activities were significantly affected by treatment temperature (F8,105 = 25.786; P \ 0.001), duration (F3,105 = 12.244; P \ 0.001), and their interaction (F24,105 = 3.382; P \ 0.001). Generally, cold stress had more influence on CAT activities than heat stress. For example, exposed to 5 °C for 2, 3 and 5 h, CAT activities significantly increased compared to the controls, whereas it enhanced only slightly among heat treatments (and these differences were not significant).
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11.09 11.09 11.09 11.09
± ± ± ±
1.01a 1.01a 1.01a 1.01a
26.13 29.66 21.81 17.76
0
± ± ± ±
1.32cde 0.47bcd 1.86b 1.12bc
23.46 28.90 19.99 18.89
5
± ± ± ±
1.20bcd 1.53bcd 1.36b 1.17bc
21.04 25.64 20.31 18.15
10 ± ± ± ±
0.73b 0.71b 1.21b 1.67bc
21.77 29.77 20.48 16.53
15 ± ± ± ±
2.86bc 0.741bcd 1.32b 1.37b
23.68 26.09 20.53 18.87
35 ± ± ± ±
1.06bcd 1.84bc 1.60b 1.52bc
Means within a row followed by different letters are significantly different (ANOVA followed by LSD: P \ 0.05)
1 2 3 5
25
Duration (h) Temperatures (°C)
25.69 31.01 23.95 21.28
38 ± ± ± ±
0.57cde 1.86d 1.77b 1.39 cd
Table 1 Malondialdehyde (MDA) concentration (mean ± SE) (nmol/mg protein) of Neoseiulus cucumeris exposed to thermal stress
27.73 36.55 28.15 23.03
41 ± ± ± ±
1.01de 1.44e 1.07c 0.73d
28.89 ± 1.33e 30.13 ± 1.25cd 22.61 ± 0.47b –
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Exp Appl Acarol
Superoxide dismutase activities were influenced by treatment temperature (F8,105 = 21.209; P \ 0.001), duration (F3,105 = 52.163; P \ 0.001) and their interaction (F24,105 = 2.838; P \ 0.001). SOD activities significantly increased in heat treatments (35–44 °C) for 1 h when compared with all cold treatments and the control (25 °C). For 2 h, SOD activity increased at all temperatures, particularly at 41 °C with the peaked value recorded as 242.9 U mg-1 protein. SOD activity decreased at all temperatures when exposure time was extended to 3 h. At 15 °C, SOD activity was significantly declined relative to the control. Glutathione S-transferases activities were affected by temperature (F8,105 = 36.604; P \ 0.001), duration (F3,105 = 28.937; P \ 0.001) and their interaction (F24,105 = 7.510; P \ 0.001). When exposure was extended to 2 h, GSTs activities showed significant increases compared with the control following exposure to 0, 5, 10, 35, 38 and 41 °C, respectively. The maximum value was recorded as 302.3 nmol min-1 mg-1 protein at 5 °C. GSTs activities of the 2-h treatment at 35–41 °C enhanced significantly compared to the control. GSTs activities were very high when exposed for 3 and 5 h to 5 or 10 °C. Peroxidase activities were significantly affected by temperature (F8,105 = 29.635; P \ 0.001), duration (F3,105 = 19.802; P \ 0.001) and their interaction (F24,105 = 4.670; P \ 0.001). POX activities were higher relative to the control after exposure for 1 h to 35, 38 and 41 °C, whereas it showed a notable decrease after cold stress at 0, 5 and 10 °C. Similar effects were found when exposure lasted 2 h, and no notable difference among treatment temperatures was seen when exposed for 3 h. Significant decreases, relative to the 25 °C control, were found after exposure for 5 h to 0, 5, 10, and 35 °C. Total antioxidant capacities were significantly influenced by temperature (F8,105 = 6.952; P \ 0.001), duration (F3,105 = 21.973; P \ 0.001) and their interaction (F24,105 = 3.430; P \ 0.001). When exposed to 0 °C for 1 h, T-AOC significantly increased compared to the control, whereas it reverted to the control level 1 h later. The same situation occurred at 44 °C. No significant difference was observed among treatment temperatures lasting 2 or 3 h. Significant decreases were observed at 5 h exposure to 10, 15, and 35 °C.
Discussion Temperature is one of the most important environmental variables that induce physiological change in organisms (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, antifreeze proteins and amino acids (Storey 1997; Wang and Kang 2005). Previous studies indicated that antioxidant enzymes and heat shock proteins are associated with responses to high temperature and other stresses in nature in a wide range of organisms (Feder and Hofmann 1999; Martindale and Holbrook 2002; Sørensen et al. 2003). The predatory mite N. cucumeris is a poikilothermic organism, and its body temperature is highly affected by the ambient temperature. In this study, we showed that LPO and antioxidant enzymes (CAT, SOD, GSTs, POX, and T-AOC) in N. cucumeris changed significantly under various thermal stresses. The mites used in the current study were mass-reared under optimal conditions for reproduction and development, and they had never been subjected to thermal stress. Commercially available mites are thought to be inherently less cold hardy and to have a lower capacity to acclimate to low temperatures than wild ones (Morewood 1993). Lipid peroxidation is a well-known mechanism of cellular injury in both vertebrates and invertebrates, and acts as a biological marker of oxidative stress in cells and tissues (Del Rio
123
123
10.08 ± 2.11ab 17.958 ± 2.45c
8.06 ± 0.74a
182.93 ± 6.17b
182.93 ± 6.17a
3
5
18.21 ± 0.80a
18.21 ± 0.80c
3
5
26.86 ± 2.34c
15.89 ± 0.56bc
15.93 ± 0.95a
19.67 ± 1.02a
185.70 ± 1.59a
11.26 ± 0.65ab
8.45 ± 0.69a
182.21 ± 6.55a
180.53 ± 6.09b 175.78 ± 5.94a
159.39 ± 2.48a
217.91 ± 3.07cd 197.56 ± 4.87b
181.60 ± 5.50a
13.39 ± 1.04b
12.04 ± 0.49bc
21.70 ± 1.82b
15.88 ± 1.20bc
16.57 ± 0.60a
19.43 ± 1.36a
192.27 ± 4.60a
243.88 ± 6.99b
229.44 ± 8.54ab
13.38 ± 1.10ab
15.31 ± 0.73a
17.96 ± 1.94a
20.67 ± 0.48b
11.01 ± 1.26a
13.62 ± 0.97a
17.13 ± 1.42a
16.41 ± 0.98ab
211.33 ± 3.37ab 227.81 ± 5.75bc
206.28 ± 8.74a
184.21 ± 8.76a
11.39 ± 0.85a
13.44 ± 3.04a
19.11 ± 1.22a
14.87 ± 0.62a
212.37 ± 6.88ab
241.20 ± 19.43b
307.02 ± 26.29c
220.20 ± 10.57ab 268.09 ± 12.46d
201.69 ± 2.2a
208.25 ± 6.85ab 238.16 ± 20.68b
205.80 ± 4.53a
262.65 ± 6.87b
206.39 ± 8.54a
213.73 ± 5.61d
230.67 ± 3.16de
212.05 ± 8.69b
8.73 ± 0.18a
8.38 ± 0.77a
11.61 ± 0.43b
8.47 ± 0.98a
38
9.99 ± 0.99abc
8.53 ± 0.92a
9.52 ± 0.70ab
8.20 ± 0.36a
238.35 ± 2.97b
221.42 ± 5.73c
196.86 ± 8.10a
180.62 ± 5.14b
242.86 ± 7.48e
213.60 ± 2.43b
41
15.35 ± 0.94bc
17.64 ± 1.75a
18.22 ± 1.17a
14.81 ± 2.00a
225.54 ± 7.25bc
248.22 ± 4.60b
292.69 ± 11.05c
313.34 ± 9.19e
187.93 ± 8.80a
15.89 ± 0.37bc
16.02 ± 1.30a
16.53 ± 0.70a
17.02 ± 2.82ab
226.90 ± 5.98bc
240.63 ± 11.69b
290.60 ± 1.75c
302.31 ± 4.71e
189.42 ± 6.34a
189.51 ± 4.60abc 196.31 ± 5.06bc
229.99 ± 10.69b
205.31 ± 9.06abc 199.15 ± 3.49ab
207.99 ± 1.62a
205.82 ± 3.42cd
221.42 ± 1.52cd
202.81 ± 3.82b
8.81 ± 0.59a
8.88 ± 0.41ab
9.03 ± 0.98ab
9.98 ± 0.93abc
35
222.90 ± 10.61d 182.13 ± 12.14ab 172.07 ± 2.59ab
231.77 ± 4.98b
Means within a row followed by different letters are significantly different (ANOVA followed by LSD: P \ 0.05)
18.20 ± 0.80ab
18.21 ± 0.80a
1
2
T-AOC (U mg-1 protein)
204.64 ± 7.45a
233.53 ± 9.78ab 205.57 ± 15.28a 205.88 ± 3.01a
233.53 ± 9.78c
3
5
203.78 ± 6.37a
233.53 ± 9.78bc 202.34 ± 18.81a 204.39 ± 5.08a
233.53 ± 9.78ab 198.21 ± 14.16a 214.67 ± 7.86ab
279.45 ± 8.78b
1
193.39 ± 8.68a
2
POX (U mg-1 protein)
194.79 ± 6.16bc 210.95 ± 6.34cd 255.71 ± 4.36e
194.79 ± 6.16a
3
5
302.31 ± 11.43c
194.80 ± 6.16ab 184.57 ± 6.44a
194.79 ± 6.16a
223.02 ± 5.68b
9.50 ± 1.21ab
15
17.96 ± 1.67cd 15.971 ± 0.76c
12.32 ± 1.32bc
10
208.25 ± 10.15bc 195.50 ± 7.33ab 185.48 ± 4.23a
191.19 ± 3.87a
189.38 ± 3.36bc
222.71 ± 3.08cd
1
192.55 ± 1.79a
188.78 ± 1.79b
215.51 ± 1.30c
197.70 ± 5.77ab 184.15 ± 4.09a
16.66 ± 1.90d
2
GSTs (nmol min-1 mg-1 protein)
182.93 ± 6.17a
182.93 ± 6.17a
1
2
SOD (U mg-1 protein)
5
12.87 ± 1.79c
8.06 ± 0.76a
10.73 ± 0.79abc 19.87 ± 1.05d
3
16.67 ± 1.40c
8.06 ± 0.74a
5
8.06 ± 0.74a
12.97 ± 1.13c
0
2
protein)
25
Temperatures (°C)
1
CAT (U mg
-1
Enzymes Durations (h)
Table 2 Antioxidant enzymes activities (mean ± SE) of Neoseiulus cucumeris exposed to thermal stress
–
15.89 ± 0.86a
17.35 ± 1.91a
27.27 ± 1.26c
–
219.27 ± 4.58ab
243.82 ± 14.82b
251.46 ± 9.73cd
–
165.70 ± 14.62a
194.75 ± 9.86a
202.77 ± 4.60abc
–
190.39 ± 7.75bc
223.76 ± 4.17cd
205.89 ± 4.78b
–
9.23 ± 0.72ab
9.67 ± 0.41ab
10.66 ± 1.03abc
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Exp Appl Acarol
Exp Appl Acarol
et al. 2005). A significantly elevated level of MDA concentration in N. cucumeris was observed in response to thermal stress. This clearly demonstrates that thermal stress in N. cucumeris is associated with the process of LPO and other responses to oxidative stress, similar to other arthropods (Yang et al. 2010; Jia et al. 2011; Jena et al. 2013). Initially, MDA concentrations in N. cucumeris induced by thermal exposure increased strongly, but after longer exposure it decreased gradually. The descending MDA with longer exposure was probably a result of the antioxidant system eliminating oxidative stress, and similar phenomenon was found in citrus red mite P. citri (Yang et al. 2010). This suggests that both N. cucumeris and P. citri may become acclimated to thermal stress with the increasing exposure duration. However, the difference between them is that LPO remained at high levels in the predatory mite with extending durations compared to the control, which suggests that this mite may be ineffective in eliminating ROS induced by thermal stress. This matches the general pattern that higher trophic levels are more sensitive to adverse environmental temperatures than lower trophic levels (Voigt et al. 2003; Montserrat et al. 2013b). Catalase, SOD, POX, and GSTs are four crucial antioxidant defense enzymes, which can work synergistically against oxidative stress generated by high concentrations of ROS inside the cell. Among these antioxidant enzymes, CAT is considered to be the principal H2O2 scavenging enzyme (Jena et al. 2013), because arthropods are deficient in seleniumdependent glutathione peroxidase, which is a scavenger in other organisms (Sohal et al. 1990). However, CAT removes H2O2 only at high cellular concentrations, whereas it is inefficient for H2O2 removal at low concentrations (Ahmad et al. 1991). In a previous study, CAT activity was too low to detect in the herbivorous citrus red mite P. citri, 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. cucumeris exposed to the same thermal stress conditions. Apparently, it enhanced the removal of H2O2 and prevented damage by oxidative stress. The difference in CAT activity between the predatory mite N. cucumeris and its prey P. citri suggests that the two species differ in antioxidant defense though they both belong to the Acari. Elevated CAT activities induced by thermal stress were also observed in the oriental fruit fly Bactrocera dorsalis (Jia et al. 2011), and in the fat body of 5th instar silkworm, Bombyx mori (Nabizadeh and Jagadeesh Kumar 2011). It has been well documented that SOD plays an important role in reducing the high levels of superoxide radical induced by low or high surrounding temperatures (Celino et al. 2011). In our study, SOD activities in N. cucumeris under thermal stress significantly increased during early exposures and no significant changes were observed during later compared with control. This suggests that SOD was induced by changes in surrounding temperatures and then protected the mites from thermal stress. In addition, N. cucumeris became acclimated to thermal stress; similar results were found in other studies (An and Choi 2010; Yang et al. 2010; Jena et al. 2013). Both SOD and CAT can directly eliminate surplus ROS in a coordinated way. SOD removes O2- through the process of dismutation to O2 and H2O2, and then H2O2 is sequentially reduced to H2O and O2 by CAT (Kashiwagi et al. 1997). However, when exposed to low temperatures, e.g. 5 and 10 °C, CAT activity was higher than SOD activity. Because CAT removes H2O2 only at high cellular concentrations (Ahmad et al. 1991), it would be attributed to the other possible processes of H2O2 being produced under such cold stresses. Glutathione S-transferases are relevant in the metabolism of LPO products in specific tissues in which they are expressed to protect organisms against oxidative stress (Sawicki et al. 2003; Nair and Choi 2011). Six classes of GSTs were found, including delta, epsilon, omega, sigma, theta, and zeta. The classes of delta and sigma GSTs have been considered
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to play a key role in antioxidant defense in various organisms (Nair and Choi 2011). Sigma GST has a dominant role in protection against oxidative damage in Drosophila melanogaster (Singh et al. 2001). In the present study, GSTs activity of crude enzyme in N. cucumeris was determined by the procedure of Habig et al. (1974), which has been shown to work well in several studies (Yang et al. 2010; Jia et al. 2011; Chen et al. 2013). In this study, the increased activity of the enzyme exposed to 5 °C for 2, 3, and 5 h, suggests that GSTs were acting in removal of toxic LPO products caused by acute thermal stress. However, no significant difference of GSTs activity was observed among treatment temperatures when the mites were exposed for 1 h except for 41 °C. This may indicate that GSTs were involved in the inactivation of accumulated toxic LPO products for 1 h, which agrees with previous findings in the oriental fruit fly B. dorsalis (Jia et al. 2011). In most cases, GSTs activities keep pace with MDA concentrations, mainly because GSTs consume the products of LPO other than H2O2 (Joanisse and Storey 1996). However, in our study, MDA concentrations in each treatment were higher than in control, indicating that the mites could not scavenge LPO products effectively, especially when exposed for a long time. Glutathione S-transferases with POX can metabolize lipid peroxides and POX can break down H2O2 (Jia et al. 2011). In our study, POX activities significantly increased after heat shock for 1–2 h compared to the control, probably as a result of a significant increase of SOD activity in response to increased H2O2 concentration. However, it decreased to control levels or (much) lower with the extension of exposure time. Our result may indicate that POX activity in N. cucumeris was efficiently induced by thermal stress, which congrues with findings in Helicoverpa armigera (Meng et al. 2009). However, in response to low-temperature stress, POX activity in N. cucumeris showed a decrease among all durations. POX activity was also decreased in oriental fruit fly (Jia et al. 2011), whereas in citrus red mite it did not change significantly (Yang et al. 2010). Because the functions of POX in response to thermal stress are not yet fully understood, further studies are needed to explain this pattern. Total antioxidant capacity is a commendable measure of the ability of all antioxidants existing in an organism, and it has been used as a tool to evaluate redox status in recent reports (Ghiselli et al. 2000). In N. cucumeris, T-AOC enhanced significantly when exposed to two extreme temperatures (0 and 44 °C) for 1 h when compared to control. This indicates that T-AOC may be sufficient to cope with oxidative stress and free radical formulation after a short shock. However, when the mites were exposed longer (2–5 h), there appeared to be no obvious change (2 and 3 h) or they were not well equipped (5 h) to defend against ROS, compared with control. Apart from enzymes, some non-enzymatic substances, e.g. trehalose (Mahmud et al. 2010) and vitamin E (a-tocopherol) (Kaur et al. 2009), can play important roles in antioxidant stress. A recent study confirmed that heat shock proteins cooperate with antioxidant enzymes to deal with ROS damage (Rosa et al. 2012). We only determined the activity of (antioxidant) enzymes that play a vital role in antioxidant defense at the physiological level. However, we did not study the synergistic effect of heat-shock proteins and antioxidant enzymes, nor the simple effect of heat shock alone in relation to thermal stress. This deserves further investigation. In conclusion, our results indicated that the redox balance in N. cucumeris was disturbed by thermal stress, which results in oxidative stress. The antioxidant enzymes are intrinsically linked and dependent upon the activity of one another and, therefore, one would expect to see correlative changes in their activity (Rosa et al. 2012). Here, this coordinated effect was confirmed in female adults of N. cucumeris. Increasing levels of SOD activity, expressed in a time-dependent manner, indicated that there was an increase of superoxide
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production. Concomitantly, CAT and POX activity also was elevated when the mites were exposed to cold and heat shock, respectively, possibly to increment the capability to catabolize H2O2 that generated from the dismutation of the O2- radical by SOD. GSTs activity, also expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. Although T-AOC activity in N. cucumeris changed slightly among treatments, it would be of importance as well in mite physiological adaptations. The higher levels of LPO in N. cucumeris serve an important signal demonstrating that antioxidant enzymes are not always adequate to counteract the production of ROS induced by thermal stresses. Further studies of the effects of thermal stress of N. cucumeris in hatch rate of eggs, survival rate and fecundity of female adults should be investigated, based on our results that surplus ROS accumulated after cold hardening or heat shock. Acknowledgments This study was supported by the National Public Benefit Industry (Agriculture) Research Projects, the Ministry of Agriculture, (200903032).
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