Effects of Cd, Zn, or Pb Stress in Populus alba berolinensis on the Antioxidant, Detoxifying, and Digestive Enzymes of Lymantria dispar

Effects of Cd, Zn, or Pb Stress in Populus alba berolinensis on the Antioxidant, Detoxifying, and... Abstract For investigating the physiological responses of herbivores to the heavy metal–stressed woody host plants, the activities of antioxidant, detoxifying, and digestive enzymes in the gypsy moth larvae, Lymantria dispar, that were fed with different heavy metal–stressed poplar seedling (Populus alba berolinensis) leaves were studied. The heavy metal treatments included Cd-treated pot soil (1.5 mg/kg), Zn-treated pot soil (500 mg/kg), and Pb-treated pot soil (500 mg/kg), plus an untreated pot soil as the control. Our results showed that compared with the untreated control, superoxide dismutase (SOD) and catalase (CAT) activities in Cd or Zn treatment group were gradually suppressed with the increases of larval ages, but Pb treatment had no significant effects on SOD activities and significantly increased the CAT activities in both fourth and fifth instar larvae; acid phosphatase (ACP) activities were gradually activated and alkaline phosphatase (AKP) activities were gradually inhibited with the increases of larval ages in Cd or Pb treatment group, but Zn treatment significantly increased the activities of ACP and AKP both in fourth and in fifth instar larvae. All three heavy metals tested did not show any significant effects on the amylase and protease activity in the fourth instar larvae but increased their activities in fifth instar larvae. These results suggest that antioxidant, detoxifying, and digestive enzymes constituted the basic defense system for gypsy moth larvae to resist the toxicity originated from the accumulated Cd, Zn, or Pb in poplar leaves, but their defense level varied with metals investigated and larval developmental stages. heavy metal, Lymantria dispar, antioxidant enzyme, detoxifying enzyme, digestive enzyme Various anthropogenic activities, such as mining, fossil fuels burning, and chemical fertilizer application, continuously bring heavy metals into the environment, causing serious ecological effect to the natural environment. Among heavy metal pollutants, Cd (cadmium) and Pb (lead), as typical nonessential elements, are unable to participate in various metabolic reactions in herbivores (Zhang et al. 2011, Shu et al. 2015). Besides, Zn (zinc) is an essential element and performs a vital function in protein, lipid, and carbohydrate metabolisms, but at high concentrations it can induce a broad range of physiological, biochemical, and behavioral dysfunctions in herbivores (Filipiak et al. 2010, Sahu et al. 2015). Toxicity of various xenobiotics including heavy metals is tightly related to their ability to catalyze oxidative reactions, in turn, generating reactive oxygen species (ROS) and oxidative stress in herbivores (Valko et al. 2016). To survive in metals contamination regions, herbivores activate an effective antioxidant defense mechanism to protect themselves against oxidative damage (Ihechiluru et al. 2015, Yuan et al. 2016). The antioxidant defense system is mainly composed of enzymes and low molecular nonenzyme compounds that have functions of inhibiting oxidative damage (Barata et al. 2005, Gauthier et al. 2016). Superoxide dismutase (SOD) and catalase (CAT), as important antioxidant enzymes, constitute the first line of defense in herbivores to remove ROS. In general, SOD catalyzes the breakdown of superoxide radical anions into hydrogen peroxide by the removal of an electron, which is then further converted into molecular dioxygen and water by CAT (Kalender et al. 2013, Sun et al. 2016). Variations of SOD and CAT activities have been well investigated for many herbivores under the heavy metal stress. For instances, the effects of heavy metal exposures on SOD and CAT activities in Oxya chinensis (Zhang et al. 2011) and Galleria mellonella (Wu and Yi 2015) varied with heavy metal types, concentrations, and insect developmental stages. Apart from the oxidative damage, herbivores fed on heavy metal–stressed plant may suffer from the heavy metal–mediated plant defense responses such as elevated secondary metabolites in leaves (Rascio and Navari-Izzo 2011, Ali and Hadi 2015). To reduce or combat these adverse effects, herbivores might activate the expression of detoxification enzymes including acid phosphatase (ACP) and alkaline phosphatase (AKP; Wouters et al. 2016, Zhang et al. 2016). Although phosphatases are not directly involved in the heavy metal detoxification, ACP and AKP may indirectly improve the ability of organisms to tolerate heavy metal exposures through catalyzing the hydrolysis of various phosphomonoesters, taking part in transphosphorylation and improving the phagocytosis responses (Calvo-Marzal et al. 2001). Moreover, these nonspecific metaloenzymes involve in digestions, carbohydrate metabolism, ion transport, excretion, and water reabsorption (Srivastava and Saxena 1967). Several studies have reported that enhanced activities of ACP and AKP in various herbivores are critical to improve the ability to cope with stressful environments such as heavy metals (Zhang et al. 2016), pesticide (Li et al. 2011), and light intensity (Lu et al. 2013). Utilizations of antioxidant enzymes to remove excessive ROS and of detoxification enzymes to resist the heavy metal–mediated plant defense responses will consume a large amount of energy (Cervera et al. 2004, van Ooik et al. 2007). The main source of energy for herbivores is through the food intakes, and to provide the necessary energy for the detoxification process, the basic strategy for herbivores survivals under heavy metal stress is to improve the efficacy of food utilization or the activity of digestive enzymes (Baghban et al. 2014). Digestive enzymes are usually made up of proteases, amylases, and lipases, which represent the digestion of proteins, starches, and lipids in foods, respectively (De Coen and Janssen 1997). Since herbivores digestive tract is the main site to ingest and accumulate exogenous toxic substances, the activity of digestive enzymes secreted by the digestive tract is tightly related to the food quality and the toxicity of the chemicals contained in the food (Silva et al. 2009, Teimouri et al. 2015). At present, a large number of studies have been conducted to study the effects of heavy metal stress on antioxidant, detoxifying, and digestive enzyme activities in herbivores by adding heavy metals to artificial diets. However, little is known about the physiological responses of herbivores to heavy metal–stressed woody plants. Lymantria dispar is one of the major forest pest insects in most parts of China with well-known knowledge on its biology, ecology, and physiology. Populus alba berolinensis, as an excellent tree species, has been widely planted to create fast-growing and high-yield forests, shelter-belt forests, and urban forests. Our previous studies found that Cd, Zn, or Pb can transfer from soils contaminated with corresponding heavy metals through P. alba berolinensis seedlings and gypsy moth, and their contents in leaves of seedlings (Jiang et al. 2018) and in gypsy moth larvae (unpublished) all were significantly higher than those of the untreated control; however, Cd-, Zn-, or Pb-stressed P. alba berolinensis did not show significant effects on larval survival, pupation, and emergence rates (Jiang and Yan 2017). We conjectured that gypsy moth larvae might activate an effective detoxification mechanism for attenuating the negative effects of plant-mediated defense responses and heavy metals in leaves. To test this hypothesis and further analyze the physiological responses of herbivores to the heavy metal–stressed woody host plants, the biological activities of antioxidant, detoxifying, and digestive enzymes in the gypsy moth larvae that were fed with different heavy metal–stressed poplar seedling (P. alba berolinensis) leaves were investigated in the current study. Materials and Methods Plant Materials P. alba berolinensis seedlings used in the experiments were generated by vegetative propagation in the Ping Shan Forest Nursery of Heilongjiang province, P. R. China. In late April 2016, cuttings were prepared and planted in about 10-liter pots (23 cm in diameter; 25 cm tall; 1 plant per pot) each filled with 5 kg of 1:1:1 mixture of sand, turf soil, and native soil. Two months later, the soils in the seedling pots were treated with cadmium chloride solution at the final Cd2+ concentration of 1.5 mg/kg (denoted as Cd), with zinc sulfate solution at the final Zn2+ concentration of 500 mg/kg (denoted as Zn) or with lead acetate solution at the final Pb2+ concentration of 500 mg/kg (denoted as Pb), and the seedling pots without heavy metal treatment were used as control group (denoted as CK). The Cd, Zn, or Pb concentration used in present experiments was set in accordance with their corresponding soil environmental quality standards for normal plant growths (Formulated by State Department of Environmental Conservation [1995], China). Insect Rearing The gypsy moth egg masses were collected from the surrounding areas of Northeast Forestry University, Harbin, China in March 2016 and kept in a refrigerator at 4°C until late June. The egg masses were incubated in a laboratory light incubator at 25 ± 1°C and 70 ± 1 relative humidity with a 16:8 (L:D) h photoperiod. Upon hatching, gypsy moth larvae were then reared on artificial diet (purchased from Institute of Forest Ecological Environment and Protection, Chinese Academy of Forestry Sciences) in laboratory under the same condition until the second instar. The newly molted second instar larvae (n = 80) were reared on the fresh poplar leaves collected from each treatment or control group, and the poplar leaves were refreshed once a day until all the larvae grew up to fifth instar or died. During the experiments, the newly molted fourth (n = 30) and fifth instar larvae (n = 30) were placed in a −80°C ultra-low temperature freezer for subsequent determination of enzyme activities (of antioxidant, detoxifying, and digestive enzymes). SOD and CAT Activity The activities of SOD and CAT were determined in 10% (w/v) homogenates in 0.05 mol/liter phosphate buffer at pH 8.8. Homogenization of three larvae from different developmental stage was performed for each replicate, and three replicates for each treatment or control group were tested. Crude homogenates were centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatants were stored on ice for enzyme activity assays. SOD activity in the supernatants was determined according to the method described by McCord and Fridovich (1968). CAT activity was assayed as the rate of H2O2 decomposition at 240 nm, based on the method described by Aebi (1984). The SOD and CAT activity levels were, respectively, expressed as units of enzymes per milligram protein (U/mg) and units of enzymes per gram protein (U/g), in which one unit of SOD is defined as the amount of sample that cause 50% inhibition of pyrogallol autoxidation, and one unit of CAT was defined as the amount of sample required to catalyze micromoles H2O2 per minute. AKP and ACP Activities The homogenizations of three larvae from different developmental stages in each group were performed in ice-cold saline 0.15 M NaCl, and three replicates for each group were used. Crude homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatants were used for AKP and ACP activity assays according to the method described by Nemec and Socha (1988). The AKP and ACP activity levels were expressed as units of enzymes per gram protein (U/g), where one unit of both AKP and ACP activities is defined as the amount of sample that released 1 μmol of p-nitrophenol per minute from phenyl phosphatase under the assay conditions. Protease and Amylase Activity The whole bodies of three larvae from different developmental stages in each group were homogenized in ice-cold deionized water, and three replicates for each group were used. Crude homogenates were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatants were used for enzyme activity assays. Protease activity was determined according to the method described by García-Carreño and Haard (1993). Amylase activity was determined based on the method described by Bernfeld (1955). The protease and amylase activity levels were, respectively, expressed as units of enzymes per gram protein (U/g) and units of enzymes per milligram protein (U/mg), where one unit of amylase activity and protease activity is defined as the amount of sample that produced 1-mg maltose in 30 min at 35°C and the amount of sample that produced 1-μg tyrosine per minute at 37°C, respectively. Total Protein Assay Protein concentration in enzyme extracting solution was assayed using the Coomassie brilliant blue G250 method described by Bradford (1976), with bovine serum albumin as a protein standard. Statistical Analysis The statistical significance of data in each treatment group and CK was determined by an independent sample t-test at the 0.05 level after testing for variance homogeneity and normal distribution or achieving variance homogeneity and normal distribution by log-transforming if necessary. Results Antioxidant Enzyme Activity As shown in Figs. 1 and 2, the SOD (t = 2.936; df = 4; P = 0.043) and CAT (t = 5.956; df = 4; P = 0.004) activities in the fourth instar L. dispar larvae that fed on the Cd-stressed poplar leaves were significantly higher than those that fed on the untreated control leaves, whereas the SOD and CAT activities in the fifth instar larvae under the Cd stress were lower than those in the control group. Fig. 1. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the SOD activities of the fourth–fifth instar gypsy moth larvae. The values presented in the graphs are the means ± standard deviations (n = 3). Asterisks (*) represent significant differences among each treatment and CK within the same developmental stage (P < 0.05). The same holds true for Figs. 2–6 below. Fig. 1. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the SOD activities of the fourth–fifth instar gypsy moth larvae. The values presented in the graphs are the means ± standard deviations (n = 3). Asterisks (*) represent significant differences among each treatment and CK within the same developmental stage (P < 0.05). The same holds true for Figs. 2–6 below. Fig. 2. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the CAT activities of the fourth–fifth instar gypsy moth larvae. Fig. 2. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the CAT activities of the fourth–fifth instar gypsy moth larvae. The SOD and CAT activities of the fourth instar larvae that fed on the Zn-stressed poplar leaves were not significantly different from those of the control group; however, the fifth instar larvae significantly showed lower SOD (t = 2.789; df = 4; P = 0.049) and CAT (t = 4.856; df = 4; P = 0.008) activities than that of untreated control (Figs.1 and 2). The SOD activities of the fourth and fifth instar larvae in Pb treatment group were not significantly different from those in controls (Fig. 1), but the CAT (t = 9.837, df = 4, P = 0.001 for fourth instar larvae; t = 4.115, df = 4, P = 0.015 for fifth instar larvae) activities in both instars larvae that fed on the Pb-stressed leaves were all significantly higher than those of the controls (Fig. 2). Detoxifying Enzyme Activity As shown in Figs. 3 and 4, the ACP and AKP activities of the fourth instar gypsy moth larvae in Cd or Pb treatment group were not significantly different from those in the control group; but ACP (t = 4.422, df = 4, P = 0.011 for Cd stress; t = 3.891, df = 4, P = 0.018 for Pb stress) and AKP (t = 3.298, df = 4, P = 0.03 for Cd stress; t = 3.993, df = 4, P = 0.016 for Pb stress) activities in the fifth instar larvae exposed to Cd or Pb stress showed an opposite pattern, with the ACP activity being significantly higher and AKP activity being significantly lower than that of their corresponding control. In contrast, Zn-stressed poplar leaves significantly increased the activities of both ACP (t = 5.412, df = 4, P = 0.006 for fourth instar larvae; t = 5.747, df = 4, P = 0.005 for fifth instar larvae) and AKP (t = 4.9482, df = 4, P = 0.008 for fourth instar larvae; t = 15.117, df = 4, P = 0.000 for fifth instar larvae) in fourth and fifth instar larvae. Fig. 3. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the ACP activities of the fourth–fifth instar gypsy moth larvae. Fig. 3. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the ACP activities of the fourth–fifth instar gypsy moth larvae. Fig. 4. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the AKP activities of the fourth–fifth instar gypsy moth larvae. Fig. 4. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the AKP activities of the fourth–fifth instar gypsy moth larvae. Digestive Enzymes Activity As shown in Figs. 5 and 6, heavy metal treatments did not show any effects on the digestive enzyme (amylase and protease) activity in the fourth instar larvae; however, they all significantly increased the amylase (t = 3.939, df = 4, P = 0.017 for Zn stress; t = 5.156, df = 4, P = 0.007 for Pb stress) and protease activities (t = 3.713, df = 4, P = 0.021 for Zn stress; t = 4.456, df = 4, P = 0.011 for Pb stress) in fifth instar larvae except the Cd-stressed leaves on protease. Fig. 5. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the amylase activities of the fourth–fifth instar gypsy moth larvae. Fig. 5. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the amylase activities of the fourth–fifth instar gypsy moth larvae. Fig. 6. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the protease activities of the fourth–fifth instar gypsy moth larvae. Fig. 6. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the protease activities of the fourth–fifth instar gypsy moth larvae. Discussion and Conclusion Antioxidant Enzyme Activity in Gypsy Moth Larvae As an important detoxification mechanism, the antioxidant defense system has been widely used by herbivores to alleviate oxidative damages under heavy metal stress (Ihechiluru et al. 2015). Functionally interconnected SOD and CAT are two of the most important antioxidant enzymes in the antioxidant defense system (Li et al. 2010). Our results showed that SOD activity in the fourth instar gypsy moth larvae that fed on the Cd-stressed poplar leaves was significantly higher than that in the control. The enhanced SOD enzyme activity might be in response to the superoxide radicals induced by the Cd stress and then converted harmful superoxide radicals into hydrogen peroxide via Haber–Weiss reaction (Valko et al. 2016). Furthermore, in the present study, the response of CAT in the fourth instar larvae to Cd stress was consistent with that of SOD and was significantly higher than that of control. The main role of enhanced CAT activity is used to remove the hydrogen peroxide catalyzed by SOD, further attenuating the oxidative damage caused by reactive oxygen to gypsy moth larvae (Krishnan and Kodrík 2006). Taken together, our findings indicate that the SOD-CAT defense system used for scavenging superoxide and peroxide plays a vital role in preventing Cd toxicity in the fourth instar gypsy moth larvae. However, with the increase of feeding duration, the SOD and CAT activities of the fifth instar larvae in the Cd treatment group were lower than those of the control group. In general, a low concentration heavy metal stress might activate the antioxidant defense mechanism, but with the increase of accumulated concentrations, the ROS levels induced by heavy metals exceed the scavenging ability of herbivores and will inhibit the activity of antioxidant enzymes, thus exacerbating the oxidative damages (Yuan et al. 2016). In addition, another possible mechanism for Cd-stressed inhibition of the antioxidant enzyme activities in herbivores might be that Cd can react directly on enzyme activity sites or replace metal cofactors, which have been well investigated in many organisms (Cuypers et al. 2010, Vlahović et al. 2015). Our results showed that the SOD and CAT activities of the fourth instar larvae were not significantly different from those of the control after feeding on the leaves of poplar leaves under Zn stress, indicating that the accumulation of Zn in the fourth instar larvae did not significantly increase the ROS formation. These data reinforce the findings reported in previous studies (Maryanski et al. 2002, Jelaska et al. 2007), demonstrating that the concentrations of nutritional metals (i.e., Zn) in the arthropods can be regulated more efficiently than that of nonessential metals. Furthermore, the second explanation for nonsignificant effects of Zn stress on antioxidant enzymes activities might be nonenzymatic cellular antioxidants can act as a substitute for antioxidant enzymes (Gauthier et al. 2016). However, the response pattern of SOD and CAT in the fifth instar larvae to the Zn stress was consistent with that to the Cd stress, and their activity was significantly lower than those of the control, suggesting that excessive Zn could also inhibit antioxidant enzyme activity as did nonessential metals. Similar observations were reported by Sahu et al. (2015), who indicated that CAT activities were inhibited in the whole body tissue of the tasar silkworm larvae Antheraea mylitta when exposed to Zn. Our results showed that effects of Pb stress on antioxidant enzymes in the gypsy moth larvae were not developmental stage specific; the SOD activities in fourth instar and fifth instar larvae were not significantly different from those in controls, but the activity of CAT was significantly higher than that of the control. These results are in line with the findings of Mirčić et al. (2013) who reported that heavy metal exposure significantly increased the CAT activity of the gypsy moth, but had no significant effects on SOD activity. They suggested that the peroxides that stimulate the increase of CAT activity are not derived from the degradation processes of superoxide by SOD, but mainly derived from the metabolic oxidoreduction processes performed by various oxidases in peroxisomes such as xanthine oxidase and cytochrome P450 monooxygenases. Detoxifying Enzyme Activity in Gypsy Moth Larvae Different from the antioxidant system that protects the organisms against exogenous substances via scavenging free radicals, the nonoxidative defense system (ACP and AKP) is mainly through direct degradation of exogenous substances to achieve the purpose of defense (Xia et al. 2000, Chen et al. 2013). Our results showed that the ACP and AKP activities of the fourth instar gypsy moth larvae under Cd and Pb stresses were not significantly different from those of the controls, suggesting that accumulations of Cd and Pb in the fourth instar larvae were insufficient to induce ACP and AKP synthesis of the nonoxidative defense system, and that the activation of antioxidant system under Cd or Pb stress can avoid the oxidative damage of ROS to ACP and AKP. Similar results were reported by Zvereva et al. (2003), who found that esterase activity in leaf beetle larvae Chrysomela lapponica did not differ between polluted and unpolluted sites. In addition, we found that ACP and AKP activities in the fifth instar larvae exposed to Cd or Pb stress presented an opposite state, showing that the ACP activity was significantly higher than that of the control and the AKP activity was significantly lower than that of the control. The main differences between ACP and AKP are their catalytic values for pH in catalytic degradation of xenobiotics. The accumulation of large amounts of Cd or Pb in insect tissues, especially in lysosomes, may therefore result in a partial acid environment that increases the activity of ACP that conducts catalyzed reactions at acidic conditions and weakens the activity of AKP that conducts catalyzed reactions at alkaline conditions (Vlahović et al. 2013, Xie et al. 2016). However, our results showed that Zn-stressed poplar leaves significantly increased the activities of both ACP and AKP in the fourth–fifth instar larvae, indicating that ACP and AKP might involve in the tolerate mechanisms of gypsy moth larvae to Zn stress, and that nutritional metals at moderate level can improve the activities of detoxifying enzymes. Our results are in line with previous studies, showing that heavy metals have the inducement effects on ACP and AKP activities in organisms such as G. mellonella and Sphaerodema urinator (Bream 2003, Wu and Yi 2015). Digestive Enzyme Activity in Gypsy Moth Larvae As the main hydrolytic enzymes of digestive function, the changes of amylase and protease activities can reflect the organism’s regulating ability to digestive physiology and adaptability to environment (Lai et al. 2011). Our results showed that after feeding on the Cd-, Zn-, or Pb-stressed poplar leaves, the amylase and protease activities of the fourth instar larvae were not significantly different from those of the control, suggesting that the fourth instar gypsy moth larvae have an effective mechanism that reduces the detrimental consequences of Cd, Zn, or Pb stress without affecting the levels of digestive enzymes (Green et al. 2003, Dar et al. 2015). In addition, we found that Cd-, Zn-, or Pb-stressed poplar leaves caused a hormesis of amylase and protease in the fifth instar larvae. Hormesis, as a widely occurring toxicological phenomenon, may be related to an adaptive response and observed at certain times during heavy metal exposures (Calabrese et al. 2005). In general, herbivores cope with heavy metal stress by improving antioxidant enzymes and detoxifying enzymes require consuming large amounts of energy substances. For example, as demonstrated by Holmstrup et al. (2011), the glycogen contents were significantly reduced when the internal heavy metal concentrations were highly regulated by the earthworms. Thus, increased activities of amylase and proteases in our present study are likely due to the needs of the gypsy moth larvae for hydrolysis of starches and proteins or for elevated utilization efficiency on the food. In summary, our results showed that antioxidant enzymes, detoxifying enzymes, and digestive enzymes constituted effective defense mechanisms for gypsy moth larvae to resist the toxicity originated from the accumulated Cd, Zn, or Pb in poplar leaves, but their defense level varied with heavy metal types and developmental stages. Acknowledgments This research was supported by Excellent academic teacher support project of Northeast forestry university (010602071). The authors declare that they have no competing interests. This article does not contain any studies with human participants or animals performed by any of the authors. 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Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Effects of Cd, Zn, or Pb Stress in Populus alba berolinensis on the Antioxidant, Detoxifying, and Digestive Enzymes of Lymantria dispar

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Abstract

Abstract For investigating the physiological responses of herbivores to the heavy metal–stressed woody host plants, the activities of antioxidant, detoxifying, and digestive enzymes in the gypsy moth larvae, Lymantria dispar, that were fed with different heavy metal–stressed poplar seedling (Populus alba berolinensis) leaves were studied. The heavy metal treatments included Cd-treated pot soil (1.5 mg/kg), Zn-treated pot soil (500 mg/kg), and Pb-treated pot soil (500 mg/kg), plus an untreated pot soil as the control. Our results showed that compared with the untreated control, superoxide dismutase (SOD) and catalase (CAT) activities in Cd or Zn treatment group were gradually suppressed with the increases of larval ages, but Pb treatment had no significant effects on SOD activities and significantly increased the CAT activities in both fourth and fifth instar larvae; acid phosphatase (ACP) activities were gradually activated and alkaline phosphatase (AKP) activities were gradually inhibited with the increases of larval ages in Cd or Pb treatment group, but Zn treatment significantly increased the activities of ACP and AKP both in fourth and in fifth instar larvae. All three heavy metals tested did not show any significant effects on the amylase and protease activity in the fourth instar larvae but increased their activities in fifth instar larvae. These results suggest that antioxidant, detoxifying, and digestive enzymes constituted the basic defense system for gypsy moth larvae to resist the toxicity originated from the accumulated Cd, Zn, or Pb in poplar leaves, but their defense level varied with metals investigated and larval developmental stages. heavy metal, Lymantria dispar, antioxidant enzyme, detoxifying enzyme, digestive enzyme Various anthropogenic activities, such as mining, fossil fuels burning, and chemical fertilizer application, continuously bring heavy metals into the environment, causing serious ecological effect to the natural environment. Among heavy metal pollutants, Cd (cadmium) and Pb (lead), as typical nonessential elements, are unable to participate in various metabolic reactions in herbivores (Zhang et al. 2011, Shu et al. 2015). Besides, Zn (zinc) is an essential element and performs a vital function in protein, lipid, and carbohydrate metabolisms, but at high concentrations it can induce a broad range of physiological, biochemical, and behavioral dysfunctions in herbivores (Filipiak et al. 2010, Sahu et al. 2015). Toxicity of various xenobiotics including heavy metals is tightly related to their ability to catalyze oxidative reactions, in turn, generating reactive oxygen species (ROS) and oxidative stress in herbivores (Valko et al. 2016). To survive in metals contamination regions, herbivores activate an effective antioxidant defense mechanism to protect themselves against oxidative damage (Ihechiluru et al. 2015, Yuan et al. 2016). The antioxidant defense system is mainly composed of enzymes and low molecular nonenzyme compounds that have functions of inhibiting oxidative damage (Barata et al. 2005, Gauthier et al. 2016). Superoxide dismutase (SOD) and catalase (CAT), as important antioxidant enzymes, constitute the first line of defense in herbivores to remove ROS. In general, SOD catalyzes the breakdown of superoxide radical anions into hydrogen peroxide by the removal of an electron, which is then further converted into molecular dioxygen and water by CAT (Kalender et al. 2013, Sun et al. 2016). Variations of SOD and CAT activities have been well investigated for many herbivores under the heavy metal stress. For instances, the effects of heavy metal exposures on SOD and CAT activities in Oxya chinensis (Zhang et al. 2011) and Galleria mellonella (Wu and Yi 2015) varied with heavy metal types, concentrations, and insect developmental stages. Apart from the oxidative damage, herbivores fed on heavy metal–stressed plant may suffer from the heavy metal–mediated plant defense responses such as elevated secondary metabolites in leaves (Rascio and Navari-Izzo 2011, Ali and Hadi 2015). To reduce or combat these adverse effects, herbivores might activate the expression of detoxification enzymes including acid phosphatase (ACP) and alkaline phosphatase (AKP; Wouters et al. 2016, Zhang et al. 2016). Although phosphatases are not directly involved in the heavy metal detoxification, ACP and AKP may indirectly improve the ability of organisms to tolerate heavy metal exposures through catalyzing the hydrolysis of various phosphomonoesters, taking part in transphosphorylation and improving the phagocytosis responses (Calvo-Marzal et al. 2001). Moreover, these nonspecific metaloenzymes involve in digestions, carbohydrate metabolism, ion transport, excretion, and water reabsorption (Srivastava and Saxena 1967). Several studies have reported that enhanced activities of ACP and AKP in various herbivores are critical to improve the ability to cope with stressful environments such as heavy metals (Zhang et al. 2016), pesticide (Li et al. 2011), and light intensity (Lu et al. 2013). Utilizations of antioxidant enzymes to remove excessive ROS and of detoxification enzymes to resist the heavy metal–mediated plant defense responses will consume a large amount of energy (Cervera et al. 2004, van Ooik et al. 2007). The main source of energy for herbivores is through the food intakes, and to provide the necessary energy for the detoxification process, the basic strategy for herbivores survivals under heavy metal stress is to improve the efficacy of food utilization or the activity of digestive enzymes (Baghban et al. 2014). Digestive enzymes are usually made up of proteases, amylases, and lipases, which represent the digestion of proteins, starches, and lipids in foods, respectively (De Coen and Janssen 1997). Since herbivores digestive tract is the main site to ingest and accumulate exogenous toxic substances, the activity of digestive enzymes secreted by the digestive tract is tightly related to the food quality and the toxicity of the chemicals contained in the food (Silva et al. 2009, Teimouri et al. 2015). At present, a large number of studies have been conducted to study the effects of heavy metal stress on antioxidant, detoxifying, and digestive enzyme activities in herbivores by adding heavy metals to artificial diets. However, little is known about the physiological responses of herbivores to heavy metal–stressed woody plants. Lymantria dispar is one of the major forest pest insects in most parts of China with well-known knowledge on its biology, ecology, and physiology. Populus alba berolinensis, as an excellent tree species, has been widely planted to create fast-growing and high-yield forests, shelter-belt forests, and urban forests. Our previous studies found that Cd, Zn, or Pb can transfer from soils contaminated with corresponding heavy metals through P. alba berolinensis seedlings and gypsy moth, and their contents in leaves of seedlings (Jiang et al. 2018) and in gypsy moth larvae (unpublished) all were significantly higher than those of the untreated control; however, Cd-, Zn-, or Pb-stressed P. alba berolinensis did not show significant effects on larval survival, pupation, and emergence rates (Jiang and Yan 2017). We conjectured that gypsy moth larvae might activate an effective detoxification mechanism for attenuating the negative effects of plant-mediated defense responses and heavy metals in leaves. To test this hypothesis and further analyze the physiological responses of herbivores to the heavy metal–stressed woody host plants, the biological activities of antioxidant, detoxifying, and digestive enzymes in the gypsy moth larvae that were fed with different heavy metal–stressed poplar seedling (P. alba berolinensis) leaves were investigated in the current study. Materials and Methods Plant Materials P. alba berolinensis seedlings used in the experiments were generated by vegetative propagation in the Ping Shan Forest Nursery of Heilongjiang province, P. R. China. In late April 2016, cuttings were prepared and planted in about 10-liter pots (23 cm in diameter; 25 cm tall; 1 plant per pot) each filled with 5 kg of 1:1:1 mixture of sand, turf soil, and native soil. Two months later, the soils in the seedling pots were treated with cadmium chloride solution at the final Cd2+ concentration of 1.5 mg/kg (denoted as Cd), with zinc sulfate solution at the final Zn2+ concentration of 500 mg/kg (denoted as Zn) or with lead acetate solution at the final Pb2+ concentration of 500 mg/kg (denoted as Pb), and the seedling pots without heavy metal treatment were used as control group (denoted as CK). The Cd, Zn, or Pb concentration used in present experiments was set in accordance with their corresponding soil environmental quality standards for normal plant growths (Formulated by State Department of Environmental Conservation [1995], China). Insect Rearing The gypsy moth egg masses were collected from the surrounding areas of Northeast Forestry University, Harbin, China in March 2016 and kept in a refrigerator at 4°C until late June. The egg masses were incubated in a laboratory light incubator at 25 ± 1°C and 70 ± 1 relative humidity with a 16:8 (L:D) h photoperiod. Upon hatching, gypsy moth larvae were then reared on artificial diet (purchased from Institute of Forest Ecological Environment and Protection, Chinese Academy of Forestry Sciences) in laboratory under the same condition until the second instar. The newly molted second instar larvae (n = 80) were reared on the fresh poplar leaves collected from each treatment or control group, and the poplar leaves were refreshed once a day until all the larvae grew up to fifth instar or died. During the experiments, the newly molted fourth (n = 30) and fifth instar larvae (n = 30) were placed in a −80°C ultra-low temperature freezer for subsequent determination of enzyme activities (of antioxidant, detoxifying, and digestive enzymes). SOD and CAT Activity The activities of SOD and CAT were determined in 10% (w/v) homogenates in 0.05 mol/liter phosphate buffer at pH 8.8. Homogenization of three larvae from different developmental stage was performed for each replicate, and three replicates for each treatment or control group were tested. Crude homogenates were centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatants were stored on ice for enzyme activity assays. SOD activity in the supernatants was determined according to the method described by McCord and Fridovich (1968). CAT activity was assayed as the rate of H2O2 decomposition at 240 nm, based on the method described by Aebi (1984). The SOD and CAT activity levels were, respectively, expressed as units of enzymes per milligram protein (U/mg) and units of enzymes per gram protein (U/g), in which one unit of SOD is defined as the amount of sample that cause 50% inhibition of pyrogallol autoxidation, and one unit of CAT was defined as the amount of sample required to catalyze micromoles H2O2 per minute. AKP and ACP Activities The homogenizations of three larvae from different developmental stages in each group were performed in ice-cold saline 0.15 M NaCl, and three replicates for each group were used. Crude homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatants were used for AKP and ACP activity assays according to the method described by Nemec and Socha (1988). The AKP and ACP activity levels were expressed as units of enzymes per gram protein (U/g), where one unit of both AKP and ACP activities is defined as the amount of sample that released 1 μmol of p-nitrophenol per minute from phenyl phosphatase under the assay conditions. Protease and Amylase Activity The whole bodies of three larvae from different developmental stages in each group were homogenized in ice-cold deionized water, and three replicates for each group were used. Crude homogenates were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatants were used for enzyme activity assays. Protease activity was determined according to the method described by García-Carreño and Haard (1993). Amylase activity was determined based on the method described by Bernfeld (1955). The protease and amylase activity levels were, respectively, expressed as units of enzymes per gram protein (U/g) and units of enzymes per milligram protein (U/mg), where one unit of amylase activity and protease activity is defined as the amount of sample that produced 1-mg maltose in 30 min at 35°C and the amount of sample that produced 1-μg tyrosine per minute at 37°C, respectively. Total Protein Assay Protein concentration in enzyme extracting solution was assayed using the Coomassie brilliant blue G250 method described by Bradford (1976), with bovine serum albumin as a protein standard. Statistical Analysis The statistical significance of data in each treatment group and CK was determined by an independent sample t-test at the 0.05 level after testing for variance homogeneity and normal distribution or achieving variance homogeneity and normal distribution by log-transforming if necessary. Results Antioxidant Enzyme Activity As shown in Figs. 1 and 2, the SOD (t = 2.936; df = 4; P = 0.043) and CAT (t = 5.956; df = 4; P = 0.004) activities in the fourth instar L. dispar larvae that fed on the Cd-stressed poplar leaves were significantly higher than those that fed on the untreated control leaves, whereas the SOD and CAT activities in the fifth instar larvae under the Cd stress were lower than those in the control group. Fig. 1. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the SOD activities of the fourth–fifth instar gypsy moth larvae. The values presented in the graphs are the means ± standard deviations (n = 3). Asterisks (*) represent significant differences among each treatment and CK within the same developmental stage (P < 0.05). The same holds true for Figs. 2–6 below. Fig. 1. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the SOD activities of the fourth–fifth instar gypsy moth larvae. The values presented in the graphs are the means ± standard deviations (n = 3). Asterisks (*) represent significant differences among each treatment and CK within the same developmental stage (P < 0.05). The same holds true for Figs. 2–6 below. Fig. 2. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the CAT activities of the fourth–fifth instar gypsy moth larvae. Fig. 2. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the CAT activities of the fourth–fifth instar gypsy moth larvae. The SOD and CAT activities of the fourth instar larvae that fed on the Zn-stressed poplar leaves were not significantly different from those of the control group; however, the fifth instar larvae significantly showed lower SOD (t = 2.789; df = 4; P = 0.049) and CAT (t = 4.856; df = 4; P = 0.008) activities than that of untreated control (Figs.1 and 2). The SOD activities of the fourth and fifth instar larvae in Pb treatment group were not significantly different from those in controls (Fig. 1), but the CAT (t = 9.837, df = 4, P = 0.001 for fourth instar larvae; t = 4.115, df = 4, P = 0.015 for fifth instar larvae) activities in both instars larvae that fed on the Pb-stressed leaves were all significantly higher than those of the controls (Fig. 2). Detoxifying Enzyme Activity As shown in Figs. 3 and 4, the ACP and AKP activities of the fourth instar gypsy moth larvae in Cd or Pb treatment group were not significantly different from those in the control group; but ACP (t = 4.422, df = 4, P = 0.011 for Cd stress; t = 3.891, df = 4, P = 0.018 for Pb stress) and AKP (t = 3.298, df = 4, P = 0.03 for Cd stress; t = 3.993, df = 4, P = 0.016 for Pb stress) activities in the fifth instar larvae exposed to Cd or Pb stress showed an opposite pattern, with the ACP activity being significantly higher and AKP activity being significantly lower than that of their corresponding control. In contrast, Zn-stressed poplar leaves significantly increased the activities of both ACP (t = 5.412, df = 4, P = 0.006 for fourth instar larvae; t = 5.747, df = 4, P = 0.005 for fifth instar larvae) and AKP (t = 4.9482, df = 4, P = 0.008 for fourth instar larvae; t = 15.117, df = 4, P = 0.000 for fifth instar larvae) in fourth and fifth instar larvae. Fig. 3. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the ACP activities of the fourth–fifth instar gypsy moth larvae. Fig. 3. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the ACP activities of the fourth–fifth instar gypsy moth larvae. Fig. 4. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the AKP activities of the fourth–fifth instar gypsy moth larvae. Fig. 4. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the AKP activities of the fourth–fifth instar gypsy moth larvae. Digestive Enzymes Activity As shown in Figs. 5 and 6, heavy metal treatments did not show any effects on the digestive enzyme (amylase and protease) activity in the fourth instar larvae; however, they all significantly increased the amylase (t = 3.939, df = 4, P = 0.017 for Zn stress; t = 5.156, df = 4, P = 0.007 for Pb stress) and protease activities (t = 3.713, df = 4, P = 0.021 for Zn stress; t = 4.456, df = 4, P = 0.011 for Pb stress) in fifth instar larvae except the Cd-stressed leaves on protease. Fig. 5. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the amylase activities of the fourth–fifth instar gypsy moth larvae. Fig. 5. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the amylase activities of the fourth–fifth instar gypsy moth larvae. Fig. 6. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the protease activities of the fourth–fifth instar gypsy moth larvae. Fig. 6. View largeDownload slide Effects of Cd-, Zn-, or Pb-stressed polar leaves on the protease activities of the fourth–fifth instar gypsy moth larvae. Discussion and Conclusion Antioxidant Enzyme Activity in Gypsy Moth Larvae As an important detoxification mechanism, the antioxidant defense system has been widely used by herbivores to alleviate oxidative damages under heavy metal stress (Ihechiluru et al. 2015). Functionally interconnected SOD and CAT are two of the most important antioxidant enzymes in the antioxidant defense system (Li et al. 2010). Our results showed that SOD activity in the fourth instar gypsy moth larvae that fed on the Cd-stressed poplar leaves was significantly higher than that in the control. The enhanced SOD enzyme activity might be in response to the superoxide radicals induced by the Cd stress and then converted harmful superoxide radicals into hydrogen peroxide via Haber–Weiss reaction (Valko et al. 2016). Furthermore, in the present study, the response of CAT in the fourth instar larvae to Cd stress was consistent with that of SOD and was significantly higher than that of control. The main role of enhanced CAT activity is used to remove the hydrogen peroxide catalyzed by SOD, further attenuating the oxidative damage caused by reactive oxygen to gypsy moth larvae (Krishnan and Kodrík 2006). Taken together, our findings indicate that the SOD-CAT defense system used for scavenging superoxide and peroxide plays a vital role in preventing Cd toxicity in the fourth instar gypsy moth larvae. However, with the increase of feeding duration, the SOD and CAT activities of the fifth instar larvae in the Cd treatment group were lower than those of the control group. In general, a low concentration heavy metal stress might activate the antioxidant defense mechanism, but with the increase of accumulated concentrations, the ROS levels induced by heavy metals exceed the scavenging ability of herbivores and will inhibit the activity of antioxidant enzymes, thus exacerbating the oxidative damages (Yuan et al. 2016). In addition, another possible mechanism for Cd-stressed inhibition of the antioxidant enzyme activities in herbivores might be that Cd can react directly on enzyme activity sites or replace metal cofactors, which have been well investigated in many organisms (Cuypers et al. 2010, Vlahović et al. 2015). Our results showed that the SOD and CAT activities of the fourth instar larvae were not significantly different from those of the control after feeding on the leaves of poplar leaves under Zn stress, indicating that the accumulation of Zn in the fourth instar larvae did not significantly increase the ROS formation. These data reinforce the findings reported in previous studies (Maryanski et al. 2002, Jelaska et al. 2007), demonstrating that the concentrations of nutritional metals (i.e., Zn) in the arthropods can be regulated more efficiently than that of nonessential metals. Furthermore, the second explanation for nonsignificant effects of Zn stress on antioxidant enzymes activities might be nonenzymatic cellular antioxidants can act as a substitute for antioxidant enzymes (Gauthier et al. 2016). However, the response pattern of SOD and CAT in the fifth instar larvae to the Zn stress was consistent with that to the Cd stress, and their activity was significantly lower than those of the control, suggesting that excessive Zn could also inhibit antioxidant enzyme activity as did nonessential metals. Similar observations were reported by Sahu et al. (2015), who indicated that CAT activities were inhibited in the whole body tissue of the tasar silkworm larvae Antheraea mylitta when exposed to Zn. Our results showed that effects of Pb stress on antioxidant enzymes in the gypsy moth larvae were not developmental stage specific; the SOD activities in fourth instar and fifth instar larvae were not significantly different from those in controls, but the activity of CAT was significantly higher than that of the control. These results are in line with the findings of Mirčić et al. (2013) who reported that heavy metal exposure significantly increased the CAT activity of the gypsy moth, but had no significant effects on SOD activity. They suggested that the peroxides that stimulate the increase of CAT activity are not derived from the degradation processes of superoxide by SOD, but mainly derived from the metabolic oxidoreduction processes performed by various oxidases in peroxisomes such as xanthine oxidase and cytochrome P450 monooxygenases. Detoxifying Enzyme Activity in Gypsy Moth Larvae Different from the antioxidant system that protects the organisms against exogenous substances via scavenging free radicals, the nonoxidative defense system (ACP and AKP) is mainly through direct degradation of exogenous substances to achieve the purpose of defense (Xia et al. 2000, Chen et al. 2013). Our results showed that the ACP and AKP activities of the fourth instar gypsy moth larvae under Cd and Pb stresses were not significantly different from those of the controls, suggesting that accumulations of Cd and Pb in the fourth instar larvae were insufficient to induce ACP and AKP synthesis of the nonoxidative defense system, and that the activation of antioxidant system under Cd or Pb stress can avoid the oxidative damage of ROS to ACP and AKP. Similar results were reported by Zvereva et al. (2003), who found that esterase activity in leaf beetle larvae Chrysomela lapponica did not differ between polluted and unpolluted sites. In addition, we found that ACP and AKP activities in the fifth instar larvae exposed to Cd or Pb stress presented an opposite state, showing that the ACP activity was significantly higher than that of the control and the AKP activity was significantly lower than that of the control. The main differences between ACP and AKP are their catalytic values for pH in catalytic degradation of xenobiotics. The accumulation of large amounts of Cd or Pb in insect tissues, especially in lysosomes, may therefore result in a partial acid environment that increases the activity of ACP that conducts catalyzed reactions at acidic conditions and weakens the activity of AKP that conducts catalyzed reactions at alkaline conditions (Vlahović et al. 2013, Xie et al. 2016). However, our results showed that Zn-stressed poplar leaves significantly increased the activities of both ACP and AKP in the fourth–fifth instar larvae, indicating that ACP and AKP might involve in the tolerate mechanisms of gypsy moth larvae to Zn stress, and that nutritional metals at moderate level can improve the activities of detoxifying enzymes. Our results are in line with previous studies, showing that heavy metals have the inducement effects on ACP and AKP activities in organisms such as G. mellonella and Sphaerodema urinator (Bream 2003, Wu and Yi 2015). Digestive Enzyme Activity in Gypsy Moth Larvae As the main hydrolytic enzymes of digestive function, the changes of amylase and protease activities can reflect the organism’s regulating ability to digestive physiology and adaptability to environment (Lai et al. 2011). Our results showed that after feeding on the Cd-, Zn-, or Pb-stressed poplar leaves, the amylase and protease activities of the fourth instar larvae were not significantly different from those of the control, suggesting that the fourth instar gypsy moth larvae have an effective mechanism that reduces the detrimental consequences of Cd, Zn, or Pb stress without affecting the levels of digestive enzymes (Green et al. 2003, Dar et al. 2015). In addition, we found that Cd-, Zn-, or Pb-stressed poplar leaves caused a hormesis of amylase and protease in the fifth instar larvae. Hormesis, as a widely occurring toxicological phenomenon, may be related to an adaptive response and observed at certain times during heavy metal exposures (Calabrese et al. 2005). In general, herbivores cope with heavy metal stress by improving antioxidant enzymes and detoxifying enzymes require consuming large amounts of energy substances. For example, as demonstrated by Holmstrup et al. (2011), the glycogen contents were significantly reduced when the internal heavy metal concentrations were highly regulated by the earthworms. Thus, increased activities of amylase and proteases in our present study are likely due to the needs of the gypsy moth larvae for hydrolysis of starches and proteins or for elevated utilization efficiency on the food. In summary, our results showed that antioxidant enzymes, detoxifying enzymes, and digestive enzymes constituted effective defense mechanisms for gypsy moth larvae to resist the toxicity originated from the accumulated Cd, Zn, or Pb in poplar leaves, but their defense level varied with heavy metal types and developmental stages. Acknowledgments This research was supported by Excellent academic teacher support project of Northeast forestry university (010602071). The authors declare that they have no competing interests. This article does not contain any studies with human participants or animals performed by any of the authors. 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Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Environmental EntomologyOxford University Press

Published: Oct 3, 2018

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