Physiological Response of Pedunculate Oak Trees to Gall-Inducing Cynipidae

Physiological Response of Pedunculate Oak Trees to Gall-Inducing Cynipidae Abstract Gall-inducing Cynipidae (Hymenoptera) manipulate the leaves of their host plants and induce local resistance, resulting in a diversity of physiological changes. In this study, three gall morphotypes caused by the asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and Neuroterus quercusbaccarum L. (Hymenoptera: Cynipidae) on pedunculate oaks (Quercus robur L. (Fagales: Fagaceae)), were used as a model to examine physiological alterations in galls and foliar tissues, compared to non-galled tissues. Our goal was to investigate whether plant physiological response to insect feeding on the same host plant varies depending on gall-wasp species. In particular, the cytoplasmic membrane condition, hydrogen peroxide (H2O2) concentration and changes in antioxidative enzyme activities, including guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were examined in this study. All cynipid species increased H2O2 levels in the leaves with galls, while the level of H2O2 in galls depended on the species. The presence of galls of all species on oak leaves caused an increase of electrolyte leakage and lipid peroxidation level. A significant induction of GPX activity was observed in the leaves with galls of all species, indicating stress induction. Conversely, the decrease in APX activity in both leaves with galls and galled tissues exposed to feeding of all cynipid species. galls, hydrogen peroxide content, membrane lipid peroxidation, electrolyte leakage, antioxidant enzyme A great number of Cynipidae species is responsible for a wide diversity of gall morphotypes that occur on all parts of plants, including roots, stems, leaves, axillary, buds, flowers, and fruits (Stone and Schönrogge 2003). Gall formation alters host characteristics and induces local resistance, which manifests in many physiological changes within the neoformed tissues and the gall structure itself (Tooker et al. 2008; Liu et al. 2010; Oliveira et al. 2011, 2016). Gall-forming insects change the normal development of plants (Stone et al. 2002), and plant cell response to galling stimuli is often associated with increased production of reactive oxygen species (ROS) (Isaias and Oliveira 2012). This is often one of the earliest responses to infestation; however, ROS is also produced in plant cells during normal developmental growth (Mittler 2002). ROS comprise both, free radicals: hydroxyl radical (OH•), superoxide anion radical (O2•–), perhydroxyl radical (HO2•) and alkoxyl radical (RO•) as well as non-radical forms: hydrogen peroxide (H2O2) and singlet oxygen (1O2). According to Sytykiewicz et al. (2014), the intensity and magnitude of ROS generation in plant tissue after insect feeding can elicit oxidative burst and lead to hypersensitivity reactions. This in turn affects nutrient concentration and enzymatic activities important for gall metabolism. ROS production occurs from the beginning of gall induction until maturation, but is highest during maturation (Isaias et al. 2015). ROS are also participating in processes such as signal transduction that regulate cell proliferation and differentiation, pathogen and insect defense and programmed cell death (PCD) (Oliveira et al. 2010). The production of H2O2 is associated with many stress conditions and recently has received considerable interest among ROS and other oxygen-derived free radicals (Hossain et al. 2015). This molecule is moderately reactive and is produced from the univalent reduction of O2•−. It has a relatively long life in relation to other ROS, such as O2•−, •OH or 1O2. In plants, it plays a dual role—at low concentrations, it acts as a signaling molecule involved in various biotic and abiotic stresses, whereas the excess H2O2 production in plant cells leads to oxidative stress and PCD (Quan et al. 2008). Concentration of ROS leads to redox-potential imbalance in cells, which causes oxidative damage to proteins, lipids, carbohydrates, and nucleic acids (Mittler 2002, Mai et al. 2013). The first symptom of excessive production and accumulation of ROS is peroxidation of lipids (LPO) at the plasma membranes, which decreases membrane fluidity, increases leakiness of the membrane and membrane protein damage (Gill and Tuteja 2010). High ROS production is buffered by enzymatic (e.g., peroxidases, dismutase, catalase) and non-enzymatic (e.g., ascorbic acid, glutathione, tocopherol, and carotenoids) scavengers (Hung et al. 2005, Gill and Tuteja 2010). Although antioxidant enzymes operate in different subcellular compartments, they respond in concert when cells are exposed to oxidative stress (Sharma et al. 2012). Among these enzymes, peroxidases seem to be crucial components of the cellular detoxification system that regulate intracellular level of H2O2 (Tayefi-Nasrabadi et al. 2011). Guaiacol peroxidase (GPX) belongs to the antioxidative defense system, that is involved in H2O2 detoxification, while ascorbate peroxidase (APX) is a key enzyme of the ascorbate-glutathione (AsA-GSH) cycle that regenerates soluble antioxidants (Caverzan et al. 2016). APX has a higher affinity for H2O2, as it catalyzes the reduction of H2O2 to water by using ascorbate as an electron donor, while GPX prefers an aromatic electron donor, such as guaiacol and pyragallol at the expense of H2O2 (Gill and Tuteja 2010, Sharma et al. 2012). Furthermore, GPX takes part in many other processes in plants, such as auxin metabolism, cell wall elongation, and protection against pathogens (Caverzan et al. 2016). The ability of gall-inducing wasps to alter indirect plant defenses and the distribution of plant defensive compounds is poorly understood. Thus, our goal was to evaluate alterations in selected physiological parameters in galls and foliar tissues and compare them with non-galled ones. The present study used three gall morphotypes caused by asexual generation (♀♀) of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and Neuroterus quercusbaccarum L. on pedunculate oaks (Quercus robur L.). They all occur between July and autumn on the underside of the oak leaf blades, but have distinct features. Galls of C. quercusfolii are large and spherical, while N. numismalis are nodular with a characteristic hollow of the upper surface and covered with silky hairs (Harper et al. 2004). In turn, galls of N. quercusbaccarum are of lenticular shape (Koncz et al. 2011). Due to the varied structure of galls, they may trigger different modes of plant cell response to insect feeding. Therefore this study was particularly interested in investigating 1) cytoplasmic membrane condition, 2) H2O2 concentration, and 3) changes in antioxidative enzyme activities, including GPX and APX. Gall formation seems to be the most specialized form of plant–insect interaction, hence our goal was to investigate whether plant physiological response to insect feeding on the same host plant varies depending on gall-wasp species. Material and Methods Study Site and Samplings Study sites in an old-growth pedunculate oaks (Quercus robur L.) forest were selected in the stands located in the vicinity of Lublin (Poland) (51.217 °N, 22.751 °E). In this forest, with an area of 272 ha, dominated stands at the age of about 70 yr. Ten oak trees were marked and samples of intact leaves (control) and leaves with galls were collected in September using the visual method. Mature galls of asexual generation (♀♀) of C. quercusfolii, N. numismalis, and N. quercusbaccarum were included. Five leaves with galls of each species and five non-galled leaves were collected from each tree within the reach of the hand. Each sample, intact leaves, leaves with galls of C. quercusfolii, N. numismalis, and N. quercusbaccarum consisted of 50 leaves. Leaves detached with scissors were kept in plastic bags and brought to the laboratory within 1 h after collection. In the laboratory, galls were cut off and dissected to remove larvae. For each gall-inducing species, three groups of samples were categorized as: control leaves (leaves without galls), leaves with galls after gall removal and galls. Leaves and galls within trees and gall-inducing species were cut into small pieces and mixed. Such plant material was weighed and used directly for physiological analysis. Physiological assays were made in three biological replicates (n = 3). H2O2 Assay H2O2 concentration was estimated by forming a titanium—hydro peroxide complex (Jena and Choudhuri 1981). Subsequently, 0.5 g of plant material was grinded in 3 ml of phosphorus buffer (50 mM, pH 6.5) in 4°C, and then the mixture was centrifuged at 6,000 × g for 25 min. Next, 1.5 ml of the supernatant was added to 0.5 ml TiO2 in 20% (v/v) H2SO4 and centrifuged again at 6,000 × g for 15 min at room temperature. The absorbance of the supernatant was measured at 410 nm against blank reagent with a Cecil CE 9500 spectrophotometer (Cecil Instruments, Cambridge, UK). The H2O2 concentration in the sample was calculated using the molar absorbance coefficient, which for H2O2 was 0.28 μM−1 cm−1, and was expressed as nanomoles per 1 g fresh weight (FW). Electrolyte Leakage Assay The status of leaf cell membranes determined by electrolyte leakage (EL), was checked in control leaves and leaves with galls of all cynipid species. It was measured using a procedure described by Kościelniak (1993), with an Elmetron CC-317 microcomputer conductometer. Ten leaf rings of 0.9 cm diameter were cut with a cork borer from each sample. The plant material was transferred to 20 cm3 deionized water and shaken at room temperature for 24 h. Then, the initial electrical conductivity (K1) was determined and the samples were autoclaved at 100°C for 15 min; after 24 h of shaking, the final conductivity of the solution was measured again to determine the total electrolyte content (K2). EL was calculated using the formula: EL (%) = (K1/K2) 100. Due to the structure of galls, EL could not be measured in their tissues. Membrane Lipid Peroxidation The level of membrane lipid peroxidation was measured as the amount of thiobarbituric acid reactive substances (TBARS), according to Heath and Packer (1968). Plant tissues (0.2 g) were homogenized in 0.1 M potassium phosphate buffer, pH 7.0, and then the homogenate was centrifuged at 12,000 × g for 20 min at room temperature. The supernatant (0.5 cm3) was mixed with 2 cm3 of 20% trichloroacetic acid (TCA) containing 0.5% thiobarbituric acid (TBA). The mixture was incubated for 30 min at 95°C, and then the samples were quickly cooled and centrifuged again at 10,000 × g for 10 min. Absorbance was measured at 532 and 600 nm with a Cecil CE 9500 spectrophotometer (Cecil Instruments). The TBARS concentration in the sample was calculated using the molar absorbance coefficient, which for TBARS was 155 nM−1 cm−1, and expressed as nanomoles per 1 g FW. Due to the structure of galls, TBARS could not be measured in their tissues. Antioxidant Enzyme Assays Leaf material (0.2 g) was homogenized with 0.05 mol dm−3 phosphate buffer (pH 7.0) containing 0.2 mol dm−3 EDTA and 2% PVP at 4°C. The homogenate was then centrifuged at 10,000 × g and 4°C for 10 min and then immediately used for GPX and APX analyses. Guaiacol peroxidase (GPX, EC 1.11.1.7) activity was measured by the method of Małolepsza et al. (1994). The reaction mixture contained 0.5 cm3 0.05 mol dm−3 phosphate buffer (pH 5.6), 0.5 cm3 0.02 mol dm−3 guaiacol, 0.5 cm3 0.06 mol dm−3 H2O2 and 0.5 cm3 of enzyme extract. The change in absorbance was measured at 480 nm for 4 min, at 1 min intervals using a Cecil CE 9500 spectrophotometer (Cecil Instruments, UK). GPX activity was determined using the absorbance coefficient for this enzyme, which was 26.6 mM cm−1 and expressed as the change of peroxidase activity per FW (U mg−1 FW). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined using a protocol described by Nakano and Asada (1981). The reaction mixture contained 1.8 ml 0.1 M phosphorus buffer pH 6.0, 20 μl of 5 mM sodium ascorbate, 100 μl of 1mM H2O2 and 100 μl of enzymatic extract. Ascorbate Oxidation was monitored at 290 nm for 5 min, measured at 1 min intervals with a Cecil CE 9500 spectrophotometer (Cecil Instruments). APX activity was determined using the absorbance coefficient for this enzyme (2800 M−1 cm−1). APX activity was expressed as the change of peroxidase activity per FW, expressed as U mg−1 FW. Statistical Analysis All data are presented as means ( x–) with standard errors values (±SE). The physiological assays were analyzed using Statistica for Windows v. 13.1 (StatSoft Inc. 2016). One-way ANOVA and Tukey’s Honestly Significant Differences (HSD) were used to compare means associated with galls, leaves with galls and control leaves. Student’s t-test or non-parametric Mann-Whitney U test were used to compare control versus leaves with galls. Student’s t-test or Mann-Whitney U test were used to compare the percentage changes in the content/activity of physiological parameters between control leaves and leaves with galls, as well as control leaves versus within galls. Results The presence of three cynipid galls on the oak leaves increased of the H2O2 content compared to the control leaves, but these changes were significant only for C. quercusfolii (Table 1). H2O2 levels in galls were highly variable and dependent on gall-making species. The level of this free radical significantly declined in galls of C. quercusfolii as compared to the leaves with galls, while in galls of two other species its level significantly increased (Table 1). A percentage change of H2O2 content was significantly different between leaves with galls of C. quercusfolii and N. quercusbaccarum versus control tissues (Table 2), an increase of 64 and 32% was detected, respectively (Fig. 1A). A significant percentage increase of H2O2 content was measured in galls of N. numismalis and N. quercusbaccarum compared to the control, its level was even a 2.4-fold higher in case of N. quercusbaccarum galls (Fig. 1B). Table 1. Changes in level/activity of physiological parameters in leaves with galls and in galls of three gall-inducing Cynipidae species (mean ± SE) Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Means within a column and species followed by the same letter are not significantly different (P < 0.05). * indicates significant difference from the control; Due to the structure of galls, EL and TBARS could not be measured in their tissues; C. quercusfolii (n = 3), N. numismalis (n = 3), N. quercusbaccarum (n = 3). View Large Table 1. Changes in level/activity of physiological parameters in leaves with galls and in galls of three gall-inducing Cynipidae species (mean ± SE) Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Means within a column and species followed by the same letter are not significantly different (P < 0.05). * indicates significant difference from the control; Due to the structure of galls, EL and TBARS could not be measured in their tissues; C. quercusfolii (n = 3), N. numismalis (n = 3), N. quercusbaccarum (n = 3). View Large Table 2. Differences in the in the content/ activity of H2O2, EL, TBARS, GPX, and APX in the leaves with galls and in galls compared to control leaves for three cynipid species Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Student’s t-test was used for normal distribution of data and non-parametric Mann-Whitney U test was used as a non-parametric alternative. View Large Table 2. Differences in the in the content/ activity of H2O2, EL, TBARS, GPX, and APX in the leaves with galls and in galls compared to control leaves for three cynipid species Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Student’s t-test was used for normal distribution of data and non-parametric Mann-Whitney U test was used as a non-parametric alternative. View Large Fig. 1. View largeDownload slide Mean (±SE) percentage change in H2O2 content, EL, TBARS content, GPX, and APX activity in the leaves with galls (A) and in galls (B) of three cynipid species (percentage change relative to the control as 100%; *represents significant difference from control at P < 0.05 and **P < 0.01). Fig. 1. View largeDownload slide Mean (±SE) percentage change in H2O2 content, EL, TBARS content, GPX, and APX activity in the leaves with galls (A) and in galls (B) of three cynipid species (percentage change relative to the control as 100%; *represents significant difference from control at P < 0.05 and **P < 0.01). The measurements of EL from the cells were used to estimate the degree of cell membrane damage during gall formation process induced by Cynipidae on oak leaves. The presence of all cynipid galls caused the significant increase of EL compared to the control (Tables 1 and 2). Data in Fig. 1A show that the presence of galls on leaves caused species-dependent increase of 22–27%. Lipid peroxidation was used as an indicator of cellular free-radical generation. It was expressed by the amount of malondialdehyde (MDA) determined by TBARS measurements. A significant increase of TBARS content was observed in leaves with N. numismalis and N. quercusbaccarum galls in comparison to control samples (Table 1), as nearly 80% difference was noted (Fig. 1A). No significant difference was detected when compare a percentage change of lipid peroxidation in control leaves and leaves with galls of C. quercusfolii (Fig. 1A, Table 2). A percentage increase in the activity of GPX was observed in leaves with galls when compared with non-galled leaves for C. quercusfolii and N. numismalis, but no significant difference was detected for N. quercusbaccarum (Fig. 1A, Table 2). Almost a sixfold higher increase compared to the control was observed in leaves with galls of N. numismalis (Fig. 1A). GPX activity levels were significantly lower in mature galls of all cynipid species compared to control leaves (Table 2), they were decreased by 66–88% depending on the species (Fig. 1B). Leaves with galls of all Cynipidae species were characterized by extremely higher GPX activity compared with galls. The highest, as a 35-fold difference between gall tissues and leaves with galls was measured for C. quercusfolii (Table 1). Leaves with galls of N. numismalis were characterized by a percentage decrease in APX activity, by 34%, compared with the control samples (Fig. 1A), but no significant differences were measured for C. quercusfolii and N. quercusbaccarum (Table 2). This enzyme showed also low activity in galls of all three species when compared to control, a decrease of 63–89% depend on the gall-inducing species was observed (Fig. 1B). The change of APX activity was similar between galls of all cynipid species versus within leaves with galls, namely significant decrease of this enzyme activity in gall tissues (Table 1). In case of N. quercusbaccarum galls almost 8.2-fold lower activity of this enzyme was observed. Discussion The group of oak gall wasps (Hymenoptera: Cynipidae) as a whole induces an incredible diversity of galls on their host plants and gall structure is highly complex and characteristic for the species (Stone et al. 2002). Galls are formed entirely by plant cells and are an expression of close relationship between gall-inducing species and host plants (Oliveira et al. 2016). Nevertheless, plant cells respond to gall-inducing insect attack by activating a wide variety of defense mechanisms (Inbar et al. 2010, Oliveira et al. 2016). Results from the current study stated that physiological response to insect feeding depends on the taxa of the gall-inducing insects even on the same host plant, what generally support previous studies regarding defense mechanisms. ROS, particularly O2• and H2O2, are the first compounds produced in plant chloroplasts, mitochondria, and peroxisomes in response to stress caused by insect feeding, and act as components of direct resistance to the insect and inducers of cell apoptosis (Kerchev et al. 2012). There are reports that infestation of mites (Farouk and Osman 2012), phloem-feeding insects (Mai et al. 2013, Czerniewicz et al. 2017) and chewing insects (Barbehenn et al. 2010) increase the content of H2O2, and its level are likely to be elevated as long as the attacks persist (Maffei et al. 2007). Our research showed that insects with similar modes of feeding can exert different effects on their host plants. Although all cynipid species increased H2O2 levels in the leaves with galls, the level of H2O2 in galls depended on the species. The role of free radicals in gall cells is not unequivocal. High ROS concentration were observed in galls of neotropical Psyllidae and Cecidomyiidae species, which according to the authors was related to oxidative stress generated in response to attack of gall-inducing species (Isaias et al. 2011, Oliveira et al. 2011). In turn, ultrastructural analyses indicate that gall cells may be involved in ROS production (Isaias et al. 2015), which may trigger gall morphogenesis and development (Isaias and Oliveira 2012). On the other hand, galls as novel plant organs may present sophisticated mechanisms of ROS scavenging (Oliveira et al. 2011). Disturbances in the integrity and stability of plasma membranes are the first evidences of existence of many plant stresses. The degree of cell membrane damage can be estimated by measuring EL from the cells, and is recommended as a plant stress tolerance test (Bajji et al. 2002). According to Demidchik et al. (2014), EL is detected almost instantaneously after the application of stress factor and lasts from a few minutes to several hours. Our results indicated that the presence of galls of all cynipid species on oak leaves caused a significant increase in EL. This suggests that EL can lasts even several weeks, as our study also included mature galls. MDA is involved in plant defense signaling against a variety of stresses (Huag et al. 2007). In our research, the increase of MDA content in leaves with galls of all cynipid species indicated the relationship between gall-wasps feeding and stress, as revealed by oxidative lipid metabolism. According to Khattab and Khattab (2005), the increase in lipid peroxidation may be due to the incapability of antioxidants to capture all ROS produced during this biotic stress. Lipid peroxidation also stimulates green leaf volatile emission in plants in response to herbivory that attracts natural enemies of herbivores (Arimura et al. 2009). Peroxidases are a large class of proteins with different functions. These include key enzymes of primary metabolic processes and ROS metabolic (Almagro et al. 2009). Stimulation of peroxidases is commonly known to play direct role in plant defense against insects (Mai et al. 2016). It has been reported that their activity increase under feeding of chewing (Singh et al. 2013) and sap sucking insects (Moloi and van der Westhuizen 2006, Franzen et al. 2007, Pierson et al. 2011, Kot et al. 2015, Kmieć et al. 2016), indicating their role in plant defense reaction. Under stress conditions, plant tissues generate phenol compounds, which together with their derivatives are oxidized to form reactive quinones. Subsequently they bind to leaf proteins, which impairs plant nutrient uptake by insects (Maffei et al. 2007, War et al. 2012), leading to a decrease in their appetite or deterring them from feeding (Mai et al. 2016). Moreover, peroxidases also mediate the oxidation of hydroxyl-cinnamyl alcohols into free radicals, cross-linking of polysaccharides and monomers, lignification and suberization (Chen et al. 2009), which in turn leads to the production of antinutritive and/or toxic compounds (Zhu-Salzman et al. 2008, He et al. 2011). In our experiment, the activity of GPX in galls of all species was low, which confirmed that galls induced by Cynipidae are an archetype of nutritive galls consisting of conspicuous nutritive tissues with cytological features associated with high metabolism of gall tissues (Oliveira et al. 2016). On the other hand, a significant induction of GPX activity was observed in the leaves with galls of all species, indicating stress induction (Biswas et al. 2014). However, this enzyme is involved in the lignification process, causing the strengthening of the plant cell wall. It is beneficial for plants, because it increases plant resistance to pathogens and insect attacks (Mai et al. 2016). APX and its various isoforms play the important role in developmental processes and are actively expressed under biotic (herbivory and pathogen attack) and abiotic stresses (e.g., heat, cold, salinity, drought). According to Pandey et al. (2017), APX level is directly positively correlated with the duration and intensity of stress. Further, the same kind of stress in different cellular sites can induce variable expression of isozymes (Shigeoka et al. 2002). Higher APX activity decreases the availability of ascorbate in plant tissue, and in turn reduces insect growth and development. Moreover, the lack of ascorbate in the insect midgut increases the oxidative stress that leads to the generation of highly unstable ROS. APX also reduces excessive H2O2 to water in the presence of electron acceptors and oxidizes phenolic compounds to quinones that inhibit insect feeding (Barbehenn et al. 2005). Our studies indicated decreased APX activity in both, leaves with galls and galled tissues exposed to feeding of all cynipid species. It indicates that gall-inducing species manipulate biochemistry of host plants for their own needs. According to Chen (2008), galls are described as a ‘zone of metabolic habitat modification’, in which the insect gains a selective advantage because of increased nutrition (by forming a sink serving as a rich source of food) and reduced plant defense. In conclusion, the direction and intensity of physiological response of host plant to gall-inducing cynipids is not unequivocal and usually insect species-dependent. For this reason, further studies are needed to fully unravel physiological and biochemical interactions between gall-inducing insects and their host plants. The production of controlled levels of ROS in the host plant seems to represent the first signaling step during the process of gall induction. In our study, the presence of galls on oak leaves increased of the H2O2 content. On the other hand, H2O2 levels in galls were highly variable and dependent on gall-inducing species. The accumulation of H2O2 can initiate the physiological and molecular response to prevent or minimize attack of gall-inducing insects. In turn, peroxidases are the vital element in the defense system involved in oak trees responses to gall-inducing Cynipidae. APX and GPX may functioning as the anti-nutritive and/or toxicological defense and trigger other signalling pathways to protect plants from insect attack. 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Physiological Response of Pedunculate Oak Trees to Gall-Inducing Cynipidae

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Abstract

Abstract Gall-inducing Cynipidae (Hymenoptera) manipulate the leaves of their host plants and induce local resistance, resulting in a diversity of physiological changes. In this study, three gall morphotypes caused by the asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and Neuroterus quercusbaccarum L. (Hymenoptera: Cynipidae) on pedunculate oaks (Quercus robur L. (Fagales: Fagaceae)), were used as a model to examine physiological alterations in galls and foliar tissues, compared to non-galled tissues. Our goal was to investigate whether plant physiological response to insect feeding on the same host plant varies depending on gall-wasp species. In particular, the cytoplasmic membrane condition, hydrogen peroxide (H2O2) concentration and changes in antioxidative enzyme activities, including guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were examined in this study. All cynipid species increased H2O2 levels in the leaves with galls, while the level of H2O2 in galls depended on the species. The presence of galls of all species on oak leaves caused an increase of electrolyte leakage and lipid peroxidation level. A significant induction of GPX activity was observed in the leaves with galls of all species, indicating stress induction. Conversely, the decrease in APX activity in both leaves with galls and galled tissues exposed to feeding of all cynipid species. galls, hydrogen peroxide content, membrane lipid peroxidation, electrolyte leakage, antioxidant enzyme A great number of Cynipidae species is responsible for a wide diversity of gall morphotypes that occur on all parts of plants, including roots, stems, leaves, axillary, buds, flowers, and fruits (Stone and Schönrogge 2003). Gall formation alters host characteristics and induces local resistance, which manifests in many physiological changes within the neoformed tissues and the gall structure itself (Tooker et al. 2008; Liu et al. 2010; Oliveira et al. 2011, 2016). Gall-forming insects change the normal development of plants (Stone et al. 2002), and plant cell response to galling stimuli is often associated with increased production of reactive oxygen species (ROS) (Isaias and Oliveira 2012). This is often one of the earliest responses to infestation; however, ROS is also produced in plant cells during normal developmental growth (Mittler 2002). ROS comprise both, free radicals: hydroxyl radical (OH•), superoxide anion radical (O2•–), perhydroxyl radical (HO2•) and alkoxyl radical (RO•) as well as non-radical forms: hydrogen peroxide (H2O2) and singlet oxygen (1O2). According to Sytykiewicz et al. (2014), the intensity and magnitude of ROS generation in plant tissue after insect feeding can elicit oxidative burst and lead to hypersensitivity reactions. This in turn affects nutrient concentration and enzymatic activities important for gall metabolism. ROS production occurs from the beginning of gall induction until maturation, but is highest during maturation (Isaias et al. 2015). ROS are also participating in processes such as signal transduction that regulate cell proliferation and differentiation, pathogen and insect defense and programmed cell death (PCD) (Oliveira et al. 2010). The production of H2O2 is associated with many stress conditions and recently has received considerable interest among ROS and other oxygen-derived free radicals (Hossain et al. 2015). This molecule is moderately reactive and is produced from the univalent reduction of O2•−. It has a relatively long life in relation to other ROS, such as O2•−, •OH or 1O2. In plants, it plays a dual role—at low concentrations, it acts as a signaling molecule involved in various biotic and abiotic stresses, whereas the excess H2O2 production in plant cells leads to oxidative stress and PCD (Quan et al. 2008). Concentration of ROS leads to redox-potential imbalance in cells, which causes oxidative damage to proteins, lipids, carbohydrates, and nucleic acids (Mittler 2002, Mai et al. 2013). The first symptom of excessive production and accumulation of ROS is peroxidation of lipids (LPO) at the plasma membranes, which decreases membrane fluidity, increases leakiness of the membrane and membrane protein damage (Gill and Tuteja 2010). High ROS production is buffered by enzymatic (e.g., peroxidases, dismutase, catalase) and non-enzymatic (e.g., ascorbic acid, glutathione, tocopherol, and carotenoids) scavengers (Hung et al. 2005, Gill and Tuteja 2010). Although antioxidant enzymes operate in different subcellular compartments, they respond in concert when cells are exposed to oxidative stress (Sharma et al. 2012). Among these enzymes, peroxidases seem to be crucial components of the cellular detoxification system that regulate intracellular level of H2O2 (Tayefi-Nasrabadi et al. 2011). Guaiacol peroxidase (GPX) belongs to the antioxidative defense system, that is involved in H2O2 detoxification, while ascorbate peroxidase (APX) is a key enzyme of the ascorbate-glutathione (AsA-GSH) cycle that regenerates soluble antioxidants (Caverzan et al. 2016). APX has a higher affinity for H2O2, as it catalyzes the reduction of H2O2 to water by using ascorbate as an electron donor, while GPX prefers an aromatic electron donor, such as guaiacol and pyragallol at the expense of H2O2 (Gill and Tuteja 2010, Sharma et al. 2012). Furthermore, GPX takes part in many other processes in plants, such as auxin metabolism, cell wall elongation, and protection against pathogens (Caverzan et al. 2016). The ability of gall-inducing wasps to alter indirect plant defenses and the distribution of plant defensive compounds is poorly understood. Thus, our goal was to evaluate alterations in selected physiological parameters in galls and foliar tissues and compare them with non-galled ones. The present study used three gall morphotypes caused by asexual generation (♀♀) of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and Neuroterus quercusbaccarum L. on pedunculate oaks (Quercus robur L.). They all occur between July and autumn on the underside of the oak leaf blades, but have distinct features. Galls of C. quercusfolii are large and spherical, while N. numismalis are nodular with a characteristic hollow of the upper surface and covered with silky hairs (Harper et al. 2004). In turn, galls of N. quercusbaccarum are of lenticular shape (Koncz et al. 2011). Due to the varied structure of galls, they may trigger different modes of plant cell response to insect feeding. Therefore this study was particularly interested in investigating 1) cytoplasmic membrane condition, 2) H2O2 concentration, and 3) changes in antioxidative enzyme activities, including GPX and APX. Gall formation seems to be the most specialized form of plant–insect interaction, hence our goal was to investigate whether plant physiological response to insect feeding on the same host plant varies depending on gall-wasp species. Material and Methods Study Site and Samplings Study sites in an old-growth pedunculate oaks (Quercus robur L.) forest were selected in the stands located in the vicinity of Lublin (Poland) (51.217 °N, 22.751 °E). In this forest, with an area of 272 ha, dominated stands at the age of about 70 yr. Ten oak trees were marked and samples of intact leaves (control) and leaves with galls were collected in September using the visual method. Mature galls of asexual generation (♀♀) of C. quercusfolii, N. numismalis, and N. quercusbaccarum were included. Five leaves with galls of each species and five non-galled leaves were collected from each tree within the reach of the hand. Each sample, intact leaves, leaves with galls of C. quercusfolii, N. numismalis, and N. quercusbaccarum consisted of 50 leaves. Leaves detached with scissors were kept in plastic bags and brought to the laboratory within 1 h after collection. In the laboratory, galls were cut off and dissected to remove larvae. For each gall-inducing species, three groups of samples were categorized as: control leaves (leaves without galls), leaves with galls after gall removal and galls. Leaves and galls within trees and gall-inducing species were cut into small pieces and mixed. Such plant material was weighed and used directly for physiological analysis. Physiological assays were made in three biological replicates (n = 3). H2O2 Assay H2O2 concentration was estimated by forming a titanium—hydro peroxide complex (Jena and Choudhuri 1981). Subsequently, 0.5 g of plant material was grinded in 3 ml of phosphorus buffer (50 mM, pH 6.5) in 4°C, and then the mixture was centrifuged at 6,000 × g for 25 min. Next, 1.5 ml of the supernatant was added to 0.5 ml TiO2 in 20% (v/v) H2SO4 and centrifuged again at 6,000 × g for 15 min at room temperature. The absorbance of the supernatant was measured at 410 nm against blank reagent with a Cecil CE 9500 spectrophotometer (Cecil Instruments, Cambridge, UK). The H2O2 concentration in the sample was calculated using the molar absorbance coefficient, which for H2O2 was 0.28 μM−1 cm−1, and was expressed as nanomoles per 1 g fresh weight (FW). Electrolyte Leakage Assay The status of leaf cell membranes determined by electrolyte leakage (EL), was checked in control leaves and leaves with galls of all cynipid species. It was measured using a procedure described by Kościelniak (1993), with an Elmetron CC-317 microcomputer conductometer. Ten leaf rings of 0.9 cm diameter were cut with a cork borer from each sample. The plant material was transferred to 20 cm3 deionized water and shaken at room temperature for 24 h. Then, the initial electrical conductivity (K1) was determined and the samples were autoclaved at 100°C for 15 min; after 24 h of shaking, the final conductivity of the solution was measured again to determine the total electrolyte content (K2). EL was calculated using the formula: EL (%) = (K1/K2) 100. Due to the structure of galls, EL could not be measured in their tissues. Membrane Lipid Peroxidation The level of membrane lipid peroxidation was measured as the amount of thiobarbituric acid reactive substances (TBARS), according to Heath and Packer (1968). Plant tissues (0.2 g) were homogenized in 0.1 M potassium phosphate buffer, pH 7.0, and then the homogenate was centrifuged at 12,000 × g for 20 min at room temperature. The supernatant (0.5 cm3) was mixed with 2 cm3 of 20% trichloroacetic acid (TCA) containing 0.5% thiobarbituric acid (TBA). The mixture was incubated for 30 min at 95°C, and then the samples were quickly cooled and centrifuged again at 10,000 × g for 10 min. Absorbance was measured at 532 and 600 nm with a Cecil CE 9500 spectrophotometer (Cecil Instruments). The TBARS concentration in the sample was calculated using the molar absorbance coefficient, which for TBARS was 155 nM−1 cm−1, and expressed as nanomoles per 1 g FW. Due to the structure of galls, TBARS could not be measured in their tissues. Antioxidant Enzyme Assays Leaf material (0.2 g) was homogenized with 0.05 mol dm−3 phosphate buffer (pH 7.0) containing 0.2 mol dm−3 EDTA and 2% PVP at 4°C. The homogenate was then centrifuged at 10,000 × g and 4°C for 10 min and then immediately used for GPX and APX analyses. Guaiacol peroxidase (GPX, EC 1.11.1.7) activity was measured by the method of Małolepsza et al. (1994). The reaction mixture contained 0.5 cm3 0.05 mol dm−3 phosphate buffer (pH 5.6), 0.5 cm3 0.02 mol dm−3 guaiacol, 0.5 cm3 0.06 mol dm−3 H2O2 and 0.5 cm3 of enzyme extract. The change in absorbance was measured at 480 nm for 4 min, at 1 min intervals using a Cecil CE 9500 spectrophotometer (Cecil Instruments, UK). GPX activity was determined using the absorbance coefficient for this enzyme, which was 26.6 mM cm−1 and expressed as the change of peroxidase activity per FW (U mg−1 FW). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined using a protocol described by Nakano and Asada (1981). The reaction mixture contained 1.8 ml 0.1 M phosphorus buffer pH 6.0, 20 μl of 5 mM sodium ascorbate, 100 μl of 1mM H2O2 and 100 μl of enzymatic extract. Ascorbate Oxidation was monitored at 290 nm for 5 min, measured at 1 min intervals with a Cecil CE 9500 spectrophotometer (Cecil Instruments). APX activity was determined using the absorbance coefficient for this enzyme (2800 M−1 cm−1). APX activity was expressed as the change of peroxidase activity per FW, expressed as U mg−1 FW. Statistical Analysis All data are presented as means ( x–) with standard errors values (±SE). The physiological assays were analyzed using Statistica for Windows v. 13.1 (StatSoft Inc. 2016). One-way ANOVA and Tukey’s Honestly Significant Differences (HSD) were used to compare means associated with galls, leaves with galls and control leaves. Student’s t-test or non-parametric Mann-Whitney U test were used to compare control versus leaves with galls. Student’s t-test or Mann-Whitney U test were used to compare the percentage changes in the content/activity of physiological parameters between control leaves and leaves with galls, as well as control leaves versus within galls. Results The presence of three cynipid galls on the oak leaves increased of the H2O2 content compared to the control leaves, but these changes were significant only for C. quercusfolii (Table 1). H2O2 levels in galls were highly variable and dependent on gall-making species. The level of this free radical significantly declined in galls of C. quercusfolii as compared to the leaves with galls, while in galls of two other species its level significantly increased (Table 1). A percentage change of H2O2 content was significantly different between leaves with galls of C. quercusfolii and N. quercusbaccarum versus control tissues (Table 2), an increase of 64 and 32% was detected, respectively (Fig. 1A). A significant percentage increase of H2O2 content was measured in galls of N. numismalis and N. quercusbaccarum compared to the control, its level was even a 2.4-fold higher in case of N. quercusbaccarum galls (Fig. 1B). Table 1. Changes in level/activity of physiological parameters in leaves with galls and in galls of three gall-inducing Cynipidae species (mean ± SE) Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Means within a column and species followed by the same letter are not significantly different (P < 0.05). * indicates significant difference from the control; Due to the structure of galls, EL and TBARS could not be measured in their tissues; C. quercusfolii (n = 3), N. numismalis (n = 3), N. quercusbaccarum (n = 3). View Large Table 1. Changes in level/activity of physiological parameters in leaves with galls and in galls of three gall-inducing Cynipidae species (mean ± SE) Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Species Sample H2O2 (nmol g−1 FW) EL (%) TBARS (nmol g−1 FW) GPX (U mg−1 FW) APX (U mg−1 FW) Control 18.09 ± 0.46 22.09 ± 0.75 22.36 ± 1.33 2.36 ± 0.40 4.87 ± 0.21 Cynips quercusfolii L. Leaves with galls 29.76 ± 3.16a* 28.17 ± 1.37a* 24.36 ± 0.52a 9.78 ± 0.19a* 4.17 ± 0.21a Galls 12.71 ± 2.21b - - 0.28 ± 0.04b* 0.84 ± 0.06b* F2, 6 = 15.07 t10 = 3.99 U = 10.0 F2, 6 = 285.76 F2, 6 = 151.62 P = 0.004 P = 0.002 P = 0.579 P < 0.001 P < 0.001 Neuroterus numismalis (Fourc.) Leaves with galls 20.38 ± 0.73b 27.63 ± 0.38a* 38.45 ± 1.56a* 13.95 ± 0.27a* 3.72 ± 0.07a* Galls 26.50 ± 1.57a* - - 0.79 ± 0.19b* 1.76 ± 0.03b* F2, 6 = 17.63 U = 0.00 t10 = 6.39 F2, 6 = 587.38 F2, 6 = 149.15 P = 0.003 P = 0.016 P < 0.001 P < 0.001 P < 0.001 Neuroterus quercusbaccarum L. Leaves with galls 23.93 ± 1.75b 27.10 ± 1.28a* 40.01 ± 0.66a* 3.84 ± 0.59a 4.17 ± 0.34a Galls 43.64 ± 1.50a* - - 0.40 ± 0.08b* 0.51 ± 0.03b* F2, 6 = 97.48 U = 2.0 t10 = 7.324 F2, 6 = 17.38 F2, 6 = 101.80 P < 0.001 P = 0.042 P < 0.001 P = 0.003 P < 0.001 Means within a column and species followed by the same letter are not significantly different (P < 0.05). * indicates significant difference from the control; Due to the structure of galls, EL and TBARS could not be measured in their tissues; C. quercusfolii (n = 3), N. numismalis (n = 3), N. quercusbaccarum (n = 3). View Large Table 2. Differences in the in the content/ activity of H2O2, EL, TBARS, GPX, and APX in the leaves with galls and in galls compared to control leaves for three cynipid species Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Student’s t-test was used for normal distribution of data and non-parametric Mann-Whitney U test was used as a non-parametric alternative. View Large Table 2. Differences in the in the content/ activity of H2O2, EL, TBARS, GPX, and APX in the leaves with galls and in galls compared to control leaves for three cynipid species Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Parameter C. quercusfolii N. numismalis N. quercusbaccarum Test df P-value Test df P-value Test df P-value Student’s t U Student’s t U Student’s t U Leaves with galls H2O2 3.65 - 4 0.02 2.64 - 4 0.06 3.23 - 4 0.03 EL 3.99 - 10 0.002 - 0.00 - 0.016 - 2.0 - 0.042 TBARS - 10.0 - 0.579 6.39 - 10 <0.001 7.324 - 10 <0.001 GPX 16.80 - 4 <0.001 24.22 - 4 <0.001 2.07 - 4 0.11 APX 2.38 - 4 0.076 5.25 - 4 0.006 1.76 - 4 0.15 Galls H2O2 - 0.00 - 0.08 5.14 - 4 0.007 16.24 - 4 <0.001 EL - - - - - - - - - - - - TBARS - - - - - - - - - - - - GPX 5.42 - 4 0.006 3.58 - 4 0.023 4.87 - 4 0.008 APX 18.46 - 4 <0.001 14.62 - 4 <0.001 20.50 - 4 <0.001 Student’s t-test was used for normal distribution of data and non-parametric Mann-Whitney U test was used as a non-parametric alternative. View Large Fig. 1. View largeDownload slide Mean (±SE) percentage change in H2O2 content, EL, TBARS content, GPX, and APX activity in the leaves with galls (A) and in galls (B) of three cynipid species (percentage change relative to the control as 100%; *represents significant difference from control at P < 0.05 and **P < 0.01). Fig. 1. View largeDownload slide Mean (±SE) percentage change in H2O2 content, EL, TBARS content, GPX, and APX activity in the leaves with galls (A) and in galls (B) of three cynipid species (percentage change relative to the control as 100%; *represents significant difference from control at P < 0.05 and **P < 0.01). The measurements of EL from the cells were used to estimate the degree of cell membrane damage during gall formation process induced by Cynipidae on oak leaves. The presence of all cynipid galls caused the significant increase of EL compared to the control (Tables 1 and 2). Data in Fig. 1A show that the presence of galls on leaves caused species-dependent increase of 22–27%. Lipid peroxidation was used as an indicator of cellular free-radical generation. It was expressed by the amount of malondialdehyde (MDA) determined by TBARS measurements. A significant increase of TBARS content was observed in leaves with N. numismalis and N. quercusbaccarum galls in comparison to control samples (Table 1), as nearly 80% difference was noted (Fig. 1A). No significant difference was detected when compare a percentage change of lipid peroxidation in control leaves and leaves with galls of C. quercusfolii (Fig. 1A, Table 2). A percentage increase in the activity of GPX was observed in leaves with galls when compared with non-galled leaves for C. quercusfolii and N. numismalis, but no significant difference was detected for N. quercusbaccarum (Fig. 1A, Table 2). Almost a sixfold higher increase compared to the control was observed in leaves with galls of N. numismalis (Fig. 1A). GPX activity levels were significantly lower in mature galls of all cynipid species compared to control leaves (Table 2), they were decreased by 66–88% depending on the species (Fig. 1B). Leaves with galls of all Cynipidae species were characterized by extremely higher GPX activity compared with galls. The highest, as a 35-fold difference between gall tissues and leaves with galls was measured for C. quercusfolii (Table 1). Leaves with galls of N. numismalis were characterized by a percentage decrease in APX activity, by 34%, compared with the control samples (Fig. 1A), but no significant differences were measured for C. quercusfolii and N. quercusbaccarum (Table 2). This enzyme showed also low activity in galls of all three species when compared to control, a decrease of 63–89% depend on the gall-inducing species was observed (Fig. 1B). The change of APX activity was similar between galls of all cynipid species versus within leaves with galls, namely significant decrease of this enzyme activity in gall tissues (Table 1). In case of N. quercusbaccarum galls almost 8.2-fold lower activity of this enzyme was observed. Discussion The group of oak gall wasps (Hymenoptera: Cynipidae) as a whole induces an incredible diversity of galls on their host plants and gall structure is highly complex and characteristic for the species (Stone et al. 2002). Galls are formed entirely by plant cells and are an expression of close relationship between gall-inducing species and host plants (Oliveira et al. 2016). Nevertheless, plant cells respond to gall-inducing insect attack by activating a wide variety of defense mechanisms (Inbar et al. 2010, Oliveira et al. 2016). Results from the current study stated that physiological response to insect feeding depends on the taxa of the gall-inducing insects even on the same host plant, what generally support previous studies regarding defense mechanisms. ROS, particularly O2• and H2O2, are the first compounds produced in plant chloroplasts, mitochondria, and peroxisomes in response to stress caused by insect feeding, and act as components of direct resistance to the insect and inducers of cell apoptosis (Kerchev et al. 2012). There are reports that infestation of mites (Farouk and Osman 2012), phloem-feeding insects (Mai et al. 2013, Czerniewicz et al. 2017) and chewing insects (Barbehenn et al. 2010) increase the content of H2O2, and its level are likely to be elevated as long as the attacks persist (Maffei et al. 2007). Our research showed that insects with similar modes of feeding can exert different effects on their host plants. Although all cynipid species increased H2O2 levels in the leaves with galls, the level of H2O2 in galls depended on the species. The role of free radicals in gall cells is not unequivocal. High ROS concentration were observed in galls of neotropical Psyllidae and Cecidomyiidae species, which according to the authors was related to oxidative stress generated in response to attack of gall-inducing species (Isaias et al. 2011, Oliveira et al. 2011). In turn, ultrastructural analyses indicate that gall cells may be involved in ROS production (Isaias et al. 2015), which may trigger gall morphogenesis and development (Isaias and Oliveira 2012). On the other hand, galls as novel plant organs may present sophisticated mechanisms of ROS scavenging (Oliveira et al. 2011). Disturbances in the integrity and stability of plasma membranes are the first evidences of existence of many plant stresses. The degree of cell membrane damage can be estimated by measuring EL from the cells, and is recommended as a plant stress tolerance test (Bajji et al. 2002). According to Demidchik et al. (2014), EL is detected almost instantaneously after the application of stress factor and lasts from a few minutes to several hours. Our results indicated that the presence of galls of all cynipid species on oak leaves caused a significant increase in EL. This suggests that EL can lasts even several weeks, as our study also included mature galls. MDA is involved in plant defense signaling against a variety of stresses (Huag et al. 2007). In our research, the increase of MDA content in leaves with galls of all cynipid species indicated the relationship between gall-wasps feeding and stress, as revealed by oxidative lipid metabolism. According to Khattab and Khattab (2005), the increase in lipid peroxidation may be due to the incapability of antioxidants to capture all ROS produced during this biotic stress. Lipid peroxidation also stimulates green leaf volatile emission in plants in response to herbivory that attracts natural enemies of herbivores (Arimura et al. 2009). Peroxidases are a large class of proteins with different functions. These include key enzymes of primary metabolic processes and ROS metabolic (Almagro et al. 2009). Stimulation of peroxidases is commonly known to play direct role in plant defense against insects (Mai et al. 2016). It has been reported that their activity increase under feeding of chewing (Singh et al. 2013) and sap sucking insects (Moloi and van der Westhuizen 2006, Franzen et al. 2007, Pierson et al. 2011, Kot et al. 2015, Kmieć et al. 2016), indicating their role in plant defense reaction. Under stress conditions, plant tissues generate phenol compounds, which together with their derivatives are oxidized to form reactive quinones. Subsequently they bind to leaf proteins, which impairs plant nutrient uptake by insects (Maffei et al. 2007, War et al. 2012), leading to a decrease in their appetite or deterring them from feeding (Mai et al. 2016). Moreover, peroxidases also mediate the oxidation of hydroxyl-cinnamyl alcohols into free radicals, cross-linking of polysaccharides and monomers, lignification and suberization (Chen et al. 2009), which in turn leads to the production of antinutritive and/or toxic compounds (Zhu-Salzman et al. 2008, He et al. 2011). In our experiment, the activity of GPX in galls of all species was low, which confirmed that galls induced by Cynipidae are an archetype of nutritive galls consisting of conspicuous nutritive tissues with cytological features associated with high metabolism of gall tissues (Oliveira et al. 2016). On the other hand, a significant induction of GPX activity was observed in the leaves with galls of all species, indicating stress induction (Biswas et al. 2014). However, this enzyme is involved in the lignification process, causing the strengthening of the plant cell wall. It is beneficial for plants, because it increases plant resistance to pathogens and insect attacks (Mai et al. 2016). APX and its various isoforms play the important role in developmental processes and are actively expressed under biotic (herbivory and pathogen attack) and abiotic stresses (e.g., heat, cold, salinity, drought). According to Pandey et al. (2017), APX level is directly positively correlated with the duration and intensity of stress. Further, the same kind of stress in different cellular sites can induce variable expression of isozymes (Shigeoka et al. 2002). Higher APX activity decreases the availability of ascorbate in plant tissue, and in turn reduces insect growth and development. Moreover, the lack of ascorbate in the insect midgut increases the oxidative stress that leads to the generation of highly unstable ROS. APX also reduces excessive H2O2 to water in the presence of electron acceptors and oxidizes phenolic compounds to quinones that inhibit insect feeding (Barbehenn et al. 2005). Our studies indicated decreased APX activity in both, leaves with galls and galled tissues exposed to feeding of all cynipid species. It indicates that gall-inducing species manipulate biochemistry of host plants for their own needs. According to Chen (2008), galls are described as a ‘zone of metabolic habitat modification’, in which the insect gains a selective advantage because of increased nutrition (by forming a sink serving as a rich source of food) and reduced plant defense. In conclusion, the direction and intensity of physiological response of host plant to gall-inducing cynipids is not unequivocal and usually insect species-dependent. For this reason, further studies are needed to fully unravel physiological and biochemical interactions between gall-inducing insects and their host plants. The production of controlled levels of ROS in the host plant seems to represent the first signaling step during the process of gall induction. In our study, the presence of galls on oak leaves increased of the H2O2 content. On the other hand, H2O2 levels in galls were highly variable and dependent on gall-inducing species. The accumulation of H2O2 can initiate the physiological and molecular response to prevent or minimize attack of gall-inducing insects. In turn, peroxidases are the vital element in the defense system involved in oak trees responses to gall-inducing Cynipidae. APX and GPX may functioning as the anti-nutritive and/or toxicological defense and trigger other signalling pathways to protect plants from insect attack. 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Environmental EntomologyOxford University Press

Published: Apr 11, 2018

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