Gall wasps change the biochemical composition of Eucalyptus leaves

Gall wasps change the biochemical composition of Eucalyptus leaves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gall wasps change the biochemical composition of Eucalyptus leaves Fatih Aytar, Yüksel Keleş This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4112070/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Gall wasps Leptocybe invasa and Ophelimus maskelli can cause loss of productivity by causing damage to Eucalyptus camaldulensis leaf tissues. Comparison of the biochemical compositions of noninfested and infested leaves may be useful to elucidate the roles of antioxidant defense compounds and antioxidant enzyme activities in protecting eucalyptus leaves against attack by insect pests. While glucose and fructose content increased in leaves infested by both gall wasps, proline content increased only in leaves infested by L. invasa . In flavonoid content, the reduction rate of 16.5% in leaves infested with L. invasa was 33.7% with O. maskelli . Total antioxidant capacity decreased with O. maskelli infestation but did not change with L. invasa infestation. Copper ion reduction capacity increased significantly with both pest infestations. Ascorbic acid increased by 87% in L. invasa infestation and 120% in O. maskelli infestation compared to control. The increase observed of superoxide dismutase activity in infested leaves was calculated as 29% for L. invasa and 20% for O. maskelli . O. maskelli infestation caused an increase in carotenoid content in non-infested leaves (total 7.29, Xan/Cds 5.0) but significantly decreased it in infested leaves (toplam 2.98, Xan/Cds 3.2). Superoxide generation rates of noninfested and infested leaves from the infested trees were found to be higher than those of the control samples. The biochemical composition of the infested and noninfested leaves of the infested plants also differs. The invasion of gall wasps triggers oxidative stress by increasing the rate of superoxide production in eucalyptus leaves. antioxidants insect pests Leptocybe invasa Ophelimus maskelli photosynthetic pigments superoxide dismutase Figures Figure 1 Figure 2 INTRODUCTION The genus Eucalyptus includes about 800 tree species common in tropical, subtropical, and temperate regions. Since it contains medicinal and aromatic compounds, in addition to those grown in the natural environment, it is also cultivated by humans (Sahin Basak and Candan 2010 ). Eucalyptus leaf extracts are of economic importance as they are used in medicine and cosmetics. Fast-growing eucalyptus trees also have an important place in the forestry industry. Eucalyptus species are grown for wood and paper production as well as for biofuel production. (Hinchee et al. 2011 ). Eucalyptus species originating from Australia can easily adapt to temperate climatic regions and can be grown with high productivity. However, these trees are adversely affected by pests and pathogens that are due to their natural characteristics or that come from the environment in which they are grown. (Wingfield et al. 2013 ). Herbivorous insects that cause damage to eucalyptus species are polyphage pests that either spread from Australia to other regions or migrated from native Myrtaceae species to eucalyptus species with similar anatomical and metabolic features (Paine et al. 2011 ). Insect pests Leptocybe invasa Fisher & La Salle (Blue gum chalcid, Hymenoptera: Eulophidae) (Billings 2011 ) and Ophelimus maskelli (Ashmead) (Hymenoptera: Eulophidae) (Floris et al. 2018 ) can cause loss of productivity by causing damage to eucalyptus leaf tissues. The single Australian insect that migrated to Asia and caused damage to eucalyptus trees is L. invasa , which emerged after 2002 (Zhang et al. 2021 ). L. invasa produces galls that inflate the stem, petiole, and midrib (Mendel et al. 2004 ). The female wasp stabs and lays her eggs on the upper part of the leaves. The larvae develop in the gall, pupate, and the adults burrow out and are released. By forming galls twice a year, at the beginning and end of summer, it causes injury, weakening, and stunting, especially of young trees. In severe infestation, wasp attacks can completely stop growth (Billings 2011 ). O. maskelli (Badmin 2008 ), one of the common species of the European wasp fauna, has been recorded as a pest of E. camaldulensis in many countries in the Mediterranean basin. In contrast to L. invasa , galls caused by O. maskelli only occur on the upper surface of the eucalyptus leaf lamina. It is typically observed as round button-like projections (Protasov et al. 2007 ). Under favorable environmental conditions, the surface of infested leaves can become completely covered with galls (Branco et al. 2016 ). O. maskelli populations in the Mediterranean basin reach their highest point in early spring (Floris et al. 2018 ). In addition to the characteristics of the invading insects and environmental factors, the established and induced defense systems of the host plant may also be decisive in the emergence of virulence (Naidoo et al. 2014 ). The lines of defense that protect plants from pests and pathogens are mechanical barriers such as bark, wall and leaf cuticles, protective secretions, and toxic secondary metabolites, respectively (Glazebrook 2005 ). When pests and pathogens overcome all these lines of defense and cause damage to cells, resistance responses are stimulated by activating PR genes through hormonal regulation and the production of signaling compounds such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Mithöfer and Boland 2008 ). Since ROS and RNS accumulation caused by pest invasion may cause structural and metabolic damage, they should be eliminated by antioxidant defense compounds (ADC) and antioxidant enzyme activities (AEA). Antioxidant defense requires the use of compounds such as ascorbate, glutathione, phenolics, carotenoids, tocopherols, and activities of enzymes such as SOD, CAT, APX, and GR (Hakiman and Maziah 2009 ). Threats from insect pests can be reduced by planting tolerant eucalyptus genotypes (hybrids) or by using biological control methods (Dittrich-Schröder et al. 2012 ). The resistance of forest trees to pests can be gained by genetic, biochemical, and physiological modifications such as the formation of genetic variations, the development of immune response, plasticity, and interaction with environmental conditions (Naidoo et al. 2014 , Oates et al. 2015 , Mhoswa et al., 2020 ). Studies are showing that eucalyptus leaves have a high antioxidant capacity (Elansary et al. 2017 ). A comparison of the biochemical compositions of uninfested and infested leaves may be useful to elucidate the roles of ADC and AEA in protecting eucalyptus leaves against attack by insect pests. This study was planned to determine the biochemical changes in the infested and uninfested leaves of E. camaldulensis infested with two different gall wasps. MATERIAL and METHODS Material Eucalyptus camaldulensis Dehnh. leaf samples were taken from the trees grown from cuttings obtained from the "Eucalyptus Clone Garden" of the Eastern Mediterranean Forestry Research Institute Karabucak / Mersin / Turkiye. Karabucak region is located on the coast of the Mediterranean. The average elevation of the region is 50 m (Geographical Coordinate: 36°52'42.25"N, 34°52'55.43" W). Mediterranean climate prevails in the region. The annual average temperature is 19˚C, the relative humidity is 67% and the annual precipitation is 503 mm. Eucalyptus leaves were collected on September 10, 2020, from trees infested by gall wasps Leptocybe invasa Fisher & La Salle and Ophelimus maskelli (Ashmead). Infested (IL) and noninfested (NL) leaf samples were collected from the same infested trees (IT). Control samples were taken from noninfested trees (NT). In the collection, leaves were sampled from 3 different individuals of each species in 3 repetitions. The leaves were randomly selected from the leaves that had completed their development on the last shoot. The leaves, which were brought to the laboratory for surface cleaning, were dried in a lyophilizer and ground in the mill, and kept at 4˚C in plastic containers until analysis. Methods 1. Soluble Carbohydrates 100 mg leaf sample was homogenized in 10 ml 80% ethanol with homogenizer for 1 min. Centrifuged at 10,000 g for 10 min and the supernatant was diluted 1/100. 2 ml of anthron reagent was added to 1 ml of the extract and incubated at 100°C for 5 min to glucose, at 40°C 30 min to fructose. The absorbance measurement of the cooled mixture was carried out at 620 nm. The amount of glucose and fructose was calculated from the curve formed with the glucose and fructose standards (Halhoul and Kleinberg 1972). 2. Free Proline 100 mg leaf material was extracted by stored for 24 h at room temperature in 10 ml of 3% 5-sulfosalicylic acid solution. The extracts were centrifuged at 5000 g for 5 min. 2 ml of each of the ninhydrin reagent, glacial acetic acid, and the extract were mixed in a test tube and incubated at 100°C for 60 min, then cooled and vortexed by adding 4 ml of cold toluene. The absorbance of the toluene phase at 520 nm was measured and the amount of proline was determined from the curve formed with the proline standard (Bates et al. 1973). 3. Soluble Phenolics 0.2 g leaf material in 20 ml of methanol and 1 ml of 1% NaHSO 3 were added and mixed with vortex for 2 min. The methanol and tissue mixture was incubated in a water bath set at 75°C for 3 min. After filtration through Whatman no. 1 filter paper, methanol was eliminated from the filtrate by evaporation in a vacuum. Total soluble phenolics in the remaining water phase were determined spectrophotometrically with the Folin-Ciocalteu reagent (prepared by 1/1 dilution with distilled water), against the chlorogenic acid standard (Ferraris et al. 1987). 4. Total Flavonoids Total flavonoid content extracted was carried out by the aluminum chloride method. 2 mL of aqueous extract (10 mg/mL) or standard solution of quercetin (25-200 µg mL -1 ) was added to 2 mL of 2% AlCl 3 solution and 2 mL of 120 mM potassium acetate. Samples were incubated for one hour at room temperature. Absorbance was measured at a wavelength of 425 nm by using a UV-Vis spectrophotometer. The total flavonoid content obtained is expressed as quercetin equivalent (Pekal and Pyrzynska 2014). 5. Total Antioxidant Capacity a) Phosphomolybdenum complex formation (PCF) method: 200 mg leaf sample was taken and extracted in 5 ml of 96% methanol and the extract was centrifuged at 5000 g for 5 min. A reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate was prepared. The final volume was adjusted to 3 ml by taking 150 μL of the sample solution and 2.85 mL of the reagent solution. The samples were incubated at 90°C for 90 min and cooled to room temperature to determine their absorbance at a wavelength of 765 nm. The ascorbic acid solution was used as a standard and the results were calculated as ascorbic acid equivalents (Prieto et al. 1999). b) Cupric ion reducing capacity (CUPRAC) method: 100 mg leaf tissue was homogenized in 10 ml of cold ethanol. The homogenized mixture was centrifuged at 5000 g for 10 min. 0.1 mL of the extract was added to the reaction mixture (containing 1 mL of 10 mM CuCl 2 , 1 mL of ammonium acetate buffer pH 7, and 1 mL of 7.5 mM solution of neocuprion). The final volume was made up to 4.1 mL with water and incubated at 50°C for 20 min. After 30 min at room temperature, absorbance was measured at 450 nm. Gallic acid dissolved in 96% ethanol was used as a standard and the results were given as gallic acid equivalents (Apak et al. 2004). 6. Total Glutathione 100 mg leaf tissue was homogenized in 3 ml of 6% meta-phosphoric acid. 0.1 mL was taken from the samples centrifuged at 5000 g for 5 min and diluted 1/30 with sodium citrate buffer containing 2 mM EDTA (pH 5.0). 0.1 mL of the diluted sample contains 2 mL of solution A (contains sodium phosphate buffer (66 mM, pH 7.0 + 2 mM EDTA + 0.3 mM 5,5' dithiobis-(2-nitrobenzoic acid) + 0.4 ml L -1 BSA) and 0.9 mL of solution B (contains sodium phosphate buffer 66 mM, pH 7.0 + 2 mM EDTA + 50 mM imidazole + 0.2 ml L -1 BSA and 1.5 units of glutathione reductase). The reaction was started with 50 µL of NADPH (8.5 mM). The increase in absorbance due to the reduction of glutathione was measured at a wavelength of 412 nm (Gosset 1994). 7. Ascorbic Acid 100 mg leaf tissue was homogenized by adding 2 ml 10% (w/v) trichloroacetic acid (TCA) cooled to 4°C and centrifuged at 5000 g for 5 min. 200 µl supernatant was mixed with 500 µl phosphate buffer (pH 7.0, 150 mM + 5 mM EDTA) and 100 µl dithiothreitol (10 mM) and incubated at room temperature for 10 min. 50 µl of the mixture was added to 2.95 ml of chlorophenol-indophenol solution (containing 13 mg L -1 DCPIP + 3 g L -1 sodium acetate) and the decrease in absorbance at 520 nm was measured with a spectrophotometer (Chen et al. 1991). 8. Superoxide dismutase (SOD, EC 1.15.1.1.) 0.5 g leaf material was homogenized in 6 mL 0.1 M potassium phosphate extraction buffer (pH 7, containing 100 mg insoluble PVP and 0.1 mM EDTA) with Ultra Turrax. The homogenate was centrifuged for 5 min at 6000 g and 4°C. The supernatant was filtered through a Whatman GF/A glass fiber disc with a vacuum filtration system (Schöner and Krause 1990). SOD activity was determined according to Beyer and Fridowich (1987). The reaction mixture (3 mL) contained potassium phosphate buffer (pH 8, 0.025% Triton X-100 and 0.1 mM EDTA), enzyme extract, 12 mM L-methionine 75 µM nitro blue tetrazolium chloride (NBT) and 2 µM riboflavin. The reaction mixture was kept under fluorescent light for 10 min at 25°C. One SOD unit was described as the amount of enzyme where the NBT reduction ratio was 50%. The NBT reduction ratio was measured with a spectrophotometer at 550 nm wavelength. 9.Chlorophyll contents Chlorophyll was extracted with 80% acetone (buffered to pH 7.8 with phosphate buffer) from dried-ground leaf material. The chlorophyll a, chlorophyll b, and total chlorophyll concentrations were measured with a spectrophotometer. The chlorophyll contents were calculated according to the equations of Porra et al. (1989). 10. Carotenoids 200 mg leaf material was homogenized in ethanol and centrifuged at 5000 g for 5 min, then the supernatant was concentrated by evaporation at 40°C in a rotary evaporator. The residue adhering to the glass surface was dissolved in 2 ml of chloroform. The obtained extract was applied with a micropipette in 100 μl on silica gel coated on a carrier layer with a thickness of 0.5 mm. Then these layers; It was placed in a chromatography tank containing hexane/diethyl ether/acetone as a solvent at a ratio of 60/35/20 by volume. The tank was kept in a dark environment so that the stains on the layers would not deteriorate. Carotene and xanthophyll stains that became evident after the running process were scraped from silica gel with a spatula, and 5 ml of acetone was added to it and centrifuged at 6000 g for 5 min. The absorbance values of the clarified supernatants were measured in a spectrophotometer adjusted to 450 nm wavelength. As standard, β-carotene and xanthophyll (lutein, Sigma) were used (Moore 1974). 11. Hidrogene peroxide H 2 O 2 content was analyzed in 0.2 g leaf tissue Tian et al. (2015) as described. Leaf tissue was homogenized in 10 ml of 0.1% TCA at 4°C in a mortar and centrifuged at 10,000 g for 5 min. 0.5 ml of supernatant, 1 ml of phosphate buffer (100 mM, pH 7), and 1 ml of potassium iodide (1 M KI) were mixed and incubated at 25°C for 60 min in the dark. A separate control was prepared for each sample to determine the absorbance due to the color of the extracts. The absorbance was measured at 390 nm and H 2 O 2 serial solution was used as a standard and results are given as a percentage of control. 12. Superoxide generation rate The rate of superoxide formation was determined by hydroxylamine oxidation in 0.2 g leaf tissue. (Tian et al. 2015). Leaf tissue was homogenized in a mortar in 2 mL of potassium phosphate buffer (50 mM, pH 7.8, containing 1% PVP and 0.1 mM EDTA) at 4°C and centrifuged at 10,000 x g for 5 min. 0.5 mL of supernatant, 0.5 mL of phosphate buffer, and 1 mL of hydroxylamine chloride (1 mM) were mixed and incubated at 25°C for 60 min. 1 mL of sulfanilic acid (17 mM) and 1 mL of α-naphthylamine (7 mM) was added to the mixture and incubated at 25°C for 20 min and absorbance was measured at 530 nm. Results are given as a percentage of control. 13. Statistics This study was planned to investigate the effects of O. maskelli and L. invasa pests invading E. cameldulensis trees on leaf biochemistry and antioxidant composition. Leaf samples from non-infested trees were used as controls and compared with intact and damaged leaves from infested trees. All analyses and measurements were performed in at least three replicates. Whether there was a difference between the groups was determined with the Kruskal-Wallis (KW) test and between which groups the difference was determined with the Least Significant Difference (LSD) test. The results of KW and LSD tests are shown in tables and graphs. RESULTS To determine the effects of insect pests on carbohydrate metabolism in eucalyptus leaves, glucose, and fructose contents were analyzed by the antron method. In addition to the free glucose and fructose contents, this method also determines those that depend on the sucrose structure. Noninfested and infested leaf samples from trees infested with L. invasa and O. maskelli were compared with leaf samples from non-infested trees as controls. Glucose and fructose contents in leaves of infested trees by both gall wasps were higher than in control samples. The effect of L. invasa and O. maskelli infestation on glucose contents was quite similar (Table 1). Noninfested leaves of the infested trees had glucose values approximately 10% higher than the infested leaves. The fructose content of all samples was measured as 2/3 of the glucose content. Fructose contents were also low in control samples and high in noninfested leaves of infested trees (Table 1). Free proline is the most characteristic indicator of direct or indirect osmotic stress. It is expected to increase in case of lack of water caused by insect damage on the leaves. The free proline content of leaves infested with L. invasa was found to be significantly higher than control samples. This increase was not observed in the noninfested leaves of the infested trees. On the other hand, the proline content in the damaged leaves of the infested trees by O. maskelli was found close to the control samples, while it was found to be significantly higher in the healthy leaves (Table 1). Insect pests did significantly affect the total soluble phenolic content of eucalyptus leaves. The soluble phenolic content of leaves from infested trees by L. invasa was found to be significantly higher. It was higher in healthy leaves of infested trees than in damaged leaves. A change in the ratio of soluble phenolic compounds in the healthy leaves of trees infested by O.maskelli could not be determined compared to the control samples. It was observed that the phenolic content of the infested leaves of the infested trees with O. maskelli was 28.3% lower than the infested leaf (Table 1). Analysis of the total flavonoid content of eucalyptus leaves revealed that the flavonoid content decreased with insect infestations. It was determined that O. maskelli was more effective than L. invasa in the decrease observed in flavonoid contents. The reduction in flavonoid content of infested trees was observed in the entire tree, although more pronounced in the infested leaves. The reduction rate of 16.5% in leaves infested with L. invasa was 33.7% with O. maskelli (Table 1). Table 1 Metabolites in eucalyptus leaves infested and noninfested by gall wasps, L. invasa, and O. maskelli (N noninfested, I infested, T tree, L leaf, KW Kruskal Wallis test results). Pests Glucose mg g -1 Fructose mg g -1 Proline µmol g -1 Phenolics mg g -1 Flavonoids mg g -1 Control NT 83.2±17.6 b 55.5±09.4 b 9.4±1.0 ab 22,0±2.1 b 5.2±0.7 a L. invasa ITNL 98.7±08.2 a 67.4±13.1 a 7.9±0.8 b 33.5±5.3 a 4.4±1.1 ab ITIL 87.8±12.0 ab 60.6±10.9 ab 13.6±1.4 ab 29.4±4.0 ab 4.3±0.9 ab O. maskelli ITNL 95.9±07.7 ab 65.5±12.9 ab 15.6±1.1 a 22.0±5.0 b 3.5±0.8 b ITIL 88.3±07.8 ab 60.1±11.3 ab 10.4±0.8 ab 28.0±3.4 ab 3.4±0.4 b KW test ** ** ** ** ** Infestation of eucalyptus leaves by insect pests can cause disruptions in metabolic processes. If the damage in metabolism is due to the formation of free radicals, stimulation of the antioxidant defense system can be expected. In this study, antioxidant defense capacity was determined by two different methods. According to the PCF method findings, the total antioxidant capacity of trees infested with L. invasa showed values close to the control samples. Whereas, antioxidant capacity was found to be significantly lower in infested trees with O. maskelli (Table 2). The CUPRAC method findings showed that both gall wasps increased the total antioxidant capacity in eucalyptus leaves. Moreover, this increase was observed in all leaves, not just infested leaves. Glutathione is a water-soluble antioxidant compound that has an important role in protecting the aqueous phases of the cell against oxidation. Extremely high glutathione values (6.72 mg g -1 ) were determined in control samples taken from eucalyptus trees that were not infested by insect pests. Glutathione levels were significantly reduced in the leaves of trees infested by both L. invasa and O. maskelli . The lowest values were determined in the noninfested leaves of the infested plants (Table 2). It was determined that the concentrations of ascorbic acid, one of the water-soluble antioxidant compounds of plants, increased significantly in response to insect pests. The increase in ascorbic acid concentrations is particularly higher in directly-infested leaves. It was determined that the amount of ascorbic acid increased by 87% in L. invasa infestation and 120% in O. maskelli infestation compared to control (Table 2). While the highest total SOD activity was found in control samples, lower SOD activity was detected in non-infested leaves of infested trees by insect pests. It was observed that the total SOD activity was higher in the infested leaves of the infested trees than in the noninfested leaves (Table 2). The increase observed in damaged leaves was calculated as 29% for L. invasa and 20% for O. maskelli . Table 2 Antioxidant capacity, antioxidant compounds and SOD in eucalyptus leaves infested and noninfested by gall wasps, L. invasa and O. maskelli (N noninfested, I infested, T tree, L leaf, KW Kruskal Wallis test results). Pests PCF mg AAE g -1 CUPRAC mg GAE g -1 Glutathione mg g -1 Ascorbate mg g -1 SOD U g -1 Control NT 111±23.5 ab 13.2±1.7 b 6.72±1.4 a 8.0±1.5 b 223±30 b L. invasa ITNL 111±12.6 ab 27.1±3.1 a 1.23±0.3 b 9.0±1.6 ab 311±57 ab ITIL 114±17.5 a 19.5±2.4 ab 5.44±1.3 ab 14.9±1.7 ab 401±74 a O. maskelli ITNL 92±18.6 ab 21.6±2.4 ab 1.99±0.2 ab 14.2±1.8 ab 290±87 ab ITIL 85±17.8 b 22.1±2.7 ab 4.18±0.6 ab 18.5±3.2 a 348±52 ab KW test ** ** ** ** ** The total chlorophyll content in leaf samples taken from non-infested trees was determined as 4.53 mg g -1 . Chlorophyll content was found to be higher in both noninfested (5.54) and infested (5.07) leaves of trees infested with L. invasa . While the non-infested leaves (total 6.20, Chl a/b 1.5) of the infested trees by O. maskelli had very high chlorophyll values, the damaged leaves (total 3.22, Chl a/b 2.1) had significant chlorophyll loss. The total carotenoid content in control samples was determined as 5.73 mg g -1 . Although the infestation of L. invasa caused a slight decrease in the carotenoid content of E. cameldulensis leaves, the carotenoid values of noninfested and infested leaves were found to be close to each other. O. maskelli infestation caused an increase in total carotenoid content in noninfested leaves (total 7.29, Xan/Cds 5.0) but significantly decreased it in infested leaves (total 2.98, Xan/Cds 3.2) (Figure 1). The hydrogen peroxide content in leaves from non-infested trees was higher than in all samples from infested trees. L. invasa infestation caused lower H 2 O 2 values than O. maskelli infestation. The values found in terms of superoxide formation rate were lower in the control samples, in contrast to the values found for the H 2 O 2 content. Superoxide generation rates of non-infested and infested leaves from the infested trees were found to be higher than those of the control samples (Figure 2). DISCUSSION Eucalyptus trees are widespread in temperate regions with their rapid growth and high adaptability. Its cultivation is also widespread due to its use as an industrial raw material or energy source. Even a single insect can cause widespread damage due to the use of eucalyptus breeding clones (Wingfield et al. 2013 ). Eucalyptus species can be damaged by a large number of pathogens and pests, despite their success in adapting to their new habitats. Two of these insect pests, L. invasa and O. maskelli , invade the leaves of E. camaldulensis trees and cause yield loss (Wingfield et al. 2008 ). Studies investigating the susceptibility and resistance properties of E. cameldulensis trees to insect pests at the genomic level Naidoo et al. ( 2014 ) compiled by the resistance of plants to harmful insect pests depends on the effectiveness of the plant defense system. Anatomical barriers play a role in the first stage of the plant defense system, physiological changes in the second stage, and biochemical regulations in the third stage (Franceschi et al. 2005 , Eyles et al. 2010 ). The antioxidant defense system, which constitutes an important part of biochemical regulation, is the focus of this research. Studies on the biochemical responses of plant cells are scarce, as research on the interaction between insect pests and plants mostly focuses on pests. Insect pests can affect the metabolism of plant cells by causing physiological and biochemical changes (Bari and Jones 2009 ). The accumulation of sugars in plants infested by insect pests may have resulted from the inhibition of the transport of photosynthetic products from the leaves to the storage organs or their reduced use (Table 1 ). It has been reported that reducing sugar levels in eucalyptus leaves infested with L. invasa are highly variable (22.3-112.7 mg/g), but not related to tolerance (Vastrad and Ramanagouda 2014 ). Proline accumulation is the result of primary or secondary water stresses. Damage caused by L. invasa , which produces gall in leaf veins, can cause leaves to become water-stressed. It was determined that the damage caused by O. maskelli on the leaf surface was less effective on water transmission. Pathogen and pest invasions cause oxidative damage by accelerating the formation of reactive oxygen species (ROS) in plants. The increase in ROS acts as secondary messengers that stimulate the antioxidant defense system that protects plants against oxidative damage (Singh et al. 2016 ). Some of the important elements of the antioxidant defense system, both enzymatic and non-enzymatic, were discussed in this study. Ascorbate, one of the water-soluble antioxidant compounds, increased significantly in infested plants, while glutathione levels decreased compared to control samples. Ascorbate and glutathione levels of damaged leaves of trees infested with L.invasa and O.maskelli were higher than healthy leaves. This finding can be interpreted as ascorbate synthesis is stimulated in damaged leaves and the stimulus is transmitted to healthy leaves as well. It can be thought that glutathione synthesis is not involved in the defense mechanism of eucalyptus trees against pest invasion. One of the defense elements of plants against oxidative stress is phenolic compounds with their strong antioxidant effects. According to Vastrad and Ramanagouda ( 2014 ), the total phenol content is 115.0 mg g − 1 in the E. cameldulensis (C-2045) clone infested with L. invasa , while it is 141 mg g − 1 in the non-infested clone (C-526). Of the 48 eucalyptus genotypes of the 3 species screened, 13 were classified as highly susceptible, 2 as susceptible, 22 as tolerant, 2 as resistant, and 9 as immune. Invasion of L. invasa in E. cameldulensis trees increased phenolic synthesis in the whole plant, but more in healthy leaves. O maskelli infestation induced phenolic synthesis only in damaged leaves (Table 1 ). Such differences can be attributed to the level of damage and stimulation of the synthesis of signal carrier molecules. In addition to studies showing that compounds such as jasmonic acid and salicylic acid are synthesized and initiate defense responses in pathogen and pest invasions, there are also studies showing the roles of secondary message carrier compounds (Orozco-Cárdenas et al. 2001 ). The increase determined in phenolic compounds was not observed in flavonoids, a subgroup of phenolic compounds (Table 1 ). There are many tests used to determine the antioxidant defense capacity of plants. These tests may give different results as they better measure the effects of some of the compounds that contribute to antioxidant defense (Pisoschi et al. 2016 ). Of the tests used in this study, PCF better represents the antioxidant capacity resulting from phenolic compounds, while CUPRAC rather measures the reducing capacity. The PCF test showed that the invasion of L. invasa did not affect the total antioxidant capacity, but the invasion of O. maskelli weakened the antioxidant capacity. The CUPRAC test showed that both insect infestations increased the total reducing capacity compared to the control samples (Table 2 ). The difference between these two test results shows that while pest attacks stimulate the synthesis of some antioxidant compounds in plant cells, they do not affect or suppress others. One of the defense responses induced by ROS damage is the synthesis of pathogen-related (PR) proteins. While PR-15 proteins from this protein family are required for oxidase activity in the production of H 2 O 2 , which is toxic to pathogens and pests (Ali et al. 2018 , van Loon 2009 ), PR-9 proteins are peroxidase enzymes and protect plant cells from H 2 O 2 damage. (Passardi et al. 2004 ). Studies show that essential oils produced and stored in eucalyptus tissues protect against insect pests (Chen et al. 2002 ). The results of this study showed decreased SOD activity in the leaves of eucalyptus trees infested with both L. invasa and O. maskelli . The SOD activity of the damaged leaves of the infested plants was higher than the healthy leaves (Table 2 ). Decreased H 2 O 2 levels of leaves and increased rate of superoxide production in infested plants may be consequences of decreased SOD activity (Fig. 2 ). Some effects of pest infestations on the pigmentation of eucalyptus leaves were observed. L. invasa infestation did not have a significant effect on pigmentation, whereas O. maskelli infestation significantly reduced the pigment content of infested leaves, while higher pigment content was detected in healthy leaves of infested trees. Since the invasion of L. invasa was observed in the leaf veins and stem, no adverse effects were observed in the photosynthesizing mesophyll cells. Whereas, O. maskelli invasion directly affects photosynthetic mesophyll cells and reduces pigment synthesis. To reduce the effects of pigment loss in leaves directly affected by the invasion, trees synthesize more pigment in their unaffected leaves. Pigment analysis results show that the major pigment of eucalyptus leaves is xanthophylls. Zeaxanthin, one of the xanthophyll molecules, is more effective than β-carotene in protecting from the harmful effects of high light and heat (Mortensen et al. 2001 ). This shows that the daily xanthophyll cycle (X-cycle) plays an important role in protecting eucalyptus trees from photoinhibition under high temperature and light conditions. Oates et al. ( 2015 ) identified significant differences in mono and sesquiterpene profiles both between genotypes and between control and invaded material. Ultimately, they proposed a model based on transcriptomic and chemical data for the interaction between E. cameldulensis and L. invasa . According to this model, the release of terpenoids into the atmosphere may be intended to attract predator insects. The observed increase in terpenoid synthesis in response to L. invasa infestations should also be evaluated in terms of its contribution to carotenoid synthesis. The effects of these two insects on leaf biochemistry were different from each other. This difference may be because L. invasa affects the veins and O. maskelli affects the leaf blades. Different localities on the leaf may affect water and solute transport and accumulation. Significant differences were found in the biochemistry of the infested leaf and the non-infested leaf from infested trees for some parameters. For example, proline was high in L. invasa and ITIL, while O. maskelli was high in ITNL. In terms of phenolic content, the situation was the opposite. The infestation of L. invasa and O. maskelli , which are two of the gall bees that caused damage to the leaves of E. cameldulensis , caused significant changes in leaf biochemistry. L. invasa produces gall on the petiole and veins, and O.maskelli on the leaf blade. Different gall formation sites caused different effects on leaf biochemistry. L. invasa caused osmotic stress and promoted the accumulation of proline, while the invasion of O. maskelli led to the loss of pigment. The biochemical composition of the damaged and intact leaves of the infested plants also differs. The invasion of gall bees triggers oxidative stress by increasing the rate of superoxide production in eucalyptus leaves. The protective roles of ADC and AEA may be critical in reducing the damage of insect pests. Declarations Author Contribution All authors contributed to the study's conception. Fatih Aytar: designed the study, collection and identification of plant samples; Yuksel Keles: performed chemical analysis, evaluated the data, and wrote the manuscript. All authors read and approved the manuscript. References Ali S, Bashir BA, Kamili AN, Bhat AA, Mir ZA, Bhat JA, Tyagi A, Islam ST, Mushtaq M, Yadav P, Rawat S, Grover A (2018) Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance. 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Tree Physiology 34, 931–943 https://doi.org/10.1093/treephys/tpu075 Oates CN, Ku¨lheim C, Myburg AA, Slippers B and Naidoo S (2015) The Transcriptome and Terpene Profile of Eucalyptus grandis Reveals Mechanisms of Defense Against the Insect Pest, Leptocybe invasa . Plant Cell Physiol. 56(7): 1418–1428 https://doi.org/10.1093/pcp/pcv064 Orozco-Cárdenas ML, Narváez-Vásquez J and Ryan CA. (2001) Hydrogen Peroxide Acts as a Second Messenger for the Induction of Defense Genes in Tomato Plants in Response to Wounding, Systemin, and Methyl Jasmonate. The Plant Cell, 13 (1) 179-191 https://doi.org/10.2307/3871162 Paine TD, Steinbauer MJ and Lawson SA. (2011) Native and Exotic Pests of Eucalyptus : A Worldwide Perspective. Annu. Rev. Entomol. 56: 181–201. https://doi.org/10.1146/annurev-ento-120709-144817 Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases modify the cell wall. Trends Plant Sci 9:534–540. PMID: 15501178, https://doi.org/10.1016/j.tplants.2004.09.002 Pisoschi AM, Pop A, Cimpeanu C. and Predoi G. (2016) Antioxidant Capacity Determination in Plants and Plant-Derived Products: A Review. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 9130976, 36 pages http://dx.doi.org/10.1155/2016/9130976 Protasov A, La Salle J, Blumberg D, Brand D, Saphir N, Assael F, Fisher N, Mendel Z (2007) Biology, revised taxonomy and impact on host plants of Ophelimus maskelli , an invasive gall inducer on Eucalyptus spp. in the Mediterranean Area. – Phytoparasitica, 35: 50-76. https://doi.org/10.1007/BF02981061 Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Analytical Biochemistry 269 (2): 337-341. https://doi.org/10.1006/abio.1999.4019 Pekal A, Pyrzynska K (2014) Evaluation of Aluminium Complexation Reaction for Flavonoid Content Assay. Food Anal. Methods 7: 1776–1782. https://doi.org/10.1007/s12161-014-9814-x Porra RJ, Thompson RA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvent verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975: 384-394 https://doi.org/10.1016/S0005-2728(89)80347-0 Schöner S, Heinrich Krause G (1990) Protective systems against active oxygen species in spinach: response to cold acclimation in excess light. Planta 180: 383–389. https://doi.org/10.1007/BF00198790 Sahin Basak S, Candan F, (2010) Chemical composition and In vitro antioxidant and antidiabetic activities of Eucalyptus camaldulensis Dehnh. essential oil. J Irn Chem Soc 7: 216–226. https://doi.org/10.1007/BF03245882 Singh R, Singh S, Parihar P, Mishra RK, Tripathi DK, Singh VP, Chauhan DK, Prasad SM (2016) Reactive Oxygen Species (ROS): Beneficial Companions of Plants' Developmental Processes. Front Plant Sci. 2016 Sep 27;7:1299. https://doi.org/10.3389/fpls.2016.01299 PMID: 27729914; PMCID: PMC5037240. Tian J, Jiang F, Wu Z (2015) The apoplastic oxidative burst as a key factor of hyperhydricity in garlic plantlet in vitro. Plant Cell Tiss Organ Cult 120: 571-584. https://doi.org/10.1007/s11240-014-0623-0 Wingfield MJ, Slippers B, Hurley BP, Coutinho TA, Wingfield BD, Roux J (2008) Eucalypt pests and diseases: growing threats to plantation productivity. South For 70: 139–144. https://doi.org/10.2989/SOUTH.FOR.2008.70.2.9.537 Wingfield MJ, Roux J, Slippers B, Hurley BP, Garnas J, Myburg AA, Wingfield BD (2013) Established and new technologies reduce increasing pest and pathogen threats to eucalypt plantations. For Ecol Manag 301: 35–42. https://doi.org/10.1016/j.foreco.2012.09.002 van Loon LC (2009) Advances in botanical research—plant innate immunity. eBook ISBN: 9780080888798 Elsevier, Oxford, UK. Vastrad S, Ramanagouda H (2014) Invasive Gall Wasp ( Leptocybe invasa ) in Eucalypt and Its Management. https://doi.org/10.13140/RG.2.1.1561.7449. Zhang H, Song J, Zhao H, Li M, Han W (2021) Predicting the Distribution of the Invasive Species Leptocybe invasa : Combining MaxEnt and Geodetector Models. Insects. 12(2):92. https://doi.org/10.3390/insects12020092. PMID: 33494404; PMCID: PMC7911618. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4112070","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282245564,"identity":"79549d54-7d22-4a5f-b7f3-562f351be386","order_by":0,"name":"Fatih Aytar","email":"","orcid":"","institution":"General Directorate of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Fatih","middleName":"","lastName":"Aytar","suffix":""},{"id":282245565,"identity":"6f77178c-3036-4319-83ab-cd873cb72c98","order_by":1,"name":"Yüksel Keleş","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACHgbGAwwMNmA2kMHA2ECEFpDKNGQtzERpOQznE9ZicObwgQMf/pyP5pc+/vBwAYON7IYD/Mc+4NVyti3h4My227kz+3IMDs9gSDPecICZeQZeLed5DA7zNtzO3XCGh+EwECWCtOB32Hn+D4f//DmXu/8M+wOglv9EaDnbA/Q724HcDTwMBkAtBwhrkTxzzOBgb1ty7owzQBfyGCQbzzzMbIxXC9+Z5IcPfvyxy+3vYX/8mafCTrbveONjvFoUDqC6E4gJxaR8AwEFo2AUjIJRMAoYAFSfUlHbCdFoAAAAAElFTkSuQmCC","orcid":"","institution":"Mersin University","correspondingAuthor":true,"prefix":"","firstName":"Yüksel","middleName":"","lastName":"Keleş","suffix":""}],"badges":[],"createdAt":"2024-03-16 08:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4112070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4112070/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53381201,"identity":"fa884325-bb92-4dc5-a754-0ee04b3872ba","added_by":"auto","created_at":"2024-03-25 10:09:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47929,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic pigments in eucalyptus leaves infested and noninfested by gall wasps, \u003cem\u003eL. invasa\u003c/em\u003e (Li) and \u003cem\u003eO. Maskelli \u003c/em\u003e(Om). N noninfested, I infested, T tree, L leaf, (Li) \u003cem\u003eL. invasa\u003c/em\u003e, (Om) \u003cem\u003eO.maskelli\u003c/em\u003e, KW test: Chl a (**), Chl b (**), β-Car (**), Xan (**).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4112070/v1/f423297e02f1dd32fe7990c8.png"},{"id":53381202,"identity":"b78fac82-3a97-499b-81b6-99d465320f5f","added_by":"auto","created_at":"2024-03-25 10:09:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24684,"visible":true,"origin":"","legend":"\u003cp\u003eSuperoxide generation rates (SGR), superoxide dismutase (SOD), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in eucalyptus leaves infested and noninfested by gall wasps, \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. Maskelli.\u003c/em\u003e N noninfested, I infested, T tree, L leaf, (Li) \u003cem\u003eL. invasa\u003c/em\u003e, (Om) \u003cem\u003eO.maskelli\u003c/em\u003e, KW test: SGR (**), SOD (**), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (**).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4112070/v1/9b9ef7bcc97257aa12a657d2.png"},{"id":53381917,"identity":"6cda4946-d4b4-4353-9d34-63cc60103ee8","added_by":"auto","created_at":"2024-03-25 10:17:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":254676,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4112070/v1/921d2c9b-7438-4e34-9c87-2434c7dfc2f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gall wasps change the biochemical composition of Eucalyptus leaves","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe genus \u003cem\u003eEucalyptus\u003c/em\u003e includes about 800 tree species common in tropical, subtropical, and temperate regions. Since it contains medicinal and aromatic compounds, in addition to those grown in the natural environment, it is also cultivated by humans (Sahin Basak and Candan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Eucalyptus leaf extracts are of economic importance as they are used in medicine and cosmetics. Fast-growing eucalyptus trees also have an important place in the forestry industry. Eucalyptus species are grown for wood and paper production as well as for biofuel production. (Hinchee et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEucalyptus species originating from Australia can easily adapt to temperate climatic regions and can be grown with high productivity. However, these trees are adversely affected by pests and pathogens that are due to their natural characteristics or that come from the environment in which they are grown. (Wingfield et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Herbivorous insects that cause damage to eucalyptus species are polyphage pests that either spread from Australia to other regions or migrated from native Myrtaceae species to eucalyptus species with similar anatomical and metabolic features (Paine et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsect pests \u003cem\u003eLeptocybe invasa\u003c/em\u003e Fisher \u0026amp; La Salle (Blue gum chalcid, Hymenoptera: Eulophidae) (Billings \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and \u003cem\u003eOphelimus maskelli\u003c/em\u003e (Ashmead) (Hymenoptera: Eulophidae) (Floris et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) can cause loss of productivity by causing damage to eucalyptus leaf tissues. The single Australian insect that migrated to Asia and caused damage to eucalyptus trees is \u003cem\u003eL. invasa\u003c/em\u003e, which emerged after 2002 (Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eL. invasa\u003c/em\u003e produces galls that inflate the stem, petiole, and midrib (Mendel et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The female wasp stabs and lays her eggs on the upper part of the leaves. The larvae develop in the gall, pupate, and the adults burrow out and are released. By forming galls twice a year, at the beginning and end of summer, it causes injury, weakening, and stunting, especially of young trees. In severe infestation, wasp attacks can completely stop growth (Billings \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eO. maskelli\u003c/em\u003e (Badmin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), one of the common species of the European wasp fauna, has been recorded as a pest of \u003cem\u003eE. camaldulensis\u003c/em\u003e in many countries in the Mediterranean basin. In contrast to \u003cem\u003eL. invasa\u003c/em\u003e, galls caused by \u003cem\u003eO. maskelli\u003c/em\u003e only occur on the upper surface of the eucalyptus leaf lamina. It is typically observed as round button-like projections (Protasov et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Under favorable environmental conditions, the surface of infested leaves can become completely covered with galls (Branco et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eO. maskelli\u003c/em\u003e populations in the Mediterranean basin reach their highest point in early spring (Floris et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to the characteristics of the invading insects and environmental factors, the established and induced defense systems of the host plant may also be decisive in the emergence of virulence (Naidoo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The lines of defense that protect plants from pests and pathogens are mechanical barriers such as bark, wall and leaf cuticles, protective secretions, and toxic secondary metabolites, respectively (Glazebrook \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). When pests and pathogens overcome all these lines of defense and cause damage to cells, resistance responses are stimulated by activating PR genes through hormonal regulation and the production of signaling compounds such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Mith\u0026ouml;fer and Boland \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Since ROS and RNS accumulation caused by pest invasion may cause structural and metabolic damage, they should be eliminated by antioxidant defense compounds (ADC) and antioxidant enzyme activities (AEA). Antioxidant defense requires the use of compounds such as ascorbate, glutathione, phenolics, carotenoids, tocopherols, and activities of enzymes such as SOD, CAT, APX, and GR (Hakiman and Maziah \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThreats from insect pests can be reduced by planting tolerant eucalyptus genotypes (hybrids) or by using biological control methods (Dittrich-Schr\u0026ouml;der et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The resistance of forest trees to pests can be gained by genetic, biochemical, and physiological modifications such as the formation of genetic variations, the development of immune response, plasticity, and interaction with environmental conditions (Naidoo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Oates et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Mhoswa et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies are showing that eucalyptus leaves have a high antioxidant capacity (Elansary et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A comparison of the biochemical compositions of uninfested and infested leaves may be useful to elucidate the roles of ADC and AEA in protecting eucalyptus leaves against attack by insect pests. This study was planned to determine the biochemical changes in the infested and uninfested leaves of \u003cem\u003eE. camaldulensis\u003c/em\u003e infested with two different gall wasps.\u003c/p\u003e"},{"header":"MATERIAL and METHODS","content":"\u003cp\u003e\u003cstrong\u003eMaterial\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEucalyptus camaldulensis\u003c/em\u003e Dehnh. leaf samples were taken from the trees grown from cuttings obtained from the \u0026quot;Eucalyptus Clone Garden\u0026quot; of the Eastern Mediterranean Forestry Research Institute Karabucak / Mersin / Turkiye. Karabucak region is located on the coast of the Mediterranean. The average elevation of the region is 50 m (Geographical Coordinate: 36\u0026deg;52\u0026apos;42.25\u0026quot;N, 34\u0026deg;52\u0026apos;55.43\u0026quot; W). Mediterranean climate prevails in the region. The annual average temperature is 19˚C, the relative humidity is 67% and the annual precipitation is 503 mm. Eucalyptus leaves were collected on September 10, 2020, from trees infested by gall wasps \u003cem\u003eLeptocybe invasa\u003c/em\u003e Fisher \u0026amp; La Salle and \u003cem\u003eOphelimus maskelli\u003c/em\u003e (Ashmead). Infested (IL) and noninfested (NL) leaf samples were collected from the same infested trees (IT). Control samples were taken from noninfested trees (NT). In the collection, leaves were sampled from 3 different individuals of each species in 3 repetitions. The leaves were randomly selected from the leaves that had completed their development on the last shoot. The leaves, which were brought to the laboratory for surface cleaning, were dried in a lyophilizer and ground in the mill, and kept at 4˚C in plastic containers until analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e1. Soluble Carbohydrates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e100 mg leaf sample was homogenized in 10 ml 80% ethanol with homogenizer for 1 min. Centrifuged at 10,000 g for 10 min and the supernatant was diluted 1/100. 2 ml of anthron reagent was added to 1 ml of the extract and incubated at 100\u0026deg;C for 5 min to glucose, at 40\u0026deg;C 30 min to fructose. The absorbance measurement of the cooled mixture was carried out at 620 nm. The amount of glucose and fructose was calculated from the curve formed with the glucose and fructose standards (Halhoul and Kleinberg 1972).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2. Free Proline\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e100 mg leaf material was extracted by stored for 24 h at room temperature in 10 ml of 3% 5-sulfosalicylic acid solution. The extracts were centrifuged at 5000 g for 5 min. 2 ml of each of the ninhydrin reagent, glacial acetic acid, and the extract were mixed in a test tube and incubated at 100\u0026deg;C for 60 min, then cooled and vortexed by adding 4 ml of cold toluene. The absorbance of the toluene phase at 520 nm was measured and the amount of proline was determined from the curve formed with the proline standard (Bates et al. 1973).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3. Soluble Phenolics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e0.2 g leaf material in 20 ml of methanol and 1 ml of 1% NaHSO\u003csub\u003e3\u003c/sub\u003e were added and mixed with vortex for 2 min. The methanol and tissue mixture was incubated in a water bath set at 75\u0026deg;C for 3 min. After filtration through Whatman no. 1 filter paper, methanol was eliminated from the filtrate by evaporation in a vacuum. Total soluble phenolics in the remaining water phase were determined spectrophotometrically with the Folin-Ciocalteu reagent (prepared by 1/1 dilution with distilled water), against the chlorogenic acid standard (Ferraris et al. 1987).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4. Total Flavonoids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal flavonoid content extracted was carried out by the aluminum chloride method. 2 mL of aqueous extract (10 mg/mL) or standard solution of quercetin (25-200 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) was added to 2 mL of 2% AlCl\u003csub\u003e3\u003c/sub\u003e solution and 2 mL of 120 mM potassium acetate. Samples were incubated for one hour at room temperature. Absorbance was measured at a wavelength of 425 nm by using a UV-Vis spectrophotometer. The total flavonoid content obtained is expressed as quercetin equivalent (Pekal and Pyrzynska 2014).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5. Total Antioxidant Capacity\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ea) Phosphomolybdenum complex formation (PCF) method:\u003c/em\u003e 200 mg leaf sample was taken and extracted in 5 ml of 96% methanol and the extract was centrifuged at 5000 g for 5 min. A reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate was prepared. The final volume was adjusted to 3 ml by taking 150 \u0026mu;L of the sample solution and 2.85 mL of the reagent solution. The samples were incubated at 90\u0026deg;C for 90 min and cooled to room temperature to determine their absorbance at a wavelength of 765 nm. The ascorbic acid solution was used as a standard and the results were calculated as ascorbic acid equivalents (Prieto et al. 1999).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eb) Cupric ion reducing capacity (CUPRAC) method:\u0026nbsp;\u003c/em\u003e100 mg leaf tissue was homogenized in 10 ml of cold ethanol. The homogenized mixture was centrifuged at 5000 g for 10 min. 0.1 mL of the extract was added to the reaction mixture (containing 1 mL of 10 mM CuCl\u003csub\u003e2\u003c/sub\u003e, 1 mL of ammonium acetate buffer pH 7, and 1 mL of 7.5 mM solution of neocuprion). The final volume was made up to 4.1 mL with water and incubated at 50\u0026deg;C for 20 min. After 30 min at room temperature, absorbance was measured at 450 nm. Gallic acid dissolved in 96% ethanol was used as a standard and the results were given as gallic acid equivalents (Apak et al. 2004).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e6. Total Glutathione\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e100 mg leaf tissue was homogenized in 3 ml of 6% meta-phosphoric acid. 0.1 mL was taken from the samples centrifuged at 5000 g for 5 min and diluted 1/30 with sodium citrate buffer containing 2 mM EDTA (pH 5.0). 0.1 mL of the diluted sample contains 2 mL of solution A (contains sodium phosphate buffer (66 mM, pH 7.0 + 2 mM EDTA + 0.3 mM 5,5\u0026apos; dithiobis-(2-nitrobenzoic acid) + 0.4 ml L\u003csup\u003e-1\u003c/sup\u003e BSA) and 0.9 mL of solution B (contains sodium phosphate buffer 66 mM, pH 7.0 + 2 mM EDTA + 50 mM imidazole + 0.2 ml L\u003csup\u003e-1\u003c/sup\u003e BSA and 1.5 units of glutathione reductase). The reaction was started with 50 \u0026micro;L of NADPH (8.5 mM). The increase in absorbance due to the reduction of glutathione was measured at a wavelength of 412 nm (Gosset 1994).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e7. Ascorbic Acid\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e100 mg leaf tissue was homogenized by adding 2 ml 10% (w/v) trichloroacetic acid (TCA) cooled to 4\u0026deg;C and centrifuged at 5000 g for 5 min. 200 \u0026micro;l supernatant was mixed with 500 \u0026micro;l phosphate buffer (pH 7.0, 150 mM + 5 mM EDTA) and 100 \u0026micro;l dithiothreitol (10 mM) and incubated at room temperature for 10 min. 50 \u0026micro;l of the mixture was added to 2.95 ml of chlorophenol-indophenol solution (containing 13 mg L\u003csup\u003e-1\u003c/sup\u003e DCPIP + 3 g L\u003csup\u003e-1\u003c/sup\u003e sodium acetate) and the decrease in absorbance at 520 nm was measured with a spectrophotometer (Chen et al. 1991).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e8. Superoxide dismutase (SOD, EC 1.15.1.1.)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e0.5 g leaf material was homogenized in 6 mL 0.1 M potassium phosphate extraction buffer (pH 7, containing 100 mg insoluble PVP and 0.1 mM EDTA) with Ultra Turrax. The homogenate was centrifuged for 5 min at 6000 g and 4\u0026deg;C. The supernatant was filtered through a Whatman GF/A glass fiber disc with a vacuum filtration system (Sch\u0026ouml;ner and Krause 1990). SOD activity was determined according to Beyer and Fridowich (1987). The reaction mixture (3 mL) contained potassium phosphate buffer (pH 8, 0.025% Triton X-100 and 0.1 mM EDTA), enzyme extract, 12 mM L-methionine 75 \u0026micro;M nitro blue tetrazolium chloride (NBT) and 2 \u0026micro;M riboflavin. The reaction mixture was kept under fluorescent light for 10 min at 25\u0026deg;C. One SOD unit was described as the amount of enzyme where the NBT reduction ratio was 50%. The NBT reduction ratio was measured with a spectrophotometer at 550 nm wavelength.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e9.Chlorophyll contents\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll was extracted with 80% acetone (buffered to pH 7.8 with phosphate buffer) from dried-ground leaf material. The chlorophyll a, chlorophyll b, and total chlorophyll concentrations were measured with a spectrophotometer. The chlorophyll contents were calculated according to the equations of Porra et al. (1989).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e10. Carotenoids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e200 mg leaf material was homogenized in ethanol and centrifuged at 5000 g for 5 min, then the supernatant was concentrated by evaporation at 40\u0026deg;C in a rotary evaporator. The residue adhering to the glass surface was dissolved in 2 ml of chloroform. The obtained extract was applied with a micropipette in 100 \u0026mu;l on silica gel coated on a carrier layer with a thickness of 0.5 mm. Then these layers; It was placed in a chromatography tank containing hexane/diethyl ether/acetone as a solvent at a ratio of 60/35/20 by volume. The tank was kept in a dark environment so that the stains on the layers would not deteriorate. Carotene and xanthophyll stains that became evident after the running process were scraped from silica gel with a spatula, and 5 ml of acetone was added to it and centrifuged at 6000 g for 5 min. The absorbance values of the clarified supernatants were measured in a spectrophotometer adjusted to 450 nm wavelength. As standard, \u0026beta;-carotene and xanthophyll (lutein, Sigma) were used (Moore 1974).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e11. Hidrogene peroxide\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was analyzed in 0.2 g leaf tissue Tian et al. (2015)\u0026nbsp;as described. Leaf tissue was homogenized in 10 ml of 0.1% TCA at 4\u0026deg;C in a mortar and centrifuged at 10,000 g for 5 min. 0.5 ml of supernatant, 1 ml of phosphate buffer (100 mM, pH 7), and 1 ml of potassium iodide (1 M KI) were mixed and incubated at 25\u0026deg;C for 60 min in the dark. A separate control was prepared for each sample to determine the absorbance due to the color of the extracts. The absorbance was measured at 390 nm and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e serial solution was used as a standard and results are given as a percentage of control.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e12. Superoxide generation rate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe rate of superoxide formation was determined by hydroxylamine oxidation in 0.2 g leaf tissue. (Tian et al. 2015).\u0026nbsp;Leaf tissue was homogenized in a mortar in 2 mL of potassium phosphate buffer (50 mM, pH 7.8, containing 1% PVP and 0.1 mM EDTA) at 4\u0026deg;C and centrifuged at 10,000 x g for 5 min. 0.5 mL of supernatant, 0.5 mL of phosphate buffer, and 1 mL of hydroxylamine chloride (1 mM) were mixed and incubated at 25\u0026deg;C for 60 min. 1 mL of sulfanilic acid (17 mM) and 1 mL of \u0026alpha;-naphthylamine (7 mM) was added to the mixture and incubated at 25\u0026deg;C for 20 min and absorbance was measured at 530 nm. Results are given as a percentage of control.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e13. Statistics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was planned to investigate the effects of \u003cem\u003eO. maskelli\u003c/em\u003e and \u003cem\u003eL. invasa\u003c/em\u003e pests invading \u003cem\u003eE. cameldulensis\u003c/em\u003e trees on leaf biochemistry and antioxidant composition. Leaf samples from non-infested trees were used as controls and compared with intact and damaged leaves from infested trees. All analyses and measurements were performed in at least three replicates. Whether there was a difference between the groups was determined with the Kruskal-Wallis (KW) test and between which groups the difference was determined with the Least Significant Difference (LSD) test. The results of KW and LSD tests are shown in tables and graphs.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTo determine the effects of insect pests on carbohydrate metabolism in eucalyptus leaves, glucose, and fructose contents were analyzed by the antron method. In addition to the free glucose and fructose contents, this method also determines those that depend on the sucrose structure. Noninfested and infested leaf samples from trees infested with \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e were compared with leaf samples from non-infested trees as controls. Glucose and fructose contents in leaves of infested trees by both gall wasps were higher than in control samples. The effect of \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e infestation on glucose contents was quite similar (Table 1). Noninfested leaves of the infested trees had glucose values approximately 10% higher than the infested leaves. The fructose content of all samples was measured as 2/3 of the glucose content. Fructose contents were also low in control samples and high in noninfested leaves of infested trees (Table 1).\u003c/p\u003e\n\u003cp\u003eFree proline is the most characteristic indicator of direct or indirect osmotic stress. It is expected to increase in case of lack of water caused by insect damage on the leaves. The free proline content of leaves infested with \u003cem\u003eL. invasa\u003c/em\u003e was found to be significantly higher than control samples. This increase was not observed in the noninfested leaves of the infested trees. On the other hand, the proline content in the damaged leaves of the infested trees by \u003cem\u003eO. maskelli\u003c/em\u003e was found close to the control samples, while it was found to be significantly higher in the healthy leaves (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsect pests did significantly affect the total soluble phenolic content of eucalyptus leaves. The soluble phenolic content of leaves from infested trees by \u003cem\u003eL. invasa\u003c/em\u003e was found to be significantly higher. It was higher in healthy leaves of infested trees than in damaged leaves. A change in the ratio of soluble phenolic compounds in the healthy leaves of trees infested by \u003cem\u003eO.maskelli\u003c/em\u003e could not be determined compared to the control samples. It was observed that the phenolic content of the infested leaves of the infested trees with \u003cem\u003eO. maskelli\u003c/em\u003e was 28.3% lower than the infested leaf (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of the total flavonoid content of eucalyptus leaves revealed that the flavonoid content decreased with insect infestations. It was determined that \u003cem\u003eO. maskelli\u003c/em\u003e was more effective than \u003cem\u003eL. invasa\u003c/em\u003e in the decrease observed in flavonoid contents. The reduction in flavonoid content of infested trees was observed in the entire tree, although more pronounced in the infested leaves. The reduction rate of 16.5% in leaves infested with \u003cem\u003eL. invasa\u003c/em\u003e was 33.7% with \u003cem\u003eO. maskelli\u003c/em\u003e (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1 Metabolites in eucalyptus leaves infested and noninfested by gall wasps, \u003cem\u003eL. invasa,\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e (N noninfested, I infested, T tree, L leaf, KW Kruskal Wallis test results).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003ePests\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003eGlucose\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003eFructose\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003cp\u003e\u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolics\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoids\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e83.2\u0026plusmn;17.6 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e55.5\u0026plusmn;09.4 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 9.4\u0026plusmn;1.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e22,0\u0026plusmn;2.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e5.2\u0026plusmn;0.7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eL. invasa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003eITNL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e98.7\u0026plusmn;08.2 a\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e67.4\u0026plusmn;13.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 7.9\u0026plusmn;0.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e33.5\u0026plusmn;5.3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e4.4\u0026plusmn;1.1 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003eITIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e87.8\u0026plusmn;12.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e60.6\u0026plusmn;10.9 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e13.6\u0026plusmn;1.4 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e29.4\u0026plusmn;4.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e4.3\u0026plusmn;0.9 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eO. maskelli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003eITNL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e95.9\u0026plusmn;07.7 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e65.5\u0026plusmn;12.9 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e15.6\u0026plusmn;1.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e22.0\u0026plusmn;5.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e3.5\u0026plusmn;0.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003eITIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e88.3\u0026plusmn;07.8 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e60.1\u0026plusmn;11.3 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e10.4\u0026plusmn;0.8 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e28.0\u0026plusmn;3.4 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e3.4\u0026plusmn;0.4 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.947189097103918%\" valign=\"top\"\u003e\n \u003cp\u003eKW test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.006814310051107%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.480408858603067%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.013628620102214%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1839863713799%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eInfestation of eucalyptus leaves by insect pests can cause disruptions in metabolic processes. If the damage in metabolism is due to the formation of free radicals, stimulation of the antioxidant defense system can be expected. In this study, antioxidant defense capacity was determined by two different methods. According to the PCF method findings, the total antioxidant capacity of trees infested with \u003cem\u003eL. invasa\u003c/em\u003e showed values close to the control samples. Whereas, antioxidant capacity was found to be significantly lower in infested trees with \u003cem\u003eO. maskelli\u003c/em\u003e (Table 2). The CUPRAC method findings showed that both gall wasps increased the total antioxidant capacity in eucalyptus leaves. Moreover, this increase was observed in all leaves, not just infested leaves.\u003c/p\u003e\n\u003cp\u003eGlutathione is a water-soluble antioxidant compound that has an important role in protecting the aqueous\u0026nbsp;phases of the cell against oxidation. Extremely high glutathione values (6.72 mg g\u003csup\u003e-1\u003c/sup\u003e) were determined in control samples taken from eucalyptus trees that were not infested by insect pests. Glutathione levels were significantly reduced in the leaves of trees infested by both \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e. The lowest values were determined in the noninfested leaves of the infested plants (Table 2).\u003c/p\u003e\n\u003cp\u003eIt was determined that the concentrations of ascorbic acid, one of the water-soluble antioxidant compounds of plants, increased significantly in response to insect pests. The increase in ascorbic acid concentrations is particularly higher in directly-infested leaves. It was determined that the amount of ascorbic acid increased by 87% in \u003cem\u003eL. invasa\u003c/em\u003e infestation and 120% in \u003cem\u003eO. maskelli\u003c/em\u003e infestation compared to control (Table 2).\u003c/p\u003e\n\u003cp\u003eWhile the highest total SOD activity was found in control samples, lower SOD activity was detected in non-infested leaves of infested trees by insect pests. It was observed that the total SOD activity was higher in the infested leaves of the infested trees than in the noninfested leaves (Table 2). The increase observed in damaged leaves was calculated as 29% for \u003cem\u003eL. invasa\u003c/em\u003e and 20% for \u003cem\u003eO. maskelli\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Antioxidant capacity, antioxidant compounds and SOD in eucalyptus leaves infested and noninfested by gall wasps, \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e (N noninfested, I infested, T tree, L leaf, KW Kruskal Wallis test results).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003ePests\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003ePCF\u003c/p\u003e\n \u003cp\u003emg AAE g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003eCUPRAC\u003c/p\u003e\n \u003cp\u003emg GAE g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003eGlutathione\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003eAscorbate\u003c/p\u003e\n \u003cp\u003emg g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003eSOD\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eU g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e111\u0026plusmn;23.5 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e13.2\u0026plusmn;1.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e6.72\u0026plusmn;1.4 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e8.0\u0026plusmn;1.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e223\u0026plusmn;30 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eL. invasa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003eITNL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e111\u0026plusmn;12.6 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e27.1\u0026plusmn;3.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e1.23\u0026plusmn;0.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e9.0\u0026plusmn;1.6 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e311\u0026plusmn;57 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003eITIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e114\u0026plusmn;17.5 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e19.5\u0026plusmn;2.4 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e5.44\u0026plusmn;1.3 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e14.9\u0026plusmn;1.7 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e401\u0026plusmn;74 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eO. maskelli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003eITNL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 92\u0026plusmn;18.6 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e21.6\u0026plusmn;2.4 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e1.99\u0026plusmn;0.2 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e14.2\u0026plusmn;1.8 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e290\u0026plusmn;87 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003eITIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; 85\u0026plusmn;17.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e22.1\u0026plusmn;2.7 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e4.18\u0026plusmn;0.6 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e18.5\u0026plusmn;3.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e348\u0026plusmn;52 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003eKW test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.43598615916955%\" valign=\"top\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe total chlorophyll content in leaf samples taken from non-infested trees was determined as 4.53 mg g\u003csup\u003e-1\u003c/sup\u003e. Chlorophyll content was found to be higher in both noninfested (5.54) and infested (5.07) leaves of trees infested with \u003cem\u003eL. invasa\u003c/em\u003e. While the non-infested leaves (total 6.20, Chl a/b 1.5) of the infested trees by \u003cem\u003eO. maskelli\u003c/em\u003e had very high chlorophyll values, the damaged leaves (total 3.22, Chl a/b 2.1) had significant chlorophyll loss.\u003c/p\u003e\n\u003cp\u003eThe total carotenoid content in control samples was determined as 5.73 mg g\u003csup\u003e-1\u003c/sup\u003e. Although the infestation of \u003cem\u003eL. invasa\u003c/em\u003e caused a slight decrease in the carotenoid content of \u003cem\u003eE. cameldulensis\u003c/em\u003e leaves, the carotenoid values of noninfested and infested leaves were found to be close to each other. \u003cem\u003eO. maskelli\u003c/em\u003e infestation caused an increase in total carotenoid content in noninfested leaves (total 7.29, Xan/Cds 5.0) but significantly decreased it in infested leaves (total 2.98, Xan/Cds 3.2) (Figure 1).\u003c/p\u003e\n\u003cp\u003eThe hydrogen peroxide content in leaves from non-infested trees was higher than in all samples from infested trees. \u003cem\u003eL. invasa\u003c/em\u003e infestation caused lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e values than \u003cem\u003eO. maskelli\u003c/em\u003e infestation. The values found in terms of superoxide formation rate were lower in the control samples, in contrast to the values found for the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content. Superoxide generation rates of non-infested and infested leaves from the infested trees were found to be higher than those of the control samples (Figure 2).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eEucalyptus trees are widespread in temperate regions with their rapid growth and high adaptability. Its cultivation is also widespread due to its use as an industrial raw material or energy source. Even a single insect can cause widespread damage due to the use of eucalyptus breeding clones (Wingfield et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Eucalyptus species can be damaged by a large number of pathogens and pests, despite their success in adapting to their new habitats. Two of these insect pests, \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e, invade the leaves of \u003cem\u003eE. camaldulensis\u003c/em\u003e trees and cause yield loss (Wingfield et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies investigating the susceptibility and resistance properties of \u003cem\u003eE. cameldulensis\u003c/em\u003e trees to insect pests at the genomic level Naidoo et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) compiled by the resistance of plants to harmful insect pests depends on the effectiveness of the plant defense system. Anatomical barriers play a role in the first stage of the plant defense system, physiological changes in the second stage, and biochemical regulations in the third stage (Franceschi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Eyles et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The antioxidant defense system, which constitutes an important part of biochemical regulation, is the focus of this research. Studies on the biochemical responses of plant cells are scarce, as research on the interaction between insect pests and plants mostly focuses on pests.\u003c/p\u003e \u003cp\u003eInsect pests can affect the metabolism of plant cells by causing physiological and biochemical changes (Bari and Jones \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The accumulation of sugars in plants infested by insect pests may have resulted from the inhibition of the transport of photosynthetic products from the leaves to the storage organs or their reduced use (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It has been reported that reducing sugar levels in eucalyptus leaves infested with \u003cem\u003eL. invasa\u003c/em\u003e are highly variable (22.3-112.7 mg/g), but not related to tolerance (Vastrad and Ramanagouda \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Proline accumulation is the result of primary or secondary water stresses. Damage caused by \u003cem\u003eL. invasa\u003c/em\u003e, which produces gall in leaf veins, can cause leaves to become water-stressed. It was determined that the damage caused by \u003cem\u003eO. maskelli\u003c/em\u003e on the leaf surface was less effective on water transmission.\u003c/p\u003e \u003cp\u003ePathogen and pest invasions cause oxidative damage by accelerating the formation of reactive oxygen species (ROS) in plants. The increase in ROS acts as secondary messengers that stimulate the antioxidant defense system that protects plants against oxidative damage (Singh et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Some of the important elements of the antioxidant defense system, both enzymatic and non-enzymatic, were discussed in this study. Ascorbate, one of the water-soluble antioxidant compounds, increased significantly in infested plants, while glutathione levels decreased compared to control samples. Ascorbate and glutathione levels of damaged leaves of trees infested with \u003cem\u003eL.invasa\u003c/em\u003e and \u003cem\u003eO.maskelli\u003c/em\u003e were higher than healthy leaves. This finding can be interpreted as ascorbate synthesis is stimulated in damaged leaves and the stimulus is transmitted to healthy leaves as well. It can be thought that glutathione synthesis is not involved in the defense mechanism of eucalyptus trees against pest invasion.\u003c/p\u003e \u003cp\u003eOne of the defense elements of plants against oxidative stress is phenolic compounds with their strong antioxidant effects. According to Vastrad and Ramanagouda (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the total phenol content is 115.0 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the \u003cem\u003eE. cameldulensis\u003c/em\u003e (C-2045) clone infested with \u003cem\u003eL. invasa\u003c/em\u003e, while it is 141 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the non-infested clone (C-526). Of the 48 eucalyptus genotypes of the 3 species screened, 13 were classified as highly susceptible, 2 as susceptible, 22 as tolerant, 2 as resistant, and 9 as immune. Invasion of \u003cem\u003eL. invasa\u003c/em\u003e in \u003cem\u003eE. cameldulensis\u003c/em\u003e trees increased phenolic synthesis in the whole plant, but more in healthy leaves. \u003cem\u003eO maskelli\u003c/em\u003e infestation induced phenolic synthesis only in damaged leaves (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Such differences can be attributed to the level of damage and stimulation of the synthesis of signal carrier molecules. In addition to studies showing that compounds such as jasmonic acid and salicylic acid are synthesized and initiate defense responses in pathogen and pest invasions, there are also studies showing the roles of secondary message carrier compounds (Orozco-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The increase determined in phenolic compounds was not observed in flavonoids, a subgroup of phenolic compounds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are many tests used to determine the antioxidant defense capacity of plants. These tests may give different results as they better measure the effects of some of the compounds that contribute to antioxidant defense (Pisoschi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Of the tests used in this study, PCF better represents the antioxidant capacity resulting from phenolic compounds, while CUPRAC rather measures the reducing capacity. The PCF test showed that the invasion of \u003cem\u003eL. invasa\u003c/em\u003e did not affect the total antioxidant capacity, but the invasion of \u003cem\u003eO. maskelli\u003c/em\u003e weakened the antioxidant capacity. The CUPRAC test showed that both insect infestations increased the total reducing capacity compared to the control samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The difference between these two test results shows that while pest attacks stimulate the synthesis of some antioxidant compounds in plant cells, they do not affect or suppress others.\u003c/p\u003e \u003cp\u003eOne of the defense responses induced by ROS damage is the synthesis of pathogen-related (PR) proteins. While PR-15 proteins from this protein family are required for oxidase activity in the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which is toxic to pathogens and pests (Ali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, van Loon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), PR-9 proteins are peroxidase enzymes and protect plant cells from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e damage. (Passardi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Studies show that essential oils produced and stored in eucalyptus tissues protect against insect pests (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The results of this study showed decreased SOD activity in the leaves of eucalyptus trees infested with both \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e. The SOD activity of the damaged leaves of the infested plants was higher than the healthy leaves (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Decreased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels of leaves and increased rate of superoxide production in infested plants may be consequences of decreased SOD activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome effects of pest infestations on the pigmentation of eucalyptus leaves were observed. \u003cem\u003eL. invasa\u003c/em\u003e infestation did not have a significant effect on pigmentation, whereas \u003cem\u003eO. maskelli\u003c/em\u003e infestation significantly reduced the pigment content of infested leaves, while higher pigment content was detected in healthy leaves of infested trees. Since the invasion of \u003cem\u003eL. invasa\u003c/em\u003e was observed in the leaf veins and stem, no adverse effects were observed in the photosynthesizing mesophyll cells. Whereas, \u003cem\u003eO. maskelli\u003c/em\u003e invasion directly affects photosynthetic mesophyll cells and reduces pigment synthesis. To reduce the effects of pigment loss in leaves directly affected by the invasion, trees synthesize more pigment in their unaffected leaves. Pigment analysis results show that the major pigment of eucalyptus leaves is xanthophylls. Zeaxanthin, one of the xanthophyll molecules, is more effective than β-carotene in protecting from the harmful effects of high light and heat (Mortensen et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This shows that the daily xanthophyll cycle (X-cycle) plays an important role in protecting eucalyptus trees from photoinhibition under high temperature and light conditions. Oates et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) identified significant differences in mono and sesquiterpene profiles both between genotypes and between control and invaded material. Ultimately, they proposed a model based on transcriptomic and chemical data for the interaction between \u003cem\u003eE. cameldulensis\u003c/em\u003e and \u003cem\u003eL. invasa\u003c/em\u003e. According to this model, the release of terpenoids into the atmosphere may be intended to attract predator insects. The observed increase in terpenoid synthesis in response to \u003cem\u003eL. invasa\u003c/em\u003e infestations should also be evaluated in terms of its contribution to carotenoid synthesis.\u003c/p\u003e \u003cp\u003eThe effects of these two insects on leaf biochemistry were different from each other. This difference may be because \u003cem\u003eL. invasa\u003c/em\u003e affects the veins and \u003cem\u003eO. maskelli\u003c/em\u003e affects the leaf blades. Different localities on the leaf may affect water and solute transport and accumulation. Significant differences were found in the biochemistry of the infested leaf and the non-infested leaf from infested trees for some parameters. For example, proline was high in \u003cem\u003eL. invasa\u003c/em\u003e and ITIL, while \u003cem\u003eO. maskelli\u003c/em\u003e was high in ITNL. In terms of phenolic content, the situation was the opposite.\u003c/p\u003e \u003cp\u003eThe infestation of \u003cem\u003eL. invasa\u003c/em\u003e and \u003cem\u003eO. maskelli\u003c/em\u003e, which are two of the gall bees that caused damage to the leaves of \u003cem\u003eE. cameldulensis\u003c/em\u003e, caused significant changes in leaf biochemistry. \u003cem\u003eL. invasa\u003c/em\u003e produces gall on the petiole and veins, and \u003cem\u003eO.maskelli\u003c/em\u003e on the leaf blade. Different gall formation sites caused different effects on leaf biochemistry. \u003cem\u003eL. invasa\u003c/em\u003e caused osmotic stress and promoted the accumulation of proline, while the invasion of \u003cem\u003eO. maskelli\u003c/em\u003e led to the loss of pigment. The biochemical composition of the damaged and intact leaves of the infested plants also differs. The invasion of gall bees triggers oxidative stress by increasing the rate of superoxide production in eucalyptus leaves. The protective roles of ADC and AEA may be critical in reducing the damage of insect pests.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study's conception. Fatih Aytar: designed the study, collection and identification of plant samples; Yuksel Keles: performed chemical analysis, evaluated the data, and wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAli S, Bashir BA, Kamili AN, Bhat AA, Mir ZA, Bhat JA, Tyagi A, Islam ST, Mushtaq M, Yadav P, Rawat S, Grover A (2018) Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance. Microbiol Res, Vol 212\u0026ndash;213: 29-37, https://doi.org/10.1016/j.micres.2018.04.008\u003c/li\u003e\n \u003cli\u003eApak R, G\u0026uuml;\u0026ccedil;l\u0026uuml; K, Ozyurek M, Kandemir SE (2004) Novel Total Antioxidant Capacity Index for Dietary Polyphenols and Vitamins C and E, Using Their Cupric Ion Reducing Capability in the Presence of Neocuproine: CUPRAC Method. J. Agric. 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For Ecol Manag 301: 35\u0026ndash;42. https://doi.org/10.1016/j.foreco.2012.09.002\u003c/li\u003e\n \u003cli\u003evan Loon LC (2009) Advances in botanical research\u0026mdash;plant innate immunity. eBook ISBN: 9780080888798 Elsevier, Oxford, UK.\u003c/li\u003e\n \u003cli\u003eVastrad S, Ramanagouda H (2014) Invasive Gall Wasp (\u003cem\u003eLeptocybe invasa\u003c/em\u003e) in Eucalypt and Its Management. https://doi.org/10.13140/RG.2.1.1561.7449.\u003c/li\u003e\n \u003cli\u003eZhang H, Song J, Zhao H, Li M, Han W (2021) Predicting the Distribution of the Invasive Species \u003cem\u003eLeptocybe invasa\u003c/em\u003e: Combining MaxEnt and Geodetector Models. Insects. 12(2):92. https://doi.org/10.3390/insects12020092. PMID: 33494404; PMCID: PMC7911618.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antioxidants, insect pests, Leptocybe invasa, Ophelimus maskelli, photosynthetic pigments, superoxide dismutase","lastPublishedDoi":"10.21203/rs.3.rs-4112070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4112070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGall wasps \u003cem\u003eLeptocybe invasa\u003c/em\u003e and \u003cem\u003eOphelimus maskelli\u003c/em\u003e can cause loss of productivity by causing damage to \u003cem\u003eEucalyptus camaldulensis\u003c/em\u003e leaf tissues. Comparison of the biochemical compositions of noninfested and infested leaves may be useful to elucidate the roles of antioxidant defense compounds and antioxidant enzyme activities in protecting eucalyptus leaves against attack by insect pests. While glucose and fructose content increased in leaves infested by both gall wasps, proline content increased only in leaves infested by \u003cem\u003eL. invasa\u003c/em\u003e. In flavonoid content, the reduction rate of 16.5% in leaves infested with \u003cem\u003eL. invasa\u003c/em\u003e was 33.7% with \u003cem\u003eO. maskelli\u003c/em\u003e. Total antioxidant capacity decreased with \u003cem\u003eO. maskelli\u003c/em\u003e infestation but did not change with \u003cem\u003eL. invasa\u003c/em\u003e infestation. Copper ion reduction capacity increased significantly with both pest infestations. Ascorbic acid increased by 87% in \u003cem\u003eL. invasa\u003c/em\u003e infestation and 120% in \u003cem\u003eO. maskelli\u003c/em\u003e infestation compared to control. The increase observed of superoxide dismutase activity in infested leaves was calculated as 29% for \u003cem\u003eL. invasa\u003c/em\u003e and 20% for \u003cem\u003eO. maskelli\u003c/em\u003e. \u003cem\u003eO. maskelli\u003c/em\u003e infestation caused an increase in carotenoid content in non-infested leaves (total 7.29, Xan/Cds 5.0) but significantly decreased it in infested leaves (toplam 2.98, Xan/Cds 3.2). Superoxide generation rates of noninfested and infested leaves from the infested trees were found to be higher than those of the control samples. The biochemical composition of the infested and noninfested leaves of the infested plants also differs. The invasion of gall wasps triggers oxidative stress by increasing the rate of superoxide production in eucalyptus leaves.\u003c/p\u003e","manuscriptTitle":"Gall wasps change the biochemical composition of Eucalyptus leaves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 10:08:57","doi":"10.21203/rs.3.rs-4112070/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-03-25T15:40:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-21T12:00:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Pest Science","date":"2024-03-16T08:19:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec542d4a-51d4-4588-801e-d0b9b605cb85","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T10:08:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 10:08:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4112070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4112070","identity":"rs-4112070","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}