Plant Science 178 (2010) 350–358 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Cytological and histochemical gradients induced by a sucking insect in galls of Aspidosperma australe Arg. Muell (Apocynaceae) Denis Coelho de Oliveira, Rosy Mary dos Santos Isaias * Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Botânica, ICB/UFMG, Av Antonio Carlos 6627, Pampulha, Cep: 31270-901, Belo Horizonte, MG, Brazil A R T I C L E I N F O A B S T R A C T Article history: Received 19 November 2009 Received in revised form 29 January 2010 Accepted 3 February 2010 Available online 11 February 2010 The storage of carbohydrates and lipids was previously investigated in nutritive tissues of galls of Cecidomyiidae and Cynipidae. Unexpectedly, starch accumulation has been detected in non-nutritive galls induced by Hemiptera, which feed directly from phloem bundles. Samples of non-galled leaves and galls induced by Pseudophacopteron sp. in Aspidosperma australe were processed for light and electron microscopy. Histochemical tests detected sites of ROS (reactive oxygen species), carbohydrates, and enzymes. PCD (programmed cell death) evidenced by plastoglobules and ROS formation also occurred. Phosphorylase and sucrose synthase activity indicated the steps of starch storage. The sites of glucose-6phosphatase activity were related to the provision of sucrose for gall growth and nutrition of Pseudophacopteron. Acid phosphatase took part in the metabolism of starch and degradation of some organelles during the main trophic phase of the insect. The invertases were related to the sites of hyperplasia, and regulation of cell growth and intense respiration. The cytological and histochemical gradients validate the storage of starch as a pattern in galls induced by sucking insects. The detection of enzymes related to carbohydrate metabolism and sites of ROS production is described for the first time for galls induced by sucking insects in the Neotropical region. ß 2010 Elsevier Ireland Ltd. All rights reserved. Keywords: Aspidosperma australe Invertase Nutritive tissue Pseudophacopteron ROS Sucrose synthase 1. Introduction Insect galls commonly store substances that provide nourishment to the gall inducer, and take part in regulating the morphogenesis of the gall itself. These substances are located in specialized cells [1]. Lipids accumulate in the galls induced by Cynipidae; whereas carbohydrate accumulation prevails in galls induced by Cecidomyiidae [1]. Variations in these patterns may occur, as demonstrated by the detection of lipid droplets in nutritive tissues of galls induced by Schismatodiplosis lantanae (Cecidomyiidae) in Lantana camara, a lipid-producing Verbenaceae [2]. This may be evidence that the storage of lipids and carbohydrates is potentially constrained by the host plant metabolism. Studies of cellular differentiation and histochemical detection of reserve substances in galls induced by insects with sucking feeding habits are few, especially in the Neotropical region, and so, to the best of our knowledge, no pattern of reserve metabolism has been proposed for these gall systems. Bronner [1] used histochemical techniques to propose reserve metabolism patterns in Cynipid and Cecidomyiid galls. Histo- * Corresponding author. Tel.: +55 31 34092687; fax: +55 31 34092671. E-mail address: rosy@icb.ufmg.br (R.M.S. Isaias). 0168-9452/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.02.002 chemical techniques are precise methods of localizing metabolites, and consequently of assessing the metabolism patterns of plant cells related to the nutrition of galling herbivores. Moreover, histochemical and cytological techniques may diagnose some stresses generated by the presence of the gall inducer within plant tissues [3,4], as well as the impacts of their different feeding habits. Among the host plant’s responses to oxidative and respiratory stresses is the generation of reactive oxygen species (ROS) with the breakdown of membrane systems in chloroplasts and mitochondria, as well as the formation of plastoglobules [5,6]. The generation of ROS in gall tissues can be easily assessed by cytological analysis and confirmed by histochemical techniques [7]. Sucking gall-inducing insects are common in the Neotropical fauna [8], and their feeding site may be restricted to phloem cells where they introduce their stylets. This behavior may cause the deposition of ‘‘wound callose’’, which forms a physical barrier at the plasma membrane, and consequently may be the first step in the plant’s reaction to the presence of the herbivore [9]. In the Pseudophacopteron sp. (Hemiptera)–Aspidosperma australe (Apocynaceae) gall system, due to the use of phloem as the feeding site [10], a nutritive tissue should be absent, and the storage of nutritional reserves and the enzyme activity should be related to maintaining the gall structure, as proposed by Oliveira et al. [11] D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 for the Euphalerus ostreoides–Lonchocarpus muehlbergianus gall system. Because of the amplitude of the cecidogenic field [12,13], the highest metabolic activity should be expected in the tissues near the nymphal chamber, where respiratory and oxidative stresses are greater. These assumptions should be confirmed by cytological and histochemical analyses. By studying the cytological and histochemical features of the Pseudophacopteron sp.–A. australe gall system, this investigation aimed to answer the following questions: (i) does the feeding activity of Pseudophacopteron sp. alter the storage of metabolites in its host plant? (ii) Is there a gradient of storage substances and enzymes in A. australe gall tissues? (iii) Do gall cytological features indicate oxidative stress? (iv) Do galls induced by Pseudophacopteron sp. in A. australe follow the patterns described in the literature for sucking gall inducers? 2. Materials and methods 2.1. Plant material collection Tissue samples of non-galled leaves (n  12) and of galls in three developmental stages, sorted by size, (immature galls 1.0  0.3 mm wide, mature galls, 5.0  0.5 mm wide, and senescent galls, 5.0  0.7 wide and open) (n  12 per stage), were collected from A. australe individuals (n = 10) located on the Pampulha campus of the Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. The collections were made from January through December 2008, at intervals of 2 months, and the samples obtained were submitted to cytological and histochemical analyses. 2.2. Cytological analysis The samples were fixed in 4% Karnovsky in 0.1 M phosphate buffer (pH 7.2) for 24 h [14], post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2), dehydrated in an ethanol series [15], and embedded in Araldite1 [16]. The material was crosssectioned in an Ultramicrotome Reichert-Jung – Ultracut, contrasted in uranyl acetate and lead citrate according to Reynolds [17], and examined in a Zeiss EM 109 transmission electron microscope. 2.3. Histochemical analysis 2.3.1. Nucleic acids Samples of non-galled leaves and mature galls were fixed in Carnoy for 48 h, dehydrated in an ethanol series, embedded in Leica1 historesin, and cross-sectioned (5–10 mm) in a Reichert Jung1 rotating microtome [15]. The material was stained with 0.15% methyl green and 0.25% pyronin B in acetate buffer (pH 4.7) [18]. For the controls, RNA and DNA were extracted by incubating the slides in 0.1% ribonuclease (pH 6.8) for 1 h at 40 8C, and in 0.2 mg deoxyribonuclease ml 1 in 0.003 M magnesium sulphate (pH 6.5) for 1 h at 25 8C, respectively. 2.3.2. Primary metabolites Handmade sections of fresh material were used for detection of proteins, lipids, starch, and reducing sugars. For proteins, the sections were stained in 0.1% bromophenol blue in a saturated solution of magnesium chloride in ethanol for 15 min, and then washed in acetic acid and water [19]. For starch detection, the sections were immersed in Lugol reagent (iodine potassium iodide) for 15 min [15]. For reducing sugars, sections were immersed in Fehling reagent [20]. For lipids, the sections were immersed in a saturated solution of Sudan III (CI 26100) in 708 GL ethanol [21]. For the controls, lipids were extracted with methanol:chloroform (1:1, v/v), and starch with salivary amylase. Blank sections were used for comparative analysis. 351 2.3.3. Callose The samples were fixed in FAA for 48 h, washed in distilled water followed by 70% ethanol, and stained in 0.1% aniline blue (pH 9.5) for 10 min [15]. The material was mounted with the stain and observed in a scanning confocal laser microscope (Zeiss, LSM 510, Germany). The controls were treated in the same manner but without the stain. 2.3.4. Enzyme activity Sections of fresh material were immersed in the appropriate detection solutions for acid phosphatase, phosphorylase, glucose6-phosphatase, invertase, and sucrose synthase. For the detection of acid-phosphatase activity, the sections were incubated in 0.012% lead nitrate and 0.1 M potassium sodium glycerophosphate in 0.5 M acetate buffer (pH 4.5) for 24 h, at room temperature (25 8C). Subsequently, sections were washed in distilled water and immersed in 1% ammonia for 5 min [22]. For the control, the samples were incubated in the same solution without potassium sodium glycerophosphate. For the detection of phosphorylase activity, the sections were incubated in 1% glucose-1-phosphate in 0.1 acetate buffer (pH 6.0) for 2 h at room temperature [23]. After the incubation, the sections were immersed in Lugol reagent [15]. For the control, samples were not incubated in glucose-1phosphate. For the detection of glucose-6-phosphatase activity, the sections were incubated in a solution containing 20 mg of potassium glucose-6-phosphate in 125 ml of 0.2 M Tris–maleate buffer (pH 6.7), and 3 ml of 2% lead nitrate in 7 ml of distilled water, for 15 min to 2 h, at 37 8C. Following the incubation, the material was washed in distilled water, immersed in 1% ammonium sulphate for 5 min, and mounted in glycerin jelly [23]. For the control, the samples were not incubated in potassium glucose-6phosphate. For the detection of invertase activity, the sections were incubated in a neutral reaction buffered medium containing 0.38 mM sodium phosphate (pH 7.5), 0.024% tetrazolium blue, 0.014% phenazin metasulphate, 30 U of glucose oxidase, and 30 mM of sucrose at room temperature for 3 h [24,25]. For the control, an incubation medium without sucrose was used. For detection of sucrose synthase (SuSy) activity, handmade sections of fresh samples and of samples fixed in 2% paraformaldehyde with 2% polyvinylpyrrolidone 40 and 0.005 M dithiothreitol were placed in incubation medium for 30 min, at 30 8C. The incubation medium contained 5 ml of 150 mM NADH, 5 ml (1 U) of phosphoglucomutase from rabbit muscle, 5 ml of 3 mM glucose-1,6-biphosphate, 5 ml (1 U) of glucose-6-phosphate dehydrogenase from Leuconostoc, 5 ml (1 U) of UDPG–pyrophosphorylase from beef liver, 280 ml of 0.07% aqueous nitro-blue tetrazolium (NBT), 350 ml of buffer, and 50 ml of substrate. The buffer consisted of 100 mM HEPES, 10 mM MgCl2, 2 mM EDTA, 0.2% BSA, and 2 mM EGTA at pH 7.4. The substrate contained 0.75 M sucrose, 15 mM UDP, and 15 mM pyrophosphate. For one of the controls, glucose-1,6-biphosphate and pyrophosphate were not used. In a second control, sucrose was not used [26]. 2.3.5. Reactive oxygen species (ROS) For DAB (3,30 -diaminobenzidine) staining, handmade sections of fresh material were immersed in 0.5% DAB (Sigma1) solution for 20–60 min, in the dark [7]. The intensity of the reaction was examined every 15 min. 3. Results 3.1. General features The leaf gall induced by Pseudophacopteron sp. in A. australe forms a slight projection of the adaxial surface and a more prominent projection of the abaxial surface of its host leaf (Fig. 1a 352 D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 Fig. 1. (a) Leaf of Aspidosperma australe showing gall induced by Pseudophacopteron sp. (arrow). (b) Detail of gall projection. (c) Cross-section showing nymphal chamber, inner cortex (ic), outer cortex (ec), and nymph (n). and b). The nymphal chamber is central, and shelters one individual of Pseudophacopteron sp. from its first instar until the adult phase (Fig. 1c). The gall is parenchymatic, with vascular tissues only at the top of the chamber, which is permanently opened to the abaxial leaf surface. This gall morphotype has two distinct tissue zones, an inner cortex, around the nymphal chamber, and an outer cortex with larger cells (Fig. 1c). 3.2. Cytological features The cells of the non-galled mesophyll have small nuclei, thin cytoplasm, large vacuoles, chloroplasts with numerous primary starch grains, and plastids associated with mitochondria (Fig. 2a and b). The palisade parenchyma cells contain osmiophilic inclusions (Fig. 2c). In immature galls, the cells near the nymphal chamber have lobed or large nuclei surrounded by numerous mitochondria associated with plastids and abundant starch grains (Fig. 2d and e). In the inner cortex, cells with transverse sinuous and thin cell walls indicate sites of hyperplasia (Fig. 2e). These cells have endoplasmic reticulum with electron-dense granules and oleosomes associated with their chloroplast thylakoids (Fig. 2f). Osmiophilic inclusions (Fig. 2d) can be observed throughout the cells over the entire gall cortex, but they concentrate in the cells around the nymphal chamber. In mature galls, sites of hyperplasia are observed (Fig. 3a) in both the outer and inner cortex. The cells around the nymphal chamber have dense and hypertrophied nuclei, conspicuous nucleoli (Fig. 3b), and a developed vacuome (Fig. 3c). The chloroplasts of the outer cortex cells have abundant grouped and peripheral plastoglobules, and are associated with mitochondria (Fig. 3d). After the eclosion of the adult of Pseudophacopteron sp., in the senescent gall, the parenchyma cells of the outer and inner cortices enter cell death and degenerate. The nuclei and the organelles of the cells immediately surrounding the nymphal chamber are encapsulated and degenerate (Fig. 3e and f). These cytological events proceed towards the gall outer cortex. 3.3. Histochemical analyses The non-galled and the galled tissues of A. australe in different stages of development are histochemically distinct in their qualitative aspects as well as in the sites of reaction (Table 1). DNA was detected by red staining of the nuclei in both galled and non-galled tissues. In non-galled tissues, the DNA staining was detected in the nuclei of the cells of the palisade parenchyma. In mature galls, DNA staining was detected in the hypertrophied Table 1 Histochemical tests in non-galled and galled tissues of the Aspidosperma australe– Pseudophacopteron sp. gall system. Tests Non-galled tissues DNA RNA Proteins Starch Sugars Lipids Callose Acid phosphatase Phosphorylase Glucose-6-phosphatase Invertase Sucrose synthase ROS + + = positive detection; + + + + + + + Galls Young Mature Senescent N.E. N.E. + + N.E. N.E. + + + + + + + + + + + + + + + + + + + + + + + = negative detection; N.E. = not evaluated. + D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 353 Fig. 2. Transmission electron micrographs of non-galled and galled tissues of Aspidosperma australe. (a–c) Non-galled leaf tissues. (a) Plastids of a mesophyll cell containing starch grains. (b) Detail of chloroplast starch grain and thylakoid membrane. (c) Detail of osmiophilic inclusions. (d–f) Immature galls. (d) Cell of the inner cortex tissue layer containing plastids with lipid droplets, numerous mitochondria, hypertrophied nucleus, and small vacuoles. (e) Detail of a cell of the inner cortex with a large lobed nucleus, and numerous plastids with starch grains. (f) Detail of a chloroplast with oleosome and associated plastoglobules. ch, chloroplast; osm, osmiophilic inclusion; m, mitochondria; n, nucleus; nu, nucleolus; ld, lipid droplet; s, starch; v, vacuole. nuclei of the cells surrounding the nymphal chamber. RNA activity was also detected in the nucleoli of these cells (Fig. 4a). In non-galled tissues, the histochemical reaction to proteins was more intense in the palisade parenchyma cells. In immature and mature galls, a centripetal gradient of proteins was established (Fig. 4b), but in senescent galls, proteins are equally distributed. Starch was detected in the palisade parenchyma cells of the nongalled mesophyll. In the cortices of the immature and mature galls, starch grains formed a centripetal gradient. In the mature galls, starch grains were sparse in the cells of the adaxial portion of the gall outer cortex (Fig. 4c), but during senescence, they dispersed throughout the gall cortex. Reducing sugars were detected in the cells next to the vascular bundles of the non-galled tissues, and of the mature (Fig. 4d) and senescent galls. Small lipid droplets were observed in the cells of the palisade parenchyma of the non-galled mesophyll. In both immature and mature galls, these droplets were only detected in the cells of the vascular parenchyma (Fig. 4e). The reaction for callose was negative for all of the samples. Acid-phosphatase activity was not detected in the non-galled tissues, but was detected in the galls in all developmental stages. This enzyme activity was more intense in the cells surrounding the nymphal chamber of the mature galls (Fig. 4f). During senescence, the gradient of this enzyme activity extended to the cells of the gall outer cortex. Phosphorylase and glucose 6-phosphatase activity was not detected in the non-galled tissues or in the senescent galls, but in the immature and mature galls, the activity of these enzymes was detected in the cells of the inner cortex, next to the nymphal chamber (Fig. 5a). Slight invertase activity was detectable in the cells around the vascular bundles of the non-galled tissues. In the immature, mature, and senescent galls, the activity of this enzyme was detected by the formation of a salt of nitrous blue tetrazolium in the cytosol (Fig. 5b). The reaction was more intense 354 D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 Fig. 3. Transmission electron micrographs of galled tissues of Aspidosperma australe. (a–d) Mature galls. (a) Cells of the outer cortex with thin sinuous cell walls (arrow), indicating a site of hyperplasia. (b and c) Cells near the nymphal chamber. (b) Hypertrophied nucleus containing evident nucleolus. (c) Intense vacuome, hypertrophied nucleus, and chloroplast with plastoglobules (arrow). (d) Plastoglobules (arrow) in cells of the outer cortex, with associated mitochondria. (e and f) Senescent galls. (e) Nucleus of a cell near the nymphal chamber, in the final stage of degradation. (f) Plastid encapsulation and degradation (arrow) in cells of the outer cortex. ch, chloroplast; m, mitochondria; n, nucleus; nu, nucleolus; s, starch; v, vacuole. in the cells of the gall inner cortex, next to the nymphal chamber, and became less intense outwards, in a centripetal gradient. Marked sucrose synthase activity was detectable in the cells of the vascular bundles in non-galled tissues. In the immature and mature galls, this activity was localized in the cells of the vascular bundles next to the adaxial surface and in the cells surrounding the nymphal chamber (Fig. 5c and d). The gradients of sucrose synthase and invertase were similar to those of proteins and starch in the galled tissues. The positive reaction to DAB appeared as a diffuse brownish color, and revealed the formation of hydrogen peroxide, among other free radicals. The reactive oxygen species (ROS) were detected in the cells of non-galled mesophyll. In the immature, mature, and senescent galls, the ROS were more concentrated in the cells of the inner cortex, next to the nymphal chamber (Fig. 5e and f), and became less intense outwards. Also, ROS was detected in chlorophyll parenchyma outwards from the galled tissues. The gradients of the reserve substances and enzyme activities varied during the development of the galls of A. australe induced by Pseudophacopteron sp., and were established in the mature galls (Fig. 6). 4. Discussion 4.1. Cytological gradients and oxidative stress Most hemipteran galls are relatively simple, both morphologically and anatomically [27]. Cytological, histochemical, and physiological analyses in gall tissues have revealed important differences from non-galled tissues [28,21,4,11], and have been commonly related to the feeding habits of the gall-inducing insects [27]. However, the storage of substances in the gall site may also be subject to constraints imposed by the metabolism of the host plant. D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 355 Fig. 4. Histochemistry of mature galls of Aspidosperma australe. (a) Test for the detection of DNA activity indicated by the red staining of nuclei, and RNA activity by the blue staining of nucleoli (arrow) near the nymphal chamber. (b) Protein gradient increasing towards nymphal chamber, indicating high metabolic activity. (c) Starch gradient (arrow) increasing towards the nymphal chamber on the abaxial surface. (d) Reducing sugars detected next to a vascular bundle located in the gall outer cortex. (e) Lipid droplets in cells of the vascular parenchyma in the gall outer cortex. (f) Phosphatase activity, detected mainly in the innermost layers of the gall tissue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) In the immature galls induced by Pseudophacopteron sp. in A. australe, the cells surrounding the nymphal chamber are redifferentiated from chlorophyll parenchyma, which may confer physiological youth on the inner cortical cells of the gall. In the mature galls, the cytological features concord with Bronner’s observation [1] for nutritive tissues of galls induced by Diptera: Cecidomyiidae, although Pseudophacopteron sp. is an hemipteran. This seems to be a novelty in studies on the development of galls induced by sucking insects, and has been detected for the first time in the Neotropical flora. Indeed, comparative studies on the histochemistry of immature and mature galls are not commonly performed. Moreover, during gall maturation, cellular redifferentiation is completed, and the synthesis of ROS seems to be increased. A cytological diagnostic feature of the ROS production and consequent oxidative stress in the galls of A. australe is the formation of plastoglobules in the chloroplasts. According to Rosseti and Bonatti [7] and Zentgraf [5], one of the early events activated by the hypersensitive response (HR) is the production of ROS, including hydrogen peroxide (H2O2) and the superoxide anion (O2 ). The HR is a mechanism employed by plants to counteract pathogen growth by causing a localized cell death [29]. The positive reaction to DAB indicates the sites of ROS production, and is also a strong indication of the localized response to gall induction in A. australe. This response also leads to a disruption of membrane systems and consequently to cell death in the gall senescent phase. During the processes of senescence, thylakoid membranes are the first to degrade, followed by mitochondria membranes and the chloroplast envelope [30]. In the galls of A. australe, these symptoms are the latest cytological events under the influence of the cecidogenetic field, and occur just after the eclosion of the imago. 4.2. Histochemical gradients The chemical impact caused by the feeding activity of Pseudophacopteron sp. results in the storage of primary metabolites, and is also under the direct influence of the cecidogenetic field. This storage of metabolites indicates the differentiation of a nutritive tissue, which is not typical of psyllid galls but has been previously described in two hemipteran galls in Picea excelsa [31]. Also, the redifferentiation of parenchyma cells may allow the establishment of the larvae without provoking hypersensitive reactions [27]. The high metabolic activity detected in the cells of the inner cortex is strongly influenced by the feeding activity of the insect, as stated by Bronner [1] and Schönrogge [4]. Distinct histochemical gradients were established in the galls of A. australe as products of metabolic changes induced by Pseudophacopteron sp., and must be more related to the growth and development of the gall structure than to the feeding requirements of the nymph. Moreover, the gradients of reserve substances and enzymes can be associated with the stresses generated by the presence of the parasitic life form within the host plant tissues. In galls induced by Hymenoptera: Cynipidae in Quercus sp. and Rosa sp., large amounts of proteins were found in the innermost 356 D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 Fig. 5. Histochemistry of mature galls of Aspidosperma australe. (a) Phosphorylase activity detected in the cells of the outer cortex. (b) Invertase detected in an increasing gradient towards the nymphal chamber. (c and d) Sucrose synthase detected in vascular bundles (arrows) and at the abaxial cortex around the nymphal chamber. (e and f) ROS detected in cells surrounding the nymphal chamber (white arrow) in a decreasing gradient towards the gall outer cortex. tissues, and in Quercus sp., a specific protein, format dehydrogenase (FDH), was expressed in correlation with the general protein gradient [4]. According to these authors, FDH is an indication of respiratory stress in the tissues surrounding the larval chamber. Similarly, the protein gradient found in the tissues of the mature galls of A. australe may indicate differential expression of proteins and must be related to high respiratory stress, mainly next to the nymphal chamber, and tends to disappear in the senescent phase. This observation is reinforced by the RNA activity, and the high production of ROS in the cells of the gall inner cortex. ROS production increased in the senescent phase. The concentration of starch in the galls of A. australe is visually higher in the cells surrounding the nymphal chamber. In general, it is possible to identify a starch-free nutritive tissue in galls of Diptera: Cecidomyiidae, which accumulate in their outer cortex [1]. However, in galls of Lonchocarpus muehlbergianus induced by E. ostreoides (Hemiptera), starch reserves were also detected throughout the gall cortex, even in the cells surrounding the chamber [11]. Álvarez et al. [31] detected the storage of starch in three galls induced by different species of Psyllidae in Pistacia terebinthus. Therefore, it seems plausible to assume that, independently of the species of host plant, starch grains accumulate in nonnutritive galls induced by sucking insects. The storage of lipids in the gall outer cortex may be inherent to the metabolism of A. australe, for in this host plant this metabolite is commonly detected in laticifers associated with the vascular bundles [32]. These substances have also been detected in other systems, such as L. muelhbergianus–E. ostreoides [11] and L. camara–Aceria lantanae [2]. In these systems, the presence of lipids were related to the maintenance of the gall structure, because lipids are molecules with high reserve capacity and energy, and are possible precursors of important components of plant metabolism [33]. 4.3. Histochemistry of enzymes Another intriguing and little-explored issue in galling insects– host plant systems is the demonstration of how storage substances become available for both the development of the gall structure and the feeding behavior of the galling insect. The histochemistry of some enzymes related to starch metabolism was first investigated by Bronner [1], and has never been assessed for galls in the Neotropical region. Because starch is not directly used either for the feeding activity of Pseudophacopteron sp. or the development of gall structure, some enzyme activity related to carbohydrate metabolism is expected to occur in gall tissues. Phosphorylase is an important enzyme responsible for the polymerization and/or the breakdown of starch molecules. In general, there is a correlation between the activity of this enzyme and the amount of accumulated amyloplasts; therefore this correlation depends on the physiological status of the plant tissue [34]. This status may also differ within the same gall, making this model of study particularly intriguing. Phosphorylase activity was detected in the outer cortex of D.C. Oliveira, R.M.S. Isaias / Plant Science 178 (2010) 350–358 357 In addition to sucrose synthase, acid phosphatase may help break down starch molecules into smaller sugars for galling insect nutrition and/or the maintenance of the gall cellular machinery. In the A. australe–Pseudophacopteron sp. system, this enzyme may be responsible for the degradation of some organelles in the cells surrounding the nymphal chamber. In senescent galls, its activity spread throughout the gall tissues. Bronner [1] attributed to acid phosphatase, some changes in the cytoplasm in galls by the formation of autophagic structures, mainly in the nutritive tissue. Acid phosphatase hydrolyzes phosphomonoesters in many plant biochemical reactions, including the formation of sucrose during photosynthesis [40], and the release of Pi (inorganic phosphate), which is important for breaking down the starch molecule [41]. So, in the current system, besides the degradation of some organelles, acid phosphatase may take part in the metabolism of starch during the main trophic phase of Pseudophacopteron sp. This proposal is reinforced by the fact that the gradient of this enzyme activity homogenizes just after the imago emerges. 4.4. Conclusions Fig. 6. Representative diagram of the histochemical gradients detected in mature galls of Aspidosperma australe. Arrow direction and intensity of gray color indicate histochemical and cytological gradients, respectively. DNA (1), RNA (2), and acidphosphatase activities (3) are more intense and exclusively detected in the cells next to the nymphal chamber. Lipids (4) and reducing sugars (5) are detected exclusively in the gall outer cortex. Proteins (6), starch (7), and invertase (8) activity become more concentrated and more intense closer to the nymphal chamber. Sucrose synthase (9) activity is more intense near the nymphal chamber and vascular bundles. For glucose 6-phosphatase (10), and phosphorylase (11), the gradients are centripetal. The gradient of ROS-reactive species of oxygen (12) is more intense centrifugally. immature and mature galls of A. australe, and was interpreted as an indication of the primary steps of starch storage in the gall tissues. The newly formed starch grains, stained red, could be distinguished from pre-existing ones, which stained blue. Another enzyme investigated was glucose-6-phosphatase because of its involvement in the synthesis of intermediate compounds during the formation of sucrose [35]. This enzyme activity may be associated with the formation of sucrose after the breakdown of starch molecules, to provide resources for gall growth and the nutritional requirements of Pseudophacopteron sp. The conversion of the products of starch metabolism into glucose and fructose, by the action of invertase, is quite important next to the nymphal chamber, a site of high stress and respiratory metabolism. In addition to catalyzing the irreversible breakdown of sucrose into glucose and fructose, the invertases take part in the biosynthesis of ABA, IAA, and cytokinins, and the perception of their levels in the formation of tumors [36,37], and consequently in cell hypertrophy. Moreover, both the cell wall and the vacuolar invertases maximize the production of hexoses, which promote cell division and expansion, and respiration [37], while the presence of sucrose favors the differentiation of storage organs and maturation of tissues [38,37]. Thus, the detection of active sites of invertases in the immature and mature gall tissues of A. australe might indicate the source of constant division and regulation of cell growth and intense respiration. The detection of sucrose synthase may be important to determine the maturation of the gall tissues, once it is involved in organ storage and maturation [37]. The role of sucrose synthase in sucrose import may involve its dual capacity to direct carbon towards both polysaccharide biosynthesis and respiration. The UDPG product of sucrose synthase has been implicated in starch formation [39], so its storage in the immature and mature galls of A. australe may be a product of the activity of sucrose synthase in the cells of vascular bundles and around the nymphal chamber. In conclusion, the effect of the feeding activity of Pseudophacopteron sp. alters the metabolism of A. australe during gall development, which was demonstrated by the histochemical and the cytological analyses. The detection of both reserve substances and enzyme activity revealed histochemical and cytological gradients, even though a nutritive tissue per se was not differentiated. The histochemical and cytological features detected in the A. australe–Pseudophacopteron sp. system validate the storage of starch as the pattern for galls induced by sucking insects. This is the first description of carbohydrate metabolic pathways and high oxidative stress for galls induced by sucking insects in the Neotropical region. The validation of this pattern will be checked by the analysis of other sucking feeding insects gall systems in comparison to those of other feeding modes herbivores. Acknowledgments The authors thank FAPEMIG CBB 782/06 for financial support; the Laboratory of Electronic Microscopy of the Universidade Federal de Lavras; Prof. Dr. Eduardo Alves, M.Sc. Eloisa A. das Graças Leite, and Thiago A. Magalhães for support in ultrastructural analyses; JP da Matta for critiquing the manuscript; and Janet Reid for language revision of the final version. References [1] R. Bronner, The role of nutritive cells in the nutrition of cynipids and cecidomyiids, in: J.D. Shorthouse, O. Rohfritsch (Eds.), Biology of Insect-induced Galls, Oxford University Press, New York, 1992, pp. 118–140. [2] M.Z.D. Moura, G.L.G. Soares, R.M.S. 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