Gradients of metabolite accumulation and redifferentiation of nutritive cells associated with vascular tissues in galls induced by sucking insects

Galls are structures entirely made of plant tissues manipulated by gall-inducing parasites. We analyzed the alterations of cell phenotypes caused by a sucking-insect, Nothotrioza cattleiani, on the leaves of Psidium cattleianum that led to the formation of a globoid gall morphotype. We found changed cell fates in the leaves compared to the galls, and different degrees of cell alterations in gall layers, which formed gradients. Surprisingly, the vascular parenchyma cells on N. cattleiani galls are nutritive. Herein, we show that even though the globoid shape is one of the most common in nature, the galls of N. cattleiani are unique entities.


Introduction
The capability of insects to induce galls depends on variable properties of plant cells, which are ultimately defined by the biotic and abiotic stimuli from the surrounding environment, and lead to different cell phenotypes (Gianoli and Valladares 2012). Plant cells under the influence of gall-inducing insects redifferentiate (sensu Lev-Yadun 2003) and often assume rapid cell cycles and new cell fates for the neo-ontogenesis of plant galls (sensu Carneiro et al. 2014a). Even though specific galling insect taxa are classically known to induce conserved, morphologically different galls (Mani 1964;Rohfritsch 1992), recent studies have documented extremely diverse aspects of cell biology in neotropical galls (Moura et al. 2009;Isaias 2009, 2010a;Formiga et al. 2011;Isaias et al. 2011;Castro et al. 2012a;Bedetti et al. 2013;Dias et al. 2013;Ferreira and Isaias 2013;Guimarães et al. 2013Guimarães et al. , 2014. The metabolism of certain neotropical gall taxa has been assessed using cytological and histochemical methods (Oliveira and Isaias 2010b;Oliveira et al. 2010Oliveira et al. , 2011aVecchi et al. 2013) in the search for gradients already described for nutritive tissues in Cynipidae and Cecidomyiidae galls (Bronner 1992). In the Neotropical galls with true nutritive tissues, some of the metabolic gradients proposed by Bronner (1992) were corroborated (Oliveira et al. 2010(Oliveira et al. , 2011b. Unexpectedly, cytological and histochemical gradients, i.e. increasing degree of cell alterations and of metabolite accumulation in the cortical cells of the gall towards the larval chamber, are also reported for the galls induced by the sucking insect, Pseudophacopteron sp. (Psyllidae), on Aspidosperma australe (Oliveira and Isaias 2010b), which have nutritivelike parenchyma cells. Despite being regarded as structurally simple and devoid of nutritive cells (Meyer 1987), galls of sucking insects, namely Euphalerus ostreoides  and Nothotrioza myrtoidis (Carneiro and Isaias 2014;Carneiro et al. 2014b), also have cytological and histochemical characteristics of metabolically active cells. These cells are evidenced in the vascular tissues and parenchyma near the nymphal chamber. Nothotrioza myrtoidis galls have gradients of primary and secondary metabolites, discrete accumulation of reactive oxygen species (ROS) and are photosynthesis deficient (Carneiro et al. 2014b). Moreover, the completion of cell cycles through standby-redifferentiation is ensured by the chemical (Carneiro et al. 2014b) and cytological ROS-scavenging apparatus (Carneiro and Isaias 2014).
Herein, we assess the cytology and histochemistry of the galls induced by Nothotrioza cattleianum Burckhardt (Triozidae)  on Psidium cattleiani Sabine (Myrtaceae). The structural -functional aspects related to cell biology in P. cattleianum galls are compared with those of other galls, especially the co-generic system of N. myrtoidis -P. myrtoides. The galls of Nothotrioza spp. on Psidium spp. are both globoid (sensu Isaias et al. 2013) and have similar phenology (Butignol and Pedrosa-Macedo 2003;Carneiro et al. 2013). We expect that the developmental cytology and histochemical profile of P. cattleianum galls should reveal conserved traits with regards to the sucking feeding habit of N. cattleiani and also distinctive ones of a unique extended phenotype (sensu Bailey et al. 2009). The following questions are addressed: (i) Do the cells of N. cattleiani galls have conserved cell fates in relation to the non-galled leaves, and in comparison to N. myrtoidis galls? (ii) Do the cytological and histochemical profiles of Nothotrioza spp. galls indicate unique neo-established tissue functionalities?

Sampling
The population of P. cattleianum Sabine (Myrtaceae) with galls induced by N. cattleiani Burckhardt is located at the Parque Estadual Pico do Marumbi, municipality of Piraquara, Paraná state, Brazil. Non-galled leaves (young and mature, n ¼ 5 per developmental stage) and galls at the phases of induction, growth and development, maturation, and senescence (n ¼ 5 per developmental stage) were collected during the years 2012 and 2013. The samples were fixed in 2.5 % glutaraldehyde (Grade I) and 4.5 % formaldehyde in phosphate buffer (0.1 M; pH 7.2) (Karnovsky 1965).

Histochemical analysis
Histochemical tests for primary and secondary plant metabolites, ROS and the activity of enzymes related to carbohydrate metabolism were performed using fresh samples of mature leaves and galls. Control tests were performed according to the references and also by comparison with blank sections. Samples were submitted to the tests/reagents, following the methods described subsequently.
Acid phosphatase activity. Incubation 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. Reaction in 1 % ammonium sulfate for 5 min, after washing in distilled water (Gomori 1956).
Treated sections were mounted on glass slides with Kaiser's glycerol gelatin (Kraus and Arduin 1997), observed and photographed with a light microscope (Leica w DM500) coupled with a digital camera (Leica w ICC50 HD).

Transmission electron microscopy
The fixed samples were post-fixed in 1 % osmium tetroxide in phosphate buffer (0.1 M; pH 7.2), dehydrated in ethanol series (O'Brien and McCully 1981) and embedded in Spurr's w resin. Ultrathin sections were obtained with a diamond knife in the Reichert -Jung Ultracut ultramicrotome (Leica, Wetzlar, Germany), attached to copper grids, contrasted with uranyl acetate and lead citrate (Reynolds 1963). The sections were analysed using a transmission electron microscope Tecnai TM G2-12-SpiritBiotwin (FEI, Hillsboro, USA) at 120 kV, at the Centro de Microscopia of the Universidade Federal de Minas Gerais (CM-UFMG).

Morphology and development of leaves and galls
The galls of N. cattleiani are globoid and protruded to the abaxial surface of the leaves of P. cattleianum (Fig. 1A). The young leaves of P. cattleianum have uniseriate epidermis, homogenous chlorophyllous parenchyma with interspaced vascular bundles undergoing differentiation. As they mature, epidermis remains uniseriate; hypodermis differentiates under the adaxial surface of the epidermis, and dorsiventral chlorophyllous parenchyma, interspaced with collateral vascular bundles, is observed (Fig. 1B). The galls of N. cattleiani are induced on the young leaves, whose cells redifferentiate to form a depression on the leaf lamina, and ultimately generate a globoid gall with an ample chamber and relatively thin wall. In mature galls, such walls have uniseriate inner and outer epidermis, hyperplasic and hypertrophied homogenous parenchyma and collateral vascular bundles near the nymphal chamber (Fig. 1C). The galls have a 1-year life cycle, with four developmental stages: induction, growth and development, maturation and senescence.

Cytological development of cell lineages
Epidermis. In the young leaves of P. cattleianum, epidermal cells have thin anticlinal walls, fragmented hyaline vacuoles, few chloroplasts and mitochondria, and large central nuclei with conspicuous nucleoli ( Fig. 2A; Table 1). In the mature leaves, the walls are thicker, and the vacuoles have confluenced into a big central vacuole with phenolics; chloroplasts and mitochondria are rare, and the nuclei are small with peripheral heterochromatin ( Fig. 2B and C). Galls are induced on young leaves, and exhibit epidermal cells with homogenously thin walls, with large hyaline vacuoles. Nuclei are peripheral and the electron-dense cytoplasm is reduced to a fine region near the cell walls (Fig. 2D). Epidermal cells in the galls at the stage of growth and development have heterogeneous thick cuticle, with protoplast similar to that of previous stage. At the phase of maturation, the inner epidermis is intermittent; cells exhibit heterogeneously thickened and polylamellate walls and thin cuticle ( Fig. 2E; Table 1). The protoplast is poorly altered in comparison to the cells of the previous stage. At senescence, the cells have vacuoles with phenolic inclusions and walls with disaggregated lamellae and end up undergoing autolysis.
Chlorophyllous parenchyma. In the young leaves, mesophyll cells have thin, homogenous primary cell walls and reduced intercellular spaces. Their vacuoles are large, sometimes fragmented around the nucleus, with electron-dense granular inclusions. Nuclei are large, with electron-dense   Table 1). In mature leaves, the palisade parenchyma cells have large phenolic-rich vacuoles, many chloroplasts with well-organized thylakoid lamellae and starch grains; nuclei are peripheral, with inconspicuous nucleoli (Fig. 3C). Chloroplasts with large and numerous plastoglobules are frequent (Fig. 3D  hyaline vacuoles, peripheral nuclei with inconspicuous nucleoli and chloroplasts with well-organized thylakoid lamellae, starch grains and rare plastoglobules ( Fig. 3E and F). Galls at induction stage have hypertrophic and hyperplasic cells, with thin primary walls, sometimes fragmented vacuoles, with phenolic inclusions. They have large nuclei with conspicuous nucleoli, small mitochondria associated with chloroplasts with well-organized thylakoid lamellae and few small plastoglobules (Fig. 4A). Polysomes, Golgi apparatus and multivesicular bodies are sometimes visualized in the periphery of the cells ( Fig. 4B and C). At the phase of growth and development, the cells have secondary polylamellated walls, phenolicrich vacuoles and electron-lucent cytoplasm poor in organelles. Nuclei are small, with little heterochromatin at the periphery; mitochondria are small, associated with chloroplasts with well-organized thylakoid lamellae, starch grains and few small plastoglobules ( Fig. 4D and E). Cells of mature galls are large, with big phenolic or hyaline vacuoles, reduced cytoplasm, sometimes with periplasmic spaces. Cell walls are secondary, either homogeneously thickened and polylamellate or irregularly thickened, sometimes polylamellate, with sites of different electron density ( Fig. 4F and G). Organelles are scarce; lamellar bodies, degraded nuclei and small chloroplasts with underdeveloped thylakoid lamellae and plastoglobules are observed at the periphery of the cells ( Fig. 4G and H). At gall senescence, the outer cortical cells exhibit thickened, polylamellate secondary walls and remain alive, with phenolic-rich vacuoles. Inner cortical cells have signs of degradation, with large periplasmic spaces, disrupted tonoplast and chloroplasts with vestigial thylakoid grana and plastoglobules ( Fig. 4I and J; Table 1).
Vascular and perivascular parenchyma. In the young leaves, the cells have thin primary walls, sometimes sinuous and with many plasmodesmata; nuclei are large, with inconspicuous nucleoli and dispersed heterochromatin (Fig. 5A). They have large, sometimes fragmented hyaline vacuoles, dense cytoplasm with rounded mitochondria, small underdeveloped chloroplasts, with few plastoglobules, little RER and lamellar bodies continuous with the plasma membrane ( Fig. 5B and C; Table 1). In mature leaves, cells have thin primary walls; cytoplasm is hyaline, with many mitochondria, scarce RER and large nuclei with dispersed heterochromatin (Fig. 5D). Phenolic-rich vacuoles and periplasmic spaces are frequent. In the galls at induction phase, cells are thin walled, with electron-dense cytoplasm, large nuclei, abundant polysomes, sometimes evident Golgi apparatus, large mitochondria, abundant plasmodesmata with evident microtubules (Fig. 5E and F). Lamellar and multivesicular bodies are frequent (Fig. 5G). At the phase of growth and development, cells have irregularly thickened walls with sites of different electron density and fragmented hyaline vacuoles. Cytoplasm is electron-lucent and organellerich, with abundant mitochondria and polysomes, Golgi apparatus, RER, large nuclei sometimes with conspicuous nucleoli and dispersed heterochromatin (Fig. 5H). Lomasomes and multivesicular bodies are frequent (Fig. 5I). Cells of mature galls have thickened and somewhat sinuous secondary walls, large vacuoles either hyaline or phenolicrich, and abundant mitochondria. Cytoplasm is electron dense, and periplasmic spaces may be observed (Fig. 5J). At gall senescence, cells are metabolically active, with organelle-rich cytoplasm. They have abundant mitochondria, polysomes and RER; nuclei are large, with peripheral heterochromatin. Lomasomes and lamellar bodies are rarely seen ( Fig. 5K; Table 1).

Histochemical profile of leaves and galls
Primary metabolites. In the leaves, reducing sugars and primary starch grains are, respectively, localized by Fehling's and Lugol's reagents throughout the chlorophyllous parenchyma and more rarely in the hypodermis. Reducing sugars are also localized in the phloem of vascular bundles. Lipid droplets and protein precipitates, respectively, stained by Sudan red B and Coomassie blue are observed more intensely in the chlorophyllous parenchyma and vascular bundles, and more scarcely in the epidermal cells and oil glands. The cuticle on both epidermal surfaces and the secretion of oil glands are also stained by Sudan red B. In the galls, reducing sugars are strongly stained by Fehling's reagent, forming a centripetal gradient of sugar accumulation from the outermost cell layers towards the inner cortex (Figs 6A and 8; Table 2). Primary starch grains are rarely stained, but they may form a discrete centrifugal gradient evidenced by the reaction to Lugol's reagent (Figs 6B and 8). Protein precipitates are stained by Coomassie blue in the vascular bundles, perivascular parenchyma and outermost cortical cell layers, forming a bidirectional gradient from the median cortex towards the inner and outer cortices (Figs 6C and 8). The cuticle of the outer epidermal cells and the secretion of oil glands were stained by Sudan red B (Fig. 6D; Table 2).
Enzyme activity. Leaves have detectable activity of acid phosphatase exclusively in the cells of the spongy parenchyma. Galls have detectable activity of glucose-6-phosphatase and invertases in the vascular bundles and perivascular parenchyma of the median and inner cortices (Figs 6E and F and 8; Table 2).
Secondary metabolites. Phenolic substances and terpenoids are, respectively, evidenced by ferric chloride and Nadi solutions intensely in the palisade parenchyma  Table 2).
Reactive oxygen species. Reactive oxygen species are detected intensely by DAB solution in the chlorophyllous parenchyma of leaves, and also in the phloem and vascular parenchyma. In the galls, ROS are detected weakly in a bidirectional gradient from the median cortex towards the inner and outer cortices. The most intense reactions are observed in the vascular bundles and perivascular parenchyma, and in the epithelium of the oil glands (Figs 7F and G and 8; Table 2).  Nevertheless, the selective pressures that affect gall morphology seem to be conserved across different gall systems, as convergent types of tissue organization and cell fates, i.e. vascular bundles interspersed with homogenous parenchyma cells, surrounded by sclerenchyma cells, are widely reported in the literature (Meyer 1987;Rohfritsch 1992;Stone and Schö nrogge 2003;Oliveira and Isaias 2009;Formiga et al. 2011;Carneiro and Isaias 2014;Carneiro et al. 2014a). Despite structural similarities, most cell lineages in N. cattleiani galls on P. cattleianum have developmental dynamics different from those of N. myrtoidis galls on P. myrtoides. Epidermal cells are the exception to this tendency, as they have conserved cytological traits in both systems, namely vacuolated cells that are well organized in the outer surface of the galls but intermittent in the inner surface (Carneiro and Isaias 2014). Chlorophyllous parenchyma cells in Nothotrioza spp. galls have different time-based development. Nothotrioza myrtoidis galls have parenchyma cells that develop by standbydifferentiation (sensu Carneiro and Isaias 2014), in which cell structure remains nearly unaltered for a long period of time, and undergoes major changes from the stage of maturation to senescence. Conversely, the cytology of N. cattleiani galls reveals that parenchyma cells gradually and constantly change from induction phase towards senescence. In galls of both Nothotrioza spp., the cell walls thicken and lignify in the outermost layers of the cortex, while the photosynthetic and respiratory apparatus of the cells are impaired. Gall structures are assumed to play defensive roles against natural enemies of the galling insects (sensu Stone and Schönrogge 2003). The changes of cell fate in the chlorophyllous parenchyma, which have thickened and lignified walls in the galls on Psidium spp., suggest the investment in a structure-based defence strategy that should increase the adaptive values of the galls to the Nothotrioza spp.

Structural -functional implications of cell fates
Contrary to the new cell fates of chlorophyllous parenchyma cells in N. cattleiani galls, vascular and  perivascular parenchyma undergo less structural alteration. The fates of these cells are not altered from nongalled leaves to galls, and they maintain a well-developed metabolic apparatus, namely large nuclei, and dense cytoplasm with abundant polysomes and mitochondria, throughout gall development in both N. myrtoidis (Carneiro and Isaias 2014) and N. cattleiani galls. The maintenance of structural -functional characteristics of vascular tissues indicates that their primordial role in plants, i.e. conducting water and solutes (Buvat 1989), is maintained in galls. Such a strategy is adaptive (sensu Stone and Schö nrogge 2003) for the gall inducers as far as their nutrition is concerned, since Nothotrioza spp. are sap feeders (Burckhardt 2005;Carneiro et al. 2013), and thus dependent on the intake of water and solutes by vascular tissues. Altogether, the cellular characteristics of epidermis, chlorophyllous parenchyma and vascular and perivascular parenchyma corroborate the adaptive nature of the gall structure (sensu Stone and Schönrogge 2003), as they directly relate to the maintenance of an adequate microenvironment, protection against natural enemies and enhancement of nutritional options for the gall inducers, as classically hypothesized by Price et al. (1987) for galls in general.
In relation to the non-galled leaves, cell fates are completely altered in galls, except for the outer epidermis, whose cell cycles are intensified, but the fates are maintained. In relation to N. myrtoidis galls, cell fates are roughly the same, but the standby-redifferentiation observed in the chlorophyllous parenchyma cells during the stage of growth and development (Carneiro and Isaias 2014) does not occur in N. cattleiani galls.

Roles of cell metabolism in gall biology
The functional aspects of plant tissues in the context of gall structure have been related to the accumulation of defensive and nutritive metabolites (Hartley 1998) and to the establishment of histochemical and cytological AoB PLANTS www.aobplants.oxfordjournals.org gradients in Cynipid (Bronner 1992), Cecidomyiidae (Bronner 1992;Oliveira et al. 2010Oliveira et al. , 2011a and Pseudophacopteronidae (Oliveira and Isaias 2010b) galls. In the galls of N. myrtoidis, such gradients are not observed in gall cortices due to their low structural complexity and metabolism (Carneiro and Isaias 2014). The galls of N. cattleiani also lack cytological gradients, as the parenchyma cells are metabolically impaired, with low protein synthesis, scarce and underdeveloped mitochondria and chloroplasts. Nevertheless, the vascular and perivascular parenchyma cells appear increasingly metabolic during gall morphogenesis, i.e. large nuclei, abundant RER, mitochondria, ribosomes and polysomes. Such cell types have also been observed in the vascular and perivascular tissues in galls of N. myrtoidis (Carneiro and Isaias 2014) and share great similarities with the nutritive cells of cynipid (Bronner 1992) and cecidomyiid galls (Bronner 1992;Oliveira et al. 2010Oliveira et al. , 2011b. Nutritive tissues have the most specialized cell types within gall structure, and they are constantly affected by the gall inducers during their feeding (Bronner 1992). Both the impact of the feeding activity of the galling insects and the high metabolism intrinsic to these cell types often lead to the accumulation of ROS. In fact, ROS were histochemically localized in the nutritive tissues of galls induced by Cecidomyiidae on Aspidosperma spruceanum (Oliveira et al. 2010) and on Copaifera langsdorffii (Oliveira et al. 2011a). Furthermore, ROS have been localized in the nutritive-like ground parenchyma around the nymphal chamber in the galls of Pseudophacopteronidae on A. australe (Oliveira and Isaias 2010b) and in the vascular bundles of N. myrtoidis (Carneiro et al. 2014a) and E. ostreoides galls . Vascular and perivascular parenchyma cells are metabolically active in the galls on P. myrtoides, with well-developed ROS-scavenging apparatus, i.e. lomasomes, lamellar and multivesicular bodies (Carneiro and Isaias 2014). Such structures were previously described in the nutritive cells of galls induced by Thysanoptera (Raman and Ananthakrishnan 1983), in the fast-dividing nutritive cells of lepidoptera galls on Marcetia taxifolia (Ferreira et al. 2015), and also herein for the galls on P. cattleianum. Lomasomes, lamellar and multivesicular bodies act together with the endoplasmic reticulum to recycle membrane systems (Staehelin 1997) to ensure the functionality of cells subjected to high oxidative stress.
In addition to the ROS-scavenging apparatus, high protein synthesis seems to be characteristic of nutritionrelated tissues in galls, i.e. true nutritive tissues in galls of cynipids (Bronner 1992), cecidomyiids (Oliveira et al. 2010(Oliveira et al. , 2011aFerreira and Isaias 2014) and lepidoptera (Vecchi et al. 2013), and of nutritive-like parenchyma cells around the nymphal chamber of Psylloidea galls (Oliveira and Isaias 2010b;Isaias et al. 2011). In fact, the histochemical detection of proteins in the galls of N. cattleiani corroborates such premise and reveals another similarity among vascular and perivascular parenchyma cells in the galls of P. myrtoides (Carneiro et al. 2014b) and P. cattleianum and those of true nutritive tissues. The well-developed ROS-scavenging apparatus and high protein synthesis seem to be a widespread trait of nutrition-related cells across different galls. More than simply feeding on the vascular bundle cells, N. cattleiani induces the redifferentiation of true nutritive cells in and around the vascular system, which is herein reported for the first time in insect galls.
Other cell types, such as those of the chlorophyllous parenchyma, may also be subjected to high oxidative stress due to photosynthesis and respiration, which are ROS-generating processes (Møller et al. 2007). Chloroplasts subjected to high oxidative stress may develop plastoglobules, which help to minimize oxidative damage to the photosynthetic apparatus (Asada 2006;Møller et al. 2007). In fact, the chlorophyllous parenchyma cells of non-galled leaves of P. myrtoides (Carneiro and Isaias 2014), and of P. cattelainum accumulate ROS, and have conspicuous and numerous plastoglobules. Also, they have low-stack grana with electron-lucent thylakoids, which is characteristic of sun-type chloroplasts previously described by Lichtenthaler et al. (1981), which photosynthesize at high levels. Galls that accumulate high amounts of ROS, such as the ones induced by the sucking insect Pseudophacopteron sp. on A. australe (Oliveira et al. 2011b), have plastoglobules in the chloroplasts. In this system, the stress imposed by the galling herbivore stimulates the differentiation of plastoglobules, which are not observed in non-galled leaves, and both galls and leaves photosynthesize at the same levels (Oliveira et al. 2011b). In the galls of N. cattleiani, the differentiation of plastoglobules is blocked, similar to the galls of N. myrtoidis (Carneiro and Isaias 2014), which are photosynthesis deficient (Carneiro et al. 2014b). In both Nothotrioza spp. galls, the chloroplasts have less lamellation throughout gall development and high-stack grana with electron-dense thylakoids, as described for shadetype leaves (Lichtenthaler et al. 1981), which photosynthesize at low levels. The cytological features of chloroplasts in the chlorophyllous parenchyma of N. cattleiani galls indicate low cellular metabolism and corroborate the hypothesis that these galls may be photosynthesis-deficient, such as the ones of the co-generic system, N. myrtoidis (Carneiro and Isaias 2014;Carneiro et al. 2014b).
Despite the low metabolism of chlorophyllous parenchyma cells in the galls of N. cattleiani, secondary and primary metabolites are histochemically localized in the cortex of mature galls, forming gradients somewhat similar to those reported for N. myrtoidis galls (Carneiro et al. 2014b). In both Nothotrioza spp. galls, the scarcity of lipid droplets and essential oils is attributed to the characteristics of the host plants, P. myrtoides and P. cattleianum. These compounds are common in Myrtaceae (Ramos et al. 2010) and represent a metabolic trait that is not changed due to gall formation, as observed for galls of Aceria lantanae on Lantana camara (Verbenaceae) (Moura et al. 2008). The centripetal gradients of reducing sugars, on the other hand, evidence the manipulation of plant cell metabolism in galls, which conspicuously accumulate sugars. Sugars are not synthesized at gall sites, but they are drained from non-galled organs (Burstein et al. 1994;Raman et al. 2006;Castro et al. 2012bCastro et al. , 2013. Herein, this premise is confirmed for the galls of N. cattleiani, which have detectable activity of glucose-6phosphatase and invertases in the vascular bundles and surrounding tissues, next to the larval chamber. These sites of enzyme activity are similar to those described in the galls on A. australe (Oliveira and Isaias 2010b), A. spruceanum (Oliveira et al. 2010) and Lonchocarpus muehlbergianus ). Glucose-6-phosphatase is involved in the formation of sucrose after starch breakdown ( Baroja-Ferná ndez et al. 2003) and invertases catalyse the irreversible conversion of sucrose into glucose and fructose (Koch 2004). The centrifugal histochemical gradient of starch in the galls of P. cattleianum indicates that starch is converted into soluble sugars in the cells surrounding the larval chamber, where enzyme activity is detected. A similar gradient of starch and related enzyme activity is described for the galls on L. muehlbergianus , which reinforces the enzyme-mediated mobilization of starch, as a pattern for inner cortical cells of galls. The activities of enzymes related to carbohydrate metabolism determine the establishment of physiological sinks in plants (Koch 1996;Koch and Zeng 2002) and are involved in tissue development and cell expansion (Rehill and Schultz 2003), crucial for gall development. In fact, the galls on Psidium spp. act as physiological sinks, but diverge in terms of the detectable carbohydrate-related enzymes. The activity of acid phosphatase is exclusive of P. myrtoidis galls (Carneiro et al. 2014b), while the activity of glucose-6-phosphatase and invertases is detected only in P. cattleianum galls.
Besides accumulating carbohydrates, the galls on P. cattleianum also have gradients of total phenolics and proanthocyanidins, similar to the galls on P. myrtoides (Carneiro et al. 2014b). The localization of such compounds in gall tissues is considered a defensive chemical strategy of gall inducers against natural enemies (Bronner 1992) for their unpalatability. In both galls, phenolic compounds are believed to control cell expansion and division by the modulation of indol-acetic acid (IAA) levels (Hori 1992). In fact, recent studies localized phenolics and IAA at the same sites in Piptadenia gonoacantha galls (Bedetti et al. 2014), thus confirming the developmental role of phenolics in galls. Also, phenolics mediate the morphogenesis of vascular tissues (Aloni 2001) and should be involved in the neoformation of vascular bundles near the nymphal chamber. Neoformed vascular bundles contribute to the establishment of the gall as a sink of photoassimilates and to the nutrition of the gall inducer, which contradicts the classical role of phenolics as plant-defensive compounds in galls, as widely attributed in literature (Hartley 1998;Nyman and Julkunen-Tiitto 2000;Formiga et al. 2009). Another group of phenolic substances, the proanthocyanidins, accumulates both in the outermost and innermost cell layers of the Psidium spp. galls, with decreasing gradients towards the median cortex. Such substances act as antioxidants (Simmonds 2003;Bouaziz et al. 2005), and their distribution along the gradient of ROS concentration possibly prevents oxidative stress in gall tissues, keeping cell alterations to a minimum.

Conclusions
The similar phenotypes of the globoid galls of the double co-generic systems, N. cattleiani -P. cattleianum and N. myrtoidis -P. myrtoides, are not extended to the cytological and histochemical levels. Nevertheless, the lack of cytological gradients and the formation of somewhat conserved histochemical profiles are common to both AoB PLANTS www.aobplants.oxfordjournals.org galls, with the centripetal accumulation of sugars and centrifugal detection of lignins, as conserved traits possibly linked to the taxonomical proximity of the involved species.
The bidirectional gradients of metabolite accumulation described for N. cattleiani galls, together with the redifferentiation of true nutritive cells associated with vascular bundles, are unique features. Also, cytological development in N. cattleiani galls is gradual from induction towards senescence, and different from the standbyredifferentiation of N. myrtoidis galls, which reinforce the species-specific subcellular aspects of these distinctive extended phenotypes.