Compartmentalization of Metabolites and Enzymatic Mediation in Nutritive Cells of Cecidomyiidae Galls on Piper Arboreum Aubl. (Piperaceae)

Galling insects commonly change the chemical profile of their host plant tissues during gall induction and establishment. As a consequence, galls accumulate a wide range of metabolites in specialized cells, which may be organized in a nutritive tissue and in outer storage cells. The nutrients compartmentalized in nutritive cells may be directly assessed or metabolized via enzymatic mediation, while the gall outer cortex may accumulate secondary metabolites. These secondary metabolites may configure a specialized chemical barrier against the attack of natural enemies. Either the nutritive inner cells or the outer cortical cells, with their specific metabolic apparatus, should differentiate under the chemical constraints of each host plant-galling herbivore interaction. This premise is herein addressed by the investigation of the histochemical profile of the non-galled leaves and galls induced by Diptera: Cecidomyiidae on Piper arboreum. The spatial compartmentalization of the nutritive and defensive metabolites indicates the new functions assumed during the redifferentiation of the host plant cells. The enzymatic mediation of the primary metabolites by sucrose synthase and invertases favors the nutritive requirements of the galling Cecidomyiidae or the structural maintenance of the gall. The accumulation of secondary metabolites is restrict to the tissue layers not involved in nutrition, and may act in the chemical protection against predators or parasitoids. Current results systematically document metabolites compartmentalization, evidence the impairment of toxic compounds storage in cells surrounding the larval chamber, as well as, detect the redirection of nutritive substances to the site of the Cecidomyiidae feeding. The activity of sucrose synthase is restrict to the nutritive tissue in the galls on Piper arboreum, and reinforces previous detection of this enzyme mediation in carbohydrate metabolism in Cecidomyiidae galls.

Proteins, lipids, reducing sugars or starch accumulate in the inner layers of the gall, i. e., the nutritive tissue, and can provide resources for the nutrition of the galling insect (Price, Waring, & Fernandes, 1986, 1987;Bronner, 1992).Nevertheless, in some systems, the availability of these metabolites to the galling insect or to the maintenance of cell metabolism depends on enzymatic activities (Bronner, 1992;Oliveira Magalhã es, Carneiro, Alvim, & Isaias, 2010;Oliveira & Isaias, 2010b;Carneiro et al., 2014).The patterns of enzymatic activities depend on the feeding behavior of the galling insects (Bronner, 1992), and on the chemical profile of the host plants (Oliveira et al. 2010(Oliveira et al. , 2014;;Oliveira & Isaias, 2010b).
The enzymatic mediation of carbohydrates metabolism has been documented just for four galling herbivore-host plant systems in the Neotropics (Oliveira et al., 2010(Oliveira et al., , 2011;;Oliveira & Isaias, 2010b;Carneiro et al., 2014).These four systems involve gall inducing Cecidomyiidae and Psylloidea with their divergent peculiarities, and demonstrated the non-exclusiveness of the carbohydrates accumulation to Cecidomyiidae galls.Among the investigated carbohydrates, sucrose is synthesized in the cytosol from photosynthetically fixed carbon, starch reserves or lipid metabolism (Wind et al., 2010), and is transported via phloem to other plant parts.The main enzymes responsible for sucrose metabolism are sucrose synthase (SuSy) and invertases, which catalyse the conversion of sucrose into glucose and fructose (Koch, 2004).In general, SuSy activity is associated to sink tissues and starch accumulation, while invertases mediate cell respiration, tissue growth and development (Koch, 2004;Wind et al., 2010).In galls, the metabolism of these enzymes was also related to the formation of histochemical gradients and maintenance of the gall tissues in Aspidosperma australe (Oliveira & Isaias, 2010b).The sites of the galling herbivore`s nutrition seems to be crucial for the redifferentiation of the nutritive cells, as documented for Nothotrioza cattleiani galls on Psidium cattleianum (Carneiro, Pacheco, & Isaias, 2015).
Cell redifferentiation and metabolism in gall outer and inner tissue layers are herein revisited in a Cecidomyiidae -Piper arboreum system.We assume that primary and secondary metabolites accumulate in distinct tissue compartments, as expected, but some metabolic steps of this gall should be dependent on the host plant carbohydrates metabolism rather than on the Cecidomyiidae feeding mode.If this is true, some similarities between the compartmentalization and metabolism of Cecidomyiidae and Psylloidea galls should be found.
The intralaminar lenticular galls on P. arboreum have nutritive cells limiting the larval chamber, where carbohydrates should accumulate, as previously observed in other two Neotropical Cecidomyiidae galls (Oliveira et al., 2010(Oliveira et al., , 2011)).Similarly, the accumulation of cabohydrates in nutritive like cells has been documented in a Psyllloidea gall (Oliveira et al., 2011;Carneiro et al., 2014).Both gall inducers should not come into contact with non-palatable secondary metabolites, and therefore, enzymes mediation and a spatial compartmentalization of nutritive and defensive metabolites in gall developmental site are expected.Current analyses focus on the following questions: (I) does the accumulation of primary and secondary metabolites follow the expected compartmentalization? (II) Is there any disruption for metabolites accumulation in response to the galling stimuli of the Cecidomyiidae on P. arboreum?(III) Are there any similarities between the enzymatic mediation of Cecidomyiidae and Psylloidea galls?And (IV) should ROS signaling involve sugar mediation during gall development on the Cecidomyiidae intralaminar leaf galls on P. arboreum?

Methods
Samples of non galled leaves (NGL) and mature leaf galls (MG) induced by an unidentified species of Diptera: Cecidomyiidae on Piper arboreum were accompanied and collected from September 2013 to March 2014 at the ecological station of Universidade Federal de Minas Gerais in Belo Horizonte, Minas Gerais state, Brazil.

Enzymatic Activity
For the detection of the activity of acid phosphatase, the sections were incubated in 0.012% lead nitrate and 0.1M potassium sodium glicerophosphate in 0.5M acetate buffer (pH 4.5) for 24 hours, at room temperature.The sections were washed in distilled water, and incubated in 1% ammonium sulfate for 5 min.As a control, the samples were not submitted to potassium sodium glicerophosphate (Gomori, 1956).For the detection of phosphorylase activity, the sections were incubated for two hours in 1% glucose-1-phosphate in 0.1M acetate buffer (pH 6.0), at room temperature, and subsequently subjected to Lugol's reagent for 5 min.For control, the samples were not incubated in glucose-1-phosphate (Jensen, 1962).For observation of invertase activity, the sections were incubated for 3 hours at room temperature in a solution containing 0.024% tetrazolium blue (NBT), 0.014% phenazin methosulfate, 30U of glucose oxidase and 30 mM of sucrose, 0.38mM sodium phosphate buffer (pH 7.5).The control was subjected to the reaction media without sucrose (Zrenner, Salanoubat, Willmitzer, & Sonnewald, 1995;Doehlert & Felker 1987).For detection of sucrose synthase activity, the sections were fixed in 2% paraformaldehyde with 2% polyvinylpyrrolidone and 0.005M of dithiothreitol (pH 7.0) for 1 hour at 4°C.Later, they were incubated in a solution containing 5µL of 150 mM NADH, 5µl (1U) of phosphoglucomutase, 5µl of 3mM glucose 1,6-bisphosphate, 5µl (1U) of glucose-6-phosphate dehydrogenase, 5µl (1U) of UDPG pyrophosphorylase, 280µL of 0.07% aqueous solution of blue tetrazolium (NTB), 350 µl of buffer and 50µL substrate during 30 minutes.The buffer contained 10 mM MgCl2, 2 mM EDTA, 100 mM HEPES, 0.2% BSA and 2mM EGTA (pH 7.4).The substrate consisted of 15 mM UDP, 0.75 mM sucrose, 15 mM pyrophosphate.Two controls were used.In the first control, the glucose 1,6-bisphosphate and pyrophosphate were not added, and for the second control, the sucrose was suppressed (Wittich & Vreugdenhil 1998).

Histochemical Test for Reactive Oxygen Species (ROS)
ROS were detected by immersion of the sections in 0.5% 3.3'-diaminobenzidine (DAB) during 15 -60min, in the dark (Rossetti & Bonatti 2001).The sections were washed in water and photographed under an optical microscope (Zeiss Primo Star ® ) with a digital camera (Canon Power Shot A 630 ® ).Blank sections were used for the comparison of results.

General Features
Leaf galls on Piper arboreum are intralaminar and lenticular.They project to both leaf surfaces, and has a uniseriate epidermis, an outer parenchymatic cortex, an inner cortex composed of sclerenchymatic cells, and a nutritive zone involving the larval chamber, which houses the galling Cecidomyiidae (Figure 1 A-C).

Histochemical Profile of Non-Galled Leaves (NGL).
Phenolic compounds, flavonoids, alkaloids, and triterpenes were detected in the cells of hypodermis in black, dark blue, brown and red, respectively (Figure 2A-D, Table 1).Terpenoids were detected in blue in the cells of the hypodermis, and also in the idioblasts located between the palisade and spongy parenchymas (Figure 2E, Table 1).Lignins were evidenced in red in the cell walls of the xylem and of the pericyclic fibers (Figure 2F, Table 1).Lipids were detected as red droplets in the cells of the abaxial hypodermis, while reducing sugars, forming red precipitates, were observed in the adaxial and abaxial hypodermis (Figure 2G-H, Table 1).Starch grains and proteins were detected as dark blue grains or precipitates in the palisade and spongy parenchyma (Figure 2I-J, Table 1).The activity of invertases was evidenced as a dark blue precipitate in the hypoderm and parenchyma cells of the veins (Figure 2K, Table 1).The activity of sucrose synthase (SuSy) was detected as a purple precipitate in the vascular bundles (Figure2L, Table 1).The activity of phosphorylase and acid phosphatase was not observed.The ROS were detected in the epidermis and chlorophyllous parenchyma of the NGL (Figure 2M, Table 1).

Metabolites Compartmentalization in Cecidomyiidae Induced Galls on Piper Arboreum
The outer cortical cells of the mature galls (MG) developed from the hypodermis of the NGL, and accumulated phenolic compounds, flavonoids, alkaloids and terpenoids (Figure 3A-C, Table 2).Terpenoids occurred in idioblasts located between the palisade and spongy parenchyma of NGL, but they were not observed in the gall inner cortex (Figure3D, Table 2).Triterpenes were not evidenced in MG (Table 2).Lignins were detected in the cell walls of the xylem and of the pericyclic fibers both in NGL and MG.In MG, lignins were also observed in the cell walls of the sclerenchyma surrounding the nutritive tissue (Figure 3E, Table 2).Lipids were observed in the outer region, and also in the nutritive tissue of the MG (Figure 3F, Table 2).Reducing sugars were revealed in the cells of the adaxial and abaxial outer cortices (Figure 3G, Table 2).The detection of starch was more intense in the cells of the inner cortex and next to the larval chamber, increasing laterally towards the non-galled region.Starch was also detected in the lignified-walled cells (Figure 3H, Table 2).Proteins were detected in the nutritive tissue (Figure 3I, Table 2).

Enzymatic Activity
The activity of invertases was detected in the cells adjacent to the larval chamber, and formed a centrifugal gradient towards the non-galled region (Figure 3J, Table 2).The activity of SuSy was detected homogeneously throughout the nutritive tissue and vascular bundles (Figure 3K, Table 2).The activity of phosphorylase and acid phosphatase was not observed either in NGL or MG.

ROS Detection.
In MG, the ROS accumulated in a centrifugal gradient, decreasing towards the outer cortical tissue layers (Figure 3L, Table 2).

Compartimentalization of Metabolites
Two distinct compartments with accumulation of primary and secondary metabolites were observed in the leaf galls on P. arboreum.Primary metabolites have been especially detected in the inner tissues, while secondary metabolites accumulated only in the outer cortical parenchyma, which corroborates the expected spatial functional division of gall tissues.The outer cells were converted from photosynthetic and respiratory compartments towards defensive tissues, while the inner cells assumed a specialized nutritional role in redifferentiated gall tissues.
The defensive compartment, i.e. the outer cortex of P. arboreum leaf gall, accumulated alkaloids, terpenes, and phenolics, which have been considered waste products of plant metabolism (Roberts & Wink, 1998).Nevertheless, they have been contemporarily evaluated as sources of nitrogen or energetic lipids, and ROS scavenging molecules (Isaias et al., 2015), all of them necessary for the maintenance both of the host plant and gall metabolism.In spite of their involvement in antioxidant mechanisms (Blokhina, Virolainen, & Fagerstedt, 2003;Detoni, Vasconcelos, Rust, Isaias, & Soares, 2011), alkaloids, terpenes, and phenolics may secondarily deter or discourage the attack of predators, due to their toxicity ( Rhodes, 1994;Róstas, Maag, Ikegami, & Inbar, 2013).In galls, because of the high oxidative stress, the role of phenolics and flavonoid derivatives has been discussed as ROS dissipation, an efficient strategy to recover the redox-potential homeostasis (Isaias et al., 2015).Also, the phenolics are involved in IAA metabolism and consequently influence cell hypertrophy at gall site (Bedetti et al., 2014).
Even though the terpenes accumulated all over leaf mesophyll, they are detected exclusively in the outer compartment of the galls on P. arboreum, reinforcing the chemical protectiveness of the gall outer tissue layers.The impairment of the terpenic idioblasts differentiation in the nutritive tissue of the galls on P. arboreum should have favored the gall inducer, which did not come into contact with the toxic potential of the terpenes, and their anti-herbivore properties (Gershenzon, 1994).The strategy of disrupting the differentitation of terpenic idioblasts have been previously observed in the galling herbivores-Lantana camara systems (Moura, Isaias, & Soares, 2005;Moura, Isaias, & Soares, 2008).

Double Metabolites Accumulation in the Inner Compartment
As a host plant potentiality, lipid droplets were detected in the cells of the cortical parenchyma, originated from the NGL mesophyll, their intrinsic location.The lipids accumulated in the inner tissue layers may function as an energetic resource both for the galling Cecidomyiidae's nutrition and gall development.Even though attributed to Cynipidae (Bronner, 1992) and Lepidoptera galls (Vecchi, Menezes, Oliveira, Ferreira, & Isaias, 2013), lipidic droplets have been previously detected in some Cecidomyiidae galls of Aspidosperma spruceanum (Oliveira et al., 2010), Copaifera langsdorffii (Oliveira et al., 2011), and Marcetia taxifolia (Ferreira & Isaias, 2014).The accumulation of lipids in such galls has been related to the potential of the host plants for such accumulation (Oliveira et al., 2011;Ferreira & Isaias, 2014), as is true for P. arboreum.
The inner compartment of the galls on P. arboreum, i.e., the nutritive tissue, also accumulates proteins, similarly to the Cecidomyiidae galls on Aspidosperma spruceanum (Oliveira et al., 2010).Proteins are excellent nutritive resources for the galling herbivores, and may have accumulated as a cellular response to the increased oxidative and respiratory stresses established during the cecidogenetic process (Schönrogge, Harper, Lichtenstein, 2000).The increased level of proteins is followed by high levels of hexoses (Sturm & Tang, 1999), which are products of the activity of sucrose synthase and invertases (Roitsch & Gonzalez, 2004).The detection of invertases indicates the fast convertion of sucrose, and the activation of a mechanism of plant defense by increasing the synthesis of secondary metabolites (Wind et al., 2010;Sturm & Tang 1999).The double accumulation of nutritive compounds, such as lipids and proteins, in the nutritive tissue of a Cecidomyiidae gall is not the expected pattern, which should be carbohydrates storage (Bronner, 1992;Oliveira et al., 2010;Oliveira et al., 2011).This double stimuli is therefore a novelty for Cecidomyiidae galls in the Neotropics, and indicates a surplus for the galling herbivore nutrition.

Enzymatic Mediation of Carbohydrates Accumulation
The activity of sucrose synthase (SuSy) and invertases detected in the nutritive tissue of the galls on P. arboreum corroborated the metabolic similarity between Psylloidea and Cecidomyiidae galls.Both enzymes have been previously detected in galls induced by Pseudophacopteron aspidospermii (Malenovsky, Burckhardt, Queiroz, Isaias, & Oliveira, 2015) on Aspidosperma australe (Oliveira et al., 2010) and by a Cecidomyiidae on A. spruceanum (Oliveira & Isaias, 2010b).This enzymatic detection indicates a host plant metabolic requirement or potential rather than a dependence on the galling herbivore mode of feeding.
Moreover, current results demonstrate a common site for the activity of SuSy in the Cecidomyiidae galls on P. arboreum, and on A. spruceanum and Copaifera langsdorffii, which diverges in the P. aspidospermii galls on A. australe, where the activity of SuSy was restricted to the vascular bundles.Based on such comparison, we can conclude that the feeding sites of the galling herbivores does not determine the host plant cells metabolism, but may determine the sites of enzymes activity.The activity of SuSy is commonly responsible for the reversible cleavage of sucrose into fructose and UDP-glucose (Amor, Haigler, Johnson, Wainscott, & Delmer, 1995;Koch, 2004), but may be especially related to the synthesis of starch observed in the nutritive tissue of the galls on P. arboreum.Furthermore, this enzyme provides substrate (UDP-glucose) for the formation of cell wall polysaccharides (Nolte & Koch, 1993;Salnikov, Grimson, Seagull, & Haigler, 2003), whose dynamics, together with the carbohydrates metabolism, have crucial roles in the development of the gall structure (Formiga et al., 2013, Oliveira et al., 2014, Carneiro et al., 2015), and consequently in the survival of the galling herbivore.
In terms of cell metabolism, the gall on P. arboreum functions as a new organ, in a strict intralaminar continuum with its host tissues, establishing a sink of photoassimilates, which culminate in the accumulation of metabolites involved in its own development, and in the feeding activity of its associated galling herbivore (Rehill & Schultz, 2003;Castro et al., 2012).The nutrients can be redirected and compartmentalized into the nutritive tissue by two pathways.The first one is the transport of sucrose, via phloem, from the non-galled portions of the host leaf towards the gall site, where it is promptly metabolized and converted into starch.The conversion of sugars into starch occurs in a SuSy dependent via, which produces UDP-glucose as demonstrated for tubers of potato (Baroja-Ferná ndez et al., 2009).In the second pathway, the sucrose is irreversibly cleaved by the activity of invertases into glucose and fructose, which are used in cell respiration (Koch, 2004).The invertases are, indeed, the major sucrose-degrading enzyme in plants, as demonstrated in Arabidopsis, where their low levels affect plant growth (Barrat et al., 2009).In insect galls, the force of the sink towards the nutritive tissues seems to be maintained by the high cytological metabolism and the dynamics between the activity of SuSy and invertases (Oliveira et al., 2010), as can be inferred for P. arboreum galls.

ROS Accumulation and Reducing Sugars Suppression in Nutritive Cells
Due to the high activity of SuSy and invertases in the nutritive tissue, as well as the direct involvement of sugars in insect feeding, a gradient of carbohydrates should be expected (Bronner, 1992;Oliveira et al., 2014).However, the accumulation of reducing sugars is impaired in the nutritive tissue of the galls on P. arboreum, indicating that the sucrose is promptly metabolized to starch synthesis and monosaccharides used in cell respiration and gall growth.This supposed increase of cell respiration is corroborated by the accumulation of ROS in the nutritive tissue of the MG on P. arboreum.The relationship between the absence of reducing sugars and high accumulation of ROS in the inner tissues is theoretically proposed for Cecidomyiidae-induced galls.High levels of sugars are expected for Cecidomyiidae galls (Bronner, 1992), and should be crucial for disrupting the high oxidative stress detected by the presence of H 2 O 2 in the inner compartment, as hypothesized by Isaias et al. (2015).

Conclusion
The galling Cecidomyiidae promotes the compartmentalization of specific secondary and primary metabolites of its host leaves, guaranteeing benefits to its own survivorship.The outer compartmentalization of the secondary metabolites maintained the pattern of the original host tissues of P. arboreum leaves, and may confer protection against natural enemies.The inner compartmentalization of primary metabolites mediated by enzymes reflects two important metabolic steps of gall development: (1) the sink of photoassimilates and the storage of starch mediated by SuSy activity, and (2) the accumulation of proteins and lipids in nutritive tissues, as well as a high respiratory metabolism mediated by invertases activity.The conversion of sucrose into monossacharides is a common metabolic step shared by the Cecidomyiidae and the Psylloidea galls.However such conversion occurs in distinct sites of enzymes activity, which is imposed by the different feeding habits of the two taxa of galling herbivores, and their target cells.
The intralaminar galls on P. arboreum have two functional inversions regarding the compartmentalization of metabolites.The outer compartment accumulates reducing sugars, expected to occur in the nutritive cells, and terpenoids, whose synthesis is disrupted in the inner gall tissues.We theoretically propose that a consequence of these new sites of accumulation is a deviation of functions, with reducing sugars taking part in cell respiration in the outer tissue layers, and mediating ROS metabolism all over gall cortex.The terpenoids, whose presence in the nutritive cells should intoxicate the galling larvae, and consequently impair gall development, may contribute to gall chemical defense against natural enemies in the outer cortical cells.

Figure 2 .
Figure 2. Histochemical tests in non-galled leaves of Piper arboreum Aubl (Piperaceae) A: Reaction with ferric chloride evidencing the presence of phenolics in the hypodermis.B: DMACA revealing flavonoids in adaxial hypodermis.C: Alkaloids in the adaxial hypodermis.D: Reaction with Lieberman-Buchard's reagent showing triterpenes in the adaxial and abaxial hypodermis.E: NADI detecting terpenoids in adaxial hypodermis and idioblasts between the palisade and spongy parenchyma.F: Lignins in the cell walls of the xylem and pericyclic fibers.G: Sudan red demonstrating lipid droplets (arrows) in the abaxial hypodermis.H: Reducing sugars detected by Fehling`s reagent in the abaxial outer cortex.I: Lugol's reagent detecting starch in the palisade parenchyma and spongy parenchyma.J: Bromophenol blue indicating total proteins in the palisade parenchyma and spongy parenchyma.K: Activity of invertases in the adaxial hypodermis (arrow).L: Activity of sucrose synthase in the vascular bundles.M: Accumulation of ROS in the outer and inner gall cortices.Scale bars = 200µm (A-D, L), 50µm (E-I, K, M), 30µm (J).

Figure 3 .
Figure 3. Histochemical tests in Cecidomyiidae galls on Piper arboreum Aubl (Piperaceae) A: Ferric chloride evidencing phenolics in the outer cortex.B: DMACA revealing flavonoids in the outer cortex.C: Alkaloids detected by Draggendorff's reagent in the outer cortex (arrow).D: NADI detecting terpenoids in the outer cortex.E: Lignins in cell walls of the sclerenchymatic sheath.F: Sudan red demonstrating lipid droplets (arrows) in the nutritive tissue.G: Reducing sugars evidenced by Fehling`s reagent in the abaxial outer cortex.H: Lugol's reagent detecting starch in the nutritive tissue and lignified cells.I: Bromophenol blue indicating total proteins in the nutritive tissue.J: Activity of invertases in the nutritive tissue.K: Activity of sucrose synthase in the nutritive tissue.L: Accumulation of ROS in the outer and inner cortices of the gall.ab, abaxial outer cortex; ad, adaxial outer cortex; ic, inner cortex; lc, larval chamber; nt, nutritive tissue.Scale bars = 200µm (A-L), 50µm (F).

Table 1 .
Histochemical detection of metabolites and enzymes in non-galled leaves of Piper arboreum Aubl (Piperaceae)