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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.

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

The cascade of biochemical changes occurring at sites of gall development seems to involve a group of common metabolites in plants, namely, the phenolics. Phenolic accumulation has been commonly related to chemical defence, but their primary role seems to be the regulation of cell hypertrophy in galls. Such regulation implies phenolics–auxin (IAA) association at some cell re-differentiation sites, and determines final gall shapes. Herein, we investigated phenolic and auxin accumulation in four gall systems, grouped in two morphotypes, namely lenticular and globoid, induced on pinnulas of Piptadenia gonoacantha (Mart.) J.F.Macbr. Changes in the direction and type of cell expansion between non-galled pinnula and galls were also evaluated. Galling insects associated to lenticular and globoid gall morphotypes promoted changes in host plant cells, leading to the development of different cell sizes, different degrees of anisotropy, and different directions of cell expansion. The accumulation of IAA–phenolics compartmentalised on the basis of gall morphotype, i.e. in the cells of superior and lateral inferior cortices in the lenticular gall morphotypes, and throughout the outer cortex in the globoid gall morphotypes. The sites of accumulation of IAA and phenolics coincided with the most hypertrophied regions, influencing on the determination of the final gall shape.

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