Bruno G. Ferreira

Manipulation of host plant cells and tissues by gall-inducing insects and adaptive strategies used by different feeding guilds

Biologists who study insect-induced plant galls are faced with the overwhelming diversity of plant forms and insect species. A challenge is to find common themes amidst this diversity. We discuss common themes that have emerged from our cytological and histochemical studies of diverse neotropical insect-induced galls. Gall initiation begins with recognition of reactive plant tissues by gall inducers, with subsequent feeding and/or oviposition triggering a cascade of events. Besides, to induce the gall structure insects have to synchronize their life cycle with plant host phenology. We predict that reactive oxygen species (ROS) play a role in gall induction, development and histochemical gradient formation. Controlled levels of ROS mediate the accumulation of (poly)phenols, and phytohormones (such as auxin) at gall sites, which contributes to the new cell developmental pathways and biochemical alterations that lead to gall formation. The classical idea of an insect-induced gall is a chamber lined with a nutritive tissue that is occupied by an insect that directly harvests nutrients from nutritive cells via its mouthparts, which function mechanically and/or as a delivery system for salivary secretions. By studying diverse gall-inducing insects we have discovered that insects with needle-like sucking mouthparts may also induce a nutritive tissue, whose nutrients are indirectly harvested as the gall-inducing insects feeds on adjacent vascular tissues. Activity of carbohydrate-related enzymes across diverse galls corroborates this hypothesis. Our research points to the importance of cytological and histochemical studies for elucidating mechanisms of induced susceptibility and induced resistance.

Developmental pathway from leaves to galls induced by a sap-feeding insect on Schinus polygamus (Cav.) Cabrera (Anacardiaceae)

Galling sap-feeding insects are presumed to cause only minor changes in host plant tissues, because they usually do not require development of nutritive tissues for their own use. This premise was examined through comparison of the histometry, cytometry and anatomical development of non-galled leaves and galls of Calophya duvauae (Scott) (Hemiptera: Calophyidae) on Schinus polygamus (Cav.) Cabrera (Anacardiaceae). Cell fates changed from non-galled leaves to galls during the course of tissue differentiation. C. duvauae caused changes in dermal, ground, and vascular systems of the leaves of S. polygamus. Its feeding activity induced the homogenization of the parenchyma, and the neoformation of vascular bundles and trichomes. The histometric and cytometric data revealed compensatory effects of hyperplasia and cell hypertrophy in the epidermis, with hyperplasia predominating in the adaxial epidermis. There was a balance between these processes in the other tissues. Thus, we found major differences between the developmental pathways of non-galled leaves and galls. These changes were associated with phenotypic alterations related to shelter and appropriate microenvironmental conditions for the gall inducer. The nondifferentiation of a typical nutritive tissue in this case was compared to other non-phylogenetically related arthropod gall systems, and is suggested to result from convergence associated with the piercing feeding apparatus of the corresponding gall-inducer.

The redifferentiation of nutritive cells in galls induced by Lepidoptera on Tibouchina pulchra (Cham.) Cogn. reveals predefined patterns of plant development

Insect galls may present nutritive tissues with distinct cytological features related to the order of the gall inducer. Galling Lepidoptera larvae chew plant cells and induce the redifferentiation of parenchymatic cells into nutritive ones. The nutritive cells in the galls induced by a microlepidoptera on the leaves of Tibouchina pulchra (Cham.) Cogn. (Melastomataceae) are organelle-rich, with developed Golgi apparatus, endoplasmic reticulum, ribosomes, polyribosomes, mitochondria, plastids, and one great central or several fragmented vacuoles. The nonobservance of the nuclei in the nutritive cells deserves special attention, and confers a similarity between the nutritive cells and the vascular conductive ones. The great amount of rough endoplasmic reticulum, ribosomes, polyribosomes, and mitochondria is indicative of the high metabolic status of these cells. They are vascular cambium-like, with high protein synthesis and lipid storage. The proteins are essential to enzymatic metabolism, and secondarily, to larvae nutrition, similarly to the lipid droplets which confer energetic profile to these nutritive cells. The living enucleated cells receive mRNA from their neighbor ones, which may support the high metabolic profile of endoplasmic reticulum and ribosomes observed in galls. Thus, the nutritive cells are stimulated by the galling larvae activity, generating a new cell type, whose redifferentiation includes a mix of intrinsic and common plant pathways.

The role of pectic composition of cell walls in the determination of the new shape-functional design in galls of Baccharis reticularia (Asteraceae)

The pectic composition of cell wall is altered during the processes of cell differentiation, plant growth, and development. These alterations may be time-dependent, and fluctuate in distinct regions of the same cell or tissue layer, due to the biotic stress caused by the activity of the gall inducer. Among the roles of the pectins in cell wall, elasticity, rigidity, porosity, and control of cell death may be crucial during gall development. Galls on Baccharis reticularia present species-specific patterns of development leading to related morphotypes where pectins were widely detected by Ruthenium red, and the pectic epitopes were labeled with specific monoclonal antibodies (LM1, LM2, LM5, LM6, JIM5, and JIM7) in distinct sites of the non-galled and the galled tissues. In the studied system B. reticularia, the epitopes for extensins were not labeled in the non-galled tissues, as well as in those of the rolling and kidney-shaped galls. The high methyl-esterified homogalacturonans (HGA) were labeled all over the tissues either of non-galled leaves or of the three gall morphotypes, while the intense labeling for arabinogalactans was obtained just in the rolling galls. The pectic composition of non-galled leaves denotes their maturity. The kidney-shaped gall was the most similar to the non-galled leaves. The pectic dynamics in the gall tissues was particularly altered in relation to low methyl-esterified HGA, which confers elasticity and expansion, as well as porosity and adhesion to cell walls, and are related to the homogenization and hypertrophy of gall cortex, and to translocation of solutes to the larval chamber. Herein, the importance of the pectic dynamics of cell walls to the new functional design established during gall development is discussed for the first time. The repetitive developmental patterns in galls are elegant models for studies on cell differentiation.

Efficiency of the Polyethylene-Glycol (PEG) Embedding Medium for Plant Histochemistry

Histochemical analyses in plants are commonly performed on hand-made sections of fresh materials. The disadvantages of embedding in historesin, paraffin or paraplast® are the alterations to cellular contents, the high costs and few evident results, depending on the test. Polyethylene-glycol (PEG), as a low cost, hydrophilic medium that maintains most of the cellular features similar to fresh conditions, may be useful for obtaining good histochemical results in thinner and homogeneous sections. The current study aimed to compare the efficiency of PEG as an embedding medium for histochemical analyses of primary and secondary metabolites accumulation. Using hand-made sections of fresh samples (T1) as a comparison, we tested the influence of the use of Karnovsky’s solution as a fixative (T2) versus embedding in PEG (T3). The samples herein analyzed comprise leaves, stems, seeds and insect galls of different plant species. Neither the Karnovsky’s fixative nor the embedding in PEG altered the histochemical results for starch, lipids, terpenoids, proteins and reducing sugars in T1, T2, and T3. However, PEG binds to phenols, such as tannins, flavonoids and lignins, thereby presenting false negatives in T3.

Variation in the degree of pectin methylesterification during the development of Baccharis dracunculifolia kidney-shaped gall.

Insect galls may be study models to test the distribution of pectins and arabinogalactan-proteins (AGPs) and their related functions during plant cell cycles. These molecules are herein histochemically and immunocitochemically investigated in the kidney-shaped gall induced by Baccharopelma dracunculifoliae (Psyllidae) on leaves of Baccharis dracunculifolia DC. (Asteraceae) on developmental basis. The homogalacturonans (HGAs) (labeled by JIM5) and the arabinans (labeled by LM6) were detected either in non-galled leaves or in young galls, and indicated stiffening of epidermal cell walls, which is an important step for cell redifferentiation. The labeling of HGAs by JIM7 changed from young to senescent stage, with an increase in the rigidity of cell walls, which is important for the acquaintance of the final gall shape and for the mechanical opening of the gall. The variation on the degree of HGAs during gall development indicated differential PMEs activity during gall development. The epitopes recognized by LM2 (AGP glycan) and LM5 (1–4-β-D-galactans) had poor alterations from non-galled leaves towards gall maturation and senescence. Moreover, the dynamics of pectin and AGPs on two comparable mature kidney-shaped galls on B. dracunculifolia and on B. reticularia revealed specific peculiarities. Our results indicate that similar gall morphotypes in cogeneric host species may present distinct cell responses in the subcelular level, and also corroborate the functions proposed in literature for HGAs.

Elucidating the determination of the rosette galls induced by Pisphondylia brasiliensis Couri and Maia 1992 (Cecidomyiidae) on Guapira opposita (Nyctaginaceae)

The modulation of plant development has been the focus of research on insect galls because galling insects induce distinct shapes to acquire the same necessities, shelter and food. Due to the variety of gall morphotypes, it can be assumed that the key processes for their development rely on plant cells’ morphogenetical potentialities. In the present study we investigated the rosette bud galls induced by Pisphondylia brasiliensis on Guapira opposita to check whether two morphogenetical pathways – the shortening of the internodes and the over differentiation of axillary buds – are independent or whether they are concomitant events towards the morphogenesis of the galls. Biometrical measures were made to test whether the final size of the galls is correlated with the number of inducers per gall. We noted that two patterns of activity were observed in gall meristems: the first differentiated pairs of leaves with opposite phyllotaxy, and the other differentiated new buds at the base of each leafy projection, with the development of sequential leafy projections, in a disorganised phyllotaxy. This second pattern repeated until gall maturation, when a master cambium, typical of the Nyctaginaceae, differentiated in larger galls. The two morphogenetical pathways occurred concomitantly, leading to the overproduction of leafy projections. Cell responses at gall development site produce mechanical protection to P. brasiliensis individuals. The larger galls have the higher number of inducers, and the coalescence of galls allows an increase in gall size by precociously triggering the master cambium activity, a developmental peculiarity of G. opposita uncommon for Cecidomyiidae galls.

Multivesicular bodies differentiate exclusively in nutritive fast-dividing cells in Marcetia taxifolia galls.

Marcetia taxifolia (A. St.-Hil.) DC. hosts two gall morphotypes, a pistil-shaped gall induced by a Cecidomyiidae (Diptera) and a fusiform stem gall induced by a Lepidoptera. The cytological study of these galls aimed to answer how the difference in nutritive tissues of Diptera and Lepidoptera galls could be explained on cytological basis. The nutritive tissues of lepidopteran galls have a fast-dividing cell zone, the storage nutritive tissue, which replaces the cells of the typical nutritive tissue, where the larvae feed. The differentiation of multivesicular bodies in the plasma membrane occurred exclusively in these fast-dividing cells of the lepidopteran galls, evidencing the meristematic condition of such tissue. The accumulation of reactive oxygen species (ROS) analyzed in situ in the nutritive cells is not sufficient to induce programmed cell death (PCD), as the cells of M. taxifolia have plastoglobules and accumulate polyphenols and terpenoids, which are diagnostic defenses against oxidative stress. The two taxa of galling insects have different nutritional requirements, thus inducing specific cytoplasm-enriched cells on their nutritive tissues.

Plant potentialities determine anatomical and histochemical diversity in Mikania glomerata Spreng. galls

Arthropod gall super-hosts have distinct developmental responses to each gall inducer species. The taxa and feeding habits of the gall inducers determine each gall’s histological patterns, but the host plant imposes histological constraints on gall differentiation. Mikania glomerata Spreng. (Asteraceae) is a gall super-host, presenting at least six distinct gall morphotypes. The aim of current study was to evaluate how different species of Cecidomyiidae can manipulate the same host plant tissues, demonstrating the potentialities of M. glomerata under distinct external signaling. We compared M. glomerata anatomy and histochemistry in fusiform galls induced by Liodiplosis cylindrica (Gagné 2001) on petioles, globoid galls induced by L. spherica (Gagné 2001) on leaf laminae, and conic galls of Clinodiplosis sp. on leaf laminae as well as in non-galled leaves and petioles. Even though each gall presents several distinct features, they share anatomical and histochemical patterns, determined by the host plant potentialities. We found that the ground and dermal system tissues were manipulated differently by each inducer, generating distinct gall morphotypes. Alterations on epidermal cell shapes and suppression of the capitate glandular trichomes occured in all studied galls. We report for the first time the occurrence of nutritive cells containing starch in globoid galls. The anatomical diversity of the galls on M. glomerata seems to be more related to distinct differentiation pathways of the host plant than to the taxonomic relationships between the gall-inducing species.

Functional compartmentalisation of nutrients and phenolics in the tissues of galls induced by Leptocybe invasa (Hymenoptera: Eulophidae) on Eucalyptus camaldulensis (Myrtaceae)

Galling herbivores induce structural and chemical alterations in their host plants tissues. These insects have been the focus of little study in the case of Australian taxa. Leptocybe invasa, a native Australian galling hymenopteran associated with Eucalyptus species, causes economic damage to plantation eucalypts in many countries around the world. Leptocybe invasa oviposits in the midribs and petioles of expanding leaves thereby intercepting photosynthates and impairing normal expansion. We analysed the ultrastructural and chemical cellular changes in L. invasa galls on Eucalyptus camaldulensis (probably subspecies camaldulensis) to diagnose how the insect manipulates plant cells and tissues and the significance of these alterations for insect nutrition and protection. Galling stimuli induce the formation of two functionally compartmentalised types of tissue. Phenolic plant secondary metabolites and anthocyanins (plant pigments) accumulate in the outer compartment, while primary metabolites accumulate in the inner compartment. The nutritive cells (inner compartment) accumulate protein and lipids that provide food for the larvae. Total polyphenol concentrations did not differ significantly between outer and inner compartments. Nevertheless, the concentrations of quercetin and kaempferol derivatives were higher in the outer compartment than in the inner compartment. These differences could be related to the protection of plant tissues against ultraviolet rays and the maintenance of redox homeostasis. There were higher ratios of hexahydroxydiphenoyl‐containing hydrolysable tannins rather than galloyl‐containing hydrolysable tannins in the inner compartment. This shift in the oxidative capacity of the polyphenols in the inner compartment could represent a defensive plant response to the larvae.