Observations on the Population Genetic Structure of the Leaf Galling Nematode, Ditylenchus gallaeformans.

Ditylenchus gallaeformans is a plant parasitic nematode that induces galls on aboveground parts of Melastomataceae plants. It differs from most gall-inducing nematodes in that it is not an endoparasite and has been considered as a possible biological control agent against invasive species of Miconia. Little is known about D. gallaeformans biology, genetic differences among populations, and host preferences. This study examined the genetic differences among D. gallaeformans populations from different locations and host species and the phylogenetic relationships among them. Nematodes were collected from galls in plants from Costa Rica, Dominica, and Trinidad. The Cytochrome c oxidase 1 (cox1) region was sequenced from a total of 33 individual nematodes isolated from 33 different plant individuals, representing 21 species of Melastomataceae. Phylogenetic reconstructions, haplotype networks, and analysis of molecular variance showed that the species is monophyletic and has three major clades, which were mostly consistent with geographic location but not with host species. The first clade was composed by two subclades, one with individuals from Costa Rica and one with individuals from Dominica. The second and third clades comprised nematodes only from Trinidad. Overall, there is no evidence of host-species specialization in D. gallaeformans. Biocontrol efforts using the nematode against invasive Miconia could focus on geographical location matching but likely will not need to match host species.

Apoplast-symplast compartmentalization and functional traits of iron and aluminum in promeristematic tissues of nematode induced galls on Miconia spp.

The nutritive tissues of galls induced by Ditylenchus gallaeformans (Nematoda) have promeristematic capacity, which may turn these galls into sinks of Al on their Melastomataceae Al-accumulating hosts. Such a sink of Al may affect gall growth and mineral nutrient intake. Based on the fact that galls are good models for plant developmental studies, we aimed to understand how Al-accumulating host plants in the Cerrado environment deal with Al toxicity in subcellular levels. Here, we used the ICP-OES method to check the variations on mineral nutrients, and the morin, hematoxylin, and Prussian blue stainings for Al and Fe histolocalization in galls induced on four Miconia species of the Brazilian Cerrado. We confirmed the new Al-accumulating feature for two Miconia species of the Cerrado environment. Furthermore, we found that Al accumulates in lesser concentrations in gall tissues than in non-galled tissues of the Miconia hosts. Staining methods indicated that the polyphenols avoid Al-binding to the apoplast and the nucleolus of the promeristematic cells, and mediated its binding to parenchyma cell walls. As well, we inferred that Fe3+ is transported by xylem and stored in gall parenchyma, where it is reduced to Fe2+, being available in gall nutritive cells. Our results demonstrated an Al compartmentalization between the apoplast and symplast of the inner cell layers in galls, as well as indicated the phenolics action against Al-toxicity and toward Fe availability for the diet of Ditylenchus gallaeformans.

Cytological attributes of storage tissues in nematode and eriophyid galls: pectin and hemicellulose functional insights.

Cell walls and protoplast may work together or distinctly in the establishment of the functional profiles of gall tissue compartments. This presumption is herein evaluated in three gall systems by immunocytochemical and ultrastructural analyses. The common storage tissues (CSTs) of leaf galls induced by Eriophyidae on Miconia ibaguensis leaves and by Ditylenchus gallaeformans on M. ibaguensis and M. albicans have rigid and porous cell walls due to their composition of pectins. Hemicelluloses in CST cell walls are scarcer when compared to the cell walls of the control leaves, being functionally compensated by rigid pectate gels. The typical nutritive tissues (TNTs) in galls induced by Ditylenchus gallaeformans are similar to promeristematic and secretory cells regarding their enriched cytoplasm, several mitochondria, and proplastids, as well as multivesicular and prolamellar bodies in cell membranes. The cytological features of the feeding cells of Eriophyidae galls indicate that they are not as metabolically active as the cells of the TNT in nematode galls. However, their cell wall composition suggests more plasticity and porosity than the cells of the TNT, which can compensate the less production of nutrients with more transport. The ultrastructural and immunocytochemical profiles of CST cells reveal functional similarities, which are independent of the taxa of the gall inducer or of the host plant. Despite their analogous functionalities, the protoplast and cell wall features of TNT cells of nematode galls and of the feeding cells of the Eriophyidae galls are distinct, and work out through different strategies toward keeping gall developmental site active.

Enzyme-mediated metabolism in nutritive tissues of galls induced by Ditylenchus gallaeformans (Nematoda: Anguinidae).

The galls induced by Ditylenchus gallaeformans (Nematoda) on leaves of Miconia albicans have unique features when compared to other galls. The nematode colonies are surrounded by nutritive tissues with promeristematic cells, capable of originating new emergences facing the larval chamber, and providing indeterminate growth to these galls. Considering enzyme activity as essential for the translocation of energetic molecules from the common storage tissue (CST) to the typical nutritive tissue (TNT), and the major occurrence of carbohydrates in nematode galls, it was expected that hormones would mediate sink strength relationships by activating enzymes in indeterminate growth regions of the galls. Histochemical, immunocytochemical and quantitative analyses were made in order to demonstrate sites of enzyme activity and hormones, and comparative levels of total soluble sugars, water soluble polysaccharides and starch. The source-sink status, via carbohydrate metabolism, is controlled by the major accumulation of cytokinins in totipotent nutritive cells and new emergences. Thus, reducing sugars, such as glucose and fructose, accumulate in the TNT, where they supply the energy for successive cycles of cell division and for nematode feeding. The histochemical detection of phosphorylase and invertase activities indicates the occurrence of starch catabolism and sucrose transformation into reducing sugars, respectively, in the establishment of a gradient from the CST towards the TNT. Reducing sugars in the TNT are important for the production of new cell walls during the indeterminate growth of the galls, which have increased levels of water-soluble polysaccharides that corroborate such a hypothesis. Functional relationship between plant hormone accumulation, carbohydrate metabolism and cell differentiation in D. gallaeformans-induced galls is attested, providing new insights on cell development and plant metabolism.

How the activity of natural enemies changes the structure and metabolism of the nutritive tissue in galls? Evidence from the Palaeomystella oligophaga (Lepidoptera) -Macairea radula (Metastomataceae) system.

Insect-induced galls usually develop nutritional cells, which they induce and consume directly, and any metabolic modification of those cells may reflect changes of the insect's own metabolism. The system Palaeomystella oligophaga (Lepidoptera)-Macairea radula (Melastomataceae) presents a series of natural enemies, including parasitoids and cecidophages that can function as a natural experiment, respectively removing the specific galling feeding stimulus and providing a nonspecific one. Considering that the process of induction and maintenance of gall tissues strictly depends on the constant specific stimulus of galling, question I:What kind of metabolic changes these different groups of natural enemies can promote in chemical and structural composition of these galls? II: How the specialized tissues are metabolically dependent on the constant specific stimulus of galling in latter stages of gall development? Galls without natural enemies, with parasitoids or cecidophages in larvae or pupae stages were analyzed through histochemistry and cytological profiles and all compared to galls in natural senescence state. The analysis revealed the accumulation of proteins and lipids in typical nutritive tissue and starch in the storage tissue, as well a high integrity of cellular organelles and membrane systems on galls with gallings in the larval stage. Both parasitoids and cecidophages stop galling feeding activities, which resulted in the paralysis of the stimulus that maintain the metabolism of gall tissues, leading to generalized collapse. We demonstrate that the development and metabolic maintenance of a typical nutritive tissue in these galls are completely dependent on constant larval stimulus.

Antioxidant metabolism in galls due to the extended phenotypes of the associated organisms.

Animal-induced galls are considered extended phenotypes of their inducers, and therefore plant morphogenesis and metabolism may vary according to the species of gall inducers. The alterations in vacuolar and apoplastic polyphenols, carotenoids, chlorophyll fluorescence rates, PSII quantum yield, and phospholipid peroxidation were studied in galls induced by Ditylenchus gallaeformans (Nematoda) on Miconia albicans and M. ibaguensis (Melastomataceae), and by an unidentified Eriophyidae (Acarina) on M. ibaguensis. The focus currently addressed is gall metabolism as the extended phenotype of the gall inducers, and the neglected determination of gall functionalities over host plant peculiarities. Galls induced by D. gallaeformans on M. albicans and by the Eriophyidae on M. ibaguensis have increased accumulation of apoplastic and vacuolar phenolics, which is related to the control of phospholipid peroxidation and photoprotection. The galls induced by D. gallaeformans on M. ibaguensis have higher carotenoid and vacuolar polyphenol contents, which are related to excessive sunlight energy dissipation as heat, and photoprotection. Accordingly, antioxidant strategies varied according to the gall-inducing species and to the host plant species. The distinctive investments in carotenoid and/or in polyphenol concentrations in the studied galls seemed to be peculiar mechanisms to maintain oxidative homeostasis. These mechanisms were determined both by the stimuli of the gall-inducing organism and by the intrinsic physiological features of the host plant species. Therefore, the roles of both associated organisms in host plant-galling organisms systems over gall metabolism is attested.

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.

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.

A comparative study of aluminium and nutrient concentrations in mistletoes on aluminium-accumulating and non-accumulating hosts.

Mistletoes offer a unique model to study interactions among Al and nutrients in vascular plants, because they grow and reproduce on hosts with distinct Al uptake strategies. We investigated Al distribution and nutrient relations of mistletoes on Al-accumulating and non-accumulating hosts. We hypothesised that mistletoes would exhibit similar leaf nutrient and Al concentrations as their host plants, but a strong compartmentalisation of Al when growing on Al-accumulators. We measured concentrations of N, P, K, Ca, Mg, Cu, Fe, Mn, Zn in leaves and Al in leaves, seeds and branches of Phthirusa ovata and Psittacanthus robustus infecting Miconia albicans, an Al-accumulator, and Ph. ovata infecting Byrsonima verbascifolia, a non-Al-accumulator. High leaf concentrations of Al in Ph. ovata only occurred while parasitizing the Al-accumulating host; there was no accumulation in branches or seeds. In P. robustus, large concentrations of Al were found in leaves, branches and seeds. Mistletoe seed viability and leaf nutrient concentrations were not affected by Al accumulation. Passive uptake of Al, Ca, Mg, Mn and Cu in mistletoes was evidenced by significant correlations between mistletoes and host leaf concentrations, but not of N, P and K. Al was retranslocated to different plant organs in P. robustus, whereas it was mostly restricted to leaves in Ph. ovata. We suggest that Al might have some specific function in P. robustus, which only parasitizes Al-accumulator hosts, while the host generalist Ph. ovata can be considered a facultative Al-accumulator.

Lepidopterans as potential agents for the biological control of the invasive plant, Miconia calvescens.

This work investigated eight species of Lepidoptera associated with Miconia calvescens DC. (Myrtales: Melastomataceae) in Brazil, including six defoliators, Salbia lotanalis Druce (Lepidoptera: Pyralidae), Druentia inscita Schaus (Mimallonidae), Antiblemma leucocyma Hampson (Noctuidae), three Limacodidae species, a fruit borer Carposina cardinata Meyrick (Carposinidae), and a damager of flowers Pleuroprucha rudimentaria Guenée (Geometridae). Based on host specificity and the damage caused to plants, S. lotanalis and D. inscita are the most promising species for biological control of M. calvescens. Furthermore, if C. cardinata and P. rudimentaria have host specificity in future tests, these caterpillars could also be considered as appropriate biocontrol agents.

Plant Vigor Hypothesis refuted: preference-performance linkage of a gall-inducing weevil on small-sized host plant resources.

The Plant Vigor Hypothesis (PVH) predicts an oviposition preference of females and higher offspring performance for insect herbivores on longer and fast-growing plant modules. We tested the PVH predictions by investigating the effects of leaf size of Miconia prasina (Sw.) DC. (Melastomataceae) on the oviposition preference and on the offspring survival of the gall-inducing weevil Prospoliata bicolorata (Coleoptera: Curculionidae). Additionally, we analysed the effects of top-down mortality force on this system. Approximately 83% of the developed galls resulted in adults of P. bicolorata, whereas 17% of the galls successfully induced were killed by natural enemies (top-down effect). Leaves of intermediate size were more abundant while smaller and longer leaves were rare. Nevertheless, the percentage of P. bicolorata galls was higher on the smallest leaves of M. prasina, refuting the preference prediction of the PVH. Our results also refuted the performance prediction: the ratio of survival per leaf was negatively related to the leaf length. Thus, we found a link between female preference and larval performance of P. bicolorata on small-sized leaves of M. prasina. The next goal is to understand the mechanisms involved in the selection of gall-inducing weevil on short leaves of its host plant.

Climate and host plant characteristics effects on lepidopteran caterpillar abundance on Miconia ferruginata DC and Miconia pohliana Cogn (Melastomataceae).

Folivore cerrado caterpillars are found on their host species in low frequencies, which vary between plants and throughout the year. We analysed the effects of climatic variation and of the characteristics of two host species (Miconia, Melastomataceae) on caterpillar abundance. The work was carried out in a cerrado sensu stricto area in Brasília, Federal District, Brazil, from May 1998 to September 1999. One hundred plants of each species were inspected monthly. All caterpillars found were collected and raised in the laboratory with Miconia leaves as food sources. Climatic data for the study area and plant characteristics such as local density, spatial distribution, foliar phenology, leaf pilosity and nutritional content were used to test the effects on caterpillar abundance. Less than 10% of the plants had larvae. Caterpillars were more frequent and abundant in M. pohliana, which was also present at higher densities than M. ferruginata. Low caterpillar frequency in host plants and an abundance peak during the dry season are consistent patterns for different cerrado Lepidoptera larvae. This abundance peak, however, is not coincident with higher leaf production in host species. We suggest that M. pohliana's higher local density and better leaf digestibility can account for the higher abundance of caterpillars in this species.

Climate and host plant characteristics effects on lepidopteran caterpillar abundance on Miconia ferruginata DC. and Miconia pohliana Cogn (Melastomataceae) / Efeitos do clima e das características das plantas hospedeiras na abundância de lagartas de Lepidoptera em Miconia ferruginata e Miconia pohliana

Folivore cerrado caterpillars are found on their host species in low frequencies, which vary between plants and throughout the year. We analysed the effects of climatic variation and of the characteristics of two host species (Miconia, Melastomataceae) on caterpillar abundance. The work was carried out in a cerrado sensu stricto area in Brasília, Federal District, Brazil, from May 1998 to September 1999. One hundred plants of each species were inspected monthly. All caterpillars found were collected and raised in the laboratory with Miconia leaves as food sources. Climatic data for the study area and plant characteristics such as local density, spatial distribution, foliar phenology, leaf pilosity and nutritional content were used to test the effects on caterpillar abundance. Less than 10 percent of the plants had larvae. Caterpillars were more frequent and abundant in M. pohliana, which was also present at higher densities than M. ferruginata. Low caterpillar frequency in host plants and an abundance peak during the dry season are consistent patterns for different cerrado Lepidoptera larvae. This abundance peak, however, is not coincident with higher leaf production in host species. We suggest that M. pohliana's higher local density and better leaf digestibility can account for the higher abundance of caterpillars in this species.

No cerrado, as lagartas de Lepidoptera, folívoras externas, são encontradas em baixas frequências e variam entre plantas e ao longo do ano. Este trabalho teve como objetivo analisar os efeitos das variações climáticas e das características das plantas (Miconia, Melastomataceae) na abundância das lagartas. O estudo foi feito no cerrado sensu stricto, Brasília, DF, de maio de 1998 a setembro de 1999. Mensalmente, 100 plantas de cada espécie foram vistoriadas. Todas as lagartas foram coletadas e criadas. Foram correlacionados com os resultados obtidos da abundância de lagartas, os dados climáticos e as seguintes características das plantas: densidade local, distribuição espacial, fenologia, pilosidade e conteúdo nutricional foliar. Menos de 10 por cento das plantas tinham lagartas. As lagartas foram mais frequentes e abundantes em M. pohliana que também foi a espécie de planta com maior densidade na área. A frequência baixa de lagartas em plantas do cerrado é um padrão bastante consistente, assim como o pico de abundância desses imaturos na estação seca. Entretanto, esse pico de abundância não coincide com a produção de folhas em nenhuma das espécies de plantas. A maior densidade local e digestibilidade das folhas de M. pohliana são os fatores que mais explicam a abundância de lagartas nessa espécie.

Biology and host preferences of Cryptorhynchus melastomae (Coleoptera: Curculionidae), a possible biocontrol agent for Miconia calvescens (Melastomataceae) in Hawaii.

The introduced plant Miconia calvescens (Melastomataceae) poses a grave threat to Hawaii's native ecosystems and biodiversity. One potential candidate for classical biological control is Cryptorhynchus melastomae (Coleoptera: Curculionidae: Cryptorhynchinae), a stem-boring weevil from Central and South America. This weevil feeds on M. calvescens in its native Costa Rica and has been successfully reared under greenhouse conditions. Comparison of its environmental conditions in Costa Rica with those in the Miconia infested areas of Hawaii indicates the latter is a suitable habitat for C. melastomae. C. melastomae has one or two generations per year. Adults feed on new stems, petioles, leaf buds, veins, and lamina, whereas larvae mine the stem until pupation. Adults appear to prefer saplings for oviposition and feeding. Under greenhouse conditions both adults and larvae can seriously damage and kill small M. calvescens. Preliminary host testing indicates that C. melastomae may be family specific on Melastomataceae. However, because Hawaii lacks native melastomes and has many other serious melastome weeds, a family specific insect may be suitable as a biocontrol agent in this case.

Three new cecidogenous Palaeomystella Fletcher (Lepidoptera, Coleophoridae, Momphinae) associated with Melastomataceae in Brazil / Três espécies novas de Palaeomystella Fletcher (Lepidoptera, Coleophoridae, Momphinae), cecidógenas, associadas com Melastomataceae no Brasil

Three new cecidogenous Palaeomystella Fletcher (Lepidoptera, Coleophoridae, Momphinae), described herein, induce galls on stems and leaves of Melastomataceae species. They include: Palaeomystella tibouchinae sp. n., on Tibouchina barbigera (Naudin) Baillon, P. oligophaga sp. n., on Macairea radula (Bonpland) de Candolle and M. thyrsiflora de Candolle, and P. henriettiphila sp. n., on Henriettea succosa (Aublet) de Candolle. Adults, including male and female genitalia, larva, pupa, and galls are illustrated and described in detail.

As espécies de Palaeomystella aqui descritas induzem cecídeas nos ramos e folhas de espécies de Melastomataceae: Palaeomystella tibouchinae sp. n. em Tibouchina barbigera (Naudin) Baillon, P. oligophaga sp. n. em Macairea radula (Bonpland) de Candolle, e M. thyrsiflora de Candolle, P. henriettiphila sp. n. em Henriettea succosa (Aublet) de Candolle. Os adultos, incluindo venação das asas, e genitálias dos machos e fêmeas, larvas, pupas e cecídeas, são descritos e ilustrados em detalhes.

Interspecific variation in the defensive responses of obligate plant-ants: experimental tests and consequences for herbivory.

The aggressive behavior of ants that protect plants from herbivores in exchange for rewards such as shelter or food is thought to be an important form of biotic defense against herbivory, particularly in tropical systems. To date, however, no one has compared the defensive responses of different ant taxa associated with the same plant species, and attempted to relate these differences to longer-term efficacy of ant defense. We used experimental cues associated with herbivory--physical damage and extracts of chemical volatiles from leaf tissue--to compare the aggressive responses of two ant species obligately associated with the Amazonian myrmecophyte Tococa bullifera (Melastomataceae). We also conducted a colony removal experiment to quantify the level of resistance from herbivores provided to plants by each ant species. Our experiments demonstrate that some cues eliciting a strong response from one ant species elicited no response by the other. For cues that do elicit responses, the magnitude of these responses can vary interspecifically. These patterns were consistent with the level of resistance provided from herbivores to plants. The colony removal experiment showed that both ant species defend plants from herbivores: however, herbivory was higher on plants colonized by the less aggressive ant species. Our results add to the growing body of literature indicating defensive ant responses are stimulated by cues associated with herbivory. However, they also suggest the local and regional variation in the composition of potential partner taxa could influence the ecology and evolution of defensive mutualisms in ways that have previously remained unexplored.