Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ecological interactions between organisms are complex and can present dynamic results, which are dependent on environmental context. Plants with extrafloral nectaries attract a wide variety of ants species, in associations commonly considered mutualistic. In tropical environments the combination of plants and ants play an important role in communities. Turnera subulata is a ruderal shrub widely distributed in disturbed areas, which has a pair of extrafloral nectaries in the base of each leaf. In this study, we evaluated whether the ants associated with T. subulata: (i) vary spatially (between locals) and temporally (over day period); (ii) respond to simulate presence of herbivores and the signs of damage caused in different structures of the host plant (stem and leaf); (iii) reduce herbivory rates; (iv) influencing the vegetative growth and reproductive success of the host plant, along its phenology. The experiments were conducted in São Cristóvão-SE. For the initial understanding of the system studied, T. subulata individuals were selected, in which we performed simulations of the presence of herbivorous and realization of injuries in the stem and leaves of the host plants. The total number of individuals and species associated ants was quantified during three periods of the day. It was quantitated the percentage of leaf area lost and leaves attacked by sucking. They also plot experiments were performed in which were established with and without ants. In each plot were transplanted T. subulata seedlings that were followed throughout their phenological stages. Observations were made in order to quantify the vegetative growth and reproductive investment measures the host plant as well as the number of sheets to damage by sucking insects and chewing; and the number of herbivores. The number of visitors associated with T. subulata was quantified during three periods of the day, being held 5min observation / share. In all cases, data were analyzed using general linear models. 21 species of ants were found. The composition of the ant meeting showed significant variation between locations and time of day. Visitation rates and predation by ants was higher in stems than in the leaves of plants. In general, herbivory rates were not correlated with the association / activity of ants, with the exception of leaf area proportion consumed, which showed a significant reduction in plants where the ants defended the leaves. The results showed that the maturation stage there was a trade-off between growth x propagation of plants; which is favorable for plants (ex.: higher reproduction) who were in plots with ants. Plants on treatment with ants had fewer visitors, including predators and parasitoids. The number of sucking herbivores was significantly reduced in the presence of ants, during the flowering stage. Our results suggest that the benefits of the association may be dependent on context. Moreover, they can contribute to the understanding of the mechanisms involved in facultative interactions between ants and plants and to the understanding of the communities in interaction nets. / Interações ecológicas entre organismos são complexas, podendo apresentar resultados dinâmicos, os quais são dependentes do contexto ambiental. Plantas com nectários extraflorais atraem uma ampla variedade de espécies de formigas, em associações comumente consideradas mutualísticas. Nos ambientes tropicais a associação de plantas e formigas desempenha importante papel nas comunidades. Turnera subulata é um arbusto ruderal, amplamente distribuído em áreas antropizadas, que apresenta um par de nectários extraflorais na base de cada folha. Neste estudo, avaliamos se as formigas associadas a T. subulata: (i) variam espacialmente (entre locais) e temporalmente (ao longo do período do dia); (ii) respondem à simulação da presença de herbívoros e aos sinais de danos ocasionados em diferentes estruturas da planta hospedeira (caule e folha); (iii) reduzem as taxas de herbivoria; (iv) influenciam o crescimento vegetativo e sucesso reprodutivo da planta hospedeira, ao longo da sua fenologia. Os experimentos foram realizados em São Cristóvão-SE. Para o entendimento inicial do sistema estudado, foram selecionados indivíduos de T. subulata, nos quais realizamos simulações da presença de herbívoro e realização de injúrias no caule e folhas das plantas hospedeiras. O número total de indivíduos e de espécies de formigas associadas foi quantificado durante três períodos do dia. Foi quantificada a proporção de área foliar perdida e de folhas atacadas por sugador. Também foram realizados experimentos em que foram estabelecidas parcelas com e sem formigas. Em cada parcela foram transplantadas mudas de T. subulata que foram acompanhadas ao longo de seus estágios fenológicos. Foram feitas observações a fim de quantificar as medidas de crescimento vegetativo e investimento reprodutivo da planta hospedeira, assim como o número de folhas com danos por insetos sugadores e mastigadores; e o número de herbívoros. O número de visitantes associados a T. subulata foi quantificado durante três períodos do dia, durante 5min de observação/parcela. Em todos os casos, os dados foram analisados através de modelos lineares generalizados. Foram encontradas 21 espécies de formigas. A composição da assembleia de formigas apresentou variação significativa no espacialmente e temporalmente. As taxas de visitação e de predação pelas formigas foi maior no caule do que nas folhas das plantas. De forma geral, as taxas de herbivoria não foram correlacionadas com a associação/atividade das formigas, com exceção da proporção de área foliar consumida, que mostrou redução significativa em plantas onde as formigas defenderam as folhas. Os resultados mostraram que no estágio de maturação houve um trade-off entre crescimento x reprodução das plantas; sendo este favorável (ex.: maior reprodução) para as plantas que estavam em parcelas com formigas. Plantas sobre tratamento com formigas tiveram menor número de visitantes, incluindo predadores e parasitoides. O número de herbívoros sugadores foi significativamente reduzido na presença de formigas, durante o estágio de floração. Nossos resultados sugerem que os benefícios da associação podem ser dependentes de contexto. Além disso, podem contribuir para a compreensão dos mecanismos envolvidos nas interações facultativas entre formigas e plantas, e para o entendimento das redes de interações em comunidades.

The growing world demand for renewable energy production in substitution of fossil fuels has given great prominence in the culture of sugarcane (Saccharum sp.). This has been considered the most efficient crop for energy production, as Brazil being the world's largest producer, with a production of 655.6 million tons in 2015/2016 harvest in an area of 8.5 million hectares. One of the major obstacles to the production of sugarcane is still the attack by pest and estimates that about 10% of the crop losses are caused by insects, being a sugarcane borer (Diatraea Saccharalis) the most importante pest. The plants, during its evolution, to reduce the damages caused by the attack of pests, they have developed a series of defense mechanisms, among them, physical barriers, proteins and toxic metabolites and volatile metabolic flags. Many works have been developed to decipher the mechanisms of defense of sugarcane to the attack of herbivorous insects, an attempt of genetic breeding programs and information biotechnology for the development of more resistant plants, however, many of the mechanisms still remain to be clarified. These works are mostly restricted to the study of genes and proteins and global studies of transcript and proteome analyzes of the plant. The current work proposes to perform an analysis of the metabolism of two varieties of sugarcane (RB92579 and SP791011) through direct and indirect extraction methods in response to herbivory by Diatraea saccharalis using NMR spectroscopy and multivariate statistical analysis from data by principal component analysis (PCA) and least squares analysis of discrimination (OPLS-DA). In the metabolomic analysis of sugarcane of the variety RB92579 was studied using the direct extraction method (leaves) in the 4 herbivory times (24, 48, 72 and 96 hours), only after 48, 72 and 96 hours of stress by herbivory, it was possible to get differences between the control groups and herbivore groups, which the greater timing of response was obtained in 72 hours, that indicated the increase of the primary metabolites asparagine, aspartic acid, dimethylamine, glutamic acid , isoleucine, leucine, malic acid, tyrosine and phenylalanine. The variety of sugarcane SP791011, the method of indirect extraction of the leaves of the plant was applied in times 24, 48 and 72 hours by stress induced by caterpillars of Diatraea saccharalis and, coincidentally, showed in the time 72 hours a greater number of herbivory response metabolites, which caused a depletion of aconitic acid, formic acid, asparagine, alanine and elevation of acetic acid and chlorogenic acid. Despite their different metabolic profiles in response to herbivory, the elucidated metabolites suggest the metabolic pathway of shikimic acid to produce phenylpropanoids due to the increase of tyrosine, phenylalanine in the leaves of the sugarcane variety RB92579, and increase of chlorogenic acid in the leaves of the cane. In addition to the herbivory test, it was carried out a biological assay with chlorogenic acid inserted in the artificial diet of Diatraea saccharalis caterpillars, which demonstrated a decrease in the development time of the pupae comparing with pupae of Caterpillars Control, which caused an outbreak of moths with deformations at all concentrations of chlorogenic acid used in the bioassay. These results may help in the development of sugarcane varieties more resistant to the attack by Diatraea Saccharalis. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A crescente demanda mundial para a produção de energias renováveis em substituição aos combustíveis fósseis tem dado grande destaque à cultura da cana-de-açúcar (Saccharum sp.). Esta tem sido considerada a cultura mais eficiente para a produção de energia, tendo o Brasil como o maior produtor mundial, com uma produção de 655,6 milhões de toneladas na safra 2015/2016 em uma área de 8,5 milhões de hectares. Um dos grandes entraves à produção de cana-de-açúcar ainda é o ataque de pragas e doenças e estima-se que cerca de 10% das perdas para esta cultura sejam ocasionadas por insetos, sendo a broca da cana-de-açúcar (Diatraea saccharalis) a praga mais importante. As plantas, durante a evolução, para reduzir os danos causados pelo ataque de pragas, têm desenvolvido uma série de mecanismos de defesa, dentre eles, barreiras físicas, proteínas, metabólitos tóxicos e metabólitos voláteis sinalizadores. Muitos trabalhos têm sido desenvolvidos para decifrar os mecanismos de defesa da cana-de-açúcar ao ataque de insetos herbívoros, na tentativa de subsidiar programas de melhoramento genético e a biotecnologia de informação para o desenvolvimento de plantas mais resistentes, porém, muitos destes mecanismos ainda permanecem a ser esclarecidos. Estes trabalhos, em sua maioria, estão restritos ao estudo de genes e proteínas e a estudos globais de análises dos transcritos e do proteoma da planta. O presente trabalho propõe realizar a análise do metaboloma de duas variedades de cana-de-açúcar (RB92579 e SP791011) através dos métodos de extração direto e indireto na resposta a herbivoria por Diatraea saccharalis, utilizando espectroscopia de RMN, e processamento e estatística dos dados pelos métodos de análise de componentes principais (PCA) e análise dos mínimos quadrados parciais-análise discriminante (OPLS-DA). A análise metabolômica da cana-de-açúcar da variedade RB92579 foi realizada pelo método de extração direta (folhas) nos 4 tempos de herbivoria (24, 48, 72 e 96 horas), sendo que, apenas após 48, 72 e 96 horas sob herbivoria, foi possível haver diferenças entre os grupos controle e grupo herbivoria, no qual o tempo em que se obteve maior resposta da planta foi no tempo 72 horas, que indicou o aumento dos metabólitos primários asparagina, ácido aspártico, dimetilamina, ácido glutâmico, isoleucina, leucina, ácido málico, tirosina e fenilalanina. Já com relação a variedade de cana-de-açúcar SP 1011, aplicou-se o método de extração indireto das folhas da planta nos tempos 24, 48 e 72 horas sob estresse induzido pelas lagartas de Diatraea saccharalis e, coincidentemente, apresentou no tempo 72 horas o maior número de metabólitos de resposta a herbivoria, o que causou numa redução dos níveis de ácido cis e trans aconítico, ácido fórmico, asparagina, alanina e no aumento dos níveis de ácido acético e ácido clorogênico. Apesar de fornecerem perfis metabólicos diferentes em resposta a herbivoria, os metabólitos elucidados sugerem a via metabólica do ácido chiquímico devido ao aumento de tirosina, fenilalanina nas folhas da cana-de-açúcar da variedade RB92579, e aumento do ácido clorogênico nas folhas da cana-de-açúcar da variedade SP791011. Além do ensaio de herbivoria, foi realizado um ensaio biológico com o ácido clorogênico inserido na dieta artificial das lagartas de Diatraea saccharalis, que demonstrou uma diminuição do tempo de desenvolvimento das pupas em comparação com as pupas de lagartas controle, que provocou a eclosão das mariposas com deformações em todas as concentrações de ácido clorogênico usadas no bioensaio. Esses resultados podem ajudar no desenvolvimento de variedades de cana-de-açúcar mais resistentes ao ataque da Diatraea Saccharalis.

Grandes mamíferos herbívoros exercem papel fundamental para a função e estrutura dos ecossistemas terrestres, através principalmente da herbivoria e da deposição de fezes e urina, que influenciam a germinação e crescimento de plantas, a composição e diversidade de comunidades vegetais, a ciclagem e translocação de nutrientes, o estoque de carbono e a freqüência de distúrbios como o fogo. Entretanto, em florestas tropicais, o papel de grandes mamíferos herbívoros foi pouco estudado até o momento. De acordo com a Teoria de Forrageio Ótimo, a seleção de recursos alimentares é resultado do balanço entre o ganho energético e o gasto com a procura, captura e ingestão do alimento. Na escala da paisagem, um dos processos que pode gerar manchas com diferentes qualidades nutricionais para herbívoros é a sucessão da vegetação. Estádios iniciais de sucessão, por serem dominados por espécies de plantas de baixa estatura e crescimento rápido, com folhas tenras, alto conteúdo de nitrogênio, poucos compostos secundários e mais palatáveis, deveriam ser preferidos por grandes mamíferos herbívoros. No entanto, para herbívoros podadores, que incluem frutos na dieta, a disponibilidade destes itens, que são mais nutritivos que a folhagem, deve também influenciar o forrageio. Neste trabalho investigamos como varia a intensidade de forrageio e a deposição de fezes por Tapirus terrestris entre estádios sucessionais, visando contribuir para o entendimento do papel do maior herbívoro terrestre sul-americano para a regeneração da floresta e a translocação de nutrientes. Investigamos se T. Terrestris forrageia mais intensamente em manchas de vegetação em estádios mais iniciais de sucessão, se esta preferência é mais forte quando não há a oferta de um recurso alimentar muito nutritivo, os frutos de cambuci (Campomanesia phaea), e se deposita maior quantidade de fezes onde forrageia mais intensamente e assim não transloca nutrientes. Para tanto, em uma paisagem de 20.000 ha de Floresta Atlântica contínua, foram alocados 12 sítios de amostragem em manchas com diferentes proporções de vegetação em estádios mais iniciais de sucessão. Em cada sítio, a amostragem foi realizada em quatro transecções perpendiculares de 160 m cada durante seis sessões entre março e agosto de 2011. A anta foi registrada através de armadilhas fotográficas e de rastros e fezes localizados e removidos no início e no final de cada sessão. Foram também quantificados a proporção das transecções ocupada por estádios iniciais de sucessão (estádios pioneiro, inicial e médio), o número de clareiras, e o número de pontos das transecções onde a densidade do sub-bosque, do estrato herbáceo e de árvores com DAP até 5 cm foi considerada alta ou muito alta. A frutificação do cambuci foi acompanhada ao longo do período de estudo, tendo sido registrada em metade das sessões de amostragem. Através da abordagem de seleção de modelos, comparamos um conjunto de modelos candidatos para três variáveis dependentes: (a) parâmetro abundância (λ, interpretado como a intensidade de forrageio ao longo das sessões de amostragem) em modelos de abundância (que assumem que não há variações temporais na abundância), considerando apenas modelos simples com cada uma das variáveis explanatórias associadas à proporção de vegetação em estádios mais iniciais de sucessão; (b) número de setores com rastros (interpretado como a intensidade de forrageio dentro das sessões de amostragem) em modelos mistos (GLMMs), considerando estes mesmos modelos simples e mais um grupo de modelos compostos que incluem uma variável temporal relacionada à frutificação do cambuci; e (c) número total de fezes em modelos lineares (GLMs), considerando todos os modelos descritos anteriormente e um modelo com o número total de setores com rastros (interpretado como a intensidade de forrageio total). Nossos resultados indicam que, como esperado pela Teoria de Forrageio Ótimo, Tapirus terrestris seleciona manchas de vegetação em estádios mais iniciais de sucessão, particularmente áreas com maior densidade de árvores pequenas e com maior número de clareiras, e especialmente quando não há oferta de frutos de cambuci. Embora o número total de fezes seja melhor explicado pelo número total de setores com rastros, a relação do número total de fezes com a proporção de estádios iniciais tende a ser negativa, sugerindo que possa haver alguma translocação de nutrientes dos estádios jovens para os tardios. Embora inferências sobre o efeito de T. terrestris sobre a comunidade de plantas dependam de estudos complementares em escalas menores, nossos resultados sugerem que: a herbivoria afeta estádios iniciais de sucessão, onde pode resultar no aumento da diversidade de plantas; antas podem ser agentes de translocação de nutrientes de estádios ricos (iniciais) para aqueles onde nutrientes tendem a ser limitantes (tardios), e a espécie é um potencial agente dispersor do cambuci, espécie ameaçada de extinção / Large herbivorous mammals play a crucial role to the function and structure of terrestrial ecosystems, mainly through herbivory and deposition of dung and urine, which influence plant germination and growth, composition and diversity of plant communities, nutrient cycling and translocation, carbon storage and the frequency of disturbances such as fire. So far, however, the role of large herbivorous mammals in tropical forests has been poorly studied. According to the Optimal Foraging Theory, selection of food resources results from the balance between energy intake and costs to search, capture and ingest the food. At the landscape scale, a process that can generate patches with different nutritional quality for herbivores is vegetation succession. Early successional stages should be preferred by large herbivorous mammals because they are dominated by plant with low height and fast growth, with leaves that are tender, present high nitrogen content and few secondary compounds, and are more palatable. However, for herbivore browsers, which include fruits in their diet, the availability of these items that are more nutritious than foliage should also affect foraging. Here, we investigate how foraging intensity and dung deposition by Tapirus terrestris vary among successional stages, aiming at contributing to the understanding of the role of the largest South American terrestrial herbivore to forest regeneration and nutrient translocation. We investigated if T. terrestris forages more intensively in earlier successional stages, if this preference is stronger when there is no availability of a highly nutritious food resource, the fruits of cambuci (Campomanesia phaea), and if it deposits larger amounts of dung where it forages more intensively, not translocating nutrients. In a 20,000 ha landscape of continuous Atlantic Forest, we allocated 12 sampling sites in patches with different proportion of vegetation in early successional stages. At each site, samplings were conducted in four 160 m long perpendicular transects during six sessions between March and August 2011. Tapir were registered by camera traps and tracks and dung located and cleared at the beginning and end of each session. We also quantified the proportion of transects occupied by early successional stages (pioneer, initial, and mid stage), the number of gaps, and the number of points in transects where the density of the understory , of herbaceous vegetation and of trees with DBH up to 5cm was considered high or very high. The fruiting of cambuci was registered throughout the study period, and was recorded in half of the sampling sessions. Using a model selection approach, we compared a set of candidate models for three dependent variables: (a) the parameter abundance (λ, interpreted as foraging intensity along sampling sessions) in abundance models (which assume no temporal variations in abundance), considering only simple models containing each of the explanatory variables associated with the proportion of earlier successional stages, (b) the number of transect sectors with tracks (interpreted as foraging intensity within sampling sessions) in mixed-effects models (GLMMs), considering these same simple models and a group of composite models that included a temporal variable related to cambuci frutification, and (c) the total number of dung piles in linear models (GLMs), considering all models described above and a model with the total number of transect sectors with tracks (interpreted as total foraging intensity). Our results indicate that, as expected by the Optimal Foraging Theory, Tapirus terrestris selects patches of vegetation in earlier successional stages, in particular areas with higher density of small trees and higher number of gaps, and especially when there is no availability of cambuci fruits. Although the total number of dung piles is better explained by the total number of transect sectors with tracks, the relationship between the number of dung piles and the proportion of earlier successional stages tends to be negative, suggesting that there may be translocation of nutrients from initial to later successional stages. Although conclusions on the effect of T. terrestris on plant communities depend on complementary studies at smaller scales, our results suggest that: herbivory affects early successional stages, where it can result in increased plant diversity; tapirs may act as agents of nutrient translocation from nutrient-rich stages (earlier) to those where nutrients tend to be limiting (later); and tapirs are a potential disperser of cambuci, an endangered plant species

Em cana-de-açúcar, a colonização do caule por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque da lagarta de Diatraea saccharalis. As plantas respondem ao ataque de insetos pela indução e acúmulo de um grande conjunto de proteínas de defesa. Dois homólogos de uma proteína da cevada induzida por ferimento (BARWIN) são conhecidos em cana-de-açúcar, SUGARWIN1 e SUGARWIN2 (sugarcane wound-inducible proteins). Embora a função da proteína BARWIN não tenha sido totalmente estabelecida, propriedades antifúngicas foram descritas para uma série de homólogos. As SUGARWINs estão localizadas no retículo endoplasmático e no espaço extracelular. A indução de seus transcritos ocorre em resposta ao ferimento mecânico, ataque de D. saccharalis e tratamento com metil jasmonato, mas não pela infecção de patógenos. Os transcritos de SUGARWIN são induzidos tardiamente em cana-de-açúcar e são restritos aos locais onde ocorrem os danos. Apesar de transcritos de SUGARWIN1 e 2 serem altamente induzidos pelo ataque de D. saccharalis a proteína em si não possui efeito sobre o desenvolvimento do inseto. Neste trabalho demonstra-se que SUGARWIN2 recombinante causa alterações na morfologia de F. verticillioides e C. falcatum, produzindo aumento da vacuolização, vários pontos de fratura, liberação de material intracelular e formando calos na região dos séptos, culminando na morte destes fungos por apoptose. A SUGARWIN2 recombinante não apesenta efeito em outros fungos, como Saccharomyces cerevisiae e Aspergillus nidulans. Os resultados sugerem que, no curso da evolução genes de SUGARWIN foram recrutados pela cana-de-açúcar para se proteger de fungos oportunistas que, geralmente, penetram na cana após o dano causado pelo inseto. / In sugarcane fields, colonization of the stalk by opportunistic fungi, like Fusarium verticillioides and Colletotrichum falcatum, usually occurs after the attack of Diatraea saccharalis caterpillars. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN) are known in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Although BARWIN protein function has not been fully established, antifungal properties have been described for a number of homologues. The SUGARWINs are located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of SUGARWIN transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment but not to pathogen infection. SUGARWIN transcripts are late induced and are restricted to the wound site. Although SUGARWIN transcripts increased after insect attack, the protein itself did not show any effect on insect development. This work shows that recombinant SUGARWIN2 altered F. verticillioides and C. falcatum morphology increasing vacuolization, points of fractures, leaking of intracellular material, leading to the apoptosis of the germlings. Recombinant SUGARWIN2 do not show any effect in other fungus like Saccharomyces cerevisiae and Aspergillus nidulans. The results suggest that, in the course of evolution, SUGARWIN genes were recruited by sugarcane to protect the plant from fungi that typically penetrate the plant stalk after insect damage.

A cana-de-açúcar é uma gramínea C4 de alta biomassa que acumula grandes quantidades de sacarose e é utilizada para produção de etanol, um combustível de baixa emissão de carbono. Restrições bióticas e abióticas afetam significativamente a produtividade das culturas, pois elas podem prejudicar severamente o crescimento e desempenho da planta. Compreender as bases moleculares para essa perda de produtividade ajudará na investigação das estratégias de mitigação. Para estudar estes dois tipos de estresses, plantas jovens de cana-de-açúcar foram submetidas à herbivoria e à privação de água. Uma investigação foi realizada para estudar as mudanças transcricionais em cana-de-açúcar sujeita ao ataque da Diatraea saccharalis, usando macroarranjo para monitorar a seleção de transcritos, contendo sequências de ESTs de serina proteases e inibidores de serina proteases de cana-de-açúcar do banco de dados SUCEST. Análises do macroarranjo revelaram sequências diferencialmente expressas em resposta à herbivoria. PCR em tempo real confirmou que 10 ESTs homólogos à inibidores de protease (4 homólogos aos inibidores do tipo Bowman-Birk de arroz e milho (BBI), 5 homólogos à inibidores de proteinase de milho (MPI) e 1 homólogo ao inibidor de subtilisina) e 3 ESTs homólogos à serina proteases das famílias S1, S10 e S14 foram positivamente regulados pela herbivoria. Embora a função dos inibidores de protease na defesa está bem estabelecida, o envolvimento de proteases de planta na resposta à herbivoria ainda precisa ser elucidado. Neste trabalho nós mostramos que uma protease da família S14 foi induzida em resposta ao ataque da lagarta e ao ferimento mecânico em cana-de-açúcar. Curiosamente, sequências homólogas de arroz e Arabidopsis também responderam aos mesmos tratamentos, sugerindo um papel conservado desta protease S14 na defesa contra herbívoros. Uma importante aplicação destes resultados é a identificação de genes para utilização em estratégias biotecnológicas para melhorar a resistência da cana-de-açúcar a insetos. Outra investigação foi realizada para estudar os parâmetros fisiológicos e perfis transcricionais de genes responsivos ao estresse hídrico em plantas jovens de cana-de-açúcar. Resultados deste trabalho indicaram que a interrupção da irrigação resultou em efeitos fisiológicos mensuráveis e análises de expressão de genes selecionados de resposta ao estresse revelaram expressão diferencial significativa entre os grupos irrigado e não irrigado. Resultados do RNA-Seq revelaram atividade transcricional de 24.142 transcritos de folhas de cana-de-açúcar submetidas à seca. Análises de expressão gênica em resposta à seca revelaram 68 (resposta precoce) e 2.390 (resposta tardia) transcritos diferencialmente expressos no 3º e 7º dia de tratamento, respectivamente. O decréscimo em vários parâmetros fisiológicos foi observado depois de seis dias de privação de água. Reduções na taxa de fotossíntese, condutância estomática e transpiração foliar ocorreram antes que fossem observadas alterações físicas visíveis, mas estas foram precedidas por mudanças significativas na expressão de genes com papel na fotossíntese. RNA-Seq identificou novos transcritos com papéis na defesa precoce e tardia à seca e a validação por PCR em tempo real confirmou os resultados obtidos pelo RNA-Seq. Isto irá incentivar mais pesquisas sobre a eficiência do uso da água em cana-de-açúcar, levando a identificação de variedades com maior tolerância às condições ambientais adversas. / Sugarcane is a high biomass tropical C4 grass crop which accumulates large quantities of sucrose and is used for bioethanol production, a low-carbon emission fuel. Biotic and abiotic constraints significantly impact crop productivity, because they can severely impair plant growth and performance. Understanding the molecular basis for this loss in productivity will aid in identifying strategies for mitigation. To study these two types of stresses, young sugarcane plants were subject to herbivore and water privation. An investigation was undertaken to study the sugarcane transcriptional changes following Diatraea saccharalis damage, using macroarrays to monitor a selection of transcripts, containing sequences of sugarcane ESTs of serine proteases and serine proteinase inhibitors from the SUCEST (Sugarcane EST Project) database. Macroarray analyses revealed differently expressed sequences in response to herbivory. Real-Time PCR confirmed that 10 ESTs homologous to proteinase inhibitors (4 homologous to maize and rice Bowman-Birk inhibitors (BBI), 5 homologous to maize proteinase inhibitors (MPI) and 1 homologue to subtilisin inhibitor) and 3 ESTs homologous to serine proteases of the S1, S10 and S14 family were positively regulated by herbivory. While the protease inhibitor\'s function in defense is well established, the involvement of plant proteases in response to herbivory still remains to be elucidated. In this work we show that a sugarcane encoding S14 family protease member was upregulated in response to both D. saccharalis damage and wound treatment. Interestingly, homologous sequences from rice and Arabidopsis also responded to the same treatments, suggesting a conserved role of this S14 protease in defense against herbivores. One important application of these results is the identification of genes for use in biotechnological strategies to improve sugarcane insect resistance. Another investigation was undertaken to study the physiological parameters and transcriptional profiles of genes responsive to water stress in young sugarcane plants. Results of this work indicated that termination of irrigation resulted in measurable physiological effects in sugarcane plants and analysis of the expression of the chosen stress-response genes revealed significant differential expression between the control and treatment groups. RNA-Seq results revealed transcriptional activity of 24.142 transcripts from sugarcane leaf subjected to water stress. Gene expression analyses in response to water deprivation revealed 68 (early response) and 2,390 (later response) differentially expressed transcripts on day 3 and day 7, respectively. Sustained decreases in various physiological parameters were observed in water-stressed sugarcane plants after six days of water deprivation. Reductions in photosynthesis rate, stomatal conductance and leaf transpiration occurred before visible physical changes were observed, but this was preceded by significant changes in expression of genes with roles in photosynthesis. RNA-seq identified novel transcripts with roles in early and late response to drought stress and Real-Time qPCR validation confirmed the RNA-Seq results. This will inform further research on water use efficiency in sugarcane, leading to identification of varieties with improved tolerance to adverse environmental conditions.

Abstract Plant community composition and diversity are determined by the balance between rates of immigration and extinction. Processes of immigration to a local community, i.e. propagule availability and dispersal of propagules between and within habitats, set the upper limit for the pool of species potentially capable of coexisting in a community, while local biotic interactions, i.e., competition, facilitation, herbivory and interactions with below-ground ecosystem components, and environmental factors control colonisation and establishment, and determine the persistence and dynamics of already existing species. In this thesis, I studied (1) the interactions between propagule availability, biotic and environmental constraints on colonisation, and (2) the interdependence between biotic and environmental factors regulating community processes in already established resident vegetation. First, I found that both propagule availability and competition with adult plants limited the rates of colonisation and total community diversity in a relatively low-productive tundra ecosystem. Long-term exclusion of mammalian herbivores and alleviation of nutrient limitation by fertilization increased the intensity of competition with established vegetation, and diminished immigration rates. In addition, I also found that community openness to colonization depended on the initial community properties, i.e., the functional composition and the traits of dominant plants in resident vegetation, which mediate the effects of nutrient addition and biomass removal on immigration rates. Second, adult plants in the resident vegetation experienced an increased extent of neighbourhood competition and herbivory in nutrient enriched conditions and in naturally more fertile habitats. However, the effects were also species-specific. On a community level, release from heavy grazing favoured lichens over graminoids and increased species richness. Furthermore, I also showed that plant community composition was strongly linked with soil organic matter quality and microbial community composition, and that these vegetation-soil-microbe interactions varied along a gradient of soil pH. Overall, my results emphasise that propagule availability, biotic and environmental control over community processes are strongly interconnected in tundra ecosystems. Especially, my findings highlight the role of plant competition and herbivory and their dependence on soil nutrient availability in governing colonisation and resident community dynamics. My results also indicate that plant functional composition and traits of dominant plants are of great importance in channelling community responses to external alterations and dictating plant-soil interactions.

Located 100 km west of Key West, Florida, Dry Tortugas National Park (DRTO) is a largely untouched subtropical marine ecosystem that serves as an important developmental habitat, nesting ground, and foraging area for several species of sea turtles, including green turtles. The Park supports a recovering population of green turtles comprised of resident juveniles, subadults, and adults of both sexes; nesting females include residents and migrating females that only return to nest. Stable isotope analysis has been applied widely to describe the trophic ecology of green turtles, from urbanized bays with significant anthropogenic input, to relatively pristine ecosystems with healthy populations at carrying capacity. However, there is a paucity of published literature about the trophic ecology of green turtles in DRTO. This study describes the trophic ecology occupied by two distinct size groups (61 green turtles < 60 cm (SCL) and 98 green turtles > 60 cm (SCL)). Flipper tissue and plasma were analyzed for stable isotopic composition of C and N. Flipper tissue values for δ15N (3.41‰ to 9.69‰) and δ13C (-22.43‰ to -5.38‰) fall within literature values for green turtles, and the wide range of values indicated they could potentially feed at multiple trophic levels. Understanding the trophic ecology of this population of green sea turtles is instrumental to effective management and habitat preservation strategies in DRTO.

Abstract The area of traditionally managed semi-natural meadows is declining throughout Europe. Knowledge of how to restore their species richness is urgently needed. Community manipulations by means of grazing and mowing and, on the other hand, knowledge of species-level responses as well as the responses of functional plant groups to management may help in planning appropriate restoration tools. The restoration of species richness was studied in two community-level experiments: in a sheep grazing experiment and in a mowing experiment. Three species-level studies were conducted to test the compensatory capacity of monocarpic, herbaceous plants (Gentianella amarella, Erysimum strictum, Euphrasia stricta) to simulated herbivory (10–50% of stem being cut). The perennial Linaria vulgaris was used to study whether there is any benefit of clonal integration in the tolerance of simulated herbivory. This topic was studied in a growth experiment and a 13C tracer study. In both grazing and mowing experiments, small herbs gained more space, while the dominant tall herbs subsided. The species number increased by 30% on the grazed plots, but mowing did not affect species richness, probably indicating seed limitation. At sites of this kind, seed addition could be used to promote restoration. Functional plant groups appeared to be useful in predicting the effects of grazing on plant communities. Early season grazing and mowing seem to be proper management tools in overgrown/degraded meadows. In species-level studies, all the species had relatively good compensatory capacity to repair small damage. Overcompensation was observed in response to apical damage, especially when the growing conditions were favourable, supporting the compensatory continuum hypothesis. The studied monocarpic plant species may share a common unbranched growth form where incidental apical damage leads to activation of uninitialized meristems and slight overcompensation. These responses may represent consequences of adaptation to above-ground competition, rather than adaptation to predictable herbivory. In Linaria, damaged ramets were not supported, but the neighbouring ramets seemed to compete with each other for root resources. While growing in disturbed habitats, it is more profitable for this species to invest in new ramets. The present studies showed that, while the appearance and structure of a traditional grassland rich in small-growing herbs can be restored in five years, it is more difficult to increase species diversity by mere mowing or grazing. Knowledge of the tolerance of individual species for herbivory could provide useful information for planning management practices. More studies on the effects of different management tools on different meadow types are urgently needed for the maintenance of the diversity of these valuable environments. / Tiivistelmä Perinteisellä tavalla hoidettujen luonnonniittyjen pinta-ala vähenee kaikkialla Euroopassa. Tarvitsemme enemmän tietoa siitä, miten näiden elinympäristöjen lajiston monimuotoisuus voitaisiin turvata. Tietämys laidunnuksen ja niiton vaikutuksesta kasviyhteisöihin ja toisaalta yksittäisiin kasvilajeihin tai toiminnallisiin kasviryhmiin voi auttaa tehokkaiden hoito- ja ennallistamistapojen löytämisessä. Tässä työssä tutkittiin niittyjen lajirikkauden palauttamista kahdessa yhteisötason kokeessa: lammaslaidunkokeessa ja kokeessa, jossa eri niittotapojen vaikutuksia selvitettiin. Kolmessa lajitason kokeessa testattiin monokarppisten ruohokasvien (rantaukonnauris, horkkakatkero, ketosilmäruoho) kompensaatiokykyä simuloituun herbivoriaan (10–50 % varresta poistettiin). Monivuotista kannusruohoa käytettiin tutkittaessa onko klonaalisesta integraatiosta hyötyä herbivorian siedossa. Tätä selvitettiin kasvukokeella ja hiili-13-merkkiainekokeella. Sekä laidun- että niittokokeessa matalat ruohokasvit saivat kasvutilaa korkeiden ruohojen valta-aseman vähentyessä. Laidunnus lisäsi lajimäärää 30 % koealaa kohti, mutta niitto ei vaikuttanut lajirikkauteen. Molemmissa kokeissa kasviyhteisö oli luultavasti siemenrajoitteinen. Tällaisissa kohteissa siementen lisäystä voitaisiin käyttää ennallistamiskeinona. Toiminnalliset kasviryhmät olivat käyttökelpoisia ennustettaessa laidunnuksen vaikutuksia kasviyhteisöön. Aikaisin kasvukaudella tapahtuva niitto/laidunnus näyttää sopivalta hoitokeinolta umpeenkasvaneilla niityillä. Lajitason kokeissa kaikki tutkitut kasvilajit kykenivät melko hyvin kompensoimaan vähäisiä vaurioita. Ylikompensaatiota havaittiin vasteena kärkivaurioihin erityisesti, kun kasvuolot olivat edulliset. Tämä tuki ns. kompensaatiojatkumohypoteesiä. Tutkituilla lajeilla voi olla yhtenäinen haaraton kasvumuoto, jossa ajoittaiset kärkivauriot johtavat lepotilassa olevien silmujen aktivoitumiseen ja lievään ylikompensaatioon. Nämä vasteet voivat olla seurausta sopeutumisesta valokilpailuun pikemmin kuin sopeutumisesta ennustettavissa olevaan herbivoriaan. Kannusruoholla vaurioituneita versoja ei autettu, vaan kloonin sisällä versot näyttävät kilpailevan keskenään juuriresursseista. Koska laji kasvaa häirityillä paikoilla, sille näyttää olevan edullisempaa investoida uusiin versoihin. Tämä työ osoitti, että vaikka perinteisen matalakasvuisen lajirikkaan niityn ulkonäkö ja rakenne voidaan palauttaa viidessä vuodessa, on vaikeampaa lisätä lajirikkautta pelkän niiton tai laidunnuksen avulla. Tietämys yksittäisten kasvilajien vasteista biomassan menetykseen voi auttaa sopivien hoitotapojen suunnittelussa. Lisää kokeita eri hoitotapojen vaikutuksista ja eri niittytyypeillä tarvitaan pikaisesti näiden arvokkaiden elinympäristöjen monimuotoisuuden ylläpitämiseksi.

Plants and herbivores influence each other for more than million years. Most studies are focused on interspecific variability in herbivory intensity, but little i known about intraspecific variability and what controls it. Environmental conditions can determine intraspecific variation. Goal of this thesis is to examine importance of environmental conditions and interspecific variation for invertebrate herbivory preferences. Plant material that was used in experiments comes from several species from subfamily Carduoideae. Preferences of invertebrate herbivores are tested in food-choice experiments. Plastic arenas are made for these experiments, in which leaves are put in small tubes that are placed along the periphery. After herbivores are placed in to arena, arena is covered by net to ensure that herbivores can't escape. Plant material for experiments was grown in experimental garden and was also brought from field. Plants from garden differ in productivity of soil and water regime. In experiments with field material, plant leaves were collected from eight localities, four fields and four grasslands. Leaves were scanted before and after each experiment. Scans were processed and the amount of consumed leaf tissue was determined. For plant characteristics I measured plant functional traits, specific...

Abstract Mycorrhizal fungi are important contributors to the functioning of boreal forests, since they act in the bilateral carbon and nutrient transport between above- and belowground parts of the ecosystem. In ectomycorrhizal (ECM) symbiosis of woody host plants, both fungal and plant partners depend on resources provided by the other. A single tree may simultaneously host several ECM fungal partners, which greatly enhance the host's nutrient uptake. At the same time nearly 20% of host primary production is allocated to mycorrhizal fungi. Although fungi depend on host-derived carbon, it is poorly understood how reduced carbon availability, e.g., due to herbivory, affects the ECM fungal symbionts. In this thesis I studied the impact of simulated insect defoliation or mammal browsing on mycorrhizal fungi of boreal woody hosts. Quantitative and qualitative changes in biomass partitioning in different fungal compartments were detected. None of the experiments showed that defoliation or shoot clipping treatments reduced the intensity of ECM colonisation, while treatments often shifted fungal composition towards less biomass producing ECM morphotypes. Above- and belowground diversity in ECM symbionts tended to decrease due to shoot or foliar damage. In addition, in some cases defoliation also reduced fungal biomass in fine roots and decreased ECM sexual reproduction by reducing the number of sporocarps produced. Defoliation induced a similar response pattern in the host and in ECM fungi with a stronger response to increasing severity of treatment (e.g. degree of removed foliage or repeated years of defoliation). This was also confirmed when relating the effects of host and ECM fungal symbionts to defoliation using present and previously published data. The present results suggest that belowground adaptation of boreal trees to the changing environment is mediated by changes in fungal community or biomass partitioning. The lack of response in the intensity of ECM colonisation further emphasises the importance of the symbiosis to boreal trees.