Caracterização proteometabolômica dos componentes da teia da aranha Nephila clavipes utilizados na estratégia de captura de presas
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2017-03-22
Autores
Esteves, Franciele Grego [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A aranha Nephila clavipes pertence ao grupo das aranhas construtoras de teias orbitais, que desenvolveram a capacidade de sintetizar fios adesivos. Esses fios adesivos são encontrados nos círculos centrais destas teias, e apresentam gotículas oleosas contendo vesículas que aprisionam em seu interior soluções de proteínas, peptídeos e muitos compostos de baixa massa molecular. Estudos da análise química dessas gotículas identificaram toxinas, ácidos graxos saturados e até alcaloides. Especula-se que quando um inseto presa é aprisionado pela teia, os ácidos graxos dessas gotículas auxiliam no processo de desestabilização da cutícula do inseto, permitindo a difusão das toxinas para o interior do corpo da presa. Também são relatados alcaloides que atuam como repelentes a predadores em algumas teias, e como inseto-toxinas em outras. A presença dessas moléculas são evidências de que a teia não é uma simples ferramenta para captura mecânica e aprisionamento de presas, mas sim uma complexa estrutura, que parece desempenhar um papel estratégico “ativo” na captura de suas presas. Considerando isso, o objetivo deste estudo foi analisar a ultraestrutura, a disposição das gotículas sobre os fios de seda, e visualizar a presença de vesículas lipídicas extraídas das gotículas da teia orbital da aranha N. clavipes através de microscopia. Além disso explorou-se a riqueza do perfil químico dos compostos de baixas massas moleculares da seda da teia através da cromatografia gasosa bidimensional abrangente acoplada a um detector de massas; e por fim investigar a riqueza de proteínas presentes na seda da teia e nas glândulas produtoras de seda, através da digestão proteolítica em solução, cromatografia líquida, e espectrometria de massas, ressaltando as possíveis toxinas envolvidas na paralisia de presas. Primeiramente, foi realizado um estudo com microscopia eletrônica de varredura das gotículas depositadas sobre os fios de seda, e com microscopia de luz das vesículas lipídicas que se encontram em suspensão, retidas dentro do conteúdo aquoso das gotículas. Posteriormente na análise da perfilagem química foram encontrados 316 compostos, dentre esses 25 foram identificados a partir de padrões químicos, sendo a maioria hidrocarbonetos saturados e alguns ácidos graxos. Este estudo também demonstrou através de bioesaios de repelência, que alguns dos ácidos graxos e hidrocarbonetos identificados nas gotículas da teia apresentaram potencial como repelentes a formigas invasoras. Enquanto que o ácido palmítico apresentou ação potencial no processo de dissolução/desintegração da camada superficial da cutícula de abelhas, para permitir a passagem das toxinas ao interior do corpo das presas da aranha N. clavipes. Na análise proteômica foram indentificados um total de 2051 proteínas na seda da teia, sendo que 163 dessas proteínas são toxinas. Também foram identificadas um total de 927, 1961, 849, e 860 proteínas nas glândulas agregada, ampulada maior, flageliforme e ampulada menor, respectivamente; sendo que desses totais, 194, 78, 32 e 30 são toxinas pertencentes a cada glândula citada acima, respectivamente. Este estudo sugere que as glândulas de seda, principalmente a glândula agregada, podem sintetizar e depositar sobre a seda da teia toxinas importantes, que são comuns a alguns venenos animais, tornando dessa forma as teias como uma estrutura ativa na captura de presas. A partir dos resultados obtidos foi possível elaborar uma hipótese que relaciona o papel das gotículas, vesículas lipídicas e dos compostos identificados como parte da estratégia química da teia na pré digestão e paralisia da presa. Portanto, este estudo forneceu uma melhor compreensão da química-ecológica da captura de presas pela teia da aranha N. clavipes, além de informações que podem possibilitar o uso desses compostos no desenvolvimento de inseticida-seletivo, ou até mesmo em possíveis aplicações farmacológicas.
Nephila clavipes belongs to the group of orb-weavings spiders that have developed the ability to synthesize adhesive threads. Such adhesive threads are found in the core circles of the orb-webs, and present oily droplets which in turn contain many vesicles in suspension, entrapping solutions of proteins, peptides and many small-molecular mass compounds. Chemical analysis studies of these droplets identified toxins, saturated fatty acids and even alkaloids. It is speculated that when an insect-prey is trapped by the web, the fatty acids of these droplets aid in the process of destabilizing the insect cuticle allowing the diffusion of the toxins into the prey body. Some studies also reported alkaloids that act as repellents to predators in some webs, and as insect-toxins in others. The presence of these molecules is evidence that the web is not a simple tool for mechanical capture and imprisonment of prey; but rather a complex structure that seems to play a strategic "active" role in capturing its prey. Considering this, the aim of this study was to analyze the ultrastructure, the disposition of the droplets on the silk fibers, and to visualize the presence of lipid vesicles extracted from the web’s droplets of N. clavipes spider, by microscopy. In addition, the richness of the chemical profile of the small-molecular mass compounds in the web-silk was explored through comprehensive two-dimensional gas chromatography coupled to a mass detector; and finally to investigate the richness of proteins present in web-silk and silk-producing glands through in solution proteolytic digestion, liquid chromatography, and mass spectrometry, highlighting the possible toxins involved in the prey paralysis. First, was performed scanning electron microscopy study of the droplets deposited on the web-silk and light microscopy of the suspended lipid vesicles retained within the aqueous contents of the droplets. Posteriorly in the profiling chemical analysis were identified 316 compounds, among these 25 were identified from chemical standards, most of them are hydrocarbons saturated and some fatty acids. This study also demonstrated through repellence bioassays, that some of the fatty acids and hydrocarbons identified in the web's droplets presented potential as repellents to invasive ants. While the palmitic acid presented potential action in the process of dissolution / disintegration superficial layer of cuticle bees, to allow the difusion of toxins into the prey body. In the proteomic analysis a total of 2051 proteins were identified in the web-silk, of which 163 of these proteins are toxins. A total of 927, 1961, 849, and 860 proteins were also identified in the aggregate, major ampullate, flagelliform and minor ampullate glands, respectively; and of these totals, 194, 78, 32 and 30 are toxins belonging to each gland mentioned above, respectively. This study suggests that silkproducing glands, especially the aggregate gland, can synthesize and deposit on the silk-web important toxins, which are common to some animal venoms, thereby making the webs an active structure in the prey capture. From the obtained results it was possible to elaborate a hypothesis that relates the role of the droplets, lipid vesicles and the compounds identified as part of the chemical strategy of the web in the pre-digestion and prey paralysis. Thus, this study provided a better understanding of the chemical-ecological prey capture by N. clavipes spider web, in addition to information that may enable the use of these compounds in the development of insecticide-selective or even possible pharmacological applications.
Nephila clavipes belongs to the group of orb-weavings spiders that have developed the ability to synthesize adhesive threads. Such adhesive threads are found in the core circles of the orb-webs, and present oily droplets which in turn contain many vesicles in suspension, entrapping solutions of proteins, peptides and many small-molecular mass compounds. Chemical analysis studies of these droplets identified toxins, saturated fatty acids and even alkaloids. It is speculated that when an insect-prey is trapped by the web, the fatty acids of these droplets aid in the process of destabilizing the insect cuticle allowing the diffusion of the toxins into the prey body. Some studies also reported alkaloids that act as repellents to predators in some webs, and as insect-toxins in others. The presence of these molecules is evidence that the web is not a simple tool for mechanical capture and imprisonment of prey; but rather a complex structure that seems to play a strategic "active" role in capturing its prey. Considering this, the aim of this study was to analyze the ultrastructure, the disposition of the droplets on the silk fibers, and to visualize the presence of lipid vesicles extracted from the web’s droplets of N. clavipes spider, by microscopy. In addition, the richness of the chemical profile of the small-molecular mass compounds in the web-silk was explored through comprehensive two-dimensional gas chromatography coupled to a mass detector; and finally to investigate the richness of proteins present in web-silk and silk-producing glands through in solution proteolytic digestion, liquid chromatography, and mass spectrometry, highlighting the possible toxins involved in the prey paralysis. First, was performed scanning electron microscopy study of the droplets deposited on the web-silk and light microscopy of the suspended lipid vesicles retained within the aqueous contents of the droplets. Posteriorly in the profiling chemical analysis were identified 316 compounds, among these 25 were identified from chemical standards, most of them are hydrocarbons saturated and some fatty acids. This study also demonstrated through repellence bioassays, that some of the fatty acids and hydrocarbons identified in the web's droplets presented potential as repellents to invasive ants. While the palmitic acid presented potential action in the process of dissolution / disintegration superficial layer of cuticle bees, to allow the difusion of toxins into the prey body. In the proteomic analysis a total of 2051 proteins were identified in the web-silk, of which 163 of these proteins are toxins. A total of 927, 1961, 849, and 860 proteins were also identified in the aggregate, major ampullate, flagelliform and minor ampullate glands, respectively; and of these totals, 194, 78, 32 and 30 are toxins belonging to each gland mentioned above, respectively. This study suggests that silkproducing glands, especially the aggregate gland, can synthesize and deposit on the silk-web important toxins, which are common to some animal venoms, thereby making the webs an active structure in the prey capture. From the obtained results it was possible to elaborate a hypothesis that relates the role of the droplets, lipid vesicles and the compounds identified as part of the chemical strategy of the web in the pre-digestion and prey paralysis. Thus, this study provided a better understanding of the chemical-ecological prey capture by N. clavipes spider web, in addition to information that may enable the use of these compounds in the development of insecticide-selective or even possible pharmacological applications.
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Palavras-chave
Seda de aranha, Glândulas de seda, Toxinas, Abordagem proteômica shotgun, Abordagem metabolômica, Web-silk, Silk-produncing glands, Toxins, Shotgun proteomic approach, Metabolomic approach