Effect of trypsin inhibitors derived from seeds of Leucaena leucocephala (Lam) A. Witt on the development of Aedes aegypti

A dengue à considerada, hoje, a arbovirose mais importante do mundo, tendo distribuiÃÃo e ressurgÃncia em todos os continentes e aumento das Ãreas de incidÃncia devido Ãs mudanÃas climÃticas. Um dos motivos que a torna tÃo importante à ausÃncia de uma vacina eficaz para os quatro sorotipos do vÃrus. Assim, para o controle dessa doenÃa à necessÃrio que haja o combate ao vetor, o mosquito Aedes aegypti. Os atuais programas de combate ao mosquito dispÃem de inseticidas sintÃticos, contudo o surgimento de populaÃÃes resistentes a esses inseticidas representa um entrave ao combate deste mosquito. Desta forma, torna-se necessÃria a busca por novas molÃculas inseticidas ou capazes de potencializar o efeito tÃxico dos inseticidas atuais. Assim, o presente trabalho teve por objetivo a obtenÃÃo de inibidores de tripsina de sementes de Leucaena leucocephala e avaliaÃÃo do potencial inseticida dessas molÃculas sobre as larvas do mosquito Ae. aegypti. Para isso, as proteÃnas presentes nas sementes de L. leucocephala foram extraÃdas, fracionadas e submetidas à cromatografia de afinidade a anidrotripsina acoplada a sepharose 4B, para a obtenÃÃo de uma fraÃÃo rica em inibidores de tripsina denominada sLlTi, a qual foi caracterizada bioquimicamente e utilizada em concentraÃÃo de 0,3 mg/mL nos ensaios de desenvolvimento das larvas do mosquito. A caracterizaÃÃo bioquÃmica do sLlTi permitiu concluir que os inibidores sÃo termorresistentes (a atividade foi mantida apÃs aquecimento por 30 min. a 100 ÂC), apresentam considerÃvel resistÃncia ao agente redutor (DTT), tendo decaimento da sua atividade inibitÃria frente a tripsina apenas quando incubado com 100 mM de DTT por 120 min, o mesmo, tambÃm, à resistente Ãs variaÃÃes de pH na faixa de 2 a 10. Os zimogramas reversos revelaram a presenÃa de bandas proteicas com massa molecular aparente de 20 kDa. Testes biolÃgicos in vitro mostraram que o sLlTi apresenta atividade inibitÃria superior a 90% frente a tripsina e 70% frente ao homogenato intestinal de larvas de Ae. aegypti. ApÃs a realizaÃÃo de eletroforese bidimensional, foi possÃvel a visualizaÃÃo de isoformas com pI entre 4 e 7, aproximadamente. Esses inibidores sÃo capazes de inibir as proteases de larvas de Ae. aegypti in vivo em 56%, causando retardo no desenvolvimento e cerca de 70% de mortalidade. Assim, o sLlTi possui potencial aÃÃo inseticida contra o mosquito vetor da dengue.Dengue fever is currently the most important arboviral disease in the world, being widely distributed among the continents and having increased incidence areas due to climate change. One of the reasons that makes it so important is the absence of an effective vaccine against the four virus serotypes. So, to control this disease is necessary to fight the disease vector, the mosquito Aedes aegypti. Nowadays, health programs use synthetic insecticides to control the mosquito populations. However, the emergence of resistant populations to these insecticides is an obstacle to combating this mosquito. Thus, it is necessary to search for new insecticides or molecules capable of enhancing the toxic effect of current insecticides. The present study aimed to obtain trypsin inhibitors from Leucaena leucocephala seeds and evaluate the insecticidal potential of these molecules against Ae. aegypti larvae survival and development. For this, the proteins of L. leucocephala seeds were extracted, fractionated and subjected to anhydrotrypsin affinity chromatography to obtain a trypsin-enriched fraction (called sLlTi), which was biochemically characterized and used at a concentration of 0.3 mg/mL in assays of larvae development. Biochemical characterization of sLlTi concluded that the inhibitors are heat resistant (the activity was retained after heating for 30 min at 100  C), showed considerable resistance to the reducing agent DTT and reduction of their inhibitory activity against trypsin was demonstrated only when incubated with 100 mM DTT for 120 min. The inhibitor is also resistant to pH changes in a range from 2 to 10. The reverse zymograms revealed the presence of protein bands with apparent mass near 20 kDa. Biological testing in vitro showed that the sLlTi showed trypsin inhibitory activity greater than 90% and that sLlTi is also capable to inhibit Ae. aegypti digestive enzymes by 70%. After two-dimensional electrophoresis isoforms with pI ranging from 4 to 7, approximately, were observed. These inhibitors were able to inhibit proteases of Ae. aegypti in vivo by 56%, and was capable to delay the larval development after incubation for 10 days. Thus, sLlTi contains proteins with promising insecticidal action against dengue vector