Author(s): Katak, Ricardo de Melo
Date: 2021
Origin: Oasisbr
Subject(s): Amazon microbiota; Biological control; Entomopathogenic microorganisms; Arbov??rus; Aedes aegypti; CI??NCIAS BIOL??GICAS; Microbiota Amaz??nica; Metab??litos; Antagonismo; Controle bi??logico
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CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior
The Aedes aegypti mosquito is considered the primary vector of many arboviruses that cause diseases such as dengue, chikungunya and Zika. The problems caused by this vector are immeasurable, and new control strategies are needed to mitigate the impacts on public health. Bacillus and Brevibacillus genus bacteria are the main entomopathogenic microorganisms used to control these mosquitoes. Considering the metabolic and genetic diversity of these bacteria, this study aimed to evaluate the chemical and biological potential of bacteria of the genus Bacillus and Brevibacillus, isolated from different Amazonian environments, with toxicity to A. aegypti. One hundred and forty three strains of bacilli were isolated in the culture media (NA, LB and ISP2) by means of heat shock, which was characterized by Gram stain, with 136 gram positive and seven gram negative bacilli found. Through the clusters of the phenotypic profile, 77 strains were selected as representatives and identified by the 16S rRNA gene sequence, being found six bacterial genera such as Bacillus, Brevibacillus, Achromobacter, Serratia, Klebsiella and Brevundimonas. The 77 characterized lines were tested in selective bioassays against A. aegypti, of which 21 (27.2%) had larvicidal activity. The strains that showed toxicity above 50% were used for bioassays of the supernatant and pellet fractions of bacterial cultures by means of selective and quantitative bioassays. Regarding the LC50 and LC90 values of the active strains (SPa07NA-Br. halotolerans and SX15LB-B. safensis) in the supernatant fraction (SUP), no statistically significant difference was observed (p>0.05) when compared to the standard Bti strain BR101NA, at intervals of 24, 48 and 72 hours. The results of the non-autoclaved pellet (PEL) at 48h showed that the SBC13LB-B. thuringiensis strain obtained a LC50 value (0.004 mg/l) lower than that of the standard strain Bti BR101NA (0.008 mg/l), with a statistically significant difference (p = 0.05), showing greater toxicity. Considering the results of autoclaved pellet + supernatant (APEL+SUP), the strains SP06LB-B. thuringiensis and SPa22LB-B. safensis did not show statistically significant differences (p>0.05) when compared to the standard Bti BR101NA strain at intervals of 24, 48 and 72h. The results of the autoclaved pellet (APEL) showed that the strain SPa22LB-B. safensis did not present statistically significant difference between the values of LC50 and LC90 in the intervals of 24, 48 and 72h. The active strains in the supernatant (SUP) and pellet (PEL) fractions were used for the study of secondary metabolites, in which two strains (SPa07NA-Br. halotolerans and SX15LB-B. safensis) showed toxicity in the extracts obtained from the supernatant ( SUP). The SPa07NA strain showed greater toxicity in LC50 and LC90, with a statistically significant difference (p<0.05) at intervals of 24, 48 and 72h. The study of metabolites of the SPa07NA-Br. halotolerans strain, allowed the tracking of active metabolites in the extracts. The results obtained by direct insertion in electrospray ionization mass spectrometry showed that during the 10 days of culture of the strains, no changes in the ions were observed, but the prevalence of major ions was observed. Furthermore, the biological activity against A. aegypti did not show significant differences. The isolation standardization technique by semi-preparative HPLC combined with biological assays and fractionation allowed obtaining three fractions and 12 active subfractions for the larvae. The chemical profile of metabolite extracts from promising strains needs further studies that can be carried out in future work in order to characterize and elucidate the active molecules. In addition to metabolite studies, the antagonistic behavior of two active strains that showed larvicidal activity only in the pellet (PEL) was evaluated. Studies of the potential of GD02.13NA-B. toyonensis strains and SBC13NA-B. thuringiensis showed similar results to the standard Bti BR 101 strain, in which bacterial colonization in the larvae's head and trunk was evidenced, and positive amplification for Cry4 and Chi genes with potential for production of chitinase enzymes. Thus, it is considered that the isolated strains have the potential to produce primary and secondary metabolites for different control strategies for these mosquitoes.
O mosquito Aedes aegypti ?? considerado o vetor prim??rio de muitos arbov??rus causadores de doen??as como a dengue, chikungunya e zika. Os problemas ocasionados por este vetor s??o imensur??veis, sendo necess??rias novas estrat??gias de controle para amenizar os impactos na sa??de p??blica. As bact??rias do g??nero Bacillus e Brevibacillus, constituem-se como principais microrganismos entomopatog??nicos utilizados no controle destes mosquitos. Considerando a diversidade metab??lica e gen??tica destas bact??rias, o presente estudo, objetivou avaliar o potencial qu??mico e biol??gico das bact??rias do g??nero Bacillus e Brevibacillus, isolados de diferentes ambientes amaz??nicos, com toxicidade para as larvas de A. aegypti. Foram isoladas 143 linhagens de bacilos nos meios de cultivo (NA, LB e ISP2) por meio do choque t??rmico, no qual foi caracterizado por colora????o de Gram, sendo encontrados 136 bacilos gram positivos e sete gram negativos. Por meio dos clusters do perfil fenot??pico foram selecionadas 77 linhagens como representantes e identificadas pela sequ??ncia do gene 16S rRNA, sendo encontrado seis g??neros bacterianos como, Bacillus, Brevibacillus, Achromobacter, Serratia, Klebsiella e Brevundimonas. As 77 linhagens caracterizadas foram testadas em bioensaios seletivos contras larvas de A. aegypti, das quais 21 (27.2%) apresentaram atividade larvicida. As linhagens que apresentaram toxicidade acima de 50% foram utilizadas para os bioensaios das fra????es do sobrenadante e do pellet das culturas bacterianas por meio de bioensaios seletivos e quantitativos. Em rela????o aos valores da CL50 e CL90 das linhagens ativas (SPa07NA-Br. halotolerans e SX15LB-B. safensis) na fra????o do sobrenadante (SUP), n??o foi observado diferen??a estat??stica significativa (p>0.05) quando comparadas com a cepa padr??o Bti BR101NA, nos intervalos de 24, 48 e 72 horas. Os resultados do pellet n??o autoclavado (PEL) em 48h mostraram que a linhagem SBC13LB-B. thuringiensis obteve valor de CL50 (0.004 mg/l) menor que o da cepa padr??o Bti BR101NA (0.008 mg/l), sendo verificado diferen??a estat??stica significativa (p = 0.05), mostrando maior toxicidade. Considerando os resultados do pellet autoclavado + sobrenadante (APEL+SUP), as linhagens SP06LB-B. thuringiensis e SPa22LB-B. safensis n??o apresentaram diferen??as estat??sticas significativa (p>0.05) quando comparado com a cepa padr??o Bti BR101NA nos intervalos de 24, 48 e 72h. Os resultados do pellet autoclavado (APEL), mostraram que a linhagem SPa22LB-B. safensis n??o apresentou diferen??a estat??stica significativa entre os valores de CL50 e CL90 nos intervalos de 24, 48 e 72h. As linhagens ativas nas fra????es do sobrenadante (SUP) e do pellet (PEL) foram utilizadas para o estudo de metab??litos secund??rios, no qual duas linhagens (SPa07NA-Br. halotolerans e SX15LB-B. safensis) apresentaram toxicidade nos extratos obtido do sobrenadante (SUP). A linhagem SPa07NA apresentou maior toxicidade na CL50 e CL90 sendo verificado diferen??a estat??stica significativa (p<0.05) nos intervalos de 24, 48 e 72h. O estudo dos metab??litos da linhagem SPa07NA-Br. halotolerans, permitiu o rastrear os metab??litos ativos nos extratos. Os resultados obtidos pela inser????o direta em espectrometria de massas por ioniza????o em electrospray mostraram que durante os 10 dias de cultivo das linhagens n??o foram observadas mudan??as nos ??ons, mas sim a preval??ncia de ??ons majorit??rios. Al??m disso, a atividade biol??gica contra as larvas de A. aegypti n??o apresentaram diferen??as significativas. A t??cnica de padroniza????o de isolamento por HPLC semipreparativo aliados com os ensaios biol??gicos e fracionamento permitiram obter tr??s fra????es e 12 subfra????es ativas para as larvas. O perfil qu??mico dos extratos de metab??litos das linhagens promissoras necessita de estudos complementares que podem ser realizados em trabalhos futuros a fim de caracterizar e elucidar as mol??culas ativas. Al??m dos estudos dos metab??litos, foi avaliado o comportamento antag??nico de duas cepas ativas que apresentaram atividade larvicida somente no pellet (PEL). Os estudos do potencial das linhagens GD02.13NA-B. toyonensis e SBC13NA-B. thuringiensis mostraram resultados semelhantes a cepa padr??o Bti BR 101, no qual foi evidenciado coloniza????o bacteriana nas partes da cabe??a e tronco das larvas, e amplifica????o positiva para os genes Cry4 e Chi com potencial para produ????o de enzimas quitinases. Dessa forma, considera-se que as linhagens isoladas apresentam potencial para produ????o de metab??litos prim??rios e secund??rios para diversas estrat??gias de controle para esses mosquitos.
