DISSERTAÇÃO - MBT Programa de Pós-Graduação em Biotecnologia e Recursos Naturais da Amazônia
URI permanente para esta coleçãohttps://ri.uea.edu.br/handle/riuea/2048
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Resultados da Pesquisa
Item Avaliação da atividade inseticida de novos derivados de neonicotinóides em Anopheles darling Root, 1926 (Diptera: Culicidae)(Universidade do Estado do Amazonas, 2012-09-24) Mesquita, Rochelly da Silva; Tadei, Wanderli Pedro; Bastos, Ana Mena Barreto; Tadei, Wanderli PedroAmongst malaria control strategies, the use of chemical insecticides constitutes, so far, one of the most utilized methods for fighting its vector. Insect control methods call for a larger number of biological active substances for being utilized as feasible alternatives, as associated to other control procedures. The present work undertook the synthesis of new insecticide activity bearing substances derived from neonicotinoids, a new class of chemical insecticides. Compound synthesis responses occurred through addition, reduction and nitration reactions. Products were characterized according to their melting point, infrared spectroscopy, mass spectroscopy, RMN of 1H and 13C. So as to evaluate the insecticidal activity of synthesized compounds and imidacloprid (Provado® - neonicotinoid commercial product), selective and dose bioassays were carried out with A. darlingi larvae for assessing compound toxicity and subsequent calculation of the CL50, through the use of the POLO-PC® software. The toxicity against adult mosquitoes was evaluated by means of bottle biological proofs. Using data acquired through spectrophotometry, three compounds were suggested: 3- piridinecarboxilic acid 6-chloro 2-hidrazine carbotioamide, 3-hidroxipiridine 6-chloro 2- hydrazine aminetiomethyl and possible isomers of 5-nitrobenzoil 3- piridinacarboxilic acid 6-chloro 2-carbotioamide hydrazine. Amongst these, only 3- piridinacarboxilic acid 6-chloro 2-carbotioamide hydrazine failed to present larvicidal activity in the selective bioassays. Dose bioassays for synthesized compounds as well as imidacloprid presented non-significant χ2 values, indicating that data adjusted itself to the probit model, within the reading gaps. The CL50 values showed to be 503 ppm and 0.17 ppm for compound 2 and imidacloprid, respectively, following 24 h of exposure. Bottle biological proof tests detected no mosquito mortality for the synthesized products. Therefore, different structural modifications of the molecule derived from neonicotinoids showed the synthesized products low insecticidal efficiency, which may be related the molecule’s substituting radicals that contributed for altering the biological activity, when compared to the commercial compound, imidacloprid. Keywords: neonicotinoids, larvicidal activity, Anopheles darlingi, compounds synthesis, imidacloprid.Item Estudo comparativo da microbiota bacteriana cultivável associada à Anopheles darlingi Root, 1926, e seu hábitat(Universidade do Estado do Amazonas, 2015-05-29) Rocha, Elerson Matos; Tadei, Wanderli Pedro; Souza, Antonia Queiroz Lima de; Tadei, Wanderli Pedro; Silva , Ademir Castro e; Pessoa, Marcos Cézar FernandesMalaria is a disease responsible for thousands of deaths worldwide every year. The etiologic agents are protozoa of the genus Plasmodium that are transmitted by female Anopheles mosquitos, considered vector in these conditions. Despite several measures are employed to control malaria and also of many international control programs being implemented, with the focus always returned to mosquito vectors, which each decade exhibit marked resistance to conventional insecticides, the disease remains very common and It presents thousands of cases, a fact that leads to the search for new vector mosquito control methods. Research based on biological control using micro-organisms, are becoming alternatives for controlling these most frequent form of vectors. Little is known about microbial communities that live in symbiosis with the larvae of Anopheles. Know and understand the functions of the diversity of bacteria that live associated with the malaria vector, will allow to understand how the relationship between bacteria, insects and protozoa happen and extensive research may be used such information for future work in order to control this disease in the world. Molecular methodologies contribute to this development analysis of microbial diversity and can reveal a scenario of the distribution of bacteria in the natural habitat of larval breeding of the malaria vector. This study aimed to make a comparative analysis of cultivable bacterial groups associated with larvae and pupae of Anopheles darlingi and their habitat in the city of Coari and Manaus in Amazonas state. Sampling was carried out in the city of Coari and Manaus. Bacteria were inoculated and purified in selective media. DNA extractions were performed and the identification was carried out from the amplification and sequencing of the 16S rDNA fragment. In this study, 1,845 were isolated bacteria from the city of Coari and Manaus. They were identified from water samples, 3rd and 4th larval and pupal stages of A. darlingi, 46 different species of bacteria belonging to 23 genera phyla Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The species Bacillus sp., Chromobacterium sp., Chromobacterium violaceum, Bacillus thuringiensis and Pseudomonas sp had the highest percentage of isolates obtained. It was obtained a collection that makes the first step towards a promising control study involving malaria bacteria associated with A. darlingi and their habitat in the Brazilian Amazon region. The next step will be to use the isolates identified in this study work aimed at understanding the relationship of bacteria with the malaria parasite in the midgut of the mosquito A. darlingi. Keywords: Bacterial microbiota - Anopheles darlingi - Malaria - Amazon.Item Análise da expressão gênica de Anopheles darlingi (Diptera, Culicidae) em condições naturais e no laboratório sob estresse produzido pelo petróleo(Universidade do Estado do Amazonas, 2012-05-24) Rocha, Marla Raquel Pontes da; Tadei, Wanderli Pedro; Nozawa, Sergio Ricardo; Tadei, Wanderli PedroGene expression studies in mosquitoes have given relevant information on the implementation of vector control strategies. The profile of gene expression can be modified by environmental factors and one example is the petroleum which has toxic organic compounds in its composition being therefore a stressor agent. Petroleum exploration in the Amazon region is growing and in the municipality of Coari-AM is located the Urucu petroliferous province with intense oil exploration and refined petroleum products. In this work, the petroleum was analyzed as a stressor agent to Anopheles darlingi Root, 1926, the main vector of human malaria in the Amazon. For this, larvae of A. darlingi were collected from riverine habitats near the east zone of Manaus town. The stress effects of petroleum were evaluated by gene expression analyzes in larvae of A. darlingi under natural conditions and stress exposure. Toxic bioassays were developed with the objective to establish an experimental design of CL50 and then proceed to the molecular analyzes. For gene expression evaluation cDNA libraries were constructed and sequencing was established by Sanger sequencing and Transcriptome Sequencing (RNA-seq) to obtain Expressed Sequence Tags (ESTs). The data from the gene expression profiles of cDNA libraries from control (larvae in natural conditions) and treatment (larvae under petroleum stress) were analyzes by the bioinformatic program BLAST2GO. In the bioassays of CL50 the concentrations were determined at 0.32 ppm and 0.06 ppm which the larvae of A. darlingi was subjected to stress for further gene expression analyzes. The obtained results showed the representativity of ESTs from the control and treatment libraries. Among the differentially expressed genes in the treatment were the family hsp known by encoding proteins involved in thermal shock, the enzymatic group tps responsible by the terpene biosynthesis and the ctnb related to the oxidoreductase biosynthesis, being all related to stress caused by petroleum. These genes are related to detoxification of organism metabolism indicating important processes of pesticide resistance and the tps can be considered as a marker of exposure to petroleum. In addition to the genes differentially expressed in the treatment, the gly gene was observed and is related to the transcription of glycine-rich proteins (GRPs), and as it was expressed in both libraries it has the possibility of being a normalize for both conditions. Therefore, the observed parameters of gene expression in larvae of A. darlingi establish new perspectives to the species functional genomics. Key-words: Anopheles darlingi; Gene expression; Sanger-CE; NGS-RNAseq; Petroleum; Environmental stressItem Avaliação da biodiversidade de bactérias não cultiváveis associadas a anofelinos e seu habitat(Universidade do Estado do Amazonas, 2015-04-15) Oliveira, Marta Rodrigues de; Souza , Antonia Queiroz Lima de; Tadei, Wanderli Pedro; Souza , Antonia Queiroz Lima deMalaria is a parasitic disease responsible for millions of deaths annually around the world. Its main vector in Brazil is Anopheles darlingi, Root, 1926. According literature, these vectors are found associated with a great diversity of microorganisms, most of them acquired during immature phase through larvae feeding. Bacteria whose can interfere directly on vetorial capacity of the mosquito highlights these microorganism, and its interaction with immature forms from these vectors in their breeding sites is a relevant factor for stablishing important strategies on transmission control of this disease. Therefore, considering there are no studies about microbial biodiversity associated to A. darlingi larvae and its aquatic habitat, especially on Amazon region, this work has as objective characterize bacterial microbiota associated to these species and its natural and/or artificial breeding sites, in the state of Amazonas-AM. It was collected larvae samples from the water of breeding sites of A. darlingi in the State of Amazonas. DNA extraction from larvae samples was performed through innuPREP Plant DNA® kit, and water samples were submitted to heat shock for cellular lysis. After Genomic DNA extraction, amplification of 16S gene from rDNA was performed, through 27F (5’AGAGTTTGATCMTGGCTCAG-3’) and 1100R (5’-AGGGTTGCGCTCGTT-3’) primers. Amplified products were sequenced, and taxonomic identification was performed by comparison of obtained sequences versus database sequences of 16S rRNA RDP II through Classifier program.16S rRNA region sequencing from 37 samples of 4ºestadio larvae and of water from breeding sites of A. darlingi, generated 827.842 sequences which were grouped into 6.714 bacterial OTUs (Operational Taxonomic Unities). To perform Taxonomical analysis the 20 most significant OTUs were selected, and showed more than 10.000 sequences. From these, it was identified 11 genera, 11 families, 8 orders, 7 classes belonging to Actinobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia e Proteobacteria phyla. Proteobacteria phylum was the most predominant, being present in 80% of analyzed OTUs. Thus, it is shown that A. darlingi and its aquatic habitat hosts a rich bacterial diversity. Keywords: Anopheles darlingi, Biodiversity, Bacterias.Item Validação do nível de expressão gênica diferencial dos genes actina, tripsina e phosrrestin ii entre larvas e adultos de anopheles darlingi root, 1926 (diptera: culicidae)(Universidade do Estado do Amazonas, 2011-07-29) Souza, Ketlen Christine Nunes de; Tadei, Wanderli Pedro; Rafael, Míriam Silva; Carvalho-Zilse, Gislene Almeida; Lozano, Jorge Luis López; Rafael, Míriam SilvaThe Anopheles (Nyssorhynchus) darlingi Root, 1926 is considered the primary vector of malaria and the species has the sharpest endophagous and anthropophily between the South American anopheline, with the biggest records in the Amazon region. Malaria is a parasitic disease that affects more than 90 countries worldwide, where about 40% of the population live in areas at risk because they are difficult to control. In recent years, studies of alternative detection and quantification of expression gene have been carried out, as the amplification and analysis of cDNA fragments by the method of Polymerase Chain Reaction in Real Time (qRTPCR), one of the technologies employed in post-genomic age. In this sense, validated the level of genes expression sampled in silico, in advance, between two stages of development of An.darlingi (larvae and adults). An. darlingi samples were obtained in the neighborhood Puraquequara (03º 03' 06.95'' S and 59º 52' 31.38'' W), of Manaus City, Amazonas State, identified and treated individually for oviposition. Larvae of the 1st or 2nd instar were used for total RNA extraction with the Extraction and Purification Quiagen® Kit. We obtained the complementary strand of mRNA (cDNA) with the kit from Promega® . The qRT-PCR method was according to the system SYBR® Green (Applied Biosystems® ) with reactions performed in the ABI-7500 thermocycler™ Real-Time PCR System, Applied Biosystems® . Was analyzed for gene expression, four genes of interest: Actin, Trypsin, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Phosrestin II (Arrestin-A). Among these, GAPDH showed little variation in their level of gene expression in larvae and adults. Therefore, this gene was chosen as reference. Larvae (L1L2) were chosen as normalizing the amplification reactions. In relative quantification, the actin expression was higher in adult compared with larvae, validating the results normalized level of expression in silico library of ESTs from An. darlingi. Trypsin was mainly expressed in larvae. In A. darling the Phosrestin II - (Arrestin-A) expression was higher in larvae than in adults. These data validate the differential gene expression between L1L2 and adults of An. darlingi, useful for understanding the biology and evolution gene of this important malaria vector in South America.Item Análise da variabilidade genética em populações de Anopheles darlingi Root, 1926 (Diptera: Culicidae) do Estado do Amazonas, usando marcadores RAPD(Universidade do Estado do Amazonas, 2007-07-15) Silva, Ana Paula Barbosa da; Santos, Joselita Maria Mendes dos; Santos, Joselita Maria Mendes dosAnopheles darlingi populations from Manaus, Coari, São Gabriel da Cachoeira and Tabatinga were analyzed by using the RAPD molecular marker, for the purpose of assessing the genetic variability and differentiation between them. Populations captured indoor and outdoor (around the house, corral) were also analyzed in Coari and Manaus, between 17:00h and 05:00h, in January and February of 2006. Genetic variability findings were high for mosquito’s four populations, showing it to be higher in São Gabriel da Cachoeira (P= 97.37%; He= 0.3202), and lower in Manaus (P= 78.94%; He= 0.2741). The chi-square test was significant (c2= 1589.5700; GL= 304; P < 0.001). The genetic structure analysis showed significant FST value (FST= 0,0851 ± 0,0075), indicating a reduced gene flow between populations. The “bootstrapping” consistency index for 1000 replicates was 95%. The genetic distance between populations was low (D= 0.0095 – 0.0502), showing to be lower between Coari and Tabatinga, and higher between São Gabriel da Cachoeira e Tabatinga. Despite the latter presenting higher genetic and geographic distance, data showed no clear relation with the Isolation by Distance Model (IBD), since Coari and Tabatinga revealed higher genetic similarity; however the lower geographic distance was between Manaus and Coari. Genetic variability analysis based on mosquito biting activity patterns, showed mosquitoes captured indoors presented highest genetic variability, where the polymorphism and expected heterozygosity were higher in both populations (Coari: P= 84.86% e He= 0.3069; Manaus: P= 78.94% e He= 0.2741). The chi-square test for the parameters was significant (c2= 695.8958; GL= 304; P < 0.001). Genetic structure analysis also showed a significant FST value (FST= 0.0775 ± 0.0072). The genetic distance between populations was low. For Manaus, the genetic distance between indoor and outdoor sub-populations was 0.0004, there being certain homogeneity among them. For Coari, the genetic distances were slightly higher, the highest being that between the outdoor (cattle) and the indoor (D= 0.0296), and the lowest between the indoor and the outdoor (D= 0.0081). As a whole, the data showed high genetic similarity between the analyzed populations, despite the little genetic structuring found. The highest genetic variability found the indoor, in the Coari and Manaus populations, indicates a higher genetic plasticity, and so it might confer them highest adaptability to the changes occurring in the environment.