Strains of Bacillus thuringiensis active against Coleoptera were evaluated for toxicity against the adult stage of the carrot weevil, Listronotus oregonensis. Mortality and frass bioassays using a suitable semi-artificial diet showed strains A30, A429 and BTT to be highly toxic. Mortality persisted after initial exposure to the bacteria with the survivors not resuming normal feeding. Attempts to reisolate B. thuringiensis from the insects revealed B. thuringiensis-like organisms in the gut and in/on other structures. At the midgut pH of the insect (pH 8.0), the crystals of the toxic strains were significantly more soluble in vitro than were crystals of the less toxic strain A311. Proteolytic activation of the crystals with gut extracts yielded a protein band (66-67 kDa) for strains A30 and A429 which was similar to he apparent molecular weight of the toxin protein for BTT. Evidence suggests that the low toxicity of strain A311 might be due, in part, to the absence of the toxic moiety of the $ delta$-endotoxin.

The control of pest populations by using insect pathogens has been an attractive alternative to the application of chemical pesticides employed for the same purpose. As these chemicals not only damage the environment, but also trigger development of resistance by the pests and can harm other organisms together with the target pest, biological control is preferable and Bacillus thuringiensis (Bt) subspecies have been the most widely used bioinsecticides in forestry, agriculture and mosquito/ black fly control. The most important property of Bt subspecies is the synthesis of protoxins named as delta-endotoxins (crystal proteins). In this study, response surface optimization of Bt subsp. israelensis HD500 batch fermentation for high level production of its toxin proteins Cry4Ba and Cry11Aa was performed. As the interaction of the medium components as well as cultivation conditions are expected to influence the production of the toxin proteins, an experimental chart was prepared by accepting the previously reported optimal values for the most important parameters as zero points: [Mn], 10-6 M / [K2HPO4], 50 mM / C:N ratio, 20:1 and incubation temperature / 30&deg / C. When the combinations of these variables at different levels were studied at 30 batch cultures and analysed for the optimum toxin protein concentrations, temperature: 28.3&amp / #61616 / C, [Mn]: 3.3x10-7M, C:N ratio: 22.2 and [K2HPO4]: 66.1mM yielded the highest concentrations of both Cry4Ba and Cry11Aa toxin proteins.

Chitinases have been shown to be potential agents for biological control of the plant diseases caused by various phytopathogenic fungi and insect pests, because fungal cell walls and insect exoskeletons contain chitin as a major structural component. Chitinase has also been found to increase the efficacy and potency of Bacillus thuringiensis crystal (Cry) proteins toxic to larvae of insect pests. The reason of this synergy is the presence of chitin in the structure of the outer membrane of larval midgut. In this study, the gene encoding chitinase A (chiA) from Serratia marcescens Bn10, a local isolate of Trabzon province was amplified by PCR and cloned into the E.coli/Bacillus shuttle vectors, pNW33N and pHT315. For the expression in B. thuringiensis, the promoter region of cry3Aa11 gene of B. thuringiensis Mm2 was placed at the upstream of chiA. The vectors carrying both chiA and promoter site of cry3Aa11 was first introduced into E. coli and then into Bacillus subtilis 168 which were used as intermediate hosts in this study. pHT315PC carying chiA was then introduced into Coleoptera-specific B. thuringiensis cells (strain 3023) and the specific chitinase activity of the recombinant B. thuringiensis was measured as 5056 U/min/mg which was 6.3 fold higher than that of the parental strain. The specific activity corresponded to about one third of that produced by S. marcescens Bn10. The chiA gene was next sequenced and characterized. The sequence was submitted to GeneBank (Accession No. DQ165083). Chitinase A of S. marcescens Bn10 was found to be a 563 residue protein with a calculated molecular mass of 60.9 kDa. The mean G+C content of the gene is 58.75%. The deduced amino acid sequence was 99.3&ndash / 91.5% identical to those of known chitinases from S. marcescens, Burkholderia cepacia and Enterobacter sp. It was found that the chitinase of S. marcescens Bn10 has six amino acids difference from the consensus sequence of aligned chitinases. The production of chitinase by the local isolate S. marcescens Bn10 in different cultural conditions was also investigated. Optimum temperature and pH for chitinase production was found to be 30 oC and 7.5, respectively. Varying the concentration of colloidal chitin and the inclusion of NAG into the medium had no effect on chitinase production. The effect of different parameters such as temperature, pH, substrate concentration and certain inhibitory elements on enzyme activity were next assayed. The highest activity was obtained at 45 oC and in a pH range of 4.0 to 9.0. Activity of chitinase increased with increasing substrate concentration up to 35 mg/mL. Ca2+, Co2+, Cu2+, EDTA, Fe2+, Mg2+, Mn2+ and Zn2+ were tested for their effects on the activity of enzyme. The enzyme was inhibited by only 4% in the presence of 10 mM EDTA, whereas 10 mM Co2+ included in the assay mixture increased the activity by 20%.

The crystal (Cry) toxins from Bacillus thuringiensis (Bt) display high specificity and toxicity against relevant insect pests and the use of Bt-based products continues to contribute to insect pest management. To protect this investment, further its potential, and investigate possible unintended effects, various research questions have been proposed. One issue related to Bt usage is the evolution of pest resistance to Bt toxins. The midgut epithelium is targeted by Cry toxins killing enterocytes, facilitating invasion of the hemocoel, leading to septicemia and mortality. While resistance may emerge from alterations to these steps, most research efforts have been focused on reduced toxin binding to midgut receptors as resistance mechanism. Lepidopteran crop pest Heliothis virescens strains have been hypothesized to have enhanced midgut proliferation and differentiation of stem cell populations allowing for regeneration and resistance to diverse Cry toxins. However, the molecular mechanisms involved are not known. We developed a flow cytometry method to monitor stem cell proliferation and differentiation to compare midgut regenerative responses to Cry intoxication in larvae from susceptible and Bt-resistant strains of H. virescens. The structure of the epithelial healing response was studied in vivo using hematoxylin-eosin stained midguts derived from larvae fed Cry1Ac toxin. We detected less regenerative cells in midguts from a Bt-susceptible strain (YDK) compared to midguts from resistant (KCB and CXC) strains, and an overall increase in the total number of cells per unit surface area in KCB midguts. Using primary midgut cell cultures, the midgut regeneration response to Cry1Ac in CXC was an increase in available differentiated cells compared to YDK. In contrast, KCB exhibited an increased abundance of stem cells compared to both YDK and CXC. Using a differential proteomics approach we characterized the proteins secreted by H. virescens midgut cells in response to Cry1Ac and identified a relevant role for arylphorin in promoting midgut regeneration in response to Cry1Ac and DiPel intoxication in both susceptible and resistant H. virescens larvae. The potential fitness costs associated with altered hexamerin transcript expression were monitored using larval bioassays.

ix, 152 leaves : photos, graphs, tables, map. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Entomology, 1980

Thèse (M. Sc.)--Université Laval, 2004. / Bibliogr.: f. 34-36.

Techn. Hochsch., Diss., 2004--Aachen.

Universiẗat, Diss., 2006--Kassel.

Submitted by HELVIO CAMPOY COSTA JUNIOR null (helviocampoy@hotmail.com) on 2018-01-24T20:17:31Z No. of bitstreams: 1 Dissertação_Helvio_Campoy_Costa_Junior.pdf: 901555 bytes, checksum: c6e5d61c52e8062ad3355807212bd08a (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2018-01-26T10:44:07Z (GMT) No. of bitstreams: 1 costajunior_hc_me_jabo.pdf: 901532 bytes, checksum: 2d8a2d766da611d354e51bed9eacd12d (MD5) / Made available in DSpace on 2018-01-26T10:44:07Z (GMT). No. of bitstreams: 1 costajunior_hc_me_jabo.pdf: 901532 bytes, checksum: 2d8a2d766da611d354e51bed9eacd12d (MD5) Previous issue date: 2017-11-30 / O objetivo desta pesquisa foi avaliar o efeito subletal causado pelo bioinseticida Dipel em lagartas de Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Bacillus thuringiensis (Bt) é um entomopatógeno amplamente utilizado no controle de pragas, porém pouco se sabe sobre seus efeitos em concentrações subletais, que podem ocorrer em campo devido à diferença na distribuição da calda de aplicação ao longo do dossel das culturas ou diferenças na concentração da expressão de toxinas Bt em tecidos de plantas geneticamente modificada. Neste estudo foi utilizado o bioinseticida Dipel na formulação SC, nas concentrações de 3,67 x 106; 6,85 x 106; 6,85 x 107; 1,37 x 108 e 6,85 X 108 esporos.mL-1, além do tratamento controle, no qual recebeu aplicação de apenas água deionizada para instalação do bioensaio para estimativa das concentrações subletais. Para cada concentração foram utilizadas dezesseis lagartas e três repeticões. A análise de Probit, foi utilizada para estimar as concentrações letais; CL10, CL15, CL20 e CL25, que são consideradas concentrações subletais por apresentarem mortalidade abaixo de 25%. Posteriormente, foi instalado outro bioensaio para avaliar alguns parâmetros biológicos, tais como: período de duração da fase larval, peso de indivíduos na fase larval, peso de indivíduos na fase de pupa e razão sexual. Em todas as concentrações foi observado um aumento significativo no período larval, com destaque para CL25 que apresentou o maior período, chegando a mais de 36 dias. Também foram observadas diferenças significativas no peso de lagartas, peso de pupas. Apenas a concentração CL10 não apresentou diferença significativa em relação ao tratamento controle no parâmetro peso de pupas. Concentrações subletais de Bt são capazes de interferir no desenvolvimento de indivíduos de H. armigera, podendo contribuir para reduzir os prejuízos causados por essa praga. / The objective of this research was to evaluate the sublethal effect caused by Dipel bioinsecticide on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) caterpillars. Bacillus thuringiensis (Bt) is an entomopathogen widely used in pest control, but little is known about its effects on sublethal concentrations, which may occur in the field due to the difference in distribution of the application along the canopy of the crops or differences in concentration expression of Bt toxins in genetically modified plant tissues. In this study, the Dipel bioinsecticide was used in the SC formulation, at concentrations of 3.67 x 106; 6.85 x 106; 6.85 x 107; 1.37 x 108 and 6.85 X 108 spores.mL-1, in addition to the control treatment, in which it received application of only deionized water to install the bioassay to estimate the sublethal concentrations. Sixteen caterpillars and three replicates were used for each concentration. Probit analysis was used to estimate lethal concentrations; LC10, LC15, LC20 and LC25, which are considered sublethal concentrations because they present a mortality below 25%. Later, another bioassay was installed to evaluate some biological parameters, such as: duration period of the larval phase, weight of individuals in the larval stage, weight of individuals in the pupal phase and sexual ratio. All the concentrations showed a significant increase in the larval period, with highlight to LC25 that presented the longest period, reaching more than 36 days. There were also significant differences in the weight of caterpillars and pupal weight. Only LC10 concentration showed no significant difference in relation to the control treatment in the pupae weight parameter. Sublethal Bt concentrations are capable of interfering in the development of H. armigera individuals, and may contribute to reduce the damage caused by this pest.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-09-15Bitstream added on 2014-06-13T18:44:29Z : No. of bitstreams: 1 abreu_il_dr_jabo.pdf: 406152 bytes, checksum: 68b3c6cb5d92e815f953857099241db8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os países industrializados apresentam uma forte tendência em buscarem, cada vez mais, sistemas de controle biológico eficiente e específicos sendo menos tóxicos para os humanos e o ecossistema em geral. Os bioinseticidas à base de microrganismos entomopatogênicos não encontram grandes restrições quanto a sua utilização. Dentre as bactérias que possuem atividade entomopatogênica, Bacillus thuringiensis é a que apresenta maior valor potencial no controle dos insetos-praga, por apresentar vários genes cry e possuir mecanismos de recombinação genética como: conjugação e transformação. O objetivo desse estudo foi identificar e caracterizar a linhagem B. thuringiensis var. londrina. A análise morfológica dos cristais indica uma nova proteína Cry. Para comparação foram utilizadas as linhagens de B. thuringiensis var. tenebrionis, var. tolworthi e var. kurstaki - HD1 que possuem os genes cry3Aa, cry3Ba e cry1Aa, respectivamente. Foi realizada a extração do DNA total das quatro linhagens, em seguida esse material foi submetido à reação de PCR com os oligonucleotideos iniciadores específicos dos genes acima citado. Para a confirmação dos resultados utilizou-se a técnica de hibridização por Southern blotting e seqüenciamento dos amplicon. O amplicom para o gene cry3 obtido pela linhagem padrão B. thuringiensis var. tenebrionis foi igual ao tamanho esperado, porém o obtido pela linhagem var. londrina foi maior em números de nucleotídeo que o esperado, indicando uma possível recombinação. A técnica de Southern blotting confirmou a recombinação do gene mostrando diferenças entre as bandas das linhagens padrão e da linhagem var. londrina, confirmando assim um novo gene cry3. Os bioensaios executados com esse novo gene não indicaram eficiência para alguns insetos-praga de lavoura da ordem Lepidoptera e foram eficientes... / Industrialized countries show a strong tendency to look for efficient and specific biological control systems that are less toxic to humans and ecosystems. Bioinsecticides based on entomopathogenic microorganisms are well accepted by the public. Among those, bacteria that exhibit such entomopathogenic activities, Bacillus thuringiensis is the one with major potential over the pests, since it harbors several cry genes and holds genetic recombination mechanisms such as bacterial conjugation and transformation in order to accomplish these control procedures. The aim of this research was to identify and characterize the strain of B. thuringiensis var. londrina. As a comparison some other strains of B. thuringiensis such as B. thuringiensis var. tenebrionis, var. tolworthi and var. kurstaki - HD1 that holds the genes cry3Aa, cry3Ba and cry1Aa, were used respectively. Alkaline lysis procedure was used for the DNA extraction from the four bacterial strains followed by its use on PCR reactions using the specific primers for the genes. To confirm the results were utilized the techniques of Southern blotting hybridization and DNA sequencing. The obtained amplicons have shown a difference in size considering the pattern strains compared to the var. londrina. The genetic recombination that took place so as to generate the var. londrina cry gene was detected by Southern blotting analysis, the hybridization results confirm the genetic recombination showing the banding pattern differences that took place generating the new cry3 gene. The bioassays showed that the recombinant gene was efficient to control Sphenophorus levis (Coleopteran).