Hemocytes of the fifth instar European borer, Ostrinia nubilalis Hubner (Lepidoptera: Pyralidae) were examined in stained and unstained preparations. Prohemocytes, plasmatocytes, granular hemocyter, and oenocytoids, and spherule cells were found in this stage, The prohemocytes are characterized by a scant intensely basophilic cytoplasm. The highly pleomorphic plasiratocytes are characterized by a punctate nucleus and production of cytoplasmic extensions in vitro. Granular hemocytes are characterized by a small nucleus, accumulation of lipid droplets, and the production of extremely fine cytoplasmic extensions in living preparations. The oenocytoid is characterized by a small eccentric nucleus in a large amount of homogeneous basophilic cytoplasm. The spherule cell is characterized by large spherules within the cytoplasm which may tend to mask the nucleus.Treatment of larvae with per os doses of Bacillus thuringiensis did not affect the differential hemocyte count. However, there did appear to be an increase in the number of degenerating cells in Bacillus thuringiensis treated groups.Ball State UniversityMuncie, IN 47306

The insect pathogen Bacillus thuringiensis (Bt) holds great promise as an effective and friendly way for management of the pests with safety for nontarget animals and humans. However, high capital investment due to high production and formulation cost of commercial Bt preparations has caused prohibitive effect on companies. The present study mainly aimed at developing a low cost medium that supports the growth of different Bt strains and their specific bioinsecticidal &amp / #948 / -endotoxins (crystal proteins). A comparison was made between the representative members of three different subspecies of Bt to observe toxin yields in response to certain nutritional conditions. Three different Bt subspecies were Bt kurstaki (strain 81), Bt israelensis (strain HD500) and Bt tenebrionis (strain 3203), producing lepidoptera- and diptera-specific Cry1 and Cry2, diptera-specific Cry4Ba and Cry11Aa and coleoptera-specific Cry3Aa toxins, respectively. Studies were conducted to optimize glucose and inorganic phosphate concentrations in standard DSM medium for the production of these Bt-based biopesticides. General suppression of toxin yields in high glucose medium (10 g/L) thought the generality of carbon catabolite regulation for biosynthesis of different types of toxins. Inorganic phosphate (Pi) level was important for Cry4Ba, Cry11Aa and Cry3Aa biosynthesis while Cry1 and Cry2 production was not responsive to high Pi. Wastewater sludge, fruit residues and broiler litter were next tested as cheap raw materials for Bt-based biopesticide production in batch cultures. Broiler litter seemed to be a much better substrate among all since some degree of production of each toxin was observed at almost every stage of fermentation. The processing of broiler litter was found to significantly improve toxin yields. The medium prepared from processed broiler litter was successfully used to cultivate all Bt stains and obtain bioinsecticidal proteins in high yields which were comparable or higher than those that can be obtained on standard semi-synthetic media.

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.