Determination of the virulence of single nucleopolyhedrovirus occlusion bodies using a novel laser capture microdissection method.

Determination of the virulence of occlusion bodies (OBs), which are the horizontal transmission structures of nucleopolyhedroviruses (NPVs), is an important area of baculovirology. A method for inoculating an insect with an isolated OB was developed using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) infection of second instar larvae as a model NPV-host pathosystem. In this novel method, laser capture microdissection (LCM) was used to directly catapult single OBs onto the surface of insect diet in bioassay containers.

A Framework for Effective Bt Maize IRM Programs: Incorporation of Lessons Learned From Resistance Development.

Bt maize is genetically engineered to express insecticidal proteins from the bacterium . Bt maize is used extensively by South African farmers to reduce yield losses caused by lepidopteran larvae. Starting in the 2004/2005 season, severe -associated damage to Cry1Ab-expressing Bt maize was noted by South African farmers.

The compatibility of Bacillus thuringiensis Cry protein-solubilizing buffers with the droplet feeding method in fall armyworm larvae.

The volumes of alkaline (pH > 10), Bacillus thuringiensis Cry protein-solubilizing buffers imbibed by fall armyworm larvae in droplet feeding assays were determined. The buffers differed in the presence or concentration of key ingredients, including buffering agents, chelating agents, reducing agents, and protease inhibitors. For both first and second instar larvae, the buffer used had a significant effect on the volume imbibed.

Significant differences in the intra-host genetic diversity of Helicoverpa armigera nucleopolyhedrovirus dnapol after serial in vivo passages in the same insect population.

A denaturing gradient gel electrophoresis assay was used to assess the genetic diversity within a region of the DNA polymerase gene (dnapol) in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) populations over serial in vivo passages. There was no evidence of movement towards a consensus dnapol variant composition in the different host larvae after multiple per os passages. The study showed that the HearNPV variant structure after in vivo passages in the same host population is not necessarily convergent, and that it may be reasonable to expect significant differences in intra-host HearNPV genetic diversity after inoculation of larvae with a genotypically-diverse HearNPV inoculum.

The persistence and ecological impacts of a cyanobacterium genetically engineered to express mosquitocidal Bacillus thuringiensis toxins.

Background: The cyanobacterium Anabaena PCC 7120#11 has been genetically engineered to act as a delivery vehicle for Bacillus thuringiensis subspecies israelensis mosquitocidal toxins. To address ecological concerns about releasing this genetically engineered microorganism into the environment for mosquito larva control, the persistence and ecological impacts of PCC 7120#11 was evaluated using multi-species, standardized aquatic microcosms.

Methods: The microcosms were set up as described in ASTM E1366-02 (Standard Practice for Standardized Aquatic Microcosms: Fresh Water), with a few modifications.

Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae).

With the aim of identifying Cry proteins that would be useful in the management of the economically important lepidopteran pest Helicoverpa armigera, the larvicidal activities of binary combinations (1:1 ratios) of six Cry proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry2Aa and Cry9Aa) were evaluated against H. armigera neonate larvae using droplet feeding bioassays. Determination of the LD50 values of individual Cry proteins and mixtures of Cry proteins enabled assessment of the nature of the interactions between Cry proteins in H.

The effect of inoculum dose on the genetic diversity detected within Helicoverpa armigera nucleopolyhedrovirus populations.

Environmental and infection variables may affect the genetic diversity of baculovirus populations. In this study, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying the effects of a key infection variable, inoculum dose, on the genetic diversity within nucleopolyhedrovirus populations. Diversity and equitability indices were calculated from DNA polymerase-specific denaturing gradient gel electrophoresis profiles obtained from individual H.

Optimization of photobioreactor growth conditions for a cyanobacterium expressing mosquitocidal Bacillus thuringiensis Cry proteins.

An Anabaena strain (PCC 7120#11) that was genetically engineered to express Bacillus thuringiensis subsp. israelensis cry genes has shown good larvicidal activity against Anopheles arabiensis, a major vector of malaria in Africa. Response surface methodology was used to evaluate the relationship between key growth factors and the volumetric productivity of PCC 7120#11 in an indoor, flat-plate photobioreactor.

The susceptibility of five African Anopheles species to Anabaena PCC 7120 expressing Bacillus thuringiensis subsp. israelensis mosquitocidal cry genes.

Background: Malaria, one of the leading causes of death in Africa, is transmitted by the bite of an infected female Anopheles mosquito. Problems associated with the development of resistance to chemical insecticides and concerns about the non-target effects and persistence of chemical insecticides have prompted the development of environmentally friendly mosquito control agents. The aim of this study was to evaluate the larvicidal activity of a genetically engineered cyanobacterium, Anabaena PCC 7120#11, against five African Anopheles species in laboratory bioassays.

High levels of genetic variation within Helicoverpa armigera nucleopolyhedrovirus populations in individual host insects.

It has been well documented that baculovirus populations exhibit high levels of genetic variation. Due to the lack of sensitivity of the techniques currently used to study baculovirus genetic variation, relatively little is known about baculovirus genetic diversity at the individual insect level. Since denaturing gradient gel electrophoresis (DGGE) has key advantages over other methods used to study genetic variation in baculoviruses, DGGE assays were used to obtain a better understanding of the genetic variation within baculovirus populations in individual host insects.

Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa.

The susceptibility of one of the most important pests in southern Africa, Helicoverpa armigera (Lepidoptera: Noctuidae), to Bacillus thuringiensis Cry proteins was evaluated by bioassay. Cry proteins were produced in Escherichia coli BL21 cells that were transformed with plasmids containing one of six cry genes. The toxicity of each Cry protein to H.

Development of highly sensitive assays for detection of genetic variation in key Helicoverpa armigera nucleopolyhedrovirus genes.

Information on the degree of genetic variation in key Helicoverpa armigera nucleopolyhedrovirus (HearNPV) genes is limited as the currently used techniques lack the detection sensitivity required to identify multiple genetic variants within a baculovirus population. To facilitate the detection and study of genetic variation within HearNPV populations, denaturing gradient gel electrophoresis (DGGE) assays were designed for a core baculovirus gene (DNA polymerase) and two core lepidopteran-specific baculovirus genes (dbp1 and me53). The gene-specific DGGE assays were capable of producing unique, sensitive and rapid genetic fingerprints of the genetic variants within a HearNPV population and were sensitive enough to detect as many as 26 genetic variants within a single portion (<500bp) of a HearNPV gene.

High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes.

It is well documented that baculovirus populations contain many genotypic variants, but little is known about the degree of genetic variation in specific baculovirus genes. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying genetic variation in nucleopolyhedrovirus genes. Next generation sequencing (NGS) was used to identify single-nucleotide polymorphisms (SNPs) within a core baculovirus gene (DNA polymerase) and two core lepidopteran-specific baculovirus genes (dbp1 and me53) in HearNPV populations.

An improved bioassay for the detection of Bacillus thuringiensis beta-exotoxin.

Some Bacillus thuringiensis strains secrete beta-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of beta-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of beta-exotoxin production. With the aim of improving the fly beta-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel beta-exotoxin bioassay.

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates.

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated.