Gustav Bouwer

Evaluation of Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) for Control of Helicoverpa armigera (Lepidoptera: Noctuidae) on Citrus in South Africa

A concentration of 1.15×107 OBs/mL of HearNPV, sprayed using a knapsack, resulted in a 100% reduction in Helicoverpa armigera larval infestation within 7 days on tomato plants in a hot house environment. A 10-fold lower concentration, 1.15×106 OBs/mL, resulted in a 100% reduction within 16 days. These two concentrations reduced damage to tomatoes by 81 and 69%, respectively. In two field trials on navel oranges, the lower concentration and an additional even lower concentration of 7.26×105 OBs/mL, both resulted in a 100% reduction in H. armigera infestation within 14 days or longer. H. armigera damage to fruit was reduced by up to 84 and 75%, respectively, in the two trials. Rejection for export was reduced by 96 and 62%. This could imply a saving of US

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

339 per hectare compared to the untreated control. These results were better than those achieved with Bacillus thuringiensis and various standard chemical insecticides used in the same trials. Reasons for these results are discussed.

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

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. armigera. There was a more than 6000-fold difference between the LD50 values of the Cry protein mixture with the lowest larvicidal activity and the mixture with the highest larvicidal activity. Cry1Ac and Cry2Aa mixtures and Cry1Ac and Cry1Ca mixtures had the highest larvicidal activity against H. armigera, with Cry1Ac and Cry1Ca interacting synergistically. Differences in the magnitudes of the antagonistic interactions observed for different binary mixtures of Cry1A-class proteins are consistent with a model of more than one binding site for some Cry1A-class proteins in H. armigera. Binary combinations of Cry1A-class and Cry9Aa proteins showed additive interactions in neonate larvae of H. armigera, whereas combinations of Cry1Ca and Cry9Aa were statistically synergistic. The results suggest that products containing mixtures of Cry1Ac and Cry2Aa or Cry1Ac and Cry1Ca may be useful components of H. armigera pest management programs.

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

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. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. For each of the proteins, dose-mortality and dose-growth inhibition responses were analyzed and the median lethal dose (LD(50)) and median inhibitory dose (ID(50)) determined. Second instar larvae were consistently less susceptible to the evaluated Cry proteins than neonate larvae. The relative toxicity of Cry proteins ranked differently between neonate larvae and second instar larvae. On the basis of the LD(50) and ID(50) values, Cry1Ab, Cry1Ac, and Cry2Aa were the most toxic of the evaluated proteins to H. armigera larvae. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa.

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

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. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between beta-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for beta-exotoxins.

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

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. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model baculovirus system, and neonate H. armigera larvae were infected with one of two geographically distinct HearNPV isolates. DGGE assays for two lepidopteran-specific baculovirus genes, me53 and dbp1, detected many HearNPV genetic variants within individual host larvae, with up to 20 genetic variants detected in a 434-bp fragment of the dbp1 gene in a single neonate larva. High levels of HearNPV genetic diversity were detected in individual host larvae irrespective of the HearNPV isolate used to infect the larvae. This study sets a benchmark for HearNPV genetic variation in individual H. armigera larvae. The levels of HearNPV genetic diversity detected are higher than reported previously for a baculovirus population at the individual insect level.

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

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. In addition to enabling the detection of the genetic variation in key HearNPV genes, the DGGE assays allowed seven geographically distinct HearNPV populations to be differentiated on the basis of their DGGE profiles. The developed DGGE assays will be useful in studies that aim to elucidate the generation and maintenance of genetic diversity in HearNPV.

Do dung beetle larvae need microbial symbionts from their parents to feed on dung

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. Respiration rates, measured as the rate of CO(2) production (VCO(2)), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD(25) or LD(75) survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO(2) values of larvae that survived inoculation (0.0288mlh(-1)), the uninoculated controls (0.0250mlh(-1)), and the larvae that did not survive inoculation (0.0199mlh(-1)) differed significantly from one another. At 4dpi, the VCO(2) of the uninoculated controls were significantly lower than the VCO(2) of inoculation survivors, but significantly higher than the VCO(2) of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.

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

Adult dung beetles eat small particles of their food using soft mouthparts, but their larvae have biting jaws with which they chew material in their natal brood ball. It was assumed that microbes supplied by the parents help breakdown food material in the brood ball, which was thought to act as a fermentation chamber. It has been generally accepted that larval dung beetles gain access to nutrients in the dung via symbionts. Various dung treatments used here, including heat sterilisation and anti‐microbials, suggest that the maternal contribution to the brood ball is a pre‐digested meal for the newly hatched larva, and that microbes from the parents are not involved in larval feeding within this group of dung beetles. However, manipulation of the dung by the mother supplied the larva with smaller dung particles from the unsorted dung, and an additional ‘maternal gift’ secretion may provide partially digested dung, both of which affect survival of the larva. This overturns the largely untested dogma that larval dung beetles depend on microbial symbionts from the adults for feeding, but confirms that adult ‘conditioning’ of the dung is important for the larva in selecting out larger dung particles from the brood ball.

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

BackgroundMalaria, 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.FindingsThere were significant differences in the susceptibility of the anopheline species to PCC 7120#11. The ranking of the larvicidal activity of PCC 7120#11 against species in the An. gambiae complex was: An. merus < An. arabiensis < An. gambiae < An. quadriannulatus, where < indicates a statistically lower LC50. The LC50 of PCC 7120#11 against the important malaria vectors An. gambiae and An. arabiensis was 12.3 × 105 cells/ml and 8.10 × 105 cells/ml, respectively. PCC 7120#11 was not effective against An. funestus, with less than 50% mortality obtained at concentrations as high as 3.20 × 107 cells/ml.ConclusionsPCC 7120#11 exhibited good larvicidal activity against larvae of the An. gambiae complex, but relatively weak larvicidal activity against An. funestus. The study has highlighted the importance of evaluating a novel mosquitocidal agent against a range of malaria vectors so as to obtain a clear understanding of the agent’s spectrum of activity and potential as a vector control agent.