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 Helicoverpa armigera 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. Since exposure via the natural oral horizontal transmission route of each larva to a single OB was established and not subject to chance variation, the method facilitated determination of the insect mortality rate (4.8%) associated with exposure to single HearNPV OBs. Droplet feeding bioassays confirmed that the novel method did not reduce OB virulence. The LCM method sets a foundation for virulence and genetic diversity studies based on single NPV OBs.

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

Bt maize is genetically engineered to express insecticidal proteins from the bacterium Bacillus thuringiensis. Bt maize is used extensively by South African farmers to reduce yield losses caused by lepidopteran larvae. Starting in the 2004/2005 season, severe Busseola fusca-associated damage to Cry1Ab-expressing Bt maize was noted by South African farmers. The unsatisfactory pest control was eventually attributed to the development of insect resistance to the Cry1Ab protein in the Bt maize hybrids. An assessment of the historical events surrounding the development of resistance by B. fusca showed that there was room for improvement both in the insect resistance management (IRM) strategy selected and the implementation of the strategy. With the recent arrival of fall armyworm (Spodoptera frugiperda) in Africa, it is important to have IRM programs that are appropriate for all of the pests that constitute the maize lepidopteran pest complex. After the identification of shortcomings in the IRM programs implemented in South Africa, a framework is proposed for effective Bt maize IRM programs. The IRM framework integrates pre-marketing research, post-marketing monitoring, and two-level remedial action plans (RAPs). The core of the framework is a regulator-approved IRM strategy that is based on comprehensive pre-marketing research and serves to guide stakeholders during the post-marketing phase. The framework will assist technology developers and regulators, especially those with nascent regulatory systems, to select and implement IRM strategies that facilitate sustainable pest management.

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. The study showed that the droplet feeding method is compatible with Cry protein-solubilizing buffers, but that it is important to determine the volume imbibed for every buffer used in dose-dependent bioassays in order to reduce dose errors.

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. The treatment group microcosms were inoculated with PCC 7120#11 and key water quality parameters and non-target effects were compared between the treatment and control groups over a period of 35 days. RESULTS: PCC 7120#11 decreased from a concentration of 4.50 × 10(6) cells/ml (at inoculation) to 1.32 × 10(3) cells/ml after 4 weeks and larvicidal activity against third instar larvae of Anopheles arabiensis was only evident for two weeks after treatment. Both treatment and the interaction of treatment and time had a significant effect on nitrate, phosphate and photosynthetic microorganism concentrations. Treatment with PCC 7120#11 caused a temporary spike in ammonia in the microcosms a week after treatment, but the concentrations were well below acute and chronic criteria values for ammonia in freshwater ecosystems. Cyprinotus vidua concentrations were not significantly different between PCC 7120#11 and control microcosms. In PCC 7120#11 microcosms, Daphnia pulex concentrations were significantly lower than control concentrations between days 18 and 25. By the end of the experiment, none of the measured variables were significantly different between the treatment groups. CONCLUSIONS: The standard aquatic microcosm experiments provided more data on the ecological impacts of PCC 7120#11 than single-organism assessments would have. On the basis of the relatively minor, short-term effects that PCC 7120#11 had on water quality parameters and non-target invertebrates, further evaluation of PCC 7120#11 for use in integrated vector management is warranted.

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. 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.

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. armigera neonate larvae inoculated with either an LD5 or LD95 of HearNPV. Although the genetic diversity detected in larvae treated with an LD95 was not statistically different from the diversity detected in the HearNPV inoculum samples, there was a statistically significant difference in the genetic diversity detected in the LD5-inoculated larvae compared with the genetic diversity detected in the HearNPV samples used for the inoculations. The study suggests that inoculum dose needs to be considered carefully in experiments that evaluate HearNPV genetic diversity or in studies where differences in genetic diversity may have phenotypic consequences.

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 interaction of input CO2 concentration and airflow rate had a statistically significant effect on the volumetric productivity of PCC 7120#11, as did the interaction of airflow rate and photosynthetic photon flux density. Model-based numerical optimization indicated that the optimal factor level combination for maximizing PCC 7120#11 volumetric productivity was a photosynthetic photon flux density of 154 µmol m⁻² s⁻¹ and air enriched with 3.18% (v/v) CO2 supplied at a flow rate of 1.02 vessel volumes per minute. At the levels evaluated in the study, none of the growth factors had a significant effect on the median lethal concentration of PCC 7120#11 against An. arabiensis larvae. This finding is important because loss of mosquitocidal activity under growth conditions that maximize volumetric productivity would impact on the feasibility of using PCC 7120#11 in malaria vector control programs. The study showed the usefulness of response surface methodology for determination of the optimal growth conditions for a cyanobacterium that is genetically engineered to have larvicidal activity against malaria vectors.

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. FINDINGS: There 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

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. 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.

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. Analysis of the NGS data identified 60 SNPs within the 3063 bp DNA polymerase gene, 13 SNPs within the 972 bp dbp1 gene, and 25 SNPs within the 1080 bp me53 gene. Depending on the gene, between 31 and 35% of the SNPs were non-synonymous and may, thus, affect the biological functioning of the encoded proteins. The number and homogenous distribution of SNPs found suggest that nucleotide substitution is a major contributor to HearNPV genetic diversity. Denaturing gradient gel electrophoresis (DGGE) assays were used to provide an additional method for evaluating HearNPV genetic variation. Since each of the gene-specific DGGE assays produced unique banding profiles containing numerous bands of differing relative intensities, the DGGE experiments confirmed that there was a high degree of genetic variation in core HearNPV genes and provided information on the relative frequency distribution of genetic variants in the population. The amount of gene-specific genetic variation detected in this study significantly exceeds that reported in other HearNPV studies, suggesting that NGS and DGGE may be useful techniques for studying genetic variation in other baculoviruses.

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. 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.

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. 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.

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. 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.

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. 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.