A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae

Hughes, Patrick; Beek, N.A.M. van; Wood, Helen

doi:10.1016/0022-2011(86)90122-9

Cited by 177 publications

(101 citation statements)

References 8 publications

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“…To determine the pathogenicity of single and mixed genotype OBs, bioassays were performed by using the droplet feeding method (23). Batches of 25 newly molted second-instar S. frugiperda from the laboratory colony were inoculated with suspensions containing 10% (wt/vol) sucrose, 0.001% (wt/vol) fluorella blue and OBs at concentrations of 9.6 ϫ 10 2 , 6.0 ϫ 10 3 , 6.0 ϫ 10 4 , and 6.0 ϫ 10 5 OBs/ml.…”

Section: Methodsmentioning

confidence: 99%

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

Clavijo

Williams

Simón

et al. 2009

J Virol

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The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-B⌬16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-B⌬pifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-B⌬16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-B⌬16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-B⌬pifs were almost identical to the results observed with SfNIC-B⌬16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-B⌬16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of ϳ85% SfNIC-B plus ϳ15% SfNIC-B⌬16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNICB⌬pifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1-and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.

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Section: Methodsmentioning

confidence: 99%

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

Clavijo

Williams

Simón

et al. 2009

J Virol

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“…Grace's medium was used as a negative control. The oral infectivity of the recombinant viruses was detected by the droplet method (Hughes et al, 1986) with early third-instar H. armigera larvae. The OBs used for bioassay were harvested and purified from diseased larvae as described by Sun et al (1998), and 10 8 OBs ml 21 were used for the bioassay.…”

Section: Methodsmentioning

confidence: 99%

Functional studies of per os infectivity factors of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Song

Wang

Deng

et al. 2008

Journal of General Virology

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A combined functional investigation on the four per os infectivity factors (PIFs) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was conducted in this study. HearNPV bacmids with deletions of p74 (Ha20), pif1 (Ha111), pif2 (Ha132) and pif3 (Ha98) were constructed individually by homologous recombination in Escherichia coli cells. Repaired bacmids with respective pifs were also constructed. Western blot analyses revealed that all four PIFs were structural components of the envelope of HearNPV occlusion-derived virus (ODV). Electron microscopy showed that deletion of the pifs did not have any obvious effects on the morphology of the occlusion bodies (OBs). Bioassay analyses indicated that deletion of any of the above pifs resulted in loss of oral infectivity of OBs. The mixtures of the four pif-deletion mutants also resulted in deficiency of oral infectivity, implying that the four PIFs must be structural components of the same ODV to accomplish their function. Repairing of the respective genes into the pif-deletion bacmids could rescue the oral infectivity of the pif-deletion viruses. Calcofluor, which can damage the peritrophic membrane (PM), could not rescue the defects of the oral infectivity of the pif-deletion viruses, indicating that the PM is not likely to be the functional target of the PIFs.

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“…Pathogenicity was expressed as the 50% lethal concentration. Bioassays were performed in secondinstar S. frugiperda using the droplet feeding method (12). Larvae were starved for 8 to 12 h at 23°C and then allowed to drink from an aqueous suspension containing 10% (wt/vol) sucrose, 0.001% (wt/vol) Fluorella blue, and OBs at one of five different concentrations (1.92 ϫ 10 3 , 9.6 ϫ 10 3 , 4.8 ϫ 10 4 , 2.4 ϫ 10 5 , and 1.2 ϫ 10 6 OB/ml).…”

Section: Methodsmentioning

confidence: 99%

Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population

Simón

Williams

López‐Ferber

et al. 2005

Appl Environ Microbiol

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A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae

Cited by 177 publications

References 8 publications

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

Functional studies of per os infectivity factors of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population

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