Open Access
Genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and the insecticidal characteristics of selected genotypic variants
(MDPI, 2019) Aguirre Sánchez, Eduardo; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Genetic variation in baculoviruses is recognized as a key factor, not only due to the influence of such variation on pathogen transmission and virulence traits, but also because genetic variants can form the basis for novel biological insecticides. In this study, we examined the genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) present in field isolates obtained from virus-killed larvae. Different ChinNPV strains were identified by restriction endonuclease analysis, from which genetic variants were isolated by plaque assay. Biological characterization studies were based on pathogenicity, median time to death (MTD), and viral occlusion body (OB) production (OBs/larva). Nine different isolates were obtained from eleven virus-killed larvae collected from fields of soybean in Mexico. An equimolar mixture of these isolates, named ChinNPV-Mex1, showed good insecticidal properties and yielded 23 genetic variants by plaque assay, one of which (ChinNPV-R) caused the highest mortality in second instars of C. includens. Five of these variants were selected: ChinNPV-F, ChinNPV-J, ChinNPV-K, ChinNPV-R, and ChinNPV-V. No differences in median time to death were found between them, while ChinNPV-F, ChinNPV-K, ChinNPV-R and ChinNPV-V were more productive than ChinNPV-J and the original mixture of field isolates ChinNPV-Mex1. These results demonstrate the high variability present in natural populations of this virus and support the use of these new genetic variants as promising active substances for baculovirus-based bioinsecticides.
Open Access
A strain of Bacillus thuringiensis containing a novel cry7Aa2 gene that is toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)
(MDPI, 2019) Domínguez Arrizabalaga, Mikel; Villanueva San Martín, Maite; Fernández González, Ana Beatriz; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 mu g/mL, which was statistically similar to the estimated LC50 of 20.8 mu g/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 mu g/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.
Open Access
Effect of optical brighteners on the insecticidal activity of a nucleopolyhedrovirus in three instars of Spodoptera frugiperda
(Blackwell Science, 2003-10-17) Martínez Castillo, Ana Mabel; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena
Certain optical brighteners are effective UV protectants, and can improve the insecticidal activity of baculoviruses. We evaluated the effect of 10 optical brighteners, from four chemically different groups, on the insecticidal activity of a nucleopolyhedrovirus (SfMNPV) in third instar Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). The most effective optical brighteners were Blankophor BBH and Calcofluor M2R, both of which are stilbenes. The distyryl-biphenyl derivative, Tinopal CBS, had no effect, whereas the stilbenes, Blankophor CLE and Leucophor SAC and the styryl-benzenic derivative, Blankophor ER, resulted in a decrease in virus induced mortality compared to larvae infected with SfMNPV alone. Mixtures of SfMNPV + 0.1% Calcofluor M2R had relative potencies of 2.7, 6.5, and 61.6 in the second, third, and fourth instars, respectively. The mean time to death differed with instar, but was not affected by the addition of 0.1% Calcofluor M2R. Analysis of published studies indicated that the concentration of Calcofluor M2R-related stilbenes was positively correlated with the relative potency observed in mixtures with homologous NPVs. The average magnitude of optical brightener activity did not differ significantly between early instars of 10 species of Lepidoptera. We conclude that virus formulations containing optical brighteners may be valuable for control of late instar lepidopteran pests.
Open Access
Study of the bacillus thuringiensis Cry1Ia protein oligomerization promoted by midgut brush border membrane vesicles of lepidopteran and coleopteran insects, or cultured insect cells
(MDPI, 2020) Khorramnejad, Ayda; Domínguez Arrizabalaga, Mikel; Caballero Murillo, Primitivo; Escriche, Baltasar; Bel, Yolanda; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

Caballero Murillo, Primitivo

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