Comparative analysis of the susceptibility/tolerance of Spodoptera littoralis to Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af toxins of Bacillus thuringiensis.

The cotton leaf worm Spodoptera littoralis is known for causing serious damages to various crops. In this study, the susceptibility/tolerance of this larvae to four Vip3A (Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af) toxins was investigated. UnlikeVip3Ad which showed no activity to S. littoralis, Vip3Aa, Vip3Ae and Vip3Af exhibited high toxicity to this larva with LC50 of 228.42 ng/cm2, 65.71 ng/cm2, and 388.90 ng/cm2, respectively. Activation of the 90 kDa Vip3A proteins by S. littoralis larvae juice generated four major bands of sizes 62, 45, 33 and 22 kDa. Binding experiments between biotinylated Vip3A toxins and the brush border membrane vesicles (BBMV) revealed two binding proteins of 55 and 100 kDa with Vip3Aa. Vip3Ae and Vip3Af recognized one single putative receptor of 65 kDa, whereas Vip3Ad did not bind to S. littoralis BBMV. In histopathological observations, Vip3Aa, Vip3Ae and Vip3Af toxins showed approximately similar damages on S. littoralis midgut including rupture and disintegration of epithelial layer and cellular vacuolization. These findings showed that Vip3Aa, Vip3Ae and Vip3Af might be useful for controlling S. littoralis.

Quantification of Bacillus thuringiensis Vip3Aa16 Entomopathogenic Toxin Using Its Hemolytic Activity.

Vegetative insecticidal proteins produced by some Bacillus thuringiensis strains are specifically toxic to different agricultural pests such as the polyphagous Spodoptera and several other Lepidopteran insects, but one of the major problems found in the use of these biopesticides was the lack of an easy and credible method of quantification of such secreted toxins. Heterologous expression of B. thuringiensis Vip3Aa16 toxin was performed in Escherichia coli then the protein was purified by chromatography. Using blood agar as well as blood agar overlay (zymogram assay), we reported, for the first time, the capacity of Vip3Aa16 to induce hemolysis. The hemolytic activity of this protein was shown to be relatively stable after treatment at 40 °C and at a range of pH between 6.5 and 9. Moreover, a linear relationship was shown between hemolysis levels and Vip3Aa16 concentrations. The model established in the present study could quantify Vip3A toxin as a function of hemolytic activity and the assay proposed showed to be a simple and low-cost method to readily assess Vip3A toxins in liquid cultures and facilitate the use of this kind of bioinsecticides in pest management programs.

Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae.

Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.

Bacillus amyloliquefaciens AG1 biosurfactant: Putative receptor diversity and histopathological effects on Tuta absoluta midgut.

The use of biosurfactant in pest management has received much attention for the control of plant pathogens, but few studies reported their insecticidal activity. The present study describes the insecticidal activity of biosurfactant extracted from Bacillus amyloliquefaciens strain AG1. This strain produces a lipopeptide biosurfactant exhibiting an LC50 of about 180ng/cm(2) against Tuta absoluta larvae. Accordingly, the histopathologic effect of this biosurfactant on T. absoluta larvae showed serious damages of the midgut tissues including rupture and disintegration of epithelial layer and cellular vacuolization. By PCR, we showed that this biosurfactant could be formed by several lipopeptides and polyketides including iturin, fengycin, surfactin, bacyllomicin, bacillaene, macrolactin and difficidin. Binding experiment revealed that it recognized five putative receptors located in the BBMV of T. absoluta with sizes of 68, 63, 44, 30 and 19kDa. Therefore, biosurfactant AG1 hold potential for use as an environmentally friendly agent to control the tomato leaf miner.

Overproduction of the Bacillus thuringiensis Vip3Aa16 toxin and study of its insecticidal activity against the carob moth Ectomyelois ceratoniae.

The vip3Aa16 gene of Bacillus thuringiensis strain BUPM95 was cloned and expressed in Escherichia coli. Optimization of Vip3A16 protein expression was conducted using Plackett-Burman design and response surface methodology. Accordingly, the optimum Vip3A16 toxin production was 170µg/ml at 18h post-induction time and 39°C post-induction temperature. This corresponds to an improvement of 21times compared to the starting conditions. The insecticidal activity, evaluated against Ectomyelois ceratoniae, displayed an LC50 value of 40ng/cm(2) and the midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration.

Susceptibility of Agrotis segetum (noctuidae) to Bacillus thuringiensis and analysis of midgut proteinases.

Seventy-eight Bacillus thuringiensis isolates were selected for a screening against the Lepidoptera species Agrotis segetum to search the higher insecticidal activity. In a preliminary bioassay, the spore-crystal mixture of 78 B. thuringiensis isolates was tested against L1 larvae of A. segetum. Fifty-two isolates had more than 60% corrected mortality after 3 days. Seven isolates caused a corrected mortality of 100% on A. segetum. Twelve isolates were selected for a second bioassay investigating the effect of the vegetative insecticidal protein (Vip) against third-instar larvae. After 7 days, the weight gain and the larval stage of each larva were recorded. This bioassay showed an aberration in larval growth increases, morphology, and weight gain. After plasmid pattern analysis, the most active strains are most likely B. thuringiensis kurstaki strains expressing the Vip3A toxin. The absence of two proteinase activities observed in the case of Cry1Ac would be the consequence of the difference in susceptibility of A. segetum to the toxins used.

Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut.

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces many insecticidal proteins including Cry1Ab, Cry1Ca and Cry1Da. In the present study, the insecticidal activity of Cry1Da against Spodoptera littoralis was investigated. It showed toxicity with an LC(50) of 224.4 ng/cm(2) with 95% confidence limits of (178.61-270.19) and an LC(90) of 467.77 ng/cm(2) with 95% confidence limits of (392.89-542.65). The midgut histopathology of Cry1Da fed larvae showed vesicle formation in the apical region, vacuolization and destruction of epithelial cells. Biotinylated-activated Cry1Da toxin bound protein of about 65 kDa on blots of S. littoralis brush border membrane preparations. This putative receptor differs in molecular size from those recognized by Cry1C and Vip3A which are active against this polyphagous insect. This difference in midgut receptors strongly supports the use of Cry1Da as insecticidal agent, particularly in case of Cry and/or Vip-resistance management.

Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin.

Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90kDa) yielding three bands of about 62, 45, 22kDa and the activated form of the toxin was active against this pest with an LC50 of about 86ng/cm(2). To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60-65kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120kDa protein whereas the Vip3Aa16 binds to a 65kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum.

Investigation of antimicrobial activity and statistical optimization of Bacillus subtilis SPB1 biosurfactant production in solid-state fermentation.

During the last years, several applications of biosurfactants with medical purposes have been reported. Biosurfactants are considered relevant molecules for applications in combating many diseases. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Use of inexpensive substrates can drastically decrease its production cost. Here, twelve solid substrates were screened for the production of Bacillus subtilis SPB1 biosurfactant and the maximum yield was found with millet. A Plackett-Burman design was then used to evaluate the effects of five variables (temperature, moisture, initial pH, inoculum age, and inoculum size). Statistical analyses showed that temperature, inoculum age, and moisture content had significantly positive effect on SPB1 biosurfactant production. Their values were further optimized using a central composite design and a response surface methodology. The optimal conditions of temperature, inoculum age, and moisture content obtained under the conditions of study were 37°C, 14 h, and 88%, respectively. The evaluation of the antimicrobial activity of this compound was carried out against 11 bacteria and 8 fungi. The results demonstrated that this biosurfactant exhibited an important antimicrobial activity against microorganisms with multidrug-resistant profiles. Its activity was very effective against Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecalis, Klebsiella pneumonia, and so forth.

The impact of the Bacillus subtilis SPB1 biosurfactant on the midgut histology of Spodoptera littoralis (Lepidoptera: Noctuidae) and determination of its putative receptor.

SPB1 is a Bacillus subtilis strain producing a lipopeptide biosurfactant. The insecticidal activity of this biosurfactant was evaluated against the Egyptian cotton leaf worm (Spodoptera littoralis). It displayed toxicity with an LC(50) of 251 ng/cm(2). The histopathological changes occurred in the larval midgut of S. littoralis treated with B. subtilis SPB1 biosurfactant were vesicle formation in the apical region, cellular vacuolization and destruction of epithelial cells and their boundaries. Ligand-blotting experiments with S. littoralis brush border membrane vesicles showed binding of SPB1 biosurfactant to a protein of 45 kDa corresponding to its putative receptor. The latter differs in molecular size from those recognized by Bacillus thuringiensis Vip3A and Cry1C toxins, commonly known by their activity against S. littoralis. This result wires the application of B. subtilis biosurfactant for effective control of S. littoralis larvae, particularly in the cases where S. littoralis will develop resistance against B. thuringiensis toxins.

Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein.

The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae remaining in the first instar), measured at 7d, were similar or even lower than the LC(50) values of mortality at 10d. This strong growth inhibition was not observed when testing the trypsin-activated protein (62 kDa) in either species. S. exigua was less susceptible than S. frugiperda to the protoxin form, with LC(50) values around 10-fold higher. However, both species were equally susceptible to the trypsin-activated form. Processing of Vip3Aa protoxin to the activated form was faster with S. frugiperda midgut juice than with S. exigua midgut juice. The results strongly suggest that the differences in the rate of activation of the Vip3Aa protoxin between both species are the basis for the differences in susceptibility towards the protoxin form.

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis.

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin.

BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65kDa compared to the 55 and 100kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.

Characterization of Tunisian Bacillus thuringiensis strains with abundance of kurstaki subspecies harbouring insecticidal activities against the lepidopteran insect Ephestia kuehniella.

The study of 257 crystal-producing Bacillus thuringiensis isolates from bioinsecticide free soil samples collected from different sites in Tunisia, was performed by PCR amplification, using six primer pairs specific for cry1, cry2, cry3, cry4, and vip3A genes, by the investigation of strain plasmid pattern, crystal morphology and delta-endotoxin content and by the assessment of insecticidal activities against the lepidopteran insect Ephestia kuehniella. Based on plasmid pattern study, 11 representative strains of the different classes were subjected to morphological and molecular analyses. The comparison of the PFGE fingerprints confirmed the heterogeneity of these strains. B. thuringiensis kurstaki strains, harbouring at the same time the genes cry1A, cry2, cry1Ia, and vip3A, were the most abundant (65.4%). 33.34% of the new isolates showed particular delta-endotoxin profiles but no PCR products with the used primer sets. B. thuringiensis israelensis was shown to be also very rare among the Tunisian B. thuringiensis isolates diversity. These findings could have considerable impacts for the set up of new pest control biological agents.

Prays oleae midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3LB differs from that of Cry1Ac toxin.

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.

Heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata strain K122 and oral toxicity against the lepidoptera Ephestia kuehniella and Spodoptera littoralis.

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities.

Insecticidal activity, putative binding proteins and histopathological effects of Bacillus thuringiensis Vip3(459) toxin on the lepidopteran pest Ectomyelois ceratoniae.

The carob moth, Ectomyelois ceratoniae, is an important agricultural pest that is susceptible to the Vip3(459) protein. The insecticidal activity, evaluated against this lepidopteran pest, displayed an LC50 value of about 28 ng/cm2. The investigation of the mode of action of this B. thuringiensis protein demonstrated that the active form of this toxin bound to putative receptors in the BBMV of E. ceratoniae. Ligand blotting experiment proved that Vip3(459) specifically bound to two proteins of about 53 and 57 kDa, located on the midgut. This specific binding caused perturbations in midgut tissues. The histopathology of 20 midguts from Vip3(459)-feeding larvae showed cytoplasm vacuolization, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. These findings suggested that B. thuringiensis Vip3(459) could be a promising biocontrol agent to eradicate E. ceratoniae and to prevent emergence of resistance.

Combinatorial effect of Photorhabdus luminescens TT01 and Bacillus thuringiensis Vip3Aa16 toxin against Agrotis segetum.

The entomopathogenic Photorhabdus luminescens TT01 is a promoting bacterium that controls effectively many insect pests. Indeed, it exhibited a mortality rate of 32.36% against the first instar larvae of the turnip moth Agrotis segetum, when it was used at a concentration of 5 × 107 cells/ml but no toxicity against the second instar larvae in the same condition. P. luminescens TT01 oral toxicity is associated to septicaemia since cells fraction exhibited the highest mortality rate of 34%. In order to enhance P. luminescens TT01 insecticidal potential, combination with Bacillus thuringiensis Vip3Aa16 toxin was tested. An improvement of insecticidal activity was shown. Indeed, 100% mortality of A. segetum first instar larvae was obtained after 2 days of treatment, when using TT01 cells and Vip3Aa16 toxin at a concentration of 5 × 107 cells/ml and 9.025 ng/cm2, respectively. Moreover, growth inhibition rate of 45% of the second instar larvae was observed, when using the same combination. A. segetum mortality could be the result of several alterations in the midgut epithelium caused by Vip3Aa16 toxin, allowing a rapid invasion of the hemocoel by TT01 cells as demonstrated by histopathological study. Clear symptoms of intoxication were observed for all combinations tested, including swelling, vesicle formation, cytoplasm vacuolization and brush border membrane lysis. Taken together, these results promote the use of P. luminescens TT01 as a potent bioinsecticide to control effectively A. segetum by oral treatment in a mixture with Vip3Aa16 toxin.

Improvement of Vip3Aa16 Toxin Production and Efficiency Through Nitrous Acid and UV Mutagenesis of Bacillus thuringiensis (Bacillales: Bacillaceae).

Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strain BUPM95 was known by the efficiency of its vegetative insecticidal protein (Vip3Aa16) against different Lepidoptera such as Spodoptera littoralis (Lepidoptera: Noctuidae). To overcome the problem of the low quantities of Vip3 proteins secreted by B. thuringiensis strains in the culture supernatant, classical mutagenesis of vegetative cells of BUPM95 strain was operated using nitrous acid and UV rays. The survivors were screened on the basis of their hemolytic activity and classified in three groups: unaffected, overproducing, and hypo-producing mutants. Using different mutants improved in their hemolytic activity, the supernatants showed an improved toxicity toward S. littoralis larvae (83.33-100% of mortality) compared with the wild-type supernatant (76%). After Vip3 protein quantification in the different supernatants, bioassays against S. littoralis larvae demonstrated that mutants M62, M43, and M76 were improved in the efficiency of their toxin as demonstrated by the lower values of LC50 and LC90 compared with the wild-type Vip3Aa16 protein. However, M26 and M73 mutants were improved in the toxin quantities produced in the supernatant. The improvement of the production and the efficiency of B. thuringiensis Vip3 toxins should contribute to a significant reduction of the production costs of these very interesting B. thuringineis proteins and facilitate the use of these toxins in the pest control management.

A novel Vip3Aa16-Cry1Ac chimera toxin: Enhancement of toxicity against Ephestia kuehniella, structural study and molecular docking.

Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resistance to existing insecticidal Cry toxins. During current study, a fusion between vip3Aa16 and the toxic core sequence of cry1Ac was constructed in pHT Blue plasmid. Vip3Aa16-Cry1Ac protein was expressed in the supernatant of B. thuringiensis with a size of about 150 kDa. Bioassays tested on Ephestia kuehniella showed that the use of the chimera toxin as biopesticide improved the toxicity to reach 90% ±â€¯2 with an enhancement of 20% compared to the single Vip3Aa16 protein. The findings indicated that the fusion protein design opens new ways to enhance Vip3A toxicity against lepidopteran species and could avoiding insect tolerance of B. thuringiensis delta-endotoxins. Through computational study, we have predicted for the first time the whole 3D structure of a Vip3A toxin. We showed that Vip3Aa16 structure is composed by three domains like Cry toxins: an N-terminal domain containing hemolysin like fold as well as two others Carbohydrate Binding Module (CBM)-like domains. Molecular docking analysis of the chimera toxin and the single Vip3Aa16 protein against specific insect receptors revealed that residues of CBM like domains are clearly involved in the binding of the toxin to receptors.