Genetically encoded libraries and spider venoms as emerging sources for crop protective peptides.

Agricultural crops are targeted by various pathogens (fungi, bacteria, and viruses) and pests (herbivorous arthropods). Antimicrobial and insecticidal peptides are increasingly recognized as eco-friendly tools for crop protection due to their low propensity for resistance development and the fact that they are fully biodegradable. However, historical challenges have hindered their development, including poor stability, limited availability, reproducibility issues, high production costs, and unwanted toxicity. Toxicity is a primary concern because crop-protective peptides interact with various organisms of environmental and economic significance. This review focuses on the potential of genetically encoded peptide libraries like the use of two-hybrid-based methods for antimicrobial peptides identification and insecticidal spider venom peptides as two main approaches for targeting plant pathogens and pests. We discuss some key findings and challenges regarding the practical application of each strategy. We conclude that genetically encoded peptide library- and spider venom-derived crop protective peptides offer a sustainable and environmentally responsible approach for addressing modern crop protection needs in the agricultural sector.

Bioactivities and Synergistic Effect of Elsholtzia ciliata Essential Oil and Its Main Components against Lasioderma serricorne.

Investigations have shown that storage bugs seriously harm grains during storage. In the interim, essential oils (EOs) have been proven to be a good botanical pesticide. The anti-Lasioderma serricorne properties of Elsholtzia ciliata essential oil, which was obtained by steam distillation, were evaluated using DL-limonene, carvone, and their two optical isomer components using contact, repelling, and fumigation techniques. Simultaneously, the fumigation, contact, and repellent activities of carvone and its two optical isomers mixed with DL-limonene against L. serruricorne were evaluated. The results showed that E. ciliata, its main components (R-carvone, DL-limonene), and S-carvone exhibited both fumigations (LC50 = 14.47, 4.42, 20.9 and 3.78 mg/L) and contact (LD50 = 7.31, 4.03, 28.62 and 5.63 µg/adult) activity against L.serricorne. A binary mixture (1:1) of R-carvone and DL-limonene displayed an obvious synergistic effect. A binary mixture (1:1) of carvone and its two optical isomers exhibited an obvious synergistic effect, too. Furthermore, the repellent activity of the EO, carvone, and its two optical isomers, DL-limonene, and a combination of them varied. To stop insect damage during storage, E. ciliata and its components can be utilized as bio-insecticides.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Lethal and repellent effect of amitraz, eugenol and thymol against Triatoma infestans, the main vector of Trypanosoma cruzi in the southern of America.

The lethal and repellent effect of the synthetic insecticide amitraz and the botanical insecticides eugenol and thymol separately and together in binary mixtures was tested against late-stage nymphs of a susceptible strain of Triatoma infestans, the main vector of Trypanosoma cruzi, the etiological agent of Chagas disease, in the Southern Cone of America. For the lethality study, the LD50 was determined for each insecticide alone and in binary mixture by topical application. The combination index (CI) was established to quantify interactions occurring between the insecticides. The repellent effect was tested using the area preference technique. The lethal effect of amitraz was 11 and 34 times more potent than that of thymol and eugenol, respectively. Only the combination of eugenol and amitraz at high concentrations showed a synergistic effect (CI: 0.3). The repellent activity of monoterpenes after 30 min of exposure was significant at 780 and 78 µg/cm2 for eugenol and thymol, respectively. The residual repellent effect of eugenol lasted for one week at the concentrations of 1170 and 1560 µg/cm2 , whereas thymol managed to retain its repellent effect for two weeks at concentrations of 1560 and 3900 µg/cm2 .

Plant secondary metabolites as potential bioinsecticides? Study of the effects of plant-derived volatile organic compounds on the reproduction and behaviour of the pest beetle Tenebrio molitor.

Modern agriculture has many environmental consequences, such as soil contamination, accumulation of toxic compounds in the environment or risk of adverse effects on nontarget organisms and for these reasons, scientists are seeking a more environmentally friendly alternative to synthetic insecticides. This study investigated the effects of four plant secondary metabolites classified as volatile organic compounds (VOCs), which have potential as bioinsecticides, (E)-2-decenal, furfural, 2-undecanone and (E,E)-2-4-decadienal, in concentrations 10-5 and 10-7 M, on female reproductive processes and larval hatchability of the Tenebrio molitor beetle. Our study indicates proper development of ovaries after application of compounds however the volume of terminal oocytes was significantly reduced, with the strongest effect of (E)- 2-decenal which reduced the volume approximately three times. The relative vitellogenin expression level was reduced, with the strongest effect observed after application of furfural, (E,E)- 2-4-decadienal and (E)- 2-decenal in concentration 10-7 M, at the same time patency index was significantly reduced up to 2-times after application of furfural at 10-7 M. What is more important morphological changes translated into physiological ones. The number of laid eggs was affected, with the strongest inhibition after application of furfural (∼43% reduction), (E,E)- 2-4-decadienal (∼33%) and (E)- 2-decenal at concentration 10-7 M (∼33%). Moreover, we observed up to 13% (in case of 2-undecanone) decrease in larval hatchability. Tested compounds exhibited a repellent effect and caused 60% reduction of insect survivability after (E)- 2-decenal at concentration 10-5 M. Altogether, VOCs seems like potential bioactive compounds in plant protection.

Current developments in the resistance, quality, and production of entomopathogenic fungi.

There is a worldwide concern to achieve food security with a sustainable approach, including the generation and implementation of techniques for the production of high-quality chemical-free crops. This food revolution has promoted the development and consolidation of programmes for integrated pest management. Some of those programmes include the use of diverse organisms (biological control agents) to suppress populations of pests potentially harmful to the crops. Among these biological control agents are entomopathogenic fungi that are highly effective in suppressing a diversity of insects and have, therefore, been produced and marketed throughout the world. However, the bottleneck for applying entomopathogenic fungi is the production of propagules (blastospores and conidia) with resistance to environment conditions and abiotic factors, maintaining high quality in terms of virulence. Therefore, this manuscript presents recent studies related to increasing resistance and quality using different bioreactors to produce conidia. The above presents a global panorama related to current developments that contribute to improving the resistance, quality, and production of entomopathogenic fungal propagules.

Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultured Insect Cells.

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125I-DI-III. In addition, the truncated protein 125I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo.IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

In Silico Studies of Lamiaceae Diterpenes with Bioinsecticide Potential against Aphis gossypii and Drosophila melanogaster.

BACKGROUND: The growing demand for agricultural products has led to the misuse/overuse of insecticides; resulting in the use of higher concentrations and the need for ever more toxic products. Ecologically, bioinsecticides are considered better and safer than synthetic insecticides; they must be toxic to the target organism, yet with low or no toxicity to non-target organisms. Many plant extracts have seen their high insecticide potential confirmed under laboratory conditions, and in the search for plant compounds with bioinsecticidal activity, the Lamiaceae family has yielded satisfactory results. OBJECTIVE: The aim of our study was to develop computer-assisted predictions for compounds with known insecticidal activity against Aphis gossypii and Drosophila melanogaster. RESULTS AND CONCLUSION: Structure analysis revealed ent-kaurane, kaurene, and clerodane diterpenes as the most active, showing excellent results. We also found that the interactions formed by these compounds were more stable, or presented similar stability to the commercialized insecticides tested. Overall, we concluded that the compounds bistenuifolin L (1836) and bistenuifolin K (1931), were potentially active against A. gossypii enzymes; and salvisplendin C (1086) and salvixalapadiene (1195), are potentially active against D. melanogaster. We observed and highlight that the diterpenes bistenuifolin L (1836), bistenuifolin K (1931), salvisplendin C (1086), and salvixalapadiene (1195), present a high probability of activity and low toxicity against the species studied.

Amino Alcohols from Eugenol as Potential Semisynthetic Insecticides: Chemical, Biological, and Computational Insights.

A series of ß-amino alcohols were prepared by the reaction of eugenol epoxide with aliphatic and aromatic amine nucleophiles. The synthesized compounds were fully characterized and evaluated as potential insecticides through the assessment of their biological activity against Sf9 insect cells, compared with a commercial synthetic pesticide (chlorpyrifos, CHPY). Three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were identified as the most potent molecules, two of them displaying higher toxicity to insect cells than CHPY. In addition, the most promising molecules were able to increase the activity of serine proteases (caspases) pivotal to apoptosis and were more toxic to insect cells than human cells. Structure-based inverted virtual screening and molecular dynamics simulations demonstrate that these molecules likely target acetylcholinesterase and/or the insect odorant-binding proteins and are able to form stable complexes with these proteins. Encapsulation assays in liposomes of DMPG and DPPC/DMPG (1:1) were performed for the most active compound, and high encapsulation efficiencies were obtained. A thermosensitive formulation was achieved with the compound release being more efficient at higher temperatures.

Method Validation and Evaluation of Safrole Persistence in Cowpea Beans Using Headspace Solid-Phase Microextraction and Gas Chromatography.

Bioinsecticides are regarded as important alternatives for controlling agricultural pests. However, few studies have determined the persistence of these compounds in stored grains. This study aimed at optimizing and validating a fast and effective method for extraction and quantification of residues of safrole (the main component of Piper hispidinervum essential oil) in cowpea beans. It also sought to assess the persistence of this substance in the grains treated by contact and fumigation. The proposed method used headspace solid-phase microextraction (HS-SPME) and gas chromatography with a flame ionization detector (GC/FID). Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was appraised via the parameters selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The LOD and LOQ of safrole were 0.0057 and 0.019 µg kg-1, respectively and the determination coefficient (R2) was >0.99. The relative recovery ranged from 99.26 to 104.85, with a coefficient of variation <15%. The validated method was applied to assess the persistence of safrole residue in grains, where concentrations ranged from 1.095 to 0.052 µg kg-1 (contact) and from 2.16 to 0.12 µg kg -1 (fumigation). The levels measured up from the fifth day represented less than 1% of the initial concentration, proving that safrole have low persistence in cowpea beans, thus being safe for bioinsecticide use. Thus, this work is relevant not only for the extraction method developed, but also for the possible use of a natural insecticide in pest management in stored grains.

Histopathological, cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice.

Dillapiole n-butyl ether is a substance derived from dillapiole, which exhibits potential insecticidal effects on Aedes aegypti, the principal vector of the Dengue fever, Zika, and Chikungunya viruses, as well as Aedes albopictus, a vector of Dengue fever. As these mosquitoes are resistant to synthetic insecticides, dillapiole n-butyl ether may represent a valuable, plant-based alternative for their control. Dillapiole n-butyl ether has insecticidal and genotoxic effects on A. aegypti and A. albopictus, as shown by the reduction in clutch size and egg viability, and increased mortality rates, as well as a high frequency of micronuclei and chromosomal aberrations. However, the potential cytotoxic and genotoxic effects of this substance in mammals are still unknown. In Balb/C mice, structural changes were detected in hepatic, renal, and cardiac tissues, which were directly proportional to the concentration of the dose applied, in both genders. The induction of genotoxic, mutagenic, and cytotoxic effects was also observed at the highest concentrations (150 and 328 mg/kg). Further research will be necessary to better characterize the potential genotoxicity of this substance at lower concentrations, for the evaluation of the potential health risks related to its presence in environmental features, such as drinking water.

Modulation of octopaminergic and cholinergic pathways induced by Caatinga tree Manilkara rufula chemical compounds in Nauphoeta cinerea cockroaches.

The entomotoxic potential of Manilkara rufula crude extract (CEMR) and its aqueous (AFMR) and methanolic (MFMR) fractions were evaluated against Nauphoeta cinerea cockroaches. The results point out to a direct modulation of octopaminergic and cholinergic pathways in insect nervous system. CEMR induced an anti-acetylcholinesterase (AChE) effect in cockroach brain homogenates. CEMR significantly decreased the cockroach heart rate in semi-isolated heart preparations. CEMR also caused a broad disturbance in the insect behavior by reducing the exploratory activity. The decreased antennae and leg grooming activities, by different doses of CEMR, mimicked those of phentolamine activity, a selective octopaminergic receptor antagonist. The lethargy induced by CEMR was accompanied by neuromuscular failure and by a decrease of sensilla spontaneous neural compound action potentials (SNCAP) firing in in vivo and ex vivo cockroach muscle-nerve preparations, respectively. AFMR was more effective in promoting neuromuscular paralysis than its methanolic counterpart, in the same dose. These data validate the entomotoxic activity of M. rufula. The phentolamine-like modulation induced in cockroaches is the result of a potential direct inhibition of octopaminergic receptors, combined to an anti-AChE activity. In addition, the modulation of CEMR on octopaminergic and cholinergic pathways is probably the result of a synergism between AFMR and MFMR chemical compounds. Further phytochemical investigation followed by a bio-guiding protocol will improve the molecular aspects of M. rufula pharmacology and toxicology to insects.

Cytotoxic Plant Extracts towards Insect Cells: Bioactivity and Nanoencapsulation Studies for Application as Biopesticides.

The potential of plant extracts as bioinsecticides has been described as a promising field of agricultural development. In this work, the extracts of Punica granatum (pomegranate), Phytolacca americana (American pokeweed), Glandora prostrata (shrubby gromwell), Ulex europaeus (gorce), Tagetes patula (French marigold), Camellia japonica red (camellia), Ruta graveolens (rue or herb-of-grace) were obtained, purified, and their activity against Spodoptera frugiperda (Sf9) insect cells was investigated. From the pool of over twenty extracts obtained, comprising different polarities and vegetable materials, less polar samples were shown to be more toxic towards the insect cell line Sf9. Among these, a dichloromethane extract of R. graveolens was capable of causing a loss of viability of over 50%, exceeding the effect of the commercial insecticide chlorpyrifos. This extract elicited chromatin condensation and the fragmentation in treated cells. Nanoencapsulation assays of the cytotoxic plant extracts in soybean liposomes and chitosan nanostructures were carried out. The nanosystems exhibited sizes lower or around 200 nm, low polydispersity, and generally high encapsulation efficiencies. Release assays showed that chitosan nanoemulsions provide a fast and total extract release, while liposome-based systems are suitable for a more delayed release. These results represent a proof-of-concept for the future development of bioinsecticide nanoformulations based on the cytotoxic plant extracts.

Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism.

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

The role of midgut nonspecific esterase in the susceptibility of Galleria mellonella larvae to Bacillus thuringiensis.

Some mechanisms of resistance to Bacillus thuringiensis, including esterase activity in the midgut, can appear in laboratory and field insect populations. We found that B. thuringiensis infection significantly reduced the esterase activity in the midgut of greater wax moth Galleria mellonella 48 h post infection. Esterase activity was inhibited 1.5-fold by triphenyl phosphate to test the impact of the enzyme in resistance to the bacteria. Bioassays demonstrated that pre-exposure of the insects to triphenyl phosphate followed by bacterial infection resulted in two-fold elevated susceptibility of the greater wax moth larvae to B. thuringiensis.

Insecticidal effects of the Olea europaea subsp. laperrinei extracts on the flour Pyralid Ephestia kuehniella.

In the present study, the aerial parts of the Laperrine olive (Olea europaea subsp. Laperrinei) are subjected to acid extraction and the chemical composition of the extracts is determined by HPLC-DAD. The main compounds found in all of extracts are: hydroxytyrosol (30.45%), tyrosol (0.69%), oleuropein (32.76%), ferrulic acid (17.77%), quercetin (31.57%) and hesperetin (6.90%).The extracts obtained from the leafy stems of Laperrine olive tree are tested on the moth Ephestia kuehniella flour. Their administration by inhalation of newly exuviated chrysalises extends the duration of nymphalid development and disturbs the exuviated adults reproduction, by reducing the period in which the eggs are being laid. Thus, compared to the control insects, the number of eggs laid by treated females is significantly reduced after the treatment by extracts. Besides, the administration of different extracts of adult butterflies has a premature mortality effect.

Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

Molecular Targets for Components of Essential Oils in the Insect Nervous System-A Review.

Essential oils (EOs) are lipophilic secondary metabolites obtained from plants; terpenoids represent the main components of them. A lot of studies showed neurotoxic actions of EOs. In insects, they cause paralysis followed by death. This feature let us consider components of EOs as potential bioinsecticides. The inhibition of acetylcholinesterase (AChE) is the one of the most investigated mechanisms of action in EOs. However, EOs are rather weak inhibitors of AChE. Another proposed mechanism of EO action is a positive allosteric modulation of GABA receptors (GABArs). There are several papers that prove the potentiation of GABA effect on mammalian receptors induced by EOs. In contrast, there is lack of any data concerning the binding of EO components in insects GABArs. In insects, EOs act also via the octopaminergic system. Available data show that EOs can increase the level of both cAMP and calcium in nervous cells. Moreover, some EO components compete with octopamine in binding to its receptor. Electrophysiological experiments performed on Periplaneta americana have shown similarity in the action of EO components and octopamine. This suggests that EOs can modify neuron activity by octopamine receptors. A multitude of potential targets in the insect nervous system makes EO components interesting candidates for bio-insecticides.

An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals.

Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune-activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term 'toxic' is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited.

A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests.

Five Bacillus thuringiensis Vip3A proteins (Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af) and their corresponding trypsin-activated toxins were tested for their toxicity against eight lepidopteran pests: Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. Toxicity was first tested at a high dose at 7 and 10 days. No major differences were found when comparing protoxins vs. trypsin-activated toxins. The proteins that were active against most of the insect species were Vip3Aa, Vip3Ae and Vip3Af, followed by Vip3Ab. Vip3Ad was non-toxic to any of the species tested. Considering the results by insect species, A. ipsilon, S. frugiperda and S. littoralis were susceptible to Vip3Aa, Vip3Ab, Vip3Ae and Vip3Af; S. exigua was susceptible to Vip3Aa and Vip3Ae, and moderately susceptible to Vip3Ab; M. brassicae and L. botrana were susceptible to Vip3Aa, Vip3Ae and Vip3Af; H. armigera was moderately susceptible to Vip3Aa, Vip3Ae and Vip3Af, and O. nubilalis was tolerant to all Vip3 proteins tested, although it showed some susceptibility to Vip3Af. The results obtained will help to design new combinations of insecticidal protein genes in transgenic crops or in recombinant bacteria for the control of insect pests.