Cry1Fa Toxin Research Articles

The Effect of Resistance to Bt Corn on the Reproductive Output of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Simple SummaryThe use of transgenic crops expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been a successful strategy to control the fall armyworm (FAW) Spodoptera frugiperda. However, resistance to many Bt proteins has been observed, which could reduce the effectiveness of this approach as a control strategy. In this study, we compared the reproductive traits of susceptible and resistant FAW males and females to determine if there are costs associated with resistance. Our data provided clear evidence that the development of resistance to the Bt protein affected the reproductive capacity of resistant FAW males.The fall armyworm (FAW) Spodoptera frugiperda is the most significant lepidopteran corn pest in South American countries. Transgenic Bt corn, producing the Cry1Fa toxins, has been used to control this pest, but there is clear evidence that some FAW populations have developed resistance. To determine if there are costs associated with resistance, we compared the mass of adults, the duration of mating, and the mass of the first spermatophore produced, as well as the lifetime fecundity and fertility of once-mated susceptible (SS) and resistant (RR) females. Adult mass was affected by both sex and strain, with SS females being significantly larger than RR ones, while the inverse was true for males. RR pairs took significantly longer to mate than SS pairs, yet the mass of spermatophores produced by RR males was significantly less than those of SS males. The total number of eggs laid did not differ but the fertility of eggs from once-mated RR pairs was significantly lower than that of SS pairs. Our data provided clear evidence that the development of Bt resistance affected the reproductive capacity of resistant FAW.

SfABCC2 transporter extracellular loops 2 and 4 are responsible for the Cry1Fa insecticidal specificity against Spodoptera frugiperda

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that kill insect pests. Insect specificity of Cry toxins relies on their binding to larval gut membrane proteins such as cadherin and ATP-binding cassette (ABC) transporter proteins. Mutations in ABC transporters have been implicated in high levels of resistance to Cry toxins in multiple pests. Spodoptera frugiperda is an insect pest susceptible to Cry1Fa and Cry1Ab toxins while Mythimna separata is tolerant to Cry1Fa and less susceptible to Cry1Ab. Here, we analyzed the potential role of ABCC2 in determining the susceptibility of S. frugiperda to Cry1Fa and Cry1Ab, by expressing SfABCC2 or MsABCC2 in Hi5 insect cell line and by the systematic replacements of extracellular loops (ECLs) between these two proteins. Expression of SfABCC2 in Hi5 conferred susceptibility to both Cry1Fa and Cry1Ab, in contrast to the expression of MsABCC2 that mediated low toxicity to Cry1Ab and no toxicity to Cry1Fa in agreement with their larvicidal toxicities. The SfABCC2 and MsABCC2 amino acid sequences showed differential residues among ECL1, ECL2, ECL4 and ECL6 loops, while ECL3 and ECL5 share the same primary sequence. The exchange of ECLs between SfABCC2 and MsABCC2 demonstrated that ECL4 and ECL2 contribute to Cry1Fa toxicity, where ECL4 plays a major role. The medium region (named M2) of ECL4 was identified as the most important region of SfABCC2 involved in Cry1Fa toxicity as shown by point mutations in this region. These findings will be helpful to understand the mechanisms of action of Bt toxins in S. frugiperda.

Bacillus thuringiensis Cry1Ab Domain III β-16 Is Involved in Binding to Prohibitin, Which Correlates with Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera is a major insect pest of several crops worldwide. This insect is susceptible to some Bacillus thuringiensis (Bt) Cry insecticidal proteins expressed in transgenic crops or used in biopesticides. Previously, we identified H. armigera prohibitin (HaPHB) as a Cry1Ac-binding protein. Here, we further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), well recognized as a Cry1Ac receptor. HaPHB-2 midgut protein and HaCAD toxin-binding region (TBR) fragment from H. armigera were expressed in Escherichia coli cells, and binding assays with different Cry1 toxins were performed. We demonstrated that Cry1Ab, Cry1Ac, and Cry1Fa toxins bound to HaPHB-2 in a manner similar to that seen with HaCAD-TBR. Different Cry1Ab mutant toxins located in domain II (Cry1AbF371A and Cry1AbG439D) or domain III (Cry1AbL511A and Cry1AbN514A), which were previously characterized and found to be affected in receptor binding, were analyzed regarding their binding interaction with HaPHB-2 and toxicity against H. armigera One β-16 mutant (Cry1AbN514A) showed increased binding to HaPHB-2 that correlated with 6-fold-higher toxicity against H. armigera, whereas the other β-16 mutant (Cry1AbL511A) was affected in binding to HaPHB-2 and lost toxicity against H. armigera Our data indicate that β-16 from domain III of Cry1Ab is involved in interactions with HaPHB-2 and in toxicity. This report identifies a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect, suggesting that this protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigeraIMPORTANCEHelicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify β-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

Two ABC transporters are differentially involved in the toxicity of two Bacillus thuringiensis Cry1 toxins to the invasive crop-pest Spodoptera frugiperda (J. E. Smith).

The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest. Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides. We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry.

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.

Fluorescent analyses of Bacillus thuringiensis Cry1Fa and Cry1Ab toxin binding sites on brush border membrane vesicles of Ostrinia nubilalis (Hübner), Diatraea grandiosella (Dyar), and Helicoverpa zea (Boddie) larvae

Bacillus thuringiensis (Bt) Cry1Fa and Cry1Ab proteins are important Cry toxins due to their high, selective toxicity against a number of lepidopteran species, including important pests of corn and cotton. Competition binding assays are a classical tool for investigating Cry toxin interactions with target pest insects. We developed a fluorescence-based binding assay and assessed Cry1Fa and Cry1Ab toxin binding to brush border membrane preparations from lepidopteran corn pests including Ostrinia nubilalis (European corn borer, ECB), Diatraea grandiosella (south western corn borer, SWCB), and Helicoverpa zea (corn earworm, CEW). Homologous and heterologous competition binding assays with fluorophore-(Alexa488)-labeled Cry1Fa toxin showed that Cry1Fa shares binding site(s) with Cry1Ab toxin in ECB, and SWCB for which Cry1Ab has higher affinity than Cry1Fa. Apart from the shared binding sites, Cry1Ab and Cry1Fa bind an additional site(s) in ECB and SWCB. In CEW, Cry1Fa and Cry1Ab each, has a high affinity binding site(s), which binds the heterologous toxin with low affinity. The Cry1Ab-Cry1Fa toxin binding models for ECB, SWCB and CEW based on our results are considered in the context of what is known about acquired cross-resistance against Cry1Ab and Cry1Fa toxins.

CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin.

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.

ATP-Binding Cassette Subfamily A Member 2 is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, but not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins.

Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239T cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa or Cry9Aa toxins.

Bt-induced hormesis in Bt-resistant insects: Theoretical possibility or factual concern?

The biphasic dose-response of a stressor where low amounts of a toxicant may stimulate some biological processes is a recent focus of attention in insecticide ecotoxicology. Nonetheless, the importance and management consequences of this phenomenon of pesticide-induced hormesis remain largely unrecognized. Curiously, the potential induction of hormesis by insecticidal proteins such as Bacillus thuringiensis toxins (i.e., Bt toxins), a major agriculture pest management tool of widespread use, has been wholly neglected. Thus, we aimed to circumvent this shortcoming while assessing the potential occurrence of hormesis induced by the Bt toxin Cry1Fa in its main target pest species – the fall armyworm Spodoptera frugiperda. Concentration-response bioassays were carried out in a Bt-susceptible and a Bt-resistant population providing the purified Cry1Fa toxin in artificial diet and recording the insect demographic parameters. As significant hormetic effect was detected in both populations with a significant increase in the net reproductive rate and the intrinsic rate of population growth, the potential occurrence of Bt-induced hormesis was subsequently tested providing the insects with leaves from transgenic Bt maize expressing the toxic protein. The performance of the Bt-resistant insects was not different in both maize genotypes, indicating that the leaf expression of the Bt protein did not promote hormesis in the resistant insects. Thus, despite the Bt-induced hormesis detected in the purified protein bioassays, the phenomenon was not detected with current levels of Bt expression in maize minimizing the risk of this additional efficacy constraint besides that of field occurrence of Bt resistance.

Helix \u03b1-3 inter-molecular salt bridges and conformational changes are essential for toxicity of Bacillus thuringiensis 3D-Cry toxin family

Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

Comparative molecular analyses of invasive fall armyworm in Togo reveal strong similarities to populations from the eastern United States and the Greater Antilles.

The fall armyworm (Spodoptera frugiperda, J.E. Smith) is a noctuid moth that is a major and ubiquitous agricultural pest in the Western Hemisphere. Infestations have recently been identified in several locations in Africa, indicating its establishment in the Eastern Hemisphere where it poses an immediate and significant economic threat. Genetic methods were used to characterize noctuid specimens infesting multiple cornfields in the African nation of Togo that were tentatively identified as fall armyworm by morphological criteria. Species identification was confirmed by DNA barcoding and the specimens were found to be primarily of the subgroup that preferentially infests corn and sorghum in the Western Hemisphere. The mitochondrial haplotype configuration was most similar to that found in the Caribbean region and the eastern coast of the United States, identifying these populations as the likely originating source of the Togo infestations. A genetic marker linked with resistance to the Cry1Fa toxin from Bacillus thuringiensis (Bt) expressed in transgenic corn and common in Puerto Rico fall armyworm populations was not found in the Togo collections. These observations demonstrate the usefulness of genetic surveys to characterize fall armyworm populations from Africa.

Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression.

Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.

Linkage of an ABCC transporter to a single QTL that controls Ostrinia nubilalis larval resistance to the Bacillus thuringiensis Cry1Fa toxin

Field evolved resistance of insect populations to Bacillus thuringiensis (Bt) crystalline (Cry) toxins expressed by crop plants has resulted in reduced control of insect feeding damage to field crops, and threatens the sustainability of Bt transgenic technologies. A single quantitative trait locus (QTL) that determines resistance in Ostrinia nubilalis larvae capable of surviving on reproductive stage transgenic corn that express the Bt Cry1Fa toxin was previously mapped to linkage group 12 (LG12) in a backcross pedigree. Fine mapping with high-throughput single nucleotide polymorphism (SNP) anchor markers, a candidate ABC transporter (abcc2) marker, and de novo mutations predicted from a genotyping-by-sequencing (GBS) data redefined a 268.8 cM LG12. The single QTL on LG12 spanned an approximate 46.1 cM region, in which marker 02302.286 and abcc2 were ≤2.81 cM, and the GBS marker 697 was an estimated 1.89 cM distant from the causal genetic factor. This positional mapping data showed that an O. nubilalis genome region encoding an abcc2 transporter is in proximity to a single QTL involved in the inheritance of Cry1F resistance, and will assist in the future identification the mutation(s) involved with this phenotype.

Evidence of field-evolved resistance of Spodoptera frugiperda to Bt corn expressing Cry1F in Brazil that is still sensitive to modified Bt toxins.

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.

Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin

The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.

Spodoptera frugiperda (J.E. Smith) with field-evolved resistance to Bt maize are susceptible to Bt pesticides

Field-evolved resistance to maize event TC1507 expressing the Cry1Fa toxin from Bacillus thuringiensis (Bt) was detected in populations of Spodoptera frugiperda from Puerto Rico. We tested for cross-resistance to purified Cry1A toxins and commercial Bt pesticides in susceptible (Benzon) and TC1507-resistant (456) strains of S. frugiperda. Larvae from the 456 strain exhibited cross-resistance to Cry1Ab and Cry1Ac toxins, while no differences in susceptibility to XenTari WG and DiPel ES pesticides were detected. These data support cross-resistance to toxins that share binding sites with Cry1Fa and no cross-resistance to Bt pesticides in S. frugiperda with field-evolved resistance to Bt maize.

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis

Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC 50 values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and Cry1Ac were marginally toxic to all strains, whereas the rest of the toxins tested (Cry1Ba, Cry2Aa, and Cry2Ab) were non toxic. Significant differences in susceptibility among strains were also found for Cry1Da, being the FRA strain 25-fold more susceptible than the HOL strain. Growth inhibition, as an additional susceptibility parameter, was determined in the FRA strain with the 9 toxins. The toxicity profile obtained differed from that observed in mortality assays. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa toxins produced a similar larval growth inhibition. Cry2Aa had a lower but clear effect on larval growth inhibition, whereas Cry1Ba and Cry2Ab did not have any effect.

Leucine Transport Is Affected by Bacillus thuringiensis Cry1 Toxins in Brush Border Membrane Vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) Midgut

The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K(+)/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K(+) gradient. Experiments with the voltage-sensitive fluorescent dye 3,3'-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM: leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K(+) gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.