Cry1Aa Toxin Research Articles

Combining Hexanoic Acid Plant Priming with Bacillus thuringiensis Insecticidal Activity against Colorado Potato Beetle

Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction.

Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. Cells can trigger different survival mechanisms to counteract the effects of sub-lytic doses of pore forming toxins. Particularly, two signaling pathways have been demonstrated to play a role in the defense mechanism to other toxins in Caenorhabditis elegans and in mammalian cells. These are the unfolded protein response (UPR) and the sterol regulatory element binding proteins (SREBP) pathways, which are proposed to facilitate membrane repair responses. In this work we analyzed the role of these pathways in Aedes aegypti response to intoxication with Cry11Aa toxin. We show that UPR is activated upon toxin ingestion. The role of these two pathways was analyzed in vivo by using RNA interference. We silenced the expression of specific proteins in A. aegypti larvae. Gene silencing of Ire-1 and Xbp-1 proteins from UPR system, resulted in hypersensitive to Cry11Aa toxin action. In contrast, silencing of Cas-1, Scap and S2P from SREBP pathway had no affect on Cry11Aa toxicity in A. aegypti larvae. However, the role of SREBP pathway requires further studies to be conclusive. Our data indicate that the UPR pathway is involved in the insect defense against Cry toxins.

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.

Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution

Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of (439)QAAG(442) or (443)AVYT(446) of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.

Susceptibility of Spodoptera littoralis caterpillars to entomopathogenic nematode Steinernema feltiae after consumption of non-specific transgenic plants

Spodoptera littoralis caterpillars were transferred from an artificial diet to potato leaves at the start of the third or fifth instar and exposed to the infective juveniles of the nematode Steinernema feltiae since the beginning of the sixth instar until the start of pupation. Leaves were taken from the control potatoes or from genetically modified potatoes expressing either Cry3Aa toxin of Bacillus thuringiensis (Bt) or Galanthus nivalis agglutinin (GNA) which are mainly non-specific to S. littoralis larvae. The nematodes killed all the caterpillars within seven days compared with the starved larvae in the same period of exposure. The average time to death and the number of nematodes successfully invaded the larvae were affected by the period of feeding on potato leaves. In the non-starved caterpillars, which received potato leaves throughout the whole period of exposure to the nematodes, the type of potato leaves had no effect on the number of nematodes inside cadavers (p = 0.352 and F = 1.070) and also on the effect on the length of survival after exposure to the nematodes (p = 0.7892 and F = 0.596). No hazardous effect on the development and survival of entomopathogenic nematode S. feltiae which successfully invaded larvae fed on modified potato (Bt or GNA) was reported.

Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle

Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa–ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.

IDENTIFICATION OF A CALMODULIN‐BINDING SITE WITHIN THE DOMAIN I OF BACILLUS THURINGIENSISCry3Aa TOXIN

Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown that Cry3Aa toxin is proteolytically cleaved by CPB midgut membrane associated metalloproteases and that this cleavage is inhibited by ADAM metalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAM shedding substrates. In pull-down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaM in a calcium-independent manner. Furthermore, we used gel shift assays and (1)H NMR spectra to demonstrate that CaM binds to a 16-amino acid synthetic peptide corresponding to residues N256-V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPB midgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM-specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.

Comparative Proteomic Analysis of Aedes aegypti Larval Midgut after Intoxication with Cry11Aa Toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.

Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts

The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Single Molecule Fluorescence Study of the B. Thuringiensis Toxin Cry1Aa Reveals Tetramerization

Pore forming toxins compose a class of potent virulence factors that attack their host membrane in a 2- or 3-step mechanism. After binding to the membrane often aided by specific receptors, they form pores in the membrane either leading to cytolysis or providing a pathway to introduce enzymes effecting intracellular proteins. The major rearrangements of the toxins upon binding to the membrane are accompanied by oligomerization to the final pore forming construct. However, it remains often unknown how many monomers constitute the pore, if that number is fixed, and whether the toxins enter the membrane in monomeric or oligomeric form. Here, we used single subunit counting, to determine the number of oligomers contained in a Cry1Aa toxin as a function of the concentration. Cry1Aa is one of the δ-endotoxins of B. thuringiensis, a soil bacteria that is, because of its specificity for certain insect larvae, used as a biological alternative to chemical pesticides. Cry toxins were purified and fluorescently labeled in their monomeric form. Lipid vesicles of various compositions were incubated with labeled toxin monomers, and supported bilayers formed from the mixture. We recorded the fluorescence intensity over time of distinct fluorescence intensities in the supported bilayer and determined their photobleaching behaviour. As single fluorophores bleach in a single step, the number of steps corresponded to the minimal number of monomers in each oligomer. The oligomerization state showed a concentration dependence which was consistent with both a concentration dependent association rate between monomers and a concentration dependent distribution on the lipid vesicles during incubation. Statistical analysis of the results showed that the toxins enter the membrane in their monomeric form and diffuse laterally to form tetramers, which appears to be the pore forming unit.

Two disulfide mutants in domain I of &amp;lt;i&amp;gt;Bacillus thuringiensis&amp;lt;/i&amp;gt; Cry3Aa δ-endotoxin increase stability with no effect on toxicity

To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.

Bacillus thuringiensis Cry3Aa toxin increases the susceptibility of Crioceris quatuordecimpunctata to Beauveria bassiana infection

The spotted asparagus beetle, Crioceris quatuordecimpunctata (Coleoptera: Chrysomelidae), is one of the most devastating pests of asparagus in China. Sprayed synthetic pesticides have been used to control C. quatuordecimpunctata damage, but they pose problems because of residues and harm to natural enemies. Neither the microbial coleopteran-specific toxin from Bacillus thuringiensis tenebrionis, Cry3Aa, nor the fungal pathogen Beauveria bassiana have sufficient activity to effectively control C. quatuordecimpunctata damage to asparagus. However, second instar C. quatuordecimpunctata larvae exposed to a sublethal dose of Cry3Aa toxin demonstrated significantly higher larval mortality when exposed to B. bassiana. Our results suggest that a combination of Cry3Aa and B. bassiana may be effective in reducing damage by C. quatuordecimpunctata larvae to asparagus.

Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.

Comparative susceptibility of Ostrinia furnacalis, Ostrinia nubilalis and Diatraea saccharalis (Lepidoptera: Crambidae) to Bacillus thuringiensis Cry1 toxins

Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) are highly effective against the European corn borer, Ostrinia nubilalis (Hübner), and the closely related Asian corn borer, Ostrinia furnacalis (Guenée). Since the registration of Bt corn hybrids in the U.S. in 1996, there has been a great deal of information generated on O. nubilalis. However, relatively little information exists for O. furnacalis. To help determine whether the information generated for O. nubilalis can be leveraged for decisions regarding the use of transgenic Bt corn against O. furnacalis, experiments were designed to determine whether the pattern of sensitivity to various Bt Cry1 toxins is similar between the two species. Test insects included laboratory-reared O. furnacalis originating from Malaysia, a Bt-susceptible laboratory colony of O. nubilalis maintained at the University of Nebraska-Lincoln (UNL) and an out-group consisting of the sugarcane borer, Diatraea saccharalis (F.), from Louisiana which represents a different genus from the same family. O. furnacalis and O. nubilalis exhibited a similar pattern of susceptibility to all the Cry1 toxins and were highly susceptible to the range of Bt toxins tested including Cry1Aa, Cry1Ab, Cry1Ac and Cry1F. Both of the Ostrinia species were more tolerant to Cry1Ba compared with D. saccharalis, although sensitivity of O. furnacalis was intermediate and did not differ significantly from that of O. nubilalis and D. saccharalis. D. saccharalis was also susceptible to the range of toxins tested but unlike the two Ostrinia species, was more tolerant to Cry1F and more susceptible to Cry1Ba. These results indicate that both of the Ostrinia corn borer species are similar in sensitivity to the Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1F toxins, thus suggesting shared toxin receptors and mechanisms of toxicity for the two species.

Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment

Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides for efficient control of important coleopteran pests. Based on the reported increase in Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Tenebrio molitor (Coleoptera: Tenebrionidae), it was hypothesized that this cadherin peptide, rTmCad1p, would enhance Cry3Aa toxicity towards coleopteran larvae. To test this hypothesis, the relative toxicity of Cry3Aa, with or without rTmCad1p, against damaging chrysomelid vegetable pests of China was evaluated. Cry3Aa toxicity was evaluated in the spotted asparagus beetle (Crioceris quatuordecimpunctata), cabbage leaf beetle (Colaphellus bowringi) and daikon leaf beetle (Phaedon brassicae). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate larvae were fed Cry3Aa toxin alone or in combination with increasing amounts of rTmCad1p. The data demonstrated that Cry3Aa toxicity was significantly increased in all three vegetable pests, resulting in as much as a 15.3-fold increase in larval mortality. The application of rTmCad1p to enhance Cry3Aa insecticidal activity has potential for use in increasing range and activity levels against coleopteran pests displaying low susceptibility to Bt-based biopesticides.