Brenda Oppert

Protease interactions with bacillus thuringiensis insecticidal toxins

The microbe Bacillus thuringiensis (Bt) produces crystals that contain insecticidal crystal proteins (ICPs) used to control many major pests. ICPs are degraded by proteases from a variety of sources, including those endogenous to the bacterium, those purified from animals and plants, or those found in insects. Proteases in the bacterium function in protein metabolism during sporulation; in some cases they hydrolyze ICPs. Insect proteases are implicated in Bt toxin specificity, mode of action and insect adaptation to Bt. This review describes the current knowledge of protease interactions with ICPs with special emphasis on the role of proteases in insect resistance to Bt toxins. Arch. Copyright 1999 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth.

ABSTRACT Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of 125I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688s) and two resistant (198r and Dplr) to Bacillus thuringiensis. Toxicological studies showed that the 198r colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dplr colony was 4-fold more resistant to protoxin versus toxin. Binding results with 125I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dplr colony showed a drastic reduction in binding affinity (60-fold higher Kd), although they had similar concentrations of binding sites. Insects from the 198r colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higherKd and ca. three-fold-lowerRt compared with the 688s colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198r colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensisresistance genes occurring in natural populations.

Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge

To investigate gene expression in Tribolium castaneum exposed to Beauveria bassiana, reference genes for qPCR were evaluated. Of these, beta-actin, alpha-tubulin, and RPS6 were not stable. The most stable were ribosomal protein genes, RPS3, RPS18, and RPL13a. Syntaxin1, syntaxin6, and E-cadherin may be appropriate for some experimental systems.

A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin.

Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera.

Tribolium castaneum Larval Gut Transcriptome and Proteome: A Resource for the Study of the Coleopteran Gut

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum

Abstract 1. Combinations of a cysteine proteinase inhibitor (CPI) and serine proteinase inhibitors (SPI) in wheat germ diets were toxic to larvae of the red flour beetle, Tribolium castaneum , when tested at levels where individual inhibitors were nontoxic. 2. Mixtures of 0.1% (w/w) CPI (E-64) plus 1% of either of three plant SPIs (soybean Kunitz trypsin inhibitor, soybean Bowman-Birk trypsin-chymotrypsin inhibitor, or lima bean trypsin inhibitor) inhibited T. castaneum growth, resulting in 82–97% reduction in larval weight gain 17 days after hatching and 40–60% mortality. 3. Supplemention of diet containing 0.1% E-64 plus 1% soybean Kunitz trypsin inhibitor (STI) with a mixture of amino acids at 7% caused a partial reversal of the growth inhibition, with 91% of the larvae surviving. 4. Diet containing 0.1% E-64 plus either 5 or 10% STI resulted in 100% mortality of the larvae during the first or second instar. 5. Addition of a mixture ofamino acids at 20% to the 0.1% E-64 plus 10% STI diet allowed 89% of the larvae to develop into adults. 6. The synergism between different classes of proteinase inhibitors in the insects diet that enhances growth inhibition and toxicity demonstrates the potential for an insect pest management strategy involving the coordinated manipulation of two or more types of digestive enzyme inhibitor genes in plants.

Prospecting for cellulolytic activity in insect digestive fluids

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-beta-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.

Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.

Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full‐length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B‐ and L‐like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

Fitness costs of resistance to Bacillus thuringiensis in the Indianmeal moth, Plodia interpunctella

Genetic changes in insects that result in insecticide resistance can also affect their fitness. Here, we report measurements of development time and survival of the Indianmeal moth, Plodia interpunctella (Hübner), to compare the relative fitness of Bacillus thuringiensis (Bt)‐susceptible and ‐resistant colonies. Measurements of larval development time and survival indicated that a fitness cost was associated with resistance to Bt in some Bt‐resistant colonies but not others. Comparisons of geographically different populations revealed inherent differences in development time and survival. In most cases, Bt‐resistant moths suffered no disadvantage when feeding on a Bt‐treated diet. In many cases, the development of Bt‐resistant moths on Bt‐treated diet was slower than the unselected moths on untreated diet, but it is unclear whether these differences would affect the successful mating of susceptible and resistant moths.