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ARS Home Plains Area Manhattan, Kansas Center for Grain and Animal Health Research Stored Product Insect and Engineering Research Docs







**Brenda Oppert - Research Summaries**     [back]  **A digestive prolyl carboxypeptidase in *Tenebrio molitor* larvae**
Prolyl carboxypeptidase (PRCP) is a serine peptidase that cleaves specifically at proline amino acids in proteins, which are abundant in cereals. The study of PRCP is important because it has been described in the regulation of physiological processes in different organisms, but, it has not been studied extensively in insects. We discovered that PRCP is an important enzyme in the digestion of proteins in cereals for the yellow mealworm, *Tenebrio molitor*, and, we purified and characterized the enzyme, providing the first detailed analysis of PRCP in insects. Our data indicates that the complex of digestive enzymes found in this insect may be adaptations of mammalian lysozyme enzymes during evolution, in this case to allow the insect to digest cereal proteins enriched in proline amino acids. Therefore, the enzyme may be a target for the development of new control methods to prevent damage to stored grains and products.

**Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of *Ostrinia nubilalis*larvae**
Insect peptidases affect the efficacy of insecticidal microbial toxins, such as those from the bacterium *Bacillus thuringiensis* (Bt). Thus, understanding the expression of peptidase genes in a target pest is important to more effectively design and deploy Bt toxins. Larvae of the European corn borer, *Ostrinia nubilalis*, a major target of Bt transgenic corn, express a suite of peptidases at various locations in the gut. Our data suggest that peptidase genes can be grouped by genetic relatedness and expression patterns in the gut. We found that some genes were increased in expression when larvae were exposed to toxin. These data represent the most comprehensive study to date on the effect of Bt toxin on the expression of larval gut peptidase genes in the *O. nubilalis*. The data can be used to improve transgenic constructs for efficacy and durability in the field.

**Expression of an endoglucanase from *Tribolium castaneum* (TcEG1) in *Saccharomyces cerevisiae***
Some insects have enzymes that can efficiently process plant material, and thus these enzymes may have applications in the biofuels industry. One such enzyme comes from the red flour beetle, *Tribolium castaneum*, which we previously identified as a potential plant-processing enzyme in extreme conditions, such as very alkaline environments. We put the gene for this enzyme in a yeast system to further study its properties. The yeast-expressed enzyme was similar to that of the beetle enzyme, demonstrating that expression of the gene in different systems does not disrupt the activity of the protein. We speculate that this enzyme may be useful in cases where harsh alkaline conditions are necessary for processing of plant materials for applications such as biofuels.

***Bacillus thuringiensis* Cry3Aa protoxin intoxication of *Tenebrio molitor*induces widespread changes in the expression of serine peptidase transcripts**
Some insecticides are based on toxins produced by bacteria or other microbes, but these insecticides often don't work well for beetle pests. In order to make these microbial toxins more effective for controlling beetles, we need to understand how they kill the insect. Most of the microbial toxins used for insect control are produced by the bacterium *Bacillus thuringiensis*, and we used one of these toxins in our study. We know that there are enzymes in the beetle gut that are involved in activating the toxin and making it soluble so that it can bind to the lining of the insect gut. Binding of the toxin starts a chain of events that we still don't fully understand, but leads to killing the insect. To better understand this process, we determined how many enzymes are in the gut of mealworm larvae, and what changes are induced in these enzymes when mealworm larvae are fed toxin. Our results suggest that that mealworm larvae exposed to toxin are attempting to decrease toxicity of the toxin while maintaining efficient digestion. Knowledge of how the gut enzymes in mealworm larvae are affected by microbial toxins may help us to improve these important biopesticides for beetle pest control.     Poster

**Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests**
To be successful, stored product pests need to have enzymes capable of efficiently digesting their main dietary proteins, which have many glutamine and proline amino acids. We describe for the first time one enzyme, a glutamine-specific peptidase, in the gut of *Tenebrio molitor* larvae that are capable of digesting proteins by hydrolyzing at glutamine residues. Our characterization of these peptidases indicates that they are previously studied enzymes belonging to the class of cysteine peptidases, hypothesized to have evolved in storage pests to protect the insect from serine protease inhibitors found in cereals. We now propose that storage pests also have retained cysteine peptidases to efficiently digest cereal proteins. These results may be exploited to develop new control products for storage pests.

**Transcriptome profiling of the intoxication response of *Tenebrio molitor* larvae to *Bacillus thuringiensis* Cry3Aa protoxin**
Responses of coleopteran storage pests to microbial toxins has not been well-studied. We used high throughput sequencing to determine the effect of *Bacillus thuringiensis* Cry3Aa protoxin on transcript expression in the gut of *Tenebrio molitor* larvae. Transcripts associated with mitochondrial respiration, membrane restructuring, immunity, and signaling were increased, while transcripts associated with the metabolism of food and storage proteins were repressed in Cry3Aa-intoxicated larvae. Knowledge of the intricate responses to toxin in intoxicated larvae can provide information critical to the improvement of microbial toxins for coleopteran pest control.      Poster

**Microarray analysis reveals strategies of *Tribolium castaneum* larvae to compensate for cysteine and serine protease inhibitors**
Previously, our studies determined that flour beetle larvae respond to dietary inhibitors by shifting from one class of proteases to another. This response is problematic if cereal inhibitors are to be incorporated into IPM strategies for storage pest control. To study this at the gene level, whole-genome microarrays were used to evaluate the inhibitor response. The data demonstrated that the response by beetle larvae to dietary inhibitors involves a complicated adjustment of gene expression. However, the study also provided clues as to how to better utilize inhibitors for beetle pest control. Therefore, effective control of beetles may soon be possible with a strategy that can anticipate beetle responses to inhibitors.     Poster


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