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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND PROTEOMICS OF STORED-PRODUCT INSECTS FOR DEVELOPMENT OF NEW BIOPESTICIDES

Location: Stored Product Insect Research Unit

Title: Protein digestion in red aak borer larvae, Enaphalodes rufulus

Authors
item Crook, Damon - USDA APHIS PPQ
item Prabhakar, Sheila - KANSAS STATE UNIV
item Oppert, Brenda

Submitted to: Physiological Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 18, 2008
Publication Date: May 30, 2009
Citation: Crook, D.J., Prabhakar, S., Oppert, B.S. 2009. Protein digestion in red aak borer larvae, Enaphalodes rufulus. Physiological Entomology. 34(2): 152-157. Doi: http://dx.doi.org/10.1111/j.1365-3032.2008.00667.x.

Interpretive Summary: Red oak borer larvae have caused damage and threatened the survival of oak trees in the Ozark Mountains. To try to identify new control methods, we studied the digestive proteinases in larvae from trees that were transferred to artificial diet, or those that were reared in tree sections on phloem. Biochemical tests indicated that red oak borer larvae use serine proteinases to digest protein. Inhibitors of one type of serine proteinase, trypsin, were very effective in reducing total proteolytic activity from gut samples. Therefore, we suggest that use of trypsin inhibitors may be an effective way to reduce red oak borer damage to trees, either by increasing the inherent expression of trypsin inhibitors in trees, or by transgenic technologies that use genes encoding trypsin inhibitors.

Technical Abstract: In the Ozark Mountains of Arkansas, Oklahoma, and Missouri, a recent outbreak of red oak borer, Enaphalodes rufulus (Haldeman), contributed to the death of tens of thousands of red oaks. To better understand nutrient digestion in E. rufulus larvae, biochemical analyses were used to characterize digestive proteinases in larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts was approximately 6.3 in mid and foregut and decreased to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increased in buffers of pH>6.19, with a maximum hydrolysis observed in pH 10.1 buffer. Casein zymogram analysis revealed two major proteinase activities in larval midgut extracts of diet-fed larvae, with molecular masses of approximately 25 and 40-60 kDa, whereas phloem-fed larvae had proteinase activities corresponding to 40, 45, 60, 80, and >100 kDa. Substrate analysis indicated at least one major trypsin-like activity in both gut extracts with a molecular mass of >100 kDa, and two chymotrypsin-like activities of ~25 and >200 kDa were found only in diet-fed larvae. Inhibitors of serine proteinases were most effective in reducing the general proteolytic activity of midgut extracts from both larvae. Increased inhibition by trypsin inhibitors was observed when diet-fed midgut extracts were incubated in reducing buffer. The data suggest that serine proteinase inhibitors have the potential to reduce feeding damage to oaks by E. rufulus larvae. However, substrate and inhibitor analysis suggested that inhibitors of trypsin would be particularly effective in reducing protein digestion in phloem-feeding larvae, the most likely target of transgenic trees with proteinase inhibitor genes.

Last Modified: 8/27/2014
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