EFFECTS OF PROTEINASE INHIBITORS ON GROWTH AND DIGESTIVE PROTEOLYSIS OF THE REF FLOUR BEETLE, TRIBOLIUM CASTANEUM (HERBST) (COLEOPTERA: TENEBRIONIDAE)

Authors: Oppert, Brenda; Morgan, Thomas; HARTZER, KRIS - KANSAS STATE UNIVERSITY; LENARCIC, B - J STEFAN INST, SLOVENIA; GALESA, K - J STEFAN INST, SLOVENIA; BRZIN, J - J STEFAN INST, SLOVENIA; TURK, VITO - J STEFAN INST, SLOVENIA; YOZA, K - NATL FOOD RES INST,JAPAN; OHTSUBO, K - NATL FOOD RES INST, JAPAN; Kramer, Karl

Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2003
Publication Date: 5/23/2003
Citation: OPPERT,B.S., MORGAN,T.D., HARTZER,K., LENARCIC,B., GALESA,K., BRZIN,J., TURK,V., YOZA,K., OHTSUBO,K., KRAMER,K.J., EFFECTS OF PROTEINASE INHIBITORS ON GROWTH AND DIGESTIVE PROTEOLYSIS OF THE REF FLOUR BEETLE, TRIBOLIUM CASTANEUM (HERBST) (COLEOPTERA: TENEBRIONIDAE). COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART C 134 (2003) 481-490.

Interpretive Summary: The red flour beetle is a major pest of stored grains and grain products worldwide. Our previous research suggested that inhibitors of digestive enzymes, in the right combination, could be effective in controlling this beetle pest. We tested a number of protein inhibitors alone and in combination to identify those that reduced the growth of red flour beetle larvae. Although several demonstrated potency, inhibitors from potato and soybean had the greatest potential for red flour beetle control. Combining genes encoding the potato inhibitor and a soybean inhibitor in transgenic cereals may help to prevent damage to grains by storage pests.

Technical Abstract: Several proteinase inhibitors were tested in vivo with larvae of the red flour beetle, Tribolium castaneum, and in vitro with larval gut extracts to identify inhibitors with potential for use as biopesticides in transgenic plants. In vivo, the most effective inhibitor for suppressing larval growth was L-trans-epoxysuccinylleucylamide [4-guanidino] butane (E-64), followed by cysteine proteinase inhibitors from potato (PCPI), Job's tears (JCPI), and a sea anemone (equistatin). The combination of 0.1% E-64 and 1% soybean trypsin inhibitor (Kunitz) (STI)resulted in the most significant growth suppression (87% weight reduction). Maximum hydrolysis of casein by extracts of larval gut proteinases occurred at pH 4.2. T. castaneum proteinases hydrolyzed the trypsin substrate N-a-benzoyl-DL-arginine-p- nitroanilide at both acidic and alkaline pH values. With the chymotrypsin substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, however, hydrolysis by yT. castaneum proteinases was greatest at alkaline pH. In vitro, additional inhibitors were tested for their ability to inhibit casein hydrolysis by larval gut proteinases. The most effective inhibitors were leupeptin and E- 64, followed by equistatin, tosyl-L-lysine chloromethyl ketone, JCPI, PCPI, and antipain. Chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and STI only weakly inhibited T. castaneum digestive proteinases. These data indicate that cysteine proteinases have a predominate role in digestion, with serine proteinases playing a lesser role in protein digestion.