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

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 insect's 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.