Effects of proteinase inhibitors on western corn rootworm life parameters R. Bazˇok 1 , J. Igrc Barcˇic´ 1 and C. R. Edwards 2 1 Department for Agricultural Zoology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia; 2 Department of Entomology, Purdue University, West Lafayette, IN, USA Ms. received: October 20, 2004; accepted: March 7, 2005 Abstract: Plants have developed defensive mechanisms to minimize predation by insect pests. Proteinase inhibitors are an example of plant compounds synthesized as a mechanism for defence. The objective of this study was to determine the impact of trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane (E-64), phenylmethylsulfonyl fluoride (PMSF- serine protenase inhibitor) and Kunitz trypsin inhibitors on the pre-ovipositional and ovipositional periods, the mean number of eggs laid per female, and the longevity of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, adults. This study provides information on the effectiveness of proteinase inhibitors as a host-plant resistance tool for managing WCR beetles. The study was conducted in 1997, 1998, and 2000. In 1997, E-64 was added to an artificial diet at the concentrations of 0.05, 0.025 and 0.0125% (w/w), corresponding to 500, 250, and 125 ppm respectively. In 1998, PMSF was added to the artificial diet at the same concentrations. In 2000, Kunitz trypsin inhibitor was added to the artificial diet at concentrations of 0.2, 0.1 and 0.05% (w/w), corresponding to 2000, 1000, and 500 ppm respectively. The mean fecundity of beetles fed the untreated diet was between 67 and 111 eggs per female. The fecundity of beetles fed E-64 and PMSF at different concentrations, ranged between 162 and 246 eggs per female for E-64 and 61 and 80.5 eggs per female for PMSF. The fecundity of the beetles fed Kunitz trypsin inhibitor was between 155 and 225 eggs per female. When beetles fed on the diet which consisted of the lowest dosage of Kunitz trypsin inhibitor (500 ppm), fecundity was higher than that on untreated control. The proteinase inhibitors investigated did not show a negative impact on WCR adults. Beetle fecundity, the length of the pre-ovipositional and ovipositional periods and the longevity of the beetles fed with proteinase inhibitors were not lower than that of the beetles fed only the artificial diet. This study does not support the use of investigated proteinase inhibitors at applied concentrations as effective host-plant resistance tools for managing WCR beetles. Key words: E-64, Kunitz trypsin inhibitor, life parameters, PMSF, serine proteinase inhibitors, western corn rootworm 1 Introduction Insects obtain essential amino acids through the breakdown of dietary proteins by extracellular proteinases. Of the four mechanistic classes of pro- teinases (Barrett, 1986), insects possess one of three (Applebaum, 1985): aspartic, cysteine and serine. Plants have developed defensive mechanisms to minimize predation by insect pests. One such mechan- ism is the ability to be an unacceptable food source. Proteinase inhibitors are plant compounds synthesized for defensive purposes. Proteinase inhibitors are pro- teins or polypeptides that bind specifically to enzymes that split peptide bonds of proteins, resulting in the inhibition of the proteolytic activities of these enzymes (Ryan, 1979). Recent studies have shown that some insects, such as the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, could compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behaviour and over-production of insensitive proteases (Cloutier et al., 2000; Gruden et al., 2004). Pompermayer et al. (2003) reported that despite their inhibitory effect on midgut enzymes, the effectiveness of proteinase inhibitors is directly depend- ent on the quality of diet. Proteinase inhibitors are found throughout all life forms. The most well-known inhibitors are among the plant families of Leguminosae (Gatehouse et al., 1979; Weder, 1981; Hilder et al., 1987; Hines et al., 1991; Boulter et al., 1990; Pratt et al., 1990), Gramineae (Steffens et al., 1978; Liang et al., 1991; Masoud et al., 1993), and Solanaceae (Wolfson and Murdock, 1990). In addition to the plant proteinase inhibitors, there is a class of low-molecular-weight inhibitors isolated from culture filtrates of actinomycetes. Belonging to this class is an inhibitor called trans-epoxysuccinyl-l-leu- cylamido (4-guanidino) butane (E-64). E-64 is a tripeptide from the TRP-64 strain of Aspergillus japonicus Saito, a specific inhibitor of cysteine protein- ases (Hanada et al., 1978). Proteolytic enzyme inhibitors of plants may be a mechanism of plant protection against insects (Steffens et al., 1978; Gatehouse et al., 1979; Hilder et al., 1987; JEN 129(4) doi: 10.1111/j.1439-0418.2005.00951.185–190 Ó 2005 Blackwell Verlag, Berlin