The control of synthesis of the cell-wall polymer chitin has been implicated as a crucial event in yeast - hyphal dimorphism of the pathogenic fungus, <i>Candida albicans</i>. This thesis presents evidence that suggests that the activity of the zymogenic enzyme, chitin synthase, is modulated by an endogenous activatory metalloprotease <i>in vitro</i>. The proposed activating protease is characterised and partially purified. A comparison of the intracellular proteolytic activity of yeast and hyphal cells suggest that hyphal cell may be nutrient-stressed compared to yeast cells.

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Components of successful pest management programs must be complementary and not antagonistic. This project examined interactions between natural enemies of the black cutworm, Agrotis ipsilon (Hufnagel), an important turfgrass pest, and host plant resistance by endophytic grass. Agrotis ipsilon nucleopolyhedrovirus (AgipMNPV) was examined as a bio-insecticide for controlling A. ipsilon in turfgrass. Fresh (1-week-old) AgipMNPV residues killed 76−86% of neonates hatching from eggs on golf course tees, however, residual control of implanted larvae lasted no more than a few weeks. Combinations of AgipMNPV with adjuvants, such as optical brightener and lignin, failed to accelerate or extend efficacy of the virus. AgipMNPV seems better suited for targeted control of early instars than for season-long control. Several applications per growing season would likely be needed to maintain high enough titers on turfgrass to effectively control cutworms. The addition of a chitin synthesis inhibiting turfgrass fungicide failed to synergize AgipMNPV infectivity to A. ipsilon. Choice tests revealed the fungicide residues to be a mild feeding deterrent, the likely cause of slightly reduced mortality from virus infection seen in field trials. Combination applications in turfgrass might interfere with larval ingestion of a lethal virus dose, resulting in prolonged feeding in the field. I examined how feeding on perennial ryegrass (Lolium perenne) with or without Neotyphodium lolii, its alkaloid-producing fungal endophyte, affects susceptibility of A. ipsilon to AgipMNPV. Feeding on endophytic grass neither compromises nor synergizes infectivity of AgipMNPV in the cutworm midgut. However, reduced consumption or avoidance of less-palatable endophytic grass could decrease ingestion of virus and rates of subsequent mortality in the field. Host feeding on endophytic grass had differing effects on the tachinid fly, Linnaemya comta, a fast-developing solitary parasitoid, and the encyrtid wasp, Copidosoma bakeri, a slow-developing gregarious parasitoid. L. comta development did not appear to be affected when its host fed on endophytic grass; in contrast, C. bakeri suffered negative fitness effects. These results suggest that parasitoid life strategy and taxonomy play a role in endophyte mediated tritrophic interactions.

Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.