Doctor of Philosophy / Department of Entomology / C. M. Smith / Brenda Oppert / Coleopteran insects compensate for dietary protease inhibitors by a number of mechanisms. To study this compensation response at the molecular level, the digestive proteases of Tenebrio molitor were studied. Biochemical studies of the pH optima and inhibitor sensitivity of proteases indicated the cysteine proteases were mostly in the anterior and serine proteases were in the posterior midgut of T. molitor larvae. Expressed Sequence Tags (ESTs) from T. molitor larval midgut cDNA libraries contained sequences encoding putative digestive proteases. Of a total of 1,528 cDNA sequences, 92 cDNAs encoded proteases, and 50 full-length cDNAs were grouped into serine, cysteine and metallo protease classes. Sequences tmt1a, tmt1b and tmt1c were identified as genes encoding isoforms of T. molitor trypsin, and tmc1a encoded T. molitor chymotrypsin. The general distribution cysteine protease transcripts in the anterior and serine protease transcripts in the posterior midgut, of T. molitor larvae, was in agreement with the biochemically-characterized compartmentalization of proteases. Expression analyses of selected transcripts demonstrated varied expression patterns across five developmental stages of T. molitor, with maximal expression of most protease transcripts in first instar larvae. Dietary serine and cysteine protease inhibitors fed in combination to early-instar T. molitor larvae caused a significant delay in larval growth in 21-day-old larvae. Real-time quantitative PCR analysis of RNA isolated from larvae fed different protease inhibitor treatments indicated that dietary inhibitors affected the expression of serine and cysteine proteases. Larvae fed soybean trypsin inhibitor, a serine protease inhibitor, compensated by the hyperproduction of proteases from the same class, as well as the upregulation of cysteine proteases. A cysteine protease inhibitor, E-64, caused a reduction in the hyperproduction of all proteases, and, in combination with the soybean trypsin inhibitor, lowered the compensation response of T. molitor larvae to negligible levels. These data suggest that T. molitor larvae are more sensitive to the effects of cysteine protease inhibitors, perhaps because these proteases are the first line of defense for larvae against plant protease inhibitor. The bioassay and molecular studies suggested that combinations of inhibitors that target both serine and cysteine proteases are needed to effectively control larval infestations of T. molitor.

Thesis (M. Sc. Agriculture (Animal Production)) -- University of Limpopo, 2021 / Two experiments were conducted to determine the effect of replacing soya bean meal with yellow mealworm larvae meal (Tenebrio molitor) in a diet on productivity, gut morphology, carcass characteristics and bone morphometrics of Ross 308 broiler chickens aged one to 42 days. In each experiment, a total of 360 Ross 308 broiler chickens were randomly assigned to the five dietary treatments, each treatment having four replications, and 18 chickens per replicate. Five diets were formulated to contain yellow mealworm replacement levels at 0, 25, 50, 75 and 100% to meet the nutrient requirements of Ross 308 broiler chickens. Data was analysed using the General Linear Model procedures of the Statistical Analysis System, Version 9.3.1 software program. Fisher’s least significant difference (LSD) test was applied for mean separation where there were significant differences (P<0.05). A quadratic regression model was used to determine the levels for optimal responses in the variables measured. The first experiment determined the effect of replacing soya bean meal with yellow mealworm larvae meal on productivity and gut morphology of unsexed Ross 308 broiler chickens aged one to 21 days. Replacement of soya bean meal with yellow mealworm meal in a diet had no effect (p > 0.05) on feed intake, growth rate, FCR, live body weight, ME intake and nitrogen retention of unsexed Ross 308 broiler chickens aged one to 21 days. Replacing soya bean meal with yellow mealworm meal in a diet did not affect (p > 0.05) caecum weight of unsexed Ross 308 broiler chickens. However, replacing soya bean meal with yellow mealworm meal in a diet increased (p < 0.05) gastro intestinal tract, crop, ileum and large intestine weights. Crop and ileum lengths of unsexed Ross 308 broiler chickens aged 21 days were not affected (p > 0.05) by replacement of soya bean meal with yellow mealworm meal in the diet. However, replacing soya bean meal with yellow mealworm meal in a diet increased (p < 0.05) gizzard, caecum and large intestine lengths of unsexed Ross 308 broiler chickens aged 21 days. Yellow mealworm meal in a diet did not affect (p > 0.05) gut organ digesta pH values of unsexed Ross 308 broiler chickens aged 21 days. The second experiment determined the effect of replacing soya bean meal with yellow mealworm meal in a diet on productivity, gut morphology, carcass characteristics and bone morphometrics of Ross 308 broiler chickens aged 22 to 42 days. Replacement of soya bean meal with yellow mealworm meal in a diet did not affect (p > 0.05) growth rate, FCR, ME intake and nitrogen retention of male Ross 308 broiler chickens aged 22 to 42 days. However, replacing soya bean meal with yellow mealworm meal in a diet affected (p < 0.05) feed intake and live body weight of male Ross 308 broiler chickens aged 22 to 42 days. Broiler chickens on diets containing 75 or 100% yellow mealworm meal had higher (p < 0.05) intakes than those on diets containing no yellow mealworm meal. Similarly, male broiler chickens on diets having 50% yellow mealworm meal had higher (p < 0.05) live body weights than those on diets containing no yellow mealworm. Quadratic equations indicated that feed intake and live body weight of male Ross 308 broiler chickens were optimized at yellow mealworm meal replacement levels of 13 and 61%, respectively. The present study showed that replacing soya bean meal with yellow mealworm meal in a diet did not affect (p > 0.05) gut organ digesta pH values, gut organ weights, gut organ lengths, meat colour, meat pH values, bone morphometric values, carcass part weights and meat sensory attributes of male Ross 308 broiler chickens aged 42 days. However, meat from chickens on diets containing yellow mealworm meal was softer (p < 0.05) than meat from chickens on diets having 100% soya bean meal. It is concluded that soya bean meal can be replaced with yellow mealworm larvae meal in a diet at 25, 50, 75 and 100% levels without having adverse effects on production and carcass characteristics of Ross 308 broiler chickens aged one to 42 days