Embryo growth of oviparous animals depends upon utilization of nutritive proteins, primarily the glycoprotein vitellin (Vt). These proteins are usually extraovarian in origin and accumulate in the oocyte through receptor-mediated endocytosis. This event is well characterized for both insects and vertebrates. In contrast, the mechanisms of yolk protein utilization are not understood. In this study, the German cockroach Blattella germanica was used as a model insect system to explore the components that initiate and regulate Vt processing during early embryo development. In B. germanica, Vt processing is initiated at days 3-4 postoviposition at 30$\sp\circ$C. The yolk of freshly oviposited eggs assayed in vitro for protease was devoid of activity but protease specific activity increased dramatically during embryo development. This activity correlated temporally with Vt processing in vivo suggesting that the protease is necessary for Vt processing. The protease was purified from yolk at day 6 postoviposition by gel filtration and affinity chromatography and classified as a cysteine protease. Its molecular weight, estimated by SDS-PAGE and immunoblotting, was approximately 29 kDa. Its pH optimum was 5.5, within the pH range of yolk granules. The purified protease degraded Vt in vitro yielding peptides of the same apparent molecular weights as Vt processed in vivo. Acidification of day 0 yolk in vitro induced protease activity suggesting that a latent protease is present in eggs early in embryo development. The latent protease activity was purified from yolk at day 0 postoviposition by successive use of gel filtration, Mono Q FPLC, and hydrophobic interaction chromatography. The purified latent protease had a molecular weight of approximately 40 kDa and could be activated in vitro into a cysteine protease of 29 kDa. The conversion depended on acidification and was blocked by the cysteine protease inhibitor E-64, suggesting the activation is autocatalytic. Kinetic studies showed that activation occurred by intermolecular catalysis. The pH-activity and inhibitor sensitivity profile of the in vitro-activated protease matched those of the protease suggesting that the latent protease is the proenzyme of the protease. Active site derivatization of the 40 kDa proprotease revealed that its conversion to the 29 kDa protease in vivo occurs as Vt processing begins, suggesting that Vt processing is regulated through the protease. The proenzyme activation and pH optimum data of the purified protease emphasize that yolk granule acidification is an important cellular factor for the regulation of Vt processing by B. germanica in vivo. A murine polyclonal antibody against purified proprotease was made monospecific by affinity column chromatography. Using this antibody, the proprotease was detected in fat bodies and ovaries of vitellogenic females by immunoblotting. This result is consistent with the hypothesis that the protease precursor is synthesized extraovarily, probably in the fat body.

Calmodulin (CaM) is a major intracellular calcium receptor. There is abundant calmodulin (CaM) in the oocytes and eggs of B. germanica during vitellogenesis and early embryogenesis. The accumulation of CaM in oocytes may be for immediate use in the oocytes and/or in preparation for later stages of their development. Previous investigation from this laboratory suggested that maternal follicle cells are the most likely source of this CaM. Tissue culture labeling with $\sp{35}$S methionine showed a 13-fold higher rate of synthesis of CaM in the follicle cells than in oocyte preparations (Zhang & Kunkel, 1994). The high rate of biosynthesis of CaM in the follicle cells, and the absence of extracellular CaM in transit in the hemolymph suggested that CaM is made in the follicle cells and transferred to the oocytes. In order to obtain more information about the site of CaM synthesis I isolated total RNA from different tissues that could potentially contribute to the high amounts of follicular CaM and measured the amounts of CaM transcripts during development. I show that isolated whole follicles accumulate more transcripts for CaM than the fat body. The steady state levels of CaM transcripts increases 150 fold during the 4 day developmental period under study. This is in addition to a 32 fold increase in total follicle RNA during the period. Steady state levels of CaM transcripts in whole follicles also show a pattern of increase disproportionate to the increase in volume of the whole follicle. In comparison steady state levels of actin transcripts increase 35 fold during the same developmental period. At 96 hr post feeding, in a given amount of total RNA, follicle cell total RNA contains 3 times more CaM transcripts than whole follicle total RNA, and 70 times more CaM transcripts than the fat body tissue. The oocyte total RNA collected from material expelled from the whole follicle contains less than 10% of the amount of CaM transcripts available in the follicle cells. The fat body tissue preparation shows little developmental increase in steady state levels of CaM transcripts despite a 4 fold increase in total RNA. In my investigation into the control of the accumulation of this transcript I found that deprivation of JH, by head ligation, not only causes atresia of the follicles, but also reduces CaM transcript accumulation. Reconstituting JH titer by injection allows a selected population of follicles to develop to full size and also reinstates steady state CaM transcript levels above that of unligated controls. The results of my study makes the CaM gene a potentially important target for the study of JH action in follicle cells during oogenesis.

The intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, undergo two periods of developmentally programmed cell death. The first period occurs during the larval/pupal transition where half of the ISMs die and the remainder persist throughout pupal development until death upon eclosion of the adult. Both periods of ISM death employ much of the same molecular machinery even though the endocrine cues for the degeneration process is different for each period of muscle death. A gene that is dramatically increased in its expression during both periods of ISM death is polyubiquitin. This gene has been isolated and characterized in this research. Despite a large amount of allelic heterogeneity in the population, it has been determined that not only is this gene increased in response to developmental cues, but that its transcript is also increased in response to stress. Therefore, polyubiquitin is multifaceted in its regulation. Due to the diverse transcriptional regulation of this gene, it was determined to be a good candidate in which to develop a method to introduce promoter/reporter constructs into the ISMs of the hawkmoth. Even though this research does not conclusively demonstrate that reporter activity after transfection of the promoter/reporter constructs is due to the polyubiquitin promoter, the research does demonstrate that DNA mediated transfection of insect muscle tissue can be achieved.