Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3281. Adviser: Lawrence M. Hanks. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for egg production. The blood proteins are digested in the midgut to yield amino acids which are the nutritional source for oogenesis. Serine proteases are important enzymes that participate in the process of blood protein digestion. The identification of the corresponding genes may have significant implications in the control of mosquito-borne diseases. A gut-specific chymotrypsin-like cDNA was isolated and sequenced. The 938 bp clone encodes a preproenzyme with a putative 18 amino acid signal peptide sequence, a 7 amino acid activation peptide sequence rich in serine and charged residues, and a mature enzyme of 268 amino acids. The deduced amino acid sequence has a typical catalytic triad region for serine proteases (His 57, Asp 102 and Ser 195 in bovine chymotrypsin numbering system), and the hydrophobic substrate binding pocket with most features of chymotrypsins. Six cysteine residues are present in the sequence which are characteristically involved in disulfide bond formation in invertebrate serine proteases. Characterization of the gene expression and the protein synthesis, as well as the enzymatic activity in the midgut, clearly demonstrated that (1) the chymotrypsin gene is newly transcribed after eclosion and the mRNA is present almost steadily during the digestion of a meal; (2) the chymotrypsin synthesis and its corresponding activity are induced and increased significantly by the ingestion of a meal. In vitro studies of the recombinant protease derived from the cDNA clone indicated several unique properties of the mosquito chymotrypsin compared with its bovine analog.

Three vitelline membrane (envelope) genes in Aedes aegypti mosquitoes were studied. A genomic clone corresponding to a novel vitelline membrane gene (15a-3) was isolated. The predicted peptide sequence of 15a-3 is similar to those of the A. aegypti vitelline membrane genes 15a-1 and 15a-2. The deduced amino acid sequences of 15a-1, 15a-2 and 15a-3 contain a conserved region of 45 residues. This region overlaps with a conserved region in four Drosophila melanogaster vitelline membrane genes. Genomic clones corresponding to 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp region 5' of the 15a-2 coding region was identified with 72% identity to a region upstream of the A. aegypti VgA1 vitellogenin gene Sequences with similarity to a 20-hydroxyecdysone response element consensus sequence were found upstream of the three coding regions. The transcriptional regulation of three A. aegypt vitelline membrane genes was examined. The sites and timing of expression of the three transcripts were determined by Northern blot analysis and whole-mount in situ hybridization using mutually exclusive probes. The temporal pattern of 15a-1, 15a-2 and 15a-3 expression was similar. The spatial pattern of expression differed between the three genes. Only 15a-2 was expressed at the anterior region in addition to the remainder of the follicle. 15a-1 and 15a-3 wee only expressed in the mid and posterior regions of the follicle. Expression of 15a-1 was higher in ovaries that were dissected at intervals of 0, 2, 10 and 24 h after a blood meal and were cultured in medium containing 20-hydroxyecdysone, as compared to control incubations. In ovaries that were dissected at 36 h after a blood meal, incubation in 20-hydroxyecdysone had no effect on 15a-1 expression, as compared to control incubations.

There is great interest in ecology to determine the drivers of species richness. For many taxa, and in natural circumstances, temperature is often found to be a good predictor of richness. The goal of this thesis was to determine, amongst several human-related and natural, environmental and ecological variables, the most important broad-scale predictors of butterfly species richness across Canada. Also, I presented a test of whether spatial relationships are adequate surrogates for the temporal relationship between richness and predictor variables. Using precisely georeferenced and dated butterfly records across Canada, I created butterfly species richness maps for two periods (1900-1930 and 1960-1990), and then related them to candidate predictors. Natural variables such as temperature, precipitation and soil type tended to explain most of the variance in species richness, while human-related variables such as habitat fragmentation, habitat heterogeneity and pesticide density added very little. A comparison of temporal and spatial relationships showed that temperature was a consistent predictor of richness through time and space, but that the impact of human activities on richness differed. My results are consistent with the species-energy hypothesis while showing that human-related variables are not having a large measurable effect on butterfly species richness patterns in Canada at broad scales. Also of critical importance in this thesis is the difference I found between the spatial and temporal relationships of richness vs. human activity level. I show that the assumption commonly made in macro-ecology that spatial variables are appropriate surrogates for temporal ones, is not always correct.

A newly discovered, nonoccluded baculovirus GSV has been reported to be a causative agent of sterility in adult corn ear-worms, Helicoverpa zea. Previous studies conducted by Hamm et al. (1996) and our laboratory indicated that it is an unusual insect virus with strict tissue tropism and the ability to establish persistent infections in vivo. After acquiring purified virus from infected adult insects, two permissive cell culture systems, TN-368 and Ld652Y were established for GSV replication in vitro. The cell culture-derived virus was confirmed to have the same morphology, biological activity and genetic identity as that of GSV recovered from insects. Using a cell culture system, several genetically pure GSV cloned isolates were acquired by plaque purification. The replication cycle of the virus including ultrastructural studies, viral DNA replication and virus specific protein synthesis were investigated in these two cell lines and interestingly, it was found that the exact same virus isolate had a different biology in the different permissive cell lines. Difference in the molecular biology of virus replication in these two cell lines was also observed. This suggests that host factors play an important role in determining the different host-viral interaction of the virus. In addition, biochemical properties of the GSV genome were investigated. The genome size was estimated using pulse-field gel electrophoresis to be 215-235 kb. CsCl-EtBr density gradient centrifugation indicated that GSV has a supercoiled, circular genome. Purified viral structural proteins, envelope proteins and glycoproteins were analyzed by SDS-PAGE and a total 16 of viral structural proteins were identified, three of them are glycosylated and five of these proteins are likely to be virus envelope or matrix components. Studies of GSV specific protein synthesis, DNA replication and transcription in the presence of specific inhibitors suggests that as with other most baculoviruses, GSV gene expression is temporally regulated and can be separated into early and late phases based on viral DNA replication and differential responses to the cellular RNA polymerase inhibitor, alpha-amanitin. That is, early gene expression is likely mediated by cellular RNA polymerase whereas a viral encoded or viral-modified host RNA polymerase likely mediates late viral gene expression. GSV persistent infection in vitro has been investigated using a persistently infected cell line, GSVP. GSV viral sequences and a very low level of infectious virus were detected from this normal-looking, persistently infected cell line. Co-culture of GSVP cells with another permissive cell line, Ld652Y, resulted in productive replication of GSV.

No description available.

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.

acase@tulane.edu

acase@tulane.edu

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0048. Adviser: James D. Bever. "Title from dissertation home page (viewed Feb. 9, 2007)."