Physiological and Molecular Studies of the Digestive System and the Juvenile Hormone Metabolizing Enzymes in Selected Lepidoptera

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Title: Physiological and Molecular Studies of the Digestive System and the Juvenile Hormone Metabolizing Enzymes in Selected Lepidoptera
Author: Khalil, Sayed
Advisors: R. Michael Roe, Committee Chair
Abstract: Following the success using Bacillus thuringiensis protein toxins for pest control, there is great interest in finding new pesticide targets in the insect gut. To investigate possible molecular targets in the digestive system of the cabbage looper, Trichoplusia ni, we sequenced 49 expressed sequence tags (ESTs) from a cDNA library made from the digestive system of the last stadium of day 1 and day 2 larvae. Among these ESTs, 44 were high quality sequences and were subjected to amino acid and nucleic acid alignments with sequences in the GenBank database. Thirty percent of the ESTs were novel, 7% matched with sequences of unknown functions and 63% matched with sequences of known functions. As might be expected, one of the most abundant classes of ESTs (8/44 ESTs, 18%) codes for digestive enzymes. Another 18% of the ESTs code for elements required for nucleic acid or protein synthesis. The rest of the ESTs code for signal transduction molecules, other cellular structures, and enzymatic activities. A new epoxide hydrolase gene (EH) that is different from TmEH-1 and TmEH-2 previously isolated from T. ni larvae was identified. The current study is part of a larger T. ni EST project (~1000 ESTs) available on GenBank. The role of juvenile hormone esterase (JHE) and EH in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were mixed with males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence. The highest oviposition rates for mated females were noted on d 2-4 post-emergence. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d 1 to d 8 post-emergence. Maximal JHE activity for virgin females occurred on d 2 which was approximately twice that of mated females on the same day. The same results were observed for EH. By d 4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same between sexes through d 7. The developmental changes and effects of mating on JH metabolic activity were similar when measured per mg protein and per insect. The highest levels of JHE and JH EH activity in d 2 virgin and mated females was found in ovaries followed by the carcass and then hemolymph; no EH activity was found in hemolymph. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed. In insects, EHs are believed to play a role in xenobiotic transformation and JH metabolism. The Roe lab at North Carolina State University isolated two EH cDNAs, TmEH-1 and TmEH-2, from the fat body and the digestive system of the fifth stadium of T. ni larvae, respectively. To study the difference between these two EHs and their functional role in T. ni, an attempt to express the full-length cDNAs was conducted using the InsectSelectTM Glow system. In separate experiment, only the ORF of both EHs was used. PCR of genomic DNA from transformed cells, resistance to the antibiotic Zeocin, and GFP expression indicated the incorporation of the expression vector into the cell genome. Measurement of EH activity and SDS-PAGE analysis showed that the EH genes were not expressed in the transformed cells.
Date: 2005-02-01
Degree: PhD
Discipline: Entomology

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