Research Accomplishment Reports 2007

Molecular Analysis of Pest Development and Resistance to Insecticides

S. R. Palli
Department of Entomology

 

Project Description

Cloning of juvenile hormone esterase from Aedes aegypti:Juvenile hormone esterase (JHE) plays an important role in regulating juvenile hormone titers during insect life cycle. Recent sequencing and annotation of Aedes aegypti genome identified ten putative jhe gene sequences. Further analysis on these ten putative JHE gene sequences showed that only three of them, EAT43357, EAT43353 and EAT43354, had high sequence similarity with the sequences of JHE genes identified from other insect species. To determine which putative JHE gene(s) code for functional JHE, the mRNA profiles of EAT43357, EAT43353 and EAT43354 were measured during final instar larval and pupal stages by using quantitative real-time reverse transcriptase PCR. The mRNA for EAT43357 was detected during the late final instar larval stage. In contrast, the EAT43354 mRNA was detected only during the pupal stage and the EAT43353 mRNA was detected only during the larval stage. The mRNA of EAT43357 was found in both fat body and midgut tissues. The JHE enzyme levels gradually increased during the final instar larval stage and reached a peak by 42 hr after ecdysis into the final instar larval stage. The mRNA expression profiles of EAT43357 gene correlate with the developmental expression profiles of JHE enzyme activity suggesting that this gene may code for functional JHE. The EAT43357 cDNA was expressed in a baculovirus system and the recombinant protein exhibited the JHE activity confirming that EAT43357 codes for a functional JHE enzyme. Identification of broad from Tribolium castaneum:Broad (br), a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) and zinc finger domains was shown to mediate 20-hydroxyecdysone (20E) action and pupal development in Drosophila melanogaster and Manduca sexta. We determined the key roles of br during larval-pupal metamorphosis using RNA interference (RNAi) in a coleopteran insect, Tribolium castaneum. Two major peaks of T. castaneum broad (Tcbr) mRNA, one peak at the end of feeding stage prior to the larvae entering the quiescent stage and another peak during the quiescent stage were detected in the whole body and midgut tissue dissected from staged insects. Expression of br during the final instar larval stage is essential for successful larval-pupal metamorphosis, because, RNAi-mediated knock-down of Tcbr during this stage derailed larval-pupal metamorphosis and produced insects that showed larval, pupal and adult structures. Tcbr dsRNA injected into the final instar larvae caused reduction in the mRNA levels of genes known to be involved in 20E action (EcRA, E74 and E75B). Tcbr dsRNA injected into the final instar larvae also caused an increase in the mRNA levels of JH-response genes (JHE and Kr-h1b). Knock-down of Tcbr expression also affected 20E-mediated remodeling of midgut during larval-pupal metamorphosis. These data suggest that the expression of Tcbr during the final instar larval stage promotes pupal program while suppressing the larval and adult programs ensuring a transitory pupal stage in holometabolous insects.

Impact

The long-term goals of this research are to understand the molecular basis of hormone action and use this information for development of environmentally friendly pest management methods. The information and materials generated during these studies can be used to (a) understand the mode of action of existing hormone analogs, (b) develop target-site based screening assays to discover new, more potent, species-specific, environmentally benign hormone analogs, (c) interfere with insect development by over or under expressing critical genes involved in JH action through recombinant microorganisms, transgenic insects or transgenic plants and (d) develop gene switches for controlling the expression of transgenes in plants, animals and human beings.

Publications

Li Y., Robinson G.E. and Palli S.R. (2007) Identification and characterization of DmJHRE. J. Biol. Chem. 282, 37605-37617.

Panguluri S.K., Li B., Hormann R.E. and Palli S.R. (2007) Effect of ecdysone receptor gene switch ligands on endogenous gene expression in 293 cells.The Febs J. 274, 5669-5689.

Dhadialla. T.S., Dat L.., Palli S.R., Raikhel, A. and Carlson G.R. (2007) Characterization of a non-steroidal ecdysone agonist photoaffinity compound, RH-131039. Insect Biochem. Mol. Biol. 37,865-875.

Bai H., Parthasarathy R. and Palli S. R. (2007) Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 37,829-837.

Tavva V.S., Palli S.R., Dinkins R.D and Collins G.B. (2007) Applications of EcR gene switch technology in functional genomics. Archiv. Insect Physiol. Biochem. 65, 164-169.

Parthasarathy, R. and Palli, S.R. (2007) Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens. Mechanisms of Development 124, 23-34.