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Molecular Analysis of Juvenile Hormone Action in the Red Flour Beetle, Tribolium cataneum
Department of Entomology
There has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E is the most active form) are the major hormones that regulate development and reproduction in insects. Since these hormones are not present in vertebrates, they represent attractive targets for the development of insect control methods. Hindering this effort is the lack of understanding of the molecular basis of JH action. We propose to study mechanism of JH action in regulation of vitellogenin gene expression in the fat body.
Completion of whole genome sequencing, availability of microarrays, functioning of systemic RNA interference (RNAi) and excellent response to JH and its analogs make T. castaneum an excellent model system for studies on JH action. Microarray, RNAi, qRT-PCR, electrophoretic mobility shift assays and ligand-binding assays will be employed to identify and characterize receptors, transcription factors and protein modifiers involved in JH regulation of vitellogenin gene expression JH analogs are being used for pest management, but some pests are already developing resistance to these analogs.
The results from these studies will aid in understanding the mode of action of JH analogs, thus facilitating their judicious use. The genes identified will be useful to develop screening assays to identify new insect disease vector control agents. The receptors identified can also be used in agriculture by developing gene switches for various applications such as regulated expression of transgenes in crop plants.
2009 Project Description
To elucidate the endocrine regulation of vitellogenin (Vg) synthesis in the red flour beetle, Tribolium castaneum, the titers of JH and ecdysteroids in the whole body of female beetles were measured and compared with the levels of Vg mRNA.
The results showed that Vg mRNA expression was detectable by the end of third day post-adult emergence (PAE) and peaked during 4-5 days PAE. Both JH levels and the mRNA levels of JHAMT (JH-acid methyl transferase, an enzyme involved in JH biosynthesis) were high on third day PAE prior to the initiation of Vg gene expression, while the ecdysteroid titers declined steadily during 0-5 days PAE.
The expression profiling of genes coding for proteins involved in JH and 20E biosynthesis and action showed that the genes coding for involved in JH biosynthesis and action but not those coding for proteins involved in 20E biosynthesis and action have similar expression pattern as the Vg genes during 1-5 days PAE.
RNA interference (RNAi)-aided knock-down in the expression of these genes during the previtellogenic phase showed that the expression of genes coding for proteins involved in both JH and 20E biosynthesis and action are required for Vg gene expression.
However, the induction of Vg gene expression in the previtellogenic females by the application of JH but not by the injection of 20E confirmed that JH stimulates Vg synthesis in the fat body. These data suggest a direct role for JH and an indirect role for 20E in the regulation of Vg synthesis.
These studies show that beetle reproduction is regulated by juvenile hormone and juvenile hormone analogs could be developed for managing beetle populations.