Progress 05/01/05 to 04/30/07
Outputs
Nutrient reserves accumulated prior to diapause fuel diapause metabolism. It is clear that fat storage ant utilization is important for diapause metabolism and post-diapause function, what is less clear is the role of protein storage and intermediary metabolism. We have identified and biochemically characterized two critical proteins for amino acid intermediary metabolism the arylphorin/LSP-1 and LSP-2 storage proteins in larval, pupal, and adult development. Diapause and non-diapause larvae and pupae did not differ in arylphorin gene expression or protein accumulation, but arylphorin appears to be utilized during the diapause period so that newly-eclosed, post-diapause adults contain less arylphorin reserves than newly-eclosed adults that did not diapause. We have recently cloned the LSP-2 homologue and are characterizing patterns of gene expression and protein abundance in diapause and non-diapause individuals. We have also cloned the arylphorin receptor protein and
shown that there is no difference in the temporal pattern of receptor expression or of the appearance and cleavage of the functional receptor protein between diapause and non-diapause individuals, suggesting that the same mechanisms underlie storage protein processing in diapause and non-diapause individuals. Protein depletion during diapause may affect post-diapause reproduction. We have shown that unmated females can be used as an assay system for documenting the interaction of effects of nutrient income via feeding and nutritional storage in reserves on reproduction. We have shown that the quantity and temporal availability of protein during adulthood affect reproductive parameters including fecundity, timing of oogenesis, and egg size. We have also shown that there is a threshold quantity of protein that must be consumed in non-diapause individuals to complete oogenesis and we are currently testing the hypothesis that this threshold will be higher in post-diapause individuals that
have utilized more of their larvally-accumulated protein stores prior to adult eclosion. Nutrient stores are correlated with body size and we have shown that there is an advantage to larger individuals in surviving diapause and that this advantage is greater under conditions of greater metabolic demand. We are working towards investigating the effects of size and diapause metabolic demand on post-diapause reproduction. We are also investigating associations between size during diapause and cold-tolerance, which is critical to over winter survival and comparing the results to a non-diapausing insect with a wide body size range, the red imported fire ant. We have cloned several members of the flesh fly insulin-signaling cascade including the insulin receptor, the receptor substrate, and AKT. We are developing quantitative RT-PCR and western-blotting protocols for quantifying pathway activity prior to and during diapause. Our data so far suggest that the insulin signaling is
down-regulated during diapause and up-regulated at diapause break. We have also identified the flesh fly adipokinetic hormone and are working on assaying effects on nutrient mobilization during diapause. This work is ongoing.
Impacts
Because diapause is a critical lifecycle component of many pest insects, understanding the physiological mechanisms regulating the accumulation and metabolism of nutrients in diapausing insects will provide new insights about how to subvert the diapause program as a way of controlling insect pests.
Publications
- Hahn, D.A., and Denlinger, D.L. 2007. Meeting the energetic demands of insect diapause: nutrient storage and utilization. Journal of Insect Physiology. Published Online April 2007 doi:10.1016/j.jinsphys.2007.03.018. To appear in print in fall 2007.
- Hahn, D.A., Martin, A.R., and Porter, S.D. 2007. In Review. Body size, but not cooling rate affects supercooling points in the red imported fire ant Solenopsis invicta. Physiological Entomology.
- Hahn, D.A., Rourke, M.N., and Milne, K.R. 2007. Submitted. Mating and male exposure affect the magnitude but not the timing of yolk protein production and oogenesis in the flesh fly Sarcophaga crassipalpis. Journal of Comparative Physiology B.
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Progress 05/01/05 to 04/30/06
Outputs
It is clear that nutrient reserves accumulated prior to diapause fuel diapause metabolism. What is less clear is the relationship between allocation to reserves for diapause use alone versus reserves that will be used to construct the adult soma after diapause. We have developed morphometrics methodology to quantify the size and shape of the thorax, heads, and wings of flesh fly adults to determine whether diapause history affects body size or shape. In addition, we have adapted methods designed for Drosophila wings to flesh fly wings for quantifying differences in cell size and cell number as they relate to body size and diapause history. We are currently using these techniques to quantify differences in size and shape in relation to diapause history in the flesh flies. We have identified and biochemically characterized the arylphorin/LSP-1 and LSP-2 storage protein homologues in larval, pupal, and adult development in non-diapause individuals and we are currently
characterizing them in diapausing individuals with the goal of understanding how amino acid storage varies prior to and during diapause and what role depletion of storage hexamerins during pupal diapause may have on post-diapause adults. We have developed a full-length cDNA clone for the arylphorin homologue and are quantifying its expression prior to and during diapause and in adulthood in relation to reproduction. We are currently working on cloning the LSP-2 homologue to complement the arylphorin expression work. We have recently completed an experiment using early extirpation from the larval diet to determine the minimum viable size and nutrient storage for diapause entry and to determine whether size has an effect on diapause duration. We have found that the smallest individuals fail to enter diapause. We are currently monitoring diapausing individuals for the timing of diapause termination and are performing biochemical analyses to determine what levels of lipid, glycogen, and
storage hexamerins are correlated with the minimum size threshold for diapause initiation. We are developing quantitative RT-PCR protocols for quantifying the expression of the flesh fly insulin receptor. Our data so far suggest that the insulin receptor is expressed at only very low levels during larval and pharate adult development. We have not been able to detect transcripts in either direct developing or diapause pupae. In addition to the insulin receptor, we are working to develop clones from several other insulin-signaling pathway members downstream of the receptor. Furthermore, we are developing a western blot-based assay of insulin signaling downstream of the receptor using available antibodies from humans and Drosophila to the phosphorylated and unphosphorylated forms of the fork-head transcription factor FOXO, an important downstream insulin pathway component. We are developing assays for mobilization of trehalose and lipids by synthetic Sarcophaga adipokinetic hormone (AKH)
during both diapause and direct development. We are also attempting to clone the AKH receptor from S. crassipalpis, with the goal of understanding its role in hormone sensitivity in diapause and direct development.
Impacts
Because diapause is a critical lifecycle component of many pest insects, understanding the physiological mechanisms regulating the accumulation and metabolism of nutrients in diapausing insects will provide new insights about how to subvert the diapause program as a way of controlling insect pests.
Publications
- No publications reported this period
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