Progress 07/01/00 to 10/01/03
Outputs
Trypsin Modulating Oostatic Factor, a mosquito decapeptide that controls trypsin biosynthesis in the mosquito gut, was cloned and expressed in Chlorella sp., Chlorella desiccata, S. cereviciae and P. pastoris. The peptide hormone was cloned using various hormonal analogoues and as a fusion protein to green fluorescent protein. Cells expressing the hormone killed mosquito larvae after larvae ate the recombinant cells. The hormone was recently cloned into Alfalfa and stopped Heliothis virescens and diaprepes larval growth and development. Thus, the hormone has a potential in controling mosquito larvae and as a future control agent of agricultural pest insects that use trypsin as their main proteolytic enzyme.
Impacts
The hormone has a potential as a new mosquito larvicide and can complement the Bti toxin that is currently used to control mosquito larvae. The hormone can be formulated and used in the marsh to control mosquito larvae. Cloning and expressing the hormone in plants will allow future biological control of agricultural pest insects that use trypsin as their main digestive enzyme.
Publications
- Borovsky, D. 2003. Trypsin Modulating Oostatic Factor: A Potential New Larvicide for Mosquito Control. J. Experimental Biol. 206: 3869-3875.
- Borovsky, D. 2003. Biosynthesis and Control of Mosquito Gut Proteases. IUBMB Life 55(8): 435-441.
- Borovsky, D. and Meola, S. M. (2003). Biochemical and Cytoimmunological Evidence for the Control of Aedes aegypti Larval Trypsin with Aea-TMOF. Arch. Insect Biochem. Physiol. (In the Press).
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Progress 10/01/01 to 10/01/02
Outputs
TMOF and several of its analogues were cloned and expressed in Saccharomyces cerevisiae and Pichia pastoris. In S. cerevisiae we have cloned the hormone by homologous recombination resulting in the expression of one gene, or by insertion of free plasmids, up to 300 copies, carrying TMOF. In P. pastoris Southern blot hybridization showed that 10 copies of the hormone were cloned by homologous recombination using the strong promoter alcohol oxidase. TMOF_GFP and TMOF_Cry4Aa genes were also expressed in Pichia pastories. Using pKylx plasmid and Nitrate Reductase gene TMOF was cloned into Chlorella desiccata, a salt-water chlorella, using homologous recombination. Southern blot hybridization showed that the TMOF was inserted into the Chlorella chromosome. Feeding mosquito larvae with recombinant yeast cells carrying TMOF, TMOF_Bti or its homologous stopped trypsin biosynthesis in the larval gut causing starvation and death. Feeding Chlorella desiccata that were
transformed with a TMOF gene also caused cessation of trypsin biosynthesis in the larval gut, starvation and rapid death. Thus, yeast cells and chlorella cells expressing TMOF genes can be used to control mosquito larvae. This is a first step towards larval control in the field.
Impacts
This method will allow effective control of mosquito larvae in the marsh using a biorational insecticide that will not affect the environment.
Publications
- Borovsky, D., Nauen, R., Sorge, D. and Sterner, A. 2002. Characterization of trypsin modulating oostatic factor (TMOF) from the larval hemolymph of Heliothis virescens. Arthropods 2001 ( Eds. D.Konopinska, G.Coast, G.Goldsworthy, R.J.Nachamn and G.Rosinski) Wydawnictawa Uniwersytetu Wroclawskiego ( in press).
- Nauwelaers, S. and Borovsky, D. 2002. Cloning and expression of trypsin modulating oostatic factor (Aea-TMOF) and green fluorescent protein (GFP) in Saccharomyces cerevisiae. Arthropods 2001 ( Eds. D.Konopinska, G.Coast, G.Goldsworthy, R.J.Nachamn and G.Rosinski) Wydawnictawa Uniwersytetu Wroclawskiego (in press).
- Pesticidal Peptides. Inventors: D. Borovsky and R. Linderman. U.S. Patent Number 6,413,530. June 2, 2002.
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Progress 10/01/00 to 10/01/01
Outputs
TMOF and several of its analogues were cloned and expressed in Saccharomyces cerevisiae and Pichia pastoris. In S. cerevisiae we have cloned the hormone by homologous recombination resulting in the expression of one gene, or by insertion of free plasmids, up to 300 copies, carrying TMOF. In P. pastoris up to 30 copies of the hormone could be cloned by homologous rcombination using the strong promoter alcohol oxidase. Feeding mosquito larvae with recombinant yeast cells stopped trypsin biosynthesis in the larval gut causing starvation and death. Thus, yeast cells expressing a TMOF gene can be used to control msoquito larvae. This is the first step towards larval control in the field.
Impacts
This method will allow effective control of mosquito larvae in the marsh using a biorational insecticide that will not affect the environment.
Publications
- Nauen, R., Sorge, D., Sterner, A., and Borovsky, D. 2001. TMOF like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens. Arch. insect Biochem. Physiol. 47: 169-180.
- Borovsky, D., J. I. Auwrex, A. Sterner, K. Butaye, L. Vanzier, D. Sorge and R. Nauen. 2001. Cloning Characterization and regulation of Heliothis virescens trypsin and chymotrypsin genes with trypsin modulating oostatic factor. Abst C-6. III international conference on arthropods: chemical, Physiological and environmental aspects. Ladek Zdorj, Poland.
- Nauwelaers, S., D. Borovsky and C. R. Powell. 2001. Genetic engineering of TMOF into Saccharomyces cerevisiae and Pichia pastoris. Abst. C-7. III international conference on arthropods: chemical, Physiological and environmental aspects. Ladek Zdorj, Poland.
- Nauwelaers, S. and Borovsky, D. 2001. Cloning and expression of trypsin modulating oostatic factor (Aea-TMOF) and green fluorescent protein (GFP) in Saccharomyces cerevisiae. Insect Chemical, Physiological Environmental Aspects 2001 (in the press, book chapter).
- Borovsky, D., Nauen, R., Sorge, D.and Sterner, A. 2001. Characterization of trypsin modulating oostatic factor (TMOF) from the larval hemolymph of Heliothis virescens. Insect Chemical, Physiological Environmental Aspects 2001 (in the press, book chapter).
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Progress 10/01/99 to 09/30/00
Outputs
Trypsin and Chymotrypsin cDNAs of Heliothis virescens have been cloned and sequences. Both enzymes are regulated by Aea-TMOF and by a TMOF like factor that was highly purified from H. virescens by HPLC. The Heliothis factor is structurally related to mosquito TMOF and cross-reacted with mosquito TMOF antiserum. The Heliothis factor when injected into adult mosquitoes stopped egg development and trypsin biosynthesis. Injection of msoquito TMOF into Heliothis larvae stopped trypsin biosynthesis and affected the tryspin and chymotryspin transcripts as was shown by Northern analysis. Thus, trypsin and chymotrypsin biosynthesis in Heliothis virescens is controlled by TMOF like factors.
Impacts
This will allow to control H. virescens using natural factors that will starve larvae to death.
Publications
- Gelman D.B. and Borovsky, D. 2000. Aedes aegypti TMOF modulates ecdysteroid production by prothoracic glands of the gypsy moth Lymantria dispar. Arch. Insect Biochem. Physiol. (in the press).
- Sorge D., Sterner A., Nauen, R. and Borovsky, D. 2000. TMOF like Factor Controls the Biosynthesis of Serine Proteases in the Larval Gut of Heliothis virescen. Arch. Insect Biochem. Physiol. (submitted)
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