Source: UNIVERSITY OF FLORIDA submitted to
PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR ENGINEERING STUDIES OF TRYPSIN MODULATING OOSTATIC FACTOR
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0208520
Grant No.
(N/A)
Project No.
FLA-FME-04344
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2006
Project End Date
Sep 1, 2011
Grant Year
(N/A)
Project Director
Borovsky, D.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
FL MEDICAL ENTOMOLOGY LAB, VERO BEACH
Non Technical Summary
Mosquito transmit diseases to humans and animals. This project develops larvicides that controls mosquito larvae in the marsh and thus reduces the spread of diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
72131101040100%
Knowledge Area
721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
1. To clone and express Trypsin Modulating Oostatic Factor (TMOF) and its analogues into Chlorella desiccaae and Pichia pastoris. 2. To clone and express larval trypsin RNAi in E. coli and P. pastoris cells. 3. To test the potency of the TMOF and RNAi constructs on mosquito larvae as potential insecticides.
Project Methods
Trypsin modulating oostatic hormone will be cloned and expressed in chlorella desiccata and pichia pastoris using multiple insertions of synthetic genes of TMOF and its analogues. The recombinant cells will be tested on mosquito larvae to find out their potency to control mosquito larvae. Those recombinants that show promise, will be selected for future development as potential larvicides. Trypsin dsRNA will be expressed in bacterial cells and the cells will be fed to mosquito larvae to find out the potential of different larval trypsin dsRNA to degrade the trypsin message in larval gut, causing inhibition of food digestion, starvation and death. Those constructs that show promise will be selected for future larval control.

Progress 09/01/06 to 09/01/11

Outputs
OUTPUTS: This project created and characterized plasmids carrying synthetic genes optimized for Pichia pastoris. Several expressing consturcts like Cry4Aa-TMOF, GST-Cry4Aa-TMOF, Cry11Aa-TMOF or GST-Cry4Aa-TMOF fusion proteins were effective, killing Aedes aegypti larvae. Also found that Glutathion-S-transferase (GST)-Cry4Aa-TMOF and GST-Cry11Aa-TMOF enhance the heat stability of the cells allowing heat inactivation of yeast cells before they are used in field applications. The TMOF gut receptor was characterized and identified 4 putative genes. One gene indicated it is possibly producing a TMOF receptor with high affinity to the hormone. The effect of TMOF on Diaprepes abbreviatus, the citrus weevil, was studied. Both TMOF and Cathepsin L inhibitor caused starvation weight loss and mortality. dsRNAs to various genes were also synthesized and tested with Diaprepes larvae. Several of these molecules caused death. The exact mechanism of these effects are studied so they can be used as potential bio-control for Diaprepes. We have extended this project by using the current methodologies against Diaprepes towards the citrus psyllids. We fed psysllids TMOF and cysteine protease inhibitor using artificial diet and found out that both are very effective, killing 100% of the tested psyllids in 10 days. We also expressed several dsRNA moieties that have key biological control in insect development and are testing psyllids to find out the effective concentrations of these molecules. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Several new novel genes were identified and characterized that show promise as biocontrol agents for mosquitoes and several agricultural pests.

Publications

  • No publications reported this period


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: In 2009-2010 period we explored the finding that Bacillus thuriengiensis subsp. israelensis and TMOF are synergisitic. Several plasmids carrying synthetic genes which were codon optimized for Pichia pastoris were syntesized and cloned into KM71H Pichia cells alone and in tandem using various loci on the yeast genome. Cell expressing Cry4Aa-TMOF, GST-Cry4Aa-TMOF, Cry11Aa-TMOF and GST-Cry4Aa-TMOF fusion proteins alone or together were very effective, killing Aedes aegypti larvae in few days. Glutathion-S-transferase (GST)-Cry4Aa-TMOF and GST-Cry11Aa-TMOF enhance the heat stability of the cells allowing heat inactivation of yeast cells before they are used in field applications. The synthesis of the various Cry and TMOF proteins were confirmed using Mass MS/MS analyses. This is the first report that shows that Cry4Aa, Cry11Aa and Cyt1Aa can be synthesized by yeast cells. We continue to characterize the TMOF gut receptor. dsRNA identified 4 putative genes. One of the genes was expressed and the protein tested for binding of radioactively labeled TMOF. Michaelis constant for the receptor binding was determined (10 nM) proving that it is possibly a TMOF receptor with high affinity to the hormone. The effect of TMOF on Diaprepes abbreviatus, the citrus weevil, was also studied. A recombinant cathepsin L inhibitor was cloned, expressed and tested with Diaprepes abbreviatus larvae to find out the potency of the inhibitor. Both TMOF and Cathepsin L inhibitor caused starvation weight loss and mortality. dsRNAs to various genes were also synthesized and tested with Diaprepes larvae. Several of these molecules caused death. The exact mechanism of these effects are studied so they can be used as potential bio-control for Diaprepes. We have extended this project by using the current methodologies against Diaprepes towards the citrus psyllids. We fed psysllids TMOF and cysteine protease inhibitor using artificial diet and found out that both are very effective, killing 100% of the tested psyllids in 10 days. We also expressed several dsRNA moieties that have key biological control in insect development and are testing psyllids to find out the effective concentrations of these molecules. Our ultimate goal is to clone and express these molecules in citrus. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Mosquitoes carry many diseases that cause health problems, as well as, economical burden in many countries. Citrus weevils and psyllids cause devastating losses to the citrus industry. The purpose of this project is to develop the mosquito peptide hormone, Trypsin Modulating Oostatic Factor (TMOF) and RNA mediated interference (RNAi) technologies for biorational control of mosquitoes that transmit many important diseases, i.e. malaria, yellow fever and encephalitis and to control the citrus weevil and psyllids that devastate the citrus industry in Florida.

Publications

  • Zaritsky, A., Ben-Dov, E., Borovsky, D., Boussiba, S., Einav, M., Gindin, G., Horowitz, R., Kollot, M., Melnikov, O., Mendel, Z., Yagil, E. 2010. Transgenic Organisms Expressing Genes from Bacillus thuringiensis to Combat Insect Pests. Bioengineered Bugs 1:5, 1-4.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: In 2008 to 2009 period we showed that Bacillus thuriengiensis subsp. israelensis and TMOF are synergisitic. Using this observation several plasmids carrying synthetic genes which were codon optimized for Pichia pastoris were syntesized and cloned into KM71H Pichia cells. The yeast cells were fermented and induced in the presence of methanol. Cells were harvested and fed to mosquito larvae. Cell expressing Cry4Aa-TMOF, GST-Cry4Aa-TMOF, Cry11Aa-TMOF and GST-Cry4Aa-TMOF fusion proteins were very effective, killing Aedes aegypti larvae in few days. Glutathion-S-transferase (GST)-Cry4Aa-TMOF and GST-Cry11Aa-TMOF fusion proteins are heat stable at 50 degree C for 3 h. This finding allows us to heat inactivate yeast cells before they are used to control mosquito larvae in future field applications. We are planning to simultaneously express several Bti toxins and TMOF in yeast as an effective and improved biological larvicide. Mass spectra analysis of the various recombinant protein that were expressed in Pichia cell show that the cells synthesize Cry4Aa and Cry11Aa. This is the first report that shows that Cry4Aa can be synthesized by yeast cells. In parallel, we continue to characterize the TMOF gut receptor. Four putative genes have been isolated . dsRNA against these genes were transcribed and adults were tested with these molecules. A dsRNA against one of the gut receptor significantly redudce the amount of trypsin biosynthesis in the guts of treated females that took a blood meal. Presumabley, due to the disruption of TMOF receptor by the dsRNA that was fed to these female mosquitoes. The amount of trypsin synthesized by treated females is half than found in normal females after the blood meal. We continue to test the other 3 genes to find out if the dsRNA molecules against these genes also affect blood digestion. We continue to study the effect of TMOF on Diaprepes abbreviatus, the citrus weevil. We have genetically modified E. coli cells and synthesized a recombinant cathepsin L inhibitor. We also cloned and expressed cathepsin L in the yeast P. pastoris. The recombinant protein will be tested with cathepsin L inhibitor to find out the potency of the inhibitor. A 3 dimensional model was built and the potential binding of the inhibitor to the enzyme was studied. We are planning to use RNAi methodology to stop trypsin and cathepsin L biosynthesis in the citrus weevil larval gut causing annorexia, starvation and death as a potential bio-control against this agricultural pest. We have extended this project by using the current methodologies against Diaprepes towards the citrus psyllids. We fed psysllids TMOF and cysteine protease inhibitor using artificial diet and found out that both are very effective killing 100% of the tested psyllids in 10 days. We are extending these studies to include RNAi methodologies and plan to clone and express these molecules in citrus. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mosquitoes carry many diseases that cause health problems, as well as, economical burden in many countries. Citrus weevils and psyllids cause devastating losses to the citrus industry. The purpose of this project is to develop the mosquito peptide hormone, Trypsin Modulating Oostatic Factor (TMOF) and RNA mediated interference (RNAi) technologies for biorational control of mosquitoes that transmit many important diseases, i.e. malaria, yellow fever and encephalitis and to control the citrus weevil and psyllids that devastate the citrus industry in Florida.

Publications

  • Borovsky, D. 2008. Control of Aedes aegypti larvae with Aea-TMOF mimics: aromatic derivatives of enoic acid. Pestycyde/Pesticides 1-2, 27-34.
  • Borovsky, D. and Hamdaoui, A. 2008. Binding of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) to its receptor stimulates phosphorylation and protease processing of gut-membrane proteins. Pestycyde/Pesticides 1-2, 13-25.
  • Lemeire, E., Borovsky, D., Van Camp, J. and Smagghe, G. 2008. Effect of ACE inhibitors and TMOF on growth, development and trypsin activity of larval Spodoptera littoralis. Arch. Insect Biochem. Physiol. 69, 199-208.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Improvement of our yeast strains to produce both TMOF and Bti continued in 2007. Synthetic genes of TMOF and several Bti toxins including Cyt proteins were synthesized and cloned into Pichia pastoris cells using integrating plasmids. We are evaluating some of the cells to find out the amount of the toxins that are produced by the cells. During 2007 we continued to characterize TMOF's gut receptor. Micro and macro-approaches were utilized and several putative receptor proteins were identified using affinity chromatography, gel electrophoresis and mass spectrometry. After mass spectra analysis four genes were identified and characterized using RNAi methodologies. The effect of TMOF on Diaprepes abbreviatus, the citrus weevil, continued. A cathepsin L inhibitor was identified, cloned and sequenced. Since these insects synthesize both cathepsins and serine proteases we aim to characterize the weevil cathepsin and clone its specific inhibitor in alfalfa plants to study the effect of TMOF and D. abbreviatus cathepsin inhibitor on food digestion, growth and development of the citrus weevil. We aim to control the weevil larvae by shutting down their digestion. Improvement of our yeast strains to produce both TMOF and Bti continued in 2007. Synthetic genes of TMOF and several Bti toxins including Cyt proteins were synthesized and cloned into Pichia pastoris cells using integrating plasmids. We are evaluating some of the cells to find out the amount of the toxins that are produced by the cells. During 2007 we continued to characterize TMOF's gut receptor. Micro and macro-approaches were utilized and several putative receptor proteins were identified using affinity chromatography, gel electrophoresis and mass spectrometry. After mass spectra analyses four genes were identified and characterized using RNAi methodologies. The effect of TMOF on Diaprepes abbreviatus, the citrus weevil, continued. A cathepsin L inhibitor was identified, cloned and sequenced. Since these insects synthesize both cathepsins and serine proteases we aim to characterize the weevil cathepsin and clone its specific inhibitor in alfalfa plants to study the effect of TMOF and D. abbreviatus cathepsin inhibitor on food digestion, growth and development of the citrus weevil. We aim to control the weevil larvae by shutting down their digstion. Improvement of our yeast strains to produce both TMOF and Bti continued in 2007. Synthetic genes of TMOF and several Bti toxins including Cyt proteins were cloned into Pichia pastoris cells using intergrating plasmids. We are evaluating some of the cells to find out the amount of the toxins that are produced by the cells. During 2007 we continued to characterize TMOF's gut receptor. Micro and macro-approaches were utilized and several putative receptor proteins were identified using affinity chromatography, gel electrophoresis and mass spectrometry. After mass spectra analysis four genes were identified and characterized using RNAi methodologies. The effect of TMOF on Diaprepes abbreviatus continued. We aim to control the weevil larvae by shutting down their digestive enzymes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mosquitoes carry many diseases that cause health problems, as well as, economical burden in many countries. Citrus weevils cause devastating losses to the citrus industry. The purpose of this project is to develop the mosquito peptide hormone, Trypsin Modulating Oostatic Factor (TMOF) and RNA mediated interference (RNAi) technologies for biorational control of mosquitoes that transmit many important diseases, i.e. malaria, yellow fever and encephalitis and to control the citrus weevil that devastates the citrus industry in Florida.

Publications

  • 1. Borovsky, D. 2007. Trypsin modulating oostatic factor for developing resistant crops. In: Insecticides design using advanced technologies (eds. I. Ishaaya, R. Nauen and A. R. Horowitz) Springer-Verlag, Berlin Heidelberg pp. 135-149.
  • 2. Borovsky, D. and Nauen, R. 2007. Biological and biochemical effects of organo-synthetic analogues of Trypsin Modulating Oostatic Factor (TMOF) on Aedes aegypti, Heliothis virescens and Plutella xylostella. Pestycyde/Pesticides 3-4, 17-26.
  • 3. Pszczolkowski, M.A., Rhine, C., Ramaswamy, S. B. and Borovsky, D. 2007. The effect of Aea-TMOF on egg maturation in Heliothis virescens: a preliminary study. Pestycyde/Pesticides (3-4), 33-38.
  • 4. Borovsky, D., Breyssens, H., Laroye, C., Jenssen, S. and Smagghe, G. 2007. Functional analysis of juvenile hormone epoxide hydrolase promoters of Drosophila melanogaster. Abst. P30. Ninth Internatinal conference on juvenile hormones, York, England.
  • 5. Lemiere, E., Vanholme, B., Van Leeuwen, T., Borovsky, D., Van Camp, J. and Smagghe, G. 2007. Angiotensin converting enzyme characterization and inhibition in Spodoptera littoralis. Abst. O12. Vth International Conference on Arthropods: Chemical, Physiological and Environmental Aspects. Bialka Tatrzanska, Poland.
  • 6. Borovsky, D., Hamdaoui, A. and Deckers, K. 2007. Characterization of trypsin modulating oostatic factor (TMOF) gut-receptor. Abst. O17. Vth International Conference on Arthropods: Chemical, Physiological and Environmental Aspects. Bialka Tatrzanska, Poland.
  • 7. Pszczolkowski, M.A., Rhine, C., Ramaswamy, S. B. and Borovsky, D. 2007. Ae-TMOF action on egg maturation in Heliothis virescens: A preliminary study. Abst. P17. Vth International Conference on Arthropods: Chemical, Physiological and Environmental Aspects. Bialka Tatrzanska, Poland.
  • 8. Borovsky, D., Powell, C. A. & Shatters, R. G. 2007. "Identification and characterization of diaprepes abbreviatus cathepsin L protease inhibitor 1" USA: GenBank. (Accession number EU009453).


Progress 10/01/06 to 09/30/07

Outputs
Improvement to our recombinant yeast strains that produce more TMOF and its analogues continued during 2007. Bti activated toxin, Cry4Aa was cloned and expressed in bacterial cells and was shown to increase their potency by several fold in the presence of recombinant Pichia pastoris cells that produced TMOF. A synthetic gene carrying both TMOF and activated Cry4Aa gene was synthesized and expressed in Pichia pastoris. Biochemical and molecular analyses will be done in 2008 on the recombinant cells. We expect to express several activated Cry toxins and TMOF in yeast and test their potency and synergism. Several synthetic TMOF aromatic derivatives analogues of enoic acid were synthesized and tested. Several of the TMOF mimics showed exceptional good activity towards larval mosquito shutting trypsin biosynthesis in the larval gut like the decapeptide hormone, TMOF. These results indicate that the aromatic derivatives which can penetrate the insect cuticle may be used as potential good larvicides that starve and kill mosquito larvae. Some progress was made on the characterization and solubilization of Aede TMOF gut receptor. The receptor was solubilized without loosing its binding affinity to TMOF and partial purification was achieved by affinity chromatography and by SDS-PAGE. A putative membrane protein was sequenced by mass spectrometry and a putative receptor gene was identified. The receptor gene will be expressed in 2008 on the surface of CHO cells and the receptor characterized.

Impacts
Mosquitoes carry many diseases that cause health problems, as well as, economical burden in many countries. The purpose of this project is to develop the mosquito peptide hormone, Trypsin Modulating Oostatic Factor (TMOF)and its synthetic analogues as biorational insecticides to control, Anopheles, Culex, and Aedes mosquito populations and the disease that they vector (malaria, dengue, yellow fever and encephalitis including West Nile encephalitis). Because several lepidopterans also use trypsin to digest their food a potential to control agricultural pest insects using TMOF is also a possibility.

Publications

  • Borovsky, D. 2007. Trypsin modulating oostatic factor for developing resistant crops. In: Insecticides design using advanced technologies (eds. I. Ishaaya, R. Nauen and A. R. Horowitz) Springer-Verlag, Berlin Heidelberg pp. 135-149.
  • Borovsky, D. and Nauen, R. 2007. Biological and biochemical effects of organo-synthetic analogues of Trypsin Modulating Oostatic Factor (TMOF) on Aedes aegypti, Heliothis virescens and Plutella xylostella. Pestycyde/Pesticides (in the press).
  • Borovsky, D. 2007. Control of Aedes aegypti larvae with Aea-TMOF mimics: aromatic derivatives of enoic acid. Pestycyde/Pesticides (in the press).
  • Borovsky, D. and Hamdaoui, A. 2007. Binding of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) to its receptor stimulates phosphorylation and protease processing of gut-membrane proteins. Pestycyde/Pesticides (in the press).
  • Maciej A. Pszczolkowski, M.A., Rhine, C., Sonny B. Ramaswamy, S. B. and Borovsky, D. 2007. The effect of Aea-TMOF on egg maturation in Heliothis virescens: a preliminary study. Pestycyde/Pesticides (in the press).


Progress 10/01/05 to 09/30/06

Outputs
Improvement of yeast strains to produce more TMOF and its analogues continued during 2006. Bti toxins were tested with TMOF and we found that TMOF and Bti act as synergists. Thus, it is possible now to use 10 fold less Bti toxin in the presence of TMOF. To optimize the interaction of TMOF and Bti E. coli cells were transformed with both Cry4Aa and TMOF and the effect on mosquito larvae was studied. The initial results indicate that using TMOF and Bti enhances the activity of the transformed E. coli cells against mosquito larvae. The TMOF gene was also cloned and expressed in alfalfa plants. The potency of the transformed plants against Heliothis virescens and the citrus root weevil Diaprepes abbreviatus was checked. Both insects after feeding on the trnsgenic plants showed growth inhibition and significant inhibition of trypsin biosynthesis in the gut. These results indicate that TMOF can be used against mosquito larvae and against agricultural pest insects that use trypsin as their major digestive enzyme.

Impacts
Mosquitoes carry many diseases that cause health pronlems, as well as, economical burden in many countries.The purpose of this project is to develop the mosquito peptide hormone Trypsin Modulating Oostatic Factor (TMOF) and trypsin RNA mediated interference (RNAi) technologies for biorational use in controlling Anopheles, Culex and Aedes mosquito populations and the diseases they vector (malaria, dengue, yellow fever and encephalitis including West Nile encephalitis).

Publications

  • Borovsky, D. 2006. Trypsin modulating oostatic factor for developing resistant crops. In: Insecticides design using advanced technologies (eds. I. Ishaaya, R. Nauen and A. R. Horowitz) Springer-Verlag, Heidelberg Germany (in the press).
  • Borovsky, D. 2006. Control of mosquito larval trypsin with Aea-Trypsin Modulating Oostatic Factor (TMOF) and its analogues. Pestycyde/Pesticides (3): 71-77.
  • Borovsky, D., Iannotti, D.A., Shatters, R.G. Jr., and Powell, C. A. 2006. Effect of recombinant Aea-TMOF on Heliothis virescens. . Pestycyde/Pesticides (3): 79-85.