Progress 10/01/99 to 09/30/04
Outputs
We have found that scorpions use potassium ion as a prevenom and have isolated several novel toxins from scorpion venom. This information helps select insect specific toxins for use in the recombinant baculoviruses. We demonstrated that combinations of toxins can be used synergistically in baculoviruses for insect control. We looked at recombination frequencies as a possible environmental risk factor related to the release of the recombinant viruses. We characterized the apoptosis suppressor protein from an insect virus. Such reagents will be very useful for collaborators who are using the protein to block programmed cell death in several plant species. These recombinant plants expressing IAP are resistant to a variety of stressors including pathogens. We are investigating the molecular mechanism by which insect viruses gain control of the behaviour of their hosts. We have helped several countries develop recombinant baculovirus technology both for protein expression
and for the development of green pesticides.
Impacts
Recombinant baculoviruses work as fast as classical pesticides, preserve yield while not disrupting natural enemies and thus are very effective insect control agents on major insect pests. No evidence of potential harm to humans or to the environment has been found. They are a cost effective way to move away from dangerous pesticides in many third world areas.
Publications
- Koivunen, M.E., C. Morisseau, W.R. Horwath and B.D. Hammock. 2004. Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (ureaformaldehyde) as nitrogen source. Can. J. Microbiol. 50:167-174.
- Lydy, M., J. Belden, C. Wheelock, B. Hammock and D. Denton. 2004. Challenges in Regulating Pesticide Mixtures. Ecology and Society. 9:1-17.
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Progress 01/01/03 to 12/31/03
Outputs
We are continuing work on development of recombinant baculoviruses for insect control. In a recent PNAS paper we reported that scorpions use potassium ion as a prevenom and we have isolated several novel toxins from scorpion venom. This information helps select insect specific toxins for use in the recombinant baculoviruses. We have demonstrated that combinations of toxins can be used synergistically in baculoviruses for insect control We have looked at recombination frequencies as a possible environmental risk factor related to the release of the recombinant viruses. We have characterized the apoptosis suppressor protein from an insect virus. Such reagents will be very useful for collaborators who are using the protein to block programmed cell death in several plant species. These recombinant plants expressing IAP are resistant to a variety of stressors including pathogens. We are investigating the molecular mechanism by which insect viruses gain control of the
behaviour of their hosts. We are helping several countries to develop recombinant baculovirus technology both for protein expression and for the development of green pesticides.
Impacts
Field tests of recombinant baculoviruses have shown them to be very effective insect control agents on major insect pests. They work as fast as classical pesticides, preserve yield while not disrupting natural enemies. We have found no evidence of potential harm to humans or to the environment. They offer a cost effective way to move away from dangerous pesticides in many third world areas.
Publications
- Kamita, S.G., S. Maeda and B.D. Hammock. 2003. High-frequency homologous recombination between baculoviruses involves DNA replication. J. Virol. 77:13053-13061.
- Inceoglu, B., J. Lango, J. Jing, L. Chen, F. Doymaz, I.N. Pessah and B.D. Hammock. 2003. One scorpion, two venoms: Prevenom of Parabuthus transvaalicus acts as an alternative type of venom with distinct mechanism of action. PNAS 100:922-927.
- Pei, Z., G. Reske, Q. Huang, B.D. Hammock, Y. Qi and N. Chejanovsky. 2002. Characterization of the apoptosis suppressor protein P49 from the Spodoptera littoralis culeopolyhedrovirus. J. Biol. Chem. 277:48677-48684.
- Regev, A., H. Rivkin, B. Inceoglu, E. Gershburg, B.D. Hammock, M. Gurevitz and N. Chejanovsky. 2003. Further enhancement of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively. FEBS Lett. 537:106-110.
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Progress 01/01/02 to 12/31/02
Outputs
We are continuing work on development of recombinant baculoviruses for insect control. We have found that scorpions use potassium ion as a prevenom and we have isolated several novel toxins from scorpion venom. This information helps select insect specific toxins for use in the recombinant baculoviruses. Additional studies support the efficacy of the virus for insect control and its safety to humans and the environment. We have expressed the inhibitory protein of apoptosis reported last year in the baculovirus system and will develop antibodies to it. This reagent will be very useful for collaborators who are using the protein to block programmed cell death in several plant species. These recombinant plants expressing IAP are resistant to a variety of stressors including pathogens. We continue to develop the concept of metabolomics or high throughput metabolic profiling as an indicator of human health. This technology also can be used to profile natural and
recombinant cultivars.
Impacts
The baculovirus project is receiving lower priority until we can find a company in a position to develop the technology separate from European markets. The success of the IAP proteins when expressed in plants was greater than anticipated. This may offer a general system of resistance development in plants. The concept of metabolomics as a post genomic technology is becoming widely accepted.
Publications
- Hinton, A.C. and B.D. Hammock. 2001. Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta. Insect Biochem. Molec. Biol. 32:57-66. Watanabe, T. and B.D. Hammock. 2001. Rapid determination of soluble epoxide hydrolase inhibitors in rat hepatic microsomes by high performance liquid chromatography with electrospray tandem mass spectrometry. Anal. Biochem. 299:227-234.
- Rajendra, W., B. Inceoglu, R. Herrmann, M. Derbel, P.V. Choudary and B.D. Hammock. 2001. Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus. BMC Biochemistry 2:16.
- Inceoglu, B., J. Lango, J. Wu, P. Hawkins, J. Southern and B.D. Hammock. 2001. Isolation and characterization of a novel type of neurotoxic peptide from the venom of South African scorpion Parabuthus transvaalicus (Buthidae). Euro. J. Biochem. 268:5407-5413.
- Taniai, K., A.B. Inceoglu and B.D. Hammock. 2002. Expression efficiency of a scorpion neurotoxin, AaHIT, using baculovirus in insect cells. Appl. Entomol. Zool. 37(2):225-232.
- Fuxa, J.R., A.R. Richter, A.O. Ameen and B.D.Hammock. 2002. Vertical transmission of TnSNPV, TnCPV, AcMNPV, and possibly recombinant NPV in Trichoplusia ni. J. Invert. Pathol. 79:44-50.
- Newman, J.W., T. Watanabe and B.D. Hammock. 2002. The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC-MS/MS. J. Lipid Res. 43:1563-1578.
- Inceoglu, A.B., Y. Hayashida, J. Lango, A.T. Ishida and B.D. Hammock. 2002. A single charged surface residue modifies the activity of ikitoxin, a beta-type Na+ channel toxin from Parabuthus transvaalicus. Eur. J. Biochem. 269:5369-5376.
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Progress 01/01/01 to 12/31/01
Outputs
During the last year we have had 6 peer-reviewed publications related to this work. Possibly the most exciting recent work has been the cloning and expression of an apoptosis inhibiting protein from several insects including Spodoptera and Bombyx. These materials block programmed cell death or apoptosis in a variety of systems. Of particular excitement they inhibit apoptosis in several plant species. This makes the resulting plants resistant to a variety of stressors including heat and cold stress as well as infection with pathogens. We have continued research on developing recombinant baculoviruses for insect control. This work has included the isolation of a variety of insect and mammal specific scorpion toxins that allow us to predict that that the toxins used in our recombinant baculoviruses will be safe for nontarget organisms. We also have carried out studies on the environmental safety of the viruses. Finally, we are developing the concept of metabolic
profiling or metabolomics as an indicator of human health. Such metabolite profiling also can be used to test the hypothesis that recombinant plants or other organisms are similar to non-engineered controls.
Impacts
The baculovirus project is receiving lower priority until we can find a company in a position to develop the technology separate from European markets. The success of the IAP proteins when expressed in plants was greater than anticipated. This may offer a general system of resistance development in plants.
Publications
- Huang, Q., Q.L. Deveraux, S. Maeda, H.R. Stennicke, B.D. Hammock and J.C. Reed. 2001. Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori. Biochim. Biohys. Acta. 1499:191-198.
- Lee, Y., J.R. Fuxa, a.B. Inceoglu, S.A. Alaniz, A.R. Richter, L.M. Reilly and B.D. Hammock. 2001. Competition between wild-type and recombinant nucleopolyhedroviruses in a greenhouse microcosm. biol. Control. 20:84-93.
- Loret, E. and B.D. Hammock. 2001. Structure and neurotoxicity of venoms. In: Scorpion Biology and Research, (Brownel, P. and G. Polis, eds.), pp. 204-233. Oxford Univesity Press, New York, NY.
- Tavrner, P.D., R.V. Gunning, P. Kolesik, P.T. Bailey, A.B. Inceoglu, B. Hammock and R.T. Roush. 2001. Evidence for direct neural toxicity of a "light" oil the peripheral nerves of lightbrown apple moth. Pestic. Biochem. Phys. 69:153-165.
- Watkins, S.M., B.D. Hammock, J.W. Newman and J.B. German. 2001. Individual metabolism should guide agriculture toward foods for improved health and nutrition. Am. J. Clin. Nutr. 74:283-286.
- Inceoglu, A.B., S.G. Kamita, A.C. Hinton Q. Huang, T.F. Severson, K-d. Kang, and B.D. Hammock. 2001. Recombinant baculoviruses for insect control. Pest Mang. Sci. 57:981-987.
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Progress 01/01/00 to 12/31/00
Outputs
We have demonstrated that baculoviruses expressing scorpion toxins or insect enzymes can be used as genetically improved viral insecticides. The viruses kill target insects faster than wildtype viruses and offer no environmental problems. During the last year we demonstrated that the virus regulates its host by stealing genes from its host involved in the apoptotic cascade. This information may be used to develop still better viruses. We are developing techniques to identify new toxins based on protein isolation by microbore hplc and identification using electrospray and MALDI mass spectral methods
Impacts
This technology has been developed to the point where it could be of great benefit in pest control in developing countries. However, with the current worries in Europe over genetically modified organisms it is unlikely that further development will take place supported by large industry. We are looking for funding to develop altruistic technologies.
Publications
- Zhang, H., Huang, Q., Ke, N., Matsuyama, S., Hammock, B., Godzik, A. and Reed, J. C. 2000. Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms. J. Biol. Chem. 275(35):27303-27306.
- Huang, Q., Deveraux, Q. L., Maeda, S., Salvesen, G. S., Stennicke, H. R., Hammock, B. D. and Reed, J. C. 2000. Evolutionary conservation of apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9. Proc. Natl. Acad. Sci. USA 97(4):1427-1432.
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Progress 01/01/99 to 12/31/99
Outputs
We have demonstrated that viruses specific for pest insects can be engineered for rapid kill of the pests. For decades baculoviruses have been used in commercial agriculture, but the slow rate of kill has limited their use on many crops. We have demonstrated that the rate of kill of these viruses can be dramatically enhanced and crop damage diminished by inserting genes either encoding toxins or enzymes that regulate insect development. The most successful approach has been to insert the genes for insect selective scorpion toxins into baculoviruses. We have multiple levels of selectivity with the virus only active on pest insects, the promoter only active in insects and the toxins remarkably selective for insects. The viruses are competitive with modern classical insecticides on cotton in the U.S. and vegetables in S. E. Asia and far superior to BACILLUS THURINGENSIS. In addition they cause no detectable alteration in natural enemies in the field and have no effects
on nontarget lepidopterous insects unlike classical insecticides and BT. The recombinant viruses are more active on insecticide resistant insects than susceptible insects and synergize with pyrethroid insecticides. Thus they could be used effectively in resistance management programs. Laboratory studies have failed to show any negative effects on beneficial insects and the recombinant virus is out competed rapidly in the field by the wildtype virus thus it offers no environmental problems. Additional research is needed to improve the viruses further. We need to find more active toxins and synergistic combinations of toxins active in the virus system. The use of improved promoters and enhancers is improving virus efficacy. The concept needs to be extended not only to other baculoviruses but also to other insect viruses to widen the spectrum of control offered. Other laboratories are working on production and formulation which are critical for commercial success.
Impacts
Two major companies are pursuing commercial development of these biological pesticides. If the materials become commercial they will control the insect pests which are the target of most pesticide use world wide. The materials represent a sustainable alternative to pest control.
Publications
- Hammock, B.D. 1998. Status of recombinant baculoviruses in insect pest control. Reviews in Toxicology. In: Pesticides and the Future: Minimizing Chronic Exposure of Humans and the Environment, (Kuhr, R.J., and N. Motoyama, eds), pp: 205-221. IOS Press, Amsterdam, The Netherlands.
- Hammock, B.D., Inceoglu, A.B., Rajendra, W., Fuxa, J.R., Chejanovsky, N., Jarvis, D. and Hanzlik, T.N. 1999. Impact of biotechnology on pesticide delivery. In: Pesticide Chemistry and Bioscience. The Food-Environment Challenge, (Brooks G.T. and T.R. Roberts, eds.), pp 73-99. The Royal Society of Chemistry, Cambridge, United Kingdom.
- Hammock, B.D. 1999. The quest for a recombinant viral pesticide. A tribute to Susumu Maeda. Riken Review 22:9-13.
- Parrella, M.P., Kamita, S.G. and Hammock, B.D. 1999. Professor Susumu Maeda our colleague, mentor, and friend. Riken Review 22:3-8.
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Progress 01/01/98 to 12/31/98
Outputs
Baculoviruses are double stranded DNA viruses that are highly selective for individual pest species. The viruses however kill their hosts very slowly allowing the insect pest time to do serious crop damage. We have developed viruses which rapidly kill their hosts by expressing insect specific scorpion toxins. We have isolated several novel toxins from scorpion venom and modeled these toxins to determine the basis of insect selectivity. Corresponding genes have been synthesized, expressed in baculoviruses and found to increase dramatically the speed of kill of the viruses. These viruses can be used synergistically with classical pesticides such as pyrethroids and the recombinant viruses will kill insects resistant to classical pesticides faster than nonresistant insects. Recombinant viruses act as biological pesticides which kill very quickly then are rapidly out competed in the environment. There is little if any environmental risk from the recombinant viruses. We
have found there is tritrophic interaction among the host plant, pest insect and virus and which plant natural products cause the virus to become inactivated. Based on this knowledge we demonstrated that free radical scavengers can be used to stabilize viral formulations.
Impacts
(N/A)
Publications
- NAKAGAWA, Y., SADILEK, E.M., LEHMBERG, R., HERRMANN, R., MOSKOWITZ, H., YOUNG, M., THOMAS, B.A., SHIMIZU, R., KURODA, M., JONES, A.D. AND HAMMOCK, B.D. 1998. Rapid purification and molecular modeling of AaIT peptides from venom of ANDROCTONUS AUSTRALIS. Arch. Insect Biochem.
- MOSKOWITZ, H., HERRMANN, R., JONES, A.D., AND HAMMOCK, B D. 1998. A depressant insect-selective toxin analog from the venom of the scorpion LEIURUS QUINQUESTRIATUS HEBRAEUS. Purification and structure/function characterization. Eur. J. Biochem. 254:44-49.
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Progress 01/01/97 to 12/01/97
Outputs
Our work has focused on the isolation and characterization of natural toxins from scorpion venoms. These toxins can be highly selective for insects vs. other arthropods and even within insect groups. Using homology modeling systems running on a Silicon Graphics system, we have localized the principal determinants of selectivity to a single loop of the peptide toxin. We have isolated several potent new toxins by a combination of reversed phase ion pair HPLC and microbore HPLC. The materials were shown to be of high purity by capillary electrophoresis as well as MALDI and electrospray mass spectrophotometry and sequenced by Edmon degradation. Genes were then chemical synthesized to code for the toxin and these genes cloned into baculovirus expression vectors. Following expression, these recombinant viruses are evaluated as biological insecticides. Several kill pest insects very quickly. We also are examining the safety of these materials in model ecosystems and their
potential interaction with other biological control agents and classical insecticides. The recombinant viruses appear very promising for the control of pest insects in field and row crops.
Impacts
(N/A)
Publications
- Nakagawa, Y., Lee, Y., Lehmberg, E., Herrmann, R., Herrmann, R., Moskowitz, H., Jones, A.D. and Hammock, B.D. 1997. Anti-insect toxin (AaIT5) from ANDROCTONUS AUSTRALIS. Eur. J. Biochem. 246: 496-501.
- Gurevitz, M. et al. 1997. Utilization of scorpion insecticidal neurotoxins and baculoviruses for the design of novel selective biopesticides. Pp. 81-96 IN Modern Agriculture & the Environment (Rosen & Tel-Or, eds.), Kluwer Acad. Publ., U.K.
- Hoover, K., Schultz, C.M., Lane, S., Bonning, B., Hammock, B.D. and Duffey, S. 1997. Effects of diet-age and streptomycin on virulence of AUTOGRAPHA CALIFORNICA M nucleopolyhedrovirus against the tobacco budworm. J. Invert. Pathol. 69: 46-5
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Progress 01/01/96 to 12/30/96
Outputs
During the last year we have continued the development of baculovirus insecticides which express recombinant peptides. We have demonstrated the efficacy of the recombinant baculoviruses in speeding the kill on insect pests and in reducing crop damage in the laboratory and greenhouse. In collaboration with M. Parrella, we have found no deleterious effects on beneficial insects or natural enemies of insect pests. This indicates that the technology should be a vital part of pest management strategies. We have found that the venoms of scorpions are far more toxic to pest insects than the combination of the activity of their component toxins. Based on this, we have developed combinations of peptide toxins which are more active than either alone. We continue to isolate and identify new insect selective peptide toxins using a combination of microbore HPLC, electrospray mass spectrophotometry and bioassay. The genes for some of these toxins have been synthesized and are being
cloned into insect viruses. We continue to examine novel promoters to increase the efficacy of the virus. With the laboratory of S. Duffey we continue to study the tritrophic interactions among recombinant viruses, the crop plant and pest insect. During the last year this work has resulted in four peer-reviewed publications.
Impacts
(N/A)
Publications
- Hoover, K., Herrmann, R., Moskowitz, H., Bonning, B., Duffey, S., McCutchen, B. and Hammock, B. 1996. The potential of recombinant baculoviruses as enhanced biopesticides. Pesticide Outlook 7(3):21-27.
- Jarvis, D., Reilly, L., Hoover, K., Schultz, C., Hammock, B. and Guarino, L. 1996. Construction and characterization of immediate early baculovirus pesticides. Biol. Control. 7:228-235.
- Kunimi, Y. et al. 1996. Comparison of wild type & genet. engineered nuclear polyhedrosis viruses of AUTOGRAPHA CALIFORNICA for mortality, virus replication & polyhedra production of TRICHOPLUSIA NI larvae. Entomol. Exper. Appl. 81:251-257.
- McCutchen, B.F., Herrmann, R., Heinz, K.M., Parrella, M.P. and Hammock, B.D. 1996. Effects of recombinant baculoviruses on a nontarget endoparasitoid of HELIOTHIS VIRESCENS. Biol. Control. 6:45-50.
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Progress 01/01/95 to 12/30/95
Outputs
During the last year we have continued the development of baculovirus insecticides which express recombinant peptides. Two major U.S. companies are in field tests with our technology. Work has progressed along several lines. We have demonstrated the efficacy of the recombinant baculoviruses in speeding the kill on insect pests and in reducing crop damage in the laboratory and greenhouse and have found no deleterious effects on beneficial insects. This indicates that the technology should be a vital part of pest management strategies. We have found that the venoms of scorpions are far more toxic to pest insects than the combination of the activity of their component toxins. Based on this we have developed combinations of peptide toxins which are more active than either alone. We also have isolated and identified new insect selective peptide toxins based on a combination of microbore HPLC and electrospray mass spectrophotometry. The genes for some of these toxins have
been synthesized and are being cloned into insect viruses. We continue to examine new toxins alone and in combination as well as novel promoters to increase the efficacy of the virus. In collaboration with the laboratory of S. Duffey we are examining the tritrophic interactions among recombinant viruses, the crop plant and pest insect. During the last year this work has resulted in seven peer reviewed publications.
Impacts
(N/A)
Publications
- HERRMANN, R., MOSKOWITZ, H., ZLOTKIN, E. and HAMMOCK, B.D. 1995. Positive cooperativity among insecticidal scorpion neurotoxins. Toxicon. 33(8):1099-1102.
- OHKAWA, T., MCCUTCHEN, B., HANZLIK, T., KAMITA, S., SASAGAWA, H., CHOUDARY, P.V., HAMMOCK, B.D. and MAEDA, S. 1994. Stage independent insecticidal effects of a recombinant baculovirus on BOMBYX MORI larvae. App. Entom. Zool. 29(4):616-618.
- HEINZ, K.M., MCCUTCHEN, B.F., HERRMANN, R., PARRELLA, M.P. and HAMMOCK, B.D. 1995. Direct effects of recombinant nuclear polyhedrosis viruses on selected non-target organisms. J. Econ. Entomol. 88(2):259-264.
- DUFFEY, S., HOOVER, K., BONNING, B.C. and HAMMOCK, B.D. 1995. The impact of host plant on the efficacy of baculoviruses. In: Reviews in Pesticide Toxicology (Roe, R. and Kuhr, R., eds.), pp. 137-275, Toxicology Communications, Raleigh, NC.
- SUNDARAM, K., SUNDARAM, A., GEE, S., HARRISON, R. and HAMMOCK, B. 1995. Enzyme-linked immunosorbant assay for quantific. of B. THURINGIENSIS var. KURSTAKI crystall. protein. In: Pesticide Form. & Appl. Systems, ASTM-STP 1234, Philadelphia.
- HOOVER, K., SCHULTZ, C., LANE, S., BONNING, B., DUFFEY, S., MCCUTCHEN, B. and HAMMOCK, B. 1995. Reduction in damage to cotton plants: recombinant baculovirus knocks moribund larvae of HELIOTHIS VIRESCENS off plant. Bio. Contr. 5(3):419-4.
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