2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Currently, control of lepidopteran pests such as the noctuids in corn, soybeans, cotton and other crops requires expensive inputs of insecticides, fuel and labor. Occupational exposures of agricultural workers to insecticides are a serious problem. Pesticide applications are the major source of pollution to surrounding lands and waterways. Application of insecticides places additional strain on agricultural production through exposure to rising fuel costs. Resistance to insecticides is a growing problem in all areas of the world. Adoption of crops genetically engineered to express insecticidal toxins is a promising biological control technology which can reduce insecticide inputs on certain commodities, but which is not available for all crops for a variety of reasons, even aside from the issue of consumer resistance. Control of lepidopteran pests plaguing greenhouse vegetable and ornamental production and the growing popularity of certified organic produce are also growing markets in need of newer more effective biological controls. Entomopathogenic viruses and fungi, microbial biological control agents, have great potential to provide effective control in these circumstances. Mechanisms of immunity to microbials and determinants of host range must be fully understood to implement control measures based on these vulnerabilities. The growing number of crops modified for improved nutritional content to benefit humans and livestock will also be a boon to pest insects that rely on the same nutrients and antioxidants for development and reproduction. Knowledge of successful pest immunomodulatory compounds would provide an invaluable guide to what kinds of genetic modifications should be sought for crops, and which should be avoided. In the United States, mass-rearing of beneficial insects for release as biological control agents is a half billion dollar industry. Historically, artificial diets developed for the mass-rearing of lepidopteran pests and other insects have prioritized ease of rearing and cost of materials, but have not emphasized approximating field conditions. The range of nutrients provided to larvae by fresh vegetation is clearly wider than that provided by existing artificial diets (e.g., antioxidants, micronutrients, vitamins and phenolics) and the performance of larvae may differ substantially in important parameters such as immunocompetence against biological control agents. One major factor limiting the fitness of mass produced beneficial insects, predators and parasitoids, for augmentative biological control in field and greenhouse systems is the quality of artificial diets provided in the commercial insectary. Improvement of the diets currently used for mass rearing of high quality beneficials may improve the field survival rate, tipping the balance to even better control. Finally, correctly optimized diets approximating field conditions are needed for the accurate testing of insecticidal compounds (e.g. Bt, insecticides). Biological control agents act as major stressors on pest insect populations in the field. Pest immunocompetence is a major determinant of the success or failure of biological control using microbial or parasitical biological control agents. Development of methods which could impair pest immune systems to favor better control agents would benefit producers and the environment by reducing expensive inputs of materials and labor. Therefore elucidation of immune mechanisms and the discovery of immunomodulatory factors will improve the efficacy of biological control in the field. Accordingly, our work consists of two major thrusts and the links between them:. 1)Innate immunity. Resistance of a pest insect to a microbial biological control agent, e.g. entomopathogenic viruses and fungi, or to parasitoids, is mediated by cellular and humoral immune factors. Indeed many biological control agents have evolved mechanisms to circumvent host defenses, leading to host-specific countermeasures which limit the host range of biological control agents. Detailed explication of which immune effectors are key to resistance and host range, and how these effectors are deployed and coordinated, will reveal vulnerabilities for exploitation.. 2)Nutritional epidemiology. Immunocompetence of an herbivorous pest can vary between populations and individuals due to the amount and quality of food obtained. Adequate levels of plant vitamins, antioxidants and phenolic compounds may be important to optimal immune function. Soil micronutrients such as selenium and other plant secondary metabolites present in crops and weeds may modulate pest immunocompetence, thus limiting the effectiveness of biological control agents. This is the concept of functional foods, plants biofortified to contain greater amounts of essential vitamins, minerals and antioxidants, applied to pest insects. The goal of this research is to determine immunological, nutritional and physiological factors that limit the effectiveness of biological control agents, to provide a predictive measure of the success of a beneficial agent, and to test plant derived compounds as a means to improve the effectiveness of biological control agents of insects and weeds. This research will suggest methods to modify plants or management methods to impact immunocompetence of pest insects, and to enhance the fitness of biological control agents such as beneficial insects. This research is conducted under the National Program 304, Crop Protection and Quarantine, within the Insect and Mite Pest Control Technologies component, by addressing basic the biology of pest insect resistance to relevant biological control agents such as microbial entomopathogens and parasitoids. 2.List by year the currently approved milestones (indicators of research progress) FY2005 1. Initiate identification of hemocyte proteins induced by infection, parasitization. 2. Initiate construction of AcMNPVhsp70Red viruses. 3. Continue characterization of baculoviral inactivation by PO, and free radicals. 4. Initiate Se assimilation studies. 5. Initiate analysis of Se incorporation into selenoproteins. 6. Construct hemocyte cDNA library. 7. Initiate evaluation of organic forms of Se. 8. Initiate studies of other micronutrients. 9. Initiate greenhouse tests of Se. 10. Initiate micronutrient studies on antibacterial immunity. 11. Set up laboratory, purchase equipment, materials. 12. Begin work on adding chemicals to insect food stream. FY2006 1. Initiate isolation and analysis of clones from cDNA library. 2. Continue examination of viral inactivation by PO and free radicals. 3. Continue identification and isolation of induced proteins. 4. Utilize AcMNPVhsp70Red virus to study immunity. 5. Complete Se assimilation studies. 6. Continue analysis of Selenoproteins. 7. Initiate isolation and cloning of selected selenoproteins. 8. Initiate studies of phytochemicals. 9. Continue studies of other micronutrients. 10. Initiate evaluation of organic forms of Se. 11. Complete greenhouse studies. 12. Continue micronutrient studies on bacteria. 13. Complete studies on COX inhibitors. 14. Begin work on eicosanoid actions in viral infection. FY2007 1. Continue isolation and analysis of clones from cDNA library. 2. Complete examination of viral inactivation by PO and free radicals. 3. Complete isolation and analysis of induced proteins. 4. Continue immunity studies with AcMNPVhsp70Red. 5. Continue isolation and cloning of selected selenoproteins. 6. Continue analysis of phytochemicals. 7. Complete evaluation of organic forms of Se. 8. Continue studies of other micronutrients. 9. Initiate combinatorial micronutrient tests. 10. Continue micronutrient studies on bacteria. 11. Begin work on LOX inhibitors. 12. Complete at least one dose-response study on COX inhibitors. 13. Begin PLA2 biochemistry. 14. Begin hemocytes chemotaxis studies. FY2008 1. Continue studies of enzymatic activities involved in defense against biological control agents. 2. Continue isolation and analysis of clones from cDNA library. 3. Continue immunity studies with AcMNPVhsp70Red. 4. Continue isolation and cloning of selenoproteins. 5. Continue analysis of phytochemicals. 6. Continue studies of other micronutrients. 7. Continue combinatorial micronutrient tests. 8. Complete micronutrient studies on bacteria. 9. Complete work on LOX inhibitors. 10. Begin work on PLA2 inhibitors. 11. Complete dose-response study on COX inhibitors. 12. Begin end-product rescue experiments. 13. Complete PLA2 biochemistry, begin isolation and cloning PLA2 14. Complete work on influence of inhibitors on chemotaxis, begin work on eicosanoids. FY2009 1. Continue studies of enzymatic activities involved in defense against biological control agents. 2. Continue isolation and analysis of clones from cDNA library. 3. Continue studies of immunosuppression. 4. Continue immunity studies with AcMNPVhsp70red. 5. Continue isolation and cloning of selenoproteins. 6. Continue analysis of phytochemicals. 7. Complete studies of micronutrients. 8. Complete combinatorial micronutrient tests. FY2010 1. Complete studies of enzymatic activities involved in defense against biological control agents. 2. Complete isolation and analysis of clones from cDNA library. 3. Complete studies of immunosuppression. 4. Complete immunity studies with AcMNPVhsp70red. 5. Complete isolation and cloning of selenoproteins. 6. Complete analysis of phytochemicals. 7. Publish results of above milestones. 4a.List the single most significant research accomplishment during FY 2006. This work is under National Program 304, Research Component A, Insects and Mites. Using a baculovirus engineered to express a red fluorescent protein (AcMNPVhsp70Red) the entire course of infection from initial infective foci in the midgut to other tissues can now be monitored in real time in intact live larvae, or in dissected tissues. Using this innovation we demonstrated that the course of baculovirus infection in H. virescens larvae was significantly slowed by dietary selenium (Se) supplementation. 4b.List other significant research accomplishment(s), if any. Under National Program 304, Component A, this research investigates insect immune responses to infections. So far we isolated and partially identified over 130 proteins that change in expression following an infection. This is now the most detailed appreciation of insect immune responses to infection available. This information will be used to characterize insect immune reactions to infection and for discovery of plant chemicals that influence insect immunity. 4c.List significant activities that support special target populations. None. 4d.Progress report. Dietary levels of certain nutrients influence may have a substantial impact on the ability to mass rear beneficial insects. Working with larvae of the moth Heliothis virescens, we investigated the influence of three nutrients, selenium (Se), vitamin E and vitamin C, on insect immune competence. In work with selenium (Se), we found that two biological forms of Se, selenate and selenocysteine, are optimal dietary supplements that enhance insect immune competence. We conclude that selenate is an inexpensive form of Se supplementation that may be used by commercial beneficial insect producers for mass-rearing of improved beneficial insects for biological control programs. In work with vitamin E, we found that vitamin E does not influence larval development or immune reactions to viral infection. On the other hand, we found that Vitamin C is essential for normal larval development and for effective immune reactions to viral challenge. Our studies of alterations in proteins present in insect larvae are producing new information on the influence of dietary inputs and microbial infection on expression of the genes encoding these proteins. Amending insect larval diets with Se led to identification of more than 47 proteins whose expression level was altered relative to control larvae. Infection with a bacterium led to identification of more than 49 proteins whose expression level was altered compared to un-infected controls. Viral infection cause changes in the levels of 18 proteins secreted into larval blood plasma. These analyses of the impact of dietary changes on gene expression are producing important new information that can be directly applied in the logical design of improved culture media to mass rear beneficial insects with improved biological control potentials. With respect to the influence of microbial infection on insect immune reactions, our work contributes important new fundamental knowledge on insect immunity. This information will ultimately lead to discovery of new points of vulnerability for logical development of more effective biological control agents. We worked to gain new knowledge on the course of viral infection. The first goal was to discover the time-window within the course of infection when dietary Se influenced the spread of infection within insect larvae. As described in 4a, we learned that the course of baculovirus infection was slowed in H. virescens larvae reared on medium amended with two levels of Se, 10 and 25 ppm. In continued research into the roles of prostaglandins and other eicosanoids in insect immunity we began a new line of investigation. Using established insect cell cultures, we considered the influence of pharmaceutical PLA2 inhibitors and cyclooxygenase inhibitors on viral replication in insect cells. Specifically, we treated a cell line (HzAM1) that is not permissive to viral replication with inhibitors and then challenged the cells with a baculovirus engineered to express red fluorescent protein in the early phase of infection (AcMNPVhsp70Red). In the days following infection we recorded numbers of fluorescent cells and total viral production. Our data indicate that treating the cells with inhibitors of eicosanoid biosynthesis results in changing the non-permissive cells into semi-permissive cells. We expect this research will ultimately reveal insights into cellular mechanisms of viral resistance. 5.Describe the major accomplishments to date and their predicted or actual impact. This project under National Program #304, Component A began 10/1/05 and accomplishments to date are presented in 4a. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The results and discoveries of our work are published in the refereed scientific literature and presented at prominent scientific meetings, where they are effectively transferred to other scientists working in these areas. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Selenium May Boost Insect Immunity by Laura McGinnis. Published online at News & Events at ars.usda.gov May 31, 2006. Keeping selenium from insects could allow virus to infect them by Andrea Johnson. Published in the Prairie Star July 19, 2006. Review Publications Shelby, K., Popham, H.J., Morris, S.J. 2005. Larval Heliothis virescens tissue selenium levels correlate with elevated immunocompetence against viral infection [abstract]. Entomological Society of America Annual Meeting. Available: http://esa.confex.com/esa/2005/techprogram/paper_20664.htm. Shelby, K. 2006. Nutritional immunology of larval Heliothis virescens [abstract]. Molecular Insect Science International Symposium Proceedings. p. 77. Shelby, K. 2006. Effects of vitamin and micronutrient deficiencies on the immunocompetence of larval Heliothis virescens [abstract]. University of Missouri Life Sciences Week. Available: http://lifesciencesweek.missouri.edu/uploads06/formatted_abstracts/shelbyk@1.doc. Popham, H.J., Shelby, K. 2006. Uptake of dietary micronutrients from artificial diets by larval heliothis virescens. Journal of Insect Physiology. Available at http://dx.doi.org/10.1016/j.jinsphys.2006.04.005. Popham, H.J. 2006. Antiviral response in Heliothis virescens [abstract]. Molecular Insect Science International Symposium Proceedings. p. 70. Stanley, D.W. 2005. A novel pathogenic mechanism in an insect bacterium: secretion of phospholipase A2 inhibitors [abstract]. Southeast Regional Society of Toxicology. p. 16. Goodman, C.L., Mcintosh, A.H., Stanley, D.W. 2006. Eicosanoids influence insect cell-viral interactions [abstract]. Congress on In Vitro Biology. 42:2A. Stanley, D.W. 2006. Eicosanoids mediate insect cellular immunity [abstract]. 2nd International Conference on Non-Mammalian Eicosanoids, Bioactive Lipids and Plant Oxylipins. p. 32. Park, Y., Stanley, D.W. 2005. A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions. Archives of Insect Biochemistry and Physiology. 60:105-115. Stanley, D.W., Miller, J. 2006. Eicosanoids in invertebrate immunity: an in vitro approach [abstract]. Congress on In Vitro Biology. 42:A4. Park, Y., Stanley, D.W. 2006. The entomopathogenic bacterium, Xenorhabdus nematophila, impairs hemocytic immunity by inhibition of eicosanoid biosynthesis in adult crickets, Gryllus firmus. Biological Control. 38(2):247-253. Stanley, D.W., Miller, J.S. 2006. Eicosanoid actions in insect cellular immune functions. Entomologia Experimentalis et Applicata. 119:1-13. Shelby, K., Popham, H.J. 2006. Plasma phenoloxidase of the larval tobacco budworm, Heliothis virescens, is virucidal. Journal of Insect Science. 6:16.