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? Concerns about the impact of agricultural practices on environmental quality are increasing. Also, the effectiveness of traditional chemically-based insect and weed control measures has decreased due to resistance. These two major problems have increased the need for biologically-based integrated pest management strategies that include the use of beneficial insects for the control of pest insects and invasive plants. Populations of insect species that serve as natural enemies of pest insects and weeds could reduce the need for chemical inputs and facilitate sustainable pest management strategies. However, there are substantial obstacles to the successful implementation of biological control programs. One is the difficulty and expense of rearing beneficial insects in sufficient numbers for release at the appropriate times. Most beneficial insects are reared on their natural host, an inefficient approach. Consequently, artificial rearing and storage methods are needed for economical production of consistent populations of natural enemies. However, formulations of artificial diets have problems associated with the nutritional quality and other parameters. A second substantial obstacle is that no diet yields the highest quality beneficial insects. Life history parameters are valuable tools for recording the influence of artificial diets on insects. Unfortunately, life history studies are time consuming and not sufficiently sensitive to small changes in nutrition. Additionally, life history studies do not reveal the underlying biochemical processes that are affected by changes in nutrition. New technologies to sensitively measure alterations in digestive physiology hold promise of providing rapid and sensitive methods of detecting differences in gene expression and metabolic processes resulting from nutritional changes. The immediate challenge will be to translate the new technologies into genetic biomarkers for assessing the nutritional needs and fitness of natural enemies in commercial culture. At the same time, this information will also be helpful in understanding the effects that different plants in insect diets have on the effectiveness of biological control efforts. In addition to beneficial predatory insects, there is a need to develop more efficient and cost-effective techniques for the mass production of egg and pupal parasitoids, which also comprise a substantial portion of the commercial market. The goals of this research program are to advance the production, quality and effectiveness of mass-reared natural enemies (insects) for the integrated pest management of pest insects and weeds. There are three specific objectives:. 1)improve the formulation of artificial diets and diet-delivery systems;. 2)determine the impact of nutrient substitutions on the efficiency of diet utilization;. 3)develop genomic biomarkers that allow us to relate nutritional inputs to the quality of beneficial insects. This research will focus on the following insects: (A) the two spotted stink bug and the spined soldier bug, examples of valuable beneficial insects that are difficult and costly to mass rear; (B) the glassy-winged sharpshooter (GWSS), the primary vector of Pierce’s disease of grape; (C) the egg parasitoids, Gonatocerus spp. which account for most of the observed parasitism of GWSS in Texas and California and are a promising insect biological control agent(s) against GWSS (the research on GWSS and its egg parasitoids is a recent addition to this project with extramural funding to help expedite the work); (D) the pupal parasitoids, Nasonia spp. These parasitoids are one of the major regulators of insect populations in natural and agricultural insect communities. Because they parasitize blowflies and fleshflies, Nasonia spp. also are important regulators of arthropods of medical importance. Consequently, Nasonia spp. have occupied a significant market share for commercial beneficial insectaries. Within the life-time of this project it is expected that the Nasonia spp. genome will be sequenced, making Nasonia especially attractive for the development of biomarkers associated with nutritional and fitness responses. Increased availability and effectiveness of these biological control agents will facilitate their use in organic farming systems, or as a component of sustainable, integrated insect and weed management strategies. This research is an integral part of NP 304 Action Plan (100%), and contained within the Research Component A (Insect and Mites), Component II (Biology of Pests and Natural Enemies), Part II (Problems to be Addressed), section B (Rearing of Insect and Mites), and Component V (Pest Control Technologies), Goal 2 (Efficient production, delivery and utilization of beneficial organisms used in BC) and within the Research Component B (Weed Science), Component IX (BC of Weeds), Part II (Problems to be Addressed), section D (Combining BC Agents). This research fulfills two outcomes identified in the ARS Strategic Plan. The outcome targeting a safe and secure food and fiber systems is addressed by the development of new technologies that protect plants and reduce the impact of threats to agricultural production by rapidly and effectively managing pest insects and plants. The outcome targeting a greater harmony between agriculture and the environment is addressed by the development of environmentally safe methods to control pests for use in integrated pest management and sustainable agricultural systems. 2.List by year the currently approved milestones (indicators of research progress) FY2006 1. Test the performance of predatory pentatomids on diets where alterations have been made to the bovine and egg products and/or antibiotic products in current formulations. 2. Continue testing proteomic and genomic technologies to detect phenotypic and genotypic plasticity within the digestive track in response to changes in diet formulations. 3. Complete the selection of one or more biomarkers to be used for improving the formulation of the zoophytogenous diet for predatory pentatomids. FY2007 1. Test the performance of sharpshooters on artificial diet and diet-delivery systems. 2. Measure the phenotypic plasticity of the digestive enzymes in response to changes in diet formulations. 3. Test the temporal and sensitivity parameters of the biomarker(s) when used to evaluate changes to the fibrous, heat sensitive and/or antibiotic ingredients in the zoophytogenous diet. FY2008 1. Test the performance of predatory pentatomids on diets that have been improved based on biomarkers, and establish prey-fed and diet-fed colonies of Nasonia. 2. Continue digestive enzyme analyzes and initiate studies on the impact of PLA2 inhibitors on insects when delivered per os. 3. Continue to evaluate the performance of biomarkers for the zoophytogenous diet, and initiate a differentially expressed gene analysis of prey-fed vs diet-fed Nasonia. FY2009 1. Continue testing of the performance of predatory pentatomids on diets that have been improved based on biomarkers and initiate diet improvement parameters for Nasonia. 2. Initiate measurement of the impact of induced digestive enzyme activity and enzyme inhibitors on the ingestion and digestion of dietary nutrients. 3. Complete the selection of one or more biomarkers to be used for improving the formulation of an artificial diet for Nasonia. FY2010 1. Complete diet performance work with predatory pentatomids and continue testing of the performance of Nasonia on diets that have been improved based on biomarkers. 2. Continue measurement of the impact of induced digestive enzyme activity and enzyme inhibitors on the ingestion and digestion of dietary nutrients. 3. Test the temporal and sensitivity parameters of the biomarker(s) when used to evaluate changes to the artificial diet for Nasonia. 4a.List the single most significant research accomplishment during FY 2006. Identified genomic biomarkers to be used to optimize artificial insect diets. Recent developments in the area of nutrigenomics (originally coined in human biomedicine to represent nutritional genomics) hold the promise of providing valuable information about the impact of nutrition on a wide range of biochemical parameters by investigating how changes in nutrition alters gene expression patterns. The power of nutrigenomics in the development of artificial diets is found in the high degree of conservation in developmental and cellular pathways. That high degree of conservation translates into the possibility of isolating genes with known dietary functions or genes that are regulated by specific nutrients. This concept was successfully applied to a study of gene expression patterns in a beneficial insect fed optimal and sub-optimal diets and resulted in the first genomic-derived biomarkers to be used to optimize artificial diets for the cost-effect mass rearing of insects. This study identified two sub-optimal diet up-regulated and two optimal diet up-regulated genes in the predatory Pentatomid Perillus bioculatus. Of the four dietary-regulated biomarkers identified in this study two have been confirmed in other organisms to be involved in feeding behavior and digestive physiology. With an increasing knowledge base of dietary-regulated genes and the decreasing cost of screening techniques, nutrigenomics is poised to make significant contributions in the improvement of insect artificial diets. This accomplishment directly addresses a goal of NP 304 Action Plan, Part II, Section B (Rearing of Insect Mites) and Component V (Pest Control Technologies), Goal 2 (Efficient production of beneficial organisms used in BC). 4b.List other significant research accomplishment(s), if any. Characterized digestive enzymes that enable insects to feed on a wide range of plants. Digestive proteases play two critical roles in insect physiology – breaking down proteins into amino acids essential for growth and development and inactivating protein toxins ingested as a consequence of feeding. A thorough characterization of the digestive proteases found in the severe economic pest, the tarnished plant bug, demonstrated that its digestive activity is dynamic wherein the salivary gland and midgut enzyme activities complement one another, expanding the location and pH range over which digestion can occur. Possession of digestive proteases that remain active over a wide pH range and which consist of multiple protease classes may give this pest an adaptive advantage in the field and provide a mechanism for circumventing natural plant defensive mechanisms. However, more detailed knowledge of proteolytic mechanisms used by the tarnished plant bug will support the finding of new molecular sites that can be targeted by sustainable pest management practices to control this insect. This accomplishment directly addresses a goal of NP 304 Action Plan, Part II, Section B (Rearing of Insect Mites) and Component V (Pest Control Technologies). As part of a reimbursable agreement between ARS and the California Department of Agriculture (refer to the report for CRIS 3622-22000-030-01R) progress has been made on the development of an artificial rearing system for the glassy-winged sharpshooter (Homalodisca coagulata) (GWSS), the primary vector of Pierce’s Disease (Xylella fastidiosa) (PD). Effective and simplified delivery systems were developed for both immature and adult stages of development. The ability of this pest to digest protein was definitively established. This is important because it demonstrates that less costly nitrogen sources could be substituted for plant material when rearing this insect. Several digestive gene transcript levels that were evaluated remained stable over developmental time and changed very little whether the diet was cowpea or sunflower possibly indicating a minimal response to changes in nutritional quality or developmental stages. This information could provide new opportunities for a sustainable pest management approach to control this insect. This accomplishment directly addresses a goal of NP 304 Action Plan, Part II, Section B (Rearing of Insect Mites) and Component V (Pest Control Technologies). 4c.List significant activities that support special target populations. None. 4d.Progress report. Research into insect PLA2 function revealed the identity of a secretory PLA2 in insect hemocytes that acts in cellular immune reactions. Research into an insect pathogenic bacterium that inhibits lepidopteran secretory PLA2s showed that the bacterium also inhibits PLA2s in orthopterans. This led to a new hypothesis that secretory PLA2s are important in insect immunity, which has been articulated in papers and presentations in scientific meetings. 5.Describe the major accomplishments to date and their predicted or actual impact. The accomplishments listed above represent the life of the project. 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? Descriptions of the formulation and presentation of an artificial diet developed for the predatory pentatomids was made available to ARS, academic, and industry scientists through collaborations, discussions, and site visits. A private company is investigating the potential to market a powder form of the diet. Several researchers are testing the diet for rearing new pest and beneficial insect species and others are planning to test the effectiveness of the diet for the conservation of beneficial insects in field trials. Collectively, these efforts will explore the multifaceted benefits of a zoophytogenous diet. 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). None. Review Publications Coudron, T.A., Hunter, W.B., Labavitch, J.M. 2005. Finding the continuum between nutritional needs and feeding processes [abstract]. International Auchenorrhyncha Congress. p. 8-10. Coudron, T.A., Brandt, S.L. 2005. Precocious induction of vitellogenin with JH III in the two-spotted stink bug, Perillus bioculatus (Heteroptera: Pentatomidae). Comparative Biochemistry and Physiology. 142A:79-83. Wright Osment, M.M., Brandt, S.L., Coudron, T.A., Wagner, R.M., Habibi, J., Backus, E.A., Huesing, J.E. 2006. Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae). Journal of Insect Physiology. 52:717-728. Teran-Vargas, A.P., Rodriguez, J.C., Blanco, C.A., Martinez-Carrillo, J., Cibrian-Tovar, J., Sanchez-Arroyo, H., Stanley, D.W. 2005. Bollgard cotton and resistance of the tobacco budworm (Lepidoptera: Noctuidae) to conventional insecticides in southern Tamaulipas, Mexico. Journal of Economic Entomology. 98(6):2203-2209. Stanley, D.W. 2006. Prostaglandins and other eicosanoids in insects: biological significance. Annual Review Of Entomology. 51:25-44.