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? How insects feed and how they select plants for feeding and for oviposition are important in order to help develop new insect resistant plant varieties. We are characterizing the relationship between insect feeding and oviposition, and the morphology of host-plant leaves and mouthparts of phloem and xylem feeding pest insects via a specific cooperative agreement with the Department of Plant Pathology at North Dakota State University. We are studying the composition of the chemicals and determining their uniqueness to specific pests and beneficial insects. Biological control of pest insects is possible with the release of predators and/or parasitic wasps. Wasps of the genera Eretmocerus are useful for the control of whiteflies in greenhouses and in the field. Insects communicate by the use of chemicals. Often these chemicals are part of the surface of the insect cuticle and may protect the insect against desiccation. Some of these chemicals may be used by predators and parasitoids to locate insects as hosts, to stimulate feeding and to stimulate oviposition by the parasitoid/predator. Identification of these chemicals would allow them to be used to increase the attraction of parasites and predators to areas where they are needed to control pest insects and to improve rearing procedures for beneficial insects. Whiteflies became a very serious pest on vegetable crops and cotton in the southern US during the late 1980's by stressing the plants on which they feed, promoting microbial growth by excreting honeydew, and by transmitting plant viruses. Losses to crops and greenhouse plants were estimated at 200 million dollars annually. Biological control has been studied as a solution to the problem. The economic losses caused by the glassy-winged sharpshooter and the bacteria it transfers to cause Pierce's disease of grape plants have stimulated major research efforts to slow or prevent the spread of the vector, control the disease, find biological controls for the insect, and develop disease-resistant varieties of host plants. Efforts to reduce economic loss will benefit greatly from a more-complete understanding of the basic biology of the glassy-winged sharpshooter and its interaction with host plants. Providing a better understanding of the biochemical processes for beneficial pollinators as related to bee diapause, winter survival, vigor, pollinating performance, nesting site recognition and protection from pathogenic fungi, will assist in the development of species-specific artificial management protocols to maintain populations for commercial scale pollination needs, and thus protecting $10 billion in U.S. crops requiring cross-pollination. We are identifying the hydrocarbon and wax components of their surface lipids which may have an influence in attracting parasitoids. Also, cuticular and membrane lipids affect the permeability of eggs and insects to water movement and the uptake of cryopreservation agents during treatments necessary to enable them to withstand cold storage. Cuticular and internal lipids are also involved in the ability of insects to successfully overwinter. This area of research is aligned with the National Program involving Crop Protection & Quarantine (304) and has specific correlation to Component II: Biology of Pests and Natural Enemies and Component III: Plant, Pest, and Natural Enemy Interactions and Ecology, and National Program involving Crop Production (305) -- specific correlation to Research Component: Bees and Pollination. 2.List by year the currently approved milestones (indicators of research progress) Year 1 [FY 2006] Continue microscopic studies for interactions of whiteflies (WF) and glassy-winged sharpshooter (GWSS) with host plants. Determine cuticular lipids for whitefly parasitoid. Determine lipid changes for diapausing pollinator bees. Identification of nesting site materials. Develop methodologies for microscopic analyses of fungus germination in the larval alfalfa leafcutting bee (ALCB) gut. Year 2 [FY 2007] Continue microscopic studies for interactions of WF and GWSS with host plants – characterize stylet sheath material. Conduct microscopic studies on female parasitic wasp body parts. Determine internal lipid and glycerol (carbohydrate) profiles for ALCB. Continue studies on characterizing nesting site recognition chemicals. Using microscopic methods, document fungal spore germination in ALCB gut. Year 3 [FY 2008] Complete microscopic studies for interactions of WF and GWSS with host plants, continue studies on stylet sheath material and initiate studies on GWSS brochosome characterization. With collaborators, develop bioassays for parasitoid marking lipids. Continue studying internal lipid and glycerol (carbohydrate) profiles for ALCB. Complete studies on characterization of nesting site recognition chemicals. Continue to document fungal spore germination in ALCB gut using microscopic techniques. Year 4 [FY 2009] Complete studies on stylet sheath chemistry and polymerization process, and continue studies on GWSS brochosomes. Complete bioassays for parasitoid marking lipids. Complete internal lipid and glycerol (carbohydrate) studies for ALCB and initiate similar studies with blue orchard bees. Provide nesting recognition chemical for bioassays performed at ARS Bee Lab in Logan, Utah. Complete studies on fungal spore germination in ALCB gut. Year 5 [FY 2010] Complete studies on GWSS brochosomes. For blue orchard bees complete life-cycle studies on internal lipid and glycerol (carbohydrate). Complete studies on nesting chemical characterization for ALCB and blue orchard bees. 4a.List the single most significant research accomplishment during FY 2006. Whitefly feeding mechanism and interaction with host plant tissues characterized. The USDA-ARS-Biosciences Research Laboratory and the Department of Plant Pathology at North Dakota State University collaborated to investigate phloem penetration by sweet potato whiteflies, greenhouse whiteflies, and banded wing whiteflies on various crop host plants. The path of penetration through the abaxial (underside) surface of the leaf epidermis to the phloem tissue was studied using light microscopy, confocal scanning light microscopy, scanning electron microscopy, and transmission electron microscopy. We were able to make 3D movies that detail the entire path of stylets moving through the compound cell wall, and not through the cytoplasm, as they approach the sieve tube elements. We seek to document the ultrastructural characteristics of the penetration point where the stylets enter individual sieve tube elements. These findings are providing a better understanding of how the many species of whiteflies cause significant economic loss for a wide variety of field and horticultural crops. Damage to host plants can be a direct result of obligate phloem feeding, whitefly transmission of virus or non-viral plant disorders, or structural damage to vital host plant tissues. This research relates to Component II: Biology of Pests and Natural Enemies and Component III: Plant, Pest, and Natural Enemy Interactions and Ecology of the NP (304) Action Plan. 4b.List other significant research accomplishment(s), if any. Fungal spore germination in pollinator bee larvae assessed. Chalk brood disease in alfalfa leafcutting bees continues to be investigated. We have developed microscopic techniques to chemically clear and examine the digestive track (gut) of larvae to determine the presence and location of Ascosphaera aggregate fungal spores that cause chalk brood disease. It remains unclear as to how many spores are required to initiate chalk brood disease in the larvae, or where in the digestive track the spores germinate or how they invade the entire larvae. In the summer of 2006 we have been successful in obtaining feeding larvae within their nesting capsules that have ingested A. aggregate spores. Infected larvae at stages of development have been prepared for future confocal light and transmission electron microscopy in F07. This research relates to Component III: Plant, Pest, and Natural Enemy Interactions and Ecology of the NP (304) Action Plan and Research Component: Bees and Pollination of the NP (305) Action Plan. Cuticular lipid composition of female Eretmocerus mundus determined. Cuticular lipids were identified and percent composition determined by GC-MS. Major lipid constituents for female E. mundus include monomethyl- and dimethyl-branched, long-chained hydrocarbons. Findings will be correlated with the lipid composition from body parts (cuticular surfaces) as possible release sites for ovipositional marking lipids. This research relates to Component III: Plant, Pest, and Natural Enemy Interactions and Ecology of the NP (304) Action Plan. Lipid analyses of alfalfa leafcutting bee artificial nesting materials conducted. The USDA-ARS-Biosciences Research Laboratory and the USDA-ARS-Bee Biology Laboratory, Logan, UT collaborated to investigate the origin of bee nest recognition chemicals. The lipids from artificial nesting tubes have been identified for individual female alfalfa leafcutting bees and the results compared with lipids obtained from the cuticular surfaces of test bees, as well as, identified lipids from alfalfa flower pollen and leaves. This research relates to the Research Component: Bees and Pollination of the NP (305) Action Plan. 4c.List significant activities that support special target populations. None. 4d.Progress report. 58-5442-1-0331 This report serves to document the research conducted under a specific cooperative agreement (58-5442-1-0331) between ARS and North Dakota State University - Department of Plant Pathology. During the report period the cooperative project has employed confocal scanning light microscopy (CSLM), and both transmission and scanning electron microscopy to investigate whitefly feeding on host plant phloem tissue. The title of the project is "Xylem and Phloem Penetration by Plant Feeding Insects." Additional details of this research area can be found in the report for the parent CRIS 5442-22000-041-00D, "Biochemistry of Pest and Beneficial Insects and Interactions with Host Plants and Natural Enemies." A variety of host plants were examined including cotton, hibiscus, melons, and tobacco plants infested with sweet potato whiteflies, greenhouse whiteflies, and banded wing whiteflies. The path of the whitefly stylets from the point of penetration through the abaxial (underside) surface of the leaf epidermis to the phloem tissue was studied using a variety of microscopic techniques including light microscopy, confocal scanning light microscopy, scanning electron microscopy, and transmission electron microscopy. Using the techniques previously developed as part of this project, we were able to make 3D movies that detail the entire path of the stylets through the host tissues to successful penetration of individual sieve tube elements of the phloem. Scanning electron microscopy has provided high resolution images of the salivary sheaths within epidermal cells and throughout the leaf mesophyll, including attachment points on the surface of individual epidermal cells. Due to the ultra thin sections required, it is much more difficult to use transmission electron microscopy to follow the path of the salivary sheath and stylets from the point of penetration to the phloem. However, we have documented that the stylets actually move through the compound cell wall, and not through the cytoplasm, as they approach the sieve tube elements. Even though we can identify sheath material at the sieve tube elements using scanning confocal scanning light microscopy, we are still unsuccessful in documenting the ultrastructural characteristics of the penetration point where the stylets enter individual sieve tube elements. These findings provide a better understanding of the mechanisms of homopteran stylet penetration through host plant tissues toward the target sites (phloem and xylem sieve tube elements) that can adversely affected by stylet sheath polymeric materials and of plant diseases (bacteria, viruses, plant disorders) vectored by the stylet feeding pest insects. 5.Describe the major accomplishments to date and their predicted or actual impact. The accomplishments listed under 4a and 4b are the major accomplishments since this is the first year of the project. The impact of microscopic analyses of whitefly species feeding on selected crop plants provide a better understanding of the mechanisms of homopteran stylet penetration through host plant tissues toward the target sites (phloem and xylem sieve tube elements) that can adversely affected by polymeric sheaths of whitefly stylets that can transmit plant diseases (bacteria, viruses, plant disorders). The identification of lipids utilized by parasitic female wasps for marking their whitefly hosts will provide a better understanding of parasitoid biology and specifically, ovipositional behavior. Characterization of pollinator bee nesting attractants and the interaction of bees with pathological fungi will provide useful information for the better management of beneficial pollinator bees. The primary thrust of this research is relevant to the National Program 304, Action Plan Component II: Biology of Pests and Natural Enemies (Microbes), and Action Plan Component III: Plant, Pest, and Natural Enemy Interactions and Ecology, and National Program 305 Research Component: Bees and Pollination. Accomplishments of this project relate to Strategic Plan Goal #1, Objective 1.1, performance measures 1.1.1 and 1.1.2 as well as to Strategic Plan Goal #3, Objective 3.2, performance measures 3.2.5 and 3.2.6. 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 research findings on: elucidating the feeding mechanisms for glassy-winged sharpshooters and silverleaf whiteflies, and their interactions on host plant tissues; identifying parasitoid marking lipids related to ovipositional behavior; and the characterization of lipids for pollinator bees have been shared with ARS, university, and private sector scientists working on the control and management of these insects, and presented at scientific meetings. 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). Buckner, J.S., Pitts-Singer, T.L, Guedot, C.N., Kemp, W.P. 2004. Comparisons of adult cuticular lipids from the pollinator bees, Megachile rotundata (F.) and Osmia lignaria Say (Hymenoptera: Megachililidae). Annual Meeting of the Entomological Society of America, Salt Lake City, UT, November 14-17, 2004. Freeman, T.P., Leopold, R.A., Nelson, D.R., Buckner, J.S., Henneberry, T.J. 2004. Ultrastructure contributions to the study of the glassy-winged sharpshooter and Pierce's disease. Proceedings of the Pierce's Disease Research Symposium, Coronado, CA, December 8-10, 2004.