Gary J. Blomquist - US grants

Sex pheromone production in the housefly has been found to be associated with ovarian development, and further studies have demonstrated that a product from the ovary is essential for stimulating the biosynthesis of sex pheromones. Dr. Blomquist has shown that ecdysteroids from the ovary of the housefly regulate the biosynthesis of the pheromone, and he now wants to examine the regulation of the biosynthetic reactions by the ecdysteroids in more detail. These studies will increase our understanding of the biosynthesis and endocrine regulation of insect sex pheromones, and will provide essential basic information for designing specific techniques for insect control.

Vertebrates cannot synthesize and thus require a dietary source of linoleic acid, 18:2(n-6), which is used both as a structural component of membranes and as precursor for physiologically important eicosanoids. Until recently, it had been thought that insects, as is the case for vertebrates, were unable to synthesize linoleic acid. Studies have demonstrated that some, but not all, insect species possess a delta-12 desaturase and are thus able to produce linoleic acid. This proposal is designed to examine this synthesis of linoleic acid in the cockroach, Periplaneta americana, the aphid, Acyrthosiphon pisum, and the cricket, Acheta domesticus. Emphasis will be on studies designed to unambiguously rule out the contribution of microorganisms, both symbiotic and exterior, to linoleate synthesis, ie. that it is the insect tissue that is responsible for the desaturation of oleate to linoleate. In addition, the form of the substrate for the delta-12 desaturase and the further elongation and desaturation of linoleic acid will be examined. These studies will characterize the unique process by which certain insect species have gained a nutritional independence from dietary requirements for polyunsaturated fatty acids. Significant fundamental knowledge of comparative lipid metabolism will be obtained from this work. Linoleic acid is an essential fatty acid for vertebrates. It is an important membrane component and serves as a precursor for hormone-like substances that regulate many cell functions. Vertebrates lack the ability to synthesize linoleic acid and fulfill their dietary requirement by the consumption of plants, which, in addition to protozoa and fungi readily synthesize the fatty acid. Until recently, it was thought that insects, like other animals, were unable to synthesize linoleic acid. Recent evidence now suggests that insects do have this capability. This research will examine this phenomenon. It will provide a basic understanding of linoleic acid biosynthesis in insects and may have future implications for the exploitation of the insect-specific pathways of hormone regulation as a means of pest control.

This proposal requests funds to purchase a quadrupole mass spectrometer which would serve as the basis of a core mass spectrometry facility, to be located on the UNR campus. The instrument would be used to support the research of the six major users whose programs are described in this proposal, along with those listed as minor users. The demand generated by this level of research activity exceeds the capabilities of existing mass spectrometry instrumentation at our university. The university has agreed to pay 152,161 dollars towards the total cost of 304,322 dollars. The requested level of funding from NSF is the remaining 152,161 dollars. This figure reflects only the cost of the instrumentation itself. UNR will pay for the site renovation, operation, maintenance, and installation.

The goal of the proposed work is to gain a detailed understanding of the biochemical processes by which a product from the ovary initiates sex pheromone production in the common housefly. The housefly sex pheromone is a chemical attractant produced by the female to attract males for mating. A better understanding of sex pheromone production is needed to take advantage of this essential form of chemical communication in insects. The regulation of sex pheromone production in insects occurs via a brain factor in Lepidoptera, by juvenile hormones in cockroaches and beetles and by ecdysteroids in the housefly and perhaps in other Diptera. In none of these systems is it clearly understood which enzyme activities are affected to control sex pheromone production. The work proposed here is designed to gain a clear understanding of the enzymes influenced by ecdysone which result in sex pheromone production and to provide the basic information necessary to develop insect control strategies based on inhibiting sex pheromone production.

Insects in the order Coleoptera are among the most destructive pests in the United States. Bark beetles are responsible for losses of billions of cubic feet of coniferous standing timber each year in North America. The cotton boll weevil Anthonomas grandis accounts for over $300 million average annual yield loss and control costs. The bark beetles and the boll weevil are unique among animals in that both produce monoterpenes, C10 pheromone components that are required for mating and reproduction. The long term goal of this project is to develop new and effective pest management tactics based on insect pheromone systems. To fully exploit pheromone-mediated bark beetle and cotton boll weevil behavior from a management standpoint, a thorough understanding of the origin, expression and activity of these signals is important. Recent work in the Blomquist/Tittiger laboratory has demonstrated that the male pine engraver beetle, Ips pini, synthesizes ipsdienol de novo, and that production is controlled by juvenile hormone (JH) which induces 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R) and HMG-CoA synthase (HMG-S) transcript levels. Another key enzyme in monoterpenoid pheromone production is geranyl diphosphate synthase (GPPS), which controls the chain length of the isoprenoid product. Work is proposed to gain an understanding of the regulation of the activity of this enzyme in relationship to pheromone production and to clone, sequence and model the enzyme.
The studies outlined in this proposal are significant because of their impact on isoprenoid biochemistry and basic insect molecular biology. It is becoming clear that plant monoterpenes are synthesized entirely via the non-mevalonate pathway, making Ips spp. and A. grandis relatively novel systems for the study of the regulation of mevalonate-based monoterpenoid biosynthesis. Furthermore, there are few other insect models in which the molecular biology of isoprenyl diphosphate synthases have been studied, and none of these have involved GPPS or modeling studies. Also, none of these other models has a monoterpenoid end product that can be readily recovered in mg quantities as is the case with I. pini and A. grandis. Finally, Ips pini and A. grandis pheromone biosynthesis, and specifically the transcription of key isoprenoid enzymes, represents a non-developmental model system for the study of JH regulated gene expression. This work builds upon and expands the pioneering work on the molecular biological studies of insect pheromone regulation performed in the Blomquist/Tittiger laboratories.

This project is developing and implementing a Biotechnology Program at the University of Nevada, Reno. We are adapting courses and objectives from successful Biotechnology Programs at Ferris State University and Northwestern University. The program is a collaborative effort by the College of Arts and Sciences and the College of Agriculture, Biotechnology and Natural Resources, and extends from the University to two local community colleges, local middle and high schools, and the community at large. The specific goals of the biotechnology program are: 1) to meet the growing student interest in a biotechnology career-directed education via development of an intercollegiate biotechnology degree program, 2) to increase the scientific literacy of a wide diversity of undergraduate students through the addition of biotechnology into courses for science and non-science majors, 3) to enhance community interest and knowledge in the sciences by community outreach, and 4) to enhance economic development in the state by the creation of a well-trained work force of biotechnology scientists.

The surface of all insects is covered by a thin layer of very long-chain hydrocarbons that serve the critical function of restricting water loss to prevent a lethal rate of desiccation. Many species also use hydrocarbons in chemical communication. Despite their importance for an insect's survival and absence among most other animals, there have been few studies aimed at understanding the biochemistry and molecular biology of hydrocarbon formation. A clear understanding of this critical process in insects is also needed to design inhibitors of key steps that could function in novel and environmentally sound insect control techniques. The housefly, Musca domestica, is an excellent model insect with which to study the biochemistry and molecular biology of hydrocarbon formation. Ovarian produced ecdysteroids regulate hydrocarbon production, which results in the female producing (Z)-9-tricosene and a series of methyl-branched hydrocarbons, all of which function in the sex pheromone.
The work proposed herein is designed to gain an understanding of a critical process in hydrocarbon formation, the elongation of 16 and 18 carbon fatty acids to the very-long chain 24-36 carbon fatty acids that are the immediate precursors to hydrocarbons. Specifically, the objectives are to: (1) Isolate, clone and sequence key enzymes involved in hydrocarbon synthesis, (2) Determine the ecdysteroid regulation of these enzymes at the molecular level and (3) Assay the key enzymes.
In addition to training post-doctoral fellows and graduate students, undergraduates also will be trained in aspects of biochemistry and molecular biology. The proposed work will lead to a better understanding of critical processes in insects that could lead to new targets for future pest control strategies.

Juvenile hormone (JH) is critical for the development and sexual maturation of insects and other invertebrates. The mechanism(s) of JH signaling remains unknown due to the chemical properties of the molecule, the complexity of the processes it regulates, and the difficulty of identifying primary responder genes. The investigators will examine JH signaling in pine bark beetles, which have a devastating impact on US forests. The anterior midguts of adult male pine engraver beetles, Ips pini, produce large amounts of pheromone when the beetles are treated with JH. JH stimulates expression of genes in the mevalonate pathway, providing a simple and robust tool to monitor JH regulation. An expressed sequence tag (EST) database and cDNA microarrays of genes expressed in pheromone-biosynthetic midguts will be mined to identify genes that respond quickly to JH treatment. Rapid responders will in turn be used to screen a genomic library in order to isolate 5' flanking regions of mevalonate pathway genes, as well as others that react to JH. Putative JH-response elements are most likely located in this region of the genes, and will be identified by bioinformatic analyses. The investigators will also study the biochemistry and effects of JH on identified genes using short-term in vitro cultures of midgut cells. This work will fill a significant gap in JH research by bringing JH studies to the Coleoptera, an order that has otherwise been overlooked. In addition to providing new knowledge about JH signaling and pheromone biosynthesis, the investigators will uncover new molecular targets that may be used to manage these significant forestry pests. Students and postdoctoral fellows involved in the research will receive training in biochemistry, molecular biology, genomics and bioinformatics.

Bark beetles are among the most significant economic pests in North America, causing the loss of billions of board feet of timber each year and adding to the fuel load that contributed to the devastating wild fires in the western U.S. over the past several decades. The long term goal of this project is to develop new and effective pest management tactics based on pheromone systems. Except for a brief pheromone directed flight, bark beetles spend the majority of their lives protected beneath the bark of the trees they colonize and kill. Pheromones are essential for beetles to mount the 'mass attack' that leads to tree death. The work described in this proposal is designed to gain an understanding of the biochemical processes by which bark beetles produce pheromones. Bark beetles produce pheromone components in the midgut by mobilizing and up-regulating the enzymes of the mevalonate pathway and by specialization of the enzymes that detoxify tree defensive chemicals. These two processes are connected by a novel enzyme that serves two functions and produces the precursor to the major pheromone components of bark beetles. The work described in this proposal is designed to characterize this novel enzyme and to characterize other terminal steps in pheromone production. This grant will be used to train one post-doctoral fellow and one graduate student, and several undergraduates will also work on the project as they complete their senior thesis work.

Pine bark beetles can thrive beneath the bark of host trees, where the environment is poisonous to most other insects, because they have enzymes that can alter pine resin toxins into less dangerous chemicals. These "resin detoxification" reactions are thought to have evolved, in some cases, into pheromone biosynthetic reactions. Bark beetles rely on aggregation pheromones -chemicals that they release into the atmosphere that are perceived by other bark beetles- in order to coordinate colonization of new host trees. The pheromones and resin detoxification products are chemically similar monoterpenoids. Pheromone production and resin detoxification both require cytochrome P450 enzymes to chemically alter monoterpenoid structures.

The researchers will investigate the P450 enzymes involved in these two processes. They will use a combination of molecular cloning, enzyme assays, and computer modeling of P450 enzymes that modify monoterpenes in order to compare how the P450s are used and have evolved in different bark beetle species. These comparisons will allow scientists to begin to understand the evolutionary pressures at work on the beetle-host interaction. This work will contribute to long term efforts to manage destructive pine bark beetles by providing data that will guide development of pheromone-based control measures, and by identifying new, species-specific molecular targets for future management strategies.This project provides opportunities for graduate and undergraduate students to gain expertise in biochemistry, molecular biology, and molecular modeling. The researchers have a proven commitment to including members of under-represented groups in their research teams, and will continue to do so. Some of the enzymes characterized in this study may be useful as biotechnological tools to produce and/or modify monoterpenes in the food/cosmetics industries.

Reactive oxygen species (ROS) control many different processes in cells including cell death, stress responses, aging and cancer. Because the potential risk of oxidative stress is common to all aerobic organisms, elucidating the ROS-signaling network of plant cells would have a significant impact in medicine and agriculture. The long-term goal of the project is to dissect the ROS signaling network of cells and determine how ROS signals are sensed and transduced in plant cells. The experimental approach employed in the program will be to use a combination of genetic, molecular, biochemical and bioinformatic research tools to identify, clone and characterize different genes involved in ROS signaling in cells. The identification of genes involved in ROS sensing, the pathway for ROS signaling, and the collection of ROS-mutants, generated during the course of the proposed research, would serve as a valuable resource for the entire scientific community. The information generated from the project will enhance overall understanding of plant biology, and help determine to what extent and in what manner ROS are involved in different biological process. Broader impacts. To increase interest and understanding of science in young people, high school students and undergraduates, including minorities, will be recruited into the project and trained in ROS biology, molecular biology, and bioinformatics. This project will provide training to 2 graduate students, 8 undergraduates and 8 high school students. In addition, four 1 week summer workshops, 25 high school students each, are planned for the duration of the grant. The proposed project will provide interdisciplinary training that has become imperative for success in plant sciences. To enhance the awareness of high school students, teachers and undergraduates, two teaching-oriented websites targeted to high school students and undergraduates, and an online presentation targeted to high school teachers will be developed to share knowledge and enhance awareness to ROS metabolism in plants, mammals and other organisms.

Arabidopsis 2010: Abiotic stress combination: Bridging the gap between Arabidopsis stress research and agriculture

The work undertaken on this project is designed to make significant contributions to the goals of the 2010 program - to understand the networking and function of every gene in Arabidopsis. The specific focus of the project is on abiotic stress combinations and the genetic and metabolic networks that respond to stress combinations such as drought and heat, drought and salinity and salinity and heat. Abiotic stress is the primary cause of crop loss world-wide, with losses in the US estimated at 14-19 billion dollars each year. While abiotic stress is routinely studied in Arabidopsis by applying a single stress condition such as drought, salinity or heat, this type of analysis does not reflect the conditions that occur in the field where crop plants are subjected to a combination of different stresses. The central objective of the project is to identify novel genes, gene networks and metabolic pathways that specifically respond to a combination of two different abiotic stresses. The hypothesis to be tested is that dedicated genes, networks and pathways are activated in plants that are simultaneously exposed to two different stress conditions. This project is designed to bring Arabidopsis into the front line of applied research on abiotic stress tolerance, and bridge the gap between stress studies conducted with Arabidopsis in the lab and the conditions that impact crops in the field. The two key "Broader Impacts" of the proposed research are: 1) Development and maintenance of a centralized website that will bring together agronomists, breeders and Arabidopsis molecular biologists (http://www.ag.unr.edu/Stress_Combination/). 2) Educational outreach for K-12 and multidisciplinary training to postdoctoral, graduate and undergraduates trainees. Both undergraduate and K-12 outreach and training activities will target the under privileged and under represented in Science. Historically, abiotic stress combinations, such as drought and heat, had the outmost devastating economical and sociological impacts on the US, with losses of 48.4 and 61.6 billion dollars in 1980 and 1988 respectively. The proposed project will pave the way for the development of crops with enhanced abiotic stress tolerance, contributing to ameliorate the consequences of future weather disasters that are likely to increase in frequency due to anticipated climatic changes.

Bark beetles are among the most significant economic pests in North America, causing the loss of billions of board feet of timber each year and adding to the fuel load that contributed to the devastating wild fires in the western U.S. over the past several decades. The long term goal of this project is to develop new and effective pest management tactics based on pheromone systems (chemical secretions produced by the beetles). Except for a brief pheromone directed flight, bark beetles spend the majority of their lives protected beneath the bark of the trees they colonize and kill. Pheromones are essential for beetles to mount the mass attack that leads to tree death. This research project is designed to gain a better understanding of the biochemical processes by which bark beetles produce pheromones. Bark beetles produce pheromone components in the midgut by mobilizing and up-regulating the enzymes of the mevalonate pathway and by specialization of the enzymes that detoxify tree defensive chemicals. The final steps in pheromone production involve changing the stereochemistry of terpenoid alcohols to achieve the final blend that functions as the pheromone. This project is designed to characterize these enzymes. In addition, a post-doctoral fellow, a graduate student, and several undergraduates will be trained during the work on the project.

Project Summary The black legged tick, Ixodes scapularis, vectors several bacterial and viral pathogens, and is the major vector of Lyme disease in North America. Strategies that target this vector rather than individual pathogens stand to protect against several diseases. Thus, there is an urgent need to understand unique aspects of the tick?s biochemistry and physiology so that new targets can be identified for species-specific management strategies. Female ticks increase in size by approximately 100-fold during a several day long feeding period before dropping off the host to lay eggs. This phenomenon is barely understood at the biochemical or molecular level. A coordinated response modulating enzyme activity and structural protein production/ trafficking accompanies and enables the rapid expansion of a feeding adult female. As our first Aim, we will compare the proteomic profiles of the endocuticle (epithelial cell monolayer), which directs cuticular structure and remodeling, of unfed, ?slow phase? feeding, and ?rapid phase? feeding female ticks. We choose to work at the proteomic rather than transcriptomic level because protein levels often do not correlate with their cognate mRNAs, and protein levels provide a clearer idea of the biochemical response. This effort will produce the first comprehensive proteomic study of the cuticle of any tick. Our second Aim is to clarify the composition of the epicuticle ? the thin waxy outer layer that protects against desiccation. We challenge the paradigm that the epicuticle contains hydro- carbons. The literature notes instances of even-chain length n-alkanes and alkanes with unusual methyl branching patterns that contradict current understanding of biological hydrocarbon biosynthesis. We hypothesize that the epicuticle is dominated by dietary lipids transported directly from the midgut, and that ticks do not synthesize hydrocarbons. We will apply a multi-approach strategy incorporating proven as well as new survey methods to characterize epicuticle composition. Our work will clarify our understanding of how the tick cuticle is structured and remodeled during feeding, and correct our understanding of cuticle function. We will increase the range of molecular targets and provide new information that will accurately guide development of future tick management strategies.