Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: As an extension of the Jefferson Science Fellowship, I was invited by Global Knowledge Initiative, a Washington DC non-profit organization, to spend 2 weeks in Rwanda 7-22 January 2012 to consult on an insect pest of coffee of the genus Antestiopsis. The visit included several lectures on new methods of pest control including symbiotic control to National University of Rwanda students and faculty members and stakeholders in the coffee industry. We also gave a briefing to the Minister of Agriculture, Agnes Kalibata. From June 21-24th a briefing was given to the Office of Science and Technology Cooperation, Department of State in Washington DC on projects in Rwanda, Papua New Guinea, and California including projects on symbiotic control. Then from June 24-28th I was invited by King Abdullah University of Science and Technology (KAUST) near Jeddah, Saudi Arabia to take part in organizing a Workshop on new technology for control of red palm weevil. The Workshop proposal was subsequently approved and funded and the meeting is scheduled for 13-19 March 2013 at KAUST. A large section is symbiotic control. I invited about a third of the participants and determined most of the agenda. From June to September Arinder Arora and Adam Forshaw came to my lab from collaborator Ravi Durvasula at University of New Mexico to field test microencapsulated symbiotic control agents against Pierce's disease of grapevines. They are planning to return next summer to perfect the delivery system. We applied for a patent for the microencapsulation packaging concept through UC Riverside and University of New Mexico. I helped organize the scientific program and was chief consultant to the Organizing Committee of the International Congress of Entomology held in Daegu, Korea 19-24 August 2013. I gave a lecture on International entomology for pest control at a satellite conference in Yeocheon, Korea, 17-19 August 2013. I invited collaborator Adam Forshaw to the same meeting; he gave a talk on Microencapsulation as a means to extend uv resistance to Metarhizium anisopliae for desert locust control. While at the entomology congress, I met the Director General of ICIPIE (International Center for Insect Physiology and Ecology, Nairobi, Kenya), Christian Bergemeister and asked his help in the coffee pest project in Rwanda mentioned above. We attended the National Entomological Society of America meeting in Knoxville, TN 11-14 November 2012. I consulted with the chair of the new ESA Science Policy Committee, Robert Peterson. We also attended the international session on control of red palm weevil to meet participants from Saudi Arabia elsewhere in connection with the Workshop mentioned above. I was invited to, but did not attend the BIODESERT meeting in Tunisia in December that was the final meeting of a research project aimed at exploring the natural resources of the Sahara desert for pharmaceuticals and pest control agents. Their main success was symbiotic control of American Foul Brood disease of bee hives. These activities contributed to the success of the project by strengthening collaborations in symbiotic control approaches. PARTICIPANTS: Thomas A. Miller was PI, Professor of Entomology; UC Riverside received no compensation such as summer salary. He oversaw the project along with laboratory manager, Genet Tulgetske. Genet M. Tulgetske remains on the project as a postdoctoral in the Miller lab. Lauren Wong, John Jones and Andrew Guzman are undergraduates working on the project. Ravi Durvasula is Associate Professor of Medicine at University of New Mexico Medical School. Miller's former graduate student at Riverside, Arinder Arora, transferred to the PhD program at U New Mexico and returned to Riverside in summer of 2012 to conduct field trials in symbiotic control of Pierce's disease of grapevines. He was joined in Riverside by another graduate student of Ravi Durvasula, Adam Forshaw. Angray Kang is Reader in Molecular Cell Biology at Queen Mary University of London. He is an expert on antibody research and perfected the main symbiotic control reagent used in symbiotic control. Surachet Charoenkajonchai, Miller's graduate student finished his dissertation at the end of 2011 and returned to Thailand where he is teaching at Chulalongkorn University in Bangkok. All of the websites in the Miller lab were redesigned by Andrew Guzman, the undergraduate. Other collaborators are Professor Yupa Hanboonsong, Khon Kaen University, Khon Kaen, Thailand. Miller invited Yupa to participate in the red palm weevil workshop scheduled for March 2013 in Saudi Arabia to explain the symbiotic control approach. Waqas Wakil, Professor of Entomology, Agricultural University, Faisalabad, Pakistan, is a new collaborator. He too was invited to the red palm weevil workshop to discuss entomopathogenic fungi to control insect pests. Collaborator Ravi Durvasula was also invited and is planning to attend the red palm weevil workshop to describe microencapsulation of symbiotic control agents and the symbiotic control strategy. Daniel Rukazambuka, Professor of Entomology, National University of Rwanda, Butare, Rwanda, is a new collaborator guiding students with Mario Serracin, coffee buyer and Director of African programs for Rogers Family Company, Lincoln, California. Together they are directing 7 graduate students studying integrated pest management of coffee insect pests. Simon Martin is head of coffee research for Rwanda Agricultural Board. He has an MS degree and we are seeking support for Simon to enter the PhD program in association with ICIPE in Nairobi, Kenya. Simon oversees isolation of microbes from coffee beans that might be responsible for potato taste defect. The collaborator there is Professor Juliana Jaramillo, head of coffee research at ICIPE. Also collaborating in ICIPE is Regis Babin who is being stationed at ICIPE from CIRAD (Center for International Research in Agricultural Development) in Montpellier, France. Former collaborator, Charles Dewhurst, just left the Papua New Guinea Palm Oil Association in PNG. His replacement is not yet in place. Ed Whitman, head of quality control, Rogers Family Company, provides research samples of green coffee beans for analysis of potato taste defect. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
As a direct result of the microencapsulation technique perfected by collaborators, a patent was applied for by UC Riverside and University of New Mexico. The new application attracted a group of investors who desired to start a new company to exploit the method. Desert locust control in North Africa is the first application. The company will be based initially in Albuquerque, New Mexico. The first conference call describing the company was held December 2012. The organization of the company will be established in the spring of 2013. Initial financing is expected to come from the Gates Foundation that is supporting field trials in Ethiopia and Tunisia taking place now. Gates wants proof of concept established before larger funding is awarded. In the California trials, it was a little surprising that microencapsulation was able to deliver a symbiotic control agent to glassy winged sharpshooter, Homalodisca vitripennis, and the agent gained access to the buccal cavity. It will be a challenge to document the acquisition. Delivery is the largest hurdle to overcome in applying the symbiotic control method. A publication came from collaborator Yupa Hanboonsong and her graduate student Jittawadee Wangkeeree on symbiotic control of white leaf disease of sugar cane in Thailand. Wangkeeree was a Golden Jubilee graduate student who spent her research abroad at UC Riverside working on the FISH method (Fluorescent In Situ Hybridization). The publication describes isolation and identification of a candidate symbiont for symbiotic control.

Publications

• Wangkeeree, J., T. A. Miller and Y. Hanboonsong. 2012. Candidates for Symbiotic Control of Sugarcane White Leaf Disease. Appl. Exp. Microbiol. 78: 6804-6811.
• Wangkeeree, J., T. A. Miller, and Y. Hanboonsong. 2011. Predominant bacteria symbionts in the leafhopper Matsumuratettix hiroglyphicus - the vector of sugarcane white leaf phytoplasma. Bull. Insectology 64: S215-S216.
• Miller, T. A. (series editor). 2010. Molecular Biology and Genetics of the Lepidoptera, ed by M. K. Goldsmith and F. Marec. Taylor & Francis Group LLC, Boca Raton, Florida, 362 pp. Miller, T. A. (series editor). 2012. Honey Bee Colony Health, ed by D. Sammataro and J. A. Yoder, CRC Press, Taylor & Francis Group. Boca Raton, Florida. 302 pages.
• Miller, T. A. 2012 Regulatory experiences in symbiotic control of Pierce's disease. In: Chris Wozniak and Alan McHughen (eds) Regulation of Agricultural Biotechnology, Springer Dordrecht, New York, pp 103-122.
• Kim, Y.J., Han, T.S. 2011. Hvmar1, a mariner-like element from the tobacco budworm Heliothis virescens, can transpose in Drosophila melanogaster. Entomological Research. 41: p.28-33.
• Wang, J., Miller, T.A., Park, Y. 2011. Identification of mariner-like elements belonging to the cecropia subfamily in two closely related Helicoverpa species. Insect Science. 18: 619-628.
• Charoenkajonchai, S. 2011. The use of symbionts expressing pear PGIP to delay or possibly prevent development of Pierces disease. PhD dissertation, University of California, Riverside, 98 pages.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Jefferson Science Fellowship August 2010 to July 31, 2011. Fellowship spent in residence in Washington DC attached to the US Department of State, Office of Science and Technology Cooperation. Established January 2011 an interagency Discussion Group within the Department of State on the subject of genetically modified insects. IAEA experts meeting on The Role of Bacteria in Fruit Fly in SIT Applications. 7-11 February 2011, Vienna Austria. Invited talk: "Biotechnology for pest and disease control." Monday 9 May Invited talk: "Biotechnology in Agriculture," Rural Development Agency, Suwon, Korea. Morning of 11 May 2011, American Embassy, Seoul Korean, met with Science and Technology officer and Agricultural attache. Discussed biotechnology for pest and disease control, activities of Jefferson Science Fellowship and the future International Congress of Entomology in Daegu, Korea scheduled for 19-24 August 2012. Seoul National University, 4 pm, 11 May 2011, invited talk: "Symbiosis in Medicine and Agriculture." Korean Society of Applied Entomology, 12-14 May 2011, Hongchon, Korea (Vivaldi Park). Invited talk: "Current status and future of applied entomology in USA, part of 50th anniversary of establishment of KSAE, in an International Symposium on session on Current status and future of applied entomology in Asia. 3 days in Iksan, Korea to the home of Byung Jin Kim, President of the upcoming meeting of International Congress of Entomology 19-24 August 2012. With us was James Ridsdill-Smith, Secretary of the International Entomology Council that supports and sanctions the ICE organization and meetings. 17 May 2011, Invited talk: "The Korean Economy, the role of science and education in development." Daejeon University, Daejeon, Korea. 18 May 2011, "Symbiosis in Medicine and Agriculture," Korean Advanced Institute of Science & Technology, Daejeon, Korea. 17-18 June 2011, invited talk on "Symbiotic control of Pierce's disease," at 2011 Baltimore Vector Encounter meeting at Johns Hopkins University, 615 N. Wolfe Street, Sheldon Hall. Meeting subsidized by Johns Hopkins Malaria Research Institute. 31 July 2011. Finished the Jefferson Science Fellowship year at US Department of State. Drove back to California. Transportation and moving provided by National Academy of Sciences and US Department of State. 29 August - 2 September 2011; gave an invited talk: "Manipulating DNA for the solution of pest and disease problems in agriculture and public health," at the Genetics Society of Brazil during their annual meeting in Aguas de Lindoas, Sao Paulo state. Gave another talk of the same title at Federal University of Alfenas, state of Minas Gerais. 12-16 November 2011, 59th meeting of Entomological Society of America. Formal report of Jefferson Science Fellow year and Advisory Committee of International Congress of Entomology to be held 19-24 August, 2012. 11-15 December 2011, invited talk: "BIOLOCUST, biotechnology for control of desert locust," at COST BIODESERT Workshop in Tunis, December 2011. Wrote two editorials about use of genetically modified insects for crop protection and disease control, both invited (listed with publications). PARTICIPANTS: Thomas A Miller was PI, Professor of Entomology UC Riverside received no compensation such as summer salary. He oversaw the project largely via email with Genet Tulgetski. Genet M. Tulgetske joined the project as postdoctoral in fall of 2010 and remains. Genet supervised candice and Laura Wong and is responsible for the main laboratory work. Ravi Durvasula is a PI on the BRAG grant supporting this project. He is Associate Professor of Medicine at University of New Mexico Medical School. His expertise is paratransgenesis of sandflies and triatomid bugs that transmit the pathogen causing Chagas disease. He has expanded his interests to global food security, which explains his participation with us in plant protection from pathogens transmitted by insects. Ravi is pioneering the use of nanotechnology as a delivery vehicle in symbiotic control with Sandia National Laboratories in Albuquerque. Raymond St. Leger is PI on our biopesticide control of plant pests with genetically modified and entomopathogenic fungi. He is Professor of Entomology at University of Maryland in College Park, MD. I visited his labs there during the course of the Jefferson Science Fellowship in 2011 and we had him for dinner to our apartment in Rosslyn, VA. Genet Tulgetski conducts bioassays on test insect with fungal biopesticides provided by the St. Leger lab. Surachet (Lek) Charoenkajonchai worked on the project and completed a PhD one month ago. Lek was guided by his dissertation committee during the time the PI was in Washington DC as a Jefferson Science Fellow. His dissertation title is The Use of Symbionts Expressing Pear PGIP to Delay or Possibly Prevent Development of Pierce's Disease. This amounts to proof of concept of this reagent to control PD. This title was not reported in publications. The publication date is December 2011. Arinder Arora graduated with an MS degree in December of 2010 and immediately joined the research group of collaborator Ravi Durvasula at University of New Mexico in Albuquerque. He is now in the PhD program there conducting research on Pierce's disease in California. An undergraduate, Candice Sanscartier, worked part time on the project. She monitored the presence of insect pests on sticky traps in experimental vineyards in agricultural operations at UC Riverside. She also did PCR analysis of leaf samples from experimental grapevines for the presence of Pierce's disease pathogens. Another undergraduate, Laura Wong, joined the project in September 2001. She is rearing insects and mailing samples and keeping the lab clean. Global Knowledge Initiative, Washington DC, a nonprofit organization. University of Maryland University of New Mexico California State University San Bernardino University of Milan, Italy University of Ioannina, Greece National University of Rwanda International Atomic Energy Agency, Vienna, Austria US Department of State Don Cooksey, UC Riverside, Department of Plant Pathology Jacob Vasquez, UC Riverside, Department of Bioengineering Stefan Jaronski, USDA ARS, Sidney, Montana TARGET AUDIENCES: Grape and wine industry in California; fruit growers; oil palm industry in Papua New Guinea; Specialty coffee industry in Rwanda. No significant public outreach this year. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The issue of use of genetically modified insects came to a head while I was serving as a Jefferson Science Fellow at the US Department of State and convened an interagency discussion group on the topic. The USDA ARS reported it was developing genetically modified screwworms with conditional lethal genes for use in the SIT program in Panama. They reported a cost analysis showed they would save millions of dollars a year by replacing radiation-based sterile insects with genetically modified screwworms with conditional lethal genes. USDA picked a person to lead the project who is fundamentally opposed as shown by word and deed to using genetically modified insect for any practical purpose. They could spend a million dollars and wind up with nothing to show for it. Moreover, the Key West Mosquito Control District requested permission in 2009 from USDA Veterinary Services branch to use Oxitec Aedes aegypti RIDL mosquitoes to eradicate a local population of the same species to stop a Dengue fever outbreak. The Dengue virus appears to have been brought to Key West by return of an infected traveler. By 2011 there were over a hundred clinical cases reported with no fatalities and evidence of the virus was present without symptoms in over a 1000 citizens of Key West on the basis of positive tests of blood samples for Dengue serotypes. When the USDA Veterinary Services declined to regulate, FDA decided they had regulatory authority. As the request enters its third year, FDA promises a decision soon but there is no evidence as this is being written 24 February 2012 that this will actually be done. All evidence, actions and deeds suggest that the US government will not proceed with anything beyond a glacial pace on these issues. The chief reason for this is the tendency for federal actions on this topic to draw law suits from environmental advocacy groups. While permits have been issued for genetically modified insects in the past, mostly for field trials under controlled conditions, these permits were successfully issued only after the law was rigidly adhered to and a dialog with environmental groups was undertaken. As a result permits did not draw a law suit.

Publications

• Miller, T. A. 2011. Genetically modified insects as used in SIT should not require regulation. Phytoparasitica 39: 415-418
• Miller T. A. 2011. Let Hi-tech Genetically Modified Insects Counter Dengue. American Institute of Biological Sciences. 61: 586-587 In-Press: Miller T. A. 2011 Regulatory experiences in symbiotic control of Pierces disease. In: Chris Wozniak and Alan McHughen (eds.) Regulation of Agricultural Biotechnology.
• Miller TA 2012 Random Thoughts on Recombinant Insects. Nova Publishers. In Press February 2012.
• Wang, J., G. S. Simmons, T. A. Miller, B. E. Tabashnik, and Y. Park 2011. Global variation in the PiggyBac-like element of pink Bollworm, Pectinophora gossypiella J. Asia Pac Entomol. 14: 131-135.
• Gai, C. S., F. Dini-Andreote, J. R. S. Lopes, W. L. Araujo, T. A. Miller, J. L. Azevedo and P. T. Lacava. 2011. Endophytic bacteria associated to sharpshooters (Hemiptera: Cicadellidae), insect vectors of Xylella fastidiosa subsp. pauca.J Plant Patho. & Microbiol.
• Azizi, A., A. Arora, A. Markiv, D. Lampe, T. Miller, and A. Kang. 2012. Ribosome display of combinatorial antibody libraries derived from mice immunised with heat-killed Xylella fastidiosa and the selection of MopB-specific single-chain antibodies. Appl. Exp. Microbiol.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: I have written papers on the subject of biotechnology for pest and disease control in agriculture for the past 13 years. Two such opinion papers came out of the 2007 International Congress of Insect Biotechnology and Industry (ICIBI2007) that I helped organize. These position papers attracted graduate students excited by the new area. The new book, Insect Biotechnology, 2011, 250 pp., from Springer-Verlag, edited by Andreas Vilcinskas, was inspired by topics covered at the ICIBI2007 meeting that Andreas attended. I am also a supporter and participant in the European Union research organization COST project on arthropod symbiosis (Action FA0701) that started about 4 years ago. I was invited to their most recent meeting in Crete, 19-22 September 2010, "Workshop on Applied Arthropod Symbiosis: from the development of novel technologies to commercial, legal and ethical considerations." I gave a talk on "Paratransgenesis as a potential tool for pest control" to be published. Three participants of that COST meeting, Kostas Bourtzis, the organizer, Boaz Yuval, from Israel and I were invited as expert consultants by the UN's International Atomic Energy Agency, Vienna, Austria, to participate in a workshop on the question use of symbionts to improve diets of mass-reared Medflies used in Sterile Insect Technique operations. This is scheduled for the second week of February 2011. I gave an invited talk on "Genetically modified insects as a tool for biorational control" in a session on Biorational Control methods organized by Isaac Ishaaya and Rami Horowitz of Israel at the European Congress of Entomology, 22-27 August 2010 in Budapest, Hungary. I gave another invited talk on "Microbial Toxicology" as part of a Symposium on "Diversity and function of microbial symbioses in solitary and social insects." organized by Angela Douglas and Kirk Anderson. The session was heavily attended. I was told later that my presentation sparked some lively discussion afterwards on regulation of transgenic and paratransgenic insects. While at this meeting, Kris Wyckhuys from the Tropical Agricultural Research Center (CIAT) in Cali, Colombia, asked me to help him write a grant for research on symbiotic control of Huanglongbing (Citrus Greening), a major disease of citrus in Florida, Brazil and Colombia. I saw this as an opportunity to help control the disease in the USA. California has the vector insect, but not the pathogen yet. I started an Interagency Discussion Group on release of genetically engineered insects as part of my Jefferson Science Fellowship at the US State Department (2010-2011). Since the group got started after the first of the year, results will be reported next year. One of the participants from USDA reported on a genetically engineering screwworm project intended for the mass-rearing facilities in Panama. This caught the participants from regulatory agencies FDA, USDA and EPA off-guard. It seems the regulatory responsibility for insect pests of cattle falls between agencies and needs to be sorted out. PARTICIPANTS: Collaborator, Ravi Durvasula, is Associate Professor, University of Mexico Medical School. Ravi has begun a very creative association with the Sandia National Laboratory also in Albuquerque, NM on packaging nanoparticles for delivery of microbial agents to control agricultural pests and diseases. Angray Kang, Reader of Biology, Westminster University, London, announced recently he is moving to Queen's College, London. He developed antibodies to the coat protein of the pathogenic bacterium Xylella fastidiosa that causes Pierces disease in grapevines. He has trained one graduate student while working on our project and the student was given permission to switch from an MS program to the PhD. My Lab director, Genet Tulgetske, finished PhD, UC Riverside in Biological control, and took over management of research on this project last August. She writes progress reports while I am away in Washington DC. She hosted a visit to our lab by collaborator Ray St. Leger, Professor of Entomology, University of Maryland. They were able to coordinate activities. She attended the National Entomology meetings in San Diego last December, and won a student award, The Lillian and Alex Feir Graduate Student Travel Award in Insect Physiology, Biochemistry or Molecular Biology. Genet has been mentoring two undergraduate students, Tyler Bui and Candace Sanscarter. Candace has applied for an undergraduate research grant from UC Riverside. She has been conducting an independent research project directly related to the Pierces disease research project. One graduate student finished his MS degree, Arinder Arora, and applied for the PhD program at University of New Mexico, Albuquerque, NM in the lab of Ravi Durvasula. Surachet Charoenkajonchai continues in the PhD program at UC Riverside and continues working on the Pierces disease project. He had the opportunity to undergo a stringent review by the graduate program that required him to rewrite his research objectives and apparently passed the review. His graduate committee is taking a more active interest in my absence. Jacob Vasquez joined the lab in summer of 2010 at the insistence of Genet from the UCR bioengineering graduate program. They do not provide graduate student support so Jacob is paid to work on our biotechnology projects. Since he is an expert in nanotechnology, we benefit from having an instant-expert in the lab at the start of the nanoparticle delivery project; it is a cross-fertilization of engineering and entomology. I asked Genet Tulgetske to organize a program on women in entomology for the next Entomology Congress in Korea, August 2012. She appears to be rather good at that sort of thing. I am organizing the Biological Control section for the next International Congress of Entomology 19-25 August 2012 in Daegu, Korea. It affords the opportunity to contact outstanding entomologists over the world and get them involved. I am trying to get the World Bank and US AID interested in a training grant for countries afflicted by desert locust. TARGET AUDIENCES: Certain of my publications and much of my tenure as Jefferson Science Fellow at the US State Department in Washington, DC are aimed at addressing the procedures and attitudes of regulators of biotechnology in US government. This is fairly difficult since those attitudes were framed by the process of approving the original transgenic crops. The regulatory apparatus is geared to hide proprietary information to protect new technology and costs are paid for by expected seed sales carrying a "technology" fee. All small applications using new technology lack large markets and are easily discouraged by demands for expensive environmental impact or risk assessment studies. Therefore, I was surprised to learn than none of the transgenic crops now in use was the object of an environmental impact study. There are groups strongly opposed to anything being genetically modified that will not be moved. In spite of this, various other groups seek biotechnology solutions to problems with no alternative treatments and thus no options, such as Huanglongbing (previously known as citrus greening) and Pierces disease. The current controversy over release of conditional lethal GE Aedes mosquitoes to eradicate dengue fever in local areas is a good example of that dynamic at work. PROJECT MODIFICATIONS: The only new wrinkle in our approach is to initiate packaging of microbes in nanoparticles for delivery of pest and disease reagents. This is the subject of a new risk assessment grant from the BRAG program at NIFA. It is an opportunity to contain the reagents until key conditions for release are met; in other words a new delivery paradigm.

Impacts
One MS dissertation was produced last year from this project. A Risk Assessment grant was funded by USDA/NIFA for employing nanoparticles to deliver symbiotic control agents to glassy-winged sharpshooters. I was invited by a senior editor of Spring-Verlag, to become an editor of a new Spring Book Series, "Biological Inspired Systems." Currently, there are two Editors of the series, Adam Summers from University of Washington and Stanislav Gorb from the University of Kiel, Germany. They cover the biomechanics, biomaterials, biomimetics quite well, but were keen to extend the scope of the book to other biologically related technologies and thought I would be a perfect candidate to join them. Although I was interested, my current contract with Taylor & Francis overlapped a little too much and I had to turn it down. The antibodies to the pathogen, Xylella fastidiosa, that causes Pierce's disease were finally isolated by our collaborator Angray Kang in London. We will bundle these genes into the delivery symbiont, Pantoea agglomerans, for use in treating grapevines to prevent Pierce's disease. We started a new research project to evaluate the Risk Assessment of using nanoparticles to deliver symbiotic control microbes. The nanoparticles are being developed in cooperation with Sandia Laboratories and collaborator Ravi Durvasula, University of New Mexico Medical School.

Publications

• Miller, T.A. 2011. Paratransgenesis as a potential tool for pest control: review of applied arthropod symbiosis. J applied Entomology. (in press).
• Morrison, N.I., Franz, G., Koukidou, M., Miller, T.A., Saccone, G., Alphey, L.S., Beech, C.J., Nagaraju, J., Simmons, G.S., Polito, L.C. 2010. Review: Genetic Improvements to the Sterile Insect Technique for Agricultural Pests. As. Pac. J. Mol. Biol. and Biotech. 18:275-295 Arora, A. 2010. Symbiotic Control of Pireces Disease of Grapevines. MS dissertation, UC Riverside, December 2010.
• Yu Jung Kim, Y.J., Han, T.S. and Miller, T.A. 2011. Hvmar1, a mariner-like element from the tobacco budworm Heliothis virescens, can transpose in Drosophila melanogaster. Entomological Research (in press).
• Wang, J., Simmons, G.S., Miller, T.A., Tabashnik, B.E., and Park, Y-S. 2010. Global Variation in the piggyBac-like Element of Pink Bollworm, Pectinophora gossypiella. Journal of Insect Science (in press)

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: We have identified symbiotic bacteria as candidates for delivery of anti-Pierce's disease (PD) agents to control PD in California and others for control of Citrus variegated chlorosis in Brazil. Our collaborator, David Lampe at Duquesne University in Pittsburgh PA has developed a method for genetically altering the symbiotic bacteria and we are now using genetically marked bacteria to locate where in the insect they are being transmitted. The website arising from this project are still active: http://biopesticide.ucr.edu; http://www.symbiosis.ucr.edu; http://piercesdiseasetrial.ucr.edu/. The latter website shows a vineyard during symbiotic control treatment for control of Pierce's disease. It includes ELISA data of the pathogen present and condition of the plants. I gave a talk at the Volcani Center in Israel about symbiotic control of pests and diseases that was well received and a talk at the International IPM symposium in Portland Oregon in March 2009. Gave poster presentations at the International Symbiosis Society Congress in Madison WI in August 2009, gave an invited talk on the same subject at Khon Kaen University in Thailand in early July 2009. My host there, Professor Hanboonsong picked me to host her graduate student, Jureemart Wangkeeree, for PhD research on localization of symbiotic bacteria in leafhoppers that transmit pathogens causing white leaf disease of sugar cane, a major problem in Thailand. Miss Wangkeeree is winner of a Golden Jubilee Fellowship designed to identify and train future research leaders in Thailand by paying all expenses for research abroad during the PhD training and including a postdoctoral period abroad as well. The fellowship includes exchange visits between the host and foreign laboratory directors. I gave a similar talk at Chonnam National University in Korea also in July 2009. Also in July I attended the opening of the Insect Museum in Uljin, Korea where a room is dedicated to my research accomplishments. This was part of the Uljin Insect and Organic Food EXPO held there every 3 years on the ground of the County Fair. These talks and reports along with review chapters on similar topics have attracted some attention to the symbiotic control approach. PARTICIPANTS: Jennifer Parker earned a PhD on this project in the Cellular Molecular and Developmental Biology graduate program at UC Riverside. She is now at the Law School of Golden Gate University in San Francisco where she will begin legal internships next summer. She has been interviewed by the US Food and Drug Admininstration. I expect big things from her. A new collaborator is Professor Raymond St. Leger at Entomology Department, University of Maryland, College Park, MD. A series of UC Riverside undergraduate students have worked on the project including Robert Sogomonian, who left in August 2009 to enter medical school; Meng Wang who left in December to enter the work force; Rolf Gabrillo due to graduate in June and taking research units now; Candice Sanscartier is working now part time; Derek Shaibi is working part time; Maria Gutierrez and Grace Radabaugh are now taking research units and working on projects due in April. David Hawks, a senior technican in the Agricultural Experiment Station has joined the project part time. TARGET AUDIENCES: I wrote a book on the difficulty of obtaining permits for use of transgenic organisms for use to control pests and diseases in agriculture. It is being reviewed now. The intention is to influence policy. I applied for a Jefferson Fellowship to spend a year in the US State Department. I seek assignment with the economic bureau of US AID to influence biotechnology applications in developing countries. I was asked if I would be an agricultural advisor to the Governor of Hampyeong County in Korea. He plans to run for the Governorship of Chonnam Provence and is assembling a team of advisors. I find out about the Jefferson Fellowship this month. The book is being reviewed by Oxford University Press and Wiley-Blackwell. Barring major revisions, it could be published in August. I am a chief advisor to the President of the International Congress of Entomology to be held in Daegu, Korea in August of 2012. We expect to have a session there on regulatory procedures to support delivery of biotechnology applications in agriculture beyond transgenic plants. PROJECT MODIFICATIONS: We expanded our project to include development of transgenic fungi (entomopathogenic fungi) to control specific pests of crop plants.

Impacts
The conduct of this project and dissemination around the world at international meetings has had and continues to have a major impact. For example the Daegu Protocol approved at the International Congress of Insect Biotechnology & Industry in 2007 has been adapted by the Mexican government as the template for approval of transgenic organisms. The satellite symposium hosted by the Ministry of Agriculture, Rabat Morocco and UC Riverside in July of 2008 on the subject of biotechnology for control of desert locust was followed by a Workshop at the Orthoptera Congress in Antalya, Turkey in June 2009 generated a proposal to develop biotechnology for control of katydid pests of oil palm trees in Papua New Guinea (PNG). This in turn was chosen by the new NSF-Gates BREAD program for 3 years of funding and include genetic engineering of Metarhizium anisopliae fungus for specificity against the Sexava types of katydid that are only pests in PNG. It is clear that symbiotic control of Pierce's disease in California will only be possible with more movement in the regulatory aspect.

Publications

• Gai, C. S., P. T. Lacava, W. Maccheroni Jr., C. Glienke, W. L. Araujo, T. A. Miller and J. L. Azevedo (2009). Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy. J Basic Microbiol. 49: 1-11
• Gai, C. S., P. T. Lacava, M. C. Quecine, M-C. Auriac, J. R. S. Lopes, W. L. Araujo, T. A. Miller and J. L. Azevedo (2009) Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for Paratransgenic Control Strategy of Citrus Variegated Chlorosis. The Journal of Microbiology 47: 448-454.
• Mikolo B., Matos L., Massamba D., Mamonekene V. and Miller T. (2009). "Manuscript ID ENR-09-035 entitled "Extracts from the bark the Fagara heitzii (Aubr. et Pel.)(Rutaceae) tree are toxic to two weevils and the American cockroach." Entomological Research 39: 401-405.
• Wang, J., Miller, E. D. Simmons, G. S. Miller, T. A. Tabashnik, B. E. and Yoonseong Park. (2009) PiggyBac-like Elements in the pink Bollworm, Pectinophora gossypiella Insect Molecular Biology (Revision May 4, 2009, 25 manuscript pages). (IN PRESS)
• Gai, C. S., P. T. Lacava, J. R. S. Lopes, W. L. Araujo, T. A. Miller and J. L. Azevedo (2009). Bacterial community associated to sharpshooters (Hemiptera: Cicadellidae), insect vectors of Xylella fastidiosa subsp. pauca. [21 MS pages]. (IN PRESS)
• Miller TA (series editor). 2010. Molecular Biology and Genetics of the Lepidoptera, ed by M. K. Goldsmith and F. Marec. Taylor & Francis Group LLC, Boca Raton, Florida, 362 pp.
• Miller TA (series editor). 2009. Food Exploitation by Social Insects, ecological, behavioral, and theoretical approaches, ed by S. Jarau and M Hrncir. Taylor & Francis Group LLCX, Boca Raton, Florida, 348 pp.
• Lacava, P.T., J. L. Azevedo, T. A. Miller and J. S. Hartung. 2009. Citrus variegated chlorosis (CVC) and interaction with endophytic bacteria: A review. Global Science Books ltd.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Helped the Ministry of Agriculture in Morocco organize and run a meeting on biotechnology for control of desert locust,1-4 July 2008 in Rabat, Morocco. Developed a website to disseminate the results: http://biopesticide.ucr.edu. There were some 50 locust and biotechnology scientists invited to this excusive 3-day meeting discuss possible advances in biotechnology that might provide new tools for locust control. A follow up symposium was designed for the International Orthoptera Society meeting to be held in Antalya Turkey 21-25 Jun 2009 to set priorities for applied research projects. The units represented were FAO, Rome, USAID, Washington, DC, IAEA, Vienna, Austria and research groups from over the world including two from China, two from Australia, several from Europe and locust workers from across Africa including the ICIPE, Internatinal Center for Insect Physiology and Ecology in Kenya. Some of the research being reported last July is only now showing up in the world press (5 February 2009, Los Angeles Times, describing the role of serotonin in triggering aggregation phase of development. This was reported in a plenary lecture by Steve Simpson from University of Sydney. I was invited to participate in a symposium in Korea, "Trends on the development of insect resources in Korea and abroad," as part of the Hampyeong World Butterfly and Insect Expo, Korea, 26 April 2008. Also as part of that participation, I was invited to and helped in planning for an Insect Museum in Uljin County, Korea. The Uljin County officials are planning a room in their new museum to highlight my research in entomology. The dedication and opening ceremony is scheduled for July 2009. The Hampyeong lecture appeared in Entomological Research, published by the Korean Entomological Society. On 7 May 2008 we planted a demonstration vineyard in Agricultural Operations at UC Riverside. The vineyard comes with a website: http://piercesdiseasetrial.ucr.edu. This site is intended to afford access by the California grape and wine industry to trials of treatments designed to protect grapevines from Pierce's disease. I was asked in 2008 to join the Biotechnology Impacts Center at UC Riverside. The founder, Norman Ellstrand, has since stepped down and BIC is in hibernation as of this writing (February 2009). [2310 characters with spaces]. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The trial plots mentioned above at Agricultural Operations, UC Riverside are the result of isolation and characterization of microbial elements associated with Chardonnay grapevines in Temecula, CA that appeared to be resistant to the initial Pierce's dieseae epidemic that occured in 1998-2000. One trial includes apparent phage virus elements that suvive filtering that removes most bacteria. the other trial invovles a Paenibacillus bacterial strain isolated from Temecula. We are tested this "biopesticide" in the laboratory and found it conferred resistance to drough in grape seedlings. Paenibacillus is reported to confer drough resistance in the literature. A trial was also established in a large organic vineyard in Temecula,that still shows abundant signs of grapevine replacement due to Pierce's disease. Being organic, the grower cannot use the now common practice of systemic treatment with imidacloprid to control the vecotor insect, Homalodisca coagulata, the glassy-winged sharpshooter (GWSS). Thus the key to control of Pierce's disease in Temecula includes finding a method to protect the organic grower. The Paenibacillus approach should be compatible with the organic grower community.

Publications

• Ramirez, J. L., T. M. Perring and T. A. Miller. 2008. Fate of a Genetically Modified Bacterium in the Foregut of Glassy-winged sharpshooter. J Econ Entomol. 101: 1519-1525.
• Dugravot, S., E. A. Backus, B. J. Reardon and T. A. Miller. 2008. Correlations of cibarial and precibarial muscle activities of Homalodisca spp. Sharpshooters (Hemiptera: Cicadellidae) with EPG waveforms and excretion. J Insect Physiol. in press.
• Miller, T. A. 2007. Pest and disease challenges and insect biotechnology solutions. Entomological Research 38: 43-40.
• Miller, T. A. 2008. Advances in insect biotechnology for human welfare. Entomol Research. 38: S9-S14 (Special Issue).
• Miller, T. A. 2007. Research advances and lessons learned. Entomological Research XX: 000-000.
• Ramirez, J. L., P. T. Lacava and T. A. Miller. 2008. Detection of Xylella fastidiosa in GWSS saliva. J. Insect Science 8:34, available online: insectscience.org/8.34.
• Simmons, G. S., L. Alphey, T. Vasquez, N. I. Morrison, M. J. Epton, E. Miller, T. A. Miller and R. T. Staten. 2007. Potential use of a conditional lethal transgenic pink bollworm Pectinophora gossypiella in area-wide eradication or suppression programmes. In: M. J. B. Vreysen, A. S. Robinson and J. Hendrichs (eds.), Area-Wide Control of Insect Pests: From research to field implementation, Springer, Dordrecht, The Netherlands.
• Bourtzis, K. and T. A. Miller (editors). 2009. Insect Symbiosis, Volume 3. CRC Press/Taylor and Frances, Boca Raton, FL, London
• Alphey, L. K. Bourtzis and T. A. Miller (2008). Genetically Modified Insects as a Tool for Biorational Control. A chapter in the book, "Biorational Control of Arthropod Pests: Application and Resistance Management," edited by Isaac Ishaaya, A. Rami Horowitz, Springer-Verlag, Berlin and Heidelberg, Germany.

Progress 01/01/07 to 12/31/07

Outputs
Invited talks at Society for Experimental Biology in Glasgow, Scotland, 30 March to 4 April 2007; talk on "Symbiotic control applications in agriculture and medicine." Talk of same title at: Ministry of Agriculture and Fisheries, Rabat, Morocco, 27 March 2007. I am helping the Moroccans organize a Workshop on biotechnology applied to control desert locust, 1-4 July 2008, Rabat, Morocco. At a Forum on biotechnology, "Discussing Genetic Engineering in the Wine Industry" held in Napa, 20 June 2007, gave a talk on "Symbiotic Control of Grapevine Pests and Disease." I gave a plenary lecture at the annual meeting of the Korean Association for Biological Sciences, Seoul, Korea, 15 August 2007. "Pest and disease challenges and insect biotechnology solutions." The talk was very well received. I was asked to help Korea organize the International Congress of Insect Biotechnology and Industry (ICIBI), 19-24 August 2007, Daegu, Korea. At the ICIBI meeting, I gave three invited talks, the Plenary Lecture, "Applying insect transgenic technology: scientific and regulatory experiences;" a lecture on insect diapause, "Diapause research in pink bollworm aids crop protection;" and another on "Research Advances and lessons learned during the ICIBI excursion to Yecheon, Korea. Following ICIBI, I was invited to give a talk at Chonnam National University in Kwangu, Korea. I was also invited to Hampyeong, Korea and eventually asked to help organize the 2008 World Butterfly and Insect Expo at Hampyeong Ecological Park. 30 October 2007 I attended the pink bollworm review session in Maricopa, AZ as a member of the technical advisory committee. I was invited to National Research Council Workshop on Genetically Modified Organisms, Wildlife and Habitat, UC Irvine Mon-Tues 5-6 Nov 2007. I gave a talk on "Research on the effects of GE insects on wildlife and their natural habitats." Was also invited to Agricultural Biotechnology Risk Analysis Research (AGRA) in the Federal Government meeting, 5-6 December 2007, FDA Wiley building, College Park, MD. I have a talk on "Transgenic and Paratransgenic insects in crop protection." My laboratory and collaborators attended the annual Pierce's disease symposium in San Diego, CA 12-14 December 2007 hosted by the California Department of Food and Agriculture. I tried to organize a second "Regulatory Communication Workshop for June 2008, John's Hopkins University, Baltimore, but received no cooperation from the regulatory agencies and gave up the effort. The website developed following the first Workshop is still active, http://biopesticide.ucr.edu and is now also being used to organize the meeting in Rabat Morocco, 1-4 July 2008. We isolated endophytic bacteria from apparently healthy grapevines at two sites with extensive Pierce's disease (PD) in Southern California. One of these bacteria showed an ability to block growth of the PD pathogen, Xylella fastidiosa in laboratory cultures. We helped a European group, SYM4EU, develop its own Symbiotic Control project aimed at several pests and diseases including grape yellows disease of vineyards.

Impacts
The first practical example of new pest control strategy we developed called ABC, Autocidal Biological Control just finished a second year of field trials in Arizona. A Risk Assessment grant proposal is being written to study impacts of the new technology. The majority of grapevines in the wine-growing area of Temecula, CA is now being protected by systemic insecticide treatments of the Bayer product, Admire with imidacloprid insecticide. This controls the main vector insect, glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, from spreading the pathogen, Xylella fastidiosa that causes Pierce's disease (PD). We analyzed petioles from organic vineyards in Temecula last season and learned that they are acting as harborage for the pathogen because they cannot use imidacloprid as the commercial vineyards do. We will begin testing a biological control method this next season that employs endophytic bacteria that appear to confer resistance to the pathogen by pretreatment of the grapevines. The PD problem in Southern California is caused by presence of GWSS. The entire region is now under quarantine in which all nursery stock is treated prior to shipment elsewhere in the state. The quarantine is a major disruption for the large nursery industry in Southern California. Alternatives to neurotoxic insecticides to treat the vector insect would be welcome.

Publications

• Sun, Z., Wu, M., Miller, T. A. and Han Z.-J. (2008) piggyBac-like elements in cotton bollworm, Helicoverpa armigera (Hubner). J Molecular Entomology. 17: 9-18.
• Ramirez, J. L., T. M. Perring and T. A. Miller. 2008. Fate of a Genetically Modified Bacterium in the Foregut of Glassy-winged sharpshooter. J Econ Entomol. (in press)
• Ramirez, J. L., P. T. Lacava and T. A. Miller. 200X. Detection of Xylella fastidiosa in GWSS saliva. J Insect Science, June 2007, (in press).
• Lacava, P. T., J. Parker, F. D. Andreote, F. Dini-Andreote, J. L. Ramirez, T. A. Miller. 2007. Analysis of the bacterial community in glassy-winged sharpshooter heads. Entomological Research 37: 261-266.
• Miller, T. A. 2007. Applying insect transgenic technology: Scientific and regulatory experiences. Entomological Research 37: 67-75.
• Miller, T. A. 2007. Symbiotic control in agriculture and medicine. Symbiosis 44: 67-73.
• Miller, T. A. 2007. Research advances and lessons learned.Entomological Research XX: 000-000. (Invited review submitted June 2007). 20 MS pages single spaced; 10 figures, 1 table.
• Bourtzis, K. and T. A. Miller (editors). 2008. Insect Symbiosis, Volume 3. CRC Press/Taylor and Frances, Boca Raton, FL, London, (in press).
• Miller, T. A., P. Lacava and J. L. de Azevedo. 2007. The Philosophy of Symbiotic Control in Agriculture and Medicine. In: J. L. de Azevedo and I. S. Melo (eds.) Biological Control 4, (in Portuguese, Controle Simbiotico de Pragas e Doencas. In: Melo, I.S. (Org.); Azevedo, J.L. (Org.). Controle Biologico IV. Jaguariuna: Editora EMBRAPA, in press).

Progress 01/01/06 to 12/31/06

Outputs
Public hearings on an Environmental Impact Statement for transgenic pink bollworm release were conducted in Washington DC, 17 Jan; Ontario, CA, 23 Jan; Tempe, AZ, 25 Jan; Weslaco, TX, 30 Jan; and Tampa, FL, 1 February 2007. These hearings were very sparsely attended; suggesting that opposition to release of transgenic pink bollworm has largely dissipated. A formal review of the transgenic pink bollworm project was conducted on 24 January 2007 in Phoenix at the Arizona Cotton Research and Protection Council conference room. Results from field testing of the first release of transgenic pink bollworm were reported and an update was given of research strains of transgenic pink bollworm containing lethal genes by the Oxitec Company from Oxford, UK. It was decided to concentrate on release of marked pink bollworm in the near term. Funding for field operations is a problem. We hosted a Workshop on Regulatory Communication in Washington, DC 7-9 November 2006. Both our transgenic pink bollworm and our Symbiotic Control of Pierce's disease projects were prominently featured in presentations. Results of the Workshop including many of the talks were posted as video clip on the website: http://biopesticide.ucr.edu. The website remains active and will continue to serve the same purpose. Future Workshops on the same topic are intended. We established a new subcontractor for the Pierce's disease project, Angray Kang, Reader at University of Westminster, London. He is an immunologists who operates a phage antibody laboratory. He is isolating a new version of the scFv single chain antibody to bind the mopB coat protein of the pathogen Xylella fastidiosa. He also agreed to take on polygalacturonase as a new target for Pierce's disease control. It is known that if polygalacturonase is inactivated, PD symptoms are reduced or eliminated. We helped a European group, SYM4EU, develop its own Symbiotic Control project aimed at several pests and diseases including grape yellows disease of vineyards.

Impacts
We developed a new pest control strategy called ABC, Autocidal Biological Control. The first practical example of this new strategy using pink bollworm is being developed as a team effort with Luke Alphey of Oxitec in UK providing the lethal genes and USDA-APHIS in Phoenix, AZ doing the testing. Gradually APHIS in Phoenix took over doing transformations from us, so the project continues between Oxitec and APHIS, as an outstanding example of technology transfer. We continue helping with regulatory matters. We identified a symbiotic organism to deliver a cure for the Pierce's disease that currently threatens grapevines in California. The approach is termed 'Symbiotic Control,' and is a form of biological control using symbiotic microorganisms in a process of competitive displacement of pathogens. This is a major breakthrough since the only current method to protect crops from pathogens transmitted by insects is by treating the vector insect with insecticide or quarantine of the uninfected plant coupled with expensive removal of infect plants and installing a repressive and expensive quarantine program to prevent movement of vector insects on alternate hosts or on produce shipped from infected areas. Symbiotic Control is selective, acting only on the pathogen and employs symbiotic microbes already in the ecosystem. The federal regulatory system appears unable to deal with this new strategy (thus the Workshop mentioned above).

Publications

• Simmons, G. S., L. Alphey, T. Vasquez, N. I. Morrison, M. J. Epton, E. Miller, T. A. Miller and R. T. Staten. 2007. Potential use of a conditional lethal transgenic pink bollworm Pectinophora gossypiella in area-wide eradication or suppression programmes. In: M. J. B. Vreysen, A. S. Robinson and J. Hendrichs (eds.), Area-Wide Control of Insect Pests: From research to field implementation, Springer, Dordrecht, The Netherlands. (pdf galley 5 Feb 07).
• Miller, Thomas A., David J. Lampe and Carol R. Lauzon. 2006. Transgenic and paratransgenic insects in crop protection. In: Insecticide Design Using Advanced Technologies, edited by: Isaac Ishaaya, Ralf Nauen and Rami Horowitz, Springer-Verlag, Heidelberg, Germany.
• Miller, T. A., C. R. Lauzon and D. J. Lampe. 2006. Technological advances to enhance agricultural pest management. In: Transgenesis and the management of vector-borne disease, edited by Serap Aksoy, Landes Bioscience, Georgetown, Texas.
• Wang, Jian-jun, T. A. Miller, and Y. Park. 2006. Development of multiple dominant markers by using vectorette PCR-based non-radioactive transposable element display. Molec. Ecol. Notes 6: 642-645.
• Wang, Jian-jun, X. Ren, T. A. Miller, and Y. Park. 2006. piggyBac-like elements in the tobacco budworm, Heliothis virescens (Fabricius). Insect Molec. Biol. 15 (4): 435-443.
• Ren, X., Z.-J. Han and T. A. Miller. 2006. Excision and transposition of piggyBac transposable element in tobacco budworm embryos. Arch. Insect Biochem. Physiol. 63: 49-56.
• Ren, X., Y. Park and T. A. Miller. 2006. Intact mariner-like element in tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae). Insect Molec. Biol. 15: 743-748.
• Bourtzis, K. and T. A. Miller (editors). 2006. Insect Symbiosis, Volume 2. CRC Press/Taylor and Frances, Boca Raton, FL, London, 276 pp.
• Miller, T. A., C. R. Lauzon, D. Lampe, R. Durvasula and C. Matthews. 2006. Paratransgenesis applied to control insect-transmitter plant pathogens: The Pierce's disease case. In: Insect Symbiosis 2, ed by K. Bourtzis and T. A. Miller. Taylor and Francis, London/CRC Press Boca Raton, FL.

Progress 01/01/05 to 12/31/05

Outputs
The development of transgenic conditional lethal pink bollworm is virtually complete except for regulatory approval. The USDA-APHIS lab in Phoenix developed the ability to make transgenic pink bollworms themselves. Before the end of 2005 one of the lethal plasmids supplied by Oxitec, UK was inserted into pink bollworm in Phoenix and proved to be 100% lethal in the egg stage, or exactly what we wanted. This transfer of technology and production of a lethal strain is a very successful University-USDA development project bringing new biotechnology into crop protection. We are still involved because the issue of open field release under permit from the BRS part of APHIS has not yet been approved; in fact, it is stifled by inaction on the part of the regulators. We have been asked to help generate a Workshop Conference in Washington, DC scheduled Oct-Nov 2006 on the topic of improving regulatory actions on recombinant organisms used in crop protection. Both the transgenic pink bollworm and our Symbiotic Control of Pierce's disease projects will be prominently featured. Indeed, these projects are the most advance towards practical application of this type using insects. The genetically altered the endophytic bacterium, Alcaligenes xylosoxidans var. denitrificans, was found not to express a single chain antibody to control the pathogen causing Pierce's disease in grapevines in high enough titer to disrupt the disease cycle. Our collaborator, Dave Lampe at Duquesne University, decided last summer to switch to a new strategy of developing a soluble antibody. We continued to develop methods to test Dave's products in disruption of the disease cycle. New genome analysis shows the Alcaligenes endophyte to be another bacterium closely related to Pseudomonas fluorescens. Originally we thought this would ease regulatory concerns about side-effects of the organism, however, last summer our field project to test the genetically marked Alcaligenes was cancelled by the State CDFA PD program, unexpectedly. At the same time our request for field trials with the marked symbiont for multiple years without burning the grapevines was denied by EPA-TOSCA (who had oversight). We found no rational explanation for this action, which stifles progress. Again these concerns and others are the reason for the Workshop conference mentioned above. A risk assessment study of possible horizontal gene movement from Alcaligenes to other bacteria continues with support from USDA-CSREES and with two collaborators, Dave Lampe at Duquesne and Ravi Durvasula at University of New Mexico, Medical School.

Impacts
We developed a new pest control strategy called ABC, Autocidal Biological Control. The first practical example of this new strategy using pink bollworm is now ready for field release and was developed as a team effort with Luke Alphey of Oxitec in UK providing the lethal genes, us providing the genetic transformations and USDA-APHIS in Phoenix, AZ doing the testing. Gradually APHIS in Phoenix took over and this year finally achieved the ability to do transformations on their own, so the project continues between Oxitec and APHIS, as an outstanding example of technology transfer. We continue helping with regulatory matters, since they are a sticking point. We identified a symbiotic organism to deliver a cure for the Pierce's disease that currently threatens grapevines in California. The approach is termed 'Symbiotic Control' (SC), and is a form of biological control using symbiotic microorganisms. This is a major breakthrough since the only current method to protect crops from pathogens transmitted by insects is by treating the vector insect with insecticide or quarantine of the uninfected plant coupled with expensive removal of infect plants and installing a repressive and expensive quarantine program to prevent movement of vector insects on alternate hosts or on produce shipped from infected areas. Symbiotic Control is selective acting only on the pathogen and employs symbiotic microbes already in the ecosystem. The federal regulatory system appears unable to deal with this new strategy (thus the Workshop mentioned above).

Publications

• Dolezal, P, B. Bextine, R. Dolezal and T. A. Miller 2004 Novel methods of monitoring the feeding behavior of Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). Ann. Entomol. Soc. Am. 97(5): 1055-1062. (reported last year w/out title).
• Bextine, B. and T. A. Miller 2004 A laboratory-based monitoring of an insect-transmitted plant pathogen system. Biotechniques 38(2): 1-2 (reported in press last year with a different title).
• Bextine, B., M. Blua, D. Harshman and T. A. Miller 2005 A SYBR green-based real-time polymerase chain reaction protocol and novel DNA extraction technique to detect Xylella fastidiosa in Homalodisca coagulata. J. Econ. Entomol. 98(3): 667-672
• Miller, T. A. 2004 Rachel Carson and the adaptation of biotechnology to crop protection. Amer. Entomol. 50(4): 194-198.
• Wang, J., R. Staten, T. A. Miller, and Y. Park. 2005. Inactivated mariner-like transposable elements (MLE) in pink bollworm, Pectinophora gossypiella. Insect Molec. Biol. 14 (5): 547-553.
• Blake Bextine, B. Jackson, D. B. Harshman and T. A. Miller. 2005. Homalodisca coagulata (Hemiptera: Cicadellidae) feeding posture. Ann. Entomol. Soc. Am. 98(6): 000-000 Galleys corrected and returned 20 September 2005. 7 ms pp.
• Kuzina, Ludmila V., T. A. Miller, D. A. Cooksey. (200_) In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa. Letters in Applied Microbiology Vol (00): 000-000. (Accepted November 2005; in press)
• Jianjun, Wang, Xiaoxia Ren, T. A. Miller and Yoonseong Park 2006 PiggyBac-like elements (PLE) in tobacco budworm, Heliothis virescens (Fabricius). Accepted for publication in: Insect Molecular Biology 31 January 2006. (in press).

Progress 01/01/04 to 12/31/04

Outputs
The conditional lethal transgenic pink bollworm strain sent from Riverside to the USDA-APHIS pink bollworm laboratories in Phoenix last year has been evaluated for ability to control field populations. This strain carries a marker gene for identification and a lethal (RIDL) gene behind a tetracycline repressor element. In the presence of tetracycline the lethal factor is suppressed allowing mass rearing. In the absence of tetracycline mating between the RIDL strain and wild types caused up to 95% mortality in offspring. Partial radiation increases the mortality and crossing different RIDL strains increases the effect. This is the first demonstration of an Autocidal Biological Control strategy first delineated by this project in 1995. Permits for field trials are being drafted by USDA-APHIS. We genetically altered the endophytic bacterium, Alcaligenes xylosoxidans denitrificans, to express a single chain antibody to control the pathogen causing Pierce's disease in grapevines. New genome analysis shows the Alcaligenes endophyte to be another bacterium closely related to Pseudomonas fluorescens. This will ease regulatory concerns about side-effects of the organism. New single chain antibody constructs are being developed. A risk assessment of possible horizontal gene movement from Alcaligenes to other bacteria is being conducted with support from USDA-CSREES.

Impacts
We have developed a conditional lethal strain of pink bollworm with insect transformation methods. These insects are capable of being mass released to cause autocidal mating with wild-type counterparts in infested cotton fields. The improved competitive efficiency of the released insects could support eradication of pink bollworm from the western United States, which is not feasible with existing SIT technology. We have developed an organism to deliver a cure for the Pierce's disease that currently threatens grapevines in California. The approach is termed 'symbiotic control (SC),' and is a form of biological control using symbiotic microorganisms. If successful, this will be the first time an insect-transmitted pathogen causing plant disease is controlled. The new technology would save millions of dollars yearly now spent for remediation of diseased plants. Other SC projects are described at: www.symbiosis.ucr.edu.

Publications

• Bextine, B., S-J. Tuan, H. Shaikh, M. Blua, T. A. Miller. 2004. Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter. J. Econ. Entomol. 97: 757-763.
• Bextine, B. and T. A. Miller. 2004. Comparison of whole-tissue and xylem fluid collection techniques to detect Xylella fastidiosa in grapevine and oleander. Plant Disease 88: 600-604.
• Bextine, B., C. Lauzon, S. Potter, D. Lampe and T. A. Miller. 2004. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy. Current Microbiol. 48: 327-331.
• Dolezal, P, B. Bextine, R. Dolezal and T. A. Miller. 2004. Ann. Entomol. Soc. Am. 97: 1055-1062.
• Bextine, B., M. Johnson, D. Harshman and T. A. Miller. 2004. Impact of pymetrozine on glassy-winged sharpshooter feeding behavior and rate of Xylella fastidiosa transmission in the laboratory. J. Insect Science 4:34. [www.insectscience.org/4.34]
• Bextine, B., C. Lauzon, D. Lampe and T. A. Miller. 2004. Establishment of a genetically marked insect-derived symbiont in multiple host plants. Current Microbiol 49: 1-7.
• Bextine, B. and T. A. Miller. 2004. A laboratory-based Pierce's disease cycle. Biotechniques.
• Miller, T. A. 2004. Designing Insects. An Action Bioscience.org original article. [http://www.actionbioscience.org/biotech/miller.html]

Progress 01/01/03 to 12/31/03

Outputs
Permits were obtained from USDA-APHIS to ship transgenic pink bollworm from Riverside to the USDA-APHIS pink bollworm laboratories in Phoenix. This strain carries a marker gene for selection and a lethal gene behind a tetracycline repressor element. In the presence of tetracycline the lethal factor is suppressed allowing mass rearing. We genetically altered the endophytic bacterium, Alcaligenes xylosoxidans denitrificans, to contain the DsRed fluorescent protein gene and obtained a permit from EPA to monitor the movement of the endophyte in commercial grapevines at four sites in California. The endophyte did not move into the grape berry and decreased in titer to undetectable levels weeks after injection into grapevines. An antibody library was screened for a single-strand antibody that is specific for the coat protein of the pathogen, Xylella fastidiosa. Injection of the antibody on a phage into host plant stems blocked transmission of X. fastidiosa from an infected stem to grape plants by the vector insect, glassy-winged sharpshooter, Homalodisca coagulata.

Impacts
We seek to develop a conditional lethal strain of pink bollworm with insect transformation methods. The goal is to supply an Autocidal Biological Control (ABC) pink bollworm to improve the existing Sterile Insect Techniques first perfected by the USDA. If implemented, this improved strain of pink bollworm would not require radiation for sterilization. The improved competitive efficiency of the released insects could support eradication of pink bollworm from the western United States which is not feasible with existing SIT technology. We seek to find a cure for the insect-transmitted disease of grapevines called Pierce's disease that 'probiotic', and is a form of biological control using symbiotic microorganisms. If successful, this will be the first time an insect-transmitted plant disease is controlled. The new technology would save millions of dollars yearly now spent for remediation of diseased plants.

Publications

• Nirgianaki, A., G. K. Banks, D. Frohlich, Z. Veneti, H. R. Braig, T. A. Miller, I. D. Bedford, P. G. Markham, C. Savakis, and K. Bourtzis. 2003. Wolbachia infections of the whitefly Bemisia tabaci. Current Microbiology. 47(2): 93-101.
• Bourtzis, Kostas and T. A. Miller (editors). 2003. Insect Symbiosis. CRC Press, Boca Raton, Florida, 347 pp.
• Peloquin, J. J., C. R. Lauzon, S. Potter and T. A. Miller. 2002. Transformed bacterial symbionts re-introduced to and detected in host gut. Current Microbiology 45: 41-45.
• Kuzina, L. V., E. D. Miller, B. Ge and T. A. Miller. 2002. Transformation of Enterobacter gergoviae isolated from pink bollworm (Lepidoptera: Gelechiidae) gut with Bacillus thuringiensis toxin. Current Microbiology 44: 1-4.
• Miller, T. A. 2002. Lethal genes for use in insect control. In: Encyclopedia of Pest Management, ed. buy David Pimentel, Marcel Dekker, Inc., pp. 448-450.
• Miller, T. A. 2003. Circulatory System. In: Encyclopedia of Insects, (ed. by R. Carde and V. H. Resh), Academic Press/Elsevier, pp. 192-197.
• Bextine, B., S-J. Tuan, H. Shaikh, M. Blua, T. A. Miller 2004 Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter. J. Econ. Entomol. (Accepted December 2003).

Progress 01/01/02 to 12/31/02

Outputs
Permits are being obtained (pending) to ship transgenic pink bollworm from Riverside to the USDA-APHIS pink bollworm laboratories in Phoenix. This strain carries a marker gene for selection and a lethal gene behind a tetracycline repressor element. In the presence of tetracycline the lethal factor is suppressed allowing mass rearing. The conditional lethal element comes from Oxitec, a company associated with Oxford University, Oxford, England. We hope to cure grape plants of Pierce's disease by using endophytic bacteria, Alcaligenes spp., to deliver anti-Pierce's agents to the pathogenic bacterium Xylella fastidiosa. Both endophyte and pathogenic bacteria are carried in the same area of the mouthparts in the vector insect, Glassy-winged sharpshooter, GWSS, and both are transmitted to the plant and occupy the same spaces there; therefore, this paratransgenic approach is feasible. We will next test anti-Xylella agents for use in crop protection.

Impacts
In July of 2002 achieved genetic transformation of pink bollworm with a conditional lethal gene. In the past six months this strain has been back-crossed to homozygosity and is now being prepared for inverse PCR analysis to identify the transgene and insertion site. Our collaborators at USDA-APHIS have requested permits to ship the strain to their larger quarantine facilities in Phoenix, AZ. The lethal gene, a nipp or nuclear inhibitor of protein phosphorylase is help off by a tetracycline repressor system from Clontech. Preliminary results show that mortality occurs when the transgenic pink bollworms are reared on tetracycline-free diet. The lethal gene was supplied by Luke Alphey at Oxitec in England. The goal for this project is to supply an Autocidal Biological Control (ABC) pink bollworm during the pink bollworm eradication project planned for the next several years. ABC is a new method designed to improvement the Sterile Insect Technique first perfected by the USDA. We have demonstrated the feasibility of controlling Pierce's disease by use of paratransgenesis. Anti-Pierce's strategies are to be delivered by shuttle bacteria identified as typical endophytes that occupy the same space in the vector insect and host plants as the pathogenic bacterium, Xylella fastidiosa. The next step in this project is to test agents for expression in the endophyte to control Pierce's disease.

Publications

• Bextine, B., T. Miller, C. Lauzon, D. Lampe, and D. Cooksey. (2002). Pressure bomb extraction of xylem fluid to improve bacterial detection of Xylella in plants. Proceedings Pierce's Disease Research Symposium, Dec. 15-18, 2002, Coronado Island Marriott Resort, San Diego, CA, pp. 29-30.
• Lauzon C., T. Miller, D. Lampe, D. Cooksey and B. Bextine. (2002). Insect-symbiotic bacteria inhibitory to Xylella fastidiosa (Paratransgenesis for control of Pierce's disease): Identification of endophytic bacteria cycled by glass-winged sharpshooters to host plants. Proceedings Pierce's Disease Research Symposium, Dec. 15-18, 2002, Coronado Island Marriott Resort, San Diego, CA, pp. 31-32.
• Miller, T. A. and Tanja Krause. (2002). Genetic control of pink bollworm. Proceedings of Cotton Pest Work Committee, Play Real Hotel, Mazatlan, Mexico, November 21, 2002.
• Lampe, D., T. Miller, C. Lauzon, D. Cooksey and B. Bextine. (2002). Paratransgenesis for control of Pierce's disease: Manipulation of endophytic bacteria for paratransgenic control of Pierce's disease. Proceedings Pierce's Disease Research Symposium, Dec. 15-18, 2002, Coronado Island Marriott Resort, San Diego, CA, pp. 27-28.

Progress 01/01/01 to 12/31/01

Outputs
A permit was obtained from USDA-APHIS for field cage studies of the reproductive ability of transgenic pink bollworm compared to non-transgenics. Preliminary results show the transgenics to be slightly less competitive than the non-transgenics. This difference in reproductive behavior matches a slightly lower viability character of the transgenic strain in culture. The USDA-APHIS issued a Finding of No Significant Impact (FONSI) for this field test. We continue to develop conditional lethal strains of pink bollworm. A collaborator at USDA-ARS, Yakima, WA, Lisa Neven, reported genetic transformation of codling moth using a Notch-piggyBac transformation plasmid designed for pink bollworm. A new project was initiated to find methods of delivering gene products via endosymbiotic bacteria of the glassy-winged sharpshooter (GWSS) to prevent the vectoring of Pierce's disease to grapes in California. A transformation protocol was worked out for the first candidate bacterium, Alcaligenes spp., isolated from GWSS.

Impacts
The major accomplishment of this project in 2001 was to obtain a permit for conducting field cage studies comparing the reproductive ability of transgenic pink bollworm with non-transgenic strains in Phoenix, AZ. This is the first time a transgenic insect has been the subject of a field test; however confined, and this explains the large volume of media attention that has been ongoing for virtually the entire year, starting with a front page story in the Wall Street Journal, Jan 29, 2001 based on the pink bollworm work, and including an article in the October 2001 issue of Popular Science magazine, highlighting Dr. Robert T. Staten's efforts to control pink bollworm from his USDA-APHIS methods development laboratories in Phoenix, AZ. The transformation protocols and conditional lethal genes developed for pink bollworm have reportedly been exported into codling moth. The cotton industry is just now gearing up for pink bollworm eradication following on the boll weevil eradication attempts. The goal for this project is to supply an Autocidal Biological Control (ABC) pink bollworm during the pink bollworm eradication project planned for the next several years. ABC ushers in a conceptually new method of controlling insects that is the first biotechnology-based improvement to the Sterile Insect Technique was first perfected by USDA.

Publications

• Kuzina, Lyudmila V., John J. Peloquin, Don C. Vacek, and T. A. Miller. 2001. Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae). Current Microbiology (April) 42 (4): 290-294.
• Peloquin, J.J., T.A. Miller, and S. Higgs. 2001. Pink Bollworm Larvae Infection with a Double Subgenomic Sindbis (dsSIN) Virus to Express Genes of Interest. J Cotton Sci. 5: 1-8. http://www.jcotsci.org, c The Cotton Foundation 2001.
• Miller, T. and R. Staten, 2001. The pink bollworm. Tale of a transgenic tool. In: Agrichemical and Environmental News. June 2001, No. 182, pp. 1-5. http://www.tricity.wsu.edu/aenews.
• Miller, T. 2001. Control of pink bollworm. Pesticide Outlook 68-70. www.rsc.org/po.
• Lampe, D., D. Cooksey, C. Lauzon, J. Peloquin and T. A. Miller. 2001. Insect-symbiotic bacteria inhibitory to Xylella fastidiosa in sharpshooters. Proc. Pierce's disease research symposium, Dec. 5-7, 2001, Coronado Island Marriott Resort, San Diego, CA, pp. 78-80.
• Miller, E., R.T.Staten, J. Claus, M. Sledge, J. Peloquin, T. Miller. 2001. A multiple generation life history study on rearing a genetically altered (EGFP) strain of pink bollworm (Lepidoptera: Gelechiidae). 2001 Proceedings Beltwide Cotton Conferences volume 2: 1118-1120.

Progress 01/02/00 to 12/31/00

Outputs
A permit was requested for the general release of the transgenic pink bollworm for the purpose of initiating field studies of competition between the transgenic strain and wild type pink bollworms. The work is to be performed somewhere in Arizona. The head of the Department of Agriculture in Phoenix has approved of the release tests. Efforts continue to develop conditional lethal strains. We genetically transformed one of the endosymbiotic bacteria from the gut of pink bollworm. The transformed bacteria expressed the Cry 1A endotoxin protein from a gene in Bacillus thuringiensis. After finding that no permits were needed for shipment, the transgenic bacterium was sent to the Mex fly lab of USDA-APHIS in Mission, Texas where testing found a low level of toxicity to larvae of the Mexican fruit fly, ANASTREPHA LUDENS. A new project was initiated to find methods of delivering gene products via endosymbiotic bacteria of the glassy-winged sharpshooter to prevent the leafhopper from vectoring Pierce s disease.

Impacts
We are developing the new field of molecular biological control, which uses modern methods in molecular genetics to create biological control agents out of pest insects. The first application is ABC or autocidal biological control. This aims to produce conditional lethal strains of insects that are objects of the sterile insect technique (SIT) such as pink bollworm, and is expected to improve SIT by making the mass-released insect much more competitive than the current technique allows. The second application is paratransgenesis whereby the endosymbiotic bacteria carried by the gut of insects are converted into vehicles to deliver agents that prevent insects from vectoring plant diseases. We are using the approach to find a cure for Pierce's disease vectored by glassy-winged sharpshooter insects. Paratransgenesis has already been successfully applied to prevent Triatoma insects from vectoring Chagas' disease. The third area of molecular biological control is to use endosymbiotic bacteria as vehicles to deliver toxins to pest insects. Converting a pest insect into a biocontrol agent avoids all of the disadvantages inherent in introducing parasite and predator species for classical biological control. The only hurdle facing this new field is public acceptance of releasing transgenic organisms.

Publications

• Kuzina, L. V., E. D. Miller, B. A. Federici and T. A. Miller (2000). Transformation of endosymbiotic bacteria isolated from pink bollworm (Lepidoptera: Gelechiidae) gut with BACILLUS THURINGIENSIS. Current Microbiology (in press).
• Peloquin, J. J., C. R. Lauzon and T. A. Miller. 2000. ECFP expressed in transformed gut bacteria of RHAGOTETIS COMPLETA Cresson origin. Submitted to Applied and Environmental Microbiology, 29 August 2000.
• Nirgianaki, A., G. K. Banks, D. Frolich, Z. Veneti, H. R. Braig, T. A. Miller, I. D. Bedford, P. G. Markham, C. Savakis And K. Bourtzis. (2001). Characterization of WOLBACHIA in a range of agriculturally important insect species, including BEMISIA TABACI (Gennadius) and aphids. Submitted to Molecular Ecology, December 2000.

Progress 01/01/99 to 12/31/99

Outputs
Federal permits have been obtained to move the transformed pink bollworms from our laboratories in Riverside to the USDA-APHIS mass rearing facilities in Phoenix, Arizona. The Phoenix laboratory has been rearing and backcrossing the transgenic pink bollworms by selecting for the green fluorescent protein product that was part of the transgene. Analysis shows some pink bollworms with two insertion sites and others with one. The colony appears homozygous now and the transformation also appears very stable in the colony. We are now preparing an application for a permit for general release of the transgenic pink bollworm for the purpose of initiating field studies of competition between the transgenic strain and wild type pink bollworms. We achieved genetic transformation of two endosymbiotic bacteria isolated from walnut husk fly supplied by Carol Lauzon of Cal State Hayward. The transformed bacteria expressing green fluorescent protein markers were returned to Carol who reports they are stable in vivo. The transgenes were very stable in the host bacterial colonies over a range of temperatures for a month (see publications 3 and 4 below). The long anticipated publication of proof of genetic transformation of pink bollworm was finally published (see number 2 below). Production of conditionally lethal strains of pink bollworm using constructs of the Notch gene from Drosophila melanogaster continue. Lethal effects of the truncated Notch gene were demonstrated in pink bollworm embryos.

Impacts
As suggested by the Patent Disclosure statements submitted with this report, it is clear that delivery of genes via the endosymbiotic bacteria of insects is a viable strategy. This will spur the development of an entirely new way of looking at biological control, a sort of molecular biological control in which a target pest insect will carry the seeds of its own destruction into the general population of its conspecifics. Such strategies are already operating to eliminate the transmission of Chaga's disease in a process termed paratransgenesis.

Publications

• Peloquin, J. J, T. A. Miller and Carol Lauzon. 1999. Transformed symbionts for insect monitoring and improvement of Tephritid SIT colonies. Disclosure and Record of Invention submitted to the University of California Office of Technology Transfer. Date of acknowledgment: November 8, 1999. UC Case Number: 2000-175-1.

Progress 01/01/98 to 12/31/98

Outputs
Most significant achievements this period: We achieved genetic transformation of the lepidopteran, Pectinophora gossypiella, the pink bollworm. The transformation vector contained the piggyBac transposable element (originating from the cabbage looper, Trichoplusiani, a Noctuid moth), a newly modified actin A3 promoter from the silkworm, Bombyx mori, and the marker gene, green fluorescent protein (gfp). We understand that this is the fist report of genetic transformation of a lepidopteran insect. The transformed insects have been backcrossed to homozygosity and the transformation appears to be remarkably stable. Part of this stability appears to be due to the lack of any native transposable elements in the pink bollworm that remotely resemble the piggyBac transposable element used. We understand that highly respected scientists in Europe have attempted to genetically transform the commercial silkworm using constructs nearly identical to those listed above, but without success. At the moment we cannot explain the difference between our success with P. gossypiella and their failure with B. mori Publications during this period: Two patent disclosure statements have been filed with the University of California Office of Technology Transfer. The first concerns details of the transformation protocols described above; the second concerns practical uses of transformed pink bollworms.

Impacts
(N/A)

Publications

• 1. Thibault, Stephen T., 1998, Towards Genetic Transformation of the Pink Bollworm (Pectinophora gossypiella, Lepidoptera: Gelechiidae): Assembling a Vector System and Cloning of a Notch Homologue. Ph.D. dissertation, University of California, Riverside.
• 2. Thibault, S. T., H. T. Luu, N. Vann and T. A. Miller, 1998. Precise excision and transposition of piggyBac in pink bollworm embryos. Insect Molecular Biology 8: 1-5.
• 3. Peloquin, J. J., and T. A. Miller, 1999. PCR optimization for detection of EGFP (Enhanced Green Fluorescent Protein) DNA in pink bollworm (Pectinophora gossypiella). J. Cotton Science, submitted, December, 1998.
• 4. Thibault, S. T., T. A. Miller and J. J. Peloquin, 1998. Plasmid for genetic transformation of Lepidopteran insects. Record of invention disclosure submitted to University of California Office of Technology Transfer, November 23, 1998.
• 5. Peloquin, J. J. and T. A. Miller, 1998. Pink Bollworm Expression System for Commercially Valuable Protein Production. Record of invention disclosure submitted to University of California Office of Technology Transfer, December 29, 1998. Assigned UC Case number: 99-178-1.

Progress 01/01/97 to 12/01/97

Outputs
The genomic Notch gene from pink bollworm, PECTINOPHORA GOSSYPIELLA Saunders, was further sequenced. We have about 20 Kb now including 17 introns so far. There are about 2 Kb remaining to be cloned. The piggyBac transposable element was obtained from Paul Shirk USDA-Gainesville) and Mac Fraser (Notre Dame). It was combined with a truncated Notch gene from DROSOPHILA MELANOGASTER and its promoter and the green fluorescent protein gene, gfp, with hsp70 as a promoter to use as a transformation plasmid. This plasmid caused a toxic reaction in pink bollworm eggs causing high mortality following injects in attempt as transformation. A new promoter, actin 5c, is being substituted for hsp70 and this will be tested for driving the expression of gfp in pink bollworm embryoes in the next reporting period. A weather and trapping station was established in Banning Pass near Palm Springs to monitor pink bollworm. RAPD methods were developed to determine origin of feral pink bollworms caught in pheromone traps. Samples of pink bollworms from Brazil; India; Egypt; China; Brawley, CA; San Joaquin Valley, CA; and Mexicali, Mexico are all being compared to laboratory reared moths from the pink bollworm rearing faculties in Phoenix, AZ. A tree is being generated by the RAPDBIOS program.

Impacts
(N/A)

Publications

• MILLER, T. 1994. Research needs in protocols for evaluation of competitiveness of mass reared Medflies. Facilitator, Breakout Session 8, Proc. Workshop, Center for Exotic Pest Research, Nov.
• MILLER, T. A. 1995. Resistance to Pesticides: Mechanisms, Development and Management, pp. 317-375. In: Cotton Insects and Mites: Characterization and Management, edited by E. G. King and J. R. Phillips. Publ. Cotton Foundation, Memphis,.
• MILLER, T. 1995. Studies on pink bollworm, pp. 167-170. In: Insects: Chemical Physiological and Environmental Aspects, edited by D. Konopinska, et al. Proc. 1st International Conference, Sept. 26-29, 1994. Univ. of Wroclaw Press, Poland.
• MILLER, T. 1995. Progress report on autocidal biological control of pink bollworm. Proc. International Cotton Pest Work Committee, Mazatlan, Mexico, Nov. 14-17, 1996. pp. 120-123.
• MILLER, T. 1996. Progress on genetic control of pink bollworm. Proc. International Cotton Pest Work Committee, Mazatlan, Mexico, Nov. 20-22, 1996. pp. 120-129.
• MILLER, T. A. 1996. Genetic engineering of the Mediterranean fruit fly to introduce genetic markers for population monitoring and eradication. In: Proceedings of Exotic Fruit Fly Research Symposium, September 17, 1996, UC Riverside. Cal.
• MILLER, T., S. THEBAULT and M. ROBETSON. 1997. Proceedings, Beltwide Cotton Meetings, New Orleans, LA, Jan 8-10, 1997. Conditional Lethal Genes to Control Cotton Pests.

Progress 01/01/96 to 12/30/96

Outputs
The synthetic Drosophila-Notch(superscript 60g11) analog (N60g11) that was constructed has been inserted into plasmid constructs each containing either the hobo or piggyBac transposable element. Plasmids containing the mariner and hermes transposable elements are under construction. The piggyBac transposable element has been tested for precise excision in pink bollworm with positive results. Other elements are being tested for precise excision and transposition. The plasmid constructs also require a marker gene to identify transformed insects. GFP (Green Fluorescent Protein gene) has been inserted and tested in pink bollworm embryos for expression. Pink bollworm eggs injected with these plasmids show a green fluorescence under UV light demonstrating proper functioning of the marker gene and its hetrologous promoter derived from the Drosophilia hsp 70 gene. Embryos injected with the hobo constructs and piggyBac constructs are being tested for insertion of the N(superscript 60g11) -GRP into pink bollworm chromosomes.

Impacts
(N/A)

Publications

• MILLER TA, M ROBERTSON and S THIBAULT. 1997. Conditional lethal genes to control cotton pests. Proceedings of the Beltwide Cotton Meetings. New Orleans, LA, January 8-9 1997, in Press.
• PELOQUIN, JJ, ST THIBAULT, LP SCHOUEST JR., TA MILLER. 1997. Electromechanical microinjection of pink bollworm Pectinophora gossypiella embryos increases survival. BioTechniques 22:496-499.
• MILLER, TA. 1997. Control of circulation in insects. Gen. Pharmac.

Progress 01/01/95 to 12/30/95

Outputs
DROSOPHILA NOTCH clones were obtained and modified to make analogs of the 60G11 mutation. A microinjection procedure was perfected for high survival of pink bollworm. Pink bollworm eggs injected with the analog of the NOTCH mutation did not survive while controls developed to hatching. cDNA libraries were made for both pink bollworm and Mediterranean fruit fly. DROSOPHILA homozygous for the NOTCH mutation were found to be sterilized when exposed to 20 degrees centigrade. Mariner-like transposable elements were discovered and described in pink bollworm. Hobo-like transposable elements were also found. Degenerate oligonucleotide primers were used to identify PCR products from pink bollworm with homology to DROSOPHILA NOTCH.

Impacts
(N/A)

Publications

• FRYXELL, K.J. and MILLER, T.A. 1995. Autocidal biological control: a general strategy for insect control based on genetic transformation with a highly conserved gene. J. Economic Entomology 88: 1221-1232.