Comparative Pathogenesis of the Baculoviruses Acmnpv and Agmnpv in Larvae of the Soybean Pests Anticarsia Gemmatalis and Pseudoplusia Includens

<p>NON-TECHNICAL SUMMARY:<br/> While soybean ranked 2nd among U.S. crops in farm value in 2005 with over 67 million acres planted in 2007, U.S. dominance of global soybean markets has eroded, in part because of increased production in Brazil. Farmers in Brazil protect soybean using a variety of low cost pest control strategies, which include the baculovirus AgMNPV. AgMNPV as a biological control agent is inexpensive to produce, effective in limiting foliar damage to soybean by the velvetbean caterpillar (Anticarsia gemmatalis) and is safe to other organisms. Indeed, reduced pest management costs provide Brazilian soybean a competitive advantage on the world soybean market, which depreciated the value of this commodity. While the reduced value of soybean prompted U.S. farmers to limit soybean plantings, use of genetically modified (GM) soybean threatens to reduce demand
further. In the U.S., 90% of soybean is GM, and the European Union (EU) is the world's largest soybean importer. However, the EU legislates restrictions on GM organisms and with increased consumer awareness of food production practices, this trend may spread to the U.S. Recently, German universities halted field trials of GM crops because of public hostility toward genetic engineering, demonstrating that acceptance of GM foods is deteriorating. Increased consumer demand for USDA certified organic foods, restrictions on GM crops and consumer willingness to pay a premium for perceived food safety are compelling reasons to promote organic cropping practices. Because natural baculoviruses are available to organic farmers in the U.S. for controlling crop pests, they constitute an underutilized strategy for reducing economic damage to soybean by lepidopteran larvae. Additionally, increased
diversification of biological control strategies will help preserve the efficacy of Bacillus thuringiensis (Bt) toxin for intensive organic agriculture. Reduced dependence on GM soybean by U.S farmers will increase the appeal of this commodity to EU consumers and buffer the industry to further market restrictions in the U.S. and abroad. AgMNPV and AcMNPV are the most widely used baculoviruses, but little is known regarding the pathogenesis of these biological control agents in the lepidopteran pests of soybean. The goal of our research is to elucidate the pathogenesis of these two baculoviruses in the two most significant lepidopteran pests of soybean: the velvetbean caterpillar and the soybean looper (Pseudoplusia includens). For the purpose of researching AgMNPV pathogenesis, we will genetically modify AgMNPV the genome so that we may easily track the cell-to-cell movement of the virus
as it infects the insect. Additionally, we will develop research methods that permit the study of baculovirus infection within individual cells with the intent to better understand the insect's antiviral response to infection. The overall goal of the proposed research is to provide the research community with effective tools for studying baculoviruses and to increase our understanding of baculovirus infection so that more organic farmers include baculoviruses into their integrated pest management program.<p>
APPROACH: <br/>The proposed research methods are rooted in using recombinant baculoviruses that encode reporter genes, which permit histological analysis of tissues for determining timing and extent of viral infection. Specifically, our research plan is to use the lacZ reporter gene incorporated into AcMNPV and AgMNPV to identify and quantify the types of cells that become infected for A. gemmatalis and P. includens orally inoculated with viral occlusion bodies. Similar studies have been executed in other species and have led to substantial gains in understanding with regard to baculovirus infection dynamics and molecular mechanisms triggered in the host to combat infection. Where appropriate, we will make statistical analyses of our results. We will consider this element of the proposed research successful upon completing and publishing our pathogenesis studies of
AcMNPV in A. gemmatalis and P. includens larvae. Currently, there is a no AgMNPV recombinant that carries all of the wild-type genes and expresses LACZ early during infection. We will construct an AgMNPV recombinant that expresses LACZ early and constitutively under the hsp70 promoter. To aid in modifying the AgMNPV genome for this and future studies, we will first generate an AgMNPV bacmid. Bacmids have the advantage of replicating in bacteria and insect cells, permitting researchers to take advantage of molecular biology tools available in both systems. The methods for producing an AgMNPV bacmid are well established because bacmids available for at least two other baculovirus species. We expect that an AgMNPV bacmid will prove useful to other investigators for making specific mutations in the AgemMNPV genome and will accelerate research on AgMNPV. We will consider the bacmid component
of our proposed research successfully completed by producing an AgMNPV bacmid that we can disseminate to research colleagues and which we can describe in a manuscript submitted to a peer-reviewed journal. Our last research goal is to adapt flow cytometry methodologies often employed for analyzing mammalian cells to insect cells for the purpose of quantifying baculovirus infection dynamics in insect hemocytes. Flow cytometry is a well-established cornerstone methodology for vertebrate immunologists, but has not been extensively implemented for studying the insect immune system, and never to study baculovirus infection kinetics in hemocytes. We will consider our flow cytometry studies successful if we can study events of baculovirus infection in hemocytes. Our long term goal is to use flow cytometery to gain a better understanding of insect immunity so that we may enhance the effectiveness
and implementation of baculoviruses for controlling insect crop pests. The primary target audiences for the disseminating the results of the proposed research to are scientists investigating insect pathogens, agriculture policy makers and organic farmers. We will convey the results of our research to the target audiences via manuscripts submitted to peer-reviewed journals, presentations at society conferences (ex. SIP, ESA and ASV) and to members of the local community via the CSU Long Beach seminar series.</p> <p>
PROGRESS: <br/>2009/01 TO 2011/01 <br/>OUTPUTS: During the reporting period of the award (2009/01/15 - 2011/01/14; inclusive of a one-year "no-cost extension"), we conducted experiments to elucidate the pathogenesis of the baculoviruses Autographa californica M nucleopolyhedrovirus (AcMNPV) and Anticarsia gemmatalis MNPV (AgMNPV) in the larval stages of several species of Lepidoptera that are important agricultural pests, including: A. gemmatalis, Pseudoplusia includens, Trichoplusia ni, Heliothis virescens, Spodoptera exigua, and Manduca sexta. During this period, the PI mentored 3 Master's students (two graduated and enrolled in Ph.D. programs at UC Davis or UC Riverside) and 21 undergraduate researchers on projects related to the funded five Objectives of the award; four of the Objectives were completed, while one has yet to be achieved. Our first completed objective,
which was to define the pathogenesis of AcMNPV in A. gemmatalis and P. includes larvae, resulted in a peer-reviewed publication (Chikhalya et al 2009), one Master's student thesis, two conference presentations and a manuscript that will be submitted for publication during 2011. Inclusive in Chikhalya et al (2009), we quantified AcMNPV ODV binding and fusion in the midgut of A. gemmatalis larvae (Objective 4) and reported an improved higher throughput virus titer assay (Objective 5). To achieve Objective 3, we developed a flow cytometry method to quantify baculovirus infection and hemocyte proliferation kinetics. To support these studies, we adapted an advanced transplant method used by vertebrate immunologists (called adoptive transfer) to move live cells between insects for the purpose of labeling hemocytes at the molecular level to track their fate during infection and proliferation.
Results of these hemocyte studies were presented at the 2009 and 2010 annual meetings of SIP and ESA. We have been unsuccessful in constructing the AgMNPV bacmid (Objective 2). Several approaches were employed, including multiple strategies using traditional cloning with restriction enzyme digest and/or Invitrogen Gateway recombinase-based cloning. Using reagents remaining from the Award funds, we hope to succeed in constructing the AgMNPV bacmid so that it may be used to support future studies. In addition to the funded Objectives, we conducted experiments to: quantify the antiviral properties of gloverin (a protein that we discovered is upregulated in larvae exposed to baculovirus), develop vectors that express dsRNA to knock down expression of genes that regulate insect growth and study the evolution of AcMNPV using 454 genome sequencing. A web-based tutorial for baculovirus genome
annotation was constructed by the PI (published at http://tinyurl.com/3brlw5f). The research conducted aided to develop collaborations with Dr. Matthew Welch (UC Berkeley), Dr. Michael Buchmeier (UC Irvine), Dr. Eric Lyons (University of Arizona, Tucson) and Dr. Michael Strand (University of Georgia). To encourage underrepresented minorities to pursue careers in agricultural research, the PI presented the results of the funded work for RISE and Bridges to baccalaureate-sponsored lectures at local universities and community colleges. <br/>PARTICIPANTS: Individuals: Dr. Eric Haas-Stapleton (CSU Long Beach). Principal investigator. Roles: design and conduct experiments, analyze data, supervise and mentor technicians and student researchers, review results from experiments, prepare and deliver conference presentations, write reports and manuscripts. Aniska Chikhalya (CSU Long Beach).
Undergraduate student researcher. Roles: Conduct experiments, analyze data and present results at conferences. Tiffany Chen (CSU Long Beach). Undergraduate student researcher. Roles: Conduct experiments, analyze data and present results at conferences. Dee Dee Luu (CSU Long Beach).. Graduate student researcher. Roles: Conduct experiments and analyze data. Non-formal Collaborators: Dr. Michael Strand (University of Georgia). Michael provided the Pseudoplusia includens larvae used in our studies. Dr. Matthew Welch (UC Berkeley). Matthew provided expert advice on constructing the AgMNPV bacmid. Dr. Michael Buchmeier (UC Irvine). Michael is contributing to assessing the antiviral roles of gloverin for viruses that infect humans. Dr. Eric Lyons (University of Arizona, Tucson). Eric has provided computational biology services for the project. <br/>Training or professional development: Three Masters
and twenty-one undergraduate student researchers were trained with funds provided by this Award. Of these, five students have been admitted to Ph.D. programs in the biological sciences or have applications currently pending. The Award enabled potent professional development of the Principal Investigator (PI) by providing resources that facilitated his rapid integration in to the Society for Invertebrate Pathology (SIP). Consequently, during his second year of attending the annual meetings of SIP, the PI organized a Bioinformatics Workshop for the 2010 meeting, was nominated to organize and co-chair a symposium entitled "Pathology of Insect Virus Interactions" for the 2011 annual meeting, and was selected to serve as editor for the SIP Newsletter. <br/>TARGET AUDIENCES: <br/>Target audiences and Efforts: One target audience served by the project is scientists with a specific interest in baculovirus
pathogenesis and those with broad interests in insect pathogens, immunology and agricultural pests. The second target audience is students enrolled at California State University, Long Beach (CSULB; a Hispanic-serving institution (HSI)) and students from local community colleges having an interest in attending CSULB. The efforts include independent laboratory-based research that generates information that informs the scientific community of the fundamental biological processes that direct baculovirus pathogenesis and insect immune responses. As Principal Investigator, I took opportunities provided by the NIH-funded Bridges to Baccalaureate program to provide targeted lectures at community colleges for the purpose of increasing underrepresented minority student enrollment at CSULB. To increase retention of underrepresented minority students at CSULB, I invested my student researcher
recruiting efforts to support minority students enrolled in the NIH-supported Research Initiative for Scientific Enhancement (RISE) Program. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.</p>