Fred Gould

Project is in support of PSI. Drs. Huerta, Baltzegar, and Gold are pleased to submit this internal pre-proposal to NC State in support of their commitment and desire to write and submit a full-length proposal to the USDA-NIFA Equipment Grant Program, where they will lead a team of 26 other faculty requesting funds to acquire a Whole Genome Sequel IIe System sequencer from PabBio. The PacBio Sequel IIe is the current state-of-the-art technology for high-quality sequencing of genomes, transcriptomes, and epigenomes. If successful, this would be the first Sequel IIe in the University and possibly the state. The PacBio Sequel IIe system (product code 101-986-400) is priced at $525,000.00; The on-site system training (product code 100-125- 100) is $12,500; and the necessary uninterrupted power supply (UPS) (product code 100-609-000) is $7,000. The total for the instrument, installation and training for the instrument is $544,500.00. This sum is well above the funding limit for the USDA-NIFA EGP. However, Dr. Huerta has been in conversation with Dr. Nicole Newell, Sequencing Application Specialist at PacBio, to negotiate a 10% discount on the above listed sale price to bring the price down to $490,050 (see quote attached and letter of support from PacBio���s willingness to collaborate with us to make this grant and purchase a reality).

Challenges at the FEW nexus are not simply technological, but convergent in the sense of spanning technical, ecological, social, political, and ethical issues. The field of biotechnology is evolving rapidly - and with it, the potential for creating a diverse array of powerful future products that could intentionally and unintentionally impact FEW systems. Depending on what products are developed and how those products are deployed, biotechnology could have a positive or negative impact on all 3 of these systems. Wise decisions will require leaders who can integrate knowledge from engineering, design, natural sciences, and social sciences. We will train STEM graduate students to respond to these challenges by conducting convergent research aimed at development, and assessment of biotechnologies to improve services provided by FEW systems. We will train our students to engage with non-scientists to elevate societal discourse about biotechnology. We will recruit 3 cohorts with emphasis on students who have shown a passion for crossing between natural and social sciences. We will work with the NCSU Initiative for Maximizing Student Diversity in recruiting students from underrepresented minority groups. Cohorts will have 6 students who will take a minor in Genetic Engineering and Society (GES). They will receive PhDs in established graduate programs such as Plant Biol, Chem & Biomol Engr, Econ, Public Adm, Entomol, Plant Path, Communication, Rhetoric & Digital Media, Forestry & Environ Res, Crop & Soil Sci, and Genetics. For students in natural science PhD programs, at least 1 thesis committee member will be from a social sciences program and vice versa for students in social sciences. For all students, at least 1 thesis chapter will demonstrate scholarship across natural and social sciences. The disciplinary breadth of our proposed NRT is very broad, so we will focus student projects narrowly on a specific biotechnology product that impact FEW systems. When they first arrive at NCSU, cohorts will participate in a training program off campus where they will be exposed to the issues they will address. Students will carry out a group project in the focus area of the cohort to continue team development. To fulfill the GES minor, students will take 3 specially designed courses: Plant Genetics & Physiology, Science Communication & Engagement, Policy & Systems Modeling. There are no NRT-eligible institutions partnering on this project outside of an evaluation role.

We will use long-term Ae. aegypti samples from Iquitos, Peru to assess patterns of spatial and temporal change in pyrethroid resistance genes and in genomic differentiation to improve our understanding of this mosquito������������������s population biology and response to human-induced selection. We will use this information and that from ongoing collections of Ae. aegypti to gain insight into the dynamics of pyrethroid resistance evolution and to improve the accuracy of components of a comprehensive and robust simulation model of Ae. aegypti/dengue dynamics. We will use the outcomes of this work to provide research, regulatory, and management communities with information needed to predict the dynamics of a variety of gene drive strategies as well as the spread of resistance to insecticides and gene drives in this arbovirus vector

The US government viewed the insecticidal properties of Bacillus thuringiensis (Bt) as a ����������������public good��������������� and took action to ensure that Bt transgenes moved into crops were used in a manner that decreased the risk of pests evolving resistance. Furthermore, the US-EPA and USDA concluded that monitoring for resistance could improve resistance management practices. Despite these recommendations, measurable field-evolved resistance to Bt Cry toxins has been described for 6 major agricultural pest species, demonstrating that current monitoring practices are inadequate. We recently applied a new genomic monitoring approach to archived, field-collected populations of Chloridea virescens and Helicoverpa zea. Using this approach, we identified dozens of rapidly changing genomic regions in C. virescens and H. zea in the years following Bt crop commercialization. In the case of C. virescens, our approach discovered the decline of a pyrethroid resistance gene after Bt-expressing cotton replaced pyrethroid sprays as the primary C. virescens management tactic between 1997 and 2012. We also discovered major changes at markers throughout the genome of H. zea between 2012 and 2016, years during which field damage to Cry1 and Cry2 expressing Bt corn cultivars increased. As with any resistance monitoring approach, genomic monitoring likely has strengths and weaknesses. Our proposed research aims to shed light on these strengths and weaknesses using a combination of genomic approaches and laboratory assays. We will compare the results of our proposed research with those of our genomic monitoring experiments to quantify the true positive, false positive, and false negative discovery to rates that can be expected for a genomic monitoring approaches. Our work will provide a quantitative assessment of the use of genomic monitoring approaches for identifying field-evolved resistance prior to yield losses. While the proposed research primarily focuses on Cry resistance, our goal is to examine the utility of this approach for resistance monitoring of any plant-incorporated insecticidal trait.

Specific Objectives: 1) Measure the impact of RIB and structured corn refuge on H. zea pupal depth survival and adult flight parameters in corn; 2) Describe the role of cotton and soybean as a reservoir for H. zea Bt resistance alleles; 3) Propose a refined model of evolution of resistance for H. zea using these data.

The US government views insecticidal properties of Bacillus thuringiensis (Bt) as a ����������������public good��������������� and has taken actions to ensure that toxin genes from this organism that are moved into transgenic crops are used in a manner that decreases the risk of pests evolving resistance and eroding this public good. The US-EPA and USDA concluded that monitoring for resistance could improve resistance management practices and decrease resistance risk. However, current monitoring methods are inadequate. We will use new genomic tools in concert with field data to better assess both the current extent of Bt resistance in Heliothis virescens and Helicoverpa zea moths and the rate at which resistance is increasing, if at all. This will be accomplished by developing tools that detect changes in the frequencies of alleles of candidate Bt resistance genes and also detect changes in genetic sequences that confer Bt resistance but are not in genomic regions associated with currently identified Bt resistance candidate genes. The RADtag methodology will be used for this part of the research. We have annually archived thousands of samples of H. virescens and H. zea from 1993 until 2011, and will use these valuable samples to predict future changes in resistance from past patterns of change and current planting patterns of Bt cultivars. While the proposed research focuses on Bt resistance, the tools developed could also be used to improve monitoring of resistance to future insecticidal crop traits as well as for monitoring weed resistance relevant to transgenic, herbicide tolerant crops.

Issues surrounding genetic engineering, biotechnology, and synthetic biology are contentious, especially when applied to food, the environment, and industrial applications for which direct human consent and medical benefits are not present. How researchers, developers, and policy-makers communicate about and reflect upon their work is of utmost importance to the fields of bioengineering. This proposal fills an important niche by encouraging those involved in biotechnology innovation systems to reflect on the ethical dimensions of their work and what it means to responsibly innovate. At the same time, this proposal contributes to important comparative research on conceptions of responsible innovation across four types of institutions. Increased understanding about how participants within and across various professional contexts conceive of and frame the ethical dimensions of their work can assist with future cross-sector dialogue, and potentially conflict resolution.

This IGERT project will create a transformative graduate education program that trains students in technologies needed for manipulating pest genomes as well as methods needed to assess the environmental and social appropriateness of specific products of these manipulations. The concept of genetically manipulating a pest species to destroy or render it benign dates back to the 1940's, and there have been several major successes in using this approach. However, restricted tools of classical genetics limited the broader application of Genetic Pest Management. Recent advances in molecular genetics have provided much more precise techniques for manipulating the genomes of pests, and efforts are now underway for development and potential release of transgenic mosquitoes and transgenic agricultural pest species aimed at achieving Genetic Pest Management. The future of this pest management strategy will be determined by further technological advances, public attitudes to the novel technologies and products involved, and the creativity and wisdom of researchers and policy makers. Although esteemed scientific groups including the U.S. National Academy of Sciences have repeatedly emphasized that risk assessment for transgenic organisms should focus on the specific product and not the process, the legacy of genetically-engineered crop commercialization has made the logic behind this idea obscure to most people, including many scientists. For new applications of genetic engineering to be developed and judged appropriately, diverse social and cultural groups will need to more deeply examine the ramifications of each application. Broadly trained PhDs in biological and social sciences will facilitate this examination and help foster more sophisticated interactions among policy makers, academicians, and members of societies where Genetic Pest Management may be applied. Intellectual Merit of this IGERT derives from the fact that this could become the first graduate program in the world that is specifically training graduate students to understand, build, and assess impacts of transgenic organisms. All students will receive core transdisciplinary training that will encompass ethics, communication, economics, ecology, epidemiology, molecular biology, and population genetics. Each student will use expertise from at least two of these specialties in developing a dissertation. Our program is broad in integrating across diverse disciplines, but maintains the focus of students and faculty by specifically studying a small set of species that are targets for Genetic Pest Management. In each of the first years of the program, we will recruit graduate students in biological and social sciences. Each cohort of about six students, balanced across disciplines, will work together with faculty to choose a single target species as the focus of their dissertations. Focus on single species will challenge both student and faculty to work together, develop a common vocabulary, and understand how each other's disciplines operate. We are developing a set of core courses, which will provide all students with a basic toolkit for working in the field of Genetic Pest Management. Students specializing in the disciplines of a specific course will act as mentors to the other students taking the course. Broader Impacts of this IGERT fall into the following categories: 1) Improvement in the administration and extent of integrated graduate education at NCSU, 2) Impact on US integrated graduate education by evaluating a novel model of such integration, 3) Increased number of students from underrepresented groups that receive interdisciplinary education, 4) Improvement of methodologies for assessing and introducing new technologies, 5) Ph.D.s in biology and social sciences who have tools needed for future interdisciplinary, global work. 6) Education of local communities. Furthermore, most of the target pest species are of importance in poor nations, and we will use existing and newly developed partnerships to set up internships and dissertation projects in th

Our research on building and testing transgenic mosquito vectors of human pathogens offers an important opportunity to contribute at the interface of basic and applied evolutionary biology. Mathematical models have been developed that predict evolutionary outcomes when two populations of the same species differing in discrete genetic features are brought together or when a species invades a new environment. However, most tests of these theories are retrospective analyses. Genetic interactions that are expected during the spread of a deliberately released transgenic strain can be similar to those in natural systems but are likely simpler, and this provides an opportunity for prospective theoretical and empirical studies. We developed a number of transgenic strains of the Asian malaria vector mosquito, Anopheles stephensi, that carry genes designed specifically to affect the abundance and genetic structure of extant wild populations. Our modeling of the predicted outcomes of releases of these insects has returned counterintuitive results due to gene linkage and linkage disequilibrium between alleles at physically unlinked loci (see details below and in Rasgon and Gould 2005, Robert et al. 2013, Okamoto et al. in review). If verified empirically, these results will challenge a number of existing expectations among infectious disease specialists about optimal use of engineered mosquito strains and will provide new insights about evolutionary dynamics when both selection and intra-genomic interactions are strong. The impact at the applied level could be major changes in the way that resources are allocated to research on transgenic mosquitoes using specific approaches that seemed reasonable/unreasonable prior to our work. At the basic level, an understanding of how gene interactions affect evolution in face of strong selection will add to our understanding of constraints on rapid evolution in natural and human-altered landscapes.

The science of advanced biotechnology is moving faster than governance considerations and actions. For example, some second generation, genetically engineered organisms are not subject to formal regulation prior to environmental release. Gene drives are a subset of second generation genetic engineering technologies that are being developed with the aim of moving synthetic gene constructs into wild animal populations to protect, suppress or eliminate them (e.g. suppression of mosquitoes that transmit malaria and dengue). Recent legal and policy scholarship has focused on the need for governance to ����������������keep pace��������������� with technological innovation in multiple domains, including biotechnology. However, we currently lack a broad evaluation of the potential ecological, political economy, ethical, and other issues to guide research and development of gene drives. This workshop will serve to fill this gap.