Max Scott

Flies are diverse models for the study of the evolution of the parasitic habit. Multiple repeated origins of specialized feeding in flies can reveal the molecular mechanisms underlying adaptive changes that both produce and sustain the parasitic lifestyle. With 1525 described species the blow flies (family Calliphoridae), are an ideal candidate for comparative study of the genetic and behavioral determinants of feeding adaptations. In this family of flies, different species have been recorded feeding on living tissues of a vertebrates, living tissues of invertebrates; decaying organic matter-- especially carrion or wound tissue; and blood. Considering the feeding habits of their larvae, blow flies are classified in: necrosaprophagous species, which feed on decomposing tissue; facultative ectoparasites, which feed on dead organic matter (necrophagous), or infest necrotic tissues of living vertebrates; and obligate parasites, which feed only on the living tissues of their hosts. For this reason, they are important as indicator organisms in forensic cases, are major agricultural pests of livestock, and they play a major role in nearly all terrestrial ecosystems as decomposers of organic material. The origin and evolutionary history of diverse specialized feeding habits in Calliphoridae are still vastly understudied, and are limited by a lack of phylogenetic, genetic and ecological information. With this project, an established team of evolutionary, ecological and genomic scientists will collaboratively investigate the causes and consequences of trophic specialization as a driver of species diversity across three integrated dimensions of blow fly research: (1) phylogenetic: reconstructing the history of blow fly evolutionary relationships and mapping the transitions of feeding habits that occurred repeatedly in the family; (2) functional, quantifying the index of preference for different food sources, and testing the effects of selection on feeding preferences; and investigating the role of the blow fly gut microbiome in mediating diet adaptations and maintaining diverse biotic interactions; and (3) genetic/genomic, identifying genes and gene regions associated with specific specialized feeding habits and using an established CRISPR Cas9 gene editing system to test hypotheses about the lability, maintenance, and control of feeding strategies in diverse blow fly models. The project includes a novel cross laboratory training and data collection for graduate students and postdocs between NCSU and Sao Paulo, Brazil, the development of an on-line data resource of genomic, taxonomic and ecological information on blow flies, and participation in multi-institutional training and research opportunities that include resources and outreach to individuals and communities that are underrepresented in science.

Farm Bill. Title: Assessing the influence of genetic background on the efficacy of Drosophila suzukii male-only and gene drive strains Rationale and specific program alignment: Release of insects with conditional dominant female lethal genes (fsRIDL) and strains with a homing gene drive targeting a female-essential gene both offer a potentially more efficient genetic mechanism for controlling populations of insect pests compared to the sterile insect technique (SIT). We have made fsRIDL strains that in cage tests effectively suppress populations of Drosophila suzukii, an invasive pest of soft-skinned fruits. We have also made ����������������split drive��������������� strains of D. suzukii that target the D. suzukii doublesex gene. The strains show biased non-Mendelian inheritance but remain to be evaluated for their ability to suppress populations. Recent work from our lab and others have shown that differences in genetic background can profoundly influence the efficacy of fsRIDL and gene drive strains. Unpublished genomic analyses indicate that there are significant genetic differences between D. suzukii populations in the USA, particularly east and west coast populations. We will obtain D. suzukii from different areas of the USA and test the potential of the fsRIDL and gene drive strains for population suppression in the different genetic backgrounds. The information gained from these experiments will be valuable for regulators in assessing future field release(s) of the D. suzukii strains and relates directly to program area 3 part (c). Overall goal: To test the influence of genetic background on the ability of fsRIDL and homing gene strains to suppress North American D. suzukii populations. Specific objectives: 1) Evaluate the ability of homing gene drive strains targeting the doublesex gene to suppress a cage population of the local North Carolina D. suzukii strain. If necessary, optimize the gene drive strain. 2) Test the ability of the fsRIDL strains to suppress D. suzukii populations collected from different locations in the USA. 3) Test the ability of the gene drive strains to suppress D. suzukii populations collected from different locations in the USA. Approach: Cage populations of D. suzukii strains will be established and maintained continuously using our lab protocols. Population suppression cage experiments will be performed as previously by adding fsRIDL or split gene drive males to the cages. For fsRIDL, it is necessary to add a small excess (3x) of males each generation. For the split drive strain it is anticipated that fewer males will be needed. Each test cage will be matched with a control cage. The cages are self-maintained simply by adding diet every other day. In the split drive strain, the Cas9 gene is widely separated from the homing gene drive construct, which provides a means of biological containment. Consequently, it will be necessary to cross the Cas9 gene into each strain prior to performing population suppression experiments. Potential impact and expected outcomes: This research will provide information for regulators on how differences in genetic background can impact the ability of fsRIDL and gene drive strains to suppress populations of D. suzukii.

Rationale and specific program alignment: Y-linked gene editors offer a potentially very efficient genetic mechanism for controlling populations of insect pests compared to the sterile insect technique (SIT) and release of insects with conditional dominant female lethal genes (fsRIDL). Modeling indicates that Y-linked gene editor strains would have significantly less impact on non-target populations with which there is some gene flow compared to homing gene drive strains. However, to date there is no experimental support of these claims. We propose to build Y-linked editor strains targeting genes required for viability or fertility in females. We will then evaluate the potential of the strains for population suppression and also any impact on no-target populations. The information gained from these experiments will be valuable for regulators in assessing a future field release of Y-linked editor strains. Overall goal: To engineer Y-linked editor strains in spotted wing Drosophila (D. suzukii) and the New World screwworm (Cochliomyia hominivorax) and evaluate their potential for population suppression and impact on non-target populations. These dipteran species were selected as they are major agricultural pests and we have extensive experience engineering genetic systems in these species. Specific objectives: 1) Create Y-linked gene editor strains in D. suzukii 2) Create Y-linked gene editor strains in C. hominivorax 3) Evaluate the potential for population suppression in cage experiments. 4) Evaluate the potential for impacts on non-target populations in cage experiments. Approach: Cas9 expression constructs will be inserted onto sites on the Y chromosome using cas9-based homology dependent integration. Promoters will be chosen such that Cas9 is expressed in the male germline. Target sites on the Y chromosome have been confirmed for each species. In addition to Cas9, the constructs will carry a fluorescent protein marker gene and a gRNA(s) expression gene. The gRNAs will target genes on the X chromosome such that mutations are introduced into genes during spermatogenesis. The mutations could be loss or gain of function (lof or gof). For example, lof mutations could be made in X-linked haplo-insufficient genes (one copy is not sufficient for embryo development). As a consequence, only embryos that were from fertilized with a Y chromosome-bearing sperm will develop, which will eventually eliminate females from the population. Population suppression cage experiments will be performed as we have done for fsRIDL strains with each species. Two connected cages with limited movement of insects will be used to evaluate the potential for gene flow. Potential impact and expected outcomes: This research will provide information for regulators on the use of Y-linked editor strains for population suppression and gene flow. It will show if additional safeguards are needed for an open field test. The Y-linked editor strains could be significantly more cost effective than current SIT or fsRIDL strains for suppression of these major pests of livestock (C. hominvorax) and fruits (D. suzukii).

The goal of this project is to develop and test a CRISPRi system for transforming female blowflies (Lucilia cuprina) into males. Lucilia cuprina is a model organism used to develop tools for controlling the related New World Screwworm (Cochliomyia hominivorax). The screwworm is a devastating livestock pest. It is currently controlled using the Sterile Insect Technique (SIT). SIT works by releasing large numbers of male screwworms into the wild, and it is most effective if only males are released, without any accompanying females. Therefore, if females could be transformed into males during the production stage, the SIT would be more effective. To develop the female-to-male transformation system, I will first design and test CRISPRi genetic constructs that target the sex-determining gene tra. CRISPRi is a variant of the CRISPR system that blocks gene expression without editing the genome. After initial validation tests, I will insert the CRISPRi constructs into the genome, and screen CRISPRi-containing flies for signs of sex transformation. I will develop several CRISPRi fly lines so that the best-performing and healthiest line can be selected. The genetic constructs used to make this line could then be ported to the screwworm, helping to control this important pest.

The primary purpose of this agreement is to advance research in area-wide integrated pest management (AW-IPM), genetic control technology, surveillance technology, and regulatory entomology to target four principle invasive species targets: Asian citrus psyllid (ACP, Diaphorina citri) spotted-wing drosophila (SWD), Drosophila suzukii, light brown apple moth (LBAM), Epiphyas postvittana, and the European grapevine moth (EGVM), Lobesia botrana. These pests have all caused significant disruptions in specialty crop markets and caused economic losses to producers. New tools are needed for development of environmentally friendly and sustainable control options to maintain US access to export markets, reduce crop losses and to protect the environment by reducing the uses of pesticides. In addition, USDA-APHIS-PPQ and its cooperative program partners in state, tribal and territorial regions of the United States need better survey and rapid response tools if EGVM were to return. Development of new technologies to respond to these pests will also provide benefits in the form of technology transfer and support for new approaches that will be transferable to target other invasive species.

The New World screwworm is a devastating pest of livestock in the Americas. The aim of this project is to develop male-only screwworm strains that would facilitate efficient genetic control of screwworm. Strains will be made that carry conditional female lethal and female transformation (i.e. develop as males) genetic systems. The addition of doxycycline to the diet suppresses the systems and allows the strains to be reared in large numbers. To achieve these aims we will develop efficient means for making transgenic secondary screwworm, which is a close relative of primary screwworm that is common in North Carolina but is not a pest.

The New World screwworm is a blowfly that is a devastating pest of livestock in South America. Screwworm females lay their eggs in an open wound and the hatched larvae eat the animal alive. In addition to being a major animal welfare issue, economic losses from screwworm in South America were estimated in 2005 to be almost $4 billion annually. The goal of this project is to use new CRISPR/Cas9 technologies to develop screwworm strains for efficient suppression of screwworm populations in Uruguay. In the first phase of the project (years 1-2), PhD students and postdoctoral fellows from Uruguay will be based in the Scott lab in Raleigh to learn how to genetically engineer blowflies and make the specific gene constructs to be used in screwworm. In the second phase of the project (years 3 and 4) the students and postdocs will return to Uruguay to make the transgenic screwworm strains needed for the suppression program.

Several transgenic male-only New World screwworm strains have been made that produce only males on diet without tetracycline. The best strains have production characteristics comparable to the parental J06 strain. In the two component and all-in-one strains that use the DR2 driver, females die at the very early stages of development. It is necessary to add tetracycline to the larval diet for the initial stages of development as the DR2 driver has some activity in first instars. Current work includes an investigation of the diet composition, antibiotic dose and time of addition of antibiotic that is optimal for producing fit, competitive males. In this application, we seek continued support to further refine the female embryo lethal systems and to develop genetic systems that cause females to transform into males. Modeling suggests that the latter could be more efficient than releasing males carrying female lethal genes. The assembled and annotated genome for New World screwworm underpins all of the proposed research.

The Mediterranean fruit fly or medfly (Ceratitis capitata), and the New World screwworm (Cochliomyia hominivorax) are devastating pests of crops and livestock, respectively. In addition to conventional control tactics (e.g. insecticides, biological control), the sterile insect technique or SIT has been successfully used for suppression or eradication of both species. SIT involves the dispersal of large numbers of radiation-sterilized insects at regular intervals over an area. Any fertile females in the targeted area that mate with released sterile males do not produce any viable offspring. As a large excess of sterile males are released, the population dies out. SIT has been used to eradicate screwworm from North and Central America and to maintain medfly-free status in Mexico, California and Florida and for suppression to levels below economic importance in Israel. Due to costs associated with mass rearing, sterilization, dispersal and monitoring, the medfly and screwworm SIT programs are relatively expensive. This has prevented the further use of SIT for control of Medfly and screwworm in other regions (e.g. most of the Mediterranean (medfly), South America (screwworm). Consequently, several different genetic systems are under development that should be more efficient than SIT and thus less expensive to implement. Here we propose to develop ����������������X-shredder��������������� technologies for both pests. In the testes of males carrying this genetic system, the X chromosome is targeted for destruction by a nuclease. After mating with a wild type female, only the male offspring develop as females with only one X chromosome are not viable. Modelling by Fred Gould (NCSU) and others have shown that such sex ratio distortion systems are very effective for population suppression. The development of an X shredder strain will initially involve the identification of DNA repetitive sequences specific to the X chromosome. Consequently, transgenic strains are made that express a nuclease in the testes that targets the X repeat. We bring complementary strengths in bioinformatics and insect transgenesis/gene promoters to this project. The development and evaluation of X shredder strains could provide a potent new genetic approach for control of these major insect pests.

Several transgenic male-only New World screwworm strains have been made that produce only males on diet without tetracycline. The best strains have production characteristics comparable to the parental J06 strain. In the two component and all-in-one strains that use the DR2 driver, females die at the very early stages of development. It is necessary to add tetracycline to the larval diet for the initial stages of development as the DR2 driver has some activity in first instars. Current work includes an investigation of the diet composition, antibiotic dose and time of addition of antibiotic that is optimal for producing fit, competitive males. In this application, we seek continued support to further refine the female embryo lethal systems and to develop genetic systems that cause females to transform into males. Modeling suggests that the latter could be more efficient than releasing males carrying female lethal genes. To facilitate this work we propose to continue developing methods for targeting gene constructs to specific locations in the genome. The assembled and annotated genome for New World screwworm underpins all of the proposed research.