Recipient Organization
NOVOCLADE, INC.
1000 WESTGATE DR STE 127
SAINT PAUL,MN 55114
Performing Department
(N/A)
Non Technical Summary
Insects cause an estimated $7B in damage to global food production despite using the best availablecontrol methods. Current management strategies of using chemicals for their control often causeharm to non-target species. Sterile Insect Technique approaches are effective ina variety of different species but suffer from wide adoption due to the need for a laborious methodto separate males and the challenge of using radiation for sterilization. New measures have beendeveloped using genetic engineering which either introduce a dominant lethal transgene into theinsect population or rely on engineered insects rendered sterile. In either of the cases, there is a riskfor transgene spreading throughout the population and potentially unintentional consequences.There is a need for control methods that are species-specific, broadly applicable to diverse insects,cost-effective to allow scale-up for control on a regional or national scale, and allow limiting geneflow between insects.Spotted wing drosophila (SWD) is one such insect, whereSIT approaches could be used for its control. It has wreaked havoc in the soft fruit industry afterits introduction in the late 2000's in the US. One of the reasons for real concern from SWDinfestation is the loss of revenue in different fruit farming. As much as 30% loss of productioncan be directly attributed to SWD infestation despite adopting the best control measures,which is estimated to be $400 million every year from all the farmed and wild fruit revenue. Traditional practices and habits ofpest control don't work against SWD since the fly doesn't sit out in the open where it would beeasy to kill. While not very effective, the total cost of traditional control including materials,labor, and machines to apply insecticides, deploy mass trapping devices, and mount and maintainenclosure nets is about $2000/ha. This is a significant burden on farmers, who were usedto spraying insecticides just once or twice in an entire season of fruit production. The use of the SITapproach for SWD could be a very effective method of control, but requires means to sort malesand effective means of sterilization in a cost-effective manner.In this proposal, we aim to batch-produce onlySWD males that don't contain anytransgene, using geneticengineering of sex chromosomes followed by a specific mating scheme, which can then besterilized and released for population control. This approach forgoes manual sex separation and canbe adapted to any sexually reproducing organism. The introduced insects and their traits will beremoved from the population as soon as they die.This proposal, if successful would drastically simplify the use of SIT in SWD and open doors to using SIT in virtually any sexually reproducing insect species.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
The goal of this proposal is to evaluate the production oftransgene-free sex-sorted males of spotted wing drosophila (SWD) meant for sterile insect technique and evaluate parameters for the ideal deployment of the technology.1. Implement a Subtractive Transgene Sex Sorting system in SWD to produce transgene-free sex-sorted males.Two SWD strains will be engineered to carry a repressible lethal construct in the X or Y chromosomes. One strain will carry the construct in the X-chromosome and another strain will carry it in the Y-chromosome. After making the strains homozygous for the transgene under permissive conditions, staged breeding will be carried outunder non-permissive conditions to create sex-sorted non-transgenic males.2. Create a computational simulation model for SWD population control.An agent-based model will be created to simulate the efficacy of using the sex-sorted males forsterile insect control, and its comparison with other competing approaches for the control of SWD. This model will simulate the efficacy of each approach with varying degrees of fitness, and additional risk to fruit farming due to off-target effects. Finally, the model will simulate different environmental factors along with migration to provide a more realistic outcome.
Project Methods
Objective 1. Implement a Subtractive Transgene Sex Sorting system in SWD to produce transgene-free sex-sorted males.Design and construction of lethal circuit - During this task we will design and construct theconditional lethal circuit meant for the creation of X and Y-lethal strains. We will design twoversions of the circuit, one repressible by tetracycline and the other inducible by temperature. Thedesigns will be based on previously described lethal circuitswith modifications to allowexpression unique to the SWD system. Among the two versions of the conditional lethal circuit, weshould be able to find one that behaves suitably to our needs.Create conditional lethal strains of SWD - During this task, we will create GE SWD strainscontaining the conditional lethal circuit by using either piggyBac, PhiC31, or CRISPR Cas9 based integration system. To speed up the process of identifying transformants, embryomicroinjections will be carried out both in-house and at a commercial facility. Following the creation of homozygous transgenic SWD lines, fitness defects willbe measured by evaluating morphological, behavioral, and reproductive abnormalities relating tofecundity or mating success. Specific parametersthat will be measured for evaluation of fitness are, time to hatch, proportion of unhatched eggs, time to pupation, eclosion, and adult survival. Strains with near wild-type performance in the abovemeasurements will be selected for further evaluation.Measure performance of conditionallethal genetic constructs - Strainscontaining conditional lethal constructs withno apparent fitness defects will be rearedfor several generations. To test the efficiencyof toxic gene expression, virgin femalesand males will be mated in non-permissiveconditions, e.g. on media lacking tetracycline.The mating experiment will be carriedout in replicates and repeated at leastthree separate times on different days. The number of offspring produced at eachstage of development will be recordedand averaged between the replicates andstatistically analyzed.Objective 2. Create a computational simulation model for SWD population control.We will develop an agent-based simulationmodel for SWD based on field and laboratorydata gathered by us and researchers atthe University of Minnesota. The model will allowus to simulate population control using the sterilemales proposed in the current proposal. We willuse the model to develop an experimental plan foran eventual field trial incorporating the differentenvironmental factors observed in the field.Modify an existing agent-based model tosimulate SWD populations - We have recentlydeveloped an agent-based simulation modelfor SWD. The model, based on Lagille et al.,takes intoaccount development through several larval instars,pupation, and adulthood. We will estimate key parameters for SWD development, fecundity, and reproduction usingempirical data published for SWD populations, andvalidate it bysimulating already published fieldstudies. We anticipate that the model developed will serve as a foundation thatcan be continuously refined as our understanding of SWD population biology improves.Add environmental factors to the simulation model - A primary interest in developingmodeling capabilities for SWD is to guide the design of realistic field release scenarios. Migrationof wild insects into control areas and migration of sterile biocontrol agents out of the control areais a known confounding factor for sterile male-based biocontrol. We will use recent data onwind-driven dispersal of SWD to add those effects into our simulation model. The main goal ofthese models will be to predict local migration into and out of biocontrol areas; on the distancescales of hundreds of meters.Also, we will add the performance measurements for the different biocontrol approaches indiverse temperature/humidity settings. Combining both sets of environmental factors (windeffects and temperature/humidity effects) into the simulation model will provide a more nuancedexperimental design for field trials.