Performing Department
(N/A)
Non Technical Summary
A homing gene drive targeting a female-essential gene is potentially a very efficient genetic mechanism for controlling populations of insect pests. Here we are targeting three flies: Drosophila suzukii, an invasive pest of soft-skinned fruits; Drosophila hydei, a pest of stored sweetpotato; and Cochliomyia hominivorax, a devastating obligate parasite of livestock. A native of Asia, Drosophila suzukii, also known as spotted wing Drosophila, is now widespread in the USA. Females have a highly developed egg laying apparatus which means they can infest a wide range of host fruit before harvest. Growers are currently applying broad spectrum insecticides more frequently than in the past to protect fruit, but the effectiveness of the sprays is weather dependent. Resistance to insecticides has been reported in California. New control tactics are needed. In North Carolina, Drosophila hydei has emerged as a serious pest in facilities that store sweetpotatoes. In warmer months, the flies can be particularly numerous requiring the facilities to release insecticide sprays multiple times a day. Recent research by the co-PD Hulseth and colleagues at NC State has shown that D. hydei is likely to be an important vector for Ceratocyctis fimbriata, the causal agent of sweetpotato black rot. Female New World screwworm (Cochliomyia hominivorax) lay their eggs in open wounds or a natural orifice. The hatched larvae then feed on the animal's living tissue. Animals with severe screwworm infestations may die if untreated. One of the greatest achievement's of the USDA in the 20th century was the development of the sterile insect technique (SIT) for the eradication of C. hominivorax from the U.S.A initially and subsequently from Mexico all of Central America. To prevent re-infestation from South America, sterilized flies were being constantly released in a "buffer zone" in Eastern Panama and along the border with Colombia. However, due to the recent outbreak in Panama, sterile flies are currently being released in Western Panama and in Costa Rica. It would be very expensive and challenging to use SIT to suppress screwworm in countries where it remains endemic (e.g. Brazil, Colombia, Cuba, Jamaica).We have previously made efficient D. suzukii split homing gene drive strains that target the doublesex (dsx) gene, which is essential for female development. In a split gene drive the Cas9 gene is located on a different chromosome than the dsx gene. We inserted a single guide RNA (sgRNA) gene into the dsx female exon along with a red fluorescent protein gene to make it easy to identify offspring that inherited the disrupted dsx gene. When Cas9 is expressed in the germline along with the sgRNA, the wild type dsx gene is cut and the broken. The disrupted dsx gene is then used as a template to repair the broken DNA. In this way both copies of the dsx gene then have the female exon with the sgRNA gene and red fluorescent protein gene. This process is called homing. We made drive strains that were heterozygous female sterile (i.e. dominant) and homozygous female sterile (i.e. recessive). Mathematical modeling showed that the drive strains should be equally effective for population suppression at much lower release ratios than used for SIT (and thus less expensive). Further modeling showed that had we made a fully autonomous drive, the dominant sterile drive would be less invasive than the recessive sterile drive. A fully autonomous drive has both Cas9 and sgRNA genes inserted into the dsx gene. A less invasive drive would be considered lower risk for a future field release. We propose to build dominant female sterile homing gene drive strains for D. suzukii and D. hydei. Gene drive efficiencies will be measured under different environmental conditions. For D. suzukii, we also propose to build split homing gene drives that carry two or more sgRNA genes in the dsx gene. It is predicted that a gene drive that uses two or more sgRNAs would be less likely to fail due to the emergence of resistance to drive. As we are not allowed to work with C.hominivorax in North Carolina, we proposed to develop split gene drive strains in Cochliomyia macellaria, a very close relative that is not a pest and is commonly found in the USA. If the drive systems are effective, we will send the gene constructs to our collaborators in Uruguay for evaluation in C. hominivorax. Uruguay has recently completed the construction of a biosecure facility for screwworm gene drive research.Lab cage population suppression experiments will be performed with the D. suzukii and D. hydei drive strains. Mathematical models will be refined based on the gene drive and collected life history data. The models will guide lab cage suppression studies and planned future trials in large field cages and sweetpotato storage facilities.
Animal Health Component
0%
Research Effort Categories
Basic
10%
Applied
20%
Developmental
70%
Goals / Objectives
1) Create D. suzukii, D. hydei and C. macellaria homing gene drive strains and assess drive efficiencies.2) Evaluate the ability of D. suzukii, D. hydei gene drive strains to suppress lab cage populations.3) Collect data required for improved modeling, including life history traits, male competitiveness and gene drive efficiency under different environmental conditions.4) Develop models to predict the impact of future releases of gene drive strains in large field cages and confined field sites.
Project Methods
Germline transformation and homing gene drive assays.dsx homing strains. Knockin lines targeting the dsx female exon with gene constructs that express guide RNAs and the DsRed marker gene will be made as previously through injection of precellular embryos with the plasmid DNA construct and preloaded Cas9/sgRNA complex. For fully autonomous dominant female sterile drives, the construct will also contain a Cas9 gene driven by a germline promoter. Transgenic lines will be identified by expression of the DsRed gene as larvae and adults will show whole body red fluorescence. The lines will be maintained through continuous backcrossing of transgenic males with wild type virgin females.dsx homing gene assays. For split gene drive assays, the male offspring of a cross between Cas9 females and dsx knockin males will be crossed with wild type virgin females. The F2 offspring are scored for the presence of the DsRed marker. The higher the proportion the greater the gene drive. For fully autonomous gene drive, transgenic males will be crossed with wild type females and the proportion of offspring that show red fluorescence determined. To estimate the rate of resistance (r2) in selected crosses, DNA will be isolated from F2 flies that do not show red fluorescence. PCR and DNA sequence analysis will show if the flies carry mutant copies of the dsx gene due to NHEJ errors.Population Suppression ExperimentsSmall cage discrete generation population suppression experiments will be performed in standard Drosophila bottles, using 20 wild type pairs for each bottle. The three control bottles will only receive 20 wild type pairs each generation. The three test bottles will receive drive males at a ratio to be determined by modeling. Previous modeling suggested adding 5 drive males each generation. Thus, the initial release ratio of drive males to wild type males would be 1:4. Large cage continuous generation population suppression experiments will be performed by first establishing populations in the cages that are within a growth chamber that provides controlled temperature, light and humidity. Two hundred pairs of wild type flies are added to the cage to initiate the population. The population is maintained by adding trays of diet, some of which are removed after 24 hours and some left in the cage for up to two weeks. Larvae develop in the diet and then crawl out and pupate on the floor of the cage. The population size can be estimated by releasing wild type flies coated with a fluorescent powder and then recapturing flies in conical traps placed in the cage the following day. Drive males will then be added at a release ratio based on results from predictive models. The productivity of the control and test cages is monitored by counting the number of eggs laid in 24h in the tray of diet. The expectation is that the test cages will become less productive and eventually cease to produce eggs.For these experiments, a three-factor ANOVA will run using PROC GLM in SAS (Version 9.4, Cary, NC) to compare the effects of treatment, sex, and generation and all two-way interactions on the numbers of offspring. Post hoc tests will be run comparing treatments for each generation, and the familywise error rate will be controlled using a Bonferroni adjustment.Life History Trait Data for Improved ModelingLongevity and lifetime female fecundity will be measured to improve modeling. These traits are known to vary with temperature, genetics, and experimental methods in both D. suzukii and D. hydei. Since these data will parameterize models used to simulate gene drive performance, it is appropriate that we measure these life history traits specifically for our lab strains. In brief, single pairs are placed in vials with standard food and transferred to fresh diet every two days. The number of eggs laid will be recorded. The diet will contain a dark food color to facilitate egg counting. The number of adults that develop from the eggs will be recorded. The day the male and female flies die will be recorded. If a male dies early in the experiment it will be replaced with a fresh male. We will measure longevity and fecundity under our standard conditions (21°C, 14/10h, 65% RH) and also early season (17°C, 12/12h, 65% RH) and mid-summer (25°C, 16/8h, 65% RH) conditions. Twenty replicates will be set for each condition.Mathematical ModelingThe models developed will build on those previously used for our gene drive experiments. An ordinary differential equations model will couple genetic and population dynamics. We shall assume the population is well-mixed and that generations are overlapping. The model will be empirically parameterized by the life history traits (see above) and using gene drive parameters measured from the results of the small cage trials. Temperature (including extreme temperatures) will be included as this is a critical driver of population dynamics. While host fruit availability is an important driver, we will ensure that sufficient food is available throughout the experiment, allowing us to ignore density dependence (although later models for field releases would need to include this feature). Models that describe population age structure at differing levels of detail will be compared, providing additional confidence in modeling results.For D. hydei, models will simulate drive performance in a sweetpotato storage facility. In this setting, fly populations invade each year in a confined, temperature-controlled warehouse, with an explosion in population size. The setting of rapidly growing D. hydei populations in warehouses raises questions about how to effectively deploy gene drives, particularly how and when releases should occur. These will be our preliminary modeling questions. The model will consider known temperature fluctuations in the warehouse and assume overlapping generations. A well-mixed population model will be used initially, this could be refined such as by including spatial extensions (e.g. describing the two- and three-dimensional structure of the warehouse). Spatial structure could be included using multi-patch or partial differential equation models. Again, assuming that the available sweetpotatoes represent a large food source, density dependence need not be included in the model.Confidence in the mathematical modeling results will be strengthened by the use of sensitivity analysis and uncertainty quantification. These analyses will identify sensitive parameters (i.e. those whose values strongly impact model predictions) and will assess how uncertainty in parameter estimates affects outcomes such as peak infestation.