DEVELOPMENT AND EVALUATION OF Y-LINKED GENE EDITORS FOR SUPPRESSION OF POPULATIONS OF SPOTTED WING DROSOPHILA AND THE NEW WORLD SCREWWORM

drosophila suzukii, also known as spotted wing drosophilad. suzukii larvae are capable of infesting a wide range of host fruit but appear to be most significant pests in stone fruits (peach, cherry, and plum) and berries (caneberries, blueberries, and strawberries). unlike most other drosophila species, female d. suzukii are able to pierce the skin of soft fruits and lay their eggs inside the fruit. d. suzukii has a short generation time and multiple generations per year. in contrast, the larger fruit flies (rhagoletis sp.) native to north america have only one generation per year. d. suzukii is endemic in asia but in 2008 the fly was found in california. since then, d. suzukii has spread rapidly and is now found in temperate regions in north america and europe. importantly, in the usa, any fruit that contain developing d. suzukii larvae can cause an entire shipment to be rejected! growers are currently using broad spectrum insecticides to protect fruit from damage caused by d. suzukii. for example, growers in north carolina are using more frequent insecticide applications to manage this invasive fruit fly but the effectiveness of these treatments is weather dependent. it is also anticipated that d. suzukii will develop resistance to some of the more commonly used insecticides. therefore, non-chemical means for controlling d. suzukii are needed.cochliomyia hominivorax, also known as the new world screwwormc. hominivorax is adevastating pest of warm-blooded animals and causes major economic losses (>$4billion annually). female screwworm flieslay 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. however, most cases are less severe but they are economically important because the animal suffers weight loss and carcasses and hides are damaged. in the firstand arguably most successful genetic control program, c. hominivorax was eradicated from the usa, mexico and central america through use of the sterile insect technique or sit. sit involves regular releases of large numbers of sterile flies over the targeted area. female flies in the area that mate with sterile males do not produce any offspring. screwworm remains in the large caribbean islands (e.g. cuba, hispaniola, jamaica) and most of south america. to prevent re-infestation from south america, sterilized flies are currently being constantly released in a 'buffer zone' in panama along the border with colombia. the screwworm mass rearing facility is in pacora, panama and is run by the u.s.-panamanian commission for the eradication and prevention of screwworms (comisión panamá-estados unidos para la erradicación y prevención del gusano barrenador del ganado or copeg). currently about 20 million flies are reared each week. all of our work on screwworm genetic engineering has been conducted in this facility.theoutbreak of screwworm in 2016 in the florida keys shows that the usa remains vulnerable to the reintroduction of this pest species. fortunately, with regular releases of sterile flies from the panama facility, the outbreak was stopped before flies spread to the mainland, where they would have been a danger to the florida livestock industry.while successful, sit is expensive due to the large numbers of flies that need to be reared. more efficient genetic control methods are needed if screwworm is to be eradicated from the caribbean or south america.our approach: y-linked crispr/cas9 systems that cause female lethalitythe crispr revolution of gene editing could lead to several novel genetic systems for efficient methods for suppression of pest populations. one system that has received considerable attention in the popular press and scientific literature are cas9-based homing gene drives.these genetic systems have the potential to be very efficient and thus very economical for pest suppression. however, the released insects would be very difficult to contain as the system is self-propagating and designed to spread. the current systems also appear to breakdown very quickly due to resistance. we are proposing to use a different system known as y-linked editor or yle. while this system is not a gene drive, modeling has shown that yles could be significantly more efficient for population suppression than sit .thus, our goal is to make d. suzukii and c. hominivorax yle strains and then assess their potential for population suppression in laboratory cage experiments.yle strainsa yle strain has two essential components in one gene construct. the first component is the cas9 nuclease. expression of the nuclease is driven by a gene promoter that is active in the male germline (testes). the second component are guide rna(s) that area also expressed in the male germline. the guide rna guide the cas9 nuclease to a gene. cleavage of the gene by cas9 and misrepair will lead to a mutant gene that is not functional. we will identify guide rnas for x-link .......