This project will have a series of targeted experiments that will allow the project team to overcome the factors limiting the use of RNA interference (RNAi) as a control measure for invasive ants, thereby improving options for biosecurity.
The project team will use laboratory-based manipulative experiments, combined with cutting-edge assessment techniques (e.g. quantitative Polymerase Chain Reaction, Nanodrop spectrophotometry) to address why RNAi constructs are currently failing to induce desired ant death, but also to attempt to find solutions. The project team will commence experiments with constructs already used targeting the pheromone biosynthesis activating neuropeptide gene, and these constructs will be made in three designs of increasing complexity. Where applicable, experiments will be conducted using four ant species from three ant subfamilies. The project team has identified four crucial Issues that must be addressed for successful RNAi-based gene silencing, and these are addressed in separate experiments, and in sequential order. A construct will only proceed to the next Issue experiment if it successfully achieves the desired outcome of the prior Issue experiment. The project team also has additional trials to be conducted accounting for any outcomes that result in experimental dead-ends, or success overcoming all four Issues.
Objective 1 will identify constructs that have the least ultra violet degradation, and therefore the highest field utility.
Objective 2 will identify constructs that can pass ant gut linings, another requirement for field utility.
Objective 3 will identify dose concentration needed to elicit death, and if death is not induced, whether the target genes are being suppressed at all.
If needing to be assessed, Objective 4 will identify if other genes are being expressed and masking construct influence.
If needing to be assessed, Objective 5 will determine if simultaneous use of multiple constructs improves death rate.
If trials for Objective 3 or 5 are successful, Objective 6 will determine if RNAi constructs can be delivered orally to ants using hydrogels. Success at this point will result in the first RNAi construct ready for field use.
If all experiments in prior Objectives fail to produce outcomes for utility, backup Objective 7 aims to identify new crucial genes that can be targeted using RNAi.
This project is the first systematic approach the project team is aware of that attempts to identify which of the multiple reasons (individual construct coding, construct design, species-level or subfamily-level ant genetics, gene redundancy, etc.) is responsible for why RNAi constructs are predominantly failing in ant gene-silencing experiments. Not only will it be insightful for the ant species targeted in these experiments, but it will also provide great guidance for all future RNAi work, especially on new target ant species. This research also advances RNAi science by using construct designs that are more complex than others tested to date. Notably, three of the species targeted are present on US Department of Defense lands either in the Pacific or on mainland USA, and all three are of great biosecurity importance globally.