Recipient Organization
NANOSUR LLC
1202 ANN ST.
MADISON,WI 537132410
Performing Department
(N/A)
Non Technical Summary
Imported Fire Ants (Solenopsis richteri, Solenopsis invicta) are invasive pests which currently infest over 140 million ha in 14 states. Damages related to fire ants have a significant impact on many economic sectors, including agriculture and biodiversity. The estimated annual cost spent on fire ant control is 3 billion dollars. Current chemical insecticides used in fire ant control are not selective, causing unnecessary ecological damage. RNAi represents a viable option for the selective and safe control of fire ants. However, current RNAi triggers are polar, have poor permeability across biomembranes and associate poorly with the oil in broadcast bait formulations. The overall goal of this project is to develop chemically modified RNAi triggers with enhanced delivery properties and oil solubility to yield bait formulations that show improved efficacy against RIFA in colonies. To attain this goal, we will demonstrate that we can accomplish the following technical objectives: 1) Identify optimal combination of phenol ether moiety, extent of the modification, and concentration, to yield high potency modified RNAi triggers (MdsRIFAs), 2) Identify oil soluble MdsRIFA candidates, prepare bait formulations, and test their stability, and 3) Test the ability of the MdsRIFA bait formulation to induce RIFA lethality on a colony scale. The project will build on results from a prior NSF SBIR Phase I project and it will be conducted in collaboration with the Imported Fire and Household Insects Research Unit of the Agricultural Research Service in Gainesville, Florida through a CRADA with the USDA.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Goals / Objectives
Imported fire ants in general, and RIFA in particular, are very difficult-to-control insect pests that have a significant impact on many economic sectors, including agriculture and biodiversity. Current chemical insecticides used in fire ant control are not selective, causing unnecessary ecological damage. The estimated annual 3 billion dollars spent on fire ant control is indicative of the impact this ant has exerted on human activities. Toxic baits with greater fire ant specificity would be better for the environment. RNAi represents a viable option for the selective control of fire ants. However, current RNAi triggers associate poorly with the oil in broadcast bait formulations, are unstable, and quantitatively inefficient, in part due to their instability and deficient transport properties across biological membranes. NanoSUR is proposing the development of RNAi triggers efficacious against RIFA with enhanced stability and delivery properties for their use in conventional broadcast baits.Increasing stability and permeability of RNAi triggers via 2'-OH derivatizationChemical modification of RNA precursor molecules has long been used for the purpose of increasing stability towards nucleolysis of RNA prepared by synthetic procedures. Hydroxyl modifications at the 2' ribose position are among the most popular methods for making nucleic acid molecules more stable towards the actions of various ribonucleases.However, the current preparation of these RNAs by chemical synthesis is too expensive to be used in large-scale agricultural applications.Another method of creating RNA molecules modified at the 2' position consists of reacting the RNA after in-vitro transcription with fluorodinitrobenzene (FDNB) to form dinitrophenyl (DNP) ethers at the 2' position. Chen, X. et al. have used dsRNA derivatized at the 2'-OH position with FDNB. Data obtained using DNP-derivatized dsRNA showed that it is 4-fold more efficacious at inhibiting cancer cell growth in mammalian cell lines than non-derivatized dsRNA. However, this derivatization method only works on ssRNA, which cannot be prepared at cost economically attractive for large scale applications. Another type of post-transcriptional modification has been developed by Weeks et al. as a tool to probe the structure of RNA (Merino et al., 2005; Mortimer and Weeks, 2008). This method uses N-methylisatoic anhydride (NMIA) or benzoyl cyanide (BzCN) as 2'-O acylation reagents. Derivatization is restricted to single stranded positions like gaps, bulges and loops as well as adjacent nucleotides, with preference for As and Gs.In addition to stability, these types of RNA modification seem to increase the lipophilicity of the nucleic acid as it can be judged from the observed increased cell membrane permeability (Liao and Wang, 2005). Interestingly, other derivatizations leading to increased lipophilicity of RNA molecules, such as cholesterol conjugation and the use of phosphorothioates in the nucleic acid backbone, also resulted in enhanced stability and cellular delivery of the modified RNA (De Paula et al., 2007).NanoSUR InnovationThe core of NanoSUR innovation is a new class of RNAi-based pesticides comprising long dsRNA modified post-transcription at the ribose's 2′-OH position with lipophilic groups. Prior to NanoSUR's breakthrough, the standard method to obtain chemically modified dsRNA consisted of hybridizing chemically modified ssRNA sense and antisense strands prepared by in vitro transcription or by stepwise chemical synthesis. Due to cost and scale constraints, only dsRNA prepared by fermentation can be used for the commercial preparation of RNAi triggers.Proof of Concept (POC) for NanoSUR modified RNAs (MdsRNAs) efficacy was achieved in vivo with insects from three orders and in planta for a fungus, to date, in research conducted under NSF SBIR Phase I Award ID # 1647751 (P.I. J. Arhancet). In this award, we demonstrated that dsRNA targeting β-actin mRNA (GenBank: XM_011175337.1) modified with the reagent FDNB (DNP-β-actin dsRNA) had significantly increased potency against Solenopsis Invicta (Red Imported Fire Ant, RIFA) compared to unmodified β-actin dsRNA. In this project, we will build on these results and develop a modified RIFA dsRNA (MdsRIFA) with improved potency and solubility in oil-based bait formulations to control fire ants at colony scale. To attain this goal, we will demonstrate that we can accomplish the following technical objectives: 1) Identify the optimal combination of modifying phenol ether moiety, extent of the modification, i.e., % 2'-O groups per number of nucleotides, and concentration, to yield high potency M-β-actin dsRNA (i.e., MdsRIFA), 2) Identify a stable oil-based bait formulation for the MdsRIFA candidates, and 3) Test the ability of the MdsRIFA bait formulation to induce RIFA lethality on a colony scale.Specifically, the Technical Objectives are as follows:Objective 1. Identify the optimal combination of modifying phenol ether moiety, % derivatization, and concentration of MdsRIFA to yield a high potency insecticideIn this Objective, we will determine whether a more lipophilic MdsRNA resulting from a modifying ether moiety with higher cLogP and/or more extensive % derivatization will result in a more potent RNAi trigger than that demonstrated in the worker ant feeding bioassay. We will prepare MdsRIFAs by reaction of dsRIFA with FDNB and 2,4-bis(trifluoromethyl)fluorobenzene, separately, and with different % derivatization. We chose to use these two reagents as they are substantially equivalent in terms of structure and reactivity but are calculated to yield MdsRNAs with very different lipophilicity and solubility parameters. The proposed ether-modified dsRIFAs are expected to exhibit improved solubility in non-polar organic solvents such as soybean oil over the unmodified or naïve dsRNA and within the range of the traditional chemical insecticides currently used to control RIFA. Developing a dsRNA for RIFA with similar physicochemical properties as currently used insecticides is critical to allow delivery of the actives in granular bait formulations.The potency of the MsdRIFAs will be evaluated in the worker ant feeding test we previously used in the NSF SBIR at different concentrations. This evaluation will allow us to correlate concentration and % derivatization with in vivo activity and gain understanding on how derivatization might affect activity, from which we can design a potent RNAi trigger.Objective 2. Identify a stable oil-based bait formulation for the MdsRIFA candidatesThe primary question to answer from this objective is whether the modified RNA triggers have the lipophilicity to be soluble in vegetable soybean oil and, once formulated in granular broadcast bait, and whether the new materials are stable. We will perform an in vitro test to demonstrate that the MdsRIFAs from Objective 1 can be formulated in baits, formulate the MdsRIFAs, and then determine the stability of the MdsRIFA in the bait vs. time as a function of the concentration of MdsRNA, the different co-solvents used in the bait, and temperature.Objective 3. Test the ability of the MdsRIFA bait formulation to induce RIFA lethalityIn this objective we will determine the ability of the best bait from Objective 2 to control RIFA colonies. Two-hundred mg of worker ants (approximately 200 ± 20 individuals) will be introduced into a large Petri dish containing the bait. Two colonies will be used for each bait bioassay, including a control in which MdsRIFA will be replaced with dsRIFA. During the bioassay, the ants will be maintained in an insect growth chamber (27 ± 1°C, RH 70 ± 1%, L: D=12:12). Mortality will be recorded daily for 14 days.
Project Methods
Fire ant colonies of both monogyne and polygyne social forms are readily collected from the field and reared under laboratory conditions. Laboratory facilities and field sites are more than adequate to carry out evaluations of RNAi bait prototypes.Objective 1: We will react a sequence targeting RIFA's actin muscle, named dsRIFA, with fluorodinitrobenzene (FDNB) and 2,4-bis(trifluoromethyl)fluorobenzene (BTFB) to yield post transcription chemically derivatized dsRIFA at the 2'-OH position in the riboses with a dinitrophenol ether, named DNP-dsRIFA, and with bis(trifluoromethyl)phenol ether, named BTP-dsRIFA, respectively. We will prepare two samples of each, DNP-dsRIFA and BTP-dsRIFA, one with medium and another with high % derivatization.We will have the starting dsRIFA and negative control dsRNA produced by the agroRNA company. Scientists at NanoSUR have had experience in using dsRNAs made by agroRNA and have found them to be of the high level of purity required for use in our derivatization reactions. We will have agroRNA produce 100 mg of dsRIFA and control dsRNA. We will choose to have these dsRNAs purified to Grade 3, the highest purity available. This features HPLC purification resulting in up to 95% purity. This material will be used in the derivatization reactions and underivatized controls.For dsRIFA we will use a 299 bp dsRNA sequence targeting RIFA (Solenopsis invicta Buren) actin muscle (LOC105205816, GenBank: XM_011175337.1 nucleotides# 465-763). The Red Imported Fire Ant's actin gene encodes a critical component of the organism's muscle and significant suppression of this essential gene results in insect death. This sequence was used to achieve POC for the MdsRIFA during NSF Phase I project. For the control RNA, we will use a dsRNA sequence for GFP.For each derivatization reagent we will produce ≥50 ug samples of MdsRNA with medium and with high % derivatization levels. We will also modifiy dsGFP with FDNB and 2,4-bis(trifluoromethyl)fluorobenzene at high % derivatization as negative controls. Therefore, a total of 6 samples of MdsRNA will be prepared.Measuring activity of RNA triggers in a RIFA feeding bioassayA simple bioassay, similar to that described by Choi et al. (2012), will be used to evaluate the ability of ether modified dsRIFAs to suppress essential RIFA gene expression and thereby kill or otherwise generate a measurable phenotypic effect. This bioassay involved feeding for 4 days a group of 20 worker ants 50 µl of liquid per day containing the test substance at known concentration in 10% sucrose aqueous solution with 4 replicates to be conducted for each MdsRNA and dsRIFA as positive control (n = 80). Mortality will be recorded daily for 20 days.There will be 9 groups in the study, these include DNP-dsRIFA high and medium % derivatization, BTP-dsRIFA high and medium % derivatization and dsRIFA. The later will serve as a positive control to induce lethality in the worker ants. DNP-GFP-dsRNA high % derivatization, BTP-GFP-dsRNA high derivatization, and underivatized control GFP-dsRNA will be included to determine if there are any non-specific effects from derivatized or underivatized dsRNA. All dsRNAs will be tested at 3 different concentrations: 10 ng/uL, 100 ng/uL, and 1,000 ng/uL to determine if there is a dose response at these concentrations. A group will also be included on the same diet without dsRNA to serve as a reference.Contingency: If we are not able to achieve the desired increase in lethality by focusing solely on the modification of the dsRNA we will try another sequence relevant to RIFA as the starting dsRIFA, e.g. those used by Vander Meer and Choi (Vander Meer and Choi, 2013) or Liming and Chen (Liming and Chen, 2013).Objective 2: We will first dissolve the most potent MdsRIFA found in Objective 1 into soybean or corn oil, which are good fire ant phagostimulants and would be used in a typical fire ant bait formulation. These can be used to determine the stability of the MdsRIFA over time as a function of the concentration of MdsRNA. Co-solvents (oleic acid and sorbitan monooleate) may be necessary to achieve the desired active material concentration.The baits will be separated into aliquots to conduct aging studies where aliquots will be aged for 21 days at 20°C and 42°C, at 2 relative humidity (RH) levels, kept at either lower than 30% or greater than 80% RH. The aging experiments conducted at greater than 80% RH, to simulate rainy weather, will be preceded by spraying water into the bait at a 10% weight/weight basis. MdsRIFA will be extracted from the oil or bait after aging, quantified and compared to the amount of MdsRIFA recovered before aging, to determine its stability.Objective 3: To control RIFA in a laboratory sub-colony bioassay. 200 mg of bait will be placed in a cap of a Wheaton 20-mL glass scintillation vial and then placed at the center of a plastic Petri dish (100x25 mm). The inner wall of the Petri dish will be coated with Fluon®. The cap with bait will be covered with another inverted Petri dish (60x15 mm) which had an entrance hole at the edge. Two-hundred mg of worker ants (approximately 200+/- 20 individuals) will be introduced into the large Petri dish. Two colonies will be used for each bait bioassay, including a control in which MdsRIFA will be replaced with MdsGFP. During the bioassay, the ants will be maintained in an insect growth chamber (27+/- 1°C., RH 70+/-1%, L: D=12:12). Mortality will be recorded daily for 14 days. Each feeding bioassay will be repeated at least 3 times.