Recipient Organization
NANOSUR LLC
1951 NW 7TH AVE
MIAMI,FL 331361104
Performing Department
(N/A)
Non Technical Summary
The following is a Non-Technical Summary of the project:The red imported fire ant (RIFA) is an invasive pest introduced in the United States from Argentina in the 1930s). Currently, imported fire ants infest over 140 million hectaresin 14 states. Like many exotic pest species, invasive fire ants were introduced without most of the natural enemies that occur in their native South American range. As a consequence, fire ant population densities in the USA are 5-10 times higher than those in South America. Fire ants are particularly pestiferous because they: 1) prefer disturbed habitats (human activities), 2) have large population densities (18-35 kg/ha) and a huge need for resources, 3) are aggressive and have a potent sting - the venom can cause hypersensitivity in humans, and 4) have a large reproductive potential (300,000 new potential queens/ha/yr).Damages related to fire ants include household, crop and livestock losses, higher equipment repair and replacement costs, damages to the lawn, yard, farmstead, medical and veterinary expenditures, and cost of materials and equipment to control them. In addition to the most noticeable problem they cause, i.e., stinging of humans and animals, RIFA is also responsible for damages to soybeans, citrus, potatoes, corn, okra, and eggplants. Additionally, some agricultural commodities in infested areas, e.g., nursery and sod, must be quarantined because regulations mandate treatment of materials to be shipped to non-infested areas.The estimated annual 3+ billion dollars spent on fire ant control is indicative of the impact this ant has exerted on human activities.Current pesticides used to control RIFA have active ingredientswith varied modes of action; however, none of them is selective to fire ants, therefore they also kill organisms that are beneficial to agriculture, e.g., predatory and pollinating insects. For example, hydramethylnon, fenoxycarb, and pyriproxyfen, are toxic to fish, methoprene is toxic to amphibians, and spinosad and fipronil are toxic to honeybees. Furthermore, large-scale use of chemical insecticides is gradually being more restricted due to their effects on non-target organisms.Biological control of fire ants has been pursued, with most success with phorid fly (Pseudoacteon spp.) parasites of fire ants. Five species of these flies, from South America, were successfully released into USA fire ant populations over the past 25 years. They are self-sustaining. While they do not control fire ant colonies, they do reduce population densities.Thus, there is a need for novel fire ant active ingredients and formulations that have greater fire ant specificity, fewer effects on non-target species, and therefore, are safer for the environment/ecosystem. A gene-targeted insecticidedeveloped by NanoSUR's in this projectpresentsunique advantages for RIFA management and control by selectively targeting only RIFA without broader environmental effects and consequences.NanoSUR's technology applied to RIFA control is one of several applications aiming at first complementing, and eventually replacing, most chemical pesticides. We believe that NanoSUR has the best - if not the only - viable solution to the worldwide threat posed by chemical pesticides to the health of consumers and the ecosystem.
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Goals / Objectives
This project has one major goal: the development of an efficaciousinsecticide to control the red imported fire ant (RIFA) which is safe for boththe people handling it and the environment and is commercially attractive to commercial partners.Four objectives will be undertaken to achieve this goal. First, we will investigate means to optimize a) the safety of a candidate double strand ribonucleic acid material (MdsRNA) with respect to non-target organisms, aiming at accessing the largest range of applications and thus a larger market and b) the efficacy of MdsRNAs to the point where the technology is economically attractive for formulating into baits targeting RIFA (MdsRIFA). Second, we will focus on the scale up and characterization of the MdsRIFA-based baits, aiming at setting specifications required for their large-scale manufacturing, essential to make our product commercially viable. Third, we will develop oil-based broadcast baits for the top MdsRIFA candidates and test the ability of these baits to control queenright RIFA colonies. Forth, we will determine the minimum concentration of MdsRIFA in the bait to consistently achieve RIFA control under field conditions.
Project Methods
The following Methods will be used for the conduction of theproject:Chemically modified double strand RNAs (MdsRNAs) will be prepared by the reaction of at least 5% of the2'OH of the riboses comprised in double strand RNAs (dsRNAs) targeting a vital RIFA gene and having greater than 30 base pairs with reagents imparting lipophylic character to the dsRNAs or making them less susceptible to RNAses, such as N-methylisatoic anhydride and benzoyl cyanide. Reactions will be carried out in a solvent comprising aprotic solvents such as dimethylsulfoxide, dimethylformamide, urea, or acetonitrileand water, at temperatures between 30ºC and 95ºC. The derivatization extent of the dsRNAwill be estimated after complete hydrolysis of an aliquoted of the aqueous purified MdsRNA with 5M NaOH aqueous solution. A Photo Diode Arraydetector monitoring UV absorbance at 254 nm coupled to a high pressure liquid chromatography instrument will beused to quantify the resulting N-methylanthranoic acid and RNA nucleotides and thus evaluate the degree of modification of the dsRNA reactant.The increased lipophylic character of the resulting MdsRNA compared to the dsRNA reactant will be evaluated by quantifying the fraction of MdsRNA or dsRNA extracted from its aqueous solution with a non-polar solvent like n-octanol or dichloromethane. The stability of MdsRNA upon storage at 37ºC and theimproved stability of MdsRNAas compared to dsRNAtowards digestion with RNAses will be evaluated by quantifying the fraction of MdsRNA or dsRNA degraded, by incubation without and with RNAse III, using gel electrophoresis.MdsRNAs functioning as RNAi triggers in RIFAwill be screened for lethality using a bioassay involving feeding for 4 days a group of 20 worker ants 50 µl of liquid per day containing the MdsRNA at a known concentration in 10% sucrose aqueous solution and measuring the mortality rate of worker ants. Each cohort will consist on 4 replicates of 20 ants each. RIFA mortality resulting from feeding on MdsRNA-laced edible oil-based baits will be evaluated in the laboratory using colony fragments composed of brood and workers and in queenright feeding experiments. It will be also evaluated in the field at locations near Gainsville, Florida.For laboratory colony tests, acceptability, consumption, and distribution through worker trophallaxis of the baits will be monitored through the addition of a dye to the prototype baits that will allow visualization of the bait in brood and nestmates. A total of 8 baits, 4 with each MdsRNA will be evaluated. Controls will be bait formulations with the bait carrier, soybean oil, and a dye. The mortality effects of the prototype baits will be monitored by physically collecting dead workers and either counting them directly or by weighing 10 dead ants and then weighing the total dead ants to give approximate measure of mortality. Prototype baits that show more than >80% mortality in this feeding assay will be advanced to queenright feeding experiments.The size of the laboratory reared queenright colonies will vary from 1,000 workers to 50,000 workers. Initially, the prototype baits will be presented to small colonies. The feeding regime will be guided by the results from the colony fragment experiments conducted earlier. Queen weight, brood production (worker, male, female sexual) and mortality will be quantitated in treatments and controls at various time periods after treatment as measures colony health and the effectiveness of the prototype baits.In order to detect significant differences between treatments and controls in the field, we will 1) identify of all colonies (via GPS) at the field site; 2) measure the population Index (PI, colony height and diameter plus an estimate of the number of workers in each colony); 3) Colonies with similar PIs will be paired (control and treatment), 4) The same individual will calculate the PIs throughout the experiment to decrease variability, 5) The control and treatment baits will applied after the dew has evaporated 11:00 to 12:00 noon, 6) Controls and treatments will be applied as simultaneously as possible (within 10 min of each other), 7) PI values will be determined once a week for up to 8 weeks or longer, depending on the results.