Location: Natural Products Utilization Research
2020 Annual Report
Objectives
Objective 1: Discover new insecticidal active ingredients that are based on natural products and that are safe with respect to occupational exposure and with respect to the environment, including non-target effects.
Subobjective 1.1: Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies.
Subobjective 1.2: Discovery of fungal compounds and natural product analogs with activity against permethrin-resistant and susceptible mosquitoes.
Subobjective 1.3: Discover novel bioinsecticide active ingredients from crude plant and fungal extract screening and bioassay-directed fractionation.
Objective 2: Participate in development of new products based on green biopesticides.
Subobjective 2.1: Develop and optimize fatty acids as natural topical and spatial arthropod repellents.
Subobjective 2.2: Development and optimization of the chromene derivative 131-1 as a biopesticide.
Approach
A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), Nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability.
Progress Report
This project was approved and became effective on April 1, 2015. This project was termed early, short of the full 5 year cycle, to bring this project in line with the cycle for all other National Program 104 projects. This project ended on 11/18/2019 and has been replaced by the project #6060-32000-003-00D entitled “Biopesticide Discovery and Development”. Refer to project #6060-32000-003-00D for additional information.
During the 4 year duration of this project, several discoveries have been made resulting in numerous publications, patent applications, invention disclosures, and scientific presentations at national and international meetings. One of the highlighted discoveries is the patent on “Chroman and chromene compounds for use as mosquito larvicides and adulticides” which was recently awarded. A Cooperative Research and Development Agreement (CRADA) partner is currently working with this technology in collaboration with colleagues at the Center for Medical, Agricultural and Veterinary Entomology in Gainesville, Florida, to scale-up and develop this technology into a commercially viable mosquito repellent product. The compounds can be applied to bodies of water that are potential mosquito habitats, used as sprays or embedded in fabrics. The compounds may also be used to treat bed nets, as fumigants, and to kill other insects and arthropods such as mosquitoes, ticks, and fleas. The lead compound, 131-1, is substantially more effective at repelling mosquitoes than DEET (N,N-Diethyl-meta-toluamide) and it also offers a longer duration of protection.
ARS researchers attempted a series of synthetic routes to optimize the yield of 131-1. Our previous synthetic scheme was shown to be tedious and not very safe in our laboratory. ARS researchers have now developed a synthetic scheme that is more efficient, convenient and most importantly produces the isomer of interest in greater than 80% yield. This method was safe, efficient and we could make the cyclized chromene aldehyde in the scale of 200 g at a time. Thus far we have produced 3.5 kilograms of the aldehyde analog which will be purified by an outside company as we do not have the chromatographic facilities to handle such large-scale purification. The purified 131-1 will be sent to inhalation toxicity studies as part of the six-pack toxicology package needed for Environmental Protection Agency registration and enhancement of private sector interest.
A large cage in-vitro bioassay system that can effectively be used to measure repellency of compounds against mosquitoes has been described and validated. This particular bioassay fills a gap for the evaluation of the effectiveness of mosquito repellents and is intended to complement both the Klun & Debboun (K&D) module bioassay and the in vivo cloth patch bioassay. The bioassay addresses some of the limitations of existing bioassays and will serve as a tool to produce data that can directly be translated into the in vivo cloth patch bioassay. The bioassay system offers benefits over the in vivo bioassay. This bioassay is an efficient tool, which can effectively be used in screening programs by researchers. There is no need to involve human subjects, who are not easily available and are one of the biggest resources in in vivo bioassays.
Previous investigations of the American beautyberry, breadfruit inflorescences and Jatropha sp. oil by ARS researchers on this project clearly demonstrated the effectiveness of evaluating folk remedies for lead compound discovery. The latter two folk remedies led researchers to focus development on fatty acids. Specifically, the investigation on structure-activity relationships (SAR) of mosquito repellent fatty acids was complete. The research resulted in multiple manuscripts on the optimum fatty acids that should be targeted for spatial repellent development. The fatty acids with the highest level of repellency were decanoic acid (C-10:0), undecanoic acid (C11:0), and dodecanoic acid (C12:0). Structure-activity relationship studies were begun on fatty acids in an attempt to optimize the activity and improve the duration of undecanoic acid (C11:0), and dodecanoic acid (C12:0). Fatty acid esters were synthesized in an effort to both improve the mosquito repellency and improve the duration of activity of the natural C-11 and C-12 fatty acids. The optimum chain length for the esters was determined and a highly active analog has been produced and shown to be more active than DEET in the K&D and Ali and Khan (A&K) module bioassay systems. This analog will be disclosed during an upcoming publication.
Two additional folk remedy investigations were completed during this project period. One of these, a plant commonly known as sweetgrass, has documented use as an insect repellent by the Flatheads of Montana and Blackfoot of Alberta. Both the Flatheads of Montana and Blackfoot of Alberta would use braided plant material in a sachet in clothing or burn them from one end as incense, air/clothing freshener, and insect repellent. This study evaluated the insect repellent properties of this plant using an in vitro mosquito feeding bioassay directed approach to identify the compound(s) responsible for the observed activities. Phytol and coumarin were both determined to be responsible for the mosquito biting deterrency. Scientific evidence reported here validates its traditional use as a biting insect deterrent.
Accomplishments
