Location: Natural Products Utilization Research
2017 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 is the second full year of research for this project since it was newly created and approved. For objective 1, discovery of new insecticidal active ingredients that are based on natural products, milestones have been met and significant progress has been made.
Vellozia (V.) gigantea is a rare, ancient, and endemic neotropical plant present in the Brazilian Rupestrian grasslands. The dichloromethane extract of V. gigantea adventitious roots was larvicidal against Ae. aegypti. Bioassay-directed fractionation of the extract revealed four new compounds. These new structures are proposed based on interpretation of spectroscopic data. One of these compounds, 7-oxo-8,11,13-cleistanthatrien-3-ol, provided 100% mortality at a concentration of 125 ppm against one-day-old Aedes (Ae.) aegypti larvae. Our results show that ancient and unique plants, like the endangered narrowly endemic neotropical species V. gigantea present in the Rupestrian grasslands, should also be protected because they can be sources of new bioactive compounds.
The red imported fire ant, Solenopsia (S.) invicta, is an invasive ant species that was introduced into the United States from South America in the 1930’s and has become ecologically dominant in areas it infests. Several native ant species have been reported to compete effectively with S. invicta; among them is the little black ant, M. minimum. Alkaloids, including 2,5-dialkylpyrrolidines and 2,5-dialkylpyrrolines, have been reported to be components in the venom of little black ants, Monomorium (M.) minimum. Two fatty amines were recently reported as minor compounds. By analyzing the discharge collected from the stinger apparatus (milking), this study revealed the presence of an additional seven compounds in the defensive secretion of this ant species. Two of these compounds, ß-springene and neocembrene, were found only in the defensive secretion of queens. The removed poison and Dufour’s glands of the queen were analyzed, showing that all amines and alkaloids were from the poison gland and ß-springene and neocembrene from the Dufour’s gland. It demonstrated that the defensive secretion in M. minimum queen consists of components from both glands. This is also the first report on the natural occurrence of 9-decenyl-1-amine, N-methylenedecan-1-amine, and N-methyllenedodecan-1-amine.
A study was initiated in collaboration with scientists at the Brazilian Agricultural Research Corporation (EMBRAPA) in Brazil to investigate a new Streptomyces species isolated from a soil sample from a tropical dry forest (Caatinga) in the Brazilian semiarid region. Preliminary research resulted in the isolation of Streptomyces species extracts with LD50 values of 4 ppm against Ae. aegypti larvae. Subsequent fractionation provided enriched fractions with LD50 values of <1 ppm against Ae. aegypti larvae. Preliminary structure elucidation has been completed but additional confirmation is needed before the compound can be unequivocally identified.
Plant pathogenic fungi are good sources of pesticidal constituents. The fungus Diaporthe eres was isolated from a fungal pathogen-infected leaf of Hedera helix (English Ivy) exhibiting necrosis. The ethyl acetate extracts of the culture broths exhibited larvicidal activity against permethrin resistant and susceptible mosquito larvae. 3,4-Dihydro-8-hydroxy-3,5-dimethyl-isocoumarin was isolated and identified as the larvicidal constituent. Six analogs of 3,4-dihydro-isocoumarin were synthesized and tested for activity. Three analogs have shown good activity as larvicides but none of these analogs showed activity as mosquito adulticides.
Malabar spinach, Basella alba, is a popular green leafy vegetable in the Basellaceae family native to tropical Asia. From an infected leaf showing necrosis a colony of three fungi were cultured in potato dextrose agar (PDA) plates and identified via molecular techniques. One of these fungi was identified as a member of Phoma genus. The culture filtrates were extracted with ethyl acetate, dried over anhydrous Na2SO4 and evaporated to obtain brown viscous liquids. The ethyl acetate extracts were larvicidal to mosquitos. Bioassay guided isolation of active compounds is in progress.
Mosquito adulticide active fractions from Cashew nut shell liquid have been fractionated and three of these compounds have been purified for further testing.
For objective 2, participated in development of new products based on green biopesticides, milestones have been met and significant progress has been made.
We reported on a large cage in-vitro bioassay system that can effectively be used to measure repellency of compounds against mosquitoes. This particular bioassay fills a gap for the evaluation of the effectiveness of mosquito repellents and is intended to complement both the K & D module bioassay and the in vivo cloth patch bioassay. The system uses temperature as a landing and feeding stimulus, and collagen as a feeding substrate placed over the feeding solution solution. The bioassay addresses the limitations of the bioassays using small cells. The bioassay will also 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.
Delivery devices have been manufactured which contain both C11:0 and C12:0. A simple version utilized a candle and this device will be evaluated by collaborators at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) in Gainesville, Florida. An additional device has been produced by a commercial partner and this second unnamed delivery device will also be evaluated at CMAVE or directly by the commercial partner.
Accomplishments
1. Mosquitoes vector many pathogens that cause human diseases. Repellents play a significant role in reducing the risk of these diseases by preventing mosquito bites. ARS scientists in Oxford, Mississippi, are reporting a large cage in-vitro bioassay system that can effectively be used to measure repellency of compounds against mosquitoes. This particular bioassay fills a gap for the evaluation of the effectiveness of mosquito repellents and is intended to complement both the 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.
Review Publications
Meepagala, K.M., Estep, A.S., Becnel, J.J. 2016. Mosquitocidal activity of extracts from Ammi visnaga (Apiaceae) seeds. Journal of Agricultural Chemistry and Environment. 5:170-178.
Chen, Y., Li, J., Li, S., Zhao, J., Bernier, U.R., Becnel, J.J., Agramonte, N.M., Duke, S.O., Cantrell, C.L., Wedge, D.E. 2016. Identification and characterization of biopesticides from Acorus Tatarinowii and A. Calamus. American Chemical Society Symposium Series. 1218:121-143.
Ferreira, M.C., Cantrell, C.L., Duke, S.O., Ali, A., Rosa, L.H. 2017. New phytotoxic diterpenoids from Vellozia gigantea (Velloziaceae), an endemic neotropical plant living in the endangered Brazilian biome Rupestrian grasslands. Molecules. 22:175. doi:10.339/molecules22010175.
Ali, A., Cantrell, C.L., Khan, I. 2017. A new in vitro bioassay system for discovery and quantitative evaluation of mosquito repellents. Journal of Medical Entomology. 54(5):1328-1336. doi:10.1093/jme/tjx100.
Srivedavyasasri, R., White, M.B., Kustova, T.S., Gemeujiyeva, N.G., Cantrell, C.L., Ross, S.A. 2017. New tetranorlabdanoic acid from aerial parts of salvia aethiopis. Natural Product Research. doi:10.1080/14786419.2017.1324961.
Cantrell, C.L., Jones, A.P., Ali, A. 2016. Isolation and identification of mosquito (Aedes aegypti) biting- deterrent compounds from the native American ethnobotanical remedy plant Hierochloë odorata (Sweetgrass). Journal of Agricultural and Food Chemistry. 64:8352-8358. doi:10.1021/acs.jafc.6b01668.
