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- Meepagala, Kumudini
- Cantrell, Charles
- 2023
- 2022
- 2021
- 2020
- Publications
- Veterinary, Medical and Urban Entomology (104)
- Aquaculture (106)

Research Project: Biopesticide Discovery and Development **Location:
                            Natural Products Utilization Research** 2023 Annual Report **Objectives**
                                

1. Develop biopesticides to improve control of arthropods.
1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation.
1.2. Discovery of fungal compounds with activity against permethrin-resistant and susceptible mosquitoes.

2. Develop biorepellents to improve control of arthropods.
2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils.
2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide.

3. Develop natural product synthetic analogs for management and control of arthropods.
3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents.
3.2. 131-1 Development into a commercial mosquito repelling product. **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**
                                

For Objective 1, develop biopesticides to improve control of arthropods, milestones have been met and significant progress has been made.
   
Streptomyces is a type of bacteria commonly found in soil and known for its ability to produce various secondary metabolites, some of which have larvicidal properties. Metabolites serve as a defense mechanism against other microorganisms and predators in their natural habitat. Microbial larvicides derived from Streptomyces have been shown to be effective against many species of mosquitoes, including those that are major vectors of human diseases such as malaria, dengue fever, and Zika virus. They are also considered to have minimal impact on non-target organisms and the environment, making them a promising candidate for sustainable mosquito control methods. This accomplishment relates to the discovery of microbial bio-insecticide compositions for controlling Aedes mosquito larvae, which are formulated from microbial formulations and/or metabolic products of S. orinoci, including neoantimycin. The potent larvicidal activity of Streptomyces metabolites, combined with their low environmental impact and selectivity, makes them a promising source for the development of effective and sustainable tools for mosquito control. We discovered larvicidal compounds against Ae. aegypti from S. orinoci. The active compound was identified as neoantimycin, and LC50 values of the crude extract of S. orinoci and neoantimycin demonstrated 1.20 and 1.93 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, neoantimycin also exhibited a LC50 value of 2.03 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti.

This accomplishment relates to the discovery of microbial bio-active compositions for controlling Aedes mosquito larvae and/or phytopathogenic fungi, which were previously unexplored Streptomyces species and their secondary metabolites. Our recent report indicates that Streptomyces distallicus and its extracts are a promising source of microbial biolarvicides. We evaluated six additional Streptomyces species, namely, S. ardus, S. kentuckensis, S. eurocidicus, S. salmonis, S. hachijoensis, and S. septatus, which are related to S. distallicus at the DNA level, for their possible application as microbial biopesticides, plant protectants, or biochemical biopesticide precursors with agricultural implications. We subjected the culture filtrates of these Streptomyces species to ethyl acetate extraction and conducted a comprehensive analysis using cutting-edge technology. We then subjected the resulting potent bioactive compounds to various in-house assays to evaluate their effectiveness against phytopathogenic fungi and larvae of Aedes aegypti. From the initial screening, LC50 values of the crude extract of S. ardus and S. kentuckensis demonstrated 1.64 and 7.27 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, S. ardus also exhibited an LC50 value of 6.48 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. Moreover, S. ardus, S. eurocidicus and S. septatus showed portent phytopathogenic antifungal activity against Colletotrichum fragraiae.

Apiaceae (formerly Umbelliferae), commonly known as parsley or carrot family is one of the largest plant families that consists of about 3780 species distributed throughout the globe. The Apiaceae family includes a wide variety of vegetables and herbs such as celery (Apium graveolens), parsley (Petroselinum crispum), dill (Anethum graveolens), fennel (Foeniculum vulgare) and cumin (Cuminum cyminum). Apiaceae plants have storage glands for essential oils giving rise to unique aroma and flavors. Apiaceae essential oils have activity against pests and microbes. Plants produce secondary metabolites as part of defense mechanisms against biotic or abiotic stress. These secondary metabolites protect plants from pests such as fungi, insects and other plants that compete for survival. Thus, these plant extracts and secondary metabolites are known to have various bioactivities, such as herbicidal, insecticidal, and antimicrobial activities.

We have extracted seeds of Celery (Apium graveolens), parsley (Petroselinum crispum), dill (Anethum graveolens), fennel (Foeniculum vulgare), cumin (Cuminum cyminum) and shown to posses larvicide activity in our laboratory bioassay at the collaborator’s laboratory. We have isolated and identified sufficient quantities to carry out repellent studies. Cumin aldehyde, cumic alcohol, apiol, carvone, anethol, carvacrol, and cumic alcohol are some of these compounds. We have synthesized various analogs of the naturally occurring constituents and these are being evaluated for larvicide activity against permethrin susceptible and resistant Aedes aegypti mosquitoes and repellent activity against the same species of female mosquitoes.

For Objective 2, develop biorepellents to improve control of arthropods, milestones have been met and significant progress has been made.

Hypericum species and especially H. perforatum are well known for their therapeutic applications. The present study assessed the essential oils (EO) composition, antifungal and aphid suppression activity of seven Bulgarian Hypericum species. The EOs were analyzed by GC–MS-FID analyses to determine the chemical constituents present. H. perforatum EO had significant repellent and insecticidal activity against two aphid species, the bird cherry-oat aphid and the English grain aphid. The tested EOs did not show significant activity against selected economically important agricultural fungal pathogens Fusarium spp., Botrytis cinerea, Colletotrichum spp., Rhizoctonia solani and A. niger. The EO of the Hypericum species found in the Bulgarian flora could be utilized for the development of new biopesticides for aphid control.

Phlebotomus papatasi, is a major vector for Leishmania major in the Middle East that significantly impacted U.S. Military operations and personnel, demonstrating the need for additional sand fly control and repellent options. We report measurement of spatial repellency and avoidance to essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cateria (catnip), Mentha piperita (peppermint), Melaleuca alternifolia (tea tree), the 9-12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid), and the synthetic repellents DEET and IR3535 to adult female Phlebotomus papatasi sand flies in a static air olfactometer. Materials tested at 1% application exhibited varying levels of activity but were not significantly different compared to DEET and IR3535 in mean repellency and avoidance to female sand flies except for nonanoic acid. Some materials produced sand fly mortality, particularly nonanoic and undecanoic acids. Different trends were noted in mean repellency over increasing exposure time: 1) Pimenta dioica oil, tea tree oil, decanoic acid, undecanoic acid, DEET and IR3535 exhibited increasing mean repellency over time; 2) catnip oil, amyris oil, peppermint oil and dodecanoic acid exhibited relatively constant mean repellency over time; and 3) Lippia graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. Essential oils of catnip, amyris, and peppermint were effective spatial repellents at reduced concentrations compared to DEET. Additional research is needed to elucidate the modes of action and potential synergism repellents, and essential oils and their components responsible for observed activity of the oils, and chemical pesticides.
C. For Objective 3, develop natural product synthetic analogs for management and control of arthropods, milestones have been met and significant progress has been made in the previous year to meet this objective's milestone.

Plumbagin, a derivative of naphthoquinone, is widely recognized for its insecticidal properties and is commonly found in natural sources such as the Plumbago plant genus, as well as the carnivorous plant genera Drosera and Nepenthes. This accomplishment relates to synthesizing various naphthoquinone derivatives and assessing their effectiveness as larvicidal agents. Furthermore, molecular docking investigations were conducted using two Aedes aegypti proteins, namely an odor-binding protein and a saliva protein, revealing that the newly developed naphthoquinone derivatives exhibit favorable binding affinities. The most potent derivative, 6-methyl-5,8-dioxo-5,8-dihydronaphthalen-1-yl hexanoate, showed LC50 values of <6.95 ppm and <8.66 against the ORL 1952 strain and PR strain of Ae. aegypti, respectively. **Accomplishments**
                                

1. Streptomyces orinoci microbial formulations as a bioinsecticide. Streptomyces is a type of bacteria commonly found in soil and known for its ability to produce various secondary metabolites, some of which have larvicidal properties. Metabolites serve as a defense mechanism against other microorganisms and predators in their natural habitat. Microbial larvicides derived from Streptomyces have been shown to be effective against many species of mosquitoes, including those that are major vectors of human diseases such as malaria, dengue fever, and Zika virus. They are also considered to have minimal impact on non-target organisms and the environment, making them a promising candidate for sustainable mosquito control methods. This accomplishment relates to the discovery of microbial bio-insecticide compositions for controlling Aedes mosquito larvae, which are formulated from microbial formulations and/or metabolic products of S. orinoci, including neoantimycin. The active compound was identified as neoantimycin, and LC50 values of the crude extract of S. orinoci and neoantimycin demonstrated 1.20 and 1.93 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, neoantimycin also exhibited a LC50 value of 2.03 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. ARS researchers in Oxford, Mississippi, have submitted the invention disclosure based on the efficacy of neoantimycin that is mentioned above. 2. Poncirus trifolia root extract against Aedes aegypti larvae. In the search for effective and environmentally friendly mosquito control agents, ARS researchers in Oxford, Mississippi, have examined natural sources such as microbes and plants and the synthetic analogs of natural products. These plants and microbes have evolved in their ecological niches to produce defensive compounds against other competing organisms in their surroundings such as microbes, plants, and insects to enhance their survival. Thus, some of these plants and microbes have bioactive compounds against insects. In our previous research, we have been very successful in isolation, synthetic modification, and total synthesis of bioactive constituents from natural sources. We have carried out synthetic modifications and total synthesis of marginally active isolated compounds to achieve significantly higher active compounds. We have focused on plants in the Rutaceae family as the members of this family are known to possess bioactive compounds with insecticidal activities. During last year we were successful in isolation of larvicidal coumarins from Poncirus trifoliata (Rutaceae) root extract. Among the five coumarins isolated, xanthoxyletin and demetylsuberosin have shown significantly high larvicide activity against ORL1952 strain of Aedes aegypti larvae. **Review Publications**
                            *Semerdjieva, I., Zheljazkov, V.D., Dincheva, I., Piperkova, N., Maneva, V., Cantrell, C.L., Astatkie, T., Stoyanova, A., Ivanova, T. 2023. Essential oil composition of seven Bulgarian Hypericum species and its potential as biopesticide. Plants. https://doi.org/10.3390/plants12040923.*
                                *Kim, S., Cantrell, C.L., Avula, B., Chen, J., Schrader, K., Santo, S., Ali, A., Khan, I.A. 2022. Streptomyces distallicus, a potential microbial biolarvicide. Journal of Agricultural and Food Chemistry. https://pubs.acs.org/doi/10.1021/acs.jafc.2c03537.*
                                *Zulfiqar, F., Ali, A., Ali, Z., Khan, I.A. 2022. Bioassay-guided isolation of iridoid glucosides from Stenaria nigricans, their biting deterrence against aedes aegypti (Diptera: Culicidae) and repellency potential against imported fire ants (Hymenoptera: Formicidae). Molecules. https://doi.org/10.3390/molecules27207053.*
                                *Meepagala, K.M., Estep III, A.S. 2023. Larvicidal constituents from Poncirus trifoliata root extracts. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjad086.*
                                *Mccomic, S.E., Duke, S.O., Burgess Iv, E.R., Swale, D.R. 2023. Defining the toxicological profile of 4-hydroxyphenylpyruvate dioxygenase-directed herbicides to Aedes aegypti and Amblyomma americanum. Pesticide Biochemistry and Physiology. https://doi.org/10.1016/j.pestbp.2023.105532.*


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