Progress 11/07/17 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): Objective 1. Enable the commercial production of microorganisms pathogenic to mosquitoes. Objective 2. Enable the commercial production of bioactive compounds/ metabolites derived from plants and microorganisms to control mosquitoes and/or the viruses they carry. Approach (from AD-416): Our approach will focus on the discovery of novel microbial and plant- based biopesticides that could be commercialized for the control of mosquitoes or the pathogens they transmit. A variety of entomopathogenic fungi will be evaluated for their effect on survival of adult mosquitoes. Selection of candidate mosquito entomopathogens will be done initially by focusing on the isolates with known pathogenicity and strains that have previously been isolated from dipteran species. Transcriptomic analysis coupled with functional assays (through reverse-genetic techniques) will be used to identify the fungal mode of action as well as the mosquito responses to infection. Attempts will be made to isolate and identify microbe-derived molecules with biological activity against mosquitoes and selected arboviruses. We will also evaluate plant-based compounds effective for activity against mosquitoes. We will integrate standard insecticide testing bioassays with modern and conventional approaches in chemical ecology to identify the chemical compounds in selected plants that are attractive to gravid females and deleterious to mosquito larvae. This is the final report for Project 5010-22410-020-00D. This research project focused on the discovery of novel compounds from plants, bacteria, and fungi for controlling mosquitoes and the diseases they vector. The overall goal was to discover new vector control tools for application in integrated vector management. Bacterial communities that inhabit the mosquito body play an important role in mosquito survival and development and can also interfere with the mosquito⿿s ability to transmit diseases. Identifying these microbes and factors that influence their composition and diversity can facilitate identification of bacterial species that could be harnessed for mosquito control. ARS scientists in Peoria, Illinois, made significant progress in Objective 1 by determining that parental background, host sampling location, and host blood meal source are factors that influence the diversity and composition of bacterial communities associated with mosquitoes. We successfully used fluorescent proteins as markers to study the functions of bacterial communities inhabiting the mosquito gut. Additionally, the scientists identified new fungal strains that are lethal to mosquitoes, increasing the list of fungal microbes that could be developed as biopesticides for vector control. In addition, the scientists successfully identified previously unrecognized molecules that enable the mosquito to detect and overcome attack by fungal infections. These molecules allow the mosquito to survive infection by some fungal strains but not others. For Objective 2, ARS scientists demonstrated under field conditions that leaf litter of common blackberry is an ecological trap for the northern house mosquito, the primary vector of West Nile virus in northeastern United States. Storm water catch basins ⿿treated with leaves of common blackberry were highly attractive to egg-laying female mosquitoes but detrimental to the hatching larvae. The detrimental effects on mosquito larvae were not due to the presence of toxic chemicals from decaying leaves, but rather due to blackberry leaves serving as a low-quality food resource for mosquito larvae. The microbial communities present in blackberry leaf infusions served as a poor food resource for mosquito larvae yet emitted chemical cues that were attractive to egg-laying adults. Because the results could not point to specific chemical compounds from blackberry leaves that were responsible for these bioactivities, research was expanded to identify botanical sources for mosquito control by investigating the biological activity of various plant essential oils on eggs and larvae. These studies have led to the identification of novel plant-based compounds and oil emulsion formulations for further development as commercialization tools for mosquito control. Substantial progress was made at screening several fungal strains for their potential to generate compounds that can be used as biopesticides and as drugs for the treatment of human microbial pathogens (i.e., bacteria). Our efforts identified several fungal strains with quantifiable activity against bacteria. Fungal growth media were manipulated to induce and maximize fungal production of bioactive compounds. The optimum media for the initial species continues to be tested with new fungal species. Accomplishments 01 Mechanisms behind the mosquito resistance to fungal infection. Specific fungi are very effective at attacking and killing insects and are environmentally friendly alternatives to synthetic insecticides. However, mosquitoes are also able to survive attack by some of these fungi. ARS scientists in Peoria, Illinois, have identified the specific molecules that the mosquito produces to successfully detect and defend itself against fungal infection. These molecules were highly effective against some fungal strains but ineffective against other strains. Overall, ARS scientists have uncovered and published significant new findings that will be exploited in our continuing research during the selection of fungal strains with different modes of action to accelerate the rate kill of mosquitoes and prevent the development of resistance. This research provides critical knowledge that can be used to exploit the use of fungal biopesticides to limit and manage mosquito resistance to synthetic insecticides. 02 Plant-based compounds for mosquito control. Plants are well-known sources of effective and environmentally friendly compounds for mosquito control. Some plants produce essential oils that kill mosquitoes. Unfortunately, these oils tend to evaporate quickly when applied to terrestrial habitats for adult mosquito control and are insoluble in water limiting their use in larval control. ARS scientists in Peoria, Illinois, discovered components from manuka essential oil that were very toxic to mosquito larvae and could be developed as effective natural pesticides. Researchers also developed a method that enhanced the toxicity of garlic, asafoetida and manuka essential oils against mosquito larvae and improved their solubility when applied in water bodies where mosquito larvae reside. This research provides critical knowledge that can be used to develop essential oil-based biopesticides for mosquito control. 03 Microbial metabolites for mosquito control. Beneficial fungal pathogens produce molecules that allow them to infect, propagate and kill targeted insect hosts. Identification of these molecules and their biological activity against mosquitoes is a critical step towards the discovery of new biopesticides for mosquito control. ARS scientists in Peoria, Illinois, identified several fungal isolates with ability to produce compounds that inhibit bacterial growth and control mosquitoes. Researchers also enhanced the production of these compounds by optimizing the growth conditions of the fungal isolates. The optimized growth of fungal isolates with antimicrobial and insecticidal properties has the potential to yield novel agents for mosquito control and for the treatment of human/animal diseases.
Impacts
(N/A)
Publications
- Muturi, E.J., Selling, G.W., Doll, K.M., Hay, W.T., Ramirez, J.L. 2020. Leptospermum scoparium essential oil is a promising source of mosquito larvicide and its toxicity is enhanced by a biobased emulsifier. PLoS One. 15(2):e0229076.
- Barletta, A.F., Trisnadi, N., Ramirez, J.L., Barillas-Mury, C. 2019. Mosquito midgut prostaglandin release establishes systemic immune priming. iScience. 19:54-62.
- Tchouassi, D.P., Muturi, E.J., Arum, S.O., Kim, C., Fields, C., Torto, B. 2019. Host species and site of collection shape the microbiota of Rift Valley fever vectors in Kenya. PLOS Neglected Tropical Diseases. 13(6) :e0007361.
- Caceres Carrera, L., Victoria, C., Ramirez, J.L., Jackman, C., Calzada, J. E., Torres, R. 2019. Study of the epidemiological behavior of malaria in Darien Region, Panama. 2015⿿2017. PLoS ONE. 14(11):e0224508.
- Muturi, E.J., Hay, W.T., Behle, R.W., Selling, G.W. 2019. Amylose inclusion complexes as emulsifiers for garlic and asafoetida essential oils for mosquito control. Insects. 10(10):337.
- Ramirez, J.L., Muturi, E.J., Flor-Weiler, L.B., Vermillion, K., Rooney, A. P. 2020. Peptidoglycan recognition proteins (PGRPs) modulates mosquito resistance to fungal entomopathogens in a fungal-strain specific manner. Frontiers in Cellular and Infection Microbiology. 9:465.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): Objective 1. Enable the commercial production of microorganisms pathogenic to mosquitoes. Objective 2. Enable the commercial production of bioactive compounds/ metabolites derived from plants and microorganisms to control mosquitoes and/or the viruses they carry. Approach (from AD-416): Our approach will focus on the discovery of novel microbial and plant- based biopesticides that could be commercialized for the control of mosquitoes or the pathogens they transmit. A variety of entomopathogenic fungi will be evaluated for their effect on survival of adult mosquitoes. Selection of candidate mosquito entomopathogens will be done initially by focusing on the isolates with known pathogenicity and strains that have previously been isolated from dipteran species. Transcriptomic analysis coupled with functional assays (through reverse-genetic techniques) will be used to identify the fungal mode of action as well as the mosquito responses to infection. Attempts will be made to isolate and identify microbe-derived molecules with biological activity against mosquitoes and selected arboviruses. We will also evaluate plant-based compounds effective for activity against mosquitoes. We will integrate standard insecticide testing bioassays with modern and conventional approaches in chemical ecology to identify the chemical compounds in selected plants that are attractive to gravid females and deleterious to mosquito larvae. Our research group focused on interdisciplinary studies to discover novel compounds from plants, bacteria, and fungi that are effective against mosquitoes and the diseases they transmit. Substantial progress has been made in the second year of this research project. ARS scientists in Peoria, Illinois, made significant progress in Objective 1 by establishing how the type of host blood meal ingested by the mosquito influences the composition of microbial communities that inhabit the mosquito gut. Researchers also successfully completed studies that examined how the site of mosquito collection influences the composition and diversity of bacterial communities associated with mosquitoes. We also successfully evaluated the use of fluorescent proteins as markers for bacterial communities inhabiting the mosquito gut to facilitate the study of their functions. We successfully identified a previously unrecognized defense mechanism mounted by the mosquito to overcome infection by fungal entomopathogens. Additionally, we continued examining additional fungal entomopathogens for their virulence against Ae. aegypti, Ae. albopictus and Cx pipiens mosquitoes. For Objective 2, we continued with studies on the potential to exploit common blackberry (Rubus allegheniensis) leaves as a tool for controlling mosquitoes in stormwater catch basins. These man-made aquatic habitats serve as the main larval habitats for the northern house mosquito, the primary vector of West Nile virus in northeastern United States. The study integrated a variety of conventional and modern technologies to better understand why leaf infusion of common blackberry is an attractive habitat for egg-laying mosquitoes, yet harmful to mosquito larvae. Because the results could not point to specific chemical compounds from blackberry leaves that were responsible for these bioactivities, the research was expanded to investigate the biological activity of various plant essential oils on mosquito eggs and larvae as outlined in the project plan. These studies have led to the identification of novel plant- based compounds that could be investigated for further development and commercialization as tools for mosquito control. We continued with the screening of entomopathogenic fungi with the potential to generate compounds with mosquitocidal and antimicrobial activity. Current efforts are focused on maximizing the production of bioactive metabolite by candidate fungi and identifying the best growing conditions during the fermentation process. Accomplishments 01 Bacterial communities associated with mosquitoes. Bacterial communities associated with mosquitoes particularly those inhabiting the mosquito gut, play essential roles in mosquito biology. They support mosquito development, aid in blood meal digestion, and can even block transmission of disease-causing agents transmitted by mosquitoes such as malaria. ARS researchers in Peoria, Illinois, discovered that bacterial communities inhabiting the mosquito guts vary markedly based on mosquito species, site of mosquito collection and the type of host from which the mosquitoes ingested blood. Further studies revealed that fluorescent proteins could successfully be used as markers for some bacterial communities that reside in mosquito gut to allow detailed analysis of their behavior and functions in mosquito biology. Collectively, these findings advance current knowledge on microbial communities associated with mosquitoes and provide a methodology for additional studies on their functions without disturbing the normal gut microflora.
Impacts
(N/A)
Publications
- Muturi, E.J., Dunlap, C.A., Ramirez, J.L., Rooney, A.P., Kim, C. 2018. Host blood meal source has a strong impact on gut microbiota of Aedes aegypti. FEMS Microbiology Ecology. 95.
- Gardner, A.M., Muturi, E.J., Allan, B.F. 2018. Discovery and exploitation of a natural ecological trap for a mosquito disease vector. Proceedings of the Royal Society B.
- Parker, A.T., Gardner, A.M., Perez, M., Allan, B.F., Muturi, E.J. 2018. Container size alters the outcome of interspecific competition between Aedes aegypti (Diptera: Culicidae)and Aedes albopictus. Journal of Medical Entomology.
- Ramirez, J.L., Muturi, E.J., Barletta-Ferreira, A.B., Rooney, A.P. 2018. The Aedes aegypti IMD pathway is a critical component of the mosquito antifungal immune response. Developmental and Comparative Immunology.
- Muturi, E.J., Doll, K.M., Berhow, M.A., Weiler, L., Rooney, A.P. 2019. Honeysuckle essential oil as a potential source of ecofriendly larvicides for mosquito control. Pest Management Science.
- Muturi, E.J., Doll, K.M., Ramirez, J.L., Rooney, A.P. 2018. Bioactivity of wild carrot (Daucus carota, Apiaceae) essential oil against mosquito larvae. Journal of Medical Entomology.
- Muturi, E.J., Ramirez, J.L., Kim, C. 2019. Green, yellow and red fluorescent proteins as markers for bacterial isolates from mosquito midguts. Insects. 10:49.
- Kim, C., Muturi, E.J., Lee, S. 2018. Copula modeling of differential effect of leaf species on Aedes albopictus development time. Environment and Natural Resources Research. 8(4).
- Muturi, E.J., Lagos-Kutz, D.M., Dunlap, C.A., Ramirez, J.L., Rooney, A.P., Hartman, G.L., Fields, C., Rendon, G., Kim, C. 2018. Mosquito microbiota cluster by host sampling location. Parasites & Vectors. 11:468.
- Le, P.V., Kumar, P., Ruiz, M.O., Mbogo, C., Muturi, E.J. 2019. Predicting the direct and indirect impacts of climate change on malaria in coastal Kenya. PLOS One. 14:e0211258.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): Objective 1. Enable the commercial production of microorganisms pathogenic to mosquitoes. Objective 2. Enable the commercial production of bioactive compounds/ metabolites derived from plants and microorganisms to control mosquitoes and/or the viruses they carry. Approach (from AD-416): Our approach will focus on the discovery of novel microbial and plant- based biopesticides that could be commercialized for the control of mosquitoes or the pathogens they transmit. A variety of entomopathogenic fungi will be evaluated for their effect on survival of adult mosquitoes. Selection of candidate mosquito entomopathogens will be done initially by focusing on the isolates with known pathogenicity and strains that have previously been isolated from dipteran species. Transcriptomic analysis coupled with functional assays (through reverse-genetic techniques) will be used to identify the fungal mode of action as well as the mosquito responses to infection. Attempts will be made to isolate and identify microbe-derived molecules with biological activity against mosquitoes and selected arboviruses. We will also evaluate plant-based compounds effective for activity against mosquitoes. We will integrate standard insecticide testing bioassays with modern and conventional approaches in chemical ecology to identify the chemical compounds in selected plants that are attractive to gravid females and deleterious to mosquito larvae. Our research group was engaged in a variety of research activities to enable the commercial production of microbe- and plant-derived bioactive compounds that are pathogenic to mosquitoes and/or the viruses they carry. Substantial progress has been made in the first year of this research project. ARS scientists in Peoria, Illinois made significant progress in Objective 1 by characterizing the gut microbial communities of the white dotted mosquito (Culex restuans) in relation to parental background. ARS scientists in Peoria, Illinois successfully developed and optimized the protocols for studying how mosquitoes interact with entomopathogenic fungi. Additionally, ARS scientists in Peoria, Illinois successfully examined the effects of selected entomopathogenic fungal strains on mosquito survival, and identified the antifungal responses mounted by the mosquito in response to the fungal challenge. For Objective 2, ARS scientists in Peoria, Illinois successfully examined the potential to exploit common blackberry (Rubus allegheniensis) leaves as a tool for mosquito control in storm water catch basins, the primary larval habitats for the West Virus vector, Culex (Cx) pipiens. ARS scientists in Peoria, Illinois combined conventional oviposition choice bioassays with standard and current state-of-the-art microbiological and molecular techniques to examine the role of microbial communities and chemical compounds associated with leaves of common blackberry in influencing Cx. pipiens oviposition behavior and larval survival and development. ARS scientists in Peoria, Illinois also evaluated a variety of essential oils extracted from local plants or purchased from commercial vendors as potential larvicides for mosquito control. These studies have resulted in the identification of novel plant- based tools and strategies that can be used as part of integrated vector management. With regard to identification of bioactive compounds from microbes, ARS scientists in Peoria, Illinois have generated a catalog of microbes with potential for bioactive compound production and assigned their antibacterial properties using bacteria isolated directly from lab- reared and field-collected mosquitoes. This list is currently being expanded by testing additional fungal strains. Accomplishments 01 Common blackberry and mosquito control. In efforts to discover new mosquito control tools that can complement or even replace the use of toxic synthetic chemicals, this research examined whether leaves of common blackberry can be used as a tool for mosquito control in storm water catch basins. ARS researchers at Peoria, Illinois, discovered that �treating� storm water catch basins with leaf litter of common blackberry increased the number of Cx. pipiens egg rafts that were laid in these man-made habitats but reduced the number of emerging adult mosquitoes. These findings indicate that common blackberry leaf infusion acts as an ecological trap � a low quality habitat that is preferred over other available high quality habitats. Oviposition choice and larval survivorship bioassays indicate that attraction of gravid female mosquitoes to common blackberry infusion is mediated by chemical cues released as the leaves decomposed while larval survivorship is limited not by the toxic compounds present in the common blackberry leaves but by the failure of bacterial communities thriving in the common blackberry leaf infusion to meet the nutritional requirements for mosquito larvae. These findings provide an experimental proof-of-concept for a new, inexpensive, environmentally safe, and effective �attract-and-kill� integrated vector management tool for mosquito control. 02 Essential oils and mosquito control. Although essential oils are known to elicit a variety of biological activities against mosquitoes, only a limited number of essential oil-based biopesticides are commercially available. In an effort to increase this number, this research examined the potency of some essential oils and their combinations against mosquito larvae. ARS researchers at Peoria, Illinois, discovered that a combination of Manuka and oregano essential oils had synergistic effects against mosquito larvae that have the potential as an alternative to synthetic insecticides. The researchers also found that garlic and asafoetida essential oils were highly toxic to mosquito larvae but became less toxic when combined. These findings increase the number of essential oils that are known to exhibit larvicidal activity against mosquitoes. The findings also demonstrate the need for knowledge-guided decision-making process when selecting essential oil combinations for use in mosquito control since some essential oil combinations can undermine vector control programs. 03 Invasive alien plants and mosquito-borne diseases. Despite the ubiquity of invasive alien plants in many ecosystems, their impact of human risk of exposure to mosquito-borne diseases is not well understood. ARS researchers at Peoria, Illinois, in collaboration with researchers at the University of Maine and the University of Illinois, investigated how Amur honeysuckle (Lonicera maackii), an invasive alien plant species can influence the risk of mosquito-borne diseases. The researchers discovered that Amur honeysuckle supports diverse bird communities and large populations of vector mosquitoes. Removal of this invasive alien shrub resulted in significant reduction in mosquito populations and loss of bird communities that are susceptible to West Nile virus including American robin, house sparrow, blue jay, and common grackle. These results suggest that this invasive alien shrub can act as a risk factor for mosquito production which may in turn increase the risk of human and livestock exposure to mosquito-borne diseases. 04 Identification of entomopathogenic fungi with mosquitocidal activity. The rapid and widespread development of insecticide resistance in mosquitoes and the increase in the incidence of mosquito-vectored diseases calls for the development of alternative methods of mosquito control. The use of fungi to control mosquitoes is an environmentally- friendly alternative to chemical pesticides. ARS researchers at Peoria, Illinois, have identified new strains of fungi that kill the major mosquito vector Aedes aegypti. The scientists also found differences in the way each fungal pathogen kills the mosquito, revealing novel compounds produced by the fungi that the researchers are working to develop as new mosquito control technologies. In addition, the information gained from these studies improves the design of microbial control strategies, for instance, through the selection of fungal entomopathogens with previously unidentified modes of action. Strategies using a combination of fungi with different modes of action will allow us to prevent/retard the development of resistance to these entomopathogenic fungi and also to accelerate their speed of kill. 05 Molecular characterization of mosquito responses to entomopathogenic fungal infection. The ability of a mosquito to survive a fungal infection depends on how efficiently its immune system recognizes the fungus so that it can appropriately respond and control the infection. In a groundbreaking new study, ARS scientists at Peoria, Illinois, found that species of the fungal genus Isaria produce compounds that suppress the mosquito immune system. This new information is a major departure from what has been described in the literature for other types of entomopathogenic fungi that are used to control mosquitoes and other insect pests. Successful development of these new compounds will provide new technologies for the control of mosquito populations that are environmentally-friendly and target-specific.
Impacts
(N/A)
Publications
- Muturi, E.J., Ramirez, J.L., Zilkowski, B.W., Weiler, L., Rooney, A.P. 2018. Ovicidal and larvicidal effects of garlic and asafoetida essential oils against West Nile virus vectors. Journal of Insect Science. 18(2):43. doi: 10.1093/jisesa/iey036.
- Muturi, E.J., Ramirez, J.L., Rooney, A.P., Kim, C. 2017. Comparative analysis of gut microbiota of Culex restuans (Diptera: Culicidae) females from different parents. Journal of Medical Entomology. 10:163-171. doi: 10. 1093/jme/tjx199.
- Dowd, P.F., Zilkowski, B.W., Johnson, E.T., Berhow, M.A., Muturi, E.J. 2018. Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens. AGRI GENE. 7:52-58.
- Karki, S., Westcott, N.E., Muturi, E.J., Brown, W.M., Ruiz, M.O. 2017. Assessing human risk of illness with West Nile virus mosquito surveillance data to improve public health preparedness. Zoonoses and Public Health. 65:177-184. doi: 10.1111/zph.12386.
- Weiler, L., Rooney, A.P., Behle, R.W., Muturi, E.J. 2017. Characterization of Tolypocladium cylindrosporum (Hypocreales: Ophiocordycipitacea) and its impact against Aedes aegypti and Aedes albopictus eggs at low temperature. Journal of the American Mosquito Control Association. 33(3):184-192.
- Moise, I.K., Riegel, C., Muturi, E.J. 2018. Environmental and social- demographic predictors of the southern house mosquito Culex quinquefasciatus in New Orleans, Louisiana. Parasites & Vectors. 11:249. doi.org/10.1186/s13071-018-2833-5.
- Muturi, E.J., Ramirez, J.L., Doll, K.M., Bowman, M.J. 2017. Combined toxicity of three essential oils against Aedes aegypti (Diptera: Culicidae) larvae. Journal of Medical Entomology. 54:1684-1691. doi: 10.1093/jme/ tjx168.
- Ramirez, J.L., Dunlap, C.A., Muturi, E.J., Barletta-Ferreira, A.B., Rooney, A.P. 2018. Entomopathogenic fungal infection leads to temporospatial modulation of the mosquito immune system. PLOS Neglected Tropical Diseases. 12:e0006433.
- Gomes, F.M., Hixson, B.L., Tyner, M.D., Ramirez, J.L., Canepa, G.E., Alves E Silva, T.L., Molina-Cruz, A., Keita, M., Kane, F., Traore, B., Sogoba, N. , Barillas-Mury, C. 2017. Effect of naturally-occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission. Proceedings of the National Academy of Sciences. 114:12566-12571. doi: 10.1073/pnas.1716181114.
- Gardner, A.M., Muturi, E.J., Overmier, L.D., Allan, B.F. 2017. Large-scale removal of invasive honeysuckle decreases mosquito and avian host abundance. EcoHealth. 14:750:761. doi: 10.1007/s10393-017-1265-6.
- Ramirez, J.L., Muturi, E.J., Dunlap, C.A., Rooney, A.P. 2018. Strain- specific pathogenicity and subversion of phenoloxidase activity in the mosquito Aedes aegypti by members of the fungal entomopathogenic genus Isaria. Scientific Reports. doi:10.1038/s41598-018-28210-6.
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