PanK manipulation in A. stephensi mosquitoes
1. Explore how Coenzyme A biosynthesis can be manipulated in Anopheles mosquitoes to limit malaria parasite development.
Pantothenate, or vitamin B5, is an essential nutrient and substrate for coenzyme A (CoA) synthesis via the pantothenate kinase (PanK) pathway. All organisms, including humans, mosquitoes and malaria parasites, require pantothenate, but few animals can directly synthesize it. Instead, these organisms must acquire pantothenate through their diet, with some mammalian intestinal microbes synthesizing Pan that can also be utilized by the host. The malaria parasite must also acquire pantothenate from its environment to convert it into CoA. We hypothesized that increasing PanK activity, either through small molecule modulators or genetic manipulation, would starve the malaria parasite of pantothenate, while still providing the necessary CoA for the mosquito. Thus, we expect that Anopheles stephensi mosquitoes with increased PanK activity would have decreased malaria parasite loads without suffering significant fitness effects.
In fact, we were able to demonstrate that manipulation of PanK using the PanK activator PZ-2891 or genetically engineered mosquitoes with indirectly increased PanK expression led to a significant decrease in mosquito pantothenate levels, a significant increase in mosquito CoA levels and significantly reduced malaria parasite infections. We also recently demonstrated that manipulations of PanK had no impact on mosquito lifespan, reproduction or metabolism.
Ongoing work includes the generation of a transgenic mosquito line that directly overexpresses PanK in the mosquito and the screening of various pantothenamides (PanK small molecule modulators) for their ability to consistently regulate PanK activity. These lines and small molecules will be assessed for their ability to suppress pantothenate levels, decrease malaria parasite infections in the mosquito and their impact on mosquito fitness. This work is a collaborative project with Dr. Shirley Luckhart's research team at the University of Idaho.
Publications associated with this project include:
Pantothenate, or vitamin B5, is an essential nutrient and substrate for coenzyme A (CoA) synthesis via the pantothenate kinase (PanK) pathway. All organisms, including humans, mosquitoes and malaria parasites, require pantothenate, but few animals can directly synthesize it. Instead, these organisms must acquire pantothenate through their diet, with some mammalian intestinal microbes synthesizing Pan that can also be utilized by the host. The malaria parasite must also acquire pantothenate from its environment to convert it into CoA. We hypothesized that increasing PanK activity, either through small molecule modulators or genetic manipulation, would starve the malaria parasite of pantothenate, while still providing the necessary CoA for the mosquito. Thus, we expect that Anopheles stephensi mosquitoes with increased PanK activity would have decreased malaria parasite loads without suffering significant fitness effects.
In fact, we were able to demonstrate that manipulation of PanK using the PanK activator PZ-2891 or genetically engineered mosquitoes with indirectly increased PanK expression led to a significant decrease in mosquito pantothenate levels, a significant increase in mosquito CoA levels and significantly reduced malaria parasite infections. We also recently demonstrated that manipulations of PanK had no impact on mosquito lifespan, reproduction or metabolism.
Ongoing work includes the generation of a transgenic mosquito line that directly overexpresses PanK in the mosquito and the screening of various pantothenamides (PanK small molecule modulators) for their ability to consistently regulate PanK activity. These lines and small molecules will be assessed for their ability to suppress pantothenate levels, decrease malaria parasite infections in the mosquito and their impact on mosquito fitness. This work is a collaborative project with Dr. Shirley Luckhart's research team at the University of Idaho.
Publications associated with this project include:
- Thakre N, Gurge RM, Isoe J, Kivi H, Strickland J, Delacruz LR, Rodriguez AM, Haney R, Sadeghi R, Joy T, Chen M, Luckhart S, Riehle MA. Manipulation of pantothenate kinase in Anopheles stephensi suppresses pantothenate levels with minimal impacts on mosquito fitness. Insect Biochemistry and Molecular Biology. (2022)
- Simão-Gurge RM, Thakre N, Strickland J, Isoe J, Delacruz LR, Torrevillas BK, Rodriguez AM, Riehle MA, & Luckhart S. Activation of Anopheles stephensi pantothenate kinase and coenzyme A biosynthesis reduces infection with diverse Plasmodium species in the mosquito host. Biomolecules. (2021)
- Luckhart, S., & Riehle, MA. Midgut mitochondrial function as a gatekeeper for malaria parasite infection and development in the mosquito host. Frontiers in Cellular and Infection Microbiology, 10. (2020)
- Souvannaseng, L., Hun, L.V., Baker, H., Klyver, J.M., Wang, B., Pakpour, N., Bridgewater, J.M., Napoli, E., Giulivi, C., Riehle, M.A. and Luckhart, S. Inhibition of JNK signaling in the Asian malaria vector Anopheles stephensi extends mosquito longevity and improves resistance to Plasmodium falciparum infection. PLoS pathogens, 14(11), p.e1007418. (2018)
2. Explore the physiological roles of distinct insulin-like peptides in mosquitoes using CRISPR/Cas9.
The insulin signaling cascade regulates a range of critical physiologies in the mosquito, including development, innate immunity, longevity, metabolism and reproduction. The cascade is initiated through insulin-like peptides (ILPs) that bind to the insulin receptor and initiate the intracellular signaling cascade. Intriguingly, most insects examined to date, including the yellow fever mosquito Aedes aegypti and malaria vector Anopheles stephensi, express multiple ILPs, but only a single insulin receptor. This begs the fundamental question of why multiple ILPs are required to stimulate a single signaling cascade? Do the ILPs regulate discrete physiological functions? Do they act in concert to fine tune the physiological processes they regulate? Do they provide redundancy to ensure this pivotal signaling cascade functions seamlessly? To address these questions, we are systematically examining the ILPs expressed by mosquitoes.
We are using CRISPR/Cas9 to disrupt the expression of key AsteILPs and AaegILPs. Our first targets, AaegILP2 and AsteILP2, are the putative orthologs of the Drosophila ILP, DILP2, which has been implicated as the key ILP regulating a host of physiologies including longevity and reproduction. We have inserted the fluorescent marker gene EGFP into the AaegILP2 gene and have examined the physiological effects of this disruption in this mosquito line. After characterizing this line we plan to disrupt additional ILPs to identify discreet roles for the individual or combined ILPs in mosquitoes.
Publications associated with this project include:
The insulin signaling cascade regulates a range of critical physiologies in the mosquito, including development, innate immunity, longevity, metabolism and reproduction. The cascade is initiated through insulin-like peptides (ILPs) that bind to the insulin receptor and initiate the intracellular signaling cascade. Intriguingly, most insects examined to date, including the yellow fever mosquito Aedes aegypti and malaria vector Anopheles stephensi, express multiple ILPs, but only a single insulin receptor. This begs the fundamental question of why multiple ILPs are required to stimulate a single signaling cascade? Do the ILPs regulate discrete physiological functions? Do they act in concert to fine tune the physiological processes they regulate? Do they provide redundancy to ensure this pivotal signaling cascade functions seamlessly? To address these questions, we are systematically examining the ILPs expressed by mosquitoes.
We are using CRISPR/Cas9 to disrupt the expression of key AsteILPs and AaegILPs. Our first targets, AaegILP2 and AsteILP2, are the putative orthologs of the Drosophila ILP, DILP2, which has been implicated as the key ILP regulating a host of physiologies including longevity and reproduction. We have inserted the fluorescent marker gene EGFP into the AaegILP2 gene and have examined the physiological effects of this disruption in this mosquito line. After characterizing this line we plan to disrupt additional ILPs to identify discreet roles for the individual or combined ILPs in mosquitoes.
Publications associated with this project include:
- Marquez A.G., Pietri J.E., Smithers H.M., Nuss A., Antonova Y. †, Drexler A.L., Riehle M.A., Brown M.R., and Luckhart S.L. Insulin-like peptides in the mosquito Anopheles stephensi: identification and expression in response to diet and infection with Plasmodium falciparum. General and Comparative Endocrinology. 173(2). 303-12. (2011)
- Krieger M.J.B., Jahan N., Riehle M.A., Cao C., and Brown M.R. Molecular characterization of insulin-like peptide genes and their expression in the African malaria mosquito, Anopheles gambiae. Insect Molecular Biology. 13(3). 305-315. (2004)
Genetically engineered Anopheles stephensi larvae (A) and adults (B)
3. Manipulate the insulin signaling pathway to improve innate immunity and impact mosquito fitness.
The insulin/insulin growth factor 1 signaling (ISS) cascade regulates a wide range of physiological processes in invertebrates including longevity, reproduction, stress, and immunity. We are interested in understanding the role this signaling cascade has on the ability of the mosquito to transmit disease. Towards this goal we are studying two key physiologies that determine how effectively a mosquito transmits disease, lifespan and immunity.
The IIS cascade is initiated by insulin-like peptides binding to the alpha subunit of the insulin receptor. This induces autophosphorylation of the beta subunit which in turn phosphorylates insulin receptor substrate 1 (IRS-1). Phosphorylated tyrosine residues provide a binding site for the regulatory subunit of P13K and SHC which are at the branch point between the P13K/Akt and MAP cascades. P13K/Akt cascade: Upon binding to IRS-1 the catalytic subunit of P13K is released and travels to the cell membrane where it converts PI(4,5)P2 to PI(3,4,5)P3. PI(3,4,5)P3 provides binding sites for both Akt and PDK1 and brings these proteins together. Phosphorylation of Akt by PDK1 activates Akt allowing it to phosphorylate extranuclear Forkhead transcription factors. The activated transcription factors can translocate into the nucleus to activate transcription of a variety of genes. PTEN dephosphorylates PI(3,4,5)P3 to PI(4,5)P2 to downregulate the P13K cascade.
To understand the role of IIS on lifespan and immunity we have genetically engineered key components of the IIS cascade into mosquitoes in a tissue specific manner. Specifically, we have overexpressed the IIS activator Akt and the IIS inhibitor PTEN into the mosquito midgut and fat body. We then test these transgenic mosquitoes against non-transgenic control mosquitoes to determine if increased or decreased IIS in a given tissue impacts lifespan or alters the mosquito’s ability to transmit the human malaria parasite Plasmodium falciparum. This work is a collaborative project with Dr. Shirley Luckhart's research team at the University of Idaho.
Publications associated with this project include:
The insulin/insulin growth factor 1 signaling (ISS) cascade regulates a wide range of physiological processes in invertebrates including longevity, reproduction, stress, and immunity. We are interested in understanding the role this signaling cascade has on the ability of the mosquito to transmit disease. Towards this goal we are studying two key physiologies that determine how effectively a mosquito transmits disease, lifespan and immunity.
The IIS cascade is initiated by insulin-like peptides binding to the alpha subunit of the insulin receptor. This induces autophosphorylation of the beta subunit which in turn phosphorylates insulin receptor substrate 1 (IRS-1). Phosphorylated tyrosine residues provide a binding site for the regulatory subunit of P13K and SHC which are at the branch point between the P13K/Akt and MAP cascades. P13K/Akt cascade: Upon binding to IRS-1 the catalytic subunit of P13K is released and travels to the cell membrane where it converts PI(4,5)P2 to PI(3,4,5)P3. PI(3,4,5)P3 provides binding sites for both Akt and PDK1 and brings these proteins together. Phosphorylation of Akt by PDK1 activates Akt allowing it to phosphorylate extranuclear Forkhead transcription factors. The activated transcription factors can translocate into the nucleus to activate transcription of a variety of genes. PTEN dephosphorylates PI(3,4,5)P3 to PI(4,5)P2 to downregulate the P13K cascade.
To understand the role of IIS on lifespan and immunity we have genetically engineered key components of the IIS cascade into mosquitoes in a tissue specific manner. Specifically, we have overexpressed the IIS activator Akt and the IIS inhibitor PTEN into the mosquito midgut and fat body. We then test these transgenic mosquitoes against non-transgenic control mosquitoes to determine if increased or decreased IIS in a given tissue impacts lifespan or alters the mosquito’s ability to transmit the human malaria parasite Plasmodium falciparum. This work is a collaborative project with Dr. Shirley Luckhart's research team at the University of Idaho.
Publications associated with this project include:
- Hun LV, Cheung KW, Brooks E, Zudekoff R, Luckhart S, Riehle MA. Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection, Insect Biochemistry and Molecular Biology. (2021)
- Oringanje, C., Delacruz, LR., Han, Y., Luckhart, S., & Riehle, MA. Overexpression of activated AMPK in the Anopheles stephensi midgut impacts mosquito metabolism, reproduction and Plasmodium resistance. Genes, 12(1), 119. (2021)
- Hun, L., Luckhart, S. and Riehle, M.A. Increased Akt signaling in the fat body of Anopheles stephensi extends lifespan and increases lifetime fecundity through modulation of insulin-like peptides. Journal of insect physiology, p.103932. (2019)
- Pietri J.E., Pietri E.J., Potts R., Riehle M.A. & Luckhart S. Plasmodium falciparum suppresses the host immune response by inducing the synthesis of insulin-like peptides (ILPs) in the mosquito Anopheles stephensi. Developmental & Comparative Immunology, 53(1), 134-144. (2015).
- Arik A.J., Hun L.V., Quicke K., Piatt M., Ziegler R., Scaraffia P.Y., Badgandi H., Riehle M.A. Increased Akt signaling in the mosquito fat body increases adult survivorship. FASEB J. 29(4): 1404-1413. (2015)
- Pakpour N., Riehle M.A., Luckhart S., Effects of ingested vertebrate-derived factors on insect immune responses. Current Opinion in Insect Science. 3:1-5 (2014)
- Drexler AL., Pietri JE., Pakpour N., Hauck E., Wand B., Glennon EKK., Georgis M., Riehle MA., Luckhart S. Human IGF1 regulates midgut oxidative stress and epithelial homeostasis to balance lifespan and Plasmodium falciparum resistance in Anopheles stephensi. PLoS Pathog 10(6): e1004231. DOI: 10.1371/journal.ppat.1004231. (2014)
- Hauck ES., Antonova-Koch Y., Drexler A., Pietri J., Pakpour N., Liu D., Blacutt J., Riehle MA., Luckhart S. Overexpression of phosphatase and tensin homolog improves fitness and decreases Plasmodium falciparum development in Anopheles stephensi. Microbes and Infection. doi: 10.1016/j.micinf.2013.05.006. (2013)
- Luckhart S., Giulivi C., Drexler A.L., Antonova-Koch Y., Sakaguchi D., Napoli E., Wong S., Price M.S., Eigenheer R., Phinney B.S., Pakpour N., Pietri J.E., Cheung K., Georgis M., and Riehle M.A. Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito most. PLoS Pathog 9(2): e1003180. doi:10.1371/journal.ppat.1003180. (2013)
- Pakpour N., Corby-Harris V. †, Green G., Smithers H., Cheung K., Riehle M.A., and Luckhart S. Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum. Infection and Immunity. 80(6):2141-9. (2012)
- Marquez A.G., Pietri J.E., Smithers H.M., Nuss A., Antonova Y. †, Drexler A.L., Riehle M.A., Brown M.R., and Luckhart S.L. Insulin-like peptides in the mosquito Anopheles stephensi: identification and expression in response to diet and infection with Plasmodium falciparum. General and Comparative Endocrinology. 173(2). 303-12. (2011)
- Corby-Harris V., Drexler A., Watkins de Jong L., Antonova Y., Pakpour N., Ziegler R., Ramberg F., Lewis E.E., Brown J.M, Luckhart S., and Riehle M.A. Activation of Akt signaling reduces the prevalence and intensity of malaria parasite infection and lifespan in Anopeheles stephensi mosquitoes. PLoS Pathogens. 6(7). e1001003 (2010)
NIRS age grading of Aedes aegypti mosquitoes
4. Develop methods for assessing the population age structure of Ae. aegypti mosquitoes in Southern Arizona using parity, transcript expression and NIRS.
The yellow fever mosquito Aedes aegypti is an important vector of dengue and dengue hemorrhagic fever, a virus that infects and sometimes kills thousands annually. Although this mosquito is found in the Southwestern US, including Southern Arizona, the virus is not currently transmitted in Arizona. One possibility for this is that Aedes aegypti mosquitoes at the edge of their ecological range do not survive long enough to transmit the dengue virus. To examine this we are attempting to identify genes in the mosquito that can be used as markers of chronological and physiological aging. We have currently identified one such gene and verified its ability to accurately age mosquitoes under laboratory and semi-field conditions. In addition, we have verified the accuracy of this gene on field collected mosquitoes by comparing the predicted mosquito age with the reproductive status of the mosquito. Young mosquitoes that have not yet taken a blood meal and produced a clutch of eggs have tightly coiled trachea on their ovaries that unfurl as the ovaries increase in size during egg production. Mosquitoes with tightly coiled trachea were consistently aged less than six days old with our aging gene marker compared with an average of 15 days old. Finally, we are also utilizing near-infrared red spectroscopy (NIRS) to attempt to age grade field collected mosquitoes. This project is a collaborative effort with multiple researchers at UA, including Drs. Kathleen Walker, Kacey Ernst and Dawn Gouge.
Publications associated with this project include:
The yellow fever mosquito Aedes aegypti is an important vector of dengue and dengue hemorrhagic fever, a virus that infects and sometimes kills thousands annually. Although this mosquito is found in the Southwestern US, including Southern Arizona, the virus is not currently transmitted in Arizona. One possibility for this is that Aedes aegypti mosquitoes at the edge of their ecological range do not survive long enough to transmit the dengue virus. To examine this we are attempting to identify genes in the mosquito that can be used as markers of chronological and physiological aging. We have currently identified one such gene and verified its ability to accurately age mosquitoes under laboratory and semi-field conditions. In addition, we have verified the accuracy of this gene on field collected mosquitoes by comparing the predicted mosquito age with the reproductive status of the mosquito. Young mosquitoes that have not yet taken a blood meal and produced a clutch of eggs have tightly coiled trachea on their ovaries that unfurl as the ovaries increase in size during egg production. Mosquitoes with tightly coiled trachea were consistently aged less than six days old with our aging gene marker compared with an average of 15 days old. Finally, we are also utilizing near-infrared red spectroscopy (NIRS) to attempt to age grade field collected mosquitoes. This project is a collaborative effort with multiple researchers at UA, including Drs. Kathleen Walker, Kacey Ernst and Dawn Gouge.
Publications associated with this project include:
- Joy T, Chen M, Arnbrister J, Williamson D, Li S, Nair S, Brophy M, Garcia VM, Walker K, Ernst K, Gouge DH, Carrière Y, Riehle MA. Assessing Near-Infrared Spectroscopy (NIRS) for Evaluation of Aedes aegypti Population Age Structure. Insects. 2022; 13(4):360.
- Ernst, K.C., Walker, K.R., Reyes-Castro, P., Joy, T.K., Castro-Luque, A.L., Diaz-Caravantes, R.E., Gameros, M., Haenchen, S., Hayden, M.H., Monaghan, A., Jeffrey-Guttierez, E., Carrière Y., and Riehle, M.A. Aedes aegypti (Diptera: Culicidae) Longevity and Differential Emergence of Dengue Fever in Two Cities in Sonora, Mexico. Journal of Medical Entomology, 54(1), pp.204-211. (2017)
- Joy T.K., Jeffrey Gutierrez E.H., Ernst K., Walker K.R., Carriere Y., Torabi M., Riehle M.A. Aging field collected Aedes aegypti to determine their capacity for dengue transmission in the Southwestern United States. PLoS One 10.1371/journal.pone.0046946 (2012)
- Joy T, Corby-Harris V., Johnson A., and Riehle M.A. The impact of larval and adult dietary restriction on lifespan and reproduction in the mosquito Aedes aegypti. Experimental Gerontology. 45(9). 685-690 (2010)
Larval midgut staining with pH sensitive probes
5. Studying and disrupting the mosquito larval midgut using novel pH responsive probes
Current control strategies for mosquito-borne pathogens largely rely on reducing mosquito vector populations using insecticides. However, this approach is becoming less effective as insecticide resistance develops against many of the various mosquito adulticides and larvicides. The midgut of the larval mosquito is a unique environment with a high pH anterior end and a neutral posterior end. We have developed novel pH responsive probes that are capable of delivering a range of toxic and probing compounds to the larval gut under the high pH conditions of the anterior gut. This provides an opportunity to develop highly specific larvicides with minimal effects on non-target organisms and the flexibility to switch toxic compounds if resistance develops. This project is a collaborative effort with Dr. John Jewett in the Department of Chemistry and Biochemistry at UA.
Publications associated with this project include:
Current control strategies for mosquito-borne pathogens largely rely on reducing mosquito vector populations using insecticides. However, this approach is becoming less effective as insecticide resistance develops against many of the various mosquito adulticides and larvicides. The midgut of the larval mosquito is a unique environment with a high pH anterior end and a neutral posterior end. We have developed novel pH responsive probes that are capable of delivering a range of toxic and probing compounds to the larval gut under the high pH conditions of the anterior gut. This provides an opportunity to develop highly specific larvicides with minimal effects on non-target organisms and the flexibility to switch toxic compounds if resistance develops. This project is a collaborative effort with Dr. John Jewett in the Department of Chemistry and Biochemistry at UA.
Publications associated with this project include:
