Progress 05/26/16 to 04/30/21
Outputs
Target Audience:The information from the project has been included in classroom instruction. The methodology of the project has been included in laboratory instruction and class practice. The outcome/information and knowledge from the project, especially the mosquito survey and resistance survey, have been continually delivered to stockholders in Alabama, and the Alabama Department of Public Health, impacting on understanding and diagnosis of mosquito resistance in Alabama. The research information has been presented in professional meetings, such as ESA and SEB ESA. Our group has been invited to give a presentation in Physiology, Biochemistry, and toxicology Section Symposium in the 2021 ESA annual meeting with the title of "Functional Analyses of House Fly Carboxylesterases Involved in Insecticide Resistance", addressing the mechanisms and strategies of arthropod adaptation to the chemical environment. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This 5-year project has provided the opportunity to train a total of 4 postdocs and 8 graduate students. By conducting the different objectives and training, working together with PI, the individuals who participate in the project have improved their professional skills in methodology and knowledge of science, and have sharpened their critical thinking. Two graduate students have currently been professors in universities and two are postdocs in universities. Especially our Heterologous Expressionand Functional Analysis of Aedes aegypti Odorant Receptors to human odors in Xenopus Oocytes has made it possible for researchers in the field of toxicology and physiology to investigate the interaction of insects with chemicals and human odorants. How have the results been disseminated to communities of interest?Our study on the response of olfactory systems of yellow fever mosquito Aedes aegypti to human orders has attracted public attention and research has been funded by NIH 2021 to continue the study on these topics. We have gotten encouragement from NIH program director to encourage us to submit the proposal for our GPCR research. Indeed, the NIH R21 proposal has been in the second revision. Our methodology for characterizing olfactory systems of yellow fever mosquito Aedes aegypti has been published by the Journal of Visualized Experiments for public use. What do you plan to do during the next reporting period to accomplish the goals?In our next hatch project period, 2022-2027, my research group will focus on 5 objectives. Objective 1. Conduct mosquito resistance surveillance in the Alabama State, Objective 2. Determine the function of insecticide resistance-related P450, GPCR, PKA genes in insecticide resistance of mosquitoes, Objective 3. Establish GPCR null (knockout) lines of resistant Culex mosquitoes as a tool to investigate GPCR regulatory factors, resistance, Objective 4. Characterize the responses of Ae. aegypti ORs to inhibitory chemical repellents and to human odorants that may be involved in Ae. aegypti host-seeking and blood-feeding, and Objective 5. Determine the competition effects of inhibitory chemical repellents and human odorants on Ae. aegypti ORs. These research objectives are very important in the management of insect pests, especially the medically important insects, leading to a number of significant discoveries, which may be important for designing the new strategies to control insect pests. In 2022, we will conduct the research on Regulation of CYP450-mediated detoxification in the Pacific oyster by the GPCR/Gαs/AC/cAMP/PKA signaling cascade through Crispr cas9 and RNA-seq techniques, for which we are revision our NIH-R21 proposal and try to submit it by June of 2022. In 2022, we will continue our extension related program of monitoring insecticide resistance and mosquito population of Culex quinquefasciatus, Aedes aegypti, Aedes albopictus in the state of Alabama.
Impacts
What was accomplished under these goals?
Survey on mosquito populations, possible mosquito-borne Zika virus surveillance, and mosquito resistance. This is a large data collection from multiple years. We have continuously conducted surveys on mosquito populations throughout the years 2017-2021 and will Finish by 2022 (5 years). Diversity of mosquito species, and mosquito resistance to 9 insecticides with different modes of action in 10 counties in Alabama. Four species were identified in 2021 in Alabama, including Aedes albopictus, Aedes triseriatus, Ades japonicas, and Aedes Aegyptus. Aedes albopictus was tested with eight insecticides and resmethrin, permethrin, and fenitrothion were found to allow mosquitoes to survive longer than the diagnostic time. The population and resistance surveys will be continued in 2022 and the first paper will be submitted in early 2022. In addition, we also expend the project to analyze the relationship between mosquito population dynamics and environmental factors. This will be the first report impacting on underrating of how mosquitoes survive under certain environmental conditions. G-Protein coupled receptors (GPCRs): signaling pathways, characterization, and functions in insect physiology and toxicology G-protein-coupled receptors (GPCRs) are known to play central roles in the physiology of many organisms. Members of these seven α-helical transmembrane protein family transduce the extracellular signals and regulate intracellular second messengers through coupling to heterotrimeric G-proteins, adenylate cyclase, cAMPs, and protein kinases. Because of the critical function of GPCRs in cell physiology and biochemistry, they not only play important roles in cell biology and the medicines used to treat a wide range of human diseases, but also in insects' physiological functions of reproduction, growth, and development, stress responses, feeding, behaviors. Our studies have revealed the expression and function of GPCRs in insecticide resistance, improving our understanding of the molecular complexes governing the development of insecticide resistance. Our current knowledge of the expression and function of GPCRs and their downstream-regulation pathways and their roles in insecticide resistance has also provided a strong foundation that will allow us to develop new insecticides and/or environmentally sound insecticides for better insect pest control. Multiple cytochrome P450 genes - conferring high levels of permethrin resistance in mosquitoes, Culex quinquefasciatus Insecticides, especially pyrethroids, are the most important in insect pest control and preventing insect vector-borne human diseases. However, insect pests, including mosquitoes, have developed resistance to the insecticides that are used against them. Cytochrome P450s are associated with insecticide resistance through overexpression and detoxification mechanisms in insect species. In this study, we utilized a powerful tool, the RNAi technique, to determine the roles of key P450 genes overexpressed in permethrin-resistant mosquitoes that confer insecticide resistance to unravel the molecular basis of resistance mechanisms in the mosquito Culex quinquefasciatus. The results showed that knockdown of 8 key P450 genes using RNAi techniques significantly decreased resistance to permethrin in resistant mosquitoes. In silico modeling and docking analysis further revealed the potential metabolic function of overexpressed P450 genes in the development of insecticide resistance in mosquitoes. These findings not only highlighted the functional importance of these P450 genes in insecticide resistance but also revealed that overexpression of multiple P450 genes was responsible for the high levels of insecticide resistance in a mosquito population of Culex quinquefasciatus. Molecular and functional characterization of three novel carboxylesterases in the detoxification of permethrin in the mosquito, Culex quinquefasciatus Carboxylesterases (CarEs) belong to a superfamily of multifunctional enzymes associated with the degradation of endogenous and exogenous compounds. Many insect CarEs are known to play important roles in catalyzing the hydrolysis of organophosphates (OPs), carbamates, and synthetic pyrethroids (SPs). The elevation of esterase activity through gene amplification and overexpression of estα2 and estβ2 genes contributes to the development of resistance to OP insecticides in the mosquito Culex quinquefasciatus. Three additional CarE genes are up-regulated in permethrin-resistant Cx. quinquefasciatus according to RNA-seq analysis, but their function remains unknown. In this study, we, for the first time, characterized the function of these three novel genes using in vitro protein expression, an insecticide metabolism study, and molecular docking analysis. All three CarE genes were significantly over-expressed in resistant mosquito larvae, but not adults, compared to susceptible strain. Metabolisms study of these CarEs to permethrin and a docking analysis confirmed that these three CarEs from resistant strain all potentially metabolize permethrin. Taken together, these three carboxylesterase genes could play important roles in the development of permethrin resistance in Cx. quinquefasciatus through transcriptional over-expression, metabolism, and detoxification. Heterologous Expressionand Functional Analysis of Aedes aegypti Odorant Receptors to human odors in Xenopus Oocytes The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti's olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus oocytes heterologous expressionto functionally analyze Aedes aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant co-receptors (Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Tiana, J., Lia, Y., Fua, H., Rena, L., Hea, Y., Zhaia, S., Yanga, B., Li, Q., Liu, N., Liu. S. 2021. Physiological role of CYP17A1-like in cadmium detoxification and its transcriptional regulation in the Pacific oyster, Crassostrea gigas. Sci Total Environ. 149039. (IF 7.9).
2. Liu, F., Chen, Z., Ye. Zi., Liu, N. 2021. The olfactory chemosensation of hematophagous hemipteran Insects" has been approved for production and accepted for publication in Frontiers in Physiology, section Invertebrate Physiology. Front. Physiol. 12:703768. (IF 4.6).
3. Liu, N., Y. Wang, T. Li, S. Liu. 2021.G-protein Coupled Receptors (GPCRs) in Insects A Potential Target for New Insecticide Development. Molecules 26(10), 2993. (IF 4.4).
4. Liu, N., Y. Wang, T. Li, X. Feng. 2021. G-Protein coupled receptors (GPCRs): signaling pathways, characterization, and functions in insect physiology and toxicology. Int. J. Mol. Sci. 22, 5260. (IF 5.9).
5. Gong, Y., M. Li, T. Li and N. Liu. 2021. Molecular and functional characterization of three novel carboxylesterases in the detoxification of permethrin in the mosquito, Culex quinquefasciatus. Insect Sci. 116. (IF 3.3).
6. Yang, T., T. Li, Feng, X., M. Li, S. Liu and N. Liu. 2021. Multiple cytochrome P450 genes - conferring high levels of permethrin resistance in mosquitoes, Culex quinquefasciatus. Sci. Rep. 11:9041. (IF 4.4).
7. Wang, X., C. Zhou, Y. Wang1, F. Liu, S. Jiang and N. Liu. 2021. Heterologous expression and functional analysis of Aedes aegypti odorant receptors to human odors in Xenopus oocytes. J. Vis. Exp. e61813. (IF 1.3)
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The information from the project has been included into classroom instruction. Methodology of the project has been included in laboratory instruction and class practice. The outcome/information and knowledge from the project, especially the objectives 1 and 2 mosquito survey and resistance survey, have been continually delivered to extension agencies in Alabama, and Alabama Department of Public Health. The research information has been presented in the professional meetings, such as ESA 2020: N. Liu. 2020. P450-mediated insecticide detoxification and its regulation in mosquito resistance. Invited Speaker in Symposium: Addressing the Wicked Problem of Pesticide Resistance and Pest Management, ESA 2020 Online. The research information has been presented in the international invited Lecture N. Liu. 2020. Insect Toxicology in Mosquitoes: Insecticide Resistance, Mechanisms, and Neuroscience. Invited Speaker in SiChun Agricultural University, December 4, 2020, Zoom Lecture, 3 hours. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided the opportunity to train 1 postdoc and 4 graduate students. By conducting the different objectives and training, working together with PI, the individuals who participate in the project have improved their professional skills in methodology and knowledge of science, and have sharpened their critical thinking. Especially ourMethods for the generation of heritable germline mutations in the disease vectorCulex quinquefasciatususing CRISPR/Cas9has made possible forresearchers in the field of toxicology and physiology to characterize the gene function in insecticide resistance. How have the results been disseminated to communities of interest?Our study on the response of olfactory systems of yellow fever mosquito Aedes aegypti to human orders has attracted public attention. We have gotten the encouragement from NIH program director for encourage us to submit the proposal to continue the study on these topics. Indeed, the NIH R03 proposal has beenin the second revision. Our methodology forcharacterizing olfactory systems of yellow fever mosquito Aedes aegypti has been invited to be published by the Journal ofJournal of Visualized Experiments. What do you plan to do during the next reporting period to accomplish the goals?In 2021, my research group will focus on the mechanisms that lead to the development of insecticide resistance, with the ultimate pragmatic goal of improving the management of insect pests. Specifically, we will: (1) investigate the GPCR regulatory pathways in P450-mediated insecticide resistance in different insect species, including house flies and mosquitoes; and 2) investigate the neuronal response of insect olfactory sensilla to chemical and human odorants in blood feeding insects, including mosquitoes and bed bugs. These research projects are very important in the management of insect pests, especially the medially important insects, leading to a number of significant discoveries, which may be important for designing the new strategies to control insect pests. In 2021, we will conduct the research on Regulation of CYP450-mediated detoxification in the Pacific oyster by the GPCR/Gαs/AC/cAMP/PKA signaling cascade through the collaboration with Ocean University of China, for which we have submit our NIH-R21 proposal by October of 2020. It is in pending. In 2021, we will continue our extension related program of monitoring insecticide resistance and mosquito population of Culex quinquefasciatus, Aedes aegypti, Aedes albopictus in the state of Alabama.
Impacts
What was accomplished under these goals?
Survey on mosquito populations, possible mosquito-borne Zika virus surveillance, and mosquito resistanceWe have continuously conducted surveys on mosquito populations throughout the year 2020, diversity of mosquito species, and mosquito resistance to 9 insecticides with different modes of action in 10 counties in Alabama. Four species were identified in 2020 in Alabama, includingAedes albopictus,Aedes triseriatus,Ades japonicas, andAedes aegypti.Aedes aegyptiwas not found in the previous years.Aedes albopictuswas tested with eight insecticides and resmethrin, permethrin, and fenitrothion were found to allow mosquitoes to survive longer than the diagnostic time. The population and resistance surveys will be continued in 2021 and the first paper will be submitted in the early of 2021. Functional analyses of house fly carboxylesterases involved in insecticide resistance Carboxylesterase-mediated metabolism is one of major mechanisms involved in insecticide resistance. Our previous study has identified multiple carboxylesterase genes with their expression levels were significantly upregulated in pyrethroid resistant house flies. To further explore their metabolic functions, we used insectSpodoptera frugiperda(Sf9) cells to express these carboxylesterasesin vitroand measure their hydrolytic activities toward esterase substrates. Our results indicated that these carboxylesterases can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based MTT cytotoxicity assay indicated that carboxylesterase-expressing cells show enhanced tolerance to permethrin, suggesting important roles of these carboxylesterases in metabolizing permethrin and thereby protecting cells from permethrin treatments. The metabolic functions of carboxylesterases were further verified by conductingin vitrometabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic pathway of permethrin in insects, but also important roles of these candidate carboxylesterases in metabolizing permethrin and conferring resistance in house flies. Homology modeling and docking were finally conducted to reflect interactions between permethrin ligand and carboxylesterase proteins, visually confirming the metabolic functions of carboxylesterases to insecticides in house flies. Identification of spinosad receptor from the yellow fever mosquito,Aedes aegyptiSpinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit have been identified that are associated with levels of spinosad resistance. Whereas the molecular characterization of nAChR gene family and causative association between nAChR α6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. In this study, we identified 10 candidate nAChR subunits in Ae. aegypti including nAChRα1 -α9 and nAChRβ1, showing similarly orthologous relationships with Anopheles gambiae. With the application of CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 5' end of the Aaeα6 (Ae. aegypti nAChR α6) gene in a homozygous mutant strain (Aaeα6-KO). The mutation produced two successive pre-mature stop codons resulting in loss of function in the target receptor. The Aaeα6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wild type. A recessive mode of inheritance for spinosad resistance was found in Aaeα6-KO strain. CRISPR/Cas9 introduced truncated Aaeα6 receptor in Ae. aegypti resulted in an increased level of resistance of the mosquitoes to spinosad, suggesting that the conserved nAChR α6 subunit is the target for spinosad insecticide. Methods for the generation of heritable germline mutations in the disease vectorCulex quinquefasciatususing CRISPR/Cas9Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats-associated protein 9 (CRISPR-Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR-Cas9-targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector. Three-dimensional Structure of the Antennal Lobe in the Southern House MosquitoCulex quinquefasciatusThe Southern house mosquitoCulex quinquefasciatusrelies on its olfactory system to locate the human hosts for blood meals, by which several deadly diseases are transmitted. Olfactory sensory neurons (OSNs) housed in the sensilla on the olfactory appendages send their axons into the antennal lobes (ALs), the primary olfactory center in the brain, where the OSNs expressing the same olfactory receptors converge upon the same spherical structures known as glomeruli in the AL. The structure of the antennal lobe, that is, the spatial organization of the glomeruli, governs the insect's odor identification and discrimination.Drosophilastudies have demonstrated the specific connections between receptors and glomeruli based on the 3D structure of the antennal lobe, deepening our understanding of the relationships between glomerular activities and behaviors, but as yet the structure of theCx. quinquefasciatusantennal lobe remains unknown. We therefore constructed a 3D model of theCx. quinquefasciatusantennal lobe using nc82 antibody staining, identifying 62 and 44 glomeruli in the female and male mosquito antennal lobe, respectively, with a significant sexual dimorphism in terms of the antennal lobe volume and glomerulus number. These results demonstrate the structural basis of mosquito odor coding and provide a platform for future studies of the mosquito olfactory signal processing mechanism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Elzaki, M.E.A., Z.F. Li, J. Wang, L. Xu, N. Liu, R.S. Zeng and Y.Y. Song. 2020. Activiation of the nitric oxide cycle by citrulline and arginine restores susceptibility of resistant brown planthoppers to the insecticide imidacloprid. J. Hazard. Mater. 396, 122755 https://doi.org/10.1016/j.jhazmat.2020.122755. Impact Factor: 9.038
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Feng, X. and N. Liu. 2020. Functional analyses of house fly carboxylesterases involved in insecticide resistance. Front. Physiol. 11:595009. doi: 10.3389/fphys.2020.595009. Impact Factor: 3.16
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ye, Z., F. Liu and N. Liu. 2020. Three-dimensional Structure of the Antennal Lobe in the Southern House Mosquito Culex quinquefasciatus. Insect science. doi: 10.1111/1744-7917.12767. Impact Factor: 2.791
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
. Li, M., T. Li and N. Liu, R. Raban, X. Wang, O.S. Akbari. 2020. Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using CRISPR/Cas9. Insect Mol. Biol. doi: 10.1111/imb.12626. Impact Factor: 2.533
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Bui, M., M. Li, R. R. Raban, N. Liu, and O. S. Akbari. 2020. Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus. J. Vis. Exp. (159), e61375, doi:10.3791/61375. Impact Factor: 1.325
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Lan, J., Z. Wang, Z. Chen, L, Zhang, J. Zhao, Q. Guan, C. Liao, N. Liu and H. Qian. 2020. Identification of spinosad receptor from the yellow fever mosquito, Aedes aegypti. Pest Manag. Sci. DOI: 10.1002/ps.6183. Impact Factor: 3.75
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The information from the project has been included into classroom instruction and methodology of the project has been included in laboratory instruction and class prectice. The outcome/information and knowledge from the project, especially the objectives 1 and 2 mosquito survey and resistance survey, have been continually delivered to extension agencies in Alabama, and Alabama Department of Public Health. The research information has been presented in the professional meetings, such as ESA, and SEB-ESA 2019. The research information has been presented in the international conferences, such as 1) International Vector & Pest Management Workshop, Taipei, 2) Conference of Medical and Veterinary Entomology of the Entomological Society of China and the 4th Meeting of the Asian Society of Vector Ecology and Mosquito Control, and 3)The 2nd international Conference on Insect Pest Management". . Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training: This project has provide the opportunity to train 1 postdocs and 4 graduate students and 1 undergraduate student. By conducting the different objectives and training, working together with PI, the individuals who participate in the project have improved their professional skills in methodology and knowledge of science, and have sharpened their critical thinking. How have the results been disseminated to communities of interest?Our study on the response of olfactory systems of yellow fever mosquitoAedes aegypti to human orders has attracted public attention. We have gotten the encouragement from NIH program director for encourage us to submit the proposal to continue the study on these topics.The Creative Project Manager in ResearchPod showed the interesting to report our study. What do you plan to do during the next reporting period to accomplish the goals?What do you plan to do during the next reporting period to accomplish the goals? We are continuing our objectives proposed in our Hatch project. We will finish Aedes mosquito and mosquito-borne virus surveillance and insecticide resistance in the Alabama State in 2020, which will be the third year survey and two papers are in preparation. We will start a survey on Culex quinquefasciatus mosquito population survey in Alabama from 2020. We will continue the our objectives of "Determine of the function of insecticide resistance related P450, GPCR, PKA genes in insecticide resistance of mosquitoes and house flies" , "Characterize GPCR regulatory pathway(s) and downstream factors involved in regulating resistance P450 gene up-regulation and insecticide resistance" and "Investigating the antennal olfactory sensilla responses to chemical repellents and human odorants in mosquitoes and house flies".
Impacts
What was accomplished under these goals?
Insects use their olfactory systems to obtain chemical information on mating partners, oviposition sites and food. The yellow fever mosquito Aedes aegypti, an important vector of human infectious diseases, shows strong preference for human blood meals. This study investigated the chemical basis of host detection by characterizing the neuronal responses of antennal olfactory sensilla of female Ae. aegypti to 103 compounds from human skin emanations. The effect of blood feeding on the responses of olfactory sensilla to these odorants was examined as well. Sensilla SBTII, GP, and three functional subtypes of SST (SST1, SST2, and SST3) responded to most of the compounds tested. Olfactory receptor neurons (ORNs) 'A' and 'B' in the trichoid sensilla, either activated or inhibited, were involved in the odour coding process. Compounds from different chemical classes elicited responses with different temporal structures and different response patterns across the olfactory sensilla. Except for their increased responses to several odorants, blood-fed mosquitoes generally evoked reduced responses to specific aldehydes, alcohols, aliphatics/aromatics, ketones, and amines through the SST1, SST2, SBTI, SBTII and GP sensilla. The odorants eliciting diminished responses in female mosquitoes after blood feeding may be important in Ae. aegypti host-seeking activity and thus can be candidates for mosquito attractants in the process of this disease vector management. The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), Adenylate cyclase (AC), and Protein Kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
1. Xu, P., Y.M. Choo, Z. Chen, F. Zeng, K. Tan, T. Y. Chen, A.J. Cornel, N. Liu and W.S Leal. 2019. Odorant inhibition in mosquito olfaction. iScience, doi: https://doi.org/10.1016/ j.isci.2019.07.008.
2. Li, T. and N. Liu. 2019. Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance. Int. J. Mol. Sci. 20, 4300; https://doi.org/10.3390/ijms20174300.
3. Zhou, C., F. Liu and N. Liu. 2019. Human Odour Coding in the Yellow Fever Mosquito, Aedes aegypti. Sci. Rep. DOI: 10.1038/s41598-019-49753-2.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The information from the project has been included into classroom instruction and methodology of the project has been included in laboratory instruction. The outcome/information and knowledge from the project, especially the objectives 1 and 2 mosquito survey and resistance survey, have been deliver to extension agencies in Alabama, and Alabama Department of Public Health. The research information has been presented in the professional meetings, such as, Pesticide Applicator University 2018, ESA, and SEB-ESA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provide the opportunity to train 1 postdocs and 4 graduate students and 1 undergraduate student. By conducting the different objectives and training, working together with PI, the individuals who participate in the project have improved their professional skills in methodology and knowledge of science, and have sharpened their critical thinking. How have the results been disseminated to communities of interest?The data and information generated from our objective of survey on mosquito populations and mosquito resistance to different insecticides have been provide to and used by Alabama Department of Public Health and Alabama County Health Departments to make the decisions of what chemicals that will be used in the coming season. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
No systematic survey on the mosquito insecticide resistance in the field population has been conducted for the last 10 - 15 years in Alabama. No study on the dynamics of population changes and species diversity of mosquitoes in Alabama was conducted. To fill these gaps in mosquito resistance and population dynamics, address the questions in the mosquito control, and find the new ways to control mosquitoes, we have conducted surveys on mosquito populations throughout the seasons, diversity of mosquito species, and mosquito resistance to 9 insecticides with different modes of action (Objectives 1-2) in 10 counties in Alabama. Three species were identified in this survey in Alabama: Aedes albopictus, Aedes triseriatus, and Aedes japonicus. The mosquito population began to emerge in April at approximately 57°F. The suitable temperature range of mosquito is 75°F to 85°F. Aedes albopictus was tested for its resistance level to eight insecticides, chlorpyrifos, deltamethrin, etofenprox, malathion, permethrin resmethrin, fenitrothion, and β-cyfluthrin, in adult and larval bioassays. Resmethrin, permethrin, and fenitrothion were found to allow mosquitoes to survive longer than the diagnostic time. Mobile is the most suitable city for mosquitoes, with most strains demonstrating insecticide resistance (Wang and Liu, M.S. Thesis 2018). Resistance survey will be continued in 2018-2019 and data will be published in 2019. The molecular mechanisms involved in regulation of resistance genes are not yet clear and a global understanding of the complex processes responsible for resistance has not been achieved. Carboxylesterases are one of the major enzyme families involved in the detoxification of pyrethroids. Up-regulation of carboxylesterase genes is thought to be a major component of insecticide resistant mechanisms in insects (Objective 3). A total of 39 carboxylesterase genes of different functional clades have been identified in house flies, eleven of which were found to be significantly overexpressed in the resistant house fly strain. Spatial expression studies revealed these up-regulated genes to be abundantly distributed in fat bodies and genetically mapped on autosome 2 or 3 of house flies, and their expression could be regulated by factors on autosome 1, 2 and 5 Taken together, these results demonstrate that multiple carboxylesterase genes are co-upregulated in resistant house flies, providing further evidence for their involv ement in the detoxification of insecticides and development of insecticide resistance (Feng et al. 2018).. G-protein coupled receptors (GPCRs) are a large family of seven-transmembrane domain proteins that exist in plants and animals, playing critical physiological functions through intracellular cascades. However, the function of this regulation pathway in field collected and laboratory-susceptible mosquitoes is still unknown. We characterized the function of each effector (GPCR, G-protein, AC, and PKA) in this GPCR intracellular pathway in both field-collected and laboratory Cx. quinquefasciatus strains, showing that knockdown of the expression of each effector gene can cause 1) decreased expression of their downstream respected genes and 2) increased sensitivity of the mosquitoes to permethrin insecticide. These results, together with our previous findings, strongly suggest the universal function of the GPCR-regulation cascade in the mosquito's sensitivity to insecticides and its regulation of resistance development through P450 mediated detoxification. (Objective 4). Our study revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing resistance P450 gene expression and P450-mediated resistance in mosquitoes (Li and Liu 2018a, b) Culex mosquitoes, adding significantly to our understanding of the molecular basis of insecticide resistance development in mosquitoes. The yellow fever mosquito, Aedes aegypti, is a vector of many human diseases such as yellow fever virus, dengue virus, and Zika virus. As insecticide resistance has been widely reported, chemical repellents have been adopted as alternative options for mosquito and mosquito-borne diseases control. (Objective 5). This study characterized the responses of olfactory receptor neurons (ORNs) in different types of antennal olfactory sensilla in Ae. aegypti to 48 chemicals that exhibited repellent activity in various insects species. Both excitatory and inhibitory responses were observed from ORNs in response to these chemicals and differential tuning properties were also observed among ORNs. Moreover, the temporal dynamics of neuronal responses were found to be compound-specific and concentration-dependent. Hierarchical cluster analysis and principal component analysis for the response to each compound across ORNs in seven types of olfactory sensilla in Ae. aegypti revealed that odor reception depended on not only chemical class but specific chemical structure. Results of this study give new insights into the sensory physiology of Aedes mosquitoes to the chemical repellents and should contribute to the development of new repellent reagents for human protection (Zhou et al. 2018).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1. Feng, X. and N. Liu. 2018. Baculovirus-mediated cell expression system in functional characterization of carboxylesterases in insecticide resistant house flies, Musca domestica. J. Vis. Exp. 138, e58106, doi:10.3791/58106.
2. Zhou, C., F. Liu, N. Liu. 2018. Neuronal Responses of Antennal Olfactory Sensilla to Insect Chemical Repellents in the Yellow Fever Mosquito, Aedes aegypti. J. Chem. Ecol. DOI: 10.1007/s10886-018-1022-5.
3. William R. R., L. Zhang, Y. Gong, T. Li, N. Liu. 2018. Gene Expression Profiles of the Southern House Mosquito Culex quinquefasciatus During Exposure to Permethrin. Insect Sci., 25, 439-453.
4. Li, T. and N. Liu. 2018. The function of G-protein-coupled receptor-regulatory cascade in southern house mosquitoes (Diptera: Culicidae). J. Med. Entomol. 1⿿9, doi: 10.1093/jme/tjy022.
5. Feng, X., M. Li., and N. Liu. 2018. Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica. Insect Biochem. Mol. Biol. 92, 30-39.
6. Adesanya, A., D. W. Held, N. Liu. 2018. Ontogeny, sex, and adult tissues influence activities of detoxification enzymes in the Japanese beetle, Popillia japonica Newman. Physiol. Entomol. DOI: 10.1111/phen.12260.
7. Li. T. and N. Liu. 2018. G-protein-coupled receptors: their expression, function and regulation in insecticide resistance. In: Advances in the Biorational Control of Medical and Veterinary Pests, Edmund J. N., Coats. J. R., Gross, A. D., and Clark, J. M. (Eds.), ACS Publications, ISBN13: 978-084-123-359-1, pp. 153-172.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The information from the project has been included into classroom instruction and methodology of the project has been included in laboratory instruction. The outcome/information and knowledge from the project, especially the objectives 1 and 2 mosquito survey and resistance survey, have been deliver to extension agencies in Alabama and Alabama Department of Public Health. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provide the opportunity to train 2 postdocs and 3 graduate students. By conducting the different objectives and training, working together with PI, the individuals who participate in the project have improved their professional skills in methodology and knowledge of science, and have sharpened their critical thinking. How have the results been disseminated to communities of interest?The data and information generated from our objective of survey on mosquito populations and mosquito resistance to different insecticides have been provide to and used by Alabama Department of Public Health and Alabama County Health Departments to make the decisions of what chemicals that will be used in the coming season. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
No systematic survey on the mosquito insecticide resistance in the field population has been conducted for the last 10 - 15 years in Alabama. To fill the gaps in mosquito resistance, address the questions in the mosquito control, and find the new ways to control mosquitoes, we have conducted a survey on mosquito populations, possible mosquito-borne Zika virus surveillance, and mosquito resistance to 9 insecticides with different modes of action (Objectives 1-2) in 10 counties in Alabama. Three mosquito species, Aedes aegypti (the principle vector of Zika), Aedes albopictus (a potential vector of ZIKA), and Culex quinquefasciatus (the primary vector of St. Louis encephalitis virus and the vector of West Nile virus) have focused on the population and resistance Survey. No Aedes aegypti was identified in Alabama. Aedes albopictus and Culex quinquefasciatus were the predominant mosquito species. Resistance survey will be continued in 2018 and 2019 and data will be published after 2018. The molecular mechanisms involved in regulation of resistance genes are not yet clear and a global understanding of the complex processes responsible for resistance has not been achieved. We conducted research characterize GPCR regulatory pathway(s) and downstream factors, Gs alpha subunit protein (Gαs), adenylyl cyclase (ACs) and protein kinase A (PKAs), involved in regulating resistance P450 gene up-regulation and insecticide resistance in mosquitoes (Objectives 3-4). Our study revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing resistance P450 gene expression and P450-mediated resistance in mosquitoes (Li and Liu 2017, Gong et al. 2017) Culex mosquitoes, adding significantly to our understanding of the molecular basis of insecticide resistance development in mosquitoes. Recent global outbreaks of mosquito-related diseases make it imperative that chemical repellents, as potential alternatives for the control of mosquito vectors and mosquito-associated diseases, are fully characterized. We therefore initiated examination of the responses of olfactory receptor neurons (ORNs) on mosquitoes Aedes aegypti, a mosquito vector for important human diseases such as yellow fever virus and Zika virus, and bed bugs to chemical repellents (Objective 5). The analysis of ORNs in Aedes aegypti permitted a comparison with those in another mosquito species Culex quinquefasciatus and the common bed bug Cimex lectularius, which revealed the specificity and generality of chemical repellent reception among the three insect species. Our study and findings provide valuable information for predicting the responses of mosquitoes and other insects to chemical patterns and can be applied to screen new repellents for pest control and mosquito-borne disease management (Liu et al. 2017, Chen et al. in preparation).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Liu, F., Z. Chen, N. Liu. 2017. Molecular Basis of Olfactory Chemoreception in the Common Bed Bug, Cimex lectularius. Sci. Rep. 7:45531. doi: 10.1038/srep45531.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Li, T. and N. Liu. 2017. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/G?s/AC/cAMP/PKA signaling cascade. Biochem. Biophys. Rep. 12, 12-19. https://doi.org/10.1016/j.bbrep.2017.08.010.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Liu, F., X. Xia, N. Liu. 2017. Molecular Basis of N,N-Diethyl-3-Methylbenzamide (DEET) in Repelling the Common Bed Bug, Cimex lectularius. Front. Physiol. | doi: 10.3389/fphys.2017.00418.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Liu, F., C. Xiong, N. Liu. 2017. Chemoreception to Aggregation Pheromones in the Common Bed Bug, Cimex lectularius. Insect Biochem. Mol. Bio. 82, 62-73.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
William R. R., L. Zhang, Y. Gong, T. Li, N. Liu. 2017 Gene Expression Profiles of the Southern House Mosquito Culex quinquefasciatus During Exposure to Permethrin. Insect Science, 115, DOI 10.1111/1744-7917.12438.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Gong Y., T. Li, Y. Feng., N. Liu. 2017. The function of two P450s, CYP9M10 and CYP6AA7, in the permethrin resistance of Culexquinquefasciatus. Sci. Rep. 7, 587; doi:10.1038/s41598.
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Progress 05/26/16 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In 2016, we have conducted a survey on mosquito population and a mosquito-borne Zika virus surveillance (Objective 1) and mosquito resistance (Objective 2)in10 counties inAlabama. Data for 2016have beenanalyzed. The survey will be continued in 2017and 2018 and data will be published after 2018. Determine the function of insecticide resistance related P450, GPCR, PKA genes in insecticide resistance of mosquitoes and house flies (Objective 3) has been initiated in 2016 and will be continue in 2017. Investigate the antennal olfactory sensilla responses to chemical repellents and human odorants in mosquitoes and house flies (Objective 5) has also been initiated.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ye, Z., F. Liu, and N. Liu. 2016. Olfactory responses of southern house mosquito, Culex quinquefasciatus, to human odorants. Chem. Sense. doi:10.1093/chemse/bjv089.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Liu, F. and N. Liu. 2016. Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals. J. Vis. Exp. 107, e53337.
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