Broad sensory expression and selective functional roles of Ir25a in the malaria mosquito Anopheles stephensi
摘要
Mosquitoes rely heavily on their peripheral nervous system to sense various cues during host-seeking and blood-feeding. Ionotropic receptors (IRs) are a family of chemosensory receptors that play fundamental roles in mosquito chemical and physical cue detection. Among the three IR coreceptors, Ir25a is the most evolutionarily ancestral and conserved, whereas its function remains poorly understood, particularly in Anopheles mosquitoes. In this study, we integrated transgenic fluorescent labeling, single-nucleus RNA-seq (snRNA-Seq), electrophysiology, and behavioral assays to determine the functions of Ir25a in An. stephensi, an urban malaria vector. We found that Ir25a is broadly expressed in sensory neurons of multiple appendages in both female and male mosquitoes, including the antennae, maxillary palps, labella, and tarsi. Notably, Ir25a is localized in both CO2-sensing and odor-sensing neurons in the maxillary palps. Detailed snRNA-Seq profiling further supports extensive co-expression of Ir25a with CO2-sensitive gustatory receptors (GRs) and odor-sensitive odorant receptors (ORs) in chemosensory neurons, while also delineating accessory cell types not previously characterized in mosquito snRNA-seq. Surprisingly, Ir25a mutants display minimal changes in peripheral chemical responses in both antennae and maxillary palps. In contrast, Ir25a mutants exhibit severe thermosensory deficits, reduced CO₂-evoked takeoff, complete abolishment of host-seeking and blood-feeding, along with reduced mating success. Taken together, this study systematically characterizes the broad expression of Ir25a and identifies its selective functional roles in sensory physiology, as well as the profound impact on host detection in the malaria mosquito An. stephensi, contributing to our long-term effort in Anopheles mosquito control and malaria prevention.
参考文献
1. McMeniman, C. J., Corfas, R. A., Matthews, B. J., Ritchie, S. A. & Vosshall, L. B. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. Cell 156, 1060–1071 (2014).
2. Morita, T. et al. Cross-modal sensory compensation increases mosquito attraction to humans. Sci. Adv. 11, eadn5758 (2025).
3. Hawaria, D. et al. First report of Anopheles stephensi from southern Ethiopia. Malar. J. 22, 373 (2023).
4. Acosta, A. L., Castro, M. C., Laporta, G. Z., Conn, J. E. & Sallum, M. A. M. Future global distribution and climatic suitability of Anopheles stephensi. Sci. Rep. 15, 22268 (2025).
5. Liu, Q. et al. Possible potential spread of Anopheles stephensi, the Asian malaria vector. BMC Infect. Dis. 24, 333 (2024).
6. Hansson, B. S. & Stensmyr, M. C. Evolution of insect olfaction. Neuron 72, 698–711 (2011).
7. Keil, T. A. Comparative morphogenesis of sensilla: A review. Int. J. Insect Morphol. Embryol. 26, 151–160 (1997).
8. Shanbhag, S. R., Müller, B. & Steinbrecht, R. A. Atlas of olfactory organs of Drosophila melanogaster 2. Internal organization and cellular architecture of olfactory sensilla. Arthropod Struct. Dev. 29, 211–229 (2000).
9. Kim, M. S., Repp, A. & Smith, D. P. LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster. Genetics 150, 711–721 (1998).
10. Prelic, S. et al. Functional interaction between Drosophila olfactory sensory neurons and their support cells. Front. Cell. Neurosci. 15, 789086 (2022).
11. Sen, A., Shetty, C., Jhaveri, D. & Rodrigues, V. Distinct types of glial cells populate the Drosophila antenna. BMC Dev. Biol. 5, 25 (2005).
12. Konopka, J. K. et al. Olfaction in Anopheles mosquitoes. Chem. Senses 46, bjab021 (2021).
13. Zhao, J., Chen, A. Q., Ryu, J. & del Mármol, J. Structural basis of odor sensing by insect heteromeric odorant receptors. Science (80-. ). 384, 1460–1467 (2024).
14. Butterwick, J. A. et al. Cryo-EM structure of the insect olfactory receptor Orco. Nature 560, 447–452 (2018).
15. Carey, A. F., Wang, G., Su, C. Y., Zwiebel, L. J. & Carlson, J. R. Odorant reception in the malaria mosquito Anopheles gambiae. Nature 464, 66–71 (2010).
16. Ma, D. et al. Structural basis for sugar perception by Drosophila gustatory receptors. Science (80-. ). 383, 1–15 (2024).
17. Lu, T. et al. Odor Coding in the Maxillary Palp of the Malaria Vector Mosquito Anopheles gambiae. Curr. Biol. 17, 1533–1544 (2007).
18. Ni, L. The structure and function of ionotropic receptors in Drosophila. Front. Mol. Neurosci. 13, 638839 (2021).
19. Montell, C. Drosophila sensory receptors—A set of molecular swiss army knives. Genetics 217, 1–34 (2021).
20. Herre, M. et al. Non-canonical odor coding in the mosquito. Cell 185, 3104-3123.e28 (2022).
21. Task, D. et al. Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons. Elife 11, (2022).
22. Raji, J. I., Konopka, J. K. & Potter, C. J. A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito. Cell Rep. 42, 112101 (2023).
23. Fernández-Chiappe, F., Ocker, G. K. & Younger, M. A. Prospects on non-canonical olfaction in the mosquito and other organisms: why co-express? Curr. Opin. Insect Sci. 67, 101291 (2025).
24. Goldman, O. V. et al. A single-nucleus transcriptomic atlas of the adult Aedes aegypti mosquito. Cell 188, 7267-7290.e26 (2025).
25. Raji, J. I. et al. Aedes aegypti Mosquitoes Detect Acidic Volatiles Found in Human Odor Using the IR8a Pathway. Curr. Biol. 29, 1253-1262.e7 (2019).
26. Ye, Z. et al. Discrete roles of Ir76b ionotropic coreceptor impact olfaction, blood feeding, and mating in the malaria vector mosquito Anopheles coluzzii. Proc. Natl. Acad. Sci. U. S. A. 119, e2112385119 (2022).
27. Vulpe, A. & Menuz, K. Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons. Front. Cell. Neurosci. 15, 759238 (2021).
28. Stanley, M., Ghosh, B., Weiss, Z. F., Christiaanse, J. & Gordon, M. D. Mechanisms of lactic acid gustatory attraction in Drosophila. Curr. Biol. 31, 3525-3537.e6 (2021).
29. Lee, Y., Poudel, S., Kim, Y., Thakur, D. & Montell, C. Calcium taste avoidance in Drosophila. Neuron 97, 67-74.e4 (2018).
30. McDowell, S. A. T., Stanley, M. & Gordon, M. D. A molecular mechanism for high salt taste in Drosophila. Curr. Biol. 32, 3070-3081.e5 (2022).
31. Ahn, J.-E., Chen, Y. & Amrein, H. Molecular basis of fatty acid taste in Drosophila. Elife 6, e30115 (2017).
32. Chen, C. et al. Drosophila ionotropic receptor 25a mediates circadian clock resetting by temperature. Nature 527, 516–520 (2015).
33. Ni, L. et al. The ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila. Elife 5, (2016).
34. Knecht, Z. A. et al. Ionotropic receptor-dependent moist and dry cells control hygrosensation in Drosophila. Elife 6, e26654 (2017).
35. De Obaldia, M. E. et al. Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels. Cell 185, 4099-4116.e13 (2022).
36. Shrestha, B., Sang, J., Rimal, S. & Lee, Y. Pharyngeal neuronal mechanisms governing sour taste perception in Drosophila melanogaster. Elife 13, RP101439 (2024).
37. Qian, Q. et al. The influence of IR25a on electrophysiology and reproductive behaviour in the oriental fruit fly, Bactrocera dorsalis. Insect Mol. Biol. 35, 1–13 (2026).
38. Omondi, B. A., Majeed, S. & Ignell, R. Functional development of carbon dioxide detection in the maxillary palp of Anopheles gambiae. J. Exp. Biol. 218, 2482–2488 (2015).
39. Speth, Z. et al. Characterization of Anopheles stephensi odorant receptor 8, an abundant component of the mouthpart chemosensory transcriptome. Insects 12, 593 (2021).
40. Potter, C. J., Tasic, B., Russler, E. V., Liang, L. & Luo, L. The Q system: A repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141, 536–548 (2010).
41. Campesan, S. et al. The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington’s disease. Curr. Biol. 21, 961–966 (2011).
42. Mukabayire, O., Cornel, A. J., Dotson, E. M., Collins, F. H. & Besansky, N. J. The tryptophan oxygenase gene of Anopheles gambiae. Insect Biochem. Mol. Biol. 26, 525–528 (1996).
43. Besansky, N. J. et al. The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion. Insect Biochem. Mol. Biol. 27, 803–805 (1997).
44. Miller, S. W., Avidor-Reiss, T., Polyanovsky, A. & Posakony, J. W. Complex interplay of three transcription factors in controlling the tormogen differentiation program of Drosophila mechanoreceptors. Dev. Biol. 329, 386–399 (2009).
45. Loh, L. S. et al. Lepidopteran scale cells derive from sensory organ precursors through a canonical lineage. Development 152, DEV204501 (2025).
46. Ballou, S., Zlotnick, K., Tolmach, D. & Menuz, K. Identification of markers facilitating the study of support cells in adult olfactory sensilla in Drosophila. Insect Biochem. Mol. Biol. 195, 104658 (2026).
47. Li, H. et al. Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science (80-. ). 375, eabk2432 (2022).
48. Sun, H., Liu, F., Ye, Z., Baker, A. & Zwiebel, L. J. Mutagenesis of the orco odorant receptor co-receptor impairs olfactory function in the malaria vector Anopheles coluzzii. Insect Biochem. Mol. Biol. 127, (2020).
49. Degennaro, M. et al. Orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET. Nature 498, 487–491 (2013).
50. Cooke, M. M., Chembars, M. S. & Pitts, R. J. The dysregulation of tuning receptors and transcription factors in the antennae of Orco and Ir8a mutants in Aedes aegypti suggests a chemoreceptor regulatory mechanism involving the MMB/dREAM complex. Insects 16, 638 (2025).
51. Long, T., Mohapatra, P., Ballou, S. & Menuz, K. Odorant receptor co-receptors affect expression of tuning receptors in Drosophila. Front. Cell. Neurosci. 18, 1390557 (2024).
52. Yao, H. et al. Unveiling the developmental dynamics and functional role of Odorant Receptor Co-receptor (Orco) in Aedes albopictus: A novel mechanism for regulating odorant receptor expression. PLoS Negl. Trop. Dis. 19, e0013753 (2025).
53. Benton, R., Vannice, K. S., Gomez-Diaz, C. & Vosshall, L. B. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162 (2009).
54. Croset, V. et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet. 6, e1001064 (2010).
55. Ando, T. et al. Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum. Curr. Biol. 29, 1512-1520.e6 (2019).
56. Wang, Y. et al. CRISPR/Cas9 mediates efficient site-specific mutagenesis of the odorant receptor co-receptor (Orco) in the malaria vector Anopheles sinensis. Pest Manag. Sci. 78, 3294–3304 (2022).
57. Riabinina, O. et al. Organization of olfactory centres in the malaria mosquito Anopheles gambiae. Nat. Commun. 7, (2016).
58. Maekawa, E. et al. The role of proboscis of the malaria vector mosquito Anopheles stephensi in host-seeking behavior. Parasites and Vectors 4, 10 (2011).
59. Greppi, C. et al. Mosquito heat seeking is driven by an ancestral cooling receptor. Science (80-. ). 367, 681–684 (2020).
60. Laursen, W. J. et al. Humidity sensors that alert mosquitoes to nearby hosts and egg-laying sites. Neuron 111, 874-887.e8 (2023).
61. Keane, G. T. & Garrity, P. A. Mosquito heat and humidity detection in host seeking and oviposition behavior. Curr. Opin. Neurobiol. 99, 103225 (2026).
62. Wang, G. et al. Anopheles gambiae TRPA1 is a heat-activated channel expressed in thermosensitive sensilla of female antennae. Eur. J. Neurosci. 30, 967–974 (2009).
63. Corfas, R. A. & Vosshall, L. B. The cation channel TRPA1 tunes mosquito thermotaxis to host temperatures. Elife 4, e11750 (2015).
64. Chandel, A. et al. Thermal infrared directs host-seeking behaviour in Aedes aegypti mosquitoes. Nature 633, 615–623 (2024).
65. Liu, M. Z. & Vosshall, L. B. General visual and contingent thermal cues interact to elicit attraction in female Aedes aegypti mosquitoes. Curr. Biol. 29, 2250-2257.e4 (2019).
66. Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: Large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
67. Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: Computational identification of cell doublets in single-cell transcriptomic data. Cell Syst. 8, 281-291.e9 (2019).
68. Pullin, J. M. & McCarthy, D. J. A comparison of marker gene selection methods for single-cell RNA sequencing data. Genome Biol. 25, 56 (2024).
69. Blum, M., Hobbs, E., Florentino, L. & Bateman, A. InterProScan 6: a modern large-scale protein function annotation pipeline. Bioinforma. Adv. 6, vbag141 (2026).
70. Emms, D. M. & Kelly, S. OrthoFinder: Phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019).
71. Mier, P. et al. Annotation and analysis of 3902 odorant receptor protein sequences from 21 insect species provide insights into the evolution of odorant receptor gene families in solitary and social insects. Genes (Basel). 13, 919 (2022).
72. Mitchell, R. F., Moran, K. M. & McKenna, D. D. Early hexapod genomes reveal the deep origin of insect gustatory and odorant receptors. Nat. Commun. 17, 6881 (2026).
73. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
74. Steenwyk, J. L., Buida, T. J., Li, Y., Shen, X. X. & Rokas, A. ClipKIT: A multiple sequence alignment trimming software for accurate phylogenomic inference. PLoS Biol. 18, e3001007 (2020).
75. Kozlov, A. M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. RAxML-NG: A fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35, 4453–4455 (2019).
76. Barbera, P. et al. EPA-ng: Massively parallel evolutionary placement of genetic sequences. Syst. Biol. 68, 365–369 (2019).
77. Hammond, A. et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat. Biotechnol. 34, 78–83 (2016).
78. Labun, K. et al. CHOPCHOP v3: Expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).
79. Gratz, S. J. et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196, 961–971 (2014).
80. Matthews, B. J., Younger, M. A. & Vosshall, L. B. The ion channel ppk301 controls freshwater egg-laying in the mosquito aedes aegypti. Elife 8, (2019).
81. Liu, F., Chen, L., Appel, A. G. & Liu, N. Olfactory responses of the antennal trichoid sensilla to chemical repellents in the mosquito, Culex quinquefasciatus. J. Insect Physiol. 59, 1169–1177 (2013).
82. Den Otter, C. J., Behan, M. & Maes, F. W. Single cell responses in female Pieris brassicae (Lepidoptera: Pieridae) to plant volatiles and conspecific egg odours. J. Insect Physiol. 26, 465–472 (1980).
83. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10 (2011).
84. Chakraborty, M. et al. Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly. BMC Biol. 19, 28 (2021).
85. Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
86. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
87. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
指标
DOI：
Submission ID：
下载次数
已发布
如何引用
下载引用
利益冲突声明
作者声明无任何需要披露的利益冲突。
Copyright
本预印本的版权持有者为作者/资助方。
保留所有权利。本作品受版权保护。未经版权所有者事先书面许可，不得以任何形式或方式复制、分发或传播本作品的任何部分。