hybridization-chain-reaction-rna-whole-mount-fluorescence-situ

Hybridization Chain Reaction RNA Whole-Mount Fluorescence <em>In situ</em> Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

Johns Hopkins University School of Medicine

Various insect species serve as valuable model systems for investigating the cellular and molecular mechanisms by which a brain controls sophisticated behaviors. In particular, the nervous system of Drosophila melanogaster has been extensively studied, yet experiments aimed at determining the number of neurons in the Drosophila brain are surprisingly lacking. Using isotropic fractionator coupled with immunohistochemistry, we counted the total number of neuronal and non-neuronal cells in the whole brain, central brain, and optic lobe of Drosophila melanogaster. For comparison, we also counted neuronal populations in three divergent mosquito species: Aedes aegypti, Anopheles coluzzii and Culex quinquefasciatus. The average number of neurons in a whole adult brain was determined to be 199,380 ±3,400 cells in D. melanogaster, 217,910 ±6,180 cells in Ae. aegypti, 223,020 ± 4,650 cells in An. coluzzii and 225,911±7,220 cells in C. quinquefasciatus. The mean neuronal cell count in the central brain vs. optic lobes for D. melanogaster (101,140 ±3,650 vs. 107,270 ± 2,720), Ae. aegypti (109,140 ± 3,550 vs. 112,000 ± 4,280), An. coluzzii (105,130 ± 3,670 vs. 107,140 ± 3,090), and C. quinquefasciatus (108,530 ±7,990 vs. 110,670 ± 3,950) was also estimated. Each insect brain was comprised of 89% ± 2% neurons out of its total cell population. Isotropic fractionation analyses did not identify obvious sexual dimorphism in the neuronal and non-neuronal cell population of these insects. Our study provides experimental evidence for the total number of neurons in Drosophila and mosquito brains.

The number of neurons in Drosophila and mosquito brains.

Half the world's human population is at risk for mosquito-borne diseases. Mosquitoes rely mainly on their sense of smell to find a vertebrate blood host, nectar source, and a suitable oviposition site. Advances in neurogenetic tools have now aided our understanding of the receptors that mediate the detection of sensory cues that emanate from humans. Recent studies in the anthropophilic mosquito vectors, Aedes aegypti and Anopheles gambiae, have implicated the chemosensory ionotropic-receptor (IR) family in the detection of behaviorally relevant odors and uncovered functions beyond chemical sensing. Here, we highlight the multifunctional roles of the chemosensory ionotropic receptors in anthropophilic mosquito vectors and suggest future directions to improve our understanding of the IR family.

Chemosensory ionotropic receptors in human host-seeking mosquitoes.

The mosquito's antenna represents its main olfactory appendage for detecting volatile chemical cues from the environment. Whole-mount fluorescence in situ hybridization of ionotropic receptors (IRs) expressed in the antennae reveals that the antenna might be divisible into proximal and distal functional domains. The number of IR-positive cells appear stereotyped within each antennal segment (flagellomere). Highly expressed odor-tuning IRs exhibit distinct co-localization patterns with the IR coreceptors Ir8a, Ir25a, and Ir76b that might predict their functional properties. Genetic knockin and in vivo functional imaging of IR41c-expressing neurons indicate both odor-induced activation and inhibition in response to select amine compounds. Targeted mutagenesis of IR41c does not abolish behavioral responses to the amine compounds. Our study provides a comprehensive map of IR-expressing neurons in the main olfactory appendage of mosquitoes. These findings show organizing principles of Anopheles IR-expressing neurons, which might underlie their functional contribution to the detection of behaviorally relevant odors.

Joshua I. Raji

The Solomon H. Snyder Department of Neuroscience

Johns Hopkins University School of Medicine

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Joshua I. Raji

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Solomon H. Snyder Department of Neuroscience

Johns Hopkins University School of Medicine

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

The Solomon H. Snyder Department of Neuroscience