Journal: Current biology : CB
Article Title: Modulation of Host Learning in Aedes aegypti Mosquitoes
doi: 10.1016/j.cub.2017.12.015
Figure Lengend Snippet: (A) Left: Dopamine receptor antagonists (DAA) (SCH-23390, bulbocapnine, flupentixol, and fluphenazine) were injected in the thorax of 6-day-old female mosquitoes that were trained 30 min post injection and tested 24 hr later. Center: Dop1 and control dsRNA were injected in 1-day-old pupae, and after 6 days post emergence, mosquitoes were trained and tested. Right: CRISPR/Cas9 constructs were injected in embryos. Mutants were backcrossed, screened and selected by sequencing for five to eight generations before being trained at 6 days old. (B–D) Mosquito choice in the olfactometer represented as a preference index. Trained mosquitoes from the Rockefeller, Liverpool strain, saline-injected, and dsRNA-injected Rockefeller lines were not significantly different in their learning performances (p > 0.05, binomial test; black bars). By contrast, mosquitoes injected with dopamine receptor antagonists (blue-green bars), dsRNA-injected (red bar), and CRISPR mosquitoes (blue bar) showed no learning. Mosquitoes injected with dopamine receptor antagonists (SCH-23398, 10−6 M) or dsRNA, as well as CRISPR mosquitoes, were still responding to positive controls such as CO2 (C) or host odors (D). When human scents were used during training, CRISPR mosquitoes showed no learning (p = 0.79, binomial test). Each bar (mean ± SE) representing 11–29 responsive female mosquitoes; asterisks indicate distributions that are significantly different from random (p < 0.05, binomial test); # indicates p < 0.06 when the response of the trained CRISPR was compared to chance. See also Figures S2 and S4.
Article Snippet: The antibody against the D1-like dopamine receptor, Dop1 was custom made by 21st Century Biochemicals against a synthetic peptide corresponding to amino acids 138–154 of the Ae. aegypti protein, affinity purified, and used at a concentration of 1:100 for immunohistochemistry.
Techniques: Injection, Control, CRISPR, Construct, Sequencing, Saline
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![(A) Schematic of the Ae. aegypti brain superimposed on a scanned electron microscope image [34]. Highlighted regions include the AL (multicolored to represent individual glomeruli that receive input from olfactory receptor neurons) and the mushroom bodies (MB), implicated in learning and memory. The open box around the AL is used to indicate the corresponding location in panels (B)–(D). CX, central complex; OL, optic lobes. Scale bar, 500 μm. (B) Confocal micrograph of a whole Ae. aegypti brain stained with antibodies against tyrosine hydroxylase (magenta) shows immunoreactivity concentrated in the lateral protocerebrum and AL. Background fluorescence in green. Scale bar, 100 μm. (C) Confocal micrograph of a Ae. aegypti brain stained with antibodies against tyrosine hydroxylase (cyan) shows heterogeneous innervation of dopaminergic neurons across antennal lobe glomeruli. Open box bounds the right AL. Background fluorescence in green. Scale bar, 60 μm. (D) A 60 μm section of Ae. aegypti brain stained with antibodies against the mosquito dopamine-1 receptor-1 <t>Dop1</t> (yellow) shows these receptors enriched in the lateral protocerebrum around the MB as well as localized around the AL. Background fluorescence in blue (synapsin). Scale bar, 100 μm. (E) A representative section from the same brain that was preadsorbed with a synthetic peptide corresponding to amino acids 138–154 of Dop1 shows synapsin immunoreactivity (blue), but Dop1 immunoreactivity (yellow) is abolished. Scale bar, 100 mm. See also Figures S4 and S6.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1430/pmc06771430/pmc06771430__nihms-1051413-f0006.jpg)
