Fig. 1

Morphology, cellular composition, and single-cell RNA-seq atlas of Aedes larval VNC. A Structure of larval VNC and segmental nerves visualized in a fillet preparation of a brp-T2A-QF2w / +; QUAS-mcd8GFP / + larva. B Cellular composition of thoracic and abdominal ganglia. Images show maximum projections of representative confocal stacks taken from segments T1, A1, A4 and A7 of larvae stained with Hoechst to label all nuclei together with antibodies to Elav to label neuronal nuclei and GFP to label neuronal membranes. Images depict Elav+ cells (top) and a montage of all three markers (bottom). White dashed lines outline the cortical cell layers on each side of the neuropile. Here and in subsequent images, anterior is up. C-E Quantification of ganglion cell numbers. Plots depict the number of (C) Elav-positive cells, (D) Hoechst-positive cells, and (E) the proportion of cells which were Elav-positive in four representative segments (T1, A1, A4, A7) at two different larval time points (3 days after hatching and 5 days after hatching). Bars depict mean values, points represent measurements from individual ganglia, sample numbers are indicated for each segment/time point combination, and error bars depict standard deviation. *P < 0.05, two-way ANOVA followed by Fisher’s LSD test. The two-way ANOVA revealed a statistically significant interaction between the effects of developmental time and segment position on neuron number (F(3, 69) = 9.846, p < 0.0001) and the proportion of neurons (F(3, 68) = 3.756, p = 0.0148). Simple main effects analysis showed that developmental time had statistically significant effects on cell number (p < 0.0001), neuron number (p < 0.0001), and neuron proportion (p = 0.0012), and that segment position had statistically significant effects on cell number (p = 0.0112) and neuron number (p = 0.0003). F Schematic workflow for generating single cell transcriptional profiles. Mosquito larval VNCs were isolated and dissociated prior to sorting GFP-positive neurons by FACS. Following library preparation, sequencing, and quality-based filtering, clustering analysis was used to identify gene expression relationships between profiled cells. G Aedes larval VNC cell atlas. The UMAP plot depicts 6564 neurons grouped into 35 clusters. H-I Feature plots depicting neuronal marker gene expression across the Aedes VNC cell atlas. Genotype for all panels: brp-T2A-QF2w / +; QUAS-mcd8GFP / +