Fig. 7

Strong association between daily levels of dmpi8 splicing and mid-day sleep at cold temperatures. a-d Shown are group averages for the splicing efficiency of dmpi8 (expressed as the ratio of spliced to unspliced levels) throughout an LD cycle for low and high altitude flies from Cameroon and Kenya pooled according to the SNP3 variant. The same fly lines as those used for the sleep analysis shown in Fig. 6b and c (see legend to Fig. 6) were also used to measure dmpi8 splicing, allowing for comparison between the behavioral and molecular results. Briefly, for each isofemale line approximately 40 flies were placed into each of 12 vials. Half of the vials were exposed to 5 days of LD at 18 °C, whereas the other half was exposed to 5 days of LD at 25 °C. On the last day of LD, flies were collected by removing a vial every 4 h, total RNA prepared and dmpi8 splicing efficiency measured for each line separately, followed by pooling results from different lines to yield group averages. For low altitude flies, the daily dmpi8 splicing curves at 18° and 25 °C were significantly different between the SNP3/A and SNP3/G variants (18 °C; one-way ANOVA, p < 0.01; 25 °C, one-way ANOVA, p < 0.05), whereas no significant differences were observed for the dmpi8 splicing curves from high altitude flies. e, g, i Using the same splicing data shown above for flies at 18 °C (a, c), the data from individual fly lines was sorted to compare the dmpi8 splicing curves from the low and high altitude groups with the same SNP3 variant. The error bars were removed to facilitate comparison. Comparison of the daily dmpi8 splicing curves for low and high altitude flies with SNP3/A (e, g) were significantly different (one-way ANOVA, p < 0.01) but no significant difference was observed for the comparison using SNP3/G flies from Cameroon (j). Directly below each splicing panel (e, g, i) is shown the corresponding daily sleep profile (f, h, j) for the same group of flies used to generate the splicing results. Similar results were obtained in several smaller scale experiments (data not shown)