Differences inorthodenticleexpression promote ommatidial size variation betweenDrosophilaspecies

Abstract

The compound eyes of insects exhibit extensive variation in ommatidia number and size, which affects how they see and underlies adaptations in their vision to different environments and lifestyles. However, very little is known about the genetic and developmental bases underlying differences in compound eye size. We previously showed that the larger eyes of Drosophila mauritiana compared to D. simulans is caused by differences in ommatidia size rather than number. Furthermore, we identified an X-linked chromosomal region in D. mauritiana that results in larger eyes when introgressed into D. simulans. Here, we used a combination of fine-scale mapping and gene expression analysis to further investigate positional candidate genes on the X chromosome. We found that orthodenticle is expressed earlier in D. mauritiana than in D. simulans during ommatidial maturation in third instar larvae, and we further show that this gene is required for the correct organisation and size of ommatidia in D. melanogaster. Using ATAC-seq, we have identified several candidate eye enhancers of otd as well as potential direct targets of this transcription factor that are differentially expressed between D. mauritiana and D. simulans. Taken together, our results suggest that differential timing of otd expression contributes to natural variation in ommatidia size between D. mauritiana and D. simulans, which provides new insights into the mechanisms underlying the regulation and evolution of compound eye size in insects.

Measuring Compound Eye Optics with Microscope and MicroCT Images

“…Automated ommatidia counts were 100.4% of the manual count on the same D. mauritiana scan, 85% of the density-based count of the same A. mellifera scan (Taylor et al 2018; Tichit et al 2022), 112% of measurements of a different D. elpenor specimen (Stöckl, O’Carroll, and Warrant 2017), and 98% and 104% of measurements of different M. sexta specimens (Stöckl et al 2017; White 2003). These ommatidia had diameters (Figure 6 B) that were consistent with measurements in the literature: 95 – 115% for D. mauritiana (Posnien et al 2012; Torres-Oliva et al 2021), 96% for A. mellifera despite missing ~15% of ommatidia (Taylor et al 2018), 99 – 103% for D. elpenor (Stöckl et al 2017; Theobald, Warrant, and O’Carroll 2010), and 96 – 108 % for M. sexta (Stöckl et al 2017; Theobald et al 2010; White 2003). We also profiled the runtime of the ODA-3D on the 4 specimens in comparison to estimates of how long it would take to extract the clusters manually.…”

“…Once in 100% ethanol, they were stained with 1% Iodine in ethanol before scanning at the TOMCAT beamline of the Swiss Light Source (Paul Scherrer Institute, Switzerland). For more information on the scanning procedure, see Torres-Oliva et al (2021). Vouchered moth specimens from the Florida Natural History Museum were stored at −20°C in 95% ethanol, then heads were sliced, with antennae removed, and soaked in staining solution (I2+KI, equal proportions 1.25% I 2 and 2.5% KI solutions) in Eppendorf vials or falcon tubes for 36–48 hours.…”

Measuring compound eye optics with microscope and microCT images

“…These ommatidia had diameters (Fig. 3b) that were consistent with measurements in the literature: 95-115% for D. mauritiana 42,43 , 96% for A. mellifera despite missing ~15% of ommatidia 37 , 99 -103% for D. elpenor 40,44 , and 96-108 % for M. sexta 40,41,44 . We also profiled the runtime of the ODA-3D on the 4 specimens in comparison to estimates of how long it would take to extract the clusters manually.…”

“…Fly heads were placed into 10 μl pipette tips in 100% Ethanol and scanned using a 16 keV monochromatic beam with a 20 µm LuAG:Ce scintillator. For more information on the scanning procedure, see Torres-Oliva et al (2021) 43 . Vouchered moth specimens from the Florida Natural History Museum were stored at -20°C in 95% ethanol, then heads were sliced, with antennae removed, and soaked in staining solution (I 2 + KI, equal proportions 1.25% I 2 and 2.5% KI solutions) in Eppendorf vials or falcon tubes for 36-48 h. M. sexta was scanned with a Phoenix V | Tome|X M system with: a 180kv x-ray tube, a diamond-tungsten target, 80 kV tube voltage, 110 µA current, 17.8 mm source object distance, 793 mm object-detector distance, and capture time adjusted to maximize absorption range for each scan.…”

“…Grayscale heatmaps present 2D histograms and medians (red point), IQRs (half-opacity red line), and 99% C.I.s (full opacity error bars) are plotted for each orientation ±15°. 43 22.61 ± 1.96 37 * 28 40 , 29 44 30 41 , 31 40 based on vectors orthogonal to the eye surface 37 . This discrepancy was likely due to ODA-3D errors during the segmentation of highly skewed ommatidia in the periphery (for example, see the horizontal slice insets in Fig.…”