ebony affects pigmentation divergence and cuticular hydrocarbons in Drosophila americana and D. novamexicana

Abstract

Drosophila pigmentation has been a fruitful model system for understanding the genetic and developmental mechanisms underlying phenotypic evolution. For example, prior work has shown that divergence of the tan gene contributes to pigmentation differences between two members of the virilis group: Drosophila novamexicana, which has a light yellow body color, and D. americana, which has a dark brown body color. Quantitative trait locus (QTL) mapping and expression analysis has suggested that divergence of the ebony gene might also contribute to pigmentation differences between these two species. Here, we directly test this hypothesis by using CRISPR/Cas9 genome editing to generate ebony null mutants in D. americana and D. novamexicana and then using reciprocal hemizygosity testing to compare the effects of each species' ebony allele on pigmentation. We find that divergence of ebony does indeed contribute to the pigmentation divergence between species, with effects on both the overall body color as well as a difference in pigmentation along the dorsal abdominal midline. Motivated by recent work in D. melanogaster, we also used the ebony null mutants to test for effects of ebony on cuticular hydrocarbon (CHC) profiles. We found that ebony affects CHC abundance in both species, but does not contribute to qualitative differences in the CHC profiles between these two species. Additional transgenic resources for working with D. americana and D. novamexicana, such as white mutants of both species and yellow mutants in D. novamexicana, were generated in the course of this work and are also described. Taken together, this study advances our understanding of loci contributing to phenotypic divergence and illustrates how the latest genome editing tools can be used for functional testing in non-model species.

Keywords: CRISPR; Cas9; abdominal pigmentation; genome editing; melanin; nanos; virilis group.

Figure 1.. D. novamexicana shows divergent body color within the virilis group.

Phylogenetic relationships with estimated divergence times (Caletka and McAllister 2004; Cooley et al 2012) are shown for D. novamexicana, D. americana, D. lummei, and D. virilis. For each species, a dorsal view of the thorax and abdomen is shown for females (left) and males (right), with heads, wings, and legs removed.

Figure 2.. Ebony affects body, wing, and pupal pigmentation in D. novamexicana and D. americana.

(A-D) Adult body pigmentation is shown from a lateral view (top) and dorsal abdominal view (segments A2-A4, bottom) for (A) D. novamexicana, (B) D. novamexicana ebony null mutants, (C) D. americana, and (D) D. americana ebony null mutants. (E-H) Adult wing pigmentation is shown for (E) D. novamexicana, (F) D. novamexicana ebony null mutants, (G) D. americana, and (H) D. americana ebony null mutants. (I-L) Pigmentation of pupal cases is shown for (I) D. novamexicana, (J) D. novamexicana ebony null mutants, (K) D. americana, and (L) D. americana ebony null mutants. Arrows in (J) and (L) highlight the most prominent areas with dark pigmentation in ebony mutants.

Figure 3.. CRISPR/Cas9-induced mutations created null alleles of the D. novamexicana and D. americana ebony genes.

(A) A schematic of the ebony gene is shown with grey boxes indicating exons; coding sequence is indicated in the darker shade of grey. Locations of the five guide RNAs targeting the second exon of ebony are shown with solid lines below scissor symbols. Mutations observed in the two ebony mutants (e^Δ10 and e^Δ7) isolated in D. novamexicana (“N”) and the one ebony mutant (e^Δ46) isolated in D. americana (“A”) are shown. All three alleles included deletions that caused frameshifts. (B) Western blotting showed that the D. americana e^Δ46 and D. novamexicana e^Δ10 mutants (lanes 2 and 4, respectively) lacked a ~100 kDa protein (arrows) recognized by an antibody raised against D. melanogaster Ebony protein (Wittkopp et al. 2002) that is present in wild-type (wt) D. americana and D. novamexicana (lanes 1 and 3, respectively). Relative abundance of total protein loaded into each lane can be seen by the relative intensities of the shorter proteins also detected by the Ebony antibody (Wittkopp et al. 2002) as well as the relative intensities of ~55kDa bands detected by an antibody recognizing alpha Tubulin (Abcam ab52866). The solid black line shows where the membrane was cut prior to incubation with primary antibodies during the western blotting procedure; the top half was incubated with anti-Ebony antibodies whereas the bottom half was incubated with anti-Tubulin antibodies. The two halves were realigned by hand for imaging, using the shape of the cut and the ladder staining as a guide. An un-annotated image of this blot is shown in Supplementary Figure 4.

Figure 4.. Reciprocal hemizygosity testing shows effects of ebony divergence between D. americana and D. novamexicana on body pigmentation.

(A) Schematic shows representative sex chromosomes (XX and XY) and autosomes of the parents and progeny of reciprocal hemizygosity crosses, along with the genotypes of the progeny. Although a single autosome is shown for simplicity, these species have five autosomes. Superscript “A” and “N”, as well as brown and yellow colored bars, indicate alleles and chromosomes from D. americana and D. novamexicana, respectively; e⁻ indicates an ebony null allele. Although the schematic illustrates the crosses only with D. americana as the female parent, the same crosses were performed with sexes of the parental species reversed. (B-I) Dorsal thorax and abdomen phenotypes are shown for female (B-E) and male (F-I) progeny of reciprocal hemizygosity crosses. Genotypes of autosomal and sex chromosomes are shown to the left and above panels B-I, respectively, using the same schematic notation as in panel A. Individuals in B, C, F, and G carry a wild-type copy of D. novamexicana ebony allele, whereas individuals in panels D, E, H, and I carry a wild-type copy of the D. americana ebony. (J-M) Dorsal thorax and abdomen phenotypes are shown for female (J, K) and male (L, M) flies heterozygous for the ebony null allele in D. novamexicana (J, L) and D. americana (K, L) for comparison to flies shown in panels B-I, which also all carry one null and one wild-type ebony allele. Red arrowheads in panels B, C, F, G, J, and L highlight the reduced dark pigmentation in the abdomen along the dorsal midline relative to lateral regions.

Figure 5.. Cuticular hydrocarbons (CHCs) are affected by ebony and differ between D. americana and D. novamexicana.

(A-C) Abundance of individual CHC compounds (ng/fly) and summed CHCs extracted from female flies are plotted for the following genotypes: (A) D. americana and D. novamexicana, each heterozygous for an ebony null (e⁻) allele, (B) D. americana heterozygous and homozygous for an ebony null allele, (C) D. novamexicana heterozygous and homozygous for an ebony null allele. Eight biological replicates are shown for each genotype, with error bars representing 95% confidence intervals. For each comparison, the p-value from a Welch’s t-test with a Benjamini-Hochberg multiple test correction (alpha = 0.05) is shown when a significant difference in abundance was detected for a CHC present in both genotypes being compared. CHCs are shown from left to right with increasing chain length (represented by “C” followed by the chain length) with double-bond and methyl-branched structures indicated by notations after the colon or before the “C”, respectively. For example, C25:1 represents a 25-carbon monoene, C25:2 represents a 25-carbon diene, and 2Me-C28 represents a 28-carbon alkene with a methyl branch at the second carbon. (D-E) Abundance of each CHC in ebony null mutants relative to flies heterozygous for the ebony null allele is plotted by carbon chain length for (D) D. americana and (E) D. novamexicana. Black trendlines in panels D-E show linear regressions, with shaded areas representing the standard error and both Spearman’s rho and p-values indicated on each plot.

Figure 6.. ebony does not contribute to divergence of CHCs between D. americana and D. novamexicana.

(A) Abundance of individual CHC compounds (ng/fly) and summed CHCs extracted from female flies are plotted for D. americana and D. novamexicana ebony heterozygotes as well as F₁ hybrids heterozygous for wild-type alleles of ebony. (B-C) CHCs from F₁ hybrids homozygous for ebony null alleles are compared to CHCs from F₁ hybrids with wild-type D. americana and D. novamexicana ebony alleles, showing the absolute abundance of individual and summed CHC compounds (B) as well as the relative abundance of CHCs by carbon chain length (C). In panel B, p-values are shown from a Welch’s t-test with a Benjamini-Hochberg multiple test correction (alpha = 0.05) when a significant difference in abundance was detected for a CHC present in both genotypes. (D-E) CHC profiles are plotted for reciprocal F₁ hybrids that differ only by which wild-type ebony allele they carry, either D. americana (e^A) or D. novamexicana (e^N), with absolute abundance of individual and summed CHCs shown in (D) and relative abundance of CHCs by chain length shown in (E). No p-values are shown in (D) because no CHCs showed a statistically significant difference in abundance between the two F₁ hybrid genotypes from the reciprocal hemizygosity test (Welch’s t-test with Benjamini-Hochberg multiple test correction, p>0.05 for each CHC). In panels C and E, blue trendlines show linear regressions, with shaded areas representing the standard error and both Spearman’s rho and p-values indicated on each plot. In all panels, data from eight replicate flies is shown for each genotype.