Optogenetic Activation of the fruitless-Labeled Circuitry in Drosophila subobscura Males Induces Mating Motor Acts

Abstract

It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. We disrupted the D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, resulting in complete loss of mating behavior. We also generated fru^soChrimV , which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by fru^soChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.SIGNIFICANCE STATEMENT How did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior of Drosophila melanogaster, a genetic model, and its relative, D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that the subobscura fruitless circuit, which is required for male courtship behavior, was slightly but clearly different from its melanogaster counterpart, and that optogenetic activation of this circuit induced subobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within the fruitless circuit determine the species-specific differences in courtship behavior.

Keywords: CRISPR/Cas9; Drosophila; courtship; fruitless.

Figure 1.

Generation of fru mutants in D. subobscura. A, The exon-intron organization of the fru gene in D. melanogaster, with the initiation and stop codons in the second exon as well as the splicing donor and acceptor sites for the conjunction of exon 2 and exon 3, highlighting the sex difference in fru splicing, which underlies the male-specific production of the full-length FruM protein. B, A schematic representation of the targeted site for gRNA (indicated by an inverted triangle) used for mutagenesis. C, The nucleotide sequence around the targeted region (shaded) that includes the splice donor site (boxed) in the wild-type genome (WT) and the induced deletion in the mutants fru^so1, fru^so2, and fru^so3. The planned position for cutting is indicated with an arrow. D, A schematic representation of CRISPR-mediated knock-in targeting of the fru locus. The black pointed bars at the bottom indicate the position of the primers used for screening. HA_L and HA_R represent left and right homology arms, respectively. attP indicates the landing site for phiC31-integrase-mediated gene insertions. p(A) indicates a poly(A) signal sequence for the fluorescent marker gene 3xP3DsRed. E, Bright-field (left) and fluorescent (right) images of a third-instar larva expressing the DsRed protein (Knock-in) and a non-injected larva (Control). Arrowheads indicate anal pads and asterisks indicate Bolwig organs. F, Screening for CRISPR-mediated knock-in events by PCR. The left-most lane representing a sample derived from a DsRed+ fly but none of the other lanes from DsRed- flies had a band (highlighted with an asterisk) of the predicted size (905 bp) indicative of successful knock-in. G, The genomic sequence around the 5′ junction of HA_L and the transgene of the DsRed+ fly. H, Anti-FruMale antibody immunoreactivity (green) of the male (top) and female (bottom) brains of D. melanogaster wild-type flies, D. subobscura wild-type and variants, i.e., fru^so3 heterozygotes, fru^so3 homozygotes, fru^soDR heterozygotes, fru^soDR homozygotes, and fru^soDR homozygotes (from left to right), counterstained with nc82 (magenta). Scale bars, 50 μm.

Figure 2.

D. subobscura fru mutant phenotypes. A, The steps of mating behavior in D. subobscura include tapping (A1), scissoring (A2), midleg swing (A3), proboscis extension (A4), nuptial gift (A5), wing extension (A6), and attempted copulation (A7). All these steps except for the nuptial gift were included in estimating the courtship index. Shown are representative behavioral acts recorded with a wild-type D. subobscura male. The courtship indices (B; top: p = 0.0003, bottom: p < 0.0001), number of tappings (C; top: p = 0.0084, bottom: p = 0.0063), number of scissorings (D; top: p = 0.1228, bottom: p = 0.0511), number of midleg swings (E; top; p = 0.0014, bottom: p < 0.0001), proboscis extension indices (F; top; p = 0.0002, bottom: p < 0.0001), and number of attempted copulations (G; top; p = 0.2, bottom: p = 0.0137) are shown for fru^so3 heterozygous males (left, bars in the top: n = 7), fru^so3 homozygous males (right, bars in the top: n = 8), fru^soDR heterozygous males (left, bars in the bottom: n = 12), and fru^soDR homozygous males (righ, bars in the bottom: n = 11). The statistical significance of differences was evaluated by the Mann–Whitney's U test. ns, Not significant; *p < 0.05, **p < 0.01, ***p < 0.001. H, Comparisons of fertility between heterozygotes (left bars) and homozygotes (right bars) of fru^so3(left graph; p < 0.0001) and fru^soDR(right graph; p < 0.0001) males. The statistical significance of differences was evaluated by the Fisher exact test. ***p < 0.001. I, Dorsal abdominal musculature in a fru^so3 heterozygous male (left) and a fru^so3 homozygous male (right). J, Dorsal abdominal musculature in a fru^soDR heterozygous male (left) and a fru^sDR3 homozygous male (right). Inset, Enlarged views of a MOL (dotted white line) for each genotype. Scale bars, 500 μm.

Figure 3.

Generation of a fru allele that expresses csChrimson-mVenus under the native fru-P1 promoter in D. subobscura. A, Top, A schematic representation of the minigene knock-in site (indicated by an inverted triangle). Middle, The structure of the donor vector that contains homologous arms for recombination (HA_L and HA_R), UAS, the coding region of csChrimson-mVenus, attP and 3xP3-DsRed. Bottom, An expected genomic organization after the successful integration of the construct. B, Larvae with and without the minigene integration as viewed under a light field (left) and a dark field (right). 3xP3-DsRed expression in the anal pad indicates the minigene integration (arrowheads). C, Sequence around the 5′ junction of HA_L and the transgene in fru^soChrimV. D–F, Anterior view of the fru-labeled circuitry in the brain of fru^soChrimV males (D, E) and females (F) stained for mVenus (green) by the anti-GFP antibody alone (D) or together with the anti-FruMale antibody (red; E, F). Scale bars: D–F, 50 μm. The somata of mAL and mcAL neurons are encircled with a white broken line (E). The ring region is boxed with a white dotted line in E and F. G–I, Magnified images of the region boxed with a solid white line in E, showing the immunoreactivity to the anti-FruM antibody (G) and anti-GFP antibody (H), and merged images (I). Scale bars, 20 μm. J, Anterior view of the fru-labeled circuitry. The arch, lateral junction, ring, and lateral crescent are indicated. Scale bar, 50 μm. K, Posterior view of the fru-labeled circuitry in male. P1 cell bodies are encircled with a white dotted line and the primary neurites are indicated by an arrowhead, respectively. Scale bar, 50 μm. L, Enlarged image of P1 cell bodies (encircled with a white dotted line) and neurites (indicated by an arrowhead). Scale bar, 30 μm.

Figure 4.

Sexual dimorphisms in the fru-labeled circuitry in D. subobscura. A, B, Magnified images of the ring region boxed by a dotted white line in Figure 3E and F, respectively. The width of the medial portion of the inner fiber tract in the ring (indicated by horizontal bars) was measured along the lateral axis at the midpoint, which was defined as the midpoint (white dots) of the inner ring diameter along the dorsal-ventral axis (indicated by vertical broken lines). The width of the ring measured was normalized by the largest width of the dorsal central brain. Scale bars, 15 μm. C, A comparison of the normalized width of the ring fiber tract between the male (n = 5) and female (n = 3) fru^soChrimV heterozygotes. The statistical significance of differences was evaluated by the Mann–Whitney U test: p = 0.0357. D–I, Sexual dimorphisms of the mAL neurites in the fru^soChrimV heterozygous male and female. Scale bars, 30 μm. D, G, Magnified images of mAL cell bodies and neurites in the male (D) and female (G) brain. Cell bodies are encircled by a broken white line, and neurites are indicated by arrowheads. E, H, mAL neurites observed at a different plane, highlighting their tip portions (indicated by arrows) in the subesophageal ganglion (SOG) of the male (E) and female (H). The thick bilateral fiber tracts along the midline and large terminals in the SOG are of mcAL neurons. F, I, Schematic representation of the neurite projection pattern in the male (F) and female (I). Neurites are indicated by arrowheads. The dark oblique structure at the center represents the esophagus. Scale bars, 30 μm.

Figure 5.

Differences of the fru-labeled circuitry in the optic lobe between D. subobscura and D. melanogaster. A–D, The fru-labeled fibers of M neruons in the optic lobe (encircled with white broken lines) in D. melanogaster (A, C) and D. subobscura (B, D) in male (A, B) and (C, D). Scale bars, 50 μm. E, The number of anti-FruM antibody-immunoreactive cells in the mAL: p = 0.9213; mcAL: p = 0.6702; Lo: p = 0.0006; and M: p = 0.0006 clusters compared between D. melanogaster (n = 7) and D. subobscura (n = 7). The statistical significance of differences was evaluated by the Mann–Whitney's U test. ns, Not significant. ***p < 0.001. Error bars show SEM. F, G, Images of the optic lobe stained with the anti-FruM antibody in D. melanogaster (F) and D. subobscura (G) males. The region with Lo and M neuron somata is encircled with a white broken line. Scale bars, 50 μm. H, I, Scanning electron micrographs of the compound eye in D. melanogaster (H) and D. subobscura (I) males. J, The number of ommatidia composing a compound eye in D. melanogaster (n = 8) and D. subobscura (n = 8) males. The statistical significance of differences was evaluated by the Mann–Whitney's U test. p = 0.0006. Error bars show SEM.

Figure 6.

The GABAergic nature of mAL-cluster neurons in D. melanogaster and D. subobscura. A, E, Anti-GABA antibody immunoreactivity in male brains of D. subobscura (A) and D. melanogaster (E) compared with anti-FruMale-antibody immunoreactivity. B–D, F–H, Magnified images of the mAL cluster, showing the merged images (B, F) highlighting immunoreactivity to the anti-GABA antibody (C, G) and the anti-FruMale antibody (D, H).

Figure 7.

Mating motor acts induced by CsChrimson-mediated activation of the fru-labeled circuitry in D. subobscura. A, Abdominal bending (blue arrowhead) and regurgitating a droplet (orange arrowhead) as induced by CsChromson activation via light illumination in an unrestrained male placed alone in a circular chamber. B, C, The proportion of flies showing abdominal bending (B) and regurgitating a droplet (C) under unrestrained conditions in the fly groups fed on diets with or without retinal. D–K, Activation of the fru-labeled circuitry in a tethered male on the treadmill. A schematic drawing of the experimental setup (D) and a stimulation protocol for CsChrimson activation (E). Snapshots of a male fly displaying abdominal bending (F) and wing extension (I) on the treadmill. Cumulative durations of abdominal bending (G) and wing extension (J) in tethered male flies during a 10 s observation period as a function of the intensity of activation light in the fly groups fed on diets with or without retinal. Raster plots showing responses to the light stimulation in 14 test males (top) and changes in the proportion of flies exhibiting abdominal bending (H) and wing extension (K) over time, with reference to the period of red light illumination for CsChrimson activation (bottom). In the raster plot, each row represents an action record, in which a black bar appears when the male fly exhibits a behavioral act (i.e., abdominal bending and wing extension).