Journal: bioRxiv

Article Title: Male-male interactions shape mate selection in Drosophila

doi: 10.1101/2023.11.03.565582

Figure Lengend Snippet: ( A ) Schematic of the tethered behavioral setup, where a fly-sized visual target is projected onto a panorama in front of a headfixed male and oscillates back and forth, while neural activity is recorded via a two-photon microscope. A speaker is positioned near the male for the delivery of synthetic D. melanogaster pulse trains. ( B ) Representative behavior of a tethered male prior to, during, and after the playback of conspecific courtship song. Black hatch marks denote left and right wing extensions; light blue hatch marks indicate bouts of unilateral wing-extensions (singing); green hatch marks indicate bouts of bilateral wing flicks. The male’s tracking index (T.I., see methods) is plotted in blue, and his linear velocity in black as he transitions from courting the fictive female by singing to charging towards it while flicking upon song-playback. Note that wing extensions are interleaved with wing flicks during the playback. ( C ) Pseudo-colored example images of a tethered male viewed from behind, during tracking while not performing any wing behaviors (top), performing a unilateral wing-extension (middle), or performing a bilateral wingflicks (green). ( D ) Average male linear speed (top), probability of males performing unilateral wing extensions (middle), and probability of males performing bilateral wing flicks (bottom) aligned to the playback of pulse trains. Light blue box denotes period of sound playback, darker hatch marks denote individual pulse trains. ( E ) Morphology of neurons labeled by pC1-SS2 in a male’s brain with (top) and without (bottom) background neuropil staining. ( F ) Average probability of solitary males performing unilateral wing extensions (song, blue) or bilateral wing flicks (flick, green) during optogenetic activation of P1a versus pC1x neurons. ( G and H ) Average probability of males performing unilateral wing extensions (top), bilateral wing flicks (middle), or lunge attacks (bottom) towards a wingless mute male prior to, during, and after two 1 min epochs of optogenetic activation of P1a neurons (G, shaded regions) or pC1x neurons (H, shaded regions). ( I and J ) Average P1a activity (∆F/F 0 , top) and tracking index of tethered males that that courted (wing extensions, tracking) the visual target, aligned to either the onset of visual motion (I; black line on top denotes when the visual target was oscillating) or to the onset of pulse song playback (J; blue line shows period of song playback). ( K and L ) Same as I and J, but showing the average activity of pC1x neurons. ( M ) Probability of control males versus pC1x-silenced males performing bilateral wing flicks in MMF triads. ( N ) Probability of control males versus pC1x-silenced males performing unilateral wing extensions when paired with a single female. ( O ) Same as (G and H), but for males paired with conspecific virgin females during optogenetic activation of pC1x neurons. ( P and Q ) Probability of males performing bilateral wing flicks (P) and the frequency of lunge attacks (Q) during optogenetic activation of pC1x neurons in males paired with a D. melanogaster female (red), a D. simulans female (light gray), or a D. simulans female perfumed with the male pheromone cVA (dark gray). Shaded lines show mean ± s.e.m.; Lines with dots represent mean and individual animals. n.s., P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Details of statistical analyses and sample sizes are given in TableS2 . See Figures S8, S9 , and S10 and Videos S7, S11, S12, S13 , and S14 .

Article Snippet: Functional imaging experiments were performed with an Ultima Investigator or Ultima Investigator Plus two-photon laser scanning microscope (Bruker Nanosystems) with a Chameleon Ultra II Ti:Sapphire laser.

Techniques: Activity Assay, Microscopy, Labeling, Staining, Activation Assay