Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila

Abstract

The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain (fln^ΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that fln^ΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.

Keywords: Drosophila; courtship song; fibre mechanics; flight muscle; flightin; myofilament lattice.

Figure 1.

fln^ΔN62 males sing an abnormal courtship song and are outcompeted for female choice. Courtship sine song (a and b) and pulse song (c through h) of fln⁺ and fln^ΔN62 males (scale bar (thick lines below the oscillograms) = 50 ms). Compared to the fln⁺ control males, fln^ΔN62 males produce a higher-frequency sine song, SSF (b), a pulse song with similar frequency, IPF (d) but with more cycles per pulse, CPP (e), longer pulse length, PL (f), longer inter-pulse interval, IPI (g), and reduced pulse duty cycle (PDC: the ratio of pulse song duration to the total time of recording including song plus silence [6]) (h). The mean value of each song parameter is the mean of average values for each fly song for N number of fly song recordings. N= 7–8 thirty-minute fly song recordings. *Significant difference (p < 0.05) from fln⁺ control. Courtship behaviour during single pair mating (i and j) and competition mating between fln⁺ and fln^ΔN62 males (k and l). fln⁺ and fln^ΔN62 males have a similar courtship index (CI) and wing extension index (WEI) when paired singly with a wild-type (Oregon R strain) female. When competing with fln⁺ males, fln^ΔN62 males have a significantly reduced courtship index and wing extension index. Courtship index is the total time duration of courtship behaviour by a male divided by the total time of video recording, or until courtship success; wing extension index is the total time duration of wing extension to produce the courtship song by a male divided by the total time of the video recording, or until courtship success [6]. Both indices are expressed as percentages. N = 25 and 10 for mating competition assays and single pair mating assays, respectively. *Significant difference (p < 0.05) from fln⁺ control.

Figure 2.

Distribution of inter-pulse interval (IPI) of fln⁺ (white bars) and fln^ΔN62 (red bars) male pulse songs. Each bar represents the frequency at which IPIs occur among different fly songs. N= 7–8 thirty-minute fly song recordings.

Figure 3.

Reduced myofilament lattice organization in fln^ΔN62 IFM fibres. Transmission electron microscopy images of cross-sections of IFM from fln⁺ (a and b) and fln^ΔN62 (d and e) transgenic fly lines. Note that myofibrils show the characteristic cylindrical shape of normal IFM, and have similar diameters. Regions within white boxes in (a and d) are magnified in (b and e), respectively. Panel (e) shows a more compact and less ordered hexagonal lattice than (b). This is reflected in the Fourier power spectra (c and f) from the images (b and e), respectively. Scale bars = 1 µm (for a and d) and 0.1 µm (for b and e).

Figure 4.

fln^ΔN62 IFM fibres have reduced elastic and viscous moduli and reduced oscillatory work and power output. Elastic and viscous moduli of skinned IFM fibres from fln⁺ (open black circles) and fln^ΔN62 (filled red squares) in relaxing (a and c) and rigour (b and d) solutions. Horizontal lines below δ symbols denote the frequency range through which measured values are significantly different between fln⁺ and fln^ΔN62 (p < 0.05). Elastic modulus (e), viscous modulus (f), work output (g) and power output (h) for active IFM fibres from fln⁺ (open circles) and fln^ΔN62 (filled squares) strains are shown. Lines below delta (δ) denote frequency ranges where measured values are significantly different between fln⁺ and fln^ΔN62 (p < 0.05). Vertical dashed lines in (g and h) represent the frequency of maximum oscillatory work and power, occurring at 171 ± 8 Hz and 205 ± 7 Hz for fln^ΔN62 compared to 179 ± 8 Hz and 217 ± 7 Hz for fln⁺. The frequencies of maximum oscillatory work and power are not significantly different between control and mutant strains. Note the broader range of frequencies of maximum oscillatory work and power in the mutant fibres compared with that of control (boxes in (g and h), respectively).