Role of the Drosophila Melanogaster Indirect Flight Muscles in Flight and Male Courtship Song: Studies of Flightin and Myosin Light Chain-2

Complex behaviors using wings have facilitated the insect evolutionary success and diversification. The Drosophila indirect flight muscles (IFM) have evolved a highly ordered myofilament lattice structure and uses oscillatory contractions by pronounced stretch activation mechanism to drive the wings for high powered flight subject to natural selection. Moreover, the IFM is also utilized during small amplitude wing vibrations for species-specific male courtship song (sine and pulse), an important Drosophila mating behavior subject to sexual selection. Unlike flight, the contractile mechanism and contribution of any muscle gene in courtship song is not known. To gain insight into how separate selection regimes are manifested at the molecular level, we investigated the effect on flight and mating behaviors of mutations in two contractile proteins essential for IFM functions: an IFM-specific protein, flightin (FLN), known to be essential for structural and mechanical integrity of the IFM, and a ubiquitous muscle protein, myosin regulatory light chain (MLC2), known to enhance IFM stretch activation. Comparison of FLN sequences across Drosophila spp. , reveal a dual nature with the N-terminal region (63 aa) evolving faster (dN/dS=0.4) than the rest of the protein (dN/dS=0.08). A deletion of the N-terminal region (fln ΔN62 ) resulted in reduced IFM fiber stiffness, oscillatory work and power output leading to a decreased flight ability (flight score: 2.8±0.1 vs 4.2±0.4 for fln + rescued control) despite a normal wing beat frequency. Interestingly, the FLN N-terminal deletion reduced myofilament lattice spacing and order suggesting that this region is required to improve IFM lattice for enhancing power output and flight performance. Moreover, fln ΔN62 males sing the pulse song abnormally with a longer interpulse interval (IPI, 56±2.5 vs 37±0.7 ms for fln + ) and a reduced pulse duty cycle (PDC, 2.6±0.2 vs 7.3±0.2 % for fln + ) resulting in a 92% reduction in their courtship success. This suggested that FLN N-terminal region fine-tunes sexually selected song parameters in D. melanogaster, possibly explaining its hypervariability under positive selection. That FLN N-terminal region is not essential but required to optimize IFM functions of both flight and song, indicate that FLN could be an evolutionary innovation for IFM-driven behaviors, possibly through its role in lattice improvement. Mutations of the highly conserved MLC2 [N-terminal 46 aa deletion (Ext), disruption of myosin light chain kinase phosphorylations (Phos), and the two mutations put together (Dual)] are known to impair or abolish flight through severe reductions in acto-myosin contractile kinetics and magnitude of the stretch activation response. Unlike FLN, these MLC2 mutations do not show a pleitropic effect on flight and song. Flight abolished Phos and Dual mutants are capable of singing suggesting that these mutations affect song minimally compared to flight. Moreover, unlike FLN, none of these mutations affect interpulse interval, the most critical sexually selected song parameter in Drosophila. Also, in contrary to the known additive effects of Ext and Phos in the Dual mutant on flight wing beat frequency, a subtractive effect on sine song frequency is found in this study. That mutations in MLC2 are manifested differently for song and flight suggest that stretch activation plays a minimal or no role in song production. The results in this study suggest that the conserved regions of FLN and MLC2 are essential to support underlying IFM contractile structure and function necessary for flight, whereas the fast evolving FLN N-terminal region optimizes IFM’s biological performance in flight and species-specific song possibly under positive selection regime.