Magazine R309 Environmental factors play a key role in the expression of phenotypic traits and life-history decisions, specifically when they act during early development. In birds, brood size is such an important environmental factor affecting development. Experimental manipulation of brood sizes can result in reduced offspring condition, indicating that conditions during development in enlarged broods have consequences within the affected generation. But it is unclear whether stress during early development can have fitness consequences extending into the offspring of the next generation. To study such trans-generational fitness effects, a team of researchers from the University of Bielefeld, Germany, and the Museum of Natural Sciences in Madrid, report a breeding experiment with zebra finches (Taeniopygia guttata) in which mothers had been raised in different experimental brood sizes (Proceedings of the Royal Society, series B, published online). The researchers, led by Marc Naguib, found that adult females were smaller as experimental brood sizes in which their mother had been raised increased. Hatching and fledging success of daughters decreased with increasing maternal brood size. These results illustrate that early developmental stress can have long-lasting effects on reproductive success of future generations. "Such trans-generational effects can be life-history responses adapted to environmental conditions experienced in early life," the authors report. Line out: New research suggests that stress faced by female zebra finches may influence the breeding success of their daughters. (Photo: Adam Jones/Science Photo Library.) Stress lines Insect flight Michael Dickinson From Leonardo da Vinci to the Wright brothers, flight has inspired engineers more than any other form of animal behavior. Like any aircraft, an animal capable of active flight must possess three critical features: a light but powerful engine; wings capable of generating sufficient aerodynamic forces; and a control system to keep it from tumbling to the ground. The special properties of the muscles, wings, and brains that satisfy these requirements have made flying animals useful models in muscle biophysics, fluid mechanics, and neurobiology. The purpose of this primer is to provide an overview of key principles in these three salient areas of flight biology, and is motivated by recent technical advances that are beginning to unravel many long-standing problems. This progress in our understanding of flight biology illustrates the utility of integrative methods, because many key insights have emerged, not simply from a focused analysis of individual elements, but also through more comprehensive approaches that link problems across disciplines. Although flight research embraces a wide variety of different organisms, from dragonflies to flying dragons, I will focus on insects in general, and flies in particular, because they have proven particularly amenable to these interdisciplinary approaches. The engine Miniaturization is the dominant theme in insect evolution, especially within the species-rich orders that include beetles, wasps and flies. This diversification was possible only because tiny insects evolved a remarkable muscle that is capable of generating high power at high frequency. Understanding the necessity of this peculiar motor starts with a consideration of scaling and aerodynamics. Whereas the lift generated by a Primer Current Biology Vol 16 No 9 R310 flapping wing scales to the fourth power of body length, an animal's weight scales to the third power. For this reason, small insects must flap their tiny wings faster to create sufficient force to offset gravity. This need for enhanced flapping frequency is even greater than predicted by scaling laws because air viscosity causes a gradual drop in the aerodynamic performance of small wings. Accordingly, the wingbeat frequency of hovering animals ranges from roughly 30 Hz in large hawk moths and hummingbirds to over 1000 Hz in tiny midges. However, the power output of conventional skeletal muscle deteriorates at frequencies well below those used by small, and even moderate-sized, insects. How, then, do these creatures manage to get off the ground? Conventional skeletal muscle has little difficulty turning on; it is turning off that presents a problem. Contraction is regulated by Ca2+, which when released from the sarcoplasmic reticulum (SR) in response to a motorneuron spike, binds to a troponin subunit on the thin filament, which in turn moves an associated protein, tropomyosin, to uncover the binding site where myosin can bind to actin. This entire process is fast because the electrochemical force driving Ca2+ into the cytoplasm is enormous. In contrast, deactivation is a slow process because it requires the active pumping of Ca2+ into the SR against its electrochemical gradient. Muscles powering an oscillating appendage, however, must deactivate quickly so that they are ready for the next cycle and do not resist the action of their antagonists during the reciprocal stroke. Very fast oscillatory muscles, such as those on rattlesnake rattles, have enormous amounts of SR, but the hypertrophy of SR comes at the expense of contractile filaments and mitochondria, creating a fundamental trade-off between deactivation speed and power. Many flying animals, including all bats and birds and some insects, use conventional twitch muscle to fly, but the cellular biophysics of contraction limits their flapping frequency and therefore their size. At least four times within the evolutionary history of insects, a new type of `asynchronous' flight muscle emerged that can generate power at high frequency, thus permitting adaptive radiation into new niches and habitats. In principle, the solution is simple; get rid of the SR and fill the entire muscle volume with the stuff that counts -- contractile filaments and mitochondria. In asynchronous flight muscle, actin­myosin binding is regulated mechanically rather than chemically. The term asynchronous comes from the fact that individual contractions are not correlated with pre-synaptic motor neurons spikes as they are in typical skeletal muscle. Rapid stretch, not depolarization-mediated Ca2+ release, activates crossbridges to generate force, and rapid shortening de-activates crossbridges to relax the muscle. Because deactivation speed is not limited by diffusion, asynchronous muscle needs little SR. The motor neurons of asynchronous muscles fire continuously, but at a rate that is much lower than the contraction frequency. This low level of excitation is thought to maintain a tonic level of calcium that is sufficient to keep the crossbridges in a stretch-activate-able state. Recent evidence suggests, however, that by varying the spike rate of the asynchronous muscle, motorneurons can raise and lower the tonic calcium level to regulate power output as required for different flight maneuvers. In addition to its peculiar physiology, asynchronous muscle has an odd anatomical arrangement. The entire exoskeleton of an insect is topologically an uninterrupted hollow sphere. Joints, including those attaching the wings to the body, consist of flexible rubbery sections surrounded by stiffer regions. Most insect muscle inserts directly onto invaginations of the exoskeleton called apodemes, which serve as tendons. In contrast, stretch-activated power muscles are classified as indirect because they insert broadly onto the walls of the thorax, not onto apodemes at the base of the wings. At the base of the wing a complicated hinge serves as a motion-amplifying gearbox to transform the tiny strains imparted by the flight muscles into the sweeping motion of the wings. The back and forth motion of the wings is created by an orthogonal arrangement of two antagonist groups, dorso-longitudinal muscles (DLMs) and dorso-ventral muscles (DVMs) (Figure 1A). Contraction of the DLMs drives the wing forward and stretches the DVMs; this in turn activates the DVMs to drive the wings backward and stretches the DLMs to continue the self-sustaining cycle. What is the molecular basis of stretch-activation? This question has general implications in muscle physiology because vertebrate heart muscle exhibits stretch activation, as does all skeletal muscle to a small degree. Stretch activation does not require the cell membrane; it is a feature intrinsic to the protein structure of the sarcomere. Somehow the extension of two adjacent Z-disks increases the net probability that myosin heads undergo a force-generating step. Many current hypotheses emphasize the role of a direct physical link between thin and thick filaments that would serve as a stretch `sensor' to influence the probability of myosin binding. Suspects include myosin regulatory light chain, which spans the distance from thick and thin filaments adjacent to myosin heads, and projectin and kettin, molecules that tether the ends of the thick filaments to the Z-disk. A new hypothesis is based on the discovery that asynchronous flight muscle has two isoforms of troponin C, a normal type (F2), and a peculiar but more abundant type that has lost one of its Ca2+ binding sites (F1). By substituting troponin isoforms in skinned fibers, researchers have shown that F1 is necessary for stretch activation, but the F2 is not. One intriguing possibility is that the altered isoform still functions by moving tropomyosin away from target zones, but now responds mechanically to the tension along the thin filament imposed by stretch. Even if true, this mechanism is not mutually exclusive of many other current hypotheses. Further, given the likelihood of multiple Magazine R311 evolutionary origins, there may not be a single mechanism either within or across taxa. It will be both intriguing and informative to determine whether insects have made the leap to stretch activation the same way each time. Aerodynamics The myth that engineers cannot explain the aerodynamics of insect flight persists despite an extensive amount of research to the contrary. This is unfortunate, because a cohesive theory of flapping flight has emerged from a collective effort in biology, physics and aeronautics. There are several mutually compatible ways of explaining the lift created by a conventional airfoil. Most simply, as a wing translates it diverts the oncoming air downward, and the resulting change in momentum of this air is equal to upward force acting on the wing. For conventional aircraft, this is most efficient at gentle angles of attack, under which conditions the stream of oncoming air separates at the leading edge of the wing, follows the contour of the wing surface, and rejoins smoothly near the trailing edge. This flow configuration is stable, and is thus well modeled by relatively simple time-invariant models. Modern airfoils are designed with graceful contours so that the flow of air stays attached to the upper surface of the wing, rather than separating to form a turbulent wake that causes a precipitous drop in lift known as stall. At first glance, insects appear to do everything wrong. First, although slightly corrugated for rigidity, their wings are flat and lack any streamlined shape. Second, they move their wings through the air at very large angles of attack, well above the threshold for flow separation and stall. What protects insects from the disastrous consequences of this flagrant disregard of sound aerodynamic design? A critical concept in predicting the behavior of fluid is the Reynolds number (Re), a dimensionless quantity that is formally defined as the ratio of inertial to viscous forces. (From a physical perspective, both liquids and gases are considered fluids because the force required to push against them is proportional to how fast you push, not how far you push.) Each tiny volume of fluid has density and velocity and therefore also momentum, Current Biology Power muscles Steering muscles DVMs active DLMs active Phase advance Phase delay Figure 1. Functional organization of flight muscle in flies. (A) Large asynchronous flight muscles provide the power required for flight. Antagonist sets of muscles oscillate to drive the wings back and forth via their indirect insertions on the sides of the thorax. (B) Tiny steering muscles alter wing motion through their actions on apodemes at the base of the wing. Some of the muscles serve as controllable springs whose stiffness is regulated by the firing phase of their motor neurons. and thus can exert an inertial force on adjacent volumes or an immersed solid such as a wing. For an object moving in a fluid, the Re is equal to UL/, where U is velocity, L is a linear dimension and is the kinematic viscosity of the fluid. Values for insect wings fall in the range of so-called `intermediate' Re (10