JOURNAL OF MORPHOLOGY 000:000–000 (2011) Resilin in Dragonfly and Damselfly Wings and Its Implications for Wing Flexibility Seth Donoughe,1 James D. Crall,2* Rachel A. Merz,3 and Stacey A. Combes2 1Department of Cell and Developmental Biology, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 2Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 3Department of Biology, Swarthmore College, Swarthmore, Pennsylvania ABSTRACT Although there is mounting evidence that and Combes, 2002). These passive deformations may passive mechanical dynamics of insect wings play an inte- be an inevitable property of wings that are con- gral role in insect flight, our understanding of the struc- structed from flexible, biological materials, yet they tural details underlying insect wing flexibility remains often appear to be beneficial (Wootton, 1981). incomplete. Here, we use comparative morphological and Flexibility in insect wings may enhance aerody- mechanical techniques to illuminate the function and di- versity of two mechanisms within Odonata wings pre- namic performance. Previous work has revealed a sumed to affect dynamic wing deformations: flexible variety of passive deformation mechanisms in insect resilin vein-joints and cuticular spikes. Mechanical tests wings that appear to be aerodynamically useful. For show that joints with more resilin have lower rotational example, the automatic depression of the trailing stiffness and deform more in response to a load applied to edge (Wootton, 1991) and twisting of the leading an intact wing. Morphological studies of 12 species of Odo- edge under aerodynamic loading (Ennos, 1988) both nata reveal that resilin joints and cuticular spikes are create camber. The varying position of the nodus in widespread taxonomically, yet both traits display a strik- damselfly and dragonfly wings is correlated with the ing degree of morphological and functional diversity that amount of wing twisting in flight, which in turn is follows taxonomically distinct patterns. Interestingly, related to stroke plane inclination (Wootton and damselfly wings (suborder Zygoptera) are mainly charac- terized by vein-joints that are double-sided (containing Newman, 2008). Several studies have provided direct resilin both dorsally and ventrally), whereas dragonfly evidence that flexible wings that are able to produce wings (suborder Epiprocta) are largely characterized camber may generate higher peak lift forces than by single-sided vein-joints (containing resilin either rigid wings (Mountcastle and Daniel, 2009; Young ventrally or dorsally, but not both). The functional signifi- et al., 2009). By contrast, however, recent work done cance and diversity of resilin joints and cuticular spikes on model wings has shown that at low and medium could yield insight into the evolutionary relationship angles of attack, aerodynamic performance decreases between form and function of wings, as well as revealing with increased flexibility (Zhao et al., 2010). Yet, the basic principles of insect wing mechanical design. aerodynamic performance of a flexible model wing Ó J. Morphol. 000:000–000, 2011. 2011 Wiley-Liss, Inc. can match or even exceed that of rigid wings if rudi- KEY WORDS: flight; wings; wing flexural stiffness; mentary longitudinal veins are added, probably resilin; Odonata because the veins promote cambering (Zhao et al., 2010). Thus, the exact role of wing flexibility in aero- dynamic performance remains unclear. Seth Donoughe and James D. Crall should be considered primary authors. INTRODUCTION Animal fliers move using wings that deform sub- Contract grant sponsor: NSF Expeditions in Computing; Contract stantially during flapping flight (Swartz et al., 1992; grant number: CCF-0926148.; Contract grant sponsors: Swarthmore College (Meinkoth Fund), Walter Kemp’s family. Biewener and Dial, 1995; Young et al., 2009). Insect wings differ from those of vertebrates in that they *Correspondence to: James D. Crall, Concord Field Station, 100 lack internal musculature extending into the aerody- Old Causeway Rd, Bedford, MA 01730. namic surface of the wing. Thus, while birds and E-mail: [email protected] bats can actively modulate the form and flexibility of their wings, insects have little active control over Received 17 November 2010; Revised 26 April 2011; wing properties, and most deformations are a prod- Accepted 11 May 2011 uct of the passive mechanical properties of the wing Published online in (Wootton, 1992) interacting with the inertial and aer- Wiley Online Library (wileyonlinelibrary.com) odynamic forces it generates while flapping (Daniel DOI: 10.1002/jmor.10992 Ó 2011 WILEY-LISS, INC. 2 S. DONOUGHE ET AL. Fig. 1. Damselfly wing with longitudinal veins shown in black and cross-veins in orange. Veins meet to form vein-joints. Inset illustrates the corrugated cross-section of the wing, with alternating ‘‘mountain’’ (1) and ‘‘valley’’ (2) longitudinal veins. The nodus is a specialized, highly conserved vein-joint. Flexibility in insect wings may also help to protect tions between cross-veins and longitudinal veins. against permanent wing damage. Newman and Vein-joints that are morphologically specialized for Wootton (1986) first suggested that dragonfly wings flexibility were first described by Newman (1982) appear to be adapted for reversible failure in and were later shown to contain resilin (Gorb, response to excess loads, enabling them to avoid 1999), a flexible, rubberlike protein found in many permanent structural damage. Although previous insect locomotory structures (Weis-Fogh, 1960). studies have provided conflicting evidence on the Resilin functions as a spring in the flea leg general fitness consequences of wing damage (see (Bennet-Clark and Lucey, 1967) and the froghop- Cartar, 1992; Kingsolver, 1999; Hedenstro¨m et al., per pleural arch (Burrows et al., 2008), and is 2001), recent work on dragonflies has shown that found in the energy-storing tendon at the base of artificial damage decreases vertical acceleration dragonfly and locust wings (Weis-Fogh, 1960). and impairs prey capture performance (Combes Resilin’s high flexibility (Young’s modulus around et al., 2010). Thus, at least in dragonflies, there is 1 MPa, in contrast to values up to 20 GPa for scle- probably selective pressure against wing damage. rotized cuticle; Vincent and Wegst, 2004), capacity Mechanisms allowing for reversible failure of drag- for energy storage (energy loss of less than 5% onfly wings may therefore represent an important over a wide range of frequencies; Jensen and Weis- and largely overlooked aspect of wing morphology. Fogh, 1962), and near indestructibility under natu- Our goal in this study, especially pressing in ral conditions (Weis-Fogh, 1960; Anderson, 1963) light of the renewed interest in the aerodynamic make it ideally suited for its role in insect locomo- consequences of wing flexibility, was to expand on tion. Resilin has since been found embedded in the existing body of work that investigated the beetle and earwig wings (Haas et al., 2000a,b, detailed morphological mechanisms underlying respectively), where it plays a role in wing folding overall wing deformations. In particular, we exam- at rest, but to our knowledge, no work other than ined the diversity of fine-scale structures associ- Gorb (1999) has described resilin structures that ated with wing flexibility within one order of spec- may be associated with wing deformations during tacular aerial acrobats: the dragonflies and dam- flight. In this study, we use microscopy and selflies (Order: Odonata). mechanical testing to examine the diversity and Odonate wings are composed of a thin, translu- function of vein-joints to understand how they con- cent bilayer of cuticle (wing membrane) that is re- tribute to the flexibility and passive deformations inforced by a meshwork of thickened cuticular of odonate wings. veins. Longitudinal veins originate at or close to the base of the wing and run along the length of the wing (Fig. 1, black), whereas smaller cross- MATERIALS AND METHODS veins run perpendicular to and connect longitudi- Specimen Collection nal veins (Fig. 1, orange). Veins meet to form Specimens were collected from Bedford, MA in the summer of vein-joints (Fig. 1, circled)—the structures that are 2009 or Swarthmore, PA in the summers of 2008–2010, and the focus of this study. then stored and euthanized in compliance with the regulations Wootton (1991) reviewed passive mechanisms of Swarthmore College Institutional Animal Care and Use Com- that enable shape changes within the wings of mittee. Before microscopy, specimens were rehydrated in a Odonata and emphasized the importance of under- humid plastic container incubated at room temperature for at least 12 h. The forewings of each specimen were then removed standing the mechanical properties of the junc- for morphological investigations. Journal of Morphology RESILIN IN DRAGONFLY AND DAMSELFLY WINGS 3 several odonate families, including Aeshna constricta, Sympe- trum rubicundulum, Erythemis simplicicollis, Somatochlora tenebrosa, Epitheca cynosura, Ischnura posita, Enallagma diva- gans, Calopteryx augustipennis, and Lestes rectangularis. For this, we used a Zeiss Axioplan fluorescent microscope. The dor- sal and ventral sides of each vein were examined separately. A given longitudinal vein was scored as ‘‘present’’ for resilin if any of the vein-joints on that vein showed visible blue fluorescence. Out of 168 examined veins between all included species, 99 were scored as ‘‘present’’ for