- Email: [email protected]
Giant stick insects reveal unique ontogenetic changes in biological attachment devices
Arthropod Structure & Development 44 (2015) 195e199
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Short communication
Giant stick insects reveal unique ontogenetic changes in biological attachment devices Marco Gottardo a, *, Davide Vallotto b, Rolf G. Beutel c a
Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena, Italy Via Cici 26/M, 30038 Spinea, VE, Italy c €t Jena, Ebertstrasse 1, D-07743 Jena, Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universita Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Accepted 8 January 2015 Available online 17 January 2015
A strong modification of tarsal and pretarsal attachment pads during the postembryonic development is described for the first time. In the exceptionally large thorny devil stick insect Eurycantha calcarata a functional arolium is only present in the immature instars, enabling them to climb on smooth surfaces, especially leaves. Nymphs are also characterized by greyish and hairy euplantulae on tarsomeres 1e4. The gradual modifications of the arolium and the euplantula of tarsomere 5 in the nymphal development are probably mainly related to increased weight. The distinct switch in the life style between the leaf-dwelling nymphal stages and the ground-dwelling adults results in the final abrupt change of the adhesive devices, resulting in a far-reaching reduction of the arolium, the presence of a fully-developed, elongated euplantula on tarsomere 5, and white and smooth euplantulae on tarsomeres 1e4. The developmental remodelling of attachment pads also reflects a phylogenetic pattern. The attachment devices of the earlier instars are similar to those found in the basalmost lineage of extant stick insects, Timema, which is characterized by a very large pan-shaped arolium and a hairy surface of the tarsal and pretarsal attachment pads. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Insect cuticle Attachment pads Ultrastructure Adaptive developmental plasticity Phasmatodea
1. Introduction Locomotory attachment devices played an important role in the evolution of the extremely successful Insecta (Gorb, 2001; Beutel and Gorb, 2001). Especially in predominantly or exclusively herbivorous insect groups a remarkable spectrum of attachment structures has evolved (Beutel and Gorb, 2001, 2006). The function is not just adhesion but they enable insects to walk efficiently on plants through systems providing fast and reversible attachment/ detachment (Gorb et al., 2002). Modifications of plant surfaces to impede attacks by phytophagous insects and corresponding variation of insect attachment devices are two counterparts of an evolutionary arms race between the two groups of organisms (Beutel and Gorb, 2001). Tarsal attachment pads specifically designed to adhere on diverse substrates occur in two main structural types in insects. Hairy systems where the pad is covered with deformable adhesive setae are found in earwigs (Haas and Gorb, 2004), beetles (Stork,
* Corresponding author. E-mail address: [email protected] (M. Gottardo). http://dx.doi.org/10.1016/j.asd.2015.01.001 1467-8039/© 2015 Elsevier Ltd. All rights reserved.
1980), and flies (Friedemann et al., 2014a). Smooth systems without prominent microornamentation of the pad surface are characteristic of cockroaches (Clemente and Federle, 2008), katydids and grasshoppers (Beutel and Gorb, 2001), ants and bees (Federle et al., 2001), and bugs (Friedemann et al., 2014b). An intermediate type of pad structure covered with cone-shaped hairs (i.e. acanthae, unicellular epidermal outgrowths) has evolved in stick insects (Beutel and Gorb, 2008; Bußhardt et al., 2012; Labonte et al., 2014), in some stoneflies (Nelson, 2009), and in sawflies (Schulmeister, 2003). Biomechanical features of the different designs of insect attachment pads have been analysed in several studies (e.g. Gorb et al., 2002; Gorb, 2007; Bullock et al., 2008), while ontogenetic aspects have been only reported in the leaf-footed bug Coreus marginatus (Gorb and Gorb, 2004). However, distinct developmental modifications of attachment pads related to environmental adaptation were not described yet. Here, we report both gradual and 'switch-like' developmental changes in the design of these structures in Eurycantha calcarata, a giant stick insect from the rain forests of New Guinea featuring different habitat preferences during its lifetime. Our results show the unprecedented adaptive developmental plasticity of attachment devices of insect cuticle.
196
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
2. Material and methods The giant stick insect E. calcarata Lucas, 1869 belongs to the order Phasmatodea, suborder Euphasmatodea, subfamily
Lonchodinae (Bradler et al., 2014). A culture-stock of E. calcarata originating from New Britain island (Papua New Guinea) provided the specimens used in this study. The insects were laboratoryreared in a large cage (60 cm high, 50 cm wide, and 80 cm long)
Fig. 1. Dramatic remodelling of attachment devices is concomitant with life-style change in E. calcarata. Attachment structures of the leaf-dwelling first instar nymph (AeF) compared to those of the ground-dwelling adult (GeL). (A) Typical resting position of immature instars. (B) Structure of the tarsus, ventral aspect. (CeD) Adhesive microstructures of the arolium. (E) Euplantula with the hairy micropattern, ventral aspect. (F) Detailed view of the dense coverage of acanthae on the surface of the euplantula. (G) Adult male. (H) Structure of the tarsus, ventral aspect. (I) Cuticular outgrowths are found in the reduced arolium. (J) Smooth micropattern of the euplantula of tarsomere 5. (K) Euplantula with the smooth micropattern, ventral aspect. (L) Cuticular microornaments are absent in the smooth euplantula.
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
under constant environmental conditions (21 C, 12:12 h lightedark photoperiod). Bramble (Rubus fruticosus L.) and ivy (Hedera helix L.) were regularly provided as food plants. For each instar we observed the morphology of attachment devices in contact with a flat vertical glass (30 30 cm). Images of attachment structures of living insects were captured with a Nikon D200 SLR digital camera equipped with Nikon Micro-Nikkor AI-s 105 mm f/2.8 lens or with Nikon 24 mm f/2.8 AI-s lens. To investigate the ultrastructure of attachment devices, tarsi dissected from nymphs and adults were fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) for 1e2 days at 4 C, then washed in PBS and post-fixed in 1% OsO4 for 2.5 h at 4 C. The material was dehydrated in ascending ethanol series and criticalpoint dried in a Balzers CPD 030 apparatus. Samples were mounted on aluminium stubs, gold-coated in a Balzers MED 010 sputtering device, and observed with a Philips XL20 scanning electron microscope operating at an accelerating voltage of 10e12 kV. 3. Results and discussion Natural history observations show that the exceptionally large first-instar nymphs of E. calcarata (30 mm in length) are leafdwelling insects. During the day they are found in the typical resting position on the upper surface of the food-plant leaves (Fig. 1A), while feeding activity takes place at night. The small nymphs mainly sit on leaves when foraging (M.G., personal observation). Attachment to the leaf surface is achieved using specialized adhesive structures of the 5-segmented tarsus at the distal region of the legs. In this stage of development we found that the apical part of the leg (pretarsus) bears a large pan-shaped membranous attachment organ (arolium) (Fig 1B). The arolium shows diversified microstructures. A thin outer band is covered with minute star-shaped protuberances (Fig. 1C) and embraces a ventro-distal trapezoidal plate-like region with a smooth surface (Fig. 2A). The basal edge of this plate-like area appears as a
197
prominent ridge that delimits a broad median region characterized by longitudinal folds with wart-like surface modifications (Fig 1D) similar to those on the arolium of crane flies (Beutel and Gorb, 2001). The surface proximad the median region is smooth and bears four setae (Fig. 2A). When attached to the flat vertical glass (Fig. 1B), the contact zone of the arolium is apparently the plate-like area and the median region. Biomechanic investigations of the arolium in stick insects, cockroaches, and ants have shown that this organ is essential during vertical and inverted climbing on smooth substrates, since it can generate large adhesive forces of up to one body weight (Roth and Willis, 1952; Federle and Endlein, 2004; Labonte and Federle, 2013). The apical tarsomere is distally equipped with a well-developed pretarsal unguitractor apparatus and two apical claws, and it lacks an attachment pad on its ventral side (Fig. 1B). The ventral side of each of the first four tarsomeres bears a cushion-like attachment device (euplantula) with a greyish opaque appearance (Fig. 1B). The contact surface of the euplantula shows a hairy micropattern (Fig. 1E) consisting of a dense coverage of acanthae with apically rounded tips (Fig. 1F). The stick insect undergoes six leaf-dwelling nymphal instars. Larger nymphs tend to attach on both leaf stalks and blades during nocturnal feeding (M.G., personal observation). Investigations of the attachment devices during postembryonic development revealed distinct morphological changes of the tarsus and pretarsus (see Supplementary Fig. S1AeE). The relative size of the arolium decreases continuously from the second to the sixth instar when compared to the size of the pretarsal claws. Nymphs from the second to the fifth instar are still capable of climbing on flat vertical glass surfaces, while this is arduous for subadults which are only equipped with a small arolium. A peculiar transformation of the ventral side of tarsomere 5 starts from the fourth instar onwards, with the differentiation of a flexible pad-like structure on the middle region. The latter structure appears as a tapering greyish field protruding over two-thirds of the length of tarsomere 5. During the six juvenile instars the euplantulae retain the greyish
Fig. 2. Detailed view of tarsomere 5 and pretarsus of E. calcarata. Comparison between the first instar nymph (A) and the adult (B).
198
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
opaque appearance and the hairy micropattern (see Supplementary Fig. S1FeJ). Hairy euplantulae similar to those of nymphs of E. calcarata are found in adults of other stick insect species (Beutel and Gorb, 2008; Bußhardt et al., 2012; Gottardo and Heller, 2012). Biomechanical studies on the hairy euplantulae of Carausius morosus showed that, regardless of the load applied, adhesive forces are always larger compared to friction forces on different substrate types (Labonte and Federle, 2013; Bußhardt et al., 2012). Adults of E. calcarata are robust wingless insects with a very large body size (15 cm in length) (Fig. 1G) and indistinct sexual size dimorphism (Hsiung, 1987). Unlike the nymphs they are not able to attach to and to walk on a glass ceiling, and when foraging they move on food-plant branches (M.G., personal observation). They show a totally different behaviour compared to the immature instars. They are ground dwellers found inside hollows at the bases of living trees. With the last moult to adulthood, attachment devices undergo dramatic morphological changes of both male and female tarsi. The arolium is reduced to a very small whitish lobe (Fig. 1H). The diverse adhesive microstructures found on its surface in the nymphal stages are missing (Fig. 2B), even though densely set irregular cuticular outgrowths are present ventrally (Fig. 1I). The ventral surface of tarsomere 5 now bears a fully-developed elongated euplantula featuring a white glossy colour (Fig. 1H). Investigation of its surface showed that this structure represents a smooth-type attachment pad without distinct microornamentation (Fig. 1J), as it is typical for most related groups of insects (Beutel and Gorb, 2001). The presence of an attachment pad on the ventral surface of the apical tarsomere has been also reported in Timema stick insects (Tilgner et al., 1999) and in some cockroaches (van Casteren and Codd, 2010). The tarsal euplantulae 1e4 of the adults are also profoundly modified in their fine structure. The colouration is also white (Fig. 1H) and the surface smooth (Fig. 1K). The contact area of the adult euplantulae has lost the hairy coverage of acanthae found in the immature instars, and no cuticular microornament is present (Fig. 1L).
4. Conclusions Distinct ontogenetic modifications of tarsal and pretarsal attachment pads were not reported for any groups of insects before. In the case of the exceptionally large E. calcarata, the arolium is a transient adhesive structure functioning only in the immature instars adapted for climbing on leaf surfaces. In fact, the distinct switch in the life style between the leaf-dwelling nymphal stages and the ground-dwelling adults results in a final abrupt transformation of the adhesive apparatus, with the loss of function of the arolium as the most conspicuos change. During the nymphal development of E. calcarata, the increasing weight primarily results in an increased size of the attachment pads. Moreover, the gradual differentiation of an accessory euplantula on tarsomere 5 is probably mainly related to the concomitant progressive reduction of the arolium. It is conceivable that an additional attachment pad on tarsomere 5 may be a necessary adaptation to compensate for the reduction of the pretarsal arolium during growth. The functional significance of the 'switch-like' modifications that occur in the euplantulae of tarsomeres 1e4 with the transition from the last nymphal stage to the adult is less clear. In some species of canopydwelling stick insects, smooth euplantulae are present together with a functional arolium (Bußhardt et al., 2012; Gottardo and Vallotto, 2014). In the euplantulae of adult cockroaches, a colour change has been correlated with a change in pad properties during ageing (Ridgel et al., 2003). In E. calcarata, the euplantular colour change is accompanied by the loss of the acanthae. Further studies
are needed to assess the functional consequences of these modifications of the pad structure. From an evolutionary perspective, the developmental remodelling of attachment pads might reflect a phylogenetic pattern. The attachment devices of the earlier instars are similar to those found in Timema, the basalmost lineage of extant stick insects, and in various subgroups of the Euphasmatodea which are characterized by a very large pan-shaped arolium and a hairy surface of the tarsal and prestarsal attachment pads (Beutel and Gorb, 2008). This condition is unusual among the polyneopteran orders, but also occurs in the distantly related Mantophasmatodea (Beutel and Gorb, 2008). Conversely, the adults of E. calcarata show smoothtype attachment pads which also characterize adults of some other members of the Euphasmatodea (Gottardo, 2011; Bußhardt et al., 2012; Gottardo and Vallotto, 2014). Acknowledgements We thank two anonymous reviewers for helpful comments on the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.asd.2015.01.001. References Beutel, R.G., Gorb, S.N., 2001. Ultrastructure of attachment specializations of hexapods (Arthropoda): evolutionary patterns inferred from a revised ordinal phylogeny. J. Zool. Syst. Evol. Res. 39, 177e207. Beutel, R.G., Gorb, S.N., 2006. A revised interpretation of the evolution of attachment structures in Hexapoda with special emphasis on Mantophasmatodea. Arthropod Syst. Phylogeny 64, 3e25. Beutel, R.G., Gorb, S.N., 2008. Evolutionary scenarios for unusual attachment devices of Phasmatodea and Mantophasmatodea (Insecta). Syst. Entomol. 33, 501e510. Bradler, S., Robertson, J.A., Whiting, M.F., 2014. A molecular phylogeny of Phasmatodea with emphasis on Necrosciinae, the most species-rich subfamily of stick insects. Syst. Entomol. 39, 205e222. Bullock, J.M.R., Drechsler, P., Federle, W., 2008. Comparison of smooth and hairy attachment pads in insects: friction, adhesion and mechanisms for directiondependence. J. Exp. Biol. 211, 3333e3343. Bußhardt, P., Wolf, H., Gorb, S., 2012. Adhesive and frictional properties of tarsal attachment pads in two species of stick insects (Phasmatodea) with smooth and nubby euplantulae. Zoology 115, 135e141. Clemente, C.J., Federle, W., 2008. Pushing versus pulling: division of labour between tarsal attachment pads in cockroaches. Proc. R. Soc. B 275, 1329e1336. €lldobler, B., 2001. Biomechanics of the Federle, W., Brainerd, E.L., McMahon, T.A., Ho movable pretarsal adhesive organ in ants and bees. Proc. Natl. Acad. Sci. U. S. A. 98, 6215e6220. Federle, W., Endlein, T., 2004. Locomotion and adhesion: dynamic control of adhesive surface contact in ants. Arthropod Struct. Dev. 33, 67e75. Friedemann, K., Schneeberg, K., Beutel, R.G., 2014a. Fly on the wall e attachment structures in lower Diptera. Syst. Entomol. 39, 460e473. Friedemann, K., Spangenberg, R., Yoshizawa, K., Beutel, R.G., 2014b. Evolution of attachment structures in the highly diverse Acercaria (Hexapoda). Cladistics 30, 170e201. Gorb, S., 2001. Attachment Devices of Insect Cuticle. Kluwer Academic Publishers, Dordrecht. Gorb, S.N., 2007. Smooth attachment devices in insects: functional morphology and biomechanics. Adv. Insect Physiol. 34, 81e115. Gorb, S.N., Beutel, R.G., Gorb, E.V., Jiao, Y., Kastner, V., Niederegger, S., Popov, V.L., €tsch, W., 2002. Structural design and biomechanics Scherge, M., Schwarz, U., Vo of friction-based releasable attachment devices in insects. Integr. Comp. Biol. 42, 1127e1139. Gorb, S.N., Gorb, E.V., 2004. Ontogenesis of the attachment ability in the bug Coreus marginatus (Heteroptera, Insecta). J. Exp. Biol. 207, 2917e2924. Gottardo, M., 2011. A new genus and new species of Philippine stick insects (Insecta: Phasmatodea) and phylogenetic considerations. C. R. Biol. 334, 555e563. Gottardo, M., Heller, P., 2012. An enigmatic new stick insect from the Philippine Islands (Insecta: Phasmatodea). C. R. Biol. 335, 594e601. Gottardo, M., Vallotto, D., 2014. External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations. C. R. Biol. 337, 258e268.
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199 Haas, F., Gorb, S., 2004. Evolution of locomotory attachment pads in the Dermaptera (Insecta). Arthropod Struct. Dev. 33, 45e66. Hsiung, C.-C., 1987. Aspects of the biology of the Melanesian stick-insect Eurycantha calcarata Lucas (Cheleutoptera: Phasmatidae). J. Nat. Hist. 21, 1241e1258. Labonte, D., Federle, W., 2013. Functionally different pads on the same foot allow control of attachment: stick insects have load-sensitive ‘heel’ pads for friction and shear-sensitive ‘toe’ pads for adhesion. PLoS One 8, e81943. Labonte, D., Williams, J.A., Federle, W., 2014. Surface contact and design of fibrillar ‘friction pads’ in stick insects (Carausius morosus): mechanisms for large friction coefficients and negligible adhesion. J. R. Soc. Interface 11, 20140034. Nelson, C., 2009. Surface ultrastructure and evolution of tarsal attachment structures in Plecoptera (Arthropoda: Hexapoda). Aquat. Insects 31, 523e545. Ridgel, A.L., Ritzmann, R.E., Schaefer, P.L., 2003. Effects of aging on behavior and leg kinematics during locomotion in two species of cockroach. J. Exp. Biol. 206, 4453e4465.
199
Roth, L., Willis, E., 1952. Tarsal structure and climbing ability of cockroaches. J. Exp. Zool. 119, 483e517. Schulmeister, S., 2003. Morphology and evolution of the tarsal plantulae in Hymenoptera (Insecta), focussing on the basal lineages. Zool. Scr. 32, 153e172. Stork, N.E., 1980. A scanning electron microscope study of tarsal adhesive setae in the Coleoptera. Zool. J. Linn. Soc. 68, 173e306. Tilgner, E.H., Kiselyova, T.G., McHugh, J.V., 1999. A morphological study of Timema cristinae Vickery with implications for the phylogenetics of Phasmida. Dtsch. Entomol. Z. 46, 149e162. van Casteren, A., Codd, J.R., 2010. Foot morphology and substrate adhesion in the Madagascan hissing cockroach, Gromphadorhina portentosa. J. Insect Sci. 10 (40), 1e12.
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Short communication
Giant stick insects reveal unique ontogenetic changes in biological attachment devices Marco Gottardo a, *, Davide Vallotto b, Rolf G. Beutel c a
Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena, Italy Via Cici 26/M, 30038 Spinea, VE, Italy c €t Jena, Ebertstrasse 1, D-07743 Jena, Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universita Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Accepted 8 January 2015 Available online 17 January 2015
A strong modification of tarsal and pretarsal attachment pads during the postembryonic development is described for the first time. In the exceptionally large thorny devil stick insect Eurycantha calcarata a functional arolium is only present in the immature instars, enabling them to climb on smooth surfaces, especially leaves. Nymphs are also characterized by greyish and hairy euplantulae on tarsomeres 1e4. The gradual modifications of the arolium and the euplantula of tarsomere 5 in the nymphal development are probably mainly related to increased weight. The distinct switch in the life style between the leaf-dwelling nymphal stages and the ground-dwelling adults results in the final abrupt change of the adhesive devices, resulting in a far-reaching reduction of the arolium, the presence of a fully-developed, elongated euplantula on tarsomere 5, and white and smooth euplantulae on tarsomeres 1e4. The developmental remodelling of attachment pads also reflects a phylogenetic pattern. The attachment devices of the earlier instars are similar to those found in the basalmost lineage of extant stick insects, Timema, which is characterized by a very large pan-shaped arolium and a hairy surface of the tarsal and pretarsal attachment pads. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Insect cuticle Attachment pads Ultrastructure Adaptive developmental plasticity Phasmatodea
1. Introduction Locomotory attachment devices played an important role in the evolution of the extremely successful Insecta (Gorb, 2001; Beutel and Gorb, 2001). Especially in predominantly or exclusively herbivorous insect groups a remarkable spectrum of attachment structures has evolved (Beutel and Gorb, 2001, 2006). The function is not just adhesion but they enable insects to walk efficiently on plants through systems providing fast and reversible attachment/ detachment (Gorb et al., 2002). Modifications of plant surfaces to impede attacks by phytophagous insects and corresponding variation of insect attachment devices are two counterparts of an evolutionary arms race between the two groups of organisms (Beutel and Gorb, 2001). Tarsal attachment pads specifically designed to adhere on diverse substrates occur in two main structural types in insects. Hairy systems where the pad is covered with deformable adhesive setae are found in earwigs (Haas and Gorb, 2004), beetles (Stork,
* Corresponding author. E-mail address: [email protected] (M. Gottardo). http://dx.doi.org/10.1016/j.asd.2015.01.001 1467-8039/© 2015 Elsevier Ltd. All rights reserved.
1980), and flies (Friedemann et al., 2014a). Smooth systems without prominent microornamentation of the pad surface are characteristic of cockroaches (Clemente and Federle, 2008), katydids and grasshoppers (Beutel and Gorb, 2001), ants and bees (Federle et al., 2001), and bugs (Friedemann et al., 2014b). An intermediate type of pad structure covered with cone-shaped hairs (i.e. acanthae, unicellular epidermal outgrowths) has evolved in stick insects (Beutel and Gorb, 2008; Bußhardt et al., 2012; Labonte et al., 2014), in some stoneflies (Nelson, 2009), and in sawflies (Schulmeister, 2003). Biomechanical features of the different designs of insect attachment pads have been analysed in several studies (e.g. Gorb et al., 2002; Gorb, 2007; Bullock et al., 2008), while ontogenetic aspects have been only reported in the leaf-footed bug Coreus marginatus (Gorb and Gorb, 2004). However, distinct developmental modifications of attachment pads related to environmental adaptation were not described yet. Here, we report both gradual and 'switch-like' developmental changes in the design of these structures in Eurycantha calcarata, a giant stick insect from the rain forests of New Guinea featuring different habitat preferences during its lifetime. Our results show the unprecedented adaptive developmental plasticity of attachment devices of insect cuticle.
196
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
2. Material and methods The giant stick insect E. calcarata Lucas, 1869 belongs to the order Phasmatodea, suborder Euphasmatodea, subfamily
Lonchodinae (Bradler et al., 2014). A culture-stock of E. calcarata originating from New Britain island (Papua New Guinea) provided the specimens used in this study. The insects were laboratoryreared in a large cage (60 cm high, 50 cm wide, and 80 cm long)
Fig. 1. Dramatic remodelling of attachment devices is concomitant with life-style change in E. calcarata. Attachment structures of the leaf-dwelling first instar nymph (AeF) compared to those of the ground-dwelling adult (GeL). (A) Typical resting position of immature instars. (B) Structure of the tarsus, ventral aspect. (CeD) Adhesive microstructures of the arolium. (E) Euplantula with the hairy micropattern, ventral aspect. (F) Detailed view of the dense coverage of acanthae on the surface of the euplantula. (G) Adult male. (H) Structure of the tarsus, ventral aspect. (I) Cuticular outgrowths are found in the reduced arolium. (J) Smooth micropattern of the euplantula of tarsomere 5. (K) Euplantula with the smooth micropattern, ventral aspect. (L) Cuticular microornaments are absent in the smooth euplantula.
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
under constant environmental conditions (21 C, 12:12 h lightedark photoperiod). Bramble (Rubus fruticosus L.) and ivy (Hedera helix L.) were regularly provided as food plants. For each instar we observed the morphology of attachment devices in contact with a flat vertical glass (30 30 cm). Images of attachment structures of living insects were captured with a Nikon D200 SLR digital camera equipped with Nikon Micro-Nikkor AI-s 105 mm f/2.8 lens or with Nikon 24 mm f/2.8 AI-s lens. To investigate the ultrastructure of attachment devices, tarsi dissected from nymphs and adults were fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) for 1e2 days at 4 C, then washed in PBS and post-fixed in 1% OsO4 for 2.5 h at 4 C. The material was dehydrated in ascending ethanol series and criticalpoint dried in a Balzers CPD 030 apparatus. Samples were mounted on aluminium stubs, gold-coated in a Balzers MED 010 sputtering device, and observed with a Philips XL20 scanning electron microscope operating at an accelerating voltage of 10e12 kV. 3. Results and discussion Natural history observations show that the exceptionally large first-instar nymphs of E. calcarata (30 mm in length) are leafdwelling insects. During the day they are found in the typical resting position on the upper surface of the food-plant leaves (Fig. 1A), while feeding activity takes place at night. The small nymphs mainly sit on leaves when foraging (M.G., personal observation). Attachment to the leaf surface is achieved using specialized adhesive structures of the 5-segmented tarsus at the distal region of the legs. In this stage of development we found that the apical part of the leg (pretarsus) bears a large pan-shaped membranous attachment organ (arolium) (Fig 1B). The arolium shows diversified microstructures. A thin outer band is covered with minute star-shaped protuberances (Fig. 1C) and embraces a ventro-distal trapezoidal plate-like region with a smooth surface (Fig. 2A). The basal edge of this plate-like area appears as a
197
prominent ridge that delimits a broad median region characterized by longitudinal folds with wart-like surface modifications (Fig 1D) similar to those on the arolium of crane flies (Beutel and Gorb, 2001). The surface proximad the median region is smooth and bears four setae (Fig. 2A). When attached to the flat vertical glass (Fig. 1B), the contact zone of the arolium is apparently the plate-like area and the median region. Biomechanic investigations of the arolium in stick insects, cockroaches, and ants have shown that this organ is essential during vertical and inverted climbing on smooth substrates, since it can generate large adhesive forces of up to one body weight (Roth and Willis, 1952; Federle and Endlein, 2004; Labonte and Federle, 2013). The apical tarsomere is distally equipped with a well-developed pretarsal unguitractor apparatus and two apical claws, and it lacks an attachment pad on its ventral side (Fig. 1B). The ventral side of each of the first four tarsomeres bears a cushion-like attachment device (euplantula) with a greyish opaque appearance (Fig. 1B). The contact surface of the euplantula shows a hairy micropattern (Fig. 1E) consisting of a dense coverage of acanthae with apically rounded tips (Fig. 1F). The stick insect undergoes six leaf-dwelling nymphal instars. Larger nymphs tend to attach on both leaf stalks and blades during nocturnal feeding (M.G., personal observation). Investigations of the attachment devices during postembryonic development revealed distinct morphological changes of the tarsus and pretarsus (see Supplementary Fig. S1AeE). The relative size of the arolium decreases continuously from the second to the sixth instar when compared to the size of the pretarsal claws. Nymphs from the second to the fifth instar are still capable of climbing on flat vertical glass surfaces, while this is arduous for subadults which are only equipped with a small arolium. A peculiar transformation of the ventral side of tarsomere 5 starts from the fourth instar onwards, with the differentiation of a flexible pad-like structure on the middle region. The latter structure appears as a tapering greyish field protruding over two-thirds of the length of tarsomere 5. During the six juvenile instars the euplantulae retain the greyish
Fig. 2. Detailed view of tarsomere 5 and pretarsus of E. calcarata. Comparison between the first instar nymph (A) and the adult (B).
198
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199
opaque appearance and the hairy micropattern (see Supplementary Fig. S1FeJ). Hairy euplantulae similar to those of nymphs of E. calcarata are found in adults of other stick insect species (Beutel and Gorb, 2008; Bußhardt et al., 2012; Gottardo and Heller, 2012). Biomechanical studies on the hairy euplantulae of Carausius morosus showed that, regardless of the load applied, adhesive forces are always larger compared to friction forces on different substrate types (Labonte and Federle, 2013; Bußhardt et al., 2012). Adults of E. calcarata are robust wingless insects with a very large body size (15 cm in length) (Fig. 1G) and indistinct sexual size dimorphism (Hsiung, 1987). Unlike the nymphs they are not able to attach to and to walk on a glass ceiling, and when foraging they move on food-plant branches (M.G., personal observation). They show a totally different behaviour compared to the immature instars. They are ground dwellers found inside hollows at the bases of living trees. With the last moult to adulthood, attachment devices undergo dramatic morphological changes of both male and female tarsi. The arolium is reduced to a very small whitish lobe (Fig. 1H). The diverse adhesive microstructures found on its surface in the nymphal stages are missing (Fig. 2B), even though densely set irregular cuticular outgrowths are present ventrally (Fig. 1I). The ventral surface of tarsomere 5 now bears a fully-developed elongated euplantula featuring a white glossy colour (Fig. 1H). Investigation of its surface showed that this structure represents a smooth-type attachment pad without distinct microornamentation (Fig. 1J), as it is typical for most related groups of insects (Beutel and Gorb, 2001). The presence of an attachment pad on the ventral surface of the apical tarsomere has been also reported in Timema stick insects (Tilgner et al., 1999) and in some cockroaches (van Casteren and Codd, 2010). The tarsal euplantulae 1e4 of the adults are also profoundly modified in their fine structure. The colouration is also white (Fig. 1H) and the surface smooth (Fig. 1K). The contact area of the adult euplantulae has lost the hairy coverage of acanthae found in the immature instars, and no cuticular microornament is present (Fig. 1L).
4. Conclusions Distinct ontogenetic modifications of tarsal and pretarsal attachment pads were not reported for any groups of insects before. In the case of the exceptionally large E. calcarata, the arolium is a transient adhesive structure functioning only in the immature instars adapted for climbing on leaf surfaces. In fact, the distinct switch in the life style between the leaf-dwelling nymphal stages and the ground-dwelling adults results in a final abrupt transformation of the adhesive apparatus, with the loss of function of the arolium as the most conspicuos change. During the nymphal development of E. calcarata, the increasing weight primarily results in an increased size of the attachment pads. Moreover, the gradual differentiation of an accessory euplantula on tarsomere 5 is probably mainly related to the concomitant progressive reduction of the arolium. It is conceivable that an additional attachment pad on tarsomere 5 may be a necessary adaptation to compensate for the reduction of the pretarsal arolium during growth. The functional significance of the 'switch-like' modifications that occur in the euplantulae of tarsomeres 1e4 with the transition from the last nymphal stage to the adult is less clear. In some species of canopydwelling stick insects, smooth euplantulae are present together with a functional arolium (Bußhardt et al., 2012; Gottardo and Vallotto, 2014). In the euplantulae of adult cockroaches, a colour change has been correlated with a change in pad properties during ageing (Ridgel et al., 2003). In E. calcarata, the euplantular colour change is accompanied by the loss of the acanthae. Further studies
are needed to assess the functional consequences of these modifications of the pad structure. From an evolutionary perspective, the developmental remodelling of attachment pads might reflect a phylogenetic pattern. The attachment devices of the earlier instars are similar to those found in Timema, the basalmost lineage of extant stick insects, and in various subgroups of the Euphasmatodea which are characterized by a very large pan-shaped arolium and a hairy surface of the tarsal and prestarsal attachment pads (Beutel and Gorb, 2008). This condition is unusual among the polyneopteran orders, but also occurs in the distantly related Mantophasmatodea (Beutel and Gorb, 2008). Conversely, the adults of E. calcarata show smoothtype attachment pads which also characterize adults of some other members of the Euphasmatodea (Gottardo, 2011; Bußhardt et al., 2012; Gottardo and Vallotto, 2014). Acknowledgements We thank two anonymous reviewers for helpful comments on the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.asd.2015.01.001. References Beutel, R.G., Gorb, S.N., 2001. Ultrastructure of attachment specializations of hexapods (Arthropoda): evolutionary patterns inferred from a revised ordinal phylogeny. J. Zool. Syst. Evol. Res. 39, 177e207. Beutel, R.G., Gorb, S.N., 2006. A revised interpretation of the evolution of attachment structures in Hexapoda with special emphasis on Mantophasmatodea. Arthropod Syst. Phylogeny 64, 3e25. Beutel, R.G., Gorb, S.N., 2008. Evolutionary scenarios for unusual attachment devices of Phasmatodea and Mantophasmatodea (Insecta). Syst. Entomol. 33, 501e510. Bradler, S., Robertson, J.A., Whiting, M.F., 2014. A molecular phylogeny of Phasmatodea with emphasis on Necrosciinae, the most species-rich subfamily of stick insects. Syst. Entomol. 39, 205e222. Bullock, J.M.R., Drechsler, P., Federle, W., 2008. Comparison of smooth and hairy attachment pads in insects: friction, adhesion and mechanisms for directiondependence. J. Exp. Biol. 211, 3333e3343. Bußhardt, P., Wolf, H., Gorb, S., 2012. Adhesive and frictional properties of tarsal attachment pads in two species of stick insects (Phasmatodea) with smooth and nubby euplantulae. Zoology 115, 135e141. Clemente, C.J., Federle, W., 2008. Pushing versus pulling: division of labour between tarsal attachment pads in cockroaches. Proc. R. Soc. B 275, 1329e1336. €lldobler, B., 2001. Biomechanics of the Federle, W., Brainerd, E.L., McMahon, T.A., Ho movable pretarsal adhesive organ in ants and bees. Proc. Natl. Acad. Sci. U. S. A. 98, 6215e6220. Federle, W., Endlein, T., 2004. Locomotion and adhesion: dynamic control of adhesive surface contact in ants. Arthropod Struct. Dev. 33, 67e75. Friedemann, K., Schneeberg, K., Beutel, R.G., 2014a. Fly on the wall e attachment structures in lower Diptera. Syst. Entomol. 39, 460e473. Friedemann, K., Spangenberg, R., Yoshizawa, K., Beutel, R.G., 2014b. Evolution of attachment structures in the highly diverse Acercaria (Hexapoda). Cladistics 30, 170e201. Gorb, S., 2001. Attachment Devices of Insect Cuticle. Kluwer Academic Publishers, Dordrecht. Gorb, S.N., 2007. Smooth attachment devices in insects: functional morphology and biomechanics. Adv. Insect Physiol. 34, 81e115. Gorb, S.N., Beutel, R.G., Gorb, E.V., Jiao, Y., Kastner, V., Niederegger, S., Popov, V.L., €tsch, W., 2002. Structural design and biomechanics Scherge, M., Schwarz, U., Vo of friction-based releasable attachment devices in insects. Integr. Comp. Biol. 42, 1127e1139. Gorb, S.N., Gorb, E.V., 2004. Ontogenesis of the attachment ability in the bug Coreus marginatus (Heteroptera, Insecta). J. Exp. Biol. 207, 2917e2924. Gottardo, M., 2011. A new genus and new species of Philippine stick insects (Insecta: Phasmatodea) and phylogenetic considerations. C. R. Biol. 334, 555e563. Gottardo, M., Heller, P., 2012. An enigmatic new stick insect from the Philippine Islands (Insecta: Phasmatodea). C. R. Biol. 335, 594e601. Gottardo, M., Vallotto, D., 2014. External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations. C. R. Biol. 337, 258e268.
M. Gottardo et al. / Arthropod Structure & Development 44 (2015) 195e199 Haas, F., Gorb, S., 2004. Evolution of locomotory attachment pads in the Dermaptera (Insecta). Arthropod Struct. Dev. 33, 45e66. Hsiung, C.-C., 1987. Aspects of the biology of the Melanesian stick-insect Eurycantha calcarata Lucas (Cheleutoptera: Phasmatidae). J. Nat. Hist. 21, 1241e1258. Labonte, D., Federle, W., 2013. Functionally different pads on the same foot allow control of attachment: stick insects have load-sensitive ‘heel’ pads for friction and shear-sensitive ‘toe’ pads for adhesion. PLoS One 8, e81943. Labonte, D., Williams, J.A., Federle, W., 2014. Surface contact and design of fibrillar ‘friction pads’ in stick insects (Carausius morosus): mechanisms for large friction coefficients and negligible adhesion. J. R. Soc. Interface 11, 20140034. Nelson, C., 2009. Surface ultrastructure and evolution of tarsal attachment structures in Plecoptera (Arthropoda: Hexapoda). Aquat. Insects 31, 523e545. Ridgel, A.L., Ritzmann, R.E., Schaefer, P.L., 2003. Effects of aging on behavior and leg kinematics during locomotion in two species of cockroach. J. Exp. Biol. 206, 4453e4465.
199
Roth, L., Willis, E., 1952. Tarsal structure and climbing ability of cockroaches. J. Exp. Zool. 119, 483e517. Schulmeister, S., 2003. Morphology and evolution of the tarsal plantulae in Hymenoptera (Insecta), focussing on the basal lineages. Zool. Scr. 32, 153e172. Stork, N.E., 1980. A scanning electron microscope study of tarsal adhesive setae in the Coleoptera. Zool. J. Linn. Soc. 68, 173e306. Tilgner, E.H., Kiselyova, T.G., McHugh, J.V., 1999. A morphological study of Timema cristinae Vickery with implications for the phylogenetics of Phasmida. Dtsch. Entomol. Z. 46, 149e162. van Casteren, A., Codd, J.R., 2010. Foot morphology and substrate adhesion in the Madagascan hissing cockroach, Gromphadorhina portentosa. J. Insect Sci. 10 (40), 1e12.