“ULTRASTRUCTURE AND FUNCTIONAL MORPHOLOGY OF ADHESIVE ORGANS AND ANTI-ADHESIVE PLANT SURFACES” Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Ingo Scholz aus Hameln Berichter: Universitätsprofessor Dr. Werner Baumgartner Universitätsprofessor Dr. Peter Bräunig Tag der mündlichen Prüfung: 01.12.2009 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar CONTENTS 1. INTRODUCTION ............................................................................................ 1 1.1 Biology of animal attachment.................................................................................... 4 1.1.1 Adhesive structures of insects ............................................................................ 4 1.1.2 Adhesive structures of tree frogs ........................................................................ 6 1.1.3 The influence of tarsal secretion on adhesion .................................................... 7 1.2 Biology of anti-adhesive surfaces............................................................................ 10 1.2.1 Anti-adhesive surfaces in pitcher plants ........................................................... 10 2. MATERIALS AND METHODS ....................................................................... 13 2.1 Study animals and plants ......................................................................................... 13 2.1.1 Stick insects (Carausius morosus) ................................................................... 13 2.1.2 Tree Frogs (Litoria caerulea) ........................................................................... 13 2.1.3 Ants (Lasius niger) ........................................................................................... 13 2.1.4 Pitcher plants (Nepenthes alata) ....................................................................... 14 2.1.5 Artificial surfaces ............................................................................................. 14 2.2 Morphology and ultrastructure ................................................................................ 15 2.2.1 Scanning Electron Microscopy (SEM) ............................................................. 15 2.2.2 Transmission Electron Microscopy (TEM) ...................................................... 16 2.2.3 Light Microscopy (LM) .................................................................................... 17 2.2.4 Confocal laser scanning microscopy (CLSM).................................................. 17 2.2.5 Focused ion beam treatment (FIB) ................................................................... 18 2.2.6 Confocal Multi-Pinhole microscopy (CMP) .................................................... 18 i 2.3 In vivo imaging and mechanical properties ............................................................. 19 2.3.1 Atomic force microscopy measurements on Carausius morosus .................... 24 2.3.2 Atomic force microscopy measurements on Litoria caerulea ......................... 27 2.3.3 Atomic force measurements on Nepenthes alata ............................................. 28 2.4 Interaction of adhesive organs and surfaces ............................................................ 29 2.4.1 Slip-off experiments ......................................................................................... 29 2.4.2 Adhesion and friction measurements by centrifugation ................................... 30 2.5. Chemical composition of the tarsal secretion ......................................................... 30 3. RESULTS .................................................................................................... 35 3.1 Morphology and ultrastructure ................................................................................ 35 3.1.1 The adhesive organ of Carausius morosus ...................................................... 35 3.1.2 The adhesive organ of Litoria caerulea ........................................................... 40 3.1.3 The conductive surface of Nepenthes alata ...................................................... 44 3.2. In vivo imaging and mechanical properties ............................................................ 48 3.2.1 Atomic force microscopy on Carausius morosus ............................................ 48 3.2.2 Atomic force microscopy of Litoria caerulea .................................................. 53 3.2.3 Atomic force microscopy of Nepenthes alata .................................................. 61 3.3 Interaction of adhesive organs and surfaces ............................................................ 64 3.3.1 Slip-off experiments ......................................................................................... 64 3.3.2 Adhesion and friction measurements ............................................................... 67 3.4 Chemical composition of tarsal secretion ................................................................ 70 4. DISCUSSION ............................................................................................... 73 4.1 Morphology and ultrastructure ................................................................................ 73 4.1.1 The adhesive organ of Carausius morosus ...................................................... 73 4.1.2 The adhesive organ of Litoria caerulea ........................................................... 75 ii 4.2 Mechanical properties ............................................................................................. 77 4.2.1 The adhesive organ of Carausius morosus ...................................................... 77 4.2.2 The adhesive organ of Litoria caerulea ........................................................... 80 4.3 The conductive surface of Nepenthes alata ............................................................. 83 4.3.1 General ............................................................................................................. 83 4.3.2 Influence of surface roughness on adhesion ..................................................... 84 4.4 Chemical composition of tarsal secretion ................................................................ 90 5. SUMMARY ................................................................................................. 93 REFERENCES ................................................................................................... 97 DANKSAGUNG .............................................................................................. 105 CURRICULUM VITAE ..................................................................................... 107 iii LIST OF FIGURES Figure 2.1: Principal setup of an atomic force microscope (AFM). ............................. 19 Figure 2.2: Vertical section of a 4-sided pyramidal indenter of an AFM. .................... 20 Figure 2.3: AFM measurements on Carausius morosus .............................................. 25 Figure 2.4: AFM measurements on Litoria caerulea ................................................... 27 Figure 3.1: SEM images of the tarsus and the arolium of Carausius morosus ............ 36 Figure 3.2: SEM images of the ultrastructure of the arolium cuticle ........................... 37 Figure 3.3: Section of the arolium cuticle in Carausius morosus, TEM images. ......... 38 Figure 3.4: Longitudinal rows of principal rods in the arolium cuticle (CLSM).......... 39 Figure 3.5: Litoria caerulea, overview and SEM images of the toe pad. ..................... 41 Figure 3.6: Nanostructural features of the toe pad epithelial cells................................ 42 Figure 3.7: Freeze-fracture image of a toe pad of Litoria caerulea .............................. 43 Figure 3.8: Morphology of a pitcher of the carnivorous plant Nepenthes alata.. ......... 44 Figure 3.9: FIB-cutting of pitcher plant material. ......................................................... 46 Figure 3.10: High resolution image of a FIB-polished Nepenthes alata surface. .......... 47 Figure 3.11: Topographic AFM images of the arolium in Carausius morosus. ............ 50 Figure 3.12: Force-distance curve of the indentation experiment on an arolium ......... 51 Figure 3.13: Thickness of the epicuticle as estimated by AFM measurements. ............ 52 Figure 3.14: 3D-reconstruction of the surfaces of toe pad epithelial cells. ................... 56 Figure 3.15: AFM height profiling of toe pad epithelium. ............................................ 57 Figure 3.16: Material stiffness of the toe epithelium as measured by the AFM. ........... 59 Figure 3.17: AFM images of the wax surface of Nepenthes alata. ............................... 62 Figure 3.18: AFM-tip scanning a wall-like wax structure. ............................................ 63 Figure 3.19: Carausius morosus tarsi after slipping off the waxy Nepenthes surface. .. 65 iv Figure 3.20: L. niger tarsi after slipping off the waxy Nepenthes surface. .................... 66 Figure 3.21: Adhesion force of Carausius morosus on different artificial surfaces. ..... 69 Figure 3.22: Gas chromatogram of a footprint sample .................................................. 71 Figure 4.1: Interactions of N. alata surface and an insect arolium and claw ............... 84 LIST OF TABLES Table 3.1: Surface profile parameters of the arolium of Carausius morosus....……...48 Table 3.2: Measurements of the stiffness of tree frog toe pad epithelium…................54 Table 3.3: Surface profile parameters of Nepenthes alata and an artificial surface….68 v 1. Introduction 1. INTRODUCTION The study of animal adhesion, for many years a relatively neglected area of research, has recently undergone a resurgence of interest. Partly this is because of the promise of practical applications arising from the research (“learning from the nature”, often referred to as bionics or biomimetics) of animal adhesive devices which have many properties material scientists envy. An example are the hexagonal structures on the tip of the toes of tree frogs which are discussed as potential models for the design for tires (Persson 2007). Another example are microstructured surfaces inspired by the setae of insect feet with application as adhesive tape and for adhesive structures on robots allowing the locomotion on smooth surfaces (Daltorio et al. 2005; Daltorio et al. 2007; Gorb et al. 2007). A strong reason for the increased interest in this field might also be that the application of recent advances in methods available in both biology and engineering (tribology) has produced a quantum leap forward in our understanding of adhesive systems (Barnes 2006; Scherge & Gorb 2001). The reversible adhesion of locomoting animals is particularly interesting: they need to combine strong adhesion with easy and rapid detachment whenever the animal makes a step (Federle et al. 2001). Animals living in and on the vegetation above the ground need to resist sudden mechanical impacts as for example gusts of wind or they have to escape quickly from predators without loosing grip and falling down (Eisner & Aneshansley 2000; Federle et al. 2000). This movement can only be possible possessing specialized adhesive organs on the feet of the animals with unique mechanical properties. Only this can provide both strong adhesion and also fast reversibility. Especially the micromechanical properties have not been described sufficiently until now. 1 1. Introduction Beside the mechanical properties, a fluid that is secreted into the contact zone between the adhesive organs and a surface plays an important role in animal adhesion (Hanna & Barnes 1991; Langer et al. 2004; Lees & Hardie 1988; Walker 1993; Zhang et al. 2007). Its influence on the adhesion of the animal (Bhushan 2003; De Souza et al. 2008; Drechsler & Federle 2006; Federle et al. 2006; Federle et al. 2004; Federle et al. 2002) and especially the chemical composition of the secretion and its physical properties has been discussed in resent studies (Betz 2003; Federle et al. 2002; Gorb 2001; Ishii 1987; Vötsch et al. 2002). They provide evidence that the adhesive secretion is an emulsion consisting of two phases with different physical and chemical properties (Federle et al. 2002; Vötsch et al. 2002). Some of the components of the secretion were identified so far but the results are inconsistent and therefore need to be revised. Mannyplants have developed specialized structures on their surfaces which permit or inhibit animals’ attachment or locomotion. Several plants adapted to nutrient-poor habitats for example capture, retain and digest arthropods to acquire additional nutrients (Juniper et al. 1989). Amongst those plants several effective structures as trapping mechanisms for making prey are already described, all based on special anti-adhesive surfaces (Bohn & Federle 2004; Gaume et al. 2002; Gaume et al. 2004; Gorb et al. 2005; Juniper et al. 1989; Lloyd 1942). Plants of the genus Macaranga are a further example for effective inhibition of adhesion. They have evolved protective “wax barriers” on their stems that are slippery for nearly all insects, except ants of some few sub-species of Crematogaster decacrema (Federle & Bruening 2006). The mechanisms inhibiting adhesion for animals on such surfaces are not sufficiently understood until now. This study will try to answer some of the questions that are still left on adhesive organs and anti-adhesive surfaces. The micro- mechanical properties of adhesive organs will be analysed in two different model organisms: the Indian stick insect (Carausius morosus) and the Australian tree frog (Litoria caerulea). These animals were chosen because they both provide adhesive organs that are described as “smooth pads” on the first view, but 2 1. Introduction reveal fundamental differences comparing their mechanical properties, morphology and (ultra-)structure of the adhesive organs. Furthermore the chemical composition of the liquid secretion of C. morosus will be analysed, especially its two-phase character. As an antagonistic system to the adhesive organs the anti-adhesive properties of the conductive zone of the pitcher plant Nepenthes alata will be part of this study. The study is therefore divided into four sections: (1) Characterisation of the morphology and ultrastructure of the adhesive organs in Carausius morosus and Litoria caerulea and the anti-adhesive surface of the conductive zone of Nepenthes alata. (2) Investigation of the mechanical properties of adhesive organs and the anti- adhesive surface with high spatial resolution. (3) Interaction of adhesive organs and anti-adhesive surfaces. (4) Characterisation of the chemical composition of the secreted liquid of Carausius morosus. 3 1. Introduction 1.1 BIOLOGY OF ANIMAL ATTACHMENT 1.1.1 Adhesive structures of insects Many insects possess specialized attachment organs on their legs which enable them to climb and run upside down on various substrates. Some species are not only capable of resisting extreme pull-off and shear forces equivalent to more than 100 times their own body weight (Eisner & Aneshansley 2000; Federle et al. 2000), but they can also run rapidly. The detailed underlying mechanisms of this impressive performance are still unclear, and some may be based on the detailed ultrastructure and physical properties of adhesive organs. Adhesive structures in arthropods and vertebrates have been classified as “smooth” pads with a soft cuticle or as “hairy” systems, i.e. pads densely covered with microscopic adhesive setae. Despite their microstructural similarity, adhesive pads in different insect orders are found at different positions of the leg, providing evidence for multiple evolutionary origins of these organs (Beutel & Gorb 2001; Beutel & Gorb 2006; Beutel & Gorb 2008). They can be located on different tarsal segments (e.g. euplantulae) and/or the pretarsus (e.g. as pulvilli or arolia). The cuticle of smooth pads differs structurally from typical hard exoskeleton cuticle and from the soft and flexible cuticle found in joints and extensible body parts (Reynolds 1975; Vincent 1981); its rod-like fibres are not arranged parallel to the surface, but are oriented at some angle to it. The arolium as an adhesive pad is probably an autapomorphy of the Neoptera (Beutel & Gorb 2006) and is homologous in the Dictyoptera, Phasmatodea and Orthoptera. Its morphology differs from the derived, unfold able arolia occurring in the Hymenoptera, Mecoptera and Trichoptera (Holway 1935; Snodgrass 1956). The basic non-foldable type of arolium (as it is found in Carausius morosus and all other Phasmatodea) has been investigated morphologically by several authors (Beutel & Gorb 2001; Beutel & 4