Daphnia carapace: form, function, structure and plasticity
Dissertation
to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology Ruhr University Bochum
International Graduate School of Biosciences Ruhr University Bochum Department of Animal Ecology, Evolution & Biodiversity
Submitted by Sebastian Kruppert From Frankfurt am Main, Germany
Bochum, October 2016
First Supervisor: Prof. Dr. R. Tollrian
Second Supervisor: Prof. Dr. D. Begerow
Daphnia Carapax: Form, Funktion, Struktur und Plastizität
Dissertation
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie der Ruhr-Universität Bochum
Internationale Graduiertenschule Biowissenschaften Ruhr-Universität Bochum Angefertigt am Lehrstuhl für Evolutionsökologie und Biodiversität der Tiere
vorgelegt von Sebastian Kruppert aus Frankfurt am Main
Bochum, Oktober 2016
Referent: Prof. Dr. R. Tollrian
Korreferent: Prof. Dr. D. Begerow
„In den kleinsten Dingen zeigt die Natur die allergrößten Wunder“
Carl von Linné
Für meine Familie
Table of contents
Table of contents
Introduction ...... 8
Predators and prey ...... 9 Permanent defenses ...... 10 Inducible defenses...... 13 Inducible defenses in Daphnia ...... 17 Functionality of inducible morphological defenses ...... 20 Objectives ...... 21
1. Carapace structure ...... 22
2. Structure and shape contribution to carapace resistance ...... 23 Biomechanical methods within an ecological framework ...... 23
3. The catching basket of Chaoborus ...... 25
Materials and Methods ...... 27
1. Carapace structure ...... 28 Experimental organisms ...... 28 In vivo light microscopy ...... 28 Whole mounts stained with hematoxylin and eosin ...... 28 Scanning electron microscopy ...... 29 Thin sections ...... 30 Ultra-thin sections and scanning transmission electron microscopy ...... 30 Hemolymphatic gauge pressure...... 31
2. Structure and shape contribution to carapace resistance ...... 33 Experimental organisms ...... 33 Induction of morphological defenses in D. pulex and D. longicephala...... 33 Microscopy, modelling and simulation ...... 34
Daphnia carapace: form, function, structure and plasticity 5 Table of contents
Procuticle analysis ...... 34 Procuticle Young´s modulus ...... 35 Carapace structure and finite element analysis ...... 38 Optical imaging of carapace shape ...... 41 Carapace shape modelling and finite element analysis...... 46 Structural Young’s-modulus and critical force ...... 48
3. The catching basket of Chaoborus ...... 50 Experimental Organisms and induction of the daphniids defenses ...... 50 High speed recordings ...... 51 microCT imaging and 3D reconstruction ...... 51 Surface mesh and animation...... 52 Feeding Experiments ...... 53 Bite force estimation ...... 53
Results ...... 56
1. Carapace structure ...... 57 Morphology of the carapace and distribution of the pillars ...... 57 Ultrastructure of the pillars ...... 64 Hemolymphatic pressure ...... 64
2. Structure and shape contribution to carapace resistance ...... 66 Structure ...... 66 Shape ...... 71
3. The catching basket of Chaoborus ...... 76 The catching process ...... 76 Morphology of the catching basket ...... 78 The catching basket ...... 80 The Antennae...... 80 The labral setae and prelabral appendage ...... 81 The mandibles ...... 81 The mandible muscles ...... 82 The Mandible joint...... 82
Daphnia carapace: form, function, structure and plasticity 6 Table of contents
The mandibular fan ...... 82 The rhinopharynx...... 83 Animation of the catching event ...... 83 Efficiency of the catching basket ...... 85 Bite force estimation ...... 87
Discussion ...... 89
1. Carapace structure ...... 90 Carapace proximal and distal integument structure ...... 90 Interconnecting pillars...... 92 Pillar function ...... 93
2. Structure and shape contribution to carapace resistance ...... 96 Structure ...... 96 Shape ...... 97 Conclusion ...... 99
3. The catching basket of Chaoborus ...... 100
Concluding Discussion ...... 104 Outlook ...... 105
References ...... 106
Summary ...... 119 Zusammenfassung ...... 123
Appendix ...... 126
Erklärung ...... 127
List of Publications ...... 128
Manuscripts’ contributions ...... 129
Curriculum Vitae ...... 131
Danksagung ...... 134
Daphnia carapace: form, function, structure and plasticity 7 Introduction
Introduction
Daphnia carapace: form, function, structure and plasticity 8 Introduction
Introduction
Predators and prey Predation is a major factor driving evolution as it is a strong selective agent. Therefore, prey organisms have evolved a vast diversity of defense strategies. Defensive strategies interrupt the repetitive predation sequence of encounter, detection, attack, handling and consumption at some point (Fig 1). Some prey species permanently express their defenses, whereas some defenses are developed only when needed i.e. in the case of seasonal-dependent fluctuations of the predator population.
Figure 1: Schematic display of the predation cycle. When a predator encounters a prey organism effective detection is crucial to launch an attack. If the attack is successful the prey is handled to prepare its consumption. Any interruption of this cycle by the prey can be considered as a defense since it prevents the cycle’s final step, i.e. consumption.
Daphnia carapace: form, function, structure and plasticity 9 Introduction
Permanent defenses Prey species that are under a constant predation risk, often show permanently expressed defenses. One common form of permanent defense is predator avoidance realized in activity anticyclical to that of main predators i.e. diurnal, nocturnal or crepuscular behavior (Speakman, 1995; McCauley et al., 2012; Monterroso et al., 2013). In addition to predator avoidance other behavioral patterns can increase survival chances in prey species upon encounter. When encountering a predator, prey species often react with fight or flight (Cannon, 1932). This can be regarded as behavioral defenses. These defenses often rely on morphological adaptations to enhance either defensiveness (claws, spines, stings etc.) or agility (speed and maneuverability). Other defensive strategies operating during encounter are camouflage, mimesis and mimicry. Camouflage describes a morphological adaptation that makes an animal less detectable in its natural habitat. While this phenomenon is mostly studied in terms of visual perception (Cott, 1940; Norris and Lowe, 1964; Stevens and Merilaita, 2009) it also includes all other senses (Ruxton, 2009). Illustrative examples are animal coloration and patterning observable in e.g. grouse, stonefish, arctic fox or the countershading of marine fish. Animals using mimesis adapt to be perceived as another organism or an object less attractive or even inedible for the predator. Especially in invertebrates eye-catching examples for mimesis can be observed: caterpillars mimicking branches, grasshoppers mimicking dead or living leafs etc. (Wallace, 1870). Both, camouflage and mimeses are morphological defenses (usually combined with behavior) against the predator’s ability to detect its prey. Hence, an encounter of predator and prey does not lead to a predatory attack if camouflage or mimeses effectively protects the prey against detection. On the other hand camouflage and mimeses are also used by predators to delay their detection by prey (e.g. tigers) or even attract prey (e.g. frogfish). Finally, mimicry describes the imitation of a harmful animal and is subdivided into two major types depending on whether the imitator itself is harmful (Müllerian mimicry) or not (Batesian mimicry)(Bates, 1861; Müller, 1864). Eventually there are types of defense that operate during or even after a predatory attack. Those are generally physiological or morphological and can be developed by animals as well as plants. Physiological defenses are commonly based on toxins that render the prey unattractive, inedible or poisonous and are either produced by the prey itself, e.g. skunks (Mephitidae), fire salamander (Salamandra salamandra), true stink bugs (Pentatomidae), nettles (Urtica dioica) and giant hog weed (Heracleum mantegazzianum) or collected while
Daphnia carapace: form, function, structure and plasticity 10 Introduction food uptake e.g. poison dart frogs (Dendrobatidae), some pufferfish (Tetraodontidae), milkweed butterflies (Danainae), and many more (Thurston and Lersten, 1969; Andersen and Bernstein, 1976; Aldrich, 1988; Boppré, 1990; Daly et al., 1994; Mebs and Pogoda, 2005; Noguchi et al., 2005; Quinn et al., 2014). Morphological defenses reduces the prey’s vulnerability by rendering it hard or impossible to handle or consume. Such defenses can be realized in spines, thorns, long appendages or body armor. The most famous examples for a spine wearing animals might be the hedgehog (Erinaceinae) and the porcupine (Hystricidea/Erethizontidae) but spine/thorn- based defense is found in various organisms e.g. sea urchin (Echinoidea), porcupinefish (Diodontidae), the spiny spider crab (Maja squinado), many rosaceae and acacieae. Spines or thorns render these organisms harmful prey and enforce specialized predator adaptations (Tegner and Levin, 1983; Neal, 1986; Duarte et al., 2003). Related to spines and thorns are long appendages as a defense structure that hamper prey handling or ingestion. Lepidopteran larvae for example often possess body appendages or hair-like structures to defend against predators and parasites (Greeney et al., 2012). Long appendages as well as spines or thorns often incorporate toxins or irritants e.g. Lasiocampa quercus, saddleback caterpillar (Acharia stimulea), oak processionary (Thaumetopoea processionea), wasps (Vespinae), stonefish (Synanceiidae) and recently have been verified even for mammals (Austin et al., 1965; Mortari et al., 2005; Hossler, 2009; Kingdon et al., 2012). The presumably most prevalent form of defense against a predatory attack is body armor. Body armor is found in single cell eukaryotes like diatoms and coccolithophores, in bivalves, most fishes, since the majority possesses scales, turtles and in the whole phylum of arthropods, whose exoskeleton in most cases provides armor (for review see Naleway et al., 2016). It is realized in different ways throughout the taxa ranging from numerous, small single structures overlapping each other (coccolithophores’ coccoliths, fish scales) up to large continuous structures (turtle shells, arthropod exoskeleton) sheltering either the whole organism or vulnerable body parts. The materials used to build body armor are often a composition of an organic matrix (either a polysaccharide e.g. cellulose or chitin or fibrous protein like keratin), often crosslinked via proteins (in arthropods: sclerotization) and sometimes reinforced with minerals (biomineralization) (Meyers et al., 2008). Coccolithophores possess a body armor composed of shield like structures i.e. coccoliths. These consist of cellulose which is reinforced by calcium carbonate (Mann and Sparks, 1988). Bivalves develop their shells from calcite and/or aragonite (calcium carbontes) and some
Daphnia carapace: form, function, structure and plasticity 11 Introduction species possess an inner layer of nacre, which is a chitin matrix, crosslinked by proteins and containing plates of aragonite (Fritz and Morse, 1998; Blank et al., 2003; Rousseau et al., 2009). Turtle shells consist of the back shield (carapace) and the belly shield (plastron) that are connected via bony bridges (Burke, 1989; Cebra-Thomas et al., 2005; Nagashima et al., 2013). While the inner compartments of the turtle shell are bones, the outer compartments, lacking in some species, are made of keratin secreted from the epidermis. The phylum arthropod shares the feature of an exoskeleton that, in most species, functions as body armor. The arthropod exoskeleton is composed of a chitin matrix containing different proteins depending of the specific function of the body part (Vincent, 2002). In joints, for example, the chitin matrix is flexible and expandable due to high amounts of the protein resilin (Gorb, 1999; Haas et al., 2000; Neff et al., 2000). Whereas exoskeletal elements operating as body armor are hardened either by sclerotization (insects) or biomineralization (crustacea) (Roer and Dillaman, 1984; Hopkins and Kramer, 1992).
Daphnia carapace: form, function, structure and plasticity 12 Introduction
Inducible defenses Inducible defenses are defined as phenotypically plastic adaptations that reduce the predation risk in the presence of a predator (Tollrian and Harvell, 1999). These phenotypic changes can evolve under the selective pressure of predators, parasites, herbivores, pathogens or competitors, if following prerequisites are fulfilled (Adler and Harvell, 1990; Tollrian and Harvell, 1999):
1. The selective pressure of the inducing agent has to be variable and unpredictable, but
sometimes strong.
2. A reliable cue is necessary to indicate an explicit risk of predation, which
subsequently activates the defense.
3. The defense must be effective.
4. Inducible defenses incur costs that are saved when the defense is superfluous.
Inducible defenses are a common phenomenon found in unicellular organisms, animals and plants. In plants it is often referred to as resistances. Some plants are able to react on herbivore or pathogenic threats either chemically, like the brown seaweed (Ascophyllum nodosum) that increases cell phlorotannin concentration to effectively thwart grazing snails (Pavia and Toth, 2000), or morphologically, like the grey alder (Alnus incana) that develops leaf trichomes as a response to insect grazing (Baur et al., 1991). These reactions can occur within hours, in case of small injuries (Green and Ryan, 1972; Schmelz and Baldwin, 1996), or be expressed throughout the following year, for example as a response to massive defoliation (Haukioja, 1980). The expression of these reactions can be either localized in tissues surrounding the injury (localized response), or as a systemic response in the whole plant (Berryman, 1988). One eye-catching example of localized morphological adaptions in plants is the European holly (Ilex aquifolium), which increases leaf spinescence coherent with a decrease in leaf size at branches browsed by ungulates (Obeso, 1997). Examples for systemic morphological response to herbivores are increased thorn length in Acacia (El Din and Obeid, 1971; Young, 1987; Milewski et al., 1991), stimulated spine production in Opuntia stricta (Myers, 1987) and increased trichome density in Urtica dioica (Pullin and Gilbert, 1989). The wild parsnip (Pastinaca sativa) is well studied for its ability to produce xanthotoxin, a phototoxic furanocoumarin (a class of organic compounds that are very reactive and often toxic), to
Daphnia carapace: form, function, structure and plasticity 13 Introduction thwart herbivores (Zangerl and Berenbaum, 1994). Furthermore, many plants are able to attract predators as an indirect defense against arthropod herbivores (for review see Price et al., 1980; Dicke, 1994). In animals inducible defenses are widespread and verified for many taxa including protozoans as well as vertebrates. Protozoans, like ciliates, respond to predation with a modification of morphology and behavior. Some ciliate species develop lateral “wings” and dorsal as well as ventral projections that hamper ingestion by predators and show flight behavior during predator encounter (for review see Heckmann, 1995). In vertebrates the immune system can be considered as an inducible defense, as it reacts effectively on pathogens and parasites (for review see Frost, 1999). Generally animal inducible defenses are categorized into three types i.e. behavioral defenses, shift of life-history parameters and morphological defenses. All these defenses can act individually or in concert to effectively counter a range of predators. While behavioral defenses represent a response that can be instantly activated, morphological defenses and life- history-shifts are developed with a time lag. One well studied example of behavioral defenses is diel vertical migration of zooplankton for predator avoidance (Stich and Lampert, 1981). While many zooplankter typically stay in surface water for grazing during the day-time, predator presence induces a cyclical migration pattern. Thereby prey species avoid predator encounter by residing in the deep water strata during the day time and migrate to the nutritional strata during the night when visual predation is limited. In regimes characterized by invertebrate (non-visual) predators and vertebrate predators (e.g. fish) reverse diel vertical migration was observed (Ohman et al., 1983). In such regimes the predation pressure is independent from light availability and thus the nutrient rich surface pattern are grazed by zooplankters during day time while larger, predatory invertebrates avoids predation by fish in deep water strata. Predator avoidance behavior in zooplankton is not limited to vertical migration but also includes horizontal migration when shallow/coastal waters offer safe refuges like abundant vegetation or reefs (Timms and Moss, 1984; Benoit-Bird et al., 2001; Burks et al., 2002). Other examples of predator induced behaviors are prey-aggregation (Johannes, 1993; Reimer and Tedengren, 1997; Watt et al., 1997; Coleman et al., 2004; Kobak and Kakareko, 2011), increased prey-vigilance, e.g. increased head-lifting duration in grazing impalas (Aepyceros melampus) (Blanchard and Fritz, 2007), and reduced activity (Relyea, 2001; Strobbe et al., 2011; van Uitregt et al., 2013).
Daphnia carapace: form, function, structure and plasticity 14 Introduction
Predator induced life-history-shifts can be seen in adaptive resource allocation shifts where somatic growth is traded with reproduction, so that animals may have a smaller size at maturity (Crowl and Covich, 1990; Stibor, 1992; Lardner, 2000), early hatching time (Chivers et al., 2001) and reduced offspring size, number or altered offspring morphology (Skelly and Werner, 1990; Dixon and Agarwala, 1999). Shifts in somatic growth rate delay or outpace a predator preferred body size. Likewise changes in hatching time, offspring size or morphology operate against predator preferences, while changes in offspring number most likely represent defense’ costs. Morphological defenses are striking examples among the predator induced responses and are described for numerous taxa. Generally these defenses hamper handling and/or ingestion by coexisting predators by exceeding predator gape limits, mouthpart interference or crushing prevention. Examples for vertebrate morphological inducible defenses are anuran tadpoles of different species that increase their tail depth and pigmentation in response to the predatory dragonfly larvae. This may enhance escape chances by directing dragonfly larvae attacks away from the vulnerable head to the tail (Van Buskirk and Relyea, 1998; Relyea, 2001). Morphological defenses are also reported for the crucian carp (Carrasius carrasius) that alters its body shape thwarting predation of the gape limited pike (Esox lucius) (Brönmark and Miner, 1992) and the stickleback (Gasterosteus aculeatus) which responds to perch predation with an increased body depth as well as spine length and body asymmetry in juvenile stages (Frommen et al., 2011). In invertebrates a large variety of morphological inducible defenses is reported. The mussel Mytilus edulis as well as the snail Littorina increase their shell thickness and Mytilus additionally produces more byssal threads enhancing substrate attachment when encountering Carcinus maenus predation (Trussell, 1996; Leonard et al., 1999). Dragonfly larvae increase their abdominal spines and thicken their exoskeleton when hunted by perch (Johansson and Samuelsson, 1994; Mikolajewski and Johansson, 2004; Flenner et al., 2009), barnacles of the genus Chthamalus alter shell and operculum shape to thwart predation by rock snails (Muricidae) (Lively, 1986; Jarrett, 2009) and mayfly larvae enlarge their caudal filaments to reduce predation pressure caused by fish (Dahl and Peckarsky, 2002). Especially the genus Daphnia (Crustacae, Cladocera) shows a vast array of inducible morphological defenses. Different species of Daphnia exhibits elongated tail spines, develop head spines, “neckteeth” (little thorns in the neck region), crests, a crown of thorns or so called “helmets” (Krueger and Dodson, 1981; Dodson, 1989; Tollrian, 1990; Spitze and Sadler, 1996; Laforsch et al., 2009). Furthermore, some species are verified to accompany
Daphnia carapace: form, function, structure and plasticity 15 Introduction these morphological defenses with additional thickening of the body armor i.e. the carapace (Laforsch et al., 2004; Rabus et al., 2013; Rabus, 2015).
Daphnia carapace: form, function, structure and plasticity 16 Introduction
Inducible defenses in Daphnia The freshwater crustacean Daphnia is a dominant filter feeder in most lentic ecosystems. With its high abundances and its herbivore ecology Daphnia plays a key role being a link between primary producers and the higher trophic levels. It thus falls prey to various predators ranging from other crustaceans, insects and insect larvae up to juvenile fishes (Zaret, 1972; Dodson, 1974). Within the few predaceous cladoceran genera Leptodora and Bythotrepes prey upon Daphnia (Sprules et al., 1990; Laforsch and Tollrian, 2004). These predaceous cladocerans possess an enlarged complex eye, reduced carapace and legs optimized for prey capture. Another crustacean predator hunting for Daphnia is the “living fossil” Triops, a representative of the Notostraca populating mainly temporary ponds (Boix et al., 2006; Petrusek et al., 2009; Rabus and Laforsch, 2011). Predatory insects feeding on Daphnia are e.g. the backswimmer Notonecta, a sit-and-wait hunting heteropteran living in ponds, and larvae of the phantom midge Chaoborus (Dodson and Havel, 1988; Black, 1993). Predation of fish on Daphnia is regulated by the fish’s prey preferences which in most cases correlate with its body size (Galbraith Jr., 1967). Small fishes like Gasterosteus or Rhodeus prey for Daphnia whereas comparatively big fishes e.g. Perca or Cyprinus only in juvenile stages prey for Daphnia (Hansen and Wahl, 1981; Černý and Bytel, 1991; Mills and Reynolds, 2004; Yin et al., 2011). Daphniids thwart their coexisting predators with a set of inducible defenses including behavior, life-history-shifts and morphological alterations. Within the behavioral defenses the most popular is the diel vertical migration (Dodson, 1989) but behavioral defenses have also been reported in forms of adaptive swimming behavior (for review see Uttieri et al. 2014). Life-history-shifts reported in daphniids are summarized in the model of Taylor and Gabriel i.e. the resource investments into somatic and population growth changes with different predator regimes (Taylor and Gabriel, 1992): In dependence of the predators prey size preferences, invertebrate predators induce higher somatic growth rates at the expense of a delayed maturity, whereas vertebrate predators induce higher reproduction rates and early maturity but reduced somatic growth. This model has been confirmed for Daphnia in several predator-prey systems: D. galeata/Rutilus rutilus (Machacek, 1991), D. hyalina/Leuciscus idus, Chaoborus flavicans and Notonecta glauca (Stibor, 1992; Stibor and Lüning, 1994), D. magna/fish and Notonecta (Weider and Pijanowska, 1993) and D. pulex/Lepomis macrochirus, Chaoborus americanus and Notonecta undulata (Dodson and Havel, 1988; Dodson, 1989). Among the inducible defenses the morphological defenses are the most
Daphnia carapace: form, function, structure and plasticity 17 Introduction obvious and numerous variations are described in Daphnia, each as response to a coexisting predator (Fig 2). The morphological defenses in Daphnia reach from large morphological alterations like the “crests” in D. longicephala (Barry, 2000), “helmets” in D. cucullata (Tollrian, 1990) and D. lumholtzi (Tollrian, 1994) to minute defense structures like the “neckteeth” in D. pulex (Krueger and Dodson, 1981; Spitze and Sadler, 1996) and D. halyina (Brancelj et al., 1996).
Daphnia carapace: form, function, structure and plasticity 18 Introduction
Figure 2: Predator induced defenses in different Daphnia species. A: Predator Gasterosteus aculeatus (three-spined stickleback) that induces helmets in D. lumholtzi. B: Undefended D. lumholtzi. C: Defended D. lumholtzi with remarkably elongated head and tail-spines. D: The invertebrate predator Chaoborus obscuripes commonly described to induce defenses in D. pulex. E: Undefended D. pulex compared to F: defended D. pulex carrying neckteeth in the dorsal head region. G: Insert shows magnification of neckteeth displayed in (F). Likewise, Chaoborus induces helmet development in D. cucullata: H: Undefended D. cucullata I: Defended D. cucullata with helmet and elongated tail spine. J: The backswimmer Notonecta glauca induces morphological defenses in D. longicephala. K: The undefended D. longicephala morphotype is small and inconspicuous in comparison to the defended morphotype. L: Defended D. longicephala grow large crests as well as elongated tail spines. M: The ancient predator Triops cancriformis that induces defenses in D. barbata. N: D. barbata (here: undefended form) develops defense modalities adapted to the predation regime. O: Notonecta-defended D. barbata develop larger and straight helmets in comparison to (N) and to P: the Triops-defended morphotype which has larger and backwards-bending helmets and tail- spines. Courtesy of L.C. Weiss.
Daphnia carapace: form, function, structure and plasticity 19 Introduction
Functionality of inducible morphological defenses The morphological defense structures of Daphnia are discussed to work in an “anti-lock-and- key” manner, interfering with the predators mouth-parts or catching structures and thus hampering capture or ingestion (Dodson, 1974). While this hypothesis is quite conceivable for prominent structures like D. longicephala’s crests, it appears questionable for the unimposing neckteeth of D. pulex. Considering Chaoborus’ complex catching basket they might be interfering with the catching basket’s elements. Nevertheless, the anti-lock-and-key- hypothesis has never been confirmed experimentally. For D. pulex, D. cucullata and D. magna it has been shown, that the obvious defense structures are accompanied by alterations of the carapace structure leading to changes in material properties (Laforsch et al., 2004; Rabus et al., 2013; Rabus, 2015). The carapace is an integumental fold sheltering thorax and abdomen (Fryer, 1996). Thus, its structure is a double layer of integument composed in reverse complement manner and interconnected by so called pillars connecting the two integuments through the hemolymphatic space in between (Anderson, 1933; Fryer, 1996). The alterations in carapace material properties, i.e. stiffness, are hypothesized to act in concert with the obvious morphological defenses (Laforsch et al., 2004; Rabus et al., 2013; Rabus, 2015). However, the morphological defenses have been proven to be effective by reducing the predators success (Krueger and Dodson, 1981; Havel and Dodson, 1984; Parejko and Dodson, 1991) but the underlying mechanism is still undetermined.
Daphnia carapace: form, function, structure and plasticity 20 Introduction
Objectives This thesis aimed to analyze two well studied predator-prey systems for their morphological defenses in detail to illuminate the mechanism of the defensive effect and to verify the anti- lock-and-key hypothesis. The predator-prey systems used in my project were D. pulex/Chaoborus and D. longicephala/Notonecta. The morphological defenses of the two Daphnia species are well studied and have been proven as effective defensive strategies. However, how this is accomplished remained elusive. Even though the anti-lock-and-key hypothesis is comprehensible at least for “crests” in D. longicephala it is rather counterintuitive for the inconspicuous “neckteeth” expressed by D. pulex. In order to elucidate the mode of action of predator induced morphological defenses, I addressed the following questions:
1st Part: Carapace structure
- What is the role of the carapace structure (i.e. the pillars) in the mechanics
of its resistance?
- Are the pillars optimized for an uptake of compression forces?
2nd Part: Structure and shape contribution to carapace resistance
- Are the shape-alterations (obvious morphological defenses) involved in the
mechanical properties of carapace resistance?
- Do the shape-alterations and the structure-alterations act in concert or
independently?
3rd Part: The catching basket of Chaoborus
- What is the detailed composition of the Chaoborus feeding basket and how
do the parts act during the motion sequence of prey capture?
- Determine the defenses’ mode of action during Chaoborus predation,
testing the anti-lock key hypothesis.
Daphnia carapace: form, function, structure and plasticity 21 Introduction
1. Carapace structure1
The carapace is an evagination of the integument extending from the cephalic region (Fryer, 1996). The crustacean integument is arranged in layers (Stevenson, 1985). The most distal layer, termed epicuticle, is very thin and delimits the body from the surrounding medium. The adjacent layer is the procuticle – generally subdivided into the exo- and endocuticle in crustaceans, although these are indistinguishable in daphniids. These cuticle layers are secreted by the underlying epidermal cells (Stevenson, 1985), which are interconnected by the extracellular matrix. As the daphniid’s carapace is an integumental fold, it possesses two integumental layers piled in a reverse complement manner (Halcrow, 1976). The space between the layers is filled with hemolymph (Anderson, 1933; Halcrow, 1976), and they are interconnected by irregularly dispersed structures, so-called pillars (Anderson, 1933; Fryer, 1996; Halcrow, 1976). Laforsch et al. (2004) showed that the adapted neckteeth morphotype is also characterized by a fortification of the whole body armor as well as a measured increase in pillar diameter. This led to the hypothesis that pillars were responsible for the observed carapace fortification (Laforsch et al., 2004). The mechanism by which these pillars could provide cuticle reinforcement was not determined. However, this information is crucial to understand the protective effect of the hidden morphological defenses in D. pulex.
In order to analyze the protective capacity of the carapace structure, we aimed to determine how the pillars are linked to the integuments of the carapace and describe the integument’s morphology in detail. We used different imaging techniques (light microscopy, transmission and scanning electron microscopy) to analyze the morphology of both the proximal and distal integuments of the carapace and the interconnecting pillars.
1 Part 1 is published in: Kruppert, S; Horstmann, M.; Weiss, L.C.; Schaber, C.F.; Gorb, S.N.; Tollrian, R. “Push or pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression” Journal of Morphology, 277, pp. 1320-1328
Daphnia carapace: form, function, structure and plasticity 22 Introduction
2. Structure and shape contribution to carapace resistance
The precise contribution of shape and structural alterations to mechanical resistance against a predation event remained undetermined. In this study, we aimed to analyse the predator induced changes of overall morphological shape and carapace structure for their effects under mechanical impact of predator attacks. In addition we wanted to distinguish between the contributions of shape and structure to the carapace’ mechanical resistance and examine whether they work in concert or independently. For this purpose, we measured carapace stiffness using bioindentation at two scales i.e. the procuticle stiffness and (nanoindentation) the geometric stiffness of the shape (microindentation). While atomic force microscopy (AFM) that is normally used as surface imaging tool is capable of imaging surfaces at nm scale, it also can be used to conduct nanoindentation measurements at a µN range of forces. We determined the procuticle Young’s-moduli (a measure of stiffness) using atomic force microscopy (AFM) driven in “contact mode”. With this method we were able to prove whether D. longicephala bear an increased carapace stiffness in predator presence too. To determine the structural Young’s-moduli (the geometric stiffness) of the overall carapace shape, we used micro-indentation. These data were used together with exact morphometric measurements, to further create models for physical simulations i.e. finite element analysis (FEA). The morphometric measurements were based on images of carapace structure, conducted with scanning transmission electron microscopy (STEM), and overall morphological shape, conducted with confocal laser scanning microscopy (CLSM). With the combination of empiric results and simulation we are now able to distinguish explicit aspects of the investigated defences i.e. the shape and the material structure.
Since FEA is not very common in ecological research it will be introduced in the following section.
Biomechanical methods within an ecological framework Finite-element analysis (FEA) is a mathematical approach to solve physical problems for complex bodies. The idea is to fragment the complex body into several geometrical bodies (finite in number) with a shape easier to describe. With that fragments the physical problem can be solved step by step, the complex problem thus is transformed into several simple, but solvable, “sub”-problems. While the mathematical basis of this method lays in the late 19th century (Schellbach, 1852; Kirsch, 1868). Its commercial use started in the second half of the
Daphnia carapace: form, function, structure and plasticity 23 Introduction last century (Turner et al., 1956) and the applicability rose with the advance of computational power and accessibility. Today it is a common method in engineering and orthopedics (for review see Rayfield, 2007). Although FEA offers considerable opportunities for biological questions, especially in functional morphology, its application is still rare in biological sciences. E.g. FEA facilitates the analysis of stress distribution in bone-/ or cranial structures during mechanical impact and thus gives insights to the principles of morphological design. For bones and skulls of vertebrates some work, using FEA, has been done so far (e.g. Guillet et al., 1985; Erickson, 2001; Rayfield et al., 2001; Rayfield, 2004; Witzel and Preuschoft, 2005; McHenry et al., 2006; Scheyer et al., 2007; Wroe et al., 2008, 2010; Degrange et al., 2010; Rivera and Stayton, 2011; Sander et al., 2011) but there are just a few publications found for analysis of invertebrate morphology (Philippi and Nachtigall, 1996; Kesel et al., 1998; Hassan et al., 2002; Hamm et al., 2003; Dai and Gorb, 2009; van der Meijden et al., 2012; Lian and Wang, 2013; Bar-On et al., 2014). Although, the potential to analyze morphologic structure of invertebrates that are intricately or impossible to measure directly is eminent. Especially, when FEA is conducted using empirical determined material properties of the analyzed structure, namely Young’s-modulus and Poisson ratio.
Daphnia carapace: form, function, structure and plasticity 24 Introduction
3. The catching basket of Chaoborus
Larvae of the phantom midge Chaoborus (Diptera, Nematocera) populate lentic freshwater ecosystems and are a food source for many fish species (von Ende, 1979). After hatching from eggs they develop trough four larval instars before they pupate and leave the water as imago. During the larval instars Chaoborus feed on zooplankton as an ambush predator. One anterior and one posterior pair of air sacks enable the larvae to stay in horizontal position and change altitude by inflating or deflating gas (Krogh, 1911; Damant, 1924; Teraguchi, 1975). All instars possess fully developed complex eyes but detect incoming prey via mechano- sensory sensillae (Swift and Forward Jr., 1981; Melzer and Paulus, 1994; Wohlfrom and Melzer, 2001). While in 1st and 2nd instar the larvae are limited to small zooplankter like ciliates in the 3rd and 4th instar they mainly feed on copepods and juvenile Daphnia (Crustacea, Cladocera; Swift and Fedorenko, 1975). Daphnia pulex develops so called neckteeth in early instars in which they match the larvae’s size preferences (Krueger and Dodson, 1981; Havel and Dodson, 1984; Parejko and Dodson, 1991). Another Daphnia species developing inducible defenses in the presence of Chaoborus larvae is D. cucullata which thwart Chaoborus with prominent “helmets”. These helmets and neckteeth, like other inducible defenses described for daphniids, are discussed to work in an anti-lock-and-key manner by interfering with the predators mouthparts or prey catching devices (Dodson, 1974). In case of Chaoborus larvae the catching devices are realized in a unique catching basket build by the larvae’s head appendages. The appendages that build this catching basket are morphologically described in detail although the movements and function of the appendages in the catching basket are only hypothesized due to the extremely fast catching event (Schremmer, 1950). However, detailed knowledge about the catching motion and the appendages’ participation in the catching event is crucial to understand the predator prey relationship of D. pulex and Chaoborus. The daphniids defenses have been shown to be effective but their mode of action remained undetermined (Krueger and Dodson, 1981; Havel and Dodson, 1984; Parejko and Dodson, 1991). Therefore, we studied the larvae’s head morphology in 3D using a µCT and analyzed this 3D data in conjunction with high-speed video recordings of the catching event. We here present a high resolution 3D head morphology motion sequence during the catching event to illuminate mechanical predator/prey interactions. Furthermore, we analyzed the high speed recordings for the daphniids defense effectiveness and conducted feeding experiments to distinguish between pre- and post-catch effect of these defenses. Finally, we estimated Chaoborus bite force using
Daphnia carapace: form, function, structure and plasticity 25 Introduction morphological data from the microCT scan as well as cross sections to assess the resistance necessary to withstand this threat.
Daphnia carapace: form, function, structure and plasticity 26 Materials and Methods
Material and Methods
Daphnia carapace: form, function, structure and plasticity 27 Materials and Methods
Materials and Methods
1. Carapace structure
Experimental organisms D. pulex (Leydig, 1860), clone R9 (originating from Canada) and D. longicephala (Hebert, 1977), clone LP1 (from Lara Pond, Australia) were cultured under constant conditions with a day:night cycle of 16:8 h at 20 °C ± 0.1 °C in a climate chamber. Both species were cultured in 1 l glass beakers containing charcoal-filtered tap water and fed ad libitum with the algae Scenedesmus obliquus.
In vivo light microscopy Six adult D. pulex were analyzed using light microscopy (LM) to assess the appearance of pillars at the dorsal keel. The animals were transferred to an object slide (Menzel GmbH & Co KG, Braunschweig, Germany) with a drop of water. Images were taken with a Colorview III camera (Olympus Soft Imaging Systems, Münster, Germany) mounted on an SZX16 dissecting microscope (Olympus, Hamburg, Germany) using the software Cell’D (Olympus, Hamburg, Germany).
Whole mounts stained with hematoxylin and eosin The Hematoxylin and eosin (HE) stain is usually employed as a routine method to differentiate between tissues, cells or cell compartments with acidophilic and basophilic properties. Hematoxylin stains nucleic acids (predominantly located within nuclei) blue, while eosin stains cytoplasm and connective tissue red. The specimens were fixed in 4% formaldehyde diluted from 37% formaldehyde (J.T. Baker, Germany) with phosphate buffered saline (PBS; 0.1 M, pH 7.4). Staining of whole mount preparations (15 specimens of D. pulex) were performed according to the manufacturer’s protocol (Thermo Scientific, Waltham, United States of America, provided as SHANDON™ instant-dyes). Briefly, D. pulex were rinsed three times in PBS (pH 7.4, 0.1M) and incubated in Instant Hematoxylin (Thermo Scientific™ Shandon™ Instant Hematoxylin, Thermo Fisher Scientific Inc.,
Daphnia carapace: form, function, structure and plasticity 28 Materials and Methods
Waltham, USA) for 3 min, then rinsed for 2 s with hydrochloric acid (HCl; 0.1N) until differentiation. The process was terminated by rinsing the samples in deionized water for 1 min. Samples were transferred into Instant Eosin (Thermo Scientific™ Shandon™ Instant Eosin, Thermo Fisher Scientific Inc., Waltham, USA) for 3 min. Immediately after staining, the specimens were dehydrated in an ascending ethanol series: 80% ethanol (2×2 min), 95% ethanol (3×2 min), 100% isopropanol (2×2 min) and 100% Roti-Histol (3×3 min) (Roti®- Histol, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Finally, the samples were mounted and cover-slipped in Entellan (Entellan®, Merck KG aA, Darmstadt, Germany) on object slides (Menzel GmbH & Co KG, Braunschweig, Germany), using ringed sticky tape (Avery Zweckform; Valley, Germany; Weiss et al. 2012). LM was carried out using a Zeiss Axiovert (Zeiss, Oberkochen, Germany) with an XC10 monochrome camera (Olympus, Hamburg, Germany) and CellSens® digital imaging software (Olympus, Hamburg, Germany). The pillar density (no. of pillars per area) and base diameter were measured along a ventral-to- dorsal transect across a central region of the carapace (Fig 3). Pillars were counted in squares of 100×100 µm along the transect and the diameter of their bases recorded. Due to inter- individual variation in body width, 6–8 squares were necessary to cover the entire transect. Squares were numbered 1-7, as this represented the average number of squares. Where more or less than seven squares were required, the squares numbers were multiplied by n/7 where n is the total number of squares, normalizing all squares to point numbers between one and seven. Division by body length was used to normalize the pillar base diameter.
Scanning electron microscopy For scanning electron microscopy (SEM), the samples (19 D. pulex, second juvenile instar) were fixed in 70% ethanol and dried according to the standard procedure using hexamethyldisilazane (HMDS, Sigma-Aldrich, St. Louis, USA; Laforsch and Tollrian, 2000). Subsequently, they were mounted on aluminum blocks using ‘Leit tabs’ (“Conduction-Tab”, Plano GmbH, Wetzlar, Germany). To visualize the interconnecting pillars, the dried carapace was fractured using fine dissection needles (euromex microscopes b.v., Arnhem, The Netherlands). Some of the fractured specimens did not have a clear breaking edge but the proximal integument was removed with the dissecting needle. The samples were sputter- coated with gold (Sputter Coater SCD 050, BALZER, 180 sec, 10,000 V) and visualized with an SEM (DSM 950, Zeiss, Oberkochen, Germany).
Daphnia carapace: form, function, structure and plasticity 29 Materials and Methods
Thin sections The specimens were fixed in 1% glutaraldehyde (VWR, Radnor, USA) buffered in PBS (0.1 M, pH 7.4) overnight. After fixation, the samples (17 D. pulex, second juvenile instar) were rinsed in PBS 3×30 min and contrasted for 40 min with 2% osmium tetroxyde solution (Heraeus, Hanau, Germany). Samples were dehydrated in an ascending ethanol series of 50% (15 min), 70% (overnight), 90% (25 min), 100% (5 min) and 2 × 100% (30 min). Infiltration with Agar 100 (Agar Scientific, Essex, United Kingdom) was carried out according to the manufacturer’s protocol. The resin was polymerized at 60° C for 48 h in a Teflon mold (Sigma-Aldrich Chemie GmbH, Munich, Germany). Specimens were positioned in an anterior–posterior direction in the molds in order to produce cross sections. Blocks were trimmed and processed using an ultra-microtome (Reichert Jung Ultracut E, Leica Microsystems, Wetzlar, Germany), equipped with an glass-knife set to an angle of 7°. Sections with thicknesses of 15–30 µm were collected in a drop of water on a glass slide (VWR; Radnor, Pennsylvania) and dried on a heat plate (Medax, Neumünster, Germany) at 60° C for 15 min. The sections on the glass slides were stained with a drop of 0.1% toluidine blue staining solution (dissolved in deionized water). Excess dye was rinsed off with deionized water after 2 min. Images were taken with a XC10 monochrome digital camera (Olympus, Münster, Germany) attached to a Zeiss Axiovert (Zeiss, Oberkochen, Germany) light microscope and processed using CellSens® Digital Imaging Software (Olympus, Germany).
Ultra-thin sections and scanning transmission electron microscopy Ultra-thin cross-sections (45–70 nm thick) of Agar 100 embedded specimens were also cut with the ultra-microtome using a diamond knife with a 2.5 mm edge (Diatome 45°, Diatome, Hatfield, PA, U.S.A.) equipped with a water-filled ‘boat’ for collecting the sections, and set to an angle of 7°. Expansion of the floating sections was facilitated using xylol fumes from a wooden stick. The floating sections were transferred to copper grids with a mesh width ranging from 20 to 80 lines per cm (Stork Veco B.V., Eerbeek, Holland) by picking them up directly from the surface. Scanning transmission electron microscopy (STEM) of ultra-thin sections of 17 animals was conducted on a Zeiss Gemini (Zeiss Gemini Sigma VP, Zeiss, Oberkochen, Germany). The acceleration voltage was set to 20 kV and STEM detector was set to ‘dark field segment mode’, resulting in images comparable to dark field TEM.
Daphnia carapace: form, function, structure and plasticity 30 Materials and Methods
Measurements of the thickness of the proximal and distal procuticle, the distance between the proximal and distal procuticle at the ventral and dorsal edge, as well as the middle region of the carapace and thickness of single pillar fibers were conducted using the software Zeiss SmartTiff (Version V02.01, Carl Zeiss Microscopy Limited, Cambridge, United Kingdom).
Hemolymphatic gauge pressure The hemolymphatic gauge pressure measurements were performed in vivo using D. longicephala – taking advantage of their comparatively large size.
Animals were fixed in a lateral position on an object slide with underwater adhesive (JBL “Haru”, Neuhofen, Germany). The pressure was measured in the head capsule in the near- vicinity of the caeca (Fig 3). This region contains the biggest volume in a Daphnia’s hemolymph system, thus facilitating precise penetration and measurement. We used an invasive blood pressure system, similar to those used in medical applications. A pressure transducer (Deltran® Disposable Pressure Transducer, Utah Medical Products, Inc., Midvale, USA) was connected to a glass capillary via a silicon tube, and additionally, also via silicon tubing, to a syringe (single-use, 5 ml; Amefa; Limburg, Germany) to remove excess air from the system. The capillary (Premium Standard Wall Borosilicate Capillary Glass, OD 1.5 mm, ID 0.86 mm, L 100 mm; Harvard Apparatus; Holliston, Massachusetts, USA) was pulled with a micropipette puller (P-97 Flaming/Brown Micropipette Puller; Sutter Instrument; Novato, California, USA), with a tip-diameter of approximately 15 ± 1 µm. The transducer was linked to a data recording device including a digitizing system and a signal amplifier (Biopac, Model MP100A-CE; BIOPAC Systems Inc., Santa Barbara, California, USA), which subsequently transmitted the data to a computer. For the measurements, animals were quickly fixed and transferred into a water-filled petri-dish placed under a dissection microscope (SZX12, Olympus, Germany), this was performed carefully to avoid harm to the organism. The capillary was positioned with a micromanipulator (Prior Scientific, Rockland, USA) at an angle of approximately 30° to the surface. A rapid forward movement of the capillary was used for penetration. Once the capillary tip pierced the integument its position was maintained for 5-10 s to ensure stable pressure measurement and then it was extracted. Pressure recording began prior to penetration and continued at 200 measurements per second until extraction of the capillary was complete. In total, the hemolymphatic gauge pressure was measured in 39 animals.
Daphnia carapace: form, function, structure and plasticity 31 Materials and Methods
Figure 3: Overview of D. longicephala to illustrate the transect over which the pillar density and base diameter were determined (which is also the region of the cross-section shown in Figure 9), and the area pierced for hemolymphatic gauge pressure measurements (black X).
Daphnia carapace: form, function, structure and plasticity 32 Materials and Methods
2. Structure and shape contribution to carapace resistance
Experimental organisms Age-synchronized Daphnia pulex, clone R9 (originating from Canada) and Daphnia longicephala, clone LP1 (from Lara Pond, Australia) were cultured under constant conditions with a day:night cycle of 16:8 hours at 20° C ±0.1° C in a climate cabinet. Both species were cultured in 1 L glass beakers (WECK®; Germany) containing charcoal-filtered tap water and were fed with the algae Scenedesmus obliquus ad libitum. Population density was kept below 30 individuals per 1 L to ensure stable population growth. Chaoborus obscuripes and Notonecta glauca were captured in ponds of the botanical garden of the Ruhr-University Bochum, Germany. C. obscuripes was kept in densities of 50 individuals in 1.5 L beakers at 4 °C ±1° C. N. glauca was kept in densities of 5 individuals in 10 L buckets at 20 °C ±1° C. Both predators were fed regularly with daphniids every 48 hours.
Induction of morphological defenses in D. pulex and D. longicephala Induction of morphological defenses was performed in triplicates, by exposing D. pulex to chemical cues released from actively feeding Chaoborus larvae (C. obscuripes). We therefore transferred ten 4th instar Chaoborus into a net-cage (mesh size 100 µm) placed in 1 L beakers filled with charcoal filtered tap water. These larvae were fed with 100 juvenile D. pulex. The net-cage prevented encounter of the predator with the test animals but allowed exchange of biologically active chemical cues. Test animals were maintained outside the net-cage and fed with the green algae S. obliquus ad libitum. We introduced 10 mother daphniids carrying black-eyed embryos in their brood pouch. Once the neonates were released, mother daphniids were removed. When the offspring molted into the second juvenile instar, they were collected and analyzed. Controls were performed likewise, but in the absence of the predator.
Morphological defenses in D. longicephala were induced accordingly, but using Notonecta glauca as predator. We transferred 25 first instar D. longicephala into 1 L of charcoal-filtered tap water. Notonecta was fed with 10 additional adult D. longicephala in the net-cage. S. obliquus served as food for D. longicephala. Predator and prey were fed every 48 h. Once neonates had reached maturity, they were analyzed.
Daphnia carapace: form, function, structure and plasticity 33 Materials and Methods
Microscopy, modelling and simulation For the morphological analysis and modelling of D. pulex and D. longicephala, shape and structure of the D. pulex and D. longicephala carapace stereomicroscopy, confocal laser scanning microscopy (CLSM) and scanning transmission electron microscopy (STEM) were applied.
Procuticle analysis We used STEM imaging to analyze the Daphnia procuticle. Specimens were fixed in 1% glutaraldehyde (VWR, Radnor, USA) diluted in phosphate buffered saline (PBS, 0.1 M, pH 7.4) over night and rinsed in PBS 3 times for 30 min. We contrasted them with 2% osmiumtetroxyde solution (Heraeus, Hanau, Germany) diluted in PBS (0.1 M, pH 7.4) for 40 min and subsequently rinsed in deionized water 3 times for 30 min. Dehydration was performed with an ascending ethanol series: 15 min in 50% EtOH, 8 h in 70% EtOH, 25 min in 90% EtOH, 5 min in 100% EtOH and finally 2 times 30 min in 100% EtOH. Infiltration with Agar 100 (Agar Scientific, Essex, United Kingdom) was carried out according to the manufacturer’s protocol; in brief: 2 h in 33% Agar 100 medium diluted in EtOH, 2 h 66% Agar 100 medium diluted in EtOH, 2 h in 100% Agar 100 medium. Subsequently, the samples were transferred into Teflon molds (Sigma-Aldrich, Chemie GmbH, Munich, Germany) filled with 100% Agar. Polymerization was performed at 60 °C for 48 h. The embedded specimens were cut (45-70 nm thickness) using an ultra-microtome (Reichert Jung Ultracut E, Leica Microsystems, Wetzlar, Germany) equipped with a diamond knife with a boat and a 2.5 mm edge (Diatome 45°, Diatome, Hatfield, PA, U.S.A.); the knife angle was set to 7°. The floating sections were expanded using xylol fumes diffusing from a wooden stick. Afterwards the sections were directly transferred to sample-grids (Stork Veco B.V. Eerbeek, Holland). We used Copper TEM grids with a mesh width ranging from 20 to 80 lines per cm. STEM imaging was conducted on a Zeiss Gemini (Zeiss Gemini Sigma VP, Zeiss, Oberkochen, Germany) with acceleration voltage set to 20 kV and detector mode set to “dark field segment mode”. The acquired images were analyzed for procuticle thickness using the software Zeiss SmartTiff and number of layers were counted (Version V02.01, Carl Zeiss Microscopy Limited, Cambridge, United Kingdom).
Daphnia carapace: form, function, structure and plasticity 34 Materials and Methods
Procuticle Young´s modulus To measure the procuticle Young´s modulus we applied Atomic Force Microscopy (AFM). For this purpose, we randomly selected defended and undefended individuals from both species. The carapace of each individual was dissected and cut with a razor blade into two or three rectangular samples of approximately 500 µm edge length in case of D. longicephala and approximately 200 µm edge length in case of D. pulex (Fig 4). The samples were mounted on an object slide (Menzel GmbH & Co KG, Braunschweig, Germany) coated with a fine layer of Vaseline (Elida Fabergé, Hamburg, Germany), and transferred into a Petri dish filled with charcoal filtered tap water. A randomly chosen area (30 µm in square) of the sample was scanned using an Atomic Force Microscope (NanoWizard, JPK Instrument AG, Berlin, Germany) in phase contact mode. Based on the acquired image, measurement spots for the indentation were determined within the flat areas of the shingle structures defining the carapace’s surface pattern (Fig 4). Up to three sets of indentation measurements were performed per single shingle and up to three shingles were used for measurements for each area scanned. Measurements were conducted by defining a region of interest as a square of 5 µm. In total 25 indentation measurements were performed in a micrometer distance within the region of interest (Fig 4). In total, we analyzed 5 defended and undefended D. pulex and 7 defended and 6 undefended individuals of D. longicephala. Due to the above described multi- point data acquisition strategy, we performed a nested ANOVA to test for significant differences between defended and undefended morphotypes. Maximal indentation force was set to 10 nN and maximal indentation depth to 50 nm. For the measurements a silicon nitride cantilever with a square based pyramid tip (ORC8-10, Bruker Corporation, Billerica, MA, USA) with a nominal tip radius of 15 nm and a spring constant of 0.1 N/m was used. The cantilever was checked for its properties by regular test measurements on a glass Petri dish: we found the spring constant ranging from 0.08 to 0.12 N/m; we measured a tip radius of 33 nm. The Young´s moduli for each measured point were calculated from the resulting force- distance curves using the manufacturer’s software (JPK DP, JPK Instrument AG, Berlin, Germany).
Daphnia carapace: form, function, structure and plasticity 35 Materials and Methods
The calculations were based on the Hertz model for four-sided pyramidal indenters:
퐹 푡푎푛 훼 퐸 = 훿2 1 − 휗2 √2
E= Young´s-module
F= force
α= cantilever face angle
δ= indentation depth
ν= Poisson’s ratio
Equation 1: Hertz model for four-sided pyramidal tips
Daphnia carapace: form, function, structure and plasticity 36 Materials and Methods
Figure 4: Scheme of preparation and measurement of daphniids on the AFM. Lines indicate the position and sequence of sections tending to create flat carapace sections. Insert shows an AFM surface scan of the created sections, revealing the shingle like pattern, red dots representing an indentation measurement matrix.
Daphnia carapace: form, function, structure and plasticity 37 Materials and Methods
Carapace structure and finite element analysis Based on the collected data on carapace structure and stiffness, we modelled the material behavior upon mechanical impact in silico. We performed FEA with the help of the software ANSYS (version R15.0, ANSYS Inc., Canonsburg, PA, USA). For this purpose, we analyzed the patterning of the procuticle layers observed in the STEM cross-section images whose grey scale continuously alternates from bright to dark representing the horizontally rotating orientation of chitin fibers. Every bright-dark stripe of the procuticle represents a lamella with fiber rotation in successive monolayers of fibers between 0° and 180° and was defined as one layer (Stevenson, 1985). This complete rotation cannot be modelled using FEA since the fiber rotation through the procuticle is virtually continuous. Therefore, we chose to simulate the procuticle in a stepwise approach by successively modelling the procuticle structure in steps of 120° fiber rotation (Fig 5). Furthermore, a model for the complete procuticle thickness exceeds soft- and hardware limitations. Hence we simulated at least a fiber rotation of 720° in total. We started with the simulation of one cylinder, representing a procuticle fraction with fiber rotation of 120°. Subsequently cylinders with an offset of 120° were added up to a total number of six cylinders to simulate a 720° fiber rotation. Based on these simulations we calculated a curve of best fit for deformation in dependence of the number of cylinders. This curve was finally used to extrapolate the deformation for the complete procuticle thickness observed in STEM.
Daphnia carapace: form, function, structure and plasticity 38 Materials and Methods
-
120°) 120°) and pasted on top of the
ked cylinders. Each of these cylinders represented 120° of fibre fibre 120° of represented cylinders Each these of cylinders. ked
. First a cylinder was created, subdivided into elements with alternating material properties reflecting the chitin the reflecting properties material alternating with elements into subdivided created, was Firsta cylinder .
rotation, equal to two procuticle layers, observed in STEM. in observed layers, procuticle two to equal rotation,
-
Scheme of the procuticle modelling for FEA for modelling the procuticle of Scheme
matrix, matrix, and afterwards simulated for mechanical impact. Then the initially build cylinder was copied, superimposed, rotated (
protein of stac 6 a to repeated maximum was procedure This simulation. the by next followed formercylinder, fibre 360° represented cylinders three rotation.Thus, Figure 5: Figure5: Daphnia carapace: form, function, structure and plasticity 39 Materials and Methods
For model creation the individual cylinder thickness was adjusted to the measured procuticle thickness by determining the mean thickness of 120° fiber rotation within the procuticle defined as:
T T = 360° 120° 3
While T360° was:
procuticle thickness [µm] T = × 2 360° procuticle layers [#]
Equation 2: cylinder thickness
Cylinder diameter was set to 1.5 µm due to model size limitations. To simulate the chitin- protein matrix within the procuticle, we applied two different material-properties for the cylinders. Therefore, the cylinders were subdivided vertically with alternating material properties. Chitin fibers are represented in our model with a Young’s-modulus of 17 GPa and a Poisson’s ratio of 0.3, while to the protein matrix, in which the fibers are embedded was assigned a Young’s-modulus of 0.2 GPa and a Poisson’s ratio of 0.3. The chosen Young’s- modulus of 17 GPa lies within the range of single cellulose fibers (Mott and Shaler, 2002) which are chemically comparable to chitin and the Poisson’s ratio of 0.3 is typical for compound materials. The matrix e-module was set to a typical dimension for compound materials in which the Young’s-modulus of the implemented fibers is distinctly higher than that of the matrix.
Conditions for all simulations were as follows: all edge-nodes of the bottom surface were blocked for movement in z direction, one of these nodes lying on the y-axis was additionally blocked for movement in x direction and the node lying on the opposite side of the y-axis was blocked for movement in all directions. This represents a standard practice for loading simulations with one direction of interest. A force was applied on five nodes (in sum: 3 mN) in the center of the respective top surface area.
This workflow was conducted for defended and undefended morphotypes of both analyzed species.
Daphnia carapace: form, function, structure and plasticity 40 Materials and Methods
Optical imaging of carapace shape To measure the body outline of the undefended and defended morphotypes of both species, they were transferred onto a glass slide. All images were taken on a stereo microscope (Olympus SZX 16, Olympus, Hamburg, Germany) with mounted digital camera (Colorview III, Soft imaging Systems, Hamburg, Germany) and the software Cell^D (Olympus, Hamburg, Germany). To create representative datasets, 11 individuals of D. pulex in undefended state and 12 in defended state as well as 13 individuals of D. longicephala in undefended state and 12 in defended state, were imaged.
Daphnia carapace: form, function, structure and plasticity 41 Materials and Methods
Table 1: Set of outline landmarks for D. pulex Point Landmark definition 1 1st dorsal thorn (counting in anterior direction from spine) 2 2nd dorsal thorn 3 3rd dorsal thorn 4 4th dorsal thorn 5 5th dorsal thorn 6 Middle distance between spine and heart lower edge (point 7) 7 Heart lower edge, horizontally projected on the dorsal edge 8 Head-carapace transition (dorsal bend) 9 Heart upper edge, horizontally projected on the dorsal edge 10 Levator lower edge, horizontally projected on the dorsal edge 11 Levator upper edge, horizontally projected on the dorsal edge 12 2nd abductor lower edge, horizontally projected on the dorsal edge 13 2nd abductor upper edge, horizontally projected on the dorsal edge 14 1st abductor lower edge, horizontally projected on the dorsal edge 15 1st abductor lower edge, vertically projected on the dorsal edge 16 1st abductor upper edge, vertically projected on the head outline 17 Crest of the head outline 18 Complex eye upper edge, horizontally projected on the head outline 19 Complex eye centre, horizontally projected on the head outline (ventral) 20 Complex eye lower edge, horizontally projected on the head outline (ventral) 21 Rostrum tip 22 Head-carapace transition (ventral bend) 23 Heart upper edge, horizontally projected on the ventral edge 24 Heart lower edge, horizontally projected on the ventral edge 25 Horizontally projection of point 6 on the ventral edge 26 5th dorsal thorn, horizontally projected on the ventral edge 27 4th dorsal thorn, horizontally projected on the ventral edge 28 3rd dorsal thorn, horizontally projected on the ventral edge 29 2nd dorsal thorn, horizontally projected on the ventral edge 30 1st dorsal thorn, horizontally projected on the ventral edge 31 Carapace lower edge ventrally 32 Transition carapace-spine (ventral bend) 33 Spine tip
Daphnia carapace: form, function, structure and plasticity 42 Materials and Methods
Figure 6: Defended D. pulex in 2nd juvenile instar. The numbers indicate the landmarks for the shape reconstruction referring to S-Table 1. Scale bar = 500 µm
Daphnia carapace: form, function, structure and plasticity 43 Materials and Methods
Table 2: Set of outline landmarks for D. longicephala Point Landmark definition 1 1st dorsal thorn (counting in anterior direction from spine) 2 4th dorsal thorn 3 7th dorsal thorn 4 10th dorsal thorn 5 13th dorsal thorn 6 Middle distance between spine and heart lower edge (point 7) 7 Heart lower edge, horizontally projected on the dorsal edge 8 Heart centre, horizontally projected on the dorsal edge 9 Heart upper edge, horizontally projected on the dorsal edge 10 Levator lower edge, horizontally projected on the dorsal edge 11 Levator upper edge, horizontally projected on the dorsal edge 12 2nd abductor lower edge, horizontally projected on the dorsal edge 13 2nd abductor upper edge, horizontally projected on the dorsal edge 14 Levator upper edge, vertically projected on the head outline 15 2nd abductor upper edge, vertically projected on the head outline 16 Caecum dorsal edge, vertically projected on the head outline 17 Complex eye ventral edge, vertically projected on the head outline 18 Complex eye lower edge, horizontally projected on the head outline (ventral) 19 Rostrum tip 20 Complex eye ventral edge, vertically projected on the rostrum edge (posterior) 21 Head-carapace transition (ventral bend) 22 Heart upper edge, horizontally projected on the ventral edge 23 Heart lower edge, horizontally projected on the ventral edge 24 Horizontally projection of point 6 on the ventral edge 25 13th dorsal thorn, horizontally projected on the ventral edge 26 10th dorsal thorn, horizontally projected on the ventral edge 27 7th dorsal thorn, horizontally projected on the ventral edge 28 4th dorsal thorn, horizontally projected on the ventral edge 29 1st dorsal thorn, horizontally projected on the ventral edge 30 Transition carapace-spine (ventral bend) 31 Spine tip
Daphnia carapace: form, function, structure and plasticity 44 Materials and Methods
Figure 7: Defended sexually mature D. longicephala. The numbers indicate the landmarks for the shape reconstruction referring to S-Table 2. Scale bar = 2000 µm
Daphnia carapace: form, function, structure and plasticity 45 Materials and Methods
We used confocal laser scanning microscopy (CLSM) to image the body in all three dimensions. For this purpose, samples were stained with congo-red, which particularly well binds to the crustacean exoskeleton (Michels and Büntzow, 2010). After fixation in 3.7% formaldehyde diluted in PBS (0.1 M, pH 7.4) the samples were rinsed in PBS (0.1 M, pH 7.4) 6 times for 10 min and 2 times for 1 h. Subsequently, they were stained in congo-red diluted in PBS (0.1 M, pH 7.4, 3 mg/ml) for 8 h and finally rinsed in PBS for 1 h shielded from light. The stained samples were transferred onto object slides prepared with ringed sticky tape (Weiss et al., 2012) as spacer. Three ringed sticky tapes were mounted on top of each for D. pulex, for D. longicephala 9 ringed sticky tapes were used. Samples were mounted in a lateral position in vectashield (Vector Laboratories, Burlingame, CA, USA) and cover- slipped. Scan depth was limited to one body hemisphere. All scans were conducted with a CLSM (Leica SP5, Leica Microsystems GmbH, Wetzlar, Germany) with an excitation wavelength of 561 nm and the emission filter set to 568 nm. Due to its size D. longicephala had to be scanned in multiple image stacks for each individual. These stacks were stitched before analysis, using the ImageJ plugin TrakEM2 (Cardona et al., 2012).
Carapace shape modelling and finite element analysis To model D. pulex and D. longicephala overall shape, we applied a classical morphometric approach. In a first step, the body’s outline from a lateral view was described by a set of landmarks and semi-landmarks, using the point measurement tool of Cell^D (Olympus, Hamburg, Germany). In geometric morphometrics, landmarks are explicitly defined morphological loci e.g. the ligament insertion on a skeletal element, whereas semi-landmarks are derivatives from landmarks e.g. half the distance between two landmarks. We placed the origin of our coordinate system describing body shape measurements in the nauplius eye in case of D. pulex. In D. longicephala, we used the center of the complex eye, as here the nauplius eye is unpigmented and difficult to determine visually. We dedicated landmarks to explicit muscle-attachment points within the head region which are visible through the transparent integument, used the insertion point of the tail spine and dorsal edge’s thorns for the carapace as well as intermediated semi-landmarks (Fig 6, Table 1, Fig 7, Table 2). In the second step semi-landmarks were projected onto the carapace surface, based on the CLSM scans, using a morphologically adjusted grid. To accomplish this, a vector grid with 10 vertical and 31 horizontal lines was projected in a sagittal orientation into the image stack using Adobe Photoshop (Adobe Systems Software Ireland Limited, Dublin, Ireland). The grid
Daphnia carapace: form, function, structure and plasticity 46 Materials and Methods was adjusted to the individual sample by bringing the vertical lines in parallel to the animal’s dorsal edge. The uppermost horizontal line was fitted to the head’s highest point, the lowermost line to the carapace-spine transition. The grid was adjusted to the animal’s body width by fitting the second vertical line to the most ventral point of the carapace and the second last vertical line to the most dorsal point of the head. The grid was colored in white (grey-value 255) after adjusting. Then the image series including the grid was loaded into FIJI (Schindelin et al., 2012) as an image stack. Contrast was corrected if necessary and the stack was superimposed via 3D Viewer (Schmid et al., 2010). In the 3D viewer, the scan was oriented laterally, the surface measurement points were collected using the multipoint tool. This was done by marking a point at every intersection of the grid, starting in the most anterior dorsal corner, leading to a surface dataset of 310 points for each individual. On each horizontal line of the grid, an additional semi-landmark was marked at the intersection with the ventral edge of the carapace. These additional semi-landmarks ensured description of the ventral cleft. In a last step, three reference points were taken at each individual for subsequent alignment of the datasets. First point was the tip of the rostrum, second the transition of the carapace into the spine on the ventral bend, and the last reference point was taken at the fifth thorn on the dorsal edge, counted anterior from the dorsal spine. Finally, the outline data and the surface data were combined and averaged to reproduce D. pulex’ and D. longicephala’s shape representatively. Based on these combined coordinate data sets, surface models were created using the FEA software z88 Aurora (Rieg F., Universität Bayreuth, Germany) and supplied with the carapace thickness (using the shell plugin) as well as loading force and constraints. The loading force of 1 mN was applied to the nodes representing the highest lateral width of the carapace. As constraints the nodes forming the outline of the daphniids body, except the ventral edge of the carapace, were blocked for movement in any direction to reflect the constraints of the bilateral nature of daphniids with a carapace free moving at the ventral cleft. For the material parameters of the models we used the Young´s modulus of the procuticle determined with AFM. Poisson’s ratio was set to 0.15 and the thickness of the model was adapted to the STEM observations. Simulations were conducted for undefended and defended animals of both species. Additionally, a defended animal with procuticle characteristics of an undefended animal and vice versa was simulated for both species to determine the shape’s contribution to the defensive effect. All simulations were analyzed for maximum deformation (node displacement after simulation) and maximum stress (stress within the models volume) in force per area ([Pa]).
Daphnia carapace: form, function, structure and plasticity 47 Materials and Methods
Structural Young’s-modulus and critical force For comparison and validation of the simulations, indentations were conducted determining the structural e-module and critical force (the force needed to collapse the carapace resulting in lethal injury). For this purpose, D. pulex and D. longicephala were asphyxiated with acidulated water and directly transferred into a glass petri dish lubricated with Vaseline for animal fixation and filled with water. Measurements were conducted using a microindenter (Basalt I, TETRA GmbH, Illmenau, Germany) equipped with a cantilever with a spring constant of 330 N mm-1. For D. pulex a steel minutie pin was used as the indenting probe. With a tip radius of about 2.5 µm, it represented quite well the geometrical properties of the mandible tips of C. obscuripes. For D. longicephala a glass pipette was pulled to a capillary with a pipette puller (KE Pipetten Puller horizontal, H. Saur Laborbedarf, Reutlingen, Germany) and afterwards the tip was sealed over a flame. With a tip radius of 12.37 µm ± 1.36 SD it resembled the size of the proboscis tip of N. Glauca. The measurements were conducted by bringing the indenter in a position slightly above the middle of the carapace followed by indenting a predefined depth. In total 94 individuals of D. pulex (50 defended, 44 undefended) and 147 individuals of D. longicephala (62 defended, 85 undefended) were analyzed for their indentation properties. Based on the obtained data the structural Young´s- modulus was determined based on the Hertz model for parabolic indenters (Tramacere et al., 2013; Schaber et al., 2015) in Matlab® (MathWorks, Natick, Ma, USA):
4 √푟 퐹 퐸 = 훿3/2 3 1 − 휗2
E= effective Young´s-modulus
F= force r= cantilever tip radius
δ= indentation depth
ν= Poisson’s ratio
Equation 3: Hertz model for parabolic tips
Daphnia carapace: form, function, structure and plasticity 48 Materials and Methods
The critical force was defined as the force needed to collapse the carapace resulting in injury lethal for a living daphniid. To evaluate the critical force rapid drops in the force-distance curves of the micro-indenter data were used as indicator for structural collapse of the carapace and analyzed for the maximum force before collapse. During the experiment the indentation was visually monitored to guarantee that force drops were indeed collapses of the carapace.
Daphnia carapace: form, function, structure and plasticity 49 Materials and Methods
3. The catching basket of Chaoborus
Experimental Organisms and induction of the daphniids defenses An age-synchronized culture of Daphnia pulex, clone R9 (originating from Canada) was reared under constant conditions in a climate cabinet (day:night cycle of 16:8 hours at 20° C ±0.1° C). The culture was kept in 1 L glass beakers (WECK®; Germany) containing charcoal-filtered tap water and was fed with the algae Scenedesmus obliquus ad libitum. To ensure stable population growth, animal density was kept below 30 individuals per 1 L. Chaoborus obscuripes larvae were captured in ponds of the botanical garden of the Ruhr- University Bochum, Germany, and reared in densities of 50 individuals in 1.5 L beakers at 4 °C ±1° C. Larvae were fed with daphniids every 48 hours.
For experimental setup female daphniids were age synchronized by selecting mothers with embryos in the fifth stage of development using a stereo microscope and randomly divided into two groups i.e. “undefended group” and “defended group”. The animals were placed into 1 L beakers filled with charcoal filtered tap water. A net-cage (mesh width of 100 µm) containing 10 Chaoborus larvae and 100 juvenile daphniids as food for the larvae was added to beakers containing the “defended group”. The “undefended group” was reared in tap water without any contact to Chaoborus larvae. Both groups were kept under the same conditions as the culture. After the first molting (about 36h after hatching) juveniles in the second instar were removed from the beakers and used for the experiments.
Daphnia carapace: form, function, structure and plasticity 50 Materials and Methods
High speed recordings For high-speed video recordings a Photron Fastcam (SA1.1, Photron, Pfullingen, Germany) with a B18Z06MA-1 F2.5 / 18-108mm 2/3" lens was used. The catching events were recorded in a miniature aquarium that was built out of one object slide, two cover slips and dental wax. The object slide served as a base and the two cover slips as aquarium sides, while the two head sides were built by the dental wax. Therefore, they were glued to each other with a spacing of 3 mm and afterwards on top of the footing. The camera was set up on a table and connected to a computer. The developers Software (Photron FASTCAM Viewer ver.351, Photron) was set to live view. Using a laboratory jack the aquarium was put in front of the camera that was equipped with a macro lens. One Chaoborus larva and up to 3 juvenile daphniids were manually added into the water filled aquarium. To focus the Chaoborus larvae the aquarium was moved in the necessary direction by hand. The height was regulated by adjusting the laboratory jack. 50 catching attempts were filmed at different frame rates (3000, 5000 and 8000fps).
microCT imaging and 3D reconstruction To obtain individuals with extended catching basket, a larva was fixed between two microscope slides gradually increasing the hemolymphatic pressure. Afterwards the larva was fixated in EtOH 70% and dried chemically. Therefore, the sample was rinsed 20 min in 80% EtOH, 20 min in 90% EtOH, 20 min in 99% EtOH, 20 min in aceton, 20 min in aceton/hexamethyldisilazane (HMDS) 50/50 ratio finally the sample was covered with a small amount of HMDS that was permitted to evaporate (Laforsch and Tollrian, 2000). The dried sample was mounted on a µCT sample holder with vertically oriented body axis. The scan was performed with a Skyscan µCT (Skyscan 1172, Bruker micro CT) with an effective pixel size of 1 µm. 1441 x-ray images were recorded over a 180° sample rotation resulting in a computed image stack of 1733 monochromatic images with 1 µm resolution in each dimension. This image stack was visualized for quality check with CTvox (CTvox 2.7.0, Bruker microCT). Volume reconstruction of the head capsule, its appendages and the involved muscles was done using ImageJ (Abramoff et al., 2004). Therefore, the “Segmentation editor” plugin (J. Schindelin, F. Kusztos, B. Schmid; Department of genetics and neurobiology Würzburg) was used. With the ImageJ selection tools the respective tissues were marked within stack slices of the image stack and with the threshold tool the selection was refined according to the grey
Daphnia carapace: form, function, structure and plasticity 51 Materials and Methods values within the selection. This process was repeated for every third to sixth slice (depending on the alteration), the slices in between were automatically labelled via interpolation. Afterwards, a surface mesh (.stl file) based on the reconstruction was exported from ImageJ for the animation of a catching event.
Surface mesh and animation The animation was created with the open source 3D computer graphics software Blender (Blender 2.72b, Blender foundation, Amsterdam, the Netherlands) following the instructions of Garwood and Dunlop (Garwood and Dunlop, 2014).
Therefore, the surface mesh, exported from ImageJ, was loaded into Blender and afterwards reduced computational effort but optimized for natural appearance. That was done by firstly reducing the number of vertices using the “decimate modifier” function to decrease computational effort. Then the surface was smoothened in “sculpt mode” using the “sculpt draw brush”, while permanently comparing the surface mesh to the µCT-data to converge the surface mesh to the natural appearance. Only the external surfaces from the volume reconstruction were used to create the surface mesh.
For animation of the head appendages so called “bones” were used in Blender. These “bones” allow the animation of individual appendages involved in the catching event. Therefore, every bone was associated with the respective appendage of the surface mesh. Several bones were combined to form a movable element. When one bone is moved all attached ones follow. To define the vertices of the surface mesh associated with a specific bone the “weight paint mode” was used. The animation of the catching event was modelled by defining key positions occurring during appendages’ movement. To smooth the animation the movement between key positions was interpolated and rendered into a video.
Daphnia carapace: form, function, structure and plasticity 52 Materials and Methods
Feeding Experiments An aquarium (12cm x 1,5cm x 10cm) was illuminated with diffuse light from several directions to avoid any impact by the daphniids’ light sensitivity. Black cardboard was added behind the aquarium to better contrast the daphniids from the background. Experiments were conducted with one starved Chaoborus larva and 20 second instar daphniids (either undefended or defended), monitored over one hour. Every Chaoborus larva that did not strike within 10 minutes or made two successful catches was exchanged by an unfed animal. Every eaten D. pulex was replaced by a new one. A slr camera (Nikon D5100, Nikon Corporation, Tokyo, Japan) with a 60mm / f2.8 lens in automatic mode was used for video recording. The videos were analyzed by counting strikes, evasions, contacts, escapes and ingestions. These terms were defined according to Havel and Dodson (Havel and Dodson, 1984): a strike was defined whenever Chaoborus grasped at a daphniid, every strike resulted in either contact or evasion. Whenever Chaoborus head touched a daphniid a contact was noted, every strike without contact was classified as evasion. Contacts resulted in either escape or ingestion. The experiment was conducted 11 times for each treatment (defended daphniids/undefended daphniids). Using the t-test the number of strikes was compared between the undefended and defended treatment as well as the contacts per strike and the ingestions per contact.
Bite force estimation To estimate the bite performance of Chaoborus larvae the cross section area of the m. adductor mandibulae was determined. That was done using virtual cross sections of the m. adductor mandibulae obtained with the µCT-Scan as well as cross sections made with classical histochemical techniques. The latter were conducted with additional samples of Chaoborus larvae that were fixated in 4% paraformaldehyde (dissolved in PBS) overnight at 4 °C and dehydrated in an ascending ethanol series: 80% EtOH overnight at 4 °C, 90% EtOH overnight at 4 °C, 96% EtOH for 4 hours at 4 °C and finally 100% EtOH for 4 hours at 4 °C. After fixation the samples were embedded in paraffin: 100% tert-butyl alcohol (TBA) overnight at 20 °C, TBA/paraffin oil (1:1) for 2 hours at 20 °C, 100% paraffin oil for 3 hours at 20 °C, 2 times 100% paraffin for 2 hours at 60 °C, 100% paraffin overnight at 60 °C eventually the samples were transferred into molds embedded in paraffin and stored at 4° C to harden. The resulting blocks were trimmed and positioned on a microtome (type 1212, Leitz, Wetzlar, Germany) with body axis orthogonal to the blade edge in order to obtain muscle cross sections. The angle of the blade (R. Jung AG, Heidelberg, Germany) was adjusted
Daphnia carapace: form, function, structure and plasticity 53 Materials and Methods between 0° and 7° and sections with a thickness of 5 µm were cut and transferred to glycerol coated object slides. Afterwards the slices were dewaxed: 100% Roti-Histol (Roti®-Histol, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 10 min, 2 times 100% EtOH I for 5 min, 96% EtOH for 5 min, 70% EtOH for 5 min, 50% EtOH for 5 min, 30% EtOH for 5 min, 2 times in purified water for 5 min. Finally, the samples were stained using a combination of picrosirius red staining after Puchtler and a haematoxylin staining after Weigert. Staining was performed by covering the samples with a mixture (1:1) of the Weigert solutions A and B for 2 min, rinsing samples in running deionized water for 1 min, staining in picrosirius red for 1 hour, rinse 2 times in acidified water for 2 min, dehydrate samples in ascending ethanol series again and mount them using Entellan.
After analyzing the muscle cross section area from the thin sections and the virtual cross section the muscle performance could be estimated using literature data of invertebrate muscle performances (Heethoff and Norton, 2009; van der Meijden et al., 2012). From the µCT-data the distance between mandible joint and muscle insertion, as well as the distance between mandible joint and tip of the biggest mandible spike was determined. Using that data the bite performance could be roughly estimated using the lever principle.
The obtained cross sections were visualized on a microscope (BX 40, Olympus, Hamburg, Germany) with a mounted camera (Altra, Olympus Soft Imaging Solutions, Münster, Germany), images were taken using Cell^D (Cell^D, Olympus Soft Imaging Solutions, Münster, Germany). These images were loaded into ImageJ and the muscle cross section area was determined using the “free hand selection” tool. All taken measurements were averaged for each analyzed individual and combined to a total average later on.
Daphnia carapace: form, function, structure and plasticity 54 Materials and Methods
Publications providing direct measurements of invertebrate muscle performances are rare but some work has been published regarding invertebrate muscle stresses (i.e. performance per cross section area). The reported muscle stresses in invertebrates range from ca. 250kPa up to about 1000kPa (van der Meijden et al., 2012). By combining the measured cross-sectional area with the published muscle stress the force at the insertion of the muscle into the mandible was determined.
F1 = 휎 ∗ Am
F1 = Force at the muscle insertion σ = Muscle stress Am = Muscle cross section area Equation 4: Force at muscle insertion Afterwards the force at the tip of the tallest mandible tooth was calculated using the lever principle. d1 × F1 = d2 × F2
F2 = F1 × d1/d2 d1 = Distance muscle insertion to joint d2 = Distance mandible tip to joint F1 = Force at the muscle insertion F2 = Force at the mandible tip
Equation 5: Lever principle
Daphnia carapace: form, function, structure and plasticity 55 Results
Results
Daphnia carapace: form, function, structure and plasticity 56 Results
Results
1. Carapace structure
Morphology of the carapace and distribution of the pillars The histological analysis of the daphniid carapace focused on the dorsal keel, since it exhibits comparatively large pillars, which can be observed with light microscopy (Fig 8A). Images of HE-stained animals revealed the pillars of the dorsal keel region to be slim-waisted and connected to the proximal and distal integument via broad branched bases, which could also be observed in vivo (Fig. 8B). Particle motion in the space between distal and proximal integument indicated hemolymph flow.
Daphnia carapace: form, function, structure and plasticity 57 Results
at the dorsal keel. Pillars (plr)
D. D. pulex
the carapace, pillars are visible as spots.
. . The head region (hcp) is covered by a single layer
D. D. pulex
observation observation of an adult
In vivo
:
B
stained stained adult
-
eosin
-
Hematoxylin
:
A
6 6 roots. The bases converge into the pillar waist (double arrowheads) connecting the proximal and
-
. .
mes mes branching into 4
daphniid daphniid carapace and its structure
Overview Overview of the
integument. integument. The body behind the head is protected by the carapace (crp), which prolongates into the dorsal spine (dsp). In The inset shows a region of the dorsal keel (dkl). The arrowheads point to single pillars. have a base on either side (arrows), someti integument. distal Figure Figure 8: Daphnia carapace: form, function, structure and plasticity 58 Results
Our HE stains differentiated the nuclei (blue) and the extracellular matrix (pink). Figure 8A displays a representative whole mount preparation of D. pulex with clearly contrasted pillars distributed irregularly across the carapace. The animal’s head is covered by a single integumental layer forming the head capsule. The body and the filtering legs are enclosed by a bivalved carapace, which is an evagination of the cephalic region (Olesen, 2013). Dorsally the carapace is fused to a keel that extends into a spine. The ventral side remains unfused, leaving a gap, the so-called ventral cleft, crucial for food intake and respiration. The ventral cleft serves as an opening allowing continuous water current by articulated limb movement. This continuous stream allows the uptake of food particles and gas exchange. The distance between proximal and distal integument is wider at the ventral margin and the dorsal keel in relation to the central region (Fig. 9A). The bases of the pillars varied in density and diameter (Fig 8A, Fig 9B,C). Pillar bases in the central carapace region had narrow diameters, whereas those near the ventral cleft and dorsal keel were wider and branched into four to six roots (Fig 8B). In these distal regions the number of pillars per 10000 µm² was lower than in the central region (Fig 9C).
Daphnia carapace: form, function, structure and plasticity 59 Results
Figure 9: Pillar density and base diameter along a ventral-dorsal transect in the central region of the carapace. A: Cross section of D. pulex displaying one lateral half of the carapace between the dorsal keel (dkl) and the ventral margin (vmg). B: Body length normalized pillar base diameter along a ventral-dorsal transect (data from three animals). C: Average pillar density (pillars / 10000 µm2) along a ventral to dorsal transect (data from three animals).
Daphnia carapace: form, function, structure and plasticity 60 Results
The toluidine blue stained cross-sections displayed the epidermis in high contrast, as well as clearly contrasting the pillars and the cuticle layers. The cross-sections also revealed slim- waisted pillars with broad bases branching into roots that attached to the integuments (Fig 10A).
Similarly, SEM images also displayed pillars with slim waists and broad bases that appeared branched at the integuments (Fig 10B). Furthermore, the SEM samples clearly displayed the pillars to be fibrous structures, interconnected with the extracellular matrix (Fig 10B). The extracellular matrix was particularly visible in the electron microscopic images (Fig 10B,C), allowing a 3-D impression of its fibrous structure (Fig 10C).
Due to the thinness of the proximal integument, the epi- and procuticle could only be distinguished in the high magnification of STEM images (Fig 11A,C). Similarly, the proximal epidermis was thinner than the distal and barely visible with light-microscopy (Fig 10A,B; Fig 11A).
Daphnia carapace: form, function, structure and plasticity 61 Results
D.
SEM
: B
wheads to wheads pillar waists.
rrow rrow with an asterisk to the laminar
an asterisk the extracellular matrix. matrix. extracellular the asterisk an
int) int) layers. integumental The white arrows point to the pillar bases,
carapace. carapace. The proximal integument (pint) is peeled off revealing the pillar bases
D. pulex
image image of
-
LM image LM of a toluidine blue stained section of the (pint) proximal and distal (dint) carapace of integument
:
A
carapace connecting theconnecting carapace proximal (pint) and the distal (d
D. pulex
Scanning electron microscopy
C:
cle. cle.
Carapace Carapace structure and pillar details.
: :
10
connected connected by the (plr)pillars thebridging hemolymphatic chamber (hlc) in between. point Arrows to pillar bases, double arro
pulex image of a pillar of a fractured the white double arrowhead to the pillar waist, the white arrow with an structure asterisk of the to procuti the extracellular matrix, and the black a with arrow white the waistand a pillar indicates arrowhead double white The roots. widelybranched with arrow) (white Figure Figure Daphnia carapace: form, function, structure and plasticity 62 Results
cle.
the the central waist of the pillar
laminar structure of the procuti of the structure laminar
Higher magnification STEM Higher magnification image of
B: B:
the the pillar’s base with the procuticle (pint). These intracuticular fibers extend from within the
section of a pillar anchoring the proximal and distal integument. The proximal procuticle (pint) (pint) procuticle proximal The integument. distal and proximal the anchoring apillar of section
-
STEM STEM micrograph displaying the pillar base. Cell organelles, nucleus and mitochondrion (black
C:
STEM image of a crossof a image STEM
ich shows a laminar structure (black arrow with an asterisk). The white arrow points to the pillar base, the white double
A:
. . Note the tightly arranged fiber bundles.
A
STEM images of pillar details. details. images pillar of STEM
: :
11
Figure is notably thinner than the distal (dint), wh arrowhead to the pillar waist and the white arrow an with asterisk to the matrix. extracellular displayed in arrowheads) are located within the pillar base. Several fibers connect the asteriskanindicates with arrow black The arrow). (black procuticle the pervading thoroughly anchorit, and pillar(plr)
Daphnia carapace: form, function, structure and plasticity 63 Results
Ultrastructure of the pillars The broad bases and the extracellular matrix were clearly observable in SEM samples where the proximal integument of the carapace had been mechanically removed (Fig 10C).
STEM and SEM micrographs showed the pillars to be interconnected with the extracellular matrix (Fig 10B; Fig 11A). We found the distal integument to be much thicker than the proximal one. The mean thickness of the D. pulex distal procuticle was 0.982±0.334 µm (± standard deviation (SD)), compared with 0.581±0.195 µm for the proximal procuticle. This difference is statistically highly significant (p<0.005, t-test, t-value 4.28, 17 specimens each). The underlying epidermis generally followed similar proportions. The procuticle clearly showed a laminated structure (Fig 10B; Fig 11A). STEM-images showed that the pillars possess a fibrous structure (Fig 11B), supporting the SEM observations. The single pillar fibers are tightly arranged into fiber bundles and have an average thickness of 14.67±2.49 nm (mean±SD, n=8). Many of the visualized pillars were found to be collapsed (Fig 11B), which may be an artifact of the preparation procedure. The fibrous structures of the pillars span through the epidermis and connect to the procuticle via fine insertions. Furthermore, a variety of cell organelles including mitochondria and nuclei were visible in some pillar bases (Fig 11C).
Hemolymphatic pressure The median gauge pressure of D. longicephala’s hemolymph was 3.12 mbar (Fig 12). The measurements showed high variance and ranged from a minimum of 0.08 mbar up to 9.73 mbar. However, all measurements showed a positive gauge pressure in the hemolymphatic chamber.
Daphnia carapace: form, function, structure and plasticity 64 Results
B:
Example measurement profile ofmeasurement pressure Example gauge of the hemolymphatic animal. one individual
A:
Measurements Measurements of thehemolymphatic gauge pressure.
: : Boxplot of the hemolymphatic gauge pressure measurements. pressure gauge of the hemolymphatic Boxplot Figure 12 Daphnia carapace: form, function, structure and plasticity 65 Results
2. Structure and shape contribution to carapace resistance
Structure Our STEM investigations focused on the distal carapace’ procuticle of D. pulex and D. longicephala. In both species the procuticle is organized in chitinous layers (Fig 13A) with a thickness of about 1 µm in undefended morphology (Mann-Whitney U-test, U value = 145, p = 0.46; n (D. pulex) = 17; n (D. longicephala) = 20). We found significant differences in this organization between the undefended and defended morphology.
Daphnia carapace: form, function, structure and plasticity 66 Results
n n
19; 19;
6, 6, n
1µm 1µm ii)
bar=
- longicephala longicephala
D.
21, 21, n(defended)=
n n (undefended)=
17, 17,
Procuticle Procuticle thickness of the
:
B
undefended, undefended, scale
longicephala longicephala
(right) (right) in undefended and defended
2µm. 2µm.
n(undefended)=
pulex pulex
D.
D.
bar=
-
n n (undefended; defended)=
longicephala longicephala
sections. sections. i)
D. D. longicephala
-
D.
and
pulex: pulex:
defended, defended, scale
16; 16;
D.
)=
test, test,
-
longicephala longicephala
D. pulex (left)
D.
Procuticle number of layers of the undefended and the defended morphotype of Procuticle ofand layers morphotype number the defended the undefended
5 5 with min 770 measurements each;
13, 13, n (defended
:
C
1µm 1µm iv)
. . (Mann Whitney U
STEM STEM images of procuticle cross
bar=
:
-
A
n n (undefended)=
pulex: pulex:
D. longicephala
Procuticle Procuticle Young´s modulus of
D.
n n (undefended; defended)=
:
and
undefended, undefended, scale
D
test, test,
-
pulex: pulex:
D.
, ,
D. pulex
longicephala longicephala
D. pulex and D. longicephala.
D.
ANOVA
. . (Mann Whitney U
1µm 1µm iii)
bar= ***p≤0.005). **p≤0.01, *p≤0.05; level 20; of significance:
-
D. longicephala
7 with min 770 measurements each; level of significance: * p≤0.05; ** p≤0.01, *** p≤0.005). *** p≤0.01, ** p≤0.05; * significance: of level each; measurements 770 min with 7
and
Procuticle Procuticle characteristics in
: :
defended, defended, scale
13
pulex pulex D. pulex
Figure D. undefended and the defended morphotype of defended)= (undefended; of level of significance: *p≤0.05; **p≤0.01, ***p≤0.005) form, measured on an AFM. (nested (defended)=
Daphnia carapace: form, function, structure and plasticity 67 Results
In D. pulex the procuticle of the defended morph is significantly thicker (median (undefended) = 0.843 µm, range = 1.399, median (defended) = 1.358 µm, range = 1.098, Mann-Whitney U-test, U value = 54, p = 0.002; n (defended) = 17; n (undefended) = 17) with a significantly increased number of layers (median (undefended) = 9, range = 4, median (defended) = 11, range = 9, Mann-Whitney U- test, U value = 43, p = 0.008; n(defended) = 16; n(undefended) = 13; Fig 13B).
D. longicephala showed no significant differences in procuticle thickness (median (undefended) = 0.919 µm, range = 1.646, median (defended) = 1.041, range = 1.988, Mann-Whitney U-test, U value = 178, p = 0.56; n (defended; undefended) = 20), but the number of procuticle layers is significantly increased in the defended form (median (undefended) = 6, range = 7, median (defended) = 9, range = 10, Mann-Whitney U- test, U value = 112.5, p = 0.02; n(defended) = 19, n(undefended) = 21; Fig 13B), leading to a decreased layer-thickness in the defended state. The morphotypes of undefended D. pulex and defended D. longicephala showed no significant differences in procuticle thickness and numbers of layers (Mann-Whitney U-test, U value = 143, p = 0.42; n (D. pulex undefended) = 17; n (D. longicephala defended) = 20). Thus in the simulations they were represented by the same model.
Material properties for FE analysis were determined by Young’s-modulus measurements of the outer procuticle for defended and undefended D. pulex and D. longicephala (Fig 13C) using AFM. We measured a mean e-module of 1.66 MPa ±1.71 SD for the undefended morphology and 2.93 MPa ±1.92 SD for the defended morphology of D. pulex. For D. longicephala we measured 5.12 MPa ±12.33 SD in the undefended morphology and 15.59 MPa ±21.55 SD in the defended morphology.
Based on the observed procuticle layer thickness and number, we created FE models for the different morphotypes and tested their capability to withstand mechanical impact. Simulations of only one cylinder for each morphotype resulted in deformation that decreases with increasing cylinder thickness following a power function (Fig 14A). The models representing the undefended morphotype of D. pulex and the defended of D. longicephala showed the strongest deformation. The procuticle models of undefended D. longicephala showed the weakest maximum deformations at the models bottom sides and the models of the defended form of D. pulex showed intermediate deformation at model’s bottom side. Comparing the models representing two complete rotations of fiber orientation for undefended and defended
Daphnia carapace: form, function, structure and plasticity 68 Results
morphotypes within one species, D. pulex showed less deformation in the defended morphotype, whereas D. longicephala showed less deformation in the undefended morphotype. Considering the complete set of simulations for the different morphotypes the deformation at the model’s bottom decreased with an increasing number of cylinders also following power functions (Fig 14B). Using these curves of best fit to calculate the deformation for procuticle thicknesses observed in STEM, the deformation of D. pulex was about 2.4 times higher in the undefended morphotype than in the defended one. In D. longicephala the deformation of the undefended morphotype was about 1.6 times higher than in the defended morphotype (Table 3).
Table 3: Simulated maximum deformation of different procuticle organizations Maximum Deformation at model’s bottom side Number of D. pulex D. pulex defended D. longicephala cylinders in model undefended undefended D. longicephala defended 1 0.786 nm 0.478 nm 0.316 nm 2 0.182 nm 0.11 nm 0.072 nm 3 0.054 nm 0.033 nm 0.022 nm 4 0.017 nm 0.011 nm 0.008 nm 5 0.009 nm 0.006 nm 0.005 nm 6 0.006 nm 0.004 nm 0.003 nm Cylinders to meet procuticle thickness 13 16 9 as observed in STEM expected maximum deformation 0.0007 nm 0.0003 nm 0.0011 nm according to fitting
Daphnia carapace: form, function, structure and plasticity 69 Results
Figure 14: FEA for the different morphotypes’ procuticle. A: Simulated deformation of single cylinders used for model-creation of the different morphotypes (Side view); simulated deformation is visually amplified (factor 27). The graph shows the deformation of the single cylinders for the different morphotypes. Curve of best fit: f(x) = 93764x-2.708 (R2 = 1) B: Simulated deformation of six calculated models with increasing thickness (one to six cylinders), representing morphotype i; simulated deformation is visually amplified (factor 27). The graph shows simulations of maximum deformation at the model’s bottom for all morphotypes. Curves of best fit: i: f(x) = 0.9679x-2.822 (R2 = 0.9887); ii: f(x) = 0.5703x-2.745 (R2 = 0.9907); iii: f(x) = 0.3621x-2.641 (R2 = 0.993).
Daphnia carapace: form, function, structure and plasticity 70 Results
Shape Our CLSM investigations showed the common morphology of D. pulex with the well described morphological changes in the defended state i.e. “neckteeth” in the dorsal head region (Fig 15A). The finite element simulations for the undefended morphotype, loaded with 1 mN, showed a maximal deformation of 95.2 µm. The deformation was limited to the ventral region of the carapace (Fig 15B). The stress distribution was not limited to the carapace but also covered the fornices localized in the head region. Maximal observed stress was 22.1 kPa (Fig 15B). The simulation of the defended morphotype showed a maximal deformation of 34.4 µm and maximal stress of 11.3 kPa (Fig 15C). The area of deformation included the whole ventral cleft but high deformations were restricted to the posterior half. In comparison to the undefended model, a wider area of the carapace showed deformation in this simulation. Similarly, the stress distribution showed a larger area of the carapace with high stresses. Regions of very high stresses were in the area of applied forces and the interceptions of the carapace into the dorsal spine as well as the headshield. Furthermore, the stress in the head region was lower than in the simulation of the undefended animals.
Daphnia carapace: form, function, structure and plasticity 71 Results
modulus modulus measurement n
-
Empirical Empirical data of structural
F:
Heat maps of deformation and stress and maps of Heat deformation stress
C:
d d animal.
test, test, for structural Young’s
-
31; Level of significance: * p≤0.05; ** p≤0.01; *** p≤0.005) *** p≤0.01; ** p≤0.05; * significance: of Level 31;
. The red x indicates the region of max lateral width, where the force force where width, the of max the region lateral indicates x red The .
D.pulex
36, n (defended)= n 36,
asurement n (undefended)= asurement n
Heat Heat maps of deformation and stress distribution in an undefended model with the material properties of a defended
D: D:
Heat maps of deformation and stress distribution an animal’s in maps model. of Heat and deformation stress distribution undefended
B:
50; for critical force me force critical for 50;
CLSM projections of undefended and defended form of and form defended of undefended projections CLSM
A:
d d animal’s model.
41, n (defended)= n 41,
FEA of D. pulex D. pulex of FEA
Heat Heat maps of anddeformation stress distribution in a defended model with material properties of an undefende
: :
modulus modulus and critical force for undefended and defended animals measured using a microindenter. (t
-
E:
15
mN was mN onto loaded the models.
of 1 distribution in a defende animal. Young’s (undefended)= Figure Daphnia carapace: form, function, structure and plasticity 72 Results
In a second step we tested shape and structure for their contributions to carapace resistance. This was done by providing a model of the undefended morphotype, with the procuticle characteristics of undefended animals and vice versa. Again we applied a force of 1 mN. The simulation of the undefended shape provided with the defended procuticle characteristics resulted in a maximal deformation of 16.9 µm and a maximal stress of 11 kPa (Fig 15D). The area of deformation and the stress distribution pattern were comparable to the undefended morphotype. The simulation of the defended shape provided with the undefended procuticle characteristics resulted in a maximal deformation of 187 µm and a maximal stress of 28 kPa (Fig 15E). The area of deformations and pattern of stress distribution was comparable to the simulation for the defended morphotype. Empirical measurements for comparison and validation of the simulations showed an average structural e-module of 142.38 kPa ±167.58 SD in the undefended and 262.84 kPa ±209.33 SD in the defended form. We found significant differences between the undefended and the defended form (t-test; t- value = -2.98257; p = 0.004; Fig 15F). Additionally, we measured the critical force which results in lethal collapse of the carapace, for both morphotypes. The undefended morphotype showed an average critical force of 1.14 mN ±0.87 SD, the defended 1.84 mN ±1.50 SD, with significant difference (t-test; t-value = -2.36935; p = 0.02; Fig 15F). Thus, the micro- indentations showed an increased geometric stiffness and puncture resistance in the defended morphotype.
In D. longicephala, we observed morphological defenses in forms of an increase in body length and spine length, and changes in head morphology, the so-called “crest” (Fig 16A). The finite element simulations for the undefended morphotype, loaded with 1 mN, showed a maximal deformation of 25.7 µm. The deformation was mainly on the ventral region of the carapace but an area on the rostrum exhibited also deformation (Fig 16B). In comparison to the simulations of D. pulex the stress distribution was not limited to the carapace but included parts of the head, predominantly the fornices. Maximal stress was 4.72 kPa (Fig 16B). The simulation for the defended morphotype resulted in a maximal deformation of 7.14 µm and a maximal stress of 7.45 kPa (Fig 16C). The area of deformation was mainly located at the dorsal half of the ventral cleft. In contrast to the simulations of D. pulex the defended morphotype of D. longicephala showed a large area of deformation on the head. The stress distribution image showed a smaller area of the carapace with high stresses in comparison to the undefended morphotype. We observed regions of very high stresses around the area of the applied force, the head shield and the ventral interceptions of the carapace into the spine. In
Daphnia carapace: form, function, structure and plasticity 73 Results the defended morphotype, the stress in the head region was lower than in the simulation of the undefended animals.
We also tested whether the form and structure of the carapace are in concert or act independently: The simulation of the undefended morphotype with procuticle characteristics of defended animals showed a maximum deformation of 8.43 µm and maximum stress of 4.72 kPa (Fig 16D). The area of deformations and pattern of stress distribution was the same as in the simulation with the undefended morphotype and only differed in magnitude of deformation. The simulation of the defended morphotype provided with the undefended procuticle characteristics resulted in a maximal deformation of 21.7 µm and a maximal stress of 7.45 kPa (Fig 16E). The area of deformation and the stress distribution pattern were comparable to the defended morphotype. In D. longicephala we measured a structural Young’s-modulus of 103.96 kPa ± 114.34 SD for the undefended and 195.39 kPa ± 182.98SD for the defended morphotype (Fig 16F). As for D. pulex, the t-test showed significant differences between the undefended and the defended form (t-value= -3.69050; p = 0.0003). We determined a critical force of 2.22 mN ±0.94 SD for the undefended and 3.07 mN ±1.18 SD for the defended morphotype (t-test; t-value= -2.71480; p = 0.009; Fig 16F). Thus, the micro-indentations showed an increased geometric stiffness and puncture resistance in the defended morphotype.
Daphnia carapace: form, function, structure and plasticity 74 Results
28, 28, n
l, l, loaded with a force of
. width, region The . x of red max the lateral indicates
62; 62; for critical force measurement n (undefended)=
D. D. longicephala
Heat maps of deformation and stress distribution in an undefended model an model in undefended and stress distribution deformation maps Heat of
D:
83, 83, n (defended)=
modulus modulus and critical force for undefended and defended animals measured using a
-
ps ps of deformation and stress distribution in a defended animal’s model with the material
Heat Heat ma
E:
Heat Heat maps of deformation and stress distribution in an undefended animal mode
B:
modulus modulus measurement n (undefended)=
-
Empirical Empirical data of structural Young’s
CLSM projections of undefended and defended form form of and ofundefended defended CLSM projections
F:
A:
test, test, for structural Young’s
-
18; Level of significance: * p≤0.05; ** p≤0.01; *** p≤0.005) *** p≤0.01; ** p≤0.05; * of significance: Level 18;
er. er. (t
FEA FEA D. of longicephala
: :
Heat maps of deformation and stress distribution in a in model defended animal stress distribution and maps Heat of deformation
16
C:
mN. mN.
in in where the models were loaded for the simulations. 1 provided with the material properties of a defended animal. properties of an undefended animal. microindent (defended)= Figure Figure Daphnia carapace: form, function, structure and plasticity 75 Results
3. The catching basket of Chaoborus
The catching process The catching process starts immediately after the Chaoborus larva detects a daphniid (Fig 17). First step of the motion sequence is a retraction of the rhinopharynx towards the head (Fig 17b). The next step is a set of simultaneous, uniform movements i.e. opening of the mandibles, the labral setae and the antennae including their setae in combination with orientation of the this way formed catching basket towards the prey (Fig 17c-e). Then, just bevor contact with the prey, the mandible fan opens up (Fig 17g). In most cases prey contact in conducted via antennae. Right after prey contact the antennae and their setae retract moving the prey towards the mouth (Fig 17h). The larva’s head continues its movement until the catching basket presses the captured prey against the larva’s body (Fig 17i-j). Thereby, the only open part of the catching basket is closed by the body and the prey is trapped in between. Finally, the larva turns its head and body back into resting position and starts to handle the prey aiming to ingest it with an alternating stuffing motion of the mandibles. The whole catching process takes less than 300 ms. The folding of the head towards the body and back into resting position takes the longest time (about 250 ms) while the duration from starting the catch to grasping the prey only takes around 30 ms.
Daphnia carapace: form, function, structure and plasticity 76 Results
Figure 17: Time series of Chaoborus larvae catching a daphniid. Images of selected steps of the catching event from a video recorded with 8000fps. a) 0 ms; b) 6,9 ms; c) 10 ms; d) 13 ms; e) 14,9 ms; f) 16,5 ms; g) 19,5 ms; h) 21,8 ms; i) 28,6 ms; j) 34,8 ms; k) 57,8 ms; l) 90,2 ms; m) 123,9 ms; n) 188,3 ms; o) 287,6 ms
Daphnia carapace: form, function, structure and plasticity 77 Results
Morphology of the catching basket The conducted µCT scan of a Chaoborus larva’s head resulted in a high resolution 3D image of its morphology (Fig 18A). This data was successfully used for a volume reconstruction of the head capsule including all appendages as well as their muscles and one of the complex eyes (Fig 18B). Based on the reconstruction volume data a surface model for the outer morphology was extracted including head capsule and appendages (Fig 18C). Finally, the surface model was analyzed for the appendages’ morphology to create an animation sequence of the catching event (Fig 19).
Daphnia carapace: form, function, structure and plasticity 78 Results
Figure 18: Overview of the Chaoborus larvae head morphology. A: lateral view of head’s µCT
scan. B: Volume reconstruction based on the µCT scan. ant = antennae; pra = prelabral appendages; lse = labral setae; rhi = rhinopharynx; mnd = mandible; mx = maxillae; mndab = mandible abductor (m. abductor mandibulae); mndad = mandible adductor (m. adductor mandibulae); cey = complex eye; antad = antennae adductor (m. retractor antennae); rhiab = rhinopharynx abductor (m. abductor epipharyngis); rhiad = rhinopharynx adductor (m. adductor epipharyngis); hdc = head capsule. C: Surface model extracted from volume reconstruction. D: Detail of the mandible from proximal side of view in closed (i) and opened (ii) stage. E: Detail of the rhinopharynx from frontal (i), lateral (ii) and backside (iii) view.
Daphnia carapace: form, function, structure and plasticity 79 Results
The catching basket During attack movement the involved appendages built an extremely effective hemispherical catching basket. The anterior ventral part of this catching basket is realized by the antennae and their widely spread setae. The posterior side is enclosed by the mandibles and the mandibular fans. The dorsal space between these two elements is closed by the spread labral setae. The detailed movement and mechanisms of every part of the catching basket are explained in detail in the following.
The Antennae In larvae of Chaoborus, the antennae, primarily a sensor organs, are modified into grasping organs. They originate from the tip of the elongated clypeus and possess proximal curved setae each (Fig 17e). In resting position these setae are orientated in ventral/posterior direction lying in front of the mouth opening (Fig 18B). The opening movement of the antennae is a result of increasing hemolymph pressure which expands the joint skin and spreads the antennae basis and setae. The retraction movement executed after a catch is based on a pair of muscles. The m. retractor antennae arises as a strong bundle at the neckline and the insertion of the muscles tendon is ventral at the antenna base. The antennae joint skin is very delicate and therefore not visible in the µCT data. The articular cavity encloses the articular head more lateral than superior and inferior and the rotary axis is not orthogonal to the sagittal plane but slightly leaned dorsal. Accordingly, the stretched antennae are not parallel but have an angle of 25° peripheral (Fig 19j).
Daphnia carapace: form, function, structure and plasticity 80 Results
The labral setae and prelabral appendage The labrum is reduced to five pairs of bristles lying ventral between mouth opening and antennae. The obtained high speed videos revealed these bristles wide spread during attack movement. The labral setae can be divided into two groups, the anterior four pairs and a posterior pair. The bases of these two groups are divided therefore the gap between antennae and mandibles are closed almost completely.
The prelabral appendages originate posterior from the labral setae. In resting position they are partially covered by the labral setae. The appearance of the prelabral appendages is blade like leading to their descriptive name “knife hairs” (Messerhaare).
The mandibles The larva’s robust mandibles end in sharp chitinized spikes at the ventral anterior edge. During ingestion, the unchewed prey is stuffed into the esophagus by alternating mandible movement. Although this movement appears to have no shredding function, sometimes daphniids burst during stuffing. The mandibles’ six spines differ in shape and movability and therefore can be subdivided into two groups. Two thin dorsally laying spikes that look more like bristles close synchronously to the mandibles without a joint. They insert on a membranous proximal surface of the mandibles which expands when hemolymphatic pressure increases and this way these bristle spikes erect. The other four spikes are robust and immobile and the longest of these bifurcates into an additional smaller tip.
Daphnia carapace: form, function, structure and plasticity 81 Results
The mandible muscles The m. adductor mandibulae is massive and consists of three strands, whereof two originate dorsal of the complex eye and the third at the postoccipital rail. All three strands insert somewhat inferior from the middle of the proximal mandible joint. The m. abductor mandibulae originates dorsally of the complex eye and inserts at the middle of the distal mandible joint.
The Mandible joint The mandible joint consists of the posterior edge of the mandible and subgenal rail. The posterior edge of the mandible builds an articular head that continues from ventral to dorsal. Therein seizes an abutment built by the subgenal rail.
The mandibular fan At the mandible’s dorsal anterior edge, of the mandibular fan arises. Its eleven bristles are bifurcated at the base. The dorsal branch of the bristles inserts upon the membranous proximal part of the mandible whereas the ventral branch articulates on the mandibles robust ventral edge. This way, the bristles rest parallel aligned upon the membranous field of the mandible when hemolymphatic pressure is low (Fig 18D i) and erect splayed to the mandible fan when hemolymphatic pressure is high (Fig 18D ii).
Daphnia carapace: form, function, structure and plasticity 82 Results
The rhinopharynx The finger-shaped rhinopharynx protrudes at an angle of about 35° from the rostrum in between the prelabral appendages and the mouth. It possesses an oval cross section diameter due to a lower transversal than sagittal diameter (Fig 18E). Two muscles facilitate its movement (m. abductor epipharyngis, m. adductor epipharyngis; Fig 18B). The rhinopharynx’ tip is laterally studded with symmetrical arranged immobile bristles: Four bristles arise on each side at the posterior part of the rhinopharynx’ tip whereof one points posterior/superior, the other three point inferior. Apical on each side is an almost vertical row of five bristles which are opened fan-like and point anterior/inferior. The rhinopharynx’ tip ends anterior in a row of six spiky squamous fork thorns (Gabeldornen) that are accompanied by five smaller bristle scales (Borstenschuppen) with a basally offset. Posterior the tip has a concave shape.
Animation of the catching event With the conducted high speed recordings, the motion sequence of the involved head appendages were identified including their maximal positions in relaxed and attacking position. This data was synchronized to the surface model and resulted into a complete animation of the catching event (Fig 19).
Daphnia carapace: form, function, structure and plasticity 83 Results
Figure 19: Comparison of a high speed recording and the animation. a-b) Larva’s head in resting position. c-d) Rhinopharynx in “trigger” position just before attack motion. e-f) All appendages spread except the mandibular fan. g-h) All appendages spread to completely opened catching basket. i-j) dorsal view of completely opened catching basket.
Daphnia carapace: form, function, structure and plasticity 84 Results
Efficiency of the catching basket The analysis of the high speed recordings resulted in a tendency of less successful attacks by Chaoborus on defended than on undefended prey but no significant differences were found
(Chi-square test, x-squared = 3.0662, nundefended = 23, ndefended = 26, p = 0.08; Fig 20A). Furthermore, this effect could not be correlated with prey position in relation to the larvae, relative approach direction or prey position during handling. The feeding experiments revealed a significantly lower number of strikes by Chaoborus on defended prey than on undefended prey (t-test, t = 2.5462, n = 11, p = 0.02; Fig 20B). During the strike no significant disparity was measured in terms of contact vs. evasion (t-test, t = 1.7034, n = 11, p = 0.1; Fig 20B). After contact, during prey handling significant difference between undefended and defended prey was observed. Defended daphniids escaped more often than undefended (t-test, t = 2.6761, n = 11, p = 0.02; Fig 20B).
Daphnia carapace: form, function, structure and plasticity 85 Results
Figure 20: Chaoborus’ catching basket efficiency. A: Strike efficiency observed in the high speed recordings for undefended and defended prey. Success was counted when prey was ingested, failure when evasion or escape were observed. B: Strike efficiency observed in the feeding experiments distinguished after strike between evasion and contact as well as ingestion and escape after contact. Boxes represent median ±25% of data, whiskers represent 98% of data, outliers are visualized as dots. Statistical tests: A) chi-square test B) t- test, level of significance: * = p < 0.05
Daphnia carapace: form, function, structure and plasticity 86 Results
Bite force estimation Table 4: Muscle sections of the m. adductor mandibulae from slices of two paraffin embedded Chaoborus as well as the µCT scan. The values are averaged from several sections and the muscles of both sides. Analyzed sample Cross section area [mm2] Thin section series 1 0.009 Thin section series 2 0.0077 µCT scan cross section 0.0084 Total average 0.0083
Table 5: Measurements of the distances from the mandible joint to the insertion of the m. adductor mandibulae as well as the tip of the mandible. Morphological feature Distance to the mandible joint [µm] Muscle insertion 69.154 Mandible tip 369.081
Using the measured muscle cross section area (table 4) and the published muscle stresses the force at the muscle insertion within the mandible was computed. Afterwards, this force, together with the distance between the mandible joint and the muscle insertion as well as the distance between mandible joint and the mandible tip (table 5) was used in the lever principle to estimate the bite force. This estimation resulted in values ranging from 0.37 mN (250 kPa muscle stress) up to 1.48 mN (1000 kPa muscle stress).
Daphnia carapace: form, function, structure and plasticity 87 Results
F1 = 휎 × Am
F1 = Force at the muscle insertion σ = Muscle stress Am = Muscle cross section area
d1 × F1 = d2 × F2
F2 = F1 × d1/d2 d1 = Distance muscle insertion to joint d2 = Distance mandible tip to joint F1 = Force at the muscle insertion F2 = Force at the mandible tip
For muscle stress of 250 kPa:
F1 = 250 kPa × 0.0083 mm2
F1 = 2 mN
F2 = 2 mN × 69.154 µm/369.081 µm
F2 = 0.37 mN
For muscle stress of 1000 kPa:
F1 = 1000 kPa × 0.0083 mm2
F1 = 8.3 mN
F2 = 8.3 mN × 69.154 µm/369.081 µm
F2 = 1.48 mN
Daphnia carapace: form, function, structure and plasticity 88 Discussion
Discussion
Daphnia carapace: form, function, structure and plasticity 89 Discussion
Discussion
1. Carapace structure
We analyzed the integument of the Daphnia carapace in order to evaluate the contribution of interconnecting pillars to the carapace’s capability to withstand compressive forces. We found the proximal integument to be significantly thinner than the distal, which is in accordance with earlier descriptions (Anderson, 1933; Fryer, 1996; Halcrow, 1976; Pirow et al., 1999). The interconnecting pillars appeared fibrous, slim-waisted and tightly attached to the epidermis and anchored in the procuticle via intercuticular fibers. This observation is in contrast to their hypothesized function as load bearing structures (Anderson, 1933; Laforsch et al., 2004) and rather indicates the capability to withstand tensile forces.
Carapace proximal and distal integument structure We observed that the distal integument was clearly distinguishable into the epicuticle, multilayered procuticle, epidermis and extracellular matrix (Fig 10A,B; Fig 11A, Fig 21) confirming earlier studies (Halcrow, 1976; Stevenson, 1985). The thickness of the distal integument probably reflects a higher risk of perforation by predators’ mouthparts. Furthermore, the relative thinness of the proximal integument reflects its contribution to daphniid respiration i.e. the thin integument facilitates oxygen uptake from the permanent water current in the filtering chamber produced by the beating thoracopods (Fryer, 1991; Pirow et al., 1999a, 1999b).
Daphnia carapace: form, function, structure and plasticity 90 Discussion
The carapace is composed of two integumental layers in reverse complement manner and interconnected
Schematic Schematic of the Daphnia carapace structure.
:
21
by pillars. by Figure
Daphnia carapace: form, function, structure and plasticity 91 Discussion
Interconnecting pillars Our results show that the pillars connecting the distal and proximal integuments within Daphnia carapaces are slim-waisted with broad, branched bases. These findings were independent of the preparation method used (in vivo, Fig 8B; resin embedding, Fig 10A, Fig 11A; or HMDS drying, Fig 10B,C) and are in agreement with the description reported by Anderson, who sketched them with thin centers but assigned them a supporting function (Anderson, 1933). Our investigations revealed that the pillars consist of single fibers, packed together to form the characteristic pillar shape (Fig 11B). Anderson assumed that these pillars are chitinous (Anderson, 1933) but our stains indicate that they originate from the connective tissue of the extracellular matrix, since the HE stained the pillars a reddish color. In addition, the pillar fibers share characteristics with intermediate filaments. Their thickness is on average 14.66 nm, which lies within the range of intermediate filament thickness (10-15 nm; between microfilaments (5-8 nm) and microtubules (20-30 nm)). Furthermore, due to preparation the fibers often appeared bent in the waist region of the pillars and thus seem to be resistant against shear forces. Intermediate filaments are known for being far more resistant against shear forces than microfilaments and microtubules (Janmey, 1991). We found that some of the fibers reached through the epidermis, anchoring the pillar in the procuticle (Fig 11C). These are analogous to the anchoring fibers in arthropods that reach through the epidermis and into the procuticle to attach muscle (Bitsch and Bitsch, 2002). Znidaršič et al. showed for isopods, that these fibers even reach through the new procuticle and anchor in the old procuticle just before molting (Žnidaršič et al., 2012). In Daphnia, such attachment fibers to the exoskeleton were described for muscles by Schulz and Kennedy (Schultz and Kennedy, 1977) and in association with pillars by Halcrow (Halcrow, 1976).
Daphnia carapace: form, function, structure and plasticity 92 Discussion
Pillar function Laforsch et al. (2004) described the carapace as a light-weight construction capable of withstanding mechanical impact and compressive forces. If that were the case, then the supporting elements should be composed of solid columns without any distinct thin weak points. Chen et al. analyzed pillar structures in the elytra of Dorcus titanus (Insecta) and showed that they are optimized for uptake of compressive forces (Chen et al., 2015). Morphologically, these pillars are solid columns with broad bases and no waists. Their internal structure is characterized by chitin fibers, reinforced by a sclerous-protein matrix that continuously merges from the procuticle into the pillar. This continuous transition from the rigid procuticle fiber-matrix into the pillars reinforces the weak points of this pillar type i.e. the pillar-procuticle contact area. In contrast to the elytra pillars the Daphnia pillars have broad bases but slim waists and are most likely composed of intermediate filaments. These filaments continuously extend into the extracellular matrix and are additionally anchored vertically in the procuticle. The vertical anchoring translates tension forces, acting on the pillars, to the procuticle in proximal direction which is its optimal load angle. The pillars’ broad bases offer a larger contact surface between integuments and pillars, allowing a higher potential to withstand tensile forces due to the distribution of forces over a larger area. The slim waists do not negatively affect the capability to withstand tensile forces because the material properties, diameter and number of fibers are more important than the shape in which they are composited (Ottani et al., 2001). In fact the broad bases and slim waists mirror the mushroom-shape of adhesive structures in e.g. Chrysomelidae (Insecta, Coleoptera). These adhesive structures were found to be capable of taking high tensile loads before losing contact (Carbone et al., 2011; Heepe and Gorb, 2014). In general, biological structures responsible for taking up or even storing tensile forces are comparatively thin and fibrous, e.g. tendons or roots (Mattheck, 1998). Tensile forces imposed on the integumental layers could result from hemolymph that fills the space between the two integumental layers of the carapace. This can be observed in insect wing expansion during adult emergence. Insect wings also consist of two integuments that are closely interconnected via microtubule rich cells, each anchored by fibers in the procuticle (Nardi and Ujhelyi, 2001). For expansion, hemolymph is pumped between the layers. If the hemolymphatic pressure in Daphnia is higher than that of the surrounding medium (i.e. the water body), similar stabilizing elements are necessary to prevent the carapace integuments from drifting apart. Such gauge pressure would contribute to the animal’s resistibility against mechanical impact by providing a hydrostatic force that
Daphnia carapace: form, function, structure and plasticity 93 Discussion works against such impact (comparable to the increase in an air bed’s stiffness with increasing gauge pressure). During a local impact caused by a predatory attack the loaded pillars would buckle (not break) and the stress would be distributed over a larger area via an increase of the hemolymphatic pressure, resulting in tensile forces acting on the pillars in the surrounding areas. In addition to the protective function of the carapace, the pillars are also relevant for respiration. While the hemolymphatic chamber between the two layers would collapse in case of negative gauge pressure it would expand without the pillars in case of positive gauge pressure. Thus, the pillars’ resistibility against tension forces is crucial for the known functions of the carapace i.e. protection and respiration.
The median hemolymphatic gauge pressure measured directly in live D. longicephala was 3.12 mbar (Fig 12). Measuring the hemolymphatic gauge pressure in D. pulex would have been challenging with our experimental setup due to their smaller size. Although the variation was high, all measurements showed a positive gauge pressure and thus proved the hemolymphatic pressure to be higher than the pressure of the surrounding medium. Data variability might result from the invasiveness of the method, where sealed connection cannot be assured and capillary penetration depth cannot be kept constant per individual trial. Furthermore, the animals might have suffered from different stress levels, during the in vivo procedure, which influences the hemolymphatic pressure. The differences in pillar density and base diameter between central regions of the carapace and the dorsal keel as well as the ventral margin could be the result of a trade-off between the strength of the ties between the integument layers and hemolymphatic flow resistance. At both the dorsal keel and ventral margin, the hemolymphatic current is higher than in the central region (Pirow et al., 1999a) and the integuments are separated by a wider gap. The reduced number of pillars might be a concession to the hemolymphatic current, enabling a better flow, and compensated by a larger connection (pillar base) for each interconnection. In conclusion, the pillars operate against tensile forces resulting from hemolymphatic pressure, rather than countering superimposed compressive forces. In order to reflect the pillars’ function, we suggest using the term ‘ligaments’ in analogy to the structures that interconnect skeletal elements.
The unique structure of the daphniid carapace offers remarkable protection with minimal material investment. Our results provide a new mechanistic explanation for its high rigidity: using the hemolymphatic pressure as a supporting element, daphniids use a light-weight construction consisting of two integuments connected via flexible ligaments and inflated by a hemolymphatic hyper-pressure to protect their body from the environment. Because
Daphnia carapace: form, function, structure and plasticity 94 Discussion hemolymph has a similar density to water, this construction does not impose negative effects on swimming. In conclusion this structure seems to be an excellent compromise between protection and swimming capability that may play an important role in Daphnia success in lentic ecosystems.
Daphnia carapace: form, function, structure and plasticity 95 Discussion
2. Structure and shape contribution to carapace resistance
Daphniids are prominent for their inducible morphological defenses. Recently it has been shown that besides the distinct shape alterations, so called “hidden” defenses exist within the carapace structure that result in a higher carapace resistance (Laforsch et al., 2004; Rabus et al., 2013). We developed a method to identify structural features of the carapace architecture responsible for the stiffness increase observed in D. pulex and D. longicephala. Although alterations in daphniids carapace stiffness have been reported before for D. pulex, D. cucullata, D. middendorffiana and D. magna (Dodson, 1984; Laforsch et al., 2004; Rabus, 2015) the underlying mechanism has not been described yet. Furthermore, we here present a method to distinguish between the contributions of structure and shape to mechanical resistance of the carapace. FEA is a tool offering the possibility to analyze single elements of complex biological defense structure, particularly in small invertebrates where empiric data acquisition is limited. We used FEA to determine the physical features contributing to the protective effect of morphological defenses. Our experimental data show increased carapace stiffness in the defended morphotype of both analyzed species that are characterized by different inducible morphological traits.
Structure Predator induced changes in procuticle structure in both investigated species result in an increase of procuticle thickness and number of layers in D. pulex but a constant thickness accompanied by an increased number in layers in D. longicephala. Both defended forms show a significant increase in the procuticle Young’s-moduli in our experiments and thus higher carapace stiffness.
We simulated the different procuticle features of the different morphotypes using FEA. Our results indicate that the defended morphotype of D. pulex realizes the increase in carapace stiffness by higher procuticle thickness, as well as a higher number of layers. This type of defenses seems to be a combination of two beneficial effects where an increase of puncture resistance due to higher level of lamination is combined with an increase of flexural strength due to higher thickness. Chitin fibers are strongest against tension in their fiber direction and therefore a compound material like the procuticle is strongest if there is a fiber orientation
Daphnia carapace: form, function, structure and plasticity 96 Discussion matching every possible loading direction. A higher level of the procuticle lamination equates a higher number of continuous rotations of fiber orientation and thus more fibers for every possible loading direction. The resulting increased puncture resistance in D. pulex is accompanied by increased flexural strength i.e. crush resistance since flexural strength increases with material thickness. Chaoborus larvae ingest their prey by alternating mandible movement and an increase in flexural strength may be beneficial and reduce damage from crushing. This might increase the D. pulex’ survival chances after escaping predator capture.
In D. longicephala the defended morphotype displays an increase in procuticle layers at a constant procuticle thickness in comparison to the undefended morphotype. This indicates an increase in carapace puncture resistance only. D. longicephala counters predation of a heteropteran predator that punctures the carapace with its proboscis. Thereby, the likelihood of successful penetration may be reduced through this procuticle rearrangement. We thus anticipate that D. longicephala’s procuticle acts like a bullet proof vest against Notonecta’s proboscis.
The structural alterations in both analyzed species indicate a defensive effect that encounters consumption of the specific coexisting predator.
We could show that procuticle reorganization has crucial impact on carapace resistance but other effects may contribute too, e.g. chemical composition, fiber crosslinking or mineralization. Since the crustacean cuticle is a very versatile structure with numerous parameters defining the overall properties, it is possible that chemical analysis of the carapace composition could reveal additional strategies for enhancement of its defensiveness.
Shape We here aimed to determine whether the adaptive defense morphologies of Daphnia actually affect overall deformation during punctual force application. We modelled the D. pulex and D. longicephala undefended and defended overall shape using a classical landmark-based system and combined these with the measured procuticle Young’s-moduli and thicknesses. The extracted models of both species matched both shape outlines and thus reflected the natural appearance. The simulations in which we applied the naturally relevant force of 1 mN resulted in decreased deformation in the defended morphology compared to the undefended for both species. In D. pulex this was a 64% reduced deformation of the defended morphology in comparison to the undefended. The area of deformation as well as the distribution of stress was greater in the defended morphology model which might explain overall reduction of
Daphnia carapace: form, function, structure and plasticity 97 Discussion stress (~50%) and deformation. This is supported by the experimentally determined Young’s- modulus which increased by 85% and the critical force for lethal injury by 62%.
The simulations of D. longicephala resulted in a 72% reduced deformation of the defended morphology model in comparison to the undefended model. In contrast to D. pulex the defended morphology possesses large modifications (i.e. the “crest”) but the thickness of the procuticle is the same as in the undefended morphology. In the undefended model highest deformation was found at the ventral cleft positioned half the distance from head to tail spine. The defended morphotype’s maximum deformation was also positioned at the ventral cleft but positioned in the lower third of the head to tail spine distance. The stress distribution was more concentrated but showed higher maxima within the area of impact. Therefore, the defended morphology reduced the deformation significantly but revealed higher local stress maxima. Our experimental data of the carapace structural Young’s-modulus confirms this increase of carapace resistance. We found a Young’s-modulus increase of 88% and the critical force showed an increase of force necessary to provoke lethal injury of 72%.
To test whether the protective effect originates from the overall shape or the underlying structure we simulated the defended shape together with the procuticle Young’s-modulus and structure of an undefended animal and vice versa. In D. pulex the defended shape combined with the undefended structure showed a deformation nearly twice as high as the undefended morphology. The simulation of the undefended shape combined with the defended structure resulted in a deformation only half as high as the defended morphology. Our results indicate that the shape alteration actually negatively influences the carapace resistance in D. pulex, which is compensated by changes of the procuticle structure. In D. longicephala we observed a marginally reduced deformation in the simulation of the defended shape combined with the undefended structure in comparison to the undefended morphology. The undefended shape combined with the defended structure showed deformation almost identical to the defended morphology. In contrast to D. pulex the shape of D. longicephala has no negative effect to the carapace resistance, but the increase in carapace resistance is mainly based on the structural alterations. The lacking contribution of shape alterations to the carapace resistance in both species indicates that the defensive effect of these alterations rather is to impede predator handling or capture as it was hypothesized before (Krueger and Dodson, 1981; Spitze and Sadler, 1996).
Daphnia carapace: form, function, structure and plasticity 98 Discussion
Conclusion Overall, our results indicate that mechanical resistance originates from the structural reorganization of the procuticle rather than the overall shape. Nevertheless, one factor explaining the evolution of inducible defenses is that such traits are beneficial, consequently neckteeth and crests must incur a different protective effect (Tollrian and Dodson, 1999). We anticipate that defensive morphological traits will pose handling difficulties on the predator and therefore act during a different moment of a predation event i.e. during capture and supports the “anti-lock-and-key” hypothesis. Likewise, morphology dependent hydrodynamic advantages may prove beneficial by improving swimming performance and reducing capture risk. In contrast, increased mechanical resistance resulting from procuticle reorganization may act during predator ingestion, so that the predator’s crushing capability (in the case of Chaoborus) or successful proboscis penetration (in the case of Notonecta) is reduced.
Daphnia carapace: form, function, structure and plasticity 99 Discussion
3. The catching basket of Chaoborus
We here provide a detailed 3D animation of the Chaoborus larva’s head performing the motion sequence during prey capture. This animation was created using high speed recordings and µCT data. The produced animation gives insights into the catching event, a crucial element of the predator prey interaction of Chaoborus and Daphnia pulex, in high time and spatial resolution. Each catching event always starts from a resting position of the larva. As an ambush predator, Chaoborus rests in the water column waiting for prey to pass by. In resting position, the antennae are directed ventrally while their setae are lying in parallel. Also the labral setae point ventrally and recline in parallel. The mandibles are closed and their fans are folded towards their proximal side. When prey is detected within reach, where reach is determined by antennal length, a rapid attack is launched. In high speed recordings the first recognizable movement initiating the attack is the rhinopharynx’ adduction to the mouth (t=0 ms). The next movement is the orientation of the head towards the prey combined with abduction of the antennae (t=10 ms). When orientated towards the prey, the larva’s catching basket begins to open (t=13 ms). Simultaneously, the antennae’s setae and the labral setae that build the dorsal side of the catching basket spread wide while attack movement of the head towards the prey continues (t=15 ms). Then the mouth opens by spreading the mandibles (t=16.5 ms). Briefly before prey contact and sometimes even at prey contact the mandible fans open wide and obstruct a prey’s escape in posterior direction (t=19.5 ms). With this wide opened catching basket, the larva grasps the prey by adducting the antennae at prey contact (t=22s). The antennae’s adduction automatically moves the prey in mouth direction while the head movement towards the thorax continues until the catching basket’s open ventral side is closed by the larva’s thorax (t=35 ms). This grasping attack, from rhinopharynx adduction to the head’s retraction against the thorax, is facilitated in about 35 ms. Our morphology and motion reconstruction confirms most of the hypothesized movements and functions of the larvae’s catching basket components made by Schremmer (Schremmer, 1950). Nevertheless, Schremmer’s estimation about the labral setae’s insignificant movability could not be confirmed as the high speed recordings revealed them to spread wide during the catching event. His hypothesis about their contribution to the catching basket i.e. closing the gap between antennal setae and the mandible fan can be confirmed. Due to reduction of associated muscles it can be assumed that their movement is realized by changes in hemolymphatic
Daphnia carapace: form, function, structure and plasticity 100 Discussion pressure (Schremmer, 1950). Furthermore, his assumed function of the rhinopharynx could not be supported. Schremmer hypothesized that this appendage is used to stuff the prey into the mouth opening and close the latter to prevent prey escape. In none of our recordings movements were observed that support this function. In fact, the rhinopharynx is the first appendage to move initiating the catching event. It folds mouthwards and stays in this neutral position during prey capture and handling. Thus, we do not anticipate its involvement in prey handling or escape prevention. The two prelabral appendages are misleadingly designated as “Messerhaare” (knife hairs) since they are not involved in prey handling or ingestion. They are used as important characteristics for species identification and may have sensory functions but so far this hypothesis is not confirmed.
With a timeframe of 20 ms from start of the movement to prey contact, the attack of Chaoborus larvae ranks within the fastest attack movements reported so far: the praying mantis Coptopteryx viridis (Mantodea, Mantidae) attacks within 42 ms (Maldonado et al., 1967), for mantis shrimps Squilla empusa (Squillidae, Stomatopoda) and Hemisquilla ensigera (Hemisquillidae, Stomatopoda) attack movements within 4-8 ms have been reported (Burrows, 1969). The small beetle Stenus comma (Coleoptera; Staphylinidae) shoots its labium within 1-3 ms at its prey, the springtails (Collembola) that show an escape response within a few milliseconds (Bauer and Pfeiffer, 1991). Trap-jaw ants Odontomachus (Hymenoptera, Formicidae) close their mandibles even in less than 1 ms (Gronenberg et al., 1993). Considering the maximal reported swimming speed of Daphnia pulex which is 15 mm/sec (O’Keefe et al., 1998) a daphniid would cover a distance of 300 µm within the timeframe of a larva’s attack. This is less than half the body length of the prey. Even if attack and escape start simultaneously, a successful active escape through swimming is unlikely and was not observed in the high speed recordings. No flight reaction by the daphniids was observed before attack’s impact.
The analysis of high speed recordings in conjunction with feeding experiments confirmed the daphniids defense’s efficiency (also reported by Krueger and Dodson, 1981; Havel and Dodson, 1984; Parejko and Dodson, 1991). However, the high speed recordings could not confirm the hypothesis of mechanical interference of the neckteeth with Chaoborus’ mouthparts or catching basket components (Krueger and Dodson, 1981; Spitze and Sadler, 1996). Since the feeding experiments revealed defensive effects before as well as after predator prey contact Daphnia pulex seems to possess a complex set of defenses including predator avoidance and after contact defenses. The rather unimposing size of the neckteeth
Daphnia carapace: form, function, structure and plasticity 101 Discussion suborns to neglect a hydrodynamic effect that may reduce perceptibility for the tactile hunting Chaoborus larvae. Therefore, behavioral defenses realized in reduced activity and thus reduced contact probability may be the main factor of increased pre-contact defensiveness (Gerritsen and Strickler, 1977). This hypothesis has been supported by Weber and van Noordwijk (Weber and Van Noordwijk, 2002). The main post-contact defense remains unknown since we did not observe any interference of the neckteeth with the larvae’s catching basket. This may be due to the larvae’s size in our experiments. We conducted the experiments with larvae of the 4th instar, but also 3rd instar larvae prey on daphniids. Thus, the neckteeth may protect mainly against 3rd instar larvae. However, interference of the neckteeth with Chaoborus’ head appendages may take place in 4th instar also but remains not observable. Due to overlapping structures during handling it is unlikely to determine mechanical interference within the recordings. Another post-contact defense of Daphnia is the carapace stiffness increase reported for different species (Laforsch et al., 2004; Rabus et al., 2013) and confirmed for Daphnia pulex (Kruppert et al. unpublished). Our conducted bite force estimation resulted in a bite force of Chaoborus larvae ranging from 0.37 to 1.48 mN. This maximum force produced at the mandible tip lies within the same range of a force necessary for a lethal injury in D. pulex which was measured to be 1.14 mN±0.87 SD for the undefended and 1.84 mN ±1.50 SD for the defended morphotype (Kruppert et al. unpublished). Therefore, defended D. pulex may survive a larva’s attack and even an ingestion attempt increasing the chances to escape after being captured.
As already mentioned, the predator’s size may play an important role for the defenses’ functionality. In the experiments, larvae of the 4th instar were used, but probably the neckteeth interfere with mouthparts or components of the catching basket in earlier predator instars. An effective mechanical defense against 3rd instar larvae might be very cost efficient since it would cover a considerable substantial of predator individuals in an environment with minor costs considering the neckteeth-size. To test this hypothesis, feeding experiments are necessary using Chaoborus larvae of the 3rd instar.
With the presented catching event’s 3D animation, we illuminate details of a crucial element of the Chaoborus / Daphnia pulex predator prey relationship. With this information the long standing hypothesis assuming that morphological defenses function as an anti-lock-and-key system can now be studied in more detail. Since the 3D surface mesh can be scaled to every desired size, physical simulations (e.g. multi body simulations) can be conducted for every instar combination of predator and prey if a 3D surface mesh of Daphnia pulex is available.
Daphnia carapace: form, function, structure and plasticity 102 Discussion
This way ingestion probability could be determined for all size combinations of predator and prey as well as every possible prey position.
Daphnia carapace: form, function, structure and plasticity 103 Discussion
Concluding Discussion
This thesis has revealed the carapace structure as a crucial element of defenses in Daphnia. Its combination of a compliable foundation (i.e. the hemolymphatic chamber) and a stiff surface (i.e. the distal cuticle) conjoined by the pillars offers high resistance against mechanical impacts. This structure is comparable to other morphological traits facing high compressive forces which often combine a stiff surface with a compliable foundation e.g. teeth and mandibles (Fong et al., 2000; Michels et al., 2012). In my thesis I analyzed the predator induced reorganization of the carapace’ distal integument and found D. longicephala to counters its piercing predator (i.e. Notonecta) with increasing lamination of the carapace distal cuticle. In D. pulex I found a combination of increased lamination and thickness of the carapace’ distal cuticle as a response to the crushing predator Chaoborus. Considering these results the daphniids’ cuticle seems to be highly plastic and appears to alter in respect to species predators. The defenses of both species resulted in an overall increased resistance of the carapace and a higher force necessary to cause lethal injury. Using finite element analysis, I was able to identify the contributions of carapace’ shape and structure to the measured increased resistance. The analyses revealed only the structural alterations to be responsible for the increase in carapace resistance. Even in D. longicephala that shows major shape alterations, the carapace resistance is based only on structural reorganization. In order to determine the shape alterations mode of action I tested the anti-lock-and-key-hypothesis in D. pulex by recording high speed videos of Chaoborus’ catching events. Although I can now provide a detailed description of the catching movement including all involved head appendages and their contribution to the complex catching basket, an interference of the daphniids neckteeth with this catching basket could not be observed in any of the conducted recordings. Nevertheless, these high speed recordings, as well as additional feeding experiments, again proved the induced trait’s effectiveness. The analysis of this data revealed pre- and post-contact advantages of defended daphniids. Chaoborus larvae showed significantly less attacks when encountering defended daphniids, indicating a pre-contact defense which is most likely a behavioral shift reducing the encounter rate. Reduced activity as a response on Chaoborus predation has been hypothesized and reported before (Gerritsen and Strickler, 1977; Weber and Van Noordwijk, 2002). Increased escape rates of defended daphniids indicate an additional post-contact defense. Despite no interference of the defensive trait with the larvae’s catching basket could be observed, the anti-lock-and-key-hypothesis
Daphnia carapace: form, function, structure and plasticity 104 Discussion cannot be neglected since the neeckteeth may cause handling issues that are hard to observe. Furthermore, while my results relate to the 4th instar larvae, a defense against earlier instars of Chaoborus that also feed on daphniids is imaginable. The increased carapace resistance offers another explanation for the increased escape rates. With the data from the microCT measurements I estimated the bite force of Chaoborus and found it to range in between the force necessary to lethally injure undefended and defended daphniids’. Therefore, the defended animals’ escape probability increases as their duration of survival during handling and ingestion extends. Considering my findings that the carapace resistance is independent from shape alterations, different modes of action are likely for structure and shape alterations. However, both act against predator handling and ingestion. As the structure alterations increase resistance they most likely prolong the prey’s survival during handling and ingestion whereas the best explanation for shape alterations’ mode of action still is a predator handling issue.
The morphological defenses in Daphnia appear to be complex sets of different defenses acting on different levels against predation pressure. The description and differentiation of several components of these defenses will help to further understand the evolved interactions between predator and prey, a crucial component of ecological research. Additionally, a thorough knowledge of morphological defense traits will offer support for the analysis of the daphniids genome, the backbone from which phenotype alterations arise (Kurtenbach et al., n.d.; Spanier et al., 2010; Tollrian and Leese, 2010; Colbourne et al., 2011; Rozenberg et al., 2015).
Outlook Since the neckteeth mode of action still remains undetermined, further investigations are necessary to verify the anti-lock-and-key-hypothesis. Additional feeding experiments will clarify whether these defensive traits act against earlier instars of the same predator. The 3rd instar of Chaoborus larvae feeds, like the 4th, upon daphniids and thus an effective protection against them would exclude a relevant percentage of predators. This predator exclusion strategy for certain instars might furthermore be conferrable to other species’ defenses or may be a general effect in Daphnia defenses.
Daphnia carapace: form, function, structure and plasticity 105 References
References
Daphnia carapace: form, function, structure and plasticity 106 References
References
Abramoff MD, Magalhães PJ, Ram SJ. 2004. Image processing with ImageJ. Biophotonics Int. 11: 36-42 Adler FR, Harvell CD. 1990. Inducible defenses, phenotypic variability and biotic environments. Trends Ecol Evol 5:407–410. Aldrich JR. 1988. Chemical ecology of the Heteroptera. Annu Rev Entomol 33:211–238. Andersen KK, Bernstein DT. 1976. Some chemical constituents of the scent of the striped skunk (Mephitis mephitis). J Chem Ecol 1:493–499. Anderson BG. 1933. Regeneration in the carapace of Daphnia magna. Biol Bull 64:70–85. Austin L, Gillis RG, Youatt G. 1965. Stonefish venom: Some biochemical and chemical observations. Aust J Exp Biol Med Sci 43:79–90. Bar-On B, Barth FG, Fratzl P, Politi Y. 2014. Multiscale structural gradients enhance the biomechanical functionality of the spider fang. Nat Commun 5:3894. Barry MJ. 2000. Inducible defences in Daphnia : responses to two closely related predator species. Oecologia 124:396–401. Bates HW. 1861. XXXII. Contributions to an insect fauna of the Amazon valley (Coleoptera, Prionides). Trans Linn Soc 23:495–566. Bauer T, Pfeiffer M. 1991. “Shooting” springtails with a sticky rod: the flexible hunting behaviour of Stenus comma (Coleoptera; Staphylinidae) and the counter-strategies of its prey. Anim Behav 41:819–828. Baur R, Binder S, Benz G. 1991. Nonglandular leaf trichomes as short-term inducible defense of the grey alder, Alnus incana (L.), against the chrysomelid beetle, Agelastica alni L. Oecologia 87:219–226. Benoit-Bird KJ, Au WWL, Brainard RE, Lammers MO. 2001. Diel horizontal migration of the Hawaiian mesopelagic boundary community observed acoustically. Mar Ecol Prog Ser 217:1–14. Berryman AA. 1988. Towards a unified theory of plant defense. In: Mattson WJ,, Levieux J,, Bernard-Dagan C, editors. Mechanisms of woody plant defense against insects: Search for pattern. New York: Springer-Verlag. p. 39–55. Bitsch C, Bitsch J. 2002. The endoskeletal structures in arthropods: cytology, morphology and evolution. Arthropod Struct Dev 30:159–177. Black a. R. 1993. Predator-induced phenotypic plasticity in Daphnia pulex: Life history and morphological responses to Notonecta and Chaoborus. Limnol Oceanogr 38:986–996. Blanchard P, Fritz H. 2007. Induced or routine vigilance while foraging. Oikos 116:1603– 1608. Blank S, Arnoldi M, Khoshnavaz S, Treccani L, Kuntz M, Mann K, Grathwohl G, Fritz M. 2003. The nacre protein perlucin nucleates growth of calcium carbonate crystals. J Microsc 212:280–291.
Daphnia carapace: form, function, structure and plasticity 107 References
Boix D, Sala J, Gascón S, Brucet S. 2006. Predation in a temporary pond with special attention to the trophic role of Triops cancriformis (Crustacea: Branchiopoda: Notostraca). Hydrobiologia 571:341–353. Boppré M. 1990. Lepidoptera and pyrrolizidine alkaloids. J Chem Ecol 16:165–185. Brancelj A, Čelhar T, Šiško M. 1996. Four different head shapes in Daphnia hyalina (Leydig) induced by the presence of larvae of Chaoborus flavicans (Meigen). Hydrobiologia 339:37–45. Brönmark C, Miner JG. 1992. Predator-induced phenotypical change in body morphology in crucian carp. Science 258:1348–1350. Burke AC. 1989. Development of the turtle carapace: implications for the evolution of a novel bauplan. J Morphol 199:363–378. Burks RL, Lodge DM, Jeppesen E, Lauridsen TL. 2002. Diel horizontal migration of zooplankton: Costs and benefits of inhabiting the littoral. Freshw Biol 47:343–365. Burrows M. 1969. The mechanics and neural control of the prey capture strike in the mantid shrimps Squilla and Hemisquilla. Z Vgl Physiol 62:361–381. Cannon WB. 1932. The wisdom of the body. New York: Norton. 312 p. Carbone G, Pierro E, Gorb SN. 2011. Origin of the superior adhesive performance of mushroom-shaped microstructured surfaces. Soft Matter 7:5545–5552. Cardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M, Tomancak P, Hartenstein V, Douglas RJ. 2012. TrakEM2 software for neural circuit reconstruction. PLoS One 7:e38011. Cebra-Thomas J, Tan F, Sistla S, Estes E, Bender G, Kim C, Riccio P, Gilbert SF. 2005. How the turtle forms its shell: A paracrine hypothesis of carapace formation. J Exp Zool Part B Mol Dev Evol 304:558–569. Černý M, Bytel J. 1991. Density and size distribution of Daphnia populations at different fish predation levels. Hydrobiologia 225:199–208. Chen B, Yin D, Ye W, Lin S, Fan J, Gou J. 2015. Fiber-continuous panel-pillar structure in insect cuticle and biomimetic research. Mater Des 86:686–691. Chivers DP, Kiesecker JM, Marco A, Vito J De, Michael T, Blaustein AR. 2001. Predator- induced life history changes in amphibians : Egg Predation Induces Hatching. Oikos 92:135–142. Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S, Aerts A, Arnold GJ, Basu MK, Bauer DJ, Cáceres CE, Carmel L, Casola C, Choi J-H, Detter JC, Dong Q, Dusheyko S, Eads BD, Fröhlich T, Geiler-Samerotte KA, Gerlach D, Hatcher P, Jogdeo S, Krijgsveld J, Kriventseva E V., Kültz D, Laforsch C, Lindquist E, Lopez J, Manak JR, Muller J, Pangilinan J, Patwardhan RP, Pitluck S, Pritham EJ, Rechtsteiner A, Rho M, Rogozin IB, Sakarya O, Salamov A, Schaack S, Shapiro H, Shiga Y, Skalitzky C, Smith Z, Souvorov A, Sung W, Tang Z, Tsuchiya D, Tu H, Vos H, Wang M, Wolf YI, Yamagata H, Yamada T, Ye Y, Shaw JR, Andrews J, Crease TJ, Tang H, Lucas SM, Robertson HM, Bork P, Koonin E V., Zdobnov EM, Grigoriev I V., Lynch M, Boore JL. 2011. The ecoresponsive genome of Daphnia pulex. Science 331:555–561.
Daphnia carapace: form, function, structure and plasticity 108 References
Coleman RA, Browne M, Theobalds T. 2004. Aggregation as a defense: limpet tenacity changes in response to simulated predator attack. Ecology 85:1153–1159. Cott HB. 1940. Adaptive Coloration in Animals. Methuen, London 1–602. Crowl TA, Covich AP. 1990. Predator-induced life-history shifts in a freshwater snail. Sci Reports 247:949–951. Dahl J, Peckarsky BL. 2002. Induced morphological defenses in the wild: Predator effects on a mayfly, Drunella coloradensis. Ecology 83:1620–1634. Dai Z, Gorb SN. 2009. Contact mechanics of pad of grasshopper (Insecta: Orthoptera) by finite element methods. Chinese Sci Bull 54:549–555. Daly JW, Secunda SI, Garraffo HM, Spande TF, Wisnieski A, Cover JF. 1994. An uptake system for dietary alkaloids in poison frogs (Dendrobatidae). Toxicon 32:657–663. Damant GCC. 1924. The adjustment of the buoyancy of the larva of Corethra plumicornis. J Physiol 59:1469–7793. Degrange FJ, Tambussi CP, Moreno K, Witmer LM, Wroe S. 2010. Mechanical analysis of feeding behavior in the extinct “terror bird” Andalgalornis steulleti (Gruiformes: Phorusrhacidae). PLoS One 5:e11856. Dicke M. 1994. Local and systemic production of volatile herbivore-induced terpenoids: their role in plant-carnivore mutualism. J Plant Physiol 143:465–472. Dixon AFG, Agarwala BK. 1999. Ladybird-induced life-history changes in aphids. Proc R Soc London Ser B-biological Sci 266:1549–1553. Dodson SI. 1974. Adaptive change in plankton morphology in response to size-selective predation: A new hypothesis of cyclomorphosis. Limnol Oceanogr 19:721–729. Dodson SI. 1984. Predation of Heterocope septentrionalis on two species of Daphnia : morphological defenses and their cost. Ecology 65:1249–1257. Dodson SI. 1989. The ecological role of chemical stimuli for the zooplankton: predator- induced morphology in Daphnia. Oecologia 78:361–367. Dodson SI, Havel JE. 1988. Indirect prey effects: Some morphological and life history responses of Daphnia pulex exposed to Notonecta undulata. Limnol Oceanogr 33:1274– 1285. Duarte MR, Herpetologia L De, Butantan I, Brazil AV, Paulo S. 2003. Prickly food : snakes preying upon porcupines. Phyllomedusa 2:109–112. El Din a. S, Obeid M. 1971. Ecological studies of the vegetation of the sudan IV . the effect of simulated grazing on the growth of Acacia. J Appl Ecol 8:211–216. Erickson GM. 2001. The bite of Allosaurus. Nature 409:987–988. Flenner I, Olne K, Suhling F, Sahlén G. 2009. Predator-induced spine length and exocuticle thickness in Leucorrhinia dubia (Insecta: Odonata): A simple physiological trade-off? Ecol Entomol 34:735–740. Fong H, Sarikaya M, White SN, Snead ML. 2000. Nano-mechanical properties profiles across dentin–enamel junction of human incisor teeth. Mater Sci Eng C 7:119–128.
Daphnia carapace: form, function, structure and plasticity 109 References
Fritz M, Morse DE. 1998. The formation of highly organized biogenic polymer/ceramic composite materials: the high-performance microaluminate of molluscan nacre. Curr Opin Colloid Interface Sci 3:55–62. Frommen JG, Herder F, Engqvist L, Mehlis M, Bakker TCM, Schwarzer J, Thünken T. 2011. Costly plastic morphological responses to predator specific odour cues in three-spined sticklebacks (Gasterosteus aculeatus). Evol Ecol 25:641–656. Frost SDW. 1999. The Immune System as an Inducible Defense. In: Tollrian R,, Drew Harvell C, editors. The Ecology and Evolution of Inducible Defenses Princeton, New Jersey: Princeton University Press. p. 104–126. Fryer G. 1991. Functional morphology and the adaptive radiation of the Daphniidae (Branchiopoda: Anomopoda). Philos Trans R Soc Lond B Biol Sci 331:1–99. Fryer G. 1996. The Carapace of the branchiopod crustacea. Philos Trans R Soc B Biol Sci 351:1703–1712. Galbraith Jr. MG. 1967. Size-selective predation on Daphnia by rainbow trout and yellow perch. Trans Am Fish 96:1–10. Garwood R, Dunlop J. 2014. The walking dead: Blender as a tool for paleontologists with a case study on extinct arachnids. J Paleontol 88:735–746. Gerritsen J, Strickler JR. 1977. Encounter probabilities and community structure in zooplankton: A mathematical model. J Fish Board Canada 34:73–82. Gorb SN. 1999. Serial elastic elements in the damselfly wing: Mobile vein joints contain resilin. Naturwissenschaften 86:552–555. Green TR, Ryan CA. 1972. Wound-induced proteinase-inhibitors in plant leaves: A possible defence mechanism against insects. Science 175:776–777. Greeney HF, Dyer LA, Smilanich AM. 2012. Feeding by lepidopteran larvae is dangerous: A review of caterpillars’ chemical, physiological, morphological, and behavioral defenses against natural enemies. Invertebr Surviv J 9:7–34. Gronenberg W, Tautz J, Hölldobler B. 1993. Fast trap jaws and giant neurons in the ant Odontomachus. Science 262:561–563. Guillet A, Doyle WS, Rüther H. 1985. The combination of photogrammetry and finite elements for a fine grained functional analysis of anatomical structures. Zoomorphology 105:51–59. Haas F, Gorb SN, Blickhan R. 2000. The function of resilin in beetle wings. Proc Biol Sci 267:1375–1381.
Halcrow K. 1976. The fine structure of the carapace integument of Daphnia magna STRAUS (Crustacea Branchiopoda). Cell Tissue Res 169:267–276. Hamm CE, Merkel R, Springer O, Jurkojc P, Maier C, Prechtel K, Smetacek V. 2003. Architecture and material properties of diatom shells provide effective mechanical protection. Nature 421:841–843. Hansen MJ, Wahl DH. 1981. Selection of small Daphnia pulex by yellow perch fry in Oneida Lake, New York. Trans Am Fish Soc 110:64–71.
Daphnia carapace: form, function, structure and plasticity 110 References
Hassan MA, Westermann GEG, Hewitt RA, Dokainish MA. 2002. Finite-element analysis of simulated ammonoid septa (extinct Cephalopoda): septal and sutural complexities do not reduce strength. Paleobiology 28:113–126. Haukioja E. 1980. On the role of plant defenses in the fluctuations of herbivore populations. Oikos 35:202–213. Havel JE, Dodson SI. 1984. Chaoborus predation on typical and spined morphs of Daphnia pulex: Behavioral observations. Limnol Oceanogr 29:487–494. Hebert PDN. 1977. A revision of the taxonomy of the gennus Daphnia (Crustacea : Daphnidae) in south-eastern Australia. Aust J Zool 25:371–398. Heckmann K. 1995. Räuber-induzierte Feindabwehr bei Protozoen. Naturwissenschaften 82:107–116. Heepe L, Gorb SN. 2014. Biologically inspired mushroom-shaped adhesive microstructures. Annu Rev Mater Res 44:173–203. Heethoff M, Norton R a. 2009. A new use for synchrotron X-ray microtomography: three- dimensional biomechanical modeling of chelicerate mouthparts and calculation of theoretical bite forces. Invertebr Biol 128:332–339. Hopkins TL, Kramer KJ. 1992. Insect cuticle sclerotization. Annu Rev Entomol 37:273–302. Hossler EW. 2009. Caterpillars and moths. Dermatol Ther 22:353–366. Janmey PA. 1991. Mechanical properties of cytoskeletal polymers. Curr Opin Cell Biol 3:4– 11. Jarrett JN. 2009. Predator-induced defense in the barnacle Chthamalus fissus. J Crustac Biol 29:329–333. Johannes MRS. 1993. Prey aggregation is correlated with increased predation pressure in lake fish communities. Can J Fish Aquat Sci 50:66–73. Johansson F, Samuelsson L. 1994. Fish-induced variation in abdominal spine length of Leucorrhinia dubia (Odonata) larvae? Oecologia 100:74–79. Kesel AB, Philippi U, Nachtigall W. 1998. Biomechanical aspects of the insect wing : an analysis using the finite element method. Comput Biol Med 28:423–437. Kingdon J, Agwanda B, Kinnaird M, O’Brien T, Holland C, Gheysens T, Boulet-Audet M, Vollrath F. 2012. A poisonous surprise under the coat of the African crested rat. Proc Biol Sci 279:675–680. Kirsch EG. 1868. Die Fundamentalgleichungen der Theorie der Elasticität fester Körper, hergeleitet aus der Betrachtung eines Systems von Punkten, welche durch elastische Streben verbunden sind. Zeitschrift des Vereins Dtsch Ingenieure 7. Kobak J, Kakareko T. 2011. The effectiveness of the induced anti-predator behaviour of zebra mussel Dreissena polymorpha in the presence of molluscivorous roach Rutilus rutilus. Aquat Ecol 45:357–366. Krogh A. 1911. On the hydrostatic mechanism of the Corethra Larva with an account of methods of microscopical gas analysis. Skand Arch Physiol 25:183–203.
Daphnia carapace: form, function, structure and plasticity 111 References
Krueger DA, Dodson SI. 1981. Embryological induction and predation ecology in Daphnia pulex. Limnol Oceanogr 26:219–223. Kurtenbach S, Mayer C, Pelz T, Hatt H, Leese F, Neuhaus EM. Molecular evolution of a chordate specific family of G protein-coupled receptors. BMC Evol. 11:234 Laforsch C, Haas A, Jung N, Schwenk K, Tollrian R, Petrusek A. 2009. “Crown of thorns ” of Daphnia. Commun Integr Biol 2:379–381. Laforsch C, Ngwa W, Grill W, Tollrian R. 2004. An acoustic microscopy technique reveals hidden morphological defenses in Daphnia. Proc Natl Acad Sci U S A 101:15911– 15914. Laforsch C, Tollrian R. 2000. A new preparation technique of daphnids for scanning electron microscopy using hexamethyldisilazane. Arch für Hydrobiol 149:587–596. Laforsch C, Tollrian R. 2004. Inducible defenses in multipredator environments : cyclomorphosis in Daphnia cucullata. Ecology 85:2302–2311. Lardner B. 2000. Morphological and life history responses to predators in larvae of seven anurans. Oikos 88:169–180. Leonard GH, Bertness MD, Yund PO. 1999. Crab predation, waterborne cues, and inducible defenses in the blue mussel, Mytilus edulis. Ecology 80:1–14. Leydig F. 1860. Naturgeschichte der Daphniden. Tübingen: Verlag der H. Laupp’schen Buchhandlung. 290 p. Lian J, Wang J. 2013. Microstructure and mechanical anisotropy of crab Cancer Magister exoskeletons. Exp Mech 54:229–239. Lively CM. 1986. Predator-induced shell dimorphism in the acorn barnacle Chthamalus anisopoma. Evolution (N Y) 40:232–242. Machacek J. 1991. Indirect effect of planktivorous fish and the growth and reproduction of Daphnia galeata. Hydrobiologia 225:193–197. Maldonado H, Levin L, Pita JCB. 1967. Hit distance and the predatory strike of the praying mantis. Z Vgl Physiol 56:237–257. Mann S, Sparks NHC. 1988. Singel crystalline nature of coccolith elements of the marine alga Emiliania huxleyi as determined by electron diffraction and high-resolution transmission electron microscopy. Proc R Soc B 234:441–453. Mattheck C. 1998. Design in nature: learning from trees. 1st ed. Berlin: Springer Science & Business Media. 276 p. McCauley DJ, Hoffmann E, Young HS, Micheli F. 2012. Night shift: Expansion of temporal niche use following reductions in predator density. PLoS One 7. McHenry CR, Clausen PD, Daniel WJT, Meers MB, Pendharkar A. 2006. Biomechanics of the rostrum in crocodilians: a comparative analysis using finite-element modeling. Anat Rec A Discov Mol Cell Evol Biol 288:827–849. Mebs D, Pogoda W. 2005. Variability of alkaloids in the skin secretion of the European fire salamander (Salamandra salamadra terrestris). Toxicon 45:603–606.
Daphnia carapace: form, function, structure and plasticity 112 References
Melzer RR, Paulus HF. 1994. Post-Larval development of compound eyes and stemmata of Chaoborus crystallinus (DE GEER, 1776) (Diptera: Chaoboridae): Stage-specific reconstructions within individual organs of vision. Int J Insect Morphol Embryol 23:261–274. Meyers MA, Chen P-Y, Lin AY-M, Seki Y. 2008. Biological materials: Structure and mechanical properties. Prog Mater Sci 53:1–206. Michels J, Büntzow M. 2010. Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes. J Microsc 238:95–101. Michels J, Vogt J, Gorb SN. 2012. Tools for crushing diatoms – opal teeth in copepods feature a rubber-like bearing composed of resilin. Sci Rep 2:1–6. Mikolajewski DJ, Johansson F. 2004. Morphological and behavioral defenses in dragonfly larvae: Trait compensation and cospecialization. Behav Ecol 15:614–620. Milewski A V., Young TP, Madden D. 1991. Thorns as induced defenses : experimental evidence. Oecologia 86:70–75. Mills SC, Reynolds JD. 2004. The importance of species interactions in conservation: the endangered European bitterling Rhodeus sericeus and its freshwater mussel hosts. Anim Conserv 7:257–263. Monterroso P, Alves PC, Ferreras P. 2013. Catch me if you can: Diel activity patterns of mammalian prey and predators. Ethology 119:1044–1056. Mortari MR, Cunha AOS, de Oliveira L, Gelfuso EA, Vieira EB, dos Santos WF. 2005. Comparative toxic effects of the venoms from three Wasp species of the genus Polybia (Hymenoptera, Vespidae). J Biol Sci 5:449,454. Mott L, Shaler S. 2002. Mechanical properties of individual southern pine fibers. Wood Fiber Sci 34:13–27. Müller F. 1864. Für Darwin. Leipzig: Wilhelm Engelmann. 91 p. Myers JH. 1987. Nutrient availability and the deployment of mechanical defenses in grazed plants : A new experimental approach to the optimal defense theory. Oikos 49:350–351. Nagashima H, Kuratani S, Hirasawa T. 2013. The endoskeletal origin of the turtle carapace. Nat Commun 4:1–7. Naleway SE, Taylor JRA, Porter MM, Meyers MA, McKittrick J. 2016. Structure and mechanical properties of selected protective systems in marine organisms. Mater Sci Eng C Mater Biol Appl 59:1143–1167. Nardi JB, Ujhelyi E. 2001. Transformations of epithelial monolayers during wing development of Manduca sexta. Arthropod Struct Dev 30:145–157. Neal E. 1986. natural history of badgers. Croom Helm. London Neff D, Frazier SF, Quimby L, Wang RT, Zill S. 2000. Identification of resilin in the leg of cockroach, Periplaneta americana: Confirmation by a simple method using pH dependence of UV fluorescence. Arthropod Struct Dev 29:75–83. Noguchi T, Arakawa O, Takatani T. 2005. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comp Biochem Physiol - Part D Genomics
Daphnia carapace: form, function, structure and plasticity 113 References
Proteomics 1:153–157. Norris KS., Lowe CH. 1964. An analysis of background color-matching in amphibians and reptiles. Ecology 45:565–580. O’Keefe TC, Brewer MC, Dodson SI. 1998. Swimming behavior of Daphnia : its role in determining predation risk. J Plankton Res 20:973–984. Obeso JR. 1997. The induction of spinescence in European holly leaves by browsing ungulates. Plant Ecol 129:149–156. Ohman MD, Frost BW, Cohen EB. 1983. Reverse diel vertical migration: an escape from invertebrate predators. Science (80- ). Olesen J. 2013. The crustacean carapace: morphology, function, development, and phylogenetic history. In: Watling L,, Thiel M, editors. Functional Morphology and Diversity Oxford: Oxford University Press. p. 103–139. Ottani V, Raspanti M, Ruggeri A. 2001. Collagen structure and functional implications. Micron 32:251–260. Parejko K, Dodson SI. 1991. The evolutionary ecology of an antipredator reaction norm: Daphnia pulex and Chaoborus americanus. Evolution (N Y) 45:1665–1674. Pavia H, Toth GB. 2000. Inducible chemical resistance to herbivory in the Brown Seaweed Ascophyllum nodosum. Ecology 81:3212–3225. Petrusek A, Tollrian R, Schwenk K, Haas A, Laforsch C. 2009. A “crown of thorns” is an inducible defense that protects Daphnia against an ancient predator. Proc Natl Acad Sci U S A 106:2248–2252. Philippi U, Nachtigall W. 1996. Functional morphology of regular echinoid tests (Echinodermata, Echinoida): a finite element study. Zoomorphology 116:35–50. Pirow R, Wollinger F, Paul RJ. 1999a. The sites of respiratory gas exchange in the planktonic crustacean Daphnia magna: an in vivo study employing blood haemoglobin as an internal oxygen probe. J Exp Biol 202:3089–3099. Pirow R, Wollinger F, Paul RJ. 1999b. The importance of feedingcurrent for oxygen uptake in the water flea Daphnia magna. J Exp Biol 202:553–562. Price PW, Bouton CE, Gross P, Mcpheron BA, Thompson JN, Weis AE. 1980. Interactions among three trophic levels : Influence of plants on interactions between insect herbivores and natural enemies. Annu Rev Ecol Syst 11:41–65. Pullin AS, Gilbert JE. 1989. The stinging nettle, Urtica Dioica, increases trichome density after herbivore and mechanical damage. Oikos 54:275. Quinn JC, Kessell A, Weston LA. 2014. Secondary plant products causing photosensitization in grazing herbivores: Their structure, activity and regulation. Int J Mol Sci 15:1441– 1465. Rabus M. 2015. Triops -induced morphological defences in Daphnia magna : Combining large-scale-, micro- and ultrastructural defences. PhD thesis, Ludwig-Maximilians- Universität München. Available from: http://edoc.ub.uni-muenchen.de/18319/ Rabus M, Laforsch C. 2011. Growing large and bulky in the presence of the enemy: Daphnia
Daphnia carapace: form, function, structure and plasticity 114 References
magna gradually switches the mode of inducible morphological defences. Funct Ecol 25:1137–1143. Rabus M, Söllradl T, Clausen-Schaumann H, Laforsch C. 2013. Uncovering Ultrastructural defences in Daphnia magna - An interdisciplinary approach to assess the predator- induced fortification of the carapace. PLoS One 8:e67856. Rayfield EJ. 2004. Cranial mechanics and feeding in Tyrannosaurus rex. Proc Biol Sci 271:1451–1459. Rayfield EJ. 2007. Finite element analysis and understanding the biomechanics and evolution of living and fossil organisms. Annu Rev Earth Planet Sci 35:541–576. Rayfield EJ, Norman DB, Horner CC, Horner JR, Smith PM, Thomason JJ, Upchurch P. 2001. Cranial design and function in a large theropod dinosaur. Nature 409:1033–1037. Reimer O, Tedengren M. 1997. Predator induced changes in byssal attachment, aggregation and migration in the blue mussel, Mytilus edulis. Mar Freshw Behav Physiol 30:251– 266. Relyea RA. 2001. Morphological and behavioral plasticity of larval anurans in response to different predators. Ecology 82:523–540. Rivera G, Stayton CT. 2011. Finite element modeling of shell shape in the freshwater turtle Pseudemys concinna reveals a trade-off between mechanical strength and hydrodynamic efficiency. J Morphol 272:1192–1203. Roer R, Dillaman R. 1984. The structure and calcification of the crustacean cuticle. Am Zool 24:893–909. Rousseau M, Meibom A, Gèze M, Bourrat X, Angellier M, Lopez E. 2009. Dynamics of sheet nacre formation in bivalves. J Struct Biol 165:190–195. Rozenberg A, Parida M, Leese F, Weiss LC, Tollrian R, Manak JR. 2015. Transcriptional profiling of predator-induced phenotypic plasticity in Daphnia pulex. Front Zool 12:18. Ruxton GD. 2009. Non-visual crypsis: a review of the empirical evidence for camouflage to senses other than vision. Philos Trans R Soc B Biol Sci 364:549–557. Sander PM, Christian A, Clauss M, Fechner R, Gee CT, Griebeler E-M, Gunga H-C, Hummel J, Mallison H, Perry SF, Preuschoft H, Rauhut OWM, Remes K, Tütken T, Wings O, Witzel U. 2011. Biology of the sauropod dinosaurs: the evolution of gigantism. Biol Rev Camb Philos Soc 86:117–155. Schaber CF, Heinlein T, Keeley G, Schneider JJ, Gorb SN. 2015. Tribological properties of vertically aligned carbon nanotube arrays. Carbon N Y 94:396–404. Schellbach K. 1852. Probleme der Variationsrechnung. J für die reine und Angew Math 51:293–363. Scheyer TM, Martin Sander P, Joyce WG, Böhme W, Witzel U. 2007. A plywood structure in the shell of fossil and living soft-shelled turtles (Trionychidae) and its evolutionary implications. Org Divers Evol 7:136–144. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image
Daphnia carapace: form, function, structure and plasticity 115 References
analysis. Nat Methods 9:676–682. Schmelz EA, Baldwin IT. 1996. Immunological “memory” in the induced accumulation of nicotine in wild tobacco. Ecology 77:236–246. Schmid B, Schindelin J, Cardona A, Longair M, Heisenberg M. 2010. A high-level 3D visualization API for Java and ImageJ. BMC Bioinformatics 11:274. Schremmer F. 1950. Zur Morphologie und funktionellen Anatomie des Larvenkopfes von Chaoborus (Corethra auct.) obscuripes v. d. Wulp (Dipt., Chaoboridae). Österreichische Zool Zeitschrift 2:471–516. Schultz TW, Kennedy JR. 1977. Analyses of the integument and muscle attachment in Daphnia pulex. J Submicrosc Cytol 9:37–51. Skelly DK, Werner EE. 1990. Behavioral and life-historical responses of larval american toads to an Odonate predator. Ecology 71:2313–2322. Spanier KI, Leese F, Mayer C, Colbourne JK, Gilbert D, Pfrender ME, Tollrian R. 2010. Predator-induced defences in Daphnia pulex: selection and evaluation of internal reference genes for gene expression studies with real-time PCR. BMC Mol Biol 11:50. Speakman JR. 1995. Chiropteran nocturnality. Symp Zool Soc London 67:187–201. Spitze K, Sadler TD. 1996. Evolution of a generalist genotype: Multivariate analysis of the adaptiveness of phenotypic plasticity. Am Nat 148:S108–S123. Sprules WG, Riessen HP, Jin EH. 1990. Dynamics of the Bythotrephes invasion of the St Lawrence Great Lakes. J Great Lakes Res 16:346–351. Stevens M, Merilaita S. 2009. Animal camouflage: current issues and new perspectives. Philos Trans R Soc London B 364:423–427. Stevenson RJ. 1985. Dynamics of the integument. In: Bliss DE,, Mantel LH, editors. The Biology of Crustacea Vol.9: Integument, Pigments, and Hormonal Processes New York: Academic Press. p. 1–31. Stibor H. 1992. Predator induced life-history shifts in a freshwater cladoceran. Oecologia 92:162–165. Stibor H, Lüning J. 1994. Predator-induced phenotypic variation in the pattern of growth and reproduction in Daphnia hyalina (Crustacea: Cladocera). Funct Ecol 8:97–101. Stich H-B, Lampert W. 1981. Predator evasion as an explanation of diurnal vertical migration by zooplankton. Nature 293:396–398. Strobbe F, McPeek MA, De Block M, Stoks R. 2011. Fish predation selects for reduced foraging activity. Behav Ecol Sociobiol 65:241–247. Swift MC, Fedorenko AY. 1975. Some aspects of prey capture by Chaoborus larvae. Limnol Oceanogr 20:418–425. Swift MC, Forward Jr. RB. 1981. Chaoborus prey capture in the light and dark. Limnol Oceanogr 26:461–466. Taylor BE, Gabriel W. 1992. To grow or not to grow: Optimal resource allocation for Daphnia. Am Nat 139:248–266.
Daphnia carapace: form, function, structure and plasticity 116 References
Tegner MJ, Levin LA. 1983. Spiny lobsters and sea urchins: Analysis of a predator-prey interaction. J Exp Mar Bio Ecol 73:125–150. Teraguchi S. 1975. Correction of negative buoyancy in the phantom larva, Chaoborus americanus. J Insect Physiol 21:1659–1670. Thurston EL, Lersten NR. 1969. The morphology and toxicology of plant stinging hairs. Bot Rev 35:393–412. Timms RM, Moss B. 1984. Prevention of growth of potentially dense phytoplankton populations by zooplankton grazing, in the presence of zooplanktivorous fish, in a shallow wetland ecosystem. Limnol Oceanogr 29:472–486.
Tollrian R. 1990. Predator-induced helmet formation in Daphnia cucullata (SARS). Arch für Hydrobiol 119:191–196. Tollrian R. 1994. Fish-kairomone induced morphological changes in Daphnia lumholtzi (SARS). Arch für Hydrobiol 130:69–75. Tollrian R, Dodson SI. 1999. Inducible defenses in Cladocera: Constraints, costs, and multipredator environments. In: Tollrian R,, Harvell CD, editors. The Ecology and Evolution of Inducible Defenses Princeton, New Jersey: Princeton University Press. p. 177–202. Tollrian R, Harvell CD. 1999. The Ecology and Evolution of Inducible Defenses. New Jersey: Princeton University Press. 383 p. Tollrian R, Leese F. 2010. Ecological genomics: steps towards unraveling the genetic basis of inducible defenses in Daphnia. BMC Biol 8:51. Tramacere F, Kovalev A, Kleinteich T, Gorb SN, Mazzolai B. 2013. Structure and mechanical properties of Octopus vulgaris suckers. J R Soc Interface 11:20130816– 20130816. Trussell GC. 1996. Phenotypic plasticity in an intertidal snail: the role of a common crab predator. Soc study Evol 50:448–454. Turner MJ, Clough RW, Martin HC, Topp LJ. 1956. Stiffness and deflection analysis of complex structures. J Aeronaut Sci 23:805–854. Uttieri M, Sandulli R, Spezie G, Zambianchi E. 2014. From small to large scale: A review of the swimming behaviour of Daphnia. In Daphnia Biology and Mathematics Perspective. New York: Nova Science Publishers, Inc. p.309–322 Van Buskirk J, Relyea RA. 1998. Selection for phenotypic plasticity in Rana sylvatica tadpoles. Biol J Linn Soc 65:301–328. van der Meijden A, Langer F, Boistel R, Vagovic P, Heethoff M. 2012. Functional morphology and bite performance of raptorial chelicerae of camel spiders (Solifugae). J Exp Biol 215:3411–3418. van Uitregt VO, Hurst TP, Wilson RS. 2013. Greater costs of inducible behavioural defences at cooler temperatures in larvae of the mosquito, Aedes notoscriptus. Evol Ecol 27:13– 26. Vincent JFV. 2002. Arthropod cuticle: A natural composite shell system. Compos Part A Appl Sci Manuf 33:1311–1315.
Daphnia carapace: form, function, structure and plasticity 117 References von Ende CN. 1979. Fish predation, interspecific predation, and the distribution of two Chaoborus species. Ecology 60:119–128. Wallace AR. 1870. Contributions to the theory of natural selection. London: W. W. Head, Royal Victoria Press. 384 p. Watt P, Nottingham S, Young S. 1997. Toad tadpole aggregation behaviour: evidence for a predator avoidance function. Anim Behav 54:865–872. Weber A, Van Noordwijk A. 2002. Swimming behaviour of Daphnia clones: Differentiation through predator infochemicals. J Plankton Res 24:1335–1348. Weider LJ, Pijanowska J. 1993. Plasticity of Daphnia life histories in response to chemical cues from predators. Oikos 67:385–392. Weiss LC, Tollrian R, Herbert Z, Laforsch C. 2012. Morphology of the Daphnia nervous system: a comparative study on Daphnia pulex, Daphnia lumholtzi, and Daphnia longicephala. J Morphol 273:1392–1405. Witzel U, Preuschoft H. 2005. Finite-element model construction for the virtual synthesis of the skulls in vertebrates: case study of Diplodocus. Anat Rec A Discov Mol Cell Evol Biol 283:391–401. Wohlfrom H, Melzer RR. 2001. Development of the sensory system in larvae and pupae of Chaoborus crystallinus (DEGEER, 1776; diptera, Chaoboridae): Sensory cells, nerves and ganglia of the tail region. Dev Genes Evol 211:124–137. Wroe S, Ferrara TL, McHenry CR, Curnoe D, Chamoli U. 2010. The craniomandibular mechanics of being human. Proc Biol Sci 277:3579–3586. Wroe S, Huber DR, Lowry M, McHenry C, Moreno K, Clausen P, Ferrara TL, Cunningham E, Dean MN, Summers a. P. 2008. Three-dimensional computer analysis of white shark jaw mechanics: how hard can a great white bite? J Zool 276:336–342. Yin M, Laforsch C, Lohr JN, Wolinska J. 2011. Predator-induced defense makes Daphnia more vulnerable to parasites. Evolution (N Y) 65:1482–1488. Young TP. 1987. Increased thorn length in Acacia depranolobium- an induced response to browsing. Oecologia 71:436–438. Zangerl AR, Berenbaum MR. 1994. Spatial, temporal, and environmental limits on xanthotoxin induction in wild parsnip foliage. Chemoecology 5–6:37–42. Zaret TM. 1972. Predators, invisible prey, and the nature of polymorphism in the cladocera. Limnol Oceanogr 17:171–184. Žnidaršič N, Mrak P, Tušek-Žnidarič M, Štrus J, Znidaršič N, Mrak P, Tušek-Žnidarič M, Strus J, Žnidaršič N, Mrak P, Tušek-Žnidarič M, Štrus J, Znidaršič N, Mrak P, Tušek- Žnidarič M, Strus J, Žnidaršič N, Mrak P, Tušek-Žnidarič M, Štrus J. 2012. Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans. Zookeys 176:39– 53.
Daphnia carapace: form, function, structure and plasticity 118 Summary
Summary
Daphnia carapace: form, function, structure and plasticity 119 Summary
Summary Phenotypic plasticity is the ability of an organism with a given genotype to develop different phenotypes in response to changing environments. A special form of phenotypic plasticity are inducible defenses, an organism’s response upon predator’s presence. These responses can be realized in behavior, the organism’s life-history parameters or morphology. The freshwater crustacean Daphnia is known for its degree of plasticity and it develops inducible defenses thwarting predators. It falls prey to a broad variety of predators, e.g. juvenile and adult insects as well as fish. The different predators use different strategies for prey detection. While fish are visually orientated, most invertebrate predators are ambush predators that often rely on tactile prey detection. Daphniids possess a general defense (i.e. the carapace) against the mechanical stress of a predatory attack. The carapace encapsulates the main body, but not the head. It is formed by a double layer of the integument interconnected by small pillars and hemolymphatic space in between. A second function of the carapace is respiration, which is performed through its proximal integument.
In addition to the carapace’ general defensive effect, daphniids thwart the different predators with different sets of inducible defenses. In predator regimes dominated by fish, daphniids mainly respond with adaptive behavior and life-history shifts whereas invertebrate predators often induce additional morphological defenses. These inducible morphological defenses are realized as morphological shape alterations e.g. “neckteeth” in D. pulex facing Chaoborus larvae and “crests” in D. longicephala facing the “backswimmer” Notonecta. In addition to the obvious defensive traits, D. pulex and some other daphniids were found to develop structural alterations resulting in increased carapace’ stiffness.
In my thesis I aimed to describe the mode of action of inducible morphological defenses during a predation event. Morphological defenses are accompanied by structural changes of the carapace resulting in an increased stiffness. Therefore, morphological defenses can be regarded as complex construction countering predation. Using an interdisciplinary approach, I described the carapace structure in detail. Furthermore, I determined the contribution of the structural changes and the obvious morphological to the carapace’ mechanical resistance. I chose D. pulex and D. longicephala due to their different defensive traits countering their coexisting predators’. Chaoborus, D. pulex’ predator, is a tactile perceptive ambush predator that ingests its prey using its prominent mandibles. Notonecta, D. longicephala’s predator, is a visually orientated heteropteran and feeds by stabbing its prey with its proboscis.
Daphnia carapace: form, function, structure and plasticity 120 Summary
For the description of the carapace structure, I analyzed D. pulex using histochemistry in combination with light and electron microscopy. I found the carapace’ distal integument to be significantly thicker than the proximal. The interconnecting pillars appear fibrous with slim waists and broad, sometimes branched bases where they meet the integument layers. The fibrous structure and the slim-waisted shape of the pillars indicate a high capacity to withstand tensile rather than compressive forces. In conclusion they can be regarded as ligaments and not pillars. To foster this assumption, I determined the hemolymphatic gauge pressure in D. longicephala and found the hemocoel pressure to be above ambient. The results offer a new mechanistic explanation of the high rigidity of the daphniid carapace. This is probably the result of a light-weight construction consisting of two integuments bound together by ligaments and inflated by a hydrostatic hyper-pressure.
I further used scanning transmission electron microscopy (STEM) and confocal laser scanning microscopy to identify structural and shape alterations. In addition, I conducted nano- and micro-indentation as well as finite element analysis (FEA) to determine the contribution of structure and shape to the carapace’ mechanical resistance. I found predator induced structural changes i.e. the cuticle becomes laminated (i.e. an increased number of layers) in both species. These resulted in increased cuticle stiffness determined via nano-indentation. The micro-indentations revealed a predator induced increase in geometric stiffness, i.e. the extent to which a geometric body resists deformation in response to an applied force. Using FEA I could show that this increase is independent from shape alterations. Furthermore, the results revealed species specific structure alterations indicating different, predator specific strategies to realize stiffness increase.
Finally, I investigated the unique catching basket of Chaoborus larvae using microCT and high speed video recordings to determine whether D. pulex neckteeth interfere with the catching basket of Chaoborus larvae. I found that some of the appendages contribute more than assumed to the catching sequence or are involved in a completely different way. The analysis of the high speed recordings showed that Chaoborus is an astonishingly fast predator. With a time frame of 20 ms from the movement’s start to prey contact, the catching sequence of Chaoborus ranks within the fastest hunting movements in the animal kingdom published so far. Although I was able to describe the catching event with high temporal and spatial resolution using the recordings, it was not possible to ultimately reveal the neckteeth’ mode of action. In none of the sequences an interference of the daphniids neckteeth with one of the catching basket elements could be observed. Nevertheless, D. pulex’ neckteeth defensive
Daphnia carapace: form, function, structure and plasticity 121 Summary effectiveness was reconfirmed. Feeding experiments revealed an advantage of defended daphniids before and after the catching event. That indicates a set of defenses including predator avoidance and morphological defenses leading to increased escape probability. Thus, the anti-lock-and-key-hypothesis remains the best explanation for the defensive traits’ functionality.
In conclusion, my results revealed the inducible morphological defenses to be even more complex and predator specific than assumed so far. The revealed structural alterations specifically antagonize the respective predator’s mode of consumption. Although I could neither approve nor disprove the anti-lock-and-key-hypothesis for the Chaoborus catching event the interdisciplinary approach renders itself an outstanding method to analyze the mechanics of predator/prey interactions. The results of my thesis hopefully initiates further investigations of the predator/prey interaction mechanics that helps to embrace the capabilities of inducible defenses and expands the knowledge about predation, a crucial element in ecology.
Daphnia carapace: form, function, structure and plasticity 122 Summary
Zusammenfassung Phänotypische Plastizität bezeichnet die Fähigkeit eines Organismus mit bestimmtem Genotyp unterschiedliche Phänotypen als Reaktion auf sich ändernde Umweltbedingungen auszuprägen. Eine Form der Phänotypischen Plastizität sind induzierbare Verteidigungen, die Reaktion einer Art auf Anwesenheit von Prädatoren. Diese Reaktionen können in Form von Verhalten, Lebenszyklusparametern oder Morphologie umgesetzt werden. Die Süßwasser Krebs-Gattung Daphnia ist bekannt für ihre Fähigkeit zur Phänotypischen Plastizität und ihre induzierbare Verteidigungen gegen Prädatoren. Sie ist Beute für eine Vielzahl von Prädatoren, wie juveniler und adulter Insekten, sowie Fischen. Diese unterschiedlichen Räuber weisen sehr unterschiedliche Jagdstrategien auf. Fische sind visuell jagende Räuber, die meisten Invertebraten sind Lauerjäger, die oft haptisch ihre Beute wahrnehmen. Daphnia besitzt eine universelle Verteidigung gegen die mechanische Belastung einer Räuber Attacke: Den Carapax. Der Carapax umschließt den Körper während der Kopf frei bleibt. Er besteht aus einer Doppelschicht aus Integument, verbunden durch Säulchen, die den hämolymphatischen Raum umschließen. Eine zweite Funktion des Carapax ist Atmung, die durch das dünne proximale Integument ermöglicht wird.
Zusätzlich zur universellen Verteidigung durch den Carapax bilden Daphnien Sets aus induzierbarer Verteidigungen gegen unterschiedliche Räuber aus. In Fisch dominierten Systemen reagieren Daphnien meist mit Verhaltens- und Lebensparameter-Anpassungen, invertebrate Räuber induzieren darüber hinaus oft morphologische Verteidigungen. Diese morphologischen Verteidigungen treten in Form von Gestaltänderungen auf, wie beispielsweise „Nackenzähnen“ bei D. pulex in Anwesenheit von Chaoborus Larven oder „Kämmen“ bei D. longicephala in Anwesenheit von Rückenschwimmern der Gattung Notonecta. Zusätzlich zu diesen offensichtlichen morphologischen Verteidigungen wurde bei D. pulex und anderen Arten eine Erhöhung der Carapax-Steifigkeit festgestellt, die mit strukturellen Veränderungen des Carapax einhergeht.
In meiner Doktorarbeit habe ich versucht die Funktionsweise der morphologischen Verteidigungen zu beschreiben. Da gezeigt wurde, dass die offensichtlichen Verteidigungsanlagen mit strukturellen, die Carapax-Steifigkeit erhöhenden, einhergehen, scheinen die morphologischen Verteidigungen ein komplexes Zusammenwirken von Verteidigungen auf verschiedenen Ebenen darzustellen. Mit einem interdisziplinären Ansatz habe ich die Struktur des Carapax detailliert beschrieben und versucht aufzutrennen, welchen
Daphnia carapace: form, function, structure and plasticity 123 Summary
Anteil an der mechanischen Stabilität des Carapax auf die offensichtlichen Verteidigungen und welche auf die strukturellen Änderungen zurückgeht. Ich habe mich dabei für die Arten D. pulex und D. longicephala als Untersuchungsobjekte entschieden, da sie sich in Größe ihrer Verteidigungsanlagen sowie in den Jagd- und Fress-Strategien ihrer jeweiligen Räuber unterscheiden. Chaoborus, D. pulex‘ Räuber, ist ein haptisch wahrnehmender Lauerjäger, der seine Beute mit Hilfe seiner ausgeprägten Mandibeln verschlingt. Notonecta, D. longicephala’s Räuber, ist eine visuell jagende Wanze, die ihre Beute mit dem Proboscis ansticht und aussaugt.
Um die Struktur des Carapax detailliert zu beschreiben habe ich D. pulex mit histochemischen Methoden sowie einer Kombination aus Licht- und Elektronen-Mikroskopie untersucht. Das distale Integument des Carapax erwies sich als deutlich dicker, als das proximale. Die verbindenden Säulchen erschienen faserig, mit dünnem Mittelteil und breiten, manchmal gegabelten Basen an den Verbindungen zu den Integumenten. Die faserige Struktur und die Taillierung der Säulchen weisen auf hohe Zugbelastbarkeit, nicht auf Druckbelastbarkeit hin. Daher sollten sie eher als Ligamente, statt als Säulchen bezeichnet werden. Um dies zu testen habe ich den Hämolymphdruck in D. longicephala bestimmt, welcher über dem Umgebungsdruck lag. Diese Ergebnisse ermöglichen eine neue Erklärung für die hohe Belastbarkeit des Daphnien Carapax, der scheinbar eine Leichtbauweise darstellt, die von den zwei Integumenten gebildet, von den Säulchen verbunden und vom Hämolymphdruck aufgepumpt wird.
Weiterhin habe ich Raster-Transmissions-Elektronenmikroskopie und konfokale Laser- Scanning-Mikroskopie verwendet, um die Änderungen in Struktur und Form zu beschreiben, sowie Nano- und Mikro-Indentation und Finite-Elemente-Analyse, um ihre jeweiligen Anteile an der mechanischen Belastbarkeit des Carapax voneinander trennen zu können. Dabei zeigte sich in beiden Arten ein von Räubern induzierten strukturellen Änderungen in Form von stärker laminierter Cuticula. Mit Nano-Indentation konnte ich eine daraus resultierende erhöhte Carapax-Steifigkeit nachweisen. Die Micro-Indentationsmessungen zeigten darüber hinaus eine Erhöhung der strukturellen Steifigkeit, i.e. das Deformationsverhalten eines geometrischen Körpers unter mechanischer Belastung. Diese Erhöhung der strukturellen Steifigkeit trat bei verteidigten Tieren beider Arten. Die durchgeführte Finiter-Elemente- Analyse ergab, dass die Steifigkeits-Erhöhung unabhängig von den offensichtlichen Verteidigungsanlagen ist. Darüber hinaus zeigten die Ergebnisse artspezifische strukturelle
Daphnia carapace: form, function, structure and plasticity 124 Summary
Änderungen, die auf räuberspezifische Verteidigungsstrategien zur Steifigkeitserhöhung hinweisen.
Abschließend habe ich den einzigartigen Fangkorb der Chaoborus Larven mittels MikroCT und Highspeed Videoaufnahmen untersucht, um zu prüfen, ob die Nackenzähne von D. pulex mit dem Fangapparat von Chaoborus interferieren. Mit den aufgenommenen Daten konnten die meisten bisherigen Vermutungen, den Fangmechanismus betreffend, und die Funktionen der beteiligten Kopfanhänge bestätigt werden. Dennoch waren einige Kopfanhänge stärker am Fangmechanismus beteiligt, als vermutet, oder zeigten ganz andere Funktionsweisen. Die Highspeed-Aufnahmen ergaben zudem, dass die Fangbewegung erstaunlich schnell von statten geht. Mit einer Dauer von 20 ms von Beginn der Bewegung bis zum Beutekontakt gehört der Fangmechanismus von Chaoborus zu den schnellsten für das Tierreich bekannten. Obwohl der Fangmechanismus mit Hilfe der Aufnahmen morphologisch und zeitlich detailliert beschrieben werden konnte, konnte die Funktionsweise der Nackenzähne nicht abschließend geklärt werden. In keinem der aufgezeichneten Videos war eine behindernde oder blockierende Funktionsweise der Nackenzähne erkennbar. Dennoch konnte deren Effektivität erneut bestätigt werden. Fraßversuche zeigten einen Vorteil verteidigter Tiere vor und nach einer Räuber Attacke. Das weist auf ein Zusammenwirken mehrerer Verteidigungen hin, wobei Räubervermeidung, sowie morphologische Verteidigung zur Erhöhung der Fluchtwahrscheinlichkeit führen. Daher bleibt die „Anti-lock-and-key-hypothesis“ die beste Erklärung zur Funktionsweise dieser Verteidigungsstrukturen.
Meine Daten zusammenfassend, konnte ich zeigen, dass die morphologischen Verteidigungen von Daphnia umfangreicher und Räuber-spezifischer sind, als bisher bekannt. Die entdeckten strukturellen Änderungen wirken spezifisch gegen die Mundwerkzeuge des jeweiligen Prädators. Obwohl ich die „anti-lock-and-key-hypothesis“ für den Fangmechanismus von Chaoborus weder bestätigen, noch widerlegen konnte, hat sich der interdisziplinäre Ansatz als hervorragende Methode herausgestellt um die mechanischen Aspekte von Räuber/Beute Interaktionen zu untersuchen. Ich hoffe, dass die Ergebnisse meiner Doktorarbeit weitere Studien zu den mechanischen Aspekten von Räuber/Beute Interaktionen einleiten, und damit helfen die Vielfalt induzierbarer Verteidigungen abzuschätzen und das Verständnis von Prädation zu erweitern, einem Schlüsselelement ökologischer Systeme.
Daphnia carapace: form, function, structure and plasticity 125 Appendix
Appendix
Daphnia carapace: form, function, structure and plasticity 126 Appendix
ERKLÄRUNG
Ich versichere an Eides statt, dass ich die eingereichte Dissertation selbstständig und ohne unzulässige fremde Hilfe verfasst, andere als die in ihr angegebene Literatur nicht benutzt und dass ich alle ganz oder annähernd übernommenen Textstellen sowie verwendete Grafiken und Tabellen kenntlich gemacht habe. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten oder eine eindeutige Dokumentation von Art und Umfang der inhaltsverändernden Bildbearbeitung vorliegt. Außerdem versichere ich, dass es sich bei der von mir vorgelegten Dissertation (elektronische und gedruckte Version) um völlig übereinstimmende Exemplare handelt und die Dissertation in dieser oder ähnlicher Form noch nicht anderweitig als Promotionsleistung vorgelegt und bewertet wurde.
Bochum, den
______
Daphnia carapace: form, function, structure and plasticity 127 Appendix
List of Publications
Part 1:
Kruppert, S; Horstmann, M; Weiss, L.C.; Schaber, C.F.; Gorb, S.N.;Tollrian, R. 2016:
Push or Pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression
Journal of Morphology, 277, pp. 1320-1328
Part 2:
Kruppert, S; Horstmann, M; Weiss, L.C.; Witzel, U.; Schaber, C.F.; Gorb, S.N.;Tollrian, R.:
Biomechanical properties of a predator induced body armor in the freshwater crustacean Daphnia
(in preparation)
Part 3:
Kruppert, S; Deussen, L.; Weiss, L.C.; Horstmann, M; Wolff, J.; Kleinteich, T.; Gorb, S.N.;Tollrian, R.:
Zooplankters Nightmare: Chaoborus larvae’s fast and effective catching basket
(in preparation)
Daphnia carapace: form, function, structure and plasticity 128 Appendix
Manuscripts’ contributions
Part 1:
Sebastian Kruppert, Martin Horstmann, Linda Weiss and Ralph Tollrian designed the study. Sebastian Kruppert and Martin Horstmann performed the experiments. Linda Weiss, Sebastian Kruppert and Martin Horstmann analyzed the data. Stanislav Gorb and Clemens Schaber designed the experimental setup for the hemolymphatic pressure measurements and provided valuable ideas on the data interpretation. Sebastian Kruppert and Linda Weiss wrote the manuscript. All authors contributed to the final version of the manuscript.
Part 2:
Sebastian Kruppert and Ralph Tollrian designed the study. Sebastian Kruppert performed the measurements on the AFM and the microindenter and conducted the FEA of the carapace shape. Sebastian Kruppert and Martin Horstmann performed the STEM image acquisition and the FEA of the carapace structure. Linda Weiss, Sebastian Kruppert and Martin Horstmann analysed the data. AFM and microindentation experiments were conducted at the department of Stanislav Gorb, Stanislav Gorb and Clemens Schaber designed the experimental setup for the nano- and microindenter measurements, provided software for data processing, and contributed to the data interpretation. Ulrich Witzel contributed his expertise in FEA regarding modelling as well as interpretation of the results. Sebastian Kruppert and Linda Weiss wrote the manuscript. All authors contributed to and approved the final version of the manuscript.
Daphnia carapace: form, function, structure and plasticity 129 Appendix
Part 3:
Sebastian Kruppert designed the study. The microCT scan and the high speed recordings were conducted at the facility of Stanislav Gorb. Sebastian Kruppert, Lisa Deussen and Thomas Kleinteich performed the microCT scan. Sebastian Kruppert, Lisa Deussen and Jonas Wolff designed the experimental setup for the high speed recordings and Sebatian Kruppert and Lisa Deussen conducted the experiments. Sebastian Kruppert, Lisa Deussen and Martin Horstmann developed the reconstruction method. Lisa Deussen conducted reconstruction and animation. Sebastian Kruppert and Lisa Deussen analysed the data. Stanislav Gorb, Jonas Wolff, Thomas Kleinteich, Linda Weiss and Martin Horstmann contributed to the data interpretation. Sebastian Kruppert and Linda Weiss wrote the manuscript. All authors contributed to and approved the final version of the manuscript.
Daphnia carapace: form, function, structure and plasticity 130 Appendix
Curriculum Vitae
Sebastian Kruppert
Academic Career
Feb 2012 – Oct 2016 PhD thesis „Daphnia carapace: form, function, structure and plasticity” at the International Graduate School of Biosciences at the Department of Animal Ecology, Evolution & Biodiversity, Ruhr-University Bochum Nov 2011 – Jan 2012 Graduate assistant, Ruhr-University Bochum Mai 2011 – Oct 2011 Scientific assistant, Ruhr-University Bochum Mai 2010 – Apr 2011 Graduate assistant, Ruhr-University Bochum Mai 2010 Graduation as Dipl. Biol. In Ecology
Additional scientific qualifications
WiSe 2015/16 “Toxicology and dangerous material’s law”, Ruhr-University Bochum WiSe 2015/16 “Applied statistics for biologists”, Ruhr-University Bochum Jan 2013 Microscopy Winterschool 2013, Uni ETH Zürich Module: “Fine structure preparation for TEM” Sep 2011 Fall-Academy 2011, University Duisburg/Essen Workshop: „Scientific writing“ Workshop: „Basics in project management“
School Education
1993 – 2002 Secondary school and graduation with the Abitur-Exam: Sophie-Scholl-Gesamtschule, Remscheid 1989 – 1993 Primary school: Grundschule Ost, Wermelskirchen
Daphnia carapace: form, function, structure and plasticity 131 Appendix
Conferences
Talks
“Arthropod cuticle” Kruppert, S; Horstmann, M; Weiss, L.C.; Schaber, C.F.; Dresden, 2015 Gorb, S.N.;Tollrian, R.: “Daphnia’s carapace: force directions in a light-weight structure”
Annual Meeting of the SEB Kruppert, S.; Horstmann, M.; Schaber, L.C.; Gorb, S.N.; “Society of Experimental Biology” Tollrian, R.: Praque, 2015 “The defenses of a waterflea: simulation and empiricism“
“Size & Shape” Kruppert, S.; Rabus, M.; Weiss, L.C.; Horstmann, M.; Göttingen, 2014 Laforsch, C.; Tollrian, R.: “Geometric morphometrics of waterfleas“
Candidate Symposium Kruppert, S.; Rabus, M.; Weiss, L.C.; Horstmann, M.; „Morphology“ Laforsch, C.; Tollrian, R.: Ulm, 2013 “Geometric morphometrics of waterfleas“
Annual Meeting of the DZG Kruppert, S.; Rabus, M.; Weiss, L.C.; Horstmann, M.; „German zoologist society“ Laforsch, C.; Tollrian, R.: Munich, 2013 “Geometric morphometrics of waterfleas“
Posters
Annual Meeting of the DZG Kruppert, S; Horstmann, M; Weiss, L.C.; Schaber, C.F.; „German zoologist society“ Gorb, S.N.;Tollrian, R.: Kiel, 2016 “Push or Pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression”
Annual Meeting of the SEB Kruppert, S; Horstmann, M; Weiss, L.C.; Schaber, C.F.; “Society of Experimental Biology” Gorb, S.N.;Tollrian, R.: Brighton, 2016 “Push or Pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression” Awarded with the “R. MCNeill Alexander” third prize
“Arthropod cuticle” Horstmann, M; Kruppert, S; Weiss, L.C.; Schaber, C.F.; Dresden, 2015 Gorb, S.N.;Tollrian, R.: “Daphnia’s carapace: An adaptable shield”
Annual Meeting of the SEB Deussen, L.; Kruppert, S.; Kleinteich, T.; Wolff, J.; Gorb, “Society of Experimental Biology” S.N.; Tollrian, R.: Praque, 2015 “Morphology and Biomechanics of predatory attack: prey capture of Chaoborus spec.”
Daphnia carapace: form, function, structure and plasticity 132 Appendix
Publications
Journal of Morphology Kruppert, S; Horstmann, M; Weiss, L.C.; Schaber, C.F.; Gorb, S.N.; 2016 Tollrian, R.: “Push or Pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression”
Hydrobiologia Weiss, L.C.; Heilgenberg, E.; Deussen, L.; Becker, S.M.; Kruppert, S.; 2016 Tollrian, R.: “Onset of kairomone sensitivity and the development of inducible morphological defenses in Daphnia pulex”
PloS one Weiss, L.C.; Kruppert, S.; Laforsch, C.; Tollrian, R.: „Chaoborus and 2012 Gasterosteus Anti-Predator Responses in Daphnia pulex Are Mediated by Independent Cholinergic and Gabaergic Neuronal Signals”
Qualifications
DAAD English Exam Approx. C1/UNIcert III Very good knowledge of English language Excellent IT knowledge (hard- and software) Extensive Experience in: Microsoft Office Adobe Illustrator/Photoshop STATISTICA R Matlab LISA (FEA) ANSYS (FEA) Certified knowledge about toxicology and dangerous material’s law (Eingeschränkten Sachkunde für das Inverkehrbringen von gefährlichen Stoffen und Zubereitungen gemäß § 5 der Chemikalien-Verbotsverordnung) Driver’s license Class A, B, BE CMAS * Diver
Hobbies
Music Jogging, historical fencing Crafting
Daphnia carapace: form, function, structure and plasticity 133 Appendix
Danksagung
Mein besonderer Dank gilt Prof. Dr. Ralph Tollrian, der mir das Thema dieser Dissertation zur Bearbeitung angeboten hat, mir Freiheit in der Durchführung gegeben und Vertrauen in meine Fähigkeiten gezeigt hat. Danke Ralph, dass du an mich geglaubt, mich wenn nötig ermutigt und in jedem Fall unterstützt hast. Der Austausch mit dir, war er nun wissenschaftlich oder persönlich, hat maßgeblich dazu beigetragen, dass ich die Zeit meiner Promotion sehr genossen habe. Danke für all die Diskussionen, unkomplizierten Absprachen, amüsanten Geschichten und besonders für deine Offenheit.
Auch möchte ich mich herzlich bei Prof. Dr. Dominic Begerow bedanken, der sich bereit erklärt hat das Korreferat meiner Promotion zu übernehmen. Lieber Dominic, ich danke dir, dass du seit meinem S-Block in deiner Arbeitsgruppe Interesse an meinem wissenschaftlichen Werdegang gezeigt und mich darin bestärkt hast. Besonders danke ich dir für die ehrlichen, kritischen Fragen zu meinen Plänen und Methoden, die nicht selten wichtige Denkprozesse bei mir ausgelöst haben. Darüber hinaus möchte ich nicht versäumen dir für die Zeit und die vielen Tassen Kaffee zu danken, die du in mich investiert hast.
Liebe Linda, Schätzelein, ich kann unmöglich im Rahmen einer Danksagung zum Ausdruck bringen, wie dankbar ich für die gemeinsame Zeit bin. Es war mir ein Fest mit dir gemeinsam ein Büro und den Alltag, sowie zuweilen gemeinsame Dienstreisen zu teilen, mich mit dir zu amüsieren und zu ärgern, zu resignieren, motiviert zu arbeiten und kreativ voraus zu denken. Vielen Dank, dass du mich bis hierhin begleitet und unterstützt hast; Für alle Motivation, Schubser und Ratschläge. Ich wünsche dir für deine(/eure) Zukunft nur das Allerbeste, und mir, dass wir uns nie aus den Augen verlieren.
Unserem langjährigen Bürogenossen Seb Striewski, sowie Max Schweinsberg gilt ebenfalls mein besonderer Dank. Vor allem für die Zerstreuung, die wir uns gegenseitig beschert haben. Für alle Schweineberge, Feierabend-Bierchen, Büroscherze und was dergleichen mehr hier nicht hingehört. Die Zeit mit euch war wunderbar und wird, was mich angeht, niemals vergessen sein.
Auch möchte ich mich bei den fleißigen Studenten bedanken, die zu betreuen ich das Vergnügen hatte. Namentlich möchte ich hier Martin Horstmann, Lisa Deussen und Luxi Chen nennen, mit denen das Arbeiten herrlich unkompliziert, produktiv und zuweilen reich an exquisiten Backwaren gewesen ist.
Daphnia carapace: form, function, structure and plasticity 134 Appendix
Zum Glück dauert es zum Teil sogar noch an. Lisa, Luxi, die Zusammenarbeit mit euch war, Martin, die Zusammenarbeit mit dir ist ein Vergnügen.
Ganz herzlich möchte ich mich natürlich auch bei allen anderen Kollegen am Lehrstuhl und in direkter Nachbarschaft bedanken. Sie haben ein Arbeitsklima erzeugt und beibehalten, wie man es sich schöner nicht ausmalen kann. Danke Leonie, Sina, Ioanna, Annika, Sandra, Manette, Patrick, Dennise, Nadja und Jörg für viele schöne Stunden Arbeit, Pause und gelegentlich Freizeit. Besonders danken möchte ich auch Gabriele Strieso, Beate Hackethal, Ingo Manstedt, Claudia Brefeld, Sabine Adler, Tanja Rollnik und Elli Buschtöns, die mich in diversen Gelegenheiten unterstützt und ebenso zu dem angenehmen Arbeitsklima beigetragen haben.
Außerhalb meiner Fakultät gilt mein Dank Prof. Dr.-Ing. Witzel und Dr.-Ing. Prüfer, die mir die Finite Elemente Analyse nahegebracht haben und sich viel Zeit für Diskussionen zur Umsetzung und Auswertung genommen haben.
Über das Ruhrgebiet hinaus gilt mein besonderer Dank Prof. Dr. Stanislav N. Gorb für die Möglichkeiten, die er mir an seinem Lehrstuhl zur Verfügung gestellt hat. Ich möchte mir nicht vorstellen müssen, wie meine Promotion ausgesehen hätte, wenn ich in Kiel nicht hätte Messungen durchführen dürfen. Vielen Dank Stas für das herzliche Willkommen in Kiel, für die Unterstützung in Form von Gerätestunden und zeitintensiver Beratung, die du mir hast zu Teil werden lassen. Auch den Mitarbeitern des Biomechanik-Lehrstuhls in Kiel gilt mein herzlicher Dank für das Interesse an meiner Arbeit, für das großartige Arbeitsklima und die geduldige Einarbeitung an die Geräte. Namentlich möchte ich Clemens F. Schaber, Thomas Kleinteich, Jonas Wolff und Kirstin Dening für die Unterstützung bei den Messungen, Angela Veenendaal und Esther Appel für die Organisation meines Aufenthalts und Joachim Oesert für den technischen Beistand danken.
Mein persönlicher Dank gilt darüber hinaus meiner Familie und meinen Freunden.
Mama, Papa, vielen Dank, dass ihr es mir ermöglicht habt zu tun, was ich für richtig hielt. Ihr habt mich immer frei walten lassen, mich nie unter Druck gesetzt, sondern, im Gegenteil, mir eure Unterstützung fast selbstverständlich werden lassen. Das ist sie aber in keinem Fall, deswegen: vielen Dank! Auch meinen Geschwistern möchte ich von Herzen Danken. Ihr habt mich geprägt und prägt mich noch, ich habe viel von euch gelernt, und lerne noch. Ich danke euch für gemeinsames Lachen und Weinen. Sarah, vielen Dank, dass du mir eine verantwortungsvolle große Schwester warst und bist und in den richtigen Momenten darauf verzichtet hast. Rebecca, vielen Dank, dass du Missstände nicht stehen lässt, sondern Konflikt suchst, wo er wichtig ist. Und für harmonische, humorvolle Momente, wann immer sie möglich sind. Lucas, vielen Dank, dass wir kleine Genüsse und große Träume teilen können. Ich freue mich auf die Umsetzung letzterer.
Daphnia carapace: form, function, structure and plasticity 135 Appendix
Auch dem Rest meiner zahllosen Verwandtschaft danke ich von Herzen. Vor allem für Verständnis, dass ich diverse Dinge verpasst habe und noch immer nicht alle Namen der nächsten Runkel- Generation sicher beherrsche. Es tut gut Einer von euch zu sein.
Auch meinen Freunden gilt mein herzlicher Dank. Danke für Kurzweil, die wir gemeinsam genossen haben, danke für Geduld, wenn ich kaum auszuhalten war, danke für Zuspruch, wenn ich gar nicht mehr auszuhalten war und danke, dass ich mich auf euch verlassen kann. Besonders möchte ich jenen Freunden danken, die mich in den letzten Monaten ungefragt und liebevoll ertragen und nach der heftigen Enttäuschung geduldig aufgebaut haben. Herzlichsten Dank, Pati, Lily, Günni, Angie, Nadja, Domme, Philipp und Sarah!
Und vielen Dank an meine WG-Kollegen, mit denen zu wohnen ein Privileg ist und war.
Zum Schluss möchte ich mich bei all Jenen entschuldigen, die meinen Dank verdient, ihren Namen aber in dieser Danksagung nicht gefunden haben. Dies war mit Sicherheit keine Absicht, sondern ist meinem Gedächtnis geschuldet. Wer mich kennt weiß, dass nichts so leicht meinem Gedächtnis entkommt wie Namen…
Daphnia carapace: form, function, structure and plasticity 136