Arthropod remains in the oral cavities of fossil reptiles support inference of early insectivory

Inference of feeding preferences in fossil terrestrial vertebrates (tetrapods) has been drawn predominantly from craniodental morphology, and less so from fossil specimens preserving conclusive evidence of diet in the form of oral and/or gut contents. Recently, the pivotal role of insectivory in tetrapod evolution was emphasized by the identification of putative insectivores as the closest relatives of the oldest known herbivorous amniotes. We provide the first compelling evidence for insectivory among early tetrapods on the basis of two 280-million-year-old (late Palaeozoic) fossil specimens of a new species of acleistorhinid parareptile with preserved arthropod cuticle on their toothed palates. Their dental morphology, consisting of homodont marginal dentition with cutting edges and slightly recurved tips, is consistent with an insectivorous diet. The intimate association of arthropod cuticle with the oral region of two small reptiles, from a rich fossil locality that has otherwise not produced invertebrate remains, strongly supports the inference of insectivory in the reptiles. These fossils lend additional support to the hypothesis that the origins and earliest stages of higher vertebrate evolution are associated with relatively small terrestrial insectivores.     

The cuticle of the enigmatic arthropod Phytophilaspis and biomineralization in Cambrian arthropods

Lin, J.‐P., Ivantsov, A.Y. & Briggs, D.E.G. 2011: The cuticle of the enigmatic arthropod Phytophilaspis and biomineralization in Cambrian arthropods. Lethaia, Vol. 44, pp. 344–349. Many non‐trilobite arthropods occur in Cambrian Burgess Shale‐type (BST) biotas, but most of these are preserved in fine‐grained siliciclastics. Only one important occurrence of Cambrian non‐trilobite arthropods, the Sinsk biota (lower Sinsk Formation, Botomian) from the Siberian Platform, has been discovered in carbonates. The chemical compositions of samples of the enigmatic arthropod Phytophilaspis pergamena Ivantsov, 1999 and the co‐occurring trilobite Jakutus primigenius Ivantsov in Ponomarenko, 2005 from this deposit were analysed. The cuticle of P. pergamena is composed of mainly calcium phosphate and differs from the cuticle of J. primigenius, which contains only calcium carbonate. Phosphatized cuticles are rare among large Cambrian arthropods, except for aglaspidids and a few trilobites. Based on recent phylogenetic studies, phosphatization of arthropod cuticle is likely to have evolved several times. □arthropod cuticle, Burgess Shale‐type preservation, fossil‐diagenesis, phosphatization.     

A novel stress distribution organ in the arthropod exoskeleton

The vertebrate endoskeleton possesses a massive internal network of load-distributing trabeculae that in most locations accounts for the vast majority of bone cross sectional area. In contrast, arthropods rely on the external cuticle and its intermittent outpocketings to distribute the daily stresses of physiological loading. One of the constraints of the arthropod exoskeleton is the necessity to house the musculature involved in locomotion, feeding and etc. Because of this lack of an extensive internal load-distributing trabecular network, any load-distributing mechanism in arthropods would necessarily have to incorporate the exoskeleton. Several authors have identified structural apophysi whose functions presumably have mechanical significance, but few have been identified using quantitative analyses. This study investigates a novel stress-reducing structure arising from the articulation sites in the exoskeleton of the blue crab, Callinectes sapidus. During dissection of the merus-carpus joint and leg cuticle of the blue crab, an unique system of internal strut-like members was found radiating, both longitudinally and laterally, from the articular surface of the proximal merus segment, tapering into the diaphyseal region. This strut system, an internal outpocketing of the exoskeleton and semi-circular in cross section, mirrors the trabecular pattern seen radiating from vertebrate joint surfaces. Earlier reports of this structural system described it as a muscle attachment site and made little or no reference to potential load distribution properties. Finite element analysis (FEA) models confirm the efficacy of stress distributing properties of this articular strut system in the blue crab leg. In the models, the struts significantly reduce stress concentrations, reduce localized strains and minimize the risk of failure via buckling. Models lacking this strut system generate 94.7% larger peak von Mises stress at the articulation site, 37% higher peak displacement and 4% greater equivalent strain. The model with the struts is capable of withstanding an applied physiological load of up to 16.6 N prior to buckling, more than twice that of the model without struts (7.8 N). We suggest that this novel arthropod strut system is likely utilized at many joint surfaces at locations of high skeletal stress concentrations, is an adaptation for minimizing skeletal failure via localized buckling, and may be present in other arthropod taxa.     

Comparative Ultrastructure of Arthropod Transporting Epithelia

The general organization of arthropod epithelia is comparedto that of vertebrates. It is suggested that although ciliatedepithelia, stratified epithelia and in some cases continuousmuscle sheaths do not occur in arthropods, they have certainanalogous structures which carry out the same functions. Forexample, the arthropod cuticle is compared to the squamous layerof vertebrate stratified epithelia, and complex arthropod basementmembranes are compared to the muscle and connective tissue sheathsof certain vertebrate epithelia. The cellular organization oftransporting epithelial cells is then discussed, with particularreference to elaboration of plasma membranes, and similaritiesand differences between vertebrates and arthropods, and betweeninsects and crustaceans are pointed out. Specializations peculiarto insect cells are described, including the insertion of mitochondriainto apical membrane microvilli, and the presence along thismembrane of small particles called portasomes believed to beinvolved in active transport. Finally, it is shown that in themidgut of theinsect Manduca sexta, distinct ultrastructuralchanges accompany loss of potassium transport activity duringa larval molt and in the prepupal stage. The ultrastructuralchanges which occur include a proliferation of the basementmembrane and muscle tissue underlying the epithelium, and achange in the morphology of the potassium transporting gobletcells. Possible correlations between ultrastructural changesand loss of transport activity are discussed.     

A single spiral artefact in Arthropod cuticle

Spirals are often seen in sections transverse to the axes of bumped structures in arthropod cuticle. (Sections through arthropod cornea or exocones yield excellent examples.) As arthropod cuticle has a helicoidal architecture (Bouligand, 1965), it might be expected that the spirals are a simple consequence of that structure. According to a symmetry argument, the spirals thus predicted must be double spirals. In contrast, the observed spirals are usually single. We propose that the single spirals result from an interaction between the microtome knife and the cuticle architecture. The direction of knife travel defines an orientation within the cuticle, subverting the symmetry arguments that require double spirals. Bouligand (1972) presented a model for the interaction of the knife with the cuticle. However, we offer arguments and observations which show that Bouligand's model is incorrect. We argue from detailed observations of the single spiral that it is indeed a knifing artifact and that its explanation probably lies within a certain class of models. Two related models based on relative movements of cuticle components are examined via computer techniques.     

Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope

Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T. albus using transmission and field-emission electron microscopy, confocal μ-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures.     

CUTICULAR LIPIDS OF TERRESTRIAL PLANTS AND ARTHROPODS: A COMPARISON OF THEIR STRUCTURE, COMPOSITION, AND WATERPROOFING FUNCTION

1. Lipids deposited on the surface or embedded within the cuticle of terrestrial plants and arthropods are primarily responsible for the observed low rates of water loss through the cuticle. 2. These lipids are a mixture of long-chain compounds which include hydrocarbons (saturated, unsaturated, branched), wax esters, free fatty acids, alcohols, ketones, aldehydes, and cyclic compounds. 3. The cuticle of both plants and arthropods is a continuous, non-cellular multilayered membrane which overlies the epidermal cells. 4. In arthropods, horizontal division of the cuticle into layers is clearly visible. In plants, the layers comprising the cuticle are not sharply demarcated. 5. The substance responsible for the structural integrity of the plant cuticle (cutin) is composed of cross-esterified fatty acids; structural integrity in arthropod cuticle is provided by a chitin-protein complex. 6. Cuticular lipids are probably formed near the surface in both plants and arthropods; however, specific sites of synthesis are known for only a few species and little is known about their transport from these sites to the surface. The elaborate pore canal and wax canal system of arthropod cuticle is absent from plants. 7. The physical structure and arrangement of the lipid deposits on the cuticular surface that are so important in controlling water movement depend on both quantity and chemical composition, and are, in turn, specific to each species in relation to its environment. 8. Different lipid components are not equally efficient in reducing transpiration. Maximum waterproofing effectiveness is provided by long-chain, saturated, non-polar molecules containing few methyl branches. 9. Plants and arthropods can, within genetic constraints, alter the composition of their cuticular waxes to improve impermeability when conditions require increased water conservation. 10. None of the models proposed to explain the change in arthropod cuticular permeability which occurs at a species-specific temperature (‘transition temperature’) in many species is supported by the experimental data now available.     

The arthropod,but not the vertebrate host or its environment,dictates bacterial community composition of fleas and ticks

Bacterial community composition in blood-sucking arthropods can shift dramatically across time and space. We used 16S rRNA gene amplification and pyrosequencing to investigate the relative impact of vertebrate host-related, arthropod-related and environmental factors on bacterial community composition in fleas and ticks collected from rodents in southern Indiana (USA). Bacterial community composition was largely affected by arthropod identity, but not by the rodent host or environmental conditions. Specifically, the arthropod group (fleas vs ticks) determined the community composition of bacteria, where bacterial communities of ticks were less diverse and more dependent on arthropod traits—especially tick species and life stage—than bacterial communities of fleas. Our data suggest that both arthropod life histories and the presence of arthropod-specific endosymbionts may mask the effects of the vertebrate host and its environment.     

Deconstructing the design of a biological material

By identifying the functional conflicts in its design, the cuticle of arthropods can be shown to cope with IR and UV irradiation in the same manner as our technology-by controlling spectral properties (transmission and reflection). However, the skeletal properties of cuticle are integrated with demands for sensory transmission, movement, etc, by controlling the local properties of the material rather than by changing global parameters (which would be the technical solution). On the basis of this study, the biomimetic similarity of cuticle with technology is only about 20%, suggesting that we can learn from the design of arthropod cuticle.     

Some ultrastructural observations on the integument of a pentastomid

The cuticle of the cephalobaenid pentastomid Reighardia sternae is described at various stages of the moult-intermoult cycle. The intermoult cuticle comprises four layers: an outer epicuticle; an underlying dense layer, the protein epicuticle; a fibrillar endocuticle; and a denser subcuticle. The overall similarity between the structure and composition of these layers and those of insects is discussed. However, the orientation of the chitin-protein fibres in the endocuticle does not show the rotating structure characteristic of many arthropod species, but this does appear in the sclerotized hooks. It is suggested that this comparatively loose, poorly oriented endocuticular structure produces a highly extensible cuticle which is precisely adapted to the specialized, endoparasitic habit of this species. Events at ecdysis, particularly the secretion of moulting fluid and the deposition of cuticulin, follow the insect pattern precisely. The phyletic significance of these observations is discussed.     

Analysis of the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine, and chitin-binding cuticle proteins. Four major cuticle proteins, BMCP30, 22, 18, and 17, have been previously identified and purified from the larval cuticle of silkworm, B. mori. We analyzed the chitin-binding activity of BMCP30 by use of chitin-affinity chromatography. The pH optimum for the binding of BMCP30 to chitin is 6.4, which corresponds to hemolymph pH. Competition experiments using chitooligosaccharides suggested that BMCP30 recognizes 4-6 mer of N-acetylglucosamine in chitin fiber as a unit for binding. The comparison of the binding properties of BMCP30 with those of BMCP18 showed that their binding activities to chitin are similar in a standard buffer but that BMCP30 binds to chitin more stably than BMCP18 in the presence of urea. BMCPs possess the RR-1 form of the R&R consensus, about 70 amino acids region conserved widely among cuticle proteins mainly from the soft cuticle of many insect and arthropod species. Analysis of the binding activity using deletion mutants of BMCPs revealed that this type of conserved region also functions as the chitin-binding domain, similarly to the RR-2 region previously shown to confer chitin binding. Thus, the extended R&R consensus is the general chitin-binding domain of cuticle proteins in Arthropoda.     

Autofluorescence lifetime variation in the cuticle of the bedbug Cimex lectularius

The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius. After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime τ_m (tau-m) at 0.4 ns and 1.0–1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration.     

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

Phylogenetic relationship of muscle tissues deduced from superimposition of gene trees.

Muscle tissues can be divided into six classes; smooth, fast skeletal, slow skeletal and cardiac muscle tissues for vertebrates, and striated and smooth muscle tissues for invertebrates. We reconstructed phylogenetic trees of six protein genes that are expressed in muscle tissues and, using a newly developed program, inferred the phylogeny of muscle tissues by superimposition of five of those gene trees. The proteins used are troponin C, myosin essential light chain, myosin regulatory light chain, myosin heavy chain, actin, and muscle regulatory factor (MRF) families. Our results suggest that the emergence of skeletal-cardiac muscle type tissues preceded the vertebrate/arthropod divergence (ca. 700 MYA), while vertebrate smooth muscle seemed to evolve independent of other muscles. In addition, skeletal muscle is not monophyletic, but cardiac and slow skeletal muscles make a cluster. Furthermore, arthropod striated muscle, urochordate smooth muscle, and vertebrate muscles except for smooth muscle share a common ancestor. On the other hand, arthropod nonmuscle and vertebrate smooth muscle and nonmuscle share a common ancestor.     

The ultrastructure of the microfilaria of Brugia, Nematoda: Filarioidea

The microfilaria of Brugia pahangi is a differentiated nematode larva. The basic nematode body plan is present showing cuticle, hypodermis, dorsal, ventral, and lateral cords, muscle cells, longitudinal nerves, papillary nerves, amphids and phasmids. Secretory granules are present in ganglionic cells and in axons in the nerve ring. There is no differentiated pseudocoelom. There is only a single row of muscle cells between each pair of cords. The excretory cell complex is similar in structure to the hypodermal gland cells of other nematodes. The alimentary canal of the microfilaria is very much modified. The pharyngeal cells are attached to the pharyngeal thread which is circular in cross section and there is no pharyngeal musculature. The intestine is represented by the solid mass of the inner body within paired intestinal cells. The intestine is separated from the rectum. The three rectal cells form a syncytium of villi in the anal vesicle. The structure in Brugia is related to the ultrastructure of other microfilariae and it is concluded that the evolution of the modifications of the basic larval structure is due to the small size of these nematodes as a consequence of their adaptation to a parasitic mode of life in the capillaries of the vertebrate host with transmission through an intermediate arthropod vector.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Phylogenetic analysis of the Wnt gene family and discovery of an arthropod wnt-10 orthologue

Wnt genes encode a conserved family of secreted signaling proteins that play many roles in arthropod and vertebrate development. We have investigated both the phylogenetic history and molecular evolution of this gene family. We have identified a novel Wnt gene in a diversity of arthropods that it is likely an orthologue of the vertebrate Wnt-10 group. Wnt-10 is one of only two cases in which orthology between protostome and deuterostome genes could be consistently assigned based on our analyses. Despite difficulties in assessing orthologies, all of our trees suggest that the most recent common ancestor of protostomes and deuterostomes possessed more than the five Wnt genes known from either arthropods or nematodes. This suggests that Wnt gene loss has occurred during protostome evolution. In addition, we examined the rate of amino acid evolution in the two arthropod/deuterostome orthology groups we identified. We found little rate variation across taxa, with the exception that Drosophila Wnt-1 is evolving more rapidly than all vertebrate and most arthropod orthologues.     

Limbs and tail as evolutionarily diverging duplicates of the main body axis

SUMMARY Contrasting hypotheses have been proposed to explain the pervasive parallels in the patterning of arthropod and vertebrate appendages. These hypotheses either call for a common ancestor already provided with patterned appendages or body outgrowths, or for the recruitment in limb patterning of single genes or genetic cassettes originally used for purposes other than axis patterning. I suggest instead that body appendages such as arthropod and vertebrate limbs and chordate tails are evolutionarily divergent duplicates (paramorphs) of the main body axis, that is, its duplicates, albeit devoid of endodermal component. Thus, vertebrate limbs and arthropod limbs are not historical homologs, but homoplastic features only transitively related to real historical homologs. Thus, the main body axis and the axis of the appendages have distinct but not independent evolutionary histories and may be involved in processes of homeotic co-option producing effects of morphological assimilation. For instance, chordate segmentation may have originated in the posterior appendage (tail) and subsequently extended to the trunk.