Theory of microtomy artefacts in arthropod cuticle

Arthropod cuticles observed in section generally present alternating clear and dark bands. These have often been interpreted in terms of superimposed layers of different structure or composition. It has been shown, however that this material is homogeneous, and is formed by a twisted arrangement of microfibrils. The dense bands correspond actually to a microtomy artefact and they form dark single spirals in certain distorted areas of the cuticle. A model was proposed, involving the interaction between knife motion and microfibrils; it will be referred to as the stepped model, since the proposed mechanism results in the formation of steps at the surface of sections, on both faces. These steps are limited by structures resembling crests or cliffs, whose regular distribution produces alternating thick and thin bands in the section. This explains the observed contrast (Bouligand, (1972)). Two very interesting models were proposed later (Gordon and Winfree, (1978)) and are referred to as the cos F model and the sand model, but steps and crests are absent in these models. However, Giraud-Guille ((1986)), has shown very clearly the existence of these crests, which seem to be quite essential in this microtomy artefact. To clarify the debate, the texts defining the initial and the two new models are reproduced here and the difficulties encountered by each model are discussed. A mathematical formulation of this artefact is presented in an appendix to the present article; this leads to a more complete discussion of the possible models. Other factors are also taken into consideration: microfibril orientation and staining. The main factor of contrasts is undoubtedly the variation in thickness over a single section, as proposed in the stepped model.     

The cuticle of the crabs Cancer pagurus L. and Carcinus maenas (L.)

Fine closely-packed parallel fibres pass obliquely, at an angle of about 10° to the horizontal, through the cuticles of Cancer and Carcinus . They lie on axes parallel to the four faces of an obtuse pyramid, and have no counterpart in the model proposed by Bouligand (1965, 1971, 1972).
Replication of vertically broken surfaces of cuticle on cellulose acetate film shows that the laminae of the cuticle are discrete structural entities which preserve their identity around the angle formed by two vertical faces meeting at right-angles. This does not conform to the requirements of the Bouligand model.     

The trichurid egg-shell: Evidence in support of the Bouligand hypothesis of helicoidal architecture

Electron microscopy of thin sections and freeze etch replicas of the eggs of the nematodes Trichuris suis and T. muris is used to provide evidence in support of the Bouligand hypothesis of helicoidal architecture. The evidence presented is as follows:

1. 1. The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt.

2. 2. Sections cut normal to the plane of the laminae do not show parabolic patterning. Parabolae appear if the section is tilted—their direction depending upon the direction of tilting.

3. 3. Freeze etching allows the direct visualization of helicoidal architecture. Fibres are parallel within any one lamina but the fibre direction rotates by an angle of 9 ° in successive laminae. Parabolic arcs are made up of short lengths of straight fibres—curved fibres were not observed.

Planes of sectioning producing single and double spiral artifacts are described and the formation of these artifacts discussed. The sense of rotation of the helicoid is shown to be asymmetrical about any mid-plane through the egg.     

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 structure of the cuticle of the shore-crab Carcinus maenas (L.)

The cuticle of Carcinus has been critically examined by light microscopy and found to present features differing from the model proposed by Bouligand (1965), elaborated by Neville and his collaborators (see references), and re-stated by Bouligand (1971). The laminae have the appearance of discrete and separable sheets connected by interlaminar fibres, and between them long unorientated macrofibres pursue a sinuous course and pass from one interlaminar zone to another. Together with the interlaminar connecting fibres the macrofibres appear to account for the plumose appearance of the interlaminar zones.     

The nature of driving forces for passive water transport through arthropod cuticle

1. 1.|We demonstrate using thermodynamic arguments that water loss through arthropod epicuticle is well described by a linear law relating water flux to transmembrane vapour pressure drop.

2. 2.|The relationship applies equally to systems where the liquid or vapour exist on either side of a membrane.

3. 3.|An earlier claim by some workers that water diffusion through arthropod epicuticle is proportional to chemical potential drop across the membrane is found to be theoretically unjustified.

4. 4.|Recent measurements with Periplaneta cuticle support the prediction that flux at a given temperature is proportional to the difference in vapour pressure.

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.     

Intramolecular interference effects in dynamic light scattering: rigid double spirals and superhelical DNAs

A theory is developed for dynamic light scattering (DLS) from rigid double spirals by treating an invisible rigid cylinder with two helical scattering stripes on opposite sides of its cylindrical surface. The exact initial, or first cumulant, diffusion coefficient Dapp (K) is obtained in terms of the translational diffusion coefficients (D parallel and D perpendicular) parallel and perpendicular to the symmetry axis, the rotational diffusion coefficients (DR parallel and DR perpendicular) around the symmetry and transverse axes, the length (L) and radius (b) of the cylindrical surface bearing the stripes, and the pitch (p). Interference effects, namely geometrical antiresonances, between strands, produce deep minima in the static structure factor S (K) and corresponding prominent peaks in Dapp (K). These peaks in Dapp (K) depend sensitively on the rotational dynamics around the symmetry axis, and nearly vanish when DR parallel = 0. Some results for single spirals are also presented. A simpler model in which scattering points are attached at opposite ends of an otherwise invisible thin rigid rod is also treated, and shown to exhibit modest minima in S (K) and corresponding maxima in Dapp (K). Confining this rod to a plane containing K enhances the amplitudes of the oscillations in S (K) and Dapp (K), as expected. Rigid double spirals are employed as crude models for interwound supercoiled DNAs in order to assess the possible occurrence of interference effects. Although native supercoiled DNAs exhibit a cylinder diameter that is much too small to exhibit geometrical antiresonances in the presently accessible range of K2, nearly relaxed supercoiled DNAs are predicted to exhibit their first maximum in Dapp (K) just inside this range. Previously reported data for the effect of Escherichia coli single-strand binding (ssb) protein on the DLS of supercoiled pBR322 DNA cannot be mimicked by a gradual homogeneous reduction of superhelix density with increasing ssb, but instead can be mimicked by inhomogeneous all-or-none binding in which uncomplexed native DNAs and nearly relaxed saturated ssb/DNA complexes coexist in varying proportions. Experimental Dapp (K) and S (K) data for a sample of relaxed pUC8 dimers display, respectively, a broad maximum and a corresponding minimum, in qualitative agreement with rough theoretical predictions.     

Reentry as an Origin for Rotors

The aim of the study is to understand in depth the meaning of “reentry”, and to decipher if and how it can lead to malfunctions of the heart and possibly of the brain. A simple model is used to reveal the mechanism by which a single pulse of action potential rotating around a ring of excitable medium, the latter simulating a reentry circuit, can generate spirals (single and/or double) when the pulse can emerge from and develop outside the ring. Two mechanisms of spiral generation are demonstrated: (1) a mechanism in which a source of single spirals is created at the contact with the core soon after the pulse freeing action, their chirality being due to the sense of the preceding pulse rotation. Interestingly, these spirals, adhering to the core, become “double-spiral patterns” while leaving behind the seeds of the new single spirals. (2) A second possible mechanism, similar to the known “arms encountering methods”, in which a double spiral (a figure of eight) is repeatedly created on the other side of the core. Similar procedures are assumed to occur in the heart, leading to tachycardia and fibrillation and possibly in the brain leading to epilepsy. The exact processes of the hitherto assumed spiral generations by reentry were established. The novel deep understanding of the mechanisms involved in these processes can lead to new methods of treating heart fibrillation (e.g., by judicial ablation).     

Some observations on removable spirals inScilla ovatifolia bak

Summary From a study ofScilla ovatifolia it appears that removability of spirals from xylem conducting tissue is independent of the shape of the spiral, and also independent of the presence or absence of a thin pectin layer coating the wall of the vessel or tracheid, but that it is dependent on the structure of the threads. The removable (R-)spirals are characterized by their suppleness and the existence of a thin cuticle covering the threads. This cuticle forms a lubricating medium between the spiral and the wall of the xylem conducting element. R-spirals can be recognised by the occurrence of dislocation figures as revealed e. g. by staining with chloroiodide of zinc. With the possible exception of the cuticle the R-spirals ofScilla ovatifolia consist of almost pure cellulose with a high degree of micellar orientation.A comparison with other plant species shows that suppleness can be decreased by lignin incrustation, and the presence of cementing substances. A smooth surface, however, as provided by a cuticle, is likely to be the main factor determining removability.     

RNA sequence evolution with secondary structure constraints: comparison of substitution rate models using maximum-likelihood methods

We test models for the evolution of helical regions of RNA sequences, where the base pairing constraint leads to correlated compensatory substitutions occurring on either side of the pair. These models are of three types: 6-state models include only the four Watson-Crick pairs plus GU and UG; 7-state models include a single mismatch state that combines all of the 10 possible mismatches; 16-state models treat all mismatch states separately. We analyzed a set of eubacterial ribosomal RNA sequences with a well-established phylogenetic tree structure. For each model, the maximum-likelihood values of the parameters were obtained. The models were compared using the Akaike information criterion, the likelihood-ratio test, and Cox's test. With a high significance level, models that permit a nonzero rate of double substitutions performed better than those that assume zero double substitution rate. Some models assume symmetry between GC and CG, between AU and UA, and between GU and UG. Models that relaxed this symmetry assumption performed slightly better, but the tests did not all agree on the significance level. The most general time-reversible model significantly outperformed any of the simplifications. We consider the relative merits of all these models for molecular phylogenetics.     

Generative models versus underlying symmetries to explain biological pattern

Mathematical models play an increasingly important role in the interpretation of biological experiments. Studies often present a model that generates the observations, connecting hypothesized process to an observed pattern. Such generative models confirm the plausibility of an explanation and make testable hypotheses for further experiments. However, studies rarely consider the broad family of alternative models that match the same observed pattern. The symmetries that define the broad class of matching models are in fact the only aspects of information truly revealed by observed pattern. Commonly observed patterns derive from simple underlying symmetries. This article illustrates the problem by showing the symmetry associated with the observed rate of increase in fitness in a constant environment. That underlying symmetry reveals how each particular generative model defines a single example within the broad class of matching models. Further progress on the relation between pattern and process requires deeper consideration of the underlying symmetries.     

Molting-specific downregulation of C. elegans body-wall muscle attachment sites: The role of RNF-5 E3 ligase

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/β-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process.     

Collision-Based Spiral Acceleration in Cardiac Media: Roles of Wavefront Curvature and Excitable Gap

We have previously shown in experimental cardiac cell monolayers that rapid point pacing can convert basic functional reentry (single spiral) into a stable multiwave spiral that activates the tissue at an accelerated rate. Here, our goal is to further elucidate the biophysical mechanisms of this rate acceleration without the potential confounding effects of microscopic tissue heterogeneities inherent to experimental preparations. We use computer simulations to show that, similar to experimental observations, single spirals can be converted by point stimuli into stable multiwave spirals. In multiwave spirals, individual waves collide, yielding regions with negative wavefront curvature. When a sufficient excitable gap is present and the negative-curvature regions are close to spiral tips, an electrotonic spread of excitatory currents from these regions propels each colliding spiral to rotate faster than the single spiral, causing an overall rate acceleration. As observed experimentally, the degree of rate acceleration increases with the number of colliding spiral waves. Conversely, if collision sites are far from spiral tips, excitatory currents have no effect on spiral rotation and multiple spirals rotate independently, without rate acceleration. Understanding the mechanisms of spiral rate acceleration may yield new strategies for preventing the transition from monomorphic tachycardia to polymorphic tachycardia and fibrillation.     

First Record of Soft Tissue Preservation in the Upper Devonian of Poland

Soft tissue preservation is reported from Upper Devonian deposits of the Holy Cross Mountains, central Poland, for the first time. The preserved soft tissues are muscles associated with arthropod cuticle fragments. The muscles are phosphatized with variable states of preservation. Well-preserved specimens display the typical banding of striated muscles. Other muscle fragments are highly degraded and/or recrystallized such that their microstructure is barely visible. The phosphatized muscles and associated cuticle are fragmented, occur in patches and some are scattered on the bedding plane. Due to the state of preservation and the lack of diagnostic features, the cuticle identification is problematic; however, it may have belonged to a phyllocarid crustacean. Taphonomic features of the remains indicate that they do not represent fossilized fecal matter (coprolite) but may represent a regurgitate, but the hypothesis is difficult to test. Most probably they represent the leftover remains after arthropod or fish scavenging. The present study shows that soft tissues, which even earlier were manipulated by scavenger, may be preserved if only special microenvironmental conditions within and around the animal remains are established.     

Random Wiring,Ganglion Cell Mosaics,and the Functional Architecture of the Visual Cortex

The architecture of iso-orientation domains in the primary visual cortex (V1) of placental carnivores and primates apparently follows species invariant quantitative laws. Dynamical optimization models assuming that neurons coordinate their stimulus preferences throughout cortical circuits linking millions of cells specifically predict these invariants. This might indicate that V1’s intrinsic connectome and its functional architecture adhere to a single optimization principle with high precision and robustness. To validate this hypothesis, it is critical to closely examine the quantitative predictions of alternative candidate theories. Random feedforward wiring within the retino-cortical pathway represents a conceptually appealing alternative to dynamical circuit optimization because random dimension-expanding projections are believed to generically exhibit computationally favorable properties for stimulus representations. Here, we ask whether the quantitative invariants of V1 architecture can be explained as a generic emergent property of random wiring. We generalize and examine the stochastic wiring model proposed by Ringach and coworkers, in which iso-orientation domains in the visual cortex arise through random feedforward connections between semi-regular mosaics of retinal ganglion cells (RGCs) and visual cortical neurons. We derive closed-form expressions for cortical receptive fields and domain layouts predicted by the model for perfectly hexagonal RGC mosaics. Including spatial disorder in the RGC positions considerably changes the domain layout properties as a function of disorder parameters such as position scatter and its correlations across the retina. However, independent of parameter choice, we find that the model predictions substantially deviate from the layout laws of iso-orientation domains observed experimentally. Considering random wiring with the currently most realistic model of RGC mosaic layouts, a pairwise interacting point process, the predicted layouts remain distinct from experimental observations and resemble Gaussian random fields. We conclude that V1 layout invariants are specific quantitative signatures of visual cortical optimization, which cannot be explained by generic random feedforward-wiring models.