Lattice-like Arrangement Research Articles

Flow interactions lead to self-organized flight formations disrupted by self-amplifying waves

Collectively locomoting animals are often viewed as analogous to states of matter in that group-level phenomena emerge from individual-level interactions. Applying this framework to fish schools and bird flocks must account for visco-inertial flows as mediators of the physical interactions. Motivated by linear flight formations, here we show that pairwise flow interactions tend to promote crystalline or lattice-like arrangements, but such order is disrupted by unstably growing positional waves. Using robotic experiments on “mock flocks” of flapping wings in forward flight, we find that followers tend to lock into position behind a leader, but larger groups display flow-induced oscillatory modes – “flonons” – that grow in amplitude down the group and cause collisions. Force measurements and applied perturbations inform a wake interaction model that explains the self-ordering as mediated by spring-like forces and the self-amplification of disturbances as a resonance cascade. We further show that larger groups may be stabilized by introducing variability among individuals, which induces positional disorder while suppressing flonon amplification. These results derive from generic features including locomotor-flow phasing and nonreciprocal interactions with memory, and hence these phenomena may arise more generally in macroscale, flow-mediated collectives.

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector.

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

Bio-inspired selective nodal decoupling for ultra-compliant interwoven lattices

Architected materials such as lattices are capable of demonstrating extraordinary mechanical performance. Lattices are often used for their stretch-dominated behavior, which gives them a high degree of stiffness at low-volume fractions. At the other end of the stiffness spectrum, bending-dominated lattices tend to be more compliant and are of interest for their energy absorption performance. Here, we report a class of ultra-compliant interwoven lattices that demonstrate up to an order of magnitude improvement in compliance over their traditional counterparts at similar volume fractions. This is achieved by selectively decoupling nodes and interweaving struts in bending-dominated lattices, inspired by observations of this structural principle in the lattice-like arrangement of the Venus flower basket sea sponge. By decoupling nodes in this manner, we demonstrate a simple and near-universal design strategy for modulating stiffness in lattice structures and achieve among the most compliant lattices reported in the literature.

Simulating the dynamics of dispersal and dispersal ability in fragmented populations with mate-finding Allee effects.

We consider the spatial propagation and genetic evolution of model populations comprising multiple subpopulations, each distinguished by its own characteristic dispersal rate. Mate finding is modeled in accord with the assumption that reproduction is based on random encounters between pairs of individuals, so that the frequency of interbreeding between two subpopulations is proportional to the product of local population densities of each. The resulting nonlinear growth term produces an Allee effect, whereby reproduction rates are lower in sparsely populated areas; the distribution of dispersal rates that evolves is then highly dependent upon the population's initial spatial distribution. In a series of numerical test cases, we consider how these dynamics affect lattice-like arrangements of population fragments, and investigate how a population's initial fragmentation determines the dispersal rates that evolve as a habitat is colonized. First, we consider a case where initial population fragments coincide with habitat islands, within which death rates differ from those that apply outside; the presence of inhospitable exterior regions exaggerates Allee effect-driven reductions in dispersal ability. We then examine how greater distances separating adjacent population fragments lead to more severe reductions in dispersal ability. For populations of a fixed initial magnitude, fragmentation into smaller, denser patches leads not only to greater losses of dispersal ability, but also helps ensure the population's long-term persistence, emphasizing the trade-offs between the benefits and risks of rapid dispersal under Allee effects. Next, simulations of well-established populations disrupted by localized depopulation events illustrate how mate-finding Allee effects and spatial heterogeneity can drive a population's dispersal ability to evolve either downward or upward depending on conditions, highlighting a qualitative distinction between population fragmentation and habitat heterogeneity. A final test case compares populations that are fragmented across multiple scales, demonstrating how differences in the relative scales of micro- and macro-level fragmentation can lead to qualitatively different evolutionary outcomes.

Skeletal elements of the penguin eye and their functional and phylogenetic implications (Aves: Sphenisciformes: Spheniscidae).

Scleral ossicles and other bony elements are present in the eyes of many vertebrates, including birds. In this study, the skeletal elements present in the penguin eye and orbit were imaged using macro photographs and micro‐computed tomography (micro‐CT), to help elucidate their function and significance. A total of 36 scleral rings and three whole skulls were imaged. King (Aptenodytes patagonicus), Fiordland crested (Eudyptes pachyrhynchus), Snares crested (Eudyptes robustus), royal (Eudyptes schlegeli) and yellow‐eyed (Megadyptes antipodes) penguins had between 12 and 14 elements in their scleral ring while the gentoo (Pygoscelis papua) had 14 and 17; little penguins (Eudyptula sp.) consistently had between 10 and 12 elements. All had at least two elements that overlapped, usually totally, each neighbour, and two that were overlapped by each neighbour. The interior structure of all ossicles revealed a lattice‐like arrangement of struts typical of cancellous bone, the whole being surrounded by thick cortical bone. The scleral ring of a 10 week gentoo chick was not completely ossified but rather had multiple small holes within it on micro‐CT. A large os opticus was present in one king penguin but in another bird of the same age and gender there was no such bone. Much smaller accessory bones were found in the posterior pole of one Snares crested and one little penguin. We conclude that the penguin scleral ring not only maintains the shape of the eye but also provides protection and a site of insertion for rectus muscles. However, the extreme variability in the os opticus suggests that it is not essential to normal function.

Emergent spatial structure and pathogen epidemics: the influence of management and stochasticity in agroecosystems

Organisms susceptible to disease, from humans to crops, inevitably have spatial geometry that influence disease dynamics. Understanding how spatial structure emerges through time in ecological systems and how that structure influences disease dynamics is of practical importance for natural and human management systems. Here we use the perennial crop, coffee, Coffea arabica, along with its pathogen, the coffee leaf rust, Hemileia vastatrix, as a model system to understand how spatial structure is created in agroecosystems and its subsequent influence on the dynamics of the system. Here, we create a simple null model of the socio-ecological process of death and stochastic replanting of coffee plants on a plot. We then use spatial networks to quantify the spatial structures and make comparisons of our stochastic null model to empirically observed spatial distributions of coffee. We then present a simple model of pathogen spread on spatial networks across a range of spatial geometries emerging from our null model and show how both local and regional management of agroecosystems interact with space and time to alter disease dynamics. Our results suggest that our null model of evolving spatial structure can capture many critical features of how the spatial arrangement of plants changes through time in coffee agroecosystems. Additionally, we find small changes in management factors that can influence the scale of pathogen transmission, such as shade tree removal, and result in a rapid transition to epidemics with lattice-like spatial arrangements but not with irregular planting geometries. The results presented here may have practical implications for farmers in Latin America who are in the process of replanting and overhauling management of their coffee farms in response to a coffee leaf rust epidemic in 2013. We suggest that shade reduction in conjunction with more lattice-like planting schemes may result in coffee being more prone to epidemic-like dynamics of the coffee leaf rust in the future.

Dual catalytic DNA circuit-induced gold nanoparticle aggregation: An enzyme-free and colorimetric strategy for amplified detection of nucleic acids

An enzyme-free dual catalytic DNA circuit for amplified detection of nucleic acids has been developed. The system functions based on a cyclic self-assembly of two auxiliary hairpins (H1 and H2) and three biotinylated hairpin oligonucleotides (H3, H4 and H5), in the format of two molecular circuits. In the upstream circuit, a target initiator (I) besides H1 and H2 hairpins constructs H1-H2 duplexes that trigger the operation of a subsequent circuit. In the downstream circuit, the H1-H2 duplex initiates cascaded self-assembly reactions, produces triplex H3-H4-H5, as sensing system, and releases the H1-H2 duplex as the catalyst for the self-assembly of additional hairpins. The H3-H4-H5 triplex acts as the scaffolds for assembling and orienting the streptavidin-functionalized gold nanoparticles (SA-AuNPs) into a lattice-like arrangement that generates a DNA-SA-AuNP cross-linked network, resulting in a dramatic pale red-to-blue color change. By ingeniously engaging two catalytic circuits with feedback amplification capabilities, the system can detect the target nucleic acid with an LOD value of 5 femtomolar and unambiguously discriminate spurious targets (i.e. targets containing substitution, insertion, and deletion nucleotides) without instrumentation. Simple and convenient operation of the assay makes the DNA circuit appropriate for point-of-care monitoring in resource-constrained settings.

A Genetically Encoded Toolbox of Orthogonal Adhesins for Bacterial Self-Assembly

In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. In particular, to our knowledge there exists no convenient method for engineering cell-cell adhesion. Towards filling this gap in synthetic biology's toolbox, here we report the first 100% genetically encoded self-assembly platform, based on modular cell-cell adhesion in the bacterium Escherichia coli. In this system, adhesive selectivity is provided by a library of outer membrane-displayed peptides with orthogonal intra-library specificities, while affinity is provided by intrinsic adhesin affinity, media conditions, and inducible expression across the entire library. We demonstrate this tool by building a variety of well-defined multicellular patterns, including multiple cell types in cluster-, mesh-, and lattice-like arrangements and comparing these to a biophysical model. This orthogonal, composable adhesion system will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, in tissue engineering, and in controlled study of minimal multicellular systems.

Simulation study on the structural properties of colloidal particles with offset dipoles

A major research theme in materials science is determining how the self-assembly of new generations of colloidal particles of complex shape and surface charge is guided by their interparticle interactions. In this paper, we describe results from quasi-2D Monte Carlo simulations of systems of colloidal particles with offset transversely-oriented extended dipole-like charge distributions interacting via an intermediate-ranged Yukawa potential. The systems are cooled slowly through an annealing procedure during which the temperature is lowered in discrete steps, allowing the system to equilibrate. We perform ground state calculations for two, three and four particles at several shifts of the dipole vector from the particle center. We create state diagrams in the plane spanned by the temperature and the area fraction outlining the boundaries between fluid, string-fluid and percolated states at various values of the shift. Remarkably we find that the effective cooling rate in our simulations has an impact on the structures formed, with chains being more prevalent if the system is cooled quickly and cyclic structures more prevalent if the system is cooled slowly. As the dipole is further shifted from the center, there is an increased tendency to assemble into small cyclic structures at intermediate temperatures. These systems further self-assemble into open lattice-like arrangements at very low temperatures. The novel structures identified might be useful for photonic applications, new types of porous media for filtration and catalysis, and gel matrices with unusual properties.

Dynamic elastic moduli in magnetic gels: Normal modes and linear response.

In the perspective of developing smart hybrid materials with customized features, ferrogels and magnetorheological elastomers allow a synergy of elasticity and magnetism. The interplay between elastic and magnetic properties gives rise to a unique reversible control of the material behavior by applying an external magnetic field. Albeit few works have been performed on the time-dependent properties so far, understanding the dynamic behavior is the key to model many practical situations, e.g., applications as vibration absorbers. Here we present a way to calculate the frequency-dependent elastic moduli based on the decomposition of the linear response to an external stress in normal modes. We use a minimal three-dimensional dipole-spring model to theoretically describe the magnetic and elastic interactions on the mesoscopic level. Specifically, the magnetic particles carry permanent magnetic dipole moments and are spatially arranged in a prescribed way, before they are linked by elastic springs. An external magnetic field aligns the magnetic moments. On the one hand, we study regular lattice-like particle arrangements to compare with previous results in the literature. On the other hand, we calculate the dynamic elastic moduli for irregular, more realistic particle distributions. Our approach measures the tunability of the linear dynamic response as a function of the particle arrangement, the system orientation with respect to the external magnetic field, as well as the magnitude of the magnetic interaction between the particles. The strength of the present approach is that it explicitly connects the relaxational modes of the system with the rheological properties as well as with the internal rearrangement of the particles in the sample, providing new insight into the dynamics of these remarkable materials.

Structure of Dimeric and Tetrameric Complexes of the BAR Domain Protein PICK1 Determined by Small-Angle X-Ray Scattering

PICK1 is a neuronal scaffolding protein containing a PDZ domain and an auto-inhibited BAR domain. BAR domains are membrane-sculpting protein modules generating membrane curvature and promoting membrane fission. Previous data suggest that BAR domains are organized in lattice-like arrangements when stabilizing membranes but little is known about structural organization of BAR domains in solution. Through a small-angle X-ray scattering (SAXS) analysis, we determine the structure of dimeric and tetrameric complexes of PICK1 in solution. SAXS and biochemical data reveal a strong propensity of PICK1 to form higher-order structures, and SAXS analysis suggests an offset, parallel mode of BAR-BAR oligomerization. Furthermore, unlike accessory domains in other BAR domain proteins, the positioning of the PDZ domains is flexible, enabling PICK1 to perform long-range, dynamic scaffolding of membrane-associated proteins. Together with functional data, these structural findings are compatible with a model in which oligomerization governs auto-inhibition of BAR domain function.

The gastralial apparatus of Plateosaurus engelhardti: morphological description and soft-tissue reconstructio

The fragmentary nature of the fossil record of the gastralial apparatus of sauropodomorph dinosaurs has considerable impact on our understanding its functional morphology and evolution in this group. With the aim of increasing our knowledge on the functional morphology of the gastralial apparatus of sauropodomorph dinosaurs, remains of the gastralial apparatus of Plateosaurus engelhardti are described. Soft-tissues and their function are reconstructed applying the Extant Phylogenetic Bracket, in this instance comprising crocodiles and birds. The gastralial apparatus of Plateosaurus consists of at least 18 gastralial rows. With exception of the 1st row, which has a chevron-shaped medial and two lateral gastralia, the gastralial rows consist of two medial and two lateral gastralia. The lateral gastralia are 2.2 –– 2.8 times longer than the associated medial gastralia. The proximal ends of the medial gastralia overlap in a latticelike arrangement, forming a mid-ventral imbricating articulation. Characteristic for Plateosaurus is the sigmoid form of the medial and lateral gastralia, the increasing angle of the gastralial rows from proximal to distal, as well as the increasing robustness of the gastralia rows from proximal to distal. According to the Extant Phylogenetic Bracket, the gastralial apparatus of Plateosaurus is embedded into M. rectus abdominis, which attaches to the caudal aspect of the sternum and indirectly to the distal end of the pubis. As in extant archosaurs, m. rectus abdominis is closely related to the other muscles of the hypaxial abdominal muscle group. The muscles of the hypaxial abdominal muscle group are active during ventilation and locomotion, which suggests that the functions of the gastralial apparatus of Plateosaurus may be more complex than previously suggested. Regina Fechner. Biomechanics Research Group, Fakultat fur Maschinenbau, Ruhr-Universitat Bochum, Universitatsstrase 150, 44801 Bochum, Germany. fechner@lmk.rub.de Rainer Gosling. Biomechanics Research Group, Fakultat fur Maschinenbau, Ruhr-Universitat Bochum, Universitatsstrase 150, 44801 Bochum, Germany. goessling@lmk.rub.de

X-ray absorption near-edge structure (XANES) spectroscopy identifies differential sulfur speciation in corneal tissue

The chemical composition of tissues can influence their form and function. As a prime example, the lattice-like arrangement of collagen fibrils required for corneal transparency is controlled, in part, by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. However, to date, no studies have been able to directly identify and characterize sulfur (S) in the cornea as a function of tissue location. In this study, X-ray absorption near-edge structure spectroscopy and micro-beam X-ray fluorescence (μ-XRF) chemical contrast imaging were employed to probe the nature of the mature (bovine) cornea as a function of position from the anterior sub-epithelial region into the deep stroma. Data indicate an inhomogeneity in the composition of S species in the first ≈50μm of stromal depth. In μ-XRF chemical contrast imaging, S did not co-localize with phosphorous (P) in the deep stroma where sulfates are prominent. Rather, P is present only as isolated micrometric spots, presumably identifiable as keratocytes. This study lends novel insights into the elemental physiology of mature cornea, especially in relation to its S distribution; future studies could be applied to human tissues. Moreover, it defines an analytical protocol for the interrogation of S species in biological tissues with micrometric resolution.

The mucosal folds at the pancreaticobiliary junction

The structure and function of the mucosal folds in the terminal bile and pancreatic ducts and hepatopancreatic ampulla are poorly characterised. The distribution, muscularity, and innervation of these folds were investigated. The pancreaticobiliary junction was excised from ten cadavers (five male, 66-90years) and examined histologically by serially sectioning (4-μm thickness) along the length of the terminal bile and pancreatic ducts from the tip of the major duodenal papilla. Three surgical specimens (two male, 63-72years) were also evaluated. Sections were stained with haematoxylin and eosin, anti-actin (smooth muscle), anti-S100 (innervation), and anti-cholecystokinin (CCK)-A receptor antibodies. ImageJ software was used to compare relative radial fold projection and semi-quantitatively assess the smooth muscle and nerve content. In one additional cadaver specimen, folds were examined by scanning electron microscopy. Mucosal folds in the terminal bile duct were arranged circumferentially in a lattice-like arrangement and were distributed over an average distance of 7.3mm along the terminal bile duct compared to 4.2mm along the pancreatic duct (P=0.001), projected further into the lumen, and were more densely innervated than those in the terminal pancreatic duct. Folds in both ducts contained smooth muscle which was more prominent in folds nearest to the major duodenal papilla. Mucosal folds in cadaver and surgical specimens showed no evidence of CCK-A receptor immunoreactivity. This study demonstrates that the mucosal folds of the terminal bile and pancreatic ducts contain muscle and nerve fibres, suggesting an active rather than purely passive function.

Structural Features of the Basal Lamina in Reissner's Membrane of the Guinea Pig

The structural features of the basal lamina in Reissner's membrane were examined electron microscopically using tannic acid fixation, poly-cationic staining (ruthenium red), and mono-cationic staining (safranin O). Tannic acid fixation revealed 5-10-nm filaments which ran perpendicularly between the lamina densa and the cytoplasmic surface of squamous epithelial cells. Ruthenium red staining showed a lattice-like arrangement of anionic sites on both sides of the lamina densa. Safranin O staining revealed filamentous networks at the same sites. These structural features of the basal lamina in the Reissner's membrane were very similar to those of the glomerular and the alveolar basement membrane. Therefore, the possibility that anionic sites of Reissner's membrane may play the role of a size and charge-selective barrier between the scala media and the scala vestibuli, in much the same way that the glomerular basement membrane plays the same role of barrier, must be taken into account.

Cold chemistry with electronically excited Ca+ Coulomb crystals

Rate constants for chemical reactions of laser-cooled Ca(+) ions and neutral polar molecules (CH(3)F, CH(2)F(2), or CH(3)Cl) have been measured at low collision energies (<E(coll)>/k(B)=5-243 K). Low kinetic energy ensembles of (40)Ca(+) ions are prepared through Doppler laser cooling to form "Coulomb crystals" in which the ions form a latticelike arrangement in the trapping potential. The trapped ions react with translationally cold beams of polar molecules produced by a quadrupole guide velocity selector or with room-temperature gas admitted into the vacuum chamber. Imaging of the Ca(+) ion fluorescence allows the progress of the reaction to be monitored. Product ions are sympathetically cooled into the crystal structure and are unambiguously identified through resonance-excitation mass spectrometry using just two trapped ions. Variations of the laser-cooling parameters are shown to result in different steady-state populations of the electronic states of (40)Ca(+) involved in the laser-cooling cycle, and these are modeled by solving the optical Bloch equations for the eight-level system. Systematic variation of the steady-state populations over a series of reaction experiments allows the extraction of bimolecular rate constants for reactions of the ground state ((2)S(1/2)) and the combined excited states ((2)D(3/2) and (2)P(1/2)) of (40)Ca(+). These results are analyzed in the context of capture theories and ab initio electronic structure calculations of the reaction profiles. In each case, suppression of the ground state rate constant is explained by the presence of a submerged or real barrier on the ground state potential surface. Rate constants for the excited states are generally found to be in line with capture theories.

Substrate Recognition by AAA+ ATPases: Distinct Substrate Binding Modes in ATP-Dependent Protease Yme1 of the Mitochondrial Intermembrane Space

The energy-dependent proteolysis of cellular proteins is mediated by conserved proteolytic AAA(+) complexes. Two such machines, the m- and i-AAA proteases, are present in the mitochondrial inner membrane. They exert chaperone-like properties and specifically degrade nonnative membrane proteins. However, molecular mechanisms of substrate engagement by AAA proteases remained elusive. Here, we define initial steps of substrate recognition and identify two distinct substrate binding sites in the i-AAA protease subunit Yme1. Misfolded polypeptides are recognized by conserved helices in proteolytic and AAA domains. Structural modeling reveals a lattice-like arrangement of these helices at the surface of hexameric AAA protease ring complexes. While helices within the AAA domain apparently play a general role for substrate binding, the requirement for binding to surface-exposed helices within the proteolytic domain is determined by the folding and membrane association of substrates. Moreover, an assembly factor of cytochrome c oxidase, Cox20, serves as a substrate-specific cofactor during proteolysis and modulates the initial interaction of nonassembled Cox2 with the protease. Our findings therefore reveal the existence of alternative substrate recognition pathways within AAA proteases and shed new light on molecular mechanisms ensuring the specificity of proteolysis by energy-dependent proteases.

Deep fascia on the dorsum of the ankle and foot: Extensor retinacula revisited

This study revisits the anatomy of the deep fascia over the distal leg, ankle, and dorsum of the foot. The arrangement of the deep fascia in these regions was recorded in 14 lower limbs of adult cadavers using photographs and drawings. The fascial layer from all three sites was subsequently removed in toto, and serial thickness measurements were made along its entire length. In addition, fiber disposition was studied under polarized light, and sections were stained to demonstrate collagen. The arrangement of deep fascia is complex. A common and novel finding at all levels is a crisscross, lattice-like arrangement of fibers. There was little evidence of the clearly defined sturdy band of the superior extensor retinaculum (SER) or of the Y-shaped inferior retinaculum (IER) commonly illustrated in topographical anatomy texts. The SER is a complex area with several thickenings commencing about 3 cm proximal to the tip of the lateral malleolus and gradually increasing to reach a maximum of 270 microm about 5 cm above the malleolus, then gradually returning to original thickness, about 9 cm above the malleolus. Fibers crossing diagonally to each other are a feature of the region. The IER characteristically has two forms: either a cross-shaped band (9 specimens) or a thickened "node" with small extensions radiating toward the malleoli (5 specimens), located about 1-2 cm distal to the lateral malleolus and centred over the common tendon of extensor digitorum where it has maximum thickness (430 microm). The deep fascia is thickened and firmly attached over both malleoli and to the tarsals and metatarsals along both borders of the foot. In general, the deep fascial structures were thicker in males than those in females.

Organization of the musculature of schistosome cercariae.

Phalloidin-fluorescein isothiocyanate staining of filamentous actin was used to identify muscle systems within the cercariae of Schistosoma mansoni. Examination of labeled cercariae by confocal scanning laser microscopy revealed distinct organizational levels of myofiber arrangements within the body wall, anterior cone, acetabulum, and esophagus. The body wall throughout showed a typical latticelike arrangement of outer circular and inner longitudinal myofibers, with an additional innermost layer of diagonal fibers in the anterior portion of the body. Circular and longitudinal fibers were also evident in the anterior organ and esophagus and, to some extent, the ventral acetabulum. Most striking was the striation of the cercarial tail musculature.

Cytochemical studies of the neuromuscular systems of the diporpa and juvenile stages of Eudiplozoon nipponicum (Monogenea: diplozoidae).

Using indirect immuno- and enzyme-cytochemical techniques, interfaced with confocal scanning laser microscopy and standard optical microscopy, neuronal pathways have been demonstrated in whole-mount preparations of the unpaired diporpae and freshly paired juvenile stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae). All 3 main classes of neuronal mediators, cholinergic, aminergic and peptidergic, were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. Neural mapping revealed considerable overlap and similarity in staining of the nervous systems of the diporpa and adult worm. The main differences in the diporpa relate to the innervation of the temporary ventral sucker and dorsal papilla, structures which are unique to the larva and which enable fusion between worms but then disappear. Branches from the longitudinal nerve cords innervate these structures and appear to be involved in the process of somatic fusion, probably giving rise to the inter-specimen connections that later link the 2 central nervous systems in paired adult parasites. In the hindbody, there is extensive haptoral innervation associated with the developing clamps and small central hooks. Reactive neuronal components were found associated with the early stages of clamp development prior to connections being made with the extrinsic adductor muscle bundles. The muscle systems of the diporpa and juvenile stages comprise a lattice-like arrangement of circular, longitudinal and diagonal fibres that make up the body wall, together with buccal suckers, haptoral clamps and associated adductor muscles, and the transient ventral sucker. All have obvious importance to diporpae when they migrate over the gill and undertake body contact, torsion and fusion during the process of pairing. Behaviour during the pairing of diporpae is described.