Effects of Shape and Orientation of Pore Canals on Mechanical Behaviors of Lobster Cuticles.

The work is to investigate the relationships between the microstructures and mechanical behaviors of lobster cuticles and reveal the inner mechanisms of the anisotropic mechanical properties of the cuticles and give the helpful guidance for the design of high-performance man-made composites. First, the tensile mechanical properties of the longitudinal and transverse specimens of the cuticles of American lobsters were tested with a mechanical-testing instrument. It is was found that the fracture strength and elastic modulus of the longitudinal specimens are distinctly larger than those of the transverse specimens. Then, the microstructural characteristics of the fracture surfaces of the specimens were observed with scanning electron microscope. It was observed that the pore canals in the cuticles are elliptic and their orientations are along the longitudinal orientation of the cuticles. Furthermore, the stresses and micro-damage of the longitudinal and transverse specimens were calculated with the rule of progressive damage by finite element method. It was revealed that the shape and orientation of the pore canals in the cuticles give rise to the anisotropic mechanical property of the cuticles and ensure that the cuticles possess the largest fracture strength and elastic modulus along their largest main-stress orientation.

Retrosynthesis of CaCO3 via amorphous precursor particles using gastroliths of the Red Claw lobster (Cherax quadricarinatus).

Gastroliths are highly calcified structures formed in the cardiac stomach wall of crustaceans for the temporary storage of amorphous CaCO3 (ACC). The gastrolithic ACC is stabilized by the presence of biomolecules, and represents a novel model for research into biomineralization. For the first time, an in vitro biomimetic retrosynthesis of scaffolds of gastrolithic matrices with CaCO3 is presented. With the help of synthetic polyacrylic (PAA) and phytic (PA) acids, amorphous precursor particles were stabilized in double (DD) and gas (GD) diffusion crystallization assays. The presence of these synthetic molecules as efficient inhibitors of nucleation and growth of CaCO3, and the use of biological gastrolith scaffolds as confined reaction environments determined the kinetics of crystallization, and controlled the morphogenesis of CaCO3. The formation of ACC particles was demonstrated and their crystallization was followed by light microscopy, scanning and transmission electron microscopy, and electron diffraction.

The La Voulte-sur-Rhône Konservat-Lagerstätte reveals the male and female internal anatomy of the Middle Jurassic clawed lobster Eryma ventrosum.

The biology of extinct animals is usually reconstructed from external morphological characters and comparison with present-day analogues. Internal soft organs are very rarely preserved in fossils and require high-tech approaches for visualization. Here, we report the internal anatomy of a female and male of the ~ 162 Myr-old lobster Eryma ventrosum from the Jurassic La Voulte-sur-Rhône Konservat-Lagerstätte in France using X-ray synchrotron tomography. The Erymidae is an extinct, species-rich, widespread and ecologically important Mesozoic family of decapod crustaceans. Our investigation revealed the anatomy of the locomotory, respiratory, circulatory, excretory, digestive, nervous and sensory, and reproductive systems at a resolution resembling low-magnification histology. Particularly notable is the detailed preservation of the small brain and the fragile hepatopancreas, the main metabolic organ of decapods that decays rapidly post-mortem. The remarkable preservation shows that the internal anatomy of Eryma ventrosum is closer to that of Nephropidae (clawed lobsters) than Astacidae (freshwater crayfish), their closest living relatives based on skeletal morphology. The microanatomy of the gonads and hepatopancreas indicates that the two specimens investigated were a young, well-nourished female and male prior to sexual maturity. The analysis of the soft anatomy reveals remarkable conservatism over 160 Myr and offers new insights into feeding, reproduction, life history and lifestyle of an important component of the macrozoobenthos of Middle Jurassic seas.

Structural and mechanical properties of the arthropod cuticle: comparison between the fang of the spider Cupiennius salei and the carapace of American lobster Homarus americanus.

Most biological materials are nanocomposites characterized by a multi-level structural hierarchy. Particularly, the arthropod cuticle is a chitin-based composite material where the mechanical properties strongly depend on both molecular chitin/protein properties, and the structural arrangement of chitin-fibrils within the protein matrix. Here materials properties and structural organization of two types of cuticle from distantly related arthropods, the wandering spider Cupiennius salei and American lobster Homarus americanus were studied using nanoindentation and X-ray diffraction. The structural analysis of the two types of cuticle including the packing and alignment of chitin-fibrils is supported by Monte Carlo simulations of the experimental X-ray data, thereby regions of parallel and rotated fibril arrangement can be clearly distinguished. The tip of the spider fang which is used to inject venom into the prey was found to be considerably harder than the lobster carapace, while its stiffness is slightly lower.

Modulation of network pacemaker neurons by oxygen at the anaerobic threshold.

Previous in vitro and in vivo studies showed that the frequency of rhythmic pyloric network activity in the lobster is modulated directly by oxygen partial pressure (PO(2)). We have extended these results by (1) increasing the period of exposure to low PO(2) and by (2) testing the sensitivity of the pyloric network to changes in PO(2) that are within the narrow range normally experienced by the lobster (1 to 6 kPa). We found that the pyloric network rhythm was indeed altered by changes in PO(2) within the range typically observed in vivo. Furthermore, a previous study showed that the lateral pyloric constrictor motor neuron (LP) contributes to the O(2) sensitivity of the pyloric network. Here, we expanded on this idea by testing the hypothesis that pyloric pacemaker neurons also contribute to pyloric O(2) sensitivity. A 2-h exposure to 1 kPa PO(2), which was twice the period used previously, decreased the frequency of an isolated group of pacemaker neurons, suggesting that changes in the rhythmogenic properties of these cells contribute to pyloric O(2) sensitivity during long-term near-anaerobic (anaerobic threshold, 0.7-1.2 kPa) conditions.

Morphology and pathology of the ectoparasitic copepod, Nicothoë astaci ('lobster louse') in the European lobster, Homarus gammarus.

Ectoparasitic copepods have been reported in a wide range of aquatic animals, including crustacean shellfish. However, with the exception of the salmon louse, Lepeophtheirus salmonis, our knowledge of such parasites in commercial species is rudimentary. The current study examines the morphology and pathology of the parasitic copepod, Nicothoë astaci (the 'lobster louse') in its host, the European lobster, Homarus gammarus. Lobsters were sampled from waters surrounding Lundy Island (Bristol Channel, UK) and all individuals collected were found to harbour female adult N. astaci in their gills, with a mean of 47·3 parasites/lobster. The majority of N. astaci were found in the basal region of pleurobranch gills. The parasite was found to attach to gill filaments via its oral sucker, maxillae and maxillipeds, and to feed on host haemolymph (blood) through a funnel-like feeding channel. It caused varying degrees of damage to the host gill, including occlusion of gill filaments and disruption to the vascular system in the central axis. Although there was evidence of extensive host response (haemocytic infiltration) to the parasite, it was displaced from the parasite attachment site and thus was observed in the central gill axis below. The region of gill filament immediately underlying the parasite feeding channel was devoid of such activity suggesting that the parasite interferes with the cellular defence and haemostatic mechanisms of the lobster in order to maintain invasion of the host.

Electron-microscopical localization of gelsolin in various crustacean muscles.

Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label. Under physiological conditions, a close association of gelsolin with the thin filaments was observed for both muscle types. The preferential localization of particles in the I- and AI-region indicated that gelsolin was distributed randomly over the whole length of the thin filaments. Preincubation of muscle strips with Ringer solution containing 0.5 mM EGTA resulted in a significantly different distribution pattern; gold particles were now localized preferentially in the cell periphery close to the sarcolemma, with significantly decreased abundance in the centre of the cell. Compared with the muscle under physiological conditions, the number of gold particles over sarcomeric structures was significantly reduced. Thus, binding of gelsolin to the thin filaments is apparently reversible in vivo and depends on the presence of calcium ions. We assume a functional role for gelsolin in the actin turnover processes in invertebrate muscle systems.

Characterization of ion transport in the isolated epipodite of the lobster Homarus americanus.

Unfolded epipodite isolated from American lobsters (Homarus americanus) acclimated to dilute seawater was mounted in an Ussing-type chamber for ion transport studies. The split epipodite is an electrically polarized, one-cell-layer epithelium supported with cuticle. Under open-circuit conditions, the transepithelial potential was -4.2+/-1.0 mV (N=38). In the short-circuited epithelium, the current averaged over all of the preparations was -185.4+/-20.2 A cm(-2) (N=38) with a high conductance of 55.2+/-11.4 mS cm(-2) (N=38), typical for a leaky epithelium. The Na:Cl absorptive flux ratio was 1:1.6; ion substitution experiments indicated that the transport of Na+ and Cl- is coupled. Basolateral application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) and niflumic acid (NFA) dose-dependently inhibited short-circuit current (ISC). Secretory K+ (Rb+) fluxes exceeded influxes and were inhibited by the Na+/K+-ATPase inhibitor ouabain and the K+ channel blocker cesium. Western blot analysis showed that Na+/K+-ATPase alpha-subunit protein was more highly expressed in the epipodite of lobsters acclimated to 20 p.p.t. compared with animals acclimated to seawater (34 p.p.t.). 3-Isobutyl-1-methyl-xanthine (IBMX) stimulated a negative ISC and enhanced apical secretory K+ flux. Basolateral application of NPPB inhibited JRbB-->A fluxes, suggesting the interaction of K+ channels with NPPB-sensitive Cl- channels. The results are summarized in a transport model, suggesting apical Na+/K+/2Cl- co-transport, a dominant apical K+-secreting channel and basolaterally located Cl- and K+ channels. This study represents the first comprehensive characterization of ion transport processes across the lobster epipodite epithelium and indeed in any tissue within the branchial cavity of the American lobster.

A new polychelidan lobster preserved with its eggs in a 165 Ma nodule.

Crustacean eggs are rare in the fossil record. Here we report the exquisite preservation of a fossil polychelidan embedded within an unbroken nodule from the Middle Jurassic La Voulte-sur-Rhône Lagerstätte (France) and found with hundreds of eggs attached to the pleon. This specimen belongs to a new species, Palaeopolycheles nantosueltae sp. nov. and offers unique clues to discuss the evolution of brooding behaviour in polychelidan lobsters. In contrast to their development, which now relies on a long-lived planktic larval stage that probably did not exist in the early evolutionary steps of the group, the brood size of polychelidan lobsters seems to have remained unchanged and comparatively small since the Jurassic. This finding is at odds with reproductive strategies in other lobster groups, in which a long-lived planktic larval stage is associated with a large brood size.

Experimental transformation of muscle fiber properties in lobster.

Like the chelipeds, the claw closer muscles of the adult lobster are asymmetric (dipmorphic). In the crusher claw the closer muscle is composed entirely of slow fibers, and in the cutter claw it has 65 to 75 percent fast fibers and 25 to 35 percent slow fibers. While claw placement in the adult is essentially random, it can be demonstrated in two ways that the muscle fiber properties are not genetically fixed: (i) if one claw is removed in the fourth and early fifth stages, the remaining closer muscle develops all slow muscle fibers, and (ii) if the animals are raised in smooth-bottomed containers, both claws can become cutter types, having closer muscles with more than 50 percent fast fibers. Thus, as in vertebrate skeletal muscle, the properties of lobster closer muscle fibers can be transformed by various experimental manipulations.

Kinematics, hydrodynamics and force production of pleopods suggest jet-assisted walking in the American lobster (Homarus americanus).

The American lobster (Homarus americanus) displays a diverse set of locomotory behaviours that includes tail flips, walking and paddling. Paddling is carried out by the four pairs of paddle-shaped pleopods on the ventral abdomen. Although it is recognized that pleopod-generated fluid flows have some locomotory role in adults, reports on their relative importance in locomotion are inconsistent. This paper integrates experimental kinematics and hydrodynamics of lobster pleopod beating to determine the mechanism and magnitude of pleopod force production. A kinematic analysis of pleopod beating in live lobsters showed that the pleopods execute an adlocomotory metachronal beating pattern. We modelled in vivo pleopod kinematics with a set of simple trigonometric functions, and used these functions to program a mechanical lobster model consisting of motor-driven pleopods on a lobster abdomen exoskeleton. Based on flow visualizations obtained from applying particle image velocimetry to the lobster model, we propose that the unsteady metachronal kinematics of the pleopods can maximize thrust by exploiting forces arising from individual pleopod activity and interactions among adjacent pairs. The pleopods continuously entrain fluid surrounding the lobster and create a caudally directed fluid jet oriented parallel to the substratum. Inputting wake morphology and velocity data into a simplified model for steady jet thrust showed that the pleopods of the lobster model produced 27-54 mN of thrust, which is comparable to the propulsive forces generated by other proficient swimmers. These results suggest that lobster pleopods are capable of producing forces of a magnitude that could assist the walking legs in forward propulsion.

Non-amidated and amidated members of the C-type allatostatin (AST-C) family are differentially distributed in the stomatogastric nervous system of the American lobster, Homarus americanus.

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~ 14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~ 42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.

Functional anatomy of the female reproductive system of the American lobster (Homarus americanus).

Light microscopy studies of the female American lobster Homarus americanus reproductive system are essentially nonexistent or outdated. Based on samples taken in the spring, summer, and autumn from the southern Gulf of St. Lawrence between 1994 and 2014, and using a combination of histological and scanning electron microscope techniques, we propose an ovarian cycle with 10 stages, identifying for the first time a recovery stage. Also, an atypical resorption stage, characterized by massive reabsorption of mature oocytes, is occasionally observed during summer months. The oviducts are composed of connective tissue (elastic and collagen fibers) with no muscle or secretory activities. Their epithelium shows a cyclic pattern and phagocytosis activities linked to spawning. Although the role of the seminal receptacle is to store and protect semen, free spermatozoa (i.e., without the spermatophoric wall and the acellular gelatinous substance that constitute the semen) were also observed in its posteriolateral grooves immediately prior to spawning, which is consistent with an external fertilization mechanism at the seminal receptacle. Unexpectedly, free spermatozoa were observed externally near two pore-like structures located on the gonopore's operculum, not at the seminal receptacle, after spawning; hence, more work is needed to fully understand the fertilization mechanism for the American lobster.

Functional anatomy of the male reproductive system of the American lobster (Homarus americanus).

Despite supporting a valuable fishery, the reproductive system of the male American lobster (Homarus americanus) is poorly understood. The elongated H-shaped testis is responsible for spermatogenesis and is composed of follicles, a common collecting duct with interlaced scattered striated muscles, and a serosa as an external wall. Sertoli cells are associated with the spermatogenesis that produces spermatozoa, which are transferred to the collecting duct through a temporary passageway. Spermatogenesis is asynchronous between follicles and occurs on a continuous basis. The anterior and posterior lobes of the testes are independent and connect to the vasa deferentia through the Y-shaped collecting tubules that have a different cell anatomy and function than the two organs they connect. The vas deferens is divided into four regions. Spermatophores, produced in the proximal vas deferens, are packets of spermatozoa encapsulated in a single layer-the spermatophoric wall, which is composed of mucopolysaccharide acid. Large dense ovoid granules and the seminal fluid, composed of acidic sulfated mucosubstances, are secreted in the median vas deferens. Spermatophores within these secreted substances (i.e., semen) are stored in the distal vas deferens that, with the spermiduct (last region of the vas deferens), is responsible for the extrusion of the semen by striated muscle contractions. Smooth muscles suggest a peristaltic movement of the spermatophores within the vas deferens. Finally, the gonopores and the first pair of pleopods (i.e., gonopod) move the semen to the female seminal receptacle during copulation.

Preferred crystallographic texture of alpha-chitin as a microscopic and macroscopic design principle of the exoskeleton of the lobster Homarus americanus.

The crystallographic texture of the crystalline alpha-chitin matrix in the biological composite material forming the exoskeleton of the lobster Homarus americanus has been determined using synchrotron X-ray pole figure measurements and the calculation of orientation distribution functions. The study has two objectives. The first one is to elucidate crystallographic building principles via the preferred synthesis of certain orientations in crystalline organic tissue. The second one is to study whether a general global design principle exists for the exoskeleton which uses preferred textures relative to the local coordinate system throughout the lobster cuticle. The first point, hence, pursues the question of the extent to which and why alpha-chitin reveals preferred textures in the lobster cuticle. The second point addresses the question of why and whether such preferred textures (and the resulting anisotropy) exist everywhere in the exoskeleton. Concerning the first aspect, a strong preference of a fiber texture of the orthorhombic alpha-chitin is observed which is characterized by a 020 crystal axis normal to the exoskeleton surface for the chitin matrix. The second question is tackled by studying samples from different parts of the carapace. While the first aspect takes a microscopic perspective at the basic structure of the biological composite, the second point aims at building a bridge between an understanding of the microstructure and the macroscopic nature of a larger biological construction. We observe that the texture is everywhere in the carapace optimized in such a way that the same crystallographic axis of the chitin matrix is parallel to the normal to the local tangent plane of the carapace. Notable differences in the texture are observed between hard mineralized parts on the one hand and soft membranous parts on the other. The study shows that the complex hierarchical microstructure of the arthropod cuticle can be well described by surprisingly simple crystallographic textures.

The exoskeleton of the lobster Homarus americanus as an example of a smart anisotropic biological material.

Many biological materials are composed of fibrils arranged according to well-ordered three-dimensional patterns. These materials often show a strong anisotropy in their properties. An essential characteristic of biological structures is their hierarchical organization from the nanometer to the millimeter scale. Lobster cuticle is a good example of this and a suitable model for studying these properties. In this study the structure of untreated as well as chemically and physically treated cuticle from the exoskeleton of the American lobster (Homarus americanus) was investigated using scanning electron microscopy. Fresh samples have been chemically decalcified and deproteinated and thermally treated to evaluate their resistance to degradation. Results showed that their structure is more complex than the commonly assumed model for arthropod cuticles. Stacked chitin-protein planes create the characteristic twisted plywood pattern found in arthropod cuticles. However, due to a well-developed pore canal system these planes are not simple arrays of parallel chitin-protein fibers. In lobster cuticle, interconnected fibers bend around the continuous lenticellate cavities of the pore canals to form a planar honeycomb-like structure. The chemically and thermally treated samples showed that the organic matrix retains its shape and structure despite the attack of chemical compounds or heat. It was also possible to study the distribution of the biominerals after the removal of the organic matrix. The observed residual structure gives a good impression of how the minerals (mainly calcite) are distributed inside the polymeric network.

Identification, physiological actions, and distribution of VYRKPPFNGSIFamide (Val1)-SIFamide) in the stomatogastric nervous system of the American lobster Homarus americanus.

In this study, the peptide VYRKPPFNGSIFamide (Val(1)-SIFamide) was identified in the stomatogastric nervous system (STNS) of the American lobster, Homarus americanus, using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). When bath-applied to the stomatogastric ganglion (STG), synthetic Val(1)-SIFamide activated the pyloric motor pattern, increasing both burst amplitude and duration in the pyloric dilator (PD) neurons. To determine the distribution of this novel SIFamide isoform within the lobster STNS and neuroendocrine organs, a rabbit polyclonal antibody was generated against synthetic Val(1)-SIFamide. Whole-mount immunolabeling with this antibody showed that this peptide is widely distributed within the STNS, including extensive neuropil staining in the STG and commissural ganglia (CoGs) as well as immunopositive somata in the CoGs and the oesophageal ganglion. Labeling was also occasionally seen in the pericardial organ (PO), but not in the sinus gland. When present in the PO, labeling was restricted to fibers-of-passage and was never seen in release terminals. Adsorption of the antibody by either Val(1)-SIFamide or Gly(1)-SIFamide abolished all Val(1)-SIFamide staining within the STNS, including the STG neuropil, whereas adsorption by other lobster neuropeptides had no effect on immunolabeling. These data strongly suggest that the staining we report is a true reflection of the distribution of this peptide in the STNS. Collectively, our mass spectrometric, physiological, and anatomical data are consistent with Val(1)-SIFamide serving as a locally released neuromodulator in the lobster STG. Thus, our study provides the first direct demonstration of function for an SIFamide isoform in any species.

Distribution of two species of Nephropsis Wood-Mason, 1872 (Crustacea, Decapoda, Nephropidae) from northeastern Brazil.

The genus Nephropsis Wood-Mason, 1872 has been reported from Brazil by Tavares (1998), Tavares & Young (2002), Silva et al. (2003), Dall´Occo et al. (2007) and Serejo et al. (2007), recording Nephropsis aculeata Smith, 1881, N. rosea Bate, 1888 and N. agassizii A. Milne-Edwards, 1880, the last of which occurs in both northeastern and southeastern of Brazil.