Comparative physicochemical analysis of chitin quality of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus.

Recently, chitin biopolymer has received much attention as a wide variety of biomedical application for this and its derivatives have been reported, in fact, the study of non-conventional species as alternative sources of them compounds has taken particular interest. Here, we present a comparative physicochemical survey of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus: the prosoma and the opisthosoma, collected in Yucatán, Mexico. The characterization included CHNSO analysis, FTIR, TGA, DSC, XRD, and SEM. The CHNSO analysis revealed that C is present in the highest proportion (∼45 %) and that chemical composition did not show significant differences (P < 0.05) between the two tagmata. FTIR spectra of two tagmata presented a wide characteristic band of the chitin between 3600 and 3000 cm-1, confirming the presence of this biopolymer in the exoskeleton studied. TGA and DTGA profiles resulted very similar for both tagmata being the residual mass at 650 °C of around 30 % for both samples; these values were associated to the presence of minerals. SEM micrographs showed a porous matrix with infinite large number of irregularly shaped particles. Results show that both tagmata are made up of chitin, and they seem to have a high mineral content.

Evaluation of remodeling and geometry on the biomechanical properties of nacreous bivalve shells.

Mollusks have developed a broad diversity of shelled structures to protect against challenges imposed by biological interactions(e.g., predation) and constraints (e.g., [Formula: see text]-induced ocean acidification and wave-forces). Although the study of shell biomechanical properties with nacreous microstructure has provided understanding about the role of shell integrity and functionality on mollusk performance and survival, there are no studies, to our knowledge, that delve into the variability of these properties during the mollusk ontogeny, between both shells of bivalves or across the shell length. In this study, using as a model the intertidal mussel Perumytilus purpuratus to obtain, for the first time, the mechanical properties of its shells with nacreous microstructure; we perform uniaxial compression tests oriented in three orthogonal axes corresponding to the orthotropic directions of the shell material behavior (thickness, longitudinal, and transversal). Thus, we evaluated whether the shell material's stress and strain strength and elastic modulus showed differences in mechanical behavior in mussels of different sizes, between valves, and across the shell length. Our results showed that the biomechanical properties of the material building the P. purpuratus shells are symmetrical in both valves and homogeneous across the shell length. However, uniaxial compression tests performed across the shell thickness showed that biomechanical performance depends on the shell size (aging); and that mechanical properties such as the elastic modulus, maximum stress, and strain become degraded during ontogeny. SEM observations evidenced that compression induced a tortuous fracture with a delamination effect on the aragonite mineralogical structure of the shell. Findings suggest that P. purpuratus may become vulnerable to durophagous predators and wave forces in older stages, with implications in mussel beds ecology and biodiversity of intertidal habitats.

Angular distribution of luminescence dissymmetry observed from a random laser built upon the exocuticle of the scarab beetle Chrysina gloriosa.

We investigate the angular distribution of luminescence dissymmetry of random lasing in the mixture of rhodamine 6G and titanium dioxide nanoparticles upon a biocompatible natural material substrate, i.e., the elytron of the scarab beetle Chrysina gloriosa. We look into both green and gold-colored areas of the elytron that exhibit distinctly different circular dichroism properties. The fabricated sample asymmetrically emits both left- and right-handed circularly polarized light at 570 nm when pumped at 532 nm, depending on the direction of emission and the angle of the pump incidence. We characterize the light via measuring the angular distribution of its luminescence dissymmetry factor (g lum), which reaches an unusually high maximal value of 0.90 or -0.50 at some specific angle depending on the handedness of its polarization. This random laser source can be used in numerous potential optoelectronic applications which require light emission of distributed luminescence dissymmetry or of high luminescence dissymmetry.

Mechanical adaptation of brachiopod shells via hydration-induced structural changes.

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration.

Evolution and biomineralization of pteropod shells.

Shelled pteropods, known as sea butterflies, are a group of small gastropods that spend their entire lives swimming and drifting in the open ocean. They build thin shells of aragonite, a metastable polymorph of calcium carbonate. Pteropod shells have been shown to experience dissolution and reduced thickness with a decrease in pH and therefore represent valuable bioindicators to monitor the impacts of ocean acidification. Over the past decades, several studies have highlighted the striking diversity of shell microstructures in pteropods, with exceptional mechanical properties, but their evolution and future in acidified waters remains uncertain. Here, we revisit the body-of-work on pteropod biomineralization, focusing on shell microstructures and their evolution. The evolutionary history of pteropods was recently resolved, and thus it is timely to examine their shell microstructures in such context. We analyse new images of shells from fossils and recent species providing a comprehensive overview of their structural diversity. Pteropod shells are made of the crossed lamellar and prismatic microstructures common in molluscs, but also of curved nanofibers which are proposed to form a helical three-dimensional structure. Our analyses suggest that the curved fibres emerged before the split between coiled and uncoiled pteropods and that they form incomplete to multiple helical turns. The curved fibres are seen as an important trait in the adaptation to a planktonic lifestyle, giving maximum strength and flexibility to the pteropod thin and lightweight shells. Finally, we also elucidate on the candidate biomineralization genes underpinning the shell diversity in these important indicators of ocean health.

Heterogeneous distribution of shell matrix proteins in the pearl oyster prismatic layer.

Shell formation in molluscan bivalves is regulated by organic matrices composed of biological macromolecules, but how these macromolecules assemble in vitro remains elusive. Prismatic layer in the pearl oyster Pinctada fucata consists of polygonal prisms enveloped by thick organic matrices. In this study, we found that the organic matrices were heterogeneously distributed, with highly acidic fractions (EDTA-soluble and EDTA-insoluble) embedded inside the prism columns, while basic EDTA-insoluble faction as inter-column framework enveloping the prisms. The intra-column matrix was enriched in aspartic acid whereas the inter-column matrix was enriched in glycine, tyrosine and phenylalanine. Moreover, the intra-column matrix contained sulfo group further contributing to its acidic property. Proteomics data showed that the intra-column proteins mainly consisted of acidic proteins, while some typical matrix proteins were absent. The absent matrix proteins such as shematrin family and KRMP family were highly basic and contained aromatic amino acids, suggesting that electric charge and hydrophobic effect might play a role in the matrix heterogeneity. Interestingly, chitin metabolism related proteins were abundant in the inter-column matrix, which may be involved in reconstructing the prism organic matrix. Overall, our study suggests that each single prism grew in an enclosed organic envelope and the organic matrix undergoes rearrangement, thus leading to the peculiar growth of the prismatic layer.

On the Planar Geometric Patterns of the Exoskeletal Relief Formation in Early Vertebrates (Osteostraci, Agnatha).

A study of the diversity of sculpture and histological structure of the exoskeleton in various osteostracan taxa (Osteostraci, Agnatha) enabled the first characterization of the main elements (geometric modules) of the planar organization of the complex relief on their armor surface. The armor relief was analyzed using a circular model of the formation of exoskeletal hard structures. The model was applied to unique material, fragments of a shield of the osteostracan Oeselaspis pustulata (Patten, 1931) from the Silurian of the Severnaya Zemlya Archipelago (Russia).

Calcite crystal orientation patterns in the bilayers of laminated shells of benthic rotaliid foraminifera.

Shells of calcifying foraminifera play a major role in marine biogeochemical cycles; fossil shells form important archives for paleoenvironment reconstruction. Despite their importance in many Earth science disciplines, there is still little consensus on foraminiferal shell mineralization. Geochemical, biochemical, and physiological studies showed that foraminiferal shell formation might take place through various and diverse mineralization mechanisms. In this study, we contribute to benthic foraminiferal shell calcification through deciphering crystallite organization within the shells. We base our conclusions on results gained from electron backscattered diffraction (EBSD) measurements and describe microstructure/texture characteristics within the laminated shell walls of the benthic, symbiontic foraminifera: Ammonia tepida, Amphistegina lobifera, Amphistegina lessonii. We highlight crystallite assembly patterns obtained on differently oriented cuts and discuss crystallite sizes, morphologies, interlinkages, orientations, and co-orientation strengths. We show that: (i) crystals within benthic foraminiferal shells are mesocrystals, (ii) have dendritic-fractal morphologies and (iii) interdigitate strongly. Based on crystal size, we (iv) differentiate between the two layers that comprise the shells and demonstrate that (v) crystals in the septa have different assemblies relative to those in the shell walls. We highlight that (vi) at junctions of different shell elements the axis of crystal orientation jumps abruptly such that their assembly in EBSD maps has a bimodal distribution. We prove (vii) extensive twin-formation within foraminiferal calcite; we demonstrate (viii) the presence of two twin modes: 60°/[001] and 77°/~[6 -6 1] and visualize their distributions within the shells. In a broader perspective, we draw conclusions on processes that lead to the observed microstructure/texture patterns.

Development of a novel Artemia eggshell-zirconium nanocomposite for efficient fluoride removal.

Fluoride pollution in water has attracted widespread concern worldwide. In this study, an Artemia eggshell-zirconium (Aes-Z) nanocomposite has been used for fluoride removal. Material characterization results showed that nano-ZrO2 was immobilized on the inner surface of the Artemia eggshell, and there was no pore blockage on the composite material. Various parameters influencing on the fluoride removal, including treatment time, composite dosage, pH, initial fluoride concentration, and other anions, were analyzed. The removal efficiency of the composite material was better than that of the single zirconia material. The removal percentage of fluoride reached 93% in 30 min with an initial fluoride concentration of 10 mg/L and a nanocomposite dosage of 8.0 g/L. The composite material had a high removal efficiency for fluoride in the pH region 4.0-10.0. The adsorption of fluoride was not influenced by the common anions (e.g., Cl-, SO42-, and NO3-) in water. The regeneration revealed that the Aes-Z composite material could be reused and remove fluoride effectively in four cycles. The pseudo-second-order rate model adequately represented the adsorption kinetics of the Aes-Z composite material. A possible, defluoridation mechanism of the Aes-Z composite material was also proposed. This study demonstrates that Aes-Z is a promising adsorbent material for fluoride removal.

3D morphology of nematode encapsulation in snail shells, revealed by micro-CT imaging.

Many parasites and hosts are embroiled in an on-going arms race that affects the evolution of each participant. One such battle is between parasitic nematodes and terrestrial gastropods which have co-evolved for 90-130 MY. Recently, snails have been shown to encase and kill invading nematodes using their shell as a defence mechanism. However, there is remarkably little known about this process in terms of understanding where, when and how nematodes are fixed within the shell. Also there has never been any attempt to observe this process using methods other than light microscopy. Therefore, we used micro CT scanning of a Cepaea nemoralis shell (a common host for nematodes) to 3D visualise encased nematode parasites and quantify morphological parameters. By taking this approach future studies could use micro CT scanning of fossil shells in conchology collections to understand nematode/snail co-evolution.

Formulation of an exoskeleton degrading bacterial consortium from seafood processing effluent for the biocontrol of crustacean parasite Alitropus typus.

Parasitic infestations on cultured fish due to the crustacean isopod Alitropus typus has been on the rise in recent years, causing large scale mortality, leading to significant economic loss to the farmer. Crustaceans are encased by an exoskeleton composed of chitin, protein and lipid microfibril frameworks, in which calcium carbonate is deposited. A strategy focused on the degradation of the exoskeletal framework utilizing nonpathogenic microorganisms that produce a wide variety of hydrolytic enzymes may be an environment-friendly and safe alternative to control these pests. The present study was aimed to formulate a microbial consortium having chitinase, protease, lipase and urease producing bacteria from seafood processing effluents that can potentially degrade the exoskeleton of A. typus. Based on the qualitative and quantitative assessment of the extracellular enzymes produced by the isolates, a novel consortium was prepared with three strains that were not antagonistic to each other and were nonpathogenic. The chitinase producing - Stenotrophomonas maltophilia and Bacillus altitudinis that produced protease and lipase as well; and non-chitinase producing Klebsiella pneumoniae were taken in the ratio of 1:1:2 respectively (109 CFU/mL). The result showed 100 % mortality of the isopods within five days when applied at a concentration of 2% (v/v) of 107 CFU/mL without any adverse effect on the fish host Oreochromis niloticus. Analysis of the ultrastructural alterations of the parasites by Environmental Scanning Electron microscopy (ESEM) showed noticeable exoskeletal damages. The microbial members of the consortium displayed remarkable chemotactic properties towards A. typus. The results suggest that the microbial consortium acts as a potential parasiticide that can be used for the control of A. typus infestation in aquaculture ponds., thus benefitting the aquaculture industry especially the small-scale farmers.

Elemental Enrichment of the Exoskeleton in Three Species of Tick (Arachnida: Ixodidae).

Three species of adult hard tick (Ixodidae) were examined with scanning electron microscopy-energy dispersive X-ray spectroscopy to obtain elemental profiles of their exoskeletons and determine the presence of trace elements. The scutum, tarsal claws, chelicerae, and hypostome were examined on females and males of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. The only trace elements present included chlorine, calcium, and sodium. Chlorine was the most abundant trace element and occurred in all examined regions. The chelicerae generally possessed the highest weight percentages of Cl (up to 11.32 ± 1.36%) across all 3 species, although high weight percentages of Cl (up to 8.78 ± 2.77%) were also present in the hypostome teeth of most specimens. All 3 trace elements were present in the hypostome of A. americanum and I. scapularis, but Ca and Na appear to be absent from the teeth of D. variabilis. In general, there were few differences in the elemental profiles of the exoskeletons between the sexes of any species. This study confirms the presence of alkali metals (Na) and alkaline earth metals (Ca) in adult ticks, which are also common in other arachnids; however, the absence of transition metals such as zinc from the exoskeletons of ticks is uncommon and only shared with species of Ricinulei and Opiliones.

Ovarian development in swimming crabs: Comparative histochemistry and ultrastructure of Callinectes ornatus and Arenaeus cribrarius (Brachyura, Portunidae).

The ovarian development of Callinectes ornatus and Arenaeus cribrarius was described using histochemistry and ultrastructure. Both species shows the same ovarian stages, which are the juvenile (JUV), adult rudimentary (RUD), developing (DEV), intermediary (INT), mature (MAT), and spent (OV) stages. The JUV and RUD stages showed similar characteristics, and previtellogenesis is characterized by meiotic prophase chromosomes. In the primary vitellogenesis, the oocyte cytoplasm shows many small and large cytoplasmic glycoprotein vesicles. These vesicles correspond to the dilated cisternae of the rough endoplasmic reticulum (RER), which produces the immature (endogenous) yolk. Secondary vitellogenesis (exogenous phase) begins at the DEV stage with the fusion of pinocytic vesicles and vesicles with immature yolks to form mature yolk granules. At the INT stage, the formation of the chorion begins, and the mature yolks increase in size and number, while the RER diminishes. In the MAT stage, the oocytes are completely formed, and the cytoplasm is filled with mature yolk, lipid droplets, and glycogen. There are no significant variations between the gonadosomatic and hepatosomatic indices, which allows us to infer that the transfer of reserves from the hepatopancreas is nearly constant during ovarian development, since we observed primiparous and multiparous females in the same sampled population.

Thermal stable characteristics of acid- and pepsin-soluble collagens from the carapace tissue of Chinese soft-shelled turtle (Pelodiscus sinensis).

The carapace from the Chinese soft-shelled turtle (Pelodiscus sinensis) is used as a traditional Chinese medicine. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from turtle carapace were isolated and characterized to screen novel collagen material in this study. Yields of 1.0% and 2.8% were obtained for ASC and PSC which contained glycine as the major amino acid and had high imino acid content. Both collagens had maximum ultraviolet absorption peaks of 220 nm. SDS-PAGE revealed that the structure of both collagens was similar, belonging to type I collagen. Relative viscosities of collagens were decreased as the temperature increased. Collagens showed minimum solubility at pH 8 and maximum solubility at a salt concentration of 3%. The denaturation temperature (Td) of PSC was higher whereas the melting temperature was lower than that of ASC. Both ASC and PSC appeared to be spongy like microstructure with fibrillar pores shown by scanning electron microscopy. The results suggest that collagens isolated from turtle carapace has high thermal stability with potential uses as new substitute for mammalian collagen in medicinal, food or biomaterial fields. However, their biological or pharmacological activities are needed to be further studied.

Structure and distribution of chalky deposits in the Pacific oyster using x-ray computed tomography (CT).

Oysters are unusual among bivalves in that they possess chambers, often filled with soft, chalky calcite, that are irregularly scattered throughout the shell. Because the function of these so-called chalky deposits is still unclear, evaluating the growth and distribution of chalk is important for elucidating the ecological function of this unique shell trait. Specimens of the Pacific oyster Magallana gigas, an oyster well known for chalk expression, were grown in Bodega Harbor, Bodega Bay, CA. At the end of an 11 month growing period, specimens were culled and selected animals were submitted for x-ray computed-tomography imaging. Three-dimensional reconstructions of oyster shells were used to assess the overall distribution of chalk, and also to better understand the relationship between chalk and other structures within the shell. Results indicate that chalky deposits underly sculptural features on the shell exterior, such as external ridges and changes in growth direction, and also that there is a relationship between chalk formation and oyster processes of cementation. Overall, chalk is useful for a cementing lifestyle because it enables morphological plasticity needed to conform to irregular substrates, but also acts as a cheap building material to facilitate rapid growth.

The origin of microscopic spheres on the exoskeleton of the woodlouse Porcellionides pruinosus (Crustacea: Isopoda) and their effect on its hydrophobicity.

The dorsal surface of the woodlouse Porcellionides pruinosus is covered with minute spheres, providing its characteristic powdered appearance. Little has been known about their composition and formation. A previously suggested function of these structures was to increase the hydrophobicity of the cuticular surface. We studied the ultrastructure, composition and formation of the spheres as well as tested whether they affect the hydrophobicity of the cuticle. We determined the composition of the spheres with histochemistry and scanning electron microscopy after applying various chemicals. We studied the process of their formation with transmission electron microscopy and assessed the hydrophobicity of the cuticle by measuring contact angles of water droplets with its surface. Our results show that the spheres are largely organic. They contain proteins and glycoproteins or possibly polysaccharides without detectable amounts of lipids. By studying the formation of the spheres we established that they are epicuticular structures. They are deposited early in the premolt stage of the molt cycle around branching extensions of epidermal cells. The sphere-covered cuticle of P. pruinosus is more hydrophobic than the cuticle with experimentally removed spheres as well as the scale-covered cuticle in a related species.

Structural and functional analyses of organic molecules regulating biomineralization.

Biomineralization by living organisms are common phenomena observed everywhere. Molluskan shells are representative biominerals that have fine microstructures with controlled morphology, polymorph, and orientation of CaCO3 crystals. A few organic molecules involved in the biominerals play important roles in the formation of such microstructures. Analyses of structure-function relationships for matrix proteins in biominerals revealed that almost all matrix proteins have an acidic region for the binding of calcium ion in CaCO3 crystals and interaction domains for other organic molecules. On the other hand, biomineralization of metal nanoparticles by microorganisms were also investigated. Gold nanoparticles and quantum dots containing cadmium were successfully synthesized by bacteria or a fungus. The analyses of components revealed that glycolipids, oligosaccharides, and lactic acids have key roles to synthesize the gold nanoparticle in Lactobacillus casei as reductants and dispersants. These researches about biomineralization will give new insights for material and environmental sciences in the human society.

X-ray nano-tomography of complete scales from the ultra-white beetles Lepidiota stigma and Cyphochilus.

High resolution X-ray nano-tomography experiments are often limited to a few tens of micrometer size volumes due to detector size. It is possible, through the use of multiple overlapping tomography scans, to produce a large area scan which can encompass a sample in its entirety. Mounting and positioning regions to be scanned is highly challenging and normally requires focused ion beam approaches. In this work we have imaged intact beetle scale cells mounted on the tip of a needle using a micromanipulator stage. Here we show X-ray holotomography data for single ultra-white scales from the beetles Lepidiota stigma (L. stigma) and Cyphochilus which exhibit the most effective scattering of white light in the literature. The final thresholded matrices represent a scan area of 25 × 70 × 362.5 µm and 25 × 67.5 × 235µm while maintaining a pixel resolution of 25 nm. This tomographic approach allowed the internal structure of the scales to be captured completely intact and undistorted by the sectioning required for traditional microscopy techniques.

Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle.

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.