Persistent polyamorphism in the chiton tooth: From a new biomineral to inks for additive manufacturing.

Engineering structures that bridge between elements with disparate mechanical properties are a significant challenge. Organisms reap synergy by creating complex shapes that are intricately graded. For instance, the wear-resistant cusp of the chiton radula tooth works in concert with progressively softer microarchitectural units as the mollusk grazes on and erodes rock. Herein, we focus on the stylus that connects the ultrahard and stiff tooth head to the flexible radula membrane. Using techniques that are especially suited to probe the rich chemistry of iron at high spatial resolution, in particular synchrotron Mössbauer and X-ray absorption spectroscopy, we find that the upper stylus of Cryptochiton stelleri is in fact a mineralized tissue. Remarkably, the inorganic phase is nano disperse santabarbaraite, an amorphous ferric hydroxyphosphate that has not been observed as a biomineral. The presence of two persistent polyamorphic phases, amorphous ferric phosphate and santabarbaraite, in close proximity, is a unique aspect that demonstrates the level of control over phase transformations in C. stelleri dentition. The stylus is a highly graded material in that its mineral content and mechanical properties vary by a factor of 3 to 8 over distances of a few hundred micrometers, seamlessly bridging between the soft radula and the hard tooth head. The use of amorphous phases that are low in iron and high in water content may be key to increasing the specific strength of the stylus. Finally, we show that we can distill these insights into design criteria for inks for additive manufacturing of highly tunable chitosan-based composites.

Polarization sensitivity and decentralized visual processing in an animal with a distributed visual system.

The marine mollusc Acanthopleura granulata (Mollusca; Polyplacophora) has a distributed visual array composed of hundreds of small image-forming eyes embedded within its eight dorsal shell plates. As in other animals with distributed visual systems, we still have a poor understanding of the visual capabilities of A. granulata and we have yet to learn where and how it processes visual information. Using behavioral trials involving isoluminant looming visual stimuli, we found that A. granulata demonstrates spatial vision with an angular resolution of 6 deg. We also found that A. granulata responds to looming stimuli defined by contrasting angles of linear polarization. To learn where and how A. granulata processes visual information, we traced optic nerves using fluorescent lipophilic dyes. We found that the optic nerves innervate the underlying lateral neuropil, a neural tissue layer that circumnavigates the body. Adjacent optic nerves innervate the lateral neuropil with highly overlapping arborizations, suggesting it is the site of an integrated visuotopic map. Using immunohistochemistry, we found that the lateral neuropil of A. granulata is subdivided into two separate layers. In comparison, we found that a chiton with eyespots (Chiton tuberculatus) and two eyeless chitons (Ischnochiton papillosus and Chaetopleura apiculata) have lateral neuropil that is a singular circular layer without subdivision, findings consistent with previous work on chiton neuroanatomy. Overall, our results suggest that A. granulata effectuates its visually mediated behaviors using a unique processing scheme: it extracts spatial and polarization information using a distributed visual system, and then integrates and processes that information using decentralized neural circuits.

Radula and Shell Microstructure Variations are Congruent with a Molecular Estimate of Shallow-Water Japanese Chitons.

Variations of the radula and shell microstructures in 33 species of Japanese chiton were investigated along with molecular phylogenetic trees. The molecular phylogenetic trees indicated that Chitonida was composed of four clades, of which two clades formed Acanthochitonina and corresponded to Mopalioidea and Cryptoplacoidea, respectively, and the other clades formed Chitonina. In the radula, the shapes of the central and centro-lateral teeth and the petaloid process varied greatly among species or genera and were useful for the identification of particular species or genera. The presence of accessory and petaloid processes and the cusp shape were relatively conserved and useful for recognizing particular genera or even suborders. In the valves, four to six shell layers were found at the section, but the ventral mesostracum was not observed in Acanthochitonina. The shell microstructures in the ventral sublayer of the tegmentum varied at suborder, but those in the other layers were almost constant. The megalaesthete chamber type varied at superfamily and was helpful to identify particular families or superfamilies. The characteristics of the shell layers and shell microstructures appear to be a synapomorphy shared by the members of Acanthochitonina. The classification within Chitonina needs to be reexamined because the variations of the cusp shape and megalaesthete chamber type were relatively large and did not correspond to the current classification. Callochiton formed a sister group with Chitonida and would be equally closely related to Chitonina and Acanthochitonina because of possessing a mosaic of characteristics from both.

Concordant phylogeographic responses to large-scale coastal disturbance in intertidal macroalgae and their epibiota.

Major ecological disturbance events can provide opportunities to assess multispecies responses to upheaval. In particular, catastrophic disturbances that regionally extirpate habitat-forming species can potentially influence the genetic diversity of large numbers of codistributed taxa. However, due to the rarity of such disturbance events over ecological timeframes, the genetic dynamics of multispecies recolonization processes have remained little understood. Here, we use single nucleotide polymorphism (SNP) data from multiple coastal species to track the dynamics of cocolonization events in response to ancient earthquake disturbance in southern New Zealand. Specifically, we use a comparative phylogeographic approach to understand the extent to which epifauna (with varying ecological associations with their macroalgal hosts) share comparable spatial and temporal recolonization patterns. Our study reveals concordant disturbance-related phylogeographic breaks in two intertidal macroalgal species along with two associated epibiotic species (a chiton and an isopod). By contrast, two codistributed species, one of which is an epibiotic amphipod and the other a subtidal macroalga, show few, if any, genetic effects of palaeoseismic coastal uplift. Phylogeographic model selection reveals similar post-uplift recolonization routes for the epibiotic chiton and isopod and their macroalgal hosts. Additionally, codemographic analyses support synchronous population expansions of these four phylogeographically similar taxa. Our findings indicate that coastal paleoseismic activity has driven concordant impacts on multiple codistributed species, with concerted recolonization events probably facilitated by macroalgal rafting. These results highlight that high-resolution comparative genomic data can help reconstruct concerted multispecies responses to recent ecological disturbance.

Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation.

Chitosanase hydrolyzes ß-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.

Similar construction of spicules and shell plates: Implications for the origin of chiton biomineralization.

The hard shells of mollusks are products of biomineralization, a distinctive feature of the Cambrian explosion. Despite our understanding of shell structure and mechanical properties, their origin remains mysterious. In addition to their shell plates, most chitons have calcium deposits on their girdles. However, the similarity of these two mineralized structures still needs to be determined, limiting our comprehension of their origins. In our study, we analyzed the matrix proteins in the spicules of chiton (Acanthopleura loochooana) and compared them with the matrix proteins in the shells of the same species. Proteomics identified 96 unique matrix proteins in spicules. Comparison of biomineralization-related matrix proteins in shell plates and spicules revealed shared proteins, including carbonic anhydrases, tyrosinase-hemocyanin, von Willebrand factor type A, cadherin, and glycine-rich unknown proteins. Based on similarities in key matrix proteins, we propose that spicules and shell plates originated from a common mineralization system in their ancestral lineage, suggesting the existence of a common core or toolkit of matrix proteins among calcifying organisms. SIGNIFICANCE: In this study, we try to understand the types and diversity of matrix proteins in the biomineralization of chiton shell plates and spicules. Through a comparative analysis, we seek insights into the core biomineralization toolkit of ancestral mollusks. To achieve this, we conducted LC-MS/MS and RT-qPCR analyses to identify the types and relative expression levels of matrix proteins in both shell plates and spicules. The analysis revealed 96 matrix proteins in the spicules. A comparison of biomineralization-related matrix proteins in shell plates and spicules from the same species revealed shared proteins including many unknown proteins unique to chitons. Blast searching reveals a universal conservation of these proteins among other chitons. Hence, we propose that spicules and shell plates originated from a common mineralization system in their ancestral lineage. Our work provides a molecular basis for studying biomineralization in polyplacophoran mollusks and understanding biomineralization evolution. In addition, it identifies potential matrix proteins that could be applied to control crystal growth.

Complete mitochondrial genome of Acanthochitona defilippii (Polyplacophora: Chitonida) from South Korea.

The chiton (Polyplacophora) occupies a significant position in molluscan evolutionary history as one of the most primitive groups within the phylum Mollusca. Acanthochitona defilippii (Tapparone-Canefri 1874) (Chitonida: Acanthochitonidae) is a commonly found intertidal chiton species in South Korea. In this study, we characterized the complete mitochondrial genome of A. defilippii (14,999 bp long), comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and an A + T rich region (166 bp). The base composition is as follows: 31.82% for A, 11.63% for C, 16.69% for G, and 39.86% for T. We reconstructed a maximum likelihood (ML) tree to elucidate phylogenetic relationships among the eight chitonid families using the nucleotide sequences of all PCGs. The ML tree revealed that A. defilippii clustered with Acanthochitona avicula (BP 100) within the family Acanthochitonidae. Acanthochitonidae formed a sister group with Mopaliidae. The results could provide a valuable understanding the phylogenetic relationships of chitonid species.

Proteomic analysis of shell matrix proteins from the chiton Acanthopleura loochooana.

Most molluscs have mineralized shells to protect themselves. Although the remarkable mechanical properties of shells have been well-studied, the origin of shells is still elusive. Chitons are unique in molluscs because they are shelly Aculifera which diverged from Conchifera (comprising all the shell-bearing classes of molluscs) in the early pre-Cambrian. We developed a method to extract shell proteins from chiton shell plates (removing embedded soft tissues) and then compared the shell proteome to that of Conchifera groups. Sixteen shell matrix proteins from Acanthopleura loochooana were identified by proteomics, in which Nacrein-like, Pif-like proteins, and protocadherin were found. Additional evidences from shell proteome in another species Chiton densiliratus and comparative sequence alignment in five chitons supported a conserved biomineralization toolkit in chitons. Our findings shed light on the evolution of mineralization in chitons and pose a hypothesis that ancestral molluscs have already evolved biomineralization toolkits, which may facilitate the formation of mineralized shells.

Growth patterns and population dynamics of Chiton articulatus (Mollusca: Polyplacophora): A multi-decade study.

The edible chiton Chiton articulatus is a commercially important mollusk found in the rocky intertidal zones of the Mexican tropical Pacific. Despite the intense harvesting in Acapulco Bay, Mexico, knowledge of its growth patterns is limited, hindering the development of effective management strategies. This study investigated the growth dynamics of C. articulatus using a multi-model inference approach based on size structure data collected in four sampling periods covering four decades. Results revealed continuous recruitment throughout the year, contributing to population resilience. The species exhibited growth plasticity, highlighting its adaptive potential. We found complex temporal patterns influenced mainly by climatic events. The El Niño event sowed higher growth rates and lower asymptotic length, while La Niña events showed the opposite pattern. This research provides insights into the growth dynamics of C. articulatus, highlighting the need for holistic management strategies for this commercially important species in the face of environmental change.

Phylogenomic analyses shed light on the relationships of chiton superfamilies and shell-eye evolution.

Mollusca is the second-largest animal phylum with over 100,000 extant species representing eight classes. Across 1000 extant species in the class Polyplacophora, chitons have a relatively constrained morphology but with some notable deviations. Several genera possess "shell eyes", i.e., true eyes with a lens and retina that are embedded within the dorsal shells. The phylogeny of the major chiton clades is mostly well established, in a set of superfamily-level and higher level taxa supported by various approaches, including morphological studies, multiple gene markers, mitogenome-phylogeny, and phylotranscriptomic approaches. However, one critical lineage has remained unclear, namely Schizochiton which was controversially suggested as being the potential independent origin of chiton shell eyes. Here, with the draft genome sequencing of Schizochiton incisus (superfamily Schizochitonoidea) plus assemblies of transcriptome data from other polyplacophorans, we present phylogenetic reconstructions using both mitochondrial genomes and phylogenomic approaches with multiple methods. We found that phylogenetic trees from mitogenomic data are inconsistent, reflecting larger scale confounding factors in molluscan mitogenomes. However, a consistent and robust topology was generated with protein-coding genes using different models and methods. Our results support Schizochitonoidea as the sister group to other Chitonoidea in Chitonina, in agreement with the established classification. Combined with evidence from fossils, our phylogenetic results suggest that the earliest origin of shell eyes is in Schizochitonoidea, and that these structures were also gained secondarily in other genera in Chitonoidea. Our results have generated a holistic review of the internal relationship within Polyplacophora, and a better understanding of the evolution of Polyplacophora.

The complete mitochondrial genome and phylogenetic analysis of Lepidozona coreanica (Reeve, 1847).

In the present study, the complete mitochondrial genome of Lepidozona coreanica was sequenced and described. The complete mitogenome sequence of L. coreanica is 16,572 bp long and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The base composition was AT biased (70.1%). The 13 PCGs of L. coreanica and the other 15 species of Polyplacophora were used for phylogenetic analysis using maximum-likelihood methods. The results showed that L. coreanica, Ischnochiton hakodadensis, and Chaetopleura apiculata are sister groups of the three lineages.

Discovery of protein-based natural hydrogel from the girdle of the 'sea cockroach' Chiton articulatus (Chitonida: Chitonidae).

Hydrogels are widely used materials in biomedical, pharmaceutical, cosmetic, and agricultural fields. However, these hydrogels are usually formed synthetically via a long and complicated process involving crosslinking natural polymers. Herein, we describe a natural hydrogel isolated using a 'gentle' acid treatment from the girdle of a chiton species (Chiton articulatus). This novel hydrogel is shown to have a proliferative effect on mouse fibroblast cells (cell line, L929). The swelling capacity of this natural hydrogel was recorded as approximately 1,200% in distilled water, which is within desired levels for hydrogels. Detailed characterizations reveal that the hydrogel consists predominantly (83.93%) of protein. Considering its non-toxicity, proliferative effect and swelling properties, this natural hydrogel is an important discovery for material sciences, with potential for further applications in industry. Whether the girdle has some hydrogel activity in the living animal is unknown, but we speculate that it may enable the animal to better survive extreme environmental conditions by preventing desiccation.

Optimized Sensory Units Integrated in the Chiton Shell.

The first step for animals to interact with external environment is to sense. Unlike vertebrate animals with flexibility, it is challenging for ancient animals that are less flexible especially for mollusca with heavy shells. Chiton, as an example, has eight overlapping shells covering almost the whole body, is known to incorporate sensory units called aesthetes inside the shell. We used micro-computed tomography combined with quantitative image analysis to reveal the optimized shell geometry to resist force and the aesthetes' global distribution at the whole animal levels to facilitate sense from diverse directions both in the seawater and air. Additionally, shell proteomics combined with transcriptome reveals shell matrix proteins responsible for shell construction and potentially sensory function, highlighting unique cadherin-related proteins among mollusca. Together, this multi-level evidence of sensory units in the chiton shell may shed light on the formation of chiton shells and inspire the design of hard armor with sensory function.

Chitin from the Mollusc Chiton: Extraction, Characterization and Chitosan Preparation.

This study presents the first ever data of extracting chitin from the Chiton shell, which was then converted to the soluble chitosan by soaking in the 45% NaOH solution. The obtained chitin and chitosan were characterized by the seven different methods. Antioxidant activity of the extracted chitosan was also evaluated using the two methods. The shell content was divided into calcium carbonate (90.5 %), protein (5.2%), and chitin (4.3 %). Due to the results of element analysis and 1H NMR, the final degree of deacetylation of chitosan was 90%. Surprisingly, a significant amount of Fe was accidentally found in the shell after demineralization, and removed from the solution through the filtering. Nonetheless, remained Fe in the extracted chitin and chitosan was 20 times higher than those previously reported from the shell of shrimps and crabs. Presence of this amount of Fe could describe why the produced chitosan was darker compared to the commercial chitosan. Antioxidant activity tests showed that the IC50 of the extracted chitosan was higher than one estimated for the commercial chitosan. Antioxidant activity of the extracted chitosan is even better than the commercial version and may be used in pharmaceutical industry as a source of antioxidant.

Microstructure and self-sharpening of the magnetite cap in chiton tooth.

The magnetite cap of chiton tooth (Acanthochiton rubrolineatus) was studied with SEM. Three microstructurally distinct regions were recognized: Region I, close to the anterior surface of the cusp and composed of thick rod microstructure units; Region III, close to the posterior surface and composed of fine fibers; and Region II, sandwiched between Regions I and II and composed of thin sheets. The microstructure units in the three regions constitute a continuous and integrated architecture component. The hardness of Regions I and II was measured to be Hv270 and Hv490, respectively. Based on these microstructure observation and hardness data, the abrasive behavior and microstructural mechanism of self-sharpening of the chiton tooth were discussed in the paper. The self-sharpening was attributed to the combined effect of the hardness and the texture orientation of the microstructure units in Regions I and II.

Grazing under experimental hypercapnia and elevated temperature does not affect the radula of a chiton (Mollusca, Polyplacophora, Lepidopleurida).

Chitons (class Polyplacophora) are benthic grazing molluscs with an eight-part aragonitic shell armature. The radula, a serial tooth ribbon that extends internally more than half the length of the body, is mineralised on the active feeding teeth with iron magnetite apparently as an adaptation to constant grazing on rocky substrates. As the anterior feeding teeth are eroded they are shed and replaced with a new row. The efficient mineralisation and function of the radula could hypothetically be affected by changing oceans in two ways: changes in seawater chemistry (pH and pCO2) may impact the biomineralisation pathway, potentially leading to a weaker or altered density of the feeding teeth; rising temperatures could increase activity levels in these ectothermic animals, and higher feeding rates could increase wear on the feeding teeth beyond the animals' ability to synthesise, mineralise, and replace radular rows. We therefore examined the effects of pH and temperature on growth and integrity in the radula of the chiton Leptochiton asellus. Our experiment implemented three temperature (∼10, 15, 20 °C) and two pCO2 treatments (∼400 µatm, pH 8.0; ∼2000 µatm, pH 7.5) for six treatment groups. Animals (n = 50) were acclimated to the treatment conditions for a period of 4 weeks. This is sufficient time for growth of ca. 7-9 new tooth rows or 20% turnover of the mineralised portion. There was no significant difference in the number of new (non-mineralised) teeth or total tooth row count in any treatment. Examination of the radulae via SEM revealed no differences in microwear or breakage on the feeding cusps correlating to treatment groups. The shell valves also showed no signs of dissolution. As a lineage, chitons have survived repeated shifts in Earth's climate through geological time, and at least their radulae may be robust to future perturbations.

Lepidochitona pseudoliozonis, una nueva especie de quitón (Polyplacophora: Ischnochitonidae) del norte del Caribe / Lepidochitona pseudoliozonis, a new species of chiton (Polyplacophora: Ischnochitonidae) from the North of the Caribbean

The genus Lepidochitona (Gray 1821) contains relatively small chitons with a distinctive girdle, dorsally clothed with non-overlapping calcareous corpuscles. In the Caribbean, it is represented by four species: L. liozonis (Dall, & Simpson, 1901), L. rosea Kaas, 1972, L. rufoi García-Ríos, 2010 and L. bullocki García-Ríos, 2011. A rutinary morphological inspection of 10 specimens of a Lepidochitona species from the Florida Keys was concordant with L. liozonis (the only species of the genus informed for Florida). They did not show many morphological differences that could justify its separation from the specimens from Puerto Rico (the type locality). However, the comparison of sequences of the mitochondrial gene coding for cytochrome oxidase I (COI) of L. liozonis from Puerto Rico and the Florida specimens showed a divergence of 14%. This divergence is incompatible with a reproductively connected species. In addition to their genetic differences, the new species differs from L. liozonis in having bigger size, longer marginal spicules and a postmucronal slope very concave. Rev. Biol. Trop. 63 (2): 369-384. Epub 2015 June 01.

El género Lepidochitona (Gray, 1821) agrupa a quitones relativamente pequeños con un cinturón distintivo, por estar cubierto dorsalmente por corpúsculos calcáreos no solapados. Esta representado en el Caribe por cuatro especies: L. liozonis (Dall, & Simpson, 1901), L. rosea Kaas, 1972, L. rufoi García-Ríos, 2010 y L. bullocki García-Ríos, 2011. La inspección de una muestra de ejemplares de los cayos de la Florida permite distinguir ejemplares de lepidoquitones que podrían clasificarse como L. liozonis (la única especie del género informada para la Florida) por no presentar grandes diferencias morfológicas que justifiquen su separación de los de Puerto Rico (localidad del tipo). Sin embargo, la comparación de secuencias del gen mitocondrial que codifica para el citocromo oxidasa I (COI) de los ejemplares de la Florida con ejemplares de L. liozonis de Puerto Rico evidencia una divergencia de 14%. Esta divergencia es incompatible con especies reproductivamente conectadas. Además de sus diferencias genéticas, la nueva especie se puede distinguir de su especie gemela por ser de mayor tamaño, tener espículas marginales más largas y una pendiente posmucronal muy cóncava.

Comunidades de quitones (Mollusca: Polyplacophora) de la Bahia de La Paz, Baja California Sur, Mexico / Polyplacophoran communities (Mollusca: Polyplacophora) at Bahia de La Paz, Baja California Sur, México

Eight species of polyplacophorans have been reported from La Bahia de la Paz, Baja California Sur, Mexico. We add Lepidochitona beanii, Chaetopleura lurida, Stenoplax limaciformis, S. mariposa, Lepidozona clathrata, L. serrata and Acanthochitona arragonites, increasing the known number of species to 15. Ordination analysis of five chiton communities at the site suggests a correlation of wave exposure to species composition and diversity: communities with intermediate wave exposure have more species (richness) and higher diversity (Shannon's index).