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In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bone morphogenetic proteins (BMPs), which are members of the superfamily of transforming growth factor-β (TGF-β), are known both in vitro and in vivo for their osteoinduction properties on the osteoblastic cells. Its role in the mollusk shell formation has also been gradually established. Using Haliotis diversicolor as a model, we characterized the HdBMP2/4 gene in the mantle tissue and showed its expression in the outer fold epithelium (particularly at the periostracal groove) the epithelial site which is involved in shell formation, both prismatic and nacreous layers. Shell notching experiments following gene analysis by qPCR revealed the upregulation of the HdBMP2/4 gene up to 3.2-fold than that of the control animals. In vitro treatments of the preosteoblastic cells, MC3T3-E1 with HdBMP2/4 synthetic peptide demonstrated the enhanced effect of many osteogenic genes that are known to regulate bone and shell biomineralization including ALP, Runx2, and OCN with 2-4 fold-change throughout 14 days of culture. In addition, the increased deposition of calcium-based mineral (as assessed by Alizarin red staining) of the treated cells was comparable to the ascorbic acid (Vit C) + glycerophosphate positive control which revealed the enhanced effect of HdBMP2/4 peptide on matrix biomineralization of the preosteoblastic cells. In conclusion, these results indicated the presence of the HdBMP2/4 gene in the mantle tissue at the site involved in shell formation and the effect of the HdBMP2/4 knuckle epitope peptide in osteoinduction in vitro., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ocean acidification poses a significant threat to calcifying invertebrates by negatively influencing shell deposition and growth. An organism's performance under ocean acidification is not determined by the susceptibility of one single life-history stage, nor is it solely controlled by the direct physical consequences of ocean acidification. Shell development by one life-history stage is sometimes a function of the pH or p CO 2 levels experienced during earlier developmental stages. Furthermore, environmental factors such as access to nutrition can buffer organismal responses of calcifying invertebrates to ocean acidification, or they can function as a co-occurring stressor when access is low. We reared larvae and juveniles of the planktotrophic marine gastropod Crepidula fornicata through combined treatments of nutritional stress and low pH, and we monitored how multiple stressors endured during the larval stage affected juvenile performance. Shell growth responded non-linearly to decreasing pH, significantly declining between pH 7.6 and pH 7.5 in larvae and juveniles. Larval rearing at pH 7.5 reduced juvenile growth as a carryover effect. Larval rearing at pH 7.6 reduced subsequent juvenile growth despite the absence of a negative impact on larval growth, demonstrating a latent effect. Low larval pH magnified the impact of larval nutritional stress on competence for metamorphosis and increased carryover effects of larval nutrition on juvenile growth. Trans-life-cycle effects of larval nutrition were thus modulated by larval exposure to ocean acidification.

The present study evaluated the association among traditional biochemical biomarkers with biometric, morphometric, and elemental composition of Lottia subrugosa (patelliform gastropod) shells from three multi-impacted coastal areas in Brazil. The study was carried out in Todos os Santos Bay (TSB), Santos/São Vicente Estuarine System (SESS) and Paranaguá Estuarine Complex (CEP), using three sampling sites to seek contamination gradients in each area. Results showed that all biomarkers evaluated responded to environmental contamination, regardless the presence (SESS and CEP) or absence (TSB) of a gradient of contamination. The responses found using biometric and morphometric parameters were consistent with the traditional biomarkers of exposure and effects (lipid peroxidation and DNA damage). Indeed, changes in elemental composition of L. subrugosa shells suggest that exposure to contaminated environments is probably responsible for the alterations detected. Despite the simplicity and lower cost of biometric and morphometric analyzes, these parameters are influenced by natural environmental conditions from which biases may arise. Therefore, these tools should be evaluated through experimental studies before it can be used in future assessments. However, the findings from the present study were observed in three aquatic systems distributed over a wide range of latitudes, which indicates that gastropod shells reflect effects resulting from environmental contamination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Gastropod shell morphologies are famously diverse but generally share a common geometry, the logarithmic coil. Variations on this morphology have been modeled mathematically and computationally but the developmental biology of shell morphogenesis remains poorly understood. Here we characterize the organization and growth patterns of the shell-secreting epithelium of the larval shell of the basket whelk Tritia (also known as Ilyanassa ). Despite the larval shell's relative simplicity, we find a surprisingly complex organization of the shell margin in terms of rows and zones of cells. We examined cell division patterns with EdU incorporation assays and found two growth zones within the shell margin. In the more anterior aperture growth zone, we find that inferred division angles are biased to lie parallel to the shell edge, and these divisions occur more on the margin's left side. In the more posterior mantle epithelium growth zone, inferred divisions are significantly biased to the right, relative to the anterior-posterior axis. These growth zones, and the left-right asymmetries in cleavage patterns they display, can explain the major modes of shell morphogenesis at the level of cellular behavior. In a gastropod with a different coiling geometry, Planorbella sp., we find similar shell margin organization and growth zones as Tritia , but different left-right asymmetries than we observed in the helically coiled shell of Tritia These results indicate that differential growth patterns in the mantle edge epithelium contribute to shell shape in gastropod shells and identify cellular mechanisms that may vary to generate shell diversity in evolution., Competing Interests: The authors declare no conflict of interest.

Gastropod shell sculpture offers a novel tool for studying morphological patterning. Existing shell features may be manipulated experimentally to test how alteration affects subsequent shell growth and form. Axial sculpture occurs in many gastropod groups, and spacing of sculpture may be regular or irregular. But how gastropods control sculpture placement during shell growth is unknown. We studied the growth and positioning of axial lamellae in the muricid Nucella lamellosa, and compared these to the superficially similar axial varices seen in other muricids. First, we tested whether the feeding rate had any effect on the rate of addition or positioning of new lamellae. Second, we tested what effect previous shell sculpture had on lamellar placement, and shell growth in general, by removing all shell sculpture and allowing snails to grow over the "shaved" shell surface. Lamellar growth appeared to be relatively plastic; spacing was highly variable both within and among individual snails, and 1-2 weeks were required to complete the addition of a new lamella. Body growth rate was the primary determinant of lamellar growth; past lamellae had no effect on placement of new lamellae or rate of shell length increase. Feeding rate and body size affected only growth in shell length, and had no direct effect on spacing or on the rate of addition of new lamellae. The growth of axial lamellae in N. lamellosa differed from that of varices by exhibiting neither a) regular spacing nor b) a growth hiatus after completion of a lamella. Significantly, despite the obvious impediment of previous sculpture to future shell growth, removal of this sculpture had no observable effect on the rate of body growth or on any aspect of subsequent lamellar growth., (© 2016 Marine Biological Laboratory.)

The morphology of gastropod shells provides a record of the growth rate at the aperture of the shell, and molecular biological studies have shown that the growth rate gradient along the aperture of a gastropod shell can be closely related to gene expression at the aperture. Here, we develop a novel method for deriving microscopic growth rates from the macroscopic shapes of gastropod shells. The growth vector map of a shell provides information on the growth rate gradient as a vector field along the aperture, over the growth history. However, it is difficult to estimate the growth vector map directly from the macroscopic shape of a specimen, because the degree of freedom of the growth vector map is very high. In order to overcome this difficulty, we develop a method of estimating the growth vector map based on a growing tube model, where the latter includes fewer parameters to be estimated. In addition, we calculate an aperture map specifying the magnitude of the growth vector at each location, which can be compared with the expression levels of several genes or proteins that are important in morphogenesis. Finally, we show a concrete example of how macroscopic shell shapes evolve in a morphospace when microscopic growth rate gradient changes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The morphology of gastropod shells has been a focus of analyses in ecology and evolution. It has recently emerged as an important issue in developmental biology, thanks to recent advancements in molecular biological techniques. The growing tube model is a theoretical morphological model for describing various coiling patterns of molluscan shells, and it is a useful theoretical tool to relate local tissue growth with global shell morphology. However, the growing tube model has rarely been adopted in empirical research owing to the difficulty in estimating the parameters of the model from morphological data. In this article, I solve this problem by developing methods of parameter estimation when (1) 3D Computed Tomography (CT) data are available and (2) only 2D image data (such as photographs) are available. When 3D CT data are available, the parameters can be estimated by fitting an analytical solution of the growing tube model to the data. When only 2D image data are available, we first fit Raup׳s model to the 2D image data and then convert the parameters of Raup׳s model to those of the growing tube model. To illustrate the use of these methods, I apply them to data generated by a computer simulation of the model. Both methods work well, except when shells grow without coiling. I also demonstrate the effectiveness of the methods by applying the model to actual 3D CT data and 2D image data of land snails. I conclude that the method proposed in this article can reconstruct the coiling pattern from observed data., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mollusk shells contain a plethora of information on past climate variability. However, only a limited toolkit is currently available to reconstruct such data from the shells. The environmental data of some proxies (e.g. Sr/Ca ratios) is obscured by physiological effects, whereas other proxies, such as δ(18)O, simultaneously provide information on two or more different environmental variables. The present study investigates whether microstructures of the freshwater gastropod Viviparus viviparus provide an alternative means to reconstruct past water temperature. Cold and highly variable temperature regimes resulted in the precipitation of highly unordered first-order lamellae of simple crossed-lamellar (XLM) structures if new shell formed from scratch. However, during stable and warm conditions, well-ordered first-order lamellae were laid down irrespective of pre-existing shell material. Homogeneous first-order lamellae also formed during times of cold and highly variable temperatures if the new shell was deposited onto existing shell material with well-ordered first-order lamellae. The growth front seems to contain instructions for building specific microstructure variants, irrespective of environmental conditions. However, if this template is missing, the animal forms a deviating microstructure. Under extremely stressful situations (e.g. removal from habitat, calcein staining, extreme temperature shifts), the gastropod precipitates an evolutionarily older microstructure (irregular simple prisms) rather than XLM structures. These shell portions were macroscopically described as disturbance lines. In addition, repetitive, presumably periodic growth patterns were observed, which consisted of gradually changing third-order lamellae between consecutive faint, organic-rich growth lines. These growth patterns were probably controlled by intrinsic biological clocks and exhibited a two-daily periodicity. The results of this study may provide the basis for using changes in the microstructure of shell sections as a new sensor (environmental proxy) for past water temperature., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Two new species of Caecinae are described from the Brazilian coast. Specimens of Caecum metamorphosicum sp. nov. were collected from northern to southeastern Brazil. The teleoconch V of this species is characterized by strong, raised, quadrangular, rather closely arranged axial ribs, except the last two to three preceding the aperture, which are slightly larger and more widely separated, and a finger-shaped mucro. Specimens of Caecum trindadense sp. nov. were only collected from southeastern Brazil. The teleoconch VI of this species is characterized by conspicuous, sinuous longitudinal striae, finger-shaped mucro, and rather smooth varix. The protoconch and all growth stages of C. metamorphosicum sp. nov. and C. trindadense sp. nov. are described and figured here based on scanning electron microscopy. A brief discussion on the biodiversity of Caecidae on the Atlantic coast of South America is given. This report provides a basis for the better recognition of growth stages among members of Caecinae.

Background: Most research on Ocean Acidification (OA) has largely focused on the process of calcification and the physiological trade-offs employed by calcifying organisms to support the building of calcium carbonate structures. However, there is growing evidence that OA can also impact upon other key biological processes such as survival, growth and behaviour. On wave-swept rocky shores the ability of gastropods to self-right after dislodgement, and rapidly return to normal orientation, reduces the risk of predation., Methodology/principal Findings: The impacts of OA on this self-righting behaviour and other important parameters such as growth, survival, shell dissolution and shell deposition in Concholepas concholepas (loco) were investigated under contrasting pCO2 levels. Although no impacts of OA on either growth or net shell calcification were found, the results did show that OA can significantly affect self-righting behaviour during the early ontogeny of this species with significantly faster righting times recorded for individuals of C. concholepas reared under increased average pCO2 concentrations (± SE) (716 ± 12 and 1036 ± 14 µatm CO2) compared to those reared at concentrations equivalent to those presently found in the surface ocean (388 ± 8 µatm CO2). When loco were also exposed to the predatory crab Acanthocyclus hassleri, righting times were again increased by exposure to elevated CO2, although self-righting times were generally twice as fast as those observed in the absence of the crab., Conclusions and Significance: These results suggest that self-righting in the early ontogeny of C. concholepas will be positively affected by pCO2 levels expected by the end of the 21st century and beginning of the next one. However, as the rate of self-righting is an adaptive trait evolved to reduce lethal predatory attacks, our result also suggest that OA may disrupt prey responses to predators in nature.

Seashells grow through the local deposition of mass along the aperture. Many mathematical descriptions of the shapes of shells have been provided over the years, and the basic logarithmic coiling seen in mollusks can be simulated with few parameters. However, the developmental mechanisms underlying shell coiling are largely not understood and the ubiquitous presence of ornamentation such as ribs, tubercles, or spines presents yet another level of difficulty. Here we develop a general model for shell growth based entirely on the local geometry and mechanics of the aperture and mantle. This local description enables us to efficiently describe both arbitrary growth velocities and the evolution of the shell aperture itself. We demonstrate how most shells can be simulated within this framework. We then turn to the mechanics underlying the shell morphogenesis, and develop models for the evolution of the aperture. We demonstrate that the elastic response of the mantle during shell deposition provides a natural mechanism for the formation of three-dimensional ornamentation in shells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

How do several characteristics adapt to gravity while mutually influencing each other? Our study addresses this issue by focusing on the terrestrial gastropod shell. The geometric relationship between the spire index (shell height/diameter) and outline (cylindricality) is theoretically estimated. When the shell grows isometrically, a high-spired shell becomes conical in shape and a low-spired shell becomes cylindrical in shape. A physical model shows that the lowest- and highest-spired shells are the most balanced. In addition, a cone shape is the most balanced for a low-spired shell, and a column shape is the most balanced for a high-spired shell. Spire index and cylindricality measured for freshwater gastropods follow the relationship estimated by the model, whereas those for terrestrial gastropods deviate from this relationship. This translates to a high shell being more cylindrical than a flat shell, except in the case of extremely high or low shells. This suggests that the shape of the most balanced shells (lowest and highest shell heights) is constrained by coiling geometry but that relatively unbalanced shells (intermediate shell heights) do not follow a coiling geometry, as a result of adaptation to enable the snail to carry its shell more effectively.