Expression Pattern of Nitric Oxide Synthase during Development of the Marine Gastropod Mollusc, Crepidula fornicata.

Nitric Oxide (NO) plays a key role in the induction of larval metamorphosis in several invertebrate phyla. The inhibition of the NO synthase in Crepidula fornicata, a molluscan model for evolutionary, developmental, and ecological research, has been demonstrated to block the initiation of metamorphosis highlighting that endogenous NO is crucial in the control of this developmental and morphological process. Nitric Oxide Synthase contributes to the development of shell gland, digestive gland and kidney, being expressed in cells that presumably correspond to FMRF-amide, serotoninergic and catecolaminergic neurons. Here we identified a single Nos gene in embryonic and larval transcriptomes of C. fornicata and studied its localization during development, through whole-mount in situ hybridization, in order to compare its expression pattern with that of other marine invertebrate animal models.

Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach.

Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs' population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs' function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.

Functional morphology and post-larval development of the buccal complex in Eubranchus rupium (Nudibranchia: Aeolidida: Fionidae).

Nudibranch molluscs represent an interesting model group to study the evolution of feeding apparatus and feeding modes, being characterized by specialized buccal complex in combination with extremely diverse dietary preferences and multiply prey shifts in evolutionary history. However, the plasticity of the buccal complex morphology in response to diet and specific feeding modes remains understudied. Here we study the general morphology and ontogenesis of the buccal complex in Eubranchus rupium (Nudibranchia: Fionidae). Specific goals are to provide a detailed description of buccal structures morphology in post-larval stages, suggest the feeding mechanism and discuss the phylogenetic value of the morphological characteristics of buccal armature within the genus Eubranchus. Methods included in vivo observations of the feeding process for E. rupium, light microscopic methods, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (cLSM). According to our results, E. rupium is a mechanical driller, boring holes in hydrozoan perisarc and sucking internal content. The mechanical drilling is supplied by functionally uniserial radula with plate-like laterals teeth of exclusively supportive function and by massive buccal musculature. Comparative phylogeny-based analysis suggests that the drilling feeding mode is common for the genus Eubranchus and indicates radular characters may have a high phylogenetic signal. The buccal complex morphology and feeding mode were found to be similar in both adults and post-metamorphic specimens, its general structures occur even in settled veligers. Juveniles and adults compete for food source, but the competition is smoothed due to characteristics of prey species growth and life cycle.

The future of food from the sea.

Global food demand is rising, and serious questions remain about whether supply can increase sustainably1. Land-based expansion is possible but may exacerbate climate change and biodiversity loss, and compromise the delivery of other ecosystem services2-6. As food from the sea represents only 17% of the current production of edible meat, we ask how much food we can expect the ocean to sustainably produce by 2050. Here we examine the main food-producing sectors in the ocean-wild fisheries, finfish mariculture and bivalve mariculture-to estimate 'sustainable supply curves' that account for ecological, economic, regulatory and technological constraints. We overlay these supply curves with demand scenarios to estimate future seafood production. We find that under our estimated demand shifts and supply scenarios (which account for policy reform and technology improvements), edible food from the sea could increase by 21-44 million tonnes by 2050, a 36-74% increase compared to current yields. This represents 12-25% of the estimated increase in all meat needed to feed 9.8 billion people by 2050. Increases in all three sectors are likely, but are most pronounced for mariculture. Whether these production potentials are realized sustainably will depend on factors such as policy reforms, technological innovation and the extent of future shifts in demand.

The evo-devo of molluscs: Insights from a genomic perspective.

Molluscs represent one of ancient and evolutionarily most successful groups of marine invertebrates, with a tremendous diversity of morphology, behavior, and lifestyle. Molluscs are excellent subjects for evo-devo studies; however, understanding of the evo-devo of molluscs has been largely hampered by incomplete fossil records and limited molecular data. Recent advancement of genomics and other technologies has greatly fueled the molluscan "evo-devo" field, and decoding of several molluscan genomes provides unprecedented insights into molluscan biology and evolution. Here, we review the recent progress of molluscan genome sequencing as well as novel insights gained from their genomes, by emphasizing how molluscan genomics enhances our understanding of the evo-devo of molluscs.

Flipping Shells! Unwinding LR Asymmetry in Mirror-Image Molluscs.

In seeking to understand the establishment of left-right (LR) asymmetry, a limiting factor is that most animals are ordinarily invariant in their asymmetry, except when manipulated or mutated. It is therefore surprising that the wider scientific field does not appear to fully appreciate the remarkable fact that normal development in molluscs, especially snails, can flip between two chiral types without pathology. Here, I describe recent progress in understanding the evolution, development, and genetics of chiral variation in snails, and place it in context with other animals. I argue that the natural variation of snails is a crucial resource towards understanding the invariance in other animal groups and, ultimately, will be key in revealing the common factors that define cellular and organismal LR asymmetry.

Methods in Brain Development of Molluscs.

Representatives of the phylum Mollusca have long been important models in neurobiological research. Recently, the routine application of immunocytochemistry and gene expression analyses in combination with confocal laserscanning microscopy has allowed fast generation of highly detailed reconstructions of neural structures of even the smallest multicellular animals, including early developmental stages. As a consequence, large-scale comparative analyses of neurogenesis-an important prerequisite for inferences concerning the evolution of animal nervous systems-are now possible in a reasonable amount of time. Herein, we describe immunocytochemical staining and in situ hybridization protocols for both, whole-mount preparations of developmental stages-usually 70-300 µm in size-as well as for vibratome and cryostat sections of complex brains. Although our procedures have been optimized for marine molluscs, they may easily be adapted to other (marine) organisms by the creative neurobiologist.

A computational framework for the morpho-elastic development of molluskan shells by surface and volume growth.

Mollusk shells are an ideal model system for understanding the morpho-elastic basis of morphological evolution of invertebrates' exoskeletons. During the formation of the shell, the mantle tissue secretes proteins and minerals that calcify to form a new incremental layer of the exoskeleton. Most of the existing literature on the morphology of mollusks is descriptive. The mathematical understanding of the underlying coupling between pre-existing shell morphology, de novo surface deposition and morpho-elastic volume growth is at a nascent stage, primarily limited to reduced geometric representations. Here, we propose a general, three-dimensional computational framework coupling pre-existing morphology, incremental surface growth by accretion, and morpho-elastic volume growth. We exercise this framework by applying it to explain the stepwise morphogenesis of seashells during growth: new material surfaces are laid down by accretive growth on the mantle whose form is determined by its morpho-elastic growth. Calcification of the newest surfaces extends the shell as well as creates a new scaffold that constrains the next growth step. We study the effects of surface and volumetric growth rates, and of previously deposited shell geometries on the resulting modes of mantle deformation, and therefore of the developing shell's morphology. Connections are made to a range of complex shells ornamentations.

Evolution of limb development in cephalopod mollusks.

Cephalopod mollusks evolved numerous anatomical novelties, including arms and tentacles, but little is known about the developmental mechanisms underlying cephalopod limb evolution. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signals, which establish dorsoventral polarity in vertebrate and arthropod limbs, are similarly polarized in cuttlefish. Inhibition of Bmp2/4 dorsally caused ectopic expression of Notum, which marks the ventral sucker field, and ectopic sucker development. Cuttlefish also show proximodistal regionalization of Hth, Exd, Dll, Dac, Sp8/9, and Wnt expression, which delineates arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of a genetic program for appendage development that was present in the bilaterian common ancestor.

Deep-sea benthic communities in the largest oceanic desert are structured by the presence of polymetallic crust.

Based on the specimens collected during three deep-sea cruises, and deposited at the Muséum National d'Histoire Naturelle (MNHN) in Paris, we analysed the diversity of benthic communities within the EEZ of French Polynesia. The literature and the MNHN database allowed us to inventory 471 species of invertebrates, among which 169 were newly described. We mainly found data for Mollusca, Crustacea, Brachiopoda and Crinoidea. We also found samples from other taxa, which still remain unidentified within the collections of the MNHN. Although this inventory is incomplete, we demonstrate that the deep waters of French Polynesia host unique benthic communities and endemic species. Using diversity and multivariate analyses, we show that the deep-sea benthic communities are structured by depth, habitats, geography and also by the presence of polymetallic crust. Furthermore, by focusing on the molluscs of the central area of French Polynesia, we show that the spectrum of shell size differs among deep-sea habitats. Specifically, shells tend to be smaller on encrusted seamounts than on island slopes. Together with the size range of organisms, low abundance, rarity and endemism designate these habitats as sensitive. These results should thus be taken into account in the evaluation of the expected impact of mining activities on biological communities.

A review on the application of Bacillus as probiotics in aquaculture.

Probiotics use in aquaculture has gained attention as microbial candidates to maintain the health and the well-being of many aquaculture animals. Among the many microbial candidates, probiotic Bacillus has sporulation capacity that makes them survive harsh environmental conditions, are non-pathogenic and non-toxic when fed to fish, and can produce antimicrobial substances making them more suitable candidates compared to other probiotics. In this review, we discussed the necessity of using the probiotic Bacillus in sustainable aquaculture as a good alternative to improve feed utilization, stress response, immune response and disease resistance, maintenance of tissue integrity, and as well improvement of water quality for sustainable aquaculture. Therefore the findings of current researches about the effects of Bacillus application to improve the culture of aquatic animals for future research and development of Bacillus application in aquaculture have been summarised.

An Antarctic flock under the Thorson's rule: Diversity and larval development of Antarctic Velutinidae (Mollusca: Gastropoda).

In most marine gastropods, the duration of the larval phase is a key feature, strongly influencing species distribution and persistence. Antarctic lineages, in agreement with Thorson's rule, generally show a short pelagic developmental phase (or lack it completely), with very few exceptions. Among them is the ascidian-feeding gastropod family Velutinidae, a quite understudied group. Based on a multilocus (COI, 16S, 28S and ITS2) dataset for 182 specimens collected in Antarctica and other regions worldwide, we investigated the actual Antarctic velutinid diversity, inferred their larval development, tested species genetic connectivity and produced a first phylogenetic framework of the family. We identified 15 Antarctic Molecular Operational Taxonomic Units (MOTUs), some of which represented undescribed species, which show two different types of larval shell, indicating different duration of the Pelagic Larval Phase (PLD). Antarctic velutinids stand as an independent lineage, sister to the rest of the family, with extensive hidden diversity likely produced by rapid radiation. Our phylogenetic framework indicates that this Antarctic flock underwent repeated events of pelagic phase shortening, in agreement with Thorson's rule, yielding species with restricted geographic ranges.

Assessment of the benthic ecological status in the adjacent waters of Yangtze River Estuary using marine biotic indices.

The adjacent waters of the Yangtze River Estuary are influenced by heavy anthropogenic activities. The benthic ecological status was assessed using the Shannon-Wiener diversity index, the AZTI Marine Biotic Index (AMBI), and the multivariate AMBI (M-AMBI) based on macrofaunal data collected in this area at 51 sites in June 2013 and June 2014. In total, 321 species of macrofauna were identified. Polychaetes were the most dominant, followed by mollusks and crustaceans. The AMBI results showed that 72.55% of the sites were under slight disturbance with a decreasing disturbance trend from inshore to offshore. M-AMBI showed that most of the sites were under lower disturbance level than those shown by AMBI. The Shannon-Wiener diversity index showed that only two sites, near the Yangtze River Estuary and the Zhoushan Islands, respectively, were under moderate status. Other sites were under good or high status, which is consistent with the M-AMBI results.

Staggered Hox expression is more widespread among molluscs than previously appreciated.

Hox genes are expressed along the anterior-posterior body axis in a colinear fashion in the majority of bilaterians. Contrary to polyplacophorans, a group of aculiferan molluscs with conserved ancestral molluscan features, gastropods and cephalopods deviate from this pattern by expressing Hox genes in distinct morphological structures and not in a staggered fashion. Among conchiferans, scaphopods exhibit many similarities with gastropods, cephalopods and bivalves, however, the molecular developmental underpinnings of these similar traits remain unknown. We investigated Hox gene expression in developmental stages of the scaphopod Antalis entalis to elucidate whether these genes are involved in patterning morphological traits shared by their kin conchiferans. Scaphopod Hox genes are predominantly expressed in the foot and mantle but also in the central nervous system. Surprisingly, the scaphopod mid-stage trochophore exhibits a near-to staggered expression of all nine Hox genes identified. Temporal colinearity was not found and early-stage and late-stage trochophores, as well as postmetamorphic individuals, do not show any apparent traces of staggered expression. In these stages, Hox genes are expressed in distinct morphological structures such as the cerebral and pedal ganglia and in the shell field of early-stage trochophores. Interestingly, a re-evaluation of previously published data on early-stage cephalopod embryos and of the gastropod pre-torsional veliger shows that these developmental stages exhibit traces of staggered Hox expression. Considering our results and all gene expression and genomic data available for molluscs as well as other bilaterians, we suggest a last common molluscan ancestor with colinear Hox expression in predominantly ectodermal tissues along the anterior-posterior axis. Subsequently, certain Hox genes have been co-opted into the patterning process of distinct structures (apical organ or prototroch) in conchiferans.

Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System.

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By miRNA sequencing analysis, we identify 482 miRNAs in the regenerating central nervous system (CNS) of the mollusc Lymnaeastagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up- or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate, for the first time, that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (pedal A), whereas expression in the growth cones of another class of respiratory motorneurons (right parietal A) was absent in vitro. These findings support our hypothesis that miRNAs are important regulators of retinoic acid-induced neuronal outgrowth and regeneration in regeneration-competent species.

Attitudinal Factors and Personal Characteristics Influence Support for Shellfish Aquaculture in Rhode Island (US) Coastal Waters.

This study explores public interests associated with shellfish aquaculture development in coastal waters of Rhode Island (US). Specifically, we examine (1) the levels of public support for (or opposition to) shellfish aquaculture development and (2) factors driving the levels of support, using survey data and ordinal logistic regressions. Results of the analysis identify several key attitudinal factors affecting individual's support for shellfish aquaculture in Rhode Island (RI). The level of support is positively associated with attitudes related to shellfish aquaculture's benefits to the local economy and its role as a nutritional food option, and negatively influenced by attitudes related to aquaculture farms' effects on aesthetic quality and their interference with other uses. Findings highlight that support for (or opposition to) aquaculture in RI is driven more by attitudes associated with social impacts than by those associated with environmental impacts. The level of support is also affected by personal characteristics related to an individual's participation in recreational activities. For instance, bicycle riders tend to be supportive of shellfish aquaculture while respondents who participate in sailing and birding are less supportive. By identifying the broader public's interests in shellfish aquaculture, findings from this study and others like it can be used to address public concerns, incorporate public perceptions and attitudes into permitting decisions, and develop outreach targeted at specific stakeholder groups.

Mollusc diversity associated with the non-indigenous macroalga Asparagopsis armata Harvey, 1855 along the Atlantic coast of the Iberian Peninsula.

The aims of this study were to explore mollusc assemblages associated with the non-indigenous macroalga Asparagopsis armata, to compare them with those on other macroalgae at the study region and to explore potential differences on mollusc assemblages between two regions in the Atlantic coast of the Iberian Peninsula, where A. armata is present. To achieve this, at each region, four intertidal shores were sampled. Twenty-nine mollusc species were reported and thus, A. armata harboured similar or higher diversity than other annual macroalgae in this area. When compared with perennial macroalgae, results depend on the species and studied area. Moreover, significant differences in structure of mollusc assemblages between the two studied regions were found. However, these were due to differences in the relative abundance of species rather than the presence of exclusive species at each region.

The evolution of mollusc shells.

Molluscan shells are externally fabricated by specialized epithelial cells on the dorsal mantle. Although a conserved set of regulatory genes appears to underlie specification of mantle progenitor cells, the genes that contribute to the formation of the mature shell are incredibly diverse. Recent comparative analyses of mantle transcriptomes and shell proteomes of gastropods and bivalves are consistent with shell diversity being underpinned by a rapidly evolving mantle secretome (suite of genes expressed in the mantle that encode secreted proteins) that is the product of (a) high rates of gene co-option into and loss from the mantle gene regulatory network, and (b) the rapid evolution of coding sequences, particular those encoding repetitive low complexity domains. Outside a few conserved genes, such as carbonic anhydrase, a so-called "biomineralization toolkit" has yet to be discovered. Despite this, a common suite of protein domains, which are often associated with the extracellular matrix and immunity, appear to have been independently and often uniquely co-opted into the mantle secretomes of different species. The evolvability of the mantle secretome provides a molecular explanation for the evolution and diversity of molluscan shells. These genomic processes are likely to underlie the evolution of other animal biominerals, including coral and echinoderm skeletons. This article is categorized under: Comparative Development and Evolution > Regulation of Organ Diversity Comparative Development and Evolution > Evolutionary Novelties.

Brain regionalization genes are co-opted into shell field patterning in Mollusca.

The 'brain regionalization genes' Six3/6, Otx, Pax2/5/8, Gbx, and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx-expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx, together with Six3/6, Otx, Pax2/5/8, and Hox1, is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx-orthologs and the other 'posterior brain regionalization genes' such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.

Ancestral and novel roles of Pax family genes in mollusks.

BACKGROUND: Pax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/ß are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxß in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera). RESULTS: All investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea. CONCLUSIONS: Pax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxß expression argue for a common role in lophotrochozoan neurogenesis.