Many questions in biology benefit greatly from the use of a variety of model systems. High-throughput sequencing methods have been a triumph in the democratization of diverse model systems. They allow for the economical sequencing of an entire genome or transcriptome of interest, and with technical variations can even provide insight into genome organization and the expression and regulation of genes. The analysis and biological interpretation of such large datasets can present significant challenges that depend on the ‘scientific status’ of the model system. While high-quality genome and transcriptome references are readily available for well-established model systems, the establishment of such references for an emerging model system often requires extensive resources such as finances, expertise and computation capabilities. The de novo assembly of a transcriptome represents an excellent entry point for genetic and molecular studies in emerging model systems as it can efficiently assess gene content while also serving as a reference for differential gene expression studies. However, the process of de novo transcriptome assembly is non-trivial, and as a rule must be empirically optimized for every dataset. For the researcher working with an emerging model system, and with little to no experience with assembling and quantifying short-read data from the Illumina platform, these processes can be daunting. In this guide we outline the major challenges faced when establishing a reference transcriptome de novo and we provide advice on how to approach such an endeavor. We describe the major experimental and bioinformatic steps, provide some broad recommendations and cautions for the newcomer to de novo transcriptome assembly and differential gene expression analyses. Moreover, we provide an initial selection of tools that can assist in the journey from raw short-read data to assembled transcriptome and lists of differentially expressed genes.

Zebra and quagga mussels ( Dreissena spp. ) are invasive freshwater biofoulers that perpetrate devastating economic and ecological impact. Their success depends on their ability to anchor onto substrates with protein-based fibers known as byssal threads. Yet, compared to other mussel lineages, little is understood about the proteins comprising their fibers or their evolutionary history. Here, we investigated the hierarchical protein structure of Dreissenid byssal threads and the process by which they are fabricated. Unique among bivalves, we found that threads possess a predominantly β -sheet crystalline structure reminiscent of spider silk. Further analysis revealed unexpectedly that the Dreissenid thread protein precursors are mechanoresponsive α -helical proteins that are mechanically processed into β -crystallites during thread formation. Proteomic analysis of the byssus secretory organ and byssus fibers revealed a family of ultrahigh molecular weight (354 to 467 kDa) asparagine-rich (19 to 20%) protein precursors predicted to form α -helical coiled coils. Moreover, several independent lines of evidence indicate that the ancestral predecessor of these proteins was likely acquired via horizontal gene transfer. This chance evolutionary event that transpired at least 12 Mya has endowed Dreissenids with a distinctive and effective fiber formation mechanism, contributing significantly to their success as invasive species and possibly, inspiring new materials design.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3, while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

Catecholaminergic neurons are abundant in molluscs and are involved in a variety of behaviors such as feeding, respiration, learning, and locomotion. However, previous identification of these neurons has relied almost exclusively on immunohistochemistry using antibodies, which have not been fully validated for use in molluscs. We employed tissue-specific quantitative PCR in adults of Lymnaea stagnalis (a pulmonate gastropod) and whole-mount in situ hybridization in larvae to both quantify and visualize messenger RNA of the catecholamine synthesis enzyme, tyrosine hydroxylase (TH). TH messenger RNA was found to localize primarily in the foot and the central nervous system, with smaller quantities present in the cephalic sensory organs. Additionally, we performed western blots that validated a popular antibody used as a marker for catecholaminergic neurons in molluscs. Taken together, these data indicate that TH messenger RNA is present in the central and peripheral nervous system of L. stagnalis and support the specificity of past immunohistochemical labeling of the TH protein. These findings have potentially broad implications, given the wide range of biological processes that have been studied in L. stagnalis.

Biominerals are organo-minerals structures produced by living systems. Since the Cambrian, they contribute to the adaptation of living organisms to different environments by fulfilling a variety of functions that go along with adapted morphologies. One of the aims of biomineralization is to understand how organisms "sculpt" these complex morphologies at nano and molecular scales: we know that occluded organic matrix (OM) proteins influence the lattice parameters of biominerals [1] and that many of them strongly interact with the mineral phase in in vitro assays, but their exact roles are still to be known. The aim of my PhD is to understand the complex relationships between the organic and mineral phases. In the present study, I discuss the case of the Mediterranean fan mussel Pinna nobilis .

Background Hemocyanin is the oxygen transporter of most molluscs. Since the oxygen affinity of hemocyanin is strongly temperature-dependent, this essential protein needs to be well-adapted to the environment. In Tectipleura, a very diverse group of gastropods with > 27,000 species living in all kinds of habitats, several hemocyanin genes have already been analyzed. Multiple independent duplications of this gene have been identified and may represent potential adaptations to different environments and lifestyles. The aim of this study is to further explore the evolution of these genes by analyzing their exon–intron architectures. Results We have reconstructed the gene architectures of ten hemocyanin genes from four Tectipleura species: Aplysia californica, Lymnaea stagnalis, Cornu aspersum and Helix pomatia . Their hemocyanin genes each contain 53 introns, significantly more than in the hemocyanin genes of Cephalopoda (9–11), Vetigastropoda (15) and Caenogastropoda (28–33). The gene structures of Tectipleura hemocyanins are identical in terms of intron number and location, with the exception of one out of two hemocyanin genes of L. stagnalis that comprises one additional intron. We found that gene structures that differ between molluscan lineages most probably evolved more recently through independent intron gains. Conclusions The strict conservation of the large number of introns in Tectipleura hemocyanin genes over 200 million years suggests the influence of a selective pressure on this gene structure. While we could not identify conserved sequence motifs within these introns, it may be simply the great number of introns that offers increased possibilities of gene regulation relative to hemocyanin genes with less introns and thus may have facilitated habitat shifts and speciation events. This hypothesis is supported by the relatively high number of introns within the hemocyanin genes of Pomacea canaliculata that has evolved independently of the Tectipleura. Pomacea canaliculata belongs to the Caenogastropoda, the sister group of Heterobranchia (that encompass Tectipleura) which is also very diverse and comprises species living in different habitats. Our findings provide a hint to some of the molecular mechanisms that may have supported the spectacular radiation of one of Metazoa’s most species rich groups.

MicroRNAs (miRNAs) are a deeply conserved class of small, single stranded RNA molecules that post-transcriptionally regulate mRNA levels via several targeted degradation pathways. They are involved in a wide variety of biological processes and have been used to infer the deep evolutionary relationships of major groups such as the Metazoa. Here we have surveyed several adult tissues of the freshwater pulmonate Lymnaea stagnalis (the Great Pond Snail) for miRNAs. In addition we perform a shell regeneration assay to identify miRNAs that may be involved in regulating mRNAs directly involved in the shell-forming process. From seven mature tissues we identify a total of 370 unique precursor miRNAs that give rise to 336 unique mature miRNAs. While the majority of these appear to be evolutionarily novel, most of the 70 most highly expressed (which account for 99.8% of all reads) share sequence similarity with a miRBase or mirGeneDB2.0 entry. We also identify 10 miRNAs that are differentially regulated in mantle tissue that is actively regenerating shell material, 5 of which appear to be evolutionarily novel and none of which share similarity with any miRNA previously reported to regulate biomineralization in molluscs. One significantly down-regulated miRNA is predicted to target Lst-Dermatopontin, a previously characterized shell matrix protein from another freshwater gastropod. This survey provides a foundation for future studies that would seek to characterize the functional role of these molecules in biomineralization or other processes of interest.

RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols.

The common grove snail Cepaea nemoralis displays a stable pigmentation polymorphism in its shell that has held the attention of scientists for decades. While the details of the molecular mechanisms that generate and maintain this diversity remain elusive, it has long been employed as a model system to address questions related to ecology, population genetics and evolution. In order to contribute to the ongoing efforts to identify the genes that generate this polymorphism we have tested the long-standing assumption that melanin is the pigment that comprises the dark-brown bands. Surprisingly, using a newly established analytical chemical method, we find no evidence that eumelanin is differentially distributed within the shells of C. nemoralis . Furthermore, genes known to be responsible for melanin deposition in other metazoans are not differentially expressed within the shell-forming mantle tissue of C. nemoralis . These results have implications for the continuing search for the supergene that generates the various pigmentation morphotypes.

In previous work (Davison et al., 2016), we used both genetic mapping and a chemical knockdown to show that a frameshift mutation in one copy of a duplicated formin gene is most likely the mutation that causes changes in left-right (LR) asymmetry, or chirality, in the pond snail Lymnaea stagnalis.We also showed that the asymmetric morphology is preceded by asymmetric formin expression in snails, and that overexpressing the same gene in frogs reverses LR asymmetry.We are therefore pleased that research by Abe and Kuroda (2019) not only corroborates our own findings, but gains definitive proof for causation.The new work puts beyond any doubt that LR asymmetry in snails originates in the cellular architecture.However, we are troubled by several errors or omissions, and also that the authors dismiss previous experimental work, by ourselves and others.These points need making because they detract from what is otherwise an important step forward, causing confusion amongst colleagues in an important area of developmental biology.(1) The new work confuses the gene names, to the extent that several colleagues mistakenly believed that we misidentified the locus in 2016.In being the first to identify the causative gene (Davison et al., 2016; submitted in August 2015, published in February 2016), we named it Ldia2 because it is evidently the derived version, compared with Ldia1.Ldia2 is located on a long branch (evidence of rapid evolution), and Ldia2 transcripts are enriched in the embryo relative to Ldia1 (indicating specialized function).Despite submitting their work after ours was published (Kuroda et al., 2016; received in July 2016, published in October 2016), the Kuroda group reversed the naming of the same genes, Lsdia1 for the mutated version and Lsdia2 for the other copy.This fact is not mentioned at all in their new work (Abe and Kuroda, 2019).It is therefore important that these differences are made clear, and that, as in other fields, precedence should be used for describing the genes in future publications.(2) The authors' title is that the work 'establishes the formin Lsdia1 as the long-sought gene for snail dextral/sinistral coiling'.Notwithstanding the fact that we established the formin as the causative gene (Davison et al., 2016), and they gained definitive proof (Abe and Kuroda, 2019), all experiments prior to ∼2003 were carried out in another species, L. peregra, for which the causative gene remains

The geometric patterns that adorn the shells of many phylogenetically disparate molluscan species are comprised of pigments that span the visible spectrum. Although early chemical studies implicated melanin as a commonly employed pigment, surprisingly little evidence generated with more recent and sensitive techniques exists to support these observations. Here we present the first mass spectrometric investigations for the presence of eumelanin and pheomelanin in 13 different molluscan species from three conchiferan classes: Bivalvia, Cephalopoda and Gastropoda. In the bivalve Mytilus edulis we demonstrate that eumelanin mainly occurs in the outermost, non-mineralised and highly pigmented layer of the shell (often referred to as the periostracum). We also identified eumelanin in the shells of the cephalopod Nautilus pompilius and the marine gastropods Clanculus pharaonius and Steromphala adriatica. In the terrestrial gastropod Cepaea nemoralis we verify the presence of pheomelanin in a mollusc shell for the first time. Surprisingly, in a large number of brown/black coloured shells we did not find any evidence for either type of melanin. We recommend methods such as high-performance liquid chromatography with mass spectrometric detection for the analysis of complex biological samples to avoid potential false-positive identification of melanin. Our results imply that many molluscan species employ as yet unidentified pigments to pattern their shells. This has implications for our understanding of how molluscs evolved the ability to pigment and pattern their shells, and for the identification of the molecular mechanisms that regulate these processes.

Hemocyanin is the primary respiratory protein for the majority of the Mollusca and therefore directly interfaces with the physiological requirements of each species and the environments to which they are adapted. Hemocyanin is therefore likely to have been evolutionarily imprinted by significant habitat shifts. In the gastropod clade Panpulmonata (>30,000 species) major realm transitions have occurred multiple times independently and may have contributed to the diversification of this group. Yet, little is known about the adaptive changes linked to these habitat shifts. In order to gain deeper insight into the evolution of panpulmonate hemocyanins and to infer possible impacts associated with those scenarios, we have assembled and analysed hemocyanin isoforms from 4 panpulmonate species: (i) Helix pomatia, (ii) Cantareus aspersus (both Helicidae, Stylommatophora), (iii) Arion vulgaris (Arionidae, Stylommatophora) and (iv) Lymnaea stagnalis (Lymnaeidae, Hygrophila). Additionally, we describe a new hemocyanin isoform within the genome of the euopisthobranch Aplysia californica. Using these newly acquired hemocyanin data, we performed a phylogenetic analysis that reveals independent duplication events of hemocyanin within lineages that correlate with significant habitat shifts.

Male accessory-gland proteins are known to affect female physiology in multiple ways, maximizing a male’s reproductive success—often at a cost to the female. Due to this inherent sexual conflict, accessory gland proteins (ACPs) are generally studied in separate-sex organisms. While ACPs have also been identified in simultaneous hermaphrodites as an important part of post-copulatory sexual selection processes, their study has lagged behind that of ACPs in organisms with separate sexes. In the great pond snail, Lymnaea stagnalis, an ACP affecting egg laying, ovipostatin, is produced in the prostate gland. Based on the published partial Ovipostatin gene sequence, we now provide the complete mRNA and gene sequences, and confirm that gene expression is prostate gland-specific. More importantly we observed a significant increase in Ovipostatin expression in sperm donors after ejaculation. Ovipostatin gene expression did not differ between donors giving their ejaculate first (primary donors) and those donating an ejaculate after having been inseminated (secondary donors). These observations support a role for ovipostatin in reproduction and highlight the importance of standardizing the time point when measuring expression levels of ACPs.

Eumelanin and pheomelanin are well known and common pigments found in nature. However, their complex polymer structure and high thermostability complicate their direct chemical identification. A widely used analytical method is indirect determination using HPLC with UV detection of both types of melanin by their most abundant oxidation products: pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA), and thiazole-2,4,5-tricarboxylic acid (TTCA). An increasing interest in pigmentation in biological research led us to develop a highly sensitive and selective method to identify and quantify these melanin markers in diverse biological samples with complex matrices. By introducing solid-phase extraction (SPE, reversed-phase) following alkaline oxidation we could significantly decrease background signals while maintaining recoveries greater than 70%. Our HPLC-UV-MS method allows for confident peak identification via exact mass information in corresponding UV signals used for quantitation. In addition to synthetic melanin and Sepia officinalis ink as reference compounds eumelanin markers were detected in brown human hair and a brown bivalve shell (Mytilus edulis). Brown feathers from the common chicken (Gallus g. domesticus) yielded all four eumelanin and pheomelanin markers. The present method can be easily adapted for a wide range of future studies on biological samples with unknown melanin content.

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle). Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis.

Background: The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date. Methods: The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing. Results: We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins. Conclusions: The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

Figure S1. SDS-PAGE of shell organic matrix. This figure in jpg format shows a SDS-PAGE comparison between the nacre acid-soluble fraction obtained with different protocols A, B and C, and comparison of prismatic layer acid-insoluble fractions A and B. Similar amounts of matrix (ca. 200Â Iźg) were applied to each lane. (JPG 1284Â kb)