Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis.

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon ß and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Development of environmental DNA metabarcoding primers for marine mollusks and comparison with published primers.

Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.

Advancing responsible genomic analyses of ancient mollusc shells.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Single-cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem-cell regulators.

Schistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. Here, single cell suspensions from S. mansoni mother sporocysts were produced and sequenced using the droplet-based 10X Genomics Chromium platform. Six cell clusters comprising two tegument, muscle, neuron, parenchyma and stem/germinal cell clusters were identified and validated by in situ hybridisation. Gene Ontology term analysis predicted key biological processes for each of the clusters, including three stem/germinal sub-clusters. Furthermore, putative transcription factors predicted for stem/germinal and tegument clusters may play key roles during parasite development and interaction with the intermediate host.

The genome of the rayed Mediterranean limpet Patella caerulea (Linnaeus, 1758).

Patella caerulea (Linnaeus, 1758) is a mollusc limpet species of the class Gastropoda. Endemic to the Mediterranean Sea, it is considered a keystone species due to its primary role in structuring and regulating the ecological balance of tidal and subtidal habitats. It is currently being used as a bioindicator to assess the environmental quality of coastal marine waters and as a model species to understand adaptation to ocean acidification. Here, we provide a high-quality reference genome assembly and annotation for P. caerulea. We generated ∼30 Gb of Pacific Biosciences high-fidelity data from a single individual and provide a final 749.8 Mb assembly containing 62 contigs, including the mitochondrial genome (14,938 bp). With an N50 of 48.8 Mb and 98% of the assembly contained in the 18 largest contigs, this assembly is near chromosome-scale. Benchmarking Universal Single-Copy Orthologs scores were high (Mollusca, 87.8% complete; Metazoa, 97.2% complete) and similar to metrics observed for other chromosome-level Patella genomes, highlighting a possible bias in the Mollusca database for Patellids. We generated transcriptomic Illumina data from a second individual collected at the same locality and used it together with protein evidence to annotate the genome. A total of 23,938 protein-coding gene models were found. By comparing this annotation with other published Patella annotations, we found that the distribution and median values of exon and gene lengths was comparable with other Patella species despite different annotation approaches. The present high-quality P. caerulea reference genome, available on GenBank (BioProject: PRJNA1045377; assembly: GCA_036850965.1), is an important resource for future ecological and evolutionary studies.

Phylogenomic reconstruction of Solenogastres (Mollusca, Aplacophora) informs hypotheses on body size evolution.

Body size is a fundamental characteristic of animals that impacts every aspect of their biology from anatomical complexity to ecology. In Mollusca, Solenogastres has been considered important to understanding the group's early evolution as most morphology-based phylogenetic reconstructions placed it as an early branching molluscan lineage. Under this scenario, molluscs were thought to have evolved from a small, turbellarian-like ancestor and small (i.e., macrofaunal) body size was inferred to be plesiomorphic for Solenogastres. More recently, phylogenomic studies have shown that aplacophorans (Solenogastres + Caudofoveata) form a clade with chitons (Polyplacophora), which is sister to all other molluscs, suggesting a relatively large-bodied (i.e., megafaunal) ancestor for Mollusca. Meanwhile, recent investigations into aplacophoran phylogeny have called the assumption that the last common ancestor of Solenogastres was small-bodied into question, but sampling of meiofaunal species was limited, biasing these studies towards large-bodied taxa and leaving fundamental questions about solenogaster body size evolution unanswered. Here, we supplemented available data with transcriptomes from eight diverse meiofaunal species of Solenogastres and conducted phylogenomic analyses on datasets of up to 949 genes. Maximum likelihood analyses support the meiofaunal family Meiomeniidae as the sister group to all other solenogasters, congruent with earlier ideas of a small-bodied ancestor of Solenogastres. In contrast, Bayesian Inference analyses support the large-bodied family Amphimeniidae as the sister group to all other solenogasters. Investigation of phylogenetic signal by comparing site-wise likelihood scores for the two competing hypotheses support the Meiomeniidae-first topology. In light of these results, we performed ancestral character state reconstruction to explore the implications of both hypotheses on understanding of Solenogaster evolution and review previous hypotheses about body size evolution and its potential consequences for solenogaster biology. Both hypotheses imply that body size evolution has been highly dynamic over the course of solenogaster evolution and that their relatively static body plan has successfully allowed for evolutionary transitions between meio-, macro- and megafaunal size ranges.

Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca.

Family B1 G protein-coupled receptors (GPCRs) are one of the most well studied neuropeptide receptor families since they play a central role in many biological processes including endocrine, gastrointestinal, cardiovascular and reproduction in animals. The genes for these receptors emerged from a common ancestral gene in bilaterian genomes and evolved via gene/genome duplications and deletions in vertebrate and invertebrate genomes. Their existence and function have mostly been characterized in vertebrates and few studies exist in invertebrate species. Recently, an increased interest in molluscs, means a series of genomes have become available, and since they are less modified than insect and nematode genomes, they are ideal to explore the origin and evolution of neuropeptide gene families. This review provides an overview of Family B1 GPCRs and their peptide ligands and incorporates new data obtained from Mollusca genomes and taking a comparative approach challenges existing models on their origin and evolution.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

A chromosome-level genome assembly of a deep-sea symbiotic Aplacophora mollusc Chaetoderma sp.

The worm-shaped, shell-less Caudofoveata is one of the least known groups of molluscs. As early-branching molluscs, the lack of high-quality genomes hinders our understanding of their evolution and ecology. Here, we report a high-quality chromosome-scale genome of Chaetoderma sp. combining PacBio, Illumina, and high-resolution chromosome conformation capture sequencing. The final assembly has a size of 2.45 Gb, with a scaffold N50 length of 141.46 Mb, and is anchored to 17 chromosomes. Gene annotations showed a high level of accuracy and completeness, with 23,675 predicted protein-coding genes and 94.44% of the metazoan conserved genes by BUSCO assessment. We further present 16S rRNA gene amplicon sequencing of the gut microbiota in Chaetoderma sp., which was dominated by the chemoautotrophic bacteria (phylum Gammaproteobacteria). This chromosome-level genome assembly presents the first genome for the Caudofoveata, which constitutes an important resource for studies ranging from molluscan evolution, symposium, to deep-sea adaptation.

Hemoglobin wonders: a fascinating gas transporter dive into molluscs.

Hemoglobin (Hb) has been identified in at least 14 molluscan taxa so far. Research spanning over 130 years on molluscan Hbs focuses on their genes, protein structures, functions, and evolution. Molluscan Hbs are categorized into single-, two-, and multiple-domain chains, including red blood cell, gill, and extracellular Hbs, based on the number of globin domains and their respective locations. These Hbs exhibit variation in assembly, ranging from monomeric and dimeric to higher-order multimeric forms. Typically, molluscan Hbs display moderately high oxygen affinity, weak cooperativity, and varying pH sensitivity. Hb's potential role in antimicrobial pathways could augment the immune defense of bivalves, which may be a complement to their lack of adaptive immunity. The role of Hb as a respiratory protein in bivalves likely originated from the substitution of hemocyanin. Molluscan Hbs demonstrate adaptive evolution in response to environmental changes via various strategies (e.g. increasing Hb types, multimerization, and amino acid residue substitutions at key sites), enhancing or altering functional properties for habitat adaptation. Concurrently, an increase in Hb assembly diversity, coupled with a downward trend in oxygen affinity, is observed during molluscan differentiation and evolution. Hb in Protobranchia, Heteroconchia, and Pteriomorphia bivalves originated from separate ancestors, with Protobranchia inheriting a relative ancient molluscan Hb gene. In bivalves, extracellular Hbs share a common origin, while gill Hbs likely emerged from convergent evolution. In summary, research on molluscan Hbs offers valuable insights into the origins, biological variations, and adaptive evolution of animal Hbs.

Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an invasive species in Central Punjab Pakistan / Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): uma espécie invasora no centro de Punjab, Paquistão

Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Os físidos pertencem à classe Gastropoda; pertencem ao filo Mollusca e, sendo bioindicadores, hospedeiros intermediários de parasitas e pragas, ocupam uma posição-chave no ecossistema. Existem três espécies do gênero Physa, ou seja, P. fontinalis, Physa acuta e P. gyrina em corpos d’água do Punjab Central e foram caracterizadas com base em marcadores moleculares. Alto nível de diversidade genética foi revelado por RAPD polimórfico, no entanto os marcadores SSR não foram amplificados. A análise multivariada revelou polimorfismo variando de 9,09% a 50% entre as três espécies de Physid. Um número total de 79 loci foi observado para as três espécies em estudo e 24 loci foram observados como polimórficos. Esses fragmentos RAPD podem ser desenvolvidos em marcadores codominantes (SCAR) por clonagem e podem ser posteriormente sequenciados para o desenvolvimento de marcadores específicos da espécie Physa para identificar as espécies introduzidas e nativas no Paquistão.

Hermafroditismo funcional de la gónada de Fissurella crassa (Mollusca: Fissurellidae) / Functional hermaphrodite gonad in Fissurella crassa (Mollusca: Fissurellidae)

La costa del Pacífico sudoriental es el hábitat de las 13 especies de lapas descritas del subgénero Fissurella Brugière. En estas especies no existe dimorfismo sexual, los animales son dioicos, el sexo se reconoce explorando directa o indirectamente las gónadas y no tienen procesos de reversión sexual. La presencia de un organismo de Fissurella crassa con gónada formada por porciones de ovario y otras de testículo con capacidad para generar óvulos y espermatozoides, evidencia la potencialidad que los organismos de Fissurella poseen para desarrollar el hermafroditismo funcional. Sin embargo, el presente hallazgo no permite inferir si el agente desencadenante del desarrollo sincrónico funcional de la gónada hermafrodita es un factor endógeno y/o asociado a algún evento exógeno medio ambiental.

The coast of the Southeastern Pacific is the habitat for 13 species of described keyhole limpets of the subgenus Fissurella Brugière. In these species sexual dimorphism does not exist, the animals are dioicos, the sex is recognized exploring directly or indirectly the gonads and they do not have processes of sexual reversion. The presence of an organism Fissurella crassa with portions of ovary and testicle with ability to generate ova and sperms, demonstrates the potential that Fissurella's organisms possess to develop functional hermaphroditism. Nevertheless, the present find does not allow to infer if the trigger agent of the synchronous functional development of the hermaphrodite gonad is a factor endogenous and/or associated with any exogenous environmental event.

Schistosome/mollusk: genetic compatibility

Schistosomiasis remains one of the most prevalent parasitic infections and has significant economic and public health consequences in many developing countries. Economic development and improvement in standard of living in these countries are dependent on the elimination of this odious disease. For the control of Schistosomiasis, understanding the host/parasite association is important, since the host parasite relationship is often complex and since questions remain concerning the susceptibility of snails to infection by respective trematodes and their specificity and suitability as hosts for continued parasite development. Thus, the long term aim of this research is to learn more about the genetic basis of the snail/parasite relationship with the hope of finding novel ways to disrupt the transmission of this disease. In the current research, genetic variability among susceptible and resistant strains within and between Biomphalaria glabrata and B. tenagophila was investigated using RAPD-PCR. The results indicate great genetic variations within the two snail species using three different primers (intrapopulational variations), while specimens from the same snail species showed few individual differences between the susceptible and resistant strains (interpopulational variation).

Polymorphisms of octopine dehydrogenase (Odh) in mollusks and implications for the neutralism-selectionism hypothesis

Octopine dehydrogenase (Odh) was examined in several species of bivalves and gastropods and complemented with bibliographic data, to assess the controversy between neutralism and selectionism in explaining the maintenance of genetic variation in natural populations. This debate was the center of the molecular evolution and population genetic research in the 1970s and 1980s, but waned thereafter, without resolution. Although DNA data have been produced, implications are not understood. We examined the polymorphims of Odh in several species of bivalves and gastropods, and the kinetic properties (apparent Km) of the different isozymes in the scallop Euvola ziczac that indicates an apparent case of overdominance of the heterozygous individuals. The question "which of the two hypothesis is correct" has shifted with time to "how much influence did each factor have in the maintenance of genetic variation".

Aspectos biotecnológicos en larvas de "loco" / Biotechnological aspects in \"locus\" larvae

The biology of planktotrophic larvae of Concholepas concholepas is the main bottleneck towards developing biotechnologies to rear this muricid. Data concerning planktonic larvae development, diets and environmental signals triggering larval settlement and recuitment is scarce. We have begun the study of the molecular and cell biology of embryos, larvae and recruits having as a final goal, the development of appropriate biotechnologies to rear this gastropod. First, an inverse ratio between BuChE and AChE enzyme activities was established. This ratio may be a precise development marker for this species. Second, for the first time a phosphoinositide rlated regulatory pathway is reported in a muricid, opening a new approach to the biotechnological management of larvae. Third, the relation between sulfate in sea water and larval motility was studied. Concentration below 125 µM sulfate decreases larval motility. The sulfate is incorporated in proteoglycans which participate in different developmental phenomena. Lastly, a genomic Concholepas concholepas DNA sequence, similar to that of a human growth hormone probe was detected. This is very interesting since growth factors are key molecules during development, growth and are involved in food conversion rares in fishes and also, a in variety of marine invertebrates