Molecular cloning, characterisation and molecular modelling of two novel T-synthases from mollusc origin.

The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

Unraveling the protective role of Nrf2 in molluscs: Insights into mitochondrial and apoptosis pathways in the defense against Bap-induced oxidative stress.

Benzopyrene (Bap) is a major constituent of petroleum pollutants commonly found in aquatic environments, and its mutagenic and carcinogenic properties have adverse effects on aquatic organisms' development, growth, and reproduction. The antioxidant defense system element, NF-E2-related factor 2 (Nrf2), has been linked to the oxidative stress response in marine invertebrates exposed to toxic substances. In a previous study, a novel Nrf2 homologue, McNrf2, was identified in mussel Mytilus coruscus, a significant model marine molluscs in ecotoxicology studies. McNrf2 showed the potential to trigger an antioxidant defense against oxidative stress induced by Bap. Here, we employed an Nrf2 overexpression and inhibition model using SFN and ML385 as Nrf2 inducer and inhibitor, respectively. Next, immunofluorescence technique was used to evaluate the nuclear activation of Nrf2 induced by Bap-mediated oxidative stress. Transmission electron microscopy revealed that overexpression of Nrf2 could maintain the quantity and structural integrity of mitochondria, while flow cytometry analysis showed that Nrf2 could alleviate Bap-induced cellular apoptosis. These findings suggest that Nrf2 can protect molluscs from Bap-induced oxidative stress through the mitochondria and apoptosis pathways, providing a novel perspective on Nrf2's antioxidant function.

The AMP deaminase of the mollusk Helix pomatia is an unexpected member of the adenosine deaminase-related growth factor (ADGF) family.

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5' monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine.

Dietary Exposure to Particles of Polytetrafluoroethylene (PTFE) and Polymethylmethacrylate (PMMA) Induces Different Responses in Periwinkles Littorina brevicula.

The marine and ocean water pollution with different-sized plastic waste poses a real threat to the lives of the next generations. Plastic, including microplastics, is found in all types of water bodies and in the organisms that live in them. However, given the chemical diversity of plastic particles, data on their toxicity are currently incomplete. Moreover, it is clear that different organisms, depending on their habitat and feeding habits, are at different risks from plastic particles. Therefore, we performed a series of experiments on feeding the gastropod scraping mollusk Littorina brevicula with two types of polymeric particles-polymethylmethacrylate (PMMA) and polytetrafluoroethylene (PTFE)-using a special feeding design. In the PMMA-exposed group, changes in gastrointestinal biochemical parameters such as increases in malondialdehyde (MDA) and protein carbonyls (PC) were detected, indicating the initiation of oxidative stress. Similarly, a comet assay showed an almost twofold increase in DNA damage in digestive gland cells compared to the control group. In mollusks fed with PTFE-containing food, no similar changes were recorded.

Ocean acidification increases copper accumulation and exacerbates copper toxicity in Amphioctopus fangsiao (Mollusca: Cephalopoda): A potential threat to seafood safety.

Ocean acidification (OA) and trace metal pollutants coexist to exert combined effects on the functions and services of marine ecosystems. Increasing atmospheric carbon dioxide has caused a decrease in the pH of the ocean, affecting the bioavailability and speciation of trace metals and consequently altering metal toxicity in marine organisms. As an important trace metal functioned in hemocyanin, the richness of Copper (Cu) in octopuses is remarkable. Therefore, the biomagnification and bioaccumulation capacities of Cu in octopuses may be a non-negligible risk of contamination. Here, Amphioctopus fangsiao was continuously exposed to acidified seawater (pH 7.8) and copper (50 µg/L) to investigate the combined effect of ocean acidification and Cu exposure on marine mollusks. Our results showed that A. fangsiao could adapt well to ocean acidification after 21 days of the rearing experiment. However, the accumulation of Cu in A. fangsiao intestine increased significantly in acidified seawater under high levels of Cu stress. In addition, Cu exposure can influence the physiological function of A. fangsiao, including growth and feeding. This study also demonstrated that Cu exposure disturbed glucolipid metabolism and induced oxidative damage to intestine tissue, and ocean acidification further exacerbated these toxic effects. The obvious histological damage and microbiota alterations were also caused by Cu stress and its combined effect with ocean acidification. At the transcription level, we found numerous differentially expressed genes (DEGs) and significantly enriched KEGG pathways, involving glycolipid metabolism, transmembrane transport, glucolipid metabolism, oxidative stress, mitochondrial, protein and DNA damage, all revealing the strong toxicological synergetic effect of Cu and OA exposure and the molecular adaptation mechanism of A. fangsiao. Collectively, this study demonstrated that octopuses may withstand future ocean acidification conditions, however, the complex interactions of future OA and trace metal pollution need to be emphasized. OA can influence the toxicity of trace metals, inducing a potential threat to marine organism safety.

Pyroptotic gasdermin exists in Mollusca and is vital to eliminating bacterial infection.

Gasdermin (GSDM) is a family of proteins that execute pyroptosis in vertebrate. In invertebrate, pyroptotic GSDM was documented only in coral. Recent studies identified abundant GSDM structural homologs in Mollusca, but their functions are unclear. Herein, we report a functional GSDM from Pacific abalone Haliotis discus (HdGSDME). HdGSDME is specifically activated by abalone caspase 3 (HdCASP3) cleavage at two distinct sites, generating two active isoforms with pyroptotic and cytotoxic activities. HdGSDME possesses evolutionarily conserved residues that proved to be essential to the N-terminal pore-formation and C-terminal auto-inhibition capacities. Bacterial challenge activates the HdCASP3-HdGSDME pathway and induces pyroptosis and extracellular traps in abalone. Blockage of the HdCASP3-HdGSDME axis promotes bacterial invasion and host mortality. Collectively, this study reveals the existence of functionally conserved and yet distinct-featured GSDM in Mollusca and provides insights into the function and evolution of invertebrate GSDM.

A Putative Receptor for Ferritin in Mollusks: Characterization of the Insulin-like Growth Factor Type 1 Receptor.

The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam (SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter 'A', with the fibronectin type III domains serving as its 'legs'. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.

Proton channels in molluscs: A new bivalvian-specific minimal HV 4 channel.

Recently, three proton channels (HV ) have been identified and characterized in Aplysia californica (AcHV 1-3). Focusing on AcHV 1 and AcHV 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV 1 and to AcHV 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV 4. This new family has high homology to AcHV 1 and AcHV 2 and was identified only in bivalvian molluscs (13 species, 12 full-length sequences). Typically, these channels possess an extracellular S1-S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV 1 channels (~ 13 amino acids) and the much larger loops of molluscan HV 2 channels (> 65 amino acids). The characteristic voltage-sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV 4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV 4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH- and voltage-regulated gating. This work is the first description of a HV 4 type channel, adding a new member to the recently expanded family of proton channels.

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.

Identification, Characterization, and Expression of a ß-Galactosidase from Arion Species (Mollusca).

ß-Galactosidases (ß-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing ß-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a ß-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative ß-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose ß1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both ß-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.

Mineralize It or Not: Comparative Proteomics and Elemental Analysis Reveal Ancestral Compositions of Iron Mineralized Molluscan Radulae.

The radula is a unique foraging organ to Mollusca, which is important for their evolution and taxonomic classification. Many radulae are mineralized with metals. Although the remarkable mechanical properties of mineralized radulae are well-studied, the formation of mineralization from nonmineralized radulae is poorly understood. Taking advantage of the recently sequenced octopus and chiton genomes, we were able to identify more species-specific radular proteins by proteomics. Comparing these proteomes with the known limpet radula proteome enabled us to gain insight into the molecular components of nonmineralized and mineralized radula, highlighting that iron mineralization in the chiton radula is possibly due to the evolution of ferritins and peroxiredoxins. Through an in vitro binding assay, ferritin is shown to be important to iron accumulation into the nonmineralized radula. Moreover, radular proteomes reflect their adaption to dietary habits to some extent. The octopus radula has many scaffold modification proteins to suit flexibility while the chiton radula has abundant sugar metabolism proteins (e.g., glycosyl hydrolases) to adapt to algae feeding. This study provides a foundation for the understanding of molluscan radula formation and evolution and may inspire the synthesis of iron nanomaterials.

Multiple tachykinins and their receptors characterized in the gastropod mollusk Pacific abalone: Expression, signaling cascades, and potential role in regulating lipid metabolism.

Tachykinin (TK) families, including the first neuropeptide substance P, have been intensively explored in bilaterians. Knowledge of signaling of TK receptors (TKRs) has enabled the comprehension of diverse physiological processes. However, TK signaling systems are largely unknown in Lophotrochozoa. This study identified two TK precursors and two TKR isoforms in the Pacific abalone Haliotis discus hannai (Hdh), and characterized Hdh-TK signaling. Hdh-TK peptides harbored protostomian TK-specific FXGXRamide or unique YXGXRamide motifs at the C-termini. A phylogenetic analysis showed that lophotrochozoan TKRs, including Hdh-TKRs, form a monophyletic group distinct from arthropod TKRs and natalisin receptor groups. Although reporter assays demonstrated that all examined Hdh-TK peptides activate intracellular cAMP accumulation and Ca2+ mobilization in Hdh-TKR-expressing mammalian cells, Hdh-TK peptides with N-terminal aromatic residues and C-terminal FXGXRamide motifs were more active than shorter or less aromatic Hdh-TK peptides with a C-terminal YXGXRamide. In addition, we showed that ligand-stimulated Hdh-TKRs mediate ERK1/2 phosphorylation in HEK293 cells and that ERK1/2 phosphorylation is inhibited by PKA and PKC inhibitors. In three-dimensional in silico Hdh-TKR binding modeling, higher docking scores of Hdh-TK peptides were consistent with the lower EC50 values in the reporter assays. The transcripts for Hdh-TK precursors and Hdh-TKR were highly expressed in the neural ganglia, with lower expression levels in peripheral tissues. When abalone were starved for 3 weeks, Hdh-TK1 transcript levels, but not Hdh-TK2, were increased in the cerebral ganglia (CG), intestine, and hepatopancreas, contrasting with the decreased lipid content and transcript levels of sterol regulatory element-binding protein (SREBP). At 24 h post-injection in vivo, the lower dose of Hdh-TK1 mixture increased SREBP transcript levels in the CG and hepatopancreas and accumulative food consumption of abalone. Higher doses of Hdh-TK1 and Hdh-TK2 mixtures decreased the SREBP levels in the CG. When Hdh-TK2-specific siRNA was injected into abalone, intestinal SREBP levels were significantly increased, whereas administration of both Hdh-TK1 and Hdh-TK2 siRNA led to decreased SREBP expression in the CG. Collectively, our results demonstrate the first TK signaling system in gastropod mollusks and suggest a possible role for TK peptides in regulating lipid metabolism in the neural and peripheral tissues of abalone.

Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 µM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Strategies of Invertebrate Osmoregulation: An Evolutionary Blueprint for Transmuting into Fresh Water from the Sea.

Early marine invertebrates like the Branchiopoda began their sojourn into dilute media some 500 million years ago in the Middle Cambrian. Others like the Mollusca, Annelida, and many crustacean taxa have followed, accompanying major marine transgressions and regressions, shifting landmasses, orogenies, and glaciations. In adapting to these events and new habitats, such invertebrates acquired novel physiological abilities that attenuate the ion loss and water gain that constitute severe challenges to life in dilute media. Among these taxon-specific adaptations, selected from the subcellular to organismal levels of organization, and constituting a feasible evolutionary blueprint for invading freshwater, are reduced body permeability and surface (S) to volume (V) ratios, lowered osmotic concentrations, increased osmotic gradients, increased surface areas of interface epithelia, relocation of membrane proteins in ion-transporting cells, and augmented transport enzyme abundance, activity, and affinity. We examine these adaptations in taxa that have penetrated into freshwater, revealing diversified modifications, a consequence of distinct body plans, morpho-physiological resources, and occupation routes. Contingent on life history and reproductive strategy, numerous patterns of osmotic regulation have emerged, including intracellular isosmotic regulation in weak hyper-regulators and well-developed anisosmotic extracellular regulation in strong hyper-regulators, likely reflecting inertial adaptations to early life in an estuarine environment. In this review, we address osmoregulation in those freshwater invertebrate lineages that have successfully invaded this biotope. Our analyses show that across 66 freshwater invertebrate species from six phyla/classes that have transmuted into freshwater from the sea, hemolymph osmolalities decrease logarithmically with increasing S:V ratios. The arthropods have the highest osmolalities, from 300 to 650 mOsmoles/kg H2O in the Decapoda with 220-320 mOsmoles/kg H2O in the Insecta; osmolalities in the Annelida range from 150 to 200 mOsmoles/kg H2O, and the Mollusca showing the lowest osmolalities at 40-120 mOsmoles/kg H2O. Overall, osmolalities reach a cut-off at ∼200 mOsmoles/kg H2O, independently of increasing S:V ratio. The ability of species with small S:V ratios to maintain large osmotic gradients is mirrored in their putatively higher Na+/K+-ATPase activities that drive ion uptake processes. Selection pressures on these morpho-physiological characteristics have led to differential osmoregulatory abilities, rendering possible the conquest of freshwater while retaining some tolerance of the ancestral medium.

Dietary consumption of polypropylene microplastics alter the biochemical parameters and histological response in freshwater benthic mollusc Pomacea paludosa.

One of the most common environmental pollutant in aquatic ecosystems are polypropylene microplastics and their impacts on aquatic organisms are still scarce. The study aimed to prepare polypropylene microplastics using organic solvent (spherical and 11.86-44.62 µm) and then test their toxicity on the freshwater benthic mollusc grazer Pomaceae paludosa. The present study investigated chronic (28 days) exposure of polypropylene microplastics via dietary supplements (250 mg kg-1, 500 mg kg-1 & 750 mg kg-1) in P. paludosa, and the toxic effect was evaluated in digestive gland tissue. The FTIR results revealed no change in polypropylene microplastics during ingestion or after egestion. On the other hand, Ingestion causes accumulation in their bodies and disrupts redox homeostasis. Meanwhile, alteration occurs in oxidative stress-related biomarkers such as increased reactive oxygen species level (ROS), impaired the biochemical parameters of antioxidant system catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and glutathione - S- transferase (GST), deterioration of oxidative stress effects in lipid peroxidation (LPO) and carbonyl protein (CP) and changed the digestive enzymes such as amylase, pepsin, esterase and alkaline phosphatase that are measured in hepatopancreas tissue. The histology results revealed that ingesting these microplastics caused severe damage to the digestive gland cells. According to the findings, ingestion of polypropylene microplastics in benthic freshwater mollusc causes more serious harm and impacts energy acquisition. This finding represents the ecological risk of polypropylene microplastic pollution in the freshwater ecosystem.

Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity.

FMRFamide (Phe-Met-Arg-Phe-amide, FMRFa) and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFa activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFa potency against one of its native targets, the excitatory FMRFa-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of neuropeptide activity, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFa, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching degenerin (DEG)/epithelial sodium channel (ENaC) superfamily, incorporating mutagenesis and experimental dissection of channel function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands but enhanced by FMRFa, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFa neuropeptide activity at FaNaC receptor channels and illuminates the evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

The Role of Anti-Viral Effector Molecules in Mollusc Hemolymph.

Molluscs are major contributors to the international and Australian aquaculture industries, however, their immune systems remain poorly understood due to limited access to draft genomes and evidence of divergences from model organisms. As invertebrates, molluscs lack adaptive immune systems or 'memory', and rely solely on innate immunity for antimicrobial defence. Hemolymph, the circulatory fluid of invertebrates, contains hemocytes which secrete effector molecules with immune regulatory functions. Interactions between mollusc effector molecules and bacterial and fungal pathogens have been well documented, however, there is limited knowledge of their roles against viruses, which cause high mortality and significant production losses in these species. Of the major effector molecules, only the direct acting protein dicer-2 and the antimicrobial peptides (AMPs) hemocyanin and myticin-C have shown antiviral activity. A better understanding of these effector molecules may allow for the manipulation of mollusc proteomes to enhance antiviral and overall antimicrobial defence to prevent future outbreaks and minimize economic outbreaks. Moreover, effector molecule research may yield the description and production of novel antimicrobial treatments for a broad host range of animal species.

Multiple bacterial partners in symbiosis with the nudibranch mollusk Rostanga alisae.

The discovery of symbiotic associations extends our understanding of the biological diversity in the aquatic environment and their impact on the host's ecology. Of particular interest are nudibranchs that unprotected by a shell and feed mainly on sponges. The symbiotic association of the nudibranch Rostanga alisae with bacteria was supported by ample evidence, including an analysis of cloned bacterial 16S rRNA genes and a fluorescent in situ hybridization analysis, and microscopic observations. A total of 74 clones belonging to the phyla α-, ß-, γ-Proteobacteria, Actinobacteria, and Cyanobacteria were identified. FISH confirmed that bacteriocytes were packed with Bradyrhizobium, Maritalea, Labrenzia, Bulkholderia, Achromobacter, and Stenotrophomonas mainly in the foot and notum epidermis, and also an abundance of Synechococcus cyanobacteria in the intestinal epithelium. An ultrastructural analysis showed several bacterial morphotypes of bacteria in epidermal cells, intestine epithelium, and in mucus layer covering the mollusk body. The high proportion of typical bacterial fatty acids in R. alisae indicated that symbiotic bacteria make a substantial contribution to its nutrition. Thus, the nudibranch harbors a high diversity of specific endo- and extracellular bacteria, which previously unknown as symbionts of marine invertebrates that provide the mollusk with essential nutrients. They can provide chemical defense against predators.

Mollusc N-glycosylation: Structures, Functions and Perspectives.

Molluscs display a sophisticated N-glycan pattern on their proteins, which is, in terms of involved structural features, even more diverse than that of vertebrates. This review summarises the current knowledge of mollusc N-glycan structures, with a focus on the functional aspects of the corresponding glycoproteins. Furthermore, the potential of mollusc-derived biomolecules for medical applications is addressed, emphasising the importance of mollusc research.