Phylogenomics of Neogastropoda: the backbone hidden in the bush.

The molluscan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in early stages of diversification, generating a 'bush' at the base of their evolutionary tree, that has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1,817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, three prove problematic: First, uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade, or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major 'core Neogastropoda' grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analysed phylogenetic signal of targeted loci in relation to potential biases, and we propose most probable resolutions in the latter two recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

Characterization and Functional Analysis of Fads Reveals Δ5 Desaturation Activity during Long-Chain Polyunsaturated Fatty Acid Biosynthesis in Dwarf Surf Clam Mulinia lateralis.

Fatty acid desaturases (Fads), as key enzymes in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), catalyze the desaturation between defined carbons of fatty acyl chains and control the degree of unsaturation of fatty acids. In the present study, two Fads genes, designated MulFadsA and MulFadsB, were identified from the genome of the dwarf surf clam Mulinia lateralis (Mollusca, Mactridae), and their spatiotemporal expression was examined. MulFadsA and MulFadsB contained the corresponding conserved functional domains and clustered closely with their respective orthologs from other mollusks. Both genes were expressed in the developmental stages and all tested adult tissues of M. lateralis, with MulFadsA exhibiting significantly higher expression levels in adult tissues than MulFadsB. Subsequently, the effects of dietary microalgae on Fads expressions in the dwarf surf clam were investigated by feeding clams with two types of unialgal diets varying in fatty acid content, i.e., Chlorella pyrenoidosa (Cp) and Platymonas helgolandica (Ph). The results show that the expressions of MulFads were significantly upregulated among adult tissues in the Cp group compared with those in the Ph group. In addition, we observed the desaturation activity of MulFadsA via heterologous expression in yeasts, revealing Δ5 desaturation activity toward PUFA substrates. Taken together, these results provide a novel perspective on M. lateralis LC-PUFA biosynthesis, expanding our understanding of fatty acid synthesis in marine mollusks.

Sensing and regulation of long-chain polyunsaturated fatty acids pool in marine mollusks: Characterization of UBXD8 from the razor clam Sinonovacula constricta.

The razor clam Sinonovacula constricta is known for its richness in long-chain polyunsaturated fatty acids (LC-PUFA, C ≥ 20). Previously, we demonstrated that it possesses a complete LC-PUFA biosynthetic pathway. However, the mechanisms by which it senses the LC-PUFA pool to regulate their biosynthesis remain unclear. Here, we presented the LC-PUFA sensor UBXD8 as a critical molecule in this intriguing process. The S. constricta UBXD8 (ScUBXD8) shared all characteristic features of its mammalian counterpart and exhibited high mRNA levels in digestive tissues, suggesting its functional role in this bivalve species. By purification of ScUBXD8 protein in vitro, we discovered its ability to sense unsaturated fatty acids (UFA, C ≥ 14) but not saturated ones, as evidenced by polymerization detection. Furthermore, the intensity of ScUBXD8 polymerization increased progressively with longer acyl chain lengths, greater unsaturation degrees, and higher UFA concentrations. Exceptionally, for those located at the same node in LC-PUFA biosynthetic pathway, ScUBXD8 displayed a stronger sensitivity to n-6 UFA compared to n-3 UFA. These results suggested a critical role for ScUBXD8 in balancing fatty acids composition and ratio of n-6/n-3 UFA in S. constricta. Moreover, the UAS domain was confirmed essential for ScUBXD8 polymerization. Through knockdown of ScUbxd8 gene in vivo, there were significant shifts in expression patterns of genes related to LC-PUFA biosynthesis, concurrently influencing fatty acids compositions. These results suggested that ScUBXD8 likely plays a regulatory role in LC-PUFA biosynthesis, possibly through the INSIG-SREBP pathway. Collectively, this study proposed that S. constricta might maintain LC-PUFA homeostasis through UBXD8 to regulate their biosynthesis.

Expanded Polystyrene-Debris-Induced Genotoxic Effect in Littoral Organisms.

Expanded polystyrene (EPS) is a major component of plastic debris in the environment, including coastal and littoral zones. EPS is widely used in various industries including fish farming and aquaculture, which poses a serious potential threat not only to cultured hydrobionts but also to all living organisms, including humans. This paper presents the results of experimental studies on the effects of EPS (0.024 m2/L) on marine mollusks Mytilus trossulus and Tegula rustica, which are typical inhabitants of the upper littoral of Peter the Great Bay (Sea of Japan), belonging to different systematic groups and differing in the type of nutrition. The results of biochemical marker analysis showed the development of oxidative stress processes. Thus, increasing malondialdehyde content relative to control values was registered in the digestive glands of M. trossulus and T. rustica. In the cells of the digestive glands of M. trossulus, integral antioxidant activity decreased more than 1.5 times compared with that of the control. The change in the concentration of protein carbonyls was unchanged in M. trossulus, whereas in T. rustica, there was a 1.5-fold increase. EPS exposure also resulted in significant DNA damage in the studied mollusks-the damage level increased 2.5-fold in M. trossulus and 1.5-fold in T. rustica relative to the control, indicating the genotoxic potential of EPS litters.

Bivalve digestive epithelial virosis (DEV): A cause of disease or a natural process?

Epithelial hyperplasia and sloughing of the digestive gland in bivalve mollusks are a global phenomenon and occur in species of commercial interest and cultural significance to indigenous peoples. Where hemocytosis, hyperplasia, and necrosis of digestive tubule cells have been observed associated with electron-dense uncoated virus-like particles (VLPs) 25-45 nm in diameter, the condition has been named digestive epithelial virosis (DEV). This condition has been associated with mortalities of some bivalve species in New Zealand. Similar digestive gland alterations, but without detection of associated VLPs, have been reported in other bivalve species worldwide and are termed "DEV-like" since no virus link has been demonstrated. It remains unclear if DEV is an infectious condition and whether associated VLPs are the cause, a contributor, or simply associated with the observed condition. It is also unclear whether DEV or DEV-like conditions pose a biosecurity or economic threat, or alternatively, whether they reflect a natural cyclic event that does not require disease management. In this mini-review, we summarize the history of digestive epithelial alteration with VLPs (i.e., DEV) or without observation of VLPs (i.e., DEV-like), and we examine the evidence for and against viral-like particles as the cause of DEV in bivalves. We also explore other viral afflictions of bivalves and non-infectious agents, such as harmful algae and xenotoxins, that could elicit similar tissue alterations. Future recommendations for approaches to identify key risk factors that lead to the development of digestive epithelial alterations such as DEV include histological characterization of the digestive gland of marine mollusks; the use of metagenome analysis to design primers that could be used for detection of VLPs and to study host microbiota; disease challenges demonstrating that DEV causes pathology and the relationship between DEV intensity and morbidity/mortality.

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways.

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8­hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Effects of tralopyril on histological, biochemical and molecular impacts in Pacific oyster, Crassostrea gigas.

Recently, the toxic effects of tralopyril, as a new antifouling biocide, on aquatic organisms have aroused widespread attention about the potential toxicity. However, the mechanism of tralopyril on marine mollusks has not been elaborated clearly. In this study, the histological, biochemical and molecular impacts of tralopyril on adult Crassostrea gigas were investigated. The results indicated that the 96 h LC50 of tralopyril to adult Crassostrea gigas was 911 µg/L. After exposure to tralopyril (0, 40, 80 and 160 µg/L) for 6 days, the mantle mucus secretion coverage ratio of Crassostrea gigas was increased with a dose-dependent pattern. Catalase (CAT) activity was significantly increased, amylase (AMS) activity, acid phosphatase (ACP) activity and calcium ion (Ca2+) concentration significantly decreased. Meanwhile, integrated biomarker responses (IBR) index suggested that higher concentrations of tralopyril caused severer damage to Crassostrea gigas. In addition, the mRNA expression levels of biomineralization related genes in the mantle were significantly upregulated. Collectively, this study firstly revealed the histological, biochemical and molecular impacts of tralopyril exposure on adult Crassostrea gigas, which provided new insights for understanding the toxicity of tralopyril in marine mollusks.

Combined effects of ocean warming and acidification on the larval stages of the European abalone Haliotis tuberculata.

This study examined the physiological responses of the larval stages of Haliotis tuberculata, an economically important abalone, to combined temperature (17 °C and 19 °C) and pH (ambient pH and -0.3 units, i.e., +200% increase in seawater acidity) in a full factorial experiment. Tissue organogenesis, shell formation, and shell length significantly declined due to low pH. High temperature significantly increased the proportion of fully shelled larvae at 24 h post-fertilization (hpf), but increased the proportion of unshelled larvae at 72 hpf. Percentage of swimming larvae at 24 hpf, 72 hpf and 96 hpf significantly declined due to high temperature, but not because of low pH. Larval settlement increased under high temperature, but was not affected by low pH. Despite the fact that no interaction between temperature and pH was observed, the results provide additional evidence on the sensitivity of abalone larvae to both low pH and high temperature. This may have negative consequences for the persistence of abalone populations in natural and aquaculture environments in the near future.

Transcriptional response of key metabolic and stress response genes of a nuculanid bivalve, Lembulus bicuspidatus from an oxygen minimum zone exposed to hypoxia-reoxygenation.

Benthic animals inhabiting the edges of marine oxygen minimum zones (OMZ) are exposed to unpredictable large fluctuations of oxygen levels. Sessile organisms including bivalves must depend on physiological adaptations to withstand these conditions. However, as habitats are rather inaccessible, physiological adaptations of the OMZ margin inhabitants to oxygen fluctuations are not well understood. We therefore investigated the transcriptional responses of selected key genes involved in energy metabolism and stress protection in a dominant benthic species of the northern edge of the Namibian OMZ, the nuculanid clam Lembulus bicuspidatus,. We exposed clams to normoxia (~5.8 ml O2 l-1), severe hypoxia (36 h at ~0.01 ml O2 l-1) and post-hypoxic recovery (24 h of normoxia following 36 h of severe hypoxia). Using newly identified gene sequences, we determined the transcriptional responses to hypoxia and reoxygenation of the mitochondrial aerobic energy metabolism (pyruvate dehydrogenase E1 complex, cytochrome c oxidase, citrate synthase, and adenine nucleotide translocator), anaerobic glycolysis (hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase, and aldolase), mitochondrial antioxidants (glutaredoxin, peroxiredoxin, and uncoupling protein UCP2) and stress protection mechanisms (a molecular chaperone HSP70 and a mitochondrial quality control protein MIEAP) in the gills and the labial palps of L. bicuspidatus. Exposure to severe hypoxia transcriptionally stimulated anaerobic glycolysis (including HK and PEPCK), antioxidant protection (UCP2), and quality control mechanisms (HSP70 and MIEAP) in the gills of L. bicuspidatus. Unlike UCP2, mRNA levels of the thiol-dependent mitochondrial antioxidants were not affected by hypoxia-reoxygenation stress. Transcript levels of marker genes for aerobic energy metabolism were not responsive to oxygen fluctuations in L. bicuspidatus. Our findings highlight the probable importance of anaerobic succinate production (via PEPCK) and mitochondrial and proteome quality control mechanisms in responses to oxygen fluctuations of the OMZ bivalve L.bicuspidatus. The reaction of L.bicuspidatus to oxygen fluctuations implies parallels to that of other hypoxia-tolerant bivalves, such as intertidal species.

Molecular and functional characterization of a glycosylated Galactose-Binding lectin from Mytilus californianus.

Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/ß fold with a predominance of ß structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the ß-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding.

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.