Photoprotective mechanisms in Elysia species hosting Acetabularia chloroplasts shed light on host-donor compatibility in photosynthetic sea slugs.

Sacoglossa sea slugs have garnered attention due to their ability to retain intracellular functional chloroplasts from algae, while degrading other algal cell components. While protective mechanisms that limit oxidative damage under excessive light are well documented in plants and algae, the photoprotective strategies employed by these photosynthetic sea slugs remain unresolved. Species within the genus Elysia are known to retain chloroplasts from various algal sources, but the extent to which the metabolic processes from the donor algae can be sustained by the sea slugs is unclear. By comparing responses to high-light conditions through kinetic analyses, molecular techniques, and biochemical assays, this study shows significant differences between two photosynthetic Elysia species with chloroplasts derived from the green alga Acetabularia acetabulum. Notably, Elysia timida displayed remarkable tolerance to high-light stress and sophisticated photoprotective mechanisms such as an active xanthophyll cycle, efficient D1 protein recycling, accumulation of heat-shock proteins and α-tocopherol. In contrast, Elysia crispata exhibited absence or limitations in these photoprotective strategies. Our findings emphasize the intricate relationship between the host animal and the stolen chloroplasts, highlighting different capacities to protect the photosynthetic organelle from oxidative damage.

Metabolomic insights into the effects of seasonal shifts on the dynamic variation of non-volatile compounds of abalone (Haliotis discus hannai).

Abalone (Haliotis spp.) is a shellfish known for its exceptional nutritional value and significant economic worth. This study investigated the dynamic characteristics of non-volatile compounds over a year, including metabolites, lipids, nucleotides, and free amino acids (FAAs), which determined the nutritional quality and flavor of abalone. 174 metabolites and 371 lipids were identified and characterized, while 20 FAAs and 11 nucleotides were quantitatively assessed. These non-volatile compounds of abalone were fluctuated with months variation, which was consistent with the fluctuations of environmental factors, especially seawater temperature. Compared with seasonal variation, gender had less influence on these non-volatiles. June and July proved to be the optimal harvesting periods for abalone, with the levels of overall metabolites, lipids, FAAs, and nucleotides in abalone exhibiting a higher value in June and July over a year. Intriguingly, taurine covered 60% of the total FAAs and abalone could be used as dietary taurine supplementation.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

Heavy metal accumulation analysis using bivalve, sponge, sea urchin, and gastropod species as bioindicators.

A comparative assessment of heavy metal accumulation potential in four distinct marine benthic bioindicators: the bivalve Perna perna, the sponge Callyspongia fibrosa, the sea urchin Tripneustes gratilla, and the gastropod Purpura bufo were conducted. These organisms were collected from the same location, and the concentration of ten heavy metals was analyzed in water, sediment and various body parts of the organisms. The bioaccumulation potential was evaluated using the bio-water accumulation factor and bio-sediment accumulation factor. There was significant variation in the bioaccumulation potential of each organism with respect to different metals. The sponge proved to be a reliable indicator of Cd with a highest concentration of 2.60 µg/g. Sea urchin accumulated high concentrations of Cr (16.98 µg/g) and Pb (4.80 µg/g), whereas Cu was predominant (21.05 µg/g) in gastropod, followed by bivalve (17.67 µg/g). The concentration of metals in hard parts was found to be lower than in the tissues.

Genome-Wide Identification and Transcriptome Analysis of the Hsp70 Gene Family in Monodonta labio Reveals Its Role in Response to Nanoplastics Stress.

For marine invertebrates, the disruption of organismal physiology and behavior by nanoplastics (NPs) has been extensively reported. Heat shock proteins (Hsps) are important for redundant protein breakdown, environmental changes, and intracellular protein transport. An exhaustive identification of Hsp70 genes and an experiment where different concentrations of NPs were stressed were performed to study how Hsp70 genes respond to NPs stress in Monodonta labio. Our results identified 15 members of Hsp70 within the genome of M. labio and provided insights into their responses to different concentrations of acute NP stress. Phylogenetic analyses revealed extensive amplification of the Hsp70 genes from the Hsc70 subfamily, with gene duplication events. As a result of NP stress, five of fifteen genes showed significant upregulation or downregulation. Three Hsp70 genes were highly expressed at an NP concentration of 0.1 mg/L, and no genes were downregulated. At 10 mg/L, they showed significant upregulation of two genes and significant downregulation of two genes. At 1 mg/L treatment, three genes were significantly downregulated, and no genes were significantly upregulated. Moreover, a purifying selection was revealed using a selection test conducted on duplicate gene pairs, indicating functional redundancy. This work is the first thorough examination of the Hsp70s in Archaeogastropoda. The findings improve knowledge of Hsp70s in molluscan adaptation to NP stress and intertidal living and offer essential data for the biological study of M. labio.

Toxic effects of combined exposure to homoyessotoxin and nitrite on the survival, antioxidative responses, and apoptosis of the abalone Haliotis discus hannai.

Homoyessotoxin (homo-YTX) and nitrite (NO2-N), released during harmful dinoflagellate cell lysis adversely affect abalones. However, their toxicity mechanisms in shellfish remain unclear. This study investigated the economic abalone species Haliotis discus hannai exposed to varying concentrations of homo-YTX (0, 2, 5, and 10 µg L-1) and NO2-N (0, 3, and 6 mg L-1) on the basis of their 12 h LC50 values (5.05 µg L-1 and 4.25 mg L-1, respectively) and the environmentally relevant dissolved concentrations during severe dinoflagellate blooms, including mixtures. The test abalones were exposed to homo-YTX and NO2-N for 12 h. The mortality rate (D), reactive oxygen species (ROS) levels, antioxidant defense capabilities, and expression levels of antioxidant-related, Hsp-related, and apoptosis-related genes in abalone gills were assessed. Results showed that the combined exposure to homo-YTX and NO2-N increased the D and ROS levels and upregulated B-cell lymphoma-2 (BCL2)-associated X (BAX) and caspase3 (CASP3) expression levels while reducing glutathione peroxidase (GPx) activity and GPx, CuZnSOD, and BCL2 expression levels. High concentrations of homo-YTX (10 µg L-1) and NO2-N (6 mg L-1) solutions and the combinations of these toxicants inhibited the activities of superoxide dismutase (SOD) and catalase (CAT) and downregulated the expression levels of MnSOD, CAT, Hsp70, and Hsp90. The ROS levels were negatively correlated with the activities of SOD, CAT, and GPx and the expression levels of MnSOD, CuZnSOD, CAT, GPx, Hsp70, Hsp90, and BCL2. These results suggest that homo-YTX, in conjunction with NO2-N, induces oxidative stress, disrupts antioxidant defense systems, and triggers caspase-dependent apoptosis in the gills of abalone. ROS-mediated antioxidative and heat-shock responses and apoptosis emerge as potential toxicity mechanisms affecting the survival of H. discus hannai due to homo-YTX and NO2-N exposure.

Low-frequency noise impairs righting reflex behavior by disrupting central nervous system in the sea slug Onchidium reevesii.

Anthropogenic noise has significantly increased due to human activities, posing a threat to the health and survival of marine organisms. However, current studies have often emphasized its effects on the physiological aspects of marine organisms, while ignored the relationship between the neuroendocrine system and behavior. This study aimed to evaluate the righting behavior and relevant physiological functions of the central nervous system (CNS) in sea slug (Onchidium reevesii) exposed to low-frequency noise and subsequent noise removal. The duration of the sea slugs' righting reflex increased with longer noise exposure time. The degree of neuronal cell damage and apoptosis were significantly increased and relevant gene expressions were affected (Glu, AChE, FMRFamide and CaMKII) (P < 0.05). After the removal of noise, the righting reflex speed gradually recovered, and the degree of neuronal cell damage, apoptosis and the expression levels of genes continued to decrease. Pearson correlation analysis showed that the righting time was positively correlated with CNS tissue and DNA damage, apoptosis rate, and negatively correlated with the expression levels of genes. Therefore, low-frequency noise exposure causes damage to the CNS of sea slugs, subsequently impairing their normal behavior. Sea slugs exhibited partial recovery within 384 h after removing noise. These findings provide valuable insights into the effects of low-frequency noise on the CNS and behavior of marine invertebrates.

Molecular and structural analysis of Hdh-MIRP3 and its impact on reproductive regulation in female Pacific abalone, Haliotis discus hannai.

Molluscan insulin-related peptides (MIRP) play a crucial role in various biological processes, including reproduction and larval development in mollusk species. To investigate the involvement of MIRP in the ovarian development of Pacific abalone (Haliotis discus hannai), the Hdh-MIRP3 was cloned from cerebral ganglion (CG). Hdh-MIRP3 cDNA was 993 bp long, encoded a 13.22 kDa peptide, comprising 118 amino acids. Fluorescence in situ hybridization confirmed the localization of Hdh-MIRP3 in the CG and ovary. Molecular docking revealed that Hdh-MIRP3 binds to the N-terminal region of Hdh-IRP-R. Tissue expression analysis showed the highest Hdh-MIRP3 expression in the CG, followed by ovarian tissue. Hdh-MIRP3 expression was significantly upregulated in the CG and ovary during the ripe stage of seasonal ovarian development and in effective accumulative temperature conditioned abalone. Furthermore, siRNA silencing of Hdh-MIRP3 significantly downregulated the expression of four reproduction-related genes, including Hdh-GnRH, Hdh-GnRH-R, Hdh-IRP-R, and Hdh-VTG in both the CG and ovary, and Hdh-MIRP3 as well. These results indicate that Hdh-MIRP3 acts as a regulator of ovarian development in Pacific abalone. Additionally, expression analysis indicated that Hdh-MIRP3 plays a role in embryonic and larval development. Overall, the present findings elucidate the role of Hdh-MIRP3 in reproductive development in female Pacific abalone.

Insights into the genome of the 'Loco' Concholepas concholepas (Gastropoda: Muricidae) from low-coverage short-read sequencing: genome size, ploidy, transposable elements, nuclear RNA gene operon, mitochondrial genome, and phylogenetic placement in the family Muricidae.

BACKGROUND: The Peruvian 'chanque' or Chilean 'loco' Concholepas concholepas is an economically, ecologically, and culturally important muricid gastropod heavily exploited by artisanal fisheries in the temperate southeastern Pacific Ocean. In this study, we have profited from a set of bioinformatics tools to recover important biological information of C. concholepas from low-coverage short-read NGS datasets. Specifically, we calculated the size of the nuclear genome, ploidy, and estimated transposable elements content using an in silico k-mer approach, we discovered, annotated, and quantified those transposable elements, we assembled and annotated the 45S rDNA RNA operon and mitochondrial genome, and we confirmed the phylogenetic position of C. concholepas within the muricid subfamily Rapaninae based on translated protein coding genes. RESULTS: Using a k-mer approach, the haploid genome size estimated for the predicted diploid genome of C. concholepas varied between 1.83 Gbp (with kmer = 24) and 2.32 Gbp (with kmer = 36). Between half and two thirds of the nuclear genome of C. concholepas was composed of transposable elements. The most common transposable elements were classified as Long Interspersed Nuclear Elements and Short Interspersed Nuclear Elements, which were more abundant than DNA transposons, simple repeats, and Long Terminal Repeats. Less abundant repeat elements included Helitron mobile elements, 45S rRNA DNA, and Satellite DNA, among a few others.The 45S rRNA DNA operon of C. concholepas that encodes for the ssrRNA, 5.8S rRNA, and lsrRNA genes was assembled into a single contig 8,090 bp long. The assembled mitochondrial genome of C. concholepas is 15,449 bp long and encodes 13 protein coding genes, two ribosomal genes, and 22 transfer RNAs. CONCLUSION: The information gained by this study will inform the assembly of a high quality nuclear genome for C. concholepas and will support bioprospecting and biomonitoring using environmental DNA to advance development of conservation and management plans in this overexploited marine snail.

Effects of dietary astaxanthin on growth performance, muscle composition, non-specific immunity, gene expression, and ammonia resistance of juvenile ivory shell (Babylonia areolate).

Astaxanthin is one of the important immunopotentators in aquaculture. However, little is known about the physiological changes and stress resistance effects of astaxanthin in marine gastropods. In this study, the effects of different astaxanthin concentrations (0, 25, 50, 75, and 100 mg/kg) on the growth, muscle composition, immune function, and resistance to ammonia stress in Babylonia areolata were investigated after three months of rearing. With the increase in astaxanthin content, the weight gain rate (WGR), specific growth rate (SGR), and survival rate (SR) of B. areolata showed an increasing trend. The 75-100 mg/kg group was significantly higher than the control group (0 mg/kg). There was no significant difference in the flesh shell ratio (FSR), viscerosomatic index (VSI), and soft tissue index (STI) of the experimental groups. Astaxanthin (75 mg/kg) significantly increased muscle crude protein content and increased hepatopancreas alkaline phosphatase (AKP), superoxide dismutase (SOD), and catalase (CAT) activity. Astaxanthin (75-100 mg/kg) significantly increased the total antioxidant capacity (T-AOC) and acid phosphatase (ACP) of the hepatopancreas and decreased the malondialdehyde (MDA) content of B. areolata. Astaxanthin significantly induced the expression levels of functional genes, such as SOD, Cu/ZnSOD, ferritin, ACP, and CYC in hepatopancreas and increased the survival rate of B. areolata under ammonia stress. The addition of 75-100 mg/kg astaxanthin to the feed improved the growth performance, muscle composition, immune function, and resistance to ammonia stress of B. areolata.

Cloning, characterization, and spatio-temporal expression patterns of HdhSPARC and its responses to multiple stressors.

SPARC is an extracellular Ca2+-binding, secreted glycoprotein that plays a dynamic role in the growth and development of organisms. This study aimed to describe the isolation, characterization, and expression analysis of HdhSPARC in Pacific abalone (Haliotis discus hannai) to infer its potential functional role. The isolated HdhSPARC was 1633 bp long, encoding a polypeptide of 284 amino acid residues. Structurally, the SPARC protein in abalone is comprised of three biological domains. However, the structure of this protein varied between vertebrates and invertebrates, as suggested by their distinct clustering patterns in phylogenetic analysis. In early development, HdhSPARC was variably expressed, and higher expression was found in veliger larvae. Moreover, HdhSPARC was highly expressed in juvenile abalone with rapid growth compared to their slower-growing counterparts. Among the testicular development stages, the growth stage exhibited higher HdhSPARC expression. HdhSPARC was also upregulated during muscle remodeling and shell biomineralization, as well as in response to different stressors such as heat shock, LPS, and H2O2 exposure. However, this gene was downregulated in Cd-exposed abalone. The present study first comprehensively characterized the HdhSPARC gene, and its spatio-temporal expressions were analyzed along with its responses to various stressors.

Exposure to bisphenol A and fiber-type microplastics induce oxidative stress and cell damage in disk abalone Haliotis discus hannai: Bioaccumulation and toxicity.

Along with environmental pollution caused by rapid economic development and industrialization, plastic waste is emerging as a global concern in relation to marine ecosystems and human health. Among the microplastics, fiber-type microfibers (MF) and bisphenol A (BPA), which are widely used as plasticizers, do not decompose well in the ocean, and tend to accumulate in organisms, generating an increased oxidative stress response. This study investigated the abalones' antioxidant and cell death responses following exposure to the environmental pollutants MF and BPA. Levels of malondialdehyde (MDA) and DNA damage increased over time, demonstrating the degree of lipid peroxidation and DNA damage in abalones exposed to individual and combined environmental conditions of MF and BPA. Compared to the single MF and BPA exposure groups, the combined exposure group showed a higher expression of antioxidant enzymes. A similar pattern was seen in the expression of the apoptosis enzyme caspase-3. Both MF and BPA caused oxidative stress and antioxidant enzymes were expressed to alleviate it, but it is believed that cell damage occurred because the stress level exceeded the allowed range.

Crustacean cardioactive peptide signaling system in the gastropod mollusk Pacific abalone.

Crustacean cardioactive peptide (CCAP) signaling systems have been characterized in a diverse range of protostomes, representatively in arthropods. The cyclic CX5C-type CCAP regulates various biological activities through CCAP receptors (CCAPRs), which are orthologous to neuropeptide S receptors (NPSRs) in deuterostomes. However, the CCAPRs of the lophotrochozoa remain poorly characterized; therefore, the relationship between the CCAP, NPS, and CX4C-type oxytocin/vasopressin (OT/VP) signaling systems is unclear. In this study, we identified a CCAP precursor and two CCAPR isoforms in the Pacific abalone (Haliotis discus hannai; Hdh). The Hdh-CCAP precursor was found to harbor three CX5C-type and one CX4C-type CCAPs. The Hdh-CCAPRs displayed homology with protostome CCAPRs and deuterostome NPSRs, having characteristics of the rhodopsin-type G protein-coupled receptors. Phylogenetic analysis showed that lophotrochozoan CCAPRs, including Hdh-CCAPRs, form a monophyletic group distinct from arthropod CCAPRs. Reporter assays demonstrated that all examined Hdh-CCAPs and insect CCAP-induced intracellular Ca2+ mobilization and cAMP accumulation in Hdh-CCAPR-expressing HEK293 cells, whereas none of the CCAP peptides inhibited the forskolin-stimulated cAMP signaling pathway even at micromolar concentrations. In silico ligand-receptor docking models showed that the N-terminal FCN motifs of Hdh-CCAPs are deeply inserted inside the binding pocket of Hdh-CCAPR, forming extensive hydrophobic interactions. In mature Pacific abalone, the transcripts for Hdh-CCAP precursor and Hdh-CCAPR were highly expressed in the neural ganglia compared to the peripheral tissues. Collectively, this study characterized the first CCAP signaling system linked to both Ca2+/PKC and cAMP/PKA signal transduction pathways in gastropod mollusks and gives insights into the evolutional origins of deuterostomian NPS and OT/VP signaling systems.

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone.

INTRODUCTION: Neuropeptides regulate vital physiological processes in multicellular organisms, including growth, reproduction, metamorphosis, and feeding. Recent transcriptome analyses have revealed neuropeptide genes with potential roles in vertebrate and invertebrate growth and reproduction. Among these genes, haliotid growth-associated peptide (HGAP) was identified as a novel gene in abalone. METHODS: This study focused on HGAP in Pacific abalone (Haliotis discus hannai), where the complete cDNA sequence named Hdh-HGAP was identified and characterized. Samples from different experiments, such as metamorphosis, juvenile abalone growth, gonad development stages, muscle remodeling, and starvation, were collected for mRNA expression analysis. RESULTS: The sequence spans 552 bp, encoding 96 amino acids with a molecular weight of 10.96 kDa. Expression analysis revealed that Hdh-HGAP exhibited higher levels in muscle tissue. Notably, during metamorphosis, Hdh-HGAP exhibited greater expression in the trochophore, veliger, and juvenile stages than in the cell division stages. Regarding growth patterns, Hdh-HGAP was highly expressed during rapid growth compared to stunted, minimal, and normal growth. In gonadal development, Hdh-HGAP mRNA reached its highest expression level during the ripening stage, indicating a potential role in gonadal cell proliferation and maturation. The in vivo effects of GnRH on gonad development and the expression of the Hdh-HGAP neuropeptide indicate its involvement in regulating reproduction in Pacific abalone. While tissue remodeling is primarily governed by immune genes, Hdh-HGAP was also upregulated during muscle tissue remodeling. Conversely, Hdh-HGAP was downregulated during prolonged starvation. CONCLUSION: This study marks the first comprehensive exploration of the Hdh-HGAP neuropeptide gene in Pacific abalone, shedding light on its involvement in growth, reproduction, metamorphosis, tissue remodeling, and response to starvation, although regulatory mechanisms are mostly unknown.

Low-frequency noise aggravates the toxicity of cadmium in sea slug Onchdium reevesii.

Industrial development not only triggers heavy metal pollution but also introduces a less easily discernible disturbance: low-frequency noise pollution. Low-frequency noise can disrupt wildlife behavior, potentially exerting complex effects through interacting with heavy metals. Nevertheless, the cumulative impacts of low-frequency noise and cadmium (Cd) pollution on marine organisms remain largely unexplored. This study aimed to evaluate the immune defense response of sea slugs (Onchdium reevesii) exposed to Cd (1.32 mg/L) and low-frequency noise (500 Hz, 1000 Hz). Our results show that Cd exposure results in Cd2+ accumulation in the sea slug's hepatopancreas, leading to a decrease in total antioxidant capacity (TAC) and a significant increase in enzyme activities, including glutathione (GSH), lipid peroxidation (LPO), and aspartate transferase (AST). Additionally, there is a substantial upregulation in the expression of genes related to tumor protein p53 (p53), Cytochrome C (CytC), Caspase 3, and Caspase 9, as well as metallothionein (MT) and heat shock protein 70 (Hsp70) genes. Concurrently, an excessive production of reactive oxygen species (ROS) occurs in the hemocytes, resulting in apoptosis and subsequent diminished cell viability, with these effects positively correlating with the exposure duration. Furthermore, when sea slugs were exposed to both Cd and low-frequency noise, there was a decrease in the hepatopancreas's antioxidant capacity and an enhancement in hemocytes immune responses, which positively correlated with low-frequency noise frequency. The comprehensive assessment of biomarker responses highlights that low-frequency noise has the potential to amplify the deleterious effects of Cd on sea slug physiology, with this negative impact positively linked to noise frequency. Consequently, our study underscores that the combined influence of low-frequency noise and Cd pollution magnifies the effects on sea slug health. This could potentially disrupt the population stability of this species within its natural habitat, providing fresh insights into the evaluation of cumulative environmental pollution risks.

Transcriptomic analysis revealed the dynamic response mechanism to acute ammonia exposure in the ivory shell, Babylonia areolata.

The ivory shell (Babylonia areolata) is an economically important shellfish in tropical and subtropical regions, but its intensive culture and biological characteristic of hiding in the sandy substrate make it highly susceptible to ammonia stress. In this study, we investigated the dynamic changes in histopathology, oxidative stress, and transcriptome of the ivory shell at different time points under high concentration (60 mg/L) ammonia exposure. With prolonged exposure to stress, vacuoles appeared in the hepatopancreas while cell volume and intercellular space increased. The activities of superoxide dismutase (SOD) and catalase (CAT) decreased significantly under high concentrations of ammonia-induced stress while malondialdehyde (MDA) levels increased significantly. Integrated analysis of differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA), and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that lipid transport primarily contributed to maintaining cellular homeostasis during the early stage of stress (6 and 12 h). Subsequently, a significant upregulation of oxidation-reduction reactions occurred at the middle stage (24 h), leading to oxidative stress. Finally, during the later stage (48 h), metabolic decomposition provided energy for survival maintenance. Additionally, lysosome and apoptosis were identified as potential key pathways in response to acute ammonia toxicity. Overall, our findings suggest that ivory shells can respond to acute ammonia toxicity via immune and antioxidant defense mechanisms but sustained high concentrations may cause irreversible damage. This study provides valuable insights into the response mechanism of mollusks towards ammonia and serves as a data reference for breeding ammonia-tolerant varieties of ivory shells.

Investigation of Immune Responses in Giant African Snail, Achatina immaculata, against a Two-Round Lipopolysaccharide Challenge.

As one of the 100 most-threatening invasive alien species, the giant African snail (Achatina immaculata) has successfully invaded and established itself in most areas of southern China. Protection against recurrent pathogen infections is vital to biological invasion. Enhanced immune protection has been previously found in other invertebrates, but not in the unique immune system of the giant African snail. In the present study, the survival rate of the giant African snail was recorded following a second infection with lethal doses of Escherichia coli after a previous first injection using lipopolysaccharide (LPS), and the mechanism of immune enhancement was investigated by examining the cellular and transcriptomic response of the giant African snail after two successive stimuli using LPS. Snails injected first with LPS, sterilized physiologic (0.9%) saline (SPS), phosphate-buffered saline (PBS) or untreated (Blank) were rechallenged at 7d with E. coli (Ec), and were named as LPS + Ec, SPS + Ec, PBS + Ec, Ec, and Blank. The log-rank test shows the survival rate of the LPS + Ec group as significantly higher than that of other control groups after the second injection (p < 0.05). By performing cell counting and BrdU labeling on newly generated circulating hemocytes, we found that the total hemocyte count (THC) and the ratio of BrdU-positive cells to total cells increased significantly after primary stimulation with LPS and that they further increased after the second challenge. Then, caspase-3 of apoptosis protease and two antioxidant enzyme activities (CAT and SOD) increased significantly after infection, and were significantly higher in the second response than they had been in the first round. Moreover, transcriptome analysis results showed that 84 differentially expressed genes (DEGs) were expressed at higher levels in both the resting and activating states after the second immune response compared to the levels observed after the first challenge. Among them, some DEGs, including Toll-like receptor 4 (TLR4) and its downstream signaling molecules, were verified using qRT-PCR and were consistent with the transcriptome assay results. Based on gene expression levels, we proposed that these genes related to the TLR signaling cascade participate in enhanced immune protection. All results provide evidence that enhanced immune protection exists in the giant African snail.

A de novo evolved domain improves the cadmium detoxification capacity of limpet metallothioneins.

Metallothioneins (MTs) constitute an important family of metal binding proteins. Mollusk MTs, in particular, have been used as model systems to better understand the evolution of their metal binding features and functional adaptation. In the present study two recombinantly produced MTs, LgiMT1 and LgiMT2, and their de novo evolved γ domain, of the marine limpet Lottia gigantea, were analyzed by electronic spectroscopy and mass spectrometry. Both MT proteins, as well as their γ domains, exhibit a strong binding specificity for Cd(II), but not for Zn(II) or Cu(I). The LgiMTs' γ domain renders an MII4(SCys)10 cluster with an increased Cd stoichiometry (binding 4 instead of 3 Cd2+ ions), representing a novel structural element in the world of MTs, probably featuring an adamantane 3D structure. This cluster significantly improves the Cd(II)-binding performance of the full length proteins and thus contributes to the particularly high Cd coping capacity observed in free-living limpets.

Dynamic Multiple Reaction Monitoring of amphipod Gammarus fossarum caeca expands molecular information for understanding the impact of contaminants.

Mass spectrometry in multiple reaction monitoring (MRM) mode is a powerful technique that can provide highly selective, multiplexed, and reproducible quantification of peptides derived from proteins. Ideal for the application of molecular biomarkers in biomonitoring surveys, MRM tools have been recently developed to quantify sets of pre-selected biomarkers in freshwater sentinel species. Still limited to the validation and application phase of biomarkers, dynamic MRM (dMRM) acquisition mode has increased the multiplexing capacity of mass spectrometers, expanding opportunities to explore proteome modulations in sentinel species. This study evaluated the feasibility to propose dMRM tools for investigating sentinel species proteomes at the organ level and demonstrated its potential for screening contaminant effects and discovering new protein biomarkers. As a proof of concept, a dMRM assay was developed to comprehensively capture the functional proteome of the caeca of Gammarus fossarum, a freshwater crustacean, commonly used as a sentinel species in environmental biomonitoring. The assay was then used to assess the effects of sub-lethal concentrations of cadmium, silver, and zinc on gammarid caeca. Results showed dose-response and specific metal effects on caecal proteomes, with a slight effect of zinc compared to the two non-essential metals. Functional analyses indicated that cadmium affected proteins involved in carbohydrate metabolism, digestive and immune processes, while silver affected proteins related to oxidative stress response, chaperonin complexes and fatty acid metabolism. Based on these metal-specific signatures, several proteins modulated in a dose-dependent manner were proposed as candidate biomarkers for tracking the level of these metals in freshwater ecosystems. Overall, this study highlights the potential of dMRM to decipher the specific modulations of proteome expression induced by contaminant exposure and pinpoints specific response signatures, offering new perspectives for the de novo identification and development of biomarkers in sentinel species.

Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai.

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1-28 aa), a transforming growth factor-ß (TGF-ß) propeptide (38-272 aa), and a mature TGF-ß peptide (314-416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.