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Background: Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems)., Results: Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score., Conclusions: We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish., (© 2024. The Author(s).)

Polymesoda erosa is a mangrove clam known for its water filtration ability. This clam was investigated for its bioremediation potential and growth in synthetic wastewater during 40 days of incubation. Variation in the nutrient composition of water, biochemical composition of the clams, and metagenomic analysis of the microorganisms associated with clam tissue were carried out. Significant differences in the concentration of ammonia (p ≤ 0.01), nitrite (p ≤ 0.001), and nitrate (p ≤ 0.05) in the wastewater were observed between day 0 and day 40. A reduction of approximately 89% in ammonia concentration at the end of the experiment was recorded indicating nitrification activity. However, biochemical parameters showed negligible differences before and after the incubation experiment. Thus suggesting that the chemosynthetic-based nutrition aids in the survival of the clam as no organic matter was added to the medium. The substantial decline in levels of ammonia in the presence of clams as compared to its absence suggests the significant role of clams in improving the water quality. Furthermore, the metagenomic analysis of the gill tissue of P. erosa revealed ~ 50% of the microbial population to consist of nitrifiers. The study highlights the contribution by the nitrifers associated with the clams not only to its growth and resilience but also to bioremediation., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Yersinia enterocolitica (Ye) is a foodborne pathogen isolated from humans, food, animals, and the environment. Yersiniosis is the third most frequently reported foodborne zoonosis in the European Union. Ye species are divided into six biotypes 1A, 1B, 2, 3, 4, and 5, based on biochemical reactions and about 70 serotypes. Biotype 1A is non-pathogenic, 1B is highly pathogenic, and biotypes 2-5 have moderate or low pathogenicity. The reference analysis method for detecting pathogenic Ye species underestimates the presence of the pathogen due to similarities between Yersinia enterocolitica-like species and other Yersiniaceae and/or Enterobacteriaceae, low concentrations of distribution pathogenic strains and the heterogeneity of Yersinia enterocolitica species. In this study, the real-time PCR method ISO/TS 18867 to identify pathogenic biovars of Ye in bivalve molluscs was validated. The sensitivity, specificity and accuracy of the molecular method were evaluated using molluscs experimentally contaminated. The results fully agree with those obtained with the ISO 10273 method. Finally, we evaluated the presence of Ye in seventy commercial samples of bivalve molluscs collected in the Gulf of Naples using ISO/TS 18867. Only one sample tested resulted positive for the ail gene, which is considered the target gene for detection of pathogenic Ye according to ISO/TS 18867. Additionally, the presence of the ystB gene, used as target for Ye biotype 1A, was assessed in all samples using a real-time PCR SYBR Green platform. The results showed amplification ystB gene aim two samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Symbionts dominate planetary diversity and three primary symbiont diversification processes have been proposed: co-speciation with hosts, speciation by host-switching, and within-host speciation. The last mechanism is prevalent among members of an extraordinary marine symbiosis in the Indian River Lagoon, Florida, composed of a host mantis shrimp, Lysiosquilla scabricauda , and seven host-specific commensal vasconielline "yoyo" clams (Galeommatoidea) that collectively occupy two distinct niches: burrow-wall-attached, and host-attached/ectocommensal. This within-host symbiont radiation provides a natural experiment to test how symbiont coexistence patterns are regulated in a common ancestral habitat. The competitive exclusion principle predicts that sister taxa produced by adaptive speciation (with distinct morphologies and within-burrow niches) are most likely to coexist whereas the neutral theory predicts no difference among adaptive and non-adaptive sister taxa co-occurrence. To test these predictions, we engaged in (1) field-censusing commensal species assemblages; (2) trophic niche analyses; (3) laboratory behavioral observations. Although predicted by both models, the field census found no mixed-niche commensal assemblages: multi-species burrows were exclusively composed of burrow-wall commensals. Their co-occurrence matched random assembly process expectations, but presence of the single ectocommensal species had a highly significant negative effect on recruitment of all burrow-wall commensal species ( P  < 0.001), including on its burrow-wall commensal sister species ( P < 0.001). Our stable isotope data indicated that commensals are suspension feeders and that co-occurring burrow-wall commensals may exhibit trophic niche differentiation. The artificial burrow behavioral experiment yielded no evidence of spatial segregation among burrow-wall commensals, and it was terminated by a sudden breakdown of the host-commensal relationship resulting in a mass mortality of all commensals unattached to the host. This study system appears to contain two distinct, superimposed patterns of commensal distribution: (1) all burrow-wall commensal species; (2) the ectocommensal species. Burrow-wall commensals (the plesiomorphic condition) broadly adhere to neutral theory expectations of species assembly but the adaptive evolution of ectocommensalism has apparently led to ecological exclusion rather than coexistence, an inverse outcome of theoretical expectations. The ecological factors regulating the observed burrow-wall/ectocommensal exclusion are currently obscure but potentially include differential recruitment to host burrows and/or differential survival in "mixed" burrow assemblages, the latter potentially due to changes in host predatory behavior. Resampling host burrows during commensal recruitment peak periods and tracking burrow-wall commensal survival in host burrows with and without added ectocommensals could resolve this outstanding issue., Competing Interests: Jingchun Li is an Academic Editor for PeerJ., (©2024 Harrison et al.)

Bathymodioline mussels dominate deep-sea methane seep and hydrothermal vent habitats and obtain nutrients and energy primarily through chemosynthetic endosymbiotic bacteria in the bacteriocytes of their gill. However, the molecular mechanisms that orchestrate mussel host-symbiont interactions remain unclear. Here, we constructed a comprehensive cell atlas of the gill in the mussel Gigantidas platifrons from the South China Sea methane seeps (1100 m depth) using single-nucleus RNA-sequencing (snRNA-seq) and whole-mount in situ hybridisation. We identified 13 types of cells, including three previously unknown ones, and uncovered unknown tissue heterogeneity. Every cell type has a designated function in supporting the gill's structure and function, creating an optimal environment for chemosynthesis, and effectively acquiring nutrients from the endosymbiotic bacteria. Analysis of snRNA-seq of in situ transplanted mussels clearly showed the shifts in cell state in response to environmental oscillations. Our findings provide insight into the principles of host-symbiont interaction and the bivalves' environmental adaption mechanisms., Competing Interests: HW, KH, HZ, QZ, LC, JL, ZZ, HC, LZ, CL, MW, KC, PQ, CL No competing interests declared, (© 2023, Wang, He, Zhang et al.)

The role of the gut microbiota in host physiology has been previously elucidated for some marine organisms, but little information is available on their metabolic activity involved in transformation of environmental pollutants. This study assessed the metabolic profiles of the gut microbial cultures from grouper (Epinephelus coioides), green mussel (Perna viridis) and giant tiger prawn (Penaeus monodon) and investigated their transformation mechanisms to typical plastic additives. Community-level physiological profiling analysis confirmed the utilization profiles of the microbial cultures including carbon sources of carbohydrates, amines, carboxylic acids, phenolic compounds, polymers and amino acids, and the plastic additives of organophosphate flame retardants, tetrabromobisphenol A derivates and bisphenols. Using in vitro incubation, triphenyl phosphate (TPHP) was found to be rapidly metabolized into diphenyl phosphate by the gut microbiota as a representative ester-containing plastic additive, whereas the transformation of BPA (a representative phenol) was relatively slower. Interestingly, all three kinds of microbial cultures efficiently transformed the hepatic metabolite of BPA (BPA-G) back to BPA, thereby increasing its bioavailability in the body. The specific enzyme analysis confirmed the ability of the gut microbiota to perform the metabolic reactions. The results of 16S rRNA sequencing and network analysis revealed that the genera Escherichia-Shigella, Citrobacter, and Anaerospora were functional microbes, and their collaboration with fermentative microbes played pivotal roles in the transformation of the plastic additives. The structure-specific transformations by the gut microbiota and their distinct bioavailability deserve more attention in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Treated sewage contains a large diversity of pathogens that can be transmitted to the environment and, directly or indirectly, infect humans through water use (i.e., consumption, bathing, or irrigation). In urban environments, wastewater normally flows into wastewater treatment plants (WWTPs), where it is subjected to different processes in order to eliminate the greatest amount of waste. However, there are inequalities among European countries concerning wastewater management. In this context, we evaluate the potential of freshwater mussels to improve water quality (i.e., reduce bacterial abundance) in rivers receiving primary, secondary, or tertiary sewage-treated effluents. Additionally, because freshwater mussels are declining at a global scale and empty niches are progressively occupied by non-native counterparts, we evaluate if depauperate communities and the Asian clams, Corbicula genus, can provide equivalent ecosystem services (i.e., water quality improvement by biofiltration) formerly provided by diverse native communities. For this, an analysis of the bacterial biodiversity of the samples filtered by the different bivalve communities was carried out. The experimental approach was performed by metabarcoding the 16S rRNA gene using Illumina technologies. According to the results obtained, secondary treatment processes were effective in reducing the bacterial diversity. Furthermore, the waters filtered by the bivalves presented a lower bacterial abundance for certain genera. Biofiltration differs, however, among species, with Corbicula reducing a large number of taxa much more efficiently than native freshwater mussels in both diverse and depauperated communities. These results are likely related to Corbicula being a generalist species in front of native mussels, which may be more selective. Considering it is not possible to eradicate Corbicula from European rivers, its filtering capacity should be considered when managing freshwater ecosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Noé Ferreira-Rodriguez reports financial support was provided by Government of Galicia, Department of Culture Education and Universities. Margarita Poza reports financial support was provided by Carlos III Health Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Impact of high-pressure processing (HP-P) on microbial inactivation, protein oxidation, collagen fiber, and muscle structure of the edible portion (EP) of blood clams (BC) was investigated. Aerobic plate count, Vibrio parahaemolyticus, V. vulnificus, other Vibrio spp. and Shewanella algae counts were not detectable when HP-P pressure of ≥300 MPa was applied. Carbonyl, disulphide bond content, and surface hydrophobicity upsurged as HP-P with augmenting pressure was employed. Protein with ∼53 kDa appeared when HP-P at 100 and 200 MPa was implemented. Increased pressure enhanced gap formation and abnormal muscle cell structure arrangements. HP-P also affected connective tissue, causing size reduction and disruption of the collagen filament fibers. However, firmness and toughness of BC-EP with HP-P ≤ 300 MPa were comparable to those of the control. HP-P at 300 MPa was therefore appropriate for treatment of BC with maintained textural properties, while less protein oxidation, collagen fiber and muscle structure disruption occurred., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nanoplastics and engineering nanomaterials (ENMs) are contaminants of emerging concern (CECs), increasingly being detected in the marine environment and recognized as a potential threat for marine biota at the global level including in polar areas. Few studies have assessed the impact of these anthropogenic nanoparticles in the microbiome of marine invertebrates, however combined exposure resembling natural scenarios has been overlooked. The present study aimed to evaluate the single and combined effects of polystyrene nanoparticles (PS NP) as proxy for nanoplastics and nanoscale titanium dioxide (nano-TiO 2 ) on the prokaryotic communities associated with the gill tissue of the Antarctic soft-shell clam Laternula elliptica, a keystone species of marine benthos Wild-caught specimens were exposed to two environmentally relevant concentrations of carboxylated PS NP (PS-COOH NP, ∼62 nm size) and nano-TiO 2 (Aeroxide P25, ∼25 nm) as 5 and 50 μg/L either single and combined for 96h in a semi-static condition.Our findings show a shift in microbiome composition in gills of soft-shell clams exposed to PS NP and nano-TiO 2 either alone and in combination with a decrease in the relative abundance of OTU1 (Spirochaetaceae). In addition, an increase of gammaproteobacterial OTUs affiliated to MBAE14 and Methylophagaceae (involved in ammonia denitrification and associated with low-quality water), and the OTU Colwellia rossensis (previously recorded in polluted waters) was observed. Our results suggest that nanoplastics and nano-TiO 2 alone and in combination induce alterations in microbiome composition by promoting the increase of negative taxa over beneficial ones in the gills of the Antarctic soft-shell clam. An increase of two low abundance OTUs in PS-COOH NPs exposed clams was also observed. A predicted gene function analysis revealed that sugar, lipid, protein and DNA metabolism were the main functions affected by either PS-COOH NP and nano-TiO 2 exposure. The molecular functions involved in the altered affiliated OTUs are novel for nano-CEC exposures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, we investigate the mortality of the clam Meretrix petechialis facing a vibrio challenge under different temperatures and the underlying molecular mechanisms. Our experiment distinctly revealed that clam mortality was predominantly observed under high temperature, highlighting the critical impact of thermal stress on clam susceptibility to infection. Using RNA-seq, we further compared the global transcriptional response to vibrio in clam gills between high and low temperatures. Compared to other groups, the differentially expressed genes in vibrio-challenged group at high temperature associated with immunity, oxidative stress, and membrane transport. Key results show a weakened immune response in clams at high temperature, especially in the TNF signaling pathway, and a decrease in membrane transport efficiency, notably in SLC proteins. Additionally, high temperature enhanced pro-inflammatory related unsaturated fatty acid metabolism, leading to increased oxidative damage. This was further evidenced by our biochemical assays, which showed significantly higher levels of lipid peroxidation and protein carbonylation in clams at high temperature, indicating heightened oxidative damage. RT-PCR validation of selected DEGs corroborated the RNA-seq findings. Our findings contribute to the understanding of more frequent shellfish mortality in summer, emphasizing the role of temperature in pathogen response, elucidating the molecular mechanisms underlying the synergistic effect of pathogen and high temperature stresses. The key genes identified provide potential targets for resistance-assisted breeding. This research has significant implications for bivalve aquaculture and their physiology, particularly in light of global climate changes affecting marine ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Disease is a major threat to the economic, ecological and cultural services provided by wild bivalve populations. Over the past decade anecdotal reports on declining health of native bivalve populations around Aotearoa New Zealand have been supported by increasing observations of mass die-offs. Causes of declining health and mass die-offs of wild bivalves are not clear and could be due to a number of interactive and cumulative factors, including declining water quality, climate change, or disease. Pipi/kōkota (Paphies australis) within the Whangārei area (northern New Zealand) have suffered repeated die-offs and declining health since at least 2009. Baseline health data for wild native bivalve populations are scarce making it difficult to identify changes in pathogen infection prevalence and intensity and infer their importance to host health. This research aimed to examine and document the health of pipi in Whangārei with the objective of identifying factors that may contribute to their ill health and lack of population recovery. We sampled pipi from four sites within Whangārei, eight times across two years (total n = 640) to establish a health baseline using histopathology, general bacteriology, and qPCR for the intracellular bacteria Endozoicomonas spp. Three pipi mass die-offs occurred during the sampling window that were opportunistically sampled to compare against the health baseline established using healthy pipi. An increase in bacterial growth and a decrease in the abundance of Endozoicomonas spp. in mortality pipi was observed compared with the health baseline. Establishing a health baseline for pipi from Whangārei provided a benchmark to assess changes in a pipi population experiencing high mortality. Such data can help identify factors contributing to die-offs and to help inform what mitigation, if any, is possible in wild shellfish populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The interaction between environmental factors and Vibrio in bivalves is not well understood, despite the widely held belief that pathogen infection and seawater temperature significantly impact summer mortality. In the present study, we conducted simulated experiments to explore the effects of high temperature and Vibrio infection on the clam Meretrix petechialis. The survival curve analysis revealed that the combined challenge of high temperature and Vibrio infection (31°C-vibrio) led to significantly higher clam mortality compared to the groups exposed solely to Vibrio (27°C-vibrio), high temperature (31°C-control), and the control condition (27°C-control). Furthermore, PCoA analysis of 11 immune genes indicated that Vibrio infection predominated during the incubation period, with a gradual equilibrium between these factors emerging during the course of the infection. Additionally, our investigations into apoptosis and autophagy processes exhibited significant induction of mTOR and Bcl2 of the 31°C-vibrio group in the early challenge stage, followed by inhibition in the later stage. Oxidative stress analysis demonstrated a substantial additive effect on malondialdehyde (MDA) and glutathione (GSH) content in the combined challenge group compared to the control group. Comparative transcriptome analysis revealed a significant increase in differentially expressed genes related to immunity, such as complement C1q-like protein, C-type lectin, big defensin, and lysozyme, in the 31°C-vibrio group, suggesting that the synergistic effect of high temperature and Vibrio infection triggers more robust antibacterial immune responses. These findings provide critical insights for understanding the infection process and uncovering the causes of summer mortality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Bacterial symbionts, with their shorter generation times and capacity for horizontal gene transfer (HGT), play a critical role in allowing marine organisms to cope with environmental change. The closure of the Isthmus of Panama created distinct environmental conditions in the Tropical Eastern Pacific (TEP) and Caribbean, offering a "natural experiment" for studying how closely related animals evolve and adapt under environmental change. However, the role of bacterial symbionts in this process is often overlooked. We sequenced the genomes of endosymbiotic bacteria in two sets of sister species of chemosymbiotic bivalves from the genera Codakia and Ctena (family Lucinidae) collected on either side of the Isthmus, to investigate how differing environmental conditions have influenced the selection of symbionts and their metabolic capabilities. The lucinid sister species hosted different Candidatus Thiodiazotropha symbionts and only those from the Caribbean had the genetic potential for nitrogen fixation, while those from the TEP did not. Interestingly, this nitrogen-fixing ability did not correspond to symbiont phylogeny, suggesting convergent evolution of nitrogen fixation potential under nutrient-poor conditions. Reconstructing the evolutionary history of the nifHDKT operon by including other lucinid symbiont genomes from around the world further revealed that the last common ancestor (LCA) of Ca. Thiodiazotropha lacked nif genes, and populations in oligotrophic habitats later re-acquired the nif operon through HGT from the Sedimenticola symbiont lineage. Our study suggests that HGT of the nif operon has facilitated niche diversification of the globally distributed Ca. Thiodiazotropha endolucinida species clade. It highlights the importance of nitrogen availability in driving the ecological diversification of chemosynthetic symbiont species and the role that bacterial symbionts may play in the adaptation of marine organisms to changing environmental conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Morel-Letelier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Freshwater mussels (Mollusca: Unionidae) play a crucial role in freshwater river environments where they live in multi-species aggregations and often serve as long-lived benthic ecosystem engineers. Many of these species are imperiled and it is imperative that we understand their basic needs to aid in the reestablishment and maintenance of mussel beds in rivers. In an effort to expand our knowledge of the diet of these organisms, five species of mussel were introduced into enclosed systems in two experiments. In the first, mussels were incubated in water from the Clinch River (Virginia, USA) and in the second, water from a manmade pond at the Commonwealth of Virginia's Aquatic Wildlife Conservation Center in Marion, VA. Quantitative PCR and eDNA metabarcoding were used to determine which planktonic microbes were present before and after the introduction of mussels into each experimental system. It was found that all five species preferentially consumed microeukaryotes over bacteria. Most microeukaryotic taxa, including Stramenopiles and Chlorophytes were quickly consumed by all five mussel species. We also found that they consumed fungi but not as quickly as the microalgae, and that one species of mussel, Ortmanniana pectorosa, consumed bacteria but only after preferred food sources were depleted. Our results provide evidence that siphon feeding Unionid mussels can select preferred microbes from mixed plankton, and mussel species exhibit dietary niche differentiation., (© 2024. The Author(s).)

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea., (© 2024. The Author(s).)

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 10 5  CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy., Methods and Results: A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays., Conclusions: Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world"., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Previous studies have revealed tight metabolic complementarity between bivalves and their endosymbiotic chemosynthetic bacteria, but little is known about their interactions with ectosymbionts. Our analysis of the ectosymbiosis between a deep-sea scallop (Catillopecten margaritatus) and a gammaproteobacterium showed that bivalves could be highly interdependent with their ectosymbionts as well. Our microscopic observation revealed abundant sulfur-oxidizing bacteria (SOB) on the surfaces of the gill epithelial cells. Microbial 16S rRNA gene amplicon sequencing of the gill tissues showed the dominance of the SOB. An analysis of the SOB genome showed that it is substantially smaller than its free-living relatives and has lost cellular components required for free-living. Genomic and transcriptomic analyses showed that this ectosymbiont relies on rhodanese-like proteins and SOX multienzyme complex for energy generation, mainly on the Calvin-Benson-Bassham (CBB) cycle and peripherally on a phosphoenolpyruvate carboxylase for carbon assimilation. Besides, the symbiont encodes an incomplete tricarboxylic acid (TCA) cycle. Observation of the scallop's digestive gland and its nitrogen metabolism pathways indicates it does not fully rely on the ectosymbiont for nutrition. Analysis of the host's gene expression provided evidence that it could offer intermediates for the ectosymbiont to complete its TCA cycle and some amino acid synthesis pathways using exosomes, and its phagosomes, endosomes, and lysosomes might be involved in harvesting nutrients from the symbionts. Overall, our study prompts us to rethink the intimacy between the hosts and ectosymbionts in Bivalvia and the evolution of chemosymbiosis in general., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Importance: This study highlights diversity in iron acquisition and regulation in bacteria. The mechanisms of iron acquisition and its regulation in Teredinibacter turnerae , as well as its connection to cellulose utilization, a hallmark phenotype of T. turnerae , expand the paradigm of bacterial iron acquisition. Two of the four TonB genes identified in T. turnerae exhibit functional redundancy and play a crucial role in siderophore-mediated iron transport. Unlike typical TonB genes in bacteria, none of the TonB genes in T. turnerae are clearly iron regulated. This unusual regulation could be explained by another important finding in this study, namely, that the two TonB genes involved in iron transport are also essential for cellulose utilization as a carbon source, leading to the expression of TonB genes even under iron-rich conditions., Competing Interests: The authors declare no conflict of interest.

The rapid spread of the protozoan Haplosporidium pinnae is having a strong negative effect on Pinna nobilis populations. A case study on a residual population in Lake Faro (Sicily, Central Mediterranean), whose long-term monitoring has revealed a dramatic decline following the 2018-2020 mass mortality event, is presented. In the framework of such monitoring, we performed tissue sampling on nine living P. nobilis, detecting the pathogen in seven of them. In contrast, other pathogens associated with P. nobilis disease in other areas, i.e., Mycobacterium spp. and Vibrio mediterranei, were not recorded. The surviving individuals (approximately twenty) showed that brackish areas only weakly mitigate the effects of H. pinnae disease and might not be resolutive. Nevertheless, the results show that Lake Faro may constitute one of the last Mediterranean P. nobilis sanctuaries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell ( Pinna nobilis ), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae , although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae . Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei . The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis . The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Aim: Assessment of the fate of microbial contamination driven from treated wastewater disposal at a highly productive zone on a South European coastal lagoon (Ria Formosa)., Methods and Results: Microbial indicators of contamination (Total coliforms, Escherichia coli, and Enterococci) were evaluated monthly during September 2018-September 2020 at three study areas (Faro, Olhão, and Tavira) under different wastewater discharge flows and hydrodynamic conditions. Additional data on E. coli monitoring in bivalves, available from the national institution responsible for their surveillance was also considered. The maximum microbial contamination was found at Faro, the highest-load and less-flushed study area, contrasting the lowest contamination at Olhão, a lower-load and strongly flushed area. The wastewater impact decreased along the spatial dispersal gradients and during high water, particularly at Faro and Tavira study areas, due to a considerable dilution effect. Microbial contamination at Olhão increased during the summer, while at the other study areas seasonal evidence was not clear. Data also indicate that E. coli in bivalves from bivalve production zones next to the three study areas reflected the differentiated impact of the wastewater treatment plants effluents on the water quality of those areas., Conclusions: Effluent loads together with local hydrodynamics, water temperature, solar radiation, precipitation, and land runoff as well as seabirds populations and environmentally adapted faecal or renaturelized bacterial communities, contributed to microbial contamination of the study areas., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

There are many studies revealed that metal-based nanoparticles (NPs) possess excellent bactericidal effect on multitudinous bacteria and fungi. However, the control effect of NPs as antimicrobial agents to against Vibrio parahaemolyticus infection remain in poorly understood for blood clam, Tegillarca granosa. In order to evaluate the effect, the changes in six physiological parameters and the immune-related genes expression of clams exposed to V. parahaemolyticus alone or along with NPs (nZnO or nCuO) were investigated in present study. Results showed that both tested NPs exerted prominent redemptive or mitigative effect in an inverse dose-dependent way on physiological indexes of clam, especially in the total counts, phagocytosis and the cell viability of haemocytes, as well as the concentration and activity of lysozymes, when co-exposed with Vibrio. Gene expression analysis showed NPs at a concentration of 0.1 mg/L generally mitigated the downregulation of immune-related genes after clam exposure to V. parahaemolyticus. The combination of 0.1 mg/mL nZnO and nCuO additives has been shown to significantly enhance the humoral immunity of blood clam, suggesting its potential as a protective measure against V. parahaemolyticus infection in T. granosa., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Recently, P. nobilis populations have suffered a tremendous reduction, with pathogens potentially playing a crucial role. Considering its highly endangered status, mechanisms leading to mass mortalities were examined in one or multiple pathogens infected populations. Thus, seasonal antioxidant enzymatic activities, hsp70 and catalase mRNA levels, were investigated in two different Greek populations, during mass mortality events in summer of 2020. Samples were collected from Fthiotis and Lesvos during February (T o C 14 ± 1.2 and 15 ± 1 respectively), April (T o C 18 ± 1.2 and 17 ± 1.3 respectively), and June (T o C 24.5 ± 1.5 and 21.5 ± 1.5 respectively) 2020. In July of the same year (T o C 26.5 ± 1.7 in Fthiotis and 24.5 ± 1.7 in Lesvos), no live specimens were found. All biochemical parameters and phylogenetic analysis suggest that pathogen infection increases P. nobilis sensitivity to water temperature, subsequently leading to mass mortality. The latter was obvious in Fthiotis individuals, in which Haplosporidium pinnae was also observed with Mycobacterium spp., compared to Lesvos individuals., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Microbial pollution in marine environments is one of the critical issues with regard to the sanitary status of recreational activities and seafood harvesting due to a potential contamination by pathogenic microorganisms. This review's objectives were to identify instances of bacterial, viral and protozoan parasite pollution in the Tunisian coastal region and to make recommendations for further research. Fecal indicators such as Escherichia coli and Salmonella spp. were detected in samples of clams and mussels. Vibrionaceae species were also recorded in seawater, sediment, fish and clams in different sites from north to south with the dominance of Vibrio alginolyticus. Bivalve mollusks collected from the Tunisian coast have been revealed to harbor viruses as well as protozoan parasites. Furthermore, the isolation of multidrug-resistant bacterial strains from Tunisian coastlines proves the significant spread and circulation of antibiotic resistance caused by the massive use of antibiotics. In conclusion, we suggest intensive monitoring and cutting-edge wastewater treatment technologies to enhance seawater quality and preserve the biodiversity of aquatic life. Rapid detection techniques for the most important pathogenic microorganisms in seafood and seawater must be also developed to reduce human health risk.

Oysters and mussels are known vectors of foodborne pathogens because of their immobile and filter-feeding nature leading to the accumulation of biological particles in their tissues. Accumulated bacteria which comes from the culture environment and unsanitary handling can cause food poisoning if these shellfish are consumed raw or partially processed. This study determined the incidence of bacterial pathogen contamination along the different channels of the oyster and mussel supply chain through a time-distribution simulation analysis. First, the route of the fresh bivalve products from a local farm to its market was established through interviews. From the data gathered, a simulation experiment was conducted following the observed time-temperature conditions and the actual bulk packaging material used by the traders. The presence of target pathogens Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae were detected using standard conventional culture techniques. Initial E. coli counts in both mussels and oysters were higher than the safety limit of 330 MPN in 100 g tissue. Interestingly, E. coli counts in mussels decreased after 6 h and maintained low numbers after more than 24 h postharvest. Counts in oysters however increased to 1000 MPN in 100 g tissue. V. parahaemolyticus in mussels and oysters showed a gradual increase in counts with increasing holding time albeit in numbers that are lower than the safety limit of 1000 cfu g -1 tissue. Qualitative detection of Salmonella and V. cholerae showed the presence of both pathogens in all the sampling points. All four pathogens were also detected in the culture waters and in the sediment. Results of the study showed that the culture environment and the handling practices contribute greatly to the pathogen contamination in oysters and mussels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exogenous applications of phage lysins against Vibrio parahaemolyticus (V. parahaemolyticus) are a challenge due to the gram-negative bacteria outer membrane barrier. This study aimed to improve the antibacterial effect of V. parahaemolyticus phage lysin Lysqdvp001 (Lys), the best-characterized lysin with lytic activity against multiple species of Vibrios, by using liposome delivery. Various kinds of Lys-loaded liposome (Lys-lip) systems were designed and tested. The antibacterial activities of cationic guar gum (CGG) containing liposomes were much higher than the other liposomes, causing >5 log 10 CFU/mL of reductions of V. parahaemolyticus in buffer and severely damaging the bacterial cell structure. Moreover, some CGG liposome formulations retained high antibacterial effect after both 60-80 °C heat treatments and freeze-drying. Besides, the most stable liposome formulation killed 99 % of V. parahaemolyticus in the seawater with live clams, and its depuration rate against the bacterial contaminated clams also reached 99 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Shipworms (Bivalvia, Teredinidae) are the principal consumers of wood in marine environments. Like most wood-eating organisms, they digest wood with the aid of cellulolytic enzymes supplied by symbiotic bacteria. However, in shipworms the symbiotic bacteria are not found in the digestive system. Instead, they are located intracellularly in the gland of Deshayes, a specialized tissue found within the gills. It has been independently demonstrated that symbiont-encoded cellulolytic enzymes are present in the digestive systems and gills of two shipworm species, Bankia setacea and Lyrodus pedicellatus , confirming that these enzymes are transported from the gills to the lumen of the gut. However, the mechanism of enzyme transport from gill to gut remains incompletely understood. Recently, a mechanism was proposed by which enzymes are transported within bacterial cells that are expelled from the gill and transported to the mouth by ciliary action of the branchial or food grooves. Here we use in situ immunohistochemical methods to provide evidence for a different mechanism in the shipworm B. setacea , in which cellulolytic enzymes are transported via the ducts of Deshayes, enigmatic structures first described 174 years ago, but whose function have remained unexplained.

Two sampling approaches for the growth of common or dominant bacteria from bivalve haemolymph were compared: (1) samples processed in the field immediately after collection (field samples), and (2) samples processed in the laboratory at least 24 h after collection (laboratory samples). The sampling approaches were compared on 210 marine bivalve molluscs Paphies subtriangulata and P. australis from two shallow intertidal sites in North Island New Zealand. The approaches were evaluated for the amount of bacterial growth, type of growth, and diversity of growth. Differences in amount and type of growth between the two sampling approaches were observed. Samples processed in the field from P. subtriangulata had significantly more bacterial growth, and a higher diversity of bacteria, including more common or dominant bacterial species. Laboratory samples had a higher proportion of samples with no growth, however common or dominant bacteria were still isolated from these samples. For P. australis, field samples more often had no bacterial growth and laboratory samples had a significantly higher number of common or dominant growth present. Field samples did however contain a higher diversity of bacteria. By conducting bacteriology on bivalves in either the field or the laboratory only, there may be limitations to determining the significance of a bacterial agent isolated. Sampling of both field and laboratory samples should be carried out where possible to optimise detection of important bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites . We determined that 2052S produces the signal N- decanoyl-l-homoserine lactone (C 10 -HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms. IMPORTANCE Bacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step toward linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

The clam Ruditapes philippinarum is an important species in the marine aquaculture industry in China. However, in recent years, the aquaculture of R. philippinarum has been negatively impacted by various bacterial pathogens. In this study, the transcriptome libraries of R. philippinarum showing different levels of resistance to challenge with Vibrio anguillarum were constructed and RNA-seq was performed using the Illumina sequencing platform. Host immune factors were identified that responded to V. anguillarum infection, including C-type lectin domain, glutathione S-transferase 9, lysozyme, methyltransferase FkbM domain, heat shock 70 kDa protein, Ras-like GTP-binding protein RHO, C1q, F-box and BTB/POZ domain protein zf-C2H2. Ten genes were selected and verified by RT-qPCR, and nine of the gene expression results were consistent with those of RNA-seq. The lectin gene in the phagosome pathway was expressed at a significantly higher level after V. anguillarum infection, which might indicate the role of lectin in the immune response to V. anguillarum . Comparing the results from R. philippinarum resistant and nonresistant to V. anguillarum increases our understanding of the resistant genes and key pathways related to Vibrio challenge in this species. The results obtained here provide a reference for future immunological research focusing on the response of R. philippinarum to V. anguillarum infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yin, Nie, Jiang and Yan.)

Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline's effect, OTU threshold and rarefaction had a minimal impact on all measurements., Competing Interests: The authors have declared that no competing interests exist.

In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens., (Copyright © 2021. Published by Elsevier Ltd.)

Prevalence, impact on shellfish resources and interspecific, spatial, and temporal variabilities of domoic acid (DA) in bivalves from Galicia (NW Spain) have been studied based on more than 25 years of monitoring data. The maximum prevalence (samples in which DA was detected) (100%) and incidence (samples with DA levels above the regulatory limit) (97.4%) were recorded in Pecten maximus , and the minimum ones in Mytilus galloprovincialis (12.6 and 1.1%, respectively). The maximum DA concentrations were 663.9 mg kg -1 in P. maximus and 316 mg kg - 1 in V enerupis corrugata . After excluding scallop P. maximus data, DA was found (prevalence) in 13.3% of bivalve samples, with 1.3% being over the regulatory limit. In general, the prevalence of this toxin decreased towards the North but not the magnitude of its episodes. The seasonal distribution was characterized by two maxima, in spring and autumn, with the later decreasing in intensity towards the north. DA levels decreased slightly over the studied period, although this decreasing trend was not linear. A cyclic pattern was observed in the interannual variability, with cycles of 4 and 11 years. Intoxication and detoxification rates were slower than those expected from laboratory experiments, suggesting the supply of DA during these phases plays an important role., Competing Interests: The authors declare no conflict of interest.

Salmonellosis is one of the most common foodborne illnesses in the world. The irrational use of antibiotics in medicine and in animal nutrition has greatly favored the emergence and spread of resistant strains of non-typhoid Salmonella. This study aims the determination of the prevalence of Salmonella in bivalve mollusks in Northern Morocco, as well as the molecular typing and antibiotic susceptibility testing of the strains isolated from positive samples. In total, 150 samples from shellfish composed of mussels (Mytilus galloprovincialis), clams (Callista chione and Ruditapes descussatus) and oysters (Magallana gigas). Isolated Salmonella were characterized by Molecular techniques PCR, MLST and MLVA, phylogenetically grouped by MLSA, and susceptibilities were determined for 30 antimicrobial drugs using microdilution method by the BD Phoenix Automated Microbiology System. Prevalence of Salmonella enterica subsp. enterica was 12.67%, grouped in four serovars identified as Chester, Hadar, Typhimurium and Kentucky. Five different MLST STs (sequence types) were detected, ST1954 being the most common, which was mostly found in Chester isolates. Forty-two percent of the isolates showed resistance to more than one antibiotic, especially trimethoprim, sulfa drugs, quinolones and β-lactam. There was a marked change in the serovars and antimicrobial resistance profiles of the Salmonella isolates in this study compared to those in previous studies., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

A cultivated form of bacteria (strain 2202) was isolated from the hemal fluid of the bivalve mollusk Modiolus kurilensis. Based on the set of data collected by genetic and physiological/biochemical analyses, the strain was identified as the species Pseudoalteromonas piscicida. Strain 2202 exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans, and Bacillus subtilis but not against Escherichia coli and Pseudomonas aeruginosa. These activities characterize the behavior of strain 2202 as predator-like and classify it as a facultative predator. Being part of the normal microflora in the hemolymph of M. kurilensis, when external conditions change, strain 2202 shows features of opportunistic microflora. The strain 2202 exhibits selective toxicity towards larvae of various invertebrates: it impairs the early development of Mytilus edulis, but not of Strongylocentrotus nudus. Thus, the selective manner in which P. piscicida strains interact with various species of microorganisms and eukaryotes should be taken into consideration when using their biotechnological potential as a probiotic in aquaculture, source of antimicrobial substances, and factors that prevent fouling., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The p38 mitogen-activated protein kinase (MAPK) is one important member of MAPK family and reported to serve a predominant function in regulating innate immunity after the occurrence of certain infection. In the present study, one novel p38 MAPK gene was acquired from Cyclina sinensis based on the RNA-seq analysis and designated as Csp38 MAPK. This novel gene contained a full length of 1781 bp, 1104 bp of which was deemed as open reading frames and gave rise to a peptide of 367 amino acids with a predicted molecular weight of 42.31 KDa. A conserved serine/threonine protein kinase (S&#95;Tkc) region along with a Thr-Gly-Tyr motif was discovered in the deduced sequence. According to the phylogenetic analysis, there was a close relationship between this kinase and Meretrix petechialis p38 MAPK. As for the expression pattern, this newly-identified Csp38 MAPK was ubiquitously distributed in several tissues throughout the body but with varied abundance. After the challenge of Vibrio anguillarum, both the transcription and phosphorylation level of Csp38 MAPK in hemolymph were coordinately altered with a time-dependent manner. Besides, with the application of double strand RNA homologous to myeloid differentiation factor 88 (MyD88) of C. sinensis, the activation of Csp38 MAPK was found to obviously decrease in hemolymph after the pathogen stimulation. Hence, our experimental data presented evidence for the potential involvement of p38 MAPK in response to bacterial invaders in C. sinensis, possibly facilitating the understanding for pathogen-induced innate immunity in clams., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The formation of extracellular traps (ETs) is an important innate immune mechanism that serves to combat different invading pathogens. In this study, zymosan significantly induced the formation of ETs in the hemocytes of Ruditapes philippinarum, and this effect was accompanied by translocation of the mitochondria to the cell surface. Zymosan stimulation clearly induced an increase in intracellular ROS and MPO production and an overexpression of ROS-related genes (PI3K, AKT and HIF). In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Conversely, mitochondrial superoxide inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. In addition, we found that zymosan-induced ETs showed antibacterial activities against gram-negative and gram-positive bacteria, such as Vibrio anguillarum, Vibrio harveyi, Escherichia coli and Micrococcus luteus. Taken together, these findings elucidated a new antibacterial approach for R. philippinarum and highlighted the role of mitochondria in the formation of zymosan-induced ETs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In the ocean, most hosts acquire their symbionts from the environment. Due to the immense spatial scales involved, our understanding of the biogeography of hosts and symbionts in marine systems is patchy, although this knowledge is essential for understanding fundamental aspects of symbiosis such as host-symbiont specificity and evolution. Lucinidae is the most species-rich and widely distributed family of marine bivalves hosting autotrophic bacterial endosymbionts. Previous molecular surveys identified location-specific symbiont types that "promiscuously" form associations with multiple divergent cooccurring host species. This flexibility of host-microbe pairings is thought to underpin their global success, as it allows hosts to form associations with locally adapted symbionts. We used metagenomics to investigate the biodiversity, functional variability, and genetic exchange among the endosymbionts of 12 lucinid host species from across the globe. We report a cosmopolitan symbiont species, Candidatus Thiodiazotropha taylori, associated with multiple lucinid host species. Ca. T. taylori has achieved more success at dispersal and establishing symbioses with lucinids than any other symbiont described thus far. This discovery challenges our understanding of symbiont dispersal and location-specific colonization and suggests both symbiont and host flexibility underpin the ecological and evolutionary success of the lucinid symbiosis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Purpose: Pinna nobilis (fan mussel) is one of the most important endemic bivalve molluscs in the Mediterranean and mass mortality events were observed in these mussels in recent years. In this study, we report mass mortalities caused by Haplosporidium pinnae , which has been spreading in the Mediterranean for 3 years, and reached the Çanakkale Strait, which is the entrance of the Marmara and the Black Sea., Material and Methods: Field observations during sampling and subsequent histopathological, biochemical, genetic, and microbiological analyses were carried out., Results: These analyses showed that H. pinnae infection spread among the natural beds of P. nobilis , causing severe tissue damage and oxidative stress. Our phylogenetic analyses suggested that the parasite spread through the Mediterranean much faster than thought. The results showed that vibriosis originating from Vibrio coralliilyticus , Vibrio tubiashii , Vibrio mediterranei , and Vibrio hispanicus , acted together with H. pinnae in infected individuals and caused death., Conclusion: It is highly probable that the spread of H. pinnae to the Sea of Marmara and the Black Sea may occur earlier than expected, and it was concluded that mass deaths were caused by co-infection with H. pinnae and a geographically specific marine pathogen that can infect P. nobilis populations.

In the present study, a ubiquitin (designated as RpUbi) was identified and characterized from clam Ruditapes philippinarum. Phylogenetic analysis strongly suggested that RpUbi was a member of the ubiquitin family. In non-stimulated clams, RpUbi transcripts were constitutively expressed in all examined tissues, especially in the gills and hemocytes. After Vibrio anguillarum challenge, expression of RpUbi mRNA in hemocytes was significantly up-regulated. Recombinant RpUbi (rRpUbi) showed high antibacterial activity against Gram-positive and Gram-negative bacteria. Notably, membrane integrity and electrochemical assay indicated that rRpUbi could invade the inner layer. Moreover, DNA migration could be inhibited by rRpUbi in a concentration-dependent manner. In general, our results suggested that RpUbi played an important role in host defense against invading bacteria, perhaps through a DNA-binding process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii .

Relationships fueled by sulfide between deep-sea invertebrates and bacterial symbionts are well known, yet the diverse overlapping factors influencing symbiont specificity are complex. For animals that obtain their symbionts from the environment, both host identity and geographic location can impact the ultimate symbiont partner. Bacterial symbionts were analysed for three co-occurring species each of Bathymodiolus mussels and vestimentiferan tubeworms, from three deep methane seeps off the west coast of Costa Rica. The bacterial internal transcribed spacer gene was analysed via direct and barcoded amplicon sequencing to reveal fine-scale symbiont diversity. Each of the three mussel species (B. earlougheri, B. billschneideri and B. nancyschneideri) hosted genetically distinct thiotrophic endosymbionts, despite living nearly side-by-side in their habitat, suggesting that host identity is crucial in driving symbiont specificity. The dominant thiotrophic symbiont of co-occurring tubeworms Escarpia spicata and Lamellibrachia (L. barhami and L. donwalshi), on the other hand, was identical regardless of host species or sample location, suggesting lack of influence by either factor on symbiont selectivity in this group of animals. These findings highlight the specific, yet distinct, influences on the environmental acquisition of symbionts in two foundational invertebrates with similar lifestyles, and provide a rapid, precise method of examining symbiont identities., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Extracellular traps (ETs) have been found to be an important strategy of mammals to immobilize and kill invading microorganisms. In the present study, we observed the formation of ETs in the hemocytes of marine mollusks Ruditapes philippinarum in response to challenge from bacteria Vibrio anguillarum, and examined the potential factors and signaling pathways underling this process. We detected an increase of reactive oxygen species (ROS) and myeloperoxidase (MPO) production during ETosis, accompanied by significantly up-regulated expression of ROS-related and MPO genes. The suppression of ETs structures by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium chloride, DPI) and MPO inhibitor (aminobenzoic acid hydrazide, ABAH) further confirmed the essential roles ROS and MPO played in ETosis. Furthermore, ET production could be inhibited by phosphotidylinsitol-3-kinase (PI3K) inhibitor (LY294002) and extracellular regulated protein kinase (ERK) inhibitor (U0126), suggesting the idea that both the PI3K and ERK pathways were suggested to function during ETosis. In addition, the ETosis process was accompanied by enhancement of glycolysis-related enzymatic activities, e.g., pyruvate kinase (PK) and hexokinase (HK), and over-expression of the glycolysis-related genes, e.g., PK, HK and glucose transport protein (GLUT), indicating high involvement of glycolysis in the ETosis process. Furthermore, our scanning electron microscopy (SEM) observation and antibacterial activities test successfully showed the patterns how clam ETs entrapped and killed the invading V. anguillarum. Taken together, our results revealed that ETosis with bactericidal effect increased ROS, MPO and glycolysis level and carried out in a ROS-, MPO-, PI3K-ERK-dependent manner., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tyrosinase (EC1.14.18.1, TYR) is also called phenol oxidase, is not only involved in pigmentation but also plays an important role in modulating innate immunity in invertebrates. Tyrosinase is a copper containing metalloenzyme. The tyrosinase protein has two copper binding sites and three conserved histidines. In this study, 21 tyrosinase genes (RpTYR) were obtained from the whole genome of Ruditapes philippinarum. Their open reading frames were from 951 to 5424 aa, the range of predicted relative molecular weight from 36.72 to 203.81 kDa, and the range of isoelectric point from 4.72 to 9.88. Transcriptome analysis showed that RpTYR gene was expressed specifically in different developmental stages, adult tissues, four strains and two groups with different shell colors. Besides, the expression profiles of 21 RpTYRs were investigated against the immune response of R. philippinarum to a Vibrio challenge. The qPCR results showed that RpTYRs were involved in the immune response of R. philippinarum after Vibrio anguillarum challenge. This study provides preliminary evidence that the tyrosinases genes are involved in the immune defense and the potential immune function of R. philippinarum. Overall, these findings suggested that the expansion of TYR genes may play vital roles in larval development, the formation of shell color pattern, and immune response in R. philippinarum., (Copyright © 2020 Elsevier Inc. All rights reserved.)