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Mollusks are a major component of animal biodiversity and play a critical role in ecosystems and global food security. The Pacific oyster, Crassostrea (Magallana) gigas , is the most farmed bivalve mollusk in the world and is becoming a model species for invertebrate biology. Despite the extensive research on hemocytes, the immune cells of bivalves, their characterization remains elusive. Here, we were able to extensively characterize the diverse hemocytes and identified at least seven functionally distinct cell types and three hematopoietic lineages. A combination of single-cell RNA sequencing, quantitative cytology, cell sorting, functional assays, and pseudo-time analyses was used to deliver a comprehensive view of the distinct hemocyte types. This integrative analysis enabled us to reconcile molecular and cellular data and identify distinct cell types performing specialized immune functions, such as phagocytosis, reactive oxygen species production, copper accumulation, and expression of antimicrobial peptides. This study emphasized the need for more in depth studies of cellular immunity in mollusks and non-model invertebrates and set the ground for further comparative immunology studies at the cellular level. Pacific oysters are vital to the ecosystem. They are also a popular seafood and are increasingly used in life science research as a model to represent animals without a backbone (known as invertebrates). However, these oysters are prone to deadly infections caused by bacteria and viruses. Like humans, oysters and other invertebrates need an immune system to fight infections. Immune cells called hemocytes – which travel through the oyster’s body in a blood-like fluid called hemolymph – help eliminate bacteria, viruses and other disease-causing microbes by absorbing them, releasing toxic molecules, and producing natural antibiotics called antimicrobial peptides. However, it is still unclear how many types of hemocytes Pacific oysters have or what each type does. De La Forest Divonne et al. used single-cell RNA sequencing and other cell biology techniques to study the genetic activity and anatomy of immune cells in the Pacific oyster. The experiments confirmed that the oysters have at least seven distinct types of hemocytes, each with a specialized immune role, for example, some eat microbes while others produce antimicrobial peptides. The team also mapped how immature hemocytes develop into mature hemocytes with these specialist roles. This work provides the first detailed atlas of oyster immune cells and reveals how their immune system is organized and operates. A deeper understanding of oyster immune cells may guide the development of new strategies to reduce disease outbreaks in farmed or wild oysters. Before these benefits can be realized, future studies must test how each type of hemocyte responds to actual infections and explore whether targeted treatments or breeding programs can enhance the immune systems of farmed oysters.

The increase in marine diseases, particularly in economically important mollusks, is a growing concern. Among them, the Pacific oyster ( Crassostrea gigas ) production faces challenges from several diseases, such as the Pacific Oyster Mortality Syndrome (POMS) or vibriosis. The microbial education, which consists of exposing the host immune system to beneficial microorganisms during early life stages is a promising approach against diseases. This study explores the concept of microbial education using controlled and pathogen-free bacterial communities and assesses its protective effects against POMS and Vibrio aestuarianus infections, highlighting potential applications in oyster production. We demonstrate that it is possible to educate the oyster immune system by adding microorganisms during the larval stage. Adding culture based bacterial mixes to larvae protects only against the POMS disease while adding whole microbial communities from oyster donors protects against both POMS and vibriosis. The efficiency of immune protection depends both on oyster origin and on the composition of the bacterial mixes used for exposure. No preferential protection was observed when the oysters were stimulated with their sympatric strains. Furthermore, the added bacteria were not maintained into the oyster microbiota, but this bacterial addition induced long term changes in the microbiota composition and oyster immune gene expression. Our study reveals successful immune system education of oysters by introducing beneficial microorganisms during the larval stage. We improved the long-term resistance of oysters against critical diseases (POMS disease and Vibrio aestuarianus infections) highlighting the potential of microbial education in aquaculture.

Background In holobiont, microbiota is known to play a central role on the health and immunity of its host. Then, understanding the microbiota, its dynamic according to the environmental conditions and its link to the immunity would help to react to potential dysbiosis of aquacultured species. While the gut microbiota is highly studied, in marine invertebrates the hemolymph microbiota is often set aside even if it remains an important actor of the hemolymph homeostasis. Indeed, the hemolymph harbors the factors involved in the animal homeostasis that interacts with the microbiota, the immunity. In the Southwest Pacific, the high economical valued shrimp Penaeus stylirostris is reared in two contrasted sites, in New Caledonia (NC) and in French Polynesia (FP). Results We characterized the active microbiota inhabiting the hemolymph of shrimps while considering its stability during two seasons and at a one-month interval and evidenced an important microbial variability between the shrimps according to the rearing conditions and the sites. We highlighted specific biomarkers along with a common core microbiota composed of 6 ASVs. Putative microbial functions were mostly associated with bacterial competition, infections and metabolism in NC, while they were highly associated with the cell metabolism in FP suggesting a rearing site discrimination. Differential relative expression of immune effectors measured in the hemolymph of two shrimp populations from NC and FP, exhibited higher level of expression in NC compared to FP. In addition, differential relative expression of immune effectors was correlated to bacterial biomarkers based on their geographical location. Conclusions Our data suggest that, in Pacific shrimps, both the microbiota and the expression of the immune effectors could have undergone differential immunostimulation according to the rearing site as well as a geographical adaptative divergence of the shrimps as an holobiont, to their rearing sites. Further, the identification of proxies such as the core microbiota and site biomarkers, could be used to guide future actions to monitor the bacterial microbiota and thus preserve the productions.

Colistin is a widespread last resort antibiotic for treatment of multidrug-resistant bacteria. The recent worldwide emergence of colistin resistance (Col-R) conferred by mcr-1 in human pathogens has raised concern, but the putative sources and reservoirs of novel mcr genes in the marine environment remain underexplored. We observed a high prevalence of Col-R, particularly in Vibrio isolated from European coastal waters by using the same cohorts of oysters as bioaccumulators in three sites across Europe. The high sequence diversity found in the mcr/eptA gene family was geographically structured, particularly for three novel eptA gene variants, which were restricted to the Mediterranean (France, Spain) and occurred as a dgkA-eptA operon. The RstA/RstB two component system was shown to control both the dgkA-eptA operon and the Col-R phenotype. The analysis of 29 427 Vibrionaceae genomes revealed that this mechanism of intrinsic resistance is prevalent and specific to the Harveyi clade, which includes the human pathogens Vibrio parahaemolyticus and Vibrio alginolyticus. The operon conferred colistin-resistance when transferred to sensitive non-Vibrio strains. In general, eptA gene variants are widespread and evolved with the Vibrio lineage. They occur in clade-specific genomic environments, suggesting that eptA expression responds to distinct environmental signals across the Vibrio phylogeny. However, we also identified mobile eptA paralogues that have been recently transferred between and within Vibrio clades. This highlights Vibrio as a potential source of Col-R mechanisms, emphasizing the need for enhanced surveillance to prevent colistin-resistant infections in coastal areas.

Mollusks are a major component of animal biodiversity and play a critical role in ecosystems and global food security. The Pacific oyster, Crassostrea (Magallana) gigas , is the most farmed bivalve mollusk in the world and is becoming a model species for invertebrate biology. Despite the extensive research on hemocytes, the immune cells of bivalves, their characterization remains elusive. Here, we were able to extensively characterize the diverse hemocytes and identified at least seven functionally distinct cell types and three hematopoietic lineages. A combination of single-cell RNA sequencing, quantitative cytology, cell sorting, functional assays, and pseudo-time analyses was used to deliver a comprehensive view of the distinct hemocyte types. This integrative analysis enabled us to reconcile molecular and cellular data and identify distinct cell types performing specialized immune functions, such as phagocytosis, reactive oxygen species production, copper accumulation, and expression of antimicrobial peptides. This study emphasized the need for more in depth studies of cellular immunity in mollusks and non-model invertebrates and set the ground for further comparative immunology studies at the cellular level. Pacific oysters are vital to the ecosystem. They are also a popular seafood and are increasingly used in life science research as a model to represent animals without a backbone (known as invertebrates). However, these oysters are prone to deadly infections caused by bacteria and viruses. Like humans, oysters and other invertebrates need an immune system to fight infections. Immune cells called hemocytes – which travel through the oyster’s body in a blood-like fluid called hemolymph – help eliminate bacteria, viruses and other disease-causing microbes by absorbing them, releasing toxic molecules, and producing natural antibiotics called antimicrobial peptides. However, it is still unclear how many types of hemocytes Pacific oysters have or what each type does. De La Forest Divonne et al. used single-cell RNA sequencing and other cell biology techniques to study the genetic activity and anatomy of immune cells in the Pacific oyster. The experiments confirmed that the oysters have at least seven distinct types of hemocytes, each with a specialized immune role, for example, some eat microbes while others produce antimicrobial peptides. The team also mapped how immature hemocytes develop into mature hemocytes with these specialist roles. This work provides the first detailed atlas of oyster immune cells and reveals how their immune system is organized and operates. A deeper understanding of oyster immune cells may guide the development of new strategies to reduce disease outbreaks in farmed or wild oysters. Before these benefits can be realized, future studies must test how each type of hemocyte responds to actual infections and explore whether targeted treatments or breeding programs can enhance the immune systems of farmed oysters.

Mollusks are a major component of animal biodiversity and play a critical role in ecosystems and global food security. The Pacific oyster, Crassostrea (Magallana) gigas, is the most farmed bivalve mollusk in the world and is becoming a model species for invertebrate biology. Despite the extensive research on hemocytes, the immune cells of bivalves, their characterization remains elusive. Here we were able to extensively characterize the diverse hemocytes and identified at least seven functionally distinct cell types and three hematopoietic lineages. A combination of single-cell RNA sequencing, quantitative cytology, cell sorting, functional assays and pseudo-time analyses was used to deliver a comprehensive view of the distinct hemocyte types. This integrative analysis enabled us to reconcile molecular and cellular data and identify distinct cell types performing specialized immune functions, such as phagocytosis, reactive oxygen species production, copper accumulation, and expression of antimicrobial peptides. This study emphasized the need for more in depth studies of cellular immunity in mollusks and non-model invertebrates and set the ground for further comparative immunology studies at the cellular level.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Revision of the paper to improve the readability of the figures, enrich the introduction, and the discussion.

Polymicrobial diseases significantly impact the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease that impacts oyster production and is prevalent worldwide. Analysis of POMS-infected oysters on the French North Atlantic coast revealed that the disease involves co-infection with the endemic ostreid herpesvirus 1 (OsHV-1) and virulent bacterial species such as Vibrio crassostreae. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with the OsHV-1 virus during pathogenesis. We resolved the Vibrio population structure in oysters from a Mediterranean ecosystem and investigated their functions in POMS development. We find that Vibrio harveyi and Vibrio rotiferianus are the predominant species found in OsHV-1-diseased oysters and show that OsHV-1 is necessary to reproduce the partition of the Vibrio community observed in the field. By characterizing the interspecific interactions between OsHV-1, V. harveyi and V. rotiferianus, we find that only V. harveyi synergizes with OsHV-1. When co-infected, OsHV-1 and V. harveyi behave cooperatively by promoting mutual growth and accelerating oyster death. V. harveyi showed high virulence potential in oysters and dampened host cellular defenses, making oysters a more favorable niche for microbe colonization. We next investigated the interactions underlying the co-occurrence of diverse Vibrio species in diseased oysters. We found that V. harveyi harbors genes responsible for the biosynthesis and uptake of a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, a cheater that efficiently colonizes oysters during POMS without costly investment in host manipulation nor metabolite sharing. By connecting field-based approaches, laboratory infection assays and functional genomics, we have uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We showed that cooperative behaviors contribute to synergy between bacterial and viral co-infecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling such behaviors or countering their effects opens new avenues for mitigating polymicrobial diseases.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://doi.org/10.12770/173c0414-a3ca-4a79-b6b2-cd424ee90593

Mollusks are a major component of animal biodiversity and play a critical role in ecosystems and global food security. The Pacific oyster, Crassostrea (Magallana) gigas, is the most farmed bivalve mollusk in the world and is becoming a model species for invertebrate biology. Despite the extensive research on hemocytes, the immune cells of bivalves, their characterization remains elusive. Here we were able to extensively characterize the diverse hemocytes and identified at least seven functionally distinct cell types and three hematopoietic lineages. A combination of single-cell RNA sequencing, quantitative cytology, cell sorting, functional assays and pseudo-time analyses was used to deliver a comprehensive view of the distinct hemocyte types. This integrative analysis enabled us to reconcile molecular and cellular data and identify distinct cell types performing specialized immune functions, such as phagocytosis, reactive oxygen species production, copper accumulation, and expression of antimicrobial peptides. This study emphasized the need for more in depth studies of cellular immunity in mollusks and non-model invertebrates and set the ground for further comparative immunology studies at the cellular level.

The precise localization of postsynaptic receptors opposite neurotransmitter release sites is essential for synaptic function. This alignment relies on adhesion molecules, intracellular scaffolds, and a growing class of extracellular scaffolding proteins. However, how these secreted proteins are retained at synapses remains unclear. We addressed this question using C. elegans neuromuscular junctions, where Punctin, a conserved extracellular synaptic organizer, positions postsynaptic receptors. We identified CLE-1, the ortholog of collagens XV/XVIII, as a key stabilizer of Punctin. Punctin and CLE-1B, the main isoform present at neuromuscular junctions, form a complex and rely on each other for synaptic localization. Punctin undergoes cleavage, and in the absence of CLE-1, specific fragments are lost, resulting in the mislocalization of cholinergic receptors to GABAergic synapses. Additionally, CLE-1 regulates receptor levels independently of Punctin. These findings highlight a crucial extracellular complex that maintains synapse identity.

Oyster farming is a significant industry worldwide, but it is threatened by various diseases such as Pacific Oyster Mortality Syndrome or vibriosis. V. aestuarianus is a major cause of mortality for market-size oysters, resulting in significant economic losses for oyster farmers. Among the various control methods developed, probiotics appear to be a promising approach. More specifically, the use of the antibacterial activity of bacteria from the natural microbiota of the oyster Magallana gigas appears to be a sustainable solution against V. aestuarianus infections. Our study investigated the probiotic potential of bacteria isolated from the microbiota of M. gigas oysters. We screened a collection of 334 bacteria against eight target pathogens, including V. aestuarianus, and identified 78 bacteria with antibacterial activity for which eight retained this activity in their culture supernatants. Five strains were selected for further testing and exposed to oysters prior to V. aestuarianus infection. Our results show that four strains significantly reduced oyster mortality, with a maximum reduction of 70%. In addition, changes in oyster microbiota composition were observed following exposure, but the administered bacteria were not detected in the microbiota. Our findings demonstrate the potential of oyster microbiota-derived bacteria as probiotics for disease control in oyster farming. This approach could provide a sustainable and environmentally friendly solution for the oyster farming industry. Further research is needed to understand the underlying mechanisms and to develop effective probiotic-based strategies for preventing V. aestuarianus infection.