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Trans-generational immune priming (TGIP) refers to the transfer of the parental immunological experience to its progeny. This may result in offspring protection from repeated encounters with pathogens that persist across generations. Although extensively studied in vertebrates for over a century, this phenomenon has only been identified 20 years ago in invertebrates. Since then, invertebrate TGIP has been the focus of an increasing interest, with half of studies published during the last few years. TGIP has now been tested in several invertebrate systems using various experimental approaches and measures to study it at both functional and evolutionary levels. However, drawing an overall picture of TGIP from available studies still appears to be a difficult task. Here, we provide a comprehensive review of TGIP in invertebrates with the objective of confronting all the data generated to date to highlight the main features and mechanisms identified in the context of its ecology and evolution. To this purpose, we describe all the articles reporting experimental investigation of TGIP in invertebrates and propose a critical analysis of the experimental procedures performed to study this phenomenon. We then investigate the outcome of TGIP in the offspring and its ecological and evolutionary relevance before reviewing the potential molecular mechanisms identified to date. In the light of this review, we build hypothetical scenarios of the mechanisms through which TGIP might be achieved and propose guidelines for future investigations.

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.

In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster ( Crassostrea gigas ), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates. Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas . Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide. IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster ( Crassostrea gigas ), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.

Biomphalysins are β-Pore Forming Toxins (β-PFT) identified in the planorbid Biomphalaria glabrata that belong to the aerolysin-like protein family. Despite potentially diverse biochemical activities, very few eukaryotic aerolysin-related proteins have been extensively studied. Most of the data refers to their discovery in genomes or to transcriptional activity. The involvement of biomphalysins in the immune response of Biomphalaria glabrata has been studied previously, especially regarding biomphalysin 1, which can bind and kill Schistosoma mansoni mother sporocysts. However, the repartition of biomphalysin 1 protein in B. glabrata has yet to be defined. The transcriptional behavior of the 22 other biomphalysin genes following immune challenge also remains uncharacterized. Therefore, herein, we investigate for the first time the tissular distribution of biomphalysin 1 (and 2) in B. glabrata by histological and cytological analyses through immunofluorescence approaches, notably unveiling unexpected tissue location that are involved in biomphalysin 1 synthesis. Structural predictions of the 23 members of the family have been updated using predictions based on aminoacyl spatial pair representation (AlphaFold2), highlighting unique features of the small lobe. In addition, mass spectrometry-based proteomic data more precisely predicted the regions of post-translational cleavage of biomphalysin 1. Transcriptional activity of the biomphalysin genes was explored, after which the plasmatic presence of the biomphalysin proteins was investigated in naive and S. mansoni -infected snails. The ability of native biomphalysin 1 (and 2) to bind several cell types was also investigated and correlated with the lytic ability of plasma toward the exposed cells, highlighting the central role occupied by biomphalysin 1 (and 2) in the humoral immunity of B. glabrata .

Detection of pathogens by all living organisms is the primary step needed to implement a coherent and efficient immune response. This implies a mediation by different soluble and/or membrane-anchored proteins related to innate immune receptors called PRRs (pattern-recognition receptors) to trigger immune signaling pathways. In most invertebrates, their roles have been inferred by analogy to those already characterized in vertebrate homologs. Despite the induction of their gene expression upon challenge and the presence of structural domains associated with the detection of pathogen-associated molecular patterns in their sequence, their exact role in the induction of immune response and their binding capacity still remain to be demonstrated. To this purpose, we developed a fast interactome approach, usable on any host-pathogen couple, to identify soluble proteins capable of directly or indirectly detecting the presence of pathogens. To investigate the molecular basis of immune recognition specificity, different pathogens (Gram-positive bacterium, ; Gram-negative, ; yeast, ; and metazoan parasites, or ) were exposed to hemocyte-free hemolymph from the gastropod . Twenty-three different proteins bound to pathogens were identified and grouped into three different categories based on their primary function. Each pathogen was recognized by a specific but overlapping set of circulating proteins in mollusk's hemolymph. While known PRRs such as C-type lectins were identified, other proteins not known to be primarily involved in pathogen recognition were found, including actin, tubulin, collagen, and hemoglobin. Confocal microscopy and specific fluorescent labeling revealed that extracellular actin present in snail hemolymph was able to bind to yeasts and induce their clotting, a preliminary step for their elimination by the snail immune system. Aerolysin-like proteins (named biomphalysins) were the only ones involved in the recognition of all the five pathogens tested, suggesting a sentinel role of these horizontally acquired toxins. These findings highlight the diversity and complexity of a highly specific innate immune sensing system. It paves the way for the use of such approach on a wide range of host-pathogen systems to provide new insights into the specificity and diversity of immune recognition by innate immune systems.

Discoveries made over the past ten years have provided evidence that invertebrate antiparasitic responses may be primed in a sustainable manner, leading to the failure of a secondary encounter with the same pathogen. This phenomenon called \"immune priming\" or \"innate immune memory\" was mainly phenomenological. The demonstration of this process remains to be obtained and the underlying mechanisms remain to be discovered and exhaustively tested with rigorous functional and molecular methods, to eliminate all alternative explanations. In order to achieve this ambitious aim, the present study focuses on the Lophotrochozoan snail, Biomphalaria glabrata, in which innate immune memory was recently reported. We provide herein the first evidence that a shift from a cellular immune response (encapsulation) to a humoral immune response (biomphalysin) occurs during the development of innate memory. The molecular characterisation of this process in Biomphalaria/Schistosoma system was undertaken to reconcile mechanisms with phenomena, opening the way to a better comprehension of innate immune memory in invertebrates. This prompted us to revisit the artificial dichotomy between innate and memory immunity in invertebrate systems.

Schistosomiasis is considered as a significant public health problem, imposing a deeper understanding of the intricate interplay between parasites and their hosts. Unfortunately, current invasive methodologies employed to study the compatibility and the parasite development impose limitations on exploring diverse strains under various environmental conditions, thereby impeding progress in the field. In this study, we demonstrate the usefulness for the trematode parasite Schistosma mansoni , leveranging a fluorescence-imaging-based approach that employs fluorescein 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethylfluorescein diacetate (CMAC) as organism tracker for intramolluscan studies involving the host snail Biomphalaria glabrata. These probes represent key tools for qualitatively assessing snail infections with unmatched accuracy and precision. By monitoring the fluorescence of parasites within the snail vector, our method exposes an unprecedented glimpse into the host-parasite compatibility landscape. The simplicity and sensitivity of our approach render it an ideal choice for evolutionary studies, as it sheds light on the intricate mechanisms governing host-parasite interactions. Fluorescent probe-based methods play a pivotal role in characterizing factors influencing parasite development and phenotype of compatibility, paving the way for innovative, effective, and sustainable solutions to enhance our understanding host-parasite immunobiological interaction and compatibility.

The freshwater snail Biomphalaria glabrata is an intermediate host of Schistosoma mansoni , the agent of human intestinal schistosomiasis. However, much is to be discovered about its innate immune system that appears as a complex black box, in which the immune cells (called hemocytes) play a major role in both cellular and humoral response towards pathogens. Until now, hemocyte classification has been based exclusively on cell morphology and ultrastructural description and depending on the authors considered from 2 to 5 hemocyte populations have been described. In this study, we proposed to evaluate the hemocyte heterogeneity at the transcriptomic level. To accomplish this objective, we used single cell RNA sequencing (scRNAseq) technology coupled to a droplet-based system to separate hemocytes and analyze their transcriptome at a unique cell level in naive Biomphalaria glabrata snails. We were able to demonstrate the presence of 7 hemocyte transcriptomic populations defined by the expression of specific marker genes. As a result, scRNAseq approach showed a high heterogeneity within hemocytes, but provides a detailed description of the different hemocyte transcriptomic populations in B. glabrata supported by distinct cellular functions and lineage trajectory. As a main result, scRNAseq revealed the 3 main population as a super-group of hemocyte diversity but, on the contrary, a great hemocytes plasticity with a probable capacity of hemocytes to engage to different activation pathways. This work opens a new field of research to understand the role of hemocytes particularly in response to pathogens, and towards S. mansoni parasites.

Thioester-containing proteins (TEPs) superfamily is known to play important innate immune functions in a wide range of animal phyla. TEPs are involved in recognition, and in the direct or mediated killing of several invading organisms or pathogens. While several TEPs have been identified in many invertebrates, only one TEP (named BgTEP) has been previously characterized in the freshwater snail, Biomphalaria glabrata. As the presence of a single member of that family is particularly intriguing, transcriptomic data and the recently published genome were used to explore the presence of other BgTEP related genes in B. glabrata. Ten other TEP members have been reported and classified into different subfamilies: Three complement-like factors (BgC3-1 to BgC3-3), one α-2-macroblobulin (BgA2M), two macroglobulin complement-related proteins (BgMCR1, BgMCR2), one CD109 (BgCD109), and three insect TEP (BgTEP2 to BgTEP4) in addition to the previously characterized BgTEP that we renamed BgTEP1. This is the first report on such a level of TEP diversity and of the presence of macroglobulin complement-related proteins (MCR) in mollusks. Gene structure analysis revealed alternative splicing in the highly variable region of three members (BgA2M, BgCD109, and BgTEP2) with a particularly unexpected diversity for BgTEP2. Finally, different gene expression profiles tend to indicate specific functions for such novel family members.

Background Freshwater snails are the intermediate hosts of a large variety of trematode flukes such as Schistosoma mansoni responsible for one of the most important parasitic diseases caused by helminths, affecting 67 million people worldwide. Recently, the WHO Global Vector Control Response 2017–2030 (GVCR) programme reinforced its message for safer molluscicides as part of required strategies to strengthen vector control worldwide. Here, we present the essential oil from Eryngium triquetrum as a powerful product with molluscicide and parasiticide effect against S. mansoni and the snail intermediate host Biomphalaria glabrata. Methods In the present study, we describe using several experimental approaches, the chemical composition of E. triquetrum essential oil extract and its biological effects against the snail B. glabrata and its parasite S. mansoni . Vector and the free-swimming larval stages of the parasite were exposed to different oil concentrations to determine the lethal concentration required to produce a mortality of 50% (LC 50 ) and 90% (LC 90 ). In addition, toxic activity of this essential oil was analyzed against embryos of B. glabrata snails by monitoring egg hatching and snail development. Also, short-time exposure to sublethal molluscicide concentrations on S. mansoni miracidia was performed to test a potential effect on parasite infectivity on snails. Mortality of miracidia and cercariae of S. mansoni is complete for 5, 1 and 0.5 ppm of oil extract after 1 and 4 h exposure. Results The major chemical component found in E. triquetrum oil determined by GC-FID and GC/MS analyses is an aliphatic polyacetylene molecule, the falcarinol with 86.9–93.1% of the total composition. The LC 50 and LC 90 values for uninfected snails were 0.61 and 1.02 ppm respectively for 24 h exposure. At 0.5 ppm, the essential oil was two times more toxic to parasitized snails with a mortality rate of 88.8 ± 4.8%. Moderate embryonic lethal effects were observed at the concentration of 1 ppm. Severe surface damage in miracidia was observed with a general loss of cilia that probably cause their immobility. Miracidia exposed 30 min to low concentration of plant extract (0.1 ppm) were less infective with 3.3% of prevalence compare to untreated with a prevalence of 44%. Conclusions Essential oil extracted from E. triquetrum and falcarinol must be considered as a promising product for the development of new interventions for schistosomiasis control and could proceed to be tested on Phase II according to the WHO requirements.