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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.

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.

Biomphalaria glabrata is a freshwater Planorbidae snail. In its environment, this mollusk faces numerous microorganisms or pathogens, and has developed sophisticated innate immune mechanisms to survive. The mechanisms of recognition are quite well understood in Biomphalaria glabrata, but immune effectors have been seldom described. In this study, we analyzed a new family of potential immune effectors and characterized five new genes that were named Glabralysins. The five Glabralysin genes showed different genomic structures and the high degree of amino acid identity between the Glabralysins, and the presence of the conserved ETX/MTX2 domain, support the hypothesis that they are pore-forming toxins. In addition, tertiary structure prediction confirms that they are structurally related to a subset of Cry toxins from Bacillus thuringiensis, including Cry23, Cry45, and Cry51. Finally, we investigated their gene expression profiles in snail tissues and demonstrated a mosaic transcription. We highlight the specificity in Glabralysin expression following immune stimulation with bacteria, yeast or trematode parasites. Interestingly, one Glabralysin was found to be expressed in immune-specialized hemocytes, and two others were induced following parasite exposure.

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.