Single-cell RNA-seq uncovered hemocyte functional subtypes and their differentiational characteristics and connectivity with morphological subpopulations in Litopenaeus vannamei

Abstract

Hemocytes play central roles in shrimp immune system, whereas whose subclasses have not yet been completely defined. At present, the morphological classification of hemocytes is inadequate to classify the complete hemocyte repertoire and elucidate the functions and differentiation and maturation processes. Based on single-cell RNA sequencing (scRNA-seq) of hemocytes in healthy Litopenaeus vannamei, combined with RNA-FISH and flow cytometric sorting, we identified three hemocyte clusters including TGase+ cells, CTL+ cells and Crustin+ cells, and further determined their functional properties, potential differentiation trajectory and correspondence with morphological subpopulations. The TGase+ cells were mainly responsible for the coagulation, exhibiting distinguishable characteristics of hyalinocyte, and appeared to be developmentally arrested at an early stage of hemocyte differentiation. The CTL+ cells and Crustin+ cells arrested at terminal stages of differentiation mainly participated in recognizing foreign pathogens and initiating immune defense responses, owning distinctive features of granule-containing hemocytes. Furthermore, we have revealed the functional sub-clusters of three hemocyte clusters and their potential differentiation pathways according to the expression of genes involved in cell cycle, cell differentiation and immune response, and the successive differentiation and maturation of hyalinocytes to granule-containing hemocytes have also mapped. The results revealed the diversity of shrimp hemocytes and provide new theoretical rationale for hemocyte classification, which also facilitate systematic research on crustacean immunity.

scRNA-seq analysis of hemocytes of penaeid shrimp under virus infection

“…Silencing of CHF or its candidate receptor, laminin, has been reported to decrease the number of hemocytes, especially hyaline cells (Charoensapsri et al, 2015; Lin et al, 2011; Söderhäll, 2013). Recent studies suggest that granulocytes differentiate from hyaline cells; it is possible that viral infection suppresses CHF expression, resulting in cell death at the hyaline cell stage, reducing the cluster Hem4 and AMPs expression, or preventing the hemocyte differentiation from hyaline cells into hemocytes expressing AMPs (Cui et al, 2022; Koiwai et al, 2021; Li et al, 2021; Liu et al, 2021; Li et al, 2022). The critical function of CHFs remains unclear, but effective anti-WSSD control strategies may depend on the protection of AMPs-expressing populations by controlling CHFs expression.…”

“…Previously, authors used Drop-seq to classify hemocytes of P. japonicus , which had primarily been classified by morphology, and they proposed new cell populations and markers for these populations (Koiwai et al, 2021). The analysis is currently being conducted on aquaculture species like penaeid shrimp (Cui et al, 2022; Li et al, 2022; Yang et al, 2022; Zhu et al, 2021), crayfish (Söderhäll et al, 2022), and other shellfish (Meng et al, 2022; Meng & Wang, 2022; Sun et al, 2021). Future efforts to control diseases in aquaculture species will benefit from knowing this information.…”

A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein

“…Hemocytes play central roles in the shrimp immune system. They are mainly classified into granulocytes, semigranulocytes, and hyalinocytes depending on the morphology and intracellular granule staining characteristics [ 46 ]. These three subpopulations exhibited different functions with significantly distinct gene expression patterns.…”

Hallmarks of crustacean immune hemocytes at single-cell resolution

“…In this study, the results of single-cell RNA sequencing of shrimp and crayfish revealed that the hemocytes of crustaceans consisted of three classic hemocytes (granular hemocytes, semi-granular hemocytes and hyaline hemocytes) and novel subpopulations (macrophage-like hemocytes, adipohemocyte-like hemocytes, reproductive hemocytes, hemocytes related to phagocytosis, hemocytes related to nervous system, and natural killer T cell-like cluster) and intermediate subpopulations (intermediate hemocytes between semi-granular hemocytes and hyalinocytes, and intermediate hemocytes between semi-granular and granular hemocytes). Recently the single-cell RNA sequencing analysis of shrimp was performed by other research groups ( 20 , 37 , 54 , 55 ). Nevertheless three typical hemocytes (granular cells, semi-granular cells and hyaline cells) are not identified in the previous studies ( 20 , 37 , 54 ), showing the limitation of the previous investigations.…”

“…Recently the single-cell RNA sequencing analysis of shrimp was performed by other research groups ( 20 , 37 , 54 , 55 ). Nevertheless three typical hemocytes (granular cells, semi-granular cells and hyaline cells) are not identified in the previous studies ( 20 , 37 , 54 ), showing the limitation of the previous investigations. The immunity-associated markers, including hemocyte transglutaminase, Vago 5, ALF-like, penaeidin-II, C-type lysozyme, stylicin, penaeidin-II, hemocyte kazal-type proteinase inhibitor (KPI), crustin, copper/zinc superoxide dismutase (SOD), prophenoloxidase and alpha2-macroglobulin, revealed in the present study, were also identified in the previous studies ( 20 , 37 , 54 , 55 ).…”