Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes

  1. Peng Yang
  2. Yaohui Chen
  3. Zhiqi Huang
  4. Huidan Xia
  5. Ling Cheng
  6. Hao Wu
  7. Yueling Zhang
  8. Fan Wang  Is a corresponding author
  1. Institute of Marine Sciences, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, China
  2. Department of Biology, College of Science, Shantou University, China
  3. Guangzhou Genedenovo Biotechnology Company Limited, China
  4. Shantou University-Universiti Malaysia Terengganu Joint Shellfish Research Laboratory, Shantou University, China
  5. Southern Marine Science and Engineering Guangdong Laboratory, China

Abstract

Despite the importance of innate immunity in invertebrates, the diversity and function of innate immune cells in invertebrates are largely unknown. Using single-cell RNA-seq, we identified prohemocytes, monocytic hemocytes, and granulocytes as the three major cell-types in the white shrimp hemolymph. Our results identified a novel macrophage-like subset called monocytic hemocytes 2 (MH2) defined by the expression of certain marker genes, including Nlrp3 and Casp1. This subtype of shrimp hemocytes is phagocytic and expresses markers that indicate some conservation with mammalian macrophages. Combined, our work resolves the heterogenicity of hemocytes in a very economically important aquatic species and identifies a novel innate immune cell subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.

Editor's evaluation

This study provides a single cell transcriptomic atlas of shrimp hemocytes and identifies a subset of myeloid cells with markers that resemble mammalian macrophages. These novel phagocytic macrophage subset may be the target of future studies in diseased shrimp.

https://doi.org/10.7554/eLife.80127.sa0

Introduction

Compared with vertebrates, invertebrates do not have B- and T-cell-based adaptive immunity, which makes them primarily reliant on innate immunity as defense against various pathogens (Little et al., 2005). Although invertebrates have developed diverse forms of innate immunity in order to adapt to various environmental challenges, the innate immune cells themselves contribute significantly because they have ‘trained immunity’ (Lanz-Mendoza and Contreras-Garduño, 2022). However, the evolution of innate immune systems has diverged into many branches in the metazoan tree of life, making invertebrate immune cell-typing extremely complex. In contrast, the cells of the vertebrate immune system are conserved from chordates (Rosental et al., 2018). Recent advances in single-cell sequencing technology have shed light on this issue. For example, recent research in mosquito cellular immunity revealed that mosquito hemocytes are of four major types (prohemocytes, granulocytes, oenocytoids, and megacytes) (Kwon et al., 2021; Raddi et al., 2020). Hemocytes from another invertebrate model — Drosophila melanogaster — can be divided into eight subgroups, including crystal cells, lamellocytes, unspecified plasmatocytes, proliferative plasmatocytes, PSC-like hemocytes, antimicrobial plasmatocytes, phagocytic plasmatocytes, and secretory plasmatocytes (Cattenoz et al., 2021; Cho et al., 2020; Li et al., 2022; Tattikota et al., 2020). These classifications cover the major innate immune cell functions, including proliferation, reactive oxygen species (ROS) generation, phagocytosis, and effector secretion, suggesting some shared similarities in innate immunity between invertebrates and vertebrates.

To explore these analogies, we used the marine invertebrate Penaeus vannamei to characterize immune cell subsets using single-cell RNA sequencing (scRNA-seq). This species was selected because it is a popular mariculture species due to being fast growing and delectable (Zhang et al., 2019). Additionally, this invertebrate has an open circulatory system filled with hemolymph, which consists of plasma and hemocytes (Lin and Söderhäll, 2011). The shrimp hemolymph bears high similarity to that of the vertebrate peripheral blood in terms of functions such as transportation of nutrients and metabolic waste, maintenance of acid–base equilibrium, defense against various invading pathogens, and hemostatic effect (McNamara and Faria, 2012; Tassanakajon et al., 2018). Moreover, the circulating plasma contains more than 400 proteins, many of which are vertebrate homologs (Luo et al., 2022; Tao et al., 2019). The circulating hemocytes comprise proliferating cells, phagocytic cells, and effector secreting cells (Lin and Söderhäll, 2011), which are functionally similar to vertebrate peripheral myeloid cells. Among these cells, the phagocytes have been identified in all metazoans (Musser et al., 2021), although they appear to be different in invertebrates and vertebrates. Some recently performed functional assays suggest that the bacterial engulfment in phagocytes may be conserved from invertebrates to vertebrates (Kokhanyuk et al., 2021). In this study, we attempted to analyze shrimp hemocytes via single-cell sequencing and redefined their classification according to their functional marker gene distribution. We also attempted to understand evolutionary phagocyte development along the metazoan tree of life.

Results

Major cell-types among the circulating hemocytes in shrimp

Previously, we identified a lipopolysaccharide (LPS)-induced shrimp plasma protein CREG and noticed that recombinant CREG (rCREG) was more effective than recombinant EGFP (rEGFP) in activating the shrimp hemocytes (Huang, Yang, & Wang, 2021). Based on this observation, we performed scRNA-seq for rCREG-treated shrimp hemocytes to further explore their function. To maximize the collection of circulating hemocytes from shrimp, we applied iodixanol gradient centrifugation to concentrate the hemocytes for the Gel Bead-In-Emulsion (GEM) preparation (Tattikota et al., 2020; Figure 1A). A total of 34,693 cells including control (12544), rEGFP- (12640), and rCREG-treated (9509) cells were retained for further analyses, and these cells exhibited a median of 5656, 6837, and 7916 transcripts and 1089.5, 1245, and 1364 genes per cell, respectively (Figure 1—figure supplement 1). The rCREG-treated samples had the highest unique molecular identifiers (UMIs) and detected genes compared with that of the other two groups. This observation is consistent with our previous conclusion that CREG is a hemocyte activation factor (Huang et al., 2021).

Figure 1 with 2 supplements see all
Major cell types identified in shrimp hemolymph.

(A) A schematic workflow of sample preparation. The hemocytes were collected from non-treated, rEGFP-treated, and rCREG-treated shrimps (n=15 for each treatment) and subjected to iodixanol gradient centrifugation and single-cell RNA sequencing (ScRNA-seq) using 10 X Genomics. (B) A t-SNE plot showing five major cell types identified in scRNA-seq dataset (n=34,693 in total; Control, 12544; rEGFP treated, 12640; rCREG treated, 9509 cells). The count of each cell type is indicated in parentheses. (C) A heatmap showing five representative marker genes for each major cluster. The gene name and its NCBI GeneID is listed (left) and its expression level in each cell is shown with different colors (right). (D) Two-dimensional projections, and proportions of the cell types for each treatment. Proportions of prohemocytes (red), monocytic hemocytes (brown), and granulocytes (blue) are indicated (left). Proportions of all five major cell types in each treatment are indicated (right).

To further define the major cell types of circulating shrimp hemocytes, we combined all 34,693 cells from different treatments and applied canonical correlation analysis (Stuart et al., 2019) to perform batch correction. We then aggregated the cell clusters and identified five major groups of isolated hemocytes, including prohemocytes (PHs) (6838, 19.7%), granulocytes (GHs) (13871, 40%), monocytic hemocytes (MHs) (11112, 32%), transitional cells (TCs) (2090, 6%), and germ-like cells (GCs) (782, 2.3%), which were annotated according to their potential functions implied by the marker genes (Figure 1B). Granulocytes are characterized by their ProPO system (Sun et al., 2020). Here, we found that prophenoloxidase activating factors 1 and 2 (PPAF1 and PPAF2) were highly expressed in this population. In addition to ProPO system genes, secreted proteins, including crustin-like protein (CRUL), penaeidin 3 a.1 (PEN-3), and crustacean hematopoietic factor-like protein (CHF), were also highly expressed in this population (Figure 1C, Figure 1—figure supplement 2A). The MHs were named thus because they shared some critical genes with mammalian monocytes. For example, NOD-like receptor protein 3 (Nlrp3) is a key component of the inflammasome and is highly expressed in monocytes and macrophages for processing of IL-1ß (He et al., 2016). Lysosome (Lyz1), another canonical antibacterial enzyme, is a well-known macrophage-secreting hydrolase (Short et al., 1996). Vago5 encodes an IFN-like antiviral cytokine and plays a role in the anti-white spot syndrome virus (WSSV) resistance (C. Li, Yang, Hong, Zhao, & Wang, 2021) (Figure 1C, Figure 1—figure supplement 2B). Generally, these cells secrete antibacterial and antiviral effectors. In PHs, histone1 (h1f0) is the key component of heterochromatin assembly, and its high expression is associated with reduced gene expression and cell stemness properties (Pan and Fan, 2016). In a previous study, hemocyte transglutaminase (TGM1) was identified as an immature hemocyte marker (Koiwai et al., 2021). The gene Igfbp7 has been shown to promote hemocyte proliferation in the small abalone Haliotis diversicolor (Wang et al., 2015; Figure 1C, Figure 1—figure supplement 2C). TCs were difficult to characterize due to the lack of exclusively expressed genes. Hence, we identify the top five significantly upregulated genes and analyzed the distribution of the top three (Figure 1C, Figure 1—figure supplement 2D); we found that this group of cells had no significant marker genes, thus the name. We found some reproduction-related genes in the GC group. For example, lhx9 encodes a key transcription factor in gonadal development (Balasubramanian et al., 2014), and spir localization is critical for mouse oocyte asymmetric division (Jo et al., 2019; Figure 1C, Figure 1—figure supplement 2E). Hence, we annotated this group of cells as GCs.

To further determine whether CREG is a differentiation factor for shrimp hemocytes, we examined the ratio of the five annotated major cell types in different treatments. Recombinant protein injection slightly increased the proportion of GHs and PHs and decreased the ratio of MHs (Figure 1D). However, there were no significant differences between the rEGFP and rCREG treatments, which suggests that CREG is probably an activation factor rather than a differentiation factor for shrimp hemocytes.

Subtyping of shrimp immune cell clusters and construction of their differentiation trajectory

While classifying the immune cells in shrimp hemolymph, the TC group that lacks gene markers and GC group that is not a typical immune cell are not further explored in this study (Supplementary file 1, Supplementary file 2). To further trace the immune cell lineages in shrimp hemolymph, we subtyped the remaining three major classes of cells—PHs, MHs, and GHs; each major class type was divided into two subtypes and labelled PH1 (1577, 4.5%) and PH2 (5261, 15.2%), MH1 (10463, 30.2%) and MH2 (649, 1.9%), and GH1 (10353, 29.8%) and GH2 (3518, 10.1%), respectively (Figure 2A). We identified some unique marker genes located at the edge of the t-SNE map for the subpopulations PH1, GH2, and MH2 (Figure 2A, Figure 2—figure supplement 1, Supplementary file 3, Supplementary file 4 and Supplementary file 5), but could not identify exclusive marker genes for the subpopulations PH2, GH1, and MH1, which constituted the main body of the t-SNE map. The marker genes for PH2, GH1, and MH1 were also highly expressed in PH1, GH2, and MH2, respectively (Figure 2B, Supplementary file 6, Supplementary file 7 and Supplementary file 8). Thus, these six subtypes of hemocytes might have lineage differentiation relationships. To explore this, we performed cell cycle analyses of the six subtypes of hemocytes. PH1 was characterized by high expression of all marker genes for the G1, G2, and M stages (Figure 2C). This observation was consistent with a previous report that approximately 2–5% of circulating hemocytes were proliferating hemocytes and could be labelled with BrdU (Sun et al., 2013). Thus, we set PH1 as the initiating cell and applied a Monocle to construct differentiation trajectories for PHs, GHs, and MHs. Two major branches—MH lineage and GH lineage—were identified to have differentiated from one common PH (Figure 2D–E). This is similar to the development of human myeloid cells, in which granulocyte–monocyte progenitor (GMP) differentiates into monocyte and granulocyte (Bassler et al., 2019). To further compare innate immune cell differentiation between shrimp and humans, we screened shrimp homologs of human myeloid differentiation-related transcription factors (TFs) because TFs are the key regulators of cell fate determination (Friedman, 2002). In total, 3790 differentially expressed genes among different branches were identified and are shown as a specific heatmap on which three shrimp homologs of human TFs were labeled (Figure 2F, Supplementary file 9). Fli1 is specifically expressed in the granulocyte lineage, which is consistent with previous observations that Fli1 deletion decreases granulocytic cell number in mice (Starck et al., 2010). MAF and c-Rel were highly expressed in the MH lineage (Figure 2G). c-Rel is a key TF in the NF-κB pathway and plays important roles in monocyte differentiation (Li et al., 2020). MAF is a bZip TF that could induce monocytic differentiation (Kelly et al., 2000). In general, our data suggest that some myeloid regulators may be conserved between shrimp and humans.

Figure 2 with 1 supplement see all
Subclustering and pseudotime trajectory analyses of three major hemocyte types in shrimp hemolymph.

(A) Subclusters of hemocytes–prohemocytes (PH), monocytic hemocytes (MH), granulocytes (GH) are projected onto two-dimensional t-SNE plots. The numbers in the plots represent the subcluster number. (B) Dot plot showing corresponding expression of cluster marker genes. The color indicates mean expression and dot size represents the percentage of cells within the cluster expressing the marker. Last nine digits of each marker gene are the NCBI GeneID. (C) Expression of cell-cycle regulating genes in the six subtypes. Dot color indicates average expression levels and dot size displays the average percentage of cells with cell cycle controlling genes (Cdk1(ncbi_113818305), CycD(ncbi_113814652), and CycE(ncbi_113822658) for G1; stg(ncbi_113800052), CycA(ncbi_113821735), and CycB(ncbi_113803283) for G2; polo(ncbi_113805901), aurB(ncbi_113827838), and birc5(ncbi_113828653) for M) in each subcluster. (D) A differentiation trajectory of PH, GH, and MH subpopulation using Monocle2 (n=31821). (E) Differentiation trajectory reconstruction with 6 subclusters. PH lineage, GH lineage, and MH lineage were labelled with red, blue, and brown circles respectively. (F) A heatmap showing differentially expressed gene dynamics during hemocyte differentiation process. (G) Spline plots showing the expression dynamics of Fli1, c-Rel, and MAF. Imaginary line, monocytic hemocyte lineage; Full line, granulocyte lineage.

Identification of a macrophage-like phagocytic cell population in shrimp hemolymph

Next, we analyzed the similarities that MH2 might be sharing with terminally differentiated monocyte-like macrophages or dendritic cells. Recently, the Human Cell Atlas mapped the expression of most genes across major human cell types (Karlsson et al., 2021). We compared MH2 marker genes with that in the human database and found human homologs for nine MH2 marker genes including chitotriosidase (CHIT1), lysozyme (Lyz1), lipase (LIPF), legumain (LGMN), Nlrp3, alpha-N-acetylgalactosaminidase (NAGA), zinc finger E-box-binding homeobox 1(zfh1), caspase1 (Casp1), and NPC intracellular cholesterol transporter 2 (NPC2). These genes were specifically expressed in human macrophages, including in some tissue-specific macrophages such as Kupffer cells and Hofbauer cells (Figure 3A, Figure 3—figure supplement 1, Figure 1—figure supplement 2B; Karlsson et al., 2021). This suggests that MH2 might be the invertebrate homolog of human macrophages, and various tissue-specific macrophages could have evolved from a common primitive cell type.

Figure 3 with 1 supplement see all
Identification of MH2 as macrophage-like phagocytic cells.

(A) Comparison between MH2 and human macrophage marker genes. (B) A representative contour plot of shrimp hemocytes against FITC-VP. Threshold intensity (FITC-A) was set to <103 representing control hemocytes (R2), and >2 × 103 representing phagocytic hemocytes (R1). R1 and R2 were sorted based on the forward scatter (FSC) and fluorescence intensity (FITC) two-dimensional space. (C) Confocal microscopy of sorted hemocytes (R1) with ingested FITC-labelled Vibrio Parahemolyticus. Green, ingested Vibrio Parahemolyticus; Blue, nuclei. Scale bar: 10 μM. (D) Efficiency of the phagocytosis inhibitor on the Vibrio Parahemolyticus uptake of shrimp hemocytes. The results are presented as mean ± SD of 6–8 replicates. Asterisks denote statistical significance (**p=0.007) between the control and different treatments. (E) Differential gene expression analysis (CHIT1 (**p=0.004), Lyz1 (*p=0.049), and NAGA (*p=0.032)) between R1 and R2 sorted using FACS and analyzed using qPCR. (F) Differential protein expression analysis (NAGA, LYZ1, and NLRP3) between R1 and R2 sorted using FACS. The immunoblot signals were quantified with ImageJ. The relative immunoblot signal intensities of NAGA (*p=0.011), LYZ1 (*p=0.022), and NLRP3 (**p=0.009) compared with that of ß-actin were recorded with bar chart. Both qPCR and immunoblot data were analyzed using the student t test.

To further prove this hypothesis, we labeled phagocytes via injection of fluorescein isothiocyanate-conjugated Vibrio parahaemolyticus (FITC-VP). The hemocytes which engulfed FITC-VP were isolated using a cell sorter and labelled as phagocytic hemocytes (R1) (fluorescence intensity >2 × 103). The hemocytes with low fluorescence (<103) were labelled control hemocytes (R2) (Figure 3B). To characterize these phagocytes, we observed them using confocal microscopy. These cells had round nuclei with a highly vacuolated cytoplasm similar to that in vertebrate macrophages (Figure 3C). To further compare these cells with vertebrate macrophage, we performed a phagocytosis inhibition assay using an actin polymerization inhibitor—cytochalasin D (Kokhanyuk et al., 2021)—that effectively suppressed the shrimp hemocyte phagocytosis rate (Figure 3D). To characterize the R1 cells, we quantified CHIT1, Lyz1, and NAGA expression in R1 and R2 using qPCR. The results indicated that these three genes were expressed at higher levels in R1 than in R2 (Figure 3E). In addition, we examined LYZ1, NAGA, and NLRP3 using immunoblotting and found that these three proteins were expressed at significantly higher levels in R1 than in R2 (Figure 3F). Thus, our results indicated that phagocytic cells in shrimp hemolymph specifically expressed MH2 marker genes. Our data suggest that MH2 may be an invertebrate homolog of human macrophages.

Comparison between hyalinocytes, semi-granulocytes, and granulocytes and their classifications in this study

Next, we compared our classification with the traditional classification. Previously, shrimp hemocytes have been divided into three major types: hyalinocytes, semi-granulocytes, and granulocytes based on morphological criteria and functional properties (Söderhäll, 2016). Recently, these three major types were separated using cell sorting or Percoll density gradient centrifugation and their marker genes were identified and validated using qPCR (Sun et al., 2020; Yang et al., 2015). Here, we analyzed the distribution of previous published marker genes: for hyalinocytes—lysosome membrane protein2 (LIMP2, ncbi_113826216), tubulin beta chain (TUBB4B, ncbi_113826677), dipeptidyl peptidase 1 (CTSC, ncbi_113824311), transglutaminase 1 (TGM1, ncbi_113823934) (Figure 4A, Figure 1—figure supplement 2C); for semi-granulocytes—beta-arrestin-1 (ARRB1, ncbi_113804686), ADP-ribosylation factor 6 (ARF6, ncbi_113820333), lysozyme (Lyz1, ncbi_113802295), Penaeid-3a (PEN-3, ncbi_113808997) (Figure 4B, Figure 1—figure supplement 2A and B); and for granulocytes—clone ZAP 18 putative antimicrobial peptide (CRU, ncbi_113801825), phenoloxidase-activating factor 3 (PPAF3, ncbi_113800184), phenoloxidase 3-like (PPO3, ncbi_113827090), peroxinectin (Pxt, ncbi_113820150) (Figure 4C, Figure 2—figure supplement 1C). Hyalinocyte marker genes were highly expressed in PH1, PH2, MH1, and MH2 groups (Figure 4A and D). Semi-granulocyte marker genes were highly expressed in GH2 and MH2 groups (Figure 4B and D). These data are consistent with previous observations that hyalinocytes contain both proliferating progenitors and phagocytic cells (Söderhäll, 2016). It also explained the observation of phagocytic activities in semi-granulocytes in some studies (Sun et al., 2020). The granulocyte marker genes were consistent with our observations and were highly expressed in GH2 (Figure 4C–D), which indicates that granulocytes are indeed the largest cell-type with internal condensed granules (Söderhäll, 2016).

Comparison between the traditional classification and the classification in this study.

(A) Dot plot showing corresponding expression of previously reported hyalinocyte marker genes in eight subclusters. (B) Dot plot showing corresponding expression of previously reported semi-granulocyte marker genes in eight subclusters. (C) Dot plot showing corresponding expression of previously reported granulocyte marker genes in eight subclusters. The color indicates mean expression, and dot size represents the percentage of cells within the cluster expressing the marker. (D) A proposed model for comparison between two classifications. The hyalinocyte, semi-granulocyte, and granulocyte were labelled on the t-SNE map with red, brown, and blue circles, respectively.

Discussion

Innate immune cells play an important role in the adaptation of animals to complex and volatile environments. Their ability for fast response protects animals from various pathogenic invasions. However, invertebrates have experienced a long evolution in a diversified environment, which has led to the fact that invertebrate immunity is extremely complex and immune cell typing in various invertebrates seems quite different (Supplementary file 10). Fortunately, proteins seem to have evolved at a much slower rate (Jayaraman et al., 2022), and cell-specific functional proteins are therefore the key to define cell subsets. Thus, in this study, we compared shrimp immune cell marker genes with their human homologs to identify evolutionary traces of innate immune cells between invertebrates and vertebrates (Supplementary file 11). Our data revealed macrophage-like phagocytes in shrimp hemolymph. This group of cells exhibited phagocytic activity. Additionally, Nlrp3 and Casp1, two well-known mammalian macrophage inflammasome components were identified in these cells, suggesting that inflammasome-mediated anti-pathogenic processes might exist in invertebrate innate immunity and play a role in microbial restriction (Wang et al., 2021). NAGA, another enzyme found in macrophages, could inhibit macrophage activation via deglycosylation of macrophage activators (Saburi et al., 2017). The existence of NAGA in shrimp phagocytes implies that this subset of cells might share similar negative regulatory mechanisms recently partially uncovered in vertebrate macrophages (Luo et al., 2022). In addition, VEGF3, a well-known angiogenic factor, was also identified in this subtype (Eswarappa and Fox, 2015). Shrimp have an open circulation system with partial blood vessels; whether this factor plays a role in wound healing needs to be answered by future studies (Söderhäll, 2016).

Phagocytic ability is one of the fundamental functions in an organism. Unicellular organisms employ phagocytosis for this purpose. Cells in multicellular organisms have functional specializations that increase their adaptability. Thus, the phagocytic ability of metazoans is limited to certain cells. The ratio of phagocytic cells in the different species varies. For example, in the primitive oyster Crassostrea gigas, approximately 40–60% of hemocytes engulf pathogens (Sun et al., 2021). In fish, most immune cells, including monocytes, granulocytes, B cells, and red blood cells, possess phagocytic activity against invading pathogens (Heimroth et al., 2021; Li et al., 2006; Xu et al., 2021). In humans, most myeloid cells, including monocytes, macrophages, dendritic cells, and neutrophils, engulf pathogens (Bassler et al., 2019). Shrimps contain fewer phagocytes than do other species (Alenton et al., 2019; Huang et al., 2021; Li et al., 2021). Our data suggest that at least a part of the phagocytes in the shrimp hemolymph originated from a macrophage-like subset. Moreover, vertebrate macrophages include large populations of cells that reside in different tissues with diversified roles. Whether invertebrate macrophages have similar tissue-specific functions is yet unknown and future studied are needed to elucidate the evolution of these helper cells in maintaining homeostasis.

Over the past decades, crustacean hemocytes have been classified into hyalinocytes, semi-granulocytes, and granulocytes based on their morphology and function (Söderhäll, 2016). However, morphology-based classification has caused several problems. For example, crustacean hyalinocytes are generally regarded as small phagocytes with few granules (Lin and Söderhäll, 2011), but some researchers believe that these cells may be immature or prematurely released prohemocytes of the semi-granulocyte or granulocyte lineage (van de Braak et al., 2002). In this study, our data clearly indicated that the small cells though similar in morphology include both prohemocytes and phagocytic hemocytes, and these two subtypes of cells have different marker genes and varied functional roles in the shrimp hemolymph. In addition, semi-granulocytes were considered a major component of circulating hemocytes that were involved in both melanization and phagocytosis. This conclusion cannot be accurate because less than 20% of circulating hemocytes are phagocytic cells, whereas approximately 65% of total hemocytes are considered to be semi-granulocytes (Alenton et al., 2019; Lin and Söderhäll, 2011). Our results indicated that the cells sorted as semi-granulocytes contain both monocytic hemocytes and cells of granulocyte lineage.

In this study, we selected Penaeus vannamei as a crustacean model and proposed a novel crustacean hemocyte classification system. A new functional monocytic hemocyte lineage for circulating shrimp hemocytes was identified. The terminally differentiated cells of this lineage share several functional genes with mammalian macrophages, which suggests that this monocytic hemocyte lineage might be an invertebrate evolutionary homolog of the vertebrate monocyte lineage. Although crustaceans contain diverse species with different morphologies and evolutionary histories, our data, to some extent, provide a different interpretation for the current crustacean immune cell subtyping. This classification is far from complete but might provide insights about crustacean cellular immunity in the future. Moreover, this study is at an early stage of investigating cellular immunity in shrimp. For example, we could not systematically validate our conclusion at this moment due to the lack of specific monoclonal antibodies to label the three proposed major immune cell subtypes. Some fundamental questions remain unresolved, such as: can shrimp macrophage-like phagocytes infiltrate various tissues and where do the circulating shrimp prohemocytes originate? In future studies, we plan to find answers to such questions to improve our understanding of this important mariculture species and find solutions to serious shrimp diseases that have caused tremendous economic losses worldwide.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-ß-ACTIN
(Rabbit monoclonal)
BeyotimeCat#AF5003WB (1:200)
AntibodyAnti-NAGA (Rabbit polyclonal)SinoBiologicalCat#13686-T24WB (1:200)
AntibodyAnti-LYZ1 (Rabbit polyclonal)Bioss AntibodiesCat#bs-0816RWB (1:200)
AntibodyAnti-NLRP3 (Rabbit polyclonal)GenScriptpolypeptide (aa29-42)
WB (1:200)
Strain, strain background (Vibrio parahaemolyticus)FITC-VPShantou University2×106 particles/g
Chemical compound, drugOptiPrepAxis-shieldCat# AS1114542
Chemical compound, drugTrypan blueSolarbioCat# C0040
Chemical compound, drugFITCBiossCat# D-9801
Chemical compound, drugHoechst 33342 stainBeyotimeCat# C1028100×
Peptide, recombinant proteinrEGFPHuang et al., 2021 (https://doi.org/10.3389/fimmu.2021.707770)recombinant plasmid, prokaryotic expression, purification
Peptide, recombinant proteinrCREGHuang et al., 2021 (https://doi.org/10.3389/fimmu.2021.707770)recombinant plasmid, prokaryotic expression, purification
Biological sample (Penaeus vannamei)HaemolymphShantou local farmsFreshly isolated from Penaeus vannamei
Commercial assay, kitRNAprep Pure Micro KitTIANGENCat#DP420
Commercial assay, kitFirst Strand cDNA Synthesis KitBeyotimeCat#D7168M
Commercial assay, kit3’Reagent Kits v3.110 X Genomics1000268
Sequence-based reagentCHIT1_FThis paperqPCR primersGTCGAAATTCCGGCCAAAGA
Sequence-based reagentCHIT1_RThis paperqPCR primersGGCCCGTTCTTGTTTGACTT
Sequence-based reagentLyz1_FThis paperqPCR primersCAAGAACTGGGAGTGCATCG
Sequence-based reagentLyz1_RThis paperqPCR primersTCTGGAAGATGCCGTAGTCC
Sequence-based reagentNAGA _FThis paperqPCR primersCTACGAGGACTACGGCAACT
Sequence-based reagentNAGA _RThis paperqPCR primersCGAACTCTGGGTAGCCTTCA
Sequence-based reagentEF-1α_FThis paperqPCR primersGTATTGGAACAGTGCCCGTG
Sequence-based reagentEF-1α_RThis paperqPCR primersACCAGGGACAGCCTCAGTAAG

Experimental organisms

Request a detailed protocol

Shrimp was purchased from Shantou local farms. Upon delivery, the shrimp were cultured in water tanks filled with aerated seawater at 20 °C and acclimatized for 2–3 days before the experiments. All animal-related experiments were conducted in accordance with Shantou University guidelines.

Collection of shrimp hemocytes with different treatments

Request a detailed protocol

Recombinant EGFP and CREG were purified, as previously described (Huang et al., 2021). Sixty shrimp were divided equally into three groups. One group was left untreated and labelled as control. The other two groups were injected with rEGFP or rCREG (1 μg/g), respectively. The hemolymph was collected 8 hr post-injection from each group and mixed well. Hemolymph (1.5 mL) was loaded onto an OptiPrep (Axis-shield, NO) separation solution (1.09 g/mL) and centrifuged at 2000 rpm for 10 min at 4 °C. Circulating hemocytes were concentrated between the hemolymph and the separation solution and carefully collected with a pipettor. The collected hemocytes were stained with 0.4% trypan blue to estimate cell viability. Next, cells with >85% viability were subjected to further scRNA-seq experiments.

Library preparation and ScRNA sequencing

Request a detailed protocol

The hemocyte suspensions were loaded onto a 10 X Genomics GemCode Single-cell instrument to generate single-cell GEMs. Libraries were generated using Chromium Next GEM Single Cell 3’Reagent Kits v3.1 (10X Genomics, USA). Upon dissolution of the GEM, primers containing (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10 X Barcode, (iii) a 10 nt UMI, and (iv) a poly-dT primer sequence were released and mixed with the cell lysate and Master Mix. Barcoded full-length cDNAs were then reverse-transcribed from poly adenylated mRNA and amplified using PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) was added during the GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via end repair, A-Tailing, adaptor ligation, and PCR. Reads 1 and 2 were standard Illumina sequencing primers used for paired-end sequencing. All raw sequencing data were stored in the genome sequence archive of the Beijing Institute of Genomics, Chinese Academy of Sciences, gsa.big.ac.cn (accession nos. PRJCA006297).

Bioinformatics analysis of single-cell RNA sequencing data

Request a detailed protocol

Raw BCL files were converted into FASTQ files using 10 X Genomics Cell Ranger software (version 5.0). The reads were then mapped to the shrimp genome (taxid:6689), and the reads that uniquely intersected at least 50% of an exon were considered for UMI counting. Valid barcodes were identified using the EmptyDrops method (Lun et al., 2019). The hemocytes by gene matrices for control, rEGFP treatment and rCREG treatment were individually imported to Seurat version 3.1.1 for the following analyses (Butler et al., 2018).

Cells with UMIs (≥17,000), mitochondrial genes (≥10%), ≤230 detected genes, or ≥2200 detected genes were excluded. The qualified cells were normalized via ‘LogNormalize’ method, which normalizes the gene expression for each cell by the total expression. The formula is as follows:

Ageneexpressionlevel=log(1+UMIA/UMITotal×10000)

The batch effect was corrected using a canonical correlation analysis (Stuart et al., 2019). The integrated expression matrix was then scaled and subjected to principal component analysis (PCA) for dimensional reduction. Subsequently, the significant principal components (PCs) were identified as those with a strong enrichment of low-p-value genes (Chung and Storey, 2015).

Seurat was used for cell clusters based on principal component analysis (PCA) scores with a subset of the data (1% by default), constructing a ‘null distribution’ of gene scores. A graph-based approach that calculates the distance based on previously identified PCs was implemented for cell clustering (Chung and Storey, 2015). Finally, a resolution of 0.2 was chosen as the clustering parameter, which identified 8 clusters. The t-SNE was then performed to visualize the data in a two-dimensional space.

Differentially expressed gene (up-regulation) analysis: The median expression patterns across all cells in each cluster were calculated to identify genes that were enriched in a specific cluster. The expression value of each gene in the given clusters was compared against the rest of the cells using the Wilcoxon rank-sum test (Camp et al., 2017). The significantly upregulated genes were identified using several criteria. First, genes had to be at least 1.28-fold overexpressed in the target cluster. Second, genes had to be expressed in more than 25% of the cells belonging to the target cluster. Third, the p-value had to be less than 0.05.

Cell trajectory analysis: Single-cell trajectories were analyzed using a matrix of cells and gene expression by Monocle (Version2.10.1) (Trapnell et al., 2014). Monocle reduced the space to one with two dimensions and ordered the cells (sigma = 0.001, lambda = NULL, param.gamma=10, tol = 0.001) (Qiu et al., 2017). Once the cells were ordered, we visualized the trajectory in reduced dimensional space. The trajectory had a tree-like structure, including tips and branches. Monocle was used to identify genes that were differentially expressed between the groups of cells. The key genes were identified as having a false discovery rate (FDR)<1e-5. Additionally, genes with similar trends in expression such as shared common biological functions and regulators were grouped. Finally, the Monocle developed BEAM to analyze branch-dependent gene expression by formulating the problem as a contrast between the two negative binomial GLMs.

Phagocytic cell labeling and sorting

Request a detailed protocol

Shrimp phagocytic cells were labeled as previously described (Huang et al., 2021). In brief, FITC labeled Vibrio parahaemolyticus (VP) (2×106 particles/g) were injected into the shrimp. The hemocytes were collected from 30 to 40 shrimp 2 hr post-injection. Each sorting was performed on a FACSMelody cell sorter (BD Biosciences, USA). The fluorescence boundary was set based on the detection of shrimp hemocyte self-fluorescence without VP injection.

Morphological analysis of sorted hemocyte and phagocytosis inhibition assay

Request a detailed protocol

Phagocytic hemocytes (R1) were collected, stained with Hoechst 33342 (Beyotime, Shanghai, China), and observed using an LSM800 confocal microscope (Zeiss, Germany). The phagocytosis inhibition assay was performed according to a previously described method with some modifications (Kokhanyuk et al., 2021). In brief, each shrimp was injected with either FITC-VP (2×106 particles/g) or FITC-VP (2×106 particles/g)+cytochalasin D (5 μM/g). The hemocytes were collected 2 hr post-injection and immediately analyzed with a BD Accuri C6 Plus Flow Cytometer (Becton Dickinson, USA). The phagocytic hemocytes were quantified based on fluorescence intensity, and the fluorescence boundary was set based on the detection of self-fluorescence of untreated hemocytes.

Collection of sorted hemocyte RNA and proteins for RT-qPCR and immunoblot analyses

Request a detailed protocol

For each experiment, 50–100 k events from phagocytic hemocytes (R1) and control hemocytes (R2) were collected. Total RNA from the collected samples was purified using the RNAprep Pure Micro Kit (TIANGEN, Beijing, China) and reverse-transcribed into cDNA using a First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China). qPCR was performed as previously described (Luo et al., 2022; Supplementary file 10), and the gene expression level was recorded as relative expression to EF-1α. This experiment was repeated five times. Total proteins from sorted hemocytes were precipitated by adding 1/100 volume of 2% sodium deoxycholate (Macklin, Shanghai, China) and 1/10 volume of 100% trichloroacetic acid (Macklin), followed by vortexing and centrifugation at 15,000×g for 15 min at 4 °C. The pellet was collected for performing SDS-PAGE and immunoblotting, as described before (Luo et al., 2022). This experiment was repeated thrice. The following antibodies were used: β-actin (AF5003; Beyotime, Shanghai, China), anti-NAGA (13686-T24; SinoBiological, Beijing, China), and anti-LYZ1 (bs-0816R; Bioss Antibodies, MA, USA). The polypeptide antibody against shrimp NLRP3 (aa29-42) was prepared by GenScript (Nanjing, China).

Statistical Analyses

Request a detailed protocol

The data in this study are presented as the results of at least three independent experiments. Statistical analyses were performed using the GraphPad Prism 8.0. Two-tailed unpaired Student’s t-tests were used to calculate the significance at *p<0.05, **p<0.01, and ***p<0.001.

Data availability

The sequence data reported in this paper have been deposited in the Genome Sequence Archive of the Beijing Institute of Genomics, Chinese Academy of Sciences, accession no. PRJCA006297. All other data are available in this manuscript and online in the Supplementary Material.

The following data sets were generated
    1. Wang Fan
    (2022) Genome Sequence Archive
    ID PRJCA006297. Single-cell RNA sequencing for shrimp (Penaeus vannamei) hemocytes treated with recombinant CREG.

References

    1. Li H
    2. Janssens J
    3. De Waegeneer M
    4. Kolluru SS
    5. Davie K
    6. Gardeux V
    7. Saelens W
    8. David FPA
    9. Brbić M
    10. Spanier K
    11. Leskovec J
    12. McLaughlin CN
    13. Xie Q
    14. Jones RC
    15. Brueckner K
    16. Shim J
    17. Tattikota SG
    18. Schnorrer F
    19. Rust K
    20. Nystul TG
    21. Carvalho-Santos Z
    22. Ribeiro C
    23. Pal S
    24. Mahadevaraju S
    25. Przytycka TM
    26. Allen AM
    27. Goodwin SF
    28. Berry CW
    29. Fuller MT
    30. White-Cooper H
    31. Matunis EL
    32. DiNardo S
    33. Galenza A
    34. O’Brien LE
    35. Dow JAT
    36. Jasper H
    37. Oliver B
    38. Perrimon N
    39. Deplancke B
    40. Quake SR
    41. Luo L
    42. Aerts S
    43. Agarwal D
    44. Ahmed-Braimah Y
    45. Arbeitman M
    46. Ariss MM
    47. Augsburger J
    48. Ayush K
    49. Baker CC
    50. Banisch T
    51. Birker K
    52. Bodmer R
    53. Bolival B
    54. Brantley SE
    55. Brill JA
    56. Brown NC
    57. Buehner NA
    58. Cai XT
    59. Cardoso-Figueiredo R
    60. Casares F
    61. Chang A
    62. Clandinin TR
    63. Crasta S
    64. Desplan C
    65. Detweiler AM
    66. Dhakan DB
    67. Donà E
    68. Engert S
    69. Floc’hlay S
    70. George N
    71. González-Segarra AJ
    72. Groves AK
    73. Gumbin S
    74. Guo Y
    75. Harris DE
    76. Heifetz Y
    77. Holtz SL
    78. Horns F
    79. Hudry B
    80. Hung R-J
    81. Jan YN
    82. Jaszczak JS
    83. Jefferis GSXE
    84. Karkanias J
    85. Karr TL
    86. Katheder NS
    87. Kezos J
    88. Kim AA
    89. Kim SK
    90. Kockel L
    91. Konstantinides N
    92. Kornberg TB
    93. Krause HM
    94. Labott AT
    95. Laturney M
    96. Lehmann R
    97. Leinwand S
    98. Li J
    99. Li JSS
    100. Li K
    101. Li K
    102. Li L
    103. Li T
    104. Litovchenko M
    105. Liu H-H
    106. Liu Y
    107. Lu T-C
    108. Manning J
    109. Mase A
    110. Matera-Vatnick M
    111. Matias NR
    112. McDonough-Goldstein CE
    113. McGeever A
    114. McLachlan AD
    115. Moreno-Roman P
    116. Neff N
    117. Neville M
    118. Ngo S
    119. Nielsen T
    120. O’Brien CE
    121. Osumi-Sutherland D
    122. Özel MN
    123. Papatheodorou I
    124. Petkovic M
    125. Pilgrim C
    126. Pisco AO
    127. Reisenman C
    128. Sanders EN
    129. Dos Santos G
    130. Scott K
    131. Sherlekar A
    132. Shiu P
    133. Sims D
    134. Sit RV
    135. Slaidina M
    136. Smith HE
    137. Sterne G
    138. Su Y-H
    139. Sutton D
    140. Tamayo M
    141. Tan M
    142. Tastekin I
    143. Treiber C
    144. Vacek D
    145. Vogler G
    146. Waddell S
    147. Wang W
    148. Wilson RI
    149. Wolfner MF
    150. Wong Y-CE
    151. Xie A
    152. Xu J
    153. Yamamoto S
    154. Yan J
    155. Yao Z
    156. Yoda K
    157. Zhu R
    158. Zinzen RP
    159. FCA Consortium§
    (2022) Fly cell atlas: a single-nucleus transcriptomic atlas of the adult fruit fly
    Science 375:eabk2432.
    https://doi.org/10.1126/science.abk2432
    1. Saburi E
    2. Tavakol-Afshari J
    3. Biglari S
    4. Mortazavi Y
    (2017)
    Is α-N-acetylgalactosaminidase the key to curing cancer? A mini-review and hypothesis
    Journal of B.U.ON 22:1372–1377.

Decision letter

  1. Irene Salinas
    Reviewing Editor; University of New Mexico, United States
  2. Carla V Rothlin
    Senior Editor; Yale University, United States
  3. Irene Salinas
    Reviewer; University of New Mexico, United States
  4. Beatriz Novoa
    Reviewer; CSIC, Spain

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Myeloid cell evolution uncovered by shrimp immune cell analysis at single-cell resolution" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Irene Salinas as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Carla Rothlin as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Beatriz Novoa (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The title needs to be revised according to the reviewers' comments.

2) Please revise the hypothesis and goal of the study in the introduction so that the experimental work actually matches the hypothesis and goals. This is very important because all three reviewers agree that the evolutionary hypothesis presented is vague and not the main point of the work.

3) It is critical to address all the experimental concerns regarding the phagocytosis assays and data presented in Figure 3 (see reviewer #1 comments).

4) Please include light microscopy or electron microscopy images of sorted phagocytes.

5) Please include in a supplementary table the % nucleotide and % amino acid identity scores between vertebrate and shrimp gene markers used in the single cell RNA-Seq dataset.

6) As brought up by all reviewers, data interpretation and discussion need to be completely reorganized. The discussion should be much more thorough and include very careful interpretation of the findings.

Reviewer #1 (Recommendations for the authors):

The present study provides a single cell transcriptional atlas of the white shrimp, P. vannamei, immune cells in the hemolymph, known as hemocytes. White shrimp ScRNA-Seq studies uncovered two macrophage-like populations, one of them with markers similar to mammalian macrophages. The study also shows that this new population is phagocytic and expresses proteins such as NLRP3, LYS, and NAGA. These findings redefine the current classification of shrimp immune cells which has been done using morphological approaches and via targeted qPCR studies but never using single cell transcriptomics.

Currently, the manuscript is heavily focused on the single cell dataset which should be the start to formulate hypotheses, not the main focus of the paper. The authors do a good job at identifying the MH2 subset and suggesting that they are phagocytic but further experiments are needed to substantiate this observation and increase the significance of these results.

Abstract:

1. The following sentence does not make any sense "How myeloid cells evolved from invertebrate to vertebrate is still a mystery." I suggest rephrasing to: Despite the importance of innate immunity in invertebrates, the diversity and function of myeloid cells in invertebrates are largely unknown.

2. The abstract needs to be rewritten to just say: Using single-cell RNA-Seq we uncover XXX in the white shrimp hemolymph. Our results identified a novel macrophage-like subset defined by the expression of the XXX gene MH2. MH2+ hemocytes are phagocytic and express markers that indicate some conservation with mammalian macrophages.

3. Combined, our work resolves the heterogeneity of hemocytes in the very economically important aquatic species and identifies a novel myeloid subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.

Introduction:

The introduction should start by stating the importance of innate immunity in invertebrates since there is no B and T cell-based adaptive immunity. Innate immune cells in invertebrates are also very important because they have trained immunity.

After the introductory paragraph, the authors can describe what is known based on single-cell RNA-Seq from invertebrate hemocytes, basically what is already written in the current version of the manuscript from lines 66-79.

Line 80-81: I do not think the question is properly formulated. Of course, there is a common ancestor between vertebrates and invertebrates, and therefore there may be a conservation of many myeloid-like characteristics in invertebrate and vertebrate myeloid subsets. However, invertebrates may have also evolved their own subsets of myeloid cells over evolutionary time. Some may have co-opted some of the same molecules that vertebrate myeloid cells also use but many new functions may have been acquired from other genes/gene families that may have been expanded. I really beg authors to ask themselves again if this is the real question they want to answer.

Line 82: Intrigued by this question, we here use the marine invertebrate XXX to characterize immune cell subsets using scRNA-Seq. The reason to select this species is because of XXX.

Line 91: remove various.

Lines 94-99 read very strangely. If this is the end of the introduction, the authors need to frame the conceptual question and the goal of the study. For instance, if single-cell RNA-Seq has not been done before in P. vannamei please state so.

Line 204: dendritic, not dendric.

Results:

– According to Figure 1C, MH cells express not only lyz1, nlrp3 but also VEGF5. The authors do not comment on this very interesting finding. VEGF expression may confer MH cells the ability to penetrate tissues and induce angiogenesis. I do not see any VEGF5 expression data in Figure 2 so I do not know if it was differentially expressed in MH1 and MH2 populations. Can the authors please clarify? If VEGF5 is, in fact, a marker for MH2 it needs to be shown and discussed in the discussion given that VEGF is also a marker for Hofbauer cells (see my comments below). Lastly, the VEGF pathway plays a very important role in the immune response of shrimp to infection with the very important virus WSSV (doi: 10.3389/fimmu.2017.01457). Because of this, this finding is important and deserves careful examination and discussion.

– There are three unknown genes that chiefly differentiate the MH1 and MH2 clusters. These three genes are highly expressed in MH1 but not MH2 according to Figure 2B. Did the authors try to blast these three unknown genes and identify motifs/superfamilies that may suggest function? I highly suggest trying to do that and add that to the manuscript if any interesting findings emerge.

– In figure 3A: gene markers for MH1 not only point towards classical human macrophages but also Hofbauer cells, which is a unique villous macrophage population in the placenta with fetal origin. This finding is however not further commented on by the authors in the text and is something that needs to be highlighted in the manuscript. These placental macrophages also have phagocytic capabilities and therefore this finding deserves careful examination.

– Figure 3B: please show an FSC/SSC plot to show the gating strategy based on morphology prior to the FITC gating strategy.

– Interpretation of phagocytosis assays: do authors suggest that only MH2 cells are phagocytic or does the GFP+ phagocytic population include cells from other single-cell clusters?

– Phagocytosis assays: please show IF images of the in vivo assay following isolation of hemocytes and imaging of cells with ingested Vibrio GFP in the cytoplasm to confirm true phagocytosis as well as to visualize the morphology of the MH2 population.

– The morphology of the sorted phagocytic population used for qPCR and western blot studies is very important since it may show heterogeneity. It is also very important for the last figure where the authors attempt to compare the past hemocyte classification with their findings. Finally, it is important because of the Hofbauer cell villous morphology. Hofbauer cells have small nuclei and large, highly vacuolated cytoplasm so let's see if this matches with MH2 morphology.

– Phagocytosis control: please conduct ex vivo phagocytosis assays with hemocytes exposed to vibrio GFP in the presence or absence of phagocytosis inhibitors such as inhibitors of actin polymerization.

Key experiments need to be performed pertaining to increased experimental evidence to characterize the MH2 subset.

For instance, really important questions are whether this subset is found in tissues (resident macrophages), when they first appear during shrimp ontogeny, whether dietary immunostimulation or infection alters the numbers of MH2, and their transcriptional profile or even whether they display trained immunity. I do not think the authors need to answer all these questions in this one study but at least one more functional experiment needs to be done to elevate the paper's significance.

Discussion:

– The discussion is very short, lacks structure, and is poorly written. There are grammar errors in the first paragraph. For example: for animals, not animal. Please check the grammar.

– Discussions must always acknowledge the limitations of the study. For instance, here, the authors did not evaluate whether MH2 populations expand during the course of an infection/experimental challenge, which would have made the paper a lot better.

– Another limitation of the paper is whether or not MH2 cells are able to reside within tissues or they are only a circulating population. Please discuss.

– The discussion does not cover in depth any of the findings of the manuscript. For instance, there is a sentence on the NLPR3 expression, but there is no deep discussion of what it means. NLPR3 expression appears to define MH cells even at the steady state but it increases upon phagocytosis according to figure 3. Please discuss these findings. The same goes for NAGA expression, it needs to be further interpreted in the discussion.

– Line 271-273: the citations for teleost RBC being phagocytic need to be more comprehensive, many old papers had shown that not just the 2021 citation provided by the authors.

Reviewer #2 (Recommendations for the authors):

The authors have treated the shrimps with recombinant CREG, however, with the exception of showing that it did not induce cell differentiation, it is not mentioned in the article, nor in the abstract. Even the name of the protein has not been included.

I do not understand the relevance of fish red blood cells in the discussion.

The authors mention that they: "explained some debates in this field". This should be clarified and better explained. Maybe they forgot to include this information.

Also, the questions included in the discussion are not answered or discussed: "For example, why hyalinocytes have both proliferating activity and phagocytic activity (Soderhall, 2016). Why semi-granulocytes have phagocytic activity(M. Sun et al., 2020)."

In summary, the article is correct, but the declared general aim compared with the results is overrated.

Reviewer #3 (Recommendations for the authors):

I have a number of comments that require clarification before I would be willing to endorse the publication of this manuscript.

1. The written manuscript, particularly the introduction, does not align with the expectations set by the manuscript title. As it is written, this paper seems as though it is an investigation into the role of CREG in hemocyte development/differentiation. It seems as though the expectations that CREG would influence hemocyte development did not pan out, and the myeloid evolution angle became the focus. The manuscript introduction and discussion should be refocused to better guide the reader through other invertebrate studies, some of which are single-cell analyses, that contribute to our current understanding of myeloid cell evolution.

2. The methods are generally vague and at times, seem conflicting. For example, DropSeq is mentioned in the Figure 1 caption whereas 10x is mentioned elsewhere. More detail could be included for nearly all method sections.

3. Based on the information provided, primarily in the supplemental tables, it is unclear how similar the vertebrate homologs are to the shrimp genes. These vertebrate factors are used to assign a function to the shrimp transcripts, however, without a % nucleotide or amino acid identity, it is not possible to evaluate how appropriate those putative functions are. This is a cruz of the manuscript, as the conclusions that these factors can be used to define hemocyte subtypes as evolutionarily related to vertebrate myeloid cells appear to be hinged on the fact that they are functionally the same. The % nucleotide and % amino acid identity scores should be included in the supplementary tables.

4. I do not think it is sufficient to suggest that expression patterns of marker genes are strong evidence of developmental relationships between hemocyte subtypes. The authors even use the word 'probably' on line 177 when explaining this analysis. To demonstrate the differentiation relationships being proposed, the authors should sort the hemocyte subtypes using the markers identified and then experimentally show that those subtypes proposed as being 'progenitor cells' can in fact differentiate into effector subtypes.

5. The discussion is incredibly brief and speculative. Statements such as "Crustacean seems possess less phagocytic cells compared with other species, which may be due to its unique endosymbiosis with microbes in its hemolymph, which has been partially unveiled by recent studies" are unsupported and seem tangential to the stated theme of the manuscript. In other instances, the use of terms such as 'a lot' (line 171) seems sloppy and does little to inform the reader. Substantial effort should be directed at focusing the manuscript towards the intended theme.

6. Given the breadth of invertebrate species out there, it seems inappropriate to suggest that shrimp hemocytes are the only invertebrate key to unlocking myeloid cell evolution. The authors should, at the least, create a summary table of other single-cell analyses and hematopoietic hemocyte studies undertaken in invertebrates, highlighting whether there is consistency with the findings of this study in other vertebrate phyla. This type of analysis would lend itself to supporting a primarily sequence-based conclusion of myeloid cell evolution from an invertebrate origin.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Single cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes" for further consideration by eLife. Your revised article has been evaluated by Carla Rothlin (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below.

Specifically, reviewers have still identified issues with the English grammar which needs further improvement, the comparison with other invertebrate single-cell data sets beyond arthropods, and the tone down of some of the conclusions.

Reviewer #5 (Recommendations for the authors):

1) In the manuscript entitled: "Single cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes" by Peng Yang et al. The authors describe the characterization on a single cell level of hemocytes and immunocytes of the Shrimp Penaeus vannamei. Moreover, they find on the molecular level similarities to vertebrate myeloid lineage, specifically macrophage-like cells. They show that those cells are functionally phagocytic cells and have macrophage markers on the RNA and protein levels. Moreover, they compare their findings with the previous characterization of shrimp hemocytes.

2) This is important work since the authors use a commercially important species. The single-cell analysis is laying the base for future cellular understanding. The authors validate their sequencing data for the macrophage-like population using phagocytic function and RT-PCR and protein validation. The manuscript is well written and explained.

There are some overstatements that should be addressed. While the authors are very couscous in making homology overstatement regarding the macrophage-like cells, they are using the myeloid cell lineage as a given to exist and be homolog between vertebrates and invertebrates. Due to that please revise the second sentence in the abstract (line 19) to "innate immune cells" or something like that instead of myeloid. The same is also true for the third line of the discussion (lines 247-248). Since myeloid is a specifically defined subpopulation in vertebrates' hematopoietic system, it is not parallel to phagocytes or innate immune cells. The authors by the end of the manuscript can discuss their findings and that the myeloid lineage might evolve in arthropods already.

An additional issue related to that is to do with the first paragraph of the introduction, while the authors are trying to make a general statement of myeloid lineage in invertebrates, they give only examples from other arthropods. I think to make it a general discussion they should discuss additional works, such as the work of Rosental et al., Nature 2018, where myeloid lineage was shown in tunicates (Botryllus) on the molecular and functional levels. This way the authors can make a more general statement about the earlier emergence of the myeloid lineage.

https://doi.org/10.7554/eLife.80127.sa1

Author response

Essential revisions:

1) The title needs to be revised according to the reviewers' comments.

Thanks for your kindly suggestion. The new title has been focused on the major finding of this manuscript: “Single cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes”.

2) Please revise the hypothesis and goal of the study in the introduction so that the experimental work actually matches the hypothesis and goals. This is very important because all three reviewers agree that the evolutionary hypothesis presented is vague and not the main point of the work.

Thanks for all three reviewers’ valuable suggestion. We have carefully revised the introduction mainly according to reviewer1’s suggestion. In general, we have weakened the evolution hypothesis and addressed that we identified a macrophage-like phagocyte in shrimp hemolymph.

3) It is critical to address all the experimental concerns regarding the phagocytosis assays and data presented in Figure 3 (see reviewer #1 comments).

Thanks for your kindly suggestion. We carefully revised this part and added Figure 3C (confocal microscopy) and Figure 3D (phagocytosis inhibition assay) to justify that the sorted phagocytes were the cells which engulf bacteria.

4) Please include light microscopy or electron microscopy images of sorted phagocytes.

Thanks for your kindly suggestion. We have added Figure 3C (light microscopy). For electron microscopy, we have tried hard but didn’t get enough amount for TEM sample preparation. This group of phagocytes are extremely fragile and collection rate is very low even with fixation.

5) Please include in a supplementary table the % nucleotide and % amino acid identity scores between vertebrate and shrimp gene markers used in the single cell RNA-Seq dataset.

Thanks for your kindly suggestion. We have added supplementary file 11 to indicate the % nucleotide and % amino acid identity scores between vertebrate and shrimp gene markers used in this study.

6) As brought up by all reviewers, data interpretation and discussion need to be completely reorganized. The discussion should be much more thorough and include very careful interpretation of the findings.

Thanks for your kindly suggestion. We have carefully revised the whole discussion including comprehensive understanding of the findings.

Reviewer #1 (Recommendations for the authors):

The present study provides a single cell transcriptional atlas of the white shrimp, P. vannamei, immune cells in the hemolymph, known as hemocytes. White shrimp ScRNA-Seq studies uncovered two macrophage-like populations, one of them with markers similar to mammalian macrophages. The study also shows that this new population is phagocytic and expresses proteins such as NLRP3, LYS, and NAGA. These findings redefine the current classification of shrimp immune cells which has been done using morphological approaches and via targeted qPCR studies but never using single cell transcriptomics.

Thanks for your kindly comment. We have uncovered one macrophage-like population and another population is granulocyte.

Currently, the manuscript is heavily focused on the single cell dataset which should be the start to formulate hypotheses, not the main focus of the paper. The authors do a good job at identifying the MH2 subset and suggesting that they are phagocytic but further experiments are needed to substantiate this observation and increase the significance of these results.

Thanks for your kindly comments. We have added confocal microscopy image (Figure 3C) and phagocytosis inhibition assay (Figure 3D) for verification of these sorted phagocytes.

Abstract:

1. The following sentence does not make any sense "How myeloid cells evolved from invertebrate to vertebrate is still a mystery." I suggest rephrasing to: Despite the importance of innate immunity in invertebrates, the diversity and function of myeloid cells in invertebrates are largely unknown.

Thanks for your valuable comments, we have followed your suggestion and revised this part in abstract (Line18-19).

2. The abstract needs to be rewritten to just say: Using single-cell RNA-Seq we uncover XXX in the white shrimp hemolymph. Our results identified a novel macrophage-like subset defined by the expression of the XXX gene MH2. MH2+ hemocytes are phagocytic and express markers that indicate some conservation with mammalian macrophages.

Thanks for your valuable comments, we have followed your suggestion and revised this part in abstract (Line19-25).

3. Combined, our work resolves the heterogeneity of hemocytes in the very economically important aquatic species and identifies a novel myeloid subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.

Thanks for your valuable comments, we have followed your suggestion and revised this part in abstract (Line25-28).

Introduction:

The introduction should start by stating the importance of innate immunity in invertebrates since there is no B and T cell-based adaptive immunity. Innate immune cells in invertebrates are also very important because they have trained immunity.

After the introductory paragraph, the authors can describe what is known based on single-cell RNA-Seq from invertebrate hemocytes, basically what is already written in the current version of the manuscript from lines 66-79.

Line 80-81: I do not think the question is properly formulated. Of course, there is a common ancestor between vertebrates and invertebrates, and therefore there may be a conservation of many myeloid-like characteristics in invertebrate and vertebrate myeloid subsets. However, invertebrates may have also evolved their own subsets of myeloid cells over evolutionary time. Some may have co-opted some of the same molecules that vertebrate myeloid cells also use but many new functions may have been acquired from other genes/gene families that may have been expanded. I really beg authors to ask themselves again if this is the real question they want to answer.

Thanks for your kindly suggestion. We recognize that the evolution is so complex and my study in this manuscript could not lead to such a conclusion. Thus, we have deleted line80-81.

Line 82: Intrigued by this question, we here use the marine invertebrate XXX to characterize immune cell subsets using scRNA-Seq. The reason to select this species is because of XXX.

Line 91: remove various.

Lines 94-99 read very strangely. If this is the end of the introduction, the authors need to frame the conceptual question and the goal of the study. For instance, if single-cell RNA-Seq has not been done before in P. vannamei please state so.

Line 204: dendritic, not dendric.

Thanks for your valuable comments, we have revised this part as you suggestion.

Results:

– According to Figure 1C, MH cells express not only lyz1, nlrp3 but also VEGF5. The authors do not comment on this very interesting finding. VEGF expression may confer MH cells the ability to penetrate tissues and induce angiogenesis. I do not see any VEGF5 expression data in Figure 2 so I do not know if it was differentially expressed in MH1 and MH2 populations. Can the authors please clarify? If VEGF5 is, in fact, a marker for MH2 it needs to be shown and discussed in the discussion given that VEGF is also a marker for Hofbauer cells (see my comments below). Lastly, the VEGF pathway plays a very important role in the immune response of shrimp to infection with the very important virus WSSV (doi: 10.3389/fimmu.2017.01457). Because of this, this finding is important and deserves careful examination and discussion.

Thanks for your kindly reminder. Figure 1C shows that MH cells express VEGF3. Below are the reasons that why we don’t talk about VEGF:

1. Currently, five VEGF subtypes (VEGF1-5) have been identified in shrimp (doi: 10.1016/j.fsi.2015.10.026 doi: 10.1016/j.dci.2016.05.020 doi: 10.1016/j.fsi.2018.10.019). However, shrimp holds an open circulation system with partial blood vessel. Until now it is not quite sure that whether angiogenesis in adult is important or not.

2. Besides VEGF3, other VEGFs could express almost all tissues in shrimp (doi: 10.1016/j.fsi.2015.10.026 doi: 10.1016/j.dci.2016.05.020 doi: 10.1016/j.fsi.2018.10.019) although different subtypes may have some tissue preference. People don’t know what’s the difference for all these VEGF subtypes in shrimp. Here, we used bubble chart to characterize the expression of VEGF3 in different types of blood cells of shrimp (see Author response image 1).

Author response image 1
This Figure indicates that VEGF3 has no significant differentially expression in MH1 and MH2 populations.

Thus, we don’t list this gene in Figure 2B and give a discussion for this marker. Moreover, it is difficult to address this point because VEGFs in shrimp have five subtypes. All five subtypes play some roles during WSSV infection (doi: 10.1016/j.fsi.2015.10.026 doi: 10.1016/j.dci.2016.05.020 doi: 10.1016/j.fsi.2018.10.019). Only VEGF3 was highly expressed in MH subtype. This system is too complicated and far away from being clarified at this moment. Thus, it is very difficult to discussion this part now.

– There are three unknown genes that chiefly differentiate the MH1 and MH2 clusters. These three genes are highly expressed in MH1 but not MH2 according to Figure 2B. Did the authors try to blast these three unknown genes and identify motifs/superfamilies that may suggest function? I highly suggest trying to do that and add that to the manuscript if any interesting findings emerge.

Thanks for your valuable suggestion. Author response image 2 is the blast results in Pubmed.

Author response image 2
This Image represents the top three genes are quite similar and only conserved in shrimp.

The blast results don’t show much information about these proteins’ function.

– In figure 3A: gene markers for MH1 not only point towards classical human macrophages but also Hofbauer cells, which is a unique villous macrophage population in the placenta with fetal origin. This finding is however not further commented on by the authors in the text and is something that needs to be highlighted in the manuscript. These placental macrophages also have phagocytic capabilities and therefore this finding deserves careful examination.

Thanks for your valuable suggestion. we have added some discussion about this point (Line189-Line190). Currently very few information about invertebrate phagocytes has been studied. Further study is needed after preparation of monoclonal antibodies recognizing this subtype, which is under the way in my lab.

– Figure 3B: please show an FSC/SSC plot to show the gating strategy based on morphology prior to the FITC gating strategy.

Thanks for your valuable comments. We have sorted out a typical FSC/SSC plot for the gating strategy (Author response image 3).

Author response image 3
These images show that setting a suitable gating strategy to excluded interference factors to visualize FITC.

– Interpretation of phagocytosis assays: do authors suggest that only MH2 cells are phagocytic or does the GFP+ phagocytic population include cells from other single-cell clusters?

Thanks for your question, we cannot say only MH2 cells are phagocytic at this moment. In this study MH2 cell is 1.9% of total hemocytes. According to our previous study and other group study (doi: 10.3389/fimmu.2021.707770; doi: 10.4049/jimmunol.1900156.), shrimp phagocyte rate is around 1% to 10% of total hemocytes with huge individual variation. GFP+ phagocytic population may include cells from other single-cell clusters.

– Phagocytosis assays: please show IF images of the in vivo assay following isolation of hemocytes and imaging of cells with ingested Vibrio GFP in the cytoplasm to confirm true phagocytosis as well as to visualize the morphology of the MH2 population.

Thanks for your kindly suggestion. We have added the confocal image for isolated phagocytes which ingested FITC-VP (Figure 3C).

– The morphology of the sorted phagocytic population used for qPCR and western blot studies is very important since it may show heterogeneity. It is also very important for the last figure where the authors attempt to compare the past hemocyte classification with their findings. Finally, it is important because of the Hofbauer cell villous morphology. Hofbauer cells have small nuclei and large, highly vacuolated cytoplasm so let's see if this matches with MH2 morphology.

Thanks for your kindly comment. We have added confocal microscopy image in this manuscript (Figure 3C). As shown in Figure 3C, the shrimp phagocytes have round nuclei with highly vacuolated cytoplasm which is a typical macrophage morphology (Line 198-200).

– Phagocytosis control: please conduct ex vivo phagocytosis assays with hemocytes exposed to vibrio GFP in the presence or absence of phagocytosis inhibitors such as inhibitors of actin polymerization.

Key experiments need to be performed pertaining to increased experimental evidence to characterize the MH2 subset.

For instance, really important questions are whether this subset is found in tissues (resident macrophages), when they first appear during shrimp ontogeny, whether dietary immunostimulation or infection alters the numbers of MH2, and their transcriptional profile or even whether they display trained immunity. I do not think the authors need to answer all these questions in this one study but at least one more functional experiment needs to be done to elevate the paper's significance.

Thanks for your kindly suggestion. We have added both confocal image (Figure 3C) and phagocytosis inhibition assay (Figure 3D) to characterize the phagocytes which ingested FITC-VP. For the question about resident macrophages, how it changes with immunostimulation or infection. All these important questions required high quality antibodies for certain MH2 marker genes, which we are working hard to screen out.

Discussion:

– The discussion is very short, lacks structure, and is poorly written. There are grammar errors in the first paragraph. For example: for animals, not animal. Please check the grammar.

Thanks for your kindly comment. We have rewritten the discussion.

– Discussions must always acknowledge the limitations of the study. For instance, here, the authors did not evaluate whether MH2 populations expand during the course of an infection/experimental challenge, which would have made the paper a lot better.

Thanks for your kindly comment. We are working hard to screen monoclonal antibodies for MH2 surface marker and examine these issues in future studies.

– Another limitation of the paper is whether or not MH2 cells are able to reside within tissues or they are only a circulating population. Please discuss.

Thanks for your kindly suggestion. We have added line 279-284 to discuss this issue.

– The discussion does not cover in depth any of the findings of the manuscript. For instance, there is a sentence on the NLPR3 expression, but there is no deep discussion of what it means. NLPR3 expression appears to define MH cells even at the steady state but it increases upon phagocytosis according to figure 3. Please discuss these findings. The same goes for NAGA expression, it needs to be further interpreted in the discussion.

Thanks for your kindly suggestion. Because we cannot isolate MH2 subset at this moment, whether Nlrp3 expression is upregulated upon phagocytosis is not clear. We have added line256 -line 267 to further discuss this finding.

– Line 271-273: the citations for teleost RBC being phagocytic need to be more comprehensive, many old papers had shown that not just the 2021 citation provided by the authors.

Thanks for your kindly suggestion. We have rewritten this part and added several references.

Reviewer #2 (Recommendations for the authors):

The authors have treated the shrimps with recombinant CREG, however, with the exception of showing that it did not induce cell differentiation, it is not mentioned in the article, nor in the abstract. Even the name of the protein has not been included.

Thanks for your kindly suggestion. Indeed, this experiment was designed originally to explore shrimp plasma CREG function. However, recombinant CREG treatment doesn’t induce hemocyte differentiation and just activate the hemocytes in general which we have mentioned in line75-77 and line86-90. For this reason, we focused on identification of novel shrimp hemocyte subtype in this study.

I do not understand the relevance of fish red blood cells in the discussion.

Thanks for your kindly comment. We have rewritten the discussion of this manuscript and put focus on identification of a novel macrophage-like phagocyte in shrimp hemolymph. Thus, we tried to compare different phagocytic cells from various species and discuss their evolution(line273-284).

The authors mention that they: "explained some debates in this field". This should be clarified and better explained. Maybe they forgot to include this information.

Thanks for your kindly suggestion. We have rewritten this part and explained the debates about hyalinocytes and semi-granulocytes (Line287-300).

Also, the questions included in the discussion are not answered or discussed: "For example, why hyalinocytes have both proliferating activity and phagocytic activity (Soderhall, 2016). Why semi-granulocytes have phagocytic activity (M. Sun et al., 2020)."

Thanks for your kindly comments. I have deleted these two sentences and rewritten this part.

In summary, the article is correct, but the declared general aim compared with the results is overrated.

Thanks for your kindly suggestion. We have realized this problem and rewritten the whole manuscript in which we have shifted the manuscript focus from myeloid cell evolution to identification of an invertebrate macrophage-like phagocyte.

Reviewer #3 (Recommendations for the authors):

I have a number of comments that require clarification before I would be willing to endorse the publication of this manuscript.

1. The written manuscript, particularly the introduction, does not align with the expectations set by the manuscript title. As it is written, this paper seems as though it is an investigation into the role of CREG in hemocyte development/differentiation. It seems as though the expectations that CREG would influence hemocyte development did not pan out, and the myeloid evolution angle became the focus. The manuscript introduction and discussion should be refocused to better guide the reader through other invertebrate studies, some of which are single-cell analyses, that contribute to our current understanding of myeloid cell evolution.

Thanks for your kindly comment. Yes, we designed this experiment to explore CREG differentiation function at the beginning. However, CREG doesn’t show such a function so that we put focus on shrimp hemocyte subtype identification in this study. I have carefully rewritten the introduction and Discussion section to address identification of a novel microphage-like phagocyte instead of myeloid cell evolution which is too broad for this study.

2. The methods are generally vague and at times, seem conflicting. For example, DropSeq is mentioned in the Figure 1 caption whereas 10x is mentioned elsewhere. More detail could be included for nearly all method sections.

Thanks for your kindly comment. I have carefully revised the method sections which was labelled with red (Line334-345, Line364-389) and deleted the DropSeq in Figure 1 caption. The study was completely performed in 10×Genomics platform.

3. Based on the information provided, primarily in the supplemental tables, it is unclear how similar the vertebrate homologs are to the shrimp genes. These vertebrate factors are used to assign a function to the shrimp transcripts, however, without a % nucleotide or amino acid identity, it is not possible to evaluate how appropriate those putative functions are. This is a cruz of the manuscript, as the conclusions that these factors can be used to define hemocyte subtypes as evolutionarily related to vertebrate myeloid cells appear to be hinged on the fact that they are functionally the same. The % nucleotide and % amino acid identity scores should be included in the supplementary tables.

Thanks for your kindly suggestion. We have added supplementary file 11 to address this issue.

4. I do not think it is sufficient to suggest that expression patterns of marker genes are strong evidence of developmental relationships between hemocyte subtypes. The authors even use the word 'probably' on line 177 when explaining this analysis. To demonstrate the differentiation relationships being proposed, the authors should sort the hemocyte subtypes using the markers identified and then experimentally show that those subtypes proposed as being 'progenitor cells' can in fact differentiate into effector subtypes.

Thanks for your kindly suggestion. I recognized that I have over-interpreted the data shown in this manuscript. Thus, I have carefully rewritten the whole manuscript and shifted my major claim from myeloid cell evolution to identification of a novel invertebrate macrophage-like phagocytes. We are trying hard to screen the markers’ monoclonal antibodies for major subtypes listed in this study.

5. The discussion is incredibly brief and speculative. Statements such as "Crustacean seems possess less phagocytic cells compared with other species, which may be due to its unique endosymbiosis with microbes in its hemolymph, which has been partially unveiled by recent studies" are unsupported and seem tangential to the stated theme of the manuscript. In other instances, the use of terms such as 'a lot' (line 171) seems sloppy and does little to inform the reader. Substantial effort should be directed at focusing the manuscript towards the intended theme.

Thanks for your kindly comment. we have deleted the sentence” Crustacean seems possess less phagocytic cells compared with other species, which may be due to its unique endosymbiosis with microbes in its hemolymph, which has been partially unveiled by recent studies”. Moreover, we have changed “a lot of” to “some” (line147). In general, we have carefully rewritten the whole manuscript.

6. Given the breadth of invertebrate species out there, it seems inappropriate to suggest that shrimp hemocytes are the only invertebrate key to unlocking myeloid cell evolution. The authors should, at the least, create a summary table of other single-cell analyses and hematopoietic hemocyte studies undertaken in invertebrates, highlighting whether there is consistency with the findings of this study in other vertebrate phyla. This type of analysis would lend itself to supporting a primarily sequence-based conclusion of myeloid cell evolution from an invertebrate origin.

Thanks for your kindly suggestion. I have added Supplementary file 10 to compare our study with other invertebrate cellular immunity studies.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Reviewer #5 (Recommendations for the authors):

1) In the manuscript entitled: "Single cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes" by Peng Yang et al. The authors describe the characterization on a single cell level of hemocytes and immunocytes of the Shrimp Penaeus vannamei. Moreover, they find on the molecular level similarities to vertebrate myeloid lineage, specifically macrophage-like cells. They show that those cells are functionally phagocytic cells and have macrophage markers on the RNA and protein levels. Moreover, they compare their findings with the previous characterization of shrimp hemocytes.

2) This is important work since the authors use a commercially important species. The single-cell analysis is laying the base for future cellular understanding. The authors validate their sequencing data for the macrophage-like population using phagocytic function and RT-PCR and protein validation. The manuscript is well written and explained.

There are some overstatements that should be addressed. While the authors are very couscous in making homology overstatement regarding the macrophage-like cells, they are using the myeloid cell lineage as a given to exist and be homolog between vertebrates and invertebrates. Due to that please revise the second sentence in the abstract (line 19th) to "innate immune cells" or something like that instead of myeloid. The same is also true for the third line of the discussion (lines 247-248). Since myeloid is a specifically defined subpopulation in vertebrates' hematopoietic system, it is not parallel to phagocytes or innate immune cells. The authors by the end of the manuscript can discuss their findings and that the myeloid lineage might evolve in arthropods already.

Thank you for this suggestion, I have replaced “myeloid cell” with “innate immune cell” for invertebrate.

An additional issue related to that is to do with the first paragraph of the introduction, while the authors are trying to make a general statement of myeloid lineage in invertebrates, they give only examples from other arthropods. I think to make it a general discussion they should discuss additional works, such as the work of Rosental et al., Nature 2018, where myeloid lineage was shown in tunicates (Botryllus) on the molecular and functional levels. This way the authors can make a more general statement about the earlier emergence of the myeloid lineage.

Thank you for this suggestion, I have weakened the myeloid lineage claim for invertebrate and replace them with “innate immune cell”. In addition, I have mentioned the work of Rosental et al., Nature 2018 in line 39.

https://doi.org/10.7554/eLife.80127.sa2

Article and author information

Author details

  1. Peng Yang

    Institute of Marine Sciences, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China
    Contribution
    Validation, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8298-8926
  2. Yaohui Chen

    Department of Biology, College of Science, Shantou University, Shantou, China
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4044-4373
  3. Zhiqi Huang

    Department of Biology, College of Science, Shantou University, Shantou, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Huidan Xia

    Department of Biology, College of Science, Shantou University, Shantou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Ling Cheng

    Guangzhou Genedenovo Biotechnology Company Limited, Guangzhou, China
    Contribution
    Software, Formal analysis
    Competing interests
    are employees of Guangzhou Genedenovo Biotechnology Company Limited
  6. Hao Wu

    Guangzhou Genedenovo Biotechnology Company Limited, Guangzhou, China
    Contribution
    Software, Formal analysis
    Competing interests
    are employees of Guangzhou Genedenovo Biotechnology Company Limited
  7. Yueling Zhang

    1. Institute of Marine Sciences, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China
    2. Shantou University-Universiti Malaysia Terengganu Joint Shellfish Research Laboratory, Shantou University, Shantou, China
    3. Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China
    Contribution
    Funding acquisition, Writing – review and editing
    Competing interests
    No competing interests declared
  8. Fan Wang

    1. Institute of Marine Sciences, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China
    2. Department of Biology, College of Science, Shantou University, Shantou, China
    3. Shantou University-Universiti Malaysia Terengganu Joint Shellfish Research Laboratory, Shantou University, Shantou, China
    Contribution
    Conceptualization, Data curation, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing – review and editing
    For correspondence
    wangfan@stu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6059-6956

Funding

National Natural Science Foundation of China (41976123)

  • Fan Wang

Guangdong Science and Technology Department (14600703)

  • Fan Wang

Li Ka Shing Foundation (2020LKSFG01E)

  • Yueling Zhang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (41976123 to FW), Sail Plan Program for the Introduction of Outstanding Talents of Guangdong Province of China (14600703 to FW), and 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (2020LKSFG01E to YZ).

Ethics

All the animal-related experiments were in accordance with Shantou University guidelines.

Senior Editor

  1. Carla V Rothlin, Yale University, United States

Reviewing Editor

  1. Irene Salinas, University of New Mexico, United States

Reviewers

  1. Irene Salinas, University of New Mexico, United States
  2. Beatriz Novoa, CSIC, Spain

Publication history

  1. Received: May 9, 2022
  2. Preprint posted: May 18, 2022 (view preprint)
  3. Accepted: October 5, 2022
  4. Accepted Manuscript published: October 6, 2022 (version 1)
  5. Version of Record published: October 20, 2022 (version 2)

Copyright

© 2022, Yang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 913
    Page views
  • 331
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Peng Yang
  2. Yaohui Chen
  3. Zhiqi Huang
  4. Huidan Xia
  5. Ling Cheng
  6. Hao Wu
  7. Yueling Zhang
  8. Fan Wang
(2022)
Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes
eLife 11:e80127.
https://doi.org/10.7554/eLife.80127
  1. Further reading

Further reading

    1. Chromosomes and Gene Expression
    2. Immunology and Inflammation
    Allison R Wagner, Chi G Weindel ... Kristin L Patrick
    Research Article Updated

    To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage’s response to pathogens.

    1. Computational and Systems Biology
    2. Immunology and Inflammation
    Harry Kane, Nelson M LaMarche ... Lydia Lynch
    Research Article

    Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated murine iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.