Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process.

Keiichiro Koiwai,Kiyoshi Kikuchi,Hiroaki Suzuki,Ryuji Kawano,Soichiro Tsuda,Atsushi Toyoda,Takashi Koyama

doi:10.7554/elife.66954

Abstract

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

Highlights

Aquaculture is an important source of animal protein and is considered one of the most important long-term growth areas of food production, providing 60% of fish for human consumption (FAO, 2020)
We identified 10 cell growth-related genes from Ex50 that are predicted to be involved in cell growth and differentiation: extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase kinase 4 (MKK4), insulin-like growth factor-binding protein 4 (IGFBP-4), vascular endothelial growth factor 1 (VEGF-1), VEGF-3, astakine, crustacean hematopoietic factor-like protein (CHF-like), PDGF/VEGF-related factor 1, growth hormone secretagogue receptor type 1 (GHSR), and platelet-derived growth factor receptor alpha-like (PDGFRA-like) (Figure 7A; Supplementary file 1)
The results showed that the average DDCt values of the transcripts of HemTGase were higher in region 1 (R1) hemocytes than in region 2 (R2) hemocytes, while the values of penaeidin, crustin, stylicin, proPO, and superoxide dismutase (SOD) were higher in R2 hemocytes (Figure 9F)

Summary

Introduction

Aquaculture is an important source of animal protein and is considered one of the most important long-term growth areas of food production, providing 60% of fish for human consumption (FAO, 2020) (http://www.fao.org/fishery/statistics/en). As the morphology and dye staining properties of shrimp hemocytes are not absolute indicators, no unified understanding of their role has been established yet. Molecular markers, such as specific mRNAs, antibodies, or lectins, are usually available for characterizing the subpopulations of cells in model organisms, but this is not often the case for non-model organisms. To perform scRNA-seq, a high-quality gene reference is essential; such reference genomes are scarce for crustaceans because of the extremely high proportion of simple sequence repeats (Zhang et al, 2019). Our results present a unified classification of shrimp hemocytes and a deeper understanding of the immune system of shrimp

Results

Discussion

Materials and methods

Funding Funder