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

  • Curated by eLife

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    Evaluation Summary:

    The shrimp market is growing globally, with 8.12 tons produced in 2020. The market size is thought to reach $55 billion by 2027. Most of the market comes from farms, the white shrimp, Penaeus vannamei, being one of the most commonly farmed species worldwide. 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 single cell RNA sequencing studies uncovered two macrophage-like populations, one of them with markers similar to mammalian macrophages. 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.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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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.

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  1. Version published to 10.7554/elife.80127 on eLife
  2. eLife

    Evaluation Summary:

    The shrimp market is growing globally, with 8.12 tons produced in 2020. The market size is thought to reach $55 billion by 2027. Most of the market comes from farms, the white shrimp, Penaeus vannamei, being one of the most commonly farmed species worldwide. 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 single cell RNA sequencing studies uncovered two macrophage-like populations, one of them with markers similar to mammalian macrophages. 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.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The present study aims to define the main immune cell subsets found in the hemolymph of the white shrimp, P. vannamei. This is significant because this species is heavily farmed around the world to meet the demand of the human consumption market. Yet, farmed shrimp suffer from infectious diseases and therefore we need to understand how their immune system works to design strategies that decrease infection losses.

    Classification of crustacean (and other invertebrates) hemocytes is difficult due to the lack of antibodies to use traditional flow cytometry approaches. Furthermore, hemocyte purification is not easy, cells die and clump, again precluding flow cytometry studies. Thus, the majority of what we know about shrimp hemocytes is based on morphological classification. This study contributes significantly to advancing our knowledge of shrimp Immunobiology by defining hemocyte subsets based on their transcriptional profiles.

    Another strength of the paper is that some function in vivo assays (phagocytosis) are presented in an attempt to validate the single-cell data. The authors frame their question or try to frame their question with a more evolutionary angle, such as whether the macrophage-like cell is the evolutionary precursor of human macrophages. I think that this question is not really achievable because the evolution of innate immune systems may have diverged in many branches of the metazoan tree of life. The authors, however, identify gene markers that are conserved in macrophages from shrimp and humans and that is a fair conclusion. There are some methodological caveats to the study and the manuscript needs to be heavily edited to improve language as well as to increase the depth of the interpretation.

    In summary, there are interesting findings in this manuscript but the manuscript needs to be significantly improved so that its quality and impact are elevated.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript, the authors have studied the hemocytes from Penaeus vannamei by single-cell transcriptomics. They redefined the shrimp hemocyte classification based on functional marker genes. One of the interesting findings was the identification of the monocytic hemocyte MH2 population, with macrophage-like phagocytic properties.

    The article is interesting because it improves the characterization of shrimp hemocytes and it compares the new classification with the traditional one. However, in my opinion, the manuscript would need some reorganization. There is a considerable mix-up of information that belongs to other sections included in the Results section. Also, the authors did not answer their question: How do myeloid cells evolve from invertebrate to vertebrate? This "evolution" is present also in the title. However, there are no clear results that explain this evolution. Moreover, the discussion is too brief and out of focus.

  5. eLife

    Reviewer #3 (Public Review):

    Yang et al have undertaken a single cell transcriptomic analysis of circulating immune cells from the shrimp, Penes vannamei. They set out to characterize transcriptional differences between circulating immune cell subsets following immune stimulation. Their investigation reveals that shrimp immune cells can be classified into a number of specific subsets defined by unique transcriptional profiles. Using specific marker genes for each cell subset, the authors provide evidence suggesting that shrimp immune cells share transcription factors that define myeloid cell development in mammalian (human) systems.

    This study follows an investigative path that is shared by numerous single-cell transcriptomic studies. The authors do an admirable job of synthesizing a complex single-cell transcriptomic analysis into a focused report that highlights important transcripts that define the hemocyte subsets of the shrimp. While I disagree with some of the claims being made related to the evolutionary connection between shrimp hemocytes and mammalian myeloid cells, this dataset will undoubtedly contribute to our understanding of invertebrate immune cell complexity and the relationships these cells have to other invertebrate hemocytes and immune cell evolution.

  6. Version published to 10.1101/2022.05.17.492277v1 on bioRxiv