Abstract
The classification of cells in non-model organisms has lagged behind the classification of cells in model organisms that have established cluster of differentiation marker sets. To reduce fish diseases, research is needed to better understand immune-related cells, or hemocytes, in non-model organisms like shrimp and other marine invertebrates. In this study, we used Drop-seq to examine how virus infection affected the populations of hemocytes in kuruma shrimp, Penaeus japonicus, which had been artificially infected with a virus. The findings demonstrated that virus infection reduced particular cell populations in circulating hemolymph and inhibited the expression of antimicrobial peptides. We also identified the gene sets that are likely to be responsible for this reduction. Additionally, we identified functionally unknown genes as novel antimicrobial peptides, and we supported this assumption by the fact that these genes were expressed in the population of hemocytes that expressed other antimicrobial peptides. In addition, we aimed to improve the operability of the experiment by conducting Drop-seq with fixed cells as a source and discussed the impact of methanol fixation on Drop-seq data in comparison to previous results obtained without fixation. These results not only deepen our understanding of the immune system of crustaceans but also demonstrate that single-cell analysis can accelerate research on non-model organisms.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
Sequencing data have been deposited in DDBJ under accession codes DRA015407. Data code can be accessed at https://github.com/KeiichiroKOIWAI/Drop-seq-PjHem
The following dataset was generated:
Author(s) | Year | Dataset title | Dataset URL | Database and identifier |
|---|---|---|---|---|
Koiwai K, Kondo H, Hirono I | 2022 | Raw sequence data of drop-seq | https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA015/DRA015407/ | DDBJ Sequence Read Archive, DRA015407 |
The following previously published dataset was used:
Author(s) | Year | Dataset title | Dataset URL | Database and identifier |
|---|---|---|---|---|
Koiwai K, Koyama T, Tsuda S, Toyoda A, Kikuchi K, Suzuki H, Kawano R | 2020 | Raw sequence data of drop-seq on shrimp 1 | https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA010/DRA010950/ | DDBJ Sequence Read Archive, DRA010950 |
Koiwai K, Koyama T, Tsuda S, Toyoda A, Kikuchi K, Suzuki H, Kawano R | 2020 | Raw sequence data of drop-seq on shrimp 2 | https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA010/DRA010951/ | DDBJ Sequence Read Archive, DRA010951 |
Koiwai K, Koyama T, Tsuda S, Toyoda A, Kikuchi K, Suzuki H, Kawano R | 2020 | Raw sequence data of drop-seq on shrimp 3 | https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA010/DRA010952/ | DDBJ Sequence Read Archive, DRA010952 |
References
Alles J, Karaiskos N, Praktiknjo SD, Grosswendt S, Wahle P, Ruffault PL, Ayoub S, Schreyer L, Boltengagen A, Birchmeier C, Zinzen R, Kocks C, Rajewsky N (2017) Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol 15:44.
Biočanin M, Bues J, Dainese R, Amstad E, Deplancke B (2019) Simplified Drop-seq workflow with minimized bead loss using a bead capture and processing microfluidic chip. Lab Chip 19:1610–1620.
Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420.
Charoensapsri W, Sangsuriya P, Lertwimol T, Gangnonngiw W, Phiwsaiya K, Senapin S (2015) Laminin receptor protein is implicated in hemocyte homeostasis for the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Dev Comp Immunol 51:39–47.
Cui C, Liang Q, Tang X, Xing J, Sheng X, Zhan W (2020) Differential apoptotic responses of hemocyte subpopulations to white spot syndrome virus infection in Fenneropenaeus chinensis. Front Immunol 11:594390. https://doi.org/10.3389/fimmu.2020.594390
Cui C, Tang X, Xing J, Sheng X, Chi H, Zhan W (2022) Single-cell RNA-seq uncovered hemocyte functional subtypes and their differentiational characteristics and connectivity with morphological subpopulations in Litopenaeus vannamei. Front Immunol 13:980021.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21.
Elbahnaswy S, Koiwai K, Zaki VH, Shaheen AA, Kondo H, Hirono I (2017) A novel viral responsive protein (MjVRP) from Marsupenaeus japonicus haemocytes is involved in white spot syndrome virus infection. Fish Shellfish Immunol 70:638–647.
Flegel TW (2012) Historic emergence, impact and current status of shrimp pathogens in Asia. J Invertebr Pathol 110:166–173.
Flegel TW (2019) A future vision for disease control in shrimp aquaculture. J World Aquaculture Soc 50:249–266
Garcia-Castro H, Kenny NJ, Iglesias M, Alvarez-Campos P, Mason V, Elek A, Schonauer A, Sleight VA, Neiro J, Aboobaker A, Permanyer J, Irimia M, Sebe-Pedros A, Solana J (2021) ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. Genome Biol 22:89.
Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, Papalexi E, Mimitou EP, Jain J, Srivastava A, Stuart T, Fleming LM, Yeung B, Satija R (2021) Integrated analysis of multimodal single-cell data. Cell 184:3573-3587.e29
Huan Y, Kong Q, Mou H, Yi H (2020) Antimicrobial peptides: classification, design, application and research progress in multiple fields. Front Microbiol 11:582779.
Kawato S, Nishitsuji K, Arimoto A, Hisata K, Kawamitsu M, Nozaki R, Kondo H, Shinzato C, Ohira T, Satoh N, Shoguchi E, Hirono I (2021) Genome and transcriptome assemblies of the kuruma shrimp, Marsupenaeus japonicus. G3 11(11). https://doi.org/10.1093/g3journal/jkab268
Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, Peshkin L, Weitz DA, Kirschner MW (2015) Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161:1187–1201.
Koiwai K, Koyama T, Tsuda S, Toyoda A, Kikuchi K, Suzuki H, Kawano R (2021) Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process. eLife 10 e66954. https://doi.org/10.7554/eLife.66954
Li H, Xu H, Zhao C, Sulaiman Y, Wu C (2011) A PCR amplification method without DNA extraction. Electrophoresis 32:394–397.
Li C, Li H, Chen Y, Chen Y, Wang S, Weng S-P, Xu X, He J (2015) Activation of Vago by interferon regulatory factor (IRF) suggests an interferon system-like antiviral mechanism in shrimp. Sci Rep 5:15078.
Li F, Zheng Z, Li H, Fu R, Xu L, Yang F (2021) Crayfish hemocytes develop along the granular cell lineage. Sci Rep 11:13099. https://doi.org/10.1038/s41598-021-92473-9
Li Y, Zhou F, Yang Q, Jiang S, Huang, J, Yang L, Ma Z, Jiang S (2022) Single-cell sequencing reveals types of hepatopancreatic cells and haemocytes in black tiger shrimp (Penaeus monodon) and their molecular responses to ammonia stress. Front Immunol 13. https://doi.org/10.3389/fimmu.2022.883043
Lin X, Söderhäll K, Söderhäll I (2011) Invertebrate hematopoiesis: an astakine-dependent novel hematopoietic factor. J Immunol 186:2073–2079.
Lin Z, Akin H, Rao R, Hie B, Zhu Z, Lu W, Smetanin N, Verkuil R, Kabeli O, Shmueli Y, dos Santos Costa A, Fazel-Zarandi M, Sercu T, Candido S, Rives A (2023) Evolutionary-scale prediction of atomic-level protein structure with a language model. Science 379:1123–1130.
Lin TT, Yang LY, Lu IH, Cheng WC, Hsu ZR, Chen S-H, Lin, CY (2021) AI4AMP: an Antimicrobial peptide predictor using physicochemical property-based encoding method and deep learning. mSystems 6:e0029921.
Liu MJ, Liu S, Liu HP (2021) Recent insights into hematopoiesis in crustaceans. Fish and Shellfish Immunol Reports 2:100040.
Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161:1202–1214.
Meng J, Wang W-X (2022) Highly sensitive and specific responses of oyster hemocytes to copper exposure: single-cell transcriptomic analysis of different cell populations. Environ Sci Technol 56:2497–2510.
Meng J, Zhang G, Wang W-X (2022) Functional heterogeneity of immune defenses in molluscan oysters Crassostrea hongkongensis revealed by high-throughput single-cell transcriptome. Fish Shellfish Immunol 120:202–213.
Naylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger DH, Little DC, Lubchenco J, Shumway SE, Troell M (2021) A 20-year retrospective review of global aquaculture. Nature 591:551–563.
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612.
Ren Q, Huang X, Cui Y, Sun J, Wang W, Zhang X (2017. Two white spot syndrome virus microRNAs target the dorsal gene to promote virus infection in Marsupenaeus japonicus shrimp. J Virol 91(8). https://doi.org/10.1128/JVI.02261-16
De Rop F, Ismail JN, Bravo González-Blas C, Hulselmans GJ, Flerin CC, Janssens J, Theunis K, Christiaens VM, Wouters J, Marcassa G, de Wit J, Poovathingal S, Aerts S (2022) HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads. eLife 11. https://doi.org/10.7554/eLife.73971
Satija R, Farrell JA, Gennert D, Schier AF, Regev A (2015) Spatial reconstruction of single-cell gene expression data. Nat Biotechnol 33:495–502.
Saucedo-Vázquez JP, Gushque F, Vispo NS, Rodriguez J, Gudiño-Gomezjurado ME, Albericio F, Tellkamp MP, Alexis F (2022) Marine arthropods as a source of antimicrobial peptides. Mar Drugs 20(8). https://doi.org/10.3390/md20080501
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.
Söderhäll I (2013) Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity. Cytotechnology 65:691–695.
Söderhäll I, Fasterius E, Ekblom C, Söderhäll K (2022) Characterization of hemocytes and hematopoietic cells of a freshwater crayfish based on single-cell transcriptome analysis. iScience 104850. https://doi.org/10.1016/j.isci.2022.104850
Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM III, Hao Y, Stoeckius M, Smibert P, Satija R (2019) Comprehensive integration of single-cell data. Cell 177:1888-1902 e21.
Sun Y, Li F, Xiang J (2013) Analysis on the dynamic changes of the amount of WSSV in Chinese shrimp Fenneropenaeus chinensis during infection. Aquaculture 376–379:124–132.
Sun X, Li L, Wu B, Ge J, Zheng Y, Yu T, Zhou L, Zhang T, Yang A, Liu Z (2021) Cell type diversity in scallop adductor muscles revealed by single-cell RNA-Seq. Genomics. https://doi.org/10.1016/j.ygeno.2021.08.015
Sun Z, Li S, Li F, Xiang J (2014) Bioinformatic prediction of WSSV-host protein-protein interaction. BioMed Res Int 2014 416543 https://doi.org/10.1155/2014/416543
Tassanakajon A, Somboonwiwat K, Supungul P, Tang S (2013) Discovery of immune molecules and their crucial functions in shrimp immunity. Fish Shellfish Immunol 34:954–967.
Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H (2022) SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40:1023–1025
Tincu JA, Taylor SW (2004) Antimicrobial peptides from marine invertebrates. Antimicrob Agents Chemother 48:3645–3654.
van de Braak CBT, Botterblom MHA, Huisman EA, Rombout JHWM, van der Knaap WPW (2002) Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon. Dis Aquat Org 51:149–155.
Van Phan H, van Gent M, Drayman N, Basu A, Gack MU, Tay S (2021) High-throughput RNA sequencing of paraformaldehyde-fixed single cells. Nat Commun 12:5636.
Veltri D, Kamath U, Shehu A (2018) Deep learning improves antimicrobial peptide recognition. Bioinformatics 34:2740–2747.
Wang YT, Liu W, Seah JN, Lam CS, Xiang JH, Korzh V, Kwang J (2002) White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis. Dis Aquat Org 52:249–259.
Wang F, Li S, Xiang J, Li F (2019) Transcriptome analysis reveals the activation of neuroendocrine-immune system in shrimp hemocytes at the early stage of WSSV infection. BMC Genomics 20:247.
Wang X, Yu L, Wu AR (2021) The effect of methanol fixation on single-cell RNA sequencing data. BMC Genomics 22:420.
Wohnhaas CT, Leparc GG, Fernandez-Albert F, Kind D, Gantner F, Viollet C, Hildebrandt T, Baum P (2019) DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing. Sci Rep 9:10699.
Wongprasert K, Khanobdee K, Glunukarn SS, Meeratana P, Withyachumnarnkul B (2003) Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus. Dis Aquat Org 55:3–10.
Xin F, Zhang X (2023) Hallmarks of crustacean immune hemocytes at single-cell resolution. Front Immunol 14:1121528.
Xue S, Liu Y, Zhang Y, Sun Y, Geng X, Sun J (2013) Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection. PloS One 8:e76718.
Yang H, Li S, Li F, Wen R, Xiang J (2015) Analysis on the expression and function of syndecan in the Pacific white shrimp Litopenaeus vannamei. Dev Comp Immunol 51:278–286.
Yang P, Chen Y, Huang Z, Xia H, Cheng L, Wu H, Zhang Y, Wang F (2022) Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes. eLife 11. https://doi.org/10.7554/eLife.80127
Zhang K, Koiwai K, Kondo H, Hirono I (2018) White spot syndrome virus (WSSV) suppresses penaeidin expression in Marsupenaeus japonicus hemocytes. Fish Shellfish Immunol 78:233–237.
Zhu W, Yang C, Chen X, Liu Q, Li Q, Peng M, Wang H, Chen X, Yang Q, Liao Z, Li M, Pan C, Feng P, Zeng D, Zhao Y (2021) Single-cell ribonucleic acid sequencing clarifies cold tolerance mechanisms in the Pacific white shrimp (Litopenaeus vannamei). Front Gene 12:792172.
Acknowledgements
We would like to thank On-chip Biotechnologies Co., Ltd. for providing their pressure pump On-chip Droplet Generator; Hiroaki Suzuki (Department of Precision Mechanics, Faculty of Science and Engineering, Chuo University, Bunkyo, Japan) and Ryuji Kawano (Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Japan) for their technical support in the fabrication of the Drop-seq microfluidic devices, and GenomeLead for the sequencing of Drop-seq libraries by NovaSeq 600.
Funding
This work was supported by Japan Society for Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists Grant Number 20K15603 and Japan Science and Technology Agency (JST) ACT-X Grant Number JPMJAX21B5 to Keiichiro Koiwai; JSPS KAKENHI Grant Numbers 22H00379 and Science and Technology Research Partnership for Sustainable Development (SATREPS) in collaboration between JST and Japan International Cooperation Agency (JICA) Grant Number JPMJSA1806 to Ikuo Hirono.
Author information
Authors and Affiliations
Contributions
K. Koiwai designed the experiments, performed the experiments, analyzed the data, and wrote the manuscript; H. Kondo and I. Hirono supervised the research.
Corresponding author
Ethics declarations
Ethics Approval
Not applicable.
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Koiwai, K., Kondo, H. & Hirono, I. scRNA-seq Analysis of Hemocytes of Penaeid Shrimp Under Virus Infection. Mar Biotechnol 25, 488–502 (2023). https://doi.org/10.1007/s10126-023-10221-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-023-10221-8