Elsevier

Aquaculture

Volume 533, 25 February 2021, 735943
Aquaculture

Differentially expressed genes in hemocytes of red swamp crayfish Procambarus clarkii following lipopolysaccharide challenge

https://doi.org/10.1016/j.aquaculture.2020.735943Get rights and content

Highlights

  • The hemocytes transcriptomes of Procambarus clarkii were constructed using high-throughput sequencing.

  • Totally 53,910 unigenes was obtained from the transcriptome.

  • Totally 589 DGEs were identified including 310 up- and 279 down-regulated unigenes.

  • The qRT-PCR results of the transcriptome validation candidate gene are consistent with the transcriptome.

Abstract

Procambarus clarkii is an important aquatic organism in China, but its aquaculture has suffered great economic losses due to pathogen infections. To better understand the P. clarkii immune response, we used RNA sequencing (RNA-seq) to examine the expression responses of its hemocyte transcriptome to lipopolysaccharide (LPS). Through assembly and annotation, a total of 53,910 unigenes were identified, with an average length of 1246 bp. 589 differentially in total expressed genes (DEGs) were obtained by the injection of LPS with 310 upregulated genes and 279 downregulated genes. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified several immune response pathways. Additionally, a lot of DEGs to do with the Ras signaling pathway, lysosome, Rap1 signaling pathway, and mitogen-activated protein kinase signaling pathway were upregulated after LPS challenge. Results of Real-time quantitative reverse transcription PCR revealed that 11 randomly selected immune response genes were upregulated after LPS stimulation compared to phosphate-buffered saline stimulation, and this result also validated the RNA-seq data. Our data further enrich the transcriptome databases of P. clarkii and provide a basis for further analysis of the immune system and defense mechanisms of P. clarkii against LPS challenge.

Introduction

Procambarus clarkii, native to northeastern Mexico and southern Unites States, has been diffusely distributed in the natural environment. It is one of the most successful invasive species worldwide due to its strong environmental adaptability (Skelton, 2010; Qin et al., 2018). P. clarkii is a rich source of high quality proteins with all of the essential amino acids required for human nutrition and has recently become the most economically important freshwater crustacean species in inland China due to its high commercial value and palatability (Zhou et al., 2017; Fernándezcisnal et al., 2018). However, P. clarkii aquaculture has been threatened by severe outbreaks of infectious disease caused by parasites, bacteria, and viruses, resulting in significant economic losses (Sun et al., 2017). In addition, P. clarkii is an invertebrate model organism for the study of the molecular mechanisms belonging the innate immune system (Liao et al., 2018; Liu et al., 2018a). Therefore, studying the innate immune processes of P. clarkii can develop new strategies for disease prevention and control.

As an integrated organ of immune processes and metabolic transport, hemocytes are involved in several defense reactions including recognition, phagocytosis, encapsulation, cytotoxicity and melanization (Zhang et al., 2018). The crustacean innate immune system is the only line to defend against foreign antigens and invading pathogens, which is divided into humoral and cellular defense responses (Li et al., 2019). Humoral defenses include antimicrobial peptides, the cascades regulate coagulation and melanization of hemolymph and the production of reactive intermediates of oxygen and nitrogen (Rowley and Powell, 2007). Cellular defenses refer to hemocyte-mediated responsessuch as phagocytosis and encapsulation (Parsons and Foley, 2016). The zebrafish is a suitable experimental model for immunity to study developmental immunity, mucosal immunity and related host-microbe interactions (Galindo-Villegas et al., 2012; Montalban-Arques et al., 2015; Galindo-Villegas, 2016). Similar to vertebrates, mucosal immunity also plays vital roles in immune response. Also, the proper functioning of non-vertebrate gut defense mechanisms requires the presence of a resident microbiota (Garcia-Garcia et al., 2013). Meanwhile, hemocytes are the main players in cellular immunity; they can recognize a variety of foreign targets against various infectious pathogens with alterations to self ensuring efficient defense responses (Johnson, 1987; Ohta et al., 2006; Ng et al., 2013). Lipopolysaccharide (LPS) is a bacterial endotoxin as a major constituent of the outer membrane of Gram-negative bacteria and has been used as an immune stimulator to study immune recognition and defense (Wu et al., 2017). However, little information is available on the hemocyte transcriptome of P. clarkii.

High-throughput sequencing technology is a powerful, effective tool to generate hundreds of millions of short reads from RNA molecules for comparative analysis of tissue data in plants, microorganisms, paleontology and animals. The technology is also widely used in crustacean genomics (Liu et al., 2018a; Chu et al., 2019; Meng et al., 2019; Jiao et al., 2019). RNA sequencing (RNA-seq) has been increasingly applied in various fields including physiology, ecology, evolution and genetics. It plays key role in gene discovery, gene expression profiling and genetic marker mining in crustaceans (Wang et al., 2009). In the present study, researcher injected hemocytes of P. clarkii with LPS or phosphate-buffered saline (PBS; control) to construct the transcriptome sequencing libraries for the purpose of screening for differentially expressed genes (DEGs) involved in the immune response. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to identify immune-related DEGs. Our study provides a comprehensive examination of the immune response and defense mechanisms against LPS stimulation in P. clarkii based on transcriptome analysis.

Section snippets

Experimental preparation and immune challenge of P. clarkii

Healthy red swamp crayfish were purchased from a farming pond in Yancheng, Jiangsu Province, China and cultured at 24 °C for 2 weeks in filtered, aerated freshwater. More than 20 crayfish were divided into two groups: the LPS group and PBS (control) group. Three crayfish were randomly selected to be injected with 10 μL LPS of 1 mg/mL (Escherichia coli, serotype O26:B6; Cat. No. L-2880, Sigma, USA) or 10 μL PBS. At 6 h after injection, hemolymph was drawn from the hemocoel in the arthrodial

De novo assembly and splicing

To identify genes about the response of P. clarkii to LPS challenge, two cDNA libraries, a control hemocyte library (PBS injection) and an LPS-challenged library were constructed and assembled de novo with raw paired-end reads from the Illumina HiSeq 2000 sequencing platform. As shown in Table S1, 49,093,266 raw reads were obtained from the PBS-challenged group and 49,630,440 raw reads were obtained from the LPS-challenged group. After removing the low-quality reads, short sequences and

Conclusion

We analyzed the P. clarkii hemocyte transcriptome after LPS and PBS injection. A total of 53,910 unigenes were annotated in different functional databases.589 DEGs were acquired after injection of LPS, including 310 upregulated genes and 279 downregulated genes. In addition, several genes and pathways involved in immune responses were identified and functionally annotated. This study provides P. clarkii transcriptome information and extends our understanding of immune-related genes and the

Declaration of Competing Interest

The authors declare no competing interests.

Acknowledgements

This work was supported by the Natural Science Foundation of Zhejiang Province (LQ20C190009), the National Key R&D Program of China (2019YFD0900404), the Natural Science Foundation of Jiangsu Province (BK20160444), the National Natural Science Foundation of China (31640074), the China Postdoctoral Science Foundation (2018M642105), the Jiangsu Agriculture Science and Technology Innovation Fund (CX(18)3027), the Scientific and Technological Innovation Special Project for Seed and Seedling of

References (55)

  • T. Jiao et al.

    Characterization and expression analysis of immune-related genes in the red swamp crayfish, Procambarus clarkii in response to lipopolysaccharide challenge

    Fish Shellfish Immunol.

    (2019)
  • P.T. Johnson

    A review of fixed phagocytic and pinocytotic cells of decapod crustaceans, with remarks on hemocytes

    Dev. Comp. Immunol.

    (1987)
  • M.R. Kanost

    Serine proteinase inhibitors in arthropod immunity

    Dev. Comp. Immunol.

    (1999)
  • H. Li et al.

    Growth performance, non-specific immunity, intestinal histology and disease resistance of Litopenaeus vannamei fed on a diet supplemented with live cells of Clostridium butyricum

    Aquaculture

    (2019)
  • T.J. Liao et al.

    Chicken-type lysozyme functions in the antibacterial immunity in red swamp crayfish, Procambarus clarkii

    Dev. Comp. Immunol.

    (2018)
  • Y. Liu et al.

    Transcriptomic analysis of immune-related genes in the lipopolysaccharide-stimulated hepatopancreas of the mudflat crab Helice tientsinensis

    Fish Shellfish Immunol.

    (2018)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method

    Methods

    (2001)
  • X. Meng et al.

    Transcriptome-wide identification of differentially expressed genes in Procambarus clarkii in response to chromium challenge

    Fish Shellfish Immunol.

    (2019)
  • T.H. Ng et al.

    Shrimp hemocytes release extracellular traps that kill bacteria

    Dev. Comp. Immunol.

    (2013)
  • W. Ni et al.

    Hemocytin facilitates host immune responses against Nosema bombycis

    Dev. Comp. Immunol.

    (2020)
  • M. Ohta et al.

    Mechanism by which Bombyx mori hemocytes recognize microorganisms: direct and indirect recognition systems for PAMPs

    Dev. Comp. Immunol.

    (2006)
  • J.T. Ou et al.

    Transcriptome-wide identification and characterization of the Procambarus clarkii microRNAs potentially related to immunity against Spiroplasma eriocheiris infection

    Fish Shellfish Immunol.

    (2013)
  • B. Parsons et al.

    Cellular immune defenses of Drosophila melanogaster

    Dev. Comp. Immunol.

    (2016)
  • Z.D. Qin et al.

    Antibacterial activity of hemocyanin from red swamp crayfish (Procambarus clarkii)

    Fish Shellfish Immunol.

    (2018)
  • X. Ren et al.

    Comparative proteomic investigation of Marsupenaeus japonicus hepatopancreas challenged with Vibrio parahaemolyticus and white spot syndrome virus

    Fish Shellfish Immunol.

    (2019)
  • S. Somnuk et al.

    Gene expression and characterization of a serine proteinase inhibitor PmSERPIN8 from the black tiger shrimp Penaeus monodon

    Fish Shellfish Immunol.

    (2012)
  • X. Song et al.

    A single-CRD C-type lectin (CgCLec-3) with novel DIN motif exhibits versatile immune functions in Crassostrea gigas

    Fish Shellfish Immunol.

    (2019)
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