Differentially expressed genes in hemocytes of red swamp crayfish Procambarus clarkii following lipopolysaccharide challenge
Graphical abstract
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
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These authors contributed equally to this work.