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Transcriptome analysis and immune-related genes expression reveals the immune responses of Macrobrachium rosenbergii infected by Enterobacter cloacae

https://doi.org/10.1016/j.fsi.2020.03.042Get rights and content

Highlights

  • The immune response of M. rosenbergii against E. cloacae was determined by transcriptome analysis.

  • DEGs were enriched in many immune-related pathways.

  • E. cloacae triggers a wide defensive response of various immune related genes in M. rosenbergii.

Abstract

Macrobrachium rosenbergii is an important cultural species in China and other Southeast Asian countries. However, Enterobacter cloacae infection has caused a great economic loss in M. rosenbergii culture industry. The immune responses of M. rosenbergii to the E. cloacae infection is not fully characterized. To investigate the immune response of M. rosenbergii against E. cloacae, we performed transcriptome analysis of the M. rosenbergii hepatopancreas with and without E. cloacae infection using RNA-seq. After assembly and annotation, 29,731 high quality unigenes were obtained from RNA-seq data. Differential expression analysis revealed the existence of 2498 significantly differently expressed genes (DEGs) at 12 h post infection, with 1365 up-regulated and 1133 down-regulated genes. Among these DEGs, some well-known immune-related genes were up-regulated significantly, including C-type lectin 1, lectin 3, anti-lipopolysaccharide factor 2, Cu/Zn superoxide dismutase and heat shock protein 70. GO analysis demonstrated 24 biological process subcategories, 14 cellular component subcategories, and 12 molecular function subcategories that were enriched among these DEGs, and some DEGs were clustered into immune related subcategories such as immune system process, response to stimulus, biological adhesion, and antioxidant activity. These DEGs were enriched into 216 KEGG pathways including a core set of immune correlated pathways notably in phagosome and lysosome. In addition, 5 up-regulated and 5 down-regulated immune-related DEGs were selected for further validation by quantitative real-time PCR and the results showed consistence with the RNA-seq data. Additionally, the expression level of six selected immune-related genes (ALF2, CLEC1, LEC3, hemocyanin1, HSP70 and SOD) based on the transcriptomic data were monitored at different point of time in hepatopancreas, gill, hemolymph and intestine. Results revealed these immune-related genes were significantly up-regulated in different tissues from 6 to 24 h after E. cloacae infection. Overall, these results provided valuable information for further studying the immune response of M. rosenbergii against E. cloacae infection.

Introduction

The giant freshwater prawn Macrobrachium rosenbergii is one of the most economically important crustaceans widely farmed in China and other Southeast Asian countries and the production was 233,898 tons in 2016 [1]. Its significance as a culture species stems from the fact that it is highly tolerable to different environments and has high growth performance. In China, M. rosenbergii was first introduced from Japan in 1976 and since then its aquaculture industry kept expanding due to its high value [2]. Unfortunately, M. rosenbergii culture industry is facing serious economic losses due to the pathogenic microbial invasion such as M. rosenbergii nodavirus (MrNV) [3], the extra small virus (XSV) [4], infectious hypodermal and hematopoietic necrosis virus (IHHNV) [5], Vibrio cholera [6], Vibrio alginolyticus [7], Vibrio vulnificus [8], Aeromonas hydrophila [9], E. cloacae [10]. Of these pathogenic microorganism, our previous study identified E. cloacae as a bacterial pathogen causing drastic decreases in production and catastrophic economic losses of M. rosenbergii larva in Gaoyou country, Jiangsu province, China [10].

E. cloacae is widely distributed in the environment, and an important opportunistic pathogen in the intestinal tracts of humans and animal [11,12]. The organism can cause a wide variety of infections, sepsis, infections of the respiratory tract and urinary tract, wound infections, and meningitis [13]. Prior research mainly focused on pathogenicity to human and drug resistance of E. cloacae, and there are only limited reports of infection due to E. cloacae in aquatic animals [14]. The pathogenesis and pathogenic mechanism of E. cloacae infection in M. rosenbergii is not clear. Furthermore, there is scarce information about the gene expression or pathways involved in the immune response of M. rosenbergii under E. cloacae infection. Therefore, understanding the immune behavior mechanism of M. rosenbergii to E. cloacae infection may contribute to developing new strategies for management and therapy of this disease.

Transcriptome profiling using high-throughput sequencing technologies have recently provided new insights into immune responses against bacterial infections in various aquaculture animals [[15], [16], [17], [18]]. The enormous advantages of these genomics research technologies make them admirably suited for better understand the pathways and mechanisms of life events [19]. The progress of transcriptome high-throughput sequencing technologies has laid the foundation for genome research, immune and developmental gene identification, and DGE (digital gene expression) analysis. Many studies show that the transcriptome sequencing is a powerful tool to better understand the pathways or gene expression involved in the immune response against microbe invasion. By using transcriptome sequencing tool, a large number of immune-related genes in Litopenaeus vannamei during Vibrio parahaemolyticus infection were obtained [20,21]. Ren et al. revealed a large number of immune-related genes in Marsupenaeus japonicas during V. parahaemolyticus and white spot syndrome virus infection by transcriptome analysis [22].

As crustaceans, M. rosenbergii lack typical adaptive immunity and instead rely on their innate immune systems to combat pathogenic invasion. Hepatopancreas, hemolymph, intestine and gill play important roles in their innate immune. The crustacean hepatopancreas in nutrient metabolism and regulation in aquatic animals and plays a significant role in immune function [23]. Hemocytes are cell fractions in the hemolymph that play important roles in the immune response, like recognition, phagocytosis, encapsulation, nodule formation, cytotoxicity, and cellular communication [24]. The intestine is the main organ of nutrient absorption and metabolism, and acts as the first line of defense against pathogens and environmental stress [25]. The crustaceans gill is important for respiratory and osmotic adjustment, and also involved in the immune response for eliminating pathogens [26]. The expression of immune-related genes in these immune-related organs is considered a potential marker of a prawn's health status, as it provides relevant information about the activation and modulation of the immune system.

In the present study, the transcriptomic sequencing of M. rosenbergii with and without E. cloacae infection was performed, and the differently expressed immune-related genes were identified. In addition, Gene Ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) analysis of differentially expressed genes were showed that many immune-related GO categories and pathways were significantly enriched. Moreover, the expression level of six selected immune-related genes (ALF2, CLEC1, LEC3, hemocyanin1, HSP70 and SOD) based on the transcriptomic data were monitored at different time post infection in hepatopancreas, gill, hemolymph and intestine. The results will provide a better understanding of the M. rosenbergii and E. cloacae interactions, and provide a substantial dataset for further research and may deliver the basis for preventing the bacterial diseases.

Section snippets

Experimental prawns and E. cloacae challenge

Healthy M. rosenbergii (2.5 ± 0.4 g) were obtained from a commercial aquaculture farm of Gaoyou county, Jiangsu Province, China. The prawns were maintained in the aerated recirculating fresh water system at constant temperature 30 °C and acclimatized for two weeks prior to experiments. The prawns were fed twice daily with commercial pellet feed and checked randomly to confirm that no bacterial infection occurred before experiment. Then the prawns were randomly assigned to the challenge and

Transcriptome sequencing and de novo assembly

To study gene expression pattern of hepatopancreas of M. rosenbergii in response to E. cloacae, a total RNA was extracted from E. cloacae infected and control samples. The cDNA libraries from the hepatopancreas of M. rosenbergi with or without E. cloacae infection were sequenced on Illumina sequencing platform. The Illumina Hiseq X Ten platform generated 43,615,888, 40,402,548 and 40,896,336 raw reads from three biological replicates in E. cloacae infection group, and 44,192,872, 48,959,522,

Discussion

M. rosenbergii is an economically and nutritionally important aquaculture species in China and Southeast Asian countries. However, the cultivation of this prawn has been restricted due to epidemic infectious diseases in recent years [32]. Recently, E. cloacae infection can cause considerable business losses to the local economy [10], and knowledge about the interaction between M. rosenbergii and E. cloacae is limited. Toward to this end, here we utilized RNA-seq to explore the global expression

Ethics statement

All animals' treatments were strictly in accordance with the guidelines of Animal Experiment Ethics Committee of Yangzhou University. The protocol was approved by Animal Experiment Ethics Committee of Yangzhou University.

CRediT authorship contribution statement

Xiaojian Gao: Data curation, Formal analysis, Methodology, Validation, Writing - original draft, Writing - review & editing. Ziyan Jiang: Data curation, Investigation, Methodology, Software, Writing - original draft. Shuangming Zhang: Data curation, Investigation, Methodology. Qiyun Chen: Methodology. Shuaiqi Tong: Data curation, Methodology. Xiaodan Liu: Investigation, Methodology. Qun Jiang: Methodology. Hui Yang: Investigation. Wanhong Wei: Supervision. Xiaojun Zhang: Funding acquisition,

Acknowledgments

This work was supported by the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS 2019-463), National Natural Science Foundation of China (31972830), the National Key Research and Development Project (2019YFD0900305), Fishery Science and Technology Innovation Projects of Jiangsu Province (D2017-3), and the Postgraduate Research & Practical Innovation Program of Jiangsu Province (XKYCX18_096).

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