- Email: [email protected]
Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipopolysaccharide (LPS) infection
Accepted Manuscript Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipolysaccharide (LPS) infection Miao Zhou, Muhammad Nadeem Abbas, Saima Kausar, Cheng-Xi Jiang, Li-Shang Dai PII:
S1050-4648(17)30637-X
DOI:
10.1016/j.fsi.2017.10.030
Reference:
YFSIM 4901
To appear in:
Fish and Shellfish Immunology
Received Date: 29 July 2017 Revised Date:
5 October 2017
Accepted Date: 16 October 2017
Please cite this article as: Zhou M, Abbas MN, Kausar S, Jiang C-X, Dai L-S, Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipolysaccharide (LPS) infection, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.10.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Transcriptome profiling of red swamp crayfish (Procambarus clarkii)
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hepatopancreas in response to lipolysaccharide (LPS) infection
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Miao Zhou a,1 , Muhammad Nadeem Abbas b,1 , Saima Kausar b,1, Cheng-Xi Jiang c,*,
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Li-Shang Dai a,* a
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325035, P.R. China
Department of Zoology and Fisheries, University of Agriculture, Faisalabad 38000,
SC
b
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School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou
Pakistan c
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Life Sciences Institute, Wenzhou University, Wenzhou 325035, P.R. China
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* Corresponding author: Cheng-xi Jiang and Li-Shang Dai
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Tel/fax: +86 577 86699572
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E-mail: [email protected] (Cheng-Xi Jiang), [email protected]
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(Li-Shang Dai)
Address: School of Pharmaceutical Sciences, Wenzhou Medical University,
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Wenzhou 325035, P.R. China.
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These authors contributed equally to this work.
ACCEPTED MANUSCRIPT Abstract
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The RNA-sequencing followed by de novo assembly generated 61,912 unigene
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sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS
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challenged and PBS control samples revealed 2,552 differentially expressed genes
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(DEGs). Of these sequences, 1,162 DEGs were differentially up-regulated and 1,360
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DEGs differentially down-regulated. The DEGs were then annotated against gene
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ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG)
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database. Some immune-related pathways such as PPAR signaling pathway, lysosome,
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Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis.
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The reliability of transcriptome data was validated by quantitative real time
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polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here
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shed light into antibacterial immune responses of crayfish. In addition, these results
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suggest that transcriptomic data provides valuable sequence resource for
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immune-related gene identification and helps to understand P. clarkii immune
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functions.
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Keywords: Crayfish; LPS; Immunity; Differential expressed genes; Arthropods
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1. Introduction The red-swamp crayfish (Procambarus clarkii) is a rich source of high quality
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proteins, which constitutes all the essential amino acids required for human nutrition.
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P. clarkii is a fresh water species, native to Mexico and United States [1]. It is
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extremely tolerant to adverse environmental conditions such as temperature
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fluctuations, poor water quality, and low oxygen concentrations. Therefore, the
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species is considered highly suitable for culture and extraordinary farm production
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[2,3]. Thus far, this species has been introduced and successfully cultured in many
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countries world-wide [4]. In China, it was introduced from Japan in 1929 [5], since
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then it has become one of the most economically important species in China.
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Vertebrates and invertebrates through the process of evolution have developed an
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innate immune system, and innate immune responses are considered first line of
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defense against variety of pathogens [6-8]. The innate immune system is stimulated
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by microbial infections, thereby transcribing variety of immune related genes to
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perform their specific roles in host defense [9]. Hence, to understand the immune
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mechanisms in an organism, it is crucially important to explore the genes that are
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probably implicated in the immune functions.
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In spite of crayfish economic importance and being cultured worldwide, our
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knowledge on its innate immune system is limited. In crayfish hepatopancreas is a
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vital organ for nutrient storage and it is also involved in a variety of immune
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responses and detoxification processes [10]. Hepatopancreas in crustaceans
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transcribes various immune related genes and proteins, which play crucial role to
ACCEPTED MANUSCRIPT maintain physiological processes. For instance, hemocyanin that is converted to
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antimicrobial proteins, antiviral agent, hemolysin, agglutinin and phenoxidase like
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enzyme, and is considered to be involved in immune responses of crustaceans,
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mollusks and arthropods [11-13]. In addition, many immune related genes such as
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cathepsin B, cathepsin L, interferongamma inducible lysosomal thiol reductases
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(GILT) are also produced in hepatopancreas [14,15]. Besides this, lectins are
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important immune responsive genes, and known to have tissue specific expression
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and some lectins (hsL and FLec2) are only expressed in hepatopancreas [16-18].
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Although, these fragmentary studies are available on the immune responses of
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hepatopancreas in crustacean, however, the anti-bacterial immune mechanism of
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crayfish hepatopancreas has not been explored previously. Therefore study on its
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immune responses and exploration of immune-related genes will contribute to
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understand anti-bacterial immune mechanism in the crayfish.
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The transcriptome is an entire set of RNA transcripts produced by the genome at
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any one time, and the knowledge of the transcriptome is critically important for
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interpreting the functional factors of the genome [5]. In the present study, we prepared
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and sequenced two libraries of P. clarkii hepatopancreas after LPS stimulation and
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PBS administration using Illumina sequencing platform. First, the obtained reads were
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assembled to unigene sequences by RNA-Seq de novo programs. Subsequently, the
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transcriptome data was subjected to GO, KOG, and KEGG databases to investigate
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the involvement of unigene sequences in different canonical pathways. Furthermore,
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enrichment analysis was executed following the identification of differentially
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ACCEPTED MANUSCRIPT expressed genes (DEGs) from two P. clarkii libraries. The main purpose of this study
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was to identify and align immune-related genes in the hepatopancreas. The
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transcriptomic data will also provide sequence resources for future studies on
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crustaceans and may help to understand antibacterial immune mechanisms of P.
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clarkii.
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2. Materials and methods
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2.1. Experimental animal and LPS treatment
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P. clarkii individuals were obtained from local market; the specimens were
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ranged 7-8 cm in size and approximately an average weight of 10 g. They were kept
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in spacious tanks, supplied with high-quality freshwater, and fed at 23±2 °C with
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natural (small invertebrates) and artificial diet. The specimens were divided into
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experimental group and control group, each of which contained 10 individuals. The
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experimental group was administrated with LPS (1 µg/g of body weight) in abdominal
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part and PBS was used as control group. Following 12 h of treatment hepatopancreas
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samples were collected from both the experimental and control group and
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immediately frozen in liquid nitrogen and then stored at −80 °C till the extraction of
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RNA.
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2.2. RNA extraction, library preparation and sequencing
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The collected hepatopancreas samples were used to isolate RNA by the Trizol
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Reagent (Invitrogen, USA) according to manufacturer’s instructions (Supplementary
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Fig. 1). The integrity and quality of extracted RNA was analyzed by agarose gel
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electrophoresis
(1%),
and
NanoDrop
1000
spectrophotometer
(NanoDrop
ACCEPTED MANUSCRIPT Technologies, Wilmington, DE, USA), respectively. After DNase I treatment, the
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messenger RNA (mRNA) was purified from 20 µg mixed total RNA using oligo (dT)
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magnetic beads. Following purification, the mRNA was fragmented into short
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fragments by mixing in fragmentation buffer in a thermomixer at a suitable
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temperature. First-strand cDNA was then synthesized using the mRNA fragments as
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templates, followed by second-strand cDNA synthesis using DNA polymerase I and
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RNase H. After the end repair process and connection with adapters, suitable
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fragments were used as templates for PCR amplification to create the final cDNA
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library. cDNA libraries were constructed using SMART cDNA library construction kit
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(Clontech, CA, USA) in accordance with the manufacturer’s protocol. Following the
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examination of quality and quantity, the libraries for transcriptome sequencing was
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prepared
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Solexa/Illumina sequencing was carried out by Novogene, Beijing, China.
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2.3. Quality control and assembly of reads
Illumina’s
kit
following
manufacturer’s
recommendations.
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The assembly of LPS and PBS administrated hepatopancreas samples were
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executed using RNA-Seq de novo programs Trinity and SOAP de novo assembler [19]
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with default parameters. To obtain clean reads, short reads (Read length >10bp) and
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low quality reads (Quality score >20) were discarded. The clean reads were processed
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to form contigs, and then these contigs were clustered to obtain unigene sequences,
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which were finally connected to obtain transcript [20].
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2.4. Annotation and gene ontology of transcriptome
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After transcriptome assembly, the obtained unigene and transcript sequences
ACCEPTED MANUSCRIPT were annotated on the basis of sequence similarity with known genes. Briefly, the
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contigs were aligned against available sequence in the National Center of
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Biotechnology and Information (NCBI) database [21]. The unigene sequences were
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aligned with NCBI protein data bases such as Nr, KOG, KEGG and Swiss-Prot using
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BLASTx [22]. While the remaining unigene sequences (none of the BLASTx hits)
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were annotated with Nt database of NCBI. To determine sequence homology,
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alignments were executed with previously known genes (cutoff E-value≤ 10−5). The
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alignments results were used to identify sequence direction and prediction of protein
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coding region (CDS). Functional annotation was performed with gene function
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classification
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(http://www.blast2go.com/b2ghome) [23]. The pathway annotation was executed to
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determine the biological behavior of the genes using the KEGG database.
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2.5. Identification of differentially expressed genes
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(www.geneontology.org)
using
Blast2GO
software
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To identify differentially expressed genes from hepatopancreas samples of P.
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clarkii following LPS treatment (PBS as control). cDNA libraries were constructed,
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sequenced and transcriptome was assembled according to sections 2.2 and 2.3. The
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differentially expressed genes (DEGs) were identified based on the number of
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fragments per kilo base of exon per million fragments mapped (FPKM) of the genes,
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and False Discovery Rate (FDR) was used to find the P-value threshold [24]. The
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DEGs were characterized as those having FDR less than 0.05 and a fold change
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between treated and control groups of greater than 2 (log2 fold change P1). Further,
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the DEGs were mapped to the GO database (http://www.geneontology.org/) and then
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ACCEPTED MANUSCRIPT their functions were determined using WEGO and Blast2GO programs [25]. The
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KEGG public database was used to predict enriched metabolic and signal transduction
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pathways [26]. The pathway categories classification was determined using Fisher’s
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exact test, and the FDR was used to correct the P values. Pathways with a P-value less
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than 0.05 were considered as significantly enriched [27].
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2.6. Quantitative RT-PCR assay
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Quantitative real time PCR (qRT-PCR) was performed to analyze mRNA levels
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of immune related genes. Hepatopancreas samples were collected from crayfish (7-8
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cm in size, and average weight of 10 g) purchased from local market, further the
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number of specimens in each experiment and time of hepatopancreas collection was
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same as mentioned in the section 2.1. Total RNA was isolated from LPS treated and
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control group (PBS), then reverse transcribed into cDNA. The gene specific primers
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were designed based on the identified transcript sequences by primer premier 5.0
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software (Table 1). The primers were used to analyze the expression of immune
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related genes, and 18S RNA was used as internal control. The qRT-PCR was
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performed in 25 µL reactions including 12.5 µL of 2× SYBR Premix Ex TaqII
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(Takara), 1 µL each of the forward and reverse primers, 2 µL of template (cDNA), and
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8.5 µL of RNase-free H2O. The amplification program was as follows: 95°C for 30 s;
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39 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. At the end of the reaction,
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a melting curve was produced by monitoring fluorescence continuously, while slowly
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heating the sample from 65°C to 95°C. The relative expression levels of the selected
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genes were calculated according to the 2−△△Ct method [28]. All qRT-PCR experiments
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were repeated five times.
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3. Results and Discussion
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3.1. Sequencing and assembly of trancriptome To determine gene expression profile of P. clarkii, total RNA was extracted and
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cDNA libraries were constructed from hepatopancreas following LPS treatment and
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PBS was used as control. The Illumina sequencing of two libraries generated
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52,414,336 and 47,592,460 raw reads for treated and control group, respectively.
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After discarding ambiguous nucleotides, low quality reads and short reads, treated
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group contained 50,310,480 clean reads, representing 7.55G nucleotides. The Q20
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percentage and Q30 percentage (percentage of nucleotide whose quality was more
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than 20 and 30 clean reads) were 96.21% and 90.78%, respectively and GC contents
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was47.53%. The control group comprised 45,706,720 clean reads representing 6.86G
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nucleotides. The Q20 percentage, Q30 percentage and GC percentage were 96.63%
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and 91.77%, and 43.99%, respectively (Table 2).
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The ‘de novo’ transcriptome assembly strategy is most commonly used to
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assemble RNA-seq data. It does not depend on reference genome and can provide an
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initial set of transcripts, allowing for RNA-seq expression studies [29]. Currently,
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Trinity, SOAP de novo, trans-ABySS, ABySS, and Oases assemblers have been
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documented to be used for the de novo assemblage of short read RNA-seq data into
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transcripts [30]. In the present study, we used Trinity and SOAP de novo assemblers
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to assemble high quality clean reads of two libraries [19]. Trinity is an important de
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novo assembly program that has been reported to be sensitive and efficient in
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ACCEPTED MANUSCRIPT recovering full-length transcripts/isoforms in various organisms. It constructs de
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Bruijn graph from huge amounts of short-read sequence data, then use an enumeration
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algorithm to score all possible paths and branches, and retain only those plausible
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ones as transcripts and isoforms. Additionally, it is specially programmed to recover
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paths supported by actual reads and eradicate ambiguous edges, thus ensure correct
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transcript reconstruction [31]. Likewise, the SOAP de novo program is a novel
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short-read assembler that can build draft assemblies de novo for large sized genomes
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such as animal and plant genomes. It builds full-length transcripts/isoforms and
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allowing to carry out accurate analyses of unexplored genomes [32].
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novo programs (SOAP de novo and Trinity) are highly reliable and widely used to
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recover transcripts and unigenes of unexplored species. In the present study, the
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assembly of high quality clean reads generated 61,912 unigene sequences and 11,3398
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transcripts. Both the unigene sequences and transcripts were ranged in size from <301
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bp to > 2000 bp. The maximum number of unigene sequences were between 500
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-1000 bp range, and transcripts were in <301 bp (Fig. 1).
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3.2. Functional annotation of clustered unigene sequences
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To evaluate the functions of unigene sequences, a total of 61,912 unigene
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sequences were aligned with six public databases (NR, Swiss-Port, KO, GO, KOG
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and PFAM). All of these sequences are annotated in at least one Database except
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36,091 unigene sequences. The results revealed that 20,363 unigene sequences were
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significantly best hit with the GO database, 20,264 unigene sequences were
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significantly annotated with the PFAM database, and 19,672 unigene sequences were
ACCEPTED MANUSCRIPT significantly aligned with the NR database. While 16,369, 14,515 and 10,174 unigene
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sequences were mapped against SwissProt database, KO database and KOG database,
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respectively (Table 3). Of these aligned sequences, almost 32% unigene sequences
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had more than 1e-30 E-value (Fig. 2A). The similarity distribution pattern revealed
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that 61.8% unigene sequences shared greater than 60% homology (Fig. 2B). The
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species specific distribution patterns revealed that 16.7% of the unigene sequences
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most closely matched with Zootermopsis nevadensis, 6.7% of the unigene sequences
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resembled Daphnia pulex, 3.5% unigene sequences best hits with Tribolium
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castaneum (3.5%) and so on (Fig. 2C).
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3.3. Functional analysis of transcriptome
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The putative functions of unigene sequences in LPS treated and control group
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were analyzed using Gene Ontology (GO) and Eukaryotic Orthologous Groups of
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proteins (KOG). The analysis of GO classification revealed that most of unigene
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sequences mapped to biological process, followed by cellular component and
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molecular function. The three categories were further divided into 55 functional
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groups. In the two libraries, most of the corresponding biological process unigene
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sequences were implicated in cellular processes, metabolic processes and single
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organism process. Most of the cellular component unigene sequences were associated
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with cell, cell junction, organelle, macromolecular complex and membrane. Most of
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the molecular function unigene sequences encoded protein involved in binding,
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followed by catalytic activity, and transporter activity (Fig. 3).
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Eukaryotic Orthologous Groups of proteins (KOG) database classifies gene
ACCEPTED MANUSCRIPT products into different categories, and these categories are critically important to
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understand gene functions and their evolutionary relationships [33]. A total of 10,174
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genes were aligned with KOG database and divided into 26 functional categories.
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Among the 26 KOG categories, general function prediction only and signal
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transduction mechanism were the most abundant category, followed by post
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translational modification, protein turnover, chaperones and transcription categories
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(Fig. 4).
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Kyoto Encyclopedia of Genes and Genomes (KEGG) is a valuable tool for
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canonical pathways based classification of orthologous genes, and provides important
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information to understand biological functional profiles of gene [34]. In the present
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study, 14,515 genes were annotated against the 229 biological pathways in the KEGG
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database. Among these biological pathways, the top 20 statistically significant
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pathways are shown in Table 4. Of these some important immune related biological
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pathways were predicted in the KEGG database, including Rap1 signaling pathway,
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Focal adhesion, MAPK signaling pathway PI3K-Akt signaling pathway, cAMP
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signaling pathway and Ubiquitin mediated proteolysis. In addition, lysosome,
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endocytosis, RNA transport, spliceosome, protein processing endoplasmic reticulumn,
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and so on were predicted (Table 4).
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Furthermore, the unigene sequences were classified into environmental
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information processing, genetic information processing, metabolism, cellular process
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and organismal system (Fig. 5). Among the 12 categories of metabolism, carbohydrate
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metabolism (456 sequences), lipid metabolism (449 sequences) and amino acid
ACCEPTED MANUSCRIPT metabolism (328 sequences) unigene sequences were most abundant. In organismal
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system, Endocrine system and Immune system were the most abundant clusters with
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654 sequences and 478 sequences, respectively. Interestingly, in environmental
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information processing, Signal transduction (1197 sequences) was the most abundant
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cluster in all categories. While, folding, sorting and degradation (538 sequences) was
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the most unigene sequences cluster among the genetic information processing (Fig. 5).
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These biological processes play vital role in the physiological processes of P. clarkii
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and will help to understand its immune mechanisms.
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3.4. Identification and analysis of DEGs
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The gene expression pattern is utilized to predict the gene functions, and
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generally, the genes exhibiting similar expression pattern mostly have same functions,
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or involved in the same biochemical pathways. Hence, clustering of genes can be
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useful to evaluate the function of unknown genes [35]. Here, we performed gene
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clustering to determine the functional enrichment of genes exhibiting similar
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expression pattern (Fig. 6). To determine the unigene sequences expression levels of P.
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clarkii hepatopancreas following LPS treatment and PBS was used as control. Here
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we
clean
reads
using
RSEM
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|log2(foldchange)|>1)
and
then
screened
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down-regulated unigene sequences in response to LPS challenge. A 2,522 DEGs were
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identified, of these 1,162 unigene sequences significantly upregulated and 1,360
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sequences down-regulated (Fig. 7).
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3.5. GO classification of immune-related DEGs
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aligned
software
(q-value
significantly
<
0.005
up-regulated
& and
ACCEPTED MANUSCRIPT The unigene sequences were mapped against the GO and KEGG database. The
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DEGs were categorized into biological process, cellular component and molecular
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function, and then further classified into 31 subcategories (Fig. 8). The biological
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process and molecular function categories represented most of the subcategories.
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Metabolic process and single organism metabolic process were the most abundant
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among the biological process. The hepatopancreas tissues metabolic oxygen demands
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are relatively high in crustaceans similar across different species [36]. Further, in this
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study, following the LPS treatment, we observed obvious transcription changes in
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hepatopancrease under different GO terms including catalytic activity and
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oxidoreductase (Fig. 8). Furthermore the DEGs were aligned to the KEGG database to
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determine the biological pathway enrichment. Many biological pathways were (P = <
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0.05) changed in LPS treated sample compared to control sample (PBS). Among the
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biological pathways, some immune related pathways including PPAR signaling
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pathway, Rheumatoid arthritis, lysosome, Chemical carcinogenesis, Peroxisome, and
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so on were observed in the DEGs. The knowledge on crayfish antibacterial innate
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immune mechanism is limited, and the above canonical pathways information will be
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useful to explore and understand the immune mechanisms (Fig. 9 & Table 5).
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Crayfish have an efficient innate immune system to combat various microbial
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infections. Based on the KEGG classification, we identified some of the important
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mechanisms of the crayfish innate immune system that are involved in the
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antimicrobial defense, including lysosome and antibacterial peptides production.
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These results are in agreement with study of Adema et al. [37], they performed
ACCEPTED MANUSCRIPT microarray analysis and reported the up-regulation of antibacterial peptides and
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lysosome transcripts after the treatment of Echinostoma paraensei and Schistosoma
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mansoni in Biomphalaria glabrata. Further, these results are also comparable with the
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studies of Byadgi et al. [38]. Phagocytosis is the most common mechanism in animals
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(crayfish) to overcome microbial infection. Upon the attachment of pathogen to the
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surface of phagocyte, the phagocyte forms a vesicle (Phagosome) after engulfing
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pathogen. Then lysosome containing different enzymes inside the phagocyte, fuse
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with the phagosome resulting in the degradation of pathogen [39, 40].
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Further, the synthesis of antibacterial peptides in organisms is also an important
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innate immune defense mechanism against microbial agents. Antibacterial peptides
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attach to bacterial surface and break the bacterial cell membrane and consequently
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bacterial cell is lysed. In insects, antibacterial peptides have been well studied, and
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approximately 15 different antibacterial peptides have been reported that are induced
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by bacteria or bacterial products exposure in insects. For instance, cecropins are
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produced in insects and have a broad spectrum of activity against both gram negative
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and gram positive bacteria [41, 42]. However, little information is available on the
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antibacterial peptide production in crayfish [43], and the present study will provide a
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foundation to further explore formation and possible role of antibacterial peptides in
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crayfish.
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3.6. Validation of transcriptome data by qRT-PCR
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To validate the expression levels of differentially expressed genes in our
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transcriptome analysis, eleven genes were randomly selected based on altered
ACCEPTED MANUSCRIPT expression pattern after LPS treatment, and gene of interest through functional
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enrichment and canonical pathways results, later investigated by qRT-PCR.
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Quantitative RT-PCR analysis revealed that the most of differentially expressed genes
334
were in agreement with the RNA-seq data, however some genes such as cubilin had
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relatively little lower expression in RNA-seq data compared to qRT-PCR analysis.
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Although these genes expression was relatively lower, but overall both the analysis
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showed approximately similar trend (Fig. 10). This characteristic feature has
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previously been reported in transcriptomic studies of invertebrates such as
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Biomphalaria glabrata [37], Homarus americanus [44], Exopalaemon carinicauda
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[45] and so on.
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Expression analysis of immune related genes
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Red swamp crayfish is one of the most economically important crustaceans. It is
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used as model organism in various research fields such as toxicity, environmental
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stress, animal behavior and studies on pathogen infection. Despite its importance,
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little is known about crayfish genome particularly on immune related genes, immune
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mechanisms and pathways, which are involved to combat microbial infection [46].
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The identification of immune-related genes can contribute to understand the immune
348
mechanism of crayfish. Hepatopancreas in crustaceans has been reported to be
349
involved in metabolism, nutrient absorption and immune functions [47]. In this study,
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four immune-related DEGs with clearly changed expression after LPS administration
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were investigated by qRT-PCR assay. All of the immune-related genes were
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significantly up-regulated in the LPS treated sample compared to PBS control (Fig.
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ACCEPTED MANUSCRIPT 11). The interferon (IFN) system is an important cellular defense mechanism in
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mammals, and play crucial role to suppress viral infections by inhibiting multiple
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steps of viral replication, increasing antigen-presentation process and activating
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macrophages [48] (Cebulla and Miller, 1999). In the present study, component of IFN
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system was up-regulated like gamma-interferon-inducible lysosomal thiol reductase
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(GILT) in the LPS treated sample compared to control. The GILT is widely expressed
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in a variety of cell types particularly in antigen presenting cells [49-51]. Where, it
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catalyzes the reduction of disulfide bonds in exogenous antigens and facilitate in the
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unfolding of the protein and then that is cleared by cellular proteases [52, 53], in
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addition, GILT play important role in the neutralization of pathogens and removal of
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cellular debris resulting from pathogen (bacterial) infection [54]. Further, several
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studies reported the up-regulation of GILT in response to bacterial or LPS
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administration, and its involvement in immune response [55, 56]. Our data is also
366
consistent with above mentioned studies, and suggest that GILT may be regulating the
367
immune function following LPS or bacterial challenge.
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Anti-lipopolysaccharide factor (ALF) is an important member of antimicrobial
369
peptides and a key effector molecule in the crustaceans innate immune system. ALFs
370
can neutralize LPS, mediate degradation and activate coagulation cascade in cell [57].
371
So far a number of ALFs have been described in different species of crustaceans [58,
372
59], and bacterial and viral infection or LPS treatment can induce the expression of
373
ALFs in them [59, 60]. ALFs contain amphipatic loop structure that can bind a single
374
fatty acid with the phosphoglucosamine portion of lipid A that helps to bind with LPS
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ACCEPTED MANUSCRIPT and subsequently neutralize it [61]. In this study, ALF-3 appeared as second most
376
greatly expressed factor. These results are consistent with the study of Clark et al. [44],
377
they reported up-regulation of various immune related factors including ALFs in
378
hepatopancrease of Homarus americanus after bacterial infection. Likewise,
379
Cathepsin L (CL) was significantly induced in hepatopancreas following LPS
380
challenge. Lysosomal cathepsins belonging to the family of cysteine protease have
381
been documented to be involved in the innate immunity of invertebrates. In P.
382
monodon, Microarray analysis showed that cathepsins B and L expression enhanced
383
after WSSVtreatment and the upregulation in expression of cathepsin probably reflect
384
the activation of lysosomal functions against viral infection. In the hepatopancreas of
385
F. chinensis mRNA levels of cathepsin B, C and L were upregulated following V.
386
anguillarum challenge, indicating that these cathepsins were involved in the innate
387
immunity of F. chinensis [62-64]. Hu and Leung [65] reported the expression of
388
Cathepsin L in the B cells of the hepatopancreas of Metapenaeus ensis, and suggested
389
the involvement of Cathepsin L in immune responses. Our data suggest
390
exist in the crayfish and is an important effector molecule in the innate immune
391
system, which helps to deal bacterial infection. In addition, peroxiredoxin 6 (Per6)
392
also showed significant increase compared to PBS control. It belongs to the
393
peroxiredoxin family that is broadly distributed in prokaryotes and eukaryotes [66,
394
67]. Serveral studies on invertebrates reported the increase in Per6 expression
395
following the treatment of bacteria, virus and pathogen-associated molecular pattern,
396
and has been shown to have important role in host defense [68, 69]. The members of
cathepsin L
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ACCEPTED MANUSCRIPT peroxiredoxin family play crucial protective role in tissues such as detoxification and
398
reduction of hydrogen peroxide and so on [70]. In crustaceans, hepatopancreas is the
399
site for several redox reactions, thereby producing various free radicals, and the
400
increase of Per6 may have protective role in the crayfish hepatopancraes.
401
4. Conclusion
402
In this study, we sequenced the entire hepatopancreas transcriptome from LPS
403
administrated red swamp crayfish using Illumina sequencing technology. The
404
transcriptome information acquired in the present study makes substantial
405
contribution to a sequence source for future studies on P. clarkii. The functional
406
analysis of differentially expressed genes predicted the involvement of genes in
407
various immune pathways. The explored innate immunity-related genes and
408
biological pathways make a comprehensive picture of the immune network that
409
provides valuable information to understand immune mechanisms of P. clarkii against
410
LPS. Further, qRT-PCR analysis on the selected genes indicated that these immunity
411
related genes may contribute in the anti-bacterial (LPS) immune responses of P.
412
clarkii. The sequence information provided in the present study should help to
413
understand anti-bacterial immune responses in an important cultured species;
414
moreover these results could provide a foundation to clarify the complex interaction
415
between the P. clarkii and pathogenic bacteria.
416
Acknowledgements
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This work was supported by Ph.D. programs in Wenzhou Medical University
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(89216023), the Opening Project of Zhejiang Provincial Top Key Discipline of
ACCEPTED MANUSCRIPT Pharmaceutical Sciences (201721). The authors declare no competing interests.
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ACCEPTED MANUSCRIPT Figure legends
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Fig. 1 Distribution pattern of unigene sequences and transcript length. Blue and
641
Red bars indicate unigenes and transcripts, respectively.
642
Fig. 2 Homology analysis of hepatopancreas specific unigene sequences of
643
crayfish (A) E-value distribution, (B) similarity distribution and (C) species
644
classification.
645
Fig. 3 Gene functional classification (GO) of crayfish hepatopancreas. The results
646
are summarized into three ontologies (cellular component, biological function and
647
molecular function). Y-axis indicates the total number genes in each category.
648
Fig. 4 The Eukaryotic Orthologous Groups (KOG) classification of putative
649
proteins.
650
Fig. 5 The Kyoto Encyclopedia of Genes and Genomes (KEGG) classification
651
based on assembled unigene sequences.
652
Fig. 6 Hierarchical cluster analysis of differentially expressed based on the data
653
of log ratio fold change. The colour scale shows the level of differentially expressed
654
gene: red colour shows increased expression level of mRNA and blue colour shows
655
decrease in expression level. L_1 and D_1 represent LPS treated and control PBS
656
treated, respectively.
657
Fig. 7 Distribution pattern of differentially expressed unigene sequences of
658
crayfish. X-axis exhibits change in fold between two sets of samples (PBS and LPS),
659
and Y-axis shows significance of DEGs. Red (up-regulation) and green
660
(down-regulation) dots represent significantly different expression (q-value<0.005,
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ACCEPTED MANUSCRIPT |log2 (fold change)|>1), respectively, and dots in blue colour shows no significant
662
differences.
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Fig. 8 Histogram and gene ontology analysis of crayfish differentially expressed
664
genes. The results are summarized into three ontologies (cellular component,
665
biological function and molecular function). Y-axis indicates the total number
666
differentially expressed genes in each category.
667
Fig. 9 The top 20 canonical pathways with maximum numbers of differentially
668
expressed genes based on KEGG classifications.
669
Fig. 10 Comparison of gene expression pattern between RNA-Seq and qRT-PCR
670
results in crayfish. The gene expression values were normalized to the 18S RNA
671
gene. Log2FC indicates the log2 fold change. Blue bars represent expression of genes
672
in RNA-seq data and orange bars show qRT-PCR results.
673
Fig. 11 Expression profiles of the key DEGs related immune responses after LPS
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challenge. These DEGs were quantified in crayfish hepatopancreas followingLPS
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infection. 18S RNA gene was used as an internal control.
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Legends of Supplementary Data
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Supplementary Fig. 1 1.0% agrose gel electrophoresis of Procambarus clarkii
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total RNA.
ACCEPTED MANUSCRIPT Table 1 Primers used in this study Forward Primer (5’-3’)
Reverse Primer (5’-3’)
Purpose
alkaline phosphatase
CAGCAGTGGTGGAAGACAGC
GTCGCCGAGGAACAAGATG
qPCR
beta 1,4-endoglucanase
ATGTTGTGCAGTTCGCTTGG
CGGTGTGTATGGTGCTTCGT
qPCR
catalase
TGCTGAGGTGGAACAGATGG
CGATGGCGATGAGTGTCATT
qPCR
cellulase GHF9
TGCCGGTCAGACAGAATACG
CCGTGGTCAATACCTCCGTTA qPCR
cubilin
GATCTGGCGCTCAATGACAA
TAGGAGGCGGACACTCTTGG
qPCR
acyl-CoA AGCGCAAGCACTACATTCCA
GATGGAGTAGCGCAGGAAGC
qPCR
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delta-9 desaturase
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Primer name
GCCGACAGTGTGACGAAGTC
ACGTTCACCACCAGCAACAC
qPCR
hemocyanin 2
GCTCGTCCTGATGATGTCCA
CGGTGATGTAGCCAAGAGCA
qPCR
hemocyanin
TGGAACACAGCAAGGACTGG
AAGCACGATGTCTTCGGTGA
qPCR
serine carboxypeptidase 1
CCGTGGTTAAGCTGTTGACG
GGCCACTCCATGTTCTCGAT
qPCR
vitellogenin
CCGGCTGAGCTTCACTAACC
AGTCGATGCGGTTGCCTTAT
qPCR
Per6
GCCATCAAGCTGCACACCTA
TTTGCCACACATCCCAACTC
qPCR
GTLT
CTGGAAGGCCAGAACTTGCT
CCTGGCAATCTGTCACCGTA
qPCR
CL
GCAGTGTGGATCATGCTGGT
TGCCATACTTGGTGGAGCAG
qPCR
GCGCCTGCAACTACAGCTAC
TTTGGGAGTCGCCAGAGAA
qPCR
18 S
CTGTGATGCCCTTAGATGTT
GCGAGGGGTAGAACATCCAA
qPCR
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ACCEPTED MANUSCRIPT Table 2 Summary of the sequence analyses Sample
Raw Reads
Clean reads
Clean Bases
Error(%)
Q20(%)
Q30(%)
GC content(%)
PBS
47,592,460
45,706,720
6.86G
0.02
96.63
91.77
43.99
LPS
52,414,336
50,310,480
7.55G
0.02
96.21
90.78
47.53
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Clean reads: The number of reads after removing low-quality sequences. The subsequent analysis is based on clean reads. Error rate: Base error rate. Q20 and Q30, the percentage of bases with Phred values >20 and >30, respectively. GC content: the GC ratio of the total base number
Number of genes
Percentage (%)
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Table 3 The success rate of gene annotation
20,363
32.89
14,515
23.44
10,174
16.43
20,264
32.73
19,672
31.77
Annotated in SwissProt
16,369
26.44
Total Unigenes
61,912
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Annotated in GO Annotated in KO Annotated in KOG
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Annotated in PFAM
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Annotated in GO: The unigene number and annotation rate in the GO database. Annotated in KO: The unigene number and annotation rate in the KO database. Annotated in KOG: The unigene number and annotation rate in the KOG database. Annotated in PFAM: The unigene number and annotation rate in the PFAM database. Annotated in NR: The unigene number and annotation rate in the NR database. Annotated in SwissProt: The unigene number and annotation rate in the SwissPort database.
ACCEPTED MANUSCRIPT Table 4 Top 20 statistically significant Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications Number
Pathway definition
Pathway ID
Number of unigenes
Endocytosis
ko04144
264
2
Spliceosome
ko03040
238
3
Purine metabolism
ko00230
223
4
PI3K-Akt signaling pathway
ko04151
220
5
Focal adhesion
ko04510
205
6
Lysosome
7
Rap1 signaling pathway
8
Ubiquitin mediated proteolysis
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1
205
ko04015
200
ko04120
191
Protein processing in endoplasmic reticulum
ko04141
177
10
Regulation of actin cytoskeleton
ko04810
174
11
Ras signaling pathway
ko04014
170
12
cAMP signaling pathway
ko04024
169
13
RNA transport
ko03013
163
14
MAPK signaling pathway
ko04010
161
15
Tight junction
ko04530
151
16
Insulin signaling pathway
ko04910
146
Thyroid hormone signaling pathway
ko04919
146
AMPK signaling pathway
ko04152
142
19
Hippo signaling pathway
ko04390
140
20
Carbon metabolism
ko01200
137
18
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ACCEPTED MANUSCRIPT Table 5 Top 20 differentially expressed pathways between normal crayfish and LPS challenged crayfish Pathway definition
P-value
1
Ribosome
3.05E-08
ko03010
46
2
Oxidative phosphorylation
5.89E-06
ko00190
33
3
Cardiac muscle contraction
7.56E-06
ko04260
17
4
Arachidonic acid metabolism
7.23E-05
ko00590
20
5
Longevity regulating pathway – worm
0.00015111
ko04212
27
6
Parkinson's disease
0.000678484
ko05012
31
7
Non-alcoholic fatty liver disease (NAFLD)
0.001235084
ko04932
29
8
Steroid biosynthesis
0.004219732
ko00100
8
9
Linoleic acid metabolism
0.006381182
ko00591
9
10
PPAR signaling pathway
0.006525988
ko03320
15
11
Glycolysis / Gluconeogenesis
0.006669947
ko00010
17
12
Lysosome
0.007820667
ko04142
39
13
Alzheimer's disease
0.010607122
ko05010
30
14
Carbohydrate digestion and absorption
0.016187143
ko04973
11
15
Ovarian steroidogenesis
0.020214647
ko04913
12
16
Metabolism of xenobiotics by cytochrome P450
0.02282741
ko00980
10
17
Other glycan degradation
0.023003679
ko00511
12
18
Retinol metabolism
0.024717722
ko00830
11
19
Rheumatoid arthritis
0.029414928
ko05323
12
20
Fatty acid degradation
0.03063433
ko00071
14
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DEG, differentially expressed gene
Pathway ID Number of DEGs
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No.
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ACCEPTED MANUSCRIPT Highlights 1. The
Hepatopancrease
transcriptome
of
P.
clarkii
following
Lipolysaccharide (LPS) challenge was constructed for the first time.
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2. The RNA-sequencing followed by assembly generated 61,912 unigenes. 3. In total, 2522 genes differentially expressed in LPS treated group compared with the control.
differentially expressed genes were identified and
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4. Immune-related
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categorized.
S1050-4648(17)30637-X
DOI:
10.1016/j.fsi.2017.10.030
Reference:
YFSIM 4901
To appear in:
Fish and Shellfish Immunology
Received Date: 29 July 2017 Revised Date:
5 October 2017
Accepted Date: 16 October 2017
Please cite this article as: Zhou M, Abbas MN, Kausar S, Jiang C-X, Dai L-S, Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipolysaccharide (LPS) infection, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.10.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Transcriptome profiling of red swamp crayfish (Procambarus clarkii)
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hepatopancreas in response to lipolysaccharide (LPS) infection
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Miao Zhou a,1 , Muhammad Nadeem Abbas b,1 , Saima Kausar b,1, Cheng-Xi Jiang c,*,
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Li-Shang Dai a,* a
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325035, P.R. China
Department of Zoology and Fisheries, University of Agriculture, Faisalabad 38000,
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b
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School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou
Pakistan c
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Life Sciences Institute, Wenzhou University, Wenzhou 325035, P.R. China
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* Corresponding author: Cheng-xi Jiang and Li-Shang Dai
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Tel/fax: +86 577 86699572
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E-mail: [email protected] (Cheng-Xi Jiang), [email protected]
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(Li-Shang Dai)
Address: School of Pharmaceutical Sciences, Wenzhou Medical University,
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Wenzhou 325035, P.R. China.
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22
1
These authors contributed equally to this work.
ACCEPTED MANUSCRIPT Abstract
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The RNA-sequencing followed by de novo assembly generated 61,912 unigene
25
sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS
26
challenged and PBS control samples revealed 2,552 differentially expressed genes
27
(DEGs). Of these sequences, 1,162 DEGs were differentially up-regulated and 1,360
28
DEGs differentially down-regulated. The DEGs were then annotated against gene
29
ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG)
30
database. Some immune-related pathways such as PPAR signaling pathway, lysosome,
31
Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis.
32
The reliability of transcriptome data was validated by quantitative real time
33
polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here
34
shed light into antibacterial immune responses of crayfish. In addition, these results
35
suggest that transcriptomic data provides valuable sequence resource for
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immune-related gene identification and helps to understand P. clarkii immune
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functions.
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Keywords: Crayfish; LPS; Immunity; Differential expressed genes; Arthropods
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1. Introduction The red-swamp crayfish (Procambarus clarkii) is a rich source of high quality
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proteins, which constitutes all the essential amino acids required for human nutrition.
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P. clarkii is a fresh water species, native to Mexico and United States [1]. It is
49
extremely tolerant to adverse environmental conditions such as temperature
50
fluctuations, poor water quality, and low oxygen concentrations. Therefore, the
51
species is considered highly suitable for culture and extraordinary farm production
52
[2,3]. Thus far, this species has been introduced and successfully cultured in many
53
countries world-wide [4]. In China, it was introduced from Japan in 1929 [5], since
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then it has become one of the most economically important species in China.
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Vertebrates and invertebrates through the process of evolution have developed an
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innate immune system, and innate immune responses are considered first line of
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defense against variety of pathogens [6-8]. The innate immune system is stimulated
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by microbial infections, thereby transcribing variety of immune related genes to
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perform their specific roles in host defense [9]. Hence, to understand the immune
60
mechanisms in an organism, it is crucially important to explore the genes that are
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probably implicated in the immune functions.
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In spite of crayfish economic importance and being cultured worldwide, our
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knowledge on its innate immune system is limited. In crayfish hepatopancreas is a
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vital organ for nutrient storage and it is also involved in a variety of immune
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responses and detoxification processes [10]. Hepatopancreas in crustaceans
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transcribes various immune related genes and proteins, which play crucial role to
ACCEPTED MANUSCRIPT maintain physiological processes. For instance, hemocyanin that is converted to
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antimicrobial proteins, antiviral agent, hemolysin, agglutinin and phenoxidase like
69
enzyme, and is considered to be involved in immune responses of crustaceans,
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mollusks and arthropods [11-13]. In addition, many immune related genes such as
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cathepsin B, cathepsin L, interferongamma inducible lysosomal thiol reductases
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(GILT) are also produced in hepatopancreas [14,15]. Besides this, lectins are
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important immune responsive genes, and known to have tissue specific expression
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and some lectins (hsL and FLec2) are only expressed in hepatopancreas [16-18].
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Although, these fragmentary studies are available on the immune responses of
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hepatopancreas in crustacean, however, the anti-bacterial immune mechanism of
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crayfish hepatopancreas has not been explored previously. Therefore study on its
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immune responses and exploration of immune-related genes will contribute to
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understand anti-bacterial immune mechanism in the crayfish.
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The transcriptome is an entire set of RNA transcripts produced by the genome at
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any one time, and the knowledge of the transcriptome is critically important for
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interpreting the functional factors of the genome [5]. In the present study, we prepared
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and sequenced two libraries of P. clarkii hepatopancreas after LPS stimulation and
84
PBS administration using Illumina sequencing platform. First, the obtained reads were
85
assembled to unigene sequences by RNA-Seq de novo programs. Subsequently, the
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transcriptome data was subjected to GO, KOG, and KEGG databases to investigate
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the involvement of unigene sequences in different canonical pathways. Furthermore,
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enrichment analysis was executed following the identification of differentially
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ACCEPTED MANUSCRIPT expressed genes (DEGs) from two P. clarkii libraries. The main purpose of this study
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was to identify and align immune-related genes in the hepatopancreas. The
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transcriptomic data will also provide sequence resources for future studies on
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crustaceans and may help to understand antibacterial immune mechanisms of P.
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clarkii.
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2. Materials and methods
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2.1. Experimental animal and LPS treatment
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P. clarkii individuals were obtained from local market; the specimens were
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ranged 7-8 cm in size and approximately an average weight of 10 g. They were kept
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in spacious tanks, supplied with high-quality freshwater, and fed at 23±2 °C with
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natural (small invertebrates) and artificial diet. The specimens were divided into
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experimental group and control group, each of which contained 10 individuals. The
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experimental group was administrated with LPS (1 µg/g of body weight) in abdominal
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part and PBS was used as control group. Following 12 h of treatment hepatopancreas
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samples were collected from both the experimental and control group and
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immediately frozen in liquid nitrogen and then stored at −80 °C till the extraction of
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RNA.
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2.2. RNA extraction, library preparation and sequencing
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The collected hepatopancreas samples were used to isolate RNA by the Trizol
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Reagent (Invitrogen, USA) according to manufacturer’s instructions (Supplementary
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Fig. 1). The integrity and quality of extracted RNA was analyzed by agarose gel
110
electrophoresis
(1%),
and
NanoDrop
1000
spectrophotometer
(NanoDrop
ACCEPTED MANUSCRIPT Technologies, Wilmington, DE, USA), respectively. After DNase I treatment, the
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messenger RNA (mRNA) was purified from 20 µg mixed total RNA using oligo (dT)
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magnetic beads. Following purification, the mRNA was fragmented into short
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fragments by mixing in fragmentation buffer in a thermomixer at a suitable
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temperature. First-strand cDNA was then synthesized using the mRNA fragments as
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templates, followed by second-strand cDNA synthesis using DNA polymerase I and
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RNase H. After the end repair process and connection with adapters, suitable
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fragments were used as templates for PCR amplification to create the final cDNA
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library. cDNA libraries were constructed using SMART cDNA library construction kit
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(Clontech, CA, USA) in accordance with the manufacturer’s protocol. Following the
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examination of quality and quantity, the libraries for transcriptome sequencing was
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prepared
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Solexa/Illumina sequencing was carried out by Novogene, Beijing, China.
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2.3. Quality control and assembly of reads
Illumina’s
kit
following
manufacturer’s
recommendations.
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The assembly of LPS and PBS administrated hepatopancreas samples were
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executed using RNA-Seq de novo programs Trinity and SOAP de novo assembler [19]
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with default parameters. To obtain clean reads, short reads (Read length >10bp) and
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low quality reads (Quality score >20) were discarded. The clean reads were processed
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to form contigs, and then these contigs were clustered to obtain unigene sequences,
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which were finally connected to obtain transcript [20].
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2.4. Annotation and gene ontology of transcriptome
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After transcriptome assembly, the obtained unigene and transcript sequences
ACCEPTED MANUSCRIPT were annotated on the basis of sequence similarity with known genes. Briefly, the
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contigs were aligned against available sequence in the National Center of
135
Biotechnology and Information (NCBI) database [21]. The unigene sequences were
136
aligned with NCBI protein data bases such as Nr, KOG, KEGG and Swiss-Prot using
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BLASTx [22]. While the remaining unigene sequences (none of the BLASTx hits)
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were annotated with Nt database of NCBI. To determine sequence homology,
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alignments were executed with previously known genes (cutoff E-value≤ 10−5). The
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alignments results were used to identify sequence direction and prediction of protein
141
coding region (CDS). Functional annotation was performed with gene function
142
classification
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(http://www.blast2go.com/b2ghome) [23]. The pathway annotation was executed to
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determine the biological behavior of the genes using the KEGG database.
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2.5. Identification of differentially expressed genes
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(www.geneontology.org)
using
Blast2GO
software
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To identify differentially expressed genes from hepatopancreas samples of P.
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clarkii following LPS treatment (PBS as control). cDNA libraries were constructed,
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sequenced and transcriptome was assembled according to sections 2.2 and 2.3. The
149
differentially expressed genes (DEGs) were identified based on the number of
150
fragments per kilo base of exon per million fragments mapped (FPKM) of the genes,
151
and False Discovery Rate (FDR) was used to find the P-value threshold [24]. The
152
DEGs were characterized as those having FDR less than 0.05 and a fold change
153
between treated and control groups of greater than 2 (log2 fold change P1). Further,
154
the DEGs were mapped to the GO database (http://www.geneontology.org/) and then
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ACCEPTED MANUSCRIPT their functions were determined using WEGO and Blast2GO programs [25]. The
156
KEGG public database was used to predict enriched metabolic and signal transduction
157
pathways [26]. The pathway categories classification was determined using Fisher’s
158
exact test, and the FDR was used to correct the P values. Pathways with a P-value less
159
than 0.05 were considered as significantly enriched [27].
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2.6. Quantitative RT-PCR assay
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Quantitative real time PCR (qRT-PCR) was performed to analyze mRNA levels
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of immune related genes. Hepatopancreas samples were collected from crayfish (7-8
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cm in size, and average weight of 10 g) purchased from local market, further the
164
number of specimens in each experiment and time of hepatopancreas collection was
165
same as mentioned in the section 2.1. Total RNA was isolated from LPS treated and
166
control group (PBS), then reverse transcribed into cDNA. The gene specific primers
167
were designed based on the identified transcript sequences by primer premier 5.0
168
software (Table 1). The primers were used to analyze the expression of immune
169
related genes, and 18S RNA was used as internal control. The qRT-PCR was
170
performed in 25 µL reactions including 12.5 µL of 2× SYBR Premix Ex TaqII
171
(Takara), 1 µL each of the forward and reverse primers, 2 µL of template (cDNA), and
172
8.5 µL of RNase-free H2O. The amplification program was as follows: 95°C for 30 s;
173
39 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. At the end of the reaction,
174
a melting curve was produced by monitoring fluorescence continuously, while slowly
175
heating the sample from 65°C to 95°C. The relative expression levels of the selected
176
genes were calculated according to the 2−△△Ct method [28]. All qRT-PCR experiments
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ACCEPTED MANUSCRIPT 177
were repeated five times.
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3. Results and Discussion
179
3.1. Sequencing and assembly of trancriptome To determine gene expression profile of P. clarkii, total RNA was extracted and
181
cDNA libraries were constructed from hepatopancreas following LPS treatment and
182
PBS was used as control. The Illumina sequencing of two libraries generated
183
52,414,336 and 47,592,460 raw reads for treated and control group, respectively.
184
After discarding ambiguous nucleotides, low quality reads and short reads, treated
185
group contained 50,310,480 clean reads, representing 7.55G nucleotides. The Q20
186
percentage and Q30 percentage (percentage of nucleotide whose quality was more
187
than 20 and 30 clean reads) were 96.21% and 90.78%, respectively and GC contents
188
was47.53%. The control group comprised 45,706,720 clean reads representing 6.86G
189
nucleotides. The Q20 percentage, Q30 percentage and GC percentage were 96.63%
190
and 91.77%, and 43.99%, respectively (Table 2).
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The ‘de novo’ transcriptome assembly strategy is most commonly used to
192
assemble RNA-seq data. It does not depend on reference genome and can provide an
193
initial set of transcripts, allowing for RNA-seq expression studies [29]. Currently,
194
Trinity, SOAP de novo, trans-ABySS, ABySS, and Oases assemblers have been
195
documented to be used for the de novo assemblage of short read RNA-seq data into
196
transcripts [30]. In the present study, we used Trinity and SOAP de novo assemblers
197
to assemble high quality clean reads of two libraries [19]. Trinity is an important de
198
novo assembly program that has been reported to be sensitive and efficient in
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ACCEPTED MANUSCRIPT recovering full-length transcripts/isoforms in various organisms. It constructs de
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Bruijn graph from huge amounts of short-read sequence data, then use an enumeration
201
algorithm to score all possible paths and branches, and retain only those plausible
202
ones as transcripts and isoforms. Additionally, it is specially programmed to recover
203
paths supported by actual reads and eradicate ambiguous edges, thus ensure correct
204
transcript reconstruction [31]. Likewise, the SOAP de novo program is a novel
205
short-read assembler that can build draft assemblies de novo for large sized genomes
206
such as animal and plant genomes. It builds full-length transcripts/isoforms and
207
allowing to carry out accurate analyses of unexplored genomes [32].
208
novo programs (SOAP de novo and Trinity) are highly reliable and widely used to
209
recover transcripts and unigenes of unexplored species. In the present study, the
210
assembly of high quality clean reads generated 61,912 unigene sequences and 11,3398
211
transcripts. Both the unigene sequences and transcripts were ranged in size from <301
212
bp to > 2000 bp. The maximum number of unigene sequences were between 500
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-1000 bp range, and transcripts were in <301 bp (Fig. 1).
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3.2. Functional annotation of clustered unigene sequences
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To evaluate the functions of unigene sequences, a total of 61,912 unigene
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sequences were aligned with six public databases (NR, Swiss-Port, KO, GO, KOG
217
and PFAM). All of these sequences are annotated in at least one Database except
218
36,091 unigene sequences. The results revealed that 20,363 unigene sequences were
219
significantly best hit with the GO database, 20,264 unigene sequences were
220
significantly annotated with the PFAM database, and 19,672 unigene sequences were
ACCEPTED MANUSCRIPT significantly aligned with the NR database. While 16,369, 14,515 and 10,174 unigene
222
sequences were mapped against SwissProt database, KO database and KOG database,
223
respectively (Table 3). Of these aligned sequences, almost 32% unigene sequences
224
had more than 1e-30 E-value (Fig. 2A). The similarity distribution pattern revealed
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that 61.8% unigene sequences shared greater than 60% homology (Fig. 2B). The
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species specific distribution patterns revealed that 16.7% of the unigene sequences
227
most closely matched with Zootermopsis nevadensis, 6.7% of the unigene sequences
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resembled Daphnia pulex, 3.5% unigene sequences best hits with Tribolium
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castaneum (3.5%) and so on (Fig. 2C).
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3.3. Functional analysis of transcriptome
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The putative functions of unigene sequences in LPS treated and control group
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were analyzed using Gene Ontology (GO) and Eukaryotic Orthologous Groups of
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proteins (KOG). The analysis of GO classification revealed that most of unigene
234
sequences mapped to biological process, followed by cellular component and
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molecular function. The three categories were further divided into 55 functional
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groups. In the two libraries, most of the corresponding biological process unigene
237
sequences were implicated in cellular processes, metabolic processes and single
238
organism process. Most of the cellular component unigene sequences were associated
239
with cell, cell junction, organelle, macromolecular complex and membrane. Most of
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the molecular function unigene sequences encoded protein involved in binding,
241
followed by catalytic activity, and transporter activity (Fig. 3).
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Eukaryotic Orthologous Groups of proteins (KOG) database classifies gene
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understand gene functions and their evolutionary relationships [33]. A total of 10,174
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genes were aligned with KOG database and divided into 26 functional categories.
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Among the 26 KOG categories, general function prediction only and signal
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transduction mechanism were the most abundant category, followed by post
248
translational modification, protein turnover, chaperones and transcription categories
249
(Fig. 4).
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Kyoto Encyclopedia of Genes and Genomes (KEGG) is a valuable tool for
251
canonical pathways based classification of orthologous genes, and provides important
252
information to understand biological functional profiles of gene [34]. In the present
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study, 14,515 genes were annotated against the 229 biological pathways in the KEGG
254
database. Among these biological pathways, the top 20 statistically significant
255
pathways are shown in Table 4. Of these some important immune related biological
256
pathways were predicted in the KEGG database, including Rap1 signaling pathway,
257
Focal adhesion, MAPK signaling pathway PI3K-Akt signaling pathway, cAMP
258
signaling pathway and Ubiquitin mediated proteolysis. In addition, lysosome,
259
endocytosis, RNA transport, spliceosome, protein processing endoplasmic reticulumn,
260
and so on were predicted (Table 4).
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Furthermore, the unigene sequences were classified into environmental
262
information processing, genetic information processing, metabolism, cellular process
263
and organismal system (Fig. 5). Among the 12 categories of metabolism, carbohydrate
264
metabolism (456 sequences), lipid metabolism (449 sequences) and amino acid
ACCEPTED MANUSCRIPT metabolism (328 sequences) unigene sequences were most abundant. In organismal
266
system, Endocrine system and Immune system were the most abundant clusters with
267
654 sequences and 478 sequences, respectively. Interestingly, in environmental
268
information processing, Signal transduction (1197 sequences) was the most abundant
269
cluster in all categories. While, folding, sorting and degradation (538 sequences) was
270
the most unigene sequences cluster among the genetic information processing (Fig. 5).
271
These biological processes play vital role in the physiological processes of P. clarkii
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and will help to understand its immune mechanisms.
273
3.4. Identification and analysis of DEGs
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The gene expression pattern is utilized to predict the gene functions, and
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generally, the genes exhibiting similar expression pattern mostly have same functions,
276
or involved in the same biochemical pathways. Hence, clustering of genes can be
277
useful to evaluate the function of unknown genes [35]. Here, we performed gene
278
clustering to determine the functional enrichment of genes exhibiting similar
279
expression pattern (Fig. 6). To determine the unigene sequences expression levels of P.
280
clarkii hepatopancreas following LPS treatment and PBS was used as control. Here
281
we
clean
reads
using
RSEM
282
|log2(foldchange)|>1)
and
then
screened
283
down-regulated unigene sequences in response to LPS challenge. A 2,522 DEGs were
284
identified, of these 1,162 unigene sequences significantly upregulated and 1,360
285
sequences down-regulated (Fig. 7).
286
3.5. GO classification of immune-related DEGs
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aligned
software
(q-value
significantly
<
0.005
up-regulated
& and
ACCEPTED MANUSCRIPT The unigene sequences were mapped against the GO and KEGG database. The
288
DEGs were categorized into biological process, cellular component and molecular
289
function, and then further classified into 31 subcategories (Fig. 8). The biological
290
process and molecular function categories represented most of the subcategories.
291
Metabolic process and single organism metabolic process were the most abundant
292
among the biological process. The hepatopancreas tissues metabolic oxygen demands
293
are relatively high in crustaceans similar across different species [36]. Further, in this
294
study, following the LPS treatment, we observed obvious transcription changes in
295
hepatopancrease under different GO terms including catalytic activity and
296
oxidoreductase (Fig. 8). Furthermore the DEGs were aligned to the KEGG database to
297
determine the biological pathway enrichment. Many biological pathways were (P = <
298
0.05) changed in LPS treated sample compared to control sample (PBS). Among the
299
biological pathways, some immune related pathways including PPAR signaling
300
pathway, Rheumatoid arthritis, lysosome, Chemical carcinogenesis, Peroxisome, and
301
so on were observed in the DEGs. The knowledge on crayfish antibacterial innate
302
immune mechanism is limited, and the above canonical pathways information will be
303
useful to explore and understand the immune mechanisms (Fig. 9 & Table 5).
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Crayfish have an efficient innate immune system to combat various microbial
305
infections. Based on the KEGG classification, we identified some of the important
306
mechanisms of the crayfish innate immune system that are involved in the
307
antimicrobial defense, including lysosome and antibacterial peptides production.
308
These results are in agreement with study of Adema et al. [37], they performed
ACCEPTED MANUSCRIPT microarray analysis and reported the up-regulation of antibacterial peptides and
310
lysosome transcripts after the treatment of Echinostoma paraensei and Schistosoma
311
mansoni in Biomphalaria glabrata. Further, these results are also comparable with the
312
studies of Byadgi et al. [38]. Phagocytosis is the most common mechanism in animals
313
(crayfish) to overcome microbial infection. Upon the attachment of pathogen to the
314
surface of phagocyte, the phagocyte forms a vesicle (Phagosome) after engulfing
315
pathogen. Then lysosome containing different enzymes inside the phagocyte, fuse
316
with the phagosome resulting in the degradation of pathogen [39, 40].
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Further, the synthesis of antibacterial peptides in organisms is also an important
318
innate immune defense mechanism against microbial agents. Antibacterial peptides
319
attach to bacterial surface and break the bacterial cell membrane and consequently
320
bacterial cell is lysed. In insects, antibacterial peptides have been well studied, and
321
approximately 15 different antibacterial peptides have been reported that are induced
322
by bacteria or bacterial products exposure in insects. For instance, cecropins are
323
produced in insects and have a broad spectrum of activity against both gram negative
324
and gram positive bacteria [41, 42]. However, little information is available on the
325
antibacterial peptide production in crayfish [43], and the present study will provide a
326
foundation to further explore formation and possible role of antibacterial peptides in
327
crayfish.
328
3.6. Validation of transcriptome data by qRT-PCR
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329
To validate the expression levels of differentially expressed genes in our
330
transcriptome analysis, eleven genes were randomly selected based on altered
ACCEPTED MANUSCRIPT expression pattern after LPS treatment, and gene of interest through functional
332
enrichment and canonical pathways results, later investigated by qRT-PCR.
333
Quantitative RT-PCR analysis revealed that the most of differentially expressed genes
334
were in agreement with the RNA-seq data, however some genes such as cubilin had
335
relatively little lower expression in RNA-seq data compared to qRT-PCR analysis.
336
Although these genes expression was relatively lower, but overall both the analysis
337
showed approximately similar trend (Fig. 10). This characteristic feature has
338
previously been reported in transcriptomic studies of invertebrates such as
339
Biomphalaria glabrata [37], Homarus americanus [44], Exopalaemon carinicauda
340
[45] and so on.
341
Expression analysis of immune related genes
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Red swamp crayfish is one of the most economically important crustaceans. It is
343
used as model organism in various research fields such as toxicity, environmental
344
stress, animal behavior and studies on pathogen infection. Despite its importance,
345
little is known about crayfish genome particularly on immune related genes, immune
346
mechanisms and pathways, which are involved to combat microbial infection [46].
347
The identification of immune-related genes can contribute to understand the immune
348
mechanism of crayfish. Hepatopancreas in crustaceans has been reported to be
349
involved in metabolism, nutrient absorption and immune functions [47]. In this study,
350
four immune-related DEGs with clearly changed expression after LPS administration
351
were investigated by qRT-PCR assay. All of the immune-related genes were
352
significantly up-regulated in the LPS treated sample compared to PBS control (Fig.
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ACCEPTED MANUSCRIPT 11). The interferon (IFN) system is an important cellular defense mechanism in
354
mammals, and play crucial role to suppress viral infections by inhibiting multiple
355
steps of viral replication, increasing antigen-presentation process and activating
356
macrophages [48] (Cebulla and Miller, 1999). In the present study, component of IFN
357
system was up-regulated like gamma-interferon-inducible lysosomal thiol reductase
358
(GILT) in the LPS treated sample compared to control. The GILT is widely expressed
359
in a variety of cell types particularly in antigen presenting cells [49-51]. Where, it
360
catalyzes the reduction of disulfide bonds in exogenous antigens and facilitate in the
361
unfolding of the protein and then that is cleared by cellular proteases [52, 53], in
362
addition, GILT play important role in the neutralization of pathogens and removal of
363
cellular debris resulting from pathogen (bacterial) infection [54]. Further, several
364
studies reported the up-regulation of GILT in response to bacterial or LPS
365
administration, and its involvement in immune response [55, 56]. Our data is also
366
consistent with above mentioned studies, and suggest that GILT may be regulating the
367
immune function following LPS or bacterial challenge.
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Anti-lipopolysaccharide factor (ALF) is an important member of antimicrobial
369
peptides and a key effector molecule in the crustaceans innate immune system. ALFs
370
can neutralize LPS, mediate degradation and activate coagulation cascade in cell [57].
371
So far a number of ALFs have been described in different species of crustaceans [58,
372
59], and bacterial and viral infection or LPS treatment can induce the expression of
373
ALFs in them [59, 60]. ALFs contain amphipatic loop structure that can bind a single
374
fatty acid with the phosphoglucosamine portion of lipid A that helps to bind with LPS
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ACCEPTED MANUSCRIPT and subsequently neutralize it [61]. In this study, ALF-3 appeared as second most
376
greatly expressed factor. These results are consistent with the study of Clark et al. [44],
377
they reported up-regulation of various immune related factors including ALFs in
378
hepatopancrease of Homarus americanus after bacterial infection. Likewise,
379
Cathepsin L (CL) was significantly induced in hepatopancreas following LPS
380
challenge. Lysosomal cathepsins belonging to the family of cysteine protease have
381
been documented to be involved in the innate immunity of invertebrates. In P.
382
monodon, Microarray analysis showed that cathepsins B and L expression enhanced
383
after WSSVtreatment and the upregulation in expression of cathepsin probably reflect
384
the activation of lysosomal functions against viral infection. In the hepatopancreas of
385
F. chinensis mRNA levels of cathepsin B, C and L were upregulated following V.
386
anguillarum challenge, indicating that these cathepsins were involved in the innate
387
immunity of F. chinensis [62-64]. Hu and Leung [65] reported the expression of
388
Cathepsin L in the B cells of the hepatopancreas of Metapenaeus ensis, and suggested
389
the involvement of Cathepsin L in immune responses. Our data suggest
390
exist in the crayfish and is an important effector molecule in the innate immune
391
system, which helps to deal bacterial infection. In addition, peroxiredoxin 6 (Per6)
392
also showed significant increase compared to PBS control. It belongs to the
393
peroxiredoxin family that is broadly distributed in prokaryotes and eukaryotes [66,
394
67]. Serveral studies on invertebrates reported the increase in Per6 expression
395
following the treatment of bacteria, virus and pathogen-associated molecular pattern,
396
and has been shown to have important role in host defense [68, 69]. The members of
cathepsin L
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ACCEPTED MANUSCRIPT peroxiredoxin family play crucial protective role in tissues such as detoxification and
398
reduction of hydrogen peroxide and so on [70]. In crustaceans, hepatopancreas is the
399
site for several redox reactions, thereby producing various free radicals, and the
400
increase of Per6 may have protective role in the crayfish hepatopancraes.
401
4. Conclusion
402
In this study, we sequenced the entire hepatopancreas transcriptome from LPS
403
administrated red swamp crayfish using Illumina sequencing technology. The
404
transcriptome information acquired in the present study makes substantial
405
contribution to a sequence source for future studies on P. clarkii. The functional
406
analysis of differentially expressed genes predicted the involvement of genes in
407
various immune pathways. The explored innate immunity-related genes and
408
biological pathways make a comprehensive picture of the immune network that
409
provides valuable information to understand immune mechanisms of P. clarkii against
410
LPS. Further, qRT-PCR analysis on the selected genes indicated that these immunity
411
related genes may contribute in the anti-bacterial (LPS) immune responses of P.
412
clarkii. The sequence information provided in the present study should help to
413
understand anti-bacterial immune responses in an important cultured species;
414
moreover these results could provide a foundation to clarify the complex interaction
415
between the P. clarkii and pathogenic bacteria.
416
Acknowledgements
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This work was supported by Ph.D. programs in Wenzhou Medical University
418
(89216023), the Opening Project of Zhejiang Provincial Top Key Discipline of
ACCEPTED MANUSCRIPT Pharmaceutical Sciences (201721). The authors declare no competing interests.
420
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631 632 633 634 635 636 637 638
EP
630
AC C
629
TE D
628
ACCEPTED MANUSCRIPT Figure legends
640
Fig. 1 Distribution pattern of unigene sequences and transcript length. Blue and
641
Red bars indicate unigenes and transcripts, respectively.
642
Fig. 2 Homology analysis of hepatopancreas specific unigene sequences of
643
crayfish (A) E-value distribution, (B) similarity distribution and (C) species
644
classification.
645
Fig. 3 Gene functional classification (GO) of crayfish hepatopancreas. The results
646
are summarized into three ontologies (cellular component, biological function and
647
molecular function). Y-axis indicates the total number genes in each category.
648
Fig. 4 The Eukaryotic Orthologous Groups (KOG) classification of putative
649
proteins.
650
Fig. 5 The Kyoto Encyclopedia of Genes and Genomes (KEGG) classification
651
based on assembled unigene sequences.
652
Fig. 6 Hierarchical cluster analysis of differentially expressed based on the data
653
of log ratio fold change. The colour scale shows the level of differentially expressed
654
gene: red colour shows increased expression level of mRNA and blue colour shows
655
decrease in expression level. L_1 and D_1 represent LPS treated and control PBS
656
treated, respectively.
657
Fig. 7 Distribution pattern of differentially expressed unigene sequences of
658
crayfish. X-axis exhibits change in fold between two sets of samples (PBS and LPS),
659
and Y-axis shows significance of DEGs. Red (up-regulation) and green
660
(down-regulation) dots represent significantly different expression (q-value<0.005,
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ACCEPTED MANUSCRIPT |log2 (fold change)|>1), respectively, and dots in blue colour shows no significant
662
differences.
663
Fig. 8 Histogram and gene ontology analysis of crayfish differentially expressed
664
genes. The results are summarized into three ontologies (cellular component,
665
biological function and molecular function). Y-axis indicates the total number
666
differentially expressed genes in each category.
667
Fig. 9 The top 20 canonical pathways with maximum numbers of differentially
668
expressed genes based on KEGG classifications.
669
Fig. 10 Comparison of gene expression pattern between RNA-Seq and qRT-PCR
670
results in crayfish. The gene expression values were normalized to the 18S RNA
671
gene. Log2FC indicates the log2 fold change. Blue bars represent expression of genes
672
in RNA-seq data and orange bars show qRT-PCR results.
673
Fig. 11 Expression profiles of the key DEGs related immune responses after LPS
674
challenge. These DEGs were quantified in crayfish hepatopancreas followingLPS
675
infection. 18S RNA gene was used as an internal control.
EP
TE D
M AN U
SC
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661
AC C
676 677
Legends of Supplementary Data
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Supplementary Fig. 1 1.0% agrose gel electrophoresis of Procambarus clarkii
679
total RNA.
ACCEPTED MANUSCRIPT Table 1 Primers used in this study Forward Primer (5’-3’)
Reverse Primer (5’-3’)
Purpose
alkaline phosphatase
CAGCAGTGGTGGAAGACAGC
GTCGCCGAGGAACAAGATG
qPCR
beta 1,4-endoglucanase
ATGTTGTGCAGTTCGCTTGG
CGGTGTGTATGGTGCTTCGT
qPCR
catalase
TGCTGAGGTGGAACAGATGG
CGATGGCGATGAGTGTCATT
qPCR
cellulase GHF9
TGCCGGTCAGACAGAATACG
CCGTGGTCAATACCTCCGTTA qPCR
cubilin
GATCTGGCGCTCAATGACAA
TAGGAGGCGGACACTCTTGG
qPCR
acyl-CoA AGCGCAAGCACTACATTCCA
GATGGAGTAGCGCAGGAAGC
qPCR
SC
delta-9 desaturase
RI PT
Primer name
GCCGACAGTGTGACGAAGTC
ACGTTCACCACCAGCAACAC
qPCR
hemocyanin 2
GCTCGTCCTGATGATGTCCA
CGGTGATGTAGCCAAGAGCA
qPCR
hemocyanin
TGGAACACAGCAAGGACTGG
AAGCACGATGTCTTCGGTGA
qPCR
serine carboxypeptidase 1
CCGTGGTTAAGCTGTTGACG
GGCCACTCCATGTTCTCGAT
qPCR
vitellogenin
CCGGCTGAGCTTCACTAACC
AGTCGATGCGGTTGCCTTAT
qPCR
Per6
GCCATCAAGCTGCACACCTA
TTTGCCACACATCCCAACTC
qPCR
GTLT
CTGGAAGGCCAGAACTTGCT
CCTGGCAATCTGTCACCGTA
qPCR
CL
GCAGTGTGGATCATGCTGGT
TGCCATACTTGGTGGAGCAG
qPCR
GCGCCTGCAACTACAGCTAC
TTTGGGAGTCGCCAGAGAA
qPCR
18 S
CTGTGATGCCCTTAGATGTT
GCGAGGGGTAGAACATCCAA
qPCR
AC C
ALF3
EP
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glutathione peroxidase
ACCEPTED MANUSCRIPT Table 2 Summary of the sequence analyses Sample
Raw Reads
Clean reads
Clean Bases
Error(%)
Q20(%)
Q30(%)
GC content(%)
PBS
47,592,460
45,706,720
6.86G
0.02
96.63
91.77
43.99
LPS
52,414,336
50,310,480
7.55G
0.02
96.21
90.78
47.53
RI PT
Clean reads: The number of reads after removing low-quality sequences. The subsequent analysis is based on clean reads. Error rate: Base error rate. Q20 and Q30, the percentage of bases with Phred values >20 and >30, respectively. GC content: the GC ratio of the total base number
Number of genes
Percentage (%)
M AN U
SC
Table 3 The success rate of gene annotation
20,363
32.89
14,515
23.44
10,174
16.43
20,264
32.73
19,672
31.77
Annotated in SwissProt
16,369
26.44
Total Unigenes
61,912
100
Annotated in GO Annotated in KO Annotated in KOG
EP
Annotated in NR
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Annotated in PFAM
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Annotated in GO: The unigene number and annotation rate in the GO database. Annotated in KO: The unigene number and annotation rate in the KO database. Annotated in KOG: The unigene number and annotation rate in the KOG database. Annotated in PFAM: The unigene number and annotation rate in the PFAM database. Annotated in NR: The unigene number and annotation rate in the NR database. Annotated in SwissProt: The unigene number and annotation rate in the SwissPort database.
ACCEPTED MANUSCRIPT Table 4 Top 20 statistically significant Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications Number
Pathway definition
Pathway ID
Number of unigenes
Endocytosis
ko04144
264
2
Spliceosome
ko03040
238
3
Purine metabolism
ko00230
223
4
PI3K-Akt signaling pathway
ko04151
220
5
Focal adhesion
ko04510
205
6
Lysosome
7
Rap1 signaling pathway
8
Ubiquitin mediated proteolysis
9
SC
RI PT
1
205
ko04015
200
ko04120
191
Protein processing in endoplasmic reticulum
ko04141
177
10
Regulation of actin cytoskeleton
ko04810
174
11
Ras signaling pathway
ko04014
170
12
cAMP signaling pathway
ko04024
169
13
RNA transport
ko03013
163
14
MAPK signaling pathway
ko04010
161
15
Tight junction
ko04530
151
16
Insulin signaling pathway
ko04910
146
Thyroid hormone signaling pathway
ko04919
146
AMPK signaling pathway
ko04152
142
19
Hippo signaling pathway
ko04390
140
20
Carbon metabolism
ko01200
137
18
TE D
EP
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17
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ko04142
ACCEPTED MANUSCRIPT Table 5 Top 20 differentially expressed pathways between normal crayfish and LPS challenged crayfish Pathway definition
P-value
1
Ribosome
3.05E-08
ko03010
46
2
Oxidative phosphorylation
5.89E-06
ko00190
33
3
Cardiac muscle contraction
7.56E-06
ko04260
17
4
Arachidonic acid metabolism
7.23E-05
ko00590
20
5
Longevity regulating pathway – worm
0.00015111
ko04212
27
6
Parkinson's disease
0.000678484
ko05012
31
7
Non-alcoholic fatty liver disease (NAFLD)
0.001235084
ko04932
29
8
Steroid biosynthesis
0.004219732
ko00100
8
9
Linoleic acid metabolism
0.006381182
ko00591
9
10
PPAR signaling pathway
0.006525988
ko03320
15
11
Glycolysis / Gluconeogenesis
0.006669947
ko00010
17
12
Lysosome
0.007820667
ko04142
39
13
Alzheimer's disease
0.010607122
ko05010
30
14
Carbohydrate digestion and absorption
0.016187143
ko04973
11
15
Ovarian steroidogenesis
0.020214647
ko04913
12
16
Metabolism of xenobiotics by cytochrome P450
0.02282741
ko00980
10
17
Other glycan degradation
0.023003679
ko00511
12
18
Retinol metabolism
0.024717722
ko00830
11
19
Rheumatoid arthritis
0.029414928
ko05323
12
20
Fatty acid degradation
0.03063433
ko00071
14
SC
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DEG, differentially expressed gene
Pathway ID Number of DEGs
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No.
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S
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ED
M AN U
ED
M AN
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SC
M AN U
RI P
TE
D
S
M AN U
PT ED
S
M AN U
EP TE D
SC
M AN U
CE
ED
PT SC
M AN U
ED
M AN
TE
D
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EP TE D
SC
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ACCEPTED MANUSCRIPT Highlights 1. The
Hepatopancrease
transcriptome
of
P.
clarkii
following
Lipolysaccharide (LPS) challenge was constructed for the first time.
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2. The RNA-sequencing followed by assembly generated 61,912 unigenes. 3. In total, 2522 genes differentially expressed in LPS treated group compared with the control.
differentially expressed genes were identified and
SC
4. Immune-related
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categorized.