An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis

An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis

Accepted Manuscript An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis Zhengfeng Din...

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Accepted Manuscript An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis Zhengfeng Ding, Jing Pan, Hua Huang, Gongcheng Jiang, Jianqin Chen, Xueshen Zhu, Renlei Wang, Guohua Xu PII:

S1050-4648(17)30705-2

DOI:

10.1016/j.fsi.2017.11.028

Reference:

YFSIM 4962

To appear in:

Fish and Shellfish Immunology

Received Date: 25 September 2017 Revised Date:

8 November 2017

Accepted Date: 12 November 2017

Please cite this article as: Ding Z, Pan J, Huang H, Jiang G, Chen J, Zhu X, Wang R, Xu G, An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.11.028. 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.

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ACCEPTED MANUSCRIPT An integrated metabolic consequence of Hepatospora eriocheir infection in the

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Chinese mitten crab Eriocheir sinensis

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Zhengfeng Ding1*, Jing Pan1, Hua Huang2, Gongcheng Jiang1, Jianqin Chen1,

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Xueshen Zhu1, Renlei Wang1, Guohua Xu1*

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Chemistry, Jiangsu Second Normal University, 77 West Beijing Road, Nanjing, China,

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210013

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Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and

Aquatic Technology Promotion Station, Wujin District, Changzhou City, China,

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*Corresponding author at Jiangsu Second Normal University, College of life science

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and

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+86-25-83758339;

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E-mail

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[email protected] (G. Xu)

chemistry,

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addresses:

West

Beijing

Road,

[email protected];

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Nanjing,

China,

[email protected]

210013;

(Z.

Tel:

Ding);

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Abstract Despite the economic and evolutionary importance of aquatic host-infecting

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microsporidian species, at present, limited information has been provided about the

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microsporidia–host interactions. This study focused on Hepatospora eriocheir, an

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emerging microsporidian pathogen for the Chinese mitten crab Eriocheir sinensis.

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Hypertrophy of hepatopancreas cells was a common feature of H. eriocheir infection.

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More importantly, mitochondria of the hepatopancreas were drawn around the H.

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eriocheir, most likely to aid the uptake of ATP directly from the host. To better

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understand the crab anti-microsporidian response, de novo transcriptome sequencing

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of the hepatopancreas tissue was furtherly proceeded. A total of 47.84 M and 57.21 M

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clean reads were generated from the hepatopancreas of H. eriocheir infected and

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control groups respectively. Based on homology searches, functional annotation with

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6 databases (Nr, Swiss-Prot, KEGG, KOGs, Pfam and GO) for 88,168 unigenes was

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performed. 2619 genes were identified as differently up-regulated and 2541 genes as

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differently

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differentially expressed genes (DEGs) were “ATP binding”, “mitochondrion and

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extracellular region”, “oxygen transporter activity”, “oxidoreductase activity”,

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“alanine, aspartate and glutamate metabolism”, “carbohydrate metabolic process”,

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“starch and sucrose metabolism” and “fatty acid biosynthesis”. These results

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confirmed a parasite external energy supply and an integrated metabolic stress. In

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addition, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs)

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Prominent

functional

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enriched

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down-regulated.

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ACCEPTED MANUSCRIPT were also identified from the gene library. Taken together, these findings allow us to

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better understand the underlying mechanisms regulating interactions between H.

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eriocheir and the crab E. sinensis.

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Keywords: Hepatospora eriocheir, Eriocheir sinensis, metabolism, energy,

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interaction

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1. Introduction

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Microsporidia are small obligate intracellular parasites originally considered to be

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primitive eukaryotic protozoa, but are recently reclassified with the fungi [1]. Almost

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half of the known microsporidian genera infect aquatic hosts, however,

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microsporidians are probably ubiquitous and vastly under-reported in aquatic systems

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[2]. Recently, serious microsporidian outbreaks caused catastrophic losses in the

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Chinese mitten crab Eriocheir sinensis harvest in Jiangsu Province, China. A

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conspicuous sign was the color of hepatopancreas turning from yellow-golden to

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almost white, representing a significant tissue-specific infection characteristic. It has

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been confirmed that the microsporidium Hepatospora eriocheir was closely

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associated with the disease [3].

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Hepatospora, a recently erected genus, infects epithelial cells of the

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hepatopancreas of wild and farmed decapod crustaceans [4]. Hepatospora spp. was

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isolated from different crustacean hosts, inhabiting different habitats and niches:

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freshwater Chinese mitten crab (Eriocheir sinensis), the marine mussel symbiont pea

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crab (Pinnotheres pisum) and marine edible crab (Cancer pagurus) [5, 6]. In E. 3

ACCEPTED MANUSCRIPT sinensis, the microsporidian infection was first identified during the research of

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tremor disease (TD), and confirmed to be opportunistic in nature [7]. Following this

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initial discovery, (Stentiford, et al., 2011) named the microsporidium as Hepatospora

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eriocheir [4]. Early studies of this microsporidian pathogen intensively focused on the

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pathogen aspects including the histopathology and molecular phylogenetic analysis,

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development of novel detection assays, and comparative genome research [4-6, 8],

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limited information was available about the host responses, which could provide

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important information to elucidate the diverse aspects of the host–pathogen

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interactions.

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Microsporidia, as obligate intracellular parasites, are characterized by

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development in direct contact with host cytoplasm (the majority of species), strong

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minimization of cell machinery, and acquisition of unique transporters to exploit host

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metabolic system [5]. All the aforementioned features are suggestive of the ability of

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microsporidia to modify host metabolic and regulatory pathways. However, questions

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about how H. eriocheir parasitization posed the metabolic stresses in the crab E.

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sinensis still remain.

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In this study, histology examination suggested cell hypertrophy and host

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mitochondria clustering were the common features of H. eriocheir infection in the

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hepatopancreas of E. sinensis. To better understand the underlying molecular

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mechanisms of H. eriocheir interaction with hepatopancreas cell, de novo

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transcriptome sequencing was furtherly proceeded, and a global survey of

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differentially expressed genes (DEGs), annotations of signaling pathways especially 4

ACCEPTED MANUSCRIPT the metabolism pathways, were also performed. In addition, putative simple sequence

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repeats (SSRs) and single nucleotide polymorphisms (SNPs) were discussed. These

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results provided the first experimental access to tissue-specific genes involved in the

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crab anti-microsporidian response and could serve as the basis for additional in-depth

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host-parasite interaction analyses (and potential intervention strategies) for

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economically important microsporidians.

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2. Materials and methods

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2.1 Sample collection

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Ten moribund crabs (mean body weight 86.57±5.38 g; 6 males and 4 females)

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exhibiting microsporidian infection (the color of hepatopancreas turning from

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yellow-golden to almost white) (Fig 1) were collected from Nanjing city in Jiangsu

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province, China. The hepatopancreas tissue was then collected and frozen

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immediately in liquid nitrogen for total RNA extraction, fixed in 4%

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paraformaldehyde solution (pH=7.3) for histological and in situ hybridization

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examinations, and in 4% glutaraldehyde in 0.3 M sodium phosphate buffer (pH=7.3)

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for electron microscopy. Additional 10 crabs without symptoms were examined

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initially by the detection of common pathogens in E. sinensis including WSSV [9], H.

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eriocheir [8] and spiroplasma [10]. Crabs negative for these pathogens were chosen

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for the control group.

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2.2 Histopathology, in situ hybridization and electron microscopy observations

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For histology, hepatopancreas was fixed in 4% paraformaldehyde solution for 24 5

ACCEPTED MANUSCRIPT h and then transferred to 70% ethanol. After dehydration in a graded ethanol series,

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tissues were embedded in paraffin. Six 10-µm thick sections were obtained from each

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piece in different orientations. Sections were then stained with haematoxylin and

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eosin (H&E) and examined using a light microscope (Olympus, BX53).

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In situ hybridization was carried out following the protocol of (Ding et al., 2017)

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[8]. Overnight hybridization with the DIG-labeled probe was done in a humid

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chamber at 42 °C. The slides were counterstained with 3, 3’ N-Diaminobenzidine

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Tertrahydrochloride (DAB) and examined using the light microscope (Olympus,

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BX53).

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For transmission electron microscopy (TEM), the 2.5% glutaraldehyde (fixative

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solution) was removed and the hepatopancreas tissues were post-fixed in 1% (w/v)

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osmium tetroxide in 0.1 M phosphate buffer at pH 7.4 for 2 h. After being dehydrated

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through a graded series of acetone, the tissues were embedded in Epon 812. Ultrathin

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sections were double stained with uranyl acetate and lead citrate and observed with

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the Hitachi 600-2A transmission electron microscopy.

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2.3 RNA isolation and Illumina sequencing

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Total RNA was extracted using a high-purity total RNA Rapid Extraction Kit

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(BioTeke, China) according to the manufacturer's instructions. Total RNA quality was

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checked on 1% formaldehyde agarose gel via electrophoresis, and RNA concentration

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was determined through Nano Drop (Thermo Scientific, USA). Then approximately

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1 µg of DNase-treated total RNA was used to construct a cDNA library following the

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protocols of the TruSeq Stranded mRNA Sample Preparation. After necessary 6

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quantification and qualification, the library was sequenced using an Illumina HiSeq™

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2000 instrument.

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2.4 De novo assembly and data analysis SeqPrep

(http://github.com/jstjohn/SeqPrep)

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(https://github.com/najoshi/sickle) were applied to process the raw reads with default

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parameters and eliminated sequences smaller than 60 bases. Then, Trinity program

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[11] was used to do RNA assembly of clean reads. The assembled contigs were

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annotated with sequences available in the NCBI database using the BLAST

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algorithms. The unigenes were aligned by a BLASTx search against the protein

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databases of NCBI, including Swiss-Prot, non-redundant (Nr), KEGG (Kyoto

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encyclopedia of genes and genomes), eukaryote orthologous genes (KOGs), Gene

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Ontology (GO) and Pfam. Based on the highest sequence similarity and a typical

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cut-off E-value less than 1.0 e−5, the function annotations were retrieved [12]. Next,

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the best alignment results were used to determine the sequence direction and

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protein-coding-region prediction of the unigenes. The Blast2GO suite [13] was used

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to obtain GO annotations of the uniquely assembled transcripts. Based on the KEGG

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database, the complex biological behavior of the genes was analyzed through pathway

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annotation [14].

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Microsatellite search module (MISA http://pgrc.ipk-gatersleben.de/misa/) was

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used to find simple sequence repeats (SSRs) in unigenes, then design primer for each

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SSR with Primer3 [15]. All clean reads were mapped to unigenes using HISAT

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(hierarchical indexing for spliced alignment of transcripts), then call single nucleotide 7

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polymorphisms (SNPs) with Genome Analysis Toolkit (GATK). After filter out the

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unreliable sites, the final SNP was gotten in VCF format.

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2.5 Identification and analysis of differentially expressed genes (DEGs) FRKM (fragments per kilobase of transcripts per million fragments mapped) was

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used as the unit of measurement to estimate the expression level of each transcript.

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False discovery rate (FDR) was carried out to correct for E-value. Genes with

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FDR≤0.001 and an FPKM ratio larger than 2 or smaller than 0.5 were considered as

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DEGs between samples. With the DEGs, GO and KEGG pathway classifications and

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functional enrichments were also performed, as mentioned in section 2.4.

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3. Results

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3.1 Histopathology, in situ hybridization and electron microscopy observations

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Infections by H. eriocheir in the crabs were confirmed by histology with H.E

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staining, in situ hybridization and transmission electron microscopy (TEM). No

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pathological changes could be observed in the control crabs (Fig 1A, B), whereas in

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the infected crabs, the color of hepatopancreas turned from yellow-golden to almost

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white (Fig 1C). Numbers of hypertrophic epithelial cells of the hepatopancreas were

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observed (Fig 1D). Positive hybridization signals, visualized as an intense brown to

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black precipitate were also observed within the epithelial cells of the hepatopancreas

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(Fig 1E). Importantly, host mitochondria were observed closely around the

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plasmalemma of meronts of H. eriocheir, most likely to aid the uptake of ATP directly

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from the host cell (Fig 2).

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3.2 Transcriptome sequencing and de novo assembly After removal of low-quality sequences, a total of 47.84 M clean reads that

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represent a total of 6.90 Gb clean bases were generated for the H. eriocheir infected

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group. For the control group, 57.21 M clean reads that represent a total of 8.27 Gb

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clean bases were generated. 104,998 transcripts (mean length = 536 bp) from

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hepatopancreas tissues were analyzed by de novo assembly. Further search against the

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GenBank protein and nucleotide sequences yielded 88,168 unigenes with a mean

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length of 481 bp (Table 1).

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3.3 Functional annotation and classification of transcriptome sequences

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To achieve protein identification and gene annotation, a search was made on

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standard unigenes in the NCBI Nr (20,177 unigenes, 22.88%), Swiss-Prot (11,637

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unigenes, 13.20%), KEGG (9,294 unigenes, 10.54%), KOGs (11,871 unigenes,

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13.46%), Pfam (12,062 unigenes, 13.68%) and GO (10,619 unigenes, 12.04%) using

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the BLAST program (E-value <1.0 e−5).

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The standard unigenes were then aligned to the KOG database to predict and

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classify their possible roles. A total of 12,236 unigenes had KOG classifications,

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distributed among the 25 KOG categories, including “energy production and

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conversion ", “amino acid transport and metabolism”, “nucleotide transport and

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metabolism”, “carbohydrate transport and metabolism”, “lipid transport and

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metabolism”, “cell wall/membrane/envelope biogenesis”, “secondary metabolites

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biosynthesis, transport and catabolism” and “signal transduction mechanisms ”, most

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of which played key roles in metabolism-related pathways (Fig.3).

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ACCEPTED MANUSCRIPT We obtained a total of 3,528 GO assignments, where 37.39% comprised

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biological processes, 24.46% comprised cellular component, and 38.15% comprised

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molecular function. In the category of biological processes, most unigenes were

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involved in the “translation”, “proteolysis”, and “carbohydrate metabolic process”. In

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cellular component category, the most represented were “cytoplasm”, “integral to

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membrane”, “ribosome”, “extracellular space”, “lysosome”, and “mitochondrion”.

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With respect to molecular function category, “ATP binding”, “metal ion, protein, and

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DNA binding”, “structural constituent of ribosome”, “oxidoreductase activity”, and

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“oxygen transporter activity”, were the dominant groups (Fig.4).

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In addition, we also used KEGG analysis to identify potential biological

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pathways represented in the transcriptome of the crab hepatopancreas. KEGG analysis

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indicated that a greater number of genes expressed in the hepatopancreas were

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associated with organismal systems, metabolism, environmental information

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processing, genetic information processing, human diseases, and cellular processes.

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Metabolism was the dominant group (Fig.5A). Carbohydrate metabolism (16.53%),

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nucleotide metabolism (14.64%), amino acid metabolism (12.65%), lipid metabolism

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(12.46%) and energy metabolism (9.85%) were the top five metabolism pathways,

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followed by metabolism of cofactors and

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biodegradation and metabolism (7.77%), glycan biosynthesis and metabolism (6.92%),

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metabolism of other amino acids (4.31%), biosynthesis of other secondary metabolites

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(2.90%), and metabolism of terpenoids and polyketides (2.66%) (Fig.5B).

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3.4 SSRs/SNP markers identification

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vitamins (9.29%), xenobiotics

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transcriptional regulation and maintenance of complex nuclear properties [16]. Next,

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potential SSRs in all the unigenes were mined using MISA software. As a result,

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37,851 SSRs were identified from the hepatopancreas gene library. Among them,

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dinucleotide, trinucleotide and mononucleotide, repeats were the three largest SSRs,

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accounting for 56.87%, 33.70%, and 5.43% of all SSRs, respectively (Fig. 6A).

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The transcriptomes of hepatopancreas from H. eriocheir infected or control crabs

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shared similarities within the detected SNPs, transitions were much more common

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than transversions. A similar number of A/G and C/T transitions and percentages of

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the four transversion types (A/C, A/T, C/G, G/T) were found (Fig 6B).

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3.5 Identification of aberrantly expressed genes

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Previous sequence analysis and annotation for all of the unigenes in the merged

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groups provided some valuable information to analyze the hepatopancreas

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transcriptome. However, the variation in the gene expression level after H. eriocheir

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parasitization was expected. Here, FDR≤ 0.001 and an absolute value of log2 Ratio≥

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1 were used as the filtering thresholds to identify up or down-regulated genes. After H.

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eriocheir infection, 2619 genes were identified as differently up-regulated and 2541

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genes as differently down-regulated. The up-regulated DEGs were greater than

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down-regulated DEGs, indicating that the H. eriocheir infection process was

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associated with transcript accumulation.

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Based on GO analysis of DEGs, their functions could be classified into 3

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categories. In the category of biological processes, most unigenes were involved in 11

ACCEPTED MANUSCRIPT the “translation”, “proteolysis” and “carbohydrate metabolic process”. In cellular

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component category, the most represented included “cytoplasm”, “integral to

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membrane”, “extracellular space”, “lysosome”, “mitochondrion and extracellular

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region”. With respect to molecular function category, “ATP binding”, “metal ion,

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protein, and DNA binding”, “oxidoreductase activity”, “oxygen transporter activity”

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were the dominant groups (Fig.7). Statistics of GO Enrichment also indicated that

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“translation”, “structural constituent of ribosome”, “ribosome”, “proteolysis”,

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“oxygen transporter activity”, “oxidoreductase activity”, “extracellular space” and

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“carbohydrate metabolic process” were the most dominant (Fig.8). These data suggest

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that H. eriocheir infection has an influence on a wide range of gene functions in the

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crab.

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Significantly, DEGs were consistently assigned to comprehensive host defense

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signaling pathways related to various metabolic and energetic stresses, such as “starch

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and sucrose metabolism”, “ribosome”, “phagosome”, “oxidative phosphorylation”,

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“lysosome”, “fatty acid biosynthesis”, and “alanine, aspartate and glutamate

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metabolism” (Fig. 9), suggesting that crabs use a range of tactics to cope with the

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stress of H. eriocheir infection. A detailed explanation was presented in the

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discussion.

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Genes potentially involved in host metabolism or energy synthesis were also

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selected using standard criteria for pathway prediction with a P<0.05 and induced

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ratios >2 or <0.5. The detailed information for the involved genes was listed in Table

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4. Discussion Microsporidia can infect a broad range of eukaryotic hosts, and several species

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can infect crustaceans. For example, Enterocytozoon hepatopenaei is a recently

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emerged pathogen of the farmed shrimp species Penaeus monodon and Penaeus

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vannamei and severely retards the growth of the shrimp and has rapidly spread across

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south-east Asia [17, 18]. To date, H. eriocheir was the only microsporidian parasite

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infecting the crab E. sinensis. Despite microsporidia being ubiquitous and significant

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parasites, very little was known about the crustacean host’s response to

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microsporidian infection. Here, we provided the histopathology and transcriptome

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data for the economically important crab E. sinensis after the infection of major

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yield-limiting

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previously-published information on the growing body of genomic data for both the

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parasites and crustacean species [5], and the capacity to study microsporidian

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pathogen in more crustacean host [19] will undoubtedly underpin our understanding

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of host-microsporidia interactions, monitor and potentially mitigate the negative

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impacts of H. eriocheir and other microsporidian pathogen in aquatic systems.

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As intracellular parasites, microsporidia have diverse effects on their host cells.

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Metabolite import and parasite development are facilitated by manipulation of the

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host cell cytoskeleton. Hypertrophic enlargement of the host cell (Fig 1D, E),

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including sometimes the nucleus [20], was frequently observed in parasitized cells.

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The enlargement of the host cells post-invasion reflects the ability of microsporidia to 13

ACCEPTED MANUSCRIPT subvert the host-cell cytoskeleton and volume control in order to secure a sufficient

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spatial and protected niche. Hypertrophy of host cells is a common feature of

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microsporidian infections and is accompanied by several secondary effects: individual

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infected cells may dedifferentiate into a syncytium, undergo cytoplasm or nuclear

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hypertrophy and fragmentation, increase RNA synthesis, and sometimes produce an

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increased number of cell nuclei [21]. These were in accordance with our

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transcriptome data, in which the differently expressed genes after H. eriocheir

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infection were mainly enriched in the categories of “translation”, “structural

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constituent of ribosome”, “rRNA binding”, “ribosome”, “regulation of translational

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initiation”, and “extracellular space” (Fig 8).

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The intracellular life stages, as well as spores of microsporidia lack mitochondria

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and granules for nutrient storage. They are believed to utilize host-derived ATP for

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their energy needs. ATP is produced in the respiratory chain, which again could be

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harnessed by the microsporidium. In this study, intracellular stages of H. eriocheir

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were shown to occur frequently in close association with the crab hepatopancreas

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cells’ mitochondria, the site of respiratory chain (Fig 2). An enrichment of DEGs in

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“ATP binding”, “oxygen transporter activity” and “oxidoreductase activity” was also

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observed (Fig 7, 8). These results all suggested a parasite external energy supply and a

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strong enhancement of oxidative metabolism.

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Histological results suggested that hepatopancreas was the main infection targets

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of H. eriocheir and the microsporidian infection had immediate, deleterious

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consequences for hepatopancreas structure and function (Fig 1, 2). Transcriptome data 14

ACCEPTED MANUSCRIPT furtherly indicated that resulting tissue damage and subsequent siphoning of crab

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resources may significantly and negatively impact metabolic and nutritional pathways

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in the hepatopancreas cells. KEGG pathway analysis exhibited that H. eriocheir

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infection broadly altered expression of genes involved in “starch and sucrose

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metabolism”, “glycan degradation”, “fatty acid biosynthesis” and “amino acid

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metabolism”. Based on these findings, we suggested that H. eriocheir may “starve” its

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hosts through the destruction of hepatopancreas tissue and/or by appropriating host

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resources, resulting in associated changes in crab metabolism and immunity. This was

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also consistent with previous studies indicating that microsporidian infection was

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energetically costly [5].

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Recently, a modulation of the ayu Plecoglossus altivelis, phagocytic response to

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microsporidium Glugea plecoglossi was described [22]. An elevated phagocytic

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activity of Atlantic salmon could also contribute to the resistance to microsporidian

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gill disease [23]. In our study, after H. eriocheir infection in the crab E. sinensis, gene

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expressions of phagosome and lysosome pathway were also significantly changed,

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indicating the fusion of lysosomes with the phagosome was activated and

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phagocytosis played a key role in the crab innate immunity. More importantly,

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because phagocytosis is typically mediated by pathogen ligand binding to a

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phagocytic receptor, identifying and blocking the appropriate ligands may inhibit

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pathogen entry into the host cell and provide a means of controlling infection.

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DEGs were also enriched in the ribosome pathway (Fig 8, 9). Cells infected with

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some microsporidia displayed an increase in cell ribosomes and endoplasmic 15

ACCEPTED MANUSCRIPT reticulum suggesting an increase in host cell metabolism probably related to the

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requirements of the increasing number of parasites [24]. In addition, biochemically,

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microsporidia were thought to be anaerobic and to lack evidence of electron transport

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chain and oxidative phosphorylation [25]. Similarly, in this study, most DEGs were

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involved in metal ion binding and oxidative phosphorylation pathway.

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Although some genes involved in the crab metabolism were induced in response

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to H. eriocheir infection, further investigations were needed to verify the results of

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this analysis and the expression patterns of other genes, which were not identified as

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differentially expressed genes in this study.

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5. Conclusion

In summary, histology analysis indicated hypertrophic enlargement of the

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hepatopancreas epithelial cells and host mitochondria clustering were the common

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features of H. eriocheir infection. Furtherly, de novo transcriptome sequencing of the

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hepatopancreas tissue was carried out. A global survey of the DEGs involved in

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metabolism pathways was performed to better understand the influences of H.

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eriocheir infection on the crab. The functions of many differentially expressed genes

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involved in host metabolism and defense were discussed. Furthermore, putative SSRs

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and SNPs were also analyzed. Altogether, these and other recent findings could

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provide much-needed insight into the underlying mechanisms of microsporidia-host

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interactions.

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Acknowledgments We thank the anonymous reviewers for valuable comments and suggestions. This

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work was supported by grants from the Natural Sciences Foundation of China

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(31402332), Natural Sciences Foundation of Jiangsu Province (BK20171406),

357

Natural Science Fund of Colleges and Universities in Jiangsu Province

358

(16KJA180005, 16KJB180006) and project for aquaculture of Jiangsu Province

359

(Y2016-29) and Changzhou City (CT201612).

SC

M AN U

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References

362

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[16] S. Subramanian, R. Mishra, L. Singh, Genome-wide analysis of microsatellite

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[17] K.F.J. Tang, C.R. Pantoja, R.M. Redman, J.E. Han, L.H. Tran, D.V. Lightner,

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Development of in situ hybridization and PCR assays for the detection of

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shrimp, J. Invertebr. Pathol. 130 (2015) 37-41.

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[18] K.V. Rajendran, S. Shivam, P. Ezhil Praveena, J. Joseph Sahaya Rajan, T. Sathish

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Krishnan, S.V. Alavandi, K.K. Vijayan, Emergence of Enterocytozoon hepatopenaei

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(EHP) in farmed Penaeus (Litopenaeus) vannamei in India, Aquaculture 454 (2016)

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272-280.

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[19] P. Salachan, P. Jaroenlak, S. Thitamadee, O. Itsathitphaisarn, K. Sritunyalucksana,

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microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP), BMC Vet.

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Res. 13(1) (2017) 9.

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[20] G. Stentiford, K. Bateman, M. Longshaw, S. Feist, Enterospora canceri n. gen., n.

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sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer

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pagurus, Dis. Aquat. Org. 75(1) (2007) 61-72.

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[21] J. Lom, I. Dyková, Microsporidian xenomas in fish seen in wider perspective,

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Folia Parasitol. 52(1-2) (2005) 69-81.

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[22] L. Rodriguez-Tovar, D. Speare, R. Markham, Fish microsporidia: immune

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response, immunomodulation and vaccination, Fish Shellfish Immunol. 30(4-5) (2011)

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[23] J. Becker, D. Speare, Transmission of the microsporidian gill parasite, Loma

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salmonae, Anim Health Res Rev 8(1) (2007) 59-68.

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[24] C. del Aguila, F. Izquierdo, A. Granja, C. Hurtado, S. Fenoy, M. Fresno, Y.

challenge

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hepatopancreatic

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cohabitation

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ACCEPTED MANUSCRIPT Revilla, Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected

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cells, Int. J. Parasitol. 36(8) (2006) 869-876.

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[25] N.M. Fast, P.J. Keeling, Alpha and beta subunits of pyruvate dehydrogenase E1

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from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism

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in microsporidia, Mol. Biochem. Parasi. 117(2) (2001) 201-209.

446

Table and figure legends

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M AN U

448 449

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Table 1 General information of the transcriptome analysis

450

Table 2 Selected hepatopancreas-specific DEGs (differentially expressed genes)

452

potentially involved in Eriocheir sinensis metabolic response against Hepatospora

453

eriocheir infection.

454

TE D

451

Figure 1 Gross signs and histopathology in the hepatopancreas of Hepatospora

456

eriocheir infected crab Eriocheir sinensis. A, Healthy crabs with golden yellow

457

hepatopancreas; B, no pathological changes could be observed in the healthy crabs; C,

458

hepatopancreas color of the infected crabs turning to almost white; D, numbers of

459

hypertrophic epithelial cells were observed (arrows); E, positive hybridization signals

460

visualized as an intense brown to black precipitate within the epithelial cells of the

461

hepatopancreas. Sale bar B, E=50 µm, D=10 µm.

AC C

EP

455

462 21

ACCEPTED MANUSCRIPT Figure 2 Transmission electron microscopy (TEM) analysis of Hepatospora eriocheir

464

infected crab Eriocheir sinensis. Host mitochondria (M) were observed closely

465

around the plasmalemma of meronts of H. eriocheir. Clear interfacial envelopes

466

surrounded each plasmodium and contained amorphous material in the episporontal

467

space (arrows), organelle precursors (star) and nucleus (N) of a hepatopancreas cell.

468

Scale bar=1 µm.

RI PT

463

SC

469

Figure 3 Eukaryotic cluster of orthologous groups (KOG) function classification of

471

the hepatopancreas transcriptome. A total of 12,236 unigenes had KOG classifications,

472

distributed among the 25 KOG categories, most of which played key roles in

473

metabolism related pathways.

TE D

474

M AN U

470

Figure 4 Gene ontology (GO) classification of the hepatopancreas transcripts in the

476

crab Eriocheir sinensis. The left y-axis indicates the percentage of a specific category

477

of transcripts existed in the main category.

AC C

478

EP

475

479

Figure 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification

480

of the hepatopancreas transcripts in crab Eriocheir sinensis. A, a greater number of

481

genes expressed in the hepatopancreas were associated with metabolism (grey

482

shadow); B, percentages of each metabolism related pathways.

483 484

Figure 6 A summary of the simple sequence repeats (SSRs) and single nucleotide 22

ACCEPTED MANUSCRIPT 485

polymorphisms (SNP) markers identified from the Eriocheir sinensis hepatopancreas

486

transcriptome. (A) distribution of SSRs based on different motif types; (B) SNP

487

variant type distribution of Hepatospora eriocheir infected and control crabs.

RI PT

488 489

Figure 7 Gene ontology (GO) classification of differentially expressed genes (DEGs)

490

after Hepatospora eriocheir infection.

SC

491

Figure 8 Scatter diagram of Gene ontology (GO) enrichment for differentially

493

expressed genes (DEGs) following Hepatospora eriocheir infection. In this scatter

494

diagram, the top 20 categories were listed. The magnitude of the pots displays gene

495

number ranged from 10 to 50, and p-value was described by the color classification.

M AN U

492

TE D

496

Figure 9 Scatter diagram of pathway enrichment for differentially expressed genes

498

(DEGs) following Hepatospora eriocheir infection. In this scatter diagram, the top 20

499

categories were listed, and rich factor was the ratio of DEGs in this pathway to all the

500

genes in this pathway. The X-axis corresponded to rich factor of pathway, and the

501

Y-axis represented different pathway. The magnitude of the pots displays gene

502

number ranged from 10 to 50, and p-value was described by the color classification.

AC C

503

EP

497

504 505

23

ACCEPTED MANUSCRIPT

Table 1 General information of the transcriptome analysis Median

Mean

All name

Mean

Total Assembled

Length

bases

Median Length GC%

GC%

88168

46.10

47.30

307

transcript

104998

46.30

47.47

320

481

4E+07

536

6E+07

AC C

EP

TE D

M AN U

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gene

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Dataset

ACCEPTED MANUSCRIPT Table 2 Selected hepatopancreas-specific DEGs (differentially expressed genes) potentially involved in Eriocheir sinensis metabolic response against Hepatospora eriocheir infection. Gene name

Annotation

Pathway name

TRINITY_DN22029_c0_g3

Ca7

carbonic anhydrase

Nitrogen metabolism

down

TRINITY_DN29261_c1_g1

Gusb

beta-glucuronidase

Starch and sucrose metabolism

up

TRINITY_DN27867_c0_g6

Mal-B1

alpha-glucosidase

Starch and sucrose metabolism

up

TRINITY_DN29047_c0_g10

Amy1

alpha-amylase

Starch and sucrose metabolism

down

TRINITY_DN26546_c0_g3

Amy2

alpha-amylase

Starch and sucrose metabolism

down

TRINITY_DN29118_c0_g12

SI

alpha-glucosidase

Starch and sucrose metabolism

up

TRINITY_DN29416_c2_g29

Ugt2b20

glucuronosyltransfer

Starch and sucrose metabolism

down

Oxidative phosphorylation

up

Oxidative phosphorylation

down

Oxidative phosphorylation

up

TRINITY_DN27100_c1_g6

Vha36-1

SC

M AN U

TE D

ase

RI PT

Gene_ID

V-type

Regulation

Uqcrq

AC C

TRINITY_DN16277_c0_g2

EP

H+-transporting

TRINITY_DN10081_c0_g2

Ndufb8

ATPase subunit D ubiquinol-cytochrom e c reductase subunit 8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8

ACCEPTED MANUSCRIPT TRINITY_DN9074_c0_g1

CG4692

F-type

Oxidative phosphorylation

down

Oxidative phosphorylation

down

H+-transporting

sdha-a

succinate

subunit

dehydrogenase (ubiquinone) flavoprotein

TRINITY_DN22220_c0_g2

ADSL

GLUL

adenylosuccinate

Alanine, aspartate and

M AN U

TRINITY_DN28824_c0_g4

SC

TRINITY_DN29225_c0_g6

RI PT

ATPase subunit f

lyase

glutamate metabolism

glutamine synthetase

Alanine, aspartate and

up

up

glutamate metabolism

glmS

glucosamine--fructos Alanine, aspartate and

TE D

TRINITY_DN29232_c0_g1

e-6-phosphate

up

glutamate metabolism

alas2

AC C

TRINITY_DN27958_c0_g1

EP

aminotransferase

TRINITY_DN29139_c0_g1

TRINITY_DN27350_c0_g1

TRINITY_DN28416_c0_g8

Dmgdh

Sardh

GBAc

(isomerizing) 5-aminolevulinate

Glycine, serine and threonine

synthase

metabolism

dimethylglycine

Glycine, serine and threonine

dehydrogenase

metabolism

sarcosine

Glycine, serine and threonine

dehydrogenase

metabolism

glucosylceramidase

Other glycan degradation

up

up

up

down

ACCEPTED MANUSCRIPT TRINITY_DN27431_c0_g1

lcc2

ferritin heavy chain

Porphyrin and chlorophyll

up

metabolism

TRINITY_DN29416_c2_g29

TRINITY_DN15841_c0_g1

UGT2B20

A2m

protoporphyrinogen

Porphyrin and chlorophyll

oxidase

metabolism

glucuronosyltransfer

Porphyrin and chlorophyll

ase

metabolism

alpha-2-macroglobul

Complement and coagulation

TRINITY_DN26635_c0_g2

Serpinb1a

down

down

up

cascades

M AN U

in

RI PT

PPOX

SC

TRINITY_DN3141_c0_g2

antithrombin III

Complement and coagulation

down

cascades

TRINITY_DN16277_c0_g2

Uqcrq

ubiquinol-cytochrom

Cardiac muscle contraction

down

hydroxyproline

Arginine and proline

up

oxidase

metabolism

hydroxyproline

Arginine and proline

oxidase

metabolism

glutamate 5-kinase

Arginine and proline

TE D

e c reductase subunit 8

prodh2

AC C

TRINITY_DN27120_c1_g2

prodh2

EP

TRINITY_DN27120_c1_g5

TRINITY_DN26297_c0_g1

TRINITY_DN16296_c0_g1

alh-13

Fasn

down

up

metabolism fatty acid synthase,

Fatty acid biosynthesis

up

Reductive carboxylate cycle

down

animal type TRINITY_DN29354_c0_g15

ACO1

aconitate hydratase 1

ACCEPTED MANUSCRIPT (CO2) fixation TRINITY_DN26254_c0_g2

sptl-1

serine

Sphingolipid metabolism

up

Glyoxylate and dicarboxylate

down

palmitoyltransferase ACO2

aconitate hydratase 1

RI PT

TRINITY_DN26595_c0_g11

AC C

EP

TE D

M AN U

SC

metabolism

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 1 Gross signs and histopathology in the hepatopancreas of Hepatospora eriocheir infected crab Eriocheir sinensis. A, Healthy crabs with golden yellow

TE D

hepatopancreas; B, no pathological changes could be observed in the healthy crabs; C, hepatopancreas color of the infected crabs turning to almost white; D, numbers of

EP

hypertrophic epithelial cells were observed (arrows); E, positive hybridization signals visualized as an intense brown to black precipitate within the epithelial cells of the

AC C

hepatopancreas. Sale bar B, E=50 µm, D=10 µm.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 2 Transmission electron microscopy (TEM) analysis of Hepatospora eriocheir

TE D

infected crab Eriocheir sinensis. Host mitochondria (M) were observed closely around the plasmalemma of meronts of H. eriocheir. Clear interfacial envelopes surrounded each plasmodium and contained amorphous material in the episporontal

EP

space (arrows), organelle precursors (star) and nucleus (N) of a hepatopancreas cell.

AC C

Scale bar=1 µm.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 3 Eukaryotic cluster of orthologous groups (KOG) function classification of the hepatopancreas transcriptome. A total of 12,236 unigenes had KOG classifications,

TE D

distributed among the 25 KOG categories, most of which played key roles in

AC C

EP

metabolism related pathways.

ACCEPTED MANUSCRIPT

SC

RI PT

80

100

60

80

100

M AN U

60

40

cellular component (24.46%)

40

20

TE D

biological process (37.39%)

20

str uc tur

al co m AT nst eta P itu l i bin en on di t b n ox ido proof ri indi g red tei bos ng u n om ox zin ctase bind e yg c i ac ing en o tra tra DN n bi tivity ns ns A nd lat po b in ion rt in g ini nuc RNer ac ding tia le A tiv tio oti bi ity n f de nd ac bin ing tor d ac ing tiv ity

molecular function (38.15%)

EP

60

50

40

30

20

10

to cyto me pla mb sm nu rane rib cle ex tra o u ce c soms llu yt e la o pla sm ly r sp sol a m sos ace m e o G ito m me en extr olg chonbran do ac i a dr e pla ellu pp io sm la ara n ic r re tus ret gi icu on lum

0

int eg ral

0

AC C

Number of Unigenes 0

Figure 4 Gene ontology (GO) classification of the hepatopancreas transcripts in the

crab Eriocheir sinensis. The left y-axis indicates the percentage of a specific category

of transcripts existed in the main category.

ca rbo tra h ns tra ydra la te ns me pro tion cri pti tab teo on l , D olic ysis NA pro mu ce ltic -d s ell pro epen s ula de tei ro rga pro n fo nt nis tein ldin ma g t l d rans po ev elo rt pm en t

B Glycan Biosynthesis and Metabolism

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

7%

16%

Metabolism of Other Amino Acids

4% 8%

Xenobiotics Biodegradation and Metabolism Metabolism of Terpenoids and Polyketides

3%

12%

TE D

Amino Acid Metabolism

Biosynthesis of Other Secondary Metabolites Nucleotide Metabolism

EP

Energy Metabolism

13% 9%

Metabolism of Cofactors and Vitamins

3%

Lipid Metabolism

AC C

10%

Carbohydrate Metabolism

15%

Figure 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification of the hepatopancreas transcripts in crab Eriocheir sinensis. A, a greater number of genes expressed in the hepatopancreas were associated with metabolism (grey shadow); B, percentages of each metabolism related pathways.

ACCEPTED MANUSCRIPT 25000

25000

B

A

10000

5000

0

0

M AN U

Tr an sit io

5000

er Di s m er Tr s im Qu er ad s Pe mer s nt am He ers xa m er s

RI PT

10000

15000

SC

15000

n ATr G an sv C-T er sio n AC AT CG GTo T ta l

Number of SNPs

20000

M on om

Figure 6 A summary of the simple sequence repeats (SSRs) and single nucleotide

TE D

polymorphisms (SNP) markers identified from the Eriocheir sinensis hepatopancreas transcriptome. (A) distribution of SSRs based on different motif types; (B) SNP

EP

variant type distribution of Hepatospora eriocheir infected and control crabs.

AC C

Number of SSRs

20000

Infected Control

ACCEPTED MANUSCRIPT

SC

RI PT

biological process

80

100

60

80

100

M AN U

60

40

TE D

40

20

cellular component

20

EP

60

50

40

30

20

10

to cyto me pla mb sm nu rane rib cle ex tra o u ce c soms llu yt e la o pla sm ly r sp sol a m sos ace m e o G ito m me en extr olg chonbran do ac i a dr e pla ellu pp io sm la ara n ic r re tus ret gi icu on lum

0

int eg ral

0

str uc tur

al co m AT nst eta P itu l i bin en on di t b n ox ido proof ri indi g red tei bos ng u n om ox zin ctase bind e yg c i ac ing en o tra tra DN n bi tivity ns ns A nd lat po b in ion rt in g ini nuc RNer ac ding tia le A tiv tio oti bi ity n f de nd ac bin ing tor d ac ing tiv ity

molecular function

AC C

Number of Unigenes 0

Figure 7 Gene ontology (GO) classification of differentially expressed genes (DEGs)

after Hepatospora eriocheir infection.

ca rbo tra h ns tra ydra la te ns me pro tion cri pti tab teo on l , D olic ysis NA pro mu ce ltic -d s ell pro epen s ula de tei ro rga pro n fo nt nis tein ldin ma g t l d rans po ev elo rt pm en t

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 8 Scatter diagram of Gene ontology (GO) enrichment for differentially

TE D

expressed genes (DEGs) following Hepatospora eriocheir infection. In this scatter diagram, the top 20 categories were listed. The magnitude of the pots displays gene

AC C

EP

number ranged from 10 to 50, and p-value was described by the color classification.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 9 Scatter diagram of pathway enrichment for differentially expressed genes

TE D

(DEGs) following Hepatospora eriocheir infection. In this scatter diagram, the top 20 categories were listed, and rich factor was the ratio of DEGs in this pathway to all the

EP

genes in this pathway. The X-axis corresponded to rich factor of pathway, and the Y-axis represented different pathway. The magnitude of the pots displays gene

AC C

number ranged from 10 to 50, and p-value was described by the color classification.

ACCEPTED MANUSCRIPT Research Highlights 1. H. eriocheir infection increased the crab energy requirements. 2. H. eriocheir infection activated an integrated metabolic stress response.

RI PT

3. The integrated metabolic consequence may contribute to the decreased survival.

AC C

EP

TE D

M AN U

SC

4. Results could serve as the basis for in-depth host-parasite interaction analyses.