- Email: [email protected]
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 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.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT An integrated metabolic consequence of Hepatospora eriocheir infection in the
2
Chinese mitten crab Eriocheir sinensis
3
Zhengfeng Ding1*, Jing Pan1, Hua Huang2, Gongcheng Jiang1, Jianqin Chen1,
4
Xueshen Zhu1, Renlei Wang1, Guohua Xu1*
RI PT
1
5 6
1
7
Chemistry, Jiangsu Second Normal University, 77 West Beijing Road, Nanjing, China,
8
210013
9
2
SC
M AN U
10
Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and
Aquatic Technology Promotion Station, Wujin District, Changzhou City, China,
213017
11
TE D
12 13 14
EP
15
AC C
16 17
*Corresponding author at Jiangsu Second Normal University, College of life science
18
and
19
+86-25-83758339;
20
E-mail
21
[email protected] (G. Xu)
chemistry,
77
addresses:
West
Beijing
Road,
[email protected];
22 1
Nanjing,
China,
[email protected]
210013;
(Z.
Tel:
Ding);
ACCEPTED MANUSCRIPT 23 24
Abstract Despite the economic and evolutionary importance of aquatic host-infecting
26
microsporidian species, at present, limited information has been provided about the
27
microsporidia–host interactions. This study focused on Hepatospora eriocheir, an
28
emerging microsporidian pathogen for the Chinese mitten crab Eriocheir sinensis.
29
Hypertrophy of hepatopancreas cells was a common feature of H. eriocheir infection.
30
More importantly, mitochondria of the hepatopancreas were drawn around the H.
31
eriocheir, most likely to aid the uptake of ATP directly from the host. To better
32
understand the crab anti-microsporidian response, de novo transcriptome sequencing
33
of the hepatopancreas tissue was furtherly proceeded. A total of 47.84 M and 57.21 M
34
clean reads were generated from the hepatopancreas of H. eriocheir infected and
35
control groups respectively. Based on homology searches, functional annotation with
36
6 databases (Nr, Swiss-Prot, KEGG, KOGs, Pfam and GO) for 88,168 unigenes was
37
performed. 2619 genes were identified as differently up-regulated and 2541 genes as
38
differently
39
differentially expressed genes (DEGs) were “ATP binding”, “mitochondrion and
40
extracellular region”, “oxygen transporter activity”, “oxidoreductase activity”,
41
“alanine, aspartate and glutamate metabolism”, “carbohydrate metabolic process”,
42
“starch and sucrose metabolism” and “fatty acid biosynthesis”. These results
43
confirmed a parasite external energy supply and an integrated metabolic stress. In
44
addition, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs)
EP
TE D
M AN U
SC
RI PT
25
Prominent
functional
categories
enriched
with
AC C
down-regulated.
2
ACCEPTED MANUSCRIPT were also identified from the gene library. Taken together, these findings allow us to
46
better understand the underlying mechanisms regulating interactions between H.
47
eriocheir and the crab E. sinensis.
48
Keywords: Hepatospora eriocheir, Eriocheir sinensis, metabolism, energy,
49
interaction
RI PT
45
50
1. Introduction
SC
51
Microsporidia are small obligate intracellular parasites originally considered to be
53
primitive eukaryotic protozoa, but are recently reclassified with the fungi [1]. Almost
54
half of the known microsporidian genera infect aquatic hosts, however,
55
microsporidians are probably ubiquitous and vastly under-reported in aquatic systems
56
[2]. Recently, serious microsporidian outbreaks caused catastrophic losses in the
57
Chinese mitten crab Eriocheir sinensis harvest in Jiangsu Province, China. A
58
conspicuous sign was the color of hepatopancreas turning from yellow-golden to
59
almost white, representing a significant tissue-specific infection characteristic. It has
60
been confirmed that the microsporidium Hepatospora eriocheir was closely
61
associated with the disease [3].
TE D
EP
AC C
62
M AN U
52
Hepatospora, a recently erected genus, infects epithelial cells of the
63
hepatopancreas of wild and farmed decapod crustaceans [4]. Hepatospora spp. was
64
isolated from different crustacean hosts, inhabiting different habitats and niches:
65
freshwater Chinese mitten crab (Eriocheir sinensis), the marine mussel symbiont pea
66
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
68
tremor disease (TD), and confirmed to be opportunistic in nature [7]. Following this
69
initial discovery, (Stentiford, et al., 2011) named the microsporidium as Hepatospora
70
eriocheir [4]. Early studies of this microsporidian pathogen intensively focused on the
71
pathogen aspects including the histopathology and molecular phylogenetic analysis,
72
development of novel detection assays, and comparative genome research [4-6, 8],
73
limited information was available about the host responses, which could provide
74
important information to elucidate the diverse aspects of the host–pathogen
75
interactions.
M AN U
SC
RI PT
67
Microsporidia, as obligate intracellular parasites, are characterized by
77
development in direct contact with host cytoplasm (the majority of species), strong
78
minimization of cell machinery, and acquisition of unique transporters to exploit host
79
metabolic system [5]. All the aforementioned features are suggestive of the ability of
80
microsporidia to modify host metabolic and regulatory pathways. However, questions
81
about how H. eriocheir parasitization posed the metabolic stresses in the crab E.
82
sinensis still remain.
EP
AC C
83
TE D
76
In this study, histology examination suggested cell hypertrophy and host
84
mitochondria clustering were the common features of H. eriocheir infection in the
85
hepatopancreas of E. sinensis. To better understand the underlying molecular
86
mechanisms of H. eriocheir interaction with hepatopancreas cell, de novo
87
transcriptome sequencing was furtherly proceeded, and a global survey of
88
differentially expressed genes (DEGs), annotations of signaling pathways especially 4
ACCEPTED MANUSCRIPT the metabolism pathways, were also performed. In addition, putative simple sequence
90
repeats (SSRs) and single nucleotide polymorphisms (SNPs) were discussed. These
91
results provided the first experimental access to tissue-specific genes involved in the
92
crab anti-microsporidian response and could serve as the basis for additional in-depth
93
host-parasite interaction analyses (and potential intervention strategies) for
94
economically important microsporidians.
RI PT
89
2. Materials and methods
97
2.1 Sample collection
M AN U
96
SC
95
Ten moribund crabs (mean body weight 86.57±5.38 g; 6 males and 4 females)
99
exhibiting microsporidian infection (the color of hepatopancreas turning from
100
yellow-golden to almost white) (Fig 1) were collected from Nanjing city in Jiangsu
101
province, China. The hepatopancreas tissue was then collected and frozen
102
immediately in liquid nitrogen for total RNA extraction, fixed in 4%
103
paraformaldehyde solution (pH=7.3) for histological and in situ hybridization
104
examinations, and in 4% glutaraldehyde in 0.3 M sodium phosphate buffer (pH=7.3)
105
for electron microscopy. Additional 10 crabs without symptoms were examined
106
initially by the detection of common pathogens in E. sinensis including WSSV [9], H.
107
eriocheir [8] and spiroplasma [10]. Crabs negative for these pathogens were chosen
108
for the control group.
109
2.2 Histopathology, in situ hybridization and electron microscopy observations
110
AC C
EP
TE D
98
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,
112
tissues were embedded in paraffin. Six 10-µm thick sections were obtained from each
113
piece in different orientations. Sections were then stained with haematoxylin and
114
eosin (H&E) and examined using a light microscope (Olympus, BX53).
RI PT
111
In situ hybridization was carried out following the protocol of (Ding et al., 2017)
116
[8]. Overnight hybridization with the DIG-labeled probe was done in a humid
117
chamber at 42 °C. The slides were counterstained with 3, 3’ N-Diaminobenzidine
118
Tertrahydrochloride (DAB) and examined using the light microscope (Olympus,
119
BX53).
M AN U
SC
115
For transmission electron microscopy (TEM), the 2.5% glutaraldehyde (fixative
121
solution) was removed and the hepatopancreas tissues were post-fixed in 1% (w/v)
122
osmium tetroxide in 0.1 M phosphate buffer at pH 7.4 for 2 h. After being dehydrated
123
through a graded series of acetone, the tissues were embedded in Epon 812. Ultrathin
124
sections were double stained with uranyl acetate and lead citrate and observed with
125
the Hitachi 600-2A transmission electron microscopy.
126
2.3 RNA isolation and Illumina sequencing
EP
AC C
127
TE D
120
Total RNA was extracted using a high-purity total RNA Rapid Extraction Kit
128
(BioTeke, China) according to the manufacturer's instructions. Total RNA quality was
129
checked on 1% formaldehyde agarose gel via electrophoresis, and RNA concentration
130
was determined through Nano Drop (Thermo Scientific, USA). Then approximately
131
1 µg of DNase-treated total RNA was used to construct a cDNA library following the
132
protocols of the TruSeq Stranded mRNA Sample Preparation. After necessary 6
ACCEPTED MANUSCRIPT 133
quantification and qualification, the library was sequenced using an Illumina HiSeq™
134
2000 instrument.
135
2.4 De novo assembly and data analysis SeqPrep
(http://github.com/jstjohn/SeqPrep)
and
Sickle
RI PT
136
(https://github.com/najoshi/sickle) were applied to process the raw reads with default
138
parameters and eliminated sequences smaller than 60 bases. Then, Trinity program
139
[11] was used to do RNA assembly of clean reads. The assembled contigs were
140
annotated with sequences available in the NCBI database using the BLAST
141
algorithms. The unigenes were aligned by a BLASTx search against the protein
142
databases of NCBI, including Swiss-Prot, non-redundant (Nr), KEGG (Kyoto
143
encyclopedia of genes and genomes), eukaryote orthologous genes (KOGs), Gene
144
Ontology (GO) and Pfam. Based on the highest sequence similarity and a typical
145
cut-off E-value less than 1.0 e−5, the function annotations were retrieved [12]. Next,
146
the best alignment results were used to determine the sequence direction and
147
protein-coding-region prediction of the unigenes. The Blast2GO suite [13] was used
148
to obtain GO annotations of the uniquely assembled transcripts. Based on the KEGG
149
database, the complex biological behavior of the genes was analyzed through pathway
150
annotation [14].
M AN U
TE D
EP
AC C
151
SC
137
Microsatellite search module (MISA http://pgrc.ipk-gatersleben.de/misa/) was
152
used to find simple sequence repeats (SSRs) in unigenes, then design primer for each
153
SSR with Primer3 [15]. All clean reads were mapped to unigenes using HISAT
154
(hierarchical indexing for spliced alignment of transcripts), then call single nucleotide 7
ACCEPTED MANUSCRIPT 155
polymorphisms (SNPs) with Genome Analysis Toolkit (GATK). After filter out the
156
unreliable sites, the final SNP was gotten in VCF format.
157
2.5 Identification and analysis of differentially expressed genes (DEGs) FRKM (fragments per kilobase of transcripts per million fragments mapped) was
159
used as the unit of measurement to estimate the expression level of each transcript.
160
False discovery rate (FDR) was carried out to correct for E-value. Genes with
161
FDR≤0.001 and an FPKM ratio larger than 2 or smaller than 0.5 were considered as
162
DEGs between samples. With the DEGs, GO and KEGG pathway classifications and
163
functional enrichments were also performed, as mentioned in section 2.4.
M AN U
SC
RI PT
158
164
3. Results
166
3.1 Histopathology, in situ hybridization and electron microscopy observations
TE D
165
Infections by H. eriocheir in the crabs were confirmed by histology with H.E
168
staining, in situ hybridization and transmission electron microscopy (TEM). No
169
pathological changes could be observed in the control crabs (Fig 1A, B), whereas in
170
the infected crabs, the color of hepatopancreas turned from yellow-golden to almost
171
white (Fig 1C). Numbers of hypertrophic epithelial cells of the hepatopancreas were
172
observed (Fig 1D). Positive hybridization signals, visualized as an intense brown to
173
black precipitate were also observed within the epithelial cells of the hepatopancreas
174
(Fig 1E). Importantly, host mitochondria were observed closely around the
175
plasmalemma of meronts of H. eriocheir, most likely to aid the uptake of ATP directly
176
from the host cell (Fig 2).
AC C
EP
167
8
ACCEPTED MANUSCRIPT 177
3.2 Transcriptome sequencing and de novo assembly After removal of low-quality sequences, a total of 47.84 M clean reads that
179
represent a total of 6.90 Gb clean bases were generated for the H. eriocheir infected
180
group. For the control group, 57.21 M clean reads that represent a total of 8.27 Gb
181
clean bases were generated. 104,998 transcripts (mean length = 536 bp) from
182
hepatopancreas tissues were analyzed by de novo assembly. Further search against the
183
GenBank protein and nucleotide sequences yielded 88,168 unigenes with a mean
184
length of 481 bp (Table 1).
185
3.3 Functional annotation and classification of transcriptome sequences
M AN U
SC
RI PT
178
To achieve protein identification and gene annotation, a search was made on
187
standard unigenes in the NCBI Nr (20,177 unigenes, 22.88%), Swiss-Prot (11,637
188
unigenes, 13.20%), KEGG (9,294 unigenes, 10.54%), KOGs (11,871 unigenes,
189
13.46%), Pfam (12,062 unigenes, 13.68%) and GO (10,619 unigenes, 12.04%) using
190
the BLAST program (E-value <1.0 e−5).
TE D
186
The standard unigenes were then aligned to the KOG database to predict and
192
classify their possible roles. A total of 12,236 unigenes had KOG classifications,
193
distributed among the 25 KOG categories, including “energy production and
194
conversion ", “amino acid transport and metabolism”, “nucleotide transport and
195
metabolism”, “carbohydrate transport and metabolism”, “lipid transport and
196
metabolism”, “cell wall/membrane/envelope biogenesis”, “secondary metabolites
197
biosynthesis, transport and catabolism” and “signal transduction mechanisms ”, most
198
of which played key roles in metabolism-related pathways (Fig.3).
AC C
EP
191
9
ACCEPTED MANUSCRIPT We obtained a total of 3,528 GO assignments, where 37.39% comprised
200
biological processes, 24.46% comprised cellular component, and 38.15% comprised
201
molecular function. In the category of biological processes, most unigenes were
202
involved in the “translation”, “proteolysis”, and “carbohydrate metabolic process”. In
203
cellular component category, the most represented were “cytoplasm”, “integral to
204
membrane”, “ribosome”, “extracellular space”, “lysosome”, and “mitochondrion”.
205
With respect to molecular function category, “ATP binding”, “metal ion, protein, and
206
DNA binding”, “structural constituent of ribosome”, “oxidoreductase activity”, and
207
“oxygen transporter activity”, were the dominant groups (Fig.4).
M AN U
SC
RI PT
199
In addition, we also used KEGG analysis to identify potential biological
209
pathways represented in the transcriptome of the crab hepatopancreas. KEGG analysis
210
indicated that a greater number of genes expressed in the hepatopancreas were
211
associated with organismal systems, metabolism, environmental information
212
processing, genetic information processing, human diseases, and cellular processes.
213
Metabolism was the dominant group (Fig.5A). Carbohydrate metabolism (16.53%),
214
nucleotide metabolism (14.64%), amino acid metabolism (12.65%), lipid metabolism
215
(12.46%) and energy metabolism (9.85%) were the top five metabolism pathways,
216
followed by metabolism of cofactors and
217
biodegradation and metabolism (7.77%), glycan biosynthesis and metabolism (6.92%),
218
metabolism of other amino acids (4.31%), biosynthesis of other secondary metabolites
219
(2.90%), and metabolism of terpenoids and polyketides (2.66%) (Fig.5B).
220
3.4 SSRs/SNP markers identification
AC C
EP
TE D
208
10
vitamins (9.29%), xenobiotics
ACCEPTED MANUSCRIPT SSRs played important roles in imprinting, chromosome organization,
222
transcriptional regulation and maintenance of complex nuclear properties [16]. Next,
223
potential SSRs in all the unigenes were mined using MISA software. As a result,
224
37,851 SSRs were identified from the hepatopancreas gene library. Among them,
225
dinucleotide, trinucleotide and mononucleotide, repeats were the three largest SSRs,
226
accounting for 56.87%, 33.70%, and 5.43% of all SSRs, respectively (Fig. 6A).
RI PT
221
The transcriptomes of hepatopancreas from H. eriocheir infected or control crabs
228
shared similarities within the detected SNPs, transitions were much more common
229
than transversions. A similar number of A/G and C/T transitions and percentages of
230
the four transversion types (A/C, A/T, C/G, G/T) were found (Fig 6B).
231
3.5 Identification of aberrantly expressed genes
M AN U
SC
227
Previous sequence analysis and annotation for all of the unigenes in the merged
233
groups provided some valuable information to analyze the hepatopancreas
234
transcriptome. However, the variation in the gene expression level after H. eriocheir
235
parasitization was expected. Here, FDR≤ 0.001 and an absolute value of log2 Ratio≥
236
1 were used as the filtering thresholds to identify up or down-regulated genes. After H.
237
eriocheir infection, 2619 genes were identified as differently up-regulated and 2541
238
genes as differently down-regulated. The up-regulated DEGs were greater than
239
down-regulated DEGs, indicating that the H. eriocheir infection process was
240
associated with transcript accumulation.
AC C
EP
TE D
232
241
Based on GO analysis of DEGs, their functions could be classified into 3
242
categories. In the category of biological processes, most unigenes were involved in 11
ACCEPTED MANUSCRIPT the “translation”, “proteolysis” and “carbohydrate metabolic process”. In cellular
244
component category, the most represented included “cytoplasm”, “integral to
245
membrane”, “extracellular space”, “lysosome”, “mitochondrion and extracellular
246
region”. With respect to molecular function category, “ATP binding”, “metal ion,
247
protein, and DNA binding”, “oxidoreductase activity”, “oxygen transporter activity”
248
were the dominant groups (Fig.7). Statistics of GO Enrichment also indicated that
249
“translation”, “structural constituent of ribosome”, “ribosome”, “proteolysis”,
250
“oxygen transporter activity”, “oxidoreductase activity”, “extracellular space” and
251
“carbohydrate metabolic process” were the most dominant (Fig.8). These data suggest
252
that H. eriocheir infection has an influence on a wide range of gene functions in the
253
crab.
M AN U
SC
RI PT
243
Significantly, DEGs were consistently assigned to comprehensive host defense
255
signaling pathways related to various metabolic and energetic stresses, such as “starch
256
and sucrose metabolism”, “ribosome”, “phagosome”, “oxidative phosphorylation”,
257
“lysosome”, “fatty acid biosynthesis”, and “alanine, aspartate and glutamate
258
metabolism” (Fig. 9), suggesting that crabs use a range of tactics to cope with the
259
stress of H. eriocheir infection. A detailed explanation was presented in the
260
discussion.
AC C
EP
TE D
254
Genes potentially involved in host metabolism or energy synthesis were also
261 262
selected using standard criteria for pathway prediction with a P<0.05 and induced
263
ratios >2 or <0.5. The detailed information for the involved genes was listed in Table
264
2. 12
ACCEPTED MANUSCRIPT 265 266
4. Discussion Microsporidia can infect a broad range of eukaryotic hosts, and several species
268
can infect crustaceans. For example, Enterocytozoon hepatopenaei is a recently
269
emerged pathogen of the farmed shrimp species Penaeus monodon and Penaeus
270
vannamei and severely retards the growth of the shrimp and has rapidly spread across
271
south-east Asia [17, 18]. To date, H. eriocheir was the only microsporidian parasite
272
infecting the crab E. sinensis. Despite microsporidia being ubiquitous and significant
273
parasites, very little was known about the crustacean host’s response to
274
microsporidian infection. Here, we provided the histopathology and transcriptome
275
data for the economically important crab E. sinensis after the infection of major
276
yield-limiting
277
previously-published information on the growing body of genomic data for both the
278
parasites and crustacean species [5], and the capacity to study microsporidian
279
pathogen in more crustacean host [19] will undoubtedly underpin our understanding
280
of host-microsporidia interactions, monitor and potentially mitigate the negative
281
impacts of H. eriocheir and other microsporidian pathogen in aquatic systems.
SC
M AN U
H.
eriocheir.
These
data
in
conjunction
with
EP
TE D
pathogen
AC C
282
RI PT
267
As intracellular parasites, microsporidia have diverse effects on their host cells.
283
Metabolite import and parasite development are facilitated by manipulation of the
284
host cell cytoskeleton. Hypertrophic enlargement of the host cell (Fig 1D, E),
285
including sometimes the nucleus [20], was frequently observed in parasitized cells.
286
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
288
spatial and protected niche. Hypertrophy of host cells is a common feature of
289
microsporidian infections and is accompanied by several secondary effects: individual
290
infected cells may dedifferentiate into a syncytium, undergo cytoplasm or nuclear
291
hypertrophy and fragmentation, increase RNA synthesis, and sometimes produce an
292
increased number of cell nuclei [21]. These were in accordance with our
293
transcriptome data, in which the differently expressed genes after H. eriocheir
294
infection were mainly enriched in the categories of “translation”, “structural
295
constituent of ribosome”, “rRNA binding”, “ribosome”, “regulation of translational
296
initiation”, and “extracellular space” (Fig 8).
M AN U
SC
RI PT
287
The intracellular life stages, as well as spores of microsporidia lack mitochondria
298
and granules for nutrient storage. They are believed to utilize host-derived ATP for
299
their energy needs. ATP is produced in the respiratory chain, which again could be
300
harnessed by the microsporidium. In this study, intracellular stages of H. eriocheir
301
were shown to occur frequently in close association with the crab hepatopancreas
302
cells’ mitochondria, the site of respiratory chain (Fig 2). An enrichment of DEGs in
303
“ATP binding”, “oxygen transporter activity” and “oxidoreductase activity” was also
304
observed (Fig 7, 8). These results all suggested a parasite external energy supply and a
305
strong enhancement of oxidative metabolism.
AC C
EP
TE D
297
306
Histological results suggested that hepatopancreas was the main infection targets
307
of H. eriocheir and the microsporidian infection had immediate, deleterious
308
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
310
resources may significantly and negatively impact metabolic and nutritional pathways
311
in the hepatopancreas cells. KEGG pathway analysis exhibited that H. eriocheir
312
infection broadly altered expression of genes involved in “starch and sucrose
313
metabolism”, “glycan degradation”, “fatty acid biosynthesis” and “amino acid
314
metabolism”. Based on these findings, we suggested that H. eriocheir may “starve” its
315
hosts through the destruction of hepatopancreas tissue and/or by appropriating host
316
resources, resulting in associated changes in crab metabolism and immunity. This was
317
also consistent with previous studies indicating that microsporidian infection was
318
energetically costly [5].
M AN U
SC
RI PT
309
Recently, a modulation of the ayu Plecoglossus altivelis, phagocytic response to
320
microsporidium Glugea plecoglossi was described [22]. An elevated phagocytic
321
activity of Atlantic salmon could also contribute to the resistance to microsporidian
322
gill disease [23]. In our study, after H. eriocheir infection in the crab E. sinensis, gene
323
expressions of phagosome and lysosome pathway were also significantly changed,
324
indicating the fusion of lysosomes with the phagosome was activated and
325
phagocytosis played a key role in the crab innate immunity. More importantly,
326
because phagocytosis is typically mediated by pathogen ligand binding to a
327
phagocytic receptor, identifying and blocking the appropriate ligands may inhibit
328
pathogen entry into the host cell and provide a means of controlling infection.
AC C
EP
TE D
319
329
DEGs were also enriched in the ribosome pathway (Fig 8, 9). Cells infected with
330
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
332
requirements of the increasing number of parasites [24]. In addition, biochemically,
333
microsporidia were thought to be anaerobic and to lack evidence of electron transport
334
chain and oxidative phosphorylation [25]. Similarly, in this study, most DEGs were
335
involved in metal ion binding and oxidative phosphorylation pathway.
RI PT
331
Although some genes involved in the crab metabolism were induced in response
337
to H. eriocheir infection, further investigations were needed to verify the results of
338
this analysis and the expression patterns of other genes, which were not identified as
339
differentially expressed genes in this study.
M AN U
SC
336
340 341
5. Conclusion
In summary, histology analysis indicated hypertrophic enlargement of the
343
hepatopancreas epithelial cells and host mitochondria clustering were the common
344
features of H. eriocheir infection. Furtherly, de novo transcriptome sequencing of the
345
hepatopancreas tissue was carried out. A global survey of the DEGs involved in
346
metabolism pathways was performed to better understand the influences of H.
347
eriocheir infection on the crab. The functions of many differentially expressed genes
348
involved in host metabolism and defense were discussed. Furthermore, putative SSRs
349
and SNPs were also analyzed. Altogether, these and other recent findings could
350
provide much-needed insight into the underlying mechanisms of microsporidia-host
351
interactions.
AC C
EP
TE D
342
352 16
ACCEPTED MANUSCRIPT 353
Acknowledgments We thank the anonymous reviewers for valuable comments and suggestions. This
355
work was supported by grants from the Natural Sciences Foundation of China
356
(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
360
RI PT
354
References
362
[1] R. Hirt, J. Logsdon, B. Healy, M. Dorey, W. Doolittle, T. Embley, Microsporidia
363
are related to Fungi: evidence from the largest subunit of RNA polymerase II and
364
other proteins, Proc. Natl. Acad. Sci. U.S.A. 96(2) (1999) 580-585.
365
[2] G.D. Stentiford, S.W. Feist, D.M. Stone, K.S. Bateman, A.M. Dunn,
366
Microsporidia: diverse, dynamic, and emergent pathogens in aquatic systems, Trends.
367
Parasitol. 29(11) (2013) 567-578.
368
[3] Z. Ding, Q. Meng, H. Liu, S. Yuan, F. Zhang, M. Sun, Y. Zhao, M. Shen, G. Zhou,
369
J. Pan, H. Xue, W. Wang, First case of hepatopancreatic necrosis disease in
370
pond-reared Chinese mitten crab, Eriocheir sinensis, associated with microsporidian,
371
J. Fish Dis. 39(9) (2016) 1043-1051.
372
[4] G.D. Stentiford, K.S. Bateman, A. Dubuffet, E. Chambers, D.M. Stone,
373
Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive
374
Chinese mitten crabs (Eriocheir sinensis) in Europe, J. Invertebr. Pathol. 108(3) (2011)
AC C
EP
TE D
361
17
ACCEPTED MANUSCRIPT 156-166.
376
[5] D. Wiredu Boakye, P. Jaroenlak, A. Prachumwat, T. Williams, K. Bateman, O.
377
Itsathitphaisarn, K. Sritunyalucksana, K. Paszkiewicz, K. Moore, G. Stentiford, B.
378
Williams, Decay of the glycolytic pathway and adaptation to intranuclear parasitism
379
within Enterocytozoonidae microsporidia, Environ. Microbiol. 19(5) (2017)
380
2077-2089.
381
[6] K. Bateman, D. Wiredu-Boakye, R. Kerr, B. Williams, G. Stentiford, Single and
382
multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of
383
farmed and wild crustacean hosts, Parasitology 143(8) (2016) 971-982.
384
[7] W. Wang, J. Chen, Ultrastructural study on a novel microsporidian,
385
Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of
386
Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda), J. Invertebr. Pathol.
387
94(2) (2007) 77-83.
388
[8] Z.F. Ding, J.Q. Chen, J. Lin, X.S. Zhu, G.H. Xu, R.L. Wang, Q.G. Meng, W. Wang,
389
Development of In situ hybridization and real-time PCR assays for the detection of
390
Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab
391
Eriocheir sinensis, J.Fish. Dis. 40(7) (2017) 919-927.
392
[9] Z. Ding, Y. Yao, F. Zhang, J. Wan, M. Sun, H. Liu, G. Zhou, J. Tang, J. Pan, H.
393
Xue, Z. Zhao, The first detection of white spot syndrome virus in naturally infected
394
cultured Chinese mitten crabs, Eriocheir sinensis in China, J. Virol. Methods. 220
395
(2015) 49-54.
396
[10] Z. Ding, J. Tang, H. Xue, J. Li, Q. Ren, W. Gu, Q. Meng, W. Wang, Quantitative
AC C
EP
TE D
M AN U
SC
RI PT
375
18
ACCEPTED MANUSCRIPT detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in
398
the freshwater crayfish, Procambarus clarkii, J. Invertebr. Pathol. 115 (2014) 51-54.
399
[11] M.G. Grabherr, B.J. Haas, M. Yassour, J.Z. Levin, D.A. Thompson, I. Amit, X.
400
Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A.
401
Gnirke, N. Rhind, F. di Palma, B.W. Birren, C. Nusbaum, K. Lindblad-Toh, N.
402
Friedman, A. Regev, Full-length transcriptome assembly from RNA-Seq data without
403
a reference genome, Nat Biotech 29(7) (2011) 644-652.
404
[12] A. Mortazavi, B.A. Williams, K. McCue, L. Schaeffer, B. Wold, Mapping and
405
quantifying mammalian transcriptomes by RNA-Seq, Nat Meth 5(7) (2008) 621-628.
406
[13] A. Conesa, S. Götz, J. García-Gómez, J. Terol, M. Talón, M. Robles, Blast2GO: a
407
universal tool for annotation, visualization and analysis in functional genomics
408
research, Bioinformatics 21(18) (2005) 3674-3676.
409
[14] M. Kanehisa, S. Goto, KEGG: Kyoto Encyclopedia of Genes and Genomes,
410
Nucleic. Acids. Res. 28(1) (2000) 27-30.
411
[15] A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B. Faircloth, M. Remm, S.
412
Rozen, Primer3--new capabilities and interfaces, Nucleic. Acids. Res. 40(15) (2012)
413
e115.
414
[16] S. Subramanian, R. Mishra, L. Singh, Genome-wide analysis of microsatellite
415
repeats in humans: their abundance and density in specific genomic regions, Genome
416
Biol. 4(2) (2003) R13.
417
[17] K.F.J. Tang, C.R. Pantoja, R.M. Redman, J.E. Han, L.H. Tran, D.V. Lightner,
418
Development of in situ hybridization and PCR assays for the detection of
AC C
EP
TE D
M AN U
SC
RI PT
397
19
ACCEPTED MANUSCRIPT Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid
420
shrimp, J. Invertebr. Pathol. 130 (2015) 37-41.
421
[18] K.V. Rajendran, S. Shivam, P. Ezhil Praveena, J. Joseph Sahaya Rajan, T. Sathish
422
Kumar, S. Avunje, V. Jagadeesan, S.V.A.N.V. Prasad Babu, A. Pande, A. Navaneeth
423
Krishnan, S.V. Alavandi, K.K. Vijayan, Emergence of Enterocytozoon hepatopenaei
424
(EHP) in farmed Penaeus (Litopenaeus) vannamei in India, Aquaculture 454 (2016)
425
272-280.
426
[19] P. Salachan, P. Jaroenlak, S. Thitamadee, O. Itsathitphaisarn, K. Sritunyalucksana,
427
Laboratory
428
microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP), BMC Vet.
429
Res. 13(1) (2017) 9.
430
[20] G. Stentiford, K. Bateman, M. Longshaw, S. Feist, Enterospora canceri n. gen., n.
431
sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer
432
pagurus, Dis. Aquat. Org. 75(1) (2007) 61-72.
433
[21] J. Lom, I. Dyková, Microsporidian xenomas in fish seen in wider perspective,
434
Folia Parasitol. 52(1-2) (2005) 69-81.
435
[22] L. Rodriguez-Tovar, D. Speare, R. Markham, Fish microsporidia: immune
436
response, immunomodulation and vaccination, Fish Shellfish Immunol. 30(4-5) (2011)
437
999-1006.
438
[23] J. Becker, D. Speare, Transmission of the microsporidian gill parasite, Loma
439
salmonae, Anim Health Res Rev 8(1) (2007) 59-68.
440
[24] C. del Aguila, F. Izquierdo, A. Granja, C. Hurtado, S. Fenoy, M. Fresno, Y.
challenge
model
for
shrimp
hepatopancreatic
AC C
EP
TE D
cohabitation
M AN U
SC
RI PT
419
20
ACCEPTED MANUSCRIPT Revilla, Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected
442
cells, Int. J. Parasitol. 36(8) (2006) 869-876.
443
[25] N.M. Fast, P.J. Keeling, Alpha and beta subunits of pyruvate dehydrogenase E1
444
from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism
445
in microsporidia, Mol. Biochem. Parasi. 117(2) (2001) 201-209.
446
Table and figure legends
SC
447
M AN U
448 449
RI PT
441
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
SC
gene
RI PT
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.
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.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT An integrated metabolic consequence of Hepatospora eriocheir infection in the
2
Chinese mitten crab Eriocheir sinensis
3
Zhengfeng Ding1*, Jing Pan1, Hua Huang2, Gongcheng Jiang1, Jianqin Chen1,
4
Xueshen Zhu1, Renlei Wang1, Guohua Xu1*
RI PT
1
5 6
1
7
Chemistry, Jiangsu Second Normal University, 77 West Beijing Road, Nanjing, China,
8
210013
9
2
SC
M AN U
10
Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and
Aquatic Technology Promotion Station, Wujin District, Changzhou City, China,
213017
11
TE D
12 13 14
EP
15
AC C
16 17
*Corresponding author at Jiangsu Second Normal University, College of life science
18
and
19
+86-25-83758339;
20
21
[email protected] (G. Xu)
chemistry,
77
addresses:
West
Beijing
Road,
[email protected];
22 1
Nanjing,
China,
[email protected]
210013;
(Z.
Tel:
Ding);
ACCEPTED MANUSCRIPT 23 24
Abstract Despite the economic and evolutionary importance of aquatic host-infecting
26
microsporidian species, at present, limited information has been provided about the
27
microsporidia–host interactions. This study focused on Hepatospora eriocheir, an
28
emerging microsporidian pathogen for the Chinese mitten crab Eriocheir sinensis.
29
Hypertrophy of hepatopancreas cells was a common feature of H. eriocheir infection.
30
More importantly, mitochondria of the hepatopancreas were drawn around the H.
31
eriocheir, most likely to aid the uptake of ATP directly from the host. To better
32
understand the crab anti-microsporidian response, de novo transcriptome sequencing
33
of the hepatopancreas tissue was furtherly proceeded. A total of 47.84 M and 57.21 M
34
clean reads were generated from the hepatopancreas of H. eriocheir infected and
35
control groups respectively. Based on homology searches, functional annotation with
36
6 databases (Nr, Swiss-Prot, KEGG, KOGs, Pfam and GO) for 88,168 unigenes was
37
performed. 2619 genes were identified as differently up-regulated and 2541 genes as
38
differently
39
differentially expressed genes (DEGs) were “ATP binding”, “mitochondrion and
40
extracellular region”, “oxygen transporter activity”, “oxidoreductase activity”,
41
“alanine, aspartate and glutamate metabolism”, “carbohydrate metabolic process”,
42
“starch and sucrose metabolism” and “fatty acid biosynthesis”. These results
43
confirmed a parasite external energy supply and an integrated metabolic stress. In
44
addition, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs)
EP
TE D
M AN U
SC
RI PT
25
Prominent
functional
categories
enriched
with
AC C
down-regulated.
2
ACCEPTED MANUSCRIPT were also identified from the gene library. Taken together, these findings allow us to
46
better understand the underlying mechanisms regulating interactions between H.
47
eriocheir and the crab E. sinensis.
48
Keywords: Hepatospora eriocheir, Eriocheir sinensis, metabolism, energy,
49
interaction
RI PT
45
50
1. Introduction
SC
51
Microsporidia are small obligate intracellular parasites originally considered to be
53
primitive eukaryotic protozoa, but are recently reclassified with the fungi [1]. Almost
54
half of the known microsporidian genera infect aquatic hosts, however,
55
microsporidians are probably ubiquitous and vastly under-reported in aquatic systems
56
[2]. Recently, serious microsporidian outbreaks caused catastrophic losses in the
57
Chinese mitten crab Eriocheir sinensis harvest in Jiangsu Province, China. A
58
conspicuous sign was the color of hepatopancreas turning from yellow-golden to
59
almost white, representing a significant tissue-specific infection characteristic. It has
60
been confirmed that the microsporidium Hepatospora eriocheir was closely
61
associated with the disease [3].
TE D
EP
AC C
62
M AN U
52
Hepatospora, a recently erected genus, infects epithelial cells of the
63
hepatopancreas of wild and farmed decapod crustaceans [4]. Hepatospora spp. was
64
isolated from different crustacean hosts, inhabiting different habitats and niches:
65
freshwater Chinese mitten crab (Eriocheir sinensis), the marine mussel symbiont pea
66
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
68
tremor disease (TD), and confirmed to be opportunistic in nature [7]. Following this
69
initial discovery, (Stentiford, et al., 2011) named the microsporidium as Hepatospora
70
eriocheir [4]. Early studies of this microsporidian pathogen intensively focused on the
71
pathogen aspects including the histopathology and molecular phylogenetic analysis,
72
development of novel detection assays, and comparative genome research [4-6, 8],
73
limited information was available about the host responses, which could provide
74
important information to elucidate the diverse aspects of the host–pathogen
75
interactions.
M AN U
SC
RI PT
67
Microsporidia, as obligate intracellular parasites, are characterized by
77
development in direct contact with host cytoplasm (the majority of species), strong
78
minimization of cell machinery, and acquisition of unique transporters to exploit host
79
metabolic system [5]. All the aforementioned features are suggestive of the ability of
80
microsporidia to modify host metabolic and regulatory pathways. However, questions
81
about how H. eriocheir parasitization posed the metabolic stresses in the crab E.
82
sinensis still remain.
EP
AC C
83
TE D
76
In this study, histology examination suggested cell hypertrophy and host
84
mitochondria clustering were the common features of H. eriocheir infection in the
85
hepatopancreas of E. sinensis. To better understand the underlying molecular
86
mechanisms of H. eriocheir interaction with hepatopancreas cell, de novo
87
transcriptome sequencing was furtherly proceeded, and a global survey of
88
differentially expressed genes (DEGs), annotations of signaling pathways especially 4
ACCEPTED MANUSCRIPT the metabolism pathways, were also performed. In addition, putative simple sequence
90
repeats (SSRs) and single nucleotide polymorphisms (SNPs) were discussed. These
91
results provided the first experimental access to tissue-specific genes involved in the
92
crab anti-microsporidian response and could serve as the basis for additional in-depth
93
host-parasite interaction analyses (and potential intervention strategies) for
94
economically important microsporidians.
RI PT
89
2. Materials and methods
97
2.1 Sample collection
M AN U
96
SC
95
Ten moribund crabs (mean body weight 86.57±5.38 g; 6 males and 4 females)
99
exhibiting microsporidian infection (the color of hepatopancreas turning from
100
yellow-golden to almost white) (Fig 1) were collected from Nanjing city in Jiangsu
101
province, China. The hepatopancreas tissue was then collected and frozen
102
immediately in liquid nitrogen for total RNA extraction, fixed in 4%
103
paraformaldehyde solution (pH=7.3) for histological and in situ hybridization
104
examinations, and in 4% glutaraldehyde in 0.3 M sodium phosphate buffer (pH=7.3)
105
for electron microscopy. Additional 10 crabs without symptoms were examined
106
initially by the detection of common pathogens in E. sinensis including WSSV [9], H.
107
eriocheir [8] and spiroplasma [10]. Crabs negative for these pathogens were chosen
108
for the control group.
109
2.2 Histopathology, in situ hybridization and electron microscopy observations
110
AC C
EP
TE D
98
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,
112
tissues were embedded in paraffin. Six 10-µm thick sections were obtained from each
113
piece in different orientations. Sections were then stained with haematoxylin and
114
eosin (H&E) and examined using a light microscope (Olympus, BX53).
RI PT
111
In situ hybridization was carried out following the protocol of (Ding et al., 2017)
116
[8]. Overnight hybridization with the DIG-labeled probe was done in a humid
117
chamber at 42 °C. The slides were counterstained with 3, 3’ N-Diaminobenzidine
118
Tertrahydrochloride (DAB) and examined using the light microscope (Olympus,
119
BX53).
M AN U
SC
115
For transmission electron microscopy (TEM), the 2.5% glutaraldehyde (fixative
121
solution) was removed and the hepatopancreas tissues were post-fixed in 1% (w/v)
122
osmium tetroxide in 0.1 M phosphate buffer at pH 7.4 for 2 h. After being dehydrated
123
through a graded series of acetone, the tissues were embedded in Epon 812. Ultrathin
124
sections were double stained with uranyl acetate and lead citrate and observed with
125
the Hitachi 600-2A transmission electron microscopy.
126
2.3 RNA isolation and Illumina sequencing
EP
AC C
127
TE D
120
Total RNA was extracted using a high-purity total RNA Rapid Extraction Kit
128
(BioTeke, China) according to the manufacturer's instructions. Total RNA quality was
129
checked on 1% formaldehyde agarose gel via electrophoresis, and RNA concentration
130
was determined through Nano Drop (Thermo Scientific, USA). Then approximately
131
1 µg of DNase-treated total RNA was used to construct a cDNA library following the
132
protocols of the TruSeq Stranded mRNA Sample Preparation. After necessary 6
ACCEPTED MANUSCRIPT 133
quantification and qualification, the library was sequenced using an Illumina HiSeq™
134
2000 instrument.
135
2.4 De novo assembly and data analysis SeqPrep
(http://github.com/jstjohn/SeqPrep)
and
Sickle
RI PT
136
(https://github.com/najoshi/sickle) were applied to process the raw reads with default
138
parameters and eliminated sequences smaller than 60 bases. Then, Trinity program
139
[11] was used to do RNA assembly of clean reads. The assembled contigs were
140
annotated with sequences available in the NCBI database using the BLAST
141
algorithms. The unigenes were aligned by a BLASTx search against the protein
142
databases of NCBI, including Swiss-Prot, non-redundant (Nr), KEGG (Kyoto
143
encyclopedia of genes and genomes), eukaryote orthologous genes (KOGs), Gene
144
Ontology (GO) and Pfam. Based on the highest sequence similarity and a typical
145
cut-off E-value less than 1.0 e−5, the function annotations were retrieved [12]. Next,
146
the best alignment results were used to determine the sequence direction and
147
protein-coding-region prediction of the unigenes. The Blast2GO suite [13] was used
148
to obtain GO annotations of the uniquely assembled transcripts. Based on the KEGG
149
database, the complex biological behavior of the genes was analyzed through pathway
150
annotation [14].
M AN U
TE D
EP
AC C
151
SC
137
Microsatellite search module (MISA http://pgrc.ipk-gatersleben.de/misa/) was
152
used to find simple sequence repeats (SSRs) in unigenes, then design primer for each
153
SSR with Primer3 [15]. All clean reads were mapped to unigenes using HISAT
154
(hierarchical indexing for spliced alignment of transcripts), then call single nucleotide 7
ACCEPTED MANUSCRIPT 155
polymorphisms (SNPs) with Genome Analysis Toolkit (GATK). After filter out the
156
unreliable sites, the final SNP was gotten in VCF format.
157
2.5 Identification and analysis of differentially expressed genes (DEGs) FRKM (fragments per kilobase of transcripts per million fragments mapped) was
159
used as the unit of measurement to estimate the expression level of each transcript.
160
False discovery rate (FDR) was carried out to correct for E-value. Genes with
161
FDR≤0.001 and an FPKM ratio larger than 2 or smaller than 0.5 were considered as
162
DEGs between samples. With the DEGs, GO and KEGG pathway classifications and
163
functional enrichments were also performed, as mentioned in section 2.4.
M AN U
SC
RI PT
158
164
3. Results
166
3.1 Histopathology, in situ hybridization and electron microscopy observations
TE D
165
Infections by H. eriocheir in the crabs were confirmed by histology with H.E
168
staining, in situ hybridization and transmission electron microscopy (TEM). No
169
pathological changes could be observed in the control crabs (Fig 1A, B), whereas in
170
the infected crabs, the color of hepatopancreas turned from yellow-golden to almost
171
white (Fig 1C). Numbers of hypertrophic epithelial cells of the hepatopancreas were
172
observed (Fig 1D). Positive hybridization signals, visualized as an intense brown to
173
black precipitate were also observed within the epithelial cells of the hepatopancreas
174
(Fig 1E). Importantly, host mitochondria were observed closely around the
175
plasmalemma of meronts of H. eriocheir, most likely to aid the uptake of ATP directly
176
from the host cell (Fig 2).
AC C
EP
167
8
ACCEPTED MANUSCRIPT 177
3.2 Transcriptome sequencing and de novo assembly After removal of low-quality sequences, a total of 47.84 M clean reads that
179
represent a total of 6.90 Gb clean bases were generated for the H. eriocheir infected
180
group. For the control group, 57.21 M clean reads that represent a total of 8.27 Gb
181
clean bases were generated. 104,998 transcripts (mean length = 536 bp) from
182
hepatopancreas tissues were analyzed by de novo assembly. Further search against the
183
GenBank protein and nucleotide sequences yielded 88,168 unigenes with a mean
184
length of 481 bp (Table 1).
185
3.3 Functional annotation and classification of transcriptome sequences
M AN U
SC
RI PT
178
To achieve protein identification and gene annotation, a search was made on
187
standard unigenes in the NCBI Nr (20,177 unigenes, 22.88%), Swiss-Prot (11,637
188
unigenes, 13.20%), KEGG (9,294 unigenes, 10.54%), KOGs (11,871 unigenes,
189
13.46%), Pfam (12,062 unigenes, 13.68%) and GO (10,619 unigenes, 12.04%) using
190
the BLAST program (E-value <1.0 e−5).
TE D
186
The standard unigenes were then aligned to the KOG database to predict and
192
classify their possible roles. A total of 12,236 unigenes had KOG classifications,
193
distributed among the 25 KOG categories, including “energy production and
194
conversion ", “amino acid transport and metabolism”, “nucleotide transport and
195
metabolism”, “carbohydrate transport and metabolism”, “lipid transport and
196
metabolism”, “cell wall/membrane/envelope biogenesis”, “secondary metabolites
197
biosynthesis, transport and catabolism” and “signal transduction mechanisms ”, most
198
of which played key roles in metabolism-related pathways (Fig.3).
AC C
EP
191
9
ACCEPTED MANUSCRIPT We obtained a total of 3,528 GO assignments, where 37.39% comprised
200
biological processes, 24.46% comprised cellular component, and 38.15% comprised
201
molecular function. In the category of biological processes, most unigenes were
202
involved in the “translation”, “proteolysis”, and “carbohydrate metabolic process”. In
203
cellular component category, the most represented were “cytoplasm”, “integral to
204
membrane”, “ribosome”, “extracellular space”, “lysosome”, and “mitochondrion”.
205
With respect to molecular function category, “ATP binding”, “metal ion, protein, and
206
DNA binding”, “structural constituent of ribosome”, “oxidoreductase activity”, and
207
“oxygen transporter activity”, were the dominant groups (Fig.4).
M AN U
SC
RI PT
199
In addition, we also used KEGG analysis to identify potential biological
209
pathways represented in the transcriptome of the crab hepatopancreas. KEGG analysis
210
indicated that a greater number of genes expressed in the hepatopancreas were
211
associated with organismal systems, metabolism, environmental information
212
processing, genetic information processing, human diseases, and cellular processes.
213
Metabolism was the dominant group (Fig.5A). Carbohydrate metabolism (16.53%),
214
nucleotide metabolism (14.64%), amino acid metabolism (12.65%), lipid metabolism
215
(12.46%) and energy metabolism (9.85%) were the top five metabolism pathways,
216
followed by metabolism of cofactors and
217
biodegradation and metabolism (7.77%), glycan biosynthesis and metabolism (6.92%),
218
metabolism of other amino acids (4.31%), biosynthesis of other secondary metabolites
219
(2.90%), and metabolism of terpenoids and polyketides (2.66%) (Fig.5B).
220
3.4 SSRs/SNP markers identification
AC C
EP
TE D
208
10
vitamins (9.29%), xenobiotics
ACCEPTED MANUSCRIPT SSRs played important roles in imprinting, chromosome organization,
222
transcriptional regulation and maintenance of complex nuclear properties [16]. Next,
223
potential SSRs in all the unigenes were mined using MISA software. As a result,
224
37,851 SSRs were identified from the hepatopancreas gene library. Among them,
225
dinucleotide, trinucleotide and mononucleotide, repeats were the three largest SSRs,
226
accounting for 56.87%, 33.70%, and 5.43% of all SSRs, respectively (Fig. 6A).
RI PT
221
The transcriptomes of hepatopancreas from H. eriocheir infected or control crabs
228
shared similarities within the detected SNPs, transitions were much more common
229
than transversions. A similar number of A/G and C/T transitions and percentages of
230
the four transversion types (A/C, A/T, C/G, G/T) were found (Fig 6B).
231
3.5 Identification of aberrantly expressed genes
M AN U
SC
227
Previous sequence analysis and annotation for all of the unigenes in the merged
233
groups provided some valuable information to analyze the hepatopancreas
234
transcriptome. However, the variation in the gene expression level after H. eriocheir
235
parasitization was expected. Here, FDR≤ 0.001 and an absolute value of log2 Ratio≥
236
1 were used as the filtering thresholds to identify up or down-regulated genes. After H.
237
eriocheir infection, 2619 genes were identified as differently up-regulated and 2541
238
genes as differently down-regulated. The up-regulated DEGs were greater than
239
down-regulated DEGs, indicating that the H. eriocheir infection process was
240
associated with transcript accumulation.
AC C
EP
TE D
232
241
Based on GO analysis of DEGs, their functions could be classified into 3
242
categories. In the category of biological processes, most unigenes were involved in 11
ACCEPTED MANUSCRIPT the “translation”, “proteolysis” and “carbohydrate metabolic process”. In cellular
244
component category, the most represented included “cytoplasm”, “integral to
245
membrane”, “extracellular space”, “lysosome”, “mitochondrion and extracellular
246
region”. With respect to molecular function category, “ATP binding”, “metal ion,
247
protein, and DNA binding”, “oxidoreductase activity”, “oxygen transporter activity”
248
were the dominant groups (Fig.7). Statistics of GO Enrichment also indicated that
249
“translation”, “structural constituent of ribosome”, “ribosome”, “proteolysis”,
250
“oxygen transporter activity”, “oxidoreductase activity”, “extracellular space” and
251
“carbohydrate metabolic process” were the most dominant (Fig.8). These data suggest
252
that H. eriocheir infection has an influence on a wide range of gene functions in the
253
crab.
M AN U
SC
RI PT
243
Significantly, DEGs were consistently assigned to comprehensive host defense
255
signaling pathways related to various metabolic and energetic stresses, such as “starch
256
and sucrose metabolism”, “ribosome”, “phagosome”, “oxidative phosphorylation”,
257
“lysosome”, “fatty acid biosynthesis”, and “alanine, aspartate and glutamate
258
metabolism” (Fig. 9), suggesting that crabs use a range of tactics to cope with the
259
stress of H. eriocheir infection. A detailed explanation was presented in the
260
discussion.
AC C
EP
TE D
254
Genes potentially involved in host metabolism or energy synthesis were also
261 262
selected using standard criteria for pathway prediction with a P<0.05 and induced
263
ratios >2 or <0.5. The detailed information for the involved genes was listed in Table
264
2. 12
ACCEPTED MANUSCRIPT 265 266
4. Discussion Microsporidia can infect a broad range of eukaryotic hosts, and several species
268
can infect crustaceans. For example, Enterocytozoon hepatopenaei is a recently
269
emerged pathogen of the farmed shrimp species Penaeus monodon and Penaeus
270
vannamei and severely retards the growth of the shrimp and has rapidly spread across
271
south-east Asia [17, 18]. To date, H. eriocheir was the only microsporidian parasite
272
infecting the crab E. sinensis. Despite microsporidia being ubiquitous and significant
273
parasites, very little was known about the crustacean host’s response to
274
microsporidian infection. Here, we provided the histopathology and transcriptome
275
data for the economically important crab E. sinensis after the infection of major
276
yield-limiting
277
previously-published information on the growing body of genomic data for both the
278
parasites and crustacean species [5], and the capacity to study microsporidian
279
pathogen in more crustacean host [19] will undoubtedly underpin our understanding
280
of host-microsporidia interactions, monitor and potentially mitigate the negative
281
impacts of H. eriocheir and other microsporidian pathogen in aquatic systems.
SC
M AN U
H.
eriocheir.
These
data
in
conjunction
with
EP
TE D
pathogen
AC C
282
RI PT
267
As intracellular parasites, microsporidia have diverse effects on their host cells.
283
Metabolite import and parasite development are facilitated by manipulation of the
284
host cell cytoskeleton. Hypertrophic enlargement of the host cell (Fig 1D, E),
285
including sometimes the nucleus [20], was frequently observed in parasitized cells.
286
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
288
spatial and protected niche. Hypertrophy of host cells is a common feature of
289
microsporidian infections and is accompanied by several secondary effects: individual
290
infected cells may dedifferentiate into a syncytium, undergo cytoplasm or nuclear
291
hypertrophy and fragmentation, increase RNA synthesis, and sometimes produce an
292
increased number of cell nuclei [21]. These were in accordance with our
293
transcriptome data, in which the differently expressed genes after H. eriocheir
294
infection were mainly enriched in the categories of “translation”, “structural
295
constituent of ribosome”, “rRNA binding”, “ribosome”, “regulation of translational
296
initiation”, and “extracellular space” (Fig 8).
M AN U
SC
RI PT
287
The intracellular life stages, as well as spores of microsporidia lack mitochondria
298
and granules for nutrient storage. They are believed to utilize host-derived ATP for
299
their energy needs. ATP is produced in the respiratory chain, which again could be
300
harnessed by the microsporidium. In this study, intracellular stages of H. eriocheir
301
were shown to occur frequently in close association with the crab hepatopancreas
302
cells’ mitochondria, the site of respiratory chain (Fig 2). An enrichment of DEGs in
303
“ATP binding”, “oxygen transporter activity” and “oxidoreductase activity” was also
304
observed (Fig 7, 8). These results all suggested a parasite external energy supply and a
305
strong enhancement of oxidative metabolism.
AC C
EP
TE D
297
306
Histological results suggested that hepatopancreas was the main infection targets
307
of H. eriocheir and the microsporidian infection had immediate, deleterious
308
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
310
resources may significantly and negatively impact metabolic and nutritional pathways
311
in the hepatopancreas cells. KEGG pathway analysis exhibited that H. eriocheir
312
infection broadly altered expression of genes involved in “starch and sucrose
313
metabolism”, “glycan degradation”, “fatty acid biosynthesis” and “amino acid
314
metabolism”. Based on these findings, we suggested that H. eriocheir may “starve” its
315
hosts through the destruction of hepatopancreas tissue and/or by appropriating host
316
resources, resulting in associated changes in crab metabolism and immunity. This was
317
also consistent with previous studies indicating that microsporidian infection was
318
energetically costly [5].
M AN U
SC
RI PT
309
Recently, a modulation of the ayu Plecoglossus altivelis, phagocytic response to
320
microsporidium Glugea plecoglossi was described [22]. An elevated phagocytic
321
activity of Atlantic salmon could also contribute to the resistance to microsporidian
322
gill disease [23]. In our study, after H. eriocheir infection in the crab E. sinensis, gene
323
expressions of phagosome and lysosome pathway were also significantly changed,
324
indicating the fusion of lysosomes with the phagosome was activated and
325
phagocytosis played a key role in the crab innate immunity. More importantly,
326
because phagocytosis is typically mediated by pathogen ligand binding to a
327
phagocytic receptor, identifying and blocking the appropriate ligands may inhibit
328
pathogen entry into the host cell and provide a means of controlling infection.
AC C
EP
TE D
319
329
DEGs were also enriched in the ribosome pathway (Fig 8, 9). Cells infected with
330
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
332
requirements of the increasing number of parasites [24]. In addition, biochemically,
333
microsporidia were thought to be anaerobic and to lack evidence of electron transport
334
chain and oxidative phosphorylation [25]. Similarly, in this study, most DEGs were
335
involved in metal ion binding and oxidative phosphorylation pathway.
RI PT
331
Although some genes involved in the crab metabolism were induced in response
337
to H. eriocheir infection, further investigations were needed to verify the results of
338
this analysis and the expression patterns of other genes, which were not identified as
339
differentially expressed genes in this study.
M AN U
SC
336
340 341
5. Conclusion
In summary, histology analysis indicated hypertrophic enlargement of the
343
hepatopancreas epithelial cells and host mitochondria clustering were the common
344
features of H. eriocheir infection. Furtherly, de novo transcriptome sequencing of the
345
hepatopancreas tissue was carried out. A global survey of the DEGs involved in
346
metabolism pathways was performed to better understand the influences of H.
347
eriocheir infection on the crab. The functions of many differentially expressed genes
348
involved in host metabolism and defense were discussed. Furthermore, putative SSRs
349
and SNPs were also analyzed. Altogether, these and other recent findings could
350
provide much-needed insight into the underlying mechanisms of microsporidia-host
351
interactions.
AC C
EP
TE D
342
352 16
ACCEPTED MANUSCRIPT 353
Acknowledgments We thank the anonymous reviewers for valuable comments and suggestions. This
355
work was supported by grants from the Natural Sciences Foundation of China
356
(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
360
RI PT
354
References
362
[1] R. Hirt, J. Logsdon, B. Healy, M. Dorey, W. Doolittle, T. Embley, Microsporidia
363
are related to Fungi: evidence from the largest subunit of RNA polymerase II and
364
other proteins, Proc. Natl. Acad. Sci. U.S.A. 96(2) (1999) 580-585.
365
[2] G.D. Stentiford, S.W. Feist, D.M. Stone, K.S. Bateman, A.M. Dunn,
366
Microsporidia: diverse, dynamic, and emergent pathogens in aquatic systems, Trends.
367
Parasitol. 29(11) (2013) 567-578.
368
[3] Z. Ding, Q. Meng, H. Liu, S. Yuan, F. Zhang, M. Sun, Y. Zhao, M. Shen, G. Zhou,
369
J. Pan, H. Xue, W. Wang, First case of hepatopancreatic necrosis disease in
370
pond-reared Chinese mitten crab, Eriocheir sinensis, associated with microsporidian,
371
J. Fish Dis. 39(9) (2016) 1043-1051.
372
[4] G.D. Stentiford, K.S. Bateman, A. Dubuffet, E. Chambers, D.M. Stone,
373
Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive
374
Chinese mitten crabs (Eriocheir sinensis) in Europe, J. Invertebr. Pathol. 108(3) (2011)
AC C
EP
TE D
361
17
ACCEPTED MANUSCRIPT 156-166.
376
[5] D. Wiredu Boakye, P. Jaroenlak, A. Prachumwat, T. Williams, K. Bateman, O.
377
Itsathitphaisarn, K. Sritunyalucksana, K. Paszkiewicz, K. Moore, G. Stentiford, B.
378
Williams, Decay of the glycolytic pathway and adaptation to intranuclear parasitism
379
within Enterocytozoonidae microsporidia, Environ. Microbiol. 19(5) (2017)
380
2077-2089.
381
[6] K. Bateman, D. Wiredu-Boakye, R. Kerr, B. Williams, G. Stentiford, Single and
382
multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of
383
farmed and wild crustacean hosts, Parasitology 143(8) (2016) 971-982.
384
[7] W. Wang, J. Chen, Ultrastructural study on a novel microsporidian,
385
Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of
386
Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda), J. Invertebr. Pathol.
387
94(2) (2007) 77-83.
388
[8] Z.F. Ding, J.Q. Chen, J. Lin, X.S. Zhu, G.H. Xu, R.L. Wang, Q.G. Meng, W. Wang,
389
Development of In situ hybridization and real-time PCR assays for the detection of
390
Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab
391
Eriocheir sinensis, J.Fish. Dis. 40(7) (2017) 919-927.
392
[9] Z. Ding, Y. Yao, F. Zhang, J. Wan, M. Sun, H. Liu, G. Zhou, J. Tang, J. Pan, H.
393
Xue, Z. Zhao, The first detection of white spot syndrome virus in naturally infected
394
cultured Chinese mitten crabs, Eriocheir sinensis in China, J. Virol. Methods. 220
395
(2015) 49-54.
396
[10] Z. Ding, J. Tang, H. Xue, J. Li, Q. Ren, W. Gu, Q. Meng, W. Wang, Quantitative
AC C
EP
TE D
M AN U
SC
RI PT
375
18
ACCEPTED MANUSCRIPT detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in
398
the freshwater crayfish, Procambarus clarkii, J. Invertebr. Pathol. 115 (2014) 51-54.
399
[11] M.G. Grabherr, B.J. Haas, M. Yassour, J.Z. Levin, D.A. Thompson, I. Amit, X.
400
Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A.
401
Gnirke, N. Rhind, F. di Palma, B.W. Birren, C. Nusbaum, K. Lindblad-Toh, N.
402
Friedman, A. Regev, Full-length transcriptome assembly from RNA-Seq data without
403
a reference genome, Nat Biotech 29(7) (2011) 644-652.
404
[12] A. Mortazavi, B.A. Williams, K. McCue, L. Schaeffer, B. Wold, Mapping and
405
quantifying mammalian transcriptomes by RNA-Seq, Nat Meth 5(7) (2008) 621-628.
406
[13] A. Conesa, S. Götz, J. García-Gómez, J. Terol, M. Talón, M. Robles, Blast2GO: a
407
universal tool for annotation, visualization and analysis in functional genomics
408
research, Bioinformatics 21(18) (2005) 3674-3676.
409
[14] M. Kanehisa, S. Goto, KEGG: Kyoto Encyclopedia of Genes and Genomes,
410
Nucleic. Acids. Res. 28(1) (2000) 27-30.
411
[15] A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B. Faircloth, M. Remm, S.
412
Rozen, Primer3--new capabilities and interfaces, Nucleic. Acids. Res. 40(15) (2012)
413
e115.
414
[16] S. Subramanian, R. Mishra, L. Singh, Genome-wide analysis of microsatellite
415
repeats in humans: their abundance and density in specific genomic regions, Genome
416
Biol. 4(2) (2003) R13.
417
[17] K.F.J. Tang, C.R. Pantoja, R.M. Redman, J.E. Han, L.H. Tran, D.V. Lightner,
418
Development of in situ hybridization and PCR assays for the detection of
AC C
EP
TE D
M AN U
SC
RI PT
397
19
ACCEPTED MANUSCRIPT Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid
420
shrimp, J. Invertebr. Pathol. 130 (2015) 37-41.
421
[18] K.V. Rajendran, S. Shivam, P. Ezhil Praveena, J. Joseph Sahaya Rajan, T. Sathish
422
Kumar, S. Avunje, V. Jagadeesan, S.V.A.N.V. Prasad Babu, A. Pande, A. Navaneeth
423
Krishnan, S.V. Alavandi, K.K. Vijayan, Emergence of Enterocytozoon hepatopenaei
424
(EHP) in farmed Penaeus (Litopenaeus) vannamei in India, Aquaculture 454 (2016)
425
272-280.
426
[19] P. Salachan, P. Jaroenlak, S. Thitamadee, O. Itsathitphaisarn, K. Sritunyalucksana,
427
Laboratory
428
microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP), BMC Vet.
429
Res. 13(1) (2017) 9.
430
[20] G. Stentiford, K. Bateman, M. Longshaw, S. Feist, Enterospora canceri n. gen., n.
431
sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer
432
pagurus, Dis. Aquat. Org. 75(1) (2007) 61-72.
433
[21] J. Lom, I. Dyková, Microsporidian xenomas in fish seen in wider perspective,
434
Folia Parasitol. 52(1-2) (2005) 69-81.
435
[22] L. Rodriguez-Tovar, D. Speare, R. Markham, Fish microsporidia: immune
436
response, immunomodulation and vaccination, Fish Shellfish Immunol. 30(4-5) (2011)
437
999-1006.
438
[23] J. Becker, D. Speare, Transmission of the microsporidian gill parasite, Loma
439
salmonae, Anim Health Res Rev 8(1) (2007) 59-68.
440
[24] C. del Aguila, F. Izquierdo, A. Granja, C. Hurtado, S. Fenoy, M. Fresno, Y.
challenge
model
for
shrimp
hepatopancreatic
AC C
EP
TE D
cohabitation
M AN U
SC
RI PT
419
20
ACCEPTED MANUSCRIPT Revilla, Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected
442
cells, Int. J. Parasitol. 36(8) (2006) 869-876.
443
[25] N.M. Fast, P.J. Keeling, Alpha and beta subunits of pyruvate dehydrogenase E1
444
from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism
445
in microsporidia, Mol. Biochem. Parasi. 117(2) (2001) 201-209.
446
Table and figure legends
SC
447
M AN U
448 449
RI PT
441
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
SC
gene
RI PT
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.