Aquaculture Reports 18 (2020) 100533

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Metabolic mechanism of the mud crab (Scylla paramamosain) adapting to

salinity sudden drop based on GC-MS technology
Hongzhi Yao a, Xing Li a, Lei Tang a, Huan Wang a, b, *, Chunlin Wang a, b, Changkao Mu a, b,
Ce Shi a, b
a
School of Marine Science, Ningbo University, Ningbo, 315211, Zhejiang, China
b
Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo, 315211, Zhejiang, China

A R T I C L E I N F O A B S T R A C T

Keywords: Salinity impacts the respiratory metabolism, growth, and survival of marine crustaceans. Although the
Scylla paramamosain S. paramamosain is a euryhaline species, the sudden drop in salinity often causes individual death. The study
Metabolism aimed to reveal the adaptive mechanism of S. paramamosain in response to a sudden drop from 23‰ to 3‰ in
Salinity sudden drop
salinity based on GC–MS data. We identified 1264 metabolites, and 437 were differentially expressed. Of them,
Osmoregulation
71 were up-regulated (FC > 1), including taurine, L-homoserine, aspartic acid, fructose 6-phosphate, glucose 6-
phosphate, pyruvic acid, and lactic acid, and 74 were down-regulated (FC < 1), including glutamic acid, valine,
glycine, fructose, tagatose, and ribose. KEGG enrichment analysis of differential metabolites identified 57
metabolic pathways, of which 29 were statistically significant (P < 0.05), including glycine, serine, and threo­
nine metabolism, the pentose phosphate pathway, and ABC transporters. These metabolic pathways were mostly
the amino acid metabolism pathway, carbohydrate metabolism pathway, metabolism of cofactors and vitamins,
nucleotide metabolism, energy, metabolism, membrane, transport and translation. The results of this study show
that free amino acids play an important role in adaptation to a sudden decrease in salinity and that energy
metabolism involving carbohydrates and organic acids provides the energy supply during adaptation. The study
provides important information about the osmoregulation of S. paramamosain and even other crustaceans.

1. Introduction gluconeogenesis of amino acids and accumulation of energy (Ye et al.,

Salinity greatly impacts the respiratory metabolism, growth, and
survival of marine crustaceans (Sang and Fotedar, 2004; Parado-Estepa
and Quinitio, 2011). Crustaceans adapt to changes in salinity mainly
through osmoregulation, including anisosmotic extracelluar regulation
(AER) and intracellular isometric regulation (IIR). AER regulates he­
molymph osmotic pressure, ion concentration and composition, and the
volume of hemolymph via the extracellular buffer system and ion
regulation (Gilles and Péqueux, 1981). Therefore, ion channels are
closely related to osmoregulation of crustaceans. The regulatory effects
of Na+-K+-ATPase (Lucu and Flik, 1999; Horisberger, 2004; Romano &
Zeng, 2011), carbonic anhydrase (Henry, 2001; Henry et al., 2003), and
V-ATPase (Kirschner, 2004) on ion regulation have been demonstrated.
IIR regulates and maintains cell volume change and trace intracellular
balance (Gilles and Péqueux, 1981). For example, Portunus tritubercu­
latus under low salt stress achieves osmotic re-equilibrium via
2014).
The gill is an important osmoregulatory organ in crustaceans (Cas­
tilho et al., 2001; Lv et al., 2016). The epithelial tissues of gills have
multiple functions, including gas exchange (Henry, 2015), ion exchange
(Ahearn et al., 2004), and osmoregulation (Tongsaikling and Salaenoi,
2013). The epithelial tissues are categorized according to thickness: the
respiratory epithelium is involved mainly in gas exchange, whereas the
ion transport epithelium is involved mainly in ion compensation and
transport and is responsible for regulating crustacean osmotic pressure
and blood ion concentration (Whiteley, 2011).
Scylla paramamosain is a decapod that belongs to the family Portu­
nidae. It is mainly distributed in the Indian Ocean, western Pacific
Ocean, and the southeastern coastal area of China (Ma et al., 2014).
S. paramamosain is an important marine aquaculture species in China
due to its high economic value and good nutritional characteristics.
S. paramamosain prefers low-salt intertidal or estuarine areas, which

* Corresponding author at: School of Marine Science, Ningbo University, Ningbo, 315211, Zhejiang, China.
E-mail addresses: 1025801555@qq.com (H. Yao), 2780443420@qq.com (X. Li), 791888694@qq.com (L. Tang), wanghuan1@nbu.edu.cn (H. Wang),
wangchunlin@nbu.edu.cn (C. Wang), muchangkao@nbu.edu.cn (C. Mu), shice@nbu.edu.cn (C. Shi).

https://doi.org/10.1016/j.aqrep.2020.100533
Received 28 July 2020; Received in revised form 4 October 2020; Accepted 29 October 2020
Available online 11 November 2020
2352-5134/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Yao et al. Aquaculture Reports 18 (2020) 100533

often experience rapid decreases in salinity due to heavy rainfall or fresh internal standard (2-chloro-l-phenylalanine in methanol, 0.3 mg/mL)
water inflow. Although S. paramamosain is a euryhaline species (Paital and 600 μL extraction solvent with methanol /water (4/1, v/v) were
and Chainy, 2014), a sudden decrease in salinity often causes death and added to each sample. Samples were stored at -80 ℃ for 2 min and then
thus economic loss (Romano and Zeng, 2012; Wang et al., 2018a, 2018b, grinded at 60 HZ for 2 min. 120 μL of chloroform was added to the
2018c). Therefore, salinity change should be largely avoided during samples, then the samples were vigorously vortexed and followed by
aquaculture production. 10 min ultrasound-associated extraction at ambient temperature, then
To date, little is known about osmoregulation in S. paramamosain. stored at 4 ℃ for 10 min. The samples were centrifuged at 12000 rpm
Chung and Lin (2006) cloned the Na+-K+-ATPase gene of for 10 min at 4℃. QC sample was prepared by mixing aliquots of the all
S. paramamosain and found that it was most highly expressed in the samples to be a pooled sample. An aliquot of the 400 μL supernatant was
posterior gills. Wang et al. (2018a) studied the molecular mechanism of transferred to a glass sampling vial for vacuum-dry at room temperature.
S. paramamosain during adaptation to a sudden decrease in salinity at And 80 μL of methoxylamine hydrochloride (dissolved in pyridine,
the transcriptome level. They found that in addition to ion transport, 15 mg/mL) was subsequently added. The resultant mixture was vor­
free amino acids were also involved in osmoregulation. Proteomics data texed vigorously for 2 min and incubated at 37 ℃ for 90 min. 80 μL of
showed that besides ion transport, free amino acids, and energy meta­ BSTFA (with 1% TMCS) and 20 μL n-hexane were added into the
bolism regulated osmoregulation in S. paramamosain (Wang et al., mixture, which was vortexed vigorously for 2 min and then derivatized
2018b). Wang et al. (2018c) described 7 known and 43 new microRNAs at 70 ℃ for 60 min. The samples were placed at ambient temperature
(miRNAs), including 18 differentially expressed small RNAs. They con­ for30 min before GC–MS analysis.
ducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
analysis of differentially expressed miRNA target genes and found that
2.4. GC-MS
they were mostly related to amino acid metabolism and energy meta­
bolism pathways, which demonstrated the regulatory roles of amino
The derivatived samples were analyzed on an Agilent 7890B gas
acids and energy osmoregulation in S. paramamosain.
chromatography system coupled to an Agilent 5977A MSD system
Along with genomics, transcriptomics, and proteomics, metabo­
(Agilent Technologies Inc., CA, USA). A DB-5MS fused-silica capillary
nomics is an important methodology used in system biology, and it has
column (30 m ×0.25 mm ×0.25 μm, Agilent J & W Scientific, Folsom,
become a hot topic in the field of histology. Metabonomics describes the
CA, USA) was utilized to separate the derivatives. Helium (> 99.999 %)
metabolism in living bodies (Mohamed, 2012). Metabolites are the
wasused as the carrier gas at a constant flow rate of 1 mL / min through
products of cellular regulation, and the latter is a reaction to genetic or
the column. The injector temperature was maintained at 260 ℃. Injec­
environmental changes at the biological system level (Ma et al., 2017).
tion volume was 1 μL by splitless mode. The initial oven temperature
In this study, we investigated the mechanism of adaptation of
was 60 ℃, ramped to 125 ℃ at a rate of 8 ℃/min, to 210 ℃ at a rate of 5
S. paramamosain to a sudden decrease in salinity at the metabolic level
℃/min, to 270 ℃ at a rate of 10 ℃/min, to 305 ℃ at a rate of 20 ℃/min,
based on previous studies at the transcriptome (Wang et al., 2018a),
and finally held at 305 ℃ for 5 min. The temperature of MS quadrupole
proteome (Wang et al., 2018b), and miRNA (Wang et al., 2018c) levels.
and ion source (electron impact) was set to 150 and 230 ℃, respectively.
The collision energy was 70 eV. Mass spectrometric data was acquired in
2. Materials
a full-scan mode (m/z 50–500), and the solvent delay time was set to
5 min.
2.1. Animals
The QCs were injected at regular intervals (every 8 samples)
throughout the analytical run to provide a set of data from which
A total of 300 randomly selected crabs with a body weight of ~30 g
was selected and kept in a natural water environment with a salinity of
23‰ and a temperature of approximately 20 ◦ C. Every 50 crabs were
randomly selected (weight ~30 g) as a group, with a total of six groups,
housed in six cement pools under identical physical and chemical con­
ditions. The salinity of the seawater for three of the groups was adjusted
to 3‰ from 23‰, which dropped by 20‰. These three groups were
defined as the LS (low salinity) group. The other three groups were
defined as the CK groups, where the salinity of seawater was kept at
23‰. There were 24 deaths in the LS group within 7 days and 4 deaths in
the CK group. The S. paramamosain in CK and LS group at 120 h were
killed under the condition of alcohol anesthesia, and gills were quickly
rem1oved and used for metabolomics analysis (Wang et al., 2018a). All
other conditions were the same as the LS group (Wang et al., 2018a,
2018b, 2018c, 2019).

2.2. Chemicals

All chemicals and solvents were analytical or HPLC grade. Water,

methanol, pyridine, n-hexane, methoxylamine hydrochloride(97 %),
BSTFA with 1% TMCS were purchased from CNW Technologies GmbH
(Düsseldorf, Germany). Trichloromethane was from Sinopharm Chem­
ical Reagent Co., Ltd. (Shanghai, China). L-2-chlorophenylalanine was
from Shanghai Hengchuang Bio-technology Co., Ltd. (Shanghai, China).

2.3. Sample preparation

30 mg accurately weighed sample was transferred to a 1.5-mL

Eppendorf tube. Two small steel balls were added to the tube. 20 μL Fig. 1. Total ion current (TIC) of CK (A) and LS (B) S. paramamosain gills.

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H. Yao et al. Aquaculture Reports 18 (2020) 100533

Fig. 2. PCA scores plot (A), OPLS-DA scores plots (B) and response permutation testing for CK and LS Scylla paramamosain.

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H. Yao et al. Aquaculture Reports 18 (2020) 100533

repeatability could be assessed.
2.5. Data preprocessing and statistical analysis
ChemStation (version E.02.02.1431, Agilent, USA) software was
used to convert the raw data (.D format) to. CDF format, and then the.
CDF data were imported into the ChromaTOF software (version 4.34,
LECO, St Joseph, MI) for data processing. Metabolites were annotated
through Fiehn or NIST database. After alignment with Statistic Compare
component, the ‘raw data array’ (.cvs) was obtained from raw data with
three dimension data sets including sample information, peak names (or
retention time and m/z) and peak intensities. There were 1264 peaks
detected from all samples. In the ‘data array’, all internal standards and
any known pseudo positive peaks (caused by background noise, column
bleed or BSTFA derivatization procedure) were removed. The data was
normalized to the total peak area of each sample, and multiplied by
10000, and the peaks from the same metabolite were combined. The
total detectable metabolites were437.
Data were transformed by log2 in Excel 2007 (Microsoft, USA) (use
0.000001 to replace 0 before transforming), and the resulting data
matrix were then imported into SIMCA software package (14.0, Ume­
trics, Umeå, Sweden). Principle component analysis (PCA) and
(orthogonal) partial least-squares-discriminant analysis (O)PLS-DA
were performed to visualize the metabolic difference among experi­
mental groups, after mean centering and unit variance scaling. The
Hotelling’s T2 region, shown as an ellipse in score plots of the models,
defines the 95 % confidence interval of the modeled variation. Variable
importance in the projection (VIP) ranks the overall contribution of each
variable to the OPLS-DA model, and those variables with VIP > 1 are
considered relevant for group discrimination.
In this study, the default 7-round cross-validation was applied with
1/seventh of the samples being excluded from the mathematical model
in each round, in order to guard against overfitting.
2.6. Selection of differential metabolites
The differential metabolites were selected on the basis of the com­
bination of a statistically significant threshold of variable influence on
projection (VIP) values obtained from the OPLS-DA model and pvalues
from a two-tailed Student’s t-test on the normalized peak areas from
different groups, where metabolites with VIP values larger than 1.0 and
pvalues less than 0.05 were considered as differential metabolites.
3. Results
3.1. Metabolite alterations of Scylla paramamosain gills induced by
salinity sudden drop
Previous studies showed, S. paramamosain reached an adaptive state
after 120 h, when the seawater salinity dropped suddenly from 23‰ to
3‰ (Wang et al., 2018a, 2018b, 2018c, 2019). Therefore, GC–MS was
adopted to detect the metabolome changes of gills between the CK group
and the LS group. 8 repeated measurements of total ion current (TIC)
from CK (Fig. 1A) and LS (Fig. 1B) indicated that the instrument analysis
of all samples has strong signal strength, large peak capacity and good
reproducibility of retention time. After data processing, 1264 mass
spectrum features were detected, and 437 metabolites were identified.
The detectable metabolites of gill extracts contain amino acids, nucle­
otides, organic acids, sugars and so on. In order to understand the
changes of metabolites caused by the sudden drop in salinity more
specifically, multivariate data analysis and univariate data analysis were
performed on these GC–MS data.

Table 1
Models and corresponding parameters.
Models

No. Model Type A N R2X(cum) R2Y(cum) Q2(cum) R2 Q2

ALL M1 PCA-X 4 18 0.528 − 0.0466

M2 PCA-X 3 16 0.459 − 0.0158
P-CK M3 PLS-DA 2 16 0.294 0.997 0.87
M4 OPLS-DA 1+1+0 16 0.294 0.997 0.915 0.894 0.224

The unsupervised PCA was used to observe the overall distribution between the samples and the stability of the whole analysis process, and then the supervised OPLS-
DA was used to distinguish the overall differences in the metabolic contours between CK and LS group, and to find the difference metabolites between the two groups.
In OPLS-DA analysis, variables with variable important in projection (VIP) greater than 1 are considered to be differential. In order to prevent the model from over-
fitting, the qulity of the model was investigated by 7 fold cross validation and 200 times of response permutation testing (RPT).

Fig. 3. Differential metabolite classification (A) and the pie chart (B) of the differential metabolite classification in the gill tissues the Scylla paramamosain adapting
to salinity sudden drop.

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Table 2
Up-regulated metabolites in gills of Scylla paramamosain response to sudden drop in salinity from 23‰ to 3‰.
Metabolites RT(min) VIP P-Value FC(LS/CK) AVG(LS) AVG-CK

Taurine 18.38 1.96 2.78E-07 1.58 747.23 472.48

Fumaric acid 12.14 1.94 0.000188 3.08 11.21 3.63
Phosphomycin 8.95 1.92 9.63E-07 2.28 12.38 5.42
Threo-beta-hydroxyaspartate 19.38 1.89 0.003524 3.32 5.32 1.60
L-homoserine 14.21 1.88 8.16E-05 1.65 1.41 0.86
2-hydroxybutanoic acid 6.15 1.88 8.43E-06 1.61 3.62 2.24
Adenosine 5-monophosphate 35.55 1.85 6.14E-05 5.45 0.37 0.07
N-alpha-Acetyl-L-ornithine 18.20 1.84 2.86E-05 1.75 0.63 0.36
6-phosphogluconic acid 30.95 1.84 2.34E-05 3.05 8.69 2.85
Asparagine 14.36 1.81 1.94E-05 1.72 101.74 59.06
Aspartic acid 12.76 1.81 0.000138 4.06 72.53 17.85
2-aminophenol 6.29 1.80 6.36E-05 1.48 0.37 0.25
4-hydroxyphenylpyruvate 7.29 1.79 4.36E-05 1.54 0.18 0.12
Glycocyamine 5.33 1.79 3.15E-05 1.55 63.98 41.17
Pyruvic acid 6.61 1.79 1.16E-05 2.11 15.67 7.43
Alpha-ketoglutaric acid 15.24 1.78 2.74E-05 1.94 3.47 1.79
Adenine 13.03 1.77 5.35E-05 1.42 0.63 0.45
Cystine 10.15 1.74 0.000397 2.26 7.51 3.32
3-phosphoglycerate 21.86 1.71 0.000268 7.19 0.18 0.03
Lactic acid 6.79 1.70 6.43E-05 1.61 122.74 76.20
Orotic acid 14.33 1.68 0.001001 1.86 39.70 21.31
Arachidonic acid 29.49 1.67 0.000185 1.60 1.23 0.77
5,6-dihydrouracil 5.32 1.66 6.17E-05 4.79 10.23 2.14
Pipecolinic acid 11.70 1.64 0.00132 1.79 6.05 3.39
Cyclohexylamine 13.98 1.64 0.001352 2.03 0.59 0.29
3-cyanoalanine 12.52 1.63 0.000517 1.30 1.91 1.48
22-ketocholesterol 14.38 1.62 0.000278 3.08 3.43 1.11
Citrulline 22.24 1.61 0.00031 1.57 22.47 14.31
Cytidine-5’-monophosphate 11.63 1.60 0.000431 1.65 0.12 0.07
Allose 17.69 1.59 0.000593 1.39 0.20 0.14
Uridine monophosphate 33.93 1.57 0.00283 1.86 0.25 0.14
Glucose-6-phosphate 29.91 1.56 0.006783 3.79 9.91 2.62
Thymidine 5’-monophosphate 33.90 1.54 0.002133 1.61 2.07 1.29
4-hydroxycinnamic acid 25.75 1.52 0.003806 1.88 0.28 0.15
Iminodiacetic acid 8.27 1.50 0.002676 1.37 2.00 1.46
Phthalic acid 12.16 1.49 0.001877 1.32 0.07 0.05
Cis-1,2-Dihydronaphthalene-1,2-diol 8.25 1.48 0.005657 1.46 2.94 2.01
Pyrrole-2-carboxylic acid 12.09 1.47 0.013506 2.07 0.48 0.23
Mono(2-ethylhexyl)phthalate 13.10 1.46 0.000384 7.81 0.95 0.12
3-Methylamino-1,2-propanediol 5.85 1.45 0.00342 1.21 21.56 17.75
Norleucine 7.01 1.44 0.007905 1.32 1.71 1.30
Inosine 5’-monophosphate 34.45 1.44 0.000665 1.70 3.22 1.89
Histidine 24.11 1.43 0.004787 1.45 84.98 58.69
Erythrose 14.26 1.42 0.003387 1.28 8.43 6.59
Aminooxyacetic acid 8.15 1.42 0.004643 1.25 30.47 24.34
2-hydroxybiphenyl 9.57 1.41 0.004594 191091.12 0.19 0.00
Phosphate 10.52 1.38 0.006328 1.24 277.33 223.24
N-Acetyl-L-glutamic acid 15.42 1.38 0.020769 2.54 0.93 0.37
Ribulose-5-phosphate 27.37 1.36 0.003938 2.33 1.72 0.74
8-Aminocaprylic acid 8.40 1.33 0.009031 1.21 0.96 0.79
N-ethylglycine 5.17 1.32 0.008838 1.16 254.96 219.80
Urea 10.13 1.32 0.030652 2.61 23.41 8.97
Pantothenic acid 25.48 1.29 0.021091 1.35 1.53 1.14
Guanosine-5’-monophosphate 35.19 1.25 0.004066 3.19 0.15 0.05
Xylitol 17.55 1.25 0.009676 1.47 0.54 0.37
Isoxanthopterin 27.82 1.25 9.86E-05 13.57 0.53 0.04
Inosine 32.38 1.24 0.018998 1.09 16.73 15.28
Dl-anabasine 11.24 1.21 0.008957 1.33 0.89 0.67
2-aminoethanethiol 5.24 1.20 0.01881 1.14 24.12 21.16
2-Hydroxyvaleric acid 11.09 1.19 0.019717 163543.75 0.16 0.00
Farnesol 5.48 1.16 0.025652 1.13 1.07 0.95
Hypoxanthine 21.81 1.16 0.029335 1.19 36.52 30.70
1,2-cyclohexanedione 13.51 1.16 0.001477 4.88 0.37 0.08
P-benzoquinone 5.82 1.15 0.025106 1.14 39.28 34.40
N-Acetyl-L-aspartic acid 26.35 1.15 0.013151 1.66 0.37 0.22
S-carboxymethylcysteine 15.15 1.12 0.02992 1.12 3.84 3.42
Ribose-5-phosphate 27.43 1.12 0.035608 1.84 1.48 0.81
Sarcosine 8.09 1.11 0.042995 1.26 13.51 10.71
(2R)-2-amino-3-phosphonopropanoic acid 17.32 1.09 0.026425 1.74 0.34 0.19
2-ketobutyric acid 5.65 1.04 0.048558 1.11 2.84 2.56
Fructose-6-phosphate 29.89 1.01 0.00163 6.38 13.33 2.09

RT (min): retention time. VIP is from OPLS DA model, and the larger the VIP, the greater the contribution of the variable to the grouping. P value is used to evaluate
whether the difference between the two groups of samples is significant, and the p < 0.05 indicates significant difference, and the p < 0.01 indicates highly significant
difference. FC: The ratio of the average expression of metabolites in the two groups of samples, and FC > 1 indicates up-regulated metabolites, and FC < 1 indicates
down-regulated metabolites.

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Table 3
Down-regulated metabolites in gills of Scylla paramamosain response to sudden drop in salinity from 23‰ to 3‰.
Metabolites RT(min) VIP P-Value FC(P/CK) AVG(LS) AVG(CK)

Oxoproline 11.30 1.16 0.025298 0.88 403.80 458.58

Glutamic acid 16.15 1.35 0.006332 0.83 257.62 310.76
3-methyloxindole 18.32 1.14 0.036717 0.83 0.41 0.50
Dehydroabietic acid 12.04 1.17 0.02514 0.82 1.68 2.04
Valine 9.46 1.37 0.007873 0.81 173.50 214.27
Perillyl alcohol 10.77 1.36 0.004272 0.75 0.44 0.58
O-phosphonothreonine 14.58 1.61 0.00064 0.74 8.88 11.92
D-Glucoheptose 34.73 1.35 0.008189 0.74 1.91 2.59
3-hydroxypyruvate 9.03 1.52 0.000915 0.70 0.73 1.04
Guaiacol 19.53 1.34 0.013877 0.70 0.44 0.63
3,5-dihydroxyphenylglycine 8.65 1.44 0.00569 0.69 0.77 1.12
N-Methyl-L-glutamic acid 6.87 1.03 0.041417 0.68 8.77 12.91
Myo-inositol 27.11 1.74 0.000184 0.68 5.55 8.20
2’-Deoxycytidine 5’-triphosphate degr prod 5.68 1.88 4.2E-07 0.67 122.49 181.50
Maleamate 9.26 1.35 0.013625 0.66 22.29 33.72
Glycine 11.21 1.78 1.86E-05 0.66 73.70 112.28
N-2-fluorenylacetamide 25.79 1.36 0.004191 0.65 0.25 0.39
2,6-Diaminopimelic acid 12.60 1.06 0.04462 0.65 3.24 4.97
2-Keto-L-gulonic acid 13.88 1.20 0.025482 0.64 0.33 0.51
Beta-Glutamic acid 17.80 1.27 0.022455 0.64 10.86 17.01
Glycolic acid 7.05 1.86 1.38E-06 0.64 0.31 0.48
Picolinic acid 11.58 1.61 0.000203 0.63 18.60 29.54
Tagatose 23.35 1.65 0.000916 0.63 7.75 12.34
Cytosin 27.08 1.55 0.001997 0.63 1.04 1.66
Fructose 23.13 1.62 0.001224 0.62 10.53 16.87
Ethanolamine 10.42 1.92 5.94E-06 0.62 74.30 119.47
Biphenyl 5.99 1.62 0.000334 0.62 3.44 5.54
N-Acetyl-D-galactosamine 26.96 1.60 0.000787 0.62 6.03 9.75
Alpha-Aminoadipic acid 19.98 1.13 0.025667 0.62 10.29 16.73
Lysine 24.19 1.52 0.005114 0.58 105.38 180.81
Gluconic acid 25.60 1.65 0.000418 0.58 2.41 4.15
Guanosine 33.62 1.74 3.79E-05 0.58 2.02 3.48
1-kestose 21.78 1.06 0.03223 0.58 8.56 14.83
Phloroglucinol 13.16 1.15 0.022758 0.57 1.09 1.90
4-Hydroxymandelic acid 11.99 1.74 2.05E-05 0.57 0.31 0.53
Behenic acid 32.88 1.12 0.024121 0.57 0.28 0.50
Isoleucine 10.88 1.62 0.0022 0.56 90.77 161.00
Ribose 16.08 1.48 0.008056 0.56 16.43 29.28
Dehydroascorbic acid 27.21 1.56 0.000962 0.56 4.53 8.09
Tes 17.20 1.68 0.000573 0.53 3.12 5.90
2’-deoxyguanosine 33.69 1.54 0.003502 0.52 2.85 5.45
2-Deoxy-D-galactose 16.86 1.33 0.012595 0.52 0.73 1.42
Erythrose 4-phosphate 34.79 1.35 0.007189 0.51 0.03 0.06
1-Aminocyclopropanecarboxylic acid 9.61 1.41 0.003649 0.50 0.11 0.22
Glucuronic acid 25.96 1.85 2.7E-05 0.48 1.26 2.60
DL-dihydrosphingosine 25.69 1.27 0.010055 0.48 0.53 1.12
Octacosanoic acid methyl ester 34.74 1.07 0.01516 0.47 0.34 0.73
D-Glyceric acid 11.66 1.51 0.004331 0.46 0.17 0.37
Urocanic acid 14.78 1.83 2.68E-05 0.46 7.89 17.24
Indole-3-acetamide 8.64 1.93 9.07E-06 0.46 5.05 11.04
Uridine 31.30 1.85 5.82E-05 0.44 7.17 16.42
Phosphoglycolic acid 17.40 1.55 5.68E-05 0.41 0.25 0.61
Alpha-D-glucosamine 1-phosphate 34.33 1.07 0.001232 0.41 0.21 0.51
N-Methylanthranilic acid 16.71 1.83 4.18E-05 0.41 0.22 0.54
2-Amino-3-methyl-1-butanol 28.24 1.33 0.010127 0.40 0.58 1.43
Acenaphthenequinone 8.54 1.27 0.002988 0.40 0.11 0.28
Raffinose 32.70 1.35 0.032189 0.38 0.24 0.64
Pyridine-2,3-dicarboxylic acid 21.76 1.94 2.82E-07 0.36 0.46 1.28
Ribitol 20.11 1.72 0.00396 0.35 0.50 1.44
Hydrocinnamic acid 11.44 1.34 0.048809 0.31 0.04 0.12
Palatinose 26.72 1.96 9.49E-06 0.29 0.30 1.03
Purine riboside 32.57 1.14 2.12E-05 0.29 0.05 0.19
Aminomalonic acid 14.63 1.97 2.11E-05 0.29 11.08 38.44
Methyl palmitoleate 19.60 1.07 0.026764 0.27 0.03 0.09
D-alanyl-D-alanine 13.34 1.91 4.29E-07 0.26 7.10 27.09
5-dihydrocortisol 31.79 1.32 0.039987 0.24 0.01 0.03
Salicyl alcohol 32.76 1.13 0.005464 0.17 0.04 0.24
Quinoline-4-carboxylic acid 26.27 1.13 0.030568 0.08 0.01 0.12
Spermine 28.05 1.72 1.13E-06 0.08 0.05 0.59
Cytidine-monophosphate degr prod 28.45 1.61 0.00175 0.00 0.00 0.05
1-Hydroxy-2-naphthoic acid 17.06 1.65 0.002375 0.00 0.00 0.06
Trans,trans-Muconic acid 27.91 1.85 0.000699 0.00 0.00 0.08
Methyl octadecanoate 22.36 2.10 3.19E-08 0.00 0.00 0.18
Halostachine 5.31 1.42 0.006832 0.00 0.00 4.15

6
H. Yao et al.
RT (min): retention time. VIP is from OPLS DA model, and the larger the VIP, the greater the contribution of the variable to the grouping. P value is used to evaluate
whether the difference between the two groups of samples is significant, and the p < 0.05 indicates significant difference, and the p < 0.01 indicates highly significant
difference. FC: The ratio of the average expression of metabolites in the two groups of samples, and FC > 1 indicates up-regulated metabolites, and FC < 1 indicates
down-regulated metabolites.
One of most important steps of this study was to examine the quality
of the data. The use of quality control (QC) samples is required to obtain
reliable and high-quality results (Araújo et al., 2018; Stefanuto et al.,
2015). The principal component analysis (PCA) showed that with the
exception of one outlier sample, all the other samples are within the
confidence interval of 95 %. Quality control (QC) sample aggregation
indicates the obtained data is reliable and stable. The cumulative
interpretation rate (R2X (cum)) is up to 0.528, indicating that the PCA
model can be obtained to show the actual distribution of the sample.
Therefore, the PCA result reveal the CK group and the LS group had a
relatively obvious distribution rule (Fig. 2A & Table 1). Meanwhile,
OPLS-DA model shows the difference between the two groups is sig­
nificant (x-axis orientation), and all sample points are within the 95 %
confidence interval (Fig. 2B & Table 1). Through the fitting examination
of the OPLS-DA model, it can be found that the model’s interpretation
ability (R2Y (cum)) and predictive ability (Q2 (cum)) for the samples
were 0.997 and 0.915, respectively. Besides, the slope of the straight line
is large, and the intercept of Q2 is -0.224, indicating that the OPLS-DA
model does not exceed the fitting (Fig. 2C & Table 1). In conclusion,
the OPLS-DA model has good interpretation and prediction ability, and
can reflect the difference between different sample groups in real
response. All of above results showed that the metabolites of
S. paramamosain gills at 3‰ salinity changed obviously.
3.2. Screening of differential metabolites
Using multi-dimensional analysis and single-dimensional analysis,
combined with heat map to screen the metabolites between the LS group
and the CK group. The screening criteria were P value <0.05 and VIP
value> 1, combined with heat map analysis (Fig. 4). The results showed
that a total of 145 different metabolites were screened, including 71 up-
regulated metabolites (Fig. 3A & Table 2) and 74 down-regulated me­
tabolites (Fig. 3B & Table 3).
As we all know, amino acids play a key role in osmotic regulation of
aquatic animals.Among the 145 metabolic differences, including 14
common amino acids, taurine (VIP = 1.96, FC = 1.58), L-homoserine
(VIP = 1.65, FC = 1.88), asparagine (VIP = 1.81, FC = 1.72), aspartic
acid (VIP = 1.81, FC = 1.81), cystine (VIP = 1.74, FC = 2.26), citrulline
(VIP = 1.61, FC = 1.57), norleucine (VIP = 1.44, FC = 1.32), histidine
(VIP = 1.43, FC = 1.45) and sarcosine (VIP = 1.11, FC = 1.26), they are
up-regulated (FC > 1, VIP > 1). In particular, taurine’s VIP value rea­
ches 1.96, which is the highest VIP amino acid among all the up-
regulated differential metabolites, and is also the most critical osmotic
adjustment substance reported so far (Fig. 4 & Table 2); glutamic acid
(VIP = 1.35, FC = 0.83), valine (VIP = 1.37, FC = 0.81), glycine
(VIP = 1.78, FC = 0.66), lysine (VIP = 1.52, FC = 0.58) and isoleucine
(VIP = 1.62, FC = 0.56) five amino acids content lower content than the
control group (FC < 1), which is a down-regulated adjustment (Fig. 4 &
Table 3).
At the same time, osmotic regulation is a process of oxygen con­
sumption. Polysaccharides are important participants in energy meta­
bolism and also the source of energy. The 145 differential metabolites
contain polysaccharides involved in energy metabolism, among which
up-regulated fructose-6-phosphate (VIP = 1.01, FC = 6.38), glucose-6-
phosphate (VIP = 1.56, FC = 3.79) participate in TCA Cyclic reaction
and allose (VIP = 1.59, FC = 1.39), erythrose (VIP = 1.42, FC = 1.28),
ribulose-5-phosphate (VIP = 1.36, FC = 2.33) and ribose-5-phosphate
(VIP = 1.12, FC = 1.84); however, fructose (VIP = 1.62, FC = 0.62),
tagatose (VIP = 1.65, FC = 0.63) and ribose (VIP = 1.48, FC = 0.65),
etc. showed a downward adjustment in content.
In addition, fumaric acid (VIP = 1.94, FC = 3.08) and pyruvic acid
7
Aquaculture Reports 18 (2020) 100533
(VIP = 1.79, FC = 2.11), as well as the stress response substance lactic
acid (VIP = 1.70, FC = 1.61) were found in the metabolites, they are all
up-regulated, fumaric acid is the highest VIP value of all organic acids.
In addition to the above-mentioned up-regulated differential me­
tabolites, the differential metabolites in the top 20 of VIP values also
include：phosphomycin(VIP = 1.92,FC = 2.28)、threo-beta-hydrox­
yaspartate (VIP = 1.89, FC = 3.32)、2-hydroxybutanoic acid
(VIP = 1.88, FC = 1.61)、adenosine-5-monophosphate (VIP = 1.85,
FC = 5.45)、N-alpha-Acetyl-L-ornithine (VIP = 1.84, FC = 1.75)、2-
aminophenol (VIP = 1.80, FC = 1.48)、4-hydroxyphenylpyruvate
(VIP = 1.79, FC = 1.54)、glycocyamine (VIP = 1.79, FC = 1.55)、
alpha-ketoglutaric acid (VIP = 1.78, FC = 1.94)、adenine (VIP = 1.77,
FC = 1.42)、3-phosphoglycerate(VIP = 1.71, FC = 7.19) (Fig. 3A &
Table 2).
In addition to the above-mentioned down-regulated differential
metabolites, the top 20 differential metabolisms in VIP value also
include: methyl octadecanoate (VIP = 2.10, FC = 0)、aminomalonic
acid (VIP = 1.97, FC = 0.29)、palatinose (VIP = 1.96, FC = 0.29)、
pyridine-2,3-dicarboxylic acid (VIP = 1.94, FC = 0.36)、indole-3-acet­
amide (VIP = 1.93, FC = 0.46)、ethanolamine (VIP = 1.92,
FC = 0.62)、D-alanyl-D-alanine (VIP = 1.91, FC = 0.26)、2’-Deoxy­
cytidine 5’-triphosphate degr prod (VIP = 1.88, FC = 0.67)、glycolic
acid (VIP = 1.86, FC = 0.64)、uridine (VIP = 1.85, FC = 0.44)、trans,
trans-Muconic acid (VIP = 1.85, FC = 0)、glucuronic acid (VIP = 1.85,
FC = 0.48)、urocanic acid (VIP = 1.83, FC = 0.46)、N-Methylan­
thranilic acid (VIP = 1.83, FC = 0.41)、myo-inositol (VIP = 1.74,
FC = 0.68)、4-Hydroxymandelic acid (VIP = 1.74, FC = 0.57)、gua­
nosine (VIP = 1.74, FC = 0.57)、ribitol (VIP = 1.72, FC = 0.35)、
spermine (VIP = 1.72,FC = 0.08) (Fig. 3B & Table 3).
3.3. Analysis of metabolic pathways of different metabolites
Through the analysis of metabolic pathway enrichment of differen­
tial metabolites, a total of 57 metabolic pathways were obtained, of
which 29 showed significant differences (P < 0.05) and 28 showed non-
significant differences (P > 0.05). The 29 significant differential meta­
bolic pathways are classified into amino acid metabolism/metabolism of
other amino acids (glycine, serine and threonine metabolism; arginine
and proline metabolism; alanine, aspartate and glutamate metabolism,
etc.), carbohydrate metabolism (pentose phosphate pathway; citrate
cycle; galactose metabolism, etc.), metabolism of cofactors and vitamins
(pantothenate and CoA biosynthesis; nicotinate and nicotinamide
metabolism; vitamin B6 metabolism, etc.), nucleotide metabolism (pu­
rine metabolism and pyrimidine metabolism), energy metabolism
(methane metabolism and nitrogen metabolism), translation (amino­
acyl-tRNA biosynthesis), membrane transport (ABC transporters)
(Fig. 5A & Table 4). In the classification of 28 non-significant metabo­
lites, carbohydrate metabolism (inositol phosphate metabolism; buta­
noate metabolism and starch and sucrose metabolism, etc.), amino acid
metabolism/Metabolism of other amino acids (phenylalanine, tyrosine
and tryptophan biosynthesis; valine, leucine and isoleucine degradation;
tyrosine metabolism, etc.), lipid metabolism (biosynthesis of unsatu­
rated fatty acids; linoleic acid metabolism; glycerolipid metabolism,
etc.), metabolism of cofactors and vitamins (thiamine metabolism;
biotin metabolism; porphyrin and chlorophyll metabolism, etc.)), signal
transduction (mTOR signaling pathway and phosphatidylinositol
signaling system), energy metabolism (oxidative phosphorylation and
sulfur metabolism), metabolism of terpenoids and polyketides (terpe­
noid backbone biosynthesis), signaling molecules and interaction
(neuroactive ligand-receptor interaction), metabolism of terpenoids and
polyketides (limonene and pinene degradation) and Other (biosynthesis
H. Yao et al. Aquaculture Reports 18 (2020) 100533

Fig. 4. Heat map of differential metabolites in LS group and CK group. Each gill sample is visualized in a column, and each metabolite is represented by a row. Green
indicates a lower metabolite concentration, while red indicates a higher metabolite level (refer to color scale).

8
H. Yao et al.
Fig. 5. Differential metabolite classification (A) and the pie chart (B) of the differential metabolite classification in the gill tissues of the Scylla paramamosain
adapting to salinity sudden drop.
Table 4
Significantly different metabolic pathway classification.
Class Types of metabolic pathways
Glycine, serine and threonine
metabolism
Arginine and proline metabolism
Alanine, aspartate and glutamate
metabolism
Cysteine and methionine
metabolism
Valine, leucine and isoleucine
biosynthesis
Amino acid pathway/Metabolism of other
Phenylalanine metabolism
amino acids
Histidine metabolism
Glyoxylate and dicarboxylate
metabolism
Lysine degradation
beta-Alanine metabolism
Taurine and hypotaurine
metabolism
Glutathione metabolism
Cyanoamino acid metabolism
Pentose phosphate pathway
Citrate cycle (TCA cycle)
Galactose metabolism
Carbohydrate metabolism Pentose and glucuronate
interconversions
Ascorbate and aldarate metabolism
Propanoate metabolism
Pantothenate and CoA biosynthesis
Nicotinate and nicotinamide
Metabolism of cofactors and vitamins metabolis
Vitamin B6 metabolism
Riboflavin metabolism
Purine metabolism
Nucleotide metabolism
Pyrimidine metabolism
Methane metabolism
Energy metabolism
Nitrogen metabolism
Membrane transport ABC transporters
Translation Aminoacyl-tRNA biosynthesis
of secondary metabolites) (Fig. 5B & Table 5).
From the above analysis, it is found that these significant differential
metabolic pathways mainly involve amino acid metabolism, energy
metabolism and so on.The top 10 significant differential metabolic
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Aquaculture Reports 18 (2020) 100533
Table 5
Classification of non-significant differential metabolic pathways.
Class Types of metabolic pathways
Inositol phosphate metabolism
Butanoate metabolism
Starch and sucrose metabolism
Carbohydrate metabolism Glycolysis / Gluconeogenesis
Pyruvate metabolism
Amino sugar and nucleotide sugar
metabolism
Phenylalanine, tyrosine and tryptophan
biosynthesis
Valine, leucine and isoleucine
Amino acid metabolism/Metabolism degradation
of other amino acids Tyrosine metabolism
Tryptophan metabolism
D-Glutamine and D-glutamate
metabolism
Biosynthesis of unsaturated fatty acids
Linoleic acid metabolism
Lipid metabolism Glycerolipid metabolism
Glycerophospholipid metabolism
Arachidonic acid metabolism
Thiamine metabolism
Biotin metabolism
Metabolism of cofactors and vitamins Ubiquinone and other terpenoid-
quinone biosynthesis
Porphyrin and chlorophyll metabolism
mTOR signaling pathway
Signal transduction
Phosphatidylinositol signaling system
Oxidative phosphorylation
Energy metabolism
Sulfur metabolism
Metabolism of terpenoids and
Terpenoid backbone biosynthesis
polyketides
Signaling molecules and interaction Neuroactive ligand-receptor interaction
Metabolism of terpenoids and
Limonene and pinene degradation
polyketides
Others Biosynthesis of secondary metabolites
pathways (P < 0.01) are glycine, serine and threonine metabolism
(Pyruvic acid, glycine, 6-phosphogluconic acid, etc.), pentose phosphate
pathway (pyruvic acid, ribose-5-phosphate, gluconic acid, etc.), argi­
nine and proline metabolism (pyruvic acid, aspartic acid, Asparagine,
etc.), ABC transporters (phosphate, histidine, taurine, etc.), Alanine,
H. Yao et al.
aspartate and glutamate metabolism (aspartic acid, fumaric acid,
asparagine, etc.), purine metabolism (glycine, ribose-5-phosphate,
adenine, etc.), beta-Alanine metabolism (aspartic acid, histidine, pan­
tothenic acid, etc.), pantothenate and CoA biosynthesis (pyruvic acid,
aspartic acid, valine, etc.), aminoacyl-tRNA biosynthesis (glycine, his­
tidine and asparagine, etc.), pyrimidine metabolism (glycine, ribose-5-
phosphate and inosine, etc.) (Fig. 6 A & Table 6). The above results
found that 50 % of the amino acid metabolism-related pathways are
mainly involved in the regulation of amino acid metabolism such as
glycine, L-homoserine, aspartic acid, citrulline, sarcosine, histidine,
asparagine, valine, and isoleucine. Others include carbohydrate meta­
bolism, metabolism of cofactors and vitamins, nucleotide metabolism,
membrane transport, translation, the included pathways are involved in
the sugar metabolism of ribose-5-phosphate and ribose, and the regu­
lation of organic acid metabolism such as pyrucic acid and fumaric acid,
and taurine, Sarcosine, citrulline, aspartic acid, asparagine, histidine,
valine, and isoleucine are involved in the regulation of amino acid
metabolism.
4. Discussion
Salinity is an important ecological factor that affects the distribution,
abundance, and physiology of aquatic crustaceans. Changes in envi­
ronmental salinity are closely related to osmoregulation of aquatic
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Aquaculture Reports 18 (2020) 100533
crustaceans. Osmoregulation is an important physiological process in
aquatic animals (Romano and Zeng, 2012). As such, the osmoregulation
mechanism of aquatic crustaceans is a hot research topic. Studies of the
osmoregulatory organs of S. paramamosain have focused on morphology
(Wang et al., 2018a, b), ion transport regulation (Furriel et al., 2000),
hemolymph osmotic pressure regulation (Wang et al., 2004), gene
regulation (Yu et al., 2014; Wang et al., 2018a, 2018b), and proteome
data (Wang et al., 2018b, 2019), but studies at the level of metabolism
are rare.
In this study, we used GC–MS to investigate the metabolic mecha­
nism of osmoregulation in the gills of S. paramamosain exposed to a
sudden salinity decrease. We found that the levels of the following nine
amino acids in the acute low-salt group were up-regulated: taurine, L-
homoserine, asparagine, aspartic acid, cystine, citrulline, norleucine,
histidine, and sarcosine (Fig. 5, Table 2). In contrast, the levels of glu­
tamic acid, valine, glycine, lysine, and isoleucine were down-regulated
(Fig. 5, Table 3). These results indicate that free amino acids are
important osmoregulatory substances used by crustaceans under salinity
stress (Fang et al., 1992; Huong et al., 2001). Among the nine
up-regulated amino acids, taurine and aspartic acid are known to be
important osmoregulatory factors in Litopenaeus vannamei (Huang et al.,
2010) and Eriocheir sinensis (Wang et al., 2004; Long et al., 2018).
Further, histidine is an important osmoregulatory factor in the Cteno­
pharyngodon idellus (Gao et al., 2016). These results suggest that taurine,
Fig. 6. Enrichment map of the top 10 meta­
bolic pathways involved in regulating dif­
ferential metabolic differences (a) and
Taurine and hypotaurine metabolism KEGG
pathway (b). In a, the red line indicates that
the p-value is 0.01, and the blue line indicates
that the p-value is 0.05. When the top of the bar
is higher than the blue or red line, the signal
path it represents is significant. In b, "￫" in­
dicates the reaction direction, the small box
indicates the enzyme activity, the small circle
indicates the compound, the red indicates the
up-regulated compound, the blue indicates the
down-regulated compound, the large round box
indicates the other metabolic pathway, and the
dotted arrow indicates the relationship with
other metabolic pathways Relationship, small
green squares indicate enzymes unique to the
species.
H. Yao et al.
Table 6
Different metabolites regulated by the top 10 metabolic pathways.
Metabolic pathways
Glycine, serine and threonine
metabolism
Pentose phosphate pathway
Arginine and proline metabolism
ABC transporters
Alanine,aspartate and glutamate
metabolism
Purine metabolism
beta-Alanine metabolism
Pantothenate and CoA
biosynthesis
Aminoacyl-tRNA biosynthesis
Aquaculture Reports 18 (2020) 100533
Table 6 (continued )
Metabolic pathways Differential metabolites regulated by
Differential metabolites regulated by metabolic pathways
metabolic pathways
Valine (0.810)
Pyruvic acid (2.110)* Isoleucine (0.564)
Glycine (0.656) 6-phosphogluconic acid (3.051)
Aspartic acid (4.063) cytidine-5’-monophosphate (1.653)
2-ketobutyric acid (1.113) Urea (2.610)
3-Hydroxypyruvate (0.701) Uridine monophosphate (1.863)
Sarcosine(1.262) Pyrimidine metabolism Orotic acid (1.863)
D-Glyceric acid (0.461) Uridine (0.437)
L-Homoserine (1.649) Thymidine 5’-monophosphate (1.608)
Glycocyamine (1.554) 5,6-dihydrouracil (4.786)
6-phosphogluconic acid (3.051)
Note: * represents the FC value of the metabolite.
Pyruvic acid (2.110)
Ribose-5-phosphate (1.836)
Ribose (0.561) histidine, and aspartic acid are important osmoregulatory substances in
Ribulose-5-phosphate (2.328) S. paramamosain, but little is known about their metabolic mechanism
Gluconic acid (0.582)
D-Glyceric acid (0.461)
and key functions. Although the exact roles of L-homoserine, asparagine,
Erythrose-4-phosphate (0.512) cystine, citrulline, norleucine, and sarcosine remain unclear, the results
1,2-Cyclohexanedione (4.876) of our study indicate that they are also involved in osmoregulation in
Pyruvic acid (2.110) S. paramamosain. Among the down-regulated amino acids, glycine,
Aspartic acid (4.063)
valine, glutamic acid, and lysine (Somero and Bowlus, 1983; Huong
Urea (2.610)
Fumaric acid (3.085) et al., 2001; Lu et al., 2015) were also important osmoregulatory sub­
Sarcosine (1.262) stances. In particular, glycine, as the simplest amino acid, participates in
Citrulline (1.571) carbohydrate metabolism and the tricarboxylic acid (TCA) cycle (Berg­
N-alpha-Acetyl-L-ornithine (1.752) man and Ericson, 2006). During osmoregulation and energy consump­
Glycocyamine (1.554)
N-Acetyl-L-glutamic acid (2.544)
tion, glycine is very likely to be used for energy metabolism, which likely
Spermine (0.078) explains why it was down-regulated in our study. To our knowledge,
Cytosin (0.627) there are no reports on the role of isoleucine in osmoregulation.
Phosphate (1.242) Crustacean osmoregulation is an energy consuming process (Tantulo
Glycine (0.656)
and Fotedar, 2006; Ye et al., 2009; Wang et al., 2018a). Results of this
Lysine (0.583)
Aspartic acid (4.063) study showed that glucose 6-phosphate, fructose 6-phosphate, and
Urea (2.610) ribose 5-phosphate were up-regulated in the acute low-salt group,
Ribose (0.561) whereas ribose, fructose, and erythrose were down-regulated; all of
Histidine(1.448) these are involved in glucose metabolism and energy metabolism. Up-
Valine (0.810)
Taurine (1.581)
and down-regulation of sugars may be related to dynamic energy
Isoleucine (0.564) changes in S. paramamosain in response to acute low salinity. Addi­
Allose (1.390) tionally, we found that some organic acids related to energy metabolism
Pyruvic acid(2.110) were up-regulated, including fumaric acid, pyruvic acid, and lactic acid.
Alpha-ketoglutaric acid ()
Pyruvate and lactic acid are intermediates of glycolysis (Rocha et al.,
Aspartic acid (4.063)
Fumaric acid (3.085) 2011), and fumaric acid is involved in the TCA cycle (Ma et al., 2017).
Asparagine (1.723) Pyruvate enters the TCA cycle after being converted to Acetyl-CoA by
N-acetyl-L-aspartic acid (1.662) dehydrogenase, and lactic acid enters it after being converted into py­
Adenosine 5-monophosphate (5.451) ruvate as catalyzed by lactate dehydrogenase. The level of lactic acid
Glycine (0.656)
Urea (2.610)
reflects the level of anaerobic metabolism (Venkatesh and Ramalingam,
Ribose-5-phosphate (1.836) 2007). The gill is the respiratory organ of crustaceans, and gill filaments
Inosine-5’-monophosphate (1.705) may be damaged by low-salinity. This damage affects respiratory
Guanosine-5’-monophosphate (3.187) metabolism (Lucu, 1990; Wang et al., 2018a, b) and hinders the con­
Adenine (1.418)
version of non-sugar substances into sugar, causing lactic acid and py­
Hypoxanthine (1.189)
Inosine (1.095) ruvate accumulation in the gills. Glycolysis involving lactic acid and
2’-deoxyguanosine (0.523) pyruvate is an irreversible process, so glycolysis in the gills leads to
Guanosine (0.580) generation of a large amount of pyruvate and lactic acid.
Aspartic acid (4.063) In this study, KEGG enrichment analysis revealed 57 metabolic
Histidine (1.448)
5,6-dihydrouracil (4.786)
pathways, among which 29 were significantly different (P < 0.05). A
series of amino acids, including taurine, glycine, L-homoserine, aspartic
Spermine (0.078)
pantothenic acid (1.347) acid, citrulline, sarcosine, histidine, asparagine, valine, and isoleucine,
pyridine-2,3-dicarboxylic acid(0.361) are metabolism regulation pathways. An analysis of the adaptation
Pyruvic acid (2.110)
mechanism of S. paramamosain to salinity stress showed that taurine had
Aspartic acid (4.063)
Valine (0.810) the highest VIP value, indicating that taurine is the most important
osmoregulation substance in S. paramamosain. Taurine also is an
5,6-dihydrouracil (4.786)
Pantothenic acid (1.347) important osmoregulatory substance in other marine animals (Vin­
Glycine (0.656) centmarique and Gilles, 1970; Huxtable, 1992; Daikoku, 2006). Among
Lysine (0.583)
the 29 significantly differentially expressed metabolic pathways, taurine
Aspartic acid (4.063)
Histidine (1.448) and hypotaurine metabolism (P < 0.05) and ABC transporters were
Asparagine (1.723) involved in the regulation of taurine metabolism. In this study, taurine
and hypotaurine metabolism directly regulated the synthesis of taurine,
11
H. Yao et al.
which significantly increased the taurine level in the salinity group
(Fig. 6 B). ABC transporters are part of membrane transport, which is
one of the largest known protein families. The main function of ABC
transporters involves the active transport of small molecules (Ulf-Ingo &
Meer, 2006). Transcriptome- and proteome-based analysis conducted by
Wang et al. (2018a, b, Wang et al., 2018c showed that the taurine
transporter gene was significantly up-regulated during adaptation of
S. paramamosain to a sudden decrease in salinity. These results suggest
that under a sudden decrease in salinity, S. paramamosain synthesizes a
large amount of taurine through taurine and hypotaurine metabolism,
which is then transported extracellularly via ABC transporters, thereby
regulating extracellular osmotic pressure. This mechanism likely plays
an extremely important role in osmoregulation of S. paramamosain.
Tantulo and Fotedar (2006) previously showed that osmoregulation
is an oxygen- and energy-consuming process. In this study, we identified
several metabolites and metabolic pathways associated with glucose and
energy metabolism, including fructose 6-phosphate, glucose 6-phos­
phate, erythrose, ribulose 5-phosphate, ribose 5-phosphate, fructose,
tagatose, and ribose. We also discovered metabolic pathways, including
the pentose phosphate pathway, citrate cycle, galactose metabolism,
pentose and glucuronate interconversions, ascorbate and aldarate
metabolism, propanoate metabolism, methane metabolism, and nitro­
gen metabolism. These metabolic pathways accounted for 27.6 % of the
up-regulated pathways found in the low salinity groups. This result also
shows that a large amount of energy is needed for osmoregulation in
S. paramamosain when adapting to a sudden decrease in salinity.
5. Conclusion
In this study, we investigated the adaptation mechanism of
S. paramamosain to a sudden decrease in salinity at the metabolic level,
based on previous studies at the transcriptome (Wang et al., 2018a),
proteome (Wang et al., 2018b), and miRNA (Wang et al., 2018c) levels.
We identified 1264 metabolites, among which 437 were differentially
expressed. Of them, 71 were up-regulated (FC > 1), including taurine,
L-homoserine, aspartic acid, fructose 6-phosphate, glucose 6-phosphate,
pyruvic acid, and lactic acid, and 74 were down-regulated (FC < 1),
including glutamic acid, valine, glycine, fructose, tagatose, and ribose.
KEGG enrichment analysis of differential metabolites identified 57
metabolic pathways, of which 29 were statistically significant
(P < 0.05), including glycine, serine, and threonine metabolism, the
pentose phosphate pathway, and ABC transporters. These metabolic
pathways were mostly the amino acid metabolism pathway, carbohy­
drate metabolism pathway, metabolism of cofactors and vitamins,
nucleotide metabolism, energy, metabolism, membrane, transport and
translation. The results of this study show that free amino acids play an
important role in adaptation to a sudden decrease in salinity and that
energy metabolism involving carbohydrates and organic acids provides
the energy supply during adaptation. This systematic analysis of the
metabolic adaptation mechanism of S. paramamosain to a sudden
decrease in salinity provides important information about the osmo­
regulation of this species that can be applied to production of
S. paramamosain in low-salt environments.
Data availability statement support
The data of the research results are mainly the composition and
content of osmotic adjustment substances. During the experiment, the
experimental errors have been reduced as much as possible and the
variables have been well controlled. The experimental data in this paper
will not be published in other papers. The author declares to be
responsible for the reliability of the data.
Ethics approval and consent to participate
The animal subjects used in the present study are crab, which are
12
Aquaculture Reports 18 (2020) 100533
invertebrates and are exempt from this requirement. S. paramamosain is
not an endangered or protected species. All animal work has been
conducted according to the relevant national and international guide­
lines. No specific permissions are required to work with invertebrates in
China. Similarly, no specific permissions are required for the collection
of S. paramamosain from sample sites because they were not collected
from protected areas of land.
Authors’ contributions
H Wang conceived and designed the study. HZ Yao, H Wang and L
Tang took samples of experimental animals. HZ Yao, X Li, L Tang, H
Wang, CL Wang, CK Mu and C Shi performed and analyzed all the other
experiments. HZ Yao and H Wang wrote the manuscript with support
from all authors. All authors read and approved the final manuscript.
Funding
This work was supported by the National Key R & D Program of
China (2019YFD0900405); Basic Public Welfare Research Program of
Zhejiang Province (No. LY20D060001), Natural Science Foundation of
Ningbo city (2019A610417), Major Sci & Tech Special Project of Zhe­
jiang Province (no: 2016C02055-8), Ministry of Agriculture of China &
China Agriculture Research System (no: CARS-48), the K. C. Wong
Magna Fund in Ningbo University. The funders had no role in the study
design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Consent for publication
“Not applicable”.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
Aside from funding support, we also thank oebiotech (oebiotech,
Shanghai, China) for sequencing consultation and support.
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