Strategy of Metabolic Phenotype Modulation in Portunus

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Strategy of Metabolic Phenotype Modulation in Portunus trituberculatus Exposed to Low Salinity Yangfang Ye,†,∥ Yanpeng An,‡,∥ Ronghua Li,† Changkao Mu,† and Chunlin Wang*,† †

School of Marine Sciences, Ningbo University, Ningbo 315211, China State Key Laboratory of Genetic Engineering, Biospectroscopy and Metabonomics, School of Life Sciences, Fudan University, Shanghai 200433, China

‡

S Supporting Information *

ABSTRACT: Extreme low salinity influences normal crab growth, morphogenesis, and production. Some individuals of swimming crab Portunus trituberculatus have, however, an inherent ability to adapt to such a salinity fluctuation. This study investigated the dynamic metabolite alterations of two P. trituberculatus strains, namely, a wild one and a screened (low-salinity tolerant) one in response to low-salinity challenge by combined use of NMR spectroscopy and high-throughput data analysis. The dominant metabolites in crab muscle were found to comprise amino acids, sugars, carboxylic acids, betaine, trimethylamineN-oxide, 2-pyridinemethanol, trigonelline, and nucleotides. These results further showed that the strategy of metabolic modulation of P. trituberculatus after low-salinity stimulus includes osmotic rebalancing, enhanced gluconeogenesis from amino acids, and energy accumulation. These metabolic adaptations were manifested in the accumulation of trimethylamine-N-oxide, ATP, 2-pyridinemethanol, and trigonelline and in the depletion of the amino acid pool as well as in the fluctuation of inosine levels. This lends support to the fact that the low-salinity training accelerates the responses of crabs to low-salinity stress. These findings provide a comprehensive insight into the mechanisms of metabolic modulation in P. trituberculatus in response to low salinity. This work highlights the approach of NMR-based metabonomics in conjunction with multivariate data analysis and univariate data analysis in understanding the strategy of metabolic phenotype modulation against stressors. KEYWORDS: low salinity, Portunus trituberculatus, metabonomics, nuclear magnetic resonance (NMR), metabolic phenotype

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lipids, protein, and carbohydrate metabolism16 have been revealed in Panopeus herbstii and Neohelice granulata in response to low salinity. However, little information is available regarding the metabolic mechanism on the low-salinity acclimation of P. trituberculatus, which deserves further attention. The NMR-based metabonomic analysis ought to be a suitable choice for detecting and understanding the detailed metabolite alterations in crabs responding to low-salinity challenge. This is because metabonomics systemically detects the holistic and dynamic metabolic responses of an integrated biological system to the changes of both endogenous and exogenous factors.17 Such an approach has been already successfully applied in understanding the metabolic responses to stress factors18−21 and systems metabolic reprogramming underlying the tolerance and acclimation to a harsh environment.22−24 It is particularly interesting to note that the metabonomics approach has also been proved powerful in analyzing the chemical compositions of processed products of P. trituberculatus.25 In this study, we investigated the dynamic metabolite alterations of two P. trituberculatus strains, namely, a wild one and a screened (low-salinity tolerant) one, in response to lowsalinity challenge using nuclear magetic resonance (NMR)

INTRODUCTION The swimming crab Portunus trituberculatus (Crustacea, Decapoda, Brachyura) is an important fishery species and widely cultivated on a commercial scale in China.1 P. trituberculatus is a euryhaline crab species with optimal growth salinities from 20 to 35‰. However, extreme low salinity influences normal crab growth, morphogenesis, and production.2,3 Interestingly, some individuals have an inherent ability to adapt and acclimate to such a low-salinity fluctuation. Therefore, research on the biochemical mechanisms to lowsalinity stress is of great interest for marine biologists and particularly important for crab artificial propagation. A body of evidence has elucidated a series of physiological variations at the levels of gene (DNA), transcript (mRNA), protein, and metabolite in crabs in response to salinity stress. For instance, DNA microarray analysis has demonstrated that a number of genes express differentially in P. trituberculatus after salinity challenge, such as carbonic anhydrase,4 CCAAT/ enhancer-binding protein, Na/K ATPase β-subunit, and heat shock protein genes in gills.5 The transcriptional studies have further shown that low salinity causes significant changes in the mRNA level of carbonic anhydrase,6 Na/K ATPase α-subunit, cytoplasmic carbonic anhydrase, organic action transporter, sodium/glucose cotransporter, and endomembrane protein in gills of crabs.7 Moreover, proteomics analysis has revealed that salinity exposure results in the altered activities of many enzymes such as myosin ATPase,8 alkaline phosphatase,9−11 HSPs,12 lipase,13 and asparaginase and glutaminase.14 Additionally, differential metabolic adjustments in amino acids,15 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3496

December March 14, March 23, March 24,

17, 2013 2014 2014 2014

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Figure 1. Representative 600 MHz 1H NMR spectra of muscle extracts from WG (A) and SG (B). The dotted regions were vertically expanded 32 and 2 times, respectively. Resonance assignments are given in Supplementary Table S1 in the Supporting Information. Peaks: 1, isoleucine; 2, leucine; 3, valine; 4, lactate; 5, alanine; 6, methionine; 7, glutamate; 8, glutamine; 9, α-aminoadipate; 10, succinate; 11, proline; 12, asparagine; 13, lysine; 14, arginine; 15, betaine; 16, trimethylamine-N-oxide; 17, taurine; 18, glycine; 19, β-glucose; 20, α-glucose; 21, trehalose; 22, maltose; 23, fumarate; 24, histamine; 25, histidine; 26, tyrosine; 27, phenylalanine; 28, tryptophan; 29, inosine; 30, adenosine triphosphate; 31, adenosine diphosphate; 32, 2-pyridinemethanol; 33, trigonelline; 34, sugar and amino acids. extracts, a standard water-suppressed 1H NMR spectrum named NOESYGPPR1D pulse sequence (recycle delay−G1−90°−t1−90°− tm−G2−90°−acquisition) was recorded with recycle delay of 2 s, t1 of 3 ms and tm of 100 ms. Meanwhile, the 90° pulse length was adjusted to approximately 10 μs, and 64 transients were collected into 32 K data points for each spectrum with a spectral width of 20 ppm. For NMR signal assignment purposes, a series of two-dimensional NMR spectra were acquired for selected samples and processed according to a previously reported method, including 1H−1H correlation spectroscopy (COSY), 1H−1H total correlation spectroscopy (TOCSY), 1H−13C heteronuclear single-quantum correlation (HSQC), and 1H−13C heteronuclear multiple-bond correlation spectra (HMBC).26,27 NMR Data Processing and Multivariate Data Analysis. All free induction decays were multiplied by an exponential function with a line-broadening factor of 1 Hz and zero-filled to 128 K prior to Fourier transformation. After manual phase- and baseline correction with the chemical shift referenced to TSP as δ 0.00, all 1H NMR spectra (δ 0.7−9.5) without residual water signals δ 4.70−5.15 were integrated into regions with equal widths of 0.004 ppm (2.4 Hz). All of the integral regions were normalized to wet weight of muscle for each spectrum to compensate for the overall concentration differences prior to the multivariate data analysis. Multivariate data analysis was performed with the software package SIMCA-P+ (12.0, Umetrics, Umea, Sweden). Principal component analysis (PCA) was carried out using the mean-centered NMR data to obtain an overview of group clustering and search for possible outliers. Subsequently, orthogonal projection to latent structure discriminant analysis (OPLS-DA) was conducted with six-fold cross-validation using the unit-variance scaled NMR data as X-matrix and the group information as Y-matrix. All OPLS-DA models were validated by CVANOVA approach with p < 0.05 as the significant level.28 The coefficient plots of OPLS-DA models were displayed to show changed metabolites at different post-treatment time points in both WG and SG crabs against their levels at 0 h. A cutoff value of 0.755 was used in the present study, and the metabolites with r > 0.755 or r < −0.755 were considered to be statistically significant (p < 0.05). Univariate Data Analysis. The selected metabolites with significant difference at different time points of post-treatment obtained from multivariate data analysis were further analyzed with Student’s t test (if criteria met) or nonparametric tests. The concentration of these metabolites was calculated by the integral of least-overlapping NMR signals for those metabolites as described previously,29 using SPSS 13.0 software (SPSS, Chicago, IL, USA). To illustrate the dynamic metabolite changes induced by low-salinity

spectroscopy in conjunction with high-throughput data analysis. Our aims are to understand (1) the metabolic phenotype modulation in crabs responding to low-salinity exposure and (2) the physiological advantages caused by low-salinity training.

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MATERIALS AND METHODS

Chemicals and Reagents. Methanol, sodium chloride, K2HPO4· 3H2O, and NaH2PO4·2H2O (all of analytical grade) were purchased from Sinopharm Chemical Co., Ltd. (Shanghai, China), and sodium azide was bought from Fuchen Chemical Reagent Co. (Tianjin, China). Sodium 3-trimethylsilyl [2,2,3,3-2D4]propionate (TSP-d4) and deuterated water (D2O, 99.9% D) were obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Animals and Sample Treatment. Larvae of P. trituberculatus (about 3.0 g) from a commercial farm in Xiangshan, China, were put into a salinity of 4‰ for 72 h for natural selection. The surviving individuals were collected and named the screened group (SG). The larvae crabs having no experience in low salinity were named the wild group (WG). All of the crabs were fed frozen fish once daily at 5:00− 6:00 p.m. For the low-salinity tolerance study, 30 SG crabs and 30 WG crabs with average weight of 65.3 ± 5.2 g per individual were exposed to 4‰ salinity stress. Six crabs of each group were sacrificed following the sample collection at time points of 0, 12, 24, 48, and 72 h, respectively. The muscle tissue of each crab was immediately collected, homogenized, and snap-frozen in liquid nitrogen and stored at −80 °C until metabonomic analysis. Metabolite Extraction of Crab Muscle. Each of the muscle samples (200 mg) was homogenized in 1 mL of ice-cold H2O/ methanol (1:2 v/v) extraction solution followed by sonification on wet ice for 150 cycles with each cycle consisting of a 2 s sonication and a 2 s break. Supernatant for each sample was collected respectively after 10 min of centrifugation (12000 rpm, 4 °C). The remaining solid residues of each sample were further extracted once using the above extraction procedure. After centrifugation for 10 min (12000 rpm and 4 °C), the resultant two supernatants were combined and lyophilized following removal of methanol in vacuo. The extracts were then reconstituted into 600 μL of phosphate buffer (0.1 M K2HPO4/NaH2PO4, 0.1% NaN3, 0.005% TSP, 100% D2O, pH 7.4). Following centrifugation, 550 μL of the supernatant from each extract was transferred into a 5 mm NMR tube for NMR analysis. NMR Spectroscopic Analysis. All of 1H NMR spectra of muscle extracts were acquired at 298 K on a Bruker Avance III 600 MHz spectrometer, operating at 600.13 MHz for 1H equipped with an inverse cryogenic probe (Bruker Biospin, Germany). For all of the 3497

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challenge, the ratio for metabolite concentrations in both WG and SG crabs at different time points of post-treatment were also calculated as previously described20,30 in the form of (CT − C0)/C0, where CT denotes the concentration of metabolite at the time point of posttreatment and C0 denotes the concentration of metabolite at 0 h.

are illustrated in Figure 3. The dynamic changes of representative metabolites in two P. trituberculatus strains exposed to low salinity at different time points of posttreatment are shown in Figure 4. Their corresponding correlation coefficients, which are above the cutoff value, 0.755, are listed in Table S2 in the Supporting Information. To ensure the reliability of the obtained results, the selected metabolites were further studied using univariate data analysis. Our results showed that most alterations in the metabolite levels were in agreement with those from multivariate data analysis. Interestingly, the alterations of some metabolites previously shown to be nonsignificant in multivariate data analysis presented a significant difference at the level of p < 0.05. As an example, an increase in the ATP levels of WG crabs was significant from 2.05 ± 0.54 mg/g crab muscle at 0 h to 4.97 ± 1.47 mg/g crab muscle at 12 h (p = 0.003), 5.67 ± 1.37 mg/g crab muscle at 48 h (p = 0.001), and 3.76 ± 0.79 mg/g crab muscle at 72 h (p = 0.001) (Table 1 and Table S3 in the Supporting Information). Moreover, a significant elevation in the ATP levels of SG crabs was also observed from 2.49 ± 0.17 mg/g crab muscle at 0 h to 4.75 ± 1.17 mg/g crab muscle at 12 h (p = 0.001) and 3.42 ± 0.43 mg/g crab muscle at 24 h (p = 0.001) based on the univariate data analysis. For WG crabs, 12 h of low-salinity exposure resulted in significantly reduced levels of inosine and a range of amino acids, including valine, isoleucine, leucine, alanine, histidine, and glutamine, accompanied by drastically elevated levels of trigonelline and ATP. The 24 h low-salinity challenge merely led to significantly decreased levels of succinate and inosine, as well as more amino acids including valine, isoleucine, leucine, alanine, methionine, glutamate, glutamine, lysine, histidine, tyrosine, and phenylalanine. When WG crabs were exposed to low salinity for 48 h, the levels of trigonelline, TMAO, and ATP were markedly increased, whereas the levels of valine, isoleucine, leucine, alanine, glutamine, histidine, and inosine were greatly decreased. At 72 h, crabs presented dramatically elevated metabolites including inosine, 2-pyridinemethanol (2PM), trigonelline, TMAO, and ATP along with significantly reduced metabolites including valine, leucine, alanine, methionine, and glutamine at all time points of low-salinity treatment. Conversely, low salinity caused more extensive alterations of metabolites in SG crabs relative to WG crabs. At 12 h, the levels of alanine, histidine, and inosine were significantly decreased, whereas the levels of 2-PM, trigonelline, and ATP were remarkably increased in the low-salinity-exposed SG crabs. At 24 h, SG crabs exhibited a significant decrease in the levels of inosine and a range of amino acids including valine, isoleucine, leucine, alanine, methionine, glutamate, glutamine, succinate, lysine, histidine, tyrosine, and phenylalanine accompanied with a dramatic increase in the levels of 2-PM and ATP. Similar changes of metabolites occurred at 48 h, but trigonelline and TMAO were significantly elevated instead of glutamate, lysine, and succinate, for which significant alterations were detected in 24 h low-salinity-exposed SG crabs. At 72 h, a significant elevation in the levels of 2-PM, trigonelline, and TMAO was observed in low-salinity-exposed SG crabs, together with a remarkable decline in the levels of a range of amino acids, including valine, isoleucine, leucine, alanine, methionine, glutamate, glutamine, histidine, tyrosine, and phenylalanine. The dynamic metabolite changes due to low-salinity exposure by calculating the concentration changes for those metabolites with significant differences (Table 1) are illustrated in Figure 5. A range of amino acids responded vigorously after

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RESULTS Metabolites in the Crab Muscle Extracts. Figure 1 shows representative 600 MHz 1H NMR spectra of muscle extracts obtained from WG and SG crabs. Both 1H and 13C NMR resonances for detectable metabolites were assigned according to the published results25 and further confirmed by a series of 2D NMR spectra including COSY, TOCSY, HSQC, and HMBC. The detectable metabolites of muscle extracts include a range of amino acids, sugars, carboxylic acids, trimethylamine-N-oxide (TMAO), betaine, and nucleotides (Supporting Information Table S1). To obtain more details on the metabolite alterations induced by low-salinity stress, multivariate data analysis and univariate data analysis were conducted on these NMR data. Metabolite Alterations Induced by Low-Salinity Exposure. To summarize the similarities and differences between low-salinity-induced metabolic phenotypes in WG and SG crabs, the mean PCA metabolic trajectories for each crab group were plotted in the first two principal components (Figure 2). Clearly, both WG and SG crabs presented a similar

Figure 2. PCA trajectories generated from muscle extract profiles obtained from WG and SG as a function of low-salinity exposure.

tendency of metabolic alteration in response to low-salinity stress. However, the metabolic responses of SG crabs to lowsalinity stress exhibited a more direct shift of metabolic profiles than WG crabs during the 72 h treatment. The PCA scores plots of the 1H NMR spectral data also revealed a clear difference of metabonome at each time point of post-treatment compared to 0 h in both WG and SG crabs (Supporting Information Figure S1). The metabolite alterations in WG and SG induced by low salinity were extracted using OPLS-DA models with R2X and Q2, which indicate the quality of these models. The validity of each OPLS-DA model was further evaluated with the CV-ANOVA approach (p < 0.05). OPLS-DA loadings plots were obtained to show the metabolites in muscle extracts from WG and SG crabs exposed to low salinity at different time points of 12, 24, 48, and 72 h against their levels of 0 h, respectively. Although the Q2 of each OPLS-DA model in our present study was >0.5, only two of the models for SG passed the rigorous test of CV-ANOVA. These two models and their corresponding OPLS-DA coefficient plots 3498

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Figure 3. OPLS-DA scores plots (left) and corresponding color-coded correlation coefficient loadings plots (right) generated by comparisons between muscle extract spectra of SG at 0 h (black star) and those from SG exposed to low salinity at the time points of 24 h (red circle) and 48 h (blue open triangle), respectively. These models are cross-validated with CV-ANOVA, p = 1.60 × 10−2 and p = 6.59 × 10−3, respectively. Metabolite keys to the numbers are shown in Figure 1 and the Supporting Information Table S1.

PM changed significantly in SG crabs throughout low-salinity exposure with the highest increase up to 170% at 48 h of lowsalinity exposure against the level of 0 h, whereas it changed dramatically at 72 h in WG crabs. It is also worthwhile noting that the level of trigonelline increased for 35−67% in SG crabs but only 4−37% in WG crabs after low-salinity exposure.

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DISCUSSION This study showed the dynamic metabolic responses of two P. trituberculatus strains to low-salinity stress. We found that lowsalinity exposure resulted in essential metabolic changes involving many pathways including osmoregulation, gluconeogenesis, and energy production (Figure 6). The SG crabs presented a quicker metabolic modulation in response to lowsalinity exposure compared to WG crabs. Such differential characterization might result from low-salinity training, which determines their different abilities to adapt and acclimate to low salinity. Osmotic Rebalancing. In this study, we observed a significant elevation in the level of TMAO in both WG and SG crabs after 48 h of low-salinity stress (Figure 3; Table 1). It has been well documented that TMAO functions as a ubiquitous compatible osmolyte in marine crustraceans.31 Our observations suggest that low-salinity stress can cause a disturbance in the osmotic homeostasis in two crab groups. TMAO accumulation may contribute to the rebalance of the osmotic equilibrium. Moreover, a significant elevation in the trigonelline (nicotinic acid betaine) level compared to controls was also observed in muscle tissue extracts of WG and SG crabs exposed to low salinity (Figure 3; Table 1). This quaternary ammonium compound is a methylated product of nicotinic acid (vitamin B3)32 and has been reported to function as a compatible osmolyte or an osmoprotectant on enzymes when animals and plants are subjected to salinity and water deficit stress.33−38 Interestingly, the increased trigonelline accumulation has been found in muscle tissue extracts of green shore crab Carcinus maenas exposed to the two highest seawater CO2

Figure 4. Dynamic alterations of key muscle extract metabolites in response to low-salinity exposure to WG (left) and SG (right). The color indicates a correction coefficient as scaled on the right-hand side. The warm colors (e.g., red) denote an increase in the level of metabolites at the time points of 12, 24, 48, and 72 h against the level of 0 h exposed to low salinity, respectively, and the cool colors (e.g., blue) indicate a decrease. Glu, glutamate; Gln, glutamine; Val, valine; Leu, leucine; Ile, isoleucine; Ala, alanine; Met, methionine; Lys; lysine; His, histidine; Tyr, tyrosine; Phe, phenylalanine; Lac, lactate; Succ, succinate; ATP, adenosine triphosphate; TMAO, trimethylamine-Noxide; Trig, trigonelline; Ino, inosine; 2-PM, 2-pyridinemethanol.

low-salinity exposure. For example, the levels of valine, leucine, alanine, and glutamine declined throughout the post-treatment in WG crabs. Different from these amino acids, the inosine level fluctuated with a 47% decrease before the first 48 h of lowsalinity challenge and a 35% increase at 72 h for both WG and SG crabs. In addition, the level of ATP dramatically increased about 180% at 48 h against the level of 0 h in both WG and SG crabs. Interestingly, we noted that the concentration ratio of 23499

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Table 1. Content of Selected Muscle Extract Metabolites for the Wild Group (WG) and Screened Group (SG) Crabs Exposed to Low Salinity at Different Time Points of Exposure WG, mean ± SDa (mg/g) component glutamate glutamine valine leucine isoleucine alanine methionine lysine histidine tyrosine phenylalanine lactate succinate ATPb TMAO trigonelline inosine 2-PM

0h 4.88 4.83 1.87 4.70 1.05 9.55 2.21 2.67 0.49 1.30 0.94 5.20 0.23 2.05 3.76 0.05 1.05 1.63

component glutamate glutamine valine leucine isoleucine alanine methionine lysine histidine tyrosine phenylalanine lactate succinate ATP TMAO trigonelline inosine 2-PM

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12 h 0.93 1.25 0.80 1.95 0.44 2.66 0.68 1.09 0.14 0.46 0.40 2.93 0.06 0.54 1.19 0.01 0.27 0.47

4.61 3.50 0.84 2.31 0.54 4.58 1.52 1.52 0.26 1.03 0.68 5.18 0.21 4.97 4.80 0.08 0.17 1.83

1.66 0.67 0.47 1.08 0.19 1.43 0.70 0.74 0.11 0.21 0.15 2.78 0.03 0.17 0.34 0.01 0.27 0.24

4.76 2.60 1.06 2.83 0.71 5.15 2.27 1.38 0.19 1.58 0.92 4.55 0.21 4.75 5.28 0.11 0.43 2.18

0h 5.47 3.44 1.36 3.45 0.71 8.10 2.49 1.95 0.38 1.26 0.78 6.10 0.19 2.49 4.55 0.05 1.02 1.31

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.95 0.61* 0.30* 1.00* 0.30* 1.02** 0.49 1.19 0.20* 0.36 0.44 1.78 0.04 1.47** 1.20 0.02* 0.08*** 0.59

24 h

12 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

48 h

3.03 ± 0.69* 2.21 ± 0.62** 0.61 ± 0.23** 1.79 ± 0.71** 0.39 ± 0.20* 4.49 ± 0.99** 1.38 ± 0.32* 1.24 ± 0.58* 0.21 ± 0.09** 0.74 ± 0.37* 0.47 ± 0.23* 2.70 ± 2.16 0.16 ± 0.04* 2.69 ± 0.60 3.19 ± 0.35 0.05 ± 0.01 0.37 ± 0.08*** 1.69 ± 0.33 SG, mean ± SDa (mg/g)

5.19 2.01 0.77 2.19 0.52 4.73 1.65 1.95 0.27 0.95 0.59 7.70 0.23 5.67 7.26 0.10 0.55 2.03

24 h

1.96 1.34 0.68 1.75 0.49 0.80** 1.18 0.53 0.08** 1.16 0.72 3.02 0.05 1.17** 0.77 0.05* 0.20** 0.45**

2.32 1.58 0.37 1.09 0.23 3.76 1.03 0.84 0.14 0.50 0.31 3.88 0.13 3.42 4.62 0.08 0.47 1.76

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.67** 0.49*** 0.12** 0.31** 0.06** 0.81*** 0.32** 0.16** 0.05** 0.22*** 0.09*** 2.49 0.02** 0.43** 0.57 0.03 0.23** 0.39*

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.57 0.87** 0.12* 0.45* 0.12* 1.26** 0.25 0.61 0.06** 0.22 0.16 3.16 0.04 1.37** 2.38* 0.03** 0.24** 0.61

72 h 3.58 2.40 0.97 2.50 0.71 4.56 1.40 2.30 0.36 0.85 0.71 6.44 0.19 3.76 5.76 0.12 1.44 2.23

48 h 4.84 1.86 0.51 1.56 0.36 4.40 1.31 1.72 0.22 0.69 0.45 7.11 0.23 6.92 7.61 0.12 0.46 2.41

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.43 0.29*** 0.14** 0.43** 0.09** 0.81*** 0.52** 0.31 0.05** 0.31** 0.16** 3.41 0.03 1.25*** 0.79*** 0.05** 0.18** 0.68*

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.47 0.55** 0.37* 0.96* 0.36 1.03** 0.43* 0.86 0.11 0.41 0.43 5.21 0.02 0.79** 1.17* 0.03** 0.25* 0.45*

72 h 2.74 2.04 0.59 1.61 0.42 3.20 1.06 1.64 0.22 0.57 0.42 4.31 0.17 3.54 6.55 0.10 1.51 2.18

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.34** 0.38** 0.19** 0.54** 0.13** 1.07*** 0.35** 0.43 0.05** 0.16*** 0.12*** 4.18 0.03 1.54 1.04** 0.02*** 0.48 0.38**

a Average concentration and standard deviation (mean ± SD, mg/g crab muscle) were obtained from six parallel samples. Significance when compared with 0 h: *, p < 0.05; **, p < 0.01; ***, p < 0.001. bATP, adenosine triphosphate; TMAO, trimethylamine-N-oxide; 2-PM, 2pyridinemethanol.

levels.39 Taken together, trigonelline may play a role in rebalancing the osmotic pressures for low-salinity adaptation and acclimation of P. trituberculatus. This osmotic rebalance is also supported by a previous study in which membrane fluidity, lipid composition, fatty acid concentration, and intra/ extracellular ion concentration were significantly changed in estuarine mud crabs in response to salinity stress.40 It is further supported by another previous investigation on increased level of total phospholipids of posterior gills when crab Eriocheir sinensis was acclimated in a low-salinity challenge.41 In addition, we note that low-salinity-trained SG crabs contain a higher concentration of TMAO than WG crabs, especially highlighted at 24 h with an approximate 45% higher than that in SG crabs (Table 1). Such difference in the TMAO concentration between two crab groups might result from the low-salinity training. It is possible that SG crabs can obtain far-

reaching metabolic alterations and gain swifter response to low salinity than WG crabs. In fact, a PCA trajectory plot (Figure 2) clearly indicates the distinctive feature of metabolite alterations in SG crabs. It also implies a trend of metabolic changes in WG crabs toward SG crabs. Our observations are in broad agreement with previous findings in which heat hardening accelerates the reestablishment of metabolite homeostasis in Drosophila melanogaster.42 Gluconeogenesis from Amino Acids and Energy Accumulation. Another prominent finding is a rise in the ATP level in the crab muscle after low-salinity stress (Figure 3; Table 1). Consistently, an increase in energy production for ion transportation has been observed in Callinectes sapidus and Callinectes similis exposed to low salinity.43 ATP can be produced by three main pathways including glycolysis, respiration, and β-oxidation. In glycolysis, glucose is metabo3500

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Figure 5. Dynamic changes of muscle metabolites as a function of low-salinity stress. The empty symbols represent SG crabs, whereas the solid ones represent WG crabs.

Figure 6. Summary of dynamic metabolic responses of WG and SG crabs to low-salinity exposure. Metabolites with red or blue boxes represent significant increases or decreases in levels, respectively, at the significance level of p < 0.05 at this time point against the level at 0 h. NAD, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid.

crabs. However, we cannot exclude the responsibility of βoxidation of fatty acid for the elevated level of ATP due to low salinity because lipids seem to be the major source of energy in crustaceans45 and have previously been found significantly lower in gill and muscle of crabs subjected to hypo-osmotic stress.16,45 Similarly to the TMAO alteration, the more significantly accumulated ATP and depleted amino acid pool in SG crabs relative to WG crabs indicate low-salinity training enabled crabs to develop an ability for more extensive metabolic modulation (Table 1). Other Metabolic Changes. The inosine was noted to be markedly decreased in both WG and SG crabs during the first 48 h of low-salinity challenge but remarkably increased at 72 h. This nucleotide is involved in the nucleotide metabolism. The significant fluctuation in the inosine levels may result from the altered NAD cycle activity induced by low salinity.

lized to pyruvate and the latter is a substrate for the tricarboxylic acid (TCA) cycle and anaerobic respiration. Intriguingly, no significant alteration in the levels of glucose was caused by low salinity in crabs in this study. However, we noted a pronounced depletion of the amino acid pool in lowsalinity-exposed crabs, which has also been observed in crab muscle and hepatopancreas responding to low-salinity challenge.15 It is known that most amino acids are glucogenic amino acids, which can be converted into glucose through gluconeogenesis, thus likely replenishing the consumption of glucose in the ATP production in our study. This notion is consistent with previous findings of enhanced production of glucose by gluconeogenesis in the hepatopancreas of crab Chasmagnathus granulata in response to low-salinity challenge.44 Moreover, gluconeogenesis promotion is concomitant with the decreased level of succinate (Table 1),23 which is also in agreement with our observation in low-salinity-stressed 3501

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screened group; WG, wild group; TMAO, trimethylamine-Noxide; ATP, adenosine triphosphate; 2-PM, 2-pyridinemethanol; COSY, 1H−1H correlation spectroscopy; TOCSY, 1H−1H total correlation spectroscopy; HMBC, 1H−13C heteronuclear multiple-bond correlation; HSQC, 1H−13C heteronuclear signal-quantum correlation; PCA, principal component analysis; OPLS-DA, orthogonal projection to latent structure discriminant analysis; TCA, tricarboxylic acid

In addition, we observed a significant 2-PM accumulation in crabs during the low-salinity exposure. Importantly, a faster rise occurred in SG crabs than in WG crabs (Table 1; Figure 5), implying a role during the response of crabs to low salinity. 2PM is a picolinic acid-related compound that has a growthsimulating effect in rats.46 It has also been shown to improve the immune system and induce apoptosis in HL-60 cells.47,48 However, little information exists on the physiological function of 2-PM in marine crustraceans. Future studies should determine the positive role of 2-PM in crabs responding to low salinity. In this study, we found that the dominant metabolites in crab muscle comprise amino acids, sugars, carboxylic acids, betaine, TMAO, trigonelline, and nucleotides. The strategy of metabolic modulation of P. trituberculatus in response to low salinity includes osmotic rebalancing, enhanced gluconeogenesis from amino acids, and energy accumulation. These metabolic adaptions were manifested in the accumulation of TMAO, ATP, 2-PM, and trigonelline and in the depletion of the amino acid pool as well as in the fluctuation of inosine levels. This lends to support to the fact that low-salinity training accelerates the response of crabs to low-salinity stress. These findings provide a comprehensive insight into the mechanisms of metabolic modulation in P. trituberculatus in response to low salinity. Our work also highlights the approach of NMR-based metabonomics in conjunction with multivariate data analysis and univariate data analysis in understanding the strategy of metabolic phenotype modulation against stressors.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figure and tables as cited in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(C.W.) Phone: +86-(0)574-87600356. Fax: +86-(0)2787600356. E-mail: [email protected]. Author Contributions ∥

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Y.Y. and Y.A. contributed equally to this work.

Funding

Financial support was received from the National High Technology Research and Development Program of China (No. 2012AA10A409), National Natural Science Foundation of China (No. 41106123), Zhejiang Major Special Program of Breeding (2012C12907-3, 2012C12907-9), Scientific Research Fund of Zhejiang Provincial Education Department (No. Z201121258), Public Interest Program of Zhejiang Province (2013C31032), Ningbo Innovative Program of Agriculture (2012C92010), National Sparking Plan Project (2013GA7010022013, GA701041), the major program of Ningbo (2013C11017), Ningbo Agricultural Technologies R&D Project (2012C10027) Program for Science and Technology Innovative Research Team of Ningbo (No. 2011B81003), and K C Wong Magana Fund in Ningbo University. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED NMR, nuclear magnetic resonance; TSP, sodium 3-trimethylsilyl [2,2,3,3-2D4]propionate; D2O, deuterated water; SG, 3502

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Journal of Agricultural and Food Chemistry

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