Acute metabolic response of Portunus trituberculatus to Vibrio alginolyticus infection

Aquaculture 463 (2016) 201–208

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Acute metabolic response of Portunus trituberculatus to Vibrio alginolyticus infection Yangfang Ye a,b,1, Mengjie Xia a,1, Changkao Mu a, Ronghua Li a, Chunlin Wang a,⁎ a b

Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of Education, Zhejiang, Ningbo 315211, PR China State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Hubei, Wuhan 430071, PR China

a r t i c l e

i n f o

Article history: Received 5 April 2016 Received in revised form 29 May 2016 Accepted 30 May 2016 Available online 01 June 2016 Keywords: Portunus trituberculatus Vibrio alginolyticus Metabolomics Nuclear magnetic resonance Multivariate data analysis

a b s t r a c t Vibrio alginolyticus is a common pathogen that causes vibrio disease in swimming crab, Portunus trituberculatus. An understanding of the metabolic response mechanism of swimming crab to V. alginolyticus is essential for further control of this disease. In the present study, we analyzed the metabolic responses of gill, hepatopancreas, and muscle of swimming crab to an acute V. alginolyticus infection using 1H NMR spectroscopy coupled with multivariate data analysis. Our results showed that V. alginolyticus infection induced significant metabolomic alterations in crab muscle and gill within 24 h. These alterations showed tissue-specific in the infected swimming crabs. V. alginolyticus infection resulted in depression of gluconeogenesis from amino acids, improvement of energy accumulation, and enhancement of cellular immune response of muscle. These metabolic alterations were highlighted in the depletion of amino acids and inosine monophosphate as well as in the accumulation of ATP and betaine in muscle. Vibrio infection also caused energy disturbance in gill, which manifested in the depletion of glucose and in the accumulation of lactate and adenosine monophosphate. These findings provided details of acute metabolomic alterations of swimming crab after V. alginolyticus infection and demonstrated the potentiality of altered metabolites as biomarker in vibrio infection. Statement of relevance: Provide potential metabolite biomarkers for vibrio infection of crab. © 2016 Published by Elsevier B.V.

1. Introduction Vibrio alginolyticus is an opportunistic pathogen and involved in the diseases of marine animals, such as crab (Liu et al., 2007; Hao et al., 2015), fish (Castillo et al., 2015; Luo et al., 2016), abalone (Liu et al., 2001), pearl oyster (Wang and Wang, 2016), shrimp (Lee et al., 1996; Xie et al., 2016; Zhu et al., 2016), and sea turtles (Zavala-Norzagaray et al., 2015). This Gram-negative bacterium usually causes epidemic vibriosis, leading to considerable economic losses of aquacultured marine animals (Castillo et al., 2015). Swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is regarded as one of the most crucial aquacultured crab species in China (Chen et al., 2012). With increasing development of swimming crab farming recently, an “emulsification” disease caused by V. alginolyticus frequently breaks out and crab farming subsequently suffers heavy economic losses (Liu et al., 2007). Like other invertebrates, swimming crabs merely rely on innate immune responses to fight against invading pathogens for lack of an ⁎ Corresponding author at: Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of Education, 818 Fenghua Rd., Ningbo 315211, PR China. E-mail address: [email protected] (C. Wang). 1 Yangfang Ye and Mengjie Xia contributed equally to this work.

http://dx.doi.org/10.1016/j.aquaculture.2016.05.041 0044-8486/© 2016 Published by Elsevier B.V.

adaptive immune system. Consequently, the expressions of a cascade of immune-related genes have been studied in swimming crabs when facing the challenge of V. alginolyticus, including C-type lectin (Kong et al., 2008; Hao et al., 2015), lysozyme (Pan et al., 2010), crustins (Yue et al., 2010), anti-lipopolysaccharide factor (Liu et al., 2011, 2012), phenoloxidase, prophenoloxidase, and α2-macroglobulin (Chen et al., 2010; Ren et al., 2016). Moreover, antioxidant-related genes in P. trituberculatus have also been cloned and characterized such as superoxide dismutase (Li et al., 2011), and catalase (Chen et al., 2012). In general, genes are subject to epigenetic regulation whereas proteins are subject to post-translational modification. Changes in both gene and protein levels might not directly relate to phenotypes. Metabolites are downstream of both gene transcripts and proteins, thus metabolic phenotype carries rich information not only in the metabolism level but also in the gene expression and protein functioning (Fiehn, 2002; Tang and Wang, 2006). In particular, changes of metabolites can provide the most related indication of phenotypes (Malmendal et al., 2006). However, the knowledge on the metabolic response of P. trituberculatus against V. alginolyticus, especially in the metabolomic level, is still limited. Nuclear magnetic resonance (NMR) metabolomics enables the analysis of a wide range of small molecules that are involved in many aspects of physiology and pathology. Characterization of metabolomic

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differences between healthy and diseased states can improve our understanding of the mechanisms underlying pathological processes (Guma et al., 2016). Many successful applications of NMR-based metabolomics have been reported for a wide range of diseases such as schistosomiasis (Wu et al., 2010), systemic lupus erythematosus (Ouyang et al., 2011), osteoarthritis (Lamers et al., 2005), rheumatoid arthritis (Priori et al., 2015), and diabetes mellitus (Xu et al., 2012). In this study, we systematically analyzed acute metabolomic responses of gill, hepatopancreas, and muscle of P. trituberculatus to V. alginolyticus infection by using 1H NMR spectroscopy coupled with multivariate data analysis. The main objectives of this study are to (1) unravel the tissue-specific metabolic response of P. trituberculatus to V. alginolyticus infection and (2) identify potential metabolites in P. trituberculatus indicative of acute vibrio infection. 2. Materials and methods 2.1. Crab breeding and V. alginolyticus infection The vibrio infection experiment was conducted in Xinyi Corporation (Ningbo, China). Thirty male swimming crabs with a weight of 100– 120 g of each were collected and cultured in aerated seawater at 25 ° C. Crabs were fed with clam meat once every night. Cultural seawater was changed in the next morning. After one week of acclimatization, one third of crabs (n = 10) was collected and used as 0 h control group (designated group A). These crabs were kept on the ice for hypothermic anesthesia and then sacrificed for tissue samples. Another 10 crabs were each injected with 500 μL sterile phosphate buffer saline (PBS, 0.01 mol/L, pH 7.2) via arthrodial membrane of the last walking leg and used as 24 h control group (designated group B). The remaining 10 crabs were infected with 500 μL live V. alginolyticus suspended in PBS (1.14 × 107 cfu/mL) via arthrodial membrane of the last walking leg and used as vibrio-infected group (designated group C). These crabs were cultured in groups of two in cement pools (2 m × 5 m × 0.9 m, width × length × depth) and sacrificed on 24 h post-infection. The tissues including gill, hepatopancreas, and muscle were collected from these two 24 h groups. All samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C for later analysis. 2.2. Preparation of samples and acquisition of 1H NMR spectra Each of tissue samples (about 500 mg) was homogenized with 1 mL aqueous methanol (methanol/water = 2:1) using a tissue-lyser (QIAGEN TissueLyser II, Germany) at 20 Hz for 90 s. After 10 min centrifugation at 12,000 g and 4 °C, the supernatant of homogenate mixture was obtained. The same extracting procedure was repeated once on the remaining solid residues. The two supernatants were combined and condensed in vacuo for the removal of methanol. After lyophilization, each of extract was dissolved into 600 μL of phosphate buffer containing 90% D2O and 0.005% sodium 3-trimethylsilyl [2,2,3,3-d4] propionate (TSP) (Xiao et al., 2009). Following centrifugation, 550 μL supernatant of each extract was pipetted into 5 mm NMR tube. All 1H NMR spectra of tissues were recorded on a Bruker Avance III 600 MHz NMR spectrometer equipped with an inverse detection cryogenic probe (Bruker, Biospin, Germany). Samples were held at an ambient temperature (298 K) during acquisition. A standard onedimensional NMR experiment with water pre-saturation was performed for all tissue samples using a pulse sequence (recycle delay90°-t1-90°-tm-90°-acquisition). The 90° pulse length was adjusted to about 10 μs. A recycle delay (RD) was set to 2 s. A t1 of 6.5 μs and tm of 100 ms were applied. Water suppression was achieved during the RD and tm. A total of 64 scans for tissue spectra were collected into 32 k data points with a spectral width of 20 ppm centered at the water resonance. For resonance signal assignment purpose, two-dimensional (2D) NMR experiments were acquired and processed as described previously (Dai et al., 2010), including 1H\\1H correlation spectroscopy (COSY),

1

H\\1H total correlation spectroscopy (TOCSY), 1H\\13C heteronuclear multiple bond correlation (HMBC), and 1H\\13C heteronuclear single quantum correlation (HSQC) spectra. In COSY and TOCSY, a total of 128 increments and 64 scans were accumulated into 2 k data points with a spectral width of 10.5 ppm for each dimension. In HSQC, 240 scans per 128 increments were collected into 2 k data points with spectral widths of 10.5 ppm in 1H and 175 ppm in the 13C dimension. In HMBC, 400 scans per 128 increments were collected into 2 k data points with spectral widths of 10.5 ppm in 1H and 220 ppm in the 13C dimension. The data were zero-filled into 2 k data points in both dimensions. 2.3. NMR data processing and multivariate data analysis An exponential window function equivalent to a line broadening of 0.5 Hz was applied to free induction decays prior to Fourier transformation. All 1H NMR spectra were manually phased, baseline-corrected and calibrated to TSP resonance at δ 0.0 with TOPSPIN software package (v2.0, Bruker Biospin, Germany). For gill spectra, the spectral region δ 0.8–9.2 was segmented into regions of 0.004 ppm width (2.4 Hz). Regions δ 4.70–4.95 and δ 3.34–3.38 were discarded to eliminate the effects of imperfect water suppression and residual methanol signal. Spectral regions δ 0.8–9.2 and δ 0.8–9.5 for hepatopancreas and muscle, respectively, were segmented into regions of 0.004 ppm width (2.4 Hz). The disturbance from water and methanol signals in the hepatopancreas and muscle spectra was eliminated by the removal of spectral regions δ 4.70–5.15 and δ 3.34–3.38. All remaining spectral segments were then normalized to the wet weight of corresponding tissue samples respectively to reduce variations resulting from the concentration inconsistency. To generate an overall metabolic variation of crabs to vibrio infection, multivariate data analysis were carried out on the normalized NMR data using SIMCA-P+ software (Umetrics, Umeå, Sweden). To examine any inherent clustering between the samples, principal components analysis (PCA) with mean-centered scaling was firstly performed on the NMR data. In PCA scores plot, each point represented one sample. Subsequently, orthogonal projection to latent structure with discriminant analysis (OPLS-DA) was further applied to analyze NMR data scaled to unit variance (Trygg and Wold, 2002; van den Berg et al., 2006). All OPLS-DA models were validated using a 7-fold cross-validation method (Trygg et al., 2007) and a CV-ANOVA approach with p b 0.05 as the significant level (Eriksson et al., 2008) in sequence. The back-scaled transformation of loadings was performed to improve the interpretability of OPLS-DA model (Cloarec et al., 2005). The coefficient plots of OPLS-DA models were illustrated to highlight significantly changed metabolites between two groups. The color-coded correlation coefficients of metabolites were responsible for the differentiation. The coefficient plot was generated using MATLAB script (http://www. mathworks.com/) with some modifications. Each of loading was plotted as a function of respective chemical shift whereas a color code indicated a weight of discriminatory variable. In this study, a cutoff value of 0.602 was used for statistical significance based on the discrimination significance at the level of p = 0.05. The metabolite with the absolute value of a correlation coefficient less than 0.602 was considered to be statistically significant (p b 0.05). To illustrate variations caused by vibrio infection, the relative changes of typical metabolites were further calculated against the levels of untreated control group in the form of (Cv − Cc) ∕ Cc, where Cv represented metabolite level in the infection group and Cc represented that in the control group A or B. 3. Results 3.1. 1H NMR spectra of gill, hepatopancreas and muscle Examples of typical 1H NMR spectra for aqueous gill, hepatopancreas and muscle extracts obtained from 0 h control (A), 24 h control (B), and vibrio infection group (C) of swimming crabs are shown in Figs. 1, 2, and

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Fig. 1. Typical 600 MHz 1H NMR spectra of gill extracts of P. trituberculatus. A: 0 h control crabs, B: 24 h control crabs, C: vibrio-infected crabs. The spectral region δ 4.90–9.20 is displayed at 32-fold magnification compared to the region δ 0.80–4.70. Resonance assignments are given in Table S1. Keys: 1. isoleucine, 2. leucine, 3. valine, 4. lactate, 5. threonine, 6. alanine, 7. acetate, 8. methionine, 9. glutamate, 10. succinate, 11. glutamine, 12. dimethylsulphoniopropionate, 13. aspartate, 14. sarcosine, 15. succinimide, 16. trimethylamine, 17. lysine, 18. phosphorylcholine, 19. arginine, 20. betaine, 21. taurine, 22. glycine, 23. proline, 24. α-glucose, 25. β-glucose, 26. maltose, 27. fumarate, 28. tyrosine, 29. phenylalanine, 30. 2pyridinemethanol, 31. histamine, 32. histidine, 33. tryptophan, 34. hypoxanthine, 35. inosine, 36. inosine monophosphate, 37. adenosine monophosphate, 38. ATP, 39. trigonelline, 40. residual methanol, 41. sugar and amino acids α-CH resonance, 42. trehalose, 43. uracil, 44. ribose-5-phosphate, 45. trimethylamine-N-oxide.

S1. Assignments of endogenous metabolites were performed according to the homonuclear or heteronuclear correlations provided by COSY, TOCSY, HSQC, and HMBC spectra and further confirmed with previously

published work (Fan, 1996; Ye et al., 2014). A total of 38 metabolites were identified in the 1H NMR spectra of crab gill, dominated by a range of amino acids, nucleosides and nucleotides, aliphatic organic

Fig. 2. Typical 600 MHz 1H NMR spectra of muscle extracts of P. trituberculatus. A: 0 h control crabs, B: 24 h control crabs, C: vibrio-infected crabs. The spectral region δ 4.90–9.50 is displayed at 32-fold magnification compared to the region δ 0.80–4.70. Resonance assignments are given in Table S1.

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acids, sugars, and amines. Metabolites found in crab hepatopancreas were mostly amino acids, aliphatic organic acids, betaine, trigonelline, succinimide, glucose, inosine, and 2-pyridinemethanol. The crab muscle contained 26 metabolites that similar to our previous observations (Ye et al., 2014). Some metabolite variations were clearly observed from the visual inspection of the NMR spectra. For example, an elevation in the adenosine monophosphate (AMP) level and a reduction in the glucose level were observed in the gill of infected swimming crabs. 3.2. Multivariate data analysis of gill, hepatopancreas and muscle metabolites Multivariate data analysis approaches including PCA and OPLS-DA were performed for 1H NMR data obtained from crab gill, hepatopancreas and muscle extracts. For gill samples, PCA scores plot showed a clear separation between two control crabs (Fig. S2, left). Vibrio-infected crabs distributed between two control crabs, indicating an observed delay of crab metabolism due to V. alginolyticus infection. Further OPLS-DA analysis showed that three of OPLS-DA models had high Q2 values with low p-values less than 0.05 from CV-ANOVA (Fig. 3, right). Corresponding coefficient plots of OPLS-DA (Fig. 3, left) showed

statistically significant metabolites contributing to the differentiation between two groups. After 24 h of normal growth, crab gill contained more levels of fumarate, phosphorylcholine, glucose, trehalose, uracil, and inosine whereas lower levels of lactate and trimethylamine-Noxide (TMAO). However, these metabolite fluctuations were disturbed by V. alginolyticus infection. Except of the fumarate, phosphorylcholine, uracil, and inosine levels kept increasing in the vibrio-infected crab gill, maltose was accumulated rather than glucose and trehalose. Furthermore, no metabolites were significantly decreased in the vibrio-infected crab gill compared to 0 h control (group A). Notably, at matched time point, the gill metabolic response of crabs to V. alginolyticus infection was characterized by relatively higher levels of lactate and AMP while lower levels of glucose and hypoxanthine than 24 h control (group B). The same multivariate data analysis was also applied to the 1H NMR data of hepatopancreas. PCA scores plots showed no clear cluster based on neither crab growth nor vibrio infection (Fig. S2, middle). However, an OPLS-DA model constructed from the NMR data of 0 h control crabs (group A) and vibrio-infected crabs was valid with Q2 value of 0.69 and p-value of 0.002 (Fig. S3, left). The difference between the two groups was a significantly reduced alanine level in the vibrio-infected crabs (Fig. S3, right).

Fig. 3. OPLS-DA scores and coefficient-coded loading plots for the gill models discriminating 0 h control crabs (A, black stars), 24 h control crabs (B, red dots), and vibrio-infected crabs (C, navy blue diamonds) extracts (see Table S1 or Fig. 1 for metabolite identification key). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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For muscle samples, PCA analysis displayed an overlap among three groups but with a tendency of metabolic variation from 0 h control (group A), 24 h control (group B) to vibrio-infected crabs (Fig. S2, right). All of pair-wise comparisons between two controls or between control and infected crabs were further conducted using an OPLS-DA strategy. The resultant OPLS-DA models were all valid according to the Q2 values and p-values from CV-ANOVA (Fig. 4, left). OPLS-DA results showed that normal growth for 24 h caused a significant decrease in the levels of isoleucine, leucine, valine, alanine, methionine, succinate, glutamine, lysine, phosphorylcholine, tyrosine, and phenylalanine in the crab muscle (Fig. 4, right). After the vibrio infection, no significant depletions of methionine, succinate, glutamine, and phosphorylcholine levels were observed in the crab muscle except isoleucine, leucine, valine, alanine, lysine, tyrosine, and phenylalanine. Furthermore, levels of betaine and ATP significantly raised in it due to vibrio infection. Compared with 24 h control (group B), the vibrio-infected crab muscle contained a higher level of betaine whereas a lower level of inosine monophosphate (IMP). The correlation coefficients of the significantly altered metabolites in three tissues are summarized in Table 1.

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The changes of gill and muscle metabolites due to growth and vibrio infection are illustrated in Fig. 5. Relative to 24 h control (group B), vibrio infection led to 20–30% decreases for glucose, inosine, and hypoxanthine, about 10% decrease for uracil, as well as less than 5% increases for fumarate and phosphorylcholine in the vibrio-infected crab gill. These changes were highlighted with 152.9% increase for AMP. Relative to 24 h control (group B), vibrio infection also resulted in 89.4% increase for ATP as well as 20–40% increases for phosphorylcholine and betaine in the vibrio-infected crab muscle. These increases were associated with 82.4% decrease for IMP as well as more than 30% decrease for three amino acids including leucine, tyrosine, and phenylalanine in the infected crab muscle. 4. Discussion Genetic studies have shown that V. alginolyticus infection induces the expressions of a range of genes involved in innate immune response of swimming crab (Hao et al., 2015; Ren et al., 2016). However, metabolic alterations in swimming crab due to V. alginolyticus infection

Fig. 4. OPLS-DA scores and coefficient-coded loading plots for the muscle models discriminating 0 h control crabs (A, black stars), 24 h control crabs (B, red dots), and vibrio-infected crabs (C, navy blue diamonds) extracts (see Table S1 or Fig. 1 for metabolite identification key). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 The correlation coefficients of significantly altered metabolites in the aqueous extracts of three tissues of P. trituberculatus after V. alginolyticus infection. Correlation coefficients (ra) Hepatopancreas

Muscle

Metabolites

A/Bb

Gill A/C

B/C

A/C

A/B

A/C

B/C

Isoleucine Leucine Valine Lactate Alanine Methionine Succinate Glutamine Lysine Phosphorylcholine Betaine TMAO Glucose Trehalose Maltose Uracil Tyrosine Phenylalanine Fumarate Hypoxanthine Inosine Adenosine monophosphate Inosine monophosphate ATP

– – – −0.64 – – – – – 0.75 – −0.68 0.89 0.82 – 0.81 – – 0.71 – 0.80 – – –

– – – – – – – – – 0.63 – – – – 0.61 0.86 – – 0.79 – 0.77 – – –

– – – 0.74 – – – – – – – – – – – – – – – −0.70 – 0.75 – –

– – – – −0.67 – – – – – – – – – – – – – – – – – – –

−0.80 −0.75 −0.85 – −0.78 −0.74 −0.77 −0.71 −0.74 −0.88 – – – – – – −0.72 −0.76 – – – – – –

−0.78 −0.86 −0.82 – −0.75 – – – −0.83 – 0.83 – – – – – −0.81 −0.81 – – – – – 0.85

– – – – – – – – – – 0.66 – – – – – – – – – – – −0.85 –

a Correlation coefficients, positive and negative signs indicate positive and negative correlation in the concentrations, respectively. r = 0.602 was used as the corresponding cutoff value of correlation coefficient for the statistical significance based on the discrimination significance. “–” means the correlation coefficient |r| is less than cutoff value. b A, 0 h control crab; B, 24 h control crab; C, virus-infected crab.

remain unclear. In this paper, we characterized the metabolic profiles of gill, hepatopancreas and muscle of swimming crab associated with V. alginolyticus infection. This, to our best knowledge, is the first application of metabolomics to unravel the acute metabolic response of swimming crab to V. alginolyticus infection. 4.1. Metabolic response of crab muscle to V. alginolyticus Generally, gill is considered as the primary organ in response to pathogen invasion. In the study, V. alginolyticus was injected into swimming crabs via arthrodial membrane of the last walking leg. Therefore, muscle may be affected earlier than other tissues. We noted that 24 h growth caused a significant reduction in free amino acid levels in noninfected crab muscle. Amino acids can be used as energy substrates via gluconeogenesis and further consumed via the Krebs cycle for ATP production. The concomitantly reduced succinate level might indicate an active Krebs cycle. These activated pathways of energy metabolism

were previously observed in the low salinity-stressed swimming crab (Ye et al., 2014). No significant ATP accumulation occurs during 24 h growth period of control crab whereas it does in the low salinitystressed swimming crab. Similar to low salinity stress, V. alginolyticus infection also caused a significant ATP accumulation in swimming crab muscle relative to 0 h control (group A). Such an ATP accumulation was decreased in the vibrio-infected crab muscle compared to 24 control (group B), probably indicating a joint effect of vibrio infection, crab growth and PBS injection. In addition, vibrio infection does not cause a promoted gluconeogenesis because the alterations of energy-related metabolites were not observed between vibrio-infected crab muscles and 24 h control (group B). On the contrary, it seems to weaken the promotion of gluconeogenesis as less amino acids were significantly reduced in the vibrio-infection crab muscle than 0 h control (group A). An 82.4% decrease in the IMP level occurred in vibrio-infected crabs relative to 24 h control crabs (group B). IMP is a ribonucleotide and firstly formed during the synthesis of purine. Numerous studies reported

Fig. 5. Relative changes of metabolites in gill and muscle of P. trituberculatus due to growth or V. alginolyticus infection. A: 0 h control crabs, B: 24 h control crabs, C: vibrio-infected crabs. B/ A: red diamonds, C/A: brown squares, C/B: blue triangles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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that IMP has an enhancement to the growth performance, survival, immune response, and disease defense of different aquatic species such as olive flounder (Song et al., 2012) and red sea bream (Hossain et al., 2016). Immunostimulating properties of IMP enable fishes to have an increased bactericidal activity (Song et al., 2012; Hossain et al., 2016). The IMP shortage likely implies an increased consumption of IMP in the immune response of swimming crab to V. alginolyticus infection. In addition, we observed a significant elevation in the betaine level in vibrio-infected crabs relative to 24 h control crabs (group B). Betaine is a trimethylated derivative of glycine and widely distributed in nearly all organisms. Abundant evidence stated that it mainly functions as a predominant stress protectant against physical, chemical, and biological stresses (Zou et al., 2016). In this paper, the elevated betaine seems to indicate that betaine plays an osmoprotective role in swimming crabs after V. alginolyticus infection. However, recent studies have shown that betaine plays an immunostimulatory role in fish and chick (Klasing et al., 2002; Kumar et al., 2014). Notably, in sea cucumber, this alkaloid involved in the immune response and functions as a methyl donor which enables homocysteine re-methylated to form methionine via betaine homocysteine methyltransferase (Zhang et al., 2015). The accumulation of homocysteine may contribute to the increased intracellular reactive oxygen species generation and the reduced vibrio survival rate in sea cucumber (Zhang et al., 2015). In this study, the accumulated betaine might indicate the reduced re-methylation of homocysteine in the muscle, thus implying an enhanced ability of swimming crab to resist against V. alginolyticus.

4.2. Metabolic response of crab gill to V. alginolyticus For gill, metabolomic alterations induced by V. alginolyticus infection were highlighted in energy disturbance. Normally, the gill lactate level was decreased with the crab growth, which accompanied with the elevated glucose level. However, these changes were not observed in the vibrio-infected crabs. Indeed, the increment of lactate level and the decrement of glucose occurred in the vibrio-infected crabs compared to 24 h control ones (group B). These observations strongly suggest an involvement of anaerobic glycolysis which might partially cover the energy costs of basal maintenance in the vibrio-infected crabs. Such a stressinduced transition of energy metabolism from aerobic scope to anaerobiosis has been widely observed in the aquatic animals (Sokolova et al., 2012). Furthermore, glucose deprivation generally induces energy stress in cells. AMP-activated protein kinase (AMPK) that functions as part of an energy-sensing pathway (Faubert et al., 2015) is activated in order to promote net ATP conservation in the crab gill. AMP functions as a direct agonist of AMPK (Hardie, 2003). A sharply elevated AMP level in the gill of vibrio-infected crabs indicates an enhanced AMPK activity, which subsequently switches on ATP-producing catabolic metabolism and switches off ATP-consuming anabolic metabolism to restore energy homeostasis of gill cells (Andris and Leo, 2015). In conclusion, we observed that tissue-specified metabolic effects on swimming crabs were induced by V. alginolyticus. OPLS-DA analysis of 1 H NMR spectral data derived from extracts of crab gill, hepatopancreas, and muscle tissues has shown that vibrio infection initiates an acute metabolic response in gill and muscle rather than hepatopancreas. V. alginolyticus infection resulted in the depressed gluconeogenesis from amino acids, the improved energy accumulation, as well as the enhanced cellular immune response of muscle. These metabolic alterations were highlighted in the depletion of amino acids and IMP as well as in the accumulation of ATP and betaine in muscle. Vibrio infection also caused the energy disturbance in the gill which manifested in the accumulation of lactate and AMP whereas the depletion of glucose. The significant fluctuation of IMP and ATP in the muscle as well as AMP in the gill might be identified as potential biomarkers for acute V. alginolyticus infection in the swimming crab. Given that some metabolites such as IMP and betaine are engaged in the immune system, future

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work should focus on the functions of the metabolites in the immunometabolism. Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 41476124), the transformation fund for agriculture science (2014GB2C220151), the Natural Science Foundation of Ningbo (No. 2014A610183), the Sci & Tech Project for Common Wealth of Ningbo (No. 2016C10062), and the K.C. Wong Magna Fund of Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2016.05.041. References Andris, F., Leo, O., 2015. AMPK in lymphocyte metabolism and function. Int. Rev. Immunol. 34 (1), 67–81. Castillo, D., D'Alvise, P., Kalatzis, P.G., Kokkari, C., Middelboe, M., Gram, L., Liu, S., Katharios, P., 2015. Draft genome sequences of Vibrio alginolyticus strains V1 and V2, opportunistic marine pathogens. Genome Annouc. 3 (4), e00729-15. 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