Aquaculture 463 (2016) 201–208

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Acute metabolic response of Portunus trituberculatus to Vibrioalginolyticus infection

Yangfang Ye a,b,1, Mengjie Xia a,1, Changkao Mu a, Ronghua Li a, Chunlin Wang a,⁎a Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of Education, Zhejiang, Ningbo 315211, PR Chinab 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

⁎ Corresponding author at: Key Laboratory of AppliedUniversity, Ministry of Education, 818 Fenghua Rd., Ningb

E-mail address: wangchunlin@nbu.edu.cn (C. Wang).1 Yangfang Ye and Mengjie Xia contributed equally to t

http://dx.doi.org/10.1016/j.aquaculture.2016.05.0410044-8486/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 April 2016Received in revised form 29 May 2016Accepted 30 May 2016Available online 1 June 2016

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 fur-ther control of this disease. In thepresent study,we analyzed themetabolic responses of gill, hepatopancreas, andmuscle of swimming crab to an acute V. alginolyticus infection using 1H NMR spectroscopy coupled with multi-variate data analysis. Our results showed that V. alginolyticus infection induced significant metabolomic alter-ations in crab muscle and gill within 24 h. These alterations showed tissue-specific in the infected swimmingcrabs. V. alginolyticus infection resulted in depression of gluconeogenesis from amino acids, improvement of en-ergy accumulation, and enhancement of cellular immune response of muscle. These metabolic alterations werehighlighted in the depletion of amino acids and inosine monophosphate as well as in the accumulation of ATPand betaine in muscle. Vibrio infection also caused energy disturbance in gill, whichmanifested in the depletionof glucose and in the accumulation of lactate and adenosine monophosphate. These findings provided details ofacute metabolomic alterations of swimming crab after V. alginolyticus infection and demonstrated the potential-ity of altered metabolites as biomarker in vibrio infection.Statement of relevance: Provide potential metabolite biomarkers for vibrio infection of crab.

© 2016 Elsevier B.V. All rights reserved.

Keywords:Portunus trituberculatusVibrio alginolyticusMetabolomicsNuclear magnetic resonanceMultivariate data analysis

1. Introduction

Vibrio alginolyticus is an opportunistic pathogen and involved in thediseases 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-Norzagarayet al., 2015). This Gram-negative bacterium usually causes epidemicvibriosis, leading to considerable economic losses of aquacultured ma-rine animals (Castillo et al., 2015). Swimming crab, Portunustrituberculatus (Crustacea: Decapoda: Brachyura), is regarded as one ofthe most crucial aquacultured crab species in China (Chen et al.,2012).With increasing development of swimming crab farming recent-ly, an “emulsification” disease caused by V. alginolyticus frequentlybreaks out and crab farming subsequently suffers heavy economiclosses (Liu et al., 2007).

Like other invertebrates, swimming crabs merely rely on innate im-mune responses to fight against invading pathogens for lack of an

Marine Biotechnology, Ningboo 315211, PR China.

his work.

adaptive immune system. Consequently, the expressions of a cascadeof immune-related genes have been studied in swimming crabs whenfacing the challenge of V. alginolyticus, including C-type lectin (Kong etal., 2008; Hao et al., 2015), lysozyme (Pan et al., 2010), crustins (Yueet 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 super-oxide dismutase (Li et al., 2011), and catalase (Chen et al., 2012). In gen-eral, genes are subject to epigenetic regulation whereas proteins aresubject to post-translational modification. Changes in both gene andprotein levels might not directly relate to phenotypes. Metabolites aredownstream of both gene transcripts and proteins, thus metabolic phe-notype carries rich information not only in themetabolism level but alsoin the gene expression and protein functioning (Fiehn, 2002; Tang andWang, 2006). In particular, changes of metabolites can provide themost related indication of phenotypes (Malmendal et al., 2006). How-ever, the knowledge on the metabolic response of P. trituberculatusagainst V. alginolyticus, especially in the metabolomic level, is stilllimited.

Nuclearmagnetic resonance (NMR)metabolomics enables the anal-ysis of a wide range of small molecules that are involved in many as-pects of physiology and pathology. Characterization of metabolomic

202 Y. Ye et al. / Aquaculture 463 (2016) 201–208

differences between healthy and diseased states can improve our un-derstanding of the mechanisms underlying pathological processes(Guma et al., 2016). Many successful applications of NMR-based meta-bolomics have been reported for a wide range of diseases such as schis-tosomiasis (Wu et al., 2010), systemic lupus erythematosus (Ouyang etal., 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 re-sponses of gill, hepatopancreas, and muscle of P. trituberculatus to V.alginolyticus infection by using 1HNMR spectroscopy coupledwithmul-tivariate data analysis. The main objectives of this study are to (1) un-ravel 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 experimentwas 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 seawaterwas changed in the next morning. After one week of acclimatization,one third of crabs (n = 10) was collected and used as 0 h controlgroup (designated group A). These crabs were kept on the ice for hypo-thermic anesthesia and then sacrificed for tissue samples. Another 10crabs were each injected with 500 μL sterile phosphate buffer saline(PBS, 0.01 mol/L, pH 7.2) via arthrodial membrane of the last walkingleg and used as 24 h control group (designated group B). The remaining10 crabswere infectedwith 500 μL live V. alginolyticus suspended in PBS(1.14 × 107 cfu/mL) via arthrodialmembrane of the last walking leg andused as vibrio-infected group (designated group C). These crabs werecultured 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 tis-sues including gill, hepatopancreas, and muscle were collected fromthese two 24 h groups. All samples were immediately snap-frozen inliquid 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 500mg) was homogenized with 1mLaqueous methanol (methanol/water = 2:1) using a tissue-lyser(QIAGEN TissueLyser II, Germany) at 20 Hz for 90 s. After 10min centri-fugation at 12,000 g and 4 °C, the supernatant of homogenate mixturewas obtained. The same extracting procedure was repeated once onthe remaining solid residues. The two supernatants were combinedand condensed in vacuo for the removal of methanol. After lyophiliza-tion, each of extract was dissolved into 600 μL of phosphate buffer con-taining 90% D2O and 0.005% sodium 3-trimethylsilyl [2,2,3,3-d4]propionate (TSP) (Xiao et al., 2009). Following centrifugation, 550 μL su-pernatant of each extract was pipetted into 5 mm NMR tube.

All 1H NMR spectra of tissues were recorded on a Bruker Avance III600 MHz NMR spectrometer equipped with an inverse detection cryo-genic probe (Bruker, Biospin, Germany). Samples were held at an ambi-ent temperature (298 K) during acquisition. A standard one-dimensional NMR experiment with water pre-saturation was per-formed for all tissue samples using a pulse sequence (recycle delay-90°-t1-90°-tm-90°-acquisition). The 90° pulse length was adjusted toabout 10 μs. A recycle delay (RD) was set to 2 s. A t1 of 6.5 μs and tm of100 ms were applied. Water suppression was achieved during the RDand tm. A total of 64 scans for tissue spectra were collected into 32 kdata points with a spectral width of 20 ppm centered at the water reso-nance. For resonance signal assignment purpose, two-dimensional (2D)NMRexperimentswere acquired and processed as described previously(Dai et al., 2010), including 1H\\1H correlation spectroscopy (COSY),

1H\\1H total correlation spectroscopy (TOCSY), 1H\\13C heteronuclearmultiple bond correlation (HMBC), and 1H\\13C heteronuclear singlequantum correlation (HSQC) spectra. In COSY and TOCSY, a total of128 increments and 64 scans were accumulated into 2 k data pointswith a spectral width of 10.5 ppm for each dimension. In HSQC, 240scans per 128 increments were collected into 2 k data points with spec-tral widths of 10.5 ppm in 1H and 175 ppm in the 13C dimension. InHMBC, 400 scans per 128 incrementswere collected into 2 k data pointswith spectral widths of 10.5 ppm in 1H and 220 ppm in the 13C dimen-sion. 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 of0.5 Hz was applied to free induction decays prior to Fourier transforma-tion. All 1H NMR spectra were manually phased, baseline-corrected andcalibrated to TSP resonance at δ 0.0 with TOPSPIN software package(v2.0, Bruker Biospin, Germany). For gill spectra, the spectral region δ0.8–9.2was segmented into regions of 0.004ppmwidth (2.4Hz). Regionsδ 4.70–4.95 and δ 3.34–3.38were discarded to eliminate the effects of im-perfect water suppression and residual methanol signal. Spectral regionsδ0.8–9.2 and δ0.8–9.5 for hepatopancreas andmuscle, respectively,weresegmented into regions of 0.004 ppm width (2.4 Hz). The disturbancefromwater andmethanol signals in the hepatopancreas andmuscle spec-tra was eliminated by the removal of spectral regions δ 4.70–5.15 and δ3.34–3.38. All remaining spectral segments were then normalized tothe wet weight of corresponding tissue samples respectively to reducevariations resulting from the concentration inconsistency.

To generate an overall metabolic variation of crabs to vibrio infec-tion, multivariate data analysis were carried out on the normalizedNMR data using SIMCA-P+ software (Umetrics, Umeå, Sweden). To ex-amine any inherent clustering between the samples, principal compo-nents analysis (PCA) with mean-centered scaling was firstlyperformed on the NMR data. In PCA scores plot, each point representedone sample. Subsequently, orthogonal projection to latent structurewith discriminant analysis (OPLS-DA) was further applied to analyzeNMR data scaled to unit variance (Trygg and Wold, 2002; van denBerg et al., 2006). All OPLS-DA models were validated using a 7-foldcross-validationmethod (Trygg et al., 2007) and a CV-ANOVA approachwith p b 0.05 as the significant level (Eriksson et al., 2008) in sequence.The back-scaled transformation of loadings was performed to improvethe interpretability of OPLS-DA model (Cloarec et al., 2005). The coeffi-cient plots of OPLS-DAmodels were illustrated to highlight significantlychanged metabolites between two groups. The color-coded correlationcoefficients of metabolites were responsible for the differentiation.The coefficient plot was generated using MATLAB script (http://www.mathworks.com/)with somemodifications. Each of loadingwas plottedas a function of respective chemical shift whereas a color code indicateda weight of discriminatory variable. In this study, a cutoff value of 0.602was used for statistical significance based on the discrimination signifi-cance at the level of p=0.05. The metabolite with the absolute value ofa correlation coefficient less than 0.602 was considered to be statistical-ly significant (p b 0.05).

To illustrate variations caused by vibrio infection, the relative chang-es of typicalmetaboliteswere further calculated against the levels of un-treated control group in the form of (Cv − Cc) ∕ Cc, where Cv

represented metabolite level in the infection group and Cc representedthat in the control group A or B.

3. Results

3.1. 1H NMR spectra of gill, hepatopancreas and muscle

Examples of typical 1HNMR spectra for aqueous gill, hepatopancreasandmuscle extracts obtained from 0 h control (A), 24 h control (B), andvibrio infection group (C) of swimming crabs are shown in Figs. 1, 2, and

Fig. 1. Typical 600MHz 1HNMR 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 at32-foldmagnification 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. 2-pyridinemethanol, 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.

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S1. Assignments of endogenous metabolites were performed accordingto the homonuclear or heteronuclear correlations provided by COSY,TOCSY, HSQC, andHMBC spectra and further confirmedwith previously

Fig. 2. Typical 600 MHz 1H NMR spectra of muscle extracts of P. trituberculatus. A: 0 h controdisplayed at 32-fold magnification compared to the region δ 0.80–4.70. Resonance assignment

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

l crabs, B: 24 h control crabs, C: vibrio-infected crabs. The spectral region δ 4.90–9.50 iss are given in Table S1.

204 Y. Ye et al. / Aquaculture 463 (2016) 201–208

acids, sugars, and amines. Metabolites found in crab hepatopancreaswere mostly amino acids, aliphatic organic acids, betaine, trigonelline,succinimide, glucose, inosine, and 2-pyridinemethanol. The crabmusclecontained 26 metabolites that similar to our previous observations (Yeet al., 2014). Some metabolite variations were clearly observed fromthe visual inspection of the NMR spectra. For example, an elevation inthe adenosine monophosphate (AMP) level and a reduction in the glu-cose level were observed in the gill of infected swimming crabs.

3.2. Multivariate data analysis of gill, hepatopancreas and musclemetabolites

Multivariate data analysis approaches including PCA and OPLS-DAwere performed for 1HNMRdata obtained from crab gill, hepatopancre-as and muscle extracts. For gill samples, PCA scores plot showed a clearseparation between two control crabs (Fig. S2, left). Vibrio-infectedcrabs distributed between two control crabs, indicating an observeddelay of crab metabolism due to V. alginolyticus infection. FurtherOPLS-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

Fig. 3.OPLS-DA scores and coefficient-coded loading plots for the gill models discriminating 0 hnavy blue diamonds) extracts (see Table S1 or Fig. 1 for metabolite identification key).

statistically significant metabolites contributing to the differentiationbetween two groups. After 24 h of normal growth, crab gill containedmore levels of fumarate, phosphorylcholine, glucose, trehalose, uracil,and inosine whereas lower levels of lactate and trimethylamine-N-oxide (TMAO). However, these metabolite fluctuations were disturbedby 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. Further-more, nometaboliteswere significantly decreased in the vibrio-infectedcrab gill compared to 0 h control (group A). Notably, at matched timepoint, the gill metabolic response of crabs to V. alginolyticus infectionwas characterized by relatively higher levels of lactate and AMP whilelower levels of glucose and hypoxanthine than 24 h control (group B).

The samemultivariate data analysis was also applied to the 1H NMRdata of hepatopancreas. PCA scores plots showed no clear cluster basedon neither crab growth nor vibrio infection (Fig. S2, middle). However,an OPLS-DA model constructed from the NMR data of 0 h controlcrabs (group A) and vibrio-infected crabs was valid with Q2 value of0.69 and p-value of 0.002 (Fig. S3, left). The difference between thetwo groupswas a significantly reduced alanine level in the vibrio-infect-ed crabs (Fig. S3, right).

control crabs (A, black stars), 24 h control crabs (B, red dots), and vibrio-infected crabs (C,

205Y. Ye et al. / Aquaculture 463 (2016) 201–208

For muscle samples, PCA analysis displayed an overlap among threegroups 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 betweencontrol and infected crabs were further conducted using an OPLS-DAstrategy. The resultant OPLS-DA models were all valid according to theQ2 values and p-values from CV-ANOVA (Fig. 4, left). OPLS-DA resultsshowed that normal growth for 24 h caused a significant decrease inthe levels of isoleucine, leucine, valine, alanine, methionine, succinate,glutamine, lysine, phosphorylcholine, tyrosine, and phenylalanine inthe crab muscle (Fig. 4, right). After the vibrio infection, no significantdepletions of methionine, succinate, glutamine, and phosphorylcholinelevels were observed in the crab muscle except isoleucine, leucine, va-line, alanine, lysine, tyrosine, and phenylalanine. Furthermore, levelsof betaine and ATP significantly raised in it due to vibrio infection. Com-pared with 24 h control (group B), the vibrio-infected crab musclecontained a higher level of betaine whereas a lower level of inosinemonophosphate (IMP). The correlation coefficients of the significantlyaltered metabolites in three tissues are summarized in Table 1.

Fig. 4.OPLS-DA scores and coefficient-coded loading plots for themusclemodels discriminating(C, navy blue diamonds) extracts (see Table S1 or Fig. 1 for metabolite identification key).

The changes of gill andmuscle metabolites due to growth and vibrioinfection are illustrated in Fig. 5. Relative to 24 h control (group B), vib-rio infection led to 20–30% decreases for glucose, inosine, and hypoxan-thine, about 10% decrease for uracil, as well as less than 5% increases forfumarate and phosphorylcholine in the vibrio-infected crab gill. Thesechanges were highlighted with 152.9% increase for AMP. Relative to24 h control (group B), vibrio infection also resulted in 89.4% increasefor ATP as well as 20–40% increases for phosphorylcholine and betainein the vibrio-infected crab muscle. These increases were associatedwith 82.4% decrease for IMP as well as more than 30% decrease forthree amino acids including leucine, tyrosine, and phenylalanine inthe infected crab muscle.

4. Discussion

Genetic studies have shown that V. alginolyticus infection inducesthe expressions of a range of genes involved in innate immune responseof swimming crab (Hao et al., 2015; Ren et al., 2016). However, meta-bolic alterations in swimming crab due to V. alginolyticus infection

0 h control crabs (A, black stars), 24 h control crabs (B, red dots), and vibrio-infected crabs

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

Metabolites

Correlation coefficients (ra)

Gill Hepatopancreas Muscle

A/Bb A/C B/C A/C A/B A/C B/C

Isoleucine – – – – −0.80 −0.78 –Leucine – – – – −0.75 −0.86 –Valine – – – – −0.85 −0.82 –Lactate −0.64 – 0.74 – – – –Alanine – – – −0.67 −0.78 −0.75 –Methionine – – – – −0.74 – –Succinate – – – – −0.77 – –Glutamine – – – – −0.71 – –Lysine – – – – −0.74 −0.83 –Phosphorylcholine 0.75 0.63 – – −0.88 – –Betaine – – – – – 0.83 0.66TMAO −0.68 – – – – – –Glucose 0.89 – – – – – –Trehalose 0.82 – – – – – –Maltose – 0.61 – – – – –Uracil 0.81 0.86 – – – – –Tyrosine – – – – −0.72 −0.81 –Phenylalanine – – – – −0.76 −0.81 –Fumarate 0.71 0.79 – – – – –Hypoxanthine – – −0.70 – – – –Inosine 0.80 0.77 – – – – –Adenosine monophosphate – – 0.75 – – – –Inosine monophosphate – – – – – – −0.85ATP – – – – – 0.85 –

a Correlation coefficients, positive and negative signs indicate positive and negative correlation in the concentrations, respectively. r= 0.602was used as the corresponding cutoff valueof 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.

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remain unclear. In this paper, we characterized themetabolic profiles ofgill, hepatopancreas and muscle of swimming crab associated with V.alginolyticus infection. This, to our best knowledge, is the first applica-tion of metabolomics to unravel the acute metabolic response of swim-ming 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 topathogen invasion. In the study, V. alginolyticuswas injected into swim-ming crabs via arthrodial membrane of the last walking leg. Therefore,muscle may be affected earlier than other tissues. We noted that 24 hgrowth caused a significant reduction in free amino acid levels in non-infected crab muscle. Amino acids can be used as energy substrates viagluconeogenesis and further consumed via the Krebs cycle for ATP pro-duction. The concomitantly reduced succinate level might indicate anactive Krebs cycle. These activated pathways of energy metabolism

Fig. 5. Relative changes ofmetabolites in gill andmuscle of P. trituberculatusdue to growth orV.A: red diamonds, C/A: brown squares, C/B: blue triangles.

were previously observed in the low salinity-stressed swimming crab(Ye et al., 2014). No significant ATP accumulation occurs during 24 hgrowth period of control crab whereas it does in the low salinity-stressed swimming crab. Similar to low salinity stress,V. alginolyticus in-fection also caused a significant ATP accumulation in swimming crabmuscle relative to 0 h control (group A). Such an ATP accumulationwas decreased in the vibrio-infected crab muscle compared to 24 con-trol (group B), probably indicating a joint effect of vibrio infection,crab growth and PBS injection. In addition, vibrio infection does notcause a promoted gluconeogenesis because the alterations of energy-re-latedmetabolites were not observed between vibrio-infected crabmus-cles and 24 h control (group B). On the contrary, it seems toweaken thepromotion of gluconeogenesis as less amino acids were significantly re-duced in the vibrio-infection crab muscle than 0 h control (group A).

An 82.4% decrease in the IMP level occurred in vibrio-infected crabsrelative to 24 h control crabs (group B). IMP is a ribonucleotide andfirst-ly formed during the synthesis of purine. Numerous studies reported

alginolyticus infection. A: 0 h control crabs, B: 24 h control crabs, C: vibrio-infected crabs. B/

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that IMP has an enhancement to the growth performance, survival, im-mune response, and disease defense of different aquatic species such asolive flounder (Song et al., 2012) and red sea bream (Hossain et al.,2016). Immunostimulating properties of IMP enable fishes to have anincreased bactericidal activity (Song et al., 2012; Hossain et al., 2016).The IMP shortage likely implies an increased consumption of IMP inthe immune response of swimming crab to V. alginolyticus infection.

In addition, we observed a significant elevation in the betaine levelin vibrio-infected crabs relative to 24 h control crabs (group B). Betaineis a trimethylated derivative of glycine and widely distributed in nearlyall organisms. Abundant evidence stated that it mainly functions as apredominant stress protectant against physical, chemical, and biologicalstresses (Zou et al., 2016). In this paper, the elevated betaine seems toindicate that betaine plays an osmoprotective role in swimming crabsafter V. alginolyticus infection. However, recent studies have shownthat 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 amethyldonor which enables homocysteine re-methylated to form methioninevia betaine homocysteine methyltransferase (Zhang et al., 2015). Theaccumulation of homocysteinemay contribute to the increased intracel-lular reactive oxygen species generation and the reduced vibrio survivalrate in sea cucumber (Zhang et al., 2015). In this study, the accumulatedbetaine might indicate the reduced re-methylation of homocysteine inthe muscle, thus implying an enhanced ability of swimming crab to re-sist against V. alginolyticus.

4.2. Metabolic response of crab gill to V. alginolyticus

For gill, metabolomic alterations induced by V. alginolyticus infectionwere highlighted in energy disturbance. Normally, the gill lactate levelwas decreased with the crab growth, which accompanied with the ele-vated glucose level. However, these changes were not observed in thevibrio-infected crabs. Indeed, the increment of lactate level and the dec-rement of glucose occurred in the vibrio-infected crabs compared to24 h control ones (group B). These observations strongly suggest an in-volvement of anaerobic glycolysis whichmight partially cover the ener-gy costs of basalmaintenance in the vibrio-infected crabs. Such a stress-induced transition of energy metabolism from aerobic scope to anaero-biosis has been widely observed in the aquatic animals (Sokolova et al.,2012). Furthermore, glucose deprivation generally induces energystress in cells. AMP-activated protein kinase (AMPK) that functions aspart of an energy-sensing pathway (Faubert et al., 2015) is activatedin order to promote net ATP conservation in the crab gill. AMP functionsas a direct agonist of AMPK (Hardie, 2003). A sharply elevated AMP levelin the gill of vibrio-infected crabs indicates an enhanced AMPK activity,which subsequently switches on ATP-producing catabolic metabolismand switches off ATP-consuming anabolic metabolism to restore energyhomeostasis of gill cells (Andris and Leo, 2015).

In conclusion,we observed that tissue-specifiedmetabolic effects onswimming crabs were induced by V. alginolyticus. OPLS-DA analysis of1HNMR spectral data derived from extracts of crab gill, hepatopancreas,and muscle tissues has shown that vibrio infection initiates an acutemetabolic response in gill and muscle rather than hepatopancreas. V.alginolyticus infection resulted in the depressed gluconeogenesis fromamino acids, the improved energy accumulation, as well as the en-hanced cellular immune response of muscle. These metabolic alter-ations were highlighted in the depletion of amino acids and IMP aswell as in the accumulation of ATP and betaine in muscle. Vibrio infec-tion also caused the energy disturbance in the gill which manifested inthe accumulation of lactate and AMP whereas the depletion of glucose.The significant fluctuation of IMP and ATP in themuscle as well as AMPin the gill might be identified as potential biomarkers for acute V.alginolyticus infection in the swimming crab. Given that some metabo-lites such as IMP and betaine are engaged in the immune system, future

work should focus on the functions of the metabolites in theimmunometabolism.

Acknowledgements

This work is supported by the National Natural Science Foundationof China (No. 41476124), the transformation fund for agriculture sci-ence (2014GB2C220151), the Natural Science Foundation of Ningbo(No. 2014A610183), the Sci & Tech Project for CommonWealth of Ning-bo (No. 2016C10062), and the K.C. Wong Magna Fund of NingboUniversity.

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.

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