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Metabolomics investigation of molecular responses of whiteleg

shrimp (Penaeus vannamei) to hypoxia

Thao Van Nguyen1, Andrea Alfaro1, Jenny Leon2, Bonny Arroyo2, and StanislausSonnenholzner2

1Auckland University of Technology2Affiliation not available

August 17, 2020

Abstract

Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studies have revealed

important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regarding this stressor at the molecular

level. In the present study, a gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was employed to

characterize the metabolic pathways underlying responses of shrimp (Penaeus vannamei) to hypoxia and to identify candidate

biomarkers. We compared metabolite profiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h).

Dissolved oxygen levels were maintained above 85% saturation in the control and before hypoxia, and 15% saturation in the

hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantly different between before

and after hypoxia exposure. These metabolites were energy-related metabolites (e.g., TCA cycle intermediates, lactic acids,

alanine), fatty acids and amino acids. Pathway analysis revealed 17 pathways that were significantly affected by hypoxia.

The changes in metabolites and pathways indicate a shift from aerobic to anaerobic metabolism, disturbance in amino acid

metabolism, osmoregulation, oxidative damage and Warburg effect-like response caused by hypoxic stress. Among the altered

metabolites, lactic acid was most different between before and after hypoxia exposure. Biomarker analysis also indicated lactic

acid as biomarker for hypoxia and model prediction. Future investigations may validate this molecule as a stress biomarker in

aquaculture. This study contributes to better understanding of hypoxia in shrimp and crustaceans at the metabolic level and

provides a base for future metabolomics investigations on hypoxia.

Metabolomics investigation of molecular responses of whiteleg shrimp (Penaeus vannamei) tohypoxia

Thao V. Nguyen1

Andrea Alfaro1

Jenny A. R. Leon2

Bonny B. Arroyo2

Stanislaus Sonnenholzner2

1Aquaculture Biotechnology Research Group, School of Science, Auckland University of Technology, Auck-land, New Zealand

2Escuela Superior Politecnica del Litoral, ESPOL. Centro Nacional de Acuicultura e Investigaciones Ma-rinas, CENAIM. Campus Gustavo Galindo Km. 30.5 Vıa Perimetral, P.O. Box 09-01-5863, Guayaquil,Ecuador

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*Corresponding author : Andrea C. Alfaro, Ph.D.

Aquaculture Biotechnology Research Group, School of Science, Faculty of Health and EnvironmentalSciences, Auckland University of Technology, Private Bag 92006, Auckland 1142, New Zealand

Email: andrea.alfaro@aut.ac.nz

Phone: 09 921 9999 ext 8197

Keywords : GC–MS, hypoxia, Penaeus vannamei , metabolomics, whiteleg shrimp

Abstract

Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studieshave revealed important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regardingthis stressor at the molecular level. In the present study, a gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was employed to characterize the metabolic pathways underlying responsesof shrimp (Penaeus vannamei ) to hypoxia and to identify candidate biomarkers. We compared metaboliteprofiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h). Dissolved oxygenlevels were maintained above 85% saturation in the control and before hypoxia, and 15% saturation inthe hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantlydifferent between before and after hypoxia exposure. These metabolites were energy-related metabolites (e.g.,TCA cycle intermediates, lactic acids, alanine), fatty acids and amino acids. Pathway analysis revealed 17pathways that were significantly affected by hypoxia. The changes in metabolites and pathways indicate ashift from aerobic to anaerobic metabolism, disturbance in amino acid metabolism, osmoregulation, oxidativedamage and Warburg effect-like response caused by hypoxic stress. Among the altered metabolites, lacticacid was most different between before and after hypoxia exposure. Biomarker analysis also indicated lacticacid as biomarker for hypoxia and model prediction. Future investigations may validate this molecule as astress biomarker in aquaculture. This study contributes to better understanding of hypoxia in shrimp andcrustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.

Introduction

The level of dissolved oxygen (DO) in water plays a key role in aquaculture production, especially in intensiveaquaculture systems with active stock such as shrimp. While the ideal DO level for most aquaculture speciesis 4 or 5 mg·L-1 or higher [1, 2], these levels may be difficult to maintain. Thus, low level of DO in water(hypoxia) are a threat to production since they may reduce growth rates and lead to high mortalities [3].To cope with these conditions, marine organisms are known to have adaptation strategies that allow themto mitigate hypoxic effects, including enhancing efficiency of oxygen uptake (e.g. increasing respiration rate,number of red blood cells, or oxygen binding capacity of hemoglobin), saving energy (e.g. lowering metabolicrate, down regulation of protein synthesis, altering the expression of genes and proteins of certain regulatoryenzymes), and eventually utilizing anaerobic metabolism [4, 5].

For the Pacific white shrimp (Penaeus vannamei ), which is the most important Penaeid species in aqua-culture, hypoxia appears to be a big concern in shrimp aquaculture. Under hypoxic conditions,P. vannameishrimp have been reported to turn to anaerobic metabolism which results in rapid increases of lactic acid andglucose in the hemolymph [6, 7]. In addition to accumulations of lactic acid, Mugnier et al. [8] also reporteddecreases of total haemocyte counts (THC) in shrimp exposed to hypoxia and reduction of total proteinconcentrations if exposed to both ammonia and hypoxia simultaneously. Lactate dehydrogenase (LDH) is akey enzyme of the anaerobic metabolism in all living cells that catalyzes the synthesis of lactate and pyruvatein a reversible reaction, as it converts NAD+ to NADH and back to NAD+. The structure of the LDH gene(LvanLDH) and its expression has been characterized as a response to hypoxia in P. vannamei [9]. The

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silencing of the transcriptional activator hypoxia inducible factor 1 (HIF-1) has been shown to block theincrease of LDH mRNA and activity produced by hypoxia in gills, suggesting expression of LvanLDH duringhypoxia via the HIF-1 pathway [9]. Under anaerobic conditions, shrimp require more glucose uptake inthe cells by GLUT proteins - glucose transporters [10, 11]. The genes encoding for these proteins and geneexpression during hypoxia have been characterized for P. vannamei which provides a better understandingof facilitative glucose transporters and gene regulation under hypoxic conditions in crustaceans. Phospho-fructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in glycolytic and gluconeogenicmetabolism. The cDNA of FBP and expression of PFK and FBP under normoxia (normal levels of oxygen)and hypoxia have been investigated in P. vannamei [12]. In that study the authors observed the expressionof PFK and FBP in hepatopancreas, but not in gill and muscle tissues, suggesting tissue-specific expressionof these genes. Although, such investigations on shrimp hypoxia have revealed important information re-garding the molecular responses of shrimp to hypoxia, there are still many unanswered questions. To addresssuch questions, new approaches, such as high throughput metabolomics (study of biochemical processes ofsmall molecules or metabolites) may be best suited to provide comprehensive mechanistic information aboutthe complex biochemical processes that underpin the animal’s responses to these physiological stresses.

Metabolomics is one of the newest and fastest growing omics. Metabolomics aims to characterize the metabo-lites, which are the end products of metabolism. Hence, metabolomics can be used as a tool to reveal insightsinto the molecular mechanisms underlying the responses of aquatic organisms to external stresses, diseasesand developmental processes. Metabolomics investigations for mollucscan species began in the early 2000,and the approach has expanded in recent years as applications continue to widen in scope. These studiescover diverse topics, including diet and nutrient [13-15], immunology and disease [16-18], environmentalstress [18-20], eco-toxicology [21, 22], and post-harvest handling [23, 24]. Several studies have also employedmetabolomics techniques to explore hypoxic effects in molluscan species, such as sea cucumbers [19, 20],abalone [25, 26], mussels [27, 28] and prawn [29]. In general, these studies have reported changes in metabo-lites from different tissues of the host under hypoxic stress conditions compared to controls. Most of thealtered metabolites have been found to be involved in energy and osmoregulation. These studies have led tosignificant progress in understanding molecular response mechanisms of animals to hypoxic stress. However,no metabolomics investigation has been reported for hypoxic effects onPenaeid shrimp, even though this is amajor production bottleneck in shrimp pond production. Hence, this study was conducted to reveal insightsinto the molecular pathways underlying the responses ofPenaeid shrimp to hypoxic stress. For this purpose,we exposed Pacific white shrimp (P. vannamei ) to different oxygen levels (85% and 15% saturation) andanalyzed the haemolymph metabolite profiles at 0 h, 1 h and 2 h after exposure using a GC–MS-basedmetabolomics approach. Changes in haemolymph metabolite profiles were also used to identify biomarkersassociated with hypoxic stress as a tool for on-farm management.

TheexperimentwascarriedoutattheCentroNacionaldeAcuiculturaeInvestigacionesMarinas(CENAIM),EscuelaSuperiorPolitecnicadelLitoral(ESPOL)inSanPedro,Ecuador.Whitelegshrimp(P.vannamei)(5.7±0.6gram)wereobtainedfromCENAIM’sExperimentalStation(Palmar,SantaElenaProvince,Ecuador)andwereacclimatizedinthelabforfivedays.Priortothestartoftheexperiment,120shrimpwererandomlytransferredto203-Lglassflasks(6shrimpperflask)containingfilteredandUVtreatedseawater(34.2ppt)(Figure1).TheDOinalltanksweremaintainedat85%saturation(5.8-6.7mg·L-1)bybubblingairintotheflasksthroughanairstone.DOwasmeasuredbyahandheldoxygenmeter(YSIEcoSenseDO200A,YSICompany,YellowSprings,Ohio,USA).Amongtheseflasks,10flaskswereusedascontrolwhileanothertenflaskswereusedforthehypoxiatreatment.Atthebeginningoftheexperiment(0h),twoshrimpfromeachtankwererandomlysampledformetabolomicsanalysis.Forthatpurpose,200μLhemolymphfromeachanimalwerewithdrawnandplacedinto2mLcyro-vialsandimmediatelyquenchedintoliquidnitrogen.Then,theDOlevelintenflasksfromthehypoxiatreatmentwasreducedto15%saturation(0.9-1.2mg·L-1)andtheDOlevelinthecontrolflaskswasmaintainedabove85%saturation(5.8-6.7mg·L-1).DOwasreducedbyaddingapre-determinedvolumeofa2,000mg·L-1sodiumsulfitestocksolutiontoall3-Lexperimentalflasks.Controlflaskswereaeratedwhiletreatmentflaskswerenot.Then,10shrimpfromeachtreatment(oneshrimpperflask)wererandomlysampledat1hand2handsnap-frozeninliquidnitrogen,asdescribedabove.Allhaemolymphsampleswerestoredat–80°CuntildryinginaSpeedVacConcentratorwithaRefrigeratedVaportrap(SavantTMSC250EXP,ThermoScientific)for4h(0°C,vacuumramp3,42torr/min).DriedsampleswerethensenttotheAucklandUniversityofTechnology(NewZealand)formetabolomicsanalysis(Permitno:2019073018issuedbyMinistryofPrimaryIndustries).Theshrimpwerenotfedduringtheexperiment.

Metabolomicsanalysis

Metabolitesfromhaemolymph(200μL)wereextractedwithcold(-20°C)methanol-waterandderivatizedwithmethylchloroformate(MCF),asdescribedbySmartetal.[30]withminormodifications[16].D4-alanine(20μLof10mM)wasaddedintoeachsamplebeforeextractionasaninternalstandard.Blanksamplescontainingonly20μLof10mMd4alaninewereextractedtogetherwithsamplesforqualitycontrol(QC)purposes.AnothertypeofQCsamplewereaminoacidmixtures(20μL,20mM)thatwerederivatizedastheprotocolforsamples.Afterderivatization,10μLfromeachsamplewerepooledtomakeapooledQCsampleforeachtissue.ThederivatizedsampleswereanalysedwithagaschromatographGC7890BcoupledtoaquadrupolemassspectrometerMSD5977A(AgilentTechnologies,CA,USA)withaquadrupolemassselectivedetector(EI)operatedat70eV.ThesystemwasequippedwithaZB-1701GCcapillarycolumn(30m×250μminternaldiameter×0.15μmfilmthicknesswitha5mguardcolumn)(Phenomenex,Torrance,CA,USA).TheinstrumentalparametersweresetaccordingtoSmartetal.[30].Heliumwasusedasthecarriergasandwasheldataconstantflowof1mLperminute.TheGCcolumnwasequilibratedfor6minutespriortoeachanalysis.Themassspectrometerwasoperatedinscanmode,startingafter6 minuteswithmassrange38–650 AMUat1.47scanspersecondanddetectionthresholdof100ioncounts.Samples(1 μL)wereinjectedunderpulsedsplitlessmodewiththeinjectortemperatureat260°C.Chloroformsolventandnon-derivatizedn-alkanes(C10-C40)wereinjectedatthebeginningoftheanalysistoensurereproducibilityofGC–

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MSmeasurements.ThiswasfollowedbypooledQCsamples,blankandstandardaminoacidmixtures.ThesampleswereinjectedrandomlyafterQCs.InjectionsofQCswererepeatedaftereverytensamples.Inaddition,thesamepooledQCsamplewasrunatthebeginningandattheendoftheanalysistocheckthestabilityofthesystem.Together,QCsamplesmadeupmorethan30%ofallinjectionsperformed.

Spectraprocessinganddataanalysis

RawspectraldataweretransformedintoAIAformat(.cdf)filesusingChemStation(AgilentTechnologies,Inc.,US)andprocessedusingAutomatedMassSpectralDeconvolutionandIdentificationSystem(AMDIS)software(onlinesoftwaredistributedbytheNationalInstituteofStandardsandTechnology,USA—http://www.amdis.net/)integratedwithanautomatedin-houseMassOmicspackage(theUniversityofAuckland)aspreviouslydescribed[31].Datawerenormalizedtosamplebiomassandtheinternalstandard(d4alanine)priortodataanalyses.Theintegratedweb-basedplatformMetaboAnalyst4.0(www.metaboanalyst.ca)wasusedforalldataanalysesinthisstudy,includingstatisticalanalysis,pathwayanalysisandbiomarkeranalysis[32].Datawerenormalizedbyautoscaling(mean-centeredanddividedbythestandarddeviationofeachvariable)tomakeindividualfeaturesmorecomparable.Multivariateanalyses,includingunsupervisedprincipalcomponentsanalysis(PCA)andsupervisedpartialleastsquares-discriminantanalysis(PLS-DA)wereusedtoassessvariabilityamongsamplesandbetweensampleclasses.Univariateanalyseswereperformedusingone-wayanalysisofvariance(ANOVA)(adjustedp-value[FDR]cutoff:0.05andpost-hocanalysis:Fisher’sLSD)toidentifydifferentmetabolitesinhaemolymphsamplesbetweendifferenttreatments.AheatmapofalteredmetabolitesidentifiedviaANOVAwasconstructedtoaccesstheabundanceofthesemetabolites(hypoxia/control)viaintuitivevisualization.Quantitativeenrichmentanalysis(QEA)usingglobaltestalgorithm[33]andnetworktopologyanalysis(NTA)usingrelative-betweenesscentrality[34]wereperformedtoinvestigatefunctionalrelationshipsamongtheannotatedmetabolitesforpathwayanalyses.ThepathwaylibraryofDrosophilamelanogaster(fruitfly)intheKyotoEncyclopediaofgenesandgenomes(KEGG)database[35]wasusedasthereferencepathwaylibrary.PathwaysinvolvingoneormoreannotatedmetabolitesthatmatchedwiththeKEGGdatabasewithsimultaneousQEAp-values<0.05,andwithNTApathwayimpact(PI)scores>0.0wereconsideredaspotentialprimarypathwaysofinterest.Classicalunivariatereceiveroperatingcharacteristic(ROC)analysesforlacticacidineachtissue(usinglinearsupportvectormachines)wereperformedtoassessthespecificityandsensitivityofthismetaboliteforbiomarkermodelsbasedontheareaundertheROCcurve(AUC).

Overall,weidentified81metabolitesincluding75annotatedand5unknownmetabolitesfrom480componentsinthemetaboliteprofilesofshrimphaemolymph.Themajorityofthesemetaboliteswereenergy-relatedmetabolites(e.g.,intermediatesofcitricacid[TCA]cycle,lacticacid),aminoacids,fattyacidsandotherorganicacids.PCAscoreplotsrevealedclearseparationsbetweenhypoxiagroupsandcontrolgroups(Figure2.A).However,PCAscoreplotsdidnotshowdifferencesbetweensamplingtimesforanytreatmentorcontrol.SimilarpatternswereobservedinPLS-DAscoreplotswithanexceptioninthecontrolgroupwherethe0hsamplingwasclearlyseparatedfromthe1hand2hsamplingpoints(Figure2.B).Consistentlywithmultivariatedataanalyses,univariatedataanalysisviaone-wayANOVArevealed39metabolitesthatweresignificantlydifferentamongthecontrolandtreatmentgroups.Thesemetaboliteswerevisualizedinaheatmaptorevealdetailsofdifferences(Figure2.C).Mostofthedifferenceswereobservedbetweenthehypoxiagroups(1and2h)andcontrol.Sincetherewasnodifferencebetweendifferentsamplingtimeswithineachtreatment,datafromdifferentsamplingtimeswerepooledtogethertoachieveabiggerdatasettocomparetheeffectsofhypoxiaonshrimp.

Effectsofhypoxiaonshrimphaemolymph

At-testanalysisofhaemolymphmetaboliteprofilesbeforehypoxia(0h)andafterhypoxiaexposure(1&2h)revealed44metabolitesthatdifferedbetweenthesetwogroups(Table1).Amongthesealteredmetabolites,therewere17increasedmetabolitesand27decreasedmetabolites.Mostoftheincreasedmetaboliteswereenergyrelatedmetabolites(e.g.,TCAcycleintermediates,lacticacids)someaminoacids(e.g.,alanine,threonine)andafewfattyacids(e.g.,2-hydroxyglutaramicacid,palmiticacid)whilethedecreasedmetabolitesincludedmostlyaminoacids(e.g.,asparagine,lysine,histidine)andfattyacids(palmitelaidicacid,conjugatedlinoleicacid,DHA).Pathwayanalysisindicated37involvedpathways.Afterfiltering(p<0.05andIP>0.01),17pathwayswereidentifiedassignificantlyaffectedbythelowoxygenstress(Figure3).Althoughglycolysis/gluconeogenesishadIPgreaterthan0.001,itspvaluewasgreatthan0.05.Hence,weincludedthispathwayasaslightlyaffectedpathwayofinterest.

Biomarkeranalysis

ClassicalunivariateROCcurveanalysesrevealedthatAUCoflacticacidinhaemolymphwasequalto1(Figure4).TheveryhighvalueofAUCsuggeststhatlacticacidcouldbeanimportantandaccuratebiomarkerforclassificationandpredictionmodels.Inadditiontolacticacid,therewere8metaboliteswithAUCequalto1and13metaboliteswithAUCgreaterthan0.9(Figure4).

Discussion

In the present study, we applied a GC–MS-based metabolomics approach to reveal insights into the metabolicresponses of P. vannamei to acute hypoxia. In the control treatment, there were significant decreases ofsome fatty acids and amino acids after 1 and 2 h sampling compared to the 0 h sampling time (Figure 2 ).Decreases of fatty acids and amino acids in this case indicate the use of these metabolite classes for energydemands to maintain the normal physiological functions of shrimp and maintain basic metabolic activities.The hypoxia exposure led to changes in many metabolites in the haemolymph of shrimp compared to thecontrol (0 h). These metabolites are involved in many pathways associated with the response of shrimp tohypoxic stress, including a shift in energetic metabolism from aerobic to anaerobic, osmoregulation, oxidativedamage, disturbance of amino acid metabolism and Warburg effect-like response.

Under hypoxic conditions, shrimp showed increases of many TCA cycle intermediates (citric acid, fumaricacid, succinic acid, cis-aconitic acid, itaconic acid). The TCA cycle is a major energy-producing catabolicpathway in all aerobic organisms, and does not function during hypoxia [36]. A number of studies in bothvertebrates and invertebrates have demonstrated that disturbances of TCA cycle by stress conditions (e.g.,pathogen infections, environmental stress) lead to accumulation of TCA cycle intermediates [16, 24, 37, 38].The accumulation of citric acid due to TCA cycle disturbance, in turn, leads to the generation of ITA via theenzyme immune-responsive gene 1 (IRG1) [39]. This has recently been observed in bivalves exposed toVibriosp. infections [31, 40], OsHV-1 virus [38] and also in shrimp under immune stimulation with prebiotic andprobiotics (Alfaro et al., 2020, unpublished data). Hence, the increases in many TCA cycle intermediates,itaconic acid and citraconic acid suggest the disturbance or inhibition of the aerobic TCA cycle pathway.

Along with the increase of metabolites involved in aerobic metabolism, there were accumulations of anaerobicend-products, including lactic acid, succinic acid, alanine and 2-hydroxyglutaramic acid (volatile fatty acid).

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Lactic acid is formed under insufficient oxygen conditions (anaerobiosis, onset of anaerobic metabolism) viaLDH that covert pyruvic acid to lactic acid in in a reversible reaction to generate ATP (Figure 5 ). Hence,lactic acid is considered to be a final product of anaerobic metabolism and it has been used as a stress index[41]. Indeed, an increase of lactic acid has often been observed in bivalves under different stress conditions[24, 31, 42-44]. In shrimp, accumulation of lactic acid has been recorded during out of water storage stress[45] and elevated temperatures [46]. Interestingly, increases of lactic acid due to hypoxia have been recentlyreported in M. nipponense [29] and P. vannamei [6, 7, 47, 48]. In addition to lactic acid, other metabolites,such as succinic acid, alanine and volatile fatty acids are also anaerobic end-products in invertebrates [49-51]. Increased levels of these metabolites in hypoxic shrimp are probably initial end products of anaerobicmetabolism. Together, our findings suggest that hypoxia inhibited aerobic energy metabolism and resultedin a shift from aerobic to anaerobic metabolism in shrimp. Among these metabolites, lactic acid was themost altered metabolite after hypoxia exposure (Table 1 ) and its AUC value was very high (= 1). Thissuggests that lactic acid could be a very accurate biomarker for hypoxia and stress responses in shrimp.

Interestingly, the shift in energetic metabolism from aerobic to anaerobic may indicate the presence of aWarburg effect-like response, which indicates an abnormal metabolic shift to facilitate the production ofmore energy and building blocks by non-oxidative breakdown of glucose (glycolysis). The Warburg effectwas first found in cancer cells [52], but has also been described in vertebrate cells infected by viruses [53-57].In addition, Warburg effect-like responses have been reported in invertebrate species, such as shrimp (P.vannamei ) infected with white spot syndrome virus (WSSV) [58, 59], prawn (Macrobrachium nipponense) exposed to hypoxia [29], oysters (Crassostrea gigas ) challenged with Oyster Herpesvirus type 1 (OsHV1)[38] and mussels (P. canaliculus ) exposed to Vibrio sp. [40].

Decreases of amino acids and fatty acids in hypoxia-exposed shrimp may be involved in energy metabolism.Fatty acids are important sources of metabolic energy, components of cell membranes and they participate inseveral biochemical pathways. Hence, the decrease of fatty acids has been a common observation in bivalvesas a consequence of high energy demands in responses to external stresses, such as pathogen infections [31,60], semi-anhydrous living-preservation [61] and aerial and heat exposure stress [24]. This metabolic processis proposed to be the result of a depression in the glycogen pathway and elevated gluconeogenesis, which is ametabolic pathway that generates glucose from non-carbohydrate carbon substrates. In this pathway, fattyacids can be oxidized to yield propionyl CoA, which enters into gluconeogenesis through Acetyl- CoA [51].Sun et al. [29] also observed the decrease of linoleic acid in hypoxic prawns compared to control animals,suggesting the use of fatty acid metabolism to supply energy. Hence, the decrease of many fatty acids in theshrimp haemolymph indicates the use of fatty acids as an energy source and the elevated gluconeogenesisunder hypoxic conditions.

Similar to fatty acids, amino acids are also a source of energy and their decreases in shrimp haemolymph mayindicated degradation of proteins to provide energy for shrimp under hypoxic conditions. Indeed, pathwayanalysis revealed 13 amino acid pathways that were significantly affected by hypoxia. This suggests thedisturbance of amino acid metabolisms by hypoxia. In agreement to this study, Sun et al. [29] observeddecreases of some amino acids (valine, leucine, isoleucine, lysine, glutamate and methionine) in prawn underhypoxia and reoxygenation conditions. In sea cucumbers under the combination of heat and hypoxia, mostmetabolites involved in amino acid metabolism were significantly decreased compared to the controls [20].Furthermore, decreases of amino acids to supply energy have been reported in mussels (P. canaliculus )exposed to pathogenic Vibrio sp. [31, 60]. Among the decreased amino acids, glutamine is an importantα-amino acid that serves as an energy substrate, and a building block of glucose, peptides and protein[62]. Under hypoxic conditions, cells use glutamine as an alternative fuel to generate citrate and supportproliferation [63]. Aspartic acid is a constituent of most proteins, and also play an important role in themetabolism of nitrogen and neurotransmission [64]. A study by Graham and Ellington [65] who investigatedthe metabolism of aspartate by in vitro preparations of the ventricle of the whelk (Busycon contrarium) under anoxic conditions found that there was substantial conversion of aspartate to succinate with nodetectable enrichment of other metabolites. Tyrosine (or 4-hydroxyphenylalanine) is an aromatic aminoacid that is used by cells to synthesize proteins. Tyrosine is also an antioxidant that can scavenge free

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radicals and contribute to reduce oxidative damage caused by hypoxic stress [66]. Lysine is an α-amino acid,and an essential amino acid for protein synthesis. In addition, lysine is a precursor for carnitine, a substancethat transports fatty acids to the mitochondria to be oxidised for the release of energy [67, 68]. Hence, thecommon observation in reduction of these amino acids in marine molluscs exposed to external stressors [19,20, 29, 60, 69] as well as in shrimp under the hypoxia in this study may suggest the key role of these aminoacids in energy metabolism and other unknown functions which require future investigations.

On the other hand, there were increases of some fatty acids (2-hydroxyglutaramic acid, 3-hydroxyoctanoicacid, stearic acid, palmitic acid, 9-heptadecenoic acid) and amino acids (threonine, cis-4-hydroxyproline,alanine, beta-alanine) in shrimp haemolymph exposed to hypoxia. It has been shown that short-term expo-sure to hypoxia can have a negative effect on shrimp osmoregulation [70, 71]. The lipid in the gill tissuesof marine molluscs is known to have an important role in osmoregulation and ion exchange [72-74] whichmay explain the increases in fatty acids observed in shrimp under hypoxic conditions in the present study.Aquatic invertebrates are able to use high concentrations of amino acids to regulate the intracellular osmo-larity with that of their surrounding environment [75-77]. Thus, the significant increase of many amino acidsin this study may also be involved in osmoregulation of the host. In addition, alanine and proline are amongthe most abundant osmolytes in molluscs and crustaceans [78]. The accumulation of alanine, beta-alanineand cis-4-hydroxyproline (a proline derivative) may also be indicative of osmoregulation in shrimp exposedto hypoxia.

We also observed decreases of many metabolites in glutathione pathway (cysteine, methionine, glutamicacid and glutathione) and an increase of 4-hydroxyproline which may be involve in oxidative stress causedby hypoxia exposure. Acute hypoxia was found to induce oxidative stress due to the excessive reactiveoxygen species (ROS) production in P. vannamei [79, 80]. Oxidative damage due to the excessive ROSproduction is considered a major contributor to oxidative injury during hypoxia-reoxygenation stress [81-83]. Glutathione is an antioxidant that effectively scavenges free radicals such as ROS and reactive nitrogenspecies (RNS) (e.g., hydroxyl radical, hydrogen peroxide, lipid peroxyl radical and superoxide anion) directlyand indirectly through the glutathione cycle [84]. The regulation of ROS by the glutathione pathway and theup-regulation of glutathione metabolism have been recently demonstrated in marine bivalves under stressconditions [40]. However, this process requires oxygen to convert glutathione in reduced form (GSH) intoits oxidized form (GSSG). Under the hypoxic condition in this study, we observed decreases of many keymetabolites in glutathione metabolism (cysteine, methionine, glutamic acid and glutathione) in hypoxia-exposed shrimp compared to the control shrimp, and glutathione metabolism itself was also identified assignificantly affected by the hypoxic conditions. This suggests that hypoxic conditions depress the GSHpathway in ROS regulation. This in turn may lead to oxidative damage caused by increases of ROS. Indeed,we observed the accumulation of 4-hydroxyproline which is known to be a major component of collagen,and marker for collagen degradation caused by oxidative stress [85, 86]. In agreement, the accumulation of4-hydroxyproline in the gill tissues of mussels after long-term (6 days) hypoxia exposure has been suggestedto be an indicator of oxidative injury [27]. Together, our results are suggestive of oxidative damage causedby oxidative stress in hypoxia-exposed shrimp and the depression of the GSH pathway in ROS regulationunder hypoxic conditions.

In conclusion, this study provides the first metabolomics investigation into acute hypoxia effects on Penaeidshrimp. The study revealed metabolic pathways underlying the shift in energetic metabolism from aerobicto anaerobic in shrimp under the hypoxic conditions. In addition, we observed changes in metabolites andpathways associated with amino acid metabolism and fatty acids metabolism, osmoregulation, oxidativedamage and Warburg effect-like response in hypoxia-exposed shrimp. The study also demonstrates thesensitivity and applicability of metabolomics approaches to investigate molecular mechanism of stress (e.g.,hypoxia) responses in aquatic organisms and to develop biomarkers for health assessment in crustaceanaquaculture. Future metabolomics studies may combine different analytical platforms to characterize moremetabolites classes for a broader and more detailed analysis of host responses to hypoxia.

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Acknowledgment

We are extremely thankful to the technical staff at CENAIM, ESPOL, Guayaquil, Ecuador who facilitatedand supported this research, as well as the Aquaculture Biotechnology Research Group (ABRG) team atthe Auckland University of Technology (AUT), Auckland, New Zealand for their intellectual and technicalsupport. We are also grateful to Laurence Massaut for her support and encouragement to undertake thisresearch. Timothy Lawrence assisted with the acquisition of transport permit no: 2019073018 issued by theMinistry of Primary Industries, New Zealand. This study is part of an AUT-CENAIM research collaboration,supported in part by the Ecuadorian Aquaculture Conference & AQUAEXPO 2019, Guayaquil, Ecuadorand the ABRG at AUT, New Zealand.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

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Tables

Table 1 . List of altered metabolites due to the effects of hypoxia on shrimp haemolymph metabolome.

Metabolites t.stat p.value Hypoxia effect Metabolites t.stat p.value Hypoxia effect

Lactic acid -22.405 0.000 — Asparagine 2.712 0.012 —2-Hydroxyglutaramic acid -17.051 0.000 — cis-Vaccenic acid 2.833 0.009 —Citramalic acid -15.999 0.000 — Palmitelaidic acid 2.902 0.008 —3-Hydroxyoctanoic acid -14.097 0.000 — 2,6-Diaminopimelic acid 3.232 0.004 —Threonine -13.137 0.000 — Oleic acid 3.295 0.003 —Succinic acid -12.032 0.000 — Tyrosine 3.303 0.003 —Fumaric acid -11.019 0.000 — Lysine 3.582 0.002 —Maleic acid -9.910 0.000 — Cysteine 3.881 0.001 —cis-4-Hydroxyproline -9.061 0.000 — Histidine 4.124 0.000 —2-Hydroxybutyric acid -6.923 0.000 — Glutamic acid 4.444 0.000 —Citraconic acid -6.411 0.000 — Methionine 4.596 0.000 —Citric acid -5.912 0.000 — Linoleic acid 4.711 0.000 —Malic acid -5.091 0.000 — Glutamine 4.772 0.000 —Alanine -4.472 0.000 — Glutathione 4.784 0.000 —Nicotinic acid -3.488 0.002 — Conjugated linoleic acid 4.850 0.000 —Malonic acid -2.939 0.007 — Benzothiazole 5.434 0.000 —beta-Alanine -2.521 0.019 — Aspartic acid 5.825 0.000 —Stearic acid 2.408 0.024 — Unknown 24.419 5.830 0.000 —Unknown 9.255 2.467 0.021 — EPA 6.310 0.000 —Palmitic acid 2.515 0.019 — Gondoic acid 6.333 0.000 —Unknown 29.597 2.667 0.013 — DHA 6.953 0.000 —9-Heptadecenoic acid 2.672 0.013 — Unknown 27.006 6.973 0.000 —

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Figure legends

Figure 1. Experimental design and metabolomics workflow.

Figure 2 . Chemometrics and cluster analysis of metabolite profiles of shrimp haemolymph under hypoxicstress and the control. A) PCA score plots. B) PLS-DA score plots. C) Heatmap of 39 metabolites identifiedas significantly different among the groups via one-way ANOVA.

Figure 3 . List of pathways that were affected by hypoxia in shrimp haemolymph.

Figure 4 . Biomarker analysis with univariate ROC curve of lactic acid and other metabolites with AUCgreater than 0.9 in shrimp haemolymph.

Figure 5 . Anaerobic glycolysis and Warburg effect-like response of shrimp under the hypoxia stress.

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