Molecular Ecology and Biotechnologyeprints.bice.rm.cnr.it/16460/1/Technical procedures... · Molecular Ecology and Biotechnology . Technical procedures for stimulation with organic

Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)

1

Technical procedures for stimulation with organic and inorganic pollutants, in acontrolledenvironment,ofmodelorganismMytilusgalloprovincialis(Lamarck1819)

C.Benniciᴬ,A.Nicosiaᴬ,G.Biondoᴬ,M.V.DiNataleᴬ,T.Masulloᴬ,C.Monasteroᴬ,G.M.Armeriᴬ,M.Tagliaviaᴬ,A.Cuttittaᴬ,

a-LaboratoryofMolecularEcologyandBiotechnology,Istitutoperl’AmbienteMarinoCostierodelConsiglioNazionaledelleRicerche(IAMC-CNR),UOSdiCapoGranitola,viadelMare3–91021,TorrettaGranitola(CampobellodiMazara,Tp),Italia;

Molecular Ecology and Biotechnology


2

Index1. 1Introduction………………………………………………………………………………………………………………………….3

1.1. CISASproject……………………………………………………………………………………………………………………3

1.2. Workpackage3,ecosystemandcontaminant…………………………………………………………………3

1.3. Task3.3,invivoexperiments…………………………………………………………………………………………..4

2. Musselsasamodelorganism………………………………………………………………………………………………….4

3. Invivoexperimentaldesign…………………………………………………………………………………………………….5

4. Acknowledgments………………………………………………………………………………………………………………….6

5. References………………………………………………………………………………………………………………………………7


3

1Introduction

1.1CISASproject

TheproceduresdescribedhavebeencarriedoutwithintheprojectCISAS“InternationalCentreofadvancedstudyinenvironment,ecosystemandhumanhealth”.

Thepresent researchprogramaimsat investigatingenvironmental pollution and its connectionwith theecosystemandhumanhealth,startingfromasignificantnumberofcasestudies(SIN’sofAugusta,MilazzoandCrotone)astruenaturallaboratoriessuitableformodernmultidisciplinaryinvestigation.Thewidespreaddevelopmentofhumanactivitiesintheindustrialfield,sincethepost-warperiod,inadditionto the creation of an economy that places our country in the eighth place among the world’s majorindustrialized Countries has been causing an important and complex phenomenon of environmentalcontamination,withcrucialeffectsonterrestrialandmarineecosystemsandhumanhealth.AdecisiveactionoftheGovernmentandtheMinistriesinchargeofenvironmentalprotectionallowedtoidentifyanumberofcontaminatedareasintheItalianterritoryinneedofanurgentandimportantrecoveryaction.Theyaretheso-calledcontaminatedSitesofNationalInterest(SIN),characterizedbysevereenvironmentaldegradationandhumanimpactphenomenaofvariousorigin,substantiallycausedbythedevelopmentofmajorindustrialactivities.Inrelationtotheenvironmentalcharacterizationoftheseareas,ithasrecentlybeenconsideredcrucialandnecessarytounderstandthespecificimpactofthesefactorsonthehealthofthepeoplelivinginproximityof theabovementionedsites.Theenvironmentandhumanhealtharegenerallyobservedandinvestigatedwithasectorialapproach:rarelysynergicactionshavebeencarriedouttostudyquantitativelycause-effectrelationshipsbetweenpollutionandpopulationhealthconditions.Thecollectionofimpactontheenvironmentandonhumanhealthisamandatorystepforthedevelopmentofmoderntechnologiesinthefieldsofchemistry,systembiology,biotechnology,medicine,etc.

1.2EcosystemandcontaminantWP3

ThisWPisfocusedonpollutantstoxicityandrelatedmolecularresponsemechanismsinmarineecosystem.Biological and toxicological responses to different contaminants, including emerging ones, will beinvestigatedinthethreestudyareas(Priolo-Augusta,Crotone,Milazzo),withtheaimtohighlightpossiblerelationshipswithhumandiseases.Biological responseswillbe investigatedthroughawidepanelofbio-markers inmodelmarineorganismswithdifferentcomplexity. Inordertoanalyzetoxiceffectsatvariouslevelsofbiologicalorganization,asystemicecotoxicologycalapproachwillbeemployed.Transcriptomicandepigeneticanalyseswillbecarriedoutinordertounveilresponsesandmodificationsinducedbyexposuretoselectedpollutants.Moreover,edible,benthicandnectobenthicmarinespecieswillbeanalyzed,withtheaimtoinvestigatepollutantsuptakeroutesbyhumansthroughseafoodconsumption.Dataobtainedbytheanalysesofnaturalsampleswillbevalidatedundercontrolledconditionsinmesocosm.

1.3Invivoexperiments,Task3.3

Task 3.3 The use of model organisms to unveil novel toxicity mechanisms (IFC) Based on critical issuesrecognizedinthethreestudyareas,possiblemechanismsinvolvedinemergentdiseaseswillbeinvestigatedin model systems (i.e. Danio rerio, Paracentrotus lividus, Dicentrarchus labrax, Octopus vulgaris, etc.)(Schnitzleretal.2011;Campinhoetal.20\13,2014;Coudercetal.2016;Marellietal.2016).Thestrategyisbased on the widely recognized and accepted knowledge of the evolutionary conservation of themostmolecularmechanismsinvolvedinbasalfunctions,inbothembryonalandadulttissues(likestressresponse,cell cycle regulation, embryodevelopment andmorphogenesis, etc.) (Piconeet al. 2016).Moreover, theoverlapping between many molecular mechanisms involved in tumoral and endocrine diseases and


4

development/differentiation is well known. Thus, embryonal/larval stages of selected species (easy tomanageandmanipulate,andwellcharacterizedatmolecularlevel)arewellsuitablebothforthepreliminarydetectionofphenotypicaleffects(i.e.teratogenicity,etc.)(Kinchetal.2016)oftheexposuretopollutantsandforfurtheranalysesfocusedtounveilmolecularmechanismsinvolved(Zhuoetal.2014;Guoetal.2014).Furthermore, transcriptomicanalyses (carriedoutbymeansofNextGenerationSequencing)willprovideglobal transcriptional profiles, suitable to detect variations involving any RNA species, including ncRNAs,inducedbyenvironmentalpollutantfoundinthethreestudyareas(Caoetal.2016;Chenetal.2016).

2Musselsasamodelorganism

Bivalves can filter large volumes of water, processingmicroalgae, bacteria, sediments, particulates, andnaturalnanoparticles,potentiallyaccumulatingdifferentchemicals in their tissues.Theseorganismshavebeen long recognized as valuable indicators of pollution, and extensive background information is nowavailableontheirbiologicalresponsestoawiderangeofbothinorganicandorganicchemicals(Dagninoetal.,2007;Mooreetal.,2006).Inparticular,themusselMytilusgalloprovincialis(Fig.1),abundantincoastalmarineandestuarineenvironments,canrepresentasuitablemodelorganismforcharacterizingthepotentialimpactinmarineMediterraneanecosystem.

Figure1Mytilusgalloprovincialis

Foodparticlestrappedbythegillsievearemovedtowardsthelabialpalpsandthemouththusenteringthegut,andreachingthedigestivegland,wheredigestionoccurs.Digestivecellshaveanextremelydevelopedlysosomalsystemforintra-cellulardigestionandnutrientaccumulationforgametogenesis(Gosling,1992).

Bivalvehemocytesresembleinstructureandfunctionthemammalianmonocyte/macrophagelineage:theyareresponsibleforcell-mediatedimmunitythroughphagocytosisandvariouscytotoxicreactions(suchaslysosomalenzymeandantimicrobialpeptidereleaseandoxyradicalproduction);hemolymphserumcontainshumoraldefensefactors,suchassolublelectins,hydrolyticenzymesandantimicrobialpeptides(Canesietal.,2002).Althoughbivalvehemocytesareextremelyheterogeneous, inMytilusspp.granularhemocytesrepresentthedominantcelltype;theyarecharacterizedbyalownucleus/cytoplasmratio,highphagocyticactivity and capacity for oxyradical production (Garcia-Garcia et al., 2008). The responses of Mytilusgalloprovincialishemocytestobacterialsignals,cytokines,hormones(Canesietal.,2006a),andtoanumberofenvironmentalcontaminantshasbeen largelycharacterized (Canesietal.,2003,2006b,2007a,2007b,2007c,2007d).Inthesecells,likeinmammalianimmunocytes,theimmuneresponseismodulatedbythe


5

activation stateof rapidpathways involving tyrosine-mediated cell signalling,with a key roleofMitogenActivatedProteinKinases(MAPKs),inparticularthestress-activatedMAPKsp38andJNKs,andofPKC(Canesietal.,2006b).Conservationofbasiccellularprocessesofinnateimmunityfrominvertebratestomammalsrepresentsanusefulbasisforstudyingcommonbiologicalresponsestoenvironmentalexposuretodifferentfamilyofcontaminants.

TheuseofMytilusinthecontextoftheCISASprojectwillfollowtwomainways:

1-.Validationofstressedconditionsthroughwidelyacceptedresponsemarkers

2-Indentificationofnovelmechanismsofmolecularresponsebymeansofcomprehensiveomictools

3Invivoexperimentaldesign

Mediterraneanmussels(M.galloprovincialis)withamaximumshell lengthof5cmwereobtainedfromacommercial shellfish farm from Ganzirri e Faro lake (Messina). They were acclimated in aquarium withartificialseawater(36perthousandsalinity)for3daysat18°Cwithcontinuousaerationandtheywerefeddailywhitcommercialmicroalgaefrozenmixture.

Fugure2:GroupofsixM.galloprovincialisinexperimentalcondition

Individualsweremaintained in the laboratory for 3 days before testing, in separate aquaria, in order torelease metals and microorganisms (Freitas et al., 2012 ; Maffei et al., 2009). During this period theorganismsweremaintainedattemperature17.0±1.0°C;pH7.80±0.10,12light:12darkphotoperiodandcontinuous aeration, in artificial seawater (salinity 35 ± 1) (TropicMarin® SEA SALT from TropicMarineCenter).Seawaterwasrenewedeverytwodays(Coppolaetal.,2017).Afterthisperiod,organismsweredistributedindifferentglassaquarium(6Lseawater,salinity35�),with6individualspercontainerand3concentrationcorrespondingtolow,intermediateandhighlevelofpollution(Biblio)for7contaminants(Cd,Mn,Ni,Pb,Zn,Cu,alltheprecedentinmixture),asreportedinTable1.Duringtheexposureperiodcontainerswerecontinuouslyaerated,temperatureandsalinity(35±1)weredailycheckedwithathermometerandrefractometer.Whennecessarysalinityvalueswereadjustedaddingwatertothecontainers,accordingtotheabovestatedconditions.Mortalitywasdailycheckedandorganismswereconsidereddeadwhentheirshellsgapedandfailedtoshutagainafterexternalstimulus.Aerationwasnotintensiveanditwassimilaramongaquaria.


6

Mortalitywasrecorded.

Thefollowingtableliststheusedcontaminantsandtherelativeconcentrations.

Low[µg/l] Medium[µg/l] High[µg/l]

Cd²⁺ 0.01µg/l 0.1µg/l 1µg/l

Mn²⁺ 0.01µg/l 0.1µg/l 1µg/l

Ni²⁺ 0.01µg/l 0.1µg/l 1µg/l

Pb²⁺ 5µg/l 10µg/l 20µg/l

Cu²⁺ 5µg/l 10µg/l 20µg/l

Zn²⁺ 5µg/l 10µg/l 20µg/l

Mix* Mix1Σlowconcentration Mix2ΣMedconcentration Mix3ΣHighconcentration

Table1.( *Mixcomposition:Cd²⁺;Mn²⁺;Ni²⁺;Pb²⁺;Cu²⁺;Zn²⁺.)

Ateachsamplingperiod(12h,2hand5days),organismsweredissectedindifferentanatomicaltissues(gills,adductormuscle,digestivegland,gonads,haemocytes)andmainteinedat-80°Cforsubsequentanalysisandposteriorlymanuallyhomogenizedwithamortarandapestleundericeinpresenceoftrizollysisbuffer.

Eachhomogenizedtissueswasdividedindifferentaliquots,fromamaximumof0.5gformuscleto0,05mgforhaemocytes,usedformolecularandbiomarkeranalyses.

6. Acknowledgments

ProjectCISAS“InternationalCentreofadvancedstudyinenvironment,ecosystemandhumanhealth”.

7. References

[1] Schnitzler JG1,CelisN, KlarenPH,BlustR,DirtuAC,CovaciA,DasK. Thyroiddysfunction in seabass(Dicentrarchuslabrax):underlyingmechanismsandeffectsofpolychlorinatedbiphenylsonthyroidhormone


7

physiologyandmetabolism.AquatToxicol.2011Oct;105(3-4):438-47.doi:10.1016/j.aquatox.2011.07.019.Epub2011Aug4.

[2] CampinhoMA, Saraiva J, Florindo C, PowerDM.Maternal thyroid hormones are essential for neuraldevelopmentinzebrafish.MolEndocrinol.2014Jul;28(7):1136-49.doi:10.1210/me.2014-1032.

[3]CoudercM1,MarchandJ2,Zalouk-VergnouxA1,KamariA1,MoreauB2,Blanchet-LetrouvéI1,LeBizecB3,MouneyracC4,PoirierL5.ThyroidendocrinestatusofwildEuropeaneels(Anguillaanguilla)intheLoire(France).Relationshipswithorganiccontaminantbodyburdens.SciTotalEnviron.2016Apr15;550:391-405.doi:10.1016/j.scitotenv.2015.12.136.Epub2016Feb2.

[4]MarelliF,CarraS,AgostiniM,CotelliF,PeetersR,ChatterjeeK,PersaniL.Patternsofthyroidhormonereceptorexpressioninzebrafishandgenerationofanovelmodelofresistancetothyroidhormoneaction.

[5]PiconeM,BergaminM,LossoC,DelaneyE,ArizziNovelliA,GhirardiniAV.AssessmentofsedimenttoxicityintheLagoonofVenice(Italy)usingamulti-speciessetofbioassays.EcotoxicolEnvironSaf.2016Jan;123:32-44.doi:10.1016/j.ecoenv.2015.09.002.

[6]KinchCD,KurraschDM,HabibiHR.Adversemorphologicaldevelopmentinembryoniczebrafishexposedto environmental concentrations of contaminants individually and in mixture. Aquat Toxicol. 2016Jun;175:286-98.doi:10.1016/j.aquatox.2016.03.021.

[7]ZhuB,WangQ,WangX,ZhouB.Impactofco-exposurewithleadanddecabromodiphenylether(BDE-209) on thyroid function in zebrafish larvae. Aquat Toxicol. 2014 Dec;157:186-95. doi:10.1016/j.aquatox.2014.10.011.

[8]GuoC,ChenX,SongH,MaynardMA,ZhouY,LobanovAV,GladyshevVN,GanisJJ,WileyD,JugoRH,LeeNY,CastronevesLA,ZonLI,ScanlanTS,FeldmanHA,HuangSA. Intrinsicexpressionofamultiexontype3deiodinase gene controls zebrafish embryo size. Endocrinology. 2014 Oct;155(10):4069-80. doi:10.1210/en.2013-2029.

[9]CaoC,WangQ,JiaoF,ZhuG.Impactofco-exposurewithbutachlorandtriadimefononthyroidendocrinesysteminlarvalzebrafish.ExpToxicolPathol.2016Sep;68(8):463-9.doi:10.1016/j.etp.2016.07.004.

[10]ChenL,ZhuB,GuoY,XuT,LeeJS,QianPY,ZhouB.High-throughputtranscriptomesequencingrevealsthe combined effects of key e-waste contaminants, decabromodiphenyl ether (BDE-209) and lead, inzebrafishlarvae.EnvironPollut.2016Jul;214:324-33.doi:10.1016/j.envpol.2016.04.040.

[11]A.Dagnino,J.I.Allen,M.N.Moore,K.Broeg,L.Canesi&A.Viarengo.Developmentofanexpertsystemfortheintegrationofbiomarkerresponsesinmusselsintoananimalhealthindex.Received28Feb2006,Publishedonline:08Oct2008Biomarkers,MarchApril2007;12(2):155172

[12]Moore, J.L., Rush, L.M., Breneman, C.,Mohideen,M.A., and Cheng, K.C. (2006) Zebrafish GenomicInstabilityMutantsandCancerSusceptibility.Genetics174(2):585-600.

[13]Gosling,E.M.,1992b.GeneticsofMytilus.In:Gosling,E.(Ed.),TheMusselMytilus:Ecology,Physiology,GeneticsandCulture.Elsevier,Amsterdam,pp.309–382.


8

[14]CanesiL,PezzatiE,StauderM,etal.VibriocholeraeinteractionswithMytilusgalloprovincialishemocytesmediatedbyserumcomponents.FrontiersinMicrobiology.2013;4:371.doi:10.3389/fmicb.2013.00371.

[15]García-García,E.,Prado-Alvarez,M.,Novoa,B.,Figueras,A.,Rosales,C.,2008. ImmuneResponsesofMusselhemocytesubpopulationsaredifferentiallyregulatedbyenzymesofthePI3-K,PKC,andERKkinasefamilies.DevelopmentandComparativeImmunology32,637e653.

[16]Canesi,L.,Ciacci,C.,Lorusso,L.C.,Betti,M.,Guarnieri,T.,Tavolari,S.,Gallo,G.,2006a.Immunomodulationby17b-Estradiolinbivalvehemocytes.TheAmericanJournalofphysiology,regulatory.IntegrativeandComparativePhysiology291,R664eR673

[17] Canesi, L., Ciacci, C., Betti, M., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2003. Effects of PCBcongeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cellsignalling.AquaticToxicology63,293e306.

[18]Canesi,L.,Betti,M.,Ciacci,C.,Lorusso,L.C.,Pruzzo,C.,Gallo,G.,2006b.Cellsignalingintheimmuneresponseofmusselhemocytes.InvertebrateSurvivalJournal3,40e49.

[19]Canesi, L.,Ciacci,C., Lorusso,L.C.,Betti,M.,2007a.Effectsofendocrinedisruptingchemicalson theimmune system of the edible bivalve mollusc Mytilus. In: Marino, M., Mita, D.G. (Eds.), The EndocrineDisruptors.TransworldResearchNetwork,Trivandrum,Kerala,India,pp.1e12.

[20] Canesi, L., Ciacci, C., Lorusso, L.C., Betti,M., Gallo, G., Poiana, G.,Marcomini, A., 2007b. Effects ofTriclosan onMytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possiblemodesofactiononnontargetorganisms.ComparativeBiochemistryandPhysiologyC145,464e472.

[21]Canesi,L.,Lorusso,L.C.,Ciacci,C.,Betti,M.,Regoli,F.,Poiana,G.,Gallo,G.,Marcomini,A.,2007c.Effectsof blood lipid lowering pharmaceuticals (bezafi- brate and gemfibrozil) on immune and digestive glandfunctionsofthebivalvemollusc,Mytilusgalloprovincialis.Chemosphere69,994e1002

[22] Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Rocchi, M., Poiana, G., Marcomini, A., 2007d.ImmunomodulationofMytilushemocytesbyindividualestrogenicchemicalsandenvironmentallyrelevantmixturesofestrogens:invitroandinvivostudies.AquaticToxicology81,36e44.

[23]Canesi,L.,Betti,M.,Ciacci,C.,Lorusso,L.C.,Pruzzo,C.,Gallo,G.,2006b.Cellsignalingintheimmuneresponseofmusselhemocytes.InvertebrateSurvivalJournal3,40e49.

[24] Freitas, R., Gómez-Marín, C., Wilson, J.M., Casares, F., and Gómez-Skarmeta, J.L. (2012) Hoxd13contributiontotheevolutionofvertebrateappendages.DevCell23(6):1219-1229.

[25]MaffeiA., LamboM.E., TurrigianoG.G. (2010).Developmental regulationofexperience-dependentinhibitoryplasticity.J.Neurosci.30,3304–3309

[26] Coppola, L., E. Diamond Riquier, and T. Carval(2016), DYFAMED observatory data, SEANOE,doi:10.17882/43749.

Documents

Molecular Ecology and Biotechnologyeprints.bice.rm.cnr.it/16460/1/Technical procedures... · Molecular Ecology and Biotechnology . Technical procedures for stimulation with organic