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

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

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

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

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

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.

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

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