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 a controlled environment, of model organism Mytilus galloprovincialis (Lamarck 1819) C. Benniciᴬ,A. Nicosiaᴬ, G. Biondoᴬ, M.V. Di Nataleᴬ,T. Masulloᴬ, C. Monasteroᴬ, G.M. Armeriᴬ, M. Tagliaviaᴬ, A. Cuttittaᴬ, a - Laboratory of Molecular Ecology and Biotechnology, Istituto per l’Ambiente Marino Costiero del Consiglio Nazionale delle Ricerche (IAMC-CNR), UOS di Capo Granitola, via del Mare 3 – 91021, Torretta Granitola (Campobello di Mazara, Tp), Italia; Molecular Ecology and Biotechnology
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Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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Technical procedures for stimulation with organic and inorganic pollutants, in acontrolledenvironment,ofmodelorganismMytilusgalloprovincialis(Lamarck1819)
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 a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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
Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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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.
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
Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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activation stateof rapidpathways involving tyrosine-mediated cell signalling,with a key roleofMitogenActivatedProteinKinases(MAPKs),inparticularthestress-activatedMAPKsp38andJNKs,andofPKC(Canesietal.,2006b).Conservationofbasiccellularprocessesofinnateimmunityfrominvertebratestomammalsrepresentsanusefulbasisforstudyingcommonbiologicalresponsestoenvironmentalexposuretodifferentfamilyofcontaminants.
Mediterraneanmussels(M.galloprovincialis)withamaximumshell lengthof5cmwereobtainedfromacommercial shellfish farm from Ganzirri e Faro lake (Messina). They were acclimated in aquarium withartificialseawater(36perthousandsalinity)for3daysat18°Cwithcontinuousaerationandtheywerefeddailywhitcommercialmicroalgaefrozenmixture.
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.
Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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Technical procedures for stimulation with organic and inorganic pollutants, in a controlled environment, of model organism Mytilus galloprovincialis(Lamarck1819)
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