Objective:Thisexperimentaimstodiscoveriftemperaturehasaneffectontheratesofseaurchinembryonicdevelopment.Byalteringtheexternalenvironment,theexperimentwilldetermineiftemperaturewillaffectthelengthofthefirstandsubsequentcellcyclesuptothepluteuslarvaestage.

Introduction:

SeaurchinembryosexhibitrADIalholoblasticcleavageinwhichthefirstandsecondcleavagesarebothmeridionalandperpendiculartoeachother.Thethirdcleavageisequatorial,andthefourthcleavagedividesunequallytoproducemesomeres,macromeres,andmicromeres(Gilbert,2003).Thesecleavagepatternsarewellunderstoodonagrouplevel,butitisnotcompletelyclearhowtheindividualcellsareregulatedonamolecularlevel.Onecanfollowtheeggfromfertilizationtoblastulatogastrulatopluteuslarvaeformation,butthisisusuallyperformedunderidealsituations.Iftheconditionschange,suchasthesaltconcentrationortemperatureofthewater,howwillthisaffectthedevelopmentoftheseaurchin?

Manyprocesses,actinginsequenceorinparallel,makeupthecellcycle.ManystudieshaveshownhowthecellcycleisregulatedatthemolecularlevelinthecyclicaccumulationanddestructionofcyclinsandthecyclicactivityofMphase-promotingfactor(Meijeretal.,1991).However,onlysomeoftheseprocessesaffectthelengthofthecycle(Nurse,1990).Otherenvironmentalfactorshavebeensuggestedasadditionalregulatorsofthecellcycle.Forexample,temperaturehasbeenshowntohaveanumberofeffectsontheroleofvariousphasesofmitoticdivision,suchastherateofoxygenconsumptionandcarbondioxideproduction(Hoadley,1937).Researchershavefoundthatcellcycleeventsdiffergreatlyintheirdegreeoftemperaturedependence.Seaurchinsareoneorganismthatisaffectedbytemperature.Atemperature-dependentperiodexistsaftereggfertilizationinwhichthedurationofthecleavagecycleandnormaldevelopmentpatternsareaffectedbythetemperatureofthesurroundingenvironment(Yamada,K,andK.Mihashi,1998).SewellandYoung"sexperiment"TemperaturelimitstofertilizationandearlydevelopmentinthetropicalseaurchinEchinometralucunter"showedthateachseaurchinspecieshasanoptimalfertilizationtemperaturebasedontheaveragetemperaturefoundinitsnaturalhABItat(Sewell,M.A.andC.M.Young,1999).Thisoptimaltemperatureisnecessaryforthesuccessfuldevelopmentoftheembryosandpluteuslarvae(Katsuyuki,Y.,andK.Mihashi,1998).Anumberofspeciesofseaurchinsarefoundaroundtheworld.Eachspeciesthrivesintheirownuniqueenvironment.ThesubjectofthisexperimentisLytechinusvariegatus,whichisfoundintheGulfofMexicoandnormallydevelopsinwatertemperaturesnear22°C.

Materials:maleandfemaleLytechinusvariegates100mLbeakerssterilesyringesandneedlespetridisheswithcoversartificialsaltwater(ASW)graduatedcylinderglassPasteurpipetsglassdepressionslidesicebucketaluminumfoil0.5MKCl14°Cincubator37°CincubatorThermometerlightmicrospcopedigitalcameraProcedure:1.Obtaingametesofseaurchins;storeegginagentlystirredsUSPensionat22°C,washingseveraltimeswithartificialseawater(ASW)2.Collectspermfromseaurchinanddiluteonedropinto10mlbeakercontainingASW.

3.PreparethreebathsofASWat14°C,22°C(control),and37°usingawaterbathorincubator.

4.Fertilizealltheeggswiththedilutedspermina100mlbeaker.

5.Oncefertilizationenvelopehasdeveloped,notetime.

6.Transferfertilizedeggsand30mlofASWintothreeglassdishesaccordingtoappropriatetemperature.

7.Dilutewith30mlofASWatappropriatetemperature(14°C,22°C,and38°C)

8.Covereachglassdishandplaceintoincubatormaintainingtemperature.

9.Monitortheprogressofdevelopmentbytakingasmallsampleandplacingitonadepressionslidetobeviewedunderalightmicroscope.

10.When90%ormoreoftheeggshavecompletedcytokinesis,notethetimeafterfertilizationandtakestillphotographs.

11.Allowthefertilizedembryostodevelopfor24hours.

12.After24hours,transfer1dropofeachofthethreesamplesontodepressionslides.

13.Observethesampleunderthelightmicroscope.Takestillphotographsandcompareembryonicdevelopment.

14.48hoursafterfertilization,repeatsteps12and13.

Results:Temperaturehadaneffectonthecleavageratesofdevelopingseaurchinembryos.Generally,thehighertheenvironmentaltemperature,thefastertheembryodivided.At37°C,theseaurchineggsreachedthefirstcellcleavageat40minutesafterfertilization,andcleavedthesecondtimeat65minutesafterfertilization.Abnormalitiesinthegastrulatingembryowereobservedat37°C.Underidealconditions,22°C,theembryosfirstcleavagecycleoccurredat60minutesafterfertilizationandthesecondcleavagecycleat100minutes.At14°C,thecleavageratewasdramaticallyslower,withthefirstdivisiontakingplaceat95minutesafterfertilization.Only50%oftheembryoscontinuedtodivide,inwhichtheyreachedthesecondcleavageat205minutesafterfertilization.Whenthecultureswerephotographed95minutesafterfertilization,theembryosincubatedat14°Chadonlydividedonce,thecontrolembryoshaddividedtwice,andtheembryosincubatedat37-38°Chadalreadydivided3times(Figure2).

Figure2.SeaUrchinembryos95minutesafterfertilization.Embryos(A.)incubatedat14°C,(B.)incubatedat22°C(Control),and(C.)incubatedat38°C.

Twenty-fourhoursafterfertilization(Day2),thecontrolgrouphaddevelopednormally.Themajorityoftheembryosdisplayedarchenteronformationandweremotile.Notallembryoskeptat14°Chadhatchedbyday2.Theonesthathadhatcheddidnothavearchenteronsandwerenotmotile.Themajorityoftheembryoskeptat37°Chaddiedandlackedarchenterons.Theremainingembryoshaddevelopedabnormally,hadnothatchedandweresmallerthanthecontrolgroup(Figure3)

Figure3.SeaUrchinembryos24hoursafterfertilization.Embryos(A.)incubatedat14°C,(B.)incubatedat22°C(Control),and(C.)incubatedat38°C.

Forty-eighthoursafterfertilization,allembryosofthecontrolgroupwereatthepluteuslarvaestage.At14°C,thevastmajorityoftheembryosweredeadandthosealivestilldidnotshowsignsofregulardevelopmentsuchasarchenteronformation.At37°C,thereweresomepluteuslarvaebutthemajorityoftheembryosweresmallandunhatched(Figure4).

Figure4.SeaUrchinembryos48hoursafterfertilization.Embryos(A.)incubatedat14°C,(B.)incubatedat22°C(Control),and(C.)incubatedat38°C.

Discussion:Seaurchindevelopmentcanbealteredbyaseriesofenvironmentalchanges.Oneofthesechangesistemperature.Inourinitialexperiment,weshowedthatseaurchinsundergocleavagemorerapidlyinhighertemperatures.Inoursecondexperimentobservationswereextendedoveralongerperiodoftime.Weobservedthatchangesintemperaturecausedchangesindevelopment.Twenty-fourhoursafterfertilization,archenteronformationwasvisIBLeinthecontrolembryoswhereastheembryosintheothertwotemperatureswereundergoingabnormalandslowdevelopment.Lastly,48hoursafterfertilizationthecontrolembryowereallatthepluteuslarvaestagewhiletheotherembryoswereeitherdeadordevelopingabnormally.Overall,seaurchinembryodevelopmentistemperature-dependent,withtheprocessoccurringatafasterrateanddemonstratingabnormaldevelopmentsatwarmerthanidealtemperatures,andaslowerrate,andevencelldeath,takingplaceatcoolerthanoptimaltemperatures