Compara'veOcularMorphologyof

TroglomorphicFish:Implica'onsforEvolu'on?

ChuckSchobert,DVM

Plan

•  Non‐vertebratechordateeyes,agnatha

•  Congoblindfish:Lamprologus lethops •  Travelogue:CongoRiverHabitats•  OcularAnatomy(also,HotOffthePress!)

•  Mexicanblindcavefish:Astyanax mexicanus •  Travelogue:Habitat•  Modelsystem•  OcularAnatomy•  Sowhat?:Conclusions,specula'ons,andques'ons

TeleostFishEyeAnatomy

TheChordatesinEvolu'on

Cladogram:Hagfish&Lampreys

Comparison

HagfishandLampreys

•  Nopairedpectoral(shoulder)orpelvic(hip)fins•  Notochordpersistsforlife.•  Theyhavenoscales.•  Theaxonsoftheirneuronsareunmyelinated.•  Lampreyshavebothaninnateimmunesystemandanadap'veimmunesystem,butthelaVerisen'relydifferentfromthatfoundinthejawedvertebrates.

Hagfish•  Eatdeadwhales•  Secretecopiousamountsofthickslime(mucous)

•  Liveindeepoceanictrenches(>100meters)

•  Recommenda'on:YouTube:

“EddieandtheHagfish”

HagfishEyes

•  Nocornea•  Nolens•  2‐layeredre'na(neuronal)•  Nomelanin•  Primi'vephotoreceptors•  Wiredtothebrainlikea

pinealgland•  Nopinealgland•  Photoreceptorssynapse

directlyontoganglioncells

Eye

TransverseSec'ons

Hagfish Specimen courtesy of Dr Ivan Schwab 

LampreyEye:Larval

•  Larvalformtoadultform:FiveYears!

•  Mostlyburiedinmud•  Nolensini'ally

•  Larvadetails–  Similartohagfisheye–  Eyecoveredinskin

LampreyLarvalEye

LampreyAdult•Cornealargelycon'nuouswiththeskin•Nomusclesofaccommoda'on•Hasmostofthestructuresofthevertebrateeye:

–Lens–3neuronallayersinre'na–Extraocularmuscles–Wiredtobrainlikeavisualeye–MelanininchoroidandRPE

Colorvisionandpinealgland

LampreyAdultEye:FiveYearsLater

PhotoreceptorEvolu'on

FromLamb,TDetal.Evolu?on of the Vertebrate eye: opsins, photoreceptors, re?na and eye cup.2007.NatureReviews:Neuroscience,8:960‐975.

Hagfish Lamprey

TakeHome?

•  Awindowtotheearliestdevelopmentofthevertebrateeye–  Hagfish:circadianfunc'on–  Lamprey:image‐formingeye,vertebrate‐like–  Evolu'onarycontroversyaboutrela'onshipbetweenhagfishandlamprey:

–  Ifhagfishdivergedearlierthanlampreysfromacommonancestor,opportunitytoseetheearliestexamplesofvertebrateocularevolu'on

–  Ifhagfishdivergedfromthelampreyclade,thenoculardifferencesareperhapsdegenerate

•  Andofcourse,weirdfood

Hagfish‐SlimeCheddar‐GruyereScones

4cupsall‐purposeflour2tablespoonsbakingpowder4teaspoonssugar1/2teaspoonsalt1cup(twos'cks)chilledunsaltedbuVer,cutinto1/2‐inchcubes2cups(packed)coarselygratedextra‐sharpyellowcheddarcheese(about9ounces),oramixof6ouncescheddarand3ouncesgruyere.1‐1/2cupschilledheavywhippingcream6tablespoonshagfishslimePreheatovento375F

Inafoodprocessor,blendflour,bakingpowder,sugar,andsalt.CutinthebuVerusingquickpulsesun'lthemixtureresemblescoarsemeal.Addcheeseandcutinusingquickpulses.Inasmallbowl,whisktogetherthecreamandhagfishslime.Withthefoodprocessorrunning,addcreammixturethroughfeedtube.Processun'ldoughjustholdstogether–don’tovermix!

Turndoughoutontoalightlyflouredworksurface.Gatherthedoughtogetheranddivideintoquarters.Pateachquarterintoaroundjustshortof1inchhigh(itshouldbeabout6‐7inchesindiameter).Usingaclean,sharpknife,cuteachroundintosixwedges.Transferhalfthewedgestoungreasedbakingsheetslinedwithparchmentpaper,spacingthemabout2inchesapart.

Bakethefirstbatchofsconesun'ltheedgesjuststarttobrownandatoothpickcomesoutclean,about20minutes.Transferthem,s'llontheirparchmentpaper,toawireracktocoolatleast10minutes,duringwhich'meputinthesecondbatchofscones.

Servewarmoratroomtemperature.Thesconeswillstandforabout8hours.Donotrefrigerate.Ifyouwanttoreheatthem,warmthemina350Fovenforabout5minutes

LampreyBordelaiseFor:6servingsTime:2h50Difficulty:medium

IngredientsforLampreyBordelaise:•  1live2kglamprey•  1dloil•  100gbuVer•  1boVleredBordeauxwine•  1smallglassArmagnac•  4shallots•  8onions•  15leeks•  4garliccloves•  600gBayonneham•  2tablespoonsflour•  1glassbroth•  2cloves•  1bouquetgarni•  6slicescountry‐stylebread•  salt•  Pepper

TechnicalstagesforLampreyBordelaise:

•  Bleedthelampreybyhangingitbytheheadandcuqngthetailoveracontainertocollecttheblood.Whenthere'snoblooddrippinganymore,dipthelampreyinboilingwaterfor1minute.

•  Takeitoutandpeelit.Cutin4cm‐thick(1,57inches)slices.Puttheslicesinthecontainerofblood.•  Cutthewhitepartoftheleeksinto7‐8cm‐long(2,76‐3,15inches)whistlesandsteamtheminbuVer.•  Addthedicedham,theshallotsandmincedonions.Sprinklewithflourandroast.•  Moistenwiththewineandbroth.Addtheclovesandbouquetgarni.Addpepper,saltaliVle.Addtwocrushedgarliccloves.Bringtoaboilingpoint,thencookfor45minutesoverlow

heat.•  45minuteslater,addthepiecesoflampreyinthesauce,coverandcookagainfor45minutes.•  Removetheslicesoffish,andflambtheminanotherpanorskilletwithArmagnac.•  Pourtheequivalentofaglassofwarmsauceoverthebloodinordertodiluteit,andpoorintheskillet.S'rwell.Putbackthelamprey,seasonandcookfor10minuteswiththelidon.

BeatthesaucewithbuVer.•  Meanwhile,toastthebreadandrubitwiththeremaininggarlic.Putthelampreyinashallowdish,ontheslicesofbread.Puttheleeksallaround.Coatwiththesauce.

L. lethops andL. ?gripic?lis 

BenthicFish:gasbubbledisease

“mondellibureau”

CongoRiver

•  Shortfilm

WhatiscoolabouttheCongoRiver?

•  Many,manymicrohabitats•  Evolu'onoccurringallthe'me!

•  Weirdriver=weirdfish

SightedvsBlind

L. Lethops histologyimages

L. ?gripic?lis 

Immunohistochemistry:opsins

Hotoffthepress!Mastacembelus brichardi 

Mastacembelus brichardi

•  onespecimenisfullydepigmentedandeyeless•  secondspecimenisaslightlypigmentedmorphwith'nyeyes:fullydevelopedlens,re'na,incompletecornea,eyesdorsallydisplacedtowardmidline

•  intraspecificcomparisonoristheves'gialeyedmorphahybridbetweentheeyelessbrichardiandaneyedcongener

•  collectedatthesamelocality,inshallowwaterunderrocksandboulders.

Astyanax mexicanus 

•  Blindandsightedinsamespecies•  Divergence1mya

•  Modelsystemfordevelopment,moleculargene'cs

•  Differencesinocularanatomy

AMatureAdult

Sighted

Blind

Cavehabitat

Modelsysteminevodevo

•  Moleculargene'cswellknown•  Embryologicaldevelopmenteasilystudied:eyebeginstodevelop,lenscellsundergoapoptosis,degenera'on

•  4‐6monthgenera'on'me•  Lenstransplantexperimentsshoweyedevelopmentgeness'llpresent:lensfromsightedintoblind,viceversa

•  Pleiotropy:singlegenecontrolstwoormorephenotypictraitsthatmayseemunrelated:on‐offswitch….

Ocularanatomy

•  Comparisonmontage

A. Mexicanus images:blind

A. mexicanus:sighted

Implica'onsforevolu'on?

•  Evolu'onnotalwaysconstruc've•  Regressiveevolu'onoccurs•  Differenthabitatsproducevariedselec'onpressures

•  Fishadaptstodarknessthroughselec'onof“numerousgrada'ons”leadingtoincreasedolfac'on,enhancedmechanorecep'on,alteredcraniofacialstructure

PhenotypicPlas'city

•  Phenotype=genotype+environment•  Light‐darkexperimentswithepigeanAstyanax fasciatuslarvae

•  Eyesoflarvaeraisedindarknessfor30daremuchlessdevelopedthanthoseraisedinlight

•  Alternatedevelopmentalpathwaysbasedonenvironmentalcondi'ons……..

Spin‐offs

• Why develop a morphological catalog of blind/par?ally‐sighted fish? 

•  Relatethesemorphologicaldifferencestogenomedifferencestoaidunderstandingofeyeevolu'on,developmentanddisease

•  Usewell‐studiedsystemssuchasZebrafishandAstyanax spp tounderstandtherela'onshipbetweengene'csandmorphologywhenlookingatwildfish

Manythanks!

•  DrDickDubielzig•  DrMelanieS'assnyandcolleagues,AMNH•  DrWilliamR.Jeffery,UMD•  DrIvanSchwab•  DrLeandroTeixeira•  DrJenDreyfus•  MrScoVEarnest•  MsKateLieber•  Ms.JaniceLokken

Ques'ons,specula'ons?