3 3 triol 3-glucuronate,5 had thelowestapparentEC tilapia weremostsensitiveto17 characteristic sigmoidalconcentration–responsecurves.However, Gambelas, 8005-139Faro, Portugal. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,4203-4212doi:10.1242/jeb.111518 Received 23July 2014; Accepted7October2014 *Author ([email protected]) for correspondence 94,7002-554Évora,Évora, Portugal. Apartado 1 endocrine changesinconspecificsthatpromptgonadalmaturation and facilitating thelocationandchoiceofsuitablemates,and/ortriggering fishes and,insomespecies,havebeenidentifiedassexpheromones, Sex steroidsandtheirconjugatescanbepotentodorantsforteleost communication, Cichlid KEY WORDS:Pheromone,Steroid,Olfaction,Receptor, Chemical concentrations (1 steroids. Stimulationoftheolfactoryepitheliumwithincreasing prostaglandins, unconjugatedsteroidsor17-20-conjugated several 3-glucuronidatedsteroids,butdidnotrespondto electro-olfactogram. of assess thesensitivity, specificityandversatilityoftheolfactorysystem and its20 release asexpheromone,5 Mozambique tilapia[ lives ofvertebratesandoftenarespeciesspecific.Dominantmale African cichlidradiation.Chemicalsignalsmediatekeyaspectsinthe speciation; chemicalcommunicationcouldbeoneofthedrivers Cichlids offer anexcitingopportunitytounderstandvertebrate Tina Keller-Costa detection suggests twodistinctandspecificreceptorsforpheromone Olfactory sensitivitytosteroidglucuronatesinMozambiquetilapia RESEARCH ARTICLE indeed, beinvolvedinspeciation. Astatotilapia burtoni perception fromthemorerecentlyderivedEastAfricancichlid signal. However, O.mossambicus detecting 17β receptor types;onedetectingamalesexpheromoneandsecond specificity to3-glucuronidatedsteroidsthroughtwodistinctolfactory Mozambique tilapiahasevolvedhigholfactorysensitivityand is detectedvia(a)distinctolfactoryreceptor(s).Inconclusion,the 5 Cross-adaptation andbinarymixtureexperimentssuggestedthat INTRODUCTION ABSTRACT common olfactoryreceptor(s),whereas17 Centre of MarineSciences (CCMAR),Universidade doAlgarve, Campusde Centre of α α β ,17α ,3α -glucuronate and17 O. mossambicus -reduced pregnan-andandrostan-3-glucuronatesshare(a) -dihydroxy-5 α -epimer, viatheirurine.Theobjectiveofthisstudywasto -estradiol 3-glucuronate,aputativefemale-derived pmol β , suggestingthatchemicalcommunicationcould, to othersteroidsandtheirconjugatesusingthe -pregnan-20-one 3-glucuronate,etiocholanolone Oreochromis mossambicus 1,2 Oreochromis mossambicus l − β 1 , AdelinoV. M.Canário -pregnan-3 to 10 μmol β β -pregnan-3 -estradiol 3-glucuronateproduced 50 2 Departamento deBiologia, Universidade de Departamento β and maximalresponseamplitude. -estradiol-3-glucuronate, whichalso differs markedlyinitsolfactory α l − ,17α 1 ) of5β α ,17α ,20α β -estradiol 3-glucuronate ,20β -pregnan-3 -triol 3-glucuronate, -triol 3-glucuronate was sensitiveto (Peters 1852)] 1 and PeterC.Hubbard α ,17α ,20β - however, constituteanadditional speciationfactor. Nevertheless, Divergent selectiononchemicalcommunicationsystems may, specialisation forparticulartrophic niches(Greenwood,1991). to lightheterogeneityinthehabitat (Seehausenetal.,2008)alongside mainly beenontheevolutionof colourpolymorphismthatislinked in Africancichlidradiation(Seehausen etal.,2008).Focussofarhas environmental conditionshasbeen suggestedasanimportantdriver adaptation ofthesensoryandsignallingsystemstodifferent Cichlidae currently 1670describedspecies(‘ListofNominalSpecies of Costa etal.,2014).Cichlidsareanextremelydiversetaxon with and Cichlidae(ColeStacey, 2006;Hubbardetal.,2014;Keller- 1980; Corkumetal.,2008;Murphy2001;Tierney etal.,2013) derive fromafewrepresentativesoftheGobiidae(Colombo et al., to, andpheromonalfunctionof,hormonalsteroidsarescarce and for Perciformes,www.fishbase.org), studiesonolfactorysensitivity (Sorensen etal.,1988). sulphated 17,20 Sorensen etal.,1995).Thepreovulatorypheromoneincludesfreeand sensitivity throughseparateolfactoryreceptors(Sorensenetal.,1988; released byfemales,aredetectedconspecificmaleswithhigh mixture testsestablishedthatthepre-andpost-ovulatorypheromones, for example,EOGrecordingsfromcross-adaptationandbinary receptor(s) (ColeandStacey, 2006).Ingoldfish( twice theconcentrationofeitherodorantindicate(a)sharedolfactory responses tothemixturethataresmallerorequivalentthoseat responses totheindividualodorantsgivenalone.Conversely, EOG response toamixtureoftwoodorantsisapproximatelythesum 1995). Inbinarymixturetests,receptorsitesareseparateiftheEOG (Caprio andByrd,1984;ColeStacey, 2006;Sorensenetal., greatly reduced,comparedtothesignalmeasuredprioradaptation to thetestodorantduringadaptationshouldbeunaffected, i.e.not odorant actthroughindependentolfactoryreceptorsites,theresponse during adaptationtoasecondodorant.Ifthe‘test’ and‘adapting’ to onetestodorantismeasuredprioradaptationandthenagain Johnson, 2002).InEOGcross-adaptiontests,theresponseamplitude electro-olfactogram (EOG)(forgeneralreviewseeScottandScott- different odorantsaredetectedbyseparateorsharedreceptors,isthe olfactory sensitivityinfreshwaterfishes,andtoexplorewhether and/or endocrineresponse.A simpleandreliablemethodtostudy integrate theinformationandtriggerappropriatebehavioural receptors fromwhichthesignalisrelayedtospecificbrainareasthat single ormultiplecomponent(s)andaredetectedbyolfactory Stacey andSorensen,2005).Pheromonesmaybecomposedofa improve fertilitytoenhancereproductivesuccess(Stacey, 2010; PGF pheromone, bycontrast,consistsofF-typeprostaglandins,mainly different receptors(Sorensenetal.,1995).Thepost-ovulatorygoldfish Within thePerciformes,largest teleostorder(OrderSummary 2 α and 15K-PGF ’, www.fishbase.org), mostlynativetoAfrica,and 1, * β -dihydroxy-4-pregnen-3-one (17,20 2 α , whichtoohavedistinctolfactoryreceptorsites Carassius auratus), β -P), actingvia 4203

The Journal of Experimental Biology 4204 of theconjugateinsteroid(ColeandStacey, 2006).Unfortunately, olfactory receptorsites,classifiedaccordingtothetypeandposition hormonal steroidconjugates(Robisonetal.,1998)withfivedistinct (Mozambique tilapia). mouth-brooders, signals acrosscichlidsislimited,withtheexceptionoftwomaternal knowledge oftheidentity, perception andfunctionsofchemical RESEARCH ARTICLE –1 L C taurochenodeoxycholicacid standardlength self-adaptedcontrol TCD SL SAC 5β-etiocholan-3 electro-ofactogram PGF 5β-etiocholan-3 ETIO-3-S 17β-estradiol3-sulphate ETIO-3-G 17β-estradiol3-glucuronate EOG bodyweight E2-3-S E2-3-G 17β-estradiol3,17-diglucuronate E2-3,17-diG 5β-pregnane-3α,17,20-triol-3 BW 5β-pregnane-3α,17,20-triol3 20β-P-3-G 20α-P-3-G 17α,20β-dihydroxypregn-4-en-3-one 3α,17-dihydroxy-5β-pregnan-,20-one-glucuronate 20one-P-3-G 17,20β-P 15K-PGF EOG amplitude normalised to 10 μmol l -serine standard abbreviations List of 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0 0 0 2α 10 100 nmoll 100 nmoll E2-3-G 20one-P-3-G 20α-P-3-G 100 nmoll –12

2α

10 –11

prostaglandin F 15-keto-prostaglandin F

–1 –1

–1 10 2 mV 2 mV 2 Astatotilapia burtoni

s 2 mV 2 –10

100 nmoll s 100 nmoll 100 nmoll

s

10 –9 A. burtoni

10 –1 –1 –1 –8

log Molarconcentration (moll 2α

α-ol-17-one 3-sulphate α-ol-17-one 3-glucuronate

10 –7 10 has olfactorysensitivitytoseveral 2α –6 10 and –5 α-glucuronate α-glucuronate Oreochromis mossambicus 10 100 nmoll 100 nmoll 100 nmoll ETIO-P-3-G 20β-P-3-G TCD –12

10

–11

2 mV –1 –1 –1 2 10 2 mV 2

s

s –10 100 nmoll 100 nmoll 2 mV 100 nmoll 2 –1

s 10 )

–9

10 –1 –1 –1 –8

10 –7 10 –6 10 ketotestosterone, 17 blood andurineofO.mossambicus common receptor(s).Incontrast,steroidsknowntobepresentinthe Both steroidsevokelarge olfactoryresponsesmediatedby(a) endocrine systemandoocytematuration(Keller-Costa etal.,2014). P-3-G) andofitsα sensitivity of diversification isadriverforAfricancichlidradiation. and thereforetoaddressthehypothesisthatchemicalsignal to assesstheolfactorysteroidreceptordiversityinAfricancichlids many different receptorsitesareinvolved.Suchinsightsnecessary urinary pregnanetriol-3-glucuronates,aredetectedand,ifso,how or othersteroids,includingsteroidsthatarestructurallyrelatedtothe (Frade etal.,2002).However, itisunknownwhetherprostaglandins Reis-Henriques, 1996),arenotdetectedbytheolfactoryepithelium glucuronate andsulphateconjugates(Oliveiraetal.,1996;Rocha concentrations of5β Keller-Costa etal.,2012).Dominantmaleurinecontainshigh spawn (Barataetal.,2008;Barata2007;Huertas2014; aggression betweenmalesand,duringcourtship,toprimefemales may be.Male these steroidconjugatesand,ifso,whattheirpheromonalfunction it isnotyetknownwhether Thus, theobjectivesofthisstudywere,firstly, toassessolfactory –5 10 μmol Fig. pheromone, 20 (semi-logarithmic plot,mean±s.e.m.)forthemaletilapiasex profiles tothesteroid3 sigmoidal (three-parameterHill)curvewasfittedtotheresponse filled circles)andfemales( glucuronates andabileacid(TCD).Responsesofmales( 100 male (solidline)andafemale(dashedline),recordedat the sameaslowesttwographs.RepresentativeEOGtracesofa females, dashedline).The

nmol .EOGconcentrationresponseprofiles. 1. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 O. mossambicus l − l O. mossambicus 1 − 1 L-serine response)EOGconcentration–responsecurves -epimer (20α odorant concentrations,arepresentedasinsets. -pregnane-3α α α -P-3-G and20 ,20β -dihydroxypregn-4-en-3-one andtheir A. burtoni α -glucuronates ofbothsexes(males,solid; to steroids;secondly, toestablish,by -P-3-G), whichstimulatethefemales’ N x =10–14; opencircles)areshown.A axes (odorantconcentration)areall ,17α use urinarysignalstomediate β -P-3-G, andothersteroid3 ,20β synthesises orreleasesanyof -triol-3-glucuronate (20 males, including11- Normalised (to N α =8–14; - β -

The Journal of Experimental Biology response the apparentEC EOG amplitudeandsaturationconcentration(1 1).For E2-3-G, boththe saturation oftheolfactoryreceptors(Fig. 7slhtdseod 9cro etseoe1-upaeT1- Insensitive T-17-S 3,17-diglucuronidated 18 17 carbon 20-glucuronidated 21 17 carbon 17 Testosterone 17-sulphate 21carbon 3-glucuronidated 21 19carbon Etiocholan-3 20-sulphated steroids 5 17-sulphated steroids 19carbon carbon 3-sulphated steroids 5 21carbon Unconjugated steroids RESEARCH ARTICLE detection thresholdwaslowestforE2-3-G(10 pmoll 1).The between theresponsesofmaleandfemalerecipients(Fig. 1),andnodifferences werefound 3-glucuronidated steroids(Table were notpursuedfurther. responses, yetonlyathighconcentrations(100 1).ETIO-3-SandE2-3,17-diGinducedsmallEOG (Table prostaglandins, evenatconcentrationsashigh1 Nor didtheyrespondtoanyunconjugatedsteroids,E2-3-Sor representatives of17-or20-glucuronidatedsulphatedsteroids. 1),buttheydidnotgiveEOGresponsesto steroids (Fig. Mozambique tilapiaconsistentlyrespondedto3-glucuronidated 2006), amorerecentlyderivedAfricancichlid. compare theseresultstofindingsfrom act viasharedorindependentolfactoryreceptors;andthirdly, to cross-adaptation andbinarymixturetests,whethersteroidodorants c a Testosterone 17-glucuronate 19carbon 17-glucuronidated Compoundname Prostaglandin F2 20carbon group Prostaglandins (PG) Bile acid Chemical class 89.62±16.15 2A,B).As fortheothersteroids,20α steroids (Fig. concentration EC lower. Accordingly, theE2-3-G apparent half-maximaleffective excluded fromfurtherconcentration–response,cross-adaptationandbinarymixturetests.Conc.,concentration. was around1 by 20one-P-3-G(100 Table reaching anapparentmaximumataround1 G, 20β 1 female, 0.14±0.08 Detected steroids and EOG concentration response tests andEOGconcentrationresponse Detected steroids RESULTS O. mossambicus Read estimatesbasedonmeanconcentrationresponsecurves. μmol steroids steroids steroids steroids Sigmoidal concentration–responsecurveswereobtainedusing all

1. Steroidstestedinthisstudy -P-3-G, 20one-P-3-GandETIO-3-Gincreasedrapidlybefore l − 1 I ). However, thelatterresponseswerenot consistent and max nmol (male, 0.38±0.05;female,0.34±0.04) werelowerthan nmol does nothaveanyolfactorysensitivitytothissteroid. 50 50 and l −

l nmol 1 − ; female,86.3±18.74 1 (mean ±s.e.m.;male,0.07±0.02 pmol . TheEOGresponseamplitudesto20 4cro Taurochenodeoxycholic acid 24 carbon Chemical 8cro 17 Etiocholan-3 18 carbon 19 carbon 8cro 17 18 carbon 17 18 carbon 17 Etiocholan-3 18 carbon 19 carbon I max l − values ofalltheother3-glucuronidated 1 l ) andapparentmaximalolfactory − 1 ), andforthemajority, thethreshold 5 3 3 5 Prostaglandin 15keto-F2 β β β α α β β β α β α β β -pregnan-3 -pregnan-3 -pregnan-3 -pregnan-3 ,17-dihydroxy-5 ,17α -estradiol-3-glucuronate etail31-ilcrnt 231-i 100 Insensitive E2-3,17-diG E2-17-G Insensitive Insensitive -estradiol 3,17-diglucuronate E2 E2-3-S -estradiol 17-glucuronate -estradiol 3-sulphate -estradiol ,20β ,20β A. burtoni -dihydroxy-5 dhdoy4penn3oe2-lcrnt 17,20 -dihydroxy-4-pregnen-3-one 20-glucuronate 17,20 -dihydroxy-4-pregnen-3-one 20-sulphate

μmol nmol α α α α α α α o-7oe3slht TO3S100 ETIO-3-S -ol-17-one 3-sulphate -ol-17-one-3-glucuronate o-7oeEI Insensitive ETIO -ol-17-one

,17,20β ,17α ,17,20α ,17α nmol l l α − − (Cole andStacey, β 1 1 ) andETIO-3-G ,20β ,20α , whichsuggests penn-0oe3guuoae2oeP3G100 20one-P-3-G -pregnane-20-one-3-glucuronate β penn2-n 0n- Insensitive 20one-P -pregnan-20-one -P-3-G (male,

l nmol til3guuoae20 -triol-3-glucuronate -triol-3-glucuronate − −1 1 til20 -triol til20 -triol α ) wasmuch ), followed b Concentration usedinelectro-olfactogram(EOG)cross-adaptationandbinarymixturetests.

nmol μmol l − α 1 -P-3- d Responses notconsistentandEOGamplitudessmall,thereforethissteroidwas and l l − − 1 1 ; and 20one-P-3-G(~26%)at1 μmol i.2A).TheapparentI Fig. apparent maximumupto10 threshold (around10 acid (TCD)showedarapidincreaseofEOGamplitudesatsupra- the olfactoryreceptorswithnocooperativity. close to1forallsteroids,suggestingasimple1:1bindingratio that inresponseto20α of male(butnotfemale)responsesto20one-P-3-Gwerelowerthan after a10 olfactory epithelium,responsestothesteroidswereagainrecorded exposure tosteroidsduringcross-adaptationdidnotdesensitise the To confirmthatthecontinuous perfusionwithandsequential (male, 54.38±18.4 amplitudes werenotedforsome steroids,i.e.20 However, significantincreasesinthe mean EOGresponse any ofthetestedsteroids,regardlessconcentration. responses. NoreductionintheEOGresponseswasobserved for similar andthehighestapparentEC 10 response to10 μmol 3-G andETIO-3-Gweresimilarnearlytwicethatofthe G (male,25.72±9.73 20one-P-3-G (male,4.78±1.01 control incross-adaptationandbinarymixturetests. act viaadistinctolfactoryreceptorandwasthereforeused asa EOG cross-adaptation tests EOG cross-adaptation initial responses recordedbeforecross-adaptation, theincrease test steroidsduringadaptation werecomparedonlywiththe time areawidelyobservedphenomenon; becauseresponsesto P =0.046, respectively).Increasing EOGresponsemagnitudesover The concentration–responsecurvefortaurochenodeoxycholic nmol l −

1 min wash-outandcomparedtotheinitialunadapted -7GInsensitive T-17-G brvainthreshold TCD Abbreviation 23G10 E2-3-G TO3G1 ETIO-3-G 20 15k-PGF2α PGF2α The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 concentrations (paired α β α β PInsensitive -P P3G1 -P-3-G P3G1 -P-3-G PInsensitive -P β β P2- Insensitive -P-20-G Insensitive -P-20-S

nmol l −

nmol nmol 1 -P-3-G. TheapparentHillcoefficients were -eie(i.2B).Theapparent L-serine (Fig. max l − l values inresponseto20α 1 10 Detection Conc. receptor Insensitive Insensitive l − μmol ; female,40.74±10.92 −

1 nmol nmol nmol 1 ) concentrations,withoutreachingan

pmol nmol ; female,30.12±12.92

nmol nmol nmol pmol l l l − − − l l l − 1 1 1 − − a 50 1 l l l 1 1 t − − − l c c c c c c c c c c c c c -tests, 1d 1 1d − Fg 1);TCDwasexpectedto (Fig. 1 values, followedby20β l ; female,3.67±0.71 − 1 and 20α –– –– –– –– 1 1 1 1 1 –– –– –– –– –– –– –– –– –– –– –– –1 Saturation μmol μmol μmol μmol μmol P =0.045, l l l l l − − − − − 1 1 1 1 1 -P-3-G (22%)at β a -P-3-G (~27%) -P-3-G, 20β

nmol

nmol P 10 1 1 1 1 tests 0 μmol μmol μmol μmol =0.021 and μmol I nmol max

b nmol l l − − l l l l 1 − − − − values 1 ) had l l 1 1 1 1 − − ) and 4205 -P-3- 1 1 l -P- − 1 ;

The Journal of Experimental Biology hypothesised that thissteroidactsviaareceptor otherthan3G-R-I. (except forETIO-3-G)thanthat oftheSAC. reduced byatleast57%,although theyremainedsignificantlyhigher to TCD,themeanresponses alladministeredtest-steroidswere Surprisingly, however, whentheolfactory epitheliumwasadapted 80% oftheunadaptedresponse, regardlessoftheadaptingsteroid. P-3-G. Incontrast,responsestoTCDcouldnotbereducedbelow reduction wasalsoobservedwhen20β significantly different from the SACs.A less-pronouncedresponse reduced theresponseto20one-P-3-Gonlypartially, toalevelstill however, with20α the self-adaptedcontrol(SAC).Someslightanomalieswerefound, adaptation toapointthattheywerenotsignificantlydifferent from reduced theEOGresponsestoallteststeroids(exceptTCD)during group onthesteroid.20 hereafter called‘3G-R-I’,referringtothepositionofglucuronate androstane ETIO-3-Garedetectedbythesameolfactoryreceptor(s), 3).Theyfurthersuggestedthat20one-P-3-G,aswellthe (Fig. steroids 20α interaction betweensexesandsteroids: 4206 (10 μmol drawn. noted hereforsomesteroidsshouldnotinfluenceanyconclusions RESEARCH ARTICLE of equalvariance. values inB–sexes: values in–sexes: Fig. interaction betweensexesandsteroids: males (N male tilapiasexpheromone20 s.e.m.) werecalculatedfromsigmoidalconcentration–responsecurvesofthe Holm–Sidak differences ( steroid weresimilar. Thedifferent lettersabovethebarsindicatesignificant glucuronates. Apparent(A)EC Given thedistinctconcentration–response curveofE2-3-G,we The resultsoftheEOGcross-adaptationstudiesat1

–1 .ComparisonofapparentEC 2. lmax EC50 (log mol l ) 10 10 (normalised EOG amplitude) 10 10 10 10 =7–14; blackbars)andfemales( 0.5 1.0 1.5 2.0 2.5 –12 –10 –11 l –9 –8 –7 0 −

P 20β-P-3-G post hoc 1 -P-3-G and20β <0.001) amongthesteroids,two-wayANOVA followedby TCD) confirmedthatthetwomaletilapiaurinary B A a,b F a 1,103 F -P-3-G andETIO-3-Gasadaptingsteroids;both test. F 1,103

=0.131, P 20α-P-3-G =0.686, P and P β 50 α -P-3-G actthrough(a)sharedreceptor(s) -P-3-G and20one-P-3-Gconsistently b -P-3-G and20 a values (inlogmol

=0.781; steroids: 20one-P-3-G values wereasfollows:apparentEC =0.409; steroids: 50 and I F F 4,103 4,103 N b =10–14; openbars)foreach c max a,b -P-3-G wasadaptedto20α =0.397, P =0.257, P β -P-3-G, andothersteroid3 values. ETIO-3-G

l F − 1 4,103 F ) and(B)I a,b 4,103 =0.810. Alldatawere =0.905. Apparent a,b Data (mean± =177.968, a =34.280, P

max E2-3-G values for μmol P c d <0.001; <0.001; 50 I max l α − - 1 - the 20β-P-3-GandE2-3-Gmix)wasI independence. Yet, onlyforthe20 mix wereabove1,whichfurthersupportsthenotionofreceptor 20β distinct olfactoryreceptor(s),hereaftercalled‘3G-R-II’ (Fig. from other3-glucuronidatedpregnanesandandrostanesvia(a) indicates thattheMozambiquetilapiaisabletodistinguishE2-3-G than theSACsandweregenerallyclosertoinitialresponse.This these results,asresponsestoE2-3-Gwereconsistentlymuchhigher significantly different ( discrimination index( from thatofthe20 for the20β group’ the 20α 3-G orETIO-3-Gwerestatisticallysimilartothoseinresponse detected byadifferent receptortype,3G-R-II.Themean the 20α were generallycloserto1andsignificantlydifferent ( P-3-G orE2-3-Gwerecloseto1(I steroids 20α 3-G aredetectedbythe3G-R-I,asurinarypheromonal consistent withthecross-adaptationtests;20one-P-3-GandETIO- The meanindependentcomponentindex( To testthis,cross-adaptationtestsincludingE2-3-G,20 ETIO-3-G asthe adaptingsteroidand20β were observedduringcross-adaption studiesthatused20 olfactory receptortype3G-R-I. Someslightanomalies,however, produce similarconcentration–response curvesandactviathesame structurally related3-glucuronidated pregnane(s)andandrostane(s) components 20α males tothepreviouslyidentified maletilapiasexpheromone EOG responsesnotonlyconfirmedthesensitivityoffemales and selects C21andC195 it detectsviatwodistinctolfactoryreceptormechanisms;3G-R-I high olfactorysensitivitytoseveral3-glucuronidatedsteroids,which This studydemonstratesthattheMozambiquetilapiapossesses a steroids mayalsobedetected. range ofsteroidstested,wecannotexcludethepossibilitythat other 6).However, giventhelimited selects C18aromaticsteroids(Fig. I respectively, andsignificantlylarger thanthemean‘within-group’ concentration, asopposedtothoseat1 EOG amplitudesinresponsetothethreesteroidsweresimilaratthis of theolfactoryepitheliumto20 4).Reciprocaladaptation the initialresponseduringadaptation(Fig. not reducetheresponsesto20 there isasharedolfactoryreceptormechanism.Themean consistent withthecross-adaptationstudies,stronglyindicatingthat 3-G and20β I mean ‘across-group’ I receptor type,andconsistentwiththecross-adaptationstudies.The G mix,supportingourassumptionthatTCDactsviaaseparate EOG binary mixture tests mixture EOG binary Cross-adaptation tests Cross-adaptation DISCUSSION MD CI Both the Mean and -P-3-G wereperformedatconcentrationsof10 values for20α I I CI -P-3-G and20β MD -P-3-G and20β I CI and -P-3-G andE2-3-Gmix,the20α I values (Mann–Whitneyranksumtests, values for20α The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 -P-3-G and20β CI -P-3-G werearound0.5and1.0,respectively, and and I MD -P-3-G and20β -P-3-G or20β α I values were0.49and0.99,respectively. Thisis MD -P-3-G and20β I MD P values ofTCDmixedwith20 CI <0.001) fromthe20α -P-3-G mix(Fig. β ) ofthebinarymixturecomprising20 -P-3-G mix,indicatingthatE2-3-Gis -P-3-G or20β ,3α and -P-3-G. -reduced steroids,whereas3G-R-II α -P-3-G or20β I -P-3-G mixedwitheither20one-P- MD α -P-3-G, buttheyalsoshowthat α -P-3-G or20 -P-3-G andE2-3-Gmix(butnot CI -P-3-G mix. values were0.77and1.45, ) orclearlyabove1( μmol -P-3-G mixedwithE2-3-G -P-3-G and/or20one-P-3- MD 5). Themean‘within- l − -P-3-G below70%of 1 statistically different -P-3-G and20β Fg 1).E2-3-Gdid (Fig. β -P-3-G andE2-3-G I -P-3-G confirmed CI ) andmixture

P nmol P =0.002). α <0.001) from -P-3-G, 20β α -P-3-G and α I l MD -P-3-G or − I 1 MD , asthe I

values CI 6). ) and -P-3- α and -P- -

The Journal of Experimental Biology the receptorbindingsite,therebygivingapartialolfactoryresponse. RESEARCH ARTICLE EC those oftheSAC.Thismaybeexplainedbylowerapparent G astestodorant;responseswerereducedbutnottotheextentof the lowerapparentEC receptor site,butoneodoranthasahigheraffinity (asindicatedby P-3-G and/orETIO-3-G.Whentwoodorantscompeteforthesame R % Ri % i 100 50 100 100 20 40 60 80 20 40 60 80 20 40 60 80

20β-P-3-G 0 0 0 values obtainedfor20β E2-3-G TO3GTCD ETIO-3-G 0--- 20one-P-3-G 20β-P-3-G 21 06 10 11 12 E2-3-G 66663 20α-P-3-G ‡‡ ‡ 667 20α-P-3-G 20one-P-3-G * * 20β-P-3-G ETIO-3-G 50 ‡ ), itislikelytoreplacetheotherodorantat *

TCD * * 8 -P-3-G and20one-P-3-Gthanfor20 20β-P-3-G 20α-P-3-G 21 06899946 4 9 9 9 8 6 10 12 12 * * 9984 E2-3-G 20α-P-3-G 0--- 20β-P-3-G 20α-P-3-G *

665 20one-P-3-G 20α-P-3-G * * * ETIO-3-G ‡ 20β-P-3-G

TCD * 9 ‡

20β-P-3-G

20α-P-3-G E2-3-G 20one-P-3-G α - * 555 20α-P-3-G ‡

TCD wasneverreducedbelow80%oftheinitialresponse,regardless with ourpreviouswork(Keller-Costa etal.,2014),theresponseto receptor typefromthatofthesteroidconjugatestested.Consistent different concentration–responsecurve. a separateolfactoryreceptor, type3G-R-II,producingamarkedly Cross-adaptation testsrevealfurtherthatE2-3-Gisdetectedthrough ETIO-3-G The bileacidTCDwasexpectedtoactthroughaseparateolfactory 20β-P-3-G

TCD * ‡ The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 d.f.=4, P control group.20 SACasthe Dunn’s method,multiplecomparisonsversus SAC (P bars indicatesamplesize.*Significantdifferences fromthe glucuronates (or10 same 1 response (mean+s.e.m.)to1 Fig. cross-adaptation. percentage oftheinitialunadaptedresponse(% 20one-P-3-G: P H <0.001. =23.243, d.f.=4,P the same10 a percentageoftheinitialunadaptedresponse(% Fig. adaptation started. 20β the controlgroup.E2-3-G: by Dunn’s method,multiple comparisons SAC (P indicate samplesize.*Significantdifference fromthe + s.e.m.)to10 estradiol-3-G (E2-3-G). H

=6.371, d.f.=2,P .EOGcross-adaptationstudies. 3. -P-3-G: H

.EOGcross-adaptationstudiesinvolving 17β 4. μmol <0.05). Kruskal–Wallis ANOVA onranksfollowedby <0.001. 20 <0.05). Kruskal–Wallis ANOVA onranksfollowed l − H 1

nmol =16.136, d.f.=4,P =9.420, d.f.=2,P steroid (10 β nmol ‡ -P-3-G: H Self-adapted control(SAC).Numbersin α μmol <0.001. TCD:H -P-3-G: H l =0.041. − ‡ 1 SAC. Thenumbersinthebars l − steroid deliveredbeforecross- 1 l steroid conjugatesexpressedas − 1 μmol Relative EOGresponse(mean =28.951, degreesoffreedom TCD) expressedasa H =38.624, d.f.=4,P μmol =11.523, d.f.=2,P l =0.009. 20 − =0.003. ETIO-3-G: 1 =26.903, d.f.=4, TCD) deliveredbefore l − 1 steroid 3α Relative EOG α versus -P-3-G: <0.001. R =0.003. - I ) tothe SAC as R 4207 I ) to -

The Journal of Experimental Biology group’ mixturesdonotalwaysreachtheperfect the sealamprey(0.97;LiandSorensen,1997).However, ‘across- values observedfromA.burtoni was belowtheexpectedvalueof1,andlowerthan‘across-group’ I present, withpartiallyoverlappingspecificities. mixture studies,itispossiblethatmultiplereceptorsubtypes are predicted andobservedvaluesforcross-adaptationbinary receptor cell.Giventhenumberofdiscrepanciesbetween the 3G-R-II receptortypesofO.mossambicus responses inbinarymixturetests.Itispossiblethatthe3G-R-I and receptor sitetypesondifferent neurones,leadingtoslightlyreduced same receptorneuronmaynotbeasindependentdifferent that studysuggestdifferent receptorsitetypespresentonthe odorants inthechannelcatfish, seen byCaprioetal.(Caprioal.,1989)uponusingamino acid 4208 RESEARCH ARTICLE earlier EOGstudies in amplitude inbothmalesand females, whichisconsistentwith All 3-glucuronidatedsteroids induced EOGresponsesofsimilar with thoseofthecross-adaptationtests.Mean‘within-group’ I The resultsofbinarymixtureexperimentsweregenerallyconsistent at the3G-R-Ireceptorsiteswhenpresenthighconcentrations. SAC. ItispossiblethatTCDmayactaspartialagonist,orantagonist, reduced (by50%ormore),althoughneveraslowthoseofthe adapting odorant,responsestotheteststeroidswereconsiderably of theadaptingodorant.Surprisingly, however, withTCDasthe independence. However, themean‘across-group’ exceeded thepredictedvalueof1,suggestingreceptor receptor groups.Themean‘across-group’ fitting nearlyperfectlytotheexpectedvalues(<1and1)forshared previously for steroids isspecificforpheromone 5 sex type3G-R-I detectingthetilapia receptor olfactory The tests mixture Binary . . . 1.0 0.8 0.5 0.3 0 A MD 9 11 9 6 9 11 9 6 9 14 values were0.49and0.99,evenlowerthanthoseobtained I CI A. burtoni * * * * * O. mossambicus E2-3-G and TCD 20α-P-3-G andETIO-3-G 20β-P-3-G andETIO-3-G 20β-P-3-G and TCD 20β-P-3-G and20one-P-3-G 20α-P-3-G and20one-P-3-G 20β-P-3-G andE2-3-G 20α-P-3-G and20β-P-3-G 20α-P-3-G and TCD 20α-P-3-G andE2-3-G (0.63 and1.26;ColeStacey, 2006)and β ,3 Ictalurus punctatus.Theauthorsof (0.94; ColeandStacey, 2006)and α reduced 3-glucuronidated reduced (Keller-Costa etal.,2014) are presentinthesame I MD I I CI CI value of1.45 value of1,as value of0.77 . . . 2.0 1.5 1.0 0.5 0 B 9 12 9 7 10 12 9 6 10 13 CI and I MD P-3-G couldbe valuableforfutureresearch, avoidingthetime in femalesas20 activate thesamesignalcascade thattriggerstheendocrineresponse remains tobeseenwhether20one-P-3-G andETIO-3-Gareableto routes; T.K.-C., A.V.M.C. and P.C.H., unpublished observations).It tilapia urine(althoughitispossible thattheyarereleasedviaother (Murphy etal.,2001). even androsten,aredetectedbythesame(ETIO-3-G)receptor site than thoseintilapia;severalunconjugatedandrostanes,pregnanes and the olfactoryreceptorsdetectingETIO-3-Gappeartobelessspecific to attractfemales(Tierney etal.,2013).However, intheroundgoby, forms ofthesesteroidsviatheirurine(Katareetal.,2011), eventually have demonstratedthatroundgobymalesreleaseseveralconjugated melanostomus, Pallas1814)(Murphyetal.,2001),andrecentstudies are alsopotentodorantsfortheroundgoby( Resink, 1991).Androstanesandpregnaneswitha5 potent testicularodorantwasfoundtobe20one-P-3-G(Lambert and and otherteleostspecies,e.g. made ofAfricancatfish( attracts ripefemales(Colomboetal.,1980).A similarobservationwas Testis-derived ETIO-3-Gfromblackgobymales( pheromones hasbeendiscussedpreviouslyinotherteleostspecies. although apparentlythiscanaffect affinity. to C17incyclopentanering‘D’ ofthesteroidligandispossible, I, somefreedominthefunctionalgrouporaliphaticchainattached detected by3G-R-Ior3G-R-II.However, atleastincaseof3G-R- cyclohexane ring‘A’ seemtodeterminewhethertheligandis at positionC3.Structureandthree-dimensionalorientationofthe for steroiddetectionin (PGF steroids, nortoE2-3-S,anditwasinsensitiveprostaglandins unconjugated steroids,nortoavarietyof17-or20-conjugated olfactory epitheliumof 2001). Inagreementwithpreviousfindings(Fradeetal.,2002),the goldfish (SorensenandGoetz,1993)orroundgoby(Murphyetal., 20one-P-3-G andETIO-3-Gare notnaturalconstituentsofmale The roleof20one-P-3-GandETIO-3-Gasputativereproductive * * * * 2 α and 15K-PGF The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 Dunn’s method,multiplecomparisons differences. Kruskal–Wallis ANOVA onranksfollowedby numbers inthebarsindicatesamplesize.*Significant the steroidsinmixtureactondifferent receptors.The and 20 I binary mixturetests.Openbars(‘within-group’):valuesfor (A) Independentcomponent( Fig. discrimination ( d.f.=9, P (‘across-group’): valuesfor mixture interactwith(a)commonreceptor(s).Blackbars CI α (~0.5) and -P-3-G and20β

.ResultsofEOGbinarymixturetests. 5. β -P-3-G mixascontrolgroup. 2 =<0.001; I α O. mossambicus ). Thissuggeststhattheolfactoryreceptors Clarias gariepinus I O. mossambicus MD I MD (~1) indicatethatthesteroidsin ) indices(mean+s.e.m.)calculatedfrom A. burtoni MD -P-3-G. Ifso,ETIO-3-Gor20one- in B:H I CI =62.672, d.f.=9,P require aglucuronategroup I CI (~1) andI (Cole andStacey, 2006), ) and(B)mixture ) males,wherethemost did notrespondto I CI versus MD β in A:H ,3α (>1) indicatethat Gobius niger) configuration the 20α <0.001. =67.6369, Neogobius -P-3-G

The Journal of Experimental Biology preovulatory reproductive condition.Males arecapableofdiscriminating that isreleasedbyfemaletilapia, providinginformationontheir produced bythegrowingfollicle, E2-3-Gcouldactasasocialcue epithelium respondingtothis odorant.Because17 may indicatearelativelysmall numberofreceptorneuronesinthe RESEARCH ARTICLE sensitive toE2-3-G,andthelowapparentEC The lowdetectionthresholdshowsthat respectively. intensive andexpensivesynthesisof20 high affinity for3G-R-II.Bycontrast,thelowapparent via a distinct olfactory receptor type3G-R-II receptor via adistinctolfactory detectsaputativeTilapia females, socialcuefrom E2-3-G, G O O HO ? 3-sulphated: Prostaglandins: Unconjugated steroids: 20-, 17-or3-sulphatedsteroids: 20- or17-glucuronidatedsteroids: 3-glucuronidated steroids: rga-Adotn Estratrien- Androsten- Pregnan- OH OH rga-Adotn Estratrien- Androstan- Pregnan- rge-Adotn Estratrien- Androsten- Pregnen- rge-Adotn Estratrien- Androsten- Pregnen- 1cro 19-carbon 21-carbon H H H S H H H H H H H H OH O H H H versus OH Pregnen- OH H Androstan- GRI3G-RII 3G-R I H OH OH OH G S HO H H COOH H post-spawning females throughthesmellof H

H G O O O PGF HO OH Androstan- OH 2α 3,17-diglucoronidated: H H H H H H H H H H OH H OH H H H OH G S OH O G O O. mossambicus α H -P-3-G and20β H G S H O H 18-carbon 50 OH COOH H Estratrien-

value suggestsa H H O 15k-PGF H H H H β H H -estradiol is H G H H H H I max H is highly G OH OH -P-3-G, H 2α OH value Some sensitivity concentrations only athigh sensitivity sensitivity sensitivity sensitivity sensitivity olfactory olfactory olfactory olfactory olfactory High preovulatory femalescontainslarge quantities(100–150 water thanpost-spawnfemales(Huertasetal.,2014),andurine from al., 2005).Preovulatoryfemalesrelease,overall,moreE2into the that isnearovulation,butnotwithpost-spawnfemales(Miranda et increase theirownurinationfrequencyinthepresenceofafemale females’ urine(Almeidaetal.,2005).Moreover, theydrastically chemical signal. females intheirurineandwhether itdoesindeedfunctionasa will determinewhetherE2-3-G isreleasedbypreovulatorytilapia males, butnotfemales(Murphy etal.,2001).Futureinvestigations it increasestheventilationrate (opercula movementsperminute)in potent odorantfortheroundgoby, Neogobiusmelanostomus,where A.V.M.C. andP.C.H., unpublished observations). E2-3-Gisalsoa 17β No No No No -estradiol (3and/or17)-glucuronate(M.Huertas,O.G.Almeida, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 Stacey, 2006).15k-PGF recently derivedEastAfricancichlid stars indicatesteroidsthataredetectedbythemore PGF allopatric relative that aredetectedbythephylogeneticallyclosebut study) (Fradeetal.,2002).Bluetrianglesindicatesteroids steroids, 17-or20-conjugatedsteroidsE2-3-S(this exhibit EOGresponsestoprostaglandins,unconjugated olfactory receptortypes,3G-R-Iand3G-R-II.Tilapia donot adaptation andbinarymixturetestssuggesttwodistinct highly sensitiveto3 G=glucuronate, S=sulphate.TheMozambiquetilapiais letters onsteroidstructuresindicateconjugationposition; specificity tosteroidsinO.mossambicus. Fig.

.Summaryofolfactorysensitivityandreceptor 6. 2 α , prostaglandinF O . α niloticus -glucuronidated steroids,andcross- 2 α 2 . α , 15-keto-prostaglandinF (Hubbard etal.,2014).Yellow A . burtoni The red

(Cole and ng ml 2 α ; 4209 − 1 ) of

The Journal of Experimental Biology that of thefivesteroidtypesthatitisabletodetect.Onestudyreports 2009). general featureoftheCichlidae(Stacey, 2010;StaceyandSorensen, whether insensitivitytoprostaglandinsandunconjugatedsteroidsisa It wouldbeinterestingtoinvestigatemorecichlidspeciesestablish even thePerciformes,suchasroundgoby(Murphyetal.,2001). Sorensen, 1996)andArcticchar(SveinssonHara,2000); and Waring, 1996),browntroutandbrook(Essington and Sorensen,2012);Salmoniformes,e.g.Atlanticsalmon(Moore 1988; Sorensenetal.,1995)andcarp(LimSorensen,2011; Lim substantially fromCypriniformes,suchasgoldfish(Sorensenetal., steroids (ofthosethathavebeentested).Inthistheydiffer cichlids isthattheyneitherdetectprostaglandinsnorunconjugated apparently lacks(ColeandStacey, 2006).Commontobothofthese a distinctolfactoryreceptortype(3G-R-II),anabilitythat other steroidconjugates.However, itisabletodistinguishE2-3-Gvia and highcost),butthisspeciesappearstobelargely insensitiveto been investigated(mainlyowingtolimitedcommercialavailability olfactory sensitivityofO.mossambicus sulphated- and3,17-disulphatedsteroids(ColeStacey, 2006).The receptor sitesrecognising17-glucururonidated-,3-sulphated-,17- to 3G-R-I,A.burtoni substantial difference inthesteroidtypesthattheydetect.Inaddition glucuronidated steroidsincommon(3G-R-I).Buttheyalsoshow A. burtoni species (Greenwood,1991),suggeststhatbothO.mossambicus burtoni, whichinLakeTanganyika co-occurswith>150othercichlid Comparison ofO.mossambicus chemical cueshavenotbeensubjecttodiffering selectivepressures. dwelling urine (Hubbardetal.,2014).Thissuggeststhat,intheallopatricriver- steroids, andbothfishreleasesimilarreproductivesignalsviatheir mossambicus, respondstothesametypesof3-glucuronidated i.e. pheromonal functionandreleaseroutesof the detectedsteroids, phylogeny. Inaddition,thebiologicalsignificanceofthesereceptors, between thediversityofsteroid receptortypes,ecologyand and cladeswithintheCichlidae toassesswhetherthereisanylink representatives –sympatricand allopatric–fromdifferent genera different species.Clearly, futurestudiesshouldincludemore indicating substantialdiversity in olfactorysteroidreceptorsamong great variabilityinthetypesofconjugatedsteroidsthattheydetect, ‘message’. complexity and/ordifferences inthemeaningofsteroidal However, thelarger numberofreceptorssuggestsgreater (a) similarpheromonalrole(s)asintheMozambiquetilapia. types thatitdetects(oratleastsomeofthem)viaitsurine,playing possible that Barata etal.,2007;MaruskaandFernald,2012).Itistherefore encounters withrivalsorwhencourtingfemales(Barataetal., 2008; hierarchies andincreaseurinationfrequencyduringaggressive Fernald andHirata,1977).Inbothspecies,malesestablish mouth-brooders andarenaspawners(BrutonBoltt,1975; mossambicus reproductive biologyandsocialorganisation of Stacey, 2003),butnotwhenpresentedwithonlyonetypealone.The to amixtureofrepresentativesthefivesteroidtypes(Coleand 4210 As shownrecently, RESEARCH ARTICLE Comparison of Comparison of derived Africancichlid, Unfortunately, itisnotyetknownwhether Comparison of(only)twoAfricancichlidsshowsthatthere is A. burtoni Oreochromis have oneolfactoryreceptortypefor5 A. burtoni,asO.mossambicusreleasesthesteroid are comparableinseveralways;bothmaternal males increaseserumtestosteronelevelsinresponse O. mossambicus O. O. niloticus possesses fourotherindependent(putative) (Lowe-McConnell, 1991),reproductive A. burtoni A. , likeitsclosebutallopatricrelative to themorerecentlyderived with a more recently with amore to disulphatedsteroidshasnot A. burtoni A. burtoni β ,3α releases any -reduced 3- A. burtoni and and O. O. A. Lda, Portugal). dark photoperiodandwerefeddailywithcommercialcichlidfeed(Sparos 1 nmoll mature fishwereexposedto4 determine whichsteroidsandprostaglandins Instruments, nowMolecularDevices,LLC,Sunnyvale,CA,USA). To Med, West Warwick, RI,USA)anddigitised(Digidata1322A,Axon Welwyn GardenCity, UKorGrassAC/DCstraingaugeCP122;Astro- current voltagesignalwasamplified(eitherNeurologNL102,Digitimer, contact withtheskinofheadnearby(referenceelectrode).The direct placed nearthecentreofrosette(recordingelectrode)andlightly in Borosilicate glassmicropipettesfilledwith4%agarin0.9%NaCl were were introducedintothisflowbyacomputer-controlled solenoidvalve. exposed toincreasing concentrationsfrom1 and male( body weight(BW)=44.1±34.7 curves thathadbeengenerated.Mature female[ all tested3-glucuronidatedsteroids andEOGconcentration–response (Table further concentration–response,cross-adaptation andbinarymixturestudies or onlyinconsistentresponsesathigh concentrations,wereexcludedfrom from 1 with 100 Aldrich). Theywerethenmaintainedinapurpose-builtpadded‘fish-box’, 3 Sigma-Aldrich. Themaletilapiasexpheromonecomponents5 bile acid,L-serine)werepurchasedfromSteraloids(Newport,RI,USA)or The odorantstestedinthisstudyaregivenTable with asandysubstratum,aeratedfreshwaterat27°C,under12 Portugal). Malesandfemaleswerekepttogetherinlarge 500 captivity fromabrood-stockmaintainedattheUniversityofAlgarve(Faro, Fisheries ofPortugal.SexuallymatureMozambiquetilapiawereraisedin General DirectorateoftheMinistryAgriculture,RuralDevelopmentand laboratory animalsundera‘Group-1’ licenceissuedbytheVeterinary Union Council(86/609/EU)andPortugueselegislationfortheuseof Fish careandexperimentationcompliedwiththeguidelinesofEuropean (3G-R-II) detecting17β male sexpheromone(i.e.20 two distinctreceptorsitesareinvolved;one(3G-R-I)detectinga sensitivity andspecificityto3-glucuronidatedsteroids.Apparently, among thedriversofAfricancichlidradiation. question ofwhetherchemicalcommunicationcouldhavebeen needs tobeexplored.Suchinsightsmayshedlightontheexciting detail (Fradeetal.,2002).Briefly, tilapiawereanaesthetisedwithNaHCO The methodforEOGrecordingintilapiahasbeendescribedpreviously 10 μmol water immediatelypriortouseinEOGrecording(seebelow).A solutionof solutions weredilutedtotheappropriatedilutionincharcoal-filteredtap- freshwater (4–6 skin andbonearoundthenostril,aglasstubewithconstantflowof Faraday cage.Theolfactoryrosettewasexposedbycuttingawayaringof (200 buffered MS222(3-aminobenzoicacidethylester, Sigma-Aldrich)inwater Odorants Fish MATERIALS ANDMETHODS EOG recording methanol at1 steroids, prostaglandinsandthebileacidweredissolvedinethanolor pregnan-20-one asdescribedpreviously(Keller-Costa etal.,2014).All glucuronate weresynthesisedfromtheprecursor3 function asa(preovulatory)femalepheromone. α ,17α In conclusion,theMozambiquetilapiahasevolvedhigholfactory mg 1). Consistentresponseswereobtained fromthebileacidTCD,and ,20β mmol −1 l l − − mg 1 N 1 to 1 μmol l ) andimmobilisedwith3 -triol 3-glucuronateand5 L-serine tonormaliseEOGresponseswassimilarlyprepared =8–14; BW=35.1±11.4 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518

l l mmol − − 1 1

ml aliquots (inwater)storedat MS222 inaeratedwaterpumpedoverthegillswithina min l − −1 1 − (stock solution)andstoredat− ). Odorantsthatdidnotevokeolfactoryresponses, 1 ) wasplacedclosetotheraphe.Stimulussolutions -estradiol 3-glucuronide,whichmay s pulsesofincreasingconcentrations(from

α g; standardlength(SL)=120.3±44.6 -P-3-G and20β

g; SL=106.4±11.5 mg kg − β 1 -pregnan-3α

pmol gallamine triethiodide(Sigma- − O. mossambicus 20°C.

1. Test odorants(steroids, l − N -P-3-G) andasecond 1 =10–14; mean±s.d.: to 10 μmol 20°C untiluse.Stock

mm) recipientswere α ,17-dihydroxy-5β ,17α ,20α

l stocktanks detects, 3–6

h light:12 β-pregnan- l − -triol 3- 1 in log

mm]

10 3 h - -

The Journal of Experimental Biology consecutively tosteroidA (response the adaptedresponseto1×1 responses tothetestsolutionsduringadaptationwereblank-subtractedusing the responsetoblankwater, thesamewaterusedtodilutestimuli.EOG responses tothesteroidsbeforecross-adaptionwereblank-subtractedusing likely toactviaadifferent olfactoryreceptormechanism.InitialEOG odorant fortilapia(Huertasetal.,2010)andissteroidalinnaturebut enormous differences existedat1 as 4 spulses,beginningwiththeadaptingsteroid(theSACat2 apparent maximalolfactoryresponse( 1 finally toamixtureofA andB(eachat (1 μmol percentage oftheinitial(unadapted)response(% responses tothetestsolutionsduringadaptationwerethenconverteda males (BW=68.7±20.6 ( existed from1 effective concentration(EC comparisons wasusedtolookforstatisticaldifferences within followed bytheHolm–Sidak mechanism(s). The mechanisms andbelow1(approximately 0.5)inthecaseofsharedreceptor RESEARCH ARTICLE Odorants at1 μmol ± s.d.:BW=46.9±18.1 solutions ofthesteroidswererecordedfrommaturemales( magnitudes weresimilarat1 ETIO-3-G wereperformedatsaturatingconcentrationbecauseresponse Cross-adaptation studiesincluding20 molar increments(plus50 because theresponseamplitudesof20 c involving E2-3-Gwereperformedat10 mature males(mean±s.d.:BW=49.5±22.9 concentrations asthoseusedincross-adaptationtestsonsixtofourteen (Kang andCaprio,1991;LiSorenson,1997;ColeStacey2006). The bileacidTCDat10 μmol [three parameter: calculated byfittingasigmoidalregressioncurveusingtheHillequation steroids A (R used tocomparethe binarymixtureresults. comparisons on ranksfollowedbytheDunn’s post hoc and >1ifthereisreceptorindependence. Kruskal–Wallis ANOVA analyses (1 μmol voltage stabilised(approximately1 adapting steroidwasthenusedtoperfusetheolfactoryepitheliumuntil The independentcomponentindex discrimination index with multiplecomparisons Kruskal–Wallis ANOVAs onranksfollowedbyDunn’s posthocmethod and EC sigmoidal curves).Firstly, EOGresponsesto4 EOG binary mixture tests mixture EOG binary tests EOG cross-adaptation comparable tothoseinresponseE2-3-G,at10 N =

min betweenexposures.Giventhesigmoidalshapeofthesecurves, For eachcross-adaptationdataset,mean% Cross-adaptation involvingE2-3-Gwasperformedseparatelyonmales The =5–7; BW=73.3±19.5 x 50 50(x x values. I is thelog CI , l l y − − )=EC 1 1 is predictedtobearound1inthe case ofindependentreceptor test steroidin1 adapting steroidin1 versus 2A 50

) andB(R nmol ] asamathematicalmodel,inwhich 10 y I stimulus concentration.Two-way ANOVA analysis a controlgroup(20 α MD = l l − I − ax 1 MD g; SL=116.1±16.4 1

is predictedtobe1inthecaseofa sharedreceptor g; SL=132.3±13.6

b g; SL=135.4±12 (10 μmol to 100 /(c μmol 2B (Eqn 2)werecalculatedasreportedpreviously I

versus nmol MD ) attwicetheconcentration(2× b 50 I CI ) andapparentHillcoefficient valueswere + l =

μmol nmol l −

μmol − post hoc = 1 l x μmol .() 0.5( 1 − those oftheSAC. b l was includedasacontrol;itispotent 1 μmol () adapting steroid)werethenadministered − ); ) of4 RR 1 α AB

R RR l α l min). Then,ablankwasrecorded a for TCD)weretestedatthesame -P-3-G, 20β R − R − A+B A2B 2A l -P-3-G and20β =max(y 1 + 1 l − A A+B x − , whereasconsiderablevariation 1 concentrations (linearpartofthe I ) andB(R -P-3-G and20β l mm) at10 1 mm). A 1 μmol CI mol s odourpulses,allowingatleast − +

. adapting steroid).Test solutions nmol 1 method formultiplepairwise mm). First,fishwereexposed I

max R g; SL=117.3±19.3 adapting steroidblank.EOG (Eqn 1)andthemixture , (1) I l − values werecomparedusing )=I ), apparenthalf-maximal 1 l ) toinduceresponseR − .(2) R -P-3-G, 20one-P-3-Gand 1 max method withmultiple

I

s pulsesof1 μmol on ninematuretilapia ). nmol B y , ) atx -P-3-G wereroughly is theEOGresponse b

nmol =Hill coefficient, -P-3-G mix)were l l − − 1 1 mol N solution ofthe concentrations x =6–12; mean l mol − mm). Tests 1 l − , whereas μmol l 1 , thento I l max − 1 ) and A+B and − l 1 − ). 1 . SFRH/BD/46192/2008] receivedaresearchfellowshipfromFCT. (FCT), Portugal[grantnumberPOCI/BIA-BDE/55463/2004].T.K.-C. [grantnumber This researchwaspartiallyfundedbytheScienceandTechnology Foundation manuscript. experiments anddataanalysis.T.K.-C., A.V.M.C. andP.C.H. prepared the T.K.-C., A.V.M.C. andP.C.H. conceivedthestudy. T.K.-C. andP.C.H. performed The authorsdeclarenocompetingfinancialinterests. in thisstudy. synthesising andprovidingthesteroid-conjugates20 from theMaxPlanckInstituteofChemicalEcologyinJena,Germanyfor help. TheauthorsaregratefultoDrC.Paetz,Y. NakamuraandDrB.Schneider Vanessa AfonsoandSebastiaanMestdaghareacknowledgedfortheirtechnical aaa .N,Hbad .C,Amia .G,Mrna .adCnro A.V. M. Canário, and A. Miranda, O.G., Almeida, P. C., Hubbard, E.N., Canário, Barata, and E.N. Barata, P. C., Hubbard, P., Frade, A., Miranda, O.G., Almeida, ura,M,Hgy . omn,A . ed,J,Cnro .V .adHubbard, and A.V. M. Canário, J., Cerdà, A.F., Hofmann, L., Hagey, M., Huertas, A.V. M. Canário, and J.P. daSilva, T., Keller-Costa, V. C., Mota, P. C., Hubbard, References Funding Author contributions Competing interests Acknowledgements rtn .N n ot,R.E. Boltt, and M.N. Bruton, P. W. Sorensen, P., Frade, O.G., Almeida, P. C., Hubbard, J.M., Fine, E.N., Barata, aae .K,Sot .P,Lfabie .J,L,W,Aysae . auo C.B., Caputo, Z., Alyasha’e, W., Li, A.J., Laframboise, A.P., Scott, Y. K., Katare, ura,M,Amia .G,Cnro .V .adHbad P. C. Hubbard, and A.V. M. Canário, O.G., Almeida, M., Huertas, okm .D,Muir . occi . ilnk,B .adSot A.P. Scott, and B.S. Zielinski, M., Moscicki, B., Meunier, L.D., Corkum, sigo,T .adSrne,P. W. Sorensen, and T. E. Essington, C. Frisco, and P. C. Belvedere, A., Marconato, L., Colombo, N.E. Stacey, and T. B. Cole, N.E. Stacey, and T. Cole, aro . ue,J n oisn .J,II J.J., Robinson, and J. Dudek, J., Caprio, aro .adBr,R . Jr R.P., Byrd, and J. Caprio, elrCsa . oe,O . led,O . ubr,P . aoel,A,Lima, A., Iacovella, P. C., Hubbard, O.G., Almeida, O.S., Lopes, T., Keller-Costa, Kang, J.andCaprio, Greenwood, N.R. Hirata, and R.D. Fernald, rd,P,Hbad .C,Brt,E .adCnro A.V. M. Canário, and E.N. Barata, P. C., Hubbard, P., Frade, mossambicus mossambicus (2007). MaleurinesignalssocialrankintheMozambiquetilapia( A. V. M. ( C. P. mossambicus (2014). Chemicalcommunicationintilapia:A comparisonof Evolution ( 449. tilapia maleslikelysignalssocialdominancetofemales. A.V. M. Canário, and those producedbyconspecificsalone. channel catfish, olfactory receptorresponsestobinaryandtrinarymixturesofaminoacidsinthe Fish Biol. Peters (Pisces:Cichlidae)inanaturalfreshwaterlake(LakeSibaya,SouthAfrica). male roundgoby( B. Zielinski, the putativepheromonalsteroid11-oxo-etiocholanolone byreproductivelymature and S.J. Loeb, punctatus. J.Gen.Physiol. responses tocomplexmixturesofaminoacidsinthechannelcatfish, Endocrinol. male urinarypheromonestimulatesfemalereproductiveaxis. Behavioural responsesoffemaleroundgobies( L. BollettinodiZoologico of teleostreproduction.A testicularsteroidpheromoneintheblackgoby, and browntrouttoputativehormonalpheromones. putative steroidalpheromones. mouth-brooding cichlid, African cichlidfish, 403-422. and neutralaminoacidolfactoryreceptorsitesinthecatfish. signals. Behaviour A.V. M. Canário, and bladders indominant tilapiafacilitatesthecontrolofaggression throughurinary E.N. Barata, M., 298. sensitivity oftheMozambiquetilapiatoconspecificodours. quantitative behaviouralobservations. 1254. Anguilla anguilla Oreochromis mossambicus (2010). OlfactorysensitivitytobilefluidandsaltsintheEuropeaneel (2005). Urineasasocialsignalinthemozambiquetilapia( 7 (ed. M.H.A.Keenleyside),pp.86-102.London:Chapman&Hall. , 423-445. 196 P The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 . K. ). Chem.Senses , Peters1852). with O.niloticus , 106-111. Ictalurus punctatus (1991). Speciation.In ), goldfish( 149 Neogobius melanostomus Haplochromis burtoni , 953-975. (1991). Electro-olfactogramandmulti-unitolfactoryreceptor Haplochromis burtoni 47, 355-364. (2008). A sterol-likeodorantintheurineofMozambique (2003). Olfactoryandendocrineresponsetosteroidsinan 98, 699-721. ) suggestsa‘broadrange’ sensitivitynotconfinedto BMC Biol. (1984). Electrophysiologicalevidenceforacidic,basic, (2006). OlfactoryresponsestosteroidsinanAfrican . Gen.Comp.Endocrinol. (1975). Aspectsofthebiology 0Suppl.1,i309-i310. 30 Behaviour (2011). Releaseoffreeandconjugated forms of Carassius auratus . J.Gen.Physiol. (1977). Field-studyof (1996). Overlappingsensitivitiesofbrooktrout Anim. Behav. J. Exp.Biol. 5 . FishPhysiol.Biochem. , 54. 145 Cichlid Fishes.Behaviour, Ecology and (2012). Muscularhypertrophyofurinary (1989). Electro-olfactogramandmultiunit (Günther). J.FishBiol. , 1347-1365. Pallas, 1814). J. FishBiol. 213 25, 964-975. 93, 245-262. α Neogobius melanostomus ) andMozambiquetilapia -P-3-G and20 , 308-317. 207 J. Chem.Ecol. , 13-20. Haplochromis burtoni J. FishBiol. Biol. Reprod. (1980). Endocrinology 48, 1027-1029. J. Gen.Physiol. Tilapia mossambica 28, 265-266. (2002). Olfactory 68, 661-680. β (2014). Tilapia -P-3-G used Gen. Comp. Oreochromis Oreochromis Oreochromis Gobius jozo 61, 1239- 34, 438- 84, 288- Ictalurus (2008). 4211 ) to 84, J. –

The Journal of Experimental Biology 4212 RESEARCH ARTICLE elrCsa . ubr,P . at,C,Nkmr,Y,d iv,J . ao A., Rato, J.P., daSilva, Y., Nakamura, C., Paetz, P. C., Hubbard, T., Keller-Costa, abr,J .D n eik J.W. Resink, and J.G.D. Lambert, i .adSrne,P. W. Sorensen, and W. Li, auk,K .adFrad R.D. Fernald, and K.P. Maruska, P. W. Sorensen, and H. Lim, oeMCnel R.H. Lowe-McConnell, iad,A,Amia .G,Hbad .C,Brt,E .adCnro A.V. M. Canário, and E.N. Barata, P. C., Hubbard, O.G., Almeida, A., Miranda, i,H n oesn P. W. Sorensen, and H. Lim, or,A n Waring, C.P. and A. Moore, lvia .F,Amd,V .adCnro A.V. M. Canario, and V. L.D. C. Almada, R.F., Corkum, Oliveira, and N.E. Stacey, C.A., Murphy, aaa .N,Shedr .adCnro A.V. M. Canario, Biol. and B. pheromone releasedbydominantmalesthatprimesfemalesforreproduction. Schneider, E.N., Barata, Physiol. A naturally occurringbileacidsinthesealamprey, review. pheromones inthereproductionofAfricancatfish an Africancichlidfish. Evolution waters, excludingtheAfricanGreatLakes.In prostaglandin F2 laboratory andfield. release asexpheromonecomplexthatattractsconspecificmalesinboththe ( (2005). Olfactorydiscriminationoffemalereproductivestatusbymaletilapia ovulated commoncarp. male Atlanticsalmon( evidence thatF-seriesprostaglandinsfunctionasprimingpheromonesinmature Horm. Behav. steroid concentrationsintheurineofmalecichlidfish responses. pheromones intheroundgoby, Oreochromis mossambicus 24, 2130-2135. J. SteroidBiochem.Mol.Biol. (ed. M.H.A.Keenleyside).London:Chapman&Hall. 180 J. Chem.Ecol. 30, 2-12. , 429-438. α inaspecies-specifichormonalsexpheromonereleasedby J. Chem.Ecol. Salmo salar J. Exp.Biol. (1991). EcologyofcichlidsinSouthAmericanandAfrican J. Chem.Ecol. 27, 443-470. ). J.Exp.Biol. (2012). CommoncarpimplantedwithprostaglandinF2 (1997). Highlyindependentolfactoryreceptorsitesfor (2011). Polarmetabolitessynergizetheactivityof Neogobius melanostomus (1996). Electrophysiologicalandendocrinological (2012). Contextualchemosensoryurinesignalingin ) parr. J.Exp.Biol. 215 38, 127-134. 40, 549-556. , 68-74. 37, 695-704. 208 (1991). Steroidglucuronidesasmale Cichlid Fishes:Behaviour, Ecologyand , 2037-2043. (1996). Socialmodulationofsex Petromyzon marinus 199 Oreochromis mossambicus Clarias gariepinus , 2307-2316. (2014). Identityofatilapia (2001). Putativesteroidal : olfactoryandbehavioral . J. Comp. – abrief Curr. α . ire,K . eeik . aae .K,Sot .P,Le,S .adZeisi B. Zielinski, and S.J. Loeb, A.P., Scott, Y. K., Katare, M., Kereliuk, K.B., Tierney, T. J. Hara, and T. Sveinsson, ct,J .adSotJhsn P. E. Scott-Johnson, and J.W. Scott, oesn .W n ot,F. W. Goetz, and P. W. Sorensen, Miyagi, H.D.J., Mrosso, K.L., Carleton, I.S., Magalhaes, Y., Terai, O., Seehausen, Robison, R.R.,Fernald,D.andStacey, N.E. tcy .adSrne,P. Sorensen, and N. Stacey, P. Sorensen, and N. Stacey, oesn .W,Hr,T . tcy .E n ot,F. W. M. Goetz, and N.E. Stacey, T. J., Hara, P. W., Sorensen, oh,M .adRi-erqe,M.A. Reis-Henriques, and M.J. Rocha, tcy N. Stacey, oesn .W,Sot .P,Sae,N .adBwi,L. Bowdin, and N.E. Stacey, A.P., Scott, P. W., Sorensen, pheromones intheirurinetoattractfemales. (2013). Invasivemaleroundgobies( Behav. Salvelinus alpinus and R.T. Rubin),pp.639-681.SanDiego,CA:AcademicPress. Brain andBehavior (Teleostei, Cichlidae). history anduses. . a e lis . cnie,M . an .E,Tcia .etal. prostaglandins andtheirmetabolitesinteleostfish. H. Tachida, M.E., Maan, M.V., Schneider, Speciation throughsensorydriveincichlidfish. I., vanderSluijs, R., and C cichlid fishrespondstosteroidalcompounds. 359-412. 169-192. SanDiego,CA:Elsevier. Reproduction ofVertebrates: Fishes postovulatory femalesexpheromoneingoldfish. prostaglandins functionaspotentolfactorystimulantsthatcomprisethe 142. stimulant withpheromonalactionsinthegoldfish. 17,20 21 β 69, 301-307. -dihydroxy-4-pregnen-3-one functionsasapotentandspecificolfactory steroids inanasynchronousfish,thetilapia (2010). Hormonallyderivedsexpheromonesinfishes.In The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111518 Microsc. Res.Tech. , toaputativemalepheromone,prostaglandinf(2)alpha. , Vol. 1(ed.D.W. Pfaff, A.P. Arnold,A.M.Etgen,S.E.Fahrbach Comp. Biochem.Physiol. (2000). OlfactorysensitivityandspecificityofArcticchar, (2005). Reproductivepheromones. (2009). Hormonalpheromonesinfish.In (1993). PheromonalandreproductivefunctionofF , Vol. 1(ed.D.O.NorrisandK.H.Lopez),pp. 58, 152-160. (2002). Theelectroolfactogram:areviewofits (1996). PlasmaandurinelevelsofC Can. J.Fish.Aquat.Sci. J. FishBiol. Neogobius melanostomus Nature 115C, 257-264. Biol. Reprod. Gen. Comp.Endocrinol. (1998). Theolfactorysystemofa J. LipidMediat. 455 Oreochromis mossambicus 53, 226-229. , 620-626. 39, 1039-1050. ihPhysiology Fish 6 (1995). Sulfated , 385-393. Hormones and 70, 393-400. Hormones, ) release (1988). F 100 Physiol. (2008). 18 , 128- , C 24, 19

The Journal of Experimental Biology