Three-spined stickleback: an emerging model in environmental endocrine disruption

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Three-Spined Stickleback: an Emerging Model in Environmental Endocrine Disruption

IoannaKatsiadaki*,MatthewSanders,MarionSebire,MasakiNagae1,KiyoshiSoyano2andAlexanderP.Scott

CefasWeymouthLaboratory,BarrackRoad,Weymouth,DorsetDT48UB,UK1FacultyofEnvironmentalStudies,NagasakiUniversity,

1-14Bunkyo-machi,Nagasaki852-8521,Japan2InstituteforEastChinaSeaResearch,NagasakiUniversity,

1551-7Taira-machi,Nagasaki851-2213,Japan

(ReceivedMarch1,2007;acceptedMarch22,2007)

*E-mail:[email protected]

Key words: stickleback,endocrinedisruption,review,modelspecies

Thethree-spinedstickleback,asmallteleostspecieswithhabitatsthatrangefromfullmarinetofreshwaterbodiesacrossthewholeNorthernhemisphere,hasanumberofadvantagesforendocrinedisruptionresearch.Itistheonlyteleostspecieswithanunambiguousbiomarkerforandrogens,thepresenceoftheglueproteinspiggininthemalekidney,whichcanbemeasuredbyenzyme-linkedimmunosorbentassay(ELISA).The androgen assay has been adopted to detect antiandrogens in two different waysandanhomologousELISAforsticklebackvitellogeninisalsoavailable.DNAmark-ersformolecularsexdeterminationareavailable;thus,sexratioscanalsobeusedforin situbiomonitoring.Inaddition,thecriticalperiodofsexualdifferentiationhasbeendetermined and the occurrence of intersex fish has been reported several times. The speciesfullgenomesequence isalmostcomplete. Allaspectsofsticklebackbiology(ecology,evolution,behavior,physiology,endocrinology)arewelldocumented.InEu-ropean waters, the stickleback is the only fish that can bring laboratory and field stud-ies together and allow the true impact of endocrine disruptors on fish populations to be evaluated.

1. Introduction

The three-spined stickleback (Gasterosteus aculeatus) is a small teleost specieswithanexceptionallywidegeographicdistributionthatshowsremarkablespeciation.Itsorigin lies in themarineenvironmentsincebothgeographicevidenceandgeneticevidenceshowthatfreshwaterpopulationshavebeenderivedrepeatedlyfromamarineancestor.(1) Theirubiquityandease toobtainandkeep in the laboratoryarefactors thathavecontributed to the use of this species as a vertebrate model for studies in behavior,physiology,evolutionandecology. Inmorerecentyears, thesticklebackhasalsoat-tractedtheinterestofmolecularbiologists,resultinginamarkedincreaseintheavail-ableresources(e.g.,expressedsequencetags,microarrays,andgenomesequence).Themain drive for such explosive research activity was the realization that sticklebacksofferedausefulmodel for thestudyof themolecularbasisofvertebrateevolution.(2)Fossil recordsshowthatalthoughthegeneralmorphologyofmarinegroupsofstick-lebackshave changedvery little over time, freshwater groups exhibit awidevarietyofmorphs indifferentenvironments. Thereappears tohavebeenrepeatedevolution

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ofsimilartraitsinmanywidelyseparatedfreshwaterenvironments.Theconsistentassociationofparticularphenotypeswithparticularecologicalconditionsprovidesastrongargumentthatthecorrespondingtraitsareadaptiveandhavebeenrepeatedlyselected in different locations. This observation formed the basis of a successfulproposal to sequence the stickleback genome. The consequent flood of information hasproducedincreasedinterestintheresearchcommunityinother(lesshistoricallypopular) fields of biological research, including ecotoxicology. The relatively small genomeofthestickleback(estimatedat675megabases)impliesthatintronsizeissmallandthatasecondgenomeduplicationeventdidnottakeplaceinthestickle-back (as it did in many other fish species), aiding to the speed of the annotation and functionalcharacterizationofgenesofinterest. Thefascinatingreproductivebehaviordisplayedbymaleshasgiventhestickle-back a special place in fish reproduction studies. The strong secondary sexual char-acteristicsof themaleareandrogen-dependentandprovideauniqueanimalmodelfor the study of the factors affecting male sexual differentiation and reproductiveperformanceinbothfundamentalbiologicalresearchandecotoxicology.

2. General Advantages of Stickleback as Environmental Sentinel

A useful sentinel species in water quality assessment needs to fulfill a number of basiccriteriasuchasubiquity,easeinbeingobtainedfromthewild,nocommercialvalue,awell-documentedbiology,andarelativelyhightolerancetopollutants.Thethree-spinedsticklebacknotonlymeetsallthesebasiccriteriaitalsopresentsanum-berofadditionaladvantages.Someofthemarelistedbelow.• Itispresentinenvironmentsatvarioussalinities(marine,estuarine,freshwater)

andisoneofthefewendemicspeciesinEuropethatoffersascopeforenviron-mentalmonitoring.

• Freshwaterpopulationsarenotknowntobemigratory,hencetheyarerepresenta-tiveoftheirplaceofresidence.

• Thethree-spinedsticklebackpresentsalltheadvantagesofasmallteleost(e.g.,smallaquariaandshortlifecycle).

• Itisverytoleranttomanipulationssuchashandlingandtransport.• It iseasy tomaintainandcan reproduceunder laboratoryconditions (eggsand

larvaedisplaycloseto100%hatchingandsurvivalrates).• Ithasuniquetraitsfor thedetectionofendocrinedisruptingchemicals(EDCs),

such as a genetic sex marker and a xenoandrogen-specific endpoint. Overtheyears,manystudieshaveshownthevalueofthesticklebackasasenti-nelforenvironmentalpollutants.(3–15)Alargenumberofmainlyacutetoxicitystud-ieshavealsobeenconductedusingthestickleback(Table1).(16–60) Itwouldbeanalmostimpossibletasktocriticallyreviewallthepublishedworkon stickleback toxicology because most of the studies have been fragmented andindependent.Inotherwords,despiteitattractiveness,therehasbeennosystematiceffort to employ the stickleback as a standard model species for toxicity testing.Instead,attentionhasbeenconcentratedonthefatheadminnow(Pimephales prome-las), the zebrafish (Danio rerio), the sheephead minnow (Cyprinodon variegates),medaka (Oryzias latipes), rainbow trout (Oncorhynchus mykiss) and the zebrafish (Brachydanio rerio).ForEuropeanscientistsinparticular,thishasposedadilemma,asnoneofthesespeciesisindigenous. Theapparentfailuretoproducesystematictoxicologicaldataonthesticklebackovertheyearsmaybepartlyduetothelargephenotypicvariationthatsticklebacksdisplay. Indeed, it remains largely unknown to date whether there are any differ-encesinsensitivitytowardpollutionbetweendifferentsticklebackmorphsorpopu-lations.Sticklebacksdisplayanextraordinaryabilitytoadaptrapidlytonovelenvi-ronmentalconditions,(61–63)butwhetherthisabilityextendstochemicalinsultsiscur-

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rentlynotknown.Ingeneral,sticklebacksareconsideredtoberelativelytoleranttopollutionincidents;however,wedonotknowifthistoleranceisadaptiveratherthaninherent.Evenifweassumethatsticklebackscaneasilyadaptunderchemicalpres-sureswestilldonotknowonwhatbasis,physiologicalorgenetic, theymake thischange. Evidence from studies of other fish suggests that there are differences in the expression levels of xenobiotic detoxification systems in chronically exposed popu-lationsandthatthesedifferencesaregeneticallydetermined.(64–67)Ifthisisthecase,thenotherquestionsarise,suchaswhataretheimplicationsofthegeneticdiversityin impacted populations and what is the overall cost of this to the fish communi-ties? Thenumerousendemicpopulationsof sticklebacksofferanexcellentmodelsystemtostudythebasisofindividualandpopulationsusceptibilitiesandadaptationtoenvironmentalpollutants.Ifweacceptthatthedifferentadaptationstopollutantsbetweenindividualsorpopulationsareofageneticnature(i.e.,polymorphicvariantsof key detoxification and repair enzymes), then genetic diversity in contaminated areasislikelytobelowerbecauseofselection.Theextenttowhichsuchascenariomay occur depends upon the initial gene pool, population dynamics of individualspecies,reproductivestrategiesandlifehistoryinteractionwithinthecontaminatedenvironments.Thisisanareaofresearchinwhichthestudyofallozymeandmicro-satellitevariationssinglyandincombination,canprovidenewinsights.TherehavebeennumerousstudiesonallozymevariationinGasterosteus aculeatus,(68)butsofara techniqueforcharacterizingsticklebackpopulations fromenvironmentswithdif-ferentpollutionhistorieshasnotbeenused. Alargenumberofmicrosatellitemarkersinthe stickleback have been developed and applied in the field and the laboratory(2,69–73)butso far have only been used to confirm the provenance of fish and to discriminate be-tweenanadromousandfreshwaterpopulationsorevenbetweendifferentsympatricmorphsofthespeciescomplex.Therefore,thesemarkersmayrequirerevisionandfurther development before they find an application in the assessment of population genetic status and restricted gene flow in relation to pollution. An interesting point tobemadehereisthatmatingsystemsandsexualselectionalsoaffectthenumberofexpectedpolymorphisms.Fishthatprovideparentalcareonnestspresumablyspawnwith fewer mates than fish that release their gametes freely in the water column dur-ing mass spawning. Female sticklebacks optimise their major histocompatibilitycomplex (MHC) allele polymorphism by sexual selection to avoid inbreeding andmaximisetheparasiticresistanceoftheiroffspring.(74)Theseandothersarefactorsthatneedtobeconsideredbeforerobustsetsofallozymeormicrosatellitemarkerscan be deployed to address the effects of pollution on fish populations. Nonetheless, this very same approachholds thekey to doprecisely this and it appears that thesticklebackoffersanewresourceforenvironmentalriskassessmentonthisbasis.

3. Stickleback as Sentinel for Endocrine Disruption

Asmentionedabove, thesticklebackhasgainedpopularityasamarker foren-docrinedisruptionowingtotwooftheusefultraitsitdisplays,namely,thepresenceof a specific androgen and an antiandrogen biomarker(13,14,42) and thepresenceof ageneticsexmarker.(75)Thesticklebackalsohassomedistinctivereproductivebehav-ioral traits that lend themselveswell to thedevelopmentofbehavioralbiomarkersforidentifyingEDCs.(76–78)

TheOrganisationforEconomicCo-operationandDevelopment(OECD)iscur-rently in the process of validating a short-term fish screening protocol for endocrine disrupters(estrogens,androgensandaromataseinhibitors)usingthreecorespecies:the fathead minnow, Japanese medaka and the zebrafish.(79) The main endpointsproposed for the first phase of validation of the screening are vitellogenin induc-tion,grossmorphology(secondarysexualcharacteristicsandgonado-somaticindex)and gonadal histopathology. A similar protocol is concurrently being developed

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Table1Toxicitystudiesconductedusingthestickleback.

Chemical Endpoint ReferenceSalts

Sodiumsulphide Oxygenconsumption 16,17Sodiumcyanide Acutetoxicity 18Ammoniumchloride Mediantolerancelimits 19Sodium fluoride Acutetoxicity 20Potassiumchromate Acutetoxicity 21

MetalsCadmium Acutetoxicity 22Cadmium Bodyburdenanddistribution 23Cadmium Gillpathology 24Cadmium Gillandkidneypathology 25Cadmium Binding-proteininduction 26Zinc Gillpathology 27Zinc(65Zn) Whole-bodyburden 28Copper Acutetoxicity 29,30Copper Oxidativestress 10Copper Acutetoxicity 31Mercury Bioaccumulation 32TBTO Reproduction,histopathology 3TBT Whole-bodyburden 33Organoarsenic CYP450,livermorphology 34

Herbicides/Pesticides/FungicidesAldrin,Azinphos-methyl,Carbaryl,Coumaphos,Chlordane,Dieldrin,Endrin,Heptachlor,Malathion,Lindane,Methoxychlor,dichloro-diphenyl-trichloroethane,p,p’-DDE

Acutetoxicity 35

Chlorpyrifos Acutetoxicity 36Chlorpyrifos Whole-bodyburden 37Endosulfan Acutetoxicity 38Lindane Acutetoxicity 39Tecnazene Acutetoxicity 40Fenitrothion Acutetoxicity 41Fenitrothion

Spiggininduction 42Vinclozolin(fungicide)Linuron(herbicide)Fieldmixtureofparathion-ethyl,fenvalerate,deltamethrin,lindane,andtriallat

Cholinesterase 43

PCBs20PCBMixture Bioaccumulation 44VariousPCBs Whole-bodyburden 45ClophenA50 Bioaccumulation 4

Polychlorinatednaphthalenes

Environmentalexposure Reproduction,livermorphology,CYP450 46

Halowax1014 Whole-bodyburden 4Polybrominateddiphenylethers

Bromkal70-5DE Reproduction,livermorphology,CYP450 4}

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in theUKusing the three-spinedstickleback,withendpoints identical to those forthecorespeciesbutwith theadditionofspiggininductionasasuperiorandrogen-specific endpoint. An intercalibration exercise has already been conducted using 17β-estradiol (E2) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibrationwith the core species. All threeparticipating laboratoriesdetectedstatistically significant increases in vitellogenin (VTG) level in males after 14 days of exposure to nominal concentrations of 100 ng/L E2 and statistically significant increasesinspiggininfemalesafter14daysofexposuretonominalconcentrationsof5000ng/Ltrenbolone.(80)Afurtherintercalibrationexerciseiscurrentlyunderwaytostudytheeffectsof theweakestrogen4-tert-pentylphenol, the antiandrogen flu-tamideandthearomataseinhibitorprochloraz.Itishopedthattheresultswillcon-tinue todemonstrate that thesticklebackshowspotentialasa testspeciesandwillfacilitate its inclusion in the final OECD Technical Guidelines.

3.1 Androgens and spiggin Thelifespanofwildsticklebacksisonlyoneyearinthewarmerreachesoftheirdistribution,extendingtotwooreventhreeyearsincolderenvironments.Asarule,sticklebacksbreedonce ayear starting in early spring. Althoughwell-establishedfreshwaterpopulationsarecommonlypresentacross theentirerangeof theirhabi-tats,thenormisthatbreedingfollowsmigrationfrommarineorestuarinewaterstofreshwater.Themaleestablishesaterritory,buildsanestusinganadhesiveproteinproducedinthekidneyanddevelopsnuptialcoloration(redthroatandblueirises).Anumberoffemaleswillbecourtedandinvitedtospawninthenestthatprovidesa

Chemical Endpoint ReferenceSolvents

Benzene Acutetoxicity 471,2,4-Trichlorobenzene Chronictoxicity 48Diphenylether Metabolism 49

FueloilmixturesArylsulphataseandacidphosphataseactivity 50

Livermorphology 51SurfactantsandOilDispersants

BerolTL-188&198,BP1100x,Corexit7664 Acutetoxicity 52

LaurylsulphateAcutetoxicity 39ArylsulphataseactivityAcidphosphataseactivity 50

Nonylphenol Bioaccumulation 53Tetrapro-pylenebenzenesulfonicacid Acutetoxicity 54

Plastics/plasticisers/phthalatesButylbenzylphthalate Behavior 55,562,4-Dinitrotoluene Acutetoxicity 543,4-Bis(2-ethylhexyl)phthalate

Industrial effluents/sedimentsContaminatedsediment Acuteeffects 57Kraft Mill effluent Survival,growthanddisease 58

Pulp Mill effluent Parasitefrequency,liverhistopathology 59

MiscellaneousDimethylammoniumchloride Acuteandchronictoxicities 602,4-Dichloroaniline

Acutetoxicity 542,6-Dimethyl-quinolinen-(1-Methyethyl)-2-propanamine

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well-protectedandcontrolledenvironmentfortherelativelysmalleggclutchestheyproduce. The sperm is strongly stimulated by the ovarian fluid surrounding the eggs, whichresultinanincreaseinfreshwaterspermmotilityfromabout1min(withnofluid) to 7 h or even longer. This might be a major factor allowing the colonization of freshwater habitats of the sea ancestor.(81) Fertilization follows within secondsof spawning and the male enters the parental phase during which the developingembryos are kept aerated by means of a constant fanning until hatching (in aboutsevendaysat18°C).Duetotheelaboratematingsystemtheyemployandtheeasewithwhichspawningoccursinthelaboratory,thereproductiveendocrinologyofthemalesticklebackhasbeenthesubjectofnumerousstudies.Theroleofandrogensinstimulatingreproductivebehaviorandsecondarysexualcharacters inbetterknownin sticklebacks than in perhaps any other fish. InEurope, spermatogenesis startsat theendof thebreedingseason in summerandiscompletedbyearlyautumnorwinter,wellbeforetheonsetofspawning.Bothgonadotropin(GTH)andandrogenlevelsarelowduringactivespermatogenesisandpeakduringthenest-buildingphase.(82) Thelackofacorrelationofspermproduc-tionwithGTHandandrogenlevelsisnotinagreementwithwhatappearstobegen-erallyacceptedinthemajorityofvertebrates(i.e.,spermatogenesisisstimulatedbyandrogens).Itiswelldocumented,however,thatboththereproductivebehaviorandthedevelopmentofsecondarysexualcharacteristics,whichaccompanythebreedingactivity,isunderthecontrolofandrogens. Inearlystudies,itwasfoundthatcastrationresultedintheabolitionorreductionofall secondarysexualcharacteristics including the reproductivebehaviorofmalesticklebacks,andthatthesecharacterisiticscanbestimulatedinbothintactandcas-tratedmalesbyandrogenadministration.(83–86) Borg(87) studied the effect of implanting sticklebacks that had been castratedwheninthebreedingconditionbutbeforenest-buildingwiththenaturally-occurringandrogens testosterone (T), androstenedione, 5α-dihydrotestosterone (5α-DHT) or 11-ketoandrostenedione (11-KA). Although reproductive behavior and kidneyhy-pertrophywereabolishedbycastration, androgen replacement restored, tovaryingdegrees, all types of reproductive behavior, including nest building, courtship andfanning(with11-KAbeingthemosteffectiveandrogenindoingso).Borget al.(88)studied steroid metabolism in the testes of both breeding and nonbreeding malesticklebacksbyincubatingtesticulartissuewiththeandrogenprecursor3H-pregnen-oloneinanattempttodeterminemorepreciselythemajorgonadalsteroidsproducedbythetestes.Theresultsshowedthatthelevelofsteroidmetabolismwasconsider-ablyhigherinthetestesofbreedingmalesthaninthetestesofnonbreedingmales,and that themajorsteroidproducedby the testesof thebreedingsticklebackswas11-KA. However, as in the majority of teleost fish, the major androgen measured inthebloodofbreedingmaleswas11-ketotestosterone(11-KT).(89,90)Thisapparentparadoxwasresolvedfollowingthedemonstrationthat11-KAcouldbereadilycon-vertedto11-KTin vitrobybloodcellsofthisspecies(91) and was further confirmed bycastration/implantationexperimentsinsticklebacks,whichshowedthatwhilecas-trationreducedplasmasteroidlevels,castratedmalesgiven11-KAimplantsshowedanelevatedlevelofplasma11-KTbutnot11-KA.(89) In teleosts generally, the physiologically relevant androgens are the 11-ketoan-drogensandinparticular11-KT(forareviewseeref.92).Thesameholdstrueforthe stickleback where a large number of studies have identified that seasonal levels of11-KTpeakinthebreedingseasonandareresponsiblefortheexpressionofbothsecondarysexualcharacters(e.g.,kidneyhypertrophy)andreproductivebehaviors,anddroptotheirlowestlevelinthepostbreedingseasonwhenspermatogenesistakesplace.(89) Despitetheevidencepresentedabove,thereisstillconfusionovertheexactiden-tityoftheandrogen,andtheandrogenreceptor,thatmediatesspigginproductionbythe kidney. The first detailed studies by Borg et al.(93)showedthat11-KTand11-KA


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werebyfarthemostpotentandrogenswheninjectedintocastratedmales.However,laterstudiesinwhichintactmalesandfemaleswereexposedtowaterbornesteroidsshowed that11-KTwasnomoreactive thanTalone (ourunpublisheddata). Thehypothesis that thismighthavesomething todowith the rateofabsorptionof thetwo different steroids from the water was rejected when it was shown thatT and11-KTare also similarly effective in stimulating spigginproduction in sticklebackliver cells in vitro.(94) Concerning the identity of the receptors, early evidence in-dicatedthesole involvementofanovelmembranereceptor.(95) However, thegeneof a classic β-isotype androgen receptor was subsequently identified that showed high binding affinity for 5α-DHT but low affinity for T and 11-KT.(96)Thishadtwosplicing variants, one of which, when incorporated into zebra fish cells interestingly showed higher activation by 11-KT than 5α-DHT. Even more recently, the gene of the α-isotype androgen receptor has also been identified (our unpublished results). Clearly,thereisstillmuchtobelearnedaboutthemechanismofactionofandrogensin fish. Theandrogen-inducedglueproteinusedforthebuildingofanestisproducedinthekidney followingamarked transformationof thekidney fromanexcretoryor-gantoasecretorygland.Thisphenomenoniswidelyknownaskidneyhypertrophyand the histological changes allowing this transformation have been described indetail.(97)TheglueiscalledspigginaftertheSwedishnameforthesticklebackandit was first characterized by a group in Sweden.(98) Joneset al.(99) were the first to provideapartialsequenceforspigginandwe(100,101)discoveredthepresenceofasec-ondspigginmolecule.Wenowknowthatthereisawholefamilyofgenesencodingdifferentspiggintypes.(102)Spigginis,todate,theonlyandrogen-inducedproteininfish; thus, it was quickly recognised(103)asapotentialandrogenbiomarkerthatcanbeusedinthesamewayasVTG,awidelyusedbiomarkerforestrogens. Usingspigginasabiomarker,wehavedeterminedthatanumberofandrogens,when administered via water, are capable of inducing spiggin synthesis in femalekidneys.(13) The relativepotencies of the androgens tested in this systemon spig-gin induction were as follows: 17α-methyltestosterone (17α-MT) > 5α-DHT > 17β-trenbolone > 11-KT ≥ T (our unpublished data). The stability of the test chemi-cal in the water and possibly its relative binding affinity for the sex hormone that binds globulin are factors other than receptor affinity that affect exposure responses. Todate,environmentalandrogenshavebeenofonlylocalconcernbecausetheydo not appear to be widely spread. They have been identified in three environments, namely, those downstream of pulp mills,(104–112) US beef-production facilities(113–115)and sewage effluents with no secondary treatment.(116) Asindicatedbythenumberofpublications,(104–112)theclearestcaseofandrogenicdisruption in aquatic environments is the masculinisation of fish living downstream of pulp mill effluent discharge. There, the female mosquitofish (Gambusiasp.)de-velop anal fin appendages (gonopodia) that are normally only found in males. The active compounds in the effluent have been tentatively identified as bacterial degra-dationproductsoftheplantsterolstigmastanol.Othereffectsattributedtopulpmillsinclude a decrease in the gonad size of female fish(117,118) and significantly male-bi-asedeelpout(Zoacres viviparous)broods.(119) Compounds in pulp mill effluent bind agonisticallytothehumanandrogenreceptor.(111) We have confirmed the androgenic activity of Swedish pulp mill effluent using the stickleback androgen assay where spiggin was induced in female fish at 10% of the concentration of the effluent.(14) Concerning androgenic activity downstream of US beef-production facilities,trenboloneacetateisananabolicsteroidusedasagrowthpromoterinbeefcattleinthe US and Canada that is hydrolysed to 17β-trenbolone, a potent environmental an-drogenbothin vitroandin vivo.(113–115)Wehavetestedtrenboloneinthesticklebackbothin vivo(80)andin vitrousingprimarykidneycellculture(94) and confirmed its an-drogenicproperties.

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The detection of androgenic compounds in domestic effluent has only been re-portedinalimitednumberofstudiesmostlikelybecauseitappearsthatsecondarytreatmentof sewage is successful in removingandrogens. In theUK, it hasbeendifficult to test the sensitivity of the stickleback androgen assay in domestic effluent because the vast majority of sewage treatment works have at a least one second-ary treatment. Nonetheless, someearlycagedeploymentsdownstreamofprimarytreated effluent did induce spiggin in female fish. Owing to the fact that only three of fifty females survived the exposure, we were not able to publish the results. The male fish however, demonstrated a much lower mortality rate (only 13%) suggesting adifferenttoxicitybetweenthesexes.

3.2 Antiandrogen tests in the stickleback Although androgenic xenobiotics are restricted to untreated sewage effluent, feedlots and pulp mills,(104–115) the vast majority of treated sewage effluent(120) andinterestingly,oilplatformsintheNorthSea(121)containahighlevelofantiandrogenicactivity.However,thenatureofthecausativeagentsisasyetunknown.Sofar,in vitro and in vivo data (the latter mainly in mammals) have indicated that variouschemicalclassesdisplayanti-androgenicactivity,includingpesticidessuchasfeni-trothion, vinclozolin, procymidone, linuron, iprodione, chlozolinate, ketoconazole,dichlorodiphenyltrichloroethane(DDT)anditsmetabolite,p,p’-dichlorodiphenyldi-chloroethylene (DDE),aswellasseveralpyrethroids.(122–127) Insticklebacks,theandrogenbioassaycanbeadaptedtodetectantiandrogensintwo different ways. In the first relatively simple system, female fish are stimulated to producespigginwithastandard,moderateconcentrationofamodelandrogenoveraperiodof21days.Atthesametime,thepotentialantiandrogenisadministeredinarangeofconcentrations.Theconcentrationoftheandrogeniscriticalwhenenviron-mental concentrationsof theantiandrogenareemployed (i.e.,notpharmacologicaldoses).Ifitexceedsacertainlevel,thentheantiandrogeniceffectisnotapparent;ifitisbelowacertainlevel,thenthestimulationofthepositivecontrolgroupfailstoproduce statistical significance. We have fine-tuned the system with both 5α-DHTand 17α-MT and revealed that they should be used at 5 µg/L and 500 ng/L, respec-tively.(42)Inthiswaytheamountsofspigginproducedinthefemalekidneyallowacleareffect tobedetected. Wehavetestedthisfemale-basedantiandrogentestforover five years in different laboratories and in intercalibration exercises and have foundittoberobustandreproducible. Inthesecondsystem,theantiandrogenbioassayisbasedoninhibitingtheonsetof natural breeding in male fish. In this case, there is no chemical stimulation by a modelandrogenaswhenusingfemalesbutratheraphotoperiodicmanipulationofthemalebreedingstatus. Male sticklebacksenterabreedingcondition thatmani-festsitselfwithterritoriality,thedevelopmentofnuptialcolorationandnestbuildingbehavior,withinafewdaysifkeptundersummertimeconditions.Whilethestimu-lationofnaturalbreedingiscarriedout,theantiandrogeninquestionisadministeredin the same fashion as in the first system. This test system fits in with the OECD guidelinesforEDCspawningtests,(79)whichdonotallowtheuseoftwochemicals.Thetestrunsoveraperiodof21daysandcanbecarriedoutinaspecialtankthattakesaccountoftheaggressivebehaviorofnestingmales,essentially,toavoidter-ritorialitythatcouldleadtoincreasedmortality,themalesarekeptinseparatedcom-partments within the same tank where five individual nesting places are provided. Asixthcompartmentthatstretchesacrossthewholelengthofthetankcontainsthefemales.Agraphicrepresentationofthetankisprovidedinanotherpaper.(128)Themalescanchemicallydetecteachother,buthavelimitedvisualcontactbecausetheseparatorsaremadeofdensestainlesssteelmesh.Eachmalehoweverhasbothvi-sualandchemicalcontactwiththefemalesinthemaincompartment.


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The fish (five males and eight females in each tank) are placed in the tanks on day0.Onday1,thephotoperiodandtemperatureareraisedto18hlight:6hdarkand 15–18°C, respectively, while the administration of the chemicals/effluent under investigationcommences.Onday10,nestingmaterialisprovidedforeachmalebymeansofsuspendingitovertheirterritory.Forthis,eithernaturalpondweed(Elodeaspp.)orsyntheticthreadscanbeused.Inaddition,gravelisprovidedononeedgeofeachmalecompartment.Fromday11today20,aphotographicrecordofeachter-ritoryistaken,fromwhichtheprogressionofbothdigging(bygravelshifting)andnestbuildingactivitiescanbeassessed.Onday16,abriefintroduction(5min)ofafemaleintoeachmalegroundtakesplace.Wehavefoundthatthisstimulationisparticularlyimportantbothforthemalesthatcompletedanestsoonaftertheonsetofthetestandforthosethatstartedlate.Intheformergroup,wherenestdestructionandreconstructioniscommonwhenmorethanaweekhaslapsedbetweennestcom-pletionandinteractionwithafemale,thisintroductionalleviatesfrustrationwhileinthelattergroupitprovidesanextrastimulustoacceleratenestbuildingactivity.Oncompletionofitsnest,themalewillactivelycourtgravidfemales.Ondays19and20 spawning is attempted. During this, a randomly selected female is allowed toremainineachmalecompartmentandinteractfor15min.Wefoundthatincreasingthistimedoesnotimprovethechancesofsuccessfulspawning. An elaborate courtshipbehavior, involving a series of rapid side-to-side jumpsknownasthezig-zagdanceandaleadingcuetothenestisthenormifboththemaleandthefemalearereadytospawn.Ifthefemaleisreceptive(fullygravid),shewillfollowthemalebacktothenestanddeposithereggs.Ifspawningtakesplacebe-foretheendofthe15minperiod,thefemaleisremovedfromthetank,asitislikelytobeattackedbythemale.Themalefertilisestheeggswithinsecondsafterdeposi-tion in the nest and enters the parental phase. Behavioral observations and videorecords of nest building, fanning and courtship behavior throughout the test arefurtherendpoints,additional tospiggin induction, thatcanaddvalueto theantian-drogen test. All the fish are sacrificed at the end of day 21. We have found that the manipulationofthemalereproductivestatusinthiswayismuchquickerandeasiertoachievethanthatofthefemale. This version of the stickleback antiandrogen bioassay is far more labor inten-sive and requires a significant amount of planning. The male fish need to be in a nonbreeding condition prior to the commencement of the test. If they are in theadvancedbreedingstate,thentheamountsofspigginalreadypresentinthekidney,thehighlevelsofcirculatingandrogensandthe largenumberofreceptorspresent,donotallowforacleareffecttobeseen.Althoughwehavenotyetdeterminedthehalf-lifeofspiggin, throughobservationsin thelaboratorywesurmisethatspigginfromafullyhypertrophiedkidneyrequiresatleastonemonthtoregresstothepointwhere it is undetectable. Besides, in wild caught fish, spiggin is detected in males untilSeptemberalthoughit isgenerallybelievedthatbreedingdoesnotextendbe-yondJulyintheUK.Itisthereforeofparamountimportancethatthehomogeneityof the male reproductive status be adequately managed. To this end, it is recom-mended that the test fish should be laboratory reared or at least collected from the wildatleastthreemonthspriortothetest.Toensurethesuppressionofsexualmat-urationincaptivity,themalef ishshouldbekeptunderwinterconditions(6hlight:18hdark,5–8°C)untilthetestbegins.Thefemalesontheotherhand,whenrearedin captivity, need a long time (at least two to three months) under a summertimephotoperiodtoensureVTGandreadinessforspawning.Whentheyarereadytolaytheir eggs, however, they display the characteristic head-up posture. Female fish that havebeencollectedfromthewildappeartorespondearlierandstrongertostimuli.Thereasonforthisdifferenceiscurrentlyunknown.Whenthestatusofreproductivedevelopment has been finely tuned in this way, the bioassay is easy to run and yields meaningfulresults.Toassessthestateofmaturityatthecommencementofthetest,10malesand10femalesaresampled.

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Wehavetestedthesystemafewtimesandfoundthat is itmucheasiertosyn-chronize the reproductive system of male fish between September and February (be-foretheonsetofthenaturalbreedingperiod).Thismaybeduetothestrongcircan-nualendogenousreproductivecycleofthestickleback.(129–131)

Using the first system that uses androgen-treated female fish, we found that the relativepotenciesof testedantiandrogenson the inhibitionofspiggin inductionby17α-MT (500 ng/L) or 5α-DHT (5 µg/L) were as follows: Flutamide > Fenitrothion > Vinclozolin > Linuron > Procymidon > DDE. Furthermore, we are the first to re-port that Linuron and Fenitrothion are antiandrogenic to fish. In addition, we have shownthatin vivoexposuretohighconcentrationsofestrogens(i.e.,E2andethinyl-estradiol at concentrations > 100 ng/L) also inhibit androgen-induced spiggin syn-thesis.(42) Estrogens have a relatively high affinity for the androgen receptor (AR), so they mayactaspureantiandrogensbyoccupyingthereceptorbutnotactivatingit.Itisalso possible that another mechanism underlies the observed inhibition of spigginsynthesis.Olssonet al.(96)foundthatARmRNAlevelsweresuppressedincastratedmalesthatreceivedestradiolimplants.Althoughitispossiblethatthehighestrogenlevelsmay triggera feedbackmechanismfrom thehypothalamuscausinggonado-tropinsecretioninhibition(andthusandrogenproductioninthetestes),wehaveob-servedspiggininhibitioninandrogen-treatedfemales;hence,thispotentialpathwayappearstobeirrelevant.Inaddition,thesameinhibitionisobservedin vitrousingsticklebackprimarykidneycellculture.(132)Lastly,itisalsopossiblethatthelowerspiggincontentofthekidneyisduetokidneyfailure,whichisinturnduetoexces-siveVTGproduction.

3.3 Other tools3.3.1 Behavior The regulation of reproductive behavior in fish is complex, and involves the interactionof physiological, environmental and social factors.The sticklebackhasplayedanimportantroleintheunderstandingofthehormonalcontrolofreproduc-tive behavior in fish.(133,134) Thereismorethanonereasonforthesticklebackbecominganimportantmodelspecies in this field of research. The most important one being the display of a vari-etyofpronouncedreproductivebehaviorsbythemale.Incommonwithotherparen-tal-caringspecies,eachnestingcycleofthemalesticklebackcanbedividedintoaninitialsexualphasefollowedbyaparentalphase.Thesexualphaseischaracterizedbyterritoriality,nestbuildingandcourtship,whilethedominantbehaviorshowndur-ingtheparentalphaseisnestfanning,duringwhichthemalefansoxygenatedwaterover the eggs using its pectoral fins. Other advantages that the stickleback provides include its readiness to breed and to display all the aspects of male reproductivebehaviorincaptivity,therelativeeaseoftotalgonadectomy,whichallowshormonereplacementstudiestotakeplace,anditsstresstolerance,sinceindividualswillre-sumefeedingandnormalbehaviorpatternswithinminutesafterhandling.(135,136) Whenthemaleenterstheparentalphase,thelevelsofboth11-KTandTdeclinesharply to12ng/ml(137) and8ng/ml,(135) respectively. Thesharpdecline inplasmaandrogenslevels(both11-KTandT)duringtheparentalphase,suggeststhatandro-gensdonotplayanall-importantroleinthecontrolofparentalbehaviorinthestick-leback.ThisissupportedbytheresultsofPállet al.,(138)whofoundthatthedegreesoffanningbehaviorinbothcastratedmalesandcastratedmalesgiven11-KA(con-vertedto11-KT)implantsdidnotdeviatefromthelevelsshownbysham-operatedmales.Hence,onceamalehasentereditsnestingcycle,parentalbehaviorsuchasfanningoccurs independentlyof thepresenceofgonadal steroids,as illustratedbythefactthatneithercastration(removalofcirculatingandrogens)norandrogenad-ministration(elevated11-KTlevels)affectedthelevelofexpressionofthisparentalbehavior.


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3.3.2 Estrogens Therearealreadymanytestspeciesusedfordetectingestrogensintheenviron-ment. Thus, in respect to screeningchemicals, there isnoparticular advantage tousingthestickleback. However, therearemanypartsof theNorthernHemisphere(i.e.,EuropeandnorthernAsia)where,becausetheyareindigenous,theyareamoreappropriate environmental sentinel than warm-water exotics such as the fatheadminnow and zebrafish. Thus, we have put effort into developing assays for VTG andspiggininthestickleback.WehavealreadycarriedoutVTGmeasurementsonover 2000 samples. When using small teleost fish, such as the stickleback, or larger fish at juvenile stages, a major challenge is the collection of adequate amounts of plasmaforthemeasurementofVTG.Typically,thesizeofafull-scalechemicaltestincludes as many as 300 fish. Clearly, to bleed and dissect these many individuals inoneor twodays requiresa relatively large,well-trained researchgroup. Inad-dition, theapplicationofaschedule1methodofhumanekilling isnotcompatiblewith bleeding the fish, since once the heart stops beating, no blood can be collected. Boththepracticalandethicalchallengespresentedabovehaveledustoinvestigatealternative tissues formeasuringVTG. One tissuewidelyusedfor thispurpose istheliver,howeverwefoundthattheamountoffatpresentparticularlyinlaboratoryanimals interfereswith the assay. We thenconsidered that if the animalhasbeensacrificed without losing any of its blood the amount of blood trapped in the heart at the time of killing could provide a good alternative. In this way, the fish can be ter-minallyanaesthetised,snapfrozeninliquidnitrogenanddissected(notnecessarilyonthesameday)whilestillonlyslightlythawed,sothattheheartcanbecollectedasonelumpoftissueinavial.Assaybufferisthenadded,followedbyvigorousmix-ingwithapellethomogeniser.Thevialisthencentrifuged,thesupernatantcollectedandanaliquotassayedforVTG. Wehave tested thismethodforover threeyearsnow,includinginterlaboratorycomparisons,andwefoundittobereliable,quickandreproducible.Therearetwodrawbacksthough:onebeingtheincompatibilitywithgonadal fixing and hence histological investigations, and the other being the lack of adirectcomparisonwithreportedVTGtitresfromotherspecies. Anotheralternativeway is theuseofwhole-bodyhomogenatesaswehave re-portedbefore,(9)butthismethodpresentsthedisadvantagethatothertissueinforma-tionislost(e.g.,liverandgonadaltranscriptomic,metabolomicandproteomicpro-filing). The stickleback appears to showmoderate sensitivity to steroidal and environ-mentalestrogens,albeititishardtocompareliteraturewithourowndataowingtothedifferentapproachesemployedinreportingconcentrationresponseresults.Someoftheavailabledataquotenominalconcentrationsoftestchemicalsduringexposureandsomemeasuredconcentrations.Theformersufferfromalackofstandardizationwhilethelattersufferfromvariationintheextractionmethodsandthesensitivityoftheassaysused. InourCefaslaboratory,weroutinelyemployradioimmunoassaysforthemeasurementofE2andethinylestradiol(EE2)andobtainarecoveryrateofca.65%.Thelowesteffectiveconcentrations(LOECs)intermsofsticklebackVTGresponsefornominalconcentrationsofE2andEE2werefoundtobe25ng/Land6ng/L,respectively(ourunpublisheddata).Thenoeffectconcentrations(NOECs)were10ng/LforE2and2.5ng/LforEE2. Inanotherstudy,(139)wetestedEE2atconcentrationsof1,5,50and200ng/LandfoundthattheLOECwas50ng/LandtheNOECwas5ng/L,leadingustospeculatethatthesensitivityofthespecieswasprobablylower thanthoseofmostcommonlyusedtestspecies. Thesensitivityofjuvenile sticklebacks appears to be equal to that of zebrafish.(9) Theeffectsexertedbyexogenoussexsteroidsandtheiranaloguescanbebroadlydivided into masculinisation or feminisation. The former is generally in responsetoandrogens(malehormones)and the latter toestrogens(femalehormones),eventhough,owingto thenatureofmetabolicendocrinepathways,androgensmayalso

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indirectlyinducefeminisingeffectsviatheirroleastheprecursorsofestrogens.(140)The overall androgen/estrogen amount ratio in an organism is thought to be moreimportant than theamountofeachsteroidon itsown.(141) This ratio is largelyde-terminedby theactionofenzymes involved insteroidogenicpathways. Themostimmediatedeterminantoftheandrogen/estrogenamountratioisP450arom(aroma-tase),anenzymethatconvertstestosteroneintoestradiol,makingitamajortargetofendocrinedisruption.However,factorsaffectingsteroidreceptorexpressionarealsoinfluential in determining where and to what degree the hormones are able to act. Fisher(142)hasdiscussedtherelativeimportanceoftheandrogen/estrogenbalanceandofhormonereceptorsasmediatorsoftheeffectsofendocrinedisruptingchemicals. In thestickleback, it ispossible todifferentiatebetweenthefeminisingandde-masculinisingnatureofachemicalbythepresenceofrobustbiomarkersforbothan-drogensandestrogens.Exposuretoanantiandrogenforexample,whichcanleadtodemasculinisation,thatisoftenperceivedasoverallfeminisation,willnotbeaccom-paniedbyVTGinductionasitwouldbeexpectedifaclassicestrogenwereused,butratherbeaccompaniedbyareductioninspigginproduction.Thisisparticularlyim-portant when there is currently no other recognised test for antiandrogens using fish whiletheproposedend-pointforandrogensisareductioninfemaleVTGtitre.(143)

3.3.3 Molecular sex determination Generally, sex determination in fish is more labile than in mammals and birds. The androgen/estrogen balance during sexual differentiation largely determineswhichgonadaltypedevelops,oftenoverridinggeneticsexifpresent.Agonochoris-ticspeciesischaracterizedbytheexistenceofovaryortestes,inwhichthetypeofgonadisdeterminedduringearlysexualdevelopment(sensitivephase)anddoesnotchangeduringitslifetime.Backinthe1970sand1980s,anglersintheUKshowedanabnormalappearanceofroachgonads,particularlyinthevicinityofsewagetreat-ment works. This was later confirmed as intersexuality or intersex (i.e., the pres-enceofbothovarianandtesticulartissuewithinasinglegonad).(144)Thisconditioncanoccurspontaneouslyorcanbeinducedundercertainenvironmentalconditions,mainlytemperatureregimes,andisnotconsideredtobepartofthenormallifecyclein gonochoristic species. In the context of sexual disruption in wild fish, intersexu-ality ismoreoftenassociatedwithestrogeniccompounds,and intersex individualsmoreoften appear tobe feminizedmales thanmasculinized females.(145) It is dif-ficult to confirm that this assumption is correct because no genetic sex identification haseverbeenmadeinwildintersexroaches.Besidesthefactthatthevastmajorityof fish species are strictly gonochoristic and sex determination seems to have a ge-neticbasis,thepresenceofmorphologicallydifferentiatedsexchromosomesorotherDNAmarkersisveryrare.Sticklebacks,alongwithmostteleosts,donothavehet-eromorphicsexchromosomes,buttheydopresentseveralproteinandDNAmarkerswith sex-specific alleles. Two such DNA markers(75,146)havebeenextensivelyusedin our laboratory for sex identification and appear to be very robust, easy to use and reliable.Bothmarkersarewithinthesex-determiningregion,whichhasbeenlinkedto a specific chromosome that appears to be an evolving Y chromosome.(146) Thus,thesticklebackappearstobemore‘hardwired’intermsofsexualdifferentiationthanotherteleostssinceitpresentsanascentYchromosomesimilarlytomammalianspe-cies.Tothisend,itisexpectedthatthedegreeofplasticityinsexualdifferentiationobservedinotherspeciessuchascyprinidsisnotpresentinthestickleback.Howev-er,thereareseveralreportsofintersexsticklebacksinthewild.(147–149)Inaddition,ithas been confirmed that estrogen and androgen treatments during the sensitive phase ofsexualdifferentiationresultinintersexortotalsexreversal.(8)Morerecently,themasculinisingeffectofperchlorate,achemicalwidelyusedinthemilitary,domesticandcommercialapplications,wasreported.(150) In this study, female fish were found tobefunctionalhermaphroditesinthattheyproducedbothspermandeggsthatwere


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capableofself-andcross-fertilization. Sinceperchlorateisnotconsideredtobeaclassic sex steroid agonist or antagonist,withoutgenetic sexing itwouldnothavebeenpossibletoclassifyitasanandrogenagonist.Thegeneticsexmarkersofthesticklebackarethereforeofgreatdiagnosticvalueinthestudyofsexualdisruptioninfish. This becomes particularly important when considering the complexity of efflu-entswhereamixtureofchemicalswithsimilarordissimilarmodesofactioncoexist.

3.3.4 Sex steroids Inthewhitesucker(Catostomus commersoni),circulatingconcentrationsofsexsteroids have been shown to be significantly lower in fish living in polluted wa-ters(151) and are associated with an increased age of maturation, a reduction in thenumber of defining secondary sexual characteristics and a reduced degree of gonadal development. There have been several studies that have focused on sex steroidconcentrations in relation to pollution generally(152,153) and EDCs specifically.(113,154)TheseallsupportadecreaseinsexsteroidconcentrationsinresponsetothepresenceofEDCs,eventhoughtheydonotindicatewhetherthisisaresultoftheEDCsact-ingdirectlyon theactivityofsteroidogenicenzymes, indirectly througheffectsonfeedback regulationwithin thehypothalamic-pituitary-gonadalaxisorbyaffectingtheircatabolismandexcretion.Whateverthemechanism,sexsteroidconcentrationsappeartobeverysensitivetopollutants.(118,153,155,156)Amajorproblemwithcarryingoutsimilarstudiesonsticklebacksisthat,beingsosmall(generally<1g),theyneedto be sacrificed in order to obtain a sufficient amount of blood for steroid analysis. However,basedonthediscoverythatsteroidsnaturally‘leak’acrossthegillsfromthebloodtothewater,(157)wehavedevelopedanewprocedurethatistotallynonin-vasive.(158) This involves putting the fish in a small beaker of water for 30 min and then returning them to their tank. The fish release sufficient amounts of steroids into the water for several to be measured. Moreover, the fish appear totally unaffected and can as a consequence be sampled many times without anaesthesia or sacrifice. We intend to apply this method to both feral animals caught in hot spots and fish exposed toEDCs inorder toobtainmore informationon themechanisms throughwhichchemicalscandisrupttheendocrinesystem.

4. Conclusion

There are very few fish species that can match the plethora of advantages that the sticklebackispresentasamodelspeciesinendocrinedisruptionresearchandeco-toxicologyingeneral.Someofthesetraitsarerelatedtotheirbiologyandlifehis-torywhileotherincentivesarerelatedtothewealthofinformationavailableintheliterature. It is expected that in the near future, this model species will significantly contributetotheassessmentoftheimpactofendocrinedisruptorsnotonlyonwild-lifebutalsohumanhealth.

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Three-spined stickleback: an emerging model in environmental endocrine disruption

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