Report on the research activities with Orcinus orca in Loro Parque

Date: May 23rd 2013

author: F. Javier alMunia Portolés, Ph.d. dePuty director oF loro Parque Fundación

RepoRt on the ReseaRch activities with Orcinus Orca - 2

1 IntroDuctIon

ThescientificresearchactivitiesofLoroParqueare based on the Research and Conservation Program 2010-2015, which has been submitted to the Spanish authorities in compliance with the Spanish Zoo Law (31/2003) and the European Zoo Directive(1999/22/CE).ThisProgramistheofficialdocument in which the fundamental criteria andguidelinesfortheresearchandconservationactivitiesare established. These criteria are based on the recommendationsofdifferentzoologicalinstitutionsrelatedwithLoroParque(WorldAssociationofzoosand aquariums -WAZA-, European Association ofZoosandAquarium-EAZA-,IberianAssociationofZoos and Aquariums -AIZA-, European Association of Aquatic Mammals -EAAM-), and severalbiodiversity conservation organizations relatedwith Loro Parque Fundación (International Union for Nature Conservation -IUCN-, Survival SpeciesComission -SSC-, Conservation Breeding Specialist Group -CSBG-, etc.).

ThemaingoalofthisProgramistooptimizethefinancial, material and human resources that LoroParque dedicates to the research and conservation activities, either directly or through Loro Parque Fundación. As no zoological institution can runresearchandconservationprojectsforeverysinglespeciesinitszoologicalcollection,theResearchandConservation Program of Loro Parque establishesthe criteria to distribute the resources among the most appropriate projects and species, based on their biodiversity conservation potential and the suitability of the group of animals hosted at LoroParque.

Theselectionofthescientificandconservationactivities is based on an analysis of the potentialvalueforresearchandconservation(either in situ or ex situ)ofeverysinglespeciesinthezoological

collection. To determine this potential value several criteriawillbeused,suchas:statusintheIUCNRedListofThreatenedSpecies,availabilityofbreedingprograms (ISB, EEP or ESB), level of knowledgeabout species biometrics, biology or physiology, number of individuals available in the collection,levelofhusbandry,suitabilityoftheholdingfacilities,etc.

Based on the potential conservation and researchanalysis,asetofresearchproposalsaresubmitted every year to the Board of Directors ofthe Loro Parque Fundación. All the proposals are evaluated in theAdvisoryBoardAnnualMeeting,using the most up-to-date scientific knowledge oftheboardof independentmembers.TheBoardofDirectors approve the research and conservation proposal (in situ and ex situ), which are published inafinaldocument.

In 2012 the LPF Advisory Board decided to fund 36 projects during 2013 with a total of1.136.036US$,ofthem30werefocusedonparrotsandterrestrialecosystems,and6ofthemfocusedoncetaceansandmarineecosystems.FouroutofthesixmarineprojectsaremainlyfocusedonOrcinus orca research activities ex situ, with applications for in situconservationof thisandothercetaceanspecies.

report on research actIvItIes

1 Introduction 2FinishedProjectsandScientificActivities 3ActiveProjectsandScientificActivities

RepoRt on the ReseaRch activities with Orcinus Orca - 3

2 FInIsheD projects anD scIentIFIc actIvItIes

2.1 Research on bioacoustic tools WhenMorganarrivedtoOrcaOceanshewasintegrated into the two ongoing research projects, fundedbyLoroParqueFundación,thatimpliedex situ research activities with Orcinus orca. One ofthem,aimedatthedevelopmentoftoolstoimprovebioacoustic research was finished in 2012. WhenMorgan arrived the project team was acquiringindividually identified sounds in order to establishindividual repertoires. As soon as Morgan couldbe held in the experimental pool (by then holding Pool 2), the setting-up of her individual repertoirewas initiated. As a result, over 250 Morganvocalizationswererecordedandarchivedin2012

forfutureanalysis.ThesedataweremadeavailabletotheresearchersoftheUniversityofSt.AndrewsstudyingtheevolutionofMorgan’sdialect.

Figure 1: Spectrogram of one of the sounds attributed to Morgan that were registered during the introduction phase.

LP01 LP01.1 LP01.2 LP02 LP03 LP04 LP06

LP11 LP11.1 LP12 LP13 LP15LP16

LP51LPwave

LP Dialect

Figure 2: Acoustic dialect of the orcas in Loro Parque before the introduction of Morgan

RepoRt on the ReseaRch activities with Orcinus Orca - 4

2.2 Genetic comparison between species of Marine Mammals Short after the arrival of Morgan to LoroParque, the Museum of Natural History ofCopenhagen requested good quality genetic materialtoperformacomparisonbetweendifferentspeciesofmarinemammals.AstheyalreadyusedMorgan’s genetic samples in a previous stage ofthe project (when they obtained the samples to determine Morgan’s original pod), they wantedto use genetic material from the same individualto continue the project. Loro Parque requested the necessary CITES permits to export the blood samples, and sent them to the research team, which received andprocessedthemaccordingly.Morgan’sgeneticmaterial was sequenced and provided a very high quality genome. Despite that the researchers are stillworkingonthepublicationofthecomparativegenetic adaptations in different marine mammalspecies, the genome sequence obtained fromMorgan has been published as reference forOrcinus orca as public domain: http://www.ncbi.nlm.nih.gov/nuccore/ANOL00000000.2

2.3AudiogramofOrcinus orca During 2012 Loro Parque requested a group of international experts to evaluate the hearingcapacity of Morgan, as the trainers inferred thatshe was inconsistent in responding to the sound bridge (whistle). The research team suggested the useofAuditoryEvokedPotential(AEP)asitallowsa quick assessment of hearing capabilities, andhas been extensively used with dolphins and other smallcetaceans.Inordertoget themostscientificinformation possible from this hearing evaluationnotonlyMorgan,butalsotheotherfourorcas,weretrained to perform the auditory evoked potentialmeasurements underwater, and the other two were madeavailabletobemeasuredoutofthewater.

As a result, the basic audiometric functionsof six killer whales were assessed, becoming thelargest testusingAuditoryEvokedPotential in thisspecies ever. This methodology was used only once before in 1999 with two killer whales to describetheaudiogramofthespecies.Beforethisresearchwasperformed,allthescientificinformationonthehearingcapacityof thespecieswasbasedjustonthree individuals.

Theresultsofthetestrevealedalackofclick-evoked response in Morgan, which confirmedthat thisanimalsuffers fromahearingdeficit.Themagnitude and frequency range over which thehearing deficit occurs couldn’t be specified with

Figure 3: Morgan desensitization training session. From left to right and top to down: 1 attaching dummy suction cups, 2 giving signal to put nose on target, 3 correct position and 4 incorrect position.

RepoRt on the ReseaRch activities with Orcinus Orca - 5

themethodologyusedin thestudy.Unfortunately,although AEP measurements allow a quickassessmentofhearingcapabilities,duetothesize-dependentlossoftheelectrophysiologicalsignal,thismethodology was revealed to be inappropriate to detectthelowerhearingthresholdofOrcinusorca.As a consequence, the research team submitted a

newproposaltoperformabehaviouralaudiogramof the same animals during 2013, the proposalwasapprovedbytheAdvisoryBoardMeetingandpartialfundinggrantedbyLoroParqueFundación.

Figure 4: Left: The research team measuring the AEP in water, this experimental arrangement is more complex and difficult to train, but gives better results as the sound reaches the animal in a more natural way. Right: Measuring AEP in a beached individual using a jawphone.

Figure 5: Results of AEP measurements in water (left) and beached (right), show a total absence of brain response to the sound stimulus in Morgan.

RepoRt on the ReseaRch activities with Orcinus Orca - 6

3 actIve projects anD scIentIFIc actIvItIes

Currently, there are five active projects andoneproposal,allofthemfocusedonexsituresearchactivitieswiththegroupofOrcinusorcahostedatLoroParque,thusallofthemincludeMorgan.

3.1 Pollution markers in European bottlenose dolphins This project is mainly focused on the effectsof pollution in the pathology of the wild dolphinpopulations in Europe. The research team collected biopsiesofwildandstrandeddolphinsaroundtheCanary Islands simultaneously with other researchers intheMediterraneanandNorthAtlantic.ThegroupofdolphinshostedatLoroParquewereusedasareferencegroup,tomeasurethebiodynamicsandbio-accumulationofpollutantsinagroupofdolphinswith a controlled diet.

When the research team started the sampling at Loro Parque, the opportunity to obtain blood samplesfromothermarinemammalswiththesamediet came up. As a consequence, the researchers initiated a parallel data collection to study a poorly understood issue: the relationship between concentrations of Persistent Organic Pollutants(POPs) in blubber and those in blood of marinemammals,asabetterindicatoroforganexposureto pollutants.

Morgan’s blood samples were obtainedregularly (as for the rest the group of orcas,dolphins and sea lions) and shipped to the research team in order to be analysed and to track anypossiblevariationofthepollutionmarkers.During2013 the research team is obtaining biopsy samples to evaluate the toxicological parameters in the blubber of the three species and compare themwith the plasmatic concentrations. The measurement ofthetoxicconcentrationsinplasma,blubberandfood should shed light on the bioaccumulationmechanisms in the three species.

3.2EvolutionofMorgan’sDialect When Morgan arrived to Loro Parque aproposal to evaluate the changes on her dialect was received from the University of St. Andrews(Scotland). This was the same research team that made the initial comparison of Morgan’s dialect

withtherecordsofpoddialectsfromthethreemajororcapopulationsintheNorthAtlantic.

TheproposaltestedtheacousticevolutionofMorgan’sdialect,asthereisahypothesisabouttheevolution of the orcas’ dialect during their entirelifetime. If this hypothesis proved to be correct,Morgan’s dialect should be modified with theincorporation of calls specific to the Orca Oceangroup.Similarly,therestoftheanimalsinthegroupwouldbeintegratingsoundsfromMorganintotheirdialect.

A researcher from St. Andrews Universityvisited Loro Parque two weeks during 2012 tocollectsoundsandmakeobservationsonMorgan’sdialect. Complimentarily, Loro Parque Fundación sharedalltheidentifiedsoundsofMorganthatwereregisteredfromtheon-goingbioacousticsresearchsince she arrived to Loro Parque, in order to provide abroadersetofdata.Acompletedescriptionofthegroup’sdialectprevioustoMorgan’sincorporationwas also provided by Loro Parque, so the research team could verify if any eventual new call inMorgan’sdialectderivedfromthere.

ThepreliminarilyanalysisofMorgan’sdialectduring 2012 did not show any incorporation ofsounds from the Orca Ocean group. Given thatby the end of 2012 the audiogram of Morganconfirmedthatshehadasignificativehearingdeficit(thatcouldevenbetotaldeafness)theexperimentcan not be considered valid, and the hypothesis ofthelifetimeevolutionofvocaldialectinOrcinusorca can not be rejected.

Alternatively, the research team from St.Andrews University submitted a continuation proposal to test the lifetime evolution of vocaldialectsinOrcinusorcaduring2013usingadifferentapproach.TheobjectiveistoanalyzetheindividualdialectsofthesixorcasatLoroParqueexceptforMorgan. As the auditory capacity of five animalshas been verified, the analysis of the evolution oftheirdialectsaftertheintroductionofMorganintothe group can be used to test the hypothesis. During 2013researchersfromtheUniversityofSt.Andrewswill be visiting Loro Parque to collect sounds and makeobservationsofthedialectofeveryindividualexcept Morgan. The recordings will be analyzedto find incorporations of Morgan’s calls into theindividualdialectsoftherestofthegroup.Inorderto provide the research team with a broader data settoanalyze,LoroParquewillmakeavailabletothe researchers all the identified calls registeredandarchivedasaresultofitsin-housebioacousticresearch activities.

RepoRt on the ReseaRch activities with Orcinus Orca - 7

3.3Biometricinformationderivedfrombioacoustic analysis of Orcinus orca calls Thisprojectisaimedtofindthewaytoobtainbiometric data from the bioacoustic signals of theorcas. This project is based on the technology developedbytheUniversityofLaLagunaandLoroParque Fundación in a previous 6 year projectfinished in 2011. Thanks to this technology, anindividualrepertoireforeachofthesevenorcasatLoro Parque is being established.

The research team is working in theparametrization of the calls as the stage prior toobtaining information related to the biometry ofthe animal producing the sound. The animals are alsocarefullymeasuredandweighedonaregularbasistocollecttheaccuratebiometricinformation.The bioacoustic records of the group (includingMorgan)havebeenmadeavailabletoresearcherswhen required, after presentation of an adequatescientificproposal.

The hydrophone system at Orca Ocean isbeingimprovedtodigitalizethesoundsignalsbeforeanyamplification.Thatwillprovidethetopquality

measurementsneededtoparameterizethesignalsand,subsequentlyfindbiometriccharacteristicsfromthe bioacoustic signals. If this methodology finallyworks, it would mean a very positive outcome inthe medium range non-invasive assessment of thebodyshapeof thewildkillerwhales,whichcouldbehelpfulintheconservationofthespecies.

3.4BehavioralaudiogramofOrcinus orca As the Auditory Evoked Potential (AEP)methodology was revealed to be inappropriate to detect the lower hearing threshold of Orcinusorca, a new proposal to perform a behaviouralaudiogram of the same animals during 2013 wassubmitted to the Advisory Board Meeting of LoroParque Fundación.

In this new research the hearing capacity ofMorgan and other four or five individuals will beaccurately established. This methodology implies an intensive training of the animals to make themrespond consistently to a discriminative stimulus (sd). The stimulus will initially be visual and acoustic and gradually shifted to only acoustic. Once theanimals respond consistently to the acoustic sd,

Figure 6: Voluntary blood extraction of an orca at Loro Parque

RepoRt on the ReseaRch activities with Orcinus Orca - 8

long and detailed testing will be initiated by playing differentsoundintensitiesatmultiplefrequenciesinorder todeterminethehearingthresholdofeveryindividual. IfMorganfails toshift fromthevisual/acoustic discriminative stimulus to the acoustic sd, thatwouldbeanindicationoftotaldeafness.

The information resulting from this study willmeanamajorimprovementintheactualknowledgeoftheauditorycapacityofthespeciesthatcomesfromastudyonasubadultmale40yearsago,andonemorerecentwithtwofemalesover20yearsold.All the animals tested in this experiment are under 17 years old, which will give a better approach to theauditorycapacityofyoungspecimensofOrcinusorca.Adetaileddescriptionoftheauditorycapacityofthespeciesisacriticalknowledgetobeachievedinordertobetterprotectthewildpopulationsfromthe noise pollution.

3.5 Bioacoustical characterization of differentbehaviouralsituationsinOrcinusorca TheAdvisoryBoardMeetingofLoroParqueFundaciónapprovedaproposalfromtheUniversityof La Laguna and the Free University of Berlin tostudy the prevalence of different types of callsrelatedwithbehaviourinOrcinusorca.Ithasbeensuggested that there is a relation between the differentproportionalcalltypeusagebythesamepodsandtheirbehaviour,despitethatfewstudieshavefocusedonthecharacterizationofcallusageand behaviour.

The goal of this proposal is to perform anethological observation of the group of orcas atLoro Parque, while simultaneously recording the sounds that they produce underwater. A random focal observation schedule has been set up, and

focal individualsarerandomlyselectedandvideorecorded for 15 minutes. Complementarily, avideo recording covering the whole main pool (SeeAnexI)willbealsomadetoprovidefurtherad libitum or focal observations. The ethologistsfrom both universities will analyse the video andaudiorecordingsinordertodetermineiftherearesignificant variations in the proportional call typeusageindifferentbehaviouralsituations.

3.6 Biometrics proposal In the beginning of 2013 Loro Parquereceived a proposal from the University of St.Andrewstoperformaresearchprojecttomeasurethedimensionsof thekillerwhales inLoroParqueusingahigh-frequencyscanningsonar.Eachwhalewill be scanned by the sonar as it swims past, and measurements taken from the sonar imagewill be related to hand-measurements taken fromthat individual. The sonar uses sound frequenciescentered at 375 and 610 kHz. The results shouldleadtoapeer-reviewedpublicationonthefeasibilityof thismethod toremotelymeasuredimensionsofwild whales. This methodology would mean a very positiveoutcomein thenon-invasiveassessmentofthe body shape of the wild killer whales, whichcould help in the management of its endangeredsubpopulationsand,thusintheconservationofthespecies.

Loro Parque has expressed its willingness to cooperate in this research as long as it does not compromise the welfare of the animals. CurrentlyLoro Parque is gathering advice from differentexperts in acoustics and cetacean hearing to ensure that the sonar will not affect the animals.If the welfare of the animals can be guaranteed,the experiments will be performed during the lastquarterof2013.

RepoRt on the ReseaRch activities with Orcinus Orca - 9

annex I