Can
soil
testate
amoebae
be
used
for
estimating
the
time
since
death?
A
field
experiment
in
a
deciduous
forest
Ildiko` Szelecz
a,b,*,
Bertrand
Fournier
a,
Christophe
Seppey
a,
Jens
Amendt
b,
Edward
Mitchell
aaLaboratoryofSoilBiology,UniversityofNeuchaˆtel,RueEmile-Argand11,2000Neuchaˆtel,Switzerland b
InstituteofForensicMedicine,Goethe-University,60596Frankfurt/Main,Germany
1. Introduction
Theestimationoftimesincedeath(orpost-morteminterval– PMI)is oneof the mostimportant tasks whenever events and circumstances of a death need to be reconstructed for legal investigations.Here,forensicmedicinereaches itslimitsalready after24–48hpost-mortem[1,2].Additionalmethodsaretherefore neededbeyondthistimeforPMIestimation.Forensicentomology isthemethodofchoiceincaseswhereinsectshadaccesstothe body,andismostusefulinthefirstweeksafterinsectcolonisation [2].Beyond4–6weeksentomologicalPMIestimatesbecomeless reliable. New forensic tools, complementary to existing ones especiallywithrespecttoalongerPMIarethereforenecessary.
Inthelastdecadesforensicresearchershavestartedtostudy changesin thesoilbeneatha decomposing cadaver.So far,soil investigations incrime sceneshave focused mainlyon locating burialsites[3,4]andontheidentificationofsoilsamplesrecovered from suspects’ footwear, clothes, vehicles or weapons [5–10]. However, a decomposing cadaver strongly modifies the soil environment and asa resultaffects thesoil organisms [11,12]. Soilabioticorbioticcharacteristicarethereforepotentiallyuseful sources of information for the presence of cadavers and PMI estimates.Ourfocushereisonacommongroupofsoilprotozoa, thetestateamoebae.
Testateamoebae,alsoknownasshelledamoebae,ortestaceans, areapolyphyleticgroupofshelledunicellularprotistswhichare foundinvarioushabitats,suchasmosses,soils,peatlands,lakes, riversandevenestuarineenvironmentsallaroundtheworld.They aresubdividedintothreemainphylogeneticgroupsaccordingto theirfeet-likeextensions(pseudopodia)andshellcharacteristics: (1)Arcellinida,withlobose(finger-shaped)pseudopodia,themost
Keywords: Forensicindicators Soilprotozoa Testateamoebae Communityecology Cadaver Post-morteminterval ABSTRACT
Estimationofthepost-morteminterval(PMI,thetimeintervalbetweendeathandrecoveryofabody) canbecrucialinsolvingcriminalcases.TodayminimumPMIcalculationsrelymainlyonmedicaland entomological evidence. However, beyond 4–6 weeks even entomological methods become less accurate.Thusadditionaltoolsareneeded.Cadavericfluidsreleasedbydecomposingcadaversmodify thesoilenvironmentandthusimpactsoilorganisms,whichmaythusbeusedtoestimatethePMI. Althoughtheresponseofbacteriaorfungitothepresenceofacorpsehasbeenstudied,tothebestofour knowledgenothingisknownaboutothersoilorganisms.Testateamoebae,agroupofshelledprotozoa, aresensitivebioindicatorsofsoilphysico-chemicalandmicro-climaticconditionsandaretherefore goodpotentialPMIindicators.Weinvestigatedtheresponseoftestateamoebaetothreedecomposing pigcadavers,andcomparedthepatterntotwocontrolseach,baresoilsandfakecadavers,inabeach-oak forestnear Neuchaˆtel,Switzerland.Forestlittersamplescollectedin thethree treatmentsover10 monthswereanalysedbymicroscopy.Thepigtreatmentsignificantlyimpactedthetestateamoeba community:after22and33daysnolivingamoebaremainedunderneaththepigcadavers.Communities subsequently recovered but 10 months after the beginning of the experiment recovery was not complete.Thefakecadavers also influenced thetestate amoebacommunitiesby alteringthesoil microclimateduringadryhotperiod,butlessthanthecadavers.Theseresultsconfirmthesensitivityof soiltestateamoebaetomicro-climaticconditionsandshowthattheyrespondfasttothepresenceof cadavers–andthatthiseffectalthoughdecreasingovertimelastsformonths,possiblyseveralyears. Thisstudythereforeconfirms thatsoil protozoacouldpotentially beusefulasforensicindicators, especiallyincaseswithalongerPMI.
* Correspondingauthorat:LaboratoryofSoilBiology,UniversityofNeuchaˆtel, RueEmile-Argand11,2000Neuchaˆtel,Switzerland.Tel.:+410327183108.
E-mailaddress:ildiko.szelecz@unine.ch(I.Szelecz).
Published in Forensic Science International 236, 90-98, 2014 which should be used for any reference to this work
diversegroup,comprisingthreequartersofallknownspecies,(2) Euglyphida,withfilose(thinandfilament-like)pseudopodia[13– 15],and(3)Amphitremida,withanastomosingpseudopodiaand symmetrical shells with two apertures [16]. Of the ca. 2000 describedtaxa[13,14,17],about300specieshavebeenfoundin soils[18].
Soiltestateamoebaecanbeveryabundantreaching 106–108
individualsperm2and102–105individualspergramdrymassin
soils and leaf litter [18]. They reproduce relatively slowly, by microbialstandards,withgenerationtimeofafewdaystoovera week[19,20].Theirshellcanpersistafterthedeathoftheorganism formonthstomillennia(e.g.inpeatorsediments),making long-termstudiespossible[21,22].Theyareabletoencyst(formacyst) under unfavourable conditions and excyst when conditions improve,a behaviourthat canindicatechanges inthe environ-mentalconditions[23,24].
Testateamoebaeareusedasindicatorsinavarietyofresearch fields(ecology, paleoecology, limnology,paleolimnology, paleo-climatology,peatland regeneration,soiland airpollution moni-toring and ecotoxicology) because they respond to biotic and abiotic factors by abundance, community composition or even shellmorphology[25,26].Despitethestrongpotentialoftestate amoebae and other soil protozoa as bioindicators in agro-ecosystemandnaturalecosystems(e.g.forsoilnutrientcontent, moisture, pH,various types of pollution, agricultural practices) [18,27],forensicapplicationoftestateamoebaanalysisiscurrently limitedtocorrelativeapproacheswherethesoilfromthecrime sceneiscomparedwithsoilattachedtoshoesfromsuspects[5,9]. Soil organisms, including testate amoebae, respond to spatial gradients[28]andtemporalchanges[29]inmicro-environmental conditionsandcanbeexpectedtorespondalsotothepresenceofa cadaver.Astheirgenerationtimesarerelativelyshort–typicallya fewdaysingoodconditions–compared totheinsectsthatare commonlyusedasindicators,thecommunitiescanbeexpectedto recoveronce thecadavers have decomposed completely,albeit withpossiblelongertermeffectsrelatedtolong-lastingchangesin soilchemistry.Fromtheseresponses,aforensicPMItoolcouldbe developed.However,inordertodevelopsuchatool,experimental work is required. We therefore conducted a field experiment aiming at assessing the spatial and temporal variation of soil testate amoeba assemblages in response to the presence and decomposition of cadavers in a beech and oak forest. We hypothesisedthattheirdensity,diversityandcommunity struc-turewould (1) bestrongly affected(i.e. theywoulddie and/or encyst)bytheeffectofdecomposingpigcadaversduringtheactive phaseofdecayand(2)wouldsubsequentlyslowlyrecoverover time.
2. Materialsandmethods 2.1. Fieldexperiment
Thestudyareawaslocatedinamixedbeechandoakforestnear the city of Neuchaˆtel, Switzerland (47800011.90–12.2600N/
685606.45–8.0500E, elevation 478m). Three ca. 25m2 sampling
siteswereselectedwithinaca.80m80mareafencedtokeep outdeerandallowtheregenerationofoaktrees.Inter-sitedistance rangedfrom15 to33m.Withineachsitethree90cm100cm surfaceswereselectedfor(1)acontrol,(2)afakecadaver(plastic bagsfilledwithsoilandcoveredwithacottoncloth)toinvestigate microclimatic effects without cadaveric fluids and (3) a pig cadaver, to investigatecombined effect of cadaveric fluids and microclimate.Theamount ofsoil usedtofill thefakecadavers correspondedtotheinitialweightofcadaversattheonsetofthe experimentandsoilwasgraduallyremovedateachsamplingday soastoapproximatelymatchthedecliningcadaverweightover
thecourseofdecomposition.Thisset-upallowedustoseparatethe influencesofthedecomposingcadaverfromthenormalseasonal changeintestateamoebacommunities(controlplots)andfrom the microclimatic effects+seasonal changes (fake cadavers). Withineachsite,thethreeplotswereatleastfourmetresdistant fromeachother.
Threepigs(SusscrofaLinnaeus)allfemalesand20kg(1kg) were killed with captive bolt stunning and were immediately delivered post-mortem to the study site. To enable sampling underneath thedecomposing cadaversandto preventthe distur-bancebylargescavengervertebrates,eachpigcadaverwaskeptina cage90cm100cm50cmbuiltofacompostframeandclosedat both ends withstrong wiremesh.The position ofthe cageswas markedwithastickateachcornertoensurethatthecagesarealways placedinthesamespot. Thecageswereliftedandplacednearby duringsamplingandplacedbackintheexactsameplaceafterwards. OneachsamplingdayfromAugust2009untilJune2010soil litterwassampled(downtothelitter-mineralsoilcontact)from thesurfaceareaofthecontrolplot,underneaththefakepigand fromthecontact area underneaththepigcadaver impactedby cadavericfluids.Thesamplesweretakeninatleastfiverandom pointswithineachsamplingareaateachsamplingtimeinorderto obtainrepresentativesamplesfromeachplotateachtime.Thefirst sampling took place at the start of the experiment (day 0, 05.08.2009)justbeforethedeadandfakepigswereputinplace. Thensamplesweretakenatdefinedintervals8,15,22,33,64,132 and309daysafterday0.Soiltemperaturewasrecordedeveryhour betweenAugust2009andJune2010usingthermologgers(HOBO Pendant1
temp/Alarm 64KUA-001-64),one pertreatment and replicate.Thedataloggerswereplacedattheinterfacebetween thelitterand themineralsoil.Precipitationdatawereobtained fromthelocalmeteoagency(www.meteosuisse.ch).
2.2. Laboratoryanalyses
Toextracttestateamoebaeeachsamplewascutwithscissors, mixed,and5gwasputintoaplasticflaskanddeionisedwaterwas added. The flaskwasclosed and shaken manually for approxi-mately3min.Thewaterwasthensievedthrougha160-
m
mmesh size in order toremove coarse particles. The filtrate was then sievedagainthrougha10-m
mmeshtoremoveclayandfinesilt particles.The10–160m
mfractionwascollectedandcentrifugedat 2500rpmfor10minandthesupernatantdiscarded.RoseBengale (50m
l; C.I. #45440, BBLTM, U.S.A.) was added to differentiate living fromdead cells (i.e. empty shells) [30] by staining. The sampleswereleftatroomtemperaturefor30mintocolourthe livingtestateamoebacells.Waterwasthenaddedupto50mlto removeexcessRoseBengaleandthesamplewascentrifugedagain. Thesupernatantwasdiscardedleavingapproximately5mlinthe tube. For fixation 1.5ml glutaraldehyde was added (2.5% final concentration). A Lycopodium spore tablet (batch no. 938934, 53394spores953pertablet,DepartmentofQuaternaryGeology, Lund, Sweden) was then added to allow the calculation of test concentration[31].Thetubewiththesporetabletwashomogenised for1minwithavortexandstoredovernighttoallowthetabletto completelydissolve.Slideswerepreparedbymixingtwodropsofthe preparationwithonedropofglycerol.Testate amoebae were identified to morpho-species and counted usinga lightmicroscopeat400magnification.Living, encysted, and dead individuals were tallied separately. When staining with Rose Bengale the living and encysted testate amoebae are coloured red and can be targeted. Dead testate amoebae(emptyshells)arenotcolouredandthereforecaneasily beseparatedfromlivingandencystedones.Acountof150testate amoebaewasaimedfor(totalofliving,encystedanddead),which isthenumberofindividualsthatmoststudiesusealthoughcounts
of100oreven 50wereshowntoalsobeinformative [32].The density of testate amoebae in the sample was calculated by multiplyingthetotalnumber oftestateamoebaeby53394(i.e. averagenumberofsporespertablet)dividedbythenumberof sporescounted.
2.3. Numericalanalyses
The temporal changes in soil testate amoeba assemblages duringdecompositionwereexaminedattwolevels.
First we used simple indices describing key aspects of biodiversity:Species richness (N0) and Simpson diversity (N2) asdefinedbyHill[33].HighN0indicatesalargenumberofspecies andhighN2alargenumberofspeciesevenlydistributed.Strong relationsbetweenN0andN2andenvironmentalconditionswere demonstratedatvariousspatialscales[34]andinabroadrangeof ecosystemssuchastropicalforest[35],marineecosystems[36] andfloodor fireprone ecosystems[28,37,38]. N0and N2 were calculated for each treatments and time step. The differences amongandwithintreatmentsinN0,N2,testateamoebadensity andcountdata wereanalysed usingTukey’s HonestSignificant Difference method (TukeyHSD) and p corrected for multiple comparisons.
Second,weinvestigatedthechangesinthespeciescomposition oftestateamoebacommunitiesusingPrincipalResponseCurves (PRC) [39]. PRC are a variant of the broadly used redundancy analysis [40] that was specifically designed for the analysis of multivariateresponsesinrepeated observation designas is the caseinthepresentstudy.PRCwereusedtoassesstheeffectofthe pigandcontroltreatmentsascomparedtothefakepigtreatment. Doingsoalloweddiscriminatingbetweeneffectsofcoveringthe soilsurface(fakepigversuscontroltreatments)fromtheeffectof thedecompositionprocess(fakepigversuspigtreatments).
Finally,thechangesinclimaticconditionswereassessedusing boththetemperature[8C] ofthesoilsincontrolplots (thermo-loggers)andthatmeasured 5cm abovegroundfromNeuchaˆtel meteostation(www.meteosuisse.ch)andprecipitation[mmh1]
tofurther discriminatetheeffect ofthe decompositionprocess fromthatofclimatictrendsthatmayinfluencealltreatments.In order to smooth the climatic trends moving averages were computedon 7daysandhourlymeasurementsfortemperature and precipitation respectively. All statistical analyses were conducted using the R software for statistical computing [41] andthe‘‘vegan’’packages[42].
3. Results
3.1. Temperatureandprecipitation
After a warmperiod inAugust witha meantemperatureof 19.63.88C(day0=startoftheexperiment:21.62.68C;day8: 19.12.98C;day15:22.63.98C)meantemperaturecontinually decreasedto15.43.98Conday33and15.50.98Conday64until December(day132:1.40.38C).Itthenincreasedagainandreached 18.93.38Conday309(Fig.1).AfterashortperiodofraininJuly 2009,afairlydryseason,meanprecipitationinAugust0.06mmh1
(0.44),withonlyshortrainintervalsfolloweduptoNovember2009 (day64).PrecipitationincreasedinNovemberandDecember.Thelast samplingday(309)tookplaceinJune2010afterarainyperiodinMay followedbylessraininJune(Fig.1).
3.2. Testateamoebataxaanddensity
Atotalof23testateamoebataxawereidentified,12Arcellinida (Amoebozoa)and 11 Euglyphida(Rhizaria) (Table1).Thethree most abundant taxa Centropyxis aerophila, Arcella arenaria and
Euglypharotundatogetheraccountedfor53.4%ofthecommunity onaverage(Table1andFig.2).
Testate amoeba density(alive, deadand encysted) averaged 3623individualspergramlitteroverallsamples.Living,encysted and deadamoebaeaccountedrespectivelyfor 32.2%, 18.0%and 49.8% inthecontrol,fakepigandpigcadaver samples.Density changed over time and among the three treatments (Fig. 3). Comparedtothebeginningoftheexperiment(day0andday8) densitydecreasedsignificantlyfromday8onwardsuptoday22in thepig treatment (TukeyHSD,p<0.01),whereas no significant differencewasobservedinthefakeandthecontroltreatment.In the pig samplesthe average density of living testate amoebae decreasedto21ind.g1atday15andto0ind.g1atdays22and
33.RecoverywasfirstobservedinOctober,twomonthsafterthe beginningoftheexperiment(i.e.betweendays33and64,Fig.3), butdensitywasstillsignificantlylowerthanatthestart(day0) (p=0.02). By contrast, the lowest densities of living testate amoebaerecordedonday64inthecontrol(207ind.g1)andon
day64inthefakepigplots(766ind.g1)werenotsignificantly
differentfromday0.Ninemonthslater,inJune2010(day309)the densityoflivingtestateamoebaehadincreasedagaininthecontrol (1725ind.g1) and reached its highest value in the fake
(8674ind.g1)(notsignificant).Bycontrastthedensityofliving
testateamoebaeinthepigtreatmentwasunsignificantlyloweron day309(64ind.g1)thanonbothday64(126ind.1)andday132
(319ind.g1)(Fig.3).
Assuming that encysted testate amoebae may excyst when conditionsimprovewe alsoshowtheresultsof theliving+ en-cystedvs.dead,hereafterL/Dratio(Fig.3).TheL/Dratiowas>1in thecontrolinthefirsttwoweeks(3samplingdays)aswellasin September and <1 at theend of August, in October and most clearlyinthewinter.Inthefakepigsamplesthepatternatthe beginningoftheexperimentwassimilarbutnotidenticaltothe control.Bycontrast,inthepigsamplestheL/Dratiowasonly>1at thebeginningoftheexperiment(day 0)and<1thereafterand untiltheendoftheexperiment.
3.3. Speciesrichnessandspeciesdiversity
Testateamoebaspeciesrichnessvariedinalltreatments(Fig.4). Inthecontrol(Fig.4)speciesrichnesswasonlysignificantlylower between day 8 and day 64 (TukeyHSD; p=0.03), and then increasedagain atdays 132 and 309but withoutreaching the valueofday0.Inthefaketreatment(Fig.4)speciesrichnesswas onlysignificantlylowerbetweenday15andday132(p=0.04)– increased until day 15, after that declined until day 132 and increasedagainonday309.Inthepigtreatment(Fig.4)species richnessdeclinedsignificantlybetweenday0today15(p=0.005), reachedthelowestpointondays22and33.Itthenincreasedat day64anduntilday132anddeclinedagainatday309(allnot significant),butremainedlowerthanonday0.
Simpsondiversity(N2)tendedtodeclineinallthreetreatments overthecourseoftheexperiment(Fig.4).Thiswasnotsignificant inthecontrolsamples.Inthefakesamplesthediversityonday132 and309wassignificantlylowerthanonday8,15and22(Fig.4).In thepigtreatmentdiversitydecreasedrapidlybeingsignificantly differentonday15,22and33comparedtothebeginningofthe experiment(day0)(Fig.4).Testateamoebacommunitiesstartedto recover from the cadaver-induced disturbance on day 64 (i.e. diversityincreasedagain),butneverreachedvaluescomparableto day0(Fig.4).
3.4. Speciesresponsetotreatments
The principal responsecurve analysis (PRC) shows how the testateamoebacommunitiesinthecontrolandthepigtreatment
variedover timein comparison tothefakepig,used hereas a reference(Fig.5).Thedifferencebetweencontrolandfakepigwas maximalatday64,whichcorrespondedtotheendofawarmand dryperiod(Fig.1).
Thisdifferencewasrelativelylowotherwise.Theeffectofthe pigtreatmentincreaseduptoday33andthendeclined,almost reachingthelevelofthecontrolonday64.Thedifferenceofboth pigandcontroltreatmentsandfakepigthendeclinedatday132. Thepigeffectincreasedagainatthelastsamplingdate(day309) (Fig.5).
4. Discussion
4.1. Testateamoebacommunitycomposition,speciesrichnessand density
Thetestateamoebaspeciesrichnessandcommunity composi-tionwefoundinthelittersamplesagreeswithpreviousstudiesin comparable habitats [15]. The dominant morpho-taxon in our study,Centropyxisaerophila,isalsooneofthemostabundantand frequenttestate amoeba taxon worldwide[43].Arcella arenaria andEuglypharotunda,thenextmostdominantmorpho-taxaare also very frequent globally. However it should be noted that thesemorphologicalspeciescertainly hidenumerous crypticor
pseudo-crypticspeciesthatmayhavemorerestricteddistribution orecologicalpreferences[44,45].
The density of testate amoebae was relatively low at the beginning of theexperiment (103–104ind.g1), but within the
rangeusuallyreportedinsoils[19].Thisrelativelylowdensityis probablyduetothefairlydryconditionsintheperiodpreceding the experiment (Fig. 1). Indeed testate amoeba density and community structurewerepreviouslyshown torespond tosoil moisturefluctuations[25,29,46–48].
4.2. Effectofdecomposingpigsonsoiltestateamoebae
Thisisthefirststudyassessingtheeffectofcadaversonsoil testateamoebae.Aspredicted, testateamoebaeresponded very clearlytothepresenceofdecomposingcadaversindensity,species richness, diversity and community structure. The strongest responsewasobserved22dayspostmortem(day22),bywhich timenolivingorencystedtestateamoebawaspresentunderthe pig.
Thechemicalcompositionofadomesticpigagedtwomonths is ca. 80% water, 26gkg1 nitrogen, 6.5gkg1 phosphorus,
2.9gkg1potassium,10gkg1calciumand0.4gkg1magnesium
and aC/Nratioof7.7 [49].Ascadavericfluidsaddammonium, calcium, chloride, magnesium, nitrogen, potassium, sodium, sulphate andvolatilefattyacids totheunderlyingground [50],
Fig.1.ClimaticdatameasuredinNeuchaˆtelandattheexperimentalsitefromAugust2009untilJune2010.Temperaturedata(leftaxis)are7daysrunningaveragesofairand soiltemperature;dottedlines:Neuchaˆtelmeteorologicalstation;plainlines:datafromtemperatureloggersplacedinthelitter/soilinterfaceintheexperimentalplots.Grey field:precipitationcorrespondsto7daysrunningaverageofhourlyprecipitation(rightaxis).Verticallinesindicatetimepointsforsampling0,8,15,22,33,64,132and309 daysafterthebeginningoftheexperiment.
thecadaveric fluids strongly modifythe soil environment. Our resultsshowthat testateamoebaeclearly donot toleratethese changes.
Although we cannot compare our results with any other forensicstudy,severalexperimentshaveassessedtheeffectsof soil chemistry on testate amoebae in Sphagnum and other mosses.Depositionofsulphate[21],nitrogen[51],nitrogenand phosphorus[52],PKCaandNPKCa[53],lead[54],andexposure tourbanpollution[55]significantlyreducedthetestateamoeba density(and, wherereported, also species richness). Sulphate depositionalso affectedthe abundance of some species, with positiveeffectrecordedforHyalospheniapapilio,Arcellaarenaria andCryptodifflugia oviformis and negative effects for Euglypha rotunda type, Corythion dubium, Trinema complanatum and Trinema lineare. [21]. Nitrogen and phosphorus addition de-creasedthedensityofAssulinamuscorumandDifflugiaoviformis [52],whilenitrogenadditionincreasedthedensityofBullinularia indica[52,56].
Changes in testate amoeba density in the control samples paralleledthechangesinclimaticconditions.Indeed,theverylow soilmoisturecontentresultingfromthewarmanddryperiodin AugustandSeptember(day64;Fig.1)inducedthelowestobserved densityoftestateamoebae.Bycontrast,environmentalconditions didnotinduceasimilardecreaseunderneaththefakepigwhereit waslessstrong thaninthecontrol,mostlikely becausethebag usedtosimulatethepresenceofafakepigreducedevaporation andthusmaintainedasomewhathighersoilmoisturecontent,as we indeed noticed during sampling. This hypothesis is further supportedbytheoverallhigheraveragedensityoftestateamoebae inthefake(5388ind.g1)ascomparedtothecontroltreatment
(3926ind.g1). This interpretation agrees with experimental
evidence for increased testate amoeba density in response to wateradditioninarelativelydryaspenforest[29].
Given thesepatterns,we comparedthe communitychanges underthepigandinthecontrolplotstothefaketreatmentinthe PrincipalResponseCurveanalysis.Thisallowedustoshow(1)the
Table1
Relativeabundance(%totaltests)andaveragespeciesdensity(numberofalive,deadandencystedindividualspergramlitter)oftestateamoebaeinall72samplesinthe3 treatments(control,fakepigandpigcadaver,3replicates,8samplingdays).
Testateamoebataxa Speciescode Phylogeneticgroup Relativeabundance(%) Averagespeciesdensity(ind.g1)
Control Fake Pig CentropyxisaerophilaDeflandre1929 CENTae Arcellinida 22.5 304.449.9 1020.3501.8 87.129.7 ArcellaarenariaGreef1866 ARCar Arcellinida 16.0 285.398.2 18641.1 77.237.8 EuglypharotundaWailes1911 EUGrot Euglyphida 14.9 84.539.1 8721.8 10.15.8 CentropyxissylvaticaDeflandre1929 CENTsy Arcellinida 7.0 9218.7 228.862.4 86.439.3 EuglyphastrigosaEhrenberg1872 EUGstri Euglyphida 6.2 101.836.6 107.530.3 46.224.7 CorythiumdubiumTara`nek1881 CORdu Euglyphida 5.4 91.923.9 94.930.6 27.920.9 AssulinamuscorumGreef1866 ASSmu Euglyphida 4.6 9033.6 231130.6 63.631.6 CyclopyxiskahliDeflandre1929 CYCka Arcellinida 4.0 51.515.6 158.3103.6 26.115 EuglyphacompressaCarter1864 EUGcom Euglyphida 3.4 54.920.1 5521.2 32.415 TrinemaenchelysEhrenberg1838 TRIenc Euglyphida 2.9 34.512.4 43.914 16.27.8 EuglyphaciliataEhrenberg1848 EUGcil Euglyphida 2.0 51.327 62.827.7 44.527 TrinemapenardiThomasandChardez1958 TRIpen Euglyphida 1.9 2112.3 21.311 12.39.8 TrinemalinearePenard1890 TRIlin Euglyphida 1.6 00 4.54.5 7.15.6 PadaungiellalageniformisPenard1890 PADlag Arcellinida 1.2 00 00 3.83.8 NebelacollarisEhrenberg1848sensu
Kosakyanetal.,2013
NEBcol Arcellinida 1.2 2.92.9 00 1.21.2 CentropyxiselongataPenard1890 CENTel Arcellinida 1.2 11.57.9 27.214.2 00 EuglyphacristataLeidy1879 EUGcris Euglyphida 1.0 9.97.8 11.68 00 TrinemacomplanatumPenard1890 TRIcom Euglyphida 0.8 4.14.1 6.14.5 4.14.1 PorosiabigibbosaPenard1890 PORbig Arcellinida 0.7 00 4.54.5 1.71.7 NebelatinctaDeflandre1936 NEBtin Arcellinida 0.6 1212 1.31.3 00 DifflugialucidaPenard1890 DIFFlu Arcellinida 0.6 3.73.7 6.85.1 00 TrigonopyxisarculaLeidy1879 TRIGarc Arcellinida 0.2 00 00 00 PlagiopyxisdeclivisBonnetetThomas1955 PLAdec Arcellinida 0.1 7.55.3 4.64.6 4.64.6
Fig.2.IllustrationofthethreemostabundanttestateamoebaspeciesinthisexperimentCentropyxisaerophila(left)Arcellaarenaria(middle)andEuglypharotunda(right). PicturesfromtheLaboratoryofSoilBiology,Neuchaˆtel,Switzerland.
effectofdifferencesinmicroclimaticconditionsbetweenthefake pig and the control samples,and (2) the effect of the pig but controllingforthesemicroclimaticeffects.Theresultsclearlyshow thatmicroclimaticconditionshad animpactontestateamoeba communities. During the course of our experiment climatic conditions were quite contrasted, with extensive dry periods andmorepredictableseasonalchangesintemperature(Fig.1).The testateamoebacommunitiesclearlyrespondedtothesepatterns inthecontroltreatment.Forexamplethedryperiodbetweendays 33and64causedachangeincommunitystructure.Nevertheless, theeffectofthedecomposingcadaverwasmuchstrongerthanthat of climatic conditions (Fig. 5). Although the effect of the pig treatment decreased over time, it remained strong during the wholedurationoftheexperimentandevenincreasedagainafter 132daysincontrasttotheeffectofclimaticconditionsthatwas
almostnullfromday132onward,inlinewiththeremovalofsoil fromtheplasticbagsusedtomimicthecadavers.
Testate amoebae are believed to be transported over long distancesmostlypassively(e.g.bywind)[57,58],buttransportby phoresy(passivetransportbyanimals),althoughnotstudiedfor testate amoebae,is well documented for other protists such as diatomstransported bybirds[59].Overshortdistances(cm-m), active migration is probably the main colonisation mechanism. Passivemigrationallowspotentiallyanyspeciestoreachagiven pointbutthesurvivalofindividualamoebaeandthebuild-upof measureablepopulationswilldependon localconditions. Active dispersalbycontrastwillonlytakeplaceifconditionsarefavourable. Wethereforeconsiderthatrecolonisationofperturbedhabitatssuch ascadaverdecomposingsitesreflectsmainlyactivedispersalatthe scaleofacadaverdecompositionsitefromthesurroundingsoil.
Fig.3.PatternsoftestateamoebadensityinresponsetoapigdecompositionexperimentinadeciduousforestinNeuchaˆtel,Switzerland.Density(a–c)(numberofindividuals pergramlitter)andliving(activeandencysted)todeadratio(greyarea,leftscale)andpercentage(rightscale)(d–f)ofliving(whitebars/plainline),encysted(greybars/short dashedline)anddead(blackbars/dashedline)testateamoebaefromcontrol(aandd),fakepig(bande)andpigcadaver(candf)treatment.
Fig.4.Temporalpatternsofalivetestateamoebaspeciesrichness(top)anddiversity(Simpson’sN2index)(bottom)inthethreetreatments(control,fakepig,pigcadaver)ofa pigdecompositionexperimentinadeciduousforestinNeuchaˆtel,Switzerland.Differentletters(aandb)indicatesignificantdifferencesamongsamplingdays.
Fig.5.Principalresponsecurve(PRC)diagramshowingthedeviationsofthepigcadavertreatment(dashedline)andthecontrol(baresoil)treatment(plainline)fromthe fakepigtreatmentusedasreference(horizontalgreyline)overtime.Theleftaxisshowsthetreatmenteffect(i.e.regressioncoefficient).Speciesscoresareshownontheright verticalaxis.HighvaluesindicatethattheresponseofthespeciesisstronglypositivelycorrelatedtothepatterninthePRC;lowvaluestheoppositeandvaluesclosetozero showthatthespeciesresponseandPRCpatternareun-related.
Forforensicpurposesitshallbehighlyinterestingtolookatthe testate amoeba community and at what time the population recoverstotallyandwhetherthisfollowsasuccessionpattern.As nutrient levels can remain high up to several years in soil influenced by cadavers [60–62], the influence of long-gone cadavers on soil communities can also be expected to remain visible for over one year, as indeed suggested by our results. Longer-termexperimentsarerequiredtostudyinfurtherdetail there-colonisationpatterns(whichspeciesre-colonisefirst,how dothesepatternsrelatetofood sourcessuchasbacteria,fungi, otherprotists,etc.)andresultsfromsuchstudiescouldpotentially leadtodevelopingatoolforextendedPMI.
Morphologicaltraitanalysiscouldalsopotentiallybeusefulto develop suchan index. Indeed as testate amoeba taxonomy is currently not satisfactory [45] species traits such as shell morphologyandbiovolumecan beused.Suchanapproach has onlybeenusedoncefortestateamoebae[28].Inthecontextof peatlandregeneration it hasbeen suggestedthat largertestate amoebaemightbeslowertore-colonisesecondaryhabitatsowing to(1) lower population densities, and (2) lower probability to travelpassivelyoverlongdistances[63].Wethereforeexpectthat longer-term studies combined with trait analysis of testate amoeba communities will allow toreconstructing thestage of activedecayandthepost-morteminterval.Ratherthanidentifying all taxa to species level simple morphological traitapproaches might prove sufficiently robust and would also allow forensic scientiststobecometrainedforsuchananalysis.Alternativelyor inadditionenvironmentalDNAapproachescouldbedeveloped.
Inthisstudywehaveshownthattestateamoebaewereclearly affectedbydecomposingcadaversincomparisontocontrolsand fakecadavers.Giventhestrengthofthecadavereffect(nolivingor encystedtestateamoebae22dayspostmortem)weexpectthat theresponsewillbesimilarina broadrangeofconditions (i.e. different forest, litter or soil types). Nevertheless it would be interestingtocomparepatternsacrossecosystemstypes,under differentclimatesorseasons.Furtherstudiesarethereforeneeded toestablishtestateamoeba analysisas anapproachin forensic sciencetoestimatethepost-mortemintervalandtoestablish user-friendlymethods.
Acknowledgements
ThisworkwasfundedbytheSwissNationalScienceFoundation (projectno.31003A_141188toEM),theUniversityofNeuchaˆtel, SwitzerlandandtheStiftungForensischesForumFrankfurt/Main, GermanyandtheVereinigungvonFreunden undFo¨rderernder Goethe-Universita¨t,Frankfurt/Main,Germany.WethankNicolas Derungs,Ce´cileGenetti,andEnriqueLaraforhelpinfieldandlab work.
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