Effects of methamphetamine and its primary human metabolite, p-hydroxymethamphetamine, on the development of the Australian blowﬂy Calliphora stygia

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CHAIRMAN FORENSIC SCIENCE INTERNATIONAL: P. Saukko (Turku, Finland)

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Content

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Dadour

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Effects of methamphetamine and its primary

human metabolite,

p-

hydroxymethamphetamine,

on the development of the Australian

blowfly

Calliphora stygia

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Utility of urinary ethyl glucuronide analysis in

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alcohol-related deaths

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DNA evidence: Current perspective and future

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importance of this species in future cases

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

Post-

o te β

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synovial fluid

Cristian Palmiere, Dominique Werner

e28

–

e30



E atu to sa pli g of illi it d ugs fo ua titative

analysis

–

Part II. Study of particle size and its

i flue e o ass edu tio (Fo e si S ie e

International 234C (2014) 174

–

180)

M. Bovens, T. Csesztregi, A. Franc, J. Nagy, L. Dujourdy

p221

Published online: June 11, 2014



Inside Front Cover- Editorial Board

IFC

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Effects

of

methamphetamine

and

its

primary

human

metabolite,

p-

hydroxymethamphetamine,

on

the

development

of

the

Australian

blow

ﬂ

y

Calliphora

stygia

Christina

Mullany

a

,

Paul

A.

Keller

b

,

Ari

S.

Nugraha

b

,

James

F.

Wallman

a,

*

a_Institute_for_Conservation_Biology_and_{Environmental}_Management,_School_of_Biological_Sciences,_University_of_Wollongong,_NSW_2522,_Australia b_School_of_Chemistry,_University_of_Wollongong,_NSW_2522,_Australia

ARTICLE INFO

Articlehistory:

Received22April2013

Receivedinrevisedform6April2014

Accepted8May2014

Availableonline20May2014

Keywords:

Forensicentomology

Entomotoxicology Calliphorastygia

Insectdevelopment

Methamphetamine

Postmorteminterval

ABSTRACT

Thelarvaeofnecrophagousflyspeciesareusedasforensictoolsforthedeterminationoftheminimum postmorteminterval(PMI).However,anyingesteddrugsincorpsesmayaffectlarvaldevelopment,thus leading to incorrect estimates of the periodof infestation. This study investigated the effects of methamphetamine and itsmetabolite, p-hydroxymethamphetamine, on theforensically important AustralianblowflyCalliphorastygia.Itwasfoundthatthepresenceofthedrugssignificantlyaccelerated larvalgrowthandincreasedthesizeofalllifestages.Furthermore,drug-exposedsamplesremainedas pupaeforupto78hlongerthancontrols.ThesefindingssuggestthatestimatesoftheminimumPMIof methamphetamine-dosedcorpsescouldbeincorrectifthealteredgrowthofC.stygiaisnotconsidered. Differenttemperatures,drugconcentrationsandsubstratetypesarealsolikelytoaffectthedevelopment ofthisblowfly.Pendingfurtherresearch,theapplicationofC.stygiatotheentomologicalanalysisof methamphetamine-relatedfatalitiesshouldbeappropriatelyqualified.

ã₂₀₁₄_Elsevier_Ireland_Ltd._All_rights_reserved.

1.Introduction

Analysis of entomologicalevidence can greatlyenhance the scopeofdeathinvestigationswheresuchevidenceispresent[1].A primaryobjectiveofforensicexaminationsuponﬁndingacorpseis thedetermination of theminimumpostmortem interval (PMI). Thisisoftenmadebystudyinganyinfestinginsects[2],especially

flies (Diptera),and involves an understandingof the biological characteristicsofthespecies present incarrion,including their development[3]. By determiningtheage ofthese insects, it is possibletodeterminetheirtime ofcolonisation,and hence,the minimum PMI [2]. Although the developmental rates of many forensically important species are known [4,5], bioclimatic influenceshavebeenshowntoaffectgrowthratesofflyspecies

[6,7].These factorsincludetemperature,atmospheric humidity, larvaldensityandbodylocation.Anotableenvironmentaleffectof forensicimportanceisthepresenceofdrugsinacorpseandtheir subsequenteffectsoninsectphysiology.Thisisknownasforensic entomotoxicologyandfailuretoconsidersuchfactorsmayleadto errorsinminimumPMIestimates[8–10].Preliminarystudieshave

shownthatillicitdrugsinfluencethedevelopmentofvariousfly species [11–15]. In particular, the larvae of some species have shownaccelerated growthupon exposuretostimulants[12,16]. However, few investigations have been undertaken into the physiologicaleffectsofdrugsofabuseonAustralianflies.Ifthese endemicspeciesaretobesuccessfullyusedtocorrectlyestimate minimum PMI, it is imperative to understand the effect of antemortem drug use and abuse on the growth rate in the postmortemperiod[17].

Theworldwideabundanceandsocialpopularityof metham-phetamine make it a prime candidate for entomotoxicological analysis.Thepotentialforaddictionoroverdoseforﬁrst-timeusers isoneofthehighestofallavailabledrugsduetoitsextremeand enduringphysiologicaleffects[18–20].Inpreviousexperiments, wholeanimalshavebeenusedtosimulatethepharmacokineticsof methamphetamineinhumans[12,20–22],however,thereisstill somedebateastowhetherthisisoptimalasithasbeenreported that drugmetabolism is species-speciﬁc[23]. Furthermore,the metabolicbreakdownandexcretion ratesof methamphetamine are also species-dependent. When compared to laboratory rodents,methamphetamineismetabolised ataslower rateand lesseffectivelyinhumans[23].Asaresult,sustained administra-tion of drugs is required to achieve plasma, urine or tissue concentrations comparable to humans, which in turn produce

*Correspondingauthor.Tel.:+61242214911;fax:+61242214135.

E-mailaddress:jwallman@uow.edu.au(J.F. Wallman).

http://dx.doi.org/10.1016/j.forsciint.2014.05.003

0379-0738/ã2014ElsevierIrelandLtd.Allrightsreserved.

ForensicScienceInternational241(2014)102–111

ContentslistsavailableatScienceDirect

Forensic

Science

International

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moremetabolites.Asallofthesemetaboliteshavethepotentialto affectlarvalgrowth, theeffects of metabolitesof interestcould easilybeoverrated.Theprimarymetabolicproductof metham-phetamine degradation in humans is p- hydroxymethamphet-amine,whichaccountsfor15–50%ofallmetabolitesexcretedin urine [23]. It is itself a potent hallucinogen, and contributes signiﬁcantlytothehalf-lifeofmethamphetamine[18].However,in non-humananimals,theprimarymetabolicproductsof metham-phetaminebreakdownhavebeenrecordedasp -hydroxynorephe-drineandp-hydroxymethamphetamine(rats),norephedrineand benzoicacidconjugates(guineapigs),andamphetamine(rabbits)

[12,23].

Withtheexceptionofamphetamine[24],nopublishedstudies haveinvestigatedtheeffectsofthesecompoundsonthegrowth ratesofblow_ﬂyspecies.Ascertainmetabolitesarealwayspresent in overdose victims [20], necrophagous insects feeding on a cadaverwillingestthem.Theeffectoftheseproductsoninsect metabolicactionneedstobesubstantiatedinordertoprovidea validminimumPMIestimatefordrug-inducedfatalities.

Theeasterngoldenhairedblowfly,Calliphorastygia(Fabricius) (Diptera:Calliphoridae),isamongthemostforensicallyimportant speciesineasternAustralia[25].Itisoneofthefirstspeciespresent at a corpse,usually arriving within hoursto oviposit.Previous studiesontheeffects ofdrugs onthis specieshavefocused on morphine [26,27], with no investigation having been done on stimulantsortheirmetabolites.Wereporthereforthefirsttime the effects of methamphetamine and p -hydroxymethamphet-amineonthegrowthrateoftheblow_flyC.stygia,withtheaimof determiningthesuitabilityofthisspeciesasamodelforestimates ofminimumPMIinscenariosinwhichthecorpseiscontaminated withtheseillicitdrugs.

2.Materialsandmethods

Methamphetamine and its primary human metabolite, p-hydroxymethamphetamine, wereappliedto thefood sourceof C.stygialarvaetosimulatepostmortemconditionsin metham-phetamine overdosevictims. Eggs werecollected fromblowﬂy cultures and assigned randomly to one of ten groups (nine treatments and a control). Calliphora stygia specimens were cultured through a minimum of one generation (maximum of

ﬁve generations) from pupae obtained from Sheldon’s Bait (Parawa,Australia). Flieswere keptin plastic cages withmesh lidsat 23_C, _and _exposed_to_a _photoperiod_of _12:12_light:dark. Waterandgranulatedsugarwereprovidedadlibitum.

Sheep'sliverwascutinto3cm3_pieces_and_supplied_to_cultures

tofacilitateovarianmaturationinfemale_ﬂies.Thecultures,once sufﬁcientlymatured,werepresentedwithfreshlivercoveredwith athinlayerofcottonwooltoencourageoviposition.Cageswere checkedeverytwohoursandeggstransferredtoaPetridish.Eggs werecountedinto90groupsof150each.Tenexperimentalgroups wereestablishedforfeedinglarvae:(1)control,no methamphet-amine (MA) or p-hydroxymethamphetamine (p-OHMA); (2) 0.1mg/kg methamphetamine (0.1 MA); (3) 1.0mg/kg metham-phetamine(1.0MA);(4)10mg/kgmethamphetamine(10MA);(5) 0.1mg/kg methamphetamine:0.015mg/kg p -hydroxymetham-phetamine(0.1MA:0.015p-OHMA);(6)1.0mg/kg methamphet-amine:0.15mg/kg p-hydroxymethamphetamine (1.0 MA:0.15 p-OHMA); (7) 10mg/kg methamphetamine:1.5mg/kg p -hydroxy-methamphetamine (10 MA:1.5 p-OHMA); (8) 0.015mg/kg p -hydroxymethamphetamine (0.015 p-OHMA); (9) 0.15mg/kg p -hydroxymethamphetamine(0.15p-OHMA);and(10)1.5mg/kg p-hydroxymethamphetamine(1.5p-OHMA).

The control batch containing no methamphetamine was prepared by mixing 15mL of distilled water into 1.5kg of kangaroo mince. Treatments of 10mg/kg,1mg/kg and 0.1mg/

kgmethamphetaminewerepreparedbydissolving15mg,1.5mg and 0.15mg, respectively,of ()-methamphetamine hydrochlo-ridein15mLdistilledwaterandmixinginto1.5kgofkangaroo mince.p-Hydroxymethamphetamineconcentrationsof1.5mg/kg, 0.15mg/kgand0.015mg/kgwerepreparedbydissolving2.25mg, 0.225mgand0.0225mgofp-hydroxymethamphetamine, respec-tively, in 15mL of distilled water and mixing into 1.5kg of kangaroomince.Concentrationsofeachratioofdrug:metabolite were prepared bymixing 2.25mg, 0.225mg, and 0.0225mg of p-hydroxymethamphetaminewith1.5mg,0.15mgand0.015mgof methamphetamine,respectively, in15mLofdistilledwater and 1.5kgofkangaroomincetosimulatethreepostmortemratiosof drugand metaboliteconcentrations.These concentrationswere deemedsuitableforinvestigationsbasedoncommonlyreported self-administereddosesof()-methamphetaminehydrochloride thathaveconsequentlyledtotoxicbodilyconcentrationsofthe druganddeathofmethamphetamineusers[18,28–30].

Each treatment was mixed byhand for 10min,followed by 5minofmixingwithablendertoensureanevendistributionof drug,liquidandmeat,orliquidandmeatonlyforcontrolbatches. Toavoidcontamination,newglovesandmixingcontainerswere usedforeachtreatment,andtheblenderheadrinsedinethanol andﬂushedwithnearboilingwaterfor10minbetweenuses.Meat batchesweresplitinto150gportionsandplacedintoplasticweigh boats.Batcheswerestoredat20_C,_and_meat_portions_defrosted asrequiredtoreplenishthelarvalfoodsource.

()-Methamphetamine hydrochloride (99.71.3%) and p-hydroxyamphetaminehydrochloride(p-OHAM)(99.71.3%)were obtainedunderlicensefromtheNationalMeasurementInstitute (NSW,Australia).p-Hydroxyamphetaminehydrochloridewas con-vertedtotheprimaryhumanmetaboliteofmethamphetamine, p-hydroxymethamphetaminehydrochloride,priortoinsectstudies.

Asolutionofp-hydroxyamphetaminehydrochloride(8.49mg) inMilli-Qwater(1mL)wasadjustedtopH12bythedrop-wise addition of sodium hydroxide (10%). The solution was then extracted with CH2Cl2 (210mL), and the combined organic

layers concentrated to give p-hydroxyamphetamine (3.4mg), which was used without further puriﬁcation. Boc2O (5.4mg)

wasaddedtoasolutionofp-hydroxyamphetamine(3.4mg)inTHF (0.004mL)andEt3N(2.7mg)andthereactionstirredatRTunder

N2 for 24h. CH2Cl2 (2mL) was then added and the mixture

sonicated and the organic layer extracted. This process was repeatedtwiceandthecombinedorganiclayersdried(MgSO4)and

concentratedtogivep-OHAM-Boc(6.3mg,80%yield).Con_ﬁ rma-tionofthesuccessofthechemicaltransformationcamefrommass spectrometricanalysis,withtheESI-MSspectrumshowingapeak at m/z374 (M+H+_), _assigned _to_the_protonated _mass _of _the

p -OHAM-Boc.

Asolutionofp-OHAMP-diBoc(14.3mg)indryTHF(0.5mL)was addeddrop-wiseover5mintoamixtureofNaH(1.45mg,2.42mg from60%NaHstock,1.5eq)indryTHF(1mL)ina5mLﬂaskunder N2gasandat0C.MeI(0.08mL,17.33mg,3eq)wasthenadded

drop-wiseintothemixture,whichwasstirredfor48hatRT,and thereactionmonitoredbyESIMS.Thereactionwasquenchedwith water (2mL), and the reaction extracted with CH2Cl2, the

combined organic layers dried (MgSO4)and then concentrated

anddriedundervacuumtoobtainN -methyl-4-hydroxymetham-phetamine(14.2mg,96%)asanUVactivepaleyellowpowder;1_H

NMR(CDCl3,500MHz),7.14(d,3J=7.3Hz,2H,H20,H40),6.84(d,3J

=7.3Hz,2H,H30_and_H50_),_3.78_(s,_3H,_N_CH

3),3.39(d,2J=15.1Hz,

H1A),3.33(m,1H,H2),2.80(d,2J=15.1Hz,H1B),1.33(m,3H,H3); 13_C_NMR_CDCl

3,125MHz),158.8(C40),130.4(C20,C60),127.8(C10),

114.3 (C30, C50), 57.4 (C2), 55.3 (NCH

3), 38.6 (C1), 15.6 (C3);

ESIMS,m/z166(M+H+_).

Each subset of 150 eggs, collected as above, was placed immediatelyontoasmallpieceofcottonwoolandallocatedto

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oneof the tengroups (ninereplicatesof eachgroup in total). Weighboats containing spiked meatwereplaced into 850mL rectangular plastic containers with mesh lids, lined with a shallowlayer(5mmdeep)ofwheatenchaff.Thechaffprovideda medium for the larvae to crawl into when they had _finished feeding. All larvae were reared at 23_C _in _a temperature-controlled incubator (Thermoline Scientific, Australia) and exposedtoaphotoperiodof12:12light:dark.Thedayonwhich eggs were laid, collected and distributed to an experimental groupwasdesignatedasday0.Thesamplingdesignofourstudy followed that of George et al. [27], in order to facilitate comparisonwiththeprevioustoxicologicalworkonthisblowfly. Sampleswerethereforecomparedatfourtimepoints.Thefirst two comparison stages took place on days 4 and 7 of experimentation, during the larval stage. Three replicates of eachgroupwereremovedattheday4and7comparisoninterval. Larvae were extracted from the meat and starved for 4h to encourage expulsionof thecontents of the crop.Larvae were killed and fixed by placing in boiling water for 60s and individually washed in near-boiling water for 30s to ensure thatalladhesivesubstratewasremoved.Larvaewerethenstored in80%ethanol,afterwhichtheirlength,widthandweightwere measured.The posteriorspiracles of eachspecimenwere also inspected to record developmental instar. To determine the persistence of methamphetamine and p- hydroxymethamphet-amineinthelarvalfoodsource,1gsamplesof kangaroomince were taken from each group for later analysis using high performanceliquidchromatography.

The third comparison, incorporating a third set of three replicates perexperimental group, was madeduring the pupal stage.Observationsweremadeevery4htoaccuratelyrecordthe dayandhour(wheresuitable)atwhichpupariation(theprocessof puparium formation) began and concluded in each replicate container.Commencementofpupariationwasrecordedatthe_ﬁrst indicationofcolourchangeoftheprepupafromwhitetoorange. Conclusion of pupariation was denoted by all samples having progressedfromorange todark brown, whereuponthe length, width and weight of all samples were measured. Following measurement,pupaewerereturnedtotheiroriginalcontainersfor eclosion.

The fourthcomparison was madeonceadults had emerged. Observations were made every 4h to record as accurately as possible when emergence began and concluded. The day and averagehourofinitialeclosionwererecorded,aswerethedayand hourofaverageeclosion(P50value).Adultswereremovedfrom plasticcontainersandplacedinafreezerfor5mintoslowtheir movement. Flies were then killed by asphyxiation with ethyl

acetateandstoredin80%ethanol.Measurementsofadultweight weremadewithin4hofcollection.Theleftwingandrearleftleg werethenremovedforlateranalysis.

Larvaeandpupaewereviewedunderadissectingmicroscope (MZ16A,LeicaMicrosystems,Germany).A_fibreopticlightsource (CLS150X,LeicaMicrosystems,Germany)wasusedtoilluminate samples on a contrasting background to assist analysis. Each specimenwasphotographedwithadigitalcamera(DFC259,Leica Microsystems, Germany) and Leica Application Suite V3.8 software(LeicaMicrosystems, Germany)employed tomeasure length andwidth parametersto thenearest 0.001mm. Larvae wereviewedlaterally,andtheirlengthsmeasuredbetweenthe mostdistalpointoftheheadandthemostposteriorabdominal segment (Fig. 1(a)). Larval width was measured across the intersectionofthe_fifthandsixthabdominalsegments. Ultrasen-sitivescales(ML204,MettlerToledo,Switzerland)andLabXdirect balance2.1software(MettlerToledo,USA)wereutilisedtorecord the weightsof each sampleto the closest 0.1mg. Pupae were viewedventrallyandtheirlengthsmeasuredbetweenthemost posterior to most anterior points. Width measurements were obtainedbymeasuringsamplesacrosstheintersectionofthe_first andsecondabdominalsegments(Fig.1(b)).Adultswereremoved fromethanolandallowedtodryfor10minbeforebeingweighed. Wing and leg samples were viewed under the dissecting microscopeand photographed. For each sample, thelength of thecosta(oneoftheperipheralwingveins)andtibia(oneofthe sectionsoftheleg)weremeasuredtogiveanindicationofadult size and to determine if any differences existed between treatments due to drug exposure during earlier life stages (Fig.1(c,d)).

Following measurement, maggots, puparia and adults were individuallygroundusingamortarandpestleandcombinedwith 800

mL

ofdistilledwater(maggotsandadultswereallowedtodry for24hpriortohomogenisation).Cellulardebriswasremovedby precipitationinmethanol(AjaxFinechem,Australia)and centri-fugation(Model5412D, EppendorfSouthPacific,Australia). The solution was run through _filter paper to ensure that all large contaminantswereremoved.Smallercontaminantswereremoved by HPLC specific 4mm syringe filters with 0.45

mm

polytetra-ﬂuoroethylenemembranes.Sampledmeatwasgroundinamortar andpestleandcombinedwith1000

mL

ofdistilledwater.Cellular debriswasremovedbyprecipitationinmethanoland centrifuga-tionandﬁlteredasforlarvae,puparialandadultsamples.Aslittle isknownaboutthepharmacokineticsofdrugsofabuseingestedby insects, two standard solutions of methamphetamine were preparedbeforeanyanalysisofsampleswasattemptedinorder todeterminewhichformofmethamphetamine,ifany,waspresent

Fig.1.(a)LarvaeofCalliphorastygia,showinglengthandwidthmeasurements;(b)C.stygiapupa,showinglengthandwidthmeasurements;(c)leftwingofC.stygia,showing

costallengthmeasurement;(d)rearleftlegofC.stygia,showingtibiallengthmeasurement.

[image:13.595.113.482.564.720.2]

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inC.stygiasamples.Thefirstwaspreparedbydissolving7mgof ()-methamphetaminehydrochloride in5mLof distilledwater, thus leaving the hydrochloride salt intact. The second was preparedbyneutralising7mgof ()-methamphetamine hydro-chloridewithsodiumhydroxide.Thesolutionwasthen _filtered with an HPLC specific syringe filter, and mixed with 5mL methanol.Standardsforp-hydroxymethamphetamine hydrochlo-ridewerepreparedinthesamemanner.Thepresenceorabsenceof methamphetamine was confirmed by high performance liquid chromatography(HPLC)underisocraticconditions.Solventswere acetonitrile(190grade)andwaterandethylamine.Solventswere sonicated in an ultrasonic cleaner (Ultrasonics, Australia) for 20minpriortouse.AnalytesweredetectedbyUVlightat254nm. Thepresenceorabsenceofmethamphetamineorp- hydroxyme-thamphetaminewasrecordedforeachsample.

StatisticalanalysiswasundertakenusingJMPv7forWindows (SAS,USA).NormalityofthedatawasevaluatedusingQ–Qplotsand Kolmogorov–Smirnov normalitytests.Measuredsample param-eters (length, width and weight) were analysed using nested ANOVAs,whichallowedidentificationofsignificancebothbetween groups,and betweenthereplicates ofgroups.Survivorship was investigatedatthelarvaland adultcomparison stages. Kruskal-Wallistestswereusedtodeterminewhethertherehadbeenany appreciablelossinsamplenumbersasaresultoftheirexposureto drugcompounds.Significantresultswerefurtherexaminedwitha Tukey–Kramertesttoassesswhereanydifferenceslay.

3.Results

HPLCchromatogramsqualitativelydeterminedtheabsenceof methamphetaminecompoundsinthecontrolmeat.Thepresence ofmethamphetamineand/orp-hydroxymethamphetamineinthe treatment groups was conﬁrmed by HPLC–UV analysis. These compoundswouldthereforehavebeeningestedbyfeedinglarvae. Developmentratesoflarvaeweredeterminedbyincreasesin thelength and width of sampled specimens. Weight measure-mentswerealsotakentoidentifyanyunusualgrowthpatterns.No obviousdifferencesinaveragetemperaturewerenotedbetween groupsorreplicates.

3.1.Comparisonofday4larvae

AnestedANOVAofmeanlarvallengthafterfourdaysofgrowth identi_ﬁed signi_ﬁcant differences between treatment types (F9, 3485=40.75,p< 0.0001)(Fig.2(a)).

AposthocTukey–Kramertestdeterminedthatthemeanlengths ofeachofthepuremethamphetaminetreatments(0.1MA,1.0MA and10MA)weresignificantly greaterthanthecontrolgroup,aswas themeanlengthoflarvaeexposedtothetwolowerconcentrations ofpuremetabolite(0.015p-OHMAand0.15p-OHMA).Similarly,the twolowermethamphetamine:metaboliteratios(0.01MA:0.015 p-OHMAand1.0MA:0.15p-OHMA)producedlarvaeofsignificantly greaterlengththanthecontrol.Thetreatmentwiththehighest concentrationofp-hydroxymethamphetamine(1.5p-OHMA)and theintermediateratioofmethamphetamine:p- hydroxymetham-phetamine(1.0MA:1.5p-OHMA)werenotsignificantlydifferentin meanlengthfromthecontrolgroup.Signi_ficantdifferenceswere also seen between replicates within groups (F20, 3485=16.96,

p< 0.0001).AposthocTukey–Kramertestrevealedtherewasno signi_ﬁcantdifferencein meanlengthbetweenreplicateswithin eachgroupexceptforinthecontroland0.15p-OHMAtreatment,for whichallreplicatesweresigniﬁcantlydifferentfromeachother.

Similartrendswereobservedinanalysesofmeanreplicatewidth (Fig.2(b)).AnestedANOVAshowedsigniﬁcantdifferencesbetween groups(F9,3485=136.29, p< 0.0001). Themean width of larvae exposedtomethamphetamine(0.1MA,1.0MAand 10MA)was

A B

A C

B B

C C C

A 17 17.5 18 18.5 19 19.5 20 20.5 21 Co ntr o l 0 .1 MA :0. 0 1 5 p-O H MA 1.0 MA :0. 1 5 p-0H MA 10 MA:1. 5 p-O HMA 0. 1 MA 1. 0 MA 10 MA 0. 015 p -OHMA 0. 15 p-O HMA 1. 5 p -O H MA Length (m m ) Group (b) (a) (c)

A AB _A E BC C D DE E A 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 Control 0. 1 MA :0.01 5 p-O HMA 1.0 M A :0 .1 5 p-0 H M A 10 MA:1.5 p

-OHMA 0.1 MA 1.0 MA 10

M A 0.015 p-OHMA 0. 15 p -O HMA 1.5 p-O HMA W idth (m m ) Group A B CD E D D B F F AC 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Co ntr o l 0.1 MA :0. 0 1 5 p-O HMA 1.0 MA :0. 1 5 p -0H MA 10 MA:1. 5 p-O H MA

0.1 MA 1.0 MA 10 MA

0.

01

5

p-OHMA

0.15 p-OHMA 1.5 p

-O H MA W eight (g) Group

Fig. 2.Meanlength(a),width(b)andweight(c)(SE)ofday4larvae,exposed

duringdevelopmenttodifferentconcentrationsofmethamphetamineand/or

p-hydroxymethamphetamine.Experimentalgroupsarecontrol;0.1mg/kg

metham-phetamine:0.015mg/kgp-OHMA(0.1MA:0.015p-OHMA);1.0mg/kg

methamphet-amine:0.15mg/kg p-OHMA (1.0 MA:0.15 p-OHMA); 10mg/kg

methamphetamine:0.015mg/kgp-OHMA(10MA:1.5p-OHMA);0.1mg/kg

meth-amphetamine (0.1 MA); 1.0mg/kg methamphetamine (1.0 MA); 10mg/kg

methamphetamine(10MA);0.015mg/kgp-OHMA(0.015p-OHMA);0.15mg/kg

p-OHMA(0.15p-OHMA);1.5mg/kgp-OHMA(1.5p-OHMA).Groupsnotconnected

bythesameletteraresigniﬁcantlydifferent.

[image:14.595.314.556.54.618.2]

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significantlygreaterthanthecontrol.Similarly,themeanwidthsof larvaeinthehighestratiotreatment(10MA:1.5p-OHMA)andthe low and intermediate p-hydroxymethamphetamine concentra-tions(0.015p-OHMAand0.15p-OHMA)weresignificantlygreater thanthecontrolgroup.Bycontrast,themeanwidthsofthelower ratiotreatments(0.1MA:0.015p-OHMAand1.0MA:0.15p-OHMA) and the highest p-hydroxymethamphetamine treatment (1.5 p-OHMA)didnotdiffersigni_ficantlyfromthecontrol.Withthe exception of the control and 0.15 p-OHMA groups, where one replicatedifferedsignificantlyfromtheothertwo(F20,3485=54.07,

p< 0.0001), a post hoc Tukey–Kramer test did not identify signiﬁcantdifferencesbetweenreplicateswithineachgroup.

A nested ANOVA analysis identiﬁed signiﬁcant differences between the larval groups for mean weight (F9, 3485=100.21,

p< 0.0001). The _post _hoc Tukey_–Kramer test revealed similar trendstolengthandwidthcomparison.Averagelarvalweightsof the methamphetamine (0.1 MA,1.0 MA and 10 MA) and ratio treatments(0.1MA:0.015p-OHMA,1.0MA:0.15p-OHMAand10 MA:1.5p-OHMA)weresignificantlygreaterthanthecontrol.The mean weights of the two lower p-hydroxymethamphetamine treatments(0.015p-OHMAand 0.15p-OHMA)werealsosigni_fi -cantlygreaterthanthecontrol.Bycontrast,themeanlarvalweight of the highest p-hydroxymethamphetamine treatment (1.5p-OHMA) did not differ signi_ficantly from the control (Fig.2(c)).Significantdifferencesbetweenreplicateswithin treat-ments(F20,3485=102.67,p< 0.0001)wereidentifiedbyaposthoc Tukey–Kramertest. However,only one replicateof each of the controland0.1MAgroupsdifferedsigni_ficantlyfromtheothertwo replicateswithinthegroup.

3.2.Comparisonofday7larvae

AnestedANOVAof meanlengthidentifiedsignificant differ-encesbetweentreatments(F9,3485=48.77,p< 0.001Theposthoc Tukey–Kramer test revealed that the mean lengths of larvae exposedtoanydrugcompoundweresignificantlygreaterthanthe control(Fig.3(a)).However,signi_ficantvariation between repli-cateswithingroupswasalsodetected(F20,3485=51.05,p< 0.001). Onereplicateofeachofthecontrol,intermediate methamphet-amineconcentration(1.0MA)andthehighestp -hydroxymetham-phetamineconcentration(1.5p-OHMA)wassigni_ficantlydifferent fromtheothertwowithineachgroup.

Substantialvariationwasalsoseenbetweenthemeanreplicate widthsofday7larvae(F9,3080=19.28,p< 0.001)(Fig.3(b)).The mean widths of larvae exposed to any drug treatment were signiﬁcantlygreaterthanthecontrolgroup.AnestedANOVAof replicatewithintreatment showedthat onereplicateofthe1.5 p-OHMA group differed signiﬁcantly from the other replicates withinthetreatment(F20,3080=19.10,p< 0.001).

Similarly,nestedANOVAdeterminedthatthelarvaeexposedto methamphetamine compounds were significantly heavier on average than control samples (F9, 3080=65.45, p< 0.001) (Fig. 3(c)). Signi_ficant differences were also detected between replicateswithingroups(F20,3080=50.37,p< 0.001).Onereplicate ofeach ofthe1.5p-OHMAand 1.0MA:0.15p-OHMAtreatment groups,andtheintermediatemethamphetaminegroup(1.0MA) weresigni_ficantlydifferentfromtheothertworeplicateswithin theirtreatments.Furthermore,eachreplicateofthecontrolwas significantlydifferentfromeveryother.

(a) (b) (c) A B C D E B E CD E F 17 18 19 20 21 22 23 24 Control 0.

1 MA:0.015 p-O

HMA 1.0 MA :0. 1 5 p -OH MA 10 MA:1.5 p-OHMA 0. 1 MA 1.

0 MA ₁₀ M

A 0.015 p-OH MA 0. 15 p -O H MA 1.5 p-O HMA Length (m m ) Group A B CD E BC CD EF CD DF FE 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 Co ntr o l 0.1 MA :0. 0 1 5 p-O HMA 1. 0 MA :0.15 p-O H MA 10 MA:1. 5 p-O H MA

0.1 MA 1.0 MA 10 MA

0. 015 p-OHMA 0. 15 p-O H MA 1. 5 p -O H MA Wi d th ( m m ) Group A B C E AB _AB

CDE CD DE F 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Co ntrol 0.1 MA :0.015 p-OHMA 1. 0 MA :0.15 p -OH MA 10 MA:1.5 p-OHMA 0. 1 MA 1.

0 MA ₁₀ MA

0.

015 p-OHMA 0.15 p-O

H MA 1. 5 p -O H MA W eight (g) Group

Fig. 3. Meanlength(a),width(b)andweight(c)(SE)ofday7larvae,exposed

duringdevelopmenttodifferentconcentrationsofmethamphetamineand/or

p-hydroxymethamphetamine.Experimentalgroupsarecontrol;0.1mg/kg

metham-phetamine:0.015mg/kgp-OHMA(0.1MA:0.015p-OHMA);1.0mg/kg

methamphet-amine:0.15mg/kg p-OHMA (1.0 MA:0.15 p-OHMA); 10mg/kg

methamphetamine:0.015mg/kgp-OHMA(10MA:1.5p-OHMA);0.1mg/kg

meth-amphetamine (0.1 MA); 1.0mg/kg methamphetamine (1.0 MA); 10mg/kg

[image:15.595.38.277.59.704.2]

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3.3.Comparisonofpupae

Statisticalsigniﬁcancebetweenthemeanpupallengthsofeach treatment was detected by a nested ANOVA (F9, 3022=87.59,

p< 0.0001).AposthocTukey–Kramertestrevealedthatthemean lengthsofpupaeexposedtoanydrugcompoundweresignificantly longerthanthecontrol(Fig.4(a)).Significantvariationwasalso observed between replicates within each of the groups (F20, 3022=20.10, p< 0.0001). One replicate of the control and eachpuremethamphetaminetreatment(0.1MA,1.0MA,10MA) variedsigni_ficantlyfromtheotherswithineachgroup.

A nested ANOVA of width showed significant differences between groups at the pupal stage (F9, 3022=94.23, p< 0.001) (Fig.4(b)).Themeanwidthsofpupaeexposedtoany metham-phetamineand/or metabolitetreatmentduring thelarval stage weresignificantgreater thanthecontrolsamples.Furthermore, nestedANOVAanalysisidentifiedsignificantvariabilitybetween the mean widths of individual replicates within groups (F20, 3022=97.87, p< 0.001). With the exception of the ratio treatments(0.1MA:0.015p-OHMA,1.0MA:0.15p-OHMAand10 MA:1.5p-OHMA),onereplicateineachtreatmentwassigni_ficantly differentfromtheothertwo. Eachreplicate ofthe controlwas significantlydifferentfromtheremainingtworeplicates.

Whenaverage weightwas compared, signi_ficantdifferences were detected between treatments using a nested ANOVA (F9,3022=3.74,p< 0.001)(Fig.4(c)).AposthocTukey_–Kramertest ofmean pupalweight showed thatallpuremethamphetamine treatments(0.1MA,1.0MAand10MA)andthetwolowerratio treatments(0.1MA:0.015p-OHMAand1.0MA:0.15p-OHMA)were significantlyheavierthanthecontrolgroup.Furthermore,signifi -cant differences between replicates within groups were also identified (F20,3022=3.68,p< 0.001). The highest methamphet-amine treatment (10 MA) had one replicate that differed signi_ficantlyfromtheothertwowithinthetreatment.

3.4.Comparisonofadults

Onceadultfliesemergedfromthepuparium,theleftwingand rearleftlegwereanalysedtoassessoverallflysize.Thecostalvein of the wingand the tibia of theleg were measured. A nested ANOVAshowed thatthereweresigni_ficantdifferencesbetween themeancostallengthin eachgroup(F9,2755=15.17,p< 0.001) (Fig.5(a)).AposthocTukey–Kramertestidentifiedthatthepure methamphetaminetreatments(0.1MA,1.0MAand10MA),and thelowandhighratiotreatments(0.1MA:0.015p-OHMAand10 MA:1.5 p-OHMA) varied significantly from the control. No signi_ficantdifference wasseen betweenthemeancostal length ofreplicateswithingroups(F20,2755=0.14,p=0.87).

Significantdifferencesbetweenmeantibiallengthswerealso detectedbyanestedANOVA(F9,2755=40.22,p< 0.001)(Fig.5(b)). A post hoc Tukey–Kramer test showed that all treatments containing drug compounds produced flies with significantly greatermeantibiallengthsthanthecontrolgroup.Afurthernested ANOVAshowedasignificanteffectofreplicatewithingroup(F20, 2755=3.84,p=0.021)withaposthocTukey–Kramerrevealingone

replicateofeachofthelowestmethamphetaminetreatment(0.1 MA)andthecontrolgroupdifferingsigni_ﬁcantlyfromtheother tworeplicateswithinthesegroups.

The average weights of adult flies were determined to be statisticallydifferentby anestedANOVA(F9,2755=244.43,p< 0.001) (Fig. 5(c)). Apost hocTukey–Kramertestshowed that allflies exposed tomethamphetamineor p-hydroxymethamphetamineas larvae were signi_ficantlyheavier than the control samples. A nested ANOVA was used to investigate variability within treatments between replicates.Significantdifferenceswereidentified(F20,2755=11.22,

p< 0.001)andidentiﬁedbyaposthocTukey–Kramertobebetween

A

B B B B

C B D E C 9.8 10 10.2 10.4 10.6 10.8 11 Contro l 0.1 MA :0. 0 15 p-OHMA 1. 0 MA :0.15 p-OH MA 10 MA:1.5 p-OHMA 0.

1 MA _1.0 MA _{10 MA}

0.01

5

p-OH

MA

0.15 p-OHMA 1.

5 p -O H MA Length (m m ) Group (a) (b) A B C D B B D E B B 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 Control 0.1 MA :0. 0 15p-OH M A 1.0 MA :0. 1 5 p-OH MA

10 MA:1.5

p-OHMA 0.1 MA _{1.0 MA} 10 MA

0. 01 5 p-OH M A 0. 15 p-OHMA 1.5 p-O HMA W idth (m m ) Group (c) A BCD CD AB BCD BCD D

AB ABC ABC

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Co n tr o l

0.1 MA:0.015 p-OHMA 1.0 MA:

0 .15 p-OHMA 10 MA:1.5 p-OHMA 0. 1 MA 1.

0 MA 10 M

A 0.01 5 p-OH MA 0. 15 p -OHMA 1. 5 p -O H MA W eight (g) Group

Fig. 4.Meanlength(a),width(b)andweight(c)(SE)ofpupae,exposedduring

developmenttodifferentconcentrationsofmethamphetamineand/or

p-hydro-xymethamphetamine.Experimentalgroupsarecontrol;0.1mg/kg

methamphet-amine:0.015mg/kg p-OHMA (0.1 MA:0.015 p-OHMA); 1.0mg/kg

methamphetamine:0.15mg/kgp-OHMA(1.0MA:0.15p-OHMA);10mg/kg

meth-amphetamine:0.015mg/kgp-OHMA (10MA:1.5 p-OHMA);0.1mg/kg

metham-phetamine (0.1 MA); 1.0mg/kg methamphetamine (1.0 MA); 10mg/kg

[image:16.595.318.556.61.649.2]

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thereplicatesofthecontrol,0.1MA,1.0MA,10MAand0.15p-OHMA groups.Withinthecontroland10MAgroups,theaverageweightof eachreplicatewasstatisticallyindependent.For0.1M,1.0MAand

0.15 p-OHMA treatments, only one replicate was statistically signi_ﬁcantfromtheothertworeplicatesofthetreatment.

3.5.Survivorship

Kruskal–Wallisanalysisofsurvivorshipshowednosigniﬁcant difference in the number of larvae surviving after four days (X92=3.54,p>0.432).However,attheday7intervaltherewasa

signiﬁcantdifferencein survivorshipbetweendrugand control groups (X92=28.01, p =0.017).When compared to the control,

larval numbers had decreased in all pure methamphetamine (0.1MA,1.0MAand10MA),andratiotreatments(0.1MA:0.015 p-OHMA,1.0MA:0.15p-OHMAand10MA:1.5p-OHMA)butnotin the treatments feeding on substrates spiked with pure p-hydroxymethamphetamine(0.015p-OHMA,0.15p-OHMAand 1.5p-OHMA).Adultsurvivorshipwasalsosigniﬁcantlydifferent betweentreatments(X92=29.00,p< 0.001).Thenumberofadult

ﬂies remaining in each drugged treatment was signi_ﬁcantly smallerthanthenumberofsamplessurvivinginthecontrol.

3.6.Developmentrates

Changesingrowth rateduetothepresenceof methamphet-amineandp-hydroxymethamphetamineinthelarvalfoodsource weredeterminedbyrecording,tothenearesthour,whensamples in each replicate commenced and completed pupariation, and began and completedadult emergence.Calliphora stygia larvae exposedtoanyconcentrationofmethamphetamineorp- hydrox-ymethamphetamine proceeded to show accelerated growth, commencing pupariation signiﬁcantly before control larvae (F2,10=144.03, p< 0.0001) (Fig. 6). This discrepancy was most obviousin the10 MAand 10MA:1.5 p-OHMA treatments. The initialcolourchangeoftheprepupaefromwhitetoorangebegan 44hearlierinthesetreatmentsthaninthecontrol.

Pupariationwasalsocompleteinallmethamphetamine-and metabolite-treated pupae significantly before the control (F2,10=293.47, p< 0.0001).Samplesthenremainedaspupaefor significantly longer in treatments containing drug compounds (F2,10=441.63,p< 0.0001),andemergedlaterthancontrolsamples (F2, 10=91.47, p< 0.0001). The 10 MA:1.5 p-OHMA treatment showedthegreatestincongruityfromthecontrol.Emergencein this treatment began 34h following the first control samples, equatingtoa78htotaldifferenceindevelopmentrate.

(a) A BC A D CE DE F

ABC ABC AB

2.8 2.85 2.9 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 Co ntr o l 0.1 MA

: 0.015 p-OH

MA

1.

0 MA

:0.15 p-OHMA

10 MA:1.5

p-OHMA 0.1 MA 1.0 MA

10 MA 0. 015 p-OHMA 0. 15 p -O H MA 1.5 p-OH MA

Costal Length (m

m ) Group (b) A B C B D _D E D D B 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 C ont ro l 0. 1 MA :0.015 p-OHMA 1.0 M A :0 .1 5 p-O H M A 10 MA:1.5 p-OHMA 0. 1 MA 1.

0 MA ₁₀ M

A 0. 01 5 p-OHMA 0.15 p-O H MA 1. 5 p-OH MA T

ibial Length (m

m ) Group (c) A B C B D _C E

B F BF

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 Control 0. 1 MA :0. 0 15 p-O HMA 1. 0 MA :0.15 p-OHMA 10 MA:1.5

p-OHMA 0.1 MA 1.0 MA 10 MA

0.01 5 p-OHMA 0. 15 p-O H MA 1.5 p-O H MA We ig h t ( g ) Group

Fig.5.Meancostallength(a),tibiallength(b)andweight(c)(SE)ofadults,

exposedduringdevelopmenttodifferentconcentrationsofmethamphetamine

and/orp-hydroxymethamphetamine.Experimentalgroupsarecontrol;0.1mg/kg

methamphetamine:0.015mg/kg p-OHMA (0.1 MA:0.015 p-OHMA); 1.0mg/kg

methamphetamine:0.15mg/kgp-OHMA(1.0MA:0.15p-OHMA);10mg/kg

meth-amphetamine:0.015mg/kg p-OHMA (10 MA:1.5 p-OHMA);0.1mg/kg

metham-phetamine (0.1 MA); 1.0mg/kg methamphetamine (1.0 MA); 10mg/kg

Fig. 6.Developmentrates of Calliphorastygia samples exposed to different

concentrationsofmethamphetamineand/orp-hydroxymethamphetamine.

Exper-imentalgroupsare control;0.1mg/kgmethamphetamine:0.015mg/kgp-OHMA

(0.1MA:0.015p-OHMA);1.0mg/kgmethamphetamine:0.15mg/kgp-OHMA(1.0

MA:0.15p-OHMA);10mg/kgmethamphetamine:0.015mg/kgp-OHMA(10MA:1.5

p-OHMA);0.1mg/kgmethamphetamine(0.1MA);1.0mg/kgmethamphetamine

(1.0MA);10mg/kgmethamphetamine(10MA);0.015mg/kgp-OHMA(0.015

p-OHMA);0.15mg/kgp-OHMA(0.15p-OHMA);1.5mg/kgp-OHMA(1.5p-OHMA).

[image:17.595.41.279.113.650.2] [image:17.595.303.556.527.667.2]

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3.7.HPLCanalysis

Standardsformethamphetaminehydrochlorideandp- hydrox-ymethamphetamine hydrochloride were established for HPLC underisocraticconditions,withtheformer showingaretention timeof27.3minandthelatter14.2min.Analysisofsamplesofeach lifestagebyHPLCwithUVdetectionqualitativelyconﬁrmedthe absenceofmethamphetamineandp-hydroxymethamphetamine inthefeedingsubstrateofthecontrol(Fig.7).

Methamphetamine could not be detected in homogenised larvalsamplesoftreatments0.1MA,1.0MAor10MA.However, HPLCchromatogramsqualitativelyconﬁrmeditspresenceinpupal and adult samples (Fig. 7). p-Hydroxymethamphetamine was detected in larval, pupal and adult preparations of treatments

0.015p-OHMA,0.15p-OHMAand1.5p-OHMA.Inratiotreatments 0.1 MA:0.015p-OHMA,1.0 MA:0.15p-OHMA and10MA:1.5 p-OHMA,onlyp-hydroxymethamphetaminecouldbedetected.

4.Discussion

Inthisstudy,methamphetamine-spikedkangaroomeatwas utilisedtosimulatethepostmortemenvironmentofa metham-phetamineoverdose.Kangaroomincewasselectedinfavourofa live laboratoryanimal as the major andminor metabolites of methamphetamine and absorption and excretion rates are knowntovarybetweenvertebratespecies[20].Theapplication of drugor metabolitedirectlytothemeatensuredthatlarvae were exposed to known concentrations and types of drug

Fig.7.SelectedexamplesofHPLCtracesqualitativelyshowingthepresenceorabsenceofmethamphetamine(MA)andp-hydroxymethamphetamine(p-OHMA):(a)control;

(b)0.1MA;(c)1.0MA;(d)10MA;and(e)1.0MA:0.15p-OHMA.

[image:18.595.52.559.235.713.2]

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compound,a recognised issuein entomotoxicological research

[31]. Sustained mixingensured a homogenous distribution of methamphetamineand/orp-hydroxymethamphetamine.

Thesecompoundswerefoundtosigniﬁcantlyalterthesizeofall lifestagesat theconcentrations investigated.After fourdaysof growth,larvaeinalltreatments,withtheexceptionofthehighest p-hydroxymethamphetamine (1.5p-OHMA) and the intermediate ratiotreatments(1.0MA:0.15p-OHMA),werelonger,widerand heavierthanthecontrolsamples.After sevendays,samples exposed toanymethamphetaminetreatmenthaddevelopedintosigniﬁ -cantlylonger,widerandheavierlarvaethanthecontrolmaggots.

Thesefindingscontrastwiththose ofGoffetal.[12] intheir studies of the flesh fly Sarcophaga ruficornis. When reared on methamphetamine-dosed rabbit tissues,individuals exposed to thehighestconcentrationsofthedrugweresigni_ficantlyshorterin lengththan control samples.This may have resulted fromthe different metabolism of methamphetamine in flesh flies and blow_fliesand highlights theinadvisabilityof extrapolating out-comesfromentomotoxicologicalstudiesbetweenspecies.

Inforensicpractice,thelengthsofthelargestlarvaesampled fromabodyareusedtoestimatetheageoftheoldestspecimens, andhence,giveanestimateoftheminimumPMI[32].Theresults ofthis studystronglysuggestthat,in amaggot-infestedcorpse containingmethamphetamine,thelengthof alarva ofC. stygia might not necessarily be a valid indication of its age. If this enhancedgrowthis notconsidered,estimatesof minimumPMI basedonthelarvalstagesofC.stygiacouldbeerroneous.

Furthermore, the presence of methamphetamine and p-hydroxymethamphetamineinthefeedingsubstratesignificantly affectedthegrowthratesofC.stygialarvaeattheconcentrations investigated. The onset of pupariation, and its duration, was significantlyalteredindrug-exposedtreatments.Calliphorastygia larvaeexposed tothese compounds as immaturesreachedthe pupalstageupto44hpriorto,andremainedaspupaeupto34h longerthan controls, a total divergenceof 78h. These findings againcontrastwiththoseofGoffetal.[12],whofoundtheduration ofthepupalperiodofS.ruficornistobesigni_ficantlyshorterthan thecontrolforallsamplesexposedtomethamphetamineduring thelarvalstage.Additionally,thepreviousstudyonC.stygiaby Georgeetal.[27]foundthatitsdevelopmentwasunaffectedby puremorphineatsub-lethal,lethal,andtwice-lethaldoses.These contradictoryfindingsaremostlikelyduetothedifferenttypeof drugusedineachstudy,andtheeasewithwhichtheyareabsorbed bythetissuesofanorganism.

While morphineisapotentanalgesic[33],andrepressesthe centralnervoussystem [34], methamphetamineis a psychosti-mulant [35]. In humans,it immediately induces an increase in metabolismandthereleaseof‘pleasure’neurotransmitters[18]. Althoughbothtypesofdrugarewatersoluble,morphineispoorly solubilisedinlipids[33],whereasbothmethamphetamineand p-hydroxymethamphetaminearehighlylipidsoluble[36]. Metham-phetamine compounds may therefore be better suited to the internal environment of blow_ﬂy larvae, which have a high fat content[37].Itispossiblethatthesecompoundsareabletocross thelipid bi-layer of larval cells and accelerate metabolism.An increaseinmetabolicratecouldmanifestasanacceleratedrateof development,orenlargedlarvae,pupaeandadults,whichwereall observedinthecurrentstudy.

ComparedwiththefindingsofGeorgeetal.[27],asignificant differenceinsurvivorshipwasrecordedbetweendrugtreatments andcontrols.Therewas asignificantdecreaseinthenumberof larvaeinall methamphetamine-spikedtreatmentsafter 7days. Similarly,overallsurvival,calculatedonceall_flieshademerged, showedthat there were significantly fewer flies in treatments exposedtomethamphetamineasimmatures. Thissuggeststhat methamphetamine compounds have a toxic effect onC. stygia

larvae at any concentration,and mayinﬂuence their ability to undergometamorphosis.

Methamphetamine and p-hydroxymethamphetamine were detected both in meat and C. stygia samples. HPLC analysis con_firmed that methamphetamine and/or p- hydroxymetham-phetaminewere absentfrom thecontrolgroup, but presentin thelarvalfoodsourceoftheremainingtreatments.Consequently, larvaewouldhaveingestedthedrugcompounds.However,when larvaesampledatthefirstandsecondcomparisonintervalswere analysed with HPLC–UV, methamphetamine could not be detected. These _findings differed from those of Wilson et al.

[38]intheirstudiesofCalliphoravicina.Whenrearedonskeletal musclefromasuicidaloverdoseofco-proxamolandamitriptyline, amitriptylineanditsactivehumanmetabolite,nortriptyline,were bothdetectedinC.vicinalarvae.

Thisnegativedetectionmayhavebeenduetothewayinwhich sampleswerepreparedforanalysis. Larvaewereground witha mortarandpestleandcombinedwithdistilledwatertosolubilise anycellularmaterialreleased.Thiswasarelativelycrudemethod of homogenization and the complete lysis of all cells, and the releaseofstoredmethamphetamine,wasnotguaranteed.Studies byKinnearetal.[39]haveshownthattheconcentrationoflipidsin C. stygia larvae increases sharply between days 3 and 6 of development, before decreasing after day 7. As larvae were sampled ondays 4 and day 7, within this period of increased lipid production, increased fat content, combined with poor homogenizationtechniques,couldberesponsiblefortheabsence ofmethamphetamineinlarvalpreparationsanalysedbyHPLC.

Itisalsopossiblethatdrugcompoundswerenotdetectedinlarval samplesduetothelowersensitivityofHPLC–UV,whencomparedto chemiluminescenceor_ﬂuorescencedetection[40].Takayamaetal., in their studieson methamphetaminedepositsin hair,wereunable to detectmethamphetamineinsmallhairsampleswhenUVdetection wasemployed.However,minuteamountsofthedruginsinglehairs were able to be isolated and detected by chemiluminescence techniques[41].Theuseofalternatedetectionmethodsmightyield moreconclusiveresultsinfuturestudies.

Although the results of the present studyare notable, it is recommended that further investigations at different temper-atures,and alternativeconcentrationsof methamphetamine,be carriedouttoformacomprehensivebankofdataagainstwhich forensiccasescan becompared.As corpsesare rarelyfoundin environmentswithstabletemperatures,agreaterunderstanding oftheeffectsofmethamphetamineatdifferenttemperaturescould assistwithinterpretingforensiccases.Similarly,thepostmortem concentrationsofdrugsinacorpsemayvaryaccordingtotissue type andlocation[42,43]and alsomaydifferfromthe concen-trationsatthetimeofdeathduetopostmortemredistributionby passivereleasesfromthedrugreservoirsof thegastrointestinal tract,lungsandmyocardium,oratlaterstages,fromtheautolysis ofcellsandputrefactionprocesses[44,45].Basiclipophilicdrugs, suchasmethamphetamine,appeartobeparticularlysusceptibleto postmortemredistributionprocesses [44].Itis alsoknownthat larvalgrowthcanbeinfluencedbytissuetypeandageinanimal models[46–48].Therefore,furtherinvestigationoftheeffectsof methamphetamine on blowfly larvae must be carried out at different concentrations and in different substrates for its influence to be conclusively understood. Of course, once a comprehensivesetofdataisavailableforC.stygia,theinfluence ofthisdrugonother_flyspeciesofforensicimportancewouldalso needtobeinvestigated.

5.Conclusions

These findings hold significance for forensic science with particular regard tominimum PMI calculations using_flies. The

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alteredgrowthexhibitedinthisstudysuggeststhatanyestimateof minimumPMIbasedonthenormalratesofC.stygiadevelopment at23_C_could_be_{overestimated}_by_up_to₄₄_h_when_based_on_the larval stage,and by up to78hwhen based onpupal samples. Indeed, because of the resultant developmental acceleration, cautionshouldbeexercisedwheneverC.stygiaisusedtoestimate the minimum PMI of methamphetamine-dosed corpses. The developmental changes of this species of blow_ﬂy are also yet unknownattemperatures,drugconcentrationsandinsubstrates otherthanthoseusedhere.Furtherresearchisthereforeessential tomorecomprehensivelyunderstandtheeffectsof methamphet-amineon blowﬂy development. Until then, C.stygia cannot be assumedtobeareliablemodelforagingcorpsescontainingthis drug.

Acknowledgements

Thisresearchwasmadepossiblebythe_ﬁnancialsupportofthe AustralianResearchCouncil,theAustralianFederalPolice,theNew SouthWales PoliceForceand UOW’sInstitutefor Conservation BiologyandEnvironmentalManagement.ASNthanksUOWforthe provision of a UPA scholarship. We thank Melanie Archer (Victorian Institute of Forensic Medicine) for useful initial discussions,ThaoDangand GregoryTarrant(National Measure-mentInstitute)foradviceandsupplyoftheMAandp-OHMA,and AidanJohnson(UOW)forstatisticalassistance.

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