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Effect of morphine on the growth rate ofCalliphora stygia (Fabricius)

(Diptera: Calliphoridae) and possible implications for forensic entomology

Kelly A. George a,*, Melanie S. Archer b, Lauren M. Green a, Xavier A. Conlan c, Tes Toop a

a School of Life and Environmental Sciences, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3217, Australiab Department of Forensic Medicine, Monash University, 57-83 Kavanagh Street, Southbank, Victoria 3006, Australiac Institute for Technology, Research and Innovation, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3217, Australia

1. Introduction

When a decomposed body is insect-infested, the most reliable

estimate of the minimum post-mortem interval (PMI) is often

obtained using entomological techniques. The minimum PMI is

described as the time between corpse infestation by insects and

corpse discovery[1]. The delay between death and infestation is

variable, so it is usually impossible to estimate the time of death

itself; however minimum PMI may provide a good indication of

actual death time given that infestation potentially occurs rapidly

under optimal warm daylight conditions[2]. Currently, two major

approaches are used to determine the minimum PMI: the first uses

insect succession patterns, while the second uses insect growth

rates[1]. The latter is the focus of the present study.

Insect growth rates can be affected by a number of variables,

including temperature[3,4], location of the body [5,6], and larval

density [7]. Importantly, the presence of certain toxicological

substances in the feeding substrate can affect larval growth rates,

thus, leading to an over- or under-estimation of minimum PMI.

Previous studies have investigated the effects of morphine [8,9],

paracetamol [10], codeine [11], and diazepam [12] on blowfly

larval growth. In particular, growth perturbations of calliphorids

caused by morphine may be of great importance due to the

frequency with which this drug is present post-mortem. Morphine

may enter the body in its pure form or as codeine or heroin and is

very stable in tissues over time [13]. Codeine and morphine

glucuronic derivatives are also produced during the metabolism of

these opiates, however, these compounds have been shown to be

unstable in decomposing tissues[14,15], and have therefore been

excluded from this investigation. Morphine was introduced to pet

mince to simulate post-mortem concentrations rather than the

alternative of using a live animal with a morphine overdose. This

was done both for ethical reasons and because we wanted to

isolate the effect of morphine on growth, and eliminate the

cumulative effect of secondary metabolites, which are produced

during morphine metabolism in animals (e.g. [16,17]).

Bourel et al.[9] found that morphine at a concentration of 0.5,

1.0 and 2.0 times the median lethal dose slowed the growth rate of

Forensic Science International 193 (2009) 2125

A R T I C L E I N F O

Article history:Received 18 December 2008

Received in revised form 27 July 2009

Accepted 26 August 2009

Available online 20 September 2009

Keywords:

Calliphoridae

Entomotoxicology

Forensic entomology

Insect growth rates

Minimum post-mortem interval

Morphine

A B S T R A C T

Insect specimens collected from decomposing bodies enable forensic entomologists to estimate theminimum post-mortem interval (PMI). Drugs and toxins within a corpse may affect the development

rate of insects that feed on them and it is vital to quantify these effects to accurately calculate minimum

PMI. This study investigated the effects of morphine on growth rates of the native Australian blowfly,

Calliphora stygia (Fabricius) (Diptera: Calliphoridae). Several morphine concentrations were incorpo-

ratedinto pet mince to simulate post-mortem concentrations in morphine,codeine and/or heroin-dosed

corpses. There were four treatments for feeding larvae; T 1: control (no morphine); T 2: 2mg/gmorphine; T 3: 10mg/g morphine; and T 4: 20mg/g morphine.Ten replicates of 50 larvaewere grown at22 8C foreach treatment and their developmentwas compared at four comparison intervals; CI 1: 4-day-

old larvae; CI 2: 7-day-old larvae; CI 3: pupae; and CI 4: adults. Length and width were measured for

larvae and pupae, and costae and tibiae were measured for adults. Additionally, day of pupariation, day

of adult eclosion, and survivorship were calculated for each replicate. The continued presence of

morphine in meat was qualitatively verified using high-performance liquid chromatography with acidic

potassium permanganate chemiluminescence detection. Growth rates ofC. stygia fed on morphine-

spiked mince did not differ significantly from those fed on control mince for any comparison interval or

parameter measured. This suggests that C. stygia is a reliable model to use to accurately age a corpsecontaining morphine at any of the concentrations investigated.

2009 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +61 3 5227 3449; fax: +61 3 5227 1040.

E-mail address: kageo@deakin.edu.au(K.A. George).

Contents lists available atScienceDirect

Forensic Science International

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o r s c i i n t

0379-0738/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2009.08.013

mailto:kageo@deakin.edu.auhttp://www.sciencedirect.com/science/journal/03790738http://dx.doi.org/10.1016/j.forsciint.2009.08.013http://dx.doi.org/10.1016/j.forsciint.2009.08.013http://www.sciencedirect.com/science/journal/03790738mailto:kageo@deakin.edu.au

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Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae)

larvae during the first 60 h of development (Table 1). Bourel et al.

[8], also found that morphine slowed the growth ofLucilia sericata

(Meigen) (Diptera: Calliphoridae) in a dose-dependent manner at

concentrations of approximately 5.8, 9.2 and 12.8 mg/g (Table 1).Interestingly, Kharbouche et al. [11] found thatwhen morphine was

present with codeine and norcodeine, the growth ofL. sericatawas

stimulated during the larval stage (Table 1), while Goff et al. [18]

showed that larvae ofBoettcherisca peregrina (Robineau-Desvoidy)

(Family: Sarcophagidae) were significantly larger when fed sub-

strate containing morphine (administered as heroin) at concentra-

tions of approximately 0.4, 1.2, 1.5 and 2.2 mg/mL (Table 1).Abuse of the illicit drug heroin results in an alarming number of

fatalities each year in Australia [19]. Legal and illicit use of

morphine and codeine also occurs, which can result in overdose

deaths[20,21]. However, Australian entomotoxicological knowl-

edge in general is poor, with only one study involving extraction of

morphine from the larvae of an Australian native blowfly[22], and

no studies to date examining potential growth rate effects of

morphine on native species. This study investigated the effects of

morphine on the growth rate of the native Australian blow fly,

Calliphora stygia (Fabricius) (Diptera: Calliphoridae).

2. Materials and methods

C. stygiacolonies were established with wild type (F0) specimens obtained from

the Victorian Institute of Forensic Medicine and were transferred to Deakin

University. Adult flies were kept in a laboratory with natural lighting and ambient

temperature (1320 8C, mean 16 8C). Adult blowflies were kept in 31 L clear

rectangular storage containers. Panels were cut from two sides

(160 mm 120 mm) and from the lid (230 mm 260 mm) of the container and

covered with mesh organza for ventilation. A circular panel 130 mm diameter was

removed from the front to attach an organza sleeve for accessing the cage interior.

Sugar cubes (CSR, Australia) and water were provided to adult flies ad libitum. Flies

were also supplied with protein biscuits (eggs, powdered milk, sugar, yeast and

water) to allow ovarian maturation.

Petmince (V.I.P. Petfoods, Australia),which containslean kangaroomince,lambs

fry and heart, was prepared for four treatment groups of larvae feeding at various

morphine concentrations. Treatments were T 1: control (no morphine); T 2: 2 mg/g

morphine; T 3: 10 mg/g morphine; and T 4: 20 mg/g morphine. These concentra-

tions were determined to be suitable for investigation based on known doses that

have caused human fatalities[2325]and on the results of previous work by Green

[26]. Morphine, obtainedunder licence (GlaxoSmithKline, Australia), was dissolved

in 50 mL deionised (DI) water containing 5 ml of sulphuric acid to prepare a

1 102 M morphine stock solution, which was diluted as required. Morphine

stock solution was then added to meat batches to produce the three treatments (as

described above). Each treatment was spikedwitha total volume of 21 mL DI water

containing the amount of morphine required to achieve the treatment concentra-

tions. The control treatment (no morphine) also had 21 mL of DI water added to

keep addition of liquid constant between groups. Each meat batch was mixed

separately, via hand manipulation for 5 min, to ensure a uniform spread of

morphine. Meatbatches werethensplit into30 100 g portions(0.3 g) andplaced

into polystyrene cups (250 mL Dart, Australia), which were then maintained at 20 8C

anddefrosted asrequired. It hadbeendetermined from a previouspilotstudy [26] that

morphine remains stable for at least 4 days in samples prepared in this manner and

stored by either refrigeration or freezing. High-performance liquid chromatography

(HPLC) with acidic potassium permanganate chemiluminescence detection [27]was

employed to verify morphine presence or absence (control) within the meat substrateas previously described by Gunn et al. [22].

Eggs were collected from C. stygiacolonies. Round plastic 70 mL egging dishes

(Genfac Plastics, Australia) with 50 g pet mince and a light covering of cotton wool

were placed into adult enclosures. Egging dishes were checked every 2 h and eggs

wereplaced into treatment cupsimmediatelyupon discovery. Cottonwool filledwith

egg batches was transferred to a Petri dish (850 mm diameter). Eggs were washed

onto damp, dark coloured card and counted into 120 groups of 50 eggs using a fine

paintbrush.Eachgroup of50 eggs wasthenplacedon a smallpieceof cottonwool, and

randomly allocated into a polystyrene cup (the experimenter was blind to the cups

treatment label during allocation). A damp square of paper towel was positioned

around the eggs to prevent desiccation. The day that eggs were laid and assigned to

treatment cups was designated as Day 0. Each filled mince cup was transferred to a

round plastic container (850 mL, Genfac Plastics, Australia) containing 20 mm of

paper cat litter (Fibre Cycle, Australia). A 50 mm2 hole was cut into the lid of the

container and covered withmesh curtain material. All replicateswere maintained in

an incubator(Thermoline,Australia)at 22 8C witha 12:12light:dark cycle,anda bowl

of water was placed at the bottom of the incubator to increase humidity.

Treatments were compared at four-time intervals. The first two comparison

intervals (CI) occurred during the larval stage. CI 1 occurred on day 4 and CI 2

occurred on day 7. Collection of CI 1 and CI 2 larvae occurred within 2 h of 17:00 h

on each specified day. Larvae were preserved in an 80% (v/v) ethanol solution (Ajax

Finechem, Australia) after they had been fixed in boiled water for 60 s, and

thoroughly rinsed with near boiling water for 30 s to remove adherent substrate.CI

3 occurred during the pupal period. Pupae were measured live (detailed below)

when all specimens had completed pupariation and were then returned to their

original container to eclose. Daily observations were made to record the day of

pupariation. This was recorded at the first appearance of orange to dark brown

colour change in the prepupa. After pupariation, meat was replaced with a small

water jar containing a wick and two sugar cubes for emerging adult. The average

dayof adult eclosionwas determined forall replicates. Thefinal growth comparison

occurred at CI 4 after adulteclosion. Adults were anaesthetised withcarbondioxide

(CO2) gas and preserved in 80% ethanol at least 24 h after eclosion.Larvae, pupae and adults were viewed under a dissecting microscope (Model

426126, Zeiss, Australia), and illumination on a contrasting background was

achieved using a fibre optic light source (Model KL1500 LCD, Zeiss, Australia). Each

specimen was photographed with a PowerShot digital camera (Model PC 1059,

Canon, Australia) andImage J v1.37 software(National Instituteof Health, USA) was

utilised to measure parameters to thenearest 0.5 mm as shown in Fig.1. CI 1 and CI

2 larval length and width were measured according to methods used by Day and

Wallman[28](Fig. 1a). Pupal length was measured from the most anterior to the

mostposterior points, andwidth wasmeasured along theanterior margin of the5th

segmentposterior spineband (Fig. 1b).Theleft wing and left rear legwere removed

from preserved adults and mounted on microscope slides. Tibia length was

measured, and costa length was measured between the intersections of the

subcosta and R2+3wing veins (Fig. 1c). Mortality rates were calculated at the end of

comparisonintervals 1,2 and 4. Interval3 specimens wererecycledfor interval 4 so

were therefore not suitable for statistical analysis. Ten cups of each treatment were

compared at the four-time intervals.

Statistical analysis of the data was conducted using SPSS v12.0.1 for WindowsTM

and a p 0.05 was considered significant for all of the following analyses. Data

normality was inspected using QQ plots and KolmogorovSmirnov normality

tests. Homogeneity of variance was determined using box plots and Levenes test.

One-way ANOVA and KruskalWallis tests were used to investigate potential

differences between treatment groups. KruskalWallis tests were used when data

showed non-normal distribution.

3. Results

HPLC chromatograms confirmed qualitatively that morphine

was absent from T 1 (control) and present in T 2T 4 and would,

therefore, be ingested by larvae. It was observed that the feeding

action of larvae stirred the meat andwould therefore have kept the

morphine content homogenous throughout the experiment. Also

previous work by Green [26] demonstrated that morphine

remained stable within minced meat for at least 7 days. Some

mortality was observed within replicates and was possibly due to

substrate desiccation within the incubator. Therefore, replicates

Table 1

Summary of previous fly larvae growth rate studies involving morphine, heroin and codeine.

Family Species Feeding substrate Drug Tissue concentration

approx. (mg/g)

Blood concentration

approx. (mg/mL)

Modification

to PMI

Ref.a

Calliphoridae Lucilia sericata Homogenized pig liver Codeine 0.1, 0.3, 2.0, 30 N/A Up to 29 h [11]

Calliphoridae Lucilia sericata Perfused rabbit carcasses Morphine 5.8, 9.2, 12.8 1.8, 3.5, 3.8 Up to 24 h [8]

Calliphoridae Calliphora vicina Perfused rabbit carcasses Morphine Not given Not given Yes [9]

Sarcophagidae Boettcherisca peregrina Per fu sed r abbit car cass es He roin Not given 0.4, 1.2, 1 .5, 2 .2 (morphine) Up to 38 h [18]

Not given 0.1, 0.2, 0.3 (codeine)

a

Ref. (Reference).

K.A. George et al./ Forensic Science International 193 (2009) 212522

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with less than four surviving specimens were excluded from the

analysis because the sample size was too small to derive a replicate

mean; less than 6% of the total replicates were excluded. Of theremaining replicates, 89% contained more than 10 surviving

specimens.

Rates of larval development were determined by increases in

length and width of specimens. There was no significant difference

between mean replicate lengths and widths of larvae in each of the

four treatment groups at CI 1 (F3,34= 0.7, p= 0.55 and F3,34= 0.8,

p= 0.49, respectively; Fig. 2a) and CI 2 (F3,32= 0.8, p= 0.52 and

F3,32= 1.5, p = 0.24, respectively; Fig. 2b).

One-way ANOVAs showed no statistically significant differences

between treatments in length (F3,35= 1.9, p= 0.15) or width

(F3,35= 1.9, p = 0.11) of pupae (Fig. 2c). Pupariation day values

showed that pupariation occurred between days 8 and 10 with no

significant difference between treatment groups (x32 = 7.3,

p= 0.07).

There was no significant difference in adult size as indicated by

replicatemean costa length (x32 = 3.4,p= 0.33) andreplicate mean

tibia length (x32

= 6.0, p= 0.11) between treatment groups(Fig. 2d). Replicate mean day of adult eclosion were found to be

between days 19 and 22 with no significant difference between

treatments (x32 = 0.3, p = 0.97).

KruskalWallis analysis of survivorship found no significant

differences in the replicatemean numberof larvae surviving at CI 1

(x32 = 0.7, p= 0.87) and CI 2 (x3

2 = 5.7, p= 0.13). Replicate mean

survival of CI 4 specimensreachingadulthood was notsignificantly

different (x32 = 2.3, p = 0.52).

4. Discussion

This study utilised morphine-spiked pet mince to simulate

post-mortem concentrations observed following morphine over-

dose. Pet mince was also chosen in preference to overdosing a live

Fig.1. C. stygia larva withlarvallengthand width measurementsindicated by solidblacklines (a), C. stygia pupawith length andwidth measurements indicatedby solidwhite

lines (b) and costal and tibial length measurements indicated by solid white lines (c).

Fig. 2.Larval growth rates for treatments T 1T 4 at comparison intervals CI 1CI 4. (a) Mean replicate length and width SD for CI 1 (day 4) larva in all treatment groups. (b)

Meanreplicatelength and width SD forCI 2 (day 7)larvain alltreatment groups. (c)Meanreplicate lengthand widthSD forCI 3 pupa in alltreatment groups. (d)Meanreplicate

costal and tibial lengths SD of CI 4 adult specimens in all treatment groups.

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animal because too little is known about metabolism of morphine

in other animals. There are well-known examples of differential

metabolism of drugs between humans and common laboratory

mammals (e.g. cats and paracetamol [29,30], and rabbits and

nicotine [31]); therefore an animal model is not guaranteed to

simulate human morphine overdose. It is also important to isolate

the effects of morphine from the potential confounding factors of

secondary metabolites (especially considering that these could

differ between species). A number of studies have shown that

common laboratory animals produce up to five morphine

metabolites that are notseen during human morphine metabolism

[17,3236]. Both the rat and the rabbit produce dihydromorphi-

none, dihydromorphine and hydroxy morphines, while the guinea

pig produces these as well as morphine N-oxide and b- or g-isomorphine[17,3236]. Any of these metabolites could affect

blowfly larval growth. However, since they are not produced in

humans, any of the above animals should be used with caution in

this research. Additionally, the rates of metabolite formation and

excretion differ considerably between humans and other animals

[16]. This could also influence the results.

With respect to insect growth rates, this study determined that

the development of the native Australian blow fly, C. stygia, is

unaffected by pure morphine at the concentrations investigated (2,

10, and 20mg/g). This findingdiffers from otherstudies which useddifferent species and different feeding substrates (Table 1). The

highest morphine concentration investigated here (20mg/g) is7 mg/g higher than the highest dose used by Bourel et al. [8], andyet no developmental changes were observed. This finding may be

due to a difference in morphine metabolism between the study

species used [812,18,3748], andemphasises theinadvisabilityof

forensic entomologists extrapolating the results of entomotox-

icological studies between species.

The study conducted by Kharbouche et al.[11]focused mainly

on the changes in growth ofL. sericata caused by codeine, however,

analytical testing indicated that the metabolites norcodeine and

morphine were also present and may have been the agents

affecting growth. Goff et al. [18]focused on the growth effects of

heroin on B. peregrina using an overdosed rabbit as the larvalfeeding source. It is most likelythat theheroin rapidly decomposed

to morphine, codeine (caused by acetylcodeine impurities within

the heroin), and glucuronic metabolites (morphine-3-glucuronide,

morphine-6-glucuronide and codeine-6-glucuronide) within the

living rabbit before death[14]. Therefore, similar to the study by

Kharbouche et al.[11], developmental alterations could have been

caused by any of the aforementioned metabolites or by their

interaction. The current study concentrated only on the effects of

morphine as the major product of heroin/codeine metabolism in

order to isolate the effect of this compound on the study species.

Pupariation begins following prepupal emigration from a food

source. Each prepupa shrinks in size and forms a dark coloured

outer skin. This colour change signals that the pupal growth period

has begun[8,9,11,18]. The presence of morphine in the feedingsubstrate ofC. stygia did not alter the onset or duration of the pupal

period. This finding differs from the results of prior studies

conducted on other fly species. Goff et al. [18] determined that

heroin in the food source ofB. peregrinareduced the time taken to

pupariate in an approximately dose-dependent manner, however,

the duration of this stage lasted longer in heroin/morphine-fed

colonies than in control colonies. While studying the effects of

morphine onL. sericata, Bourel et al.[8]found that the time taken

for morphine-fed larvae to pupariate was between 6 and 28 h

longer than the time taken by control colonies. Interestingly,

Kharbouche et al. [11] outline a contrary result that the pupal

period ofL. sericatawas between 21 and 29 h shorter in colonies

that had been codeine/morphine fed. Some of these differences

between studies could be partly caused by variation in intervals

between replicate checks, however, differences in studyspecies are

also likely to contribute.

Initial blowfly growth rate research focused almost entirely on

larval and pupal growth rates. There has been minimal research

looking at theeffects of morphineon adult fly species encountering

drugs in a larval food source. Only one study has investigated the

effects of this drug on adult fly size. Bourel et al. [8] found no

significant difference between the length and weight of emerging

L. sericata adults that were fed as larvae on either control or

morphine dosed meat. These results concur with those obtained

here. This research suggests that C. stygia is an accurate model to

use when aging bodies containing morphine at the concentrations

used and highlights the need for further research into growth

effects caused by morphine metabolites.

Acknowledgements

This study was supported by a Deakin Central Research Grant

and by the School of Life and Environmental Sciences student

funding to KG and LG.

We would like to say thank you to two anonymous referees

whose comments improved this manuscript.

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