USE OF Lucilia cuprina WIEDEMANN (1830)
LARVAE FOR DETECTION OF HEROIN
METABOLITES AND IDENTIFICATION OF
HOST DNA
by
NOORATINY BINTI ISHAK
Thesis submitted in fulfillment of the requirements
for the degree of
Doctor of Philosophy
August 2016
ACKNOWLEDGEMENT
My greatest gratitude to Allah S.W.T for His blessings that give me an
enormous strength and spirit to complete my study. I also would like to take this
opportunity to thank my supervisor Professor Abu Hassan Ahmad for his idea,
guidance and advice during my research. Special thanks to my co-supervisor
Professor Siti Azizah Mohd Nor for her patience and support. Thanks to Dr. Nur
Aida Hashim, Dr Nurita Tahir and Dr Azwandi Ahmad for their enthusiastic
collaborative support and valuable help during my study. I would like to extend my
thanks to my employer, the Department of Chemistry, Malaysia for allowing me to
further my study and thanks for the cooperation extended to me. Last but not least I
would like to thank to my beloved parent, husband and my daughter for their
understanding, encouragement, support and love that they have given me since I
started this research.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENT ...…………..………..………..……………………
ii
TABLE OF CONTENTS ...……………………………..……..……….……..
iii
LIST OF TABLES …………………………………...………..………………
ix
LIST OF FIGURES ...…………….…………..………..……………………...
x
LIST OF ABBREVIATIONS ………………………………………………...
xiv
ABSTRAK ……………………………..……...……………………………….
xv
ABSTRACT ……………………………………………………………………
xvii
CHAPTER 1 - INTRODUCTION
1.1
General Introduction………………….………………………...…………
1
1.2
General Objectives ……………………………………….…………...…..
6
CHAPTER 2 - LITERATURE REVIEW
2.1
Forensic toxicology and entomotoxicology …………………………........
7
2.2
Entomotoxicology as a tool in medicolegal or forensic investigations …..
10
2.3
Heroin …………………………………………………………………….
12
2.3.1
Origin and history ……….……..……………..…………….…..
12
2.3.2
Heroin addiction and medicolegal cases associated with heroin
in Malaysia …….………..………..……………………………..
15
Blow flies …………………………………………………………………
16
2.4.1
The importance of blow flies in forensics………………..……..
16
2.4.2
Lucilia cuprina (Diptera: Calliphoridae) ……….……………....
17
2.4.3
Blowfly life cycle in relation to development on
corpses..…....……………………………………………………
17
2.4
iii
2.5
The importance of 3rd instar larval crop ………………………………….
19
2.6
Effects of drugs on fly development ……………………………………...
22
2.7
The importance of proper sample collection and selection of
toxicological analysis…...…………………………………………………
24
2.8
Methods of toxicological analysis of insect samples………...……...…….
26
2.9
Forensic DNA analysis …………………………………………………...
27
2.9.1
Mitochondrial DNA (MtDNA)...………………………………..
27
2.9.2
Detection of human DNA from larval crop …………………….
29
Single Nucleotide Polymorphism (SNPs) ………………………………...
30
2.10
CHAPTER 3 - DETECTION OF HEROIN METABOLITES IN THE 3RD
INSTAR LARVAL CROP OF Lucilia cuprina (DIPTERA:
CALLIPHORIDAE)
3.1
Introduction …………………………….………………………………....
33
3.2
Materials and methods ………………………...…………….....................
35
3.2.1
Buffalo DNA sequence confirmation using sequencing assay ...
35
3.2.2
Lucilia cuprina DNA sequence confirmation using sequencing
assay …………………………………………………………….
35
3.2.3
Maintaining stock culture for Lucilia cuprina ………………….
37
3.2.4
Application of heroin onto minced buffalo meat ……………….
37
3.2.5
Collection, preservation, and measurement of the length of
Lucilia cuprina ………………………….……………………....
37
3.2.6
Dissecting the crop of Lucilia cuprina for toxicological analysis
37
3.2.7
Toxicological analysis……….………………………………….
38
Results....……………..…….……………………………………………...
40
3.3
iv
3.3.1
Detection of heroin metabolites from Lucilia cuprina larval
crop ……………………………………………………………..
40
3.4
Discussion…. …………………….……………………………………….
47
3.5
Conclusion ………………………………………………………………..
50
CHAPTER 4 – EFFECTS OF HEROIN ON THE DEVELOPMENTAL
TIME AND THE GROWTH OF Lucilia cuprina
(DIPTERA: CALLIPHORIDAE) UNDER LABORATORY
CONDITIONS
4.1
Introduction ……………………………………….……………………....
51
4.2
Materials and methods ……………………………………………………
53
4.2.1
Maintaining a stock culture of Lucilia cuprina …...……………
53
4.2.2
Application of heroin onto minced buffalo meat ……………….
53
4.2.3
Statistical analysis ………………………………………………
55
Results …………………………………………………………………….
56
4.3
4.3.1
4.4
Larval length as a tool in estimating the duration of Lucilia
cuprina larval life cycle …………….………………………….
56
4.3.2
Influence of heroin on Lucilia cuprina larval development….....
60
4.3.3
Development time of Lucilia cuprina larvae reared under
laboratory conditions …………………………………………...
66
Discussion ………………………………………………………...............
70
4.4.1
Detection of heroin intake based on L. cuprina larval
development …………………………………………………….
4.4.2
4.5
Effect of different heroin concentrations on Lucilia cuprina
developmental time …………………………………………….
.
Conclusion………………………………………………………………...
70
v
72
75
CHAPTER 5 – IDENTIFICATION OF BUFFALO DNA SEQUENCES
FROM LARVAE, PUPAE AND ADULT OF Lucilia
Cuprina (DIPTERA: CALLIPHORIDAE) COLLECTED
FROM MINCED BUFFALO MEAT TREATED WITH
FIVE DIFFERENT CONCENTRATIONS OF HEROIN.
5.1
Introduction ……………………………………………………………….
76
5.2
Materials and methods………….. …………………………………...…...
78
5.2.1
Application of heroin onto minced buffalo meat ………………
78
5.2.2
Sample collection ………………………………….……………
78
5.2.3
DNA extraction ………………………………………………....
78
5.2.4
Measuring concentration and purity of extracted DNA product..
79
5.2.5
Electrophoresis, amplification and sequencing ………………..
79
5.5.6
DNA analysis……………………………………………………
81
Result……………………………………………………………………...
83
The effect of heroin on the yield of DNA at the 1st instar larval
stage …………………………………………………………….
84
The effect of heroin on the yield of DNA at the 2nd instar larval
stage …………………………………………………………….
85
The effect of heroin on the yield of DNA at the 3rd instar larval
stage …………………………………………………………….
86
5.3.4
The effect of heroin on the yield of DNA at pupal stages ……...
87
5.3.5
The effect of heroin on the yield of DNA at the adult stages …..
88
5.3.6
Identification of extracted DNA product obtained from larvae,
pupae, and adult of Lucilia cuprina……………………….…….
89
5.4
Discussion ………………………………………….……………………..
91
5.5
Conclusion………………………………………..……………………….
.
95
5.3
5.3.1
5.3.2
5.3.3
vi
CHAPTER 6 – MOLECULAR IDENTIFICATION OF HOST DNA FROM
ENTOMOLOGICAL EVIDENCE COLLECTED FROM
HUMAN AND ANIMAL FORENSIC CASES
6.1
Introduction ……………………………………………………………….
96
6.2
Materials and methods…………………………...………………...……...
99
6.2.1
Sampling procedure for animal forensic cases ..…….……...…..
99
6.2.2
Sampling procedure for human forensic cases …………………
100
6.2.3
Referral blood for DNA analysis ……………………………….
106
6.2.4
DNA extraction ………………………........................................
107
6.2.5
Measuring concentration and purity of extracted DNA product..
108
6.2.6
Electrophoresis, amplification, and sequencing ………………..
108
6.2.7
Animal DNA analysis …………………………………………..
110
6.2.8
Sequencing analysis of human DNA .…………………………..
110
Results …………………………………………………………................
111
6.3.1
Preliminary report for animal forensic cases …………..……….
111
6.3.2
Preliminary report for human forensic cases ……………..…….
116
Discussion…………………………………………………………………
123
6.4.1
Animal DNA sequence analysis………………………………...
123
6.4.2
Sequencing analysis of human DNA…………………………...
124
Conclusion………………………………………………………………...
129
6.3
6.4
6.5
vii
CHAPTER 7- DETECTION OF NUCLEOTIDE CHANGES IN BUFFALO
DNA SEQUENCE TREATED WITH FIVE DIFFERENT
CONCENTRATIONS OF HEROIN CONSUMED BY 3rd
INSTAR LARVAL OF Lucilia cuprina
7.1
Introduction ……………………………………………………………….
130
7.2
Materials and methods ……………… ………….………………………..
132
7.2.1
Application of Heroin onto minced buffalo meat……………….
132
7.2.2
Sample Collection ………………………………………………
132
7.2.3
DNA extraction………………………………………………….
132
7.2.4
Measuring concentration and purity of DNA extracted product..
132
7.2.5
Electrophoresis, amplification, and sequencing ………………..
132
7.2.6
DNA analysis……………………………………………………
132
Results……………………………………………………………………..
133
7.3
7.3.1
Nucleotide composition of complete Cyt b gene of Bubalus
bubalis…………………………………………………………...
133
Sequencing analysis on the minced buffalo meat with the
presence of drugs………………………………………………..
135
7.4
Discussion…………………………………………………………………
139
7.5
Conclusion………………………………………………………………...
142
7.3.2
CHAPTER 8 – SUMMARY AND RECOMMENDATION
143
REFERENCES
145
APPENDICES
viii
LIST OF TABLES
Page
Table 3.1:
The mass to charge ratio (m/z) and quality (%) of heroin
metabolite measured in larvae, pupae and adult stages of L.
cuprina reared on minced buffalo meat containing different
concentrations of heroin. m/z represents mass divided by charge
number and the horizontal axis in a mass spectrum is expressed in
units of m/z. m = mass, z = charge number of ions. ND= Not
detected; g = Gram; mL= millimeter …………………………......
43
Mean larval length (mm) + standard deviation of L. cuprina
larvae reared on minced buffalo meat treated with different heroin
concentrations under laboratory conditions (Temperature: 30°C 37°C, 24 hours of light period, and RH = 75%) …………...……..
57
The effects of five different concentrations of heroin on larval
length of L. cuprina …………………………….………...………
60
Summary of the best fit linear regression equations for body
lengths of L. cuprina larvae reared on minced buffalo meat
treated with different concentrations of heroin over time under
laboratory conditions (Temperature: 30ºC - 37°C). y = Duration
of life cycle; x = Larvae length when x must be more than 0, R2 =
the proportion of variance in the criterion that can be accounted
for by the predictor variables in combination …………………….
62
The time (mean + SD) from larval stages until pupal stages for L.
cuprina reared on heroin-treated minced buffalo meat and
untreated minced buffalo meat under laboratory conditions
(temperature: 30°C - 37°C; 24 hours of light period, and RH =
75%) ………………………………………………………………
67
% Similarity index (with GenBank/NCBI) Bubalus bubalis
sequence analysis from DNA amplified from L. cuprina larvae,
pupae, and adult collected from heroin-treated minced buffalo
meat on five different concentrations of heroin ranging from 500
ng/µLto 10000 ng/µL.…………………………………………….
90
Table 6.1:
Summary of the animal forensic cases received in this study ..…..
112
Table 6.2:
Summary of the human forensic cases received in this study…….
118
Table 7.1:
Summary of mutation report generated from Applied Biosystem
(Seqscape 2.5) observed from L. cuprina crop collected from
heroin-treated minced buffalo meat……………………………….
135
The IUPAC/IUB Codes (Applied Biosystems SeqScape software
v2.5 User Guide)…………………………………………………..
136
Table 4.1:
Table 4.2:
Table 4.3:
Table 4.4:
Table 5.1:
Table 7.2:
ix
LIST OF FIGURES
Page
Figure 2.1:
The common heroin metabolic pathway (Reisfield et al., 2009).
The arrow in yellow colour indicates the elimination of the
chemical group of heroin resulting in hydromorphone and
morphine………………………………………………………….
14
Figure 2.2:
The anatomy of fly digestive system (Imms, 1934) ……………
21
Figure 2.3:
Examples of mutations affecting a few base pairs, including
examples of transition and transversion substitutions (Jobling
et.al. 2013)………………………………………………………..
31
Transition and transversion mutations (Jobling et al.,
2013)……………………………………………………………..
32
Total ion chromatogram of Tryptophan 2,4-dimethoxy-5ethoxyamphetamine (3.74 m/z) in the 3rd instar larvae after
hydrolysis ………………………………………………………..
44
Mass spectral characteristics of Tryptophan 2,4-dimethoxy-5ethoxyamphetamine in the 3rd instar larvae after hydrolysis…….
45
Total ion chromatogram of Morphine (4.940 m/z) in the 3rd
instar larvae after hydrolysis …………………………………….
45
Mass spectral characteristics of Morphine in the 3rd instar larvae
after hydrolysis………………………………………..………….
46
Lengths of larvae for (A) 500 ng/µL of heroin-treated minced
buffalo meat, (B) 1000 ng/µL of heroin-treated minced buffalo
meat, (C) 2500 ng/µL of heroin-treated minced buffalo meat, (D)
5000 ng/µL of heroin-treated minced buffalo meat, (E) 10000
ng/µL of heroin-treated minced buffalo meat, and (F) 0 ng/µL of
untreated minced buffalo meat. N = 90 individual 1st instar
larvae for all heroin-treated minced buffalo meat, 120 individual
2nd instar larvae for all heroin-treated minced buffalo meat, and
800 individual 3rd instar larvae for all heroin-treated minced
buffalo meat. N = 30 individual 1st instar larvae for untreated
minced buffalo meat, 40 individual 2nd instar larvae for untreated
minced buffalo meat, and 210 individual 3rd instar larvae for
untreated minced buffalo meat …………………………………..
65
Flow chart of the extraction protocol by using Epicenter
MasterpureTM Complete DNA Purification extraction kit ………
82
Figure 2.4:
Figure 3.1:
Figure 3.2:
Figure 3.3
Figure 3.4:
Figure 4.1:
Figure 5.1:
x
Figure 5.2:
Figure 5.3:
Figure 5.4:
Figure 5.5:
Figure 5.6:
Figure 6.1:
DNA obtained from the 1st instar larval crop of L. cuprina
collected from heroin-treated minced buffalo meat at five
different concentrations of heroin ranging from 500 ng/µL (lanes
3 to 5), 1000 ng/µL (lanes 6 to 8), 2500 ng/µL (lanes 9 to 11),
5000 ng/µL (lanes 12 to 14), and 10000 ng/µL (lanes 15 to 17).
100 bp DNA ladder (lane M), control extraction (lane 18),
control PCR (lane 19), water (lane 1), and blank (lane 2)……….
84
DNA obtained from the 2nd instar larval crop of L. cuprina
collected from heroin-treated minced buffalo meat at five
different concentrations of heroin ranging from 500 ng/µL (lanes
3 to 5), 1000 ng/µL (lanes 6 to 8), 2500 ng/µL (lanes 9 to 11),
5000 ng/µL (lanes 12 to 14), and 10000 ng/µL (lanes 15 to 17).
100 bp DNA ladder (lane M), control extraction (lane 18),
control PCR (lane 19), water (lane 1), and blank (lane 2)……….
85
DNA obtained from the 3rd instar larval crop of L. cuprina
collected from heroin-treated minced buffalo meat at five
different concentrations of heroin ranging from 500 ng/µL (lanes
3 to 5), 1000 ng/µL (lanes 6 to 8), 2500 ng/µL (lanes 9 to 11),
5000 ng/µL (lanes 12 to 14), and 10000 ng/µL (lanes 15 to 17).
100 bp DNA ladder (lane M), control extraction (lane 18),
control PCR (lane 19), water (lane 1), and blank (lane 2)……….
86
DNA obtained from the whole tissue of L. cuprina pupae
collected from heroin-treated minced buffalo meat at five
different concentrations of heroin ranging from 500 ng/µL (lanes
3 to 5), 1000 ng/µL (lanes 6 to 8), 2500 ng/µL (lanes 9 to 11),
5000 ng/µL (lanes 12 to 14), and 10000 ng/µL (lanes 15 to 17).
100 bp DNA ladder (lane M), control extraction (lane 18),
control PCR (lane 19), water (lane 1), and blank (lane 2) ………
87
DNA obtained from the whole tissue of L. cuprina adult
collected from heroin-treated minced buffalo meat at five
different concentrations of heroin ranging from 500 ng/µL (lanes
3 to 5), 1000 ng/µL (lanes 6 to 8), 2500 ng/µL (lanes 9 to 11),
5000 ng/µL (lanes 12 to 14), and 10000 ng/µL(lanes 15 to 17).
100 bp DNA ladder (lane M), control extraction (lane 18),
control PCR (lane 19), water (lane 1), and blank (lane 2)……….
88
Victim found in a bloated stage of decomposition.
Entomological evidences, such as larvae, were collected from
the chest and around the face. (A) Decomposed body of a female
found near the chancellery of Universiti Sains Malaysia in
Penang. (B) A blowfly adult was found at the neck of the
decomposed body. (C) Fly larval found on the hand of the
decomposed body……………………………………………….. 102
xi
Figure 6.2:
Figure 6.3:
Figure 6.4:
Figure 6.5:
Figure 6.6:
Figure 6.7:
Figure 6.8:
Figure 6.9:
Victim in a bloated stage of decomposition. (A) A decomposed
body found in the car boot. Entomological evidences, such as
larvae, were collected at the chest. (A) A decomposed body
found in the car boot. (B) Fly larvae and adult found on the chest
of the decomposed body. (C) Fly larvae found on the head of the
decomposed body………………………………………………...
104
Charred body in advanced decomposition stages. (A) A
decomposed charred body was found in a palm oil estate. (B)
Fly larvae found under the neck. (C) Blowfly adult was found at
the skull of the decomposed body………………………………..
106
The amplification of cytochrome b gene of the 3rd instar larval
crop (case 1-Asian Palm Civet). Lane M: 100 base pair ladder,
lanes 1-3: extracted product of the 3rd instar larval crop, lane 4:
extraction control (beef), lane 5: blank, lane 6: water, and lane 7:
PCR control (chicken)……………………………………………
113
The amplification of cytochrome b gene of the 3rd instar larval
crop and the pupae (case 2- Chicken). Lane M: 100 base pair
ladder, lanes 1-2: extracted product of pupae whole tissue, lanes
3-5: extracted product of the 3rd instar larval crop, lane 6:
extraction control beef, lane 7: PCR control (lamb) lane 8: blank,
and lane 9: water…………………………………………………
114
The amplification of cytochrome b gene of the 3rd instar larval
crop and the pupae (case 3-Pig). Lane M: 100 base pair ladder,
lanes 1-3: extracted product of pupae whole tissue, lanes 4-6:
extracted product of the 3rd instar larval crop, lane 7: extraction
control (beef), lane 8: PCR control (lamb), lane 9: blank, and
lane 10: water………………………………..…………………...
115
The amplification of cytochrome b gene of the 3rd instar larval
crop (case 4-Buffalo). Lane M: 100 base pair ladder, lanes 1-3:
extracted product of the 3rd instar larval crop, lane 4: extraction
control (duck), lane 5: PCR control (chicken), lane 6: blank, and
lane 7: water………………………………………………...……
116
The amplification of hypervarivale 1 gene from 3rd instar larvae
crop and the blowfly adult collected from a bloated woman (case
1). Lane L: 100 base pair ladder, lane 1: water, lane 2: blank,
lane 3: human PCR control, lanes 4-6: extracted 3rd instar crop,
lanes 7-9: extracted whole adult tissue, and lane 10: control
extraction (beef)…………………………………………...……..
119
The amplification of hypervarivale 1 gene from 3rd instar larval
crop and the blowfly adult collected from a bloated male corpse
(case 2). Lane M: 100 base pair ladder, lanes 1-2: extracted
product of the 3rd instar larval crop, lanes 3-4: extracted product
of whole adult tissue, lane 5: PCR human control, lane 6: blank,
xii
Figure 6.10:
Figure 6.11:
Figure 7.1:
Figure 7.2:
lane 7: water, and lane 8: extraction control (beef)……………....
120
The amplification of hypervarivale 1 gene from 3rd instar larval
crop and the pupae collected from a bloated female corpse (case
3). Lane L: 100 base pair ladder, lanes 1-3: extracted product of
pupae tissue, lanes 4-6: extracted product of the 3rd instar larvae
(dead), lanes 7-9: extraction product of the 3rd instar crop (dead
larvae), lane 10: blank, lane 11: water, lane 12: PCR control
(human), and lane 13: extraction control (beef)………………….
121
The amplification of hypervarivale 1 gene from the 3rd instar
larval crop and the blowfly adult collected from a female charred
body (case 4). Lane L: 100 base pair ladder, lane 1: water, lane
2: blank, lane 3: human PCR control, lanes 4-6: extracted
product of whole adult tissue, lanes 7-9: extracted 3rd instar
larval crop (found on bone), lanes 10-12: extracted 3rd instar
larval crop (found near the skull), lanes 13-15: extracted 3rd
instar larval crop (found on the sand near the body), and lane 16:
control extraction (beef)……………………………………..…...
122
The amplification of DNA obtained from the 3rd instar larval
crop of L. cuprina collected from heroin-treated minced buffalo
meat at five different concentrations of heroin ranging from 500
ng/µL(lanes 3 to 5), 1000 ng/µL(lanes 6 to 8), 2500 ng/µL(lanes
9 to 11), 5000 ng/µL(lanes 12 to 14), and 10000 ng/µL(lanes 15
to 17). 100 base pairs DNA ladder (lane M), DNA extraction
control (lane 18), PCR control (lane 19), water (lane 1), and
blank (lane 2)……………………………………………
134
Figure 7.2: DNA sequence change in the mtDNA region from
359 sequences obtained for the heroin-treated minced buffalo
meat. Each polymorphism was reported from variation to the
untreated minced buffalo meat. Reference sequence is a DNA
sequence obtained from untreated buffalo minced meat. N = 5
individuals of L. cuprina larvae. The yellow colour indicates the
nucleotide changes that occur in the DNA sequence of B.
bubalis……………………………………………………………
138
xiii
LIST OF ABBREVIATIONS
cyt
Cytochrome
DNA
Deoxyrobonucleic Acid
HV
Hypervariable
IUPAC
International Union of Pure and Applied Chemistry
xiv
PENGGUNAAN LARVA LUCILIA CUPRINA WIEDEMANN (1830) UNTUK
MENGESAN METABOLIT HEROIN DAN PENGENALPASTIAN DNA
ABSTRAK
Kajian ini dijalankan untuk mengkaji keberkesanan larva peringkat 3 Lucilia
cuprina Wiedemann (1830) dalam analisis toksikologi untuk mengesan metabolit
heroin dengan menggunakan aplikasi GCMS dan dalam analisis molekul dengan
menggunakan Cytochrome b gen (mtDNA) untuk mengenalpasti jujukan DNA
daging kerbau cincang (yang telah digunakan sebagai bahan eksperimen dalam
kajian ini) tanpa heroin. Kajian yang dilakukan juga menyiasat kesan heroin kepada
tempoh kitaran hidup Lucilia cuprina. Selepas itu kaedah yang dibangunkan ini
digunakan dalam kes-kes forensik untuk mengkaji keberkesanan kaedah tersebut
dalam mengendalikan kes-kes forensik manusia dan haiwan. Analisis toksikologi
yang dilakukan ke atas larva peringkat 3 telah memberi keputusan morfin, iaitu
metabolit heroin. Heroin yang telah dicampurkan bersama dengan daging kerbau
cincang (Bubalus bubalis, Linnaeus, 1758) di dalam kepekatan yang berbeza telah
mempercepatkan pertumbuhan larva L. cuprina berbanding dengan larva pada
daging kerbau cincang tanpa heroin yang mempunyai pertumbuhan yang lebih
panjang. Analisis molekul yang dijalankan pada sistem usus larva peringkat 3,
berjaya mengamplifikasi jujukan DNA daging kerbau cincang iaitu Bubalus bubalis.
Penambahan heroin pada daging kerbau cincang didapati memberi perubahan pada
nukleotida pada jujukan DNA daging kerbau cincang yang diperolehi. Terdapat
sebelas (11) polimorfisme DNA yang berlaku pada jujukan DNA kerbau yang
diperoleh daripada sistem usus larva peringkat 3. Perubahan yang berlaku pada
jujukan DNA kerbau juga boleh menyebabkan perubahan pada jujukan asid amino
xv
dan juga jujukan protein. Analisis molekular yang dijalankan pada larva instar
peringkat 3 yang dijumpai di dalam kes forensik berjaya mengamplifikasi jujukan
DNA Gallus gallus, Linnaeus, (1758); Sus scropa, Linnaeus, (1758); Civet
(Paradoxurus hermaphrodite, Pallas, 1777); dan Bubalus bubalis, Linnaeus, (1758).
Selain daripada dapat mengesan jujukan DNA bagi kes-kes forensik haiwan, sistem
usus larva peringkat 3 yang ditemui dalam kes-kes forensik manusia juga berjaya
mengesan jujukan DNA manusia dan bahan bukti ini telah digunakan untuk
prosiding mahkamah. Larva peringkat 3 digunakan dalam kedua-dua kes forensik ini
disebabkan oleh larva peringkat 3 mempunyai kepanjangan yang maksimum dan
semua hasil makanan akan disimpan di dalam sistem usus larva peringkat 3 sebelum
memupa. Panjang larva peingkat 3 merupakan satu kriteria penting kerana pada
peringkat ini, tanaman dengan mudah boleh dilihat di bawah mikroskop dan dibedah
keluar. Kajian yang dilakukan dalam kes-kes forensik menunjukkan bahawa bukti
entomologi terutamanya pada peringkat larva boleh digunakan dalam mengenal pasti
DNA tuan rumah dalam keadaan ketiadaan bukti-bukti biologi.
xvi
USE OF LUCILIA CUPRINA WIEDEMANN (1830) LARVAE FOR
DETECTION OF HEROIN METABOLITES AND IDENTIFICATION OF
HOST DNA
ABSTRACT
This study investigates the capability of 3rd instar larvae of Lucilia cuprina
Wiedemann (1830) in toxicology analysis to detect the heroin metabolites by using
GCMS application and in molecular analysis by utilizing the Cytochrome b gene
(mtDNA) for precise identification of minced buffalo meat (which had been used as
experimental materials in this study) in the absence of heroin. The study conducted
also investigates the effect of heroin on the Lucilia cuprina duration of life cycle.
Then the developed method was applied in exact forensic cases to examine the
effectiveness of the developed method in handling both human and animal forensic
cases. The toxicology analysis conducted on the 3rd instar larval crop was
successfully detecting a metabolite of heroin, such as morphine. The heroin that was
mixed with the minced buffalo (Bubalus bubalis, Linnaeus, 1758) meat in different
concentrations, accelerated the duration of life cycle of L. cuprina larvae compared
to the larvae that were collected from the minced buffalo meat without heroin, which
had a longer life cycle. The molecular analysis conducted on 3rd instar larval crop,
succesfully amplify desired results of minced buffalo meat (with heroin) DNA
sequence, which is Bubalus bubalis. The addition of heroin was found to give an
impact, which reflected the nucleotide changes to the buffalo DNA sequences
obtained. Eleven (11) DNA polymorphisms were detected to occur in the buffalo
DNA sequences, which derived from the 3rd instar larval crop. The nucleotide
changes in the buffalo DNA sequence also led to some changes in protein sequences.
xvii
The molecular analysis carried out on the 3rd instar larval crop found in actual
forensic cases successfully amplify DNA sequences of Gallus gallus, Linnaeus,
(1758); Sus scropa, Linnaeus, (1758); Civet (Paradoxurus hermaphrodite, Pallas,
1777); and Bubalus bubalis, Linnaeus, (1758). Besides being able to detect the
animal DNA sequences in animal forensic cases, the 3rd instar larval crop found in
human forensic cases also managed to determine human DNA sequence and this
evidence has, in fact, been used for court proceedings. The 3 rd instar larval crop was
used in both cases because the 3rd instar larvae have a maximum length and all the
food are stored in the 3rd instar larval crop before larvae start to pupate. The length of
3rd instar larvae, indeed, is an important criterion because at this stage, the crop can
easily be seen under a microscope and dissected to remove the crop. The study,
which had been conducted in forensic case, displayed that entomological evidence,
especially the larval stages, could be used in identifying the host DNA in conditions
where biological evidence was absent.
xviii
CHAPTER 1
INTRODUCTION
1.1
General Introduction.
In the decomposition process of human remains, every stage of the decaying
process is associated with specific insects, starting from flies to beetles, and finally,
moths (Adair, 1999; De Jong & Chadwick, 1999; Wickham, 1902). However, the
most important insects in this process are flies from family Calliphoridae, especially
several genera from subfamily Chrysomyinae, as well as Lucilia spp. and Hemilucilia
spp. (Reed, 1958). This is because; they are the first insects to colonize a human
body post-mortem (Wells et al. 2001a). Flies play an important role in the
decomposition of carcasses or human remains because fly larvae consume dead soft
tissue. Thus, this makes the fly larvae important in forensic investigation because the
tissue they consume can be utilized in toxicological and DNA analyses, especially in
cases where other tissue samples are not available or viable. However, larvae
specimens for toxicological and DNA analysis in forensic cases can only be viable if
live larval specimens are collected and properly preserved to ensure that the integrity
of the specimens is not tempered with during the decomposition process.
Apart from analyzing biological evidence such as blood and tissue, more
recently, forensic investigations have begun applying entomological evidence based
on the larval crop (the foregut region of the larvae digestive system that stores food)
to determine the identity of the corpse by performed the sequencing analysis on the
larval crop. Besides identify the human identity, larval crop also can assess in drug
abuse cases by determine the level and type of drug that had consume by the victim
1
through the toxicology analysis conducted on the larval crop. By using the larval
crop found at the crime scenee in both analyses is the right decision in the absent of
biological evidence because the entomological evidence can be utilized in various
way to meet the forensic investigator purposes.
Furthermore, the approach of using larval crop in DNA and toxicological
analyses complements other approaches, such as blood analysis, which is expectly
not available at crime scenes that involve decomposed corpses. Moreover, the use of
entomological evidence namely larval crop (foregut area) in DNA and toxicology
analyses is practical because of its function as a food store, which in terms of
forensic investigation provides a valuable source of information. The data obtained
from the analysis of crop, such as the sequences of the victim‘s DNA (which can be
tested with possible relatives), could help investigators solve forensic cases by
pinpointing the exact crime scene through the larvae feeding period based on the
crop. In addition, the information gained from larval crop can also be used to
determine the human identity.
In the DNA and toxicology analysis, the 3rd instar larvae are selected among
the 1st and the 2nd instar larvae. This is because; the crop (food storage) of the 3rd
instar larvae would have reached a maximum size of body length and the crop is
visible and easy to remove for purposes of analysis. Moreover, at this stage, the 3rd
instar larvae would have stopped eating and would be preparing themselves to
pupate, as these attributes are absent in the 1st and the 2nd instar larvae. When the
process occurs indirectly, all the food would be retained in the crop. This implies that
all the information desired by the authorities also remained in the larval crop.
Nonetheless, this feature is not characterised by the 1st and the 2nd instar larvae. The
2
factors mentioned above are among the reasons on why the 3rd instar larvae are
suitable to be applied in most DNA analyses.
The changes in developmental rates, the extended periods of insect feeding
and the variation in size of the insect during the different stages of development
actually enables an investigator to analyse the larval crop in order to identify the
presence of toxic substances (Introna et al., 2001). The utilization of the 3rd instar
larval crop in toxicology analysis is now being considered as a useful substitute tool
in detecting drugs consumed by the human that caused the death. A lot of literature
have reported that different drugs can have different effects on insect developmental
rates, which would result in inaccurate PMI estimations that will, in turn, jeopardize
the outcome of a forensic investigation (Goff et al., 1989; Goff et al., 1991; Goff et
al., 1992, Goff et al., 1993; Goff et al., 1994).
In this study, heroin was used to validate the effect of the drug on L. cuprina
life cycle by using minced buffalo meat as an experimental subject. Heroin was
chosen because this drug can be easily obtained in the black market and besides, it is
being used as a recreational medicine to relief pain widely in Malaysia. Additionally,
there are various methods of taking this drug, for instance, snorting or injection, and
sometimes, overdose of heroin could lead to death. When death occurs and no
biological evidence is present, entomological evidence can be applied in identifying
the drug and in aiding the forensic investigation. Hence, this study was conducted to
validate the potential of applying larval crop in toxicology analysis for drug
determination.
Traditionally, fly larvae are used in estimating PMI because forensic flies
usually complete their entire life cycle from larvae to pupa on a corpse (Ross et al.,
3
1982). During the larval stages, the corpse tissues ingested by the larvae would fill its
crop before being digested. Therefore, the larval stage is the most suitable stage for
use in molecular analysis to identify host DNA (Linville et al., 2004). This can be
done by analysing the larval crop, especially to identify the identity of a corpse,
because what the larvae have fed on is stored in the crop (Coulson et al., 1990;
Gokool et al., 1993; Lord et al., 1998; Wells et al., 2001).
Besides, based on previous studies conducted by several scientists, it can be
concluded that the 3rd instar larvae offer a suitable stage to serve as an alternative
specimen in DNA analysis for determining species identification. For this critical
criterion, the 3rd instar larvae are indeed suitable to be applied, and moreover, with
the lack of biological evidence, larval crop is definitely the right choice and it is able
to answer all questions in forensic decomposed corpses. Therefore, a study was
conducted in DNA analysis to utilise the information gained from the larval crop for
minced buffalo meat identification.
Even though the use of larval crop may help in identifying the identity of the
victim, a few factors could hinder the accuracy of the DNA sequence analysis of the
victim. These include weather, temperature, storage conditions, preservation
solution, humidity, and the presence of drugs, which may alter the sequences of
DNA. Although a number of studies have been done on the effect of these factors,
the effect of the presence of drugs on the DNA sequence of flies is lacking. Thus, in
order to investigate if DNA sequence is altered in the presence of drugs, an a study
project was designed to investigate this parameter.
Furthermore, most of the previous studies were carried out abroad, hence,
there in a basis to increase awareness on the importance of larval crop, especially in
4
Malaysia. The data obtained are valuable to the Royal Malaysian Police because they
can help to solve forensic cases, especially those cases involving decomposing
corpses. Information gained from the DNA analysis on larval crop can also be used
to build the human identification database and reconnect with the possible relatives.
In addition, it is important for investigators to determine if there are drugs or
toxic substances in corpses before estimating PMI because the presence of drug or
toxic substances will alter the life cycle of the insect, thus jeopardising the PMI
estimation. Therefore, the present study was conducted to determine the effects of
different heroin concentrations on the developmental growth rate, the life cycle
duration of Lucilia cuprina larval on minced buffalo meat, and if the method
developed could be applied in forensic investigation as a new tool in Malaysia.
Generally, the objectives outlined in this study applied the larval of L. cuprina
for detection of heroin metabolites and identification of host DNA. In addition, this
study also observed the effects of heroin onto the length of L. cuprina larvae and in
the DNA sequence of B. bubalis. Then, the application developed in this study was
demonstrated in the real forensic case to examine the efficiency of the developed
method.
5
1.2
General Objectives.
i.
To study the utility of L. cuprina larval crop in toxicological analysis
of forensic cases.
ii.
To study the effect of heroin on the L. cuprina developmental time
iii.
To study the utility of L. cuprina larval crop in molecular analysis of
forensic cases.
iv.
To study the effect of heroin on DNA sequence of Bubalus bubalis.
6
CHAPTER 2
LITERATURE REVIEW
2.1
Forensic toxicology and entomotoxicology.
Toxicology can be narrowly defined as the study of the adverse effects of
chemicals, including drugs and poisons, on living organisms (Hodgson, 2004).
However, it is actually a broad field of study that encompasses various other fields
such as biology, biochemistry, histology, pharmacology, pathology and forensics
(Goff & Lord, 1994; Williams et al., 2000; Hodgson, 2004). For example, cases of
poisoning or drug overdoses might require the expertise of forensic pathologists,
biochemists, biologists and those from various other fields of study (Williams et al.,
2000). The applications of toxicology are numerous and one of the most important
applications of toxicology is in the field of forensic science which is generally
referred to as forensic toxicology. Forensic toxicology involves the application of
toxicology to cases and issues that have administrative or medicolegal consequences
and where the results are likely to be used in court (Levine, 2006).
According to Goff and Lord (1994), drug related deaths have been on the rise
in several countries. However, a cadaver is often found in various stages of
decomposition under different conditions and this makes the forensic investigation
difficult because of the absence of biological samples such as blood. In order to
overcome this problem, there are many toxicological analyses, techniques and
methodologies available to forensic scientists and investigators (Kintz et al., 1990a;
Pounder, 1991). Despite the availability of such methods, investigators still face
problems in obtaining an appropriate sample for the toxicology analysis because of
7
the aforementioned complications involving the condition of the cadaver. Therefore,
in this context, insects especially those that are in the immature stages are often used
as alternatives in the absence of body fluids or other biological samples from the
cadaver for toxicological analysis (Beyer et al., 1980; Kintz et al., 1990b). The mere
presence of insects at a crime scene provides valuable information such as post
mortem interval (PMI) estimation which can be used to facilitate a criminal
investigation (Beyer et al., 1980).
Insects have long been known for their significant role in determining postmortem interval in forensic investigations. However, the role of insects in forensics
has been expanded, whereby they are now used as tools in toxicological analysis for
detection of drugs to proof cause of death by drugs, especially in cases where there is
a lack of other biological evidence. This role expansion has come to be known as
entomotoxicology.
Entomotoxicology refers to the use of insects in toxicological analysis to
detect the presence of drugs or poisons (Introna et al., 2001). This field also includes
the study of the side effects caused by drugs and toxins on the development of insect
life cycles which is vital in estimating post mortem interval (PMI) (Goff & Lord,
1994). In addition, carrion insects present in cadavers can also be used in
toxicological analyses if there is no other biological evidence such as blood or urine.
This is because drugs, toxins and other chemical substances that are ingested, inhaled
or injected by the deceased prior to death will be present in the body post-mortem
and will be passed on to the carrion insects colonizing the corpse, starting from the
blow fly which is the first insect that colonises a corpse, to coleopterans which are
abundant during the advanced decay stage (Tracqui et al., 2004)
8
In certain circumstances, insects are proven to be a more suitable or optimal
sample for toxicological analysis compared to human tissue samples. Studies by
Kintz et al. (1990b) and Nolte et al. (1992) both demonstrated such a circumstance
clearly. In these studies, tissue degradation or products from the decomposition
process interfered with quantification of foreign substances in the human tissue
samples. The insect samples proved to be a more suitable sample for toxicological
analysis due to the lack of any decomposition interference.
The use of insects in detecting poisons has been documented as early as 1958,
in a study by Utsumi, in which he observed different groups of flies were attracted to
carcasses of rats that were previously killed by different poisons. In the late 70s,
Sohal & Lamb (1977; 1979) discovered accumulation of metals such as copper, iron
and zinc in adult housefly, Musca domestica. Nuorteva & Nuorteva (1982)
demonstrated a similar condition of metal accumulation in larvae, pupae and adults
of Calliphoridae and Staphylinidae. Several studies have shown that drugs present in
animals or humans prior to death could be detected in the various life stages of
necrophagous insect collected post-mortem (Goff et al., 1993; Miller et al., 1994).
These findings prove that foreign substances that enter the digestive system of
insects can be passed on through to the subsequent life stages in their life cycle and
thus provide information useful to medicolegal or forensic investigations.
9
2.2
Entomotoxicology as a tool in medicolegal or forensic investigations.
Entomotoxicological analysis can be applied to detect poisons and drugs to
facilitate and support medicolegal or forensic investigations. Nuorteva (1977)
reported that entomotoxicology findings were applied to a forensic case in Finland
involving a corpse found in a state of advanced decay. Based on the results of the
entomotoxicology analysis of flies collected from the corpse, investigators were able
to trace the geographical origin of the victim. Beyer et al. (1980) reported that
entomotoxicological analysis of carrion flies colonising the corpse of a woman who
had committed suicide showed the presence of barbiturates. In another case of
suicide by poisoning, toxicological analysis using gas chromatography showed the
presence of organophosphates (Malathion) in larvae of Calliphorids, Chrysomya
megacephala and Chrysomya rufifacies (Gunatilake & Goff, 1989).
Entomotoxicology analysis can even be applied in cases involving
mummified cadavers found long after death. Miller et al., (1994), found two types of
tricyclic antidepressants, amitriptyline and nortriptyline, in Phoridae puparial cases
as well as beetle (Dermestidae) skin casts and faecal material collected from the
mummified remains of a woman whose death occurred two years prior to finding the
remains. Leclercq & Brahy (1985) showed that entomotoxicology can also be
applied in investigation of accidental death due to overdose drug consume.
An insect is a treasure trove of information, not only for entomotoxicologists
but for forensic investigators as well. In the late 20th century, a myriad of studies
detected a variety of drug usage through toxicology analysis of larval tissue extracts,
larval crop contents, pupae and puparial casings (Kintz et al., 1990a, 1990b; Manhoff
et al., 1991; Nolte et al., 1992; Goff et al., 1992; Wilson et al., 1993; Kintz et al.,
10
1994; Miller et al., 1994; Sadler et al., 1995; Hedouin et al., 1999) and even in insect
faeces (Goff, 2000). Studies involving toxicological analysis of insects have been
continuous, whereby in the 21st century, Bourel et al. (2001) and other more recent
studies have focused on furthering knowledge in the field of entomotoxicology
(Gosselin et al., 2011; Souza et al., 2013; Verma & Paul, 2013).
These and many other studies show that insects can provide valuable and vital
information for forensic investigations. Through entomotoxicological studies,
various investigators have reported drug usage including heroin or morphine (Bourel
et al., 1996; Bourel et al., 1999); morphine and cocaine (Goff et al., 1989; Goff et
al., 1991; Kintz et al., 1994; Hedouin et al., 1999; Bourel et al., 2001),
benzoylecognine (Goff et al., 1989; Nolte et al., 1992), tricyclic antidepressants
(Wilson et al., 1993; Goff et al., 1993; Miller et al., 1994; Sadler et al., 1995),
amphetamine (Goff et al., 1992; 1997), benzodiazepines and phenotiazines (Kintz et
al., 1990a, 1990b; Sadler et al., 1997;. Carvalho et al., 2001), barbiturates and other
salicylates (Beyer et al., 1980; Sadler et al., 1997), bromazepam and
levomepromazine (Kintz et al., 1990a), malathion (Gunatilake & Goff, 1989),
phenobarbital, brazotam, oxazepam (Kintz et al., 1990b) and diazepam (Calvalho et
al., 2001).
11
2.3
Heroin.
2.3.1
Origin and history.
Diacetylmorphine, systematic IUPAC name (5α, 6α)-7,8-didehydro-4,5epoxy-17-methylmorphinan-3,6-diol diacetate, was developed in 1874 by the English
chemist, C.R. Wright and is derived from morphine. The compound was later
synthesized for commercial production by chemist Felix Hoffman who was working
for the German company Aktiengesellschaft Farbenfabriken, which today is the
pharmaceutical company Bayer (United Nations, 1953; The Sunday Times, 1998).
Diacetylmorphine did not become commonly known as ―Heroin‖ until Bayer
commercially produced and marketed it under the trademark name ―Heroin‖ from
1898 to 1910 as a cough suppressant and cure for morphine addiction. Unfortunately,
it was only later discovered that the drug rapidly metabolises into a form of morphine
that was twice more addictive. Although Bayer‘s role in introducing heroin to the
world has become a lesser known fact today, this historical blunder was a source of
great embarrassment for the company (The Sunday Times, 1998). Heroin was
eventually classified as an illegal drug in the United States as well as many other
countries (United Nations, 1953).
Like other opioids, heroin is used as a recreational drug which inevitably
leads to chronic addiction. Legally, diacetylmorphine (heroin) is prescribed as a
controlled drug and can only be supplied in tablet or injectable form with the same
indications as morphine (Besacier & Chaudron-Thozet, 1999). Normally, in
medicolegal investigations relating to drugs, blood or urine will be sampled and
quantitated to search for metabolites associated with drugs such as diacetylmorphine,
12
6-MAM, morphine, morphine-3-glucuronide and morphine-6-glucuronide (Baselt,
2008). There are a few chemical steps in the conversion of heroin to morphine as
shown in Figure 2.1- one group of OH (carboxyl) in the morphine chemical structure
is eliminated and replaced with one group of O (oxygen) producing hydromorphone
metabolite. Hydromorphone is one part of the minor metabolic pathways of heroin.
13
Figure 2.1: The common heroin metabolic pathway (Reisfield et al., 2009). The
arrow in yellow colour indicates the elimination of the chemical group of heroin
resulting in hydromorphone and morphine.
14
2.3.2
Heroin addiction and medicolegal cases associated with heroin in
Malaysia.
Globally drugs have been widely used for many centuries among Arab traders
and the phenomenon started the 8th century. Around this period, Arab traders
introduced drug usage to many Asian cultures during their explorations, trading or
war activities. This problem escalated with the colonisation of the Asian region by
many European countries, especially during the British rule. Malaysia was also very
much affected. Being a British colony, many immigrants were brought in to what
was then Malaya as cheap workers. Heroin was commonly used by these labourers to
maintain their strength doing hard labour. Unfortunately, it also came with
devastating after effects. The active compound of heroin is altered during usage and
cause addiction among the workers leading to further widespread abuse (Maisto et
al., 1995; Anonymous 1997; Kalant, 1997).
As this problem grew, since 1929 to 2015 almost 52313 heroin addicts were
registered in the Federal Malay States and this is estimated to reach 250000 by year
2015. In part the rapid progress and urbanization brought brought about by the social,
economical and political changes within the country (Khairuddin, 1994; Rusdi et al.,
2008). The increasing trend in drug addiction results in economic loss due to
ineffectiveness of many employees or potential ones leading to social ills and
therefore poses a threat to the future of nation. The increase in number of drug
addicts in Malaysia also causes an increase in drug related deaths. Most of the time
when a corpse is discovered after a period of time upon death, it is already in a
highly decomposed or skeletonized stage (Chawarski & Schottenfeld, 2006). In such
particular situation, there is in insufficient tissue for toxicology analysis and it is
15
impossible to detect drug or toxic substances in the absence of biological evidence.
However, the problem can be solved by analyzing the insects such as larvae or pupal
skin that are present around the corpse.
2.4
Blowflies.
2.4.1
The importance of blow flies in forensics.
Order Diptera is a group of insects with only a single pair of wings (forewings) while the hind-wings are reduced to small club-like structures called halteres.
The order is divided into three suborders; Brachycera, Nematocera and
Cyclorrhapha. However, the suborder that is most important in the field of forensic
science is Cyclorrhapha, while the others have their own importance as vectors of
diseases. In the suborder Cyclorrhapha there are three different families of flies,
which are of forensic importance; Calliphoridae (blowflies), Sarcophagidae (flesh
flies) and Muscidae (houseflies). The family Calliphoridae, also known as carrion
breeding flies, can be found during the early stages of decomposition, while
Sarcophagidae can be found during the subsequent stages of decomposition,
followed by Muscidae (Carvalho & Linhares, 2000).
According to Introna et al. (1998), the most common fly species occurring on
human corpses are from the family Calliphoridae. Each stage of human
decomposition is often associated with a certain insect, and it begins with flies
through to beetles, but Calliphorids have the most important role in forensic
investigations, especially in estimating post-mortem interval (Greenberg, 1991).
Among the most forensically important flies of the family Calliphoridae are the
16
genera Chrysomya, Lucilia and Hemilucilia due to these species being the first
colonizers of a human body upon death (Reed, 1958). Once flies colonise a corpse,
females will lay eggs which will eventually hatch and develop into the larvae, pupal
and adult stages. Thus, forensic investigators can estimate the post-mortem interval
based on the current life stage of the flies found on a corpse.
2.4.2 Lucilia cuprina (Diptera: Calliphoridae).
There are many fly species of importance in forensic science. However, one
of the main species for this purpose is the Australian sheep blowfly, Lucilia cuprina
(formerly Phaenicia cuprina) due to it being one of the first species that colonizes a
human body post-mortem. Generally, species of the genus Lucilia have a size range
of between 4.5 - 10 mm and a shiny green or reflective bronze-copper coloured
abdomen and therefore are also collectively known as the bronze bottle flies (Dress
& Jackman, 1998). The adults are easy to differentiate due to bristles present on the
meron of the thorax, and plumose or feathery aristae and the prominent hair on the
terminal segment of each antenna. Lucilia cuprina are most easily identified by their
strong dorsal setae and black thoracic spiracle (Durden, 1999.
2.4.3
Blowfly life cycle in relation to develop on corpses.
Like all dipterans, blowflies undergo holometabolous development with an
egg, three larval instars, and a pupal and adult stage (Ross et al. 1982). The eggs,
which are about 2 mm in length and white in colour, are usually oviposited in
17
clusters, after which they undergo a short embryonic developmental stage. On fresh
corpses, these clusters are usually found in the mouth, ears, nasal openings, and any
area where mucous membranes come into contact with the outside air. Wounds and
bruises are also common sites for the eggs to be deposited (Byrd & Castner, 2001).
After hatching, the larvae undergo three larval instar stages, commonly referred to as
larvae (Parr, 1962; Skidmore, 1985). Generally, fly larvae lack functional legs, are
cream in colour and soft bodied (Ross et al. 1982). The first instar is approximately 5
mm long after 1.8 days; the second instar is approximately 10 mm long after 2.5 days
while the third instar reaches approximately 17 mm long after 4-5 days (Parr, 1962;
Skidmore, 1985).
Identifying the correct instar stage is relatively easy based on the size of the
larvae, the size of their mouthparts and morphology of the posterior spiracles. A
larva has a pair of "hooks" in its mouth to help attach itself to its food source while it
feeds. Differences in the fine structure of the mouth hooks and spiracles are used to
differentiate among species as well as to determine the number of times it has shed
its skin (Salimi et al., 2010).
The third instar larva will shed its skin and form a dark brown puparia
measuring about 12-14 mm in length. This takes place 18-24 days after oviposition.
The skin contracts to a capsule-like shape and becomes stiff and hardened (newlymolted). Inside the capsule the living insect cannot move or feed, is pale white and
has rudimentary legs and wings and antennae. The hardened skin surrounding it is
called puparium (Apperson et al., 2011).
In forensic investigation the presence of pupae on a corpse, usually in the
folds of clothing, is critical evidence because the pupal stage being the oldest stage
18
would indicate to the forensic entomologist the precise stage of decomposition which
can then be accurately linked with the time of death. However, their presence is
sometimes overlooked or the investigator fails to detect the presence of pupae
(Rueda et al., 2010).
Newly emerged adult flies are pale in colour and soft to the touch, and have
crumpled, unexpanded wings upon emerging from the puparium. They later expand
their wings while the body becomes a green, bronze or blue colour. After they had
fully hardened (tanning process), the adult flies will begin to mate and produce the
next generation (Apperson et al., 2011). Once they have mated they are among the
most mobile creatures on earth (due to their size), and can travel for great distances
to find suitable sites to lay their eggs. Adult flies are always found around a corpse
but due to their mobility fully mature adults are worthless as forensic indicators as
their source is doubtful (Salimi et al, 2010).
2.5
The importance of 3rd instar larval crop.
The usage of larval insect crop has been potentially demonstrated in DNA
analysis for forensic purposes and this includes those of the lice (Mukabana et al.,
2002), mosquito, (Mumcuoglu et al., 2004) and carrion feeding flies (Zehner et al.,
2004). Among the larval stages, the 3rd instar larva which is the last stage before
changing into a quiescent pupa is the suitable stage to be applied in molecular
analysis and it has proven successful, especially in human identification process and
toxicological analyses. This is because food materials contained in larval crop
provide are directly associated with its last food intake before it transforms into a
19
pupa (Wells et al., 2001a; Wells et al., 2001b; Wells & Steven, 2008; Zehner et al.,
2004).
Due to its function a as food storage, larval crop can also be utilized in DNA
and toxicological analyses to detect the identity of the corpse, chemicals and drugs
that are present in the body. Since the larvae are the first arthropods that come to the
bodies after death, it can be concluded that the larvae are the first to consume the
biological tissues of the corpse. Thus, important information can be extracted from
the food materials are that stored in the larval crop. Therefore, because of this factor,
the larval crop is suited to be applied in both analyses mentioned above. From these
two analyses, the identity of the corpse and the presence of drug or chemical
substances can be traced.
Referring to the anatomy of the larval crop (Figure 2.2), the crop is located
between the mouth and stomach. All the materials consumed by the larvae will pass
through the crop and be stored until pupation. Thus, all materials contained in the
crop are maintained. So the whole tissue can be removed which can facilitate the
process of DNA extraction and deriving DNA (Campobasso et al., 2005). Therefore,
it is highly useful in forensic investigation if larvae are found at a crime scene in the
absence of body and biological tissues. The Royal Malaysia Police can employ this
application to resolve a chain of custody disputes, if when there are question whether
the larvae used in the post-mortem interval estimation has been moved from another
crime scene.
20
Figure 2.2: The anatomy of fly digestive system (Taken from Imms, 1934).
21
2.6
Effects of drugs on fly development.
There is no denying the fact that insects, particularly flies, are important in
forensics. However, various studies have also shown that drugs and toxic foreign
substances present in a corpse can affect the development of insects that colonise it.
This will expectedly affect the information obtained from insects, especially when
estimating postmortem interval which depends on the insect life cycle. A study
conducted by Gunatilake & Goff (1989), showed that the presence of Malathion
interrupted the post-mortem interval (PMI) estimation of a female corpse. They
observed that based on the development of the blowflies, Chrysomya megacephala
and C. rufifacies found on the corpse, post-mortem interval was estimated to be five
days. However, based on the stage of decomposition of the corpse, it was determined
to be eight days. The difference of PMI estimation was due to the presence of a toxic
substance which is Malathion.
Besides Malathion, Goff et al. (1989) have also studied the effects of cocaine
on the development of Boettcherisca peregrina (Sarcophagidae). They discovered
that B. peregrina developed more rapidly on rabbit tissue that contained cocaine and
concluded that if cocaine was present in human tissue, the PMI estimates that are
based on larval development could be in error by up to 24 hours. Mustava et al.,
(2001) studied the effects of different concentrations of hydrocortisone and sodium
methohexital on the development of Sarcophagi tibialis (Sarcophagidae) on chicken
liver. In the study, larvae of S. tibialis reared on chicken liver injected with different
concentrations of the two drugs had significantly different effects on the pupal
developmental time. Sarcophagi tibialis exposed to hydrocortisone experienced a
longer pupal stage while those exposed to sodium methohexital displayed a shorter
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pupal stage. This shows that drugs and foreign substances can have different effects
on the developmental rate of the various life stages of flies.
In a further study, Goff et al. (1991) studied the effects of drugs, specifically
heroin on the development of B. peregrina (Sarcophagidae) and concluded that the
differences observed in the rates of development were sufficient to alter PMI
estimates based on larval development by up to 29 h and estimates based on the
pupal development by 18 to 38 h. Other studies by Goff et al., (1989, 1991, 1992)
were focused on various other types of drugs such as methamphetamine and cocaine
and their effects on the development rate and growth of Parasarcophaga ruficornis
(Sarcophagidae).
Other studies have provided further proof which supports the findings of Goff
and Gunatilake (1989) and Goff et al. (1989), that drugs can accelerate fly
development. For example, Lord (1990) have estimated the post-mortem interval of
decomposed body of a female aged 20 was 7 days based on the life cycle of Lucilia
sericata (Meigen) (Calliphoridae) and Cynomyopsis cadaverina (RobineauDesvoidy) (Calliphoridae) that were collected from the female decomposed body.
However, the estimated PMI was questionable because basically these two flies‘
species take approximately 3 weeks to complete their life cycle. After a few
investigations, they found out that the decomposed female had taken an overdose of
heroin and thus accelerated the life cycle of these two flies. Bourel et al. 1999
studied the effects of morphine on the growth rate of L. sericata (Calliphoridae) on
rabbit tissue and similar to Goff et al. (1989), concluded that an underestimation of
PMI of up to 24 h can occur if the presence of morphine is not considered.
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Many of the aforementioned research have been conducted on flies that are
predominant to a geographical region or climate other than that of Malaysia. For
example, Lucilia sericata is not found in the tropics including Malaysia. Although
they are closely related, they are inherently different species with probable differing
habitual and geographical adaptations and therefore the results of studies on the
effects of drugs on L. sericata, which is more prevalent in the literature, cannot be
simply applied to L. cuprina which is more predominant in Malaysia. Thus, in order
to develop a locally applicable baseline for estimation of vital information such as
PMI, studies on the effects of drugs on insect development needs to be not only
specific to the common drugs found in Malaysia but also to the species of flies,
especially those in relation to medicolegal and forensic cases.
2.7
The importance of proper sample collection and selection of toxicological
analysis.
When conducting sample collection for forensic toxicological analysis it is
vital that a systematic and quality-assured procedure be strictly followed (Catts &
Haskell, 1990). This is to guarantee the chain of custody of evidence and prevent
contamination of the crime scene and destruction of evidence especially when
dealing with live insect samples. While at the crime scene, forensic entomologists
should wear overalls, gloves and shoe covers or boots (Amendt et al. 2007). There
are several effective ways to avoid sample contamination and wastage of which the
investigator needs to be aware, especially during sample collections. Blood, tissue or
dead organic samples should be immediately stored in appropriate containers, for
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