Forensic Entomology E-Book

Dorothy Gennard

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 467

Veröffentlichungsjahr: 2012

Beschreibung

Forensic Entomology provides undergraduates with a concise introduction to the subject. The book is written with the clarity necessary for students starting out in entomology yet authoritative enough to prove useful for more experienced researchers.

Worked examples of the necessary mathematics, including how to use excel to process data, coupled with lab protocols and self-assessment questions make the book an essential starting point in the subject.

Assuming little prior knowledge of either biology or entomology the book provides information on identification, life cycles and ecology of insects presented in a forensic context. Information is conveyed in an accessible style with practical tasks and suggestions for further reading included in each chapter.

• Fully revised and updated to include new research in the field
• New chapter on aquatic forensic entomology
• New pictorial key to aid identification of species contributed by Dr. Krzysztof Szpila, Nicolaus Copernicus University, Poland
• Further coverage of civil applications of forensic entomology
• Practical tasks and further reading included to aid understanding
• Colour plate section and improved illustrations throughout to assist in the identification of insects associated with the corpse
• Approaches the topic from the dual perspectives of basic entomology and its forensic applications
• Covers the contributions to forensic investigations of both flies and beetles
• Provides information on culturing insects collected from crime scenes
• Guides students through the processes of writing entomological court reports and presenting in court alongside the scientific topics
• Extended coverage of PMI calculations, role of professional associations for forensic entomologists and sampling at the crime scene
• New sections to discuss the identification of traces of explosives found in larvae, puparia and pupae and DNA sampling from insects

Leseprobe

Contents

Cover

Title Page

Copyright

List of plates

List of figures

List of tables

Preface

Acknowledgements

Chapter 1: The scope of forensic entomology

1.1 Forensic Entomology in Urban Contexts

1.2 Stored Product Infestation and Forensic Entomology

1.3 Forensic Entomology in the Medico-Legal Context

1.4 The History of Forensic Entomology

1.5 Professional Associations for Forensic Entomologists

1.6 The UK Regulator for Forensic Science

1.7 Web Addresses of Relevant Organisations

Chapter 2: Forensic entomology, DNA and entomotoxicology

2.1 Preparation of Specimens for Molecular Analysis

2.2 Methods of Analysis and Sources of Information

2.3 Alternative Methods

2.4 Validity of Methodologies

2.5 The Use of Other Molecular Means of Insect Species Determination

2.6 Insects and Entomotoxicology

2.7 Forensic Applications of Arthropod Behaviour for Chemical Analysis

Chapter 3: Insects and decomposition

3.1 Indicators of ‘Time of Death’

3.2 Stages of Decomposition of a Body

3.3 Volatiles Released From the Body During Decomposition

3.4 Decomposition in Specific Circumstances

Chapter 4: Identifying flies that are important in forensic entomology

4.1 What is a fly and how do I spot one?

4.2 The fly lifecycle

4.3 Forensically important families of flies

4.4 Members of other orders that have forensic relevance in aquatic cases

4.5 Review Technique: Larval Spiracles or Mouthparts – Preparation of Whole Slide Mounts

Chapter 5: Key for the identification of European and Mediterranean blowflies (Diptera, Calliphoridae) of medical and veterinary importance – adult flies

5.1 Introduction

5.2 Key

Chapter 6: Identifying beetles that are important in forensic entomology

6.1 What do Beetles Look Like?

6.2 The Life Stages of the Beetles

6.3 Selected Forensically Relevant Families of Beetles

6.4 Features Used in Identifying Forensically Important Beetle Families

6.5 Identification of Beetle Families Using DNA

6.6 Key to Selected Forensically Relevant Families in the Order Coleoptera

Chapter 7: Sampling at the Crime Scene

7.1 Entomological Equipment to Sample from a Corpse

7.2 Catching Adult Flying Insects at the Crime Scene

7.3 The Sampling Strategy for the Body

7.4 Sampling at Aquatic Crime Scenes

7.5 Obtaining Meteorological Data at the Crime Scene

Chapter 8: Rearing Insects and Other Laboratory Investigations

8.1 Transporting Entomological Evidence to the Laboratory

8.2 Laboratory Conditions for Fly Rearing

8.3 Methods of Maintaining and Rearing Insects – Terrestrial Species

8.4 Dietary Requirements of Insects Reared in the Laboratory

8.5 Beetle Rearing in the Laboratory

8.6 Methods of Maintaining Aquatic Species

Chapter 9: Calculating the Post Mortem Interval

9.1 Working Out The Base Temperature

9.2 Accumulated Degree Data

9.3 Calculation of Accumulated Degree Hours (Or Days) From Crime-Scene Data

9.4 Sources of Error

9.5 Use of Larval Growth in Length to Determine Post Mortem Interval (Isomegalen Diagrams and Isomorphen Diagrams)

9.6 Calculating The Post Mortem Interval Using Succession

9.7 The Effects of Hymenopteran Parasitoids On Post Mortem Interval Determination

9.8 Review Technique: Interpretation of Data From A Crime Scene Case Study

9.9 Further Reading

Chapter 10: Ecology of Forensically Important Flies

10.1 Ecological Relationships of Some Forensically Relevant Families

10.2 Specific Family Features

10.3 Fly Infestation of the Living

10.4 Flies Influencing the Crime Scene

Chapter 11: The Ecology of Some Forensically Relevant Beetles

11.1 Ecology of Carrion Beetles (Silphidae)

11.2 Ecology of Skin, Hide, and Larder Beetles (Dermestidae)

11.3 Ecology of Clown Beetles (Histeridae)

11.4 Ecology of Chequered or Bone Beetles (Cleridae)

11.5 Ecology of Rove Beetles (Staphylinidae)

11.6 The Ecology of Dung Beetles and Related Families

11.7 Ecology of Ground Beetles (Carabidae)

Chapter 12: Investigations in an Aquatic Environment

12.1 Decomposition and Submergence in Water

12.2 The Nature of the Water Bodies in Which Submergence May Take Place

12.3 Methods of Establishing Time Since Corpse Submergence – Indicator Species

12.4 Attractants to the Corpse

12.5 Methods of Culturing Aquatic Insects

12.6 Algae an Alternative Source of Determining Time Since Submergence

Chapter 13: The forensic Entomologist in Court

13.1 The Expert's Report

13.2 The Content of The Expert's Report

13.3 The Forensic Expert In The Courtroom

13.4 Communicating Entomological Facts In Court

13.5 Physical Evidence: Its Continuity And Integrity

13.6 The Code of Practice For Experts*

13.7 Use of Single Joint Experts

13.8 Practical Assignment – Writing An Expert Report Using The Post Mortem Calculations Generated From Chapter 9

13.9 Further Reading On Presentation In Court

13.10 Web site addresses

Appendices

Glossary

References

Index

Colour Plates

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Library of Congress Cataloging-in-Publication Data

Gennard, Dorothy

Forensic entomology: an introduction / Dorothy Gennard; with guest chapter by Krzysztof Szpila. – 2nd ed.

p.; cm.

Includes bibliographical references and index.

ISBN 978-0-470-68902-8 (cloth) – ISBN 978-0-470-68903-5 (pbk.)

I. Title.

[DNLM: 1. Entomology–methods. 2. Forensic Sciences–methods. 3. Insects–classification. 4. Postmortem Changes. W 820]

614′.1–dc23

2011043531

List of plates

2.1 Electropherogram

3.1 Decomposition of a human body

3.2 Fresh stage of decomposition

3.3 Bloat

3.4 Active stage of decay

3.5 Advanced stage of decay

3.6 Skeletal remains

3.7 A cadaveric island surrounding the body where vegetation has died away

4.1 A Tabanid larva

4.2 Emerging calliphorid with inflated ptilinum

4.3 Transition from larval stage L2 to larval stage L3

4.4 Puparial colour changes in Calliphora vomitoria Linnaeus within the first 25 hours of pupariation

4.5 A pair of Piophilids taken ‘in cop’

4.6 Head of Piophila casei Linnaeus

4.7 The Chironomidae

5.1A–F General morphology of blowflies. A – habitus, Chrysomya albiceps, male; B – head profile, Calliphora uralensis; C – thorax, dorsal view, Lucilia sericata; D – wing, Protophormia terraenovae; E – male terminalia, Lucilia bufonivora; F – ovipositior, Lucilia illustris.

5.2A–N Details of morphology of blowflies. A – calypters, Cynomya mortuorum; B – calypters, Lucilia caesar; C – greater ampulla, Chrysomya albiceps; D – greater ampulla, Cochliomyia hominivorax; E – habitus, dorsal view, Chrysomya marginalis; F – head profile, Chrysomya albiceps; G – head profile, Chrysomya megacephala; H – head profile, Phormia regina; I – anterior part of thorax, Chrysomya albiceps; J – abdomen, Chrysomya albiceps; K – anterior part of thorax, Chrysomya putoria; L – abdomen, Chrysomya putoria; M – calypters, Chrysomya bezziana; N – calypters, Chrysomya megacephala.

5.3A–K Details of morphology of blowflies. A – head profile, Cochliomyia hominivorax; B – calypters, Phormia regina; C – calypters, Protophormia terraenovae; D – calypters, Calliphora vicina; E – wing, Lucilia sericata; F – head profile, Cynomya mortuorum; G – head profile, Calliphora vicina; H – head profile, Calliphora vomitoria; I – head profile, Calliphora loewi; J – basicosta, Lucilia sericata; K – basicosta, Lucilia caesar.

5.4A–G A – occipital area, Lucilia sericata; B – occipital area Lucilia cuprina; C – head profile, Lucilia sericata; D – head profile, Lucilia silvarum; E – head profile, Lucilia caesar; F – head profile, Lucilia illustris; G – coxopleural streak, Lucilia caesar.

5.5A–T Male genitalia of Calliphorinae. Calliphora loewi: A – cerci and surstyli, lateral view; B – cerci and surstyli, dorsal view; C – phallus, lateral view; D – pregonite and postgonite, lateral view. Calliphora subalpina: E – cerci and surstyli, lateral view; F – cerci and surstyli, dorsal view; G – phallus, lateral view; H – pregonite and postgonite, lateral view. Calliphora vicina: I – cerci and surstyli, lateral view; J – cerci and surstyli, dorsal view; K – phallus, lateral view; L – pregonite and postgonite, lateral view. Calliphora vomitoria: M – cerci and surstyli, lateral view; N – cerci and surstyli, dorsal view; O – phallus, lateral view; P – pregonite and postgonite, lateral view. Cynomya mortuorum: Q – cerci and surstyli, lateral view; R – cerci and surstyli, dorsal view; S – phallus, lateral view; T – pregonite and postgonite, lateral view.

5.6A–P Male genitalia of Chrysomyinae. Chrysomya albiceps: A – cerci and surstyli, lateral view; B – cerci and surstyli, dorsal view; C – phallus, lateral view; D – pregonite and postgonite, lateral view. Chrysomya bezziana: E – cerci and surstyli, lateral view; F – cerci and surstyli, dorsal view; G – phallus, lateral view; H – pregonite and postgonite, lateral view. Chrysomya marginalis: I – cerci and surstyli, lateral view; J – cerci and surstyli, dorsal view; K – phallus, lateral view; L – pregonite and postgonite, lateral view. Chrysomya megacephala: M – cerci and surstyli, lateral view; N – cerci and surstyli, dorsal view; O – phallus, lateral view; P – pregonite and postgonite, lateral view.

5.7A–T Male genitalia of Chrysomyinae and Luciliinae. Chrysomya putoria: A – cerci and surstyli, lateral view; B – cerci and surstyli, dorsal view; C – phallus, lateral view; D – pregonite and postgonite, lateral view. Cochliomyia hominivorax: E – cerci and surstyli, lateral view; F – cerci and surstyli, dorsal view; G – phallus, lateral view; H – pregonite and postgonite, lateral view. Phormia regina: I – cerci and surstyli, lateral view; J – cerci and surstyli, dorsal view; K – phallus, lateral view; L – pregonite and postgonite, lateral view. Protophormia terraenovae: M – cerci and surstyli, lateral view; N – cerci and surstyli, dorsal view; O – phallus, lateral view; P – pregonite and postgonite, lateral view. Lucilia cuprina: Q – cerci and surstyli, lateral view; R – cerci and surstyli, dorsal view; S – phallus, lateral view; T – pregonite and postgonite, lateral view.

5.8A–V Terminalia of Luciliinae. Lucilia ampullacea: A – cerci and surstyli, lateral view; B – cerci and surstyli, dorsal view; C – phallus, lateral view; D – pregonite and postgonite, lateral view. Lucilia caesar: E – cerci and surstyli, lateral view; F – cerci and surstyli, dorsal view; G – phallus, lateral view; H – ovipositor, lateral view; I – pregonite and postgonite, lateral view. Lucilia illustris: J – cerci and surstyli, lateral view; K – cerci and surstyli, dorsal view; L – phallus, lateral view; M – ovipositor, lateral view; N – pregonite and postgonite, lateral view. Lucilia sericata: O – cerci and surstyli, lateral view; P – cerci and surstyli, dorsal view; Q – phallus, lateral view; R – pregonite and postgonite, lateral view. Lucilia silvarum: S – cerci and surstyli, lateral view; T – cerci and surstyli, dorsal view; U – phallus, lateral view; V – pregonite and postgonite, lateral view.

5.9A–O European and Mediterranean blowflies of forensic importance, general appearance (A-C, E, F, I-N phot. by T. Klejdysz). A – Calliphora loewi, B – Calliphora vicina, C – Calliphora vomitoria, D – Calliphora subalpina, E – Cynomya mortuorum, F – Chrysomya albiceps, G – Chrysomya marginalis, H – Chrysomya megacephala, I – Phormia regina, J – Protophormia terraenovae, K – Lucilia ampullacea, L – Lucilia caesar, M – Lucilia illustris, N – Lucilia sericata, O – Lucilia silvarum.

6.1Dermestes lardarius Linnaeus

6.2Dermestes maculatus DeGeer

6.3 A clerid beetle

6.4Nicrophorus humator Gleditsch

12.1 Chironomidae – blood worms

12.2 Caddisfly – cased

12.3 River with bankside vegetation

12.4 Hydrophilid larva

12.5 Hydrophilid beetle

12.6 Dytiscid beetle larva

12.7Gammarus sp., - an example of a shredder

12.8 Culicid pupa

12.9 Rat-tailed larva (Syrphidae)

12.10 Turtle – a scavenger

12.11 Gyrinidae larva (whirligig beetles)

List of figures

1.1 Stored product pests

1.2 Insect structural damage to wood

1.3 Body in wrappings

1.4 Necrophagous insects colonising a body

1.5 Predatory beetles will consume eggs and larvae of those flies colonising the body

1.6 Omnivores such as wasps will consume both the body and any insects present

1.7 Opportunist insects – a spider

1.8 An opportunist butterfly attracted to faecal material

2.1 Insect mitochondrial genome

2.2 Electrophoresis gel for RAPD Analysis of Fly DNA

3.1 Fresh stage of decomposition

3.2 Bloat

3.3 Active decay stage

3.4 Advanced decay

3.5 Skeletal remains

4.1 General morphology of the insect

4.2 Structure of the insect leg

4.3 Insect wing venation exemplified using the Needham Comstock venation naming system

4.4 The insect wing structure illustrating the position of the calypters

4.5 Examples of some of the forms of insect antenna

4.6 Classification hierarchy

4.7 A Nematoceran fly

4.8 An example of a member of the Brachycera – a Tabanid

4.9 The wing of a member of the infraorder Muscomorpha Aschiza illustrating the long anal cell

4.10 The remains of a ptilinum as a ptilinal suture

4.11 Occipital dilation present on Calliphora uralensis Villeneuve

4.12 (a) Copulating blue bottle flies (b) Clump of eggs

4.13 The plastron runs the length of the egg

4.14 The lifecycle of the blowfly

4.15 (a) Electronmicrograph of the tubercles (b) Posterior spiracles

4.16 An example of the anterior spiracle on a larva

4.17 Electronmicrograph of the puparium with detached operculum

4.18 Generalised insect growth curve

4.19 The thorax showing the position of the acrostichal bristles

4.20 The ‘tessselation’ (chequer board) effect on the abdomen of flies such as Calliphora vicina Robineau-Desvoidy

4.21 An example of a larval ‘head skeleton’ with oral sclerites

4.22Lucilia richardsi Collin

4.23 A fly showing the characteristic features of the Sarcophagidae

4.24 An electronmicrograph of a Piophilid

4.25 Hydrotaea larva

4.26 A pre-imaginal stage of Hermetia illucens Linnaeus

4.27 An aquatic larva: a member of the Stratiomyidae

4.28 The curved second anal vein characteristic of the Trichoceridae

4.29 An example of the features of a Tipulid including the drooping mouthparts

6.1 Generalised morphology of a beetle – dorsal view

6.2 The structure of the coleopteran thorax

6.3 The front legs of a beetle that have been modified for digging (Geotrupes sp.)

6.4 Examples of the shapes of beetle larvae

6.5 The types of pupa and puparia to illustrate the relationship of appendages to the body

6.6 Ventral view of the beetle thorax to illustrate the distinction between the Polyphaga (Figure 6.6a) and the Adephaga (Figure 6.6b)

6.7Nicrophorus humator Gleditsch

6.8 An example of a member of the Staphylinidae

6.9 A hister beetle

6.10 A trogid beetle

6.11 The front coxa of the dermestid projects from the coxal cavity

6.12 The lifecycle of Dermestes maculatus DeGeer

6.13 A clerid beetle

6.14 An example of a nitulid beetle

6.15 The striations on the elytra of a carabid

6.16 Electropherogram profile to allow comparison of the RAPD of a Silphid beetle to that of a calliphorid fly

7.1 Entomological crime scene equipment in a carrying case

7.2 Sealed, labelled bag containing entomological specimens collected at the crime scene

7.3 Retrieving a fly from a net

7.4 Label for the inside and outside of the collecting tube

8.1 Larvae from a crime scene are ideally reared in a controlled environment cabinet

8.2 Aquarium with pump

9.1 Insect growth in relation to upper and lower threshold temperatures

9.2 Base temperature determination using the linear approximation method

9.3 Graph to show the justification for using the accumulation averaged temperatures over time

9.4 Regression of crime scene temperature data against the meteorological station temperature to determine a correction factor for the period after the body was found

9.5 An insect transporting mites

10.1 Electronmicrograph of the head of an adult Piophilid

10.2 Piophilid infestation of meat

10.3 Fly faeces and vomit contaminate surfaces

11.1 Dermestid beetles

11.2 Faecal remains (frass) from dermestid beetles

11.3 Electronmicrograph of the ventral side of Dermestes maculatus DeGeer female and Dermestes lardarius Linnaeus male

11.4 Geotrupid beetle

12.1 Stoneflies

12.2 Ephemeroptera

12.3 Molluscs feed as scrapers

12.4 Nonbiting midges: an example of the Chironomidae

12.5 A Caddisfly larva

12.6a Corixid bug

12.6b Notonecta nymph

12.7 The riffles and pools of a river

12.8 Dytiscid beetle – a predator

12.9Ascellus sp. a shredder

List of tables

2.1 Examples of primers for cytochrome oxidase

3.1 Speed of decomposition of encoffined bodies in graves

3.2 Odour profile interpretation of decomposition volatiles during human decomposition recognised using the advanced sensor for detecting clandestine graves (LABRADOR)

8.1 Average minimum lifecycle durations of a selection of dipteran species at fixed temperatures

9.1 Lower developmental thresholds (base temperatures)

9.2 An example of the headings and completed spread sheet to calculate the ADD for Calliphora vicina Robineau-Desvoidy

9.3 The nature of the Excel spreadsheet

9.4 Daily temperature readings from a meteorological station for the period between 7 and 20 April

11.1 Beetle succession on decomposing corpses

13.1 Contemporaneous notes checklist

13.2 Suggested headings for a forensic entomology expert's report

Preface

This book is intended as an introduction to forensic entomology, including those aspects that relate to aquatic forensic entomology, in which insects and macroinvertebrates can be used to interpret crime scenes and provide an indication of time since submergence. The book is intended to provide a basic entomological introduction to forensic entomology, using examples from a range of countries. It includes practical activities to enhance understanding of the subject.

The second edition has benefitted greatly from the comments of a number of reviewers to whom I am most grateful. I have tried to respond to their suggestions. Any omissions were due to pressures of space. Forensic entomology has matured greatly as a science and a profession in recent years and owes much to the enthusiasm and energy of those running the professional societies. The work of the taxonomists has also strengthened the identification skills of practitioners and academics alike and their generously shared enthusiasm is of great value.

Alongside this, television series increasingly emphasise the role of insects in solving crime. Hopefully this textbook will therefore also be of interest to a broader audience and trigger an enthusiasm for insects that will encourage ‘students’ in the broadest sense to explore further both the forensic and entomological literature in this subject area.

Dorothy Gennard

Acknowledgements

I am grateful to the following for permission to reproduce illustrations or quote from publications:

Dr Arpad Vass and co-workers for permission to quote information from their unpublished report Award Number 2007-DN-R.104 (presented in this book as Table 3.2).

Dr Mark Benecke for permission to use and provision of a copy of his RAPD profile of Oiceophoma thoracicum Linnaeus and Calliphora vicina (Robineau-Desvoidy).

Professor Lin Field, The Royal Entomological Society of London, for permission to reproduce the mitochondrial genome from an antenna (presented in this book as Figure 2.1).

Dr Sharon Erzinçliolu for permission to quote the case of Mike Evans and Zoe from Erzinçliolu Y. Z., 2000 Maggots, Murder and Men. Harley Books: Colchester.

Dr Jonathan Cooter, Honorary Associate Curator at Oxford University Museum of Natural History, for permission to quote his comments on the distribution of Nebria ruficollis in the UK.

Elsevier, for permission to quote details of lower temperature limits for a number of flies, published in Marchenko M. L. K. 2001. Medico-legal relevance of cadaver entomofauna for the determination of the time of death. Forensic Science International120(1–2): 89–109 and a section of Forensic Science International (presented in this book as Table 9.1).

Elsevier for permission to reproduce a section of Figure 4 from Benecke M. 1998. Random Amplified Polymorphic DNA (RAPD) Typing of Necrophagous Insects, p.164, presented in this book as Figure 6.15.

Mr Michael Cohen Chairman Emeritus, The Academy of Experts and ‘The Academy of Experts’ for kind permission to reproduce the ‘Code of Practice for Experts 2005’ presented in chapter 13 of this book as section 13.6.

Dr A. Brigham and Rentokil Initial Plc, for permission to reproduce diagrams of insect larvae and pupa (presented in this book as Figures 6.4 and 6.5) from Munro J. W. 1965. Pests of Stored Products. The Rentokil Library, Benham and Co.: Colchester.

The Regents of the University of California for permission to reproduce a modification of Figure 4A from Wilson L. T., and Barnett W. W. 1983. Degree days: an aid in crop and pest management. California Agriculture (January–February) 4–7, presented in this book as Figure 9.3.

Dr Chris Pamplin, Editor, UK Register of Expert Witnesses, for permission to quote from the article ‘Expert witness immunity abolished’ from Expert Witness e-Wire, Wednesday 30 March 2011.

Warrant Officer 1st Thierry Pasquerault and Colonel F. Daoust of the Criminal Research Institute of the French National Gendarmerie for permission to use a photograph of packaging of entomological specimens recovered from a crime scene, presented as Figure 7.2 in this book.

Mr Ian Ward for permission to reproduce his photographs of flies, a dragonfly, and a butterfly along with a river bank scene: Figures 1.7, 1.8, 4.12a, 4.23, 9.5 and Plate 12.3 in this book).

I would like to thank the following people for their discussions, comments, and advice: anonymous referees on the proposal to prepare a second edition of Forensic Entomology: An Introduction; Mr Bill Barnett; Mr Keith Butterfield; University of Lincoln; Dr Trevor Crosby, Curator New Zealand Arthropod Collection, Landcare Research, University of Auckland; Dr John Esser; Mr Alex Pickwell; Dr Brett Ratcliffe, Curator and Professor, Systematics Research Collections, University of Nebraska, Lincoln, USA; Mrs Kate Stafford; Janet L. White, Executive Editor California Agriculture; Dr Lloyd T. Wilson Texas A&M University; Dr Laura Woodcock and Dr Frank Zalom, University of California.

I am most grateful to Fiona Seymour and Jasmine Chang of John Wiley & Sons Ltd. and Prakash Naorem of Thomson Digital, for their help, encouragement, and support during the preparation of the second edition of Forensic Entomology: An Introduction.

Dr Darren Mann and Mr James Hogan of the Hope Entomological Collections, Oxford University Museum of Natural History, kindly provided dipteran specimens for photography on extended loan, for which I am extremely grateful.

Dr M.I. Saloa for permission to reproduce her photographs of a larva of Hydrotaea sp. (Figure 4.25) and a pre-imaginal stage of Hermetia illucens (Figure 4.26) in this publication.

I am grateful to Mr Alex Pickwell for allowing the use of his macroinvertebrate specimens for photography and Mr Richard Chadd for confirming identifications. To Mr James Coulter and Mr Alex Pickwell for the opportunity to return to the field of aquatic ecology, including carrying out fieldwork. I am especially grateful to Mr David Padley, formerly photographer with the Lincolnshire Police and lecturer at the University of Lincoln, for his excellent photography of the dipteran specimens, and to Mr Ian Ward for his willingness to allow his photographs to be used as illustrations in this book.

Chapter 1

The scope of forensic entomology

Forensic entomology is a branch of forensic science. Forensic entomologists use information about insect lifecycles and behaviour to help interpret evidence in a legal context relating to both humans and wildlife. On occasion, the term ‘forensic entomology’ is expanded to include other arthropods, mites, spiders, or macroinvertebrates such as freshwater shrimps. The legal contexts in which forensic entomology is of use relate to matters considered in either civil courts or criminal courts. The cases that are heard in civil courts most frequently relate to insect infestation in urban contexts or in relation to stored product pests. Where there is insect infestation of a body, either living or dead, and foul play is thought to have occurred, or a law has been broken, then the case is generally termed a medico-legal case. Such cases can relate to both humans and wildlife.

1.1 Forensic Entomology in Urban Contexts

Cases of infestation of homes or other buildings, such as hospitals, are instances in which forensic entomologists may have a role to play. For example, where structural timber is found to harbour insects such as longhorn beetles (Cerambycidae) an entomologist might be called to assist in determining the cause and source of infestation. Such insects are generally pests of sapwood, but can complete their lifecycle in dry wood that has been harvested. An example of such a beetle is Eburia quadrigeminata (Say), the ivory marked beetle, a longhorn beetle, which usually attacks living American oak trees but has been known to survive felling, wood treatment and transformation of the wood into furniture, only to emerge some 10 to 15 years later. In 2007, Cocquempot recorded an instance of this species having been caught when emerging from a bamboo stand in France.

Other examples of the urban application of forensic entomology relate to infestation of food premises and food production sites. For example, the owners of a butcher's shop in London, was closed in January 2010. The Magistrates' Court awarded costs of £560 to the council after meat on sale at the butchers had been found to be infested with maggots and fly eggs.

Poultry production units may similarly be convicted of causing fly infestations that affects residents living nearby. Such was the case in a small Lincolnshire village where, in 2009, the farm owners were fined £20 000 by Skegness Magistrates court, having pleaded guilty to breaching an abatement order, intended to reduce the numbers of flies, which had been put in place in 2008 for a similar misdemeanour.

1.2 Stored Product Infestation and Forensic Entomology

In general, only a small number of stored product pest species may be encountered by the forensic entomologist. They include flies, cockroaches, ants, and beetles. The insects that inhabit animal products and their waste include members of families such as larder beetles (Dermestidae), Moth flies (Psychodidae), Scuttle flies (Phoridae), Muscid flies (Muscidae) Blowflies (Calliphoridae) and Flesh flies (Sarcophagidae) such as Sarcophaga carnaria Linnaeus, ants (Formicidae) such as the Pharaoh ant (Monomorium pharaonis Linnaeus), or the Copra beetle (Necrobia rufipes DeGeer). These may also be cited in medico-legal cases (Figures 1.1 and 1.2).

Figure 1.1 Stored product pests

The following are examples of phytophagous insects, which may infest food, resulting in forensic entomologists contributing to court cases.

Biscuit beetles (Stegobium paniceum Linnaeus) will infest not only food items such as flour bread and biscuits but also wool, hair and leather material. The saw-toothed grain beetle (Oryzaephilus surinamensis Linnaeus) and the Indian meal moth (Plodia interpunctella Hubner) both infest dried fruit, breakfast cereals and pasta. The Indian meal moth is also known to consume dried dog food and fish food products. The rust-red flour beetle (Tribolium castaneum Herbst) and the confused flour beetle (Tribolium confusum Jacquelin du Val), infest grain and flour. The fruit fly (Drosophila melanogaster Meigen) will infest any fermenting fruit or vegetable, including tomatoes, onions, and bananas. It can also inhabit compost heaps and piles of rotting garden waste and so could be the subject of urban forensic entomology cases.

Because food-processing plants have difficulty in reducing insect infestation levels to zero, a legal tolerance level is specified in many countries, contravention of which can lead to prosecution. For example, the American Food and Drug Administration considers that, in canned citrus juice, a maximum of five or more eggs of Drosophila or other insects per 250 ml is allowable. The presence of one maggot per 250 ml of canned citrus juice is also considered acceptable (AOAC 970.72).

In the majority of instances in stored product and urban forensic entomology, the main focus of the contribution is confirmation of the identity of the insect species and interpreting its biology in the particular context in question. In such instances this aspect of forensic entomology is confirmatory and relates to the work of the Environmental Health Department or the Office of Trading Standards.

Figure 1.2 Insect structural damage to wood

1.3 Forensic Entomology in the Medico-Legal Context

Insects have a role in crime scene investigations on both land and in water (Anderson, 1995; Erzinçliolu, 2000; Keiper and Casamatta, 2001; Hobischak and Anderson, 2002; Oliveira-Costa and de Mello-Patiu, 2004: Moretti, Bonato and Godoy, 2011). The majority of medico-legal cases where entomological evidence is used are the result of illegal activities that take place on land and are discovered within a short time of being committed. In France for example, 70% of cadavers are found outdoors and, of these, 60% are discovered within less than one month (Gaudry et al., 2004).

Figure 1.3 Body in wrappings

All insects could be of potential relevance to a medico-legal question, however a number of species from several families are found more often than others. The insects of particular relevance to forensic entomological investigations include blow flies, flesh flies, cheese skippers, hide and skin beetles, rove beetles and clown beetles. In some of these families only the juvenile stages are carrion feeders and consume dead bodies. In others both the juvenile stages and the adults will feed on the body (are necrophages). Yet other families of insects are attracted to the body solely because they feed on the necrophagous insects that are present. Forensically relevant insects can be grouped into four categories based on feeding relationship. These are:

Necrophages, which feed only on the decomposing tissue of the body or body parts – for example blowflies, hide beetles and clown beetles (Figures 1.4 and 1.5).Predators (and parasites) of the necrophages – for example rove beetles and ground beetles.Omnivores that consume both the live insects inhabiting the corpse and the dead flesh – for example ants (Formicidae), and wasps (Figure 1.6).Opportunist (adventive) species, which arrive because the corpse is a part of their local environment – for example mites, hoverflies, butterflies and, on occasion, spiders (Figures 1.7 and 1.8).

Figure 1.4 Necrophagous insects colonising a body

Figure 1.5 Predatory beetles will consume eggs and larvae of those flies colonising the body

Figure 1.6 Omnivores such as wasps will consume both the body and any insects present

Figure 1.7 Opportunist insects – a spider. Source: Reproduced by permission of Mr. Ian Ward

On occasion waste material or faecal material may be the attraction (Figure 1.8). The roles of specific species which have these feeding strategies will be considered in later chapters.

Figure 1.8 An opportunist butterfly attracted to faecal material. Source: Reproduced by permission of Mr. Ian Ward

1.4 The History of Forensic Entomology

Insects are known to have been used in the detection of crimes over a long period of time and a number of researchers have written about the history of forensic entomology (Benecke, 2001; Greenberg and Kunich, 2002). The Chinese used the presence of flies and other insects as part of their crime-scene investigative armoury and instances of their use are recorded as early as the mid-tenth century (Cheng, 1890, cited in Greenberg and Kunich, 2002).

Such was the importance of insects in crime-scene investigation that in 1235, a training manual on investigating death, Washing Away of Wrongs, was written by Sung Tz'u. In this early medico-legal book it is recorded that attention paid by a number of blowflies to a particular sickle caused a murderer to confess to murdering a fellow Chinese farm worker with that sickle.

Between the thirteenth and nineteenth centuries a number of developments in biology laid the foundation for forensic entomology to become a branch of scientific study. The two most notable were, perhaps, experiments by Redi (1668), an Italian who, using the flesh of a number of different animal species, demonstrated that larvae developed from eggs laid by flies, and the work by Linnaeus (1735) developing a system of classification. In so doing, Linnaeus provided a means of insect identification (including identifying such forensically important flies as Calliphora vomitoria Linnaeus). These developments formed foundations from which determination of the length of the stages in the insect's lifecycle could be worked out and the indicators of time since death could be developed.

A particularly significant legal case, which helped establish forensic entomology as a recognised tool for investigating crime scenes, was that of a murdered newborn baby. In 1850 a baby's mummified body, encased in a chimney, was revealed behind a mantelpiece in a boarding house when, during renovation work, Dr Marcel Bergeret carried out an autopsy on the body and discovered larvae of a flesh fly, Sarcophaga carnaria and some moths. He concluded that the baby's body had been sealed into the chimney in 1848 and that the moths had gained access in 1849. As a result of this estimation of the time since death, occupiers of the house previous to 1848 were accused and the current occupiers exonerated (Bergeret, 1855).

The next significant point in the history of forensic entomology resulted from observations and conclusions made by Mégnin (1894). He related eight stages of human decomposition to the succession of insects colonising the body after death. He published his findings in La faune des cadavres: Application de l'entomologie à la médicine légale. These stages of decomposition were subsequently shown to vary in speed and to depend upon environmental conditions, including temperature and, for example, size of the corpse and whether or not the corpse was clothed. The similarity in overall decomposition sequence and the role of insect assemblages in decomposition has been demonstrated for a number of animal species.

Knowledge about insect succession and the periods of insect activity on a corpse has become the basis for forensic entomologists' estimations of time since death, although this is acknowledged as being based on assumptions of time of colonisation relative to the point of death. Research continues to be required in order to establish the accuracy levels of estimates of time since death and to interpret variation in different biotopes (Tomberlin et al., 2011).

In the twentieth century, insects were shown to be of value in court cases involving insect colonisation of body parts recovered from water and not just for entire corpses found on land. On 29 September 1935 several body parts, later identified as originating from two females, were recovered from a river near Moffatt in Scotland. The identities of the deceased were Isabella Kerr, the wife of a Dr Ruxton, and Mary Rogerson, the family's ‘nanny’. The presence of third instar larvae of the blowfly Calliphora vicina Robineau-Desvoidy indicated that the eggs had been laid prior to the bodies being dumped in the river. This information, combined with other evidence, resulted in the conviction of Dr Buck Ruxton, for the murder of his wife and Mary Rogerson.

The level of acceptance of forensic entomology by the courts has depended upon the results of scientific study. This has been carried out since the early twentieth century, both by academics and practitioners working alongside the police and legal authorities. As a result the subject base has been refined and protocols and rigorous forensic procedures have been developed to raise its level of esteem. However, there remain several areas for which accurate information and levels of error remain undetermined. These aspects of uncertainty with respect to forensic entomology will be addressed with focused research. The requirement for this to happen quickly is dictated both by the professional aspirations of the forensic entomology community and also as a result of reviews and legislation.

In the USA the report concerning the whole of forensic science produced by the National Research Council, was significant. The council recommended that an independent federal organisation – the National Institute for Forensic Science, be set up in order to establish mandatory standards for laboratories, the promotion of scholarly, peer-reviewed research and the establishment and reinforcement of methods of best practice and the use of standardised protocols. A similar approach was taken in the United Kingdom and in 2007 the Office of the Forensic Science Regulator was set up to help establish and maintain standards in forensic science in general. These national organisations influence the work and aspirations of the professional associations who respond to guidance that they provide.

Forensic entomologists in a number of countries have set up professional organisations to provide a forum for the exchange of ideas and experience and to develop and maintain professional standards in forensic entomology. These organisations include the North American Forensic Entomology Association and the European Association for Forensic Entomology (EAFE).

1.5 Professional Associations for Forensic Entomologists

The nature and aspirations of two major professional associations for forensic entomologists are described below.

1.5.1 North American Forensic Entomology Association (NAFEA)

This organisation is a charitable, non-profit-making educational organisation for the promotion of good practice and research in forensic entomology. It had its first annual meeting in 2003 and seeks to collaborate with other international societies to enhance the moral, ethical and scientific base of forensic entomology. It currently has over 60 members. The strength of the organisation is its inclusivity. To quote its web site (www.nafea.net/, accessed 26 October 2011):

NAFEA is an organization for anyone interested in the application of forensic entomology to civil or criminal matters of law, research on arthropods of forensic importance, or carrion ecology.

From a student perspective it is also a valuable source of support, and conference funding may be available to student members. The organisation seeks to promote good practice and the presentation of scientific research, casework, and cooperative ideas on forensic entomology. As such, it is a forum through which research in forensic entomology can receive peer-review and new developments in approach can be discussed.

1.5.2 European Association for Forensic Entomology (EAFE)

The European Association for Forensic Entomology (EAFE) was founded in 2002. The Association was launched in France and has a number of aims:

To seek a common protocol for forensic entomology case investigation.To foster high standards of competency in specimen collection and analysis.To create a solid scientific basis so that forensic entomology can be a valid analytical tool.

In 2006, EAFE produced a protocol of good practice in order to ensure that the methods used in forensic entomology investigations at a crime scene could be standardised and good forensic entomological practice could be developed by following standard operating procedures. Its annual meetings also provide an opportunity for dialogue, discussion and collaboration.

1.6 The UK Regulator for Forensic Science

The regulation and maintenance of standards for forensic expert witnesses is currently voluntary and based on the membership of such organisations as the Academy of Experts or the Institute of Expert Witnesses. In the UK in 2007, the Office of the Forensic Science Regulator was set up by the Home Secretary to operate on behalf of the criminal justice system.

The purpose of the Office of the Regulator is primarily to i) determine new and improved quality standards for organisations and if necessary to take the lead in their development; ii) advise and guide organisations undertaking forensic analysis to ensure that they can show compliance with the generally accepted standards that may be required by the courts; iii) ensure sure that that there are appropriate arrangements for quality assurance and standards monitoring; iv) ensure that there are procedures in place for the determination competence of the individual forensic scientist.

The Regulator is supported in this role by a Forensic Science Advisory Council (FSAC). Amongst other things this committee is responsible for offering advice on accreditation and procedures for validating and approving new technologies. They also have the responsibility ‘for tasking and overseeing the work of Expert Working Groups established to advise on or develop quality standards . . .’

Currently, forensic organisations are required to be accredited through the United Kingdom Accreditation Service (UKAS) in order to conduct their work and to observe ISO9000 guidelines. These organisations are required to quality control their work and a number of options including participation in blind trials have been proposed.

The accreditation of individuals is under discussion because it is the individual and not the organisation that appears as a witness and is responsible to the court. At present forensic entomologists are not included on the list of expertise that is being considered. Those forensic disciplines that are on the list include more laboratory-based experts such as toxicologists, fingerprint officers, and document examiners. Membership of professional organisations, for the forensic entomologist, therefore remains an important means of standardising operating procedures and ensuring and also demonstrating that good practice is maintained.

1.7 Web Addresses of Relevant Organisations

European Association for Forensic Entomology (EAFE): www.eafe.org (accessed 26 October 2011).

North American Forensic Entomology Association (NAFEA): www.nafea.net (accessed 26 October 2011).

The Forensic Science Regulator

http://www.homeoffice.gov.uk/agencies-public-bodies/fsr/ (accessed 26 October 2011).

Chapter 2

Forensic entomology, DNA and entomotoxicology

Molecular identification of insects feeding on corpses can be an important technique in forensic entomology, particularly if indeterminate larval species are recovered at a crime scene. Analysis is frequently carried out by molecular biologists, although the answers are interpreted by entomologists. In casework, the life stages collected from a corpse are reared to the adult stage in order to identify the species using morphology. This is a slow process so using molecular methods, alongside morphological identification may, on occasion, be a more rapid and accurate way of providing the basis for determining the PMI.

Many forensically relevant molecular techniques were originally developed to investigate insect phylogeny and particular genetic profiles have been constructed for individual species. An example of the combined use of molecular and morphological techniques is provided by Pai et al. (2007) who used them to identify larvae and determine the PMI for a murdered Taiwanese girl whose burned body was recovered from a sugarcane field. In combination the techniques confirmed that the colonising fly was Chrysomya megacephala Fabricius.

Potential molecular biology identification methods range from using chromosome C-banding (Angus, Kemeny and Wood, 2004), for example, on the one hand, to using genomic material on the other. Genetic material can be harvested from both the nucleus and from mitochondria. Mitochondrial DNA (mtDNA) is the more frequent source of genetic information, not least because more DNA is available.

Mitochondria are haploid structures with genetic material solely from maternal origins. No recombination occurs in its manufacture. The mitochondrial genome contains around 16 000 base pairs of double-strand DNA (Lessinger et al., 2000) and is a stable source of genetic information. Cells contain a large number of mitochondria and much is known about insect systematics as a result of their use in phylogenetic studies. Hence mtDNA is a ready source of information for use in forensic contexts (Figure 2.1).

Figure 2.1 Insect mitochondrial genome. Source: Professor Lin Field, The Royal Entomological Society of London for permission to reproduce the Mitochondrial genome from Antenna (presented in this book as Figure 2.1)

In mitochondria a stage of respiration called oxidative phosphorylation takes place, generating adenosine triphosphate (ATP) using enzyme complexes called cytochromes. These enzyme complexes include cytochrome c oxidase (Complex IV), which is found in the mitochondrial inner membrane. Cytochrome c oxidase is the third and final electron transfer chain enzyme complex involved in oxidative phosphorylation. It is made up of three subunits; the genes of two of which are useful for molecular investigation. The mitochondrial genome comprises approximately 37 genes (22 for transfer RNA, two for ribosomal RNA, and 13 for peptides). Amongst the 37 genes are those for the two subunits of cytochrome c oxidase, subunits I and II (COI and COII). Molecular biologists originally chose COI to investigate genetic profiles, because it is the biggest of the three mitochondrially encoded cytochrome oxidase subunits and the protein sequence combines both variable and highly conserved regions (Saraste, 1990; Gennis, 1992; Beard, Hamm and Collins, 1993; Morlais and Severson, 2002, quoting Clary and Wolstenholme, 1985). This allows refinement of information so that the degree of geographic variation may also be interpreted.

Species identification is based on sequences of nucleotides. These sequences are termed loci and are made up of strings of nucleotide base pairs-adenine (A), thymine (T), cytosine (C) and guanine (G). The non-coding region of insect mtDNA is called the control region or the A-T region. This region is made up of a large number of adenine and thymine nucleotides and controls mitochondrial DNA replication and RNA transcription (Avise et al., 1987). To describe the sequence of base pairs so that an individual ‘signature’ or haplotype can be specified for a particular species, a nucleotide position numbering system is used. This follows that described for the fruit fly Drosophila yakuba (Burla) (GenBank accession Number NC-001322).

The base pairs sections can be very short. Where the sections are made up of fewer than 1000 base pairs, it is necessary to artificially increase or ‘amplify’ the length of DNA before it can be interpreted. The process used to do this is called the polymerase chain reaction (PCR). To replicate the required sections of the DNA sample, previously generated regions are joined at known sites on the DNA, to enable it to be copied. These artificially generated sections are called primers. Specific primers are generated for particular insect families, for example the Calliphoridae (Table 2.1). This means that the amplified DNA product is from a known site, so the nucleotides and their position on the DNA molecule can be interpreted.

Table 2.1 Examples of primers for cytochrome oxidase.

Primer TitleCompositionReference SourceCO – I2f5′-CAG CTA CTT TAT GAG CTT TAG G-3′Vincent, Vian and Carlotti 2000CO – I 3r5′-CAT TTC AAG C/TTG TGT AAG CATC-3′Vincent, Vian and Carlotti 2000TY – J – 1460TAC AAT TTA TCG CCT AAA CTT CAG CCWells and Sperling 2001C1 – N – 1687CAA TTT CAA ATC CTC CAA TTA TWells and Sperling 2001C1 – J – 2319TAG CTA TTG GAC/TTA TTA GGWells and Sperling 2001C1 - N - 2514AAC TCC AGT TAA TCC TCC TACWells and Sperling 2001C1 – J – 2495CAG CTA CTT TAT GAG CTT TAGGAlso used by Harvey, Dadour and Gaudierie 2003C1 – N – 2800CAT TTC AAGT/CTG TGT AAG CATCAlso used by Harvey, Dadour and Gaudierie 2003

Fortunately some species ‘signatures’ are based on quite short sections (loci), often of fewer than 350 significant base pairs. This means that, although the DNA chain degrades over time, specimens that have been stored for a long time or which have dried out, can still be reliably identified. Mitochondrial DNA (mtDNA) is extremely useful; for the most part, it is resistant to degradation. Its use can provide fly species identification within a day.

Details of primer sequences specifically complementary to calliphorid mtDNA have the reference numbers L14945–7. They can be accessed from GenBank (Malgorn and Coquoz, 1999). This information can then be used to request prepared primers from biotechnology companies. These enzymes (primers) are robust at a range of temperatures, can be used with various buffers and are moderately inexpensive. They are designed with different degrees of specificity allowing amplification of, for example, only insect DNA or more selective species-specific products.

Once extracted, the mtDNA sequences for the protein-coding regions are compared with known species ‘signatures’ in a database using computer software. GenBank is an example of a database of genetic profiles which is ratified and publicly available. Software such as Blast Search (www.ncbi.nlm.gov) is used to search GenBank. Some degree of concern has been expressed about the validity of the information held in GenBank but over time the quality is improving (this aspect is discussed later under Validity of Methodologies).

2.1 Preparation of Specimens for Molecular Analysis

In all instances, specimens for molecular analysis should be killed and stored appropriately and any possibility of contamination minimised. The chemicals and/or the extraction method chosen can influence the outcome of the analysis, although there is some disagreement on good practice (Fukatsu, 1999; Dean and Ballard, 2001). For example, Dillon, Austin and Bartowsky (1996) considered that using ethyl acetate as a killing agent could reduce the amount of DNA extracted. Logan (1999) found that genomic DNA was adequately recovered from insect specimens that had been preserved in acetone. Espeland et al. (2010) expressed concern about the effects of the insecticide, Dichlorvos, on recovery of nuclear DNA, noting that COI amplification was prevented after 229 days.

The initial means of evidence storage is also important. Ideally, storage of samples in 95% alcohol at the crime scene, or by freezing, ensures reliable recovery of the genetic information. Storing specimens in 99% alcohol provided fragments of up to 1400 base pairs according to Sperling, Anderson and Hickey (1994). In contrast, flies stored dry, or preserved in 75% ethanol, provided DNA fragments reduced to up to 350 base pairs. If neither freezing nor alcohol use is possible then the specimens should be kept on ice in a cool box, or refrigerator, until they are received at the laboratory. Contamination by organisms residing on the external surface of the maggot must be removed prior to starting to extract. A 20% solution of bleach is effect for this purpose and does not interfere with the results of molecular analysis (Wells, 2002).

A further precaution against contamination is to analyse the genetic material from the head or thorax of an adult fly, or the mid-section of a larva. This allows, as is necessary in all forensic work, the retention of voucher specimens in the form of the remaining body parts. Where possible in forensic work, the post-feeding stage should be used, or the larvae should be starved so that their gut is empty of food. This ensures that only the DNA from that particular individual is investigated and that contamination by gut contents does not occur.

Where possible, insect specimens chosen for DNA extraction should be taken from live cultures and killed by freezing. Freezing adult flies immediately at −70 °C ensures that the DNA does not degrade as rapidly as it might if other preservation methods are used. However work by Lonsdale, Dixon and Gennard (2004) indicates that the length of time in frozen storage will affect the degree of degradation of DNA molecule if storage time is longer than one year.

If the crop content is required for analysis, the larval outer coat should be treated with 20% bleach solution (Linville and Wells, 2002) and the crop excised from the larval body. This makes interpretation of the analytical results easier. The rest of the larval specimen is also available for more traditional analysis or for preservation. At this point a preservative such as Kahle's solution can be used for specimen storage.

2.1.1 DNA Extraction

There are several methods for extracting DNA and the preferred choice of extraction chemicals varies between laboratories. DNA extraction using Qiagen tissue kits such as DNeasy® Tissue Kit or Chelex® can be helpful because the pre-prepared extraction chemicals ensure standardisation of the technique. More frequently in insect molecular science phenol-chloroform extraction is used. However, Junqueira, Lessinger and Azendo-Espin (2002) concluded that DNAzol® was the most effective chemical for extracting DNA, compared to extraction using either Chelex® or the phenol/chloroform method, particularly if there was a fear that the DNA could be damaged. More recently, genomic DNA has been extracted using automated processes such as the BioRobot EZ1 workstation and EZ1 DNA Forensic Cards (Qiagen) (Cainé et al., 2006).

2.1.2 DNA Concentration

The polymerase chain reaction (PCR) splits the double-stranded DNA by heating, and replicates it to increase the amount of genetic material available. Artificially generated single DNA strands kick start or ‘prime’ the DNA synthesis. The primers are designed to position either side of a chosen section of genetic coding. They bind to complimentary sequences and using them the DNA polymerase enzyme manufactures a new strand. This new strand forms the basis for repetition of the process; more DNA is generated over a number of cycles until there is sufficient material for extraction and realisation of the DNA. At this point, depending on the original source of the DNA, several further methods of analysis can be used to determine the genetic signature.

2.2 Methods of Analysis and Sources of Information

Examples of these methods include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), analysing genes for particular enzymes such as NADH, RNA analysis including sites related to ITS, ribosomal, (for example 28S RNA), as well as pyrosequencing amongst others. These will each be discussed under separate headings.

The use of PCR itself as a means of analysis has also been explored, particularly for determining the age of fly pupae from inside the puparia since this is a life stage the duration of which is not easily determined. Zehner, Mosch and Amendt (2010) used differential display PCR to examine changes in gene expression as the pupa developed inside the puparium. They found that only in later stages of development was there a significant difference and considered the procedure had some potential, but required further work.

2.2.1 Restriction Fragment Length Polymorphism (RFLP)

This type of analysis has been used to analyse the degree of variation in populations of a particular species. It provides information relevant to the interpretation of forensic samples from data provided from a wide context. For example nuclear DNA PCR-RFLP has been used to examine variation in populations of the secondary screwworm Cochliomyia macellaria (Fabricius) in Uruguay, as a means of determining the identity of a particular specimen.

In forensic cases, PCR- RFLP methods have been successfully used to assist in species identification to make a post mortem interval determination. Schroeder et al. (2003) carried out RFLP analysis, using a modification of the method described by Sperling, Anderson and Hickey (1994) for analysing mitochondrial DNA. They separated species of Calliphora vicina, Calliphora vomitoria, and Lucilia sericata Meigen using a 349 bp section of the mtDNA using subunit I (COI), the cytochrome oxidase subunit II gene (COII) and the tRNA-leucine gene. From these specific regions they clearly distinguished between the three most common corpse-infesting fly species in the vicinity of Hamburg, Germany.

PCR-RFLP has also been used to identify some Australian chironomid fly larvae that are often difficult to identify (Carew, Pettigrove and Hoffmann, 2003). An identification key has been built using the RFLP profiles of the species of non-biting midge (Chironomidae) present in the water body. This method was validated using specimens from both wetlands and streams and has application in aquatic forensic entomology.

2.2.2 Random Amplified Polymorphic DNA (RAPD)

This method uses non-specific primers and the PCR products come from many areas of the DNA of the specimens. Primer 5 and REP 1R are often used for forensic case work (Benecke, 1998). Based on a 5′-3′ sequence the RAPD primers are:

REP 1R*XIIIACGTCGICATCAGGCPrimer 5XAACGCGCAAC(* (From a primer description provided by Pharmacia Biotech, estimating the post-mortem interval of pupae and referenced in Benecke, 1998.)

Figure 2.2 Electrophoresis gel for RAPD Analysis of Fly DNA