Application of Forensic Entomology in Crime Scene Investigations in Malaysia
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APPLICATION OF FORENSIC ENTOMOLOGY IN CRIME SCENE INVESTIGATIONS IN MALAYSIA KAVITHA RAJAGOPAL FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2013 APPLICATION OF FORENSIC ENTOMOLOGY IN CRIME SCENE INVESTIGATIONS IN MALAYSIA KAVITHA RAJAGOPAL THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF BIOLOGICAL SCIENCE FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2013 UNIVERSITI MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: KAVITHA RAJAGOPAL (I.C/Passport No:) Registration/Matric No: SHC 080037 Name of Degree: DOCTOR OF PHILOSOPHY Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): APPLICATION OF FORENSIC ENTOMOLOGY IN CRIME SCENE INVESTIGATIONS IN MALAYSIA. Field of Study: FORENSIC ENTOMOLOGY I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature Date: 29.3.2013 Subscribed and solemnly declared before, Date: 29.3.2013 ABSTRACT Forensic entomology is the application and study of insect biology to criminal matters. One of the most important aspects of forensic entomology is the usage of maggot found in dead human body to determine the post-mortem interval (PMI). Based on the number of maggot sent for PMI determination, the application of forensic entomology in crime scene investigation is still unsatisfactory in Malaysia. Hence the present study was the first to conduct a questionnaire survey to determine the degree of knowledge and awareness of forensic entomology in Malaysia. A total of 402 participants comprising of the crime scene police officers, pathologists who did the post-mortem examination, scientific officers and university students who have taken forensic science as their main subjects were included. Results showed that pathologists, scientific officers and university students have better awareness and knowledge of forensic entomology than the crime scene police officers. Hence more professional training is needed particularly among the crime scene police officers. The survey identified two major obstacles that may hinder the growth of forensic entomology in Malaysia which are the lack of information on the forensically important fly as well as the lack of expertise in species identification. Nevertheless the survey revealed a bright prospect for forensic entomology as evidenced by increased awareness of its importance and interest in the younger generation. The present study was the first to apply both morphological and molecular methods for fly species identification in samples collected from crime scene investigation in Malaysia. A total of 50 cases from December 2008 to March 2010 were included. The present study confirmed the usefulness of molecular method based on cytochrome oxidase genes sequencing as a complementary tool in assisting fly species identification. Phylogenetic analyses confirmed the presence of Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Hemipyrellia ligurriens and Sarcophaga ruficornis. In addition, one ‘unknown’ species of blow fly was discovered. The application of molecular method has proven to be more advantageous in the case of immature maggot and egg. Due to the lack of experienced entomologist in Malaysia, it is recommended that molecular method should be widely applied. ii Since Chrysomya megacephala and Chrysomya rufifacies was found to be the two most prominent fly species recovered in crime scene investigation, DNA barcoding was done for each life cycle stage of both blow flies namely egg, 1st instar, 2nd instar, 3rd instar, pupae, empty puparium and adult fly. The present study confirms that all life cycle stage of a particular species of fly yield identical DNA barcode and hence all the stages can be used for accurate species identification. The present study represents an initial effort to establish a DNA barcoding for forensically important blow fly in Malaysia. However, the effective use of DNA barcoding would require an expert system of integrated information whereby species names and their respective DNA barcodes are coupled with data of life cycle and geographic distributions. iii ABSTRAK Forensic entomologi adalah aplikasi kajian biologi serangga ke atas hal-hal jenayah. Salah satu aspek yang paling penting dalam bidang forensik entomologi ialah penggunaan ulat yang dijumpai pada mayat manusia untuk menentukan selang masa kematian (PMI). Berdasarkan bilangan ulat yang dihantar untuk penentuan selang masa kematian didapati aplikasi forensik entomologi di tempat kejadian jenayah masih tidak memuaskan di Malaysia. Oleh itu kajian ini merupakan kaji selidik yang pertama dijalankan untuk menentukan tahap pengetahuan dan kesedaran tentang forensik entomologi di Malaysia. Seramai 402 peserta yang terdiri daripada pegawai polis yang menyiasat di tempat kejadian jenayah, pakar patologi yang melakukan bedah siasat mayat, pegawai sains and pelajar universiti yang mengambil matapelajaran sains forensik sebagai subjek utama dimasukkan dalam kajian. Hasil kajian menunjukkan bahawa pakar patologi, pegawai sains dan pelajar universiti mempunyai kesedaran dan pengetahuan forensik entomologi yang lebih baik jika dibandingkan dengan dengan pegawai polis yang menyiasat di tempat kejadian jenayah. Oleh itu lebih banyak latihan profesional perlu diberi khususnya kepada pegawai polis yang menyiasat di tempat kejadian jenayah. Kaji selidik ini juga mengenal pasti dua halangan utama yang mungkin menghalang perkembangan forensik entomologi di Malaysia iaitu kekurangan sumber maklumat berkenaan lalat yang mempunyai kepentingan forensik serta kekurangan kepakaran dalam mengenalpasti spesies lalat. Walau bagaimanapun, kaji selidik ini mendedahkan prospek yang cerah untuk bidang forensik entomologi yang mana dapat dibuktikan dengan peningkatan kesedaran tentang kepentingan forensik entomologi dan minat di kalangan generasi muda. Kajian ini adalah yang pertama menggunakan kedua-dua keadah morfologi dan molekul untuk mengenal pasti spesies lalat yang disampelkan di tempat kejadian jenayah di Malaysia. Sebanyak 50 kes dari Disember 2008 hingga Mac 2010 dimasukkan dalam kajian ini. Kajian ini mengesahkan kebergunaan kaedah molekul yang berdasarkan penjujukan gen cytochrome oxidase sebagai kaedah pelengkap dalam membantu pengenalpastian spesies lalat. Analisa filogenetik juga mengesahkan kehadiran lalat Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Hemipyrellia ligurriens dan Sarcophaga ruficornis. Di samping itu, satu spesies yang tidak dapat dikenalpasti telah ditemui. Kaedah molekul terbukti lebih berguna dalam iv pengenalpastian spesies lalat yang belum matang dan telur. Disebabkan kekurangan pakar entomologi yang berpengalaman di Malaysia adalah disyorkan bahawa kaedah molekul perlu digunakan secara meluas dalam proses pengenalpastian spesies lalat. Memandangkan Chrysomya megacephala dan Chrysomya rufifacies merupakan dua spesies lalat yang paling banyak ditemui di tempat kejadian jenayah maka DNA barcoding telah dilakukan bagi setiap peringkat kitaran hidup lalat tersebut iaitu telur, instar pertama, instar kedua, instar ketiga, kepompong, kepompong kosong dan lalat dewasa. Kajian ini mengesahkan bahawa semua peringkat kitaran hidup bagi satu spesies lalat tertentu mempunyai DNA barcode yang sama dan oleh itu semua peringkat hidup lalat boleh digunakan dalam proses pengenalpastian spesies lalat dengan tepat. Kajian ini merupakan satu usaha awal untuk menubuhkan DNA barcoding untuk spesies lalat yang berkepentingan secara forensik di Malaysia. Walau bagaimanapun, penggunaan DNA barcoding yang berkesan memerlukan satu sistem pakar maklumat yang bersepadu di mana nama spesies lalat dan DNA barcode masing-masing dipadankan bersama-sama dengan data kitaran hidup dan distribusi geografi. v ACKNOWLEDGEMENTS Life on earth is a journey, starts as well as ends with Almighty, like cyclic reactions. During this journey, we are blessed with invaluable teachers and well wishes. It is very difficult to forget important events, ups and downs, achievements, excellent collaborators, contributors, great inspirational minds and the land of harvest. At the end of my journey to PhD. it is a great pleasure to acknowledge people, who have supported my growth. First and above all I would like to thank my supervisor Prof. Dato’ Dr. Mohd Sofian Azirun, Dean of Faculty of Science, University of Malaya, Kuala Lumpur and my consultant Dr. Lee Han Lim, Head of Medical Entomology Unit, Institute of Medical Research (IMR),