62 June 2012 Journal of Vector Ecology Occurrence of oriental lies associated with indoor and outdoor human remains in the tropical climate of north Malaysia T.K. Kumara1, R.H.L. Disney2, A. Abu Hassan1*, Micah Flores3, Tan Siew Hwa4, Zulqarnain Mohamed4, M.R. CheSalmah1, and S. Bhupinder5 1 School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia, aahassan@usm.my 2 Department of Zoology, University of Cambridge, Cambridge, U.K. 3 Department of Entomology, Texas A & M University 2475 TAMU, College Station, TX, U.S.A. 4 Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, 50603, Universiti Malaya, Kuala Lumpur, Malaysia 5 Department of Forensic Medicine, Penang Hospital, 10990 Residensi Road, Penang, Malaysia Recieved 20 July 2011; Accepted 16 December 2011 ABSTRACT: Flies attracted to human remains during death investigations were surveyed in north Peninsular Malaysia. Six families, eight genera, and 16 species were identiied from human remains, with the greatest ly diversity occurring on remains recovered indoors. he total relative frequency of species was led by Chrysomya megacephala (Fabricius, 1794) (46%), followed by Chrysomya ruifacies (Macquart, 1842) (22%), Sarcophaga (Liopygia) ruicornis (Fabricius, 1974) (5%), Sarcophaga spp. (4%), Synthesiomyia nudiseta Wulp, 1883 (6%), Megaselia spp. (3%), Megaselia scalaris (Loew, 1866), (2%), Megaselia spiracularis Schmitz, 1938 (2%), and Chrysomya villeneuvi Patton, 1922 (2%). Hemipyrellia tagaliana (Bigot, 1877), Desmometopa sp., Megaselia curtineura (Brues, 1909), Hemipyrellia ligurriens Wiedemann 1830, Ophyra sp., Sarcophaga princeps Wiedemann 1830, Piophila casei (Linnaeus, 1758), and unidentiied pupae each represented 1%, respectively. Journal of Vector Ecology 37 (1): 62-68. 2012. Keyword Index: Forensic entomology, oriental lies, corpses, Malaysia. INTRODUCTION Forensic entomology was established in Malaysia in the 1950s (Reid 1953), but only in the last ive years has research begun to increase in frequency. From 2005 back to the inception of forensic entomology, few publications from Malaysia have been published (Baharudin et al. 1994a, b, Baharudin et al. 2003, Cheong et al. 1973, Lee 1989, 1996, Lee et al. 2004). Ater 2005, research eforts focused on describing arthropod succession and association with carrion (Azwandi and Abu Hassan 2009, Heo et al. 2007), including human remains (Chen et al. 2008a, Chen et al. 2010, Kumara et al. 2009a, Kurahashi and Tan 2009, Nazni et al. 2008, Zuha et al. 2009). Case reports (Ahmad Firdaus et al. 2007), arthropod behavior (Helmi and Jayaprakash 2009, Heo et al. 2009a, Nazni et al. 2007), development (Ahmad Firdaus et al. 2009, Chen et al. 2008b, Kumara et al. 2009b, Mohd. Iswadi et al. 2007), taxonomy (Ahmad Firdaus et al. 2010, Kurahashi and Tan 2009, Zuha et al. 2008a), as well as entomotoxicology (Mahat et al. 2009, Rumiza et al. 2008), DNA molecular identiication (Tan et al. 2009, Tan et al. 2010), and other ancillary studies (Heo et al. 2009b, Kavitha et al. 2008, Zuha et al. 2008b) have also been prominent. Species richness data for arthropods colonizing human remains or animal carcasses in closed vs open environments have been shown to difer (Caine et al. 2009, Gof 1991, Reibe and Madea 2010a). Oliveira and Vasconcelos (2010) reported lower numbers of species on human remains than would be expected based on the presence of the necrophagous insects in the area. In Malaysia, ly (Diptera) species infesting human remains have been extensively reviewed (Lee 1989, 1996, Lee et al. 2004). However, the variation in species composition on indoor and outdoor decomposing human remains in the tropics remains unexplored. he objective of this study was to determine the diferences in dipteran species composition associated with human remains indoors and outdoors. MATERIALS AND METHODS Study sites and specimen sampling he study sites involved various hospitals in north Peninsular Malaysia, covering Penang, Perak, and Kedah states. Facilities involved were Penang Hospital (5°23´N, 100°21´E), Kepala Batas Hospital (5°31´N, 100°26´E), Sungai Bakap Hospital (5°13´N, 100°29´E), Sultan Abdul Halim Hospital (5°44´N, 100°31´E), Kulim Hospital (5°26´N, 100°34´E), Gerik Hospital (5°25´N, 101°07´E), Bukit Mertajam Hospital (5°21´N, 100°27´E), and Seberang Jaya Hospital (5°24´N, 100°24´E). Specimens were collected in the presence of the forensic pathologist and investigating police oicer at the time of autopsy. Information about the decedent collected included: gender, age, decomposition stage, locality of death scene, and the manner of death. Age was then grouped into unknown, 21-30, 31-40, 4150, 51-60, and >61 years of age. Decomposition stage was categorized as fresh, bloated, active decay, advanced decay, mummiied, or burned. Locality of death was broken into Vol. 37, no. 1 urban or rural and then into indoor or outdoor, while manner of death consisted of natural, homicidal, accidental, and suicidal death. he sampling period was from July, 2007 until July, 2010. Approximately 20-50 eggs or maggots of each maggot type were collected from each decedent. he specimens were placed in plastic containers, 5.5 cm (height) x 4.0 cm (width) x 4.0 cm (wide), and then transferred into a plastic rearing container (11.5x10x10 cm). he plastic rearing container was prepared by adding approximately 2.5 cm of sterile soil at the bottom followed by boneless beef steak (25.0 ± 5.0 g) and a wet paper towel on top of the meat that simulated the skin. he beef steak was given ad libitum. he rearing container was covered with a paper towel that allowed for ventilation and secured to the top using rubber bands to prevent other lies from entering. he maggots that were found on the deceased were reared to adults. Dead maggots were slide mounted as suggested by Lee et al. (2004) for species identiication. Species identiication Emergent adults were killed by exposing them to chloroform. Specimens were then pinned. he taxonomic key used for adult Calliphoridae was Kurahashi (2002) and for the larvae of Calliphoridae and Muscidae the taxonomic key used was Baharudin (2002). Piophilidae were identiied using the taxonomic key of Wallman (2002) while the Sarcophagidae were identiied by a co-author (T.S.H.). Species identiication of Phoridae was done by a co-author (R.H.L.D.), while the Milichiidae genus conirmation was done by Dr. Laszlo Papp at he Natural History Museum in Hungary. Statistical analysis he relative frequency of forensic lies was calculated by taking the total number of lies in a given species and dividing it by the total number of lies collected and multiplying by 100. Data were also divided into ly families occurring on indoor and outdoor human remains. he frequency of occurrence (FO) and dominance (D) of lies was calculated for both indoor and outdoor environments as described by Oliveira and Vasconcelos (2010). he frequency of occurrence (FO) can be classiied as very frequent, frequent, and infrequent ly families. For the dominance, the values calculated are classiied into dominant, accessory, and occasional ly families. he formulae for FO and D are given by Oliveira and Vasconcelos (2010) and are as follows: Frequency of occurrence (FO) = 63 Journal of Vector Ecology Number of samples containing family Total number of samples X 100 An FO value ≥ 50% would be classiied as a very frequent ly family, 25% ≤ FO < 50% would be classiied as a frequent ly family, and a FO < 25% would be classiied as an infrequent ly family. Dominance (D) = Abundance of the specimens containing family Total abundance of the specimens X 100 When D ≥ 5%, the ly family would be classiied as dominant; if 2.5% ≤ D < 5%, the ly family would be accessory; and when D < 2.5% the ly family would be classiied as occasional. RESULTS AND DISCUSSION In total, 50 corpses were sampled, 27 indoor and 23 outdoor. A total of 154 ly specimens were collected with 95 specimens occurring on indoor corpses and 59 specimens occurring on outdoor corpses (Table 1). he relative frequency of species was led by Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) (46%), followed by Chrysomya ruifacies (Macquart, 1842) (Diptera: Calliphoridae) (22%), Sarcophaga (Liopygia) ruicornis (Fabricius, 1974) (Diptera: Sarcophagidae) (5%), Sarcophaga spp. (Diptera: Sarcophagidae) (4%), Synthesiomyia nudiseta Wulp, 1883 (Diptera: Muscidae) (6%) Megaselia spp. (Diptera: Phoridae) (3%), Megaselia scalaris (Loew, 1866) (Diptera: Phoridae) (2%), Megaselia spiracularis Schmitz, 1938 (Diptera: Phoridae) (2%), and Chrysomya villeneuvi Patton, 1922 (Diptera: Calliphoridae) (2%). Hemipyrellia tagaliana Bigot, 1877 (Diptera: Calliphoridae), Desmometopa sp. (Diptera: Milichiidae), Megaselia curtineura (Brues, 1909) (Diptera: Phoridae), Hemipyrellia ligurriens Wiedemann, 1830 (Diptera: Calliphoridae), Ophyra sp. (Diptera: Muscidae), Sarcophaga princeps Wiedemann, 1830 (Diptera: Sarcophagidae), Piophila casei (Linnaeus, 1758) (Diptera: Piophilidae) and unidentiied pupae each represent 1%, respectively. For statistical purposes and during the discussion, all species from the genera Megaselia and Sarcophaga were grouped as Megaselia spp. and Sarcophaga spp. Male remains (78%) and urban environments (68%) were the most frequent categories. Decedents older than 61 years of age were the most frequent at 40%, followed by the age groups of 31-40, 21-30, 41-50, and 51-60 years of age each representing 18, 10, 6, and 6% respectively. About 20% Table 1. Total number of corpses and ly specimens sampled. Corpses Fly specimens* Indoor 27 59 Outdoor 23 95 Total 50 154 Location *Each of the ly specimens consisted of approximately 2050 eggs or maggots of each type. 64 June 2012 Journal of Vector Ecology Table 2. he frequency and dominance of ly families infesting human corpses. Family Calliphoridae Sarcophagidae Muscidae Phoridae Piophilidae Milichiidae Frequency of occurrence (FO) Indoor Outdoor Very frequent Very frequent Frequent Infrequent Frequent Infrequent Frequent Infrequent Infrequent Infrequent - of these cases were categorized into the unknown age group due to the unknown identity of the deceased. he three main decomposition stages that were observed were fresh (12%), bloated (28%), and active decay (50%), while the rest were mummiied, burned, and advanced decay representing 2%, 2%, and 6%, respectively. Most of the deceased were classiied as natural deaths (52%), followed by homicide (30 %), accidental death (14%), and suicide (4%). Indoor environments consisted of four dominant families (Calliphoridae, Sarcophagidae, Muscidae, and Phoridae) with the Calliphoridae as the most frequent family, while the outdoor environment only contained one dominant family, Calliphoridae (Table 2). Table 3 represents the mode of occurrences, either as single or mixed infestations in an indoor or outdoor environment. Indoor environments had higher mixed infestations, normally more than three species. In north Peninsular Malaysia, the relative frequency of lies infesting human remains concur with the indings in the literature (Ahmad Firdaus et al. 2007, Kavitha et al. 2008, Lee et al. 2004). Previous local reviews of species reported 3 to 18 species of lies, depending on the study period (Ahmad Firdaus et al. 2007, Kavitha et al. 2008, Lee et al. 2004). his study found 16 species of lies during the threeyear study period with Calliphoridae being a dominant family for both indoor and outdoor corpses. Local studies on non-human animals reported the colonization of adult calliphorids within 30 min ater the carcasses were exposed (Azwandi and Abu Hassan 2009, Heo et al. 2007). Others found that the adults of C. megacephala arrived irst, followed by C. ruifacies, and hypothesized that C. ruifacies may be stimulated to oviposit with C. megacephala eggs or larvae so that C. ruifacies can consume C. megacephala larvae as an additional food source (Azwandi and Abu Hassan 2009). Although time of colonization was unknown, the mixed infestation of C. megacephala and C. ruifacies for both indoor and outdoor corpses was oten observed, though there were also occurrences of single infestations of either C. megacephala or C. ruifacies (Table 3). Other species encountered from the Calliphoridae were C. villeneuvi, H. ligurriens, and H. tagaliana. Chrysomya villeneuvi have been reported to infest human corpses in forested areas (Baharudin et al. 2003, Sukontason et al. 2003, Sukontason et al. 2005), and the three instances of this species occurring were on outdoor remains in mixed infestations with C. megacephala and C. ruifacies. Among Dominance (D) Indoor Outdoor Dominant Dominant Dominant Occasional Dominant Occasional Dominant Occasional Occasional Occasional - the mixed infestations, only one sample contained 3rd instar C. villeneuvi; the other two cases reared this species from egg masses and 1st instar larvae. Chrysomya villeneuvi can only be diferentiated from C. ruifacies in the 3rd instar. A similar situation was encountered with the H. ligurriens. Azwandi and Abu Hassan (2009) showed that C. villeneuvi and H. ligurreins were never found to colonize animal carcasses as early as C. megacephala, and Chen et al. (2010) found that C. villeneuvi infested animal carcasses placed indoors in forested areas. Sarcophagidae was a frequent and dominant family found in indoor environments, while an infrequent and occasional family in outdoor environments (Table 2). he family Sarcophagidae preferred indoor human corpses (Table 3) with only one case of sarcophagids sampled from an outdoor environment, which was later identiied as Sarcophaga princeps. Sarcophagid larvae occur in low numbers on corpses because sarcophagids larviposit with low fecundity. Similar indings were reported from carcass studies (Chen et al. 2010, Horenstein et al. 2010). For the Muscidae, there were two species sampled, S. nudiseta and Ophyra spp. he species contributing to the dominance of this family was S. nudiseta (Table 2). Synthesiomyia nudiseta were encountered on indoor human corpses only (Baharudin et al. 1994a, Lee et al. 2004, Sukontason et al. 2007), though it has been reported on animal carcasses within outdoor environments (CalderonArguedas et al. 2005). Previously, in Malaysia, it was reported as an uncommon species (Lee et al. 2004), but we found it was frequently encountered and a dominant family in indoor environments with all cases occurring in urban settings. In Alexandria, Egypt, it was a secondary invader of slow decaying carcasses in the fall (Tantawi et al. 1996), but the ecology and seasonal distribution of S. nudiseta merit further investigation in Malaysia. he other species from the family Muscidae was Ophyra spp. he occurrence of Ophyra spp. on human corpses in Malaysia was irst reported by Lee (1996). he Ophrya sp., especially Ophyra spinigera, was reported to predate on pupae of C. ruifacies (Heo et al. 2009a). In the tropics, the larvae and adults of Ophyra spinigera were observed to occur during the bloated, active decay and advanced decay stages of decomposition (Azwandi and Abu Hassan 2009, Chen et al. 2010). Piophilidae was an infrequent and occasional family for both indoor and outdoor environments (Table 2). Vol. 37, no. 1 65 Journal of Vector Ecology Table 3. The infestation of forensic lies for indoor and outdoor human corpses. Number of cases Species Indoor (%) Outdoor (%) C. megacephala 4 (8) 8 (16) C. ruifacies 0 (0) 1 (2) C. megacephala + C. ruifacies 4 (8) 8 (16) C. megacephala + Sarcophaga spp. 2 (4) 0 (0) C. megacephala + C. ruifacies + Sarcophaga spp. 2 (4) 1 (2) 2 (4) 0 (0) 1 (2) 0 (0) C. megacephala + C. ruifacies + C. villeneuvi 0 (0) 3 (6) C. megacephala + C. ruifacies + H. ligurriens 0 (0) 1 (2) 1 (2) 0 (0) 0 (0) 1 (2) 3 (6) 0 (0) 1 (2) 0 (0) C. ruifacies + S. nudiseta 1 (2) 0 (0) Megaselia spp. 2 (4) 0 (0) S. nudiseta 1 (2) 0 (0) H. tagaliana 1 (2) 0 (0) Sarcophaga spp. 2 (4) 0 (0) 27 (54) 23 (46) C. megacephala + C. ruifacies + S. nudiseta + Sarcophaga spp. C. megacephala + C. ruifacies + Megaselia spp. + Sarcophaga spp. C. megacephala + Ophyra sp. + Megaselia spp. + P. casei + Unidentiied pupae C. megacephala + Ophyra sp. + P. casei + Unidentiied pupae C. megacephala + C. ruifacies + Megaselia spp. + Sarcophaga spp. + S. nudiseta C. megacephala + C. ruifacies + Desmometopa sp. + Sarcophaga spp. + S. nudiseta Total 66 Journal of Vector Ecology In Malaysia, the irst report of P. casei on a corpse was reported in 2008 (Nazni et al. 2008). Piophila casei, cheese skippers, are late colonizers, associated with drier stages of decomposition, and are commonly found in both urban and rural environments (Bucheli et al. 2009, Lord 1990, Sukontason et al. 2007). Phoridae was a frequent and dominant family for indoor environments only (Table 2). he phorids can locate small openings with some species capable of burrowing through several meters of soil (Disney 2005, Turchetto et al. 2001). Studies examining arthropod succession on vertebrate remains in Malaysia observed Megaselia spp. adults on all stages of decomposition (Azwandi and Abu Hassan 2009). heir occurrence on human corpses and animal carcasses has been previously reported in Italy (Campobasso et al. 2004, Turchetto et al. 2001), Britain (Disney 2005, Disney and Manlove 2005), Germany (Boehme et al. 2010, Reibe and Madea 2010b), central Argentina (Horenstein et al. 2010), Egypt (Tantawi et al. 1996), and hailand (Sukontason et al. 2001). With regards to Megaselia scalaris, in northern Europe, they are found in indoor situations (Disney 2008), while in Britain Megaselia ruipes (Phoridae) (Meigen) is the most common species encountered on human corpses that are exposed above-ground or in shallow buried corpses (Disney 2005, Disney and Manlove 2005). Compared with the Phoridae, Milichiidae was an infrequent and occasional family for indoor corpses (Table 2). he species involved was Desmometopa sp. and although many species from this family have been described from the Australasian/Oceanian Regions, little is known of their biology (Sabrosky 2007, Smith 1986). In Malaysia, neither the life cycle nor the biology of this ly is known, especially for use in an accurate estimation of PMI. It is evident that more dominant families of lies associated with decomposed human corpses are encountered in indoor environments (Calliphoridae, Sarcophagidae, Muscidae, and Phoridae) compared to outdoor environments (Calliphoridae) in the tropical climates of Malaysia. Future study on the ecology and life cycle of these lies in Malaysia would be helpful if an accurate post-mortem interval is to be established. Acknowledgments hanks to Universiti Sains Malaysia for the funding provided under the research grant USM RU/1001/ PBiology/815009. We also thank Dr. Disney and Dr. Laszlo Papp for their help in identifying the ly specimens of their specialty. REFERENCES CITED Ahmad Firdaus, M.S., M.A. Marwi, J. Jefery, N.A.A. Hamid, R.M. Zuha, and O. Baharudin. 2007. Review of forensic entomology cases from Kuala Lumpur Hospital and Hospital Universiti Kebangsaan Malaysia, 2002. J. Trop. Med. Parasitol. 30: 51-54. Ahmad Firdaus, M.S., T. Anita, A.M. Mohamed, H.M.I. June 2012 Noor, R.A. 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