Forensic entomology in the Philippines: Establishing ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs...Isabela, Quezon City, and Marinduque in the Philippines matched with C. megacephala

*Corresponding author: [email protected]

Key words: Chrysomya megacephala, DNA barcoding, forensic entomology

Forensic entomology in the Philippines: Establishing Baseline Data on the Forensically Important Blow

Fly Species Chrysomya megacephala (Fabricius, 1794)

1Natural Sciences Research Institute, College of ScienceUniversity of the Philippines Diliman, Diliman, Quezon City 1101

2DNA Barcoding Laboratory, Institute of Biology, College of ScienceUniversity of the Philippines Diliman, Diliman, Quezon City 1101

Ronniel D.C. Pedales1,2,* and Ian Kendrich C. Fontanilla2

The Philippines is yet to adapt and implement guidelines and protocols in forensic entomology, particularly establishing local databases. Considering the efforts made by neighboring Southeast Asian countries in the field, the nation has been left behind in insect evidence-based investigations. Of utmost importance to forensic entomology are blow flies (Diptera: Calliphoridae), which are primary colonizers of carrion. Through knowledge of their distribution, identity, and growth rates, investigators are able to provide a post-mortem interval that is most accurate after the onset of putrefaction. The Philippines has a total of 83 blow fly species recorded, including the cosmopolitan species Chrysomya megacephala. This paper aims to establish a baseline reference in Philippine forensic entomology by mapping the distribution, providing DNA barcodes, and estimating larval growth rates from oviposition to pupariation of C. megacephala. Distribution data were mapped in QGIS using localities from fieldwork data in this study and those in the Key to the Philippine Calliphoridae by Kurahashi and Magpayo. DNA barcodes of specimens from Isabela, Quezon City, and Marinduque in the Philippines matched with C. megacephala from the database in GenBank and revealed a possible SNP in the fragment amplified. C. megacephala was reared from oviposition in a simple incubation set-up to estimate the duration of development to pupariation, which ranged 100-113 hours. This is the first study on the distribution, molecular identification, and development of C. megacephala in the Philippines. Further work is needed to distinguish among populations of the species and to construct more precise growth curves.

Philippine Journal of Science147 (1): 17-25, March 2018ISSN 0031 - 7683Date Received: 31 Aug 2016

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INTRODUCTIONThe oriental latrine fly, Chrysomya megacephala (Fabricius, 1794), is a forensically important species of blow fly (Diptera: Calliphoridae) (Wells & Sperling 2001). It is native to the Australasian and Pacific regions but is now distributed worldwide due to species introduction and its inherent invasive nature (Wells 1991; Williams & Villet 2006; Vasconcelos & Salgado 2014; Carmo &

Vasconcelos 2014). The species can be found around and on rotting animals or fruits, often in large groups, where they consume organic material for food and proteins for egg production (Linhares & Avancini 1989). They are characteristically large – ranging 7.5-10 mm long – with a setulose stem vein on the dorsal side of the basal section of their wings. They can be easily distinguished from other Philippine Chrysomyinae for having an orange gena clothed with yellow hairs and large eyes that are almost touching in males (Figure 1) (Kurahashi & Magpayo 2000).

Pedales and Fontanilla: Forensic Entomology in the Philippines

Philippine Journal of ScienceVol. 147 No. 1, March 2018

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Figure 1. The male imago of C. megacephala; defining characters are pointed by arrows:(a) yellow gena and post-gena covered with yellow hairs, (b) fuscous prothoracic spiracle, and (c) an infuscated thoracic squama (Kuruhashi & Magpayo 2000).

C. megacephala is one of the most useful taxa in the study of forensic entomology especially in Southeast Asia where it is native. Over the years, there has been thorough research on the development, succession, molecular identification, and morphology of larval forms of this species. Review of forensic entomology-related cases in Malaysia identified C. megacephala as the most common species, occurring in 47.99% of investigations during 1972-2002 (Lee et al. 2004) and in 38.70% of cases in 2005-2010 (Kavitha et al. 2013). Forensic cases spanning 2000-2006 were used for a composite review of entomological evidence in Thailand, which also noted the species as one of the most common species across all cases (Sukontason et al. 2007). In the Philippines, the species list made by Kurahashi & Magpayo (2000) was the most recent effort to study the Philippine Calliphoridae. In their publication, they provided distribution data through collections deposited in local and foreign repositories. However, distribution data were deficient since it does not include exact coordinates of collections and does not span the entirety of the Philippine archipelago.

C. megacephala is distributed throughout the major islands of the Philippines. This species is known to be the dominant primary colonizer – arriving first on the onset of carrion decay – in regions where it occurs (Goodbrod & Godd 1990; Harvey et al. 2008). For this reason, the species is an important flagship species for forensic entomology in the Philippines.

This study has sought to (1) map the distribution of the C. megacephala across the Philippines using localities from fieldwork data conducted in this study and those in the Key to the Philippine Calliphoridae by Kurahashi and Magpayo (2000); (2) provide DNA barcodes of the COI gene fragment for molecular identification of the species; and (3) provide an estimate of the life cycle of the species in a simple semi-controlled environment to provide a baseline data for the species. Insights and perspectives on the current state of Philippine forensic entomology are also included.

METHODOLOGY

Distribution of Philippine C. megacephalaThe occurrence of the species in islands of the Philippines was mapped in QGIS v2.18.3 (QGIS Development Team 2016) using the distribution data from localities provided for by the author’s fieldworks spanning 2013-2016 and from Kurahashi and Magpayo (2000). Due to the absence of coordinates in the latter, the authors resorted to approximation of the points using Google Earth (Google 2016).

Blowfly sampling ensued from Aug 2013 to Jun 2016 in twelve (12) localities: (LUZON) Diliman, Quezon City; Boac, Marinduque; Ilagan, Isabela; Subic, Zambales; Lake Palakpakin, San Pablo, Laguna; Mt. Guinatungan, Camarines Norte; (VISAYAS) Miag-ao, Iloilo; (MINDANAO) Sta. Cruz Island, Zamboanga; Bud Bongao, Tawi-tawi; Mt. Kansad, Klaja Karst, General Santos City; Balut Island, Davao del Sur; and Sitio Kangko, Lake Sebu, South Cotabato. The sampled sites were not included as localities for C. megacephala in the Key to the Calliphoridae of the Philippines (2000). Sampling was done using porcine liver bait traps and active collection using insect nets.

DNA Barcoding and Molecular Phylogenetics of C. megacephala from LuzonFrom each sampling lot, up to seven (7) samples each from Diliman, Quezon City; Ilagan, Isabela; and Boac, Marinduque were separated, processed for DNA barcoding, and vouchered at -80° C storage [Accession codes CmD-1 to CmD-7 (Diliman), CmI-1 to CmI7 (Isabela), and CmM-1 to CmM-7 (Marinduque)] in the DNA Barcoding Laboratory, Institute of Biology, University of the Philippines Diliman. Multiple samples were processed to give a better representation of the local fauna, but was limited to a maximum of seven (7) representatives per locality (Luo et al. 2015). The right hind leg of adult blow flies captured and a small slice of tissue in larval forms were used for DNA extraction using the Purelink®



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Genomic DNA Extraction Kit (Invitrogen), following the manufacturer’s protocol. Amplification of theCOI barcoding gene fragment was done using the primers LCO (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). A 50 µL PCR mastermix was made from 10 µL of 5x PCR Buffer with dNTPs (Bioline, UK), 1.5 µL of each primers (10 mM), 0.25 µL Taq Polymerase (5 units/uL) (Bioline, UK), 2.5 µL molecular grade DMSO (Sigma), 1 µL of 50 mM MgCl2 (Bioline, UK), 10-30 ng of genomic DNA, and diluted with DNAse free water. Thermal cycling was done in a LabNet Thermal Cycler using the following protocol: a 5 min denaturation step at 94° C followed by 43 cycles of 30 sec at 94° C, 30 sec at 45° C, and 60 sec at 65° C, and a final extension of 5 min at 72° C. The PCR products were visualized in a 1% agarose gel stained with ethidium bromide in a UV transilluminator. Positive products were excised and purified using the QIAquick® DNA Extraction Kit (Qiagen, USA). Purified samples were then sent to 1st Base Malaysia for single pass capillary sequencing.

Sequence contigs were assembled in Staden Package (Bonfield et al. 1995). Post-processing (i.e., primer sequence removal, sequence consolidation, etc.) was carried out in GAP4. Each assembled sequences was subject to a BLAST search (Altschul et al. 1990) and pairwise comparison of genetic distances in MEGA7 (Kumar et al. 2016) to confirm species identification.

To ensure a phylogenetic relationship of Philippine C. megacephala with neighboring countries with native distribution of the species, COI sequences from the studies of Nelson and colleagues (2007), Tan and colleagues (2009), and Chong and colleagues (2014) were downloaded from the GenBank database. COI sequences of C. bezziana and C. pacifica were also included to represent sister taxa and C. rufifacies were used as an outgroup. These were subsequently aligned with sequences from this study with the online version of MAFFT (Katoh & Standley 2016) using the iterative refinement G-INS method. Aligned sequences were trimmed in BioEdit (Hall 1999). Model testing and the construction of a maximum-likelihood tree was performed in MEGA7 (Kumar et al. 2016).

Estimating the Larval Development of Philippine C. megacephalaConstruction of accurate age-estimating diagrams of fly species for use in forensic entomology requires the use of growth chambers and rooms for controlled environments (e.g., temperature and humidity) (Michaud et al. 2012). A considerable amount of effort is needed to set up laboratories equipped with such facilities. In order to establish baseline data, a preliminary investigation on the

duration of development of the species from oviposition to pupariation was observed. Flies were collected in an area of dense vegetation in the University of the Philippines’ Diliman Campus (14.65°N, 121.071°E) using approximately 150 g of rotten porcine liver placed in plastic bottle traps (Figure 2a). Sampling was performed on 1-3 Sep 2013. Using a sling psychrometer, ambient temperature was recorded to be 28°C and relative humidity at 72%. Species identification from the specimen lot was done using the Key to the Philippine Calliphoridae (2000), upon which the adult flies were transferred to rearing cages (44 x 33 x 22 cm3) covered with cloth which prevented parasitoids and other flies from entering (Figure 2b).

Rearing was done in semi-controlled conditions through a simple modification of the methods by Vélez & Wolff (2008). A sponge drenched with 50% sugar-water was used to supplement the gravid flies. Six rearing containers with approximately 100 g of porcine liver were placed inside the cage to provide protein for egg formation of the females and an ovipository site for gravid individuals. The rearing containers were checked every 15-20 min until an egg mass is seen on the surface; this would then mark the start of the developmental time for the said egg mass. Once the rearing containers were observed to have one egg mass coming from a single oviposition event, they were then transferred inside a makeshift aerated incubator (44 x 33 x 22 cm3) (Figure 2d) kept indoors for the duration of the experimentation. Humidity was kept at high levels by partially immersing the rearing containers in water-drenched sawdust inside the incubator.

Eggs hatched in three rearing containers. Throughout the rearing procedure, the temperature was at a range of 27-29°C. The development of the larvae was monitored continuously while sampling and measurement was done every twelve hours post-eclosion until pupariation. Exposed larvae were sampled from the rearing containers for measurement. The number of samples ranged from five (5) to seven (7) individuals depending their location in the substrate; minimum disturbance was ensured in sampling larvae (e.g., tapping of the substrate as opposed to complete dissection to expose larvae). Larval lengths were plotted against time elapsed starting from oviposition (GraphPad Prism 7.03). All rearing containers were observed at the same time; three separate line graphs were generated due to the difference in oviposition times among samples. Sampling halted at approximately 110 h when the post-feeding larvae were starting to discolor. The containers were then observed hourly until most of the larvae had dark colored sclerotized puparia and time to this stage was recorded.



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RESULTS AND DISCUSSION

Distribution of Philippine C. megacephalaThe occurrence of C. megacephala in several regions of the Philippines was mapped using fieldwork data and those from the Key to the Philippine Calliphoridae (Figure 3). Samples examined by Kurahashi and Magpayo were concentrated to Southern Luzon, Palawan, and in parts of Mindanao. Fieldwork done during 2013-2016 added representation to Southern Mindanao, Iloilo, and islands such as Marinduque and Tawi-tawi. C. megacephala was present from 10 m.a.s.l. (Boac, Marinduque) to 1200-1500 m.a.s.l. (Mayoyao, Ifugao).

The blowfly species studied is present in all major islands sampled. Data are still lacking for other major islands such as Mindoro, while the Visayas region is still underrepresented. However, the researchers hypothesize that C. megacephala can be found in all major islands due to its inherent invasiveness and adaptability (Kurahashi 1982). With its ability to co-exist with human settlements,

taking advantage of the resource available in urban habitats, C. megacephala can easily adapt to homogeneous environments (Carvalho 2004).

DNA Barcoding and Molecular Phylogenetics of C. megacephalaA 532 bp fragment of the COI gene was amplified and sequenced to provide DNA barcodes representing specimens from Diliman, Quezon City (CmD); Boac, Marinduque (CmM); and Ilagan, Isabela (CmI). Sequences were submitted to the GenBank database to include the Philipppine C. megacephala (KY653047-KY653066). Sequences were subjected to nucleotide BLAST with 99.8-100% identity to the reference C. megacephala nearly complete mitochondrial genome sequence (KT272865) (Table 1). In contrast, the sequences demonstrated 99% and 97% identity only to C. megacephala’s sister taxa, C. pacifica and C. bezziana, respectively.

Phylogenetic analysis was done to determine clustering of Philippine samples with that of the samples from studies

Figure 2. Experimental set-up for capturing and rearing C. megacephala (Fabricius, 1794) in the University of the Philippines Diliman. Porcine liver traps (a) made with used bottles were set-up in forested areas of the university. They were then transferred to rearing cages for oviposition on liver boats (b). Upon oviposition, eggs were transferred to rearing containers with porcine liver substrate (c) and eventually placed in a makeshift aerated incubator (d) at 27-29° C and >60% humidity.



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Figure 3. Distribution of Philippine C. megacephala in the Philippines based on literature from Kurahashi and Magpayo (2000; red diamonds) and Pedales fieldworks (2013-2016; green dots).



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Table 1. BLAST search results against a 573bp fragment of the COI gene amplified from C. megacephala in Isabela, Quezon City, and Marinduque. The base at site 64 of the fragment is also indicated per sample.

Sample ID GenBank Accession BLAST Result % identity Closest Match (Accession) Site 64

CmD-1 KY653047 Chrysomya megacephala 100 KT272865 C

CmD-2 KY653048 Chrysomya megacephala 99 KT272865 T






CmI-1 KY653054 Chrysomya megacephala 100 KT272865 C







CmM-1 KY653061 Chrysomya megacephala 99 KT272865 T





CmM-6 KY653067 Chrysomya megacephala 100 KT272865 C

CmM-7 KY653066 Chrysomya megacephala 100 KT272865 C

in Malaysia and Australia (Figure 4). The aforementioned regions are also part of the native distribution of C. megacephala. The sister taxa of C. pacifica and C. bezziana were also included to determine whether barcoding of the COI gene fragment can discriminate amongst species. C.

Figure 4. Maximum-likelihood tree of the 532 bp fragment of the COI gene of Philippine C. megacephala using the T92 model with 1000 bootstrap repetitions. The clade highlighted in blue was polytomic with specimens from the studies of Nelson et al. (2007), Tan et al. (2009), and Chong et al. (2014). Sequences were compared with the sister taxa of C. megacephala (C. pacifica and C. bezziana) and C. rufifacies as the out-group.

pacifica was distinct from the C. megacephala cluster with 90 bootstrap support. Some Philippine C. megacephala was polytomic with samples from literature while C. megacephala from Marinduque (CmM-1 to CmM 5) and Diliman (CmD-2 to CmD-4) formed a separate cluster



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with 63 bootstrap support. Genetic distance within C. megacephala were 0 & 0.002. An SNP in site 64 was found to separate these clades. Albeit this, all C. megacephala were distinct from the out-group and sister taxa included in the analysis, thus affirming species confirmation through sequencing of the barcoding gene fragment.

DNA barcoding may serve as an important tool in species validation, as its larval forms are difficult to identify morphologically. As C. megacephala is a cosmopolitan species as well as a primary colonizer, it is expected to be the first species to dominate carrion at the early onset of decay especially in sites that are adjacent to one another. The utility of the forensically important insects in movement of the body from one habitat to another would then be significantly decreased as all sites would be expected to have the same species (Amendt et al. 2011). The low genetic variation seen in the COI gene fragment significantly affects the utility of the gene for population genetic analysis. This can be remedied by using a more variable gene region to differentiate among populations. Additional representatives from varying geographical regions and habitats are needed for this task as well as more molecular techniques such as SSR analysis, amplification of longer fragments of DNA, and sequencing of hypervariable regions.

Estimating the Developmental Time of Philippine C. megacephala

The growth rate curves shown in this study is the first record of its kind in the Philippines. Samples were collected and measured at seven time frames to construct each growth curve. Larval length was plotted against time (Figure 5). The total developmental time in this study from egg to pupariation is estimated to be 110-113 hours after oviposition. Post-feeding larvae started migrating out of the liver substrate 51-65 hours after oviposition; by the fifth time frame, all larvae were found in the sawdust base of the rearing containers. Sclerotization of post-feeding larvae was observed after 110 hours and observation was continued until the final surviving larvae achieved full pupariation.

Gruner and colleagues (2017) reported large variation among published developmental rates for C. megacephala. In a previous study with a controlled set-up, pupariation was reported to be onset at 120 h at 27°C (Wells & Kurahashi 1994). Studies on C. megacephala from Thailand during rainy months (Aug-Dec) revealed longer developmental times (132-168 h; average temperature: 23-28°C) in uncontrolled environments (Sukontason et al. 2008), highlighting changes in rates of development of blowflies per month due to changes in ambient temperature.

Figure 5. Mean larval length (mm) of C. megacephala (Fabricius) in three set-ups (A, B, and C) plotted against to time (hours) at temperature range of 27-29°C and relative humidity >60%. A, B, and C had different times of oviposition (12:00 pm, 2:15 pm, and 2:45 pm respectively) in the rearing cages. Set-ups were observed continuously and samples taken at the same time at 12 h intervals. Onset of observed migration of post-feeding larvae and full sclerotization to pupariation were observed at around 51 h and 110-113 h, respectively.



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The data provided in the developmental part of this study aims to create baseline information on the larval growth of Philippine C. megacephala. Several factors should be modified and included in the study to create more accurate growth curves. Determining the number of larvae/eggs in each set-up prior to starting experimentation will give insights on the effects of larval competition and increased temperatures in larval masses which are critical to larval growth (Goodbrod & Goff 1990). Analyses of growth of C. megacephala in various temperatures will also enable the construction of isomorphen diagrams that are critical to forensic entomology (Sukontason et al. 2008).

CONCLUSIONThis is the first study on the establishment of baseline data for C. megacephala on its (1) known distribution ranges in the Philippines, (2) molecular identification of the species, and (3) developmental rates at semi-controlled conditions of its larval forms. The species is present in most islands of the Philippines and is one of the first colonizers of carrion where it occurs. Molecular identification of both larval and adult forms of the taxon can be done using small amounts of tissue and universal primers present in literature. In an effort to increase global awareness on the practice of forensic entomology, studies on developmental rates of blowflies are being done. Although this is not the pioneer study on the developmental rate of the species, geographical differences still pose the probability of divergence of developmental rates within species due to varying environmental conditions. The data on the growth of larvae on temperature ranging 27-29°C during the rainy months (Aug-Sep) is presented here and can give a good estimate on the age of sampled flies in the field. A more complete picture of the development of C. megacephala can be achieved through (1) the use laboratory facilities that control temperature and humidity (environmental chambers), (2) establishment of developmental rate data in ambient conditions during the dry season, and (3) construction of isomorphen diagrams using ADH data.

ACKNOWLEDGMENTSThe authors would like to extend their gratitude to the Natural Sciences Research Institute for funding this project. The authors would also like to thank the Institute of Biology, University of the Philippines Diliman for the use of facilities, as well as Gizelle Batomalaque and Jeanmaire Molina for helping with fieldwork.

REFERENCES ALTSCHUL SF, GISH W, MILLER W, MYERS EW,

LIPMAN DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403-410.

AMENDT J, RICHARDS CS, CAMPOBASSO CP, ZEHNER R, HALL MJ. 2011. Forensic entomology: applications and limitations. Forensic Sci Med Pat 7(4):379-392.

BONFIELD JK, SMITH KF, STADEN R. 1995. A new DNA sequence assembly program. Nucleic Acids Res. 23:4992-99.

CARMO RF, VASCONCELOS SD. 2014. First record of the blow fly Chrysomya megacephala (Diptera: Calliphoridae) on a southern Atlantic island: implications for disease transmission in a protected environment. J Vector Ecol 39(1):228-230.

CARVALHO LML, THYSSEN PJ, GOFF ML, LINHARES AX. 2004. Observations on the succession patterns of necrophagous insects on a pig carcass in an urban area of Southeastern Brazil. Forensic Med Toxicol 5:33-39.

CHONG YV, CHUA TH, SONG BK, 2014. Genetic variations of Chrysomya megacephala populations in Malaysia (Diptera: Calliphoridae). Advances in Entomology, 2014.

FOLMER O, BLACK M, HOEH W, LUTZ R, VRIJENHOEK R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar BiolBiotechnol 3:294-299.

GOODBROD J, GOFF M. 1990. Effects of larval population density on rates of development and interactions between two species of Chrysomya (Diptera: Calliphoridae) in laboratory culture. J Med Entomol 27(3):338-343.

GOOGLE MAPS. 2016. Philippines. Retrieved from https://www.google.com.ph/maps/

GRUNER SV, SLONE DH, CAPINERA JL, TURCO MP. 2016. Development of the Oriental Latrine Fly, Chrysomya megacephala (Diptera: Calliphoridae), at Five Constant Temperatures. J Med Entomol p.tjw169.

HALL TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series. 41(41):95-98. London: Information Retrieval Ltd., c1979-c2000.

HARVEY M, GAUDIERI S, VILLET M, DADOUR I. 2008. A global study of forensically significant calliphorids: Implication for identification. Forensic



25

Sci Int 177(1):66-76.

KATOH K, STANDLEY DM. 2016. A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics p.btw108.

KAVITHA R, NAZNI WA, TAN TC, LEE HL, AZIRUN MS. 2013. Review of forensically important entomological specimens collected from human cadavers in Malaysia (2005–2010). J Forensic Leg Med 20(5):480-482.

KUMAR S, STECHER G, TAMURA K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular biology and evolution p.msw054.

KURAHASHI H. 1982. Probable origin of a synanthropic fly Chrysomya megacephala, in New Guinea (Diptera: Calliphoridae). In: Biogeography and Ecology of New Guinea. Gressitt JL ed. Netherlands: Springer. p. 689-698.

KURAHASHI H, MAGPAYO FR. 2000. Blow flies (Insecta: Diptera: Calliphoridae) of the Philippines. Raffles Bull Zool 48(9):1-78.

LEE HL, KRISHNASAMY M, ABDULLAH AG, JEFFERY J. 2004. Review of forensically important entomological specimens in the period of 1972-2002. Trop Biomed 21(2):69-75.

LINHARES AX, AVANCINI RPM. 1989. Ovarian development in the blowflies Chrysomya putoria and C. megacephala on natural diets. Med Vet Entomol 3(3):293-295.

LUO A, LAN H, LING C, ZHANG A, SHI L, HO SYW, ZHU C. 2015. A simulation study of sample size for DNA barcoding. Ecol Evol 5(24):5869-8579.

MICHAUD JP, SCHOENLY KG, MOREAU G. 2012. Sampling flies or sampling flaws? Experimental design and inference strength in forensic entomology. J Med Entomol 49(1):1-10.

NELSON LA, WALLMAN JF, DOWTON M. 2007. Using COI barcodes to identify forensically and medically important blowflies. Med Veterinary Entomol 21(1):44-52.

SUKONTASON K, NARONGCHAI P, KANCHAI C, VICHAIRAT K, SRIBANDITMONGKOL P, BHOOPAT T, KURAHASHI H, CHOCKJAMSAI M, PIANGJAI S, BUNCHU N, VONGVIVACH S, SAMAI W, CHAIWONG T, METHANITIKORN R, NGERN-KLUN R, SRIPAKDEE D, BOONSRIWONG W, SIRIWATTANARUNGSEE S, SRIMUANGWONG C, HANTERDSITH B, CHAIWAN K, SRISUWAN C, UPAKUT S, MOOPAYAK K, VOGTSBERGER

RC, OLSON JK, SUKONTASON K. 2007. Forensic entomology cases in Thailand: a review of cases from 2000 to 2006. Parasitol Res 101(5):1417-23.

S U K O N T A S O N K , P I A N G J A I S , SIRIWATTANARUNGSEE S, SUKONTASON KL. 2008. Morphology and developmental rate of blowflies Chrysomya megacephala and Chrysomya rufifacies in Thailand: application in forensic entomology. Parasitol Res 102(6):1207-16.

TAN SH, ARIS EM, SURIN J, OMAR B, KURAHASHI H, MOHAMED Z. 2009. Sequence variation in the cytochrome oxidase subunit I and II genes of two commonly found blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart)(Diptera: Calliphoridae) in Malaysia. Trop. Biomed 26:173-181.

VASCONCELOS SD, SALGADO RL. 2014. First record of six Calliphoridae (Diptera) species in a seasonally dry tropical forest in Brazil: evidence for the establishment of invasive species. Fla Entomol 97(2):814-816.

VÉLEZ MC, WOLFF M. 2008. Rearing five species of Diptera (Calliphoridae) of forensic importance in Colombia in semicontrolled field conditions. Pap Avulsos Zool 48(6)(2008):41-47.

WELLS JD. 1991. Chrysomya megacephala (Diptera: Calliphoridae) has reached the continental United States: review of its biology, pest status, and spread around the world. J Med Entomol 28(3):471-473.

WELLS JD, KURAHASHI H. 1994. Chrysomya megacephala (Fabricius)(Diptera: Calliphoridae) development: rate, variation and the implications for forensic entomology. Eisei Dobutsu 45:303-303.

WELLS JD, SPERLING FA. 2001. DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae). Forensic Sci Int 120(1):110-115.

WILLIAMS KA, VILLET MH. 2006. A new and earlier record of Chrysoma megacephala in South Africa, with notes on another exotic species, Calliphora vicina (Diptera: Calliphoridae). Afr Invertebr 47:347.

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Forensic entomology in the Philippines: Establishing ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs...Isabela, Quezon City, and Marinduque in the Philippines matched with C. megacephala