(Diptera: Sarcophagidae) in China Based on COI and Period Gene

中国科技论文在线 http://www.paper.edu.cn Int J Legal Med DOI 10.1007/s00414-013-0923-7

ORIGINAL ARTICLE

Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China based on COI and period gene

Yadong Guo & Lagabaiyila Zha & Weitao Yan & Pei Li & Jifeng Cai & LiXiang Wu

Received: 29 April 2013 /Accepted: 24 September 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Unequivocal identification of insect specimens is Introduction an essential requirement in forensic entomology. With the development of molecular identification, spate of discussions Insects colonize human and animal remains in a predictable about the feature of the DNA fragments have been raised. sequence over the time. Precise identification of these insects Relying solely on single DNA fragment for delimiting closely and their developmental stages plays an essential role in the related species is supposed to be dangerous. Aiming at estimation of postmortem interval (PMI) [1]. In China, the obtaining more reliable markers that might be universally occurrence of carrion-related arthropods has been known to used, we explore the utility of 700-bp COI fragment and include dipteran flies such as Sarcophagidae (flesh flies), 678-bp period gene fragment in the identification of Calliphoridae, and Muscidae [2, 3]. Representatives of Sarcophagidae (Diptera). Thirty-six sarcophagid fly speci- Sarcophagidae are found throughout the world, with most mens were collected from 19 locations in 11 Chinese prov- species occurring either in tropical or warm temperate regions inces. Phylogenetic analysis of the sequenced segments [4]. Flesh flies of forensic importance are larviparous [5]. showed that all sarcophagid specimens were properly Moreover, sarcophagids are attracted to carrion under most assigned into nine species with relatively strong supporting conditions, including sun, shade, dry, wet, indoors, and out- values, which indicated the possibility of separation conge- doors [6]. The genus Sarcophaga is known to fly under neric species with COI and period gene fragments. The inclement conditions that would prevent the flies of most other difference between intraspecific threshold and interspecific families [4]. In addition to the large size of many species divergence confirmed that the combination of nuclear and makes them particularly likely to be noticed and collected by mitochondrial genes for species identification is much more a forensic investigator [3]. accurate. The results of this research will be instrumental for The potentiality of sarcophagids for PMI estimations has implementation of the Chinese Sarcophagidae database. been well demonstrated in many studies [7, 8]. The use of sarcophagids for PMI estimations has been greatly hampered Keywords Sarcophagidae . Forensic entomology . by their highly similar morphological and inadequate documen- Cytochrome oxidase subunit I . Period gene . Species tation of their thermobiological histories [3, 5]. Sarcophagid identification adults and larvae are easily recognized at family level, but are similar subgenerically and interspecifically [9, 10]. Species- diagnostic anatomical characters of flesh flies are not known Electronic supplementary material The online version of this article (doi:10.1007/s00414-013-0923-7) contains supplementary material, for most immature stages or even adults, and an existing key which is available to authorized users. may be incomplete or difficult for nonspecialists to use [11]. To implement the use of sarcophagids for PMI estimation, a meth- Y. Gu o : L. Zha : W. Yan : P. Li : J. Cai Department of Forensic Science, School of Basic Medical Sciences, od for easy yet accurate species-level identification at any life Central South University, Changsha 410013, Hunan, China stage is required, followed by thermobiological studies [12]. As a supplemental means of morphological method, DNA-based : * Y. Gu o L. Wu ( ) method can provide a quick and reliable species determination Department of Physiology, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, China and is relatively insensitive to the preservation methods [12]. e-mail: [email protected] Meanwhile, DNA-based method can be carried out on any

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lifecycle stage (dead, preserved, or live samples) without further and 678 bp per fragments for identification of sarcophagid rearing. However, molecular data available with respect to the species under experimental conditions prior to application in sarcophagid flies are very limited in GenBank. Chinese criminal investigations. The two loci of 36 flesh fly Most forensic insect species identification papers focused specimens were sequenced and deposited in GenBank to on some portion of the genes for cytochrome oxidase subunits expand local databases. With the advantages of high accuracy, one and two (COI + COII)[13–16]. Even partial sequences rapid, and convenient, the two markers will offer a quick and of COI gene have been proven to have sufficient discrimina- reliable alternation in practice. With other DNA markers, tion power [17–21], which makes it in particular suitable for these two new markers will be helpful to achieve a 100 % forensic applications. One surprise was the discovery that all identification success for forensic entomologists. Hawaiian Lucilia cuprina that were genotyped formed a distinct COI lineage that was a sister group to the COI of Lucilia sericata, while a phylogeny based on the nuclear gene Materials and methods 28S ribosomal RNA (28S rRNA) produced separate branches corresponding to the two morphologically defined species Specimens [22, 23]. Thus, the use of single gene as species identifier should be done with care and additional markers will be Specimens of Boettcherisca peregrina (Robineau-Desvoidy), necessary to achieve a 100 % identification success [14, 24]. Boettcherisca javanica (Lopes), Helicophagella melanura The dangers of relying on a single locus are well illustrated by (Meigen), Parasarcophaga crassipalpi (Macquart), several recent studies [23–25]. Many discussions about the Parasarcophaga albiceps (Meigen), Parasarcophaga dux feature of the mitochondrial DNA and nuclear fragments have (Thompson), Sarcophaga africa (Wiedemann)[=Sarcophaga been raised. Suggested targets other than COI + COII includ- haemorrhoidalis (Fallén) or Bercaea cruentata (Meigen)], ed the gene for 28S rRNA [22], the ribosomal internal tran- Ravinia pernix (Harris), and Kramerea schuetzei (Kramer) scribed spacer regions [26], the NADH dehydrogenase sub- were obtained during the months of June to September in unit 5 [18], and the gene for 16S ribosomal RNA [27]. For all China from 2010 to 2012, and every species had four samples. their flaws, these markers are potential as discriminatory tools Another two dried adult specimens of Chrysomya in identification of forensically important flies. The gene megacephala (Fabricius) (Diptera: Calliphoridae) were ob- period (per) was discovered in Drosophila melanogaster in tained from Changsha (Hunan) and Zhengzhou (Henan) in 1971 [28] and shown to be widespread in insect [29]. All the year 2012 (Electronic Supplementary Material). All sam- available reporting techniques consistently revealed a broad ples were collected using traps baited with 9-month-old pigs distribution of per in fly tissues [30]. Roles of the clock gene (35–50 kg). Samples were subsequently air-dried at room per is well understood in relation to circadian clocks [31], temperature or stored in 70 % ethanol at −20 ° C. All adult while its potential of species identification has not been flies were identified using morphological keys [10, 36]by investigated. entomologists in Hunan Agricultural University. The use of more than one gene for phylogenetic methods is Samples were collected from 19 locations in 11 Chinese recommended as using only one gene may not give a true provinces. The southernmost collection location is Kunming picture of phylogenetic relationships [23–25]. Analyzing both located in Yunnan Province (102°50′E, 24°48′N), and the nuclear and mitochondrial genes simultaneously has northernmost location is Suihua located in Heilongjiang Prov- highlighted the difference between gene trees and species ince (126°98′E, 46°67′N). The easternmost collection location trees [32, 33]. The short COI fragments (200–300 bp) had is Yanbian located in Jilin Province (131°18′E, 42°15′N), been used to solve species identification of sarcophagid flies while the westernmost location is Urumqi in Xinjiang Munic- respectively in our previous studies [21, 27], while the data of ipality (87°36′E, 43°46′N). per gene from sarcophagids is still lacking. Frequently, the 5′ end of COI is the site of the proposed universal insect DNA DNA extraction and PCR protocol barcode [12, 15]. Recent research showed that the identification success using a mini-barcode region of 127 bp was very Total DNA was extracted from leg or thorax regions of each low (80.7–82.5 %), and the use of this region is not recom- adult fly using the CTAB method [37]. Representative COI and mended as a species identifier [14]. Although identification per gene sequences queried from GenBank according to pub- success is very high using the standard barcode region lished studies [5, 13–16, 31, 38] were aligned respectively using (658 bp), dangers of relying on single COI barcodes still exist the sequence alignment program DNASTAR (MEGALIGN [14]. Due to the recent burst in development of the forensic version 7.1.0; DNAStar Inc., Madison, WI). Conserved regions sciences, new court criteria require the evaluation of scientific of the alignment were evaluated and marked. The most com- evidence prior to its submission to the court [34, 35]. Thus, monly occurring nucleotides at each position of the conserved this study evaluated the suitability of the 700 bp 3′ end of COI sequence were selected and inputted in the primer design 中国科技论文在线 http://www.paper.edu.cn Int J Legal Med

program PRIMER PREMIER 5.0 (PREMIER Biosoft Interna- COI and per sequences were compared with Dipteran tional, Palo Alto, CA). The primer-binding site should lie en- sequences on the NCBI website via the BLAST function. tirely within the conserved region, and the general primer design Although there are plenty of COI gene data, per gene data rules were considered to avoid false priming and primer–dimer of forensically important sarcophagids is sparse. formation in cross-family PCR. The 700-bp fragment of the Evolutionary analyses were conducted in MEGA5 [39]. COI gene from all specimens was amplified using forward The evolutionary history was inferred using the neighbor- primer (5′-CTTTACCTGTACTTGCTGGAG-3′) and reverse joining method [40]. The bootstrap consensus tree inferred primer (5′-AACTTGTCGTTGTGATGCT-3′), which were from 500 replicates [41] is taken to represent the evolutionary designed directly from sarcophagid fly sequences [5, 16]. A history of the taxa analyzed [41]. Branches corresponding to portionof678-bpfragmentofper gene was amplified and partitions reproduced in less than 50 % bootstrap replicates are sequenced by using forward primer (5′-CGCTGTTA collapsed. The percentage of replicate trees in which the TCCCTAAGGTAA-3′) and reverse primer (5′-CTGGTATG associated taxa clustered together in the bootstrap test (500 AAAGGTTTGACG-3′), which were designed directly from replicates) is shown next to the branches [41]. The evolution- sarcophagid fly sequences [31, 38]. ary distances were computed using the maximum composite The PCR reaction volume was 25 μL containing 1–5 μL likelihood method [42]. Two C. megacephala samples be- (20–40 ng) of template DNA, 12.5 μL 2× GoTaq® Green longing to the family Calliphoridae were used as outgroup for Master Mix (Promega, Madison, WI, USA)—4 μLdNTP phylogenetic analyses. (1 mmol/ml), 1.0 U Taq polymerase, 2.5 μL 10× buffer (Mg2+ 1.5 mmol/L)—0.25–2.5 μL each primer (10 μM), Nuclease-Free Water (Promega, Madison, WI, USA) added Results and discussion to a total volume of 25 μL. PCR amplifications were performed in a thermocycler (Perkin-Elmer9600) and pro- All 36 samples were identified by entomologists into nine dis- grammed with the following parameters: initial step at tinct morphological species (B. peregrina, B. javanica, H. 94 °C for 5 min then continued for 30 cycles each of melanura, P. crassipalpi, P. albiceps, P. dux, S. africa, R. 94 °C for 1 min, an annealing step for 1 min, and pernix,andK. schuetzei) belonged to six genera (Boettcherisca, 72 °C for 1 min. The annealing temperatures were Parasarcophaga, Helicophagella, Sarcophaga, Ravinia,and optimized for primer pairs of COI and per at 51 and Kramerea) using traditional morphological characters. Mean- 55 °C, respectively. An elongation of PCR products by while, sequence similarity to previously identified COI dipteran 72 °C for 5 min completed the reaction. sequences was verified on the NCBI website via the BLAST function and no obvious discrepancies in the COI sequences Sequencing were found to those already published. Sampling multiple specimens from across the most known geographic range of the Vertical non-denaturing polyacrylamide gel electrophoresis species was included. B. peregrina, H. melanura, S. africa, P. was used to isolate PCR products, which were then purified crassipalpi, P. albiceps, and P. dux are widespread and common using a QiaQuick PCR Purification Kit (Qiagen, Germany). in China. Moreover, P. crassipalpi, P. albiceps, P. dux, B. Columns cycle sequencing was performed on both forward peregrina, B. javanica,andS. africa are supposed to be the and reverse strands using ABI PRISM Big Dye Terminator species of greatest forensic importance throughout the world [4, Cycle Sequencing Ready Reaction Kit by ABI PRISM 3730 5, 9–18], while K. schuetzei is distributed in Asia and Europe [9, (Applied Biosystems, Foster City, USA) with BigDye termi- 36]. Meanwhile, specimens of R. pernix were collected unex- nator v3.1 as the sequencing agent. Sequence chromatograms pectedly nearby the disposed animal carcasses and they were were edited, and discrepancies between forward and reverse included for purposes of phylogenetic comparisons. sequences were resolved using Sequence Navigator (v1.01, A700-bpfragmentoftheCOI gene and a 678-bp fragment Applied Biosystems, Foster City, USA). of the period gene were successfully sequenced for all the specimens. No indels (insertion or deletion) was detected. All Sequence analysis and phylogenetic tree construction the sequences were proofread and aligned in the program Megalign 7.1.0 included in the DNA analysis package Since the sequences did not contain any insertions or dele- DNAStar (DNAStar Inc., Madison, WI). Based on analyzed tions, all resultant sequences in this study were aligned using COI gene and analyzed per gene, 165 and 124 positions of ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The phylogenetically informative were revealed, respectively. obtained sequences have been deposited in GenBank by According to the numbers of positions that were consistently Sequin (http://www.ncbi.nlm.nih.Gov/Sequin/index.html), different for one species compared to the other ones, the and their accession numbers are KC966375–KC966450 interspecific and intraspecific difference could be identified (Electronic Supplementary Material). To identify species, the roughly. 中国科技论文在线 http://www.paper.edu.cn Int J Legal Med

The phylogeny of sarcophagine flies based on COI and per morphological method in identification of sarcophagids, as sequences were both separated into nine genetic clades (Figs. 1 the technology used DNA is easier to perform and save more and 2). As outgroup, the two C. megacephala samples clus- time for forensic scientists within their routine work. tered together and were clearly separated from the sarcophagid At the species level, Fig. 1 showed strongly supported mitotypes. The phylogenetic analysis results of these specimens monophyletic groupings for species of P. crassipalpi , P. from different regions provided us the possibility of applying albiceps, P. dux, S. africa,andK. schuetzei, while Fig. 2 these two genetic markers as an identification tool for the showed that of H. melanura, P. albiceps, S. africa, R. pernix, common Sarcophagidae species from China. Therefore, the and K. schuetzei. Although some species formed their own used technique can be used as supplementary means of distinct conspecific and monophyletic cluster with relatively

Fig. 1 Neighbor-joining (NJ) tree of maximum composite likelihood method for 36 COI gene sequences from nine species of Chinese Sarcophagidae. Voucher ID, City, morphological species identification and Accession no. are given in specimen label. Numbers on branches indicate the support value. Two C. megacephala from family Calliphoridae are included as outgroup. Evolutionary distance divergence scale bar is 0.01 中国科技论文在线 http://www.paper.edu.cn Int J Legal Med

Fig. 2 Neighbor-joining (NJ) tree of maximum composite likelihood method for Chinese Sarcophagidae species based on a 678-bp region of the Period (per) gene. Voucher ID, City, morphological species identification are consistent with that of Fig. 1. Numbers on branches indicate the support value. Two C. megacephala from family Calliphoridae are included as outgroup. Evolutionary distance divergence scale bar is 0.02

weak bootstrap values, correct genetic clades were formed the genus of Boettcherisca, clustered together with strong based on both fragments. The two trees are consistent with supporting bootstrap. Similar observations have been reported each other. In addition to the fragments, we also should for these two species [16], which indicate the danger of single consider the geographical differences in the samples. Thus, DNA marker to differentiate the species from Boettcherisca. the application of these two fragments for analysis is worth Both gene trees show that the R. pernix clade is very distinct trying. To determine whether fragment of per gene can be from other genera, suggesting that Ravinia is far from the used as routine molecular marker, other regions with different other genera. length should be studied in the future. At the genus levels, In Table 1, all values for maximum intraspecific variations both trees showed B. peregrina and B. javanica,belongedto of the sarcophagid species were no more than 2 %, except for 中国科技论文在线 http://www.paper.edu.cn Int J Legal Med

Table 1 Calculated intraspecific and interspecific variations expressed as a percentage of the 700 bp COI (below diagonal) and 678 bp per (above diagonal) data

No.SpeciesRangeandmean(%)123456789

COI period

1 P. dux 0–0 (0) 0–0.1 (0.1) – 3.4 3.4 2.9 6.0 4.3 3.9 2.9 9.6 2 P. albiceps 0–0 (0) 0–0 (0) 9.3 – 4.1 4.0 7.2 5.3 3.9 3.9 10.6 3 P. crassipalpi 0–1.4 (0.7) 0.3–1.0 (0.6) 7.6 9.5 – 3.6 6.0 3.5 4.5 3.7 10.5 4 H. melanura 0–0.1 (0.1) 0–0.6 (0.4) 8.7 8.8 7.8 – 6.4 4.5 3.6 3.5 9.7 5 B. peregrina 0–0.1 (0.1) 0–0.7 (0.6) 8.7 10.1 8.6 8.2 – 4.4 7.0 6.2 12.0 6 B. javanica 0–0.3 (0.1) 0–0.1 (0.1) 9.9 10.9 8.8 10.0 3.5 – 5.3 4.5 10.8 7 S. africa 0–4.2 (2.1) 0–0 (0) 8.1 11.3 8.2 9.8 11.3 11.4 – 3.8 10.7 8 K. schuetzei 0–0 (0) 0–0 (0) 8.1 10.2 7.4 8.1 8.8 9.7 8.5 – 10.0 9 R. pernix 0–0.4 (0.2) 0–0.4 (0.3) 10.4 9.9 10.3 10.1 11.9 11.6 10.1 10.1 –

The within-group means are in brackets Range and mean range from the minimum intraspecific variation to the maximum intraspecific variation

S. africa at 4.2 % based on COI.Noobviousintraspecific utilized in the identification of forensically important flies variations of the S. africa were found based on per. The [25]. However, the barcoding approach has its own limitations interspecific variations between sarcophagid species varied as is seen with its failure to identify some closely related from 7.4 to 11.9 % based on COI (below diagonal) and varied species of blowflies [22, 23]. Although recent research has from 2.9 to 12.0 % based on per. The interspecific variations proven that identification success of sarcophagids was very between species were larger than 3 % except for P. dux and K. high using the standard barcode region (658 bp) or using the schuetzei at 2.9 % based on per. But, it should be noted that entire COI region (1,535 bp) (98.2–99.3 %) [14], there was a the interspecific variation between P. dux and K. schuetzei low interspecific sequence divergence (<2 %) in some species based on COI was 8.7 %, which could distinguish these two groups. Thus, COI barcodes, or for that matter any single species explicitly. The small amplicon size and highly conser- gene, seems unlikely to resolve the identities of all fly species vative made this selected per gene region failing to supply of forensic importance because such phylogenies only infer sufficient amount of information within species. The interspe- the evolutionary relationships for the particular gene used. cific variation between species of some species were less than Consequently, various regions of DNA should be studied 5 %, which indicates that relying solely on single fragment for and a switch to multi-gene approach becomes necessary [24]. delimiting species is dangerous, especially for the closely The processes of obtaining the 678-bp per and 700-bp COI related species. fragments were simple, and the chance of misidentification In previous studies, the interspecific variations between B. was reduced. The identification results of this study were peregrina and B. javanica were less than 3 % [14, 16]. For the comparable to the ones using the longer fragments, which purposes of the current study, it is important to note that the B. showed potentiality of these two fragments as the discrimina- peregrina and B. javanica that belonged to the genus tory tool in identification of Sarcophagidae. All sarcophagids Boettcherisca can be reliably distinguished on the basis of collected on, or nearby, a corpse need to be identified even sequence data and that different patterns and levels of intraspe- though some may have no forensic value [34]. Species that are cific sequence variation for S. africa are apparent, which may believed to currently have no forensic interest may later turn have a bearing on the level of sequence variation accepted in out to have a forensic interest after their breeding/feeding sequences from unknown specimens for forensic typing. Mito- ecology is better understood [14, 43]. The Sarcophagidae or chondrial DNA has advantages over nuclear DNA in estimat- flesh flies (Diptera) comprise more than 276 species in more ing phylogenetic relationships among insect taxa because of its than 66 genera in China [36], while only nine species were rapid evolution and inheritance without gene recombinations included in this study. In summary, present study indicated the [22]. Accumulating evidences suggest that nuclear DNA can be potential of the two DNA markers for use in the identification used as supplemental markers at the species or genus levels due of forensically important flies. However, the per database to its many advantages such as PCR with ease and a relatively developed in this study still took a small degree of risks in short region with high information content [26]. recognizing some species identity because the sequences are Shorter fragments have many advantages, such as quick, very similar and this fact remains unknown as these species easy, and economical. COI barcodes have been successfully were not included in the study. Present results provided an 中国科技论文在线 http://www.paper.edu.cn Int J Legal Med

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