Molecular Identification of Forensically Important Indian Species of Flesh Flies (Diptera: Sarcophagidae) by Using COI Gene of Mitochondrial DNA

Entomological evidence at a crime place can supply information on time, origin, kind, and location of death [1-4]. Postmortem interval (PMI), the time elapsed since death, is classically calculated by techniques such as measurement of algor mortis, liver mortis, and rigor mortis [3,4]. Necrophagous insects have proved to be helpful in forensic investigations [5,6]. Most forensic entomological clues are reliant on unquestionable species identification. This estimation is founded upon the time taken for insects evolving on a corpse to come to the stage present when the body is discovered [7,8]. Insect species have distinct developmental lifecycle timings and thus to utilize the correct developmental data, species need to be accurately recognized. This can be done on the basis of morphological differences [7,8].


Introduction
Entomological evidence at a crime place can supply information on time, origin, kind, and location of death [1][2][3][4]. Postmortem interval (PMI), the time elapsed since death, is classically calculated by techniques such as measurement of algor mortis, liver mortis, and rigor mortis [3,4]. Necrophagous insects have proved to be helpful in forensic investigations [5,6]. Most forensic entomological clues are reliant on unquestionable species identification. This estimation is founded upon the time taken for insects evolving on a corpse to come to the stage present when the body is discovered [7,8]. Insect species have distinct developmental lifecycle timings and thus to utilize the correct developmental data, species need to be accurately recognized. This can be done on the basis of morphological differences [7,8].
The larval types and eggs of numerous forensically significant dipteran species are tough to distinguish as their morphological differences may be obscured or awfully maintained [8,9]. Definitive identification may be achieved by rearing larvae to adults but this can be time consuming and need the larvae to be assembled, reside and kept in situation optimal for proceeded development [8,9]. The members of family Sarcophagidae or flesh flies (Diptera) comprise over 2,500 species in over 100 genera globally, with numerous species being carrion breeders and initial corpse colonizers [10,11]. Such species have the potential to be utilized to estimate the PMI [1,[12][13][14].
The use of DNA methods can not only supplement the traditional morphological methods but may furthermore give information on population substructures [8,15]. This identification can be carried out on any lifecycle stage without further rearing and on dead, maintained or reside trials. DNA techniques are also relatively insensitive to the methods of preservation or age of samples [16]. Latest use of molecular markers for forensic species has focused on the use of mitochondrial DNA [8,14]. Mitochondria are present in high numbers inside cells and, therefore, mitochondrial DNA is present in a much higher exact replicate number than nuclear DNA [17]. Hence it is the favored goal when considering incomplete or old tissue samples [17][18][19][20][21].

Abstract
Identification of the fauna collected from and around the corpse is an absolutely vital prerequisite for the estimation of post mortem interval (PMI) in the field of forensic entomology. Morphological identification of flesh flies (Diptera; Sarcophagidae) can be perplexing due similar attributes of the species. So, to simplify the identification method, amplification of cytochrome oxidase I (COI) gene is often tried. We illustrate the use of 450 bp fragment of COI gene for differentiation of ten forensically significant species of flesh flies in India. The outcome displays the robustness of COI gene as a diagnostic marker, since its nucleotide variability endows reliable distinction to be drawn between species. Four new COI gene sequences have been added to GenBank which may be of interest for correct species identification for future workers.
Even partial sequences of this gene have been verified to have sufficient discrimination power, [18,19,25,26], which makes it specifically important for forensic applications. With this study we will be able to know the utility of COI for identification of ten forensically significant Indian sarcophagid fly species and build up genetic data for the database of sarcophagid flies from India.

Materials and Methods
Taxon sampling, identification and DNA extraction 30 specimens belonging to 10 species of Indian Sarcophagidae were collected from the four north Indian states. All the flies were collected from meat kept as bait by using sweep net and immediately killed by using potassium cyanide (KCN) killing jar. The identification was done by preparing the external genitalia slides followed by the keys given by Senior-White et al. [27], Pape [10] and Nandi [28]. After identification, the specimens were kept in insect boxes. DNA was extracted from the dried thorax of the fly specimens using the Coen method [29]. The extracted DNA was eluted in 200 μL elution buffer and kept at -20ºC for long term storage. The fraction of extracted DNA was spectrophotometrically quantitated and diluted to 50 ng/μL prior to PCR amplification step.

Amplification and sequencing of COI gene
A partial fragment (450 bp region) of COI gene of the mitochondrial DNA was amplified using the primer pairs 5'-CAGCTACTTTATGAGCTTTAGG-3'(forward primer) and 5'-CATTTCAAGCTGTGTAAGCATC-3'(reverse primer) [22]. The PCR reaction volume was 25 µL, containing 2.5 µL of template DNA, 2.5 µL Taq polymerase, 2.5 µL dNTP, 2.5 μL 10 × buffer, 2.5 µL of each primer and Nuclease-free water was added to a total volume of 25 μL. The thermocycler (Bio-Rad, USA) was programmed with the following parameters; initial step at 94ºC for 3 min then continued for 30 cycles each of 94ºC for 30s; 48ºC for 30s; and 72ºC for 30s. An elongation of PCR products by 72ºC for 5 min completed the reaction.

Sequence analysis
Sequencing of both sense and antisense strand was done using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit by ABI PRISM 3730 (Applied Biosystems, Foster City, U.S.A.). Big Dye terminator v3.1 was used as the sequencing agent. The sequences obtained have been deposited in GenBank by Sequin (http://www.ncbi. nlm.nih.gov/Sequin/index.html) and their accession numbers are listed in Table 1. For phylogenetic analysis and comparison, the available world sequences for seven species were downloaded from the NCBI website (Table 1). All the COI gene sequences were compared with Dipteral sequences on the NCBI website via the BLAST function for quick identification.

DNA sequence data analysis
Clustal X software [30] was used to align DNA sequences and after alignment homologous region was selected for phylogenetic investigations. Analysis of nucleotide composition, general transition;transversion ratio (ts;tv), variable and parsimony informative sites and pair wise nucleotide distances were calculated using MEGA5 [31]. The evolutionary history was estimated using the Neighborjoining procedure and Maximum parsimony procedure [32] with 1000 replications in bootstrap test. There were a total of 450 positions in the last dataset. Musca autumnalis belonging to the family Muscidae was taken as an outgroup for phylogenetic analysis.

Results and Discussion
The aim of this research was to ascertain the reliability of COI gene to differentiate between some Indian flesh flies of forensic interest. So far, the publications on the molecular identification of forensically applicable flesh flies from India are constrained to one [33]. They researched five species of flesh flies on the basis of COI gene but that work was only constrained to phylogenetic investigation and not associated to forensic entomology. In our previous paper [25], we have sequenced ten species of Sarcophagidae in which the primer pair was different than the primer pair used for the sequencing of samples used in the present study.
It is clear from the nucleotide composition data that A+T composition dominated in all the species studied. The average nucleotide composition for the ten species was T=40.58.6%, A=30.10%, C=16.08%, G=13.24%. The data showed that maximum A+T bias was seen in S. hirtipes (72.2%) and lowest in S. macroauriculata (69.3%). The A+T content observed during the present study was found out to be in agreement to the characteristics of the base composition of the mitochondrial genome of other dipteran insects (ranging from 72.6% to 82.2%) [34]. Silvestre et al. [35] suggested that one hypothesis that attempts to explain A+T bias is that the DNA polymerase could use these bases in a more efficient way during mtDNA replication [36]. Sharma et al. [25] showed average nucleotide composition for ten species as T=37.20%, A=29.19%, C=16.61% and G=17.00%.
The 450 bp region has revealed 289 conserved and 161 variable and parsimony informative positions which showed that COI contains both conserved and highly variable regions across taxa which are very useful for the species identification. The present results are in complete agreement with Sharma et al. [25] who sequenced 465 bp region from the ten flesh fly Indian species and showed 342 conserved and 123 variable and parsimony informative positions. Bajpai and Tewari [33] also studied five sarcophagid species and showed that COI showed 71 variable sites in 296 bp long sequence and only 26 sites were found to be parsimony informative. Song et al. [37] noted 151 variable sites in the 552 bp long fragment of COI gene amplified from fifteen sarcophagid species of China origin. They also concluded that out of 151, 129 variable sites were in the third codon position.
Sharma et al. [25] studied ten Indian flesh fly species and showed interspecific genetic divergence of 4-14%. Maximum genetic divergence of 14% was seen between samples of S. princeps and S. hirtipes. Minimum genetic divergence (4%) was seen between samples of S. misera and S. serciea. Guo et al. [38] investigated seven Chinese sarcophagid species from different populations on the basis of 272 bp region of COI gene. The mean levels of divergence between these seven sarcophagid species ranged from 4% to 8%. Subsequently, Guo et al. [39] showed interspecific variation varying from 7% to 10%. The maximum and minimum levels of divergence between the four sarcophagid species were similar and ranged from 7% to 11%. The minimum intraspecific values of the four species were all 0%. They further emphasized that the interspecific variation between species of different genera was higher than that from same genus, which indicated the ability of COI to identify the species from different genera of Sarcophagidae family.
The 450 bp COI fragment was successfully sequenced for all the specimens. All flies were rightly assigned into ten species with monophyletic separation in the NJ and MP trees (Figures 1 and 2). At the species level, the high bootstrap values (100%) provide percentage robust support for the monophyly of all the ten species under study. Phylogenetic trees constructed by both methods (NJ and MP) showed almost same topology. Within H. kempi, two specimens clustered together with three Malaysian samples (GenBank accession numbers GU174025, EF405946, EF405947) with a supporting bootstrap of 52-100%. Within I. martellata, two specimens (Accession numbers FJ440843 and FJ440844) clustered together with a supporting bootstrap of 38-100%. At the genus level, P. albiceps and B. peregrina cluster together, whereas H. kempi, S. princeps and I. martellata cluster together indicating the ability of this shorter COI fragment to identify the species from the same genus may not be as efficient as that of the longer fragments. More DNA fragments of different lengths and regions should be studied in the future.
All the samples belonging to species P. albiceps are clustered together with the sequences of the same species originating from France (JQ582062 and JQ582097) and Malaysia (EF405931 and EF405932) with bootstrap value ranging from 54-99%. With the high  Sharma et al. [25] phylogenetically analyzed ten Indian species of flesh flies and showed high bootstrap support for each species. All the ten species were separated with high bootstrap support (50-100%) which showed the robustness of this gene to distinguish between Indian flesh flies. Zehner et al. [40] investigated twelve species of Sarcophagidae and showed a bootstrap value of 52-90% in the NJ tree. Song et al. [36] noted ≥94% bootstrap support which strongly supported monophyly of the three clades including the Parasarcophaga clade, Boettcherisca clade and the Liopygia clade and suggested interior relationship as S. albiceps, S. misera and S. sericea for the Parasarcophaga clade. They also showed sister relationships between some of the species (with S. scopariiformis and S. iwuensis; S. polystylata and S. hui; S. brevicornis and S. dux). Our results are also in complete association with Guo et al. [21] who observed 100% bootstrap support for three species of flesh flies viz., S. similis, S. albiceps and B. peregrina. Qing et al. [41] showed 99% bootstrap support for 7 sarcophagous species of Chinese origin. Bajpai and Tewari [33] phylogenetically studied five species of Indian Sarcophagidae and demonstrated minimum evolution trees for COI and ND5 genes of mtDNA. They showed a bootstrap value of 59% to 94% which shows robustness of this gene to identify species within the genus Sarcophaga. Jordaens et al. [42] also showed a bootstrap value ranging from 99% to 100% while studying S. misera, S. albiceps, S. peregrina, S. ruficornis   The evolutionary history was inferred using the Maximum Parsimony method. Musca autumnalis of Muscidae is taken as an out group. Numbers on branches suggest the support value for that particular branch. Specimen voucher codes given in the Table 1. Evolutionary distance divergene is given by bar which indicates 10 substitutions per site. \ and S. kempi (a total 56 species were investigated in their study).

Conclusions
On the basis of this discussion, it can be concluded that COI gene of mtDNA is capable of identification and phylogenetic analysis of Indian sarcophagid flies. The sarcophagid fly fauna has remained neglected not only inside India but globally and, therefore, is unexploited as PMI indicators in forensic entomology. The primary step required to secure the use of sarcophagids in PMI estimator is the accurate species identification of a specimen from any life stage. The COI gene becomes an important marker for the fast species identification where the morphological based identification is not likely. This approach has proved thriving for the unquestionable identification of the Indian Sarcophagidae examined and is likely to be a perceptive typing scheme for sarcophagids associated with forensic casework. The present study is the first foremost try to recognize ten forensically important flesh fly species of Indian origin on the basis of COI gene of mtDNA. The resultant facts and figures will be rather helpful for the future workers in their investigations and for the advancement of specific markers for the identification purposes.