Journal of Forensic Science and Medicine

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 8  |  Issue : 1  |  Page : 11--16

Mitochondrial and nuclear DNA-based identification of some forensically important calliphoridae (diptera) in Luoyang of China


Mengzi Yang, Weiping Zhang, Adilai Tuerxun, Yaonan Mo, Xiandun Zhai 
 Department of Forensic Biology Laboratory, Forensic Medicine Institute, Henan University of Science and Technology, Luoyang, Henan, China

Correspondence Address:
Xiandun Zhai
Forensic Medicine Institute, Henan University of Science and Technology, No. 263 Kaiyuan Road, Luolong District, Luoyang, Henan, 471000
China

Abstract

Introduction: Calliphoridae plays a key role in forensic entomology research, which is one of the first insects to decompose animal carcasses.The mitochondrial cytochrome c oxidase subunit I and the ribosomal internal transcribed spacer 2 (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Aim: The aim of the study was to test the suitability of two genetic markers based on conducting the molecular identification of six necrophagous Calliphorid flies. Materials and Methods: Fourteen Calliphoridae flies were collected and classified with traditional morphological characteristics. The DNA of flies was extracted and the fragments of COI and ITS2 were amplified and sequenced. All the sequences were aligned and analyzed by MEGA 7 software for NCBI BLAST, nucleotide composition, intra- and inter-specific divergence calculation, and phylogenetic tree inference successively. Results: The results indicated that COI and ITS2 genes were robust in the identification of Calliphoridae at the species level and ITS2 gene sequence possessed a strong resolution power as it showed higher variation values between Lucilia sericata and Lucilia cuprina, Calliphora vomitoria and Triceratopyga calliphoroides, C.vomitoria and Aldrichina grahami, but inferior to COI for T. calliphoroides and A. grahami. Conclusions: Our results showed that combination of COI + ITS2 genes yields more accurate identification and diagnoses and better agreement with morphological data than the mitochondrial barcodes alone. As a supplementary method for morphological identification, we advocated for the combination of nuclear and mitochondrial gene approaches to address the taxonomy and phylogeny of forensic relevant flies, especially of closely related species and populations.



How to cite this article:
Yang M, Zhang W, Tuerxun A, Mo Y, Zhai X. Mitochondrial and nuclear DNA-based identification of some forensically important calliphoridae (diptera) in Luoyang of China.J Forensic Sci Med 2022;8:11-16


How to cite this URL:
Yang M, Zhang W, Tuerxun A, Mo Y, Zhai X. Mitochondrial and nuclear DNA-based identification of some forensically important calliphoridae (diptera) in Luoyang of China. J Forensic Sci Med [serial online] 2022 [cited 2022 Oct 2 ];8:11-16
Available from: https://www.jfsmonline.com/text.asp?2022/8/1/11/339792


Full Text



 Introduction



In recent decades, forensic entomology has become a significant tool to estimate postmortem interval (PMI) in forensic investigations worldwide.[1],[2] As one of the most related taxa on corpse, necrophagous flies are usually the first to arrive, especially Calliphoridae flies, even in a few minutes. The identification of these fly species and their developmental stages provides useful suggestion for the estimation of PMI. However, correct identification of the species to which flies belong is a critical process, due to similar morphology and the lack of keys for some taxa, especially for immature stages.[2] Fortunately, DNA-based identification method can be carried out on any type of samples and entire lifecycle without further rearing.

Some researchers have proposed identification methods based on the genes of cytochrome oxidase[3],[4],[5] and nuclear ribosomal DNA (rDNA).[6],[7] The nuclear ribosomal RNA genes of eukaryotes are a tandemly repeated multi-gene family, including three ribosomal RNA coding genes 18S rDNA, 5.8S rDNA, 28S rDNA, and two transcribed spacers in the noncoding region between the coding genes, namely internal transcribed spacer 1 (ITS1) and ITS2. The secondary structure of ITS2 plays a role in guiding shearing during the ribosomal RNA maturation process. The rDNA exons are highly conserved across eukaryotic organisms, whereas the ITS regions present length variability due to point mutations and insertions/deletions (indels). ITS sequences have been widely used as an excellent phylogenetic marker at the species or genus level[8],[9] for the advantages of easy amplification, high information content, and relatively short region.

Nucleotide variation in the COI gene was evenly spread across the length of the whole sequences, with no obvious gene associated clusters. DNA barcoding uses mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequence in particular region to identify different animal species. Sequencing these regions has helped recognize many Diptera species of forensic interest in the different areas of the world.[2],[6],[10] However, the limitation of such sequences in identification, especially of closely related species and populations, demand a multi-gene approach.[11]

Various molecular markers have been developed in the past for use in species determination. This article was determined to use ITS2 and DNA barcoding region to identify some forensically important Calliphoridae (Diptera) in Luoyang of China.

 Materials and Methods



Materials

Specimen collection and morphological identification

All flies specimens in this study were captured on traps baited with Sprague − Dawley rats or rabbits from June 2016 to May 2017, in Luoyang City, Henan Province, China. Fourteen Calliphoridae adults were obtained and morphologically identified using specific taxonomic keys and confirmed by entomologists, stored in individual 1.5 mL Axygen tubes and preserved at − 20°C after killed.

Methods

DNA extraction

The DNA was extracted from forelegs or thoracic muscle or both of them of each individual with the TaKaRa Mini BEST kit (Takara Bio Inc.) followed the manufacturer's instructions. Extracted DNA was quantified using a UV-VIS spectrophotometer FLA6000 (Flight Technology Co., Ltd.).

Polymerase chain reaction amplification

In total, 28 new sequences (from 14 specimens included in this study) were generated from one nuclear (ITS2) and one mitochondrial (COI) gene. There were approximate 327 bp of ITS2 gene amplified using primers: Forward primer: 5'-TGC TTG GAC TAC ATA TGG TTG A-3' and Reverse primer: 5'-GTA GTC CCA TAT GAG TTG AGG TT-3', modified from Song et al.;[12] approximate 700 bp of the COI DNA (barcoding region) gene amplified using primers: LCO1490-5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3' and HCO2198-5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3', mentioned in Folmer and Black.[13]

Each PCR reaction was carried out in a final volume of 50 μL that included 25 μL 2 × Premix Taq (LA Taq version 2.0 plus dye), 2 μL each primer (10 μmol/L) and 6 μL (about 20–40 ng) template DNA. Nuclease-free water added to a total volume of 50 μL.

Polymerase chain reaction (PCR) that performed in 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with the ITS2 gene program was initiated by denaturation at 95°C for 1 min, followed by 35 cycles of 45 s at 94°C for template denaturation, 40 s at 65°C for primer annealing, and 1 min at 72°C for primer extension. The reaction was terminated by a 5 min elongation cycle at 72°C. While the thermal cycler program of COI gene consisted of an initial denaturation step at 94°C for 1 min, followed by 5 cycles of 94°C for 1 min, 45°C for 1.5 min, and 72°C for 1.5 min, then continued for 35 cycles of 94°C for 1 min, 50°C for 1.5 min, and 72°C for 1 min, with a final extension step at 72°C for 8 min. PCR products were confirmed by 2% TBE Agarose gel electrophoresis stained in ethidium bromide and analyzed by gel imaging and analysis system.

Sequencing and phylogenetic analysis

Amplified fragments were sequenced in the forward and reverse directions by Thermo Fisher Scientific (Waltham, MA, USA). The sequences were then proofread by examining chromatograms by eyes.

All sequences were BLAST against GenBank files with default parameters. Alignments of the sequence libraries for the two genes and phylogenetic analysis were all conducted with MEGA 7.0 (Temple University, Philadelphia, Pennsylvania, USA): Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.[14] Analyses were conducted using the P-distance model.[15] The evolutionary history was inferred using the neighbor-joining (NJ) method.[16]

 Results



Alignment and specimens identification

The 14 specimens collected in this study represented six different species and five genera, including Lucilia sericata (Meigen) (2 specimens), Lucilia cuprina (Wiedemann) (2 specimens), Chrysomya megacephala (Fabricius) (3 specimens), Triceratopyga calliphoroides (Rohdendorf) (2 specimens), Calliphora vomitoria (Linnaeus) (2 specimens), and Aldrichina grahami (Aldrich) (3 specimens). The lengths of 637 bp COI and 327 bp ITS2 sequences were obtained from 14 samples, respectively. The identification of BLAST was well coincident with morphological identification, and the result of BLAST showed that index of similarity of COI gene was above 99%. The index of similarity of ITS2 gene was above 99% except for C. vomitoria which was about 94%. The high homology with the reference sequences registered in GenBank enables to confirm most of the specimen identification. Only for C. vomitoria species the divergence values are far greater. Considering this variability within C. vomitoria species may be explained with the different origin of specimens.[17] Details are listed in [Table 1] and [Table 2].{Table 1}{Table 2}

Nucleotide composition

The nucleotide composition of 637 bp COI and 327 bp ITS2 sequences was calculated using MEGA 7 software. It was clear that A + T composition dominated in all the species studied, which was characteristics of insect DNA. The average nucleotide composition of the 14 species 637 bp COI was A = 30.7%, G = 15.8%, C = 15.8%, T = 37.7%, A + T = 68.4%, and 327 bp ITS2 was A = 40.6%, G = 10.7%, C = 8.7%, T = 40.1%, A + T = 80.7%.

The 637 bp COI sequences had 538 conserved and 99 variable including 98 parsim-info and 1 singleton position, meanwhile the 327 bp ITS2 sequences had 235 conserved and 119 variable including 106 parsim-info and 13 singleton position, which showed that COI and ITS2 genes contained both conserved and highly variable regions across taxa which were great useful in different species identification.

Phylogenetic analysis

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown above the branches.[18] The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. NJ phylogenetic trees were generated, respectively, from COI and ITS2 sequences of 14 specimens. All taxa were clustered according to species and genera, without any species-or genus-level paraphyly [Figure 1] and [Figure 2]. The ITS2 sequences showed stronger bootstrap support, the value of A. grahami, T. calliphoroides, C. vomitoria, C. megacephala, L. sericata, and L. cuprina were all 100%. For COI, the bootstrap value was 100%, 100%, 100%, 100%, 97% and 94%.{Figure 1}{Figure 2}

Intraspecific and interspecific divergences of sequences

The intra- and inter-specific pairwise sequences divergence analysis for COI and ITS2 are shown in [Table 3] and [Table 4], respectively. The intraspecific divergence of COI indicated a strong similarity and even identity in most cases, which were between 0 and 0.002, and the interspecific divergence was ranged from 0.052 to 0.089, except for that between L. sericata and L. cuprina (0.006, 0.008). For ITS2, the intraspecific divergence was between 0 and 0.047, while the interspecific difference ranged from 0.054 to 0.213.{Table 3}{Table 4}

 Discussion



Calliphoridae (Diptera) has the ability to colonize corpses at varying temperatures in diverse geographical locations including several abundant species and is one of the most widely distributed necrophagous flies in China. For decades, it has been received close attention as primary indicator species in forensic entomology.[19] As the earliest insects developed on corpse, Calliphoridae fly species often provide the basis for estimation of PMI. PMI is critical in forensic and legal investigations and involves measuring time interval from death to discovery of the corpse. Henan Province, situating the South of the Yellow River and bordering on humid continental to the North, has a temperate climate that is humid subtropical. It has a distinct seasonal climate characterized by hot, humid summers due to the East Asian monsoon, and generally cool to cold, windy, dry winters which reflect the influence of the vast Siberian anticyclone. There have been recorded as many as 16 species of Calliphoridae necrophagous flies in Henan.

However, it is difficult for noninsect professionals to identify insects' species from the morphology. Identifications based on external features may be very challenging because of physical similarities or morphological damaged, particularly the immature stages of larvae, pupae, and eggs in multiple species. Since Sperling et al.[3] and Wells and Sperling[20] successively used DNA-based methods for the identification of necrophagous insect species as a complementary means of morphological identification, DNA molecular identification has been increasingly studied in forensic communities as a supplementary method for morphological identification.

The COI gene is broadly accepted as an animal DNA barcode for taxa identification, species delimitation, and phylogenetic placement because of multiple copies, easy isolation, and strongly conserved structure. Moreover, the DNA barcode region sequence was confirmed to be efficient to identify Diptera. It is believed to be conservative at the species level and typically displays ≥3% divergence among different species.[21] However, there are some cases for several specie groups where the use of COI for taxon characterization has delivered ambiguous results.[22] The nuclear gene ITS2 has universality and multiple copies in the biosphere and has good homogeneity in individual and population. A small number of samples can effectively represent the variation of the population, which is appropriate for phylogenetic analysis as a molecular marker. Because COI and ITS2 have different modes of evolution and transmission, some researchers suggest that it is an approach to combine mitochondrial and nuclear gene sequences to improve the overall phylogenetic signal and avoid some of the gene-specific constraints.[23]

In the present study, 14 specimens, representing 6 species and 5 genera of Calliphoridae, were successfully identified with 637 bp COI and 327 bp ITS2 sequences. For the COI sequences [Table 3], the interspecific divergence between L. sericata and L. cuprina was 0.006 and 0.008 (the minimum). Moreover, the values among the other species ranged from 0.052 to 0.089. For the ITS2 sequences [Table 4], the minimum interspecific divergence was 0.054, among L. sericata and L. cuprina, T. calliphoroides, and A. grahami. The interspecific divergence between C. vomitoria and T. calliphoroides and A. grahami was 0.109 and 0.116, respectively (not less than the COI sequences), the others varied from 0.101 to 0.213 (not less than the COI sequences). ITS2 gene sequence possessed strong resolution power as it showed higher values of variation between L. sericata and L. cuprina, C. vomitoria and T. calliphoroides, C. vomitoria and A. grahami, but inferior to COI for T. calliphoroides and A. grahami. Our results showed that combination of COI + ITS2 genes yielded more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. This gives support to the well-established idea that more than just one nuclear or mitochondrial gene needs to be used when trying to determine species and gene trees.[24] As numerous insect species have undergone hybridization and may carry mtDNA of another species, using COI alone could result in incorrect identifications, especially for closely related species.

As seen in this study, the COI and ITS2 sequences have their own advantages on different Calliphoridae necrophagous fly species identification, respectively, which indicated the necessity of combination of mitochondrial and nuclear genetic markers. Especially if modern hybridization is occurring at any appreciable rate, by using nuclear genes in conjunction with mitochondrial genes, a potentially misleading situation can be avoided.[25]

In this study, all the sample sequences of the two genetic markers were located on a specific branch, and the reliability of bootstrap test was relatively high. The clustering support of L. sericata and L. cuprina showed that ITS2 gene was higher than COI gene. The result may be suggested that ITS2 gene could better distinguish these fly species, which seemed to be an important advantage for the identification of closely related species. However, in some cases, both nuclear and mitochondrial genes are needed for reliable species identification and hybrid detection. The overall visualized structure reflected the good matching between the sequences of various flies, which made the comparison of population level possible.[9] In addition, in degraded samples, only the shortest DNA fragments remain amplifiable by PCR. Under these circumstances, ITS2 (327 bp) marker, the small size of the target sequence may be more appropriate for the accurate identification of entomological evidence.[17] However, due to the limited sample size, the geographical populations of Calliphoridae species failed to be distinguished, which needed to be further verified and validated by more samples.

 Conclusion



In summary, as a supplementary method for morphological identification, the approach appears to be reliable for identifying specimens collected in this study. It is effective to identify the six species of Calliphoridae using the COI and the ITS2 target gene fragments. We advocated for a combination of nuclear and mitochondrial gene approach to address the taxonomy and phylogeny of forensic relevant flies, especially of closely related species and populations.

Acknowledgments

The authors would like to thanks to Dr. Song Yueqin from the Insect Herbarium of Henan University of Science and Technology for her great help in the identification of flies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Amendt J, Krettek R, Zehner R. Forensic entomology. Naturwissenschaften 2004;91:51-65.
2Zajac BK, Sontigun N, Wannasan A, Verhoff MA, Sukontason K, Amendt J, et al. Application of DNA barcoding for identifying forensically relevant Diptera from northern Thailand. Parasitol Res 2016;115:2307-20.
3Sperling FA, Anderson GS, Hickey DA. A DNA-based approach to the identification of insect species used for postmortem interval estimation. J Forensic Sci 1994;39:418-27.
4Cai J, Wen J, Chang Y, Meng F, Guo Y, Yang L, et al. Identification of forensically significant beetles (Coleoptera: Staphylinoidae) based on COI gene in China. Rom J Leg Med 2011;19:211-8.
5Guo Y, Zha L, Yan W, Li P, Cai J, Wu L. Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China based on COI and period gene. Int J Legal Med 2014;128:221-8.
6Stevens JR, Wall R, Wells JD. Paraphyly in Hawaiian hybrid blowfly populations and the evolutionary history of anthropophilic species. Insect Mol Biol 2002;11:141-8.
7McDonagh LM, Stevens JR. The molecular systematics of blowflies and screwworm flies (Diptera: Calliphoridae) using 28S rRNA, COX1 and EF-1α: Insights into the evolution of dipteran parasitism. Parasitology 2011;138:1760-77.
8Gerbi SA. The evolution of eukaryotic ribosomal DNA. Biosystems 1986;19:247-58.
9Song Z, Wang X, Liang G. Species identification of some common necrophagous flies in Guangdong province, southern China based on the rDNA internal transcribed spacer 2 (ITS2). Forensic Sci Int 2008;175:17-22.
10Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci 2003;270:313-21.
11Bortolini S, Giordani G, Tuccia F, Maistrello L, Vanin S. Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species? PeerJ 2018;6:e5962.
12Song ZK, Wang XZ, Liang GQ. Molecular evolution and phylogenetic utility of the internal transcribed spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera). J Mol Evol 2008;67:448-64.
13Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 1994;3:294-9.
14Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4.
15Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 2004;101:11030-5.
16Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.
17GilArriortua M, Saloña Bordas MI, Köhnemann S, Pfeiffer H, de Pancorbo MM. Molecular differentiation of Central European blowfly species (Diptera, Calliphoridae) using mitochondrial and nuclear genetic markers. Forensic Sci Int 2014;242:274-82.
18Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985;39:783-91.
19Yusseff-Vanegas S, Agnarsson I. Molecular phylogeny of the forensically important genus Cochliomyia (Diptera: Calliphoridae). Zookeys 2016;609:107-20.
20Wells JD, Sperling FA. A DNA-based approach to the identification of insect species used for postmortem interval estimation and partial sequencing of the cytochrome oxydase b subunit gene I: A tool for the identification of European species of blow flies for postmortem interval estimation. J Forensic Sci 2000;45:1358-9.
21Wells JD, Sperling FA. DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae). Forensic Sci Int 2001;120:110-5.
22Sonet G, Jordaens K, Braet Y, Desmyter S. Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic? Forensic Sci Int 2012;223:153-9.
23Shayya S, Debruyne R, Nel A, Azar D. Forensically relevant blow flies in Lebanon survey and identification using molecular markers (Diptera: Calliphoridae). J Med Entomol 2018;55:1113-23.
24Williams K. Ancient and modern hybridization between Lucilia sericata and L. cuprina (Diptera: Calliphoridae). Eur J Entomol 2013;110:187-96.
25Tantawi TI, Williams KA, Villet MH. An accidental but safe and effective use of Lucilia cuprina (Diptera: Calliphoridae) in maggot debridement therapy in Alexandria, Egypt. J Med Entomol 2010;47:491-4.