Complete mitochondrial genomes of three species of fresh flies of forensic entomology interest from the genus Sarcophaga (Sarcophagidae) from Portugal and Brazil

Abstract

The Sarcophagidae family of fresh flies bears strong importance in the context of forensic entomology due to their application in the estimation of the Post Mortem Interval (PMI). Sarcophaga is the major genus in the Sarcophagidae family and includes cosmopolitan species, which are distributed worldwide. In this communication, we present the analysis of the complete mitochondrial genome (mtDNA) of two species from Portugal – S. melanura and S. dux – and one from Brazil – S. ruficornis. The mtDNA of these species range from 14,882 bp to 15,190 bp and have 22 tRNA genes, 13 protein-coding genes (PCG), and two rRNAs distributed along both strands. Our data include the first record of complete Sarcophaga mtDNA sequences from species collected in Portugal and in Brazil. These genomes represent an advance in the understanding about this group, expand the database, and can be used for the development of new markers for species identification.

Keywords:

• Forensic entomology
• Sarcophaga
• NGS
• complete mtDNA
• flesh flies

Sarcophagidae is one of the most important families in forensic entomology due to its widespread distribution and diversity of species (Pape 1996). The genus Sarcophaga is considered the most diverse, with 133 subgenera and 790 species described (Pape 1996). Species belonging to this genus are frequently used and represent an important source of information in forensic investigations (Moura et al. 1997; Carvalho et al. 2000; Oliveira et al., 2011; Rolo et al., 2013). The correct identification of species from the Sarcophaga genus is usually made using the male genitalia. However, in contrast with other genus, the females of some Sarcophaga species can be identified by their external morphology (Zhang et al. 2014). Nevertheless, morphological identification still presents its limitations, which hinder the identification of larval stages and dandified specimens. We show novel complete mtDNA sequences of flesh fly species of forensic importance. Our data expand the knowledge on the molecular database for the identification of these species.

In this work, we collected Sarcophaga specimens from two sites in Portugal and one in Brazil. In Portugal, S dux (38°35'02" N 9°10'30" W), and S melanura (38°42'52" N 9°11'45" W). Sarcophaga ruficornis was collected in Brazil (22°51'12" S 46°19'18" W). Each specimen was identified following the taxonomic key (Buenaventura and Pape 2015) and preserved in 70% ethanol. The mtDNA was extracted from the thorax, legs, and wings of the flies (Françoso et al. 2016), and the head and abdomen of S. ruficornis (Voucher 14) were deposited in the database of UNICAMP, whereas S. dux (Voucher 2) and S melanura (Voucher GE) were registered in the database of the Universidade de Lisboa. The mtDNA library was generated using the Nextera XT kit according to the manufacturer’s instructions and sequenced using a paired-end strategy 2 × 250 on the MiSeq platform (Illumina, CA). The genomes were constructed by mapping the reads against the Sarcophagidae mitogenomes available on the NCBI database, followed by de novo assembly of the mapped reads using the CLC Genomics Workbench. The mitogenomes were annotated using the MITOS WebServer (Bernt et al. 2013) and manually verified using the NCBI database.

The complete mtDNA of S. ruficornis (MH879755) was 14,940 bp long and presented 24.3% of CG. S. dux (MH879759) mtDNA was 14,882 bp long and showed 23.8% of CG. S. melanura (MH879758) presented the longest mtDNA, with 15,190 bp and 24.4% of CG. The annotation of these genomes revealed 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a noncoding control region (D-loop) located between the 12S rRNA and tRNA^Ile. The PCG sequences commonly start with an ATT, ATA, or ATG codon (12 PCGs), but COX1 starts with CAA. Five PCGs have the T-- stop codon completed to TAA by posttranscriptional polyadenylation (Ojala et al. 1981). The light strand codifies eight of the 22 tRNAs, five PCGs and the two rRNAs, while the remaining genes are encoded on the heavy strand. The phylogenetic analysis and comparison with others Sarcophaga species are showed in Figure 1.

Figure 1. Molecular phylogenetic inferences of Sarcophaga mitogenomes. The phylogenetic analyses were made using the MEGA 7 software (Kumar et al.2016) using the maximum likelihood model with the Nearest-Neighbour-Interchange heuristic method. The D-loop region was excluded from this analysis due to its high degree of variability (Gonder et al.,2007). The tree showed a well-structured separation of the novel species. S. melanura (collected in Lisbon, Portugal) showed few differences in comparison with the species from NCBI collected in China (KP091687.1). S. dux (Aroeira, Portugal) is more related to S. portschinskyi (China). On the other hand, S. ruficornis (Brazil) clustered with the branch including S. crassipalpis and N. bullata (both from the United States). The novel mtDNA published herein are highlighted in grey. The mitogenomes for comparison were obtained from the NCBI database: Musca domestica (KT272857.1) – used as outgroup, Sarcophaga impatiens (JN859549.1), Sarcophaga melanura (KP091687.1), Neobellieria bullata (KT272859.1), Sarcophaga crassipalpis (KC005711.1), Sarcophaga peregrina (KF921296.1), Sarcophaga formosensis (MF688648.1), Sarcophaga africa (KM881633.1), Sarcophaga portschinskyi (KM287570.1), Sarcophaga similis (KM287431.1), Sarcophaga albiceps (KT444443.1), Sarcophaga misera (MF133500.1).

Acknowledgments

We thank the Universidade de Lisboa and the Universidade Federal de Minas Gerais (UFMG) for the support during the project execution.

Disclosure statement

The authors report no potential conflict of interest.

Additional information

Funding

This work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Edital Ciências Forenses no. 25/2014, Process 23038.006839 / 2014-33), International postdoc fellowship (CAPES 0092/17-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Financial support was also provided by CESAM (UID/AMB/50017 - POCI-01-0145-FEDER-007638), and by FCT/MCTES through national funds (PIDDAC). Co-funding was provided by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.