DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region

Abstract

Correct identification of forensically important insects, such as flies in the family Calliphoridae, is a crucial step for them to be used as evidence in legal investigations. Traditional identification based on morphology has been effective, but has some limitations when it comes to identifying immature stages of certain species. DNA-barcoding, using COI, has demonstrated potential for rapid and accurate identification of Calliphoridae, however, this gene does not reliably distinguish among some recently diverged species, raising questions about its use for delimitation of species of forensic importance. To facilitate DNA based identification of Calliphoridae in the Caribbean we developed a vouchered reference collection from across the region, and a DNA sequence database, and further added the nuclear ITS2 as a second marker to increase accuracy of identification through barcoding. We morphologically identified freshly collected specimens, did phylogenetic analyses and employed several species delimitation methods for a total of 468 individuals representing 19 described species. Our results show that combination of COI + ITS2 genes yields more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. All of our results from independent and concatenated trees and most of the species delimitation methods yield considerably higher diversity estimates than the distance based approach and morphology. Molecular data support at least 24 distinct clades within Calliphoridae in this study, recovering substantial geographic variation for Lucilia eximia, Lucilia retroversa, Lucilia rica and Chloroprocta idioidea, probably indicating several cryptic species. In sum, our study demonstrates the importance of employing a second nuclear marker for barcoding analyses and species delimitation of calliphorids, and the power of molecular data in combination with a complete reference database to enable identification of taxonomically and geographically diverse insects of forensic importance.

Virus discovery in all three major lineages of terrestrial arthropods highlights the diversity of single-stranded DNA viruses associated with invertebrates

“…Two CRESS DNA viral sequences representing members of the Smacoviridae , which have been mainly recovered from feces from various mammals ( Ng et al, 2015 ; Steel et al, 2016 ; Varsani & Krupovic, 2018 ), were recovered from blow flies (Diptera: Calliphoridae) collected in the Caribbean. The novel fly associated smacoviruses, FlyACV-3 and -4, were recovered from blow flies collected using a chicken carcass as bait ( Yusseff-Vanegas & Agnarsson, 2017 ). Since blow flies are known to feed on feces and tissues from various vertebrates, including mammals, it is likely that FlyACV-3 and -4 represent dietary content.…”

Comparative Analysis of Molecular Identification and Wing Morphometrics of Forensically Important Blow Flies in China

“…In addition, many studies have proved the effectiveness of COI barcodes for the identi cation of many species of Calliphoridae [13,14]. Meanwhile, in GenBank, there are abundant molecular data about Calliphora, sequences of forensic important Calliphora have been published and uploaded online from different regions of the world, such as South Africa, the Caribbean Region, Southern European, the United States and Korea [15,16,17,18], making it possible to systematically study the sequence of Calliphora around the world. However, the molecular identi cation is a destructive approach for sample materials.…”

Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species?

“…Most of the publications about identification of forensically important species focused on the analysis of the genes coding for the cytochrome c oxidase subunit I (COI) as summarized by Tuccia and co-workers (Tuccia, Giordani & Vanin, 2016) and cytochrome c oxidase subunit II (COII) (Sperling, Anderson & Hickey, 1994; Aly, Wen & Wang, 2013; Boehme, Amendt & Zehner, 2012; Malgorn & Coquoz, 1999; Wallman & Donnellan, 2001; Xiong et al, 2012). Tested target markers other than COI and COII were: Cytb (GilArriortua et al, 2013; GilArriortua et al, 2014; GilArriortua et al, 2015; Giraldo, Uribe & Lopez, 2011), ND1 (Giraldo, Uribe & Lopez, 2011), ND5 (Zaidi et al, 2011; Zehner et al, 2004), 28S rDNA (Gibson et al, 2011; McDonagh & Stevens, 2011; Stevens & Wall, 2001; Tourle, Downie & Villet, 2009), mt16S rDNA (Guo et al, 2014; Li et al, 2010), CAD (Gibson et al, 2011; Meiklejohn et al, 2013; Schnell Schühli, Barros de Carvalho & Wiegmann, 2007), EF-1α (Gibson et al, 2011; Schnell Schühli, Barros de Carvalho & Wiegmann, 2007; McDonagh, García & Stevens, 2009), ITS1 (Zaidi et al, 2011), ITS2 (GilArriortua et al, 2014; GilArriortua et al, 2015; Zaidi et al, 2011; Ferreira et al, 2011; Nelson, Wallman & Dowton, 2008; Song, Wang & Liang, 2008; Yusseff-Vanegas & Agnarsson, 2017), PER (Guo et al, 2014) and Bicoid (Park et al, 2013). Analysis of mitochondrial DNA (mtDNA), in particular COI gene, seems to provide good species identification among Diptera, although a correct identification is still problematic for closely related species (Tourle, Downie & Villet, 2009; Harvey et al, 2008; Sonet et al, 2012).…”