DNA-based characterisation and classification of forensically important flesh flies (Diptera: Sarcophagidae) in Malaysia

Abstract

Insect larvae and adult insects found on human corpses provide important clues for the estimation of the postmortem interval (PMI). Among all necrophagous insects, flesh flies (Diptera: Sarcophagidae) are considered as carrion flies of forensic importance. DNA variations of 17 Malaysian, two Indonesian and one Japanese flesh fly species are analysed using the mitochondrial COI and COII. These two DNA regions were useful for identifying most species experimented. However, characterisation of the species was not sufficiently made in the case of Sarcophaga javanica. Seventeen Malaysian species of forensic importance were successfully clustered into distinct clades and grouped into the six species groups: peregrina, albiceps, dux, pattoni, princeps and ruficornis. These groups correspond with generic or subgeneric taxa of the subfamily Sarcophaginae: Boettcherisca, Parasarcophaga, Liosarcophaga, Sarcorohdendorfia-Lioproctia, Harpagophalla-Seniorwhitea and Liopygia. The genetic variations found in COI and COII can be applied not only to identify the species of forensic importance, but also to understand the taxonomic positions, generic or subgeneric status, of the sarcophagine species.

Introduction

A quick and accurate identification system is desirable in any forensic studies as well as ecology. In recent years, there has been an increase in the use of DNA sequence data in the studies of carrion flies as an aid to accurately identify insect species, even in the cases of immature stages [1], [2], [3]. Molecular techniques have the advantage of being applicable to any life stages and have the potential to distinguish morphologically similar species or even genera, such as in the case of sarcophagine flesh flies [4]. DNA-based identification techniques are also easily transferable between laboratories and are not limited to the requirements of specific taxonomical expertise.

In Malaysia, the occurrence of carrion-related arthopods has hitherto been known to include dipteran flies such as Sarcophagidae, Calliphoridae, Muscidae and Stratiomyidae [5], [6], [7]. Reviews of Malaysian forensic studies showed that calliphorid flies, such as Chrysomya megacephala (Fabricius) and Achoetandrus rufifacies (Macquart), were observed as the predominant species found in human cadavers, especially in the early stages of decomposition [8], [9], [10]. Although sarcophagine flesh flies are also important as forensic indicators, their identification was mostly carried out only to the genus level [9], [10]. Sarcophagine flies are notoriously difficult to identify due to their highly similar morphological appearance [11], [12], [13]. These authors suggested and demonstrated that mitochondrial DNA sequences could be successfully employed to distinguish some species of the sarcophagine flesh flies.

Despite the availability of mitochondrial DNA sequencing techniques, a robust taxonomic classification of these flies is still required. Such methods of species identification would be truly useful if more comprehensive baseline data could be established. Therefore, this study analysed the mitochondrial cytochrome oxidase gene subunits I and II (COI and COII) sequences of many oriental flesh fly species from Malaysia, Indonesia and Japan that has not been well represented in the previous studies [4], [11], [13]. The sequence data obtained would be useful as standards for future analyses and the practical identification of species in forensics. Phylogenies estimated from these DNA sequences also provide implications towards the understanding of the taxonomy and systematics of the sarcophagine flesh flies.