Species identification of Middle Eastern blowflies (Diptera: Calliphoridae) of forensic importance

The lack of reliable tools for species identification of necrophagous blowflies of the Middle East is a serious obstacle to the development of forensic entomology in the majority of countries of this region. Adding to the complexity of diagnosing the regional fauna is that species representing three different zoogeographical elements exist in sympatry. In response to this situation, a high-quality key to the adults of all species of forensically relevant blowflies of the Middle East has been prepared. Thanks to the modern technique of image-stack stereomicroscopy and high-quality entomological materials, this new key can be easily applied by investigators inexperienced in the taxonomy of blowflies. The major technical problems relating to the species identification of necrophagous blowflies of the Middle East are also discussed.


Introduction
Necrophagous blowflies are among the most ubiquitous insects occurring in anthropogenic ecosystems. They have great medical importance, which relates to their participation in carrion decomposition, facultative parasitism of vertebrate tissues, and mechanical transmission of various pathogenic microorganisms (Norris 1965;Greenberg 1973;Hall and Wall 1995). In recent decades, there has been growing interest in research into the biology and ecology of insects with potential forensic applications (Tomberlin et al. 2011). Necrophagous blowflies, the earliest visitors to carrion as adults and the main decomposers of carrion mass as larvae, play a special role in both casework and research dedicated to medico-legal applications (Byrd and Castner 2009). The reliability of forensic entomological expertise and associated experiments strongly depends on proper species identification of collected material.
The blowflies (family Calliphoridae) in their traditional, broad form are a paraphyletic taxon. This was postulated by Rognes (1997) and has been confirmed by recent molecular studies (e.g., Kutty et al. 2010;Marinho et al. 2012;Nelson et al. 2012;Singh and Wells 2013). All species of forensically important blowflies are grouped in four subfamilies in the sense of the systematics proposed by Rognes (1991Rognes ( , 1997. Three of them, the Calliphorinae, Chrysomyinae, and Luciliinae, are represented in all zoogeographic regions, whereas the distribution of the Toxotarsinae is restricted to the Neotropical Region (Rognes 1997). The fauna of necrophagous blowflies has been intensively studied in Australasia, Europe, East and South Asia, and North and South America, with notable effect in the form of a variety of published checklists, keys, and taxonomic monographs (e.g., Kano and Shinonaga 1968;Kurahashi 1987;Kurahashi et al. 1997;Rognes 1991;Fan et al. 1997;Wallman 2001;Whitworth 2006;Irish et al. (2014); Yang et al. 2014). However, the blowfly fauna of some large geographical regions, like the Afrotropics (except Namibia and the Republic of South Africa) and the Middle East (except Israel), remains poorly studied. This situation stops broad application of insects for medico-legal purposes due to a lack of proper tools for species identification of the local fauna. In the Middle East, the reliable diagnosis of species is especially complicated because the constituent countries of the region lie at the intersection of three zoogeographical zones. Most Middle Eastern countries possess a blowfly fauna simultaneously representative of the Palaearctic, Oriental, and Afrotropical regions (Büttiker et al. 1979;Deeming 1996Deeming , 2007Kurahashi and Afzal 2002;Rognes 2002). Additionally, the blowfly faunas of Bahrain, Egypt, Iran, Iraq, Jordan, Kuwait, Lebanon, Qatar, Syria, and Yemen have been studied in only a fragmentary way or not at all.
The aim of the present paper is to deliver a high-quality key for identification of the adults of all species of forensically relevant blowflies known from the Middle East. The main intention of the authors was to prepare a species identification tool that will be easily applicable to investigators inexperienced in blowfly taxonomy. This task was achieved thanks to the application of the modern technique of image-stack stereomicroscopy and the use of well-preserved entomological material. The authors hope that this publication will be a further milestone toward advancing the application of forensic entomology in Middle Eastern countries.

Material and methods
The Middle East is defined as the group of the following countries situated in south-east Asia and northern Africa: Bahrain, Egypt, Iran, Iraq, Israel, Jordan, Kuwait, Lebanon, Libya, Qatar, Oman, Pakistan, Saudi Arabia, Syria, United Arab Emirates, Yemen, and Turkey (Kort 2008). The relevant species of blowflies of forensic importance known from the Middle East, and their distributions, were determined using the catalogs of Schumann (1986) and Verves (2005), with the addition of the following publications: Büttiker et al. (1979), Deeming (1996Deeming ( , 2007, Parchami-Araghi et al. (2001), Kurahashi and Afzal (2002), Rognes (2002), Al-Mesbah (2010), Sabanoğlu and Sert (2010), Tüzün et al. (2010), Abouzied (2014) and Verves and Khrokalo (2014). However many records reported in the listed references need verification and should be treated with caution. New distribution records for some species for specific countries resulted from the inspection of regional collections by the authors. These new records are noted in the key by underlining the names of the countries concerned.
Most of the specimens used for the illustration of characters were collected personally by the authors. Specimens of other species, especially nominal elements of the Oriental and Afrotropical fauna, were obtained thanks to loans from the collection of the Natural History Museum of Denmark.
The preparation of image-stacking was done using an M205C Leica Stereomicroscope with an integrated high-resolution Leica DFC495 digital camera and associated software (Leica Application Suite 4.4.0).
The terminology follows Rognes (1991), and all morphological details are clearly marked and abbreviated on the figures.

Results
Key to the identification of forensically important blowflies of the Middle East.   anterior wing margin transparent … 9 9. Katepisternal setae 0+1 (Fig. 1k); all hairs on surface of tergite V black … Chrysomya nigripes Aubertin, 1932 Distribution: Pakistan. Possible occurrence in south-east Iran.
calypters light brown to brown, with at least the rim of upper calypter brownish (Fig. 3k); tibia of legs orange-brownish; body usually bluish-purple … Lucilia porphyrina (Walker, 1856) Distribution: Pakistan. Possible occurrence in south-east Iran.

Discussion
The present key covers 24 species of necrophagous blowflies. The undoubted forensic importance of most of them has been discussed and summarized in monographs (e.g., Byrd and Castner 2009;Gennard 2012;Rivers and Dahlem 2014) and some recent papers (Sukontason et al. 2010;Szpila 2010;Kurahashi and Kirk-Spriggs 2006;Klong-Klaew et al. 2012). The list of flies of forensic importance is not finite; new species are consequently being added based on new information (e.g., Vanin et al. 2011;Fremdt et al. 2012;Grzywacz et al. 2014). Therefore, necrophagous species like Ch. pinguis, Ch. phaonis, L. papuensis, H. pulchra, abundant in anthropogenic habitats but still without confirmed attraction to human corpses, have also been included in the key presented here. In the high mountain zones of Pakistan and Iran, it is possible to find additional species of Calliphora Robineau-Desvoidy, 1830 (Schumann 1986;Kurahashi and Afzal 2002): Calliphora chinghaiensis Van et Ma, 1978, Calliphora himalayana Kurahashi, 1994, and Calliphora uralensis Villeneuve, 1922. However, either their biology remains unknown or they do not show propensity for development in large vertebrate carrion. They have therefore not been included in the present key.
Species identification of the adults of necrophagous blowflies is usually not difficult, and numerous keys are currently available for potential users (e.g., Fan et al. 1997;Kano and Shinonaga 1968;Kurahashi 1987;Kurahashi et al. 1997;Rognes 1991;Wallman 2001;Whitworth 2006;Nazni et al. 2011;Szpila 2012). Given this situation, many tested morphological characters have been available for the preparation of a reliable key for the identification of material collected in Middle Eastern countries. However, some characters must nonetheless be treated with caution: 1. Color and dusting of particular elements of the body. This character may be difficult to observe if specimens are excessively weathered, or when they have been preserved in EtOH or stored too long in an ethyl acetate killing jar. Restoration of the colors and dusting of material preserved in EtOH can usually be achieved by brief immersion in distilled water, followed by about 5 min of exposure to the heat from the incandescent bulb of a simple table lamp. Longer drying may cause undesirable shrinkage and wrinkling of specimens. Specimens damaged by contact with ethyl acetate can be restored by placing them in a bath of xylene for 24 h. 2. Presence of setae. Strong setae on the bodies of flies are prone to damage, again, especially in specimens that are weathered or that have been preserved in EtOH. However, even if setae are broken off, their presence or absence may be checked by the presence of the clear round socket which always remains visible after removal of a seta. It is important to mention that populations of flies always contain a proportion of specimens with aberrations in size and in the position of particular setae (Rognes 1991). Therefore, to minimize such potential ambiguity, characters in the present key concerned with setulation are always supplemented by other features. 3. Genital apparatus. Features of the genitalia are always the most reliable characters for species identification. The male genital structures are easily observed in specimens preserved in 70 % EtOH. However, specimens preserved in absolute EtOH or pinned (if the genital apparatus was not exposed before drying out) need special treatment.
The procedure for such dry specimens was described in detail by Rognes (1991). Rigid (dehydrated) specimens after preservation in absolute EtOH may be softened by boiling for 5 min in a 5 % solution of KOH. An alternative is the relocation and storage of specimens to a more dilute EtOH solution. The disadvantage of the latter method is that it takes longer to achieve the softening effect.
The Middle East is a region in which three pairs of sister species occur sympatrically: L. cuprina and L. sericata, L. caesar and L. illustris and Ch. albiceps and Ch. rufifacies. The morphological separation of species of Lucilia is not especially controversial thanks to former and more recent detailed studies of these species (Rognes 1991(Rognes , 1994Williams and Villet 2014). Details of the male genital apparatus, especially the shape of the cerci and surstyli, are reliable characters for differentiation of particular species, supported by quite a large set of other characters in the case of L. cuprina and L. sericata (Williams and Villet 2014). Identification of the females of L. caesar and L. illustris is possible by checking the shape of the dorsal surface of the sixth tergite and the distribution and size of setae on the posterior margin of the same tergite (Rognes 1991).
The identification and overall taxonomy of Ch. albiceps and Ch. rufifacies are problematic and have been discussed by several past workers (e.g., Holdaway 1933;Zumpt 1956;Guimarães et al. 1978;Tantawi and Greenberg 1993;Whitworth 2006). The few available characters are not fully reliable, such as the presence of the proepimeral seta (Tantawi and Greenberg 1993), or are difficult to observe and prone to damage, like the color of inner surface of third antennal segment (Holdaway 1933). The results of intensive studies of the problem have only just been published, but the characters proposed for the differentiation of specimens of both species are still very subtle (Grella et al. 2015). For example, the color of the genal dilation and the angle formed by the branch of vein M1+2 are difficult for inexperienced researchers to interpret, and the number of proepisternal setae may only be clearly visible after removal of the head of the specimen. However, in the absence of morphological certainty, the identity of Ch. albiceps and Ch. rufifacies can be corroborated by molecular analysis.
The politically fraught conditions in many of the countries of the Middle East pose obvious difficulties for the study of the blowflies and other components of the natural history of the region. Even so, when secure opportunities present themselves, we encourage workers to continue to document the blowfly species that occur in this part of the world to help facilitate the diverse applications of these important insects.