Muscidae (Diptera) of forensic importance—an identification key to third instar larvae of the western Palaearctic region and a catalogue of the muscid carrion community

Muscid flies have been reported in numerous forensic studies that either describe the succession of insects on carrion or are inventories of local carrion faunas. Due to problems with identification, in many of these studies, Muscidae are referred to at the genus or family level only [13, 14, 32‐37], which may give the impression of low diversity. However, when authors have attempted to identify muscids to species, it often emerged that they were very numerous and diverse [12, 17, 38‐40].

The present study demonstrated that more than 150 species of Muscidae have been reported to visit either human bodies or animal carrion (Electronic supplementary material 1). We were cautious with our pooling of literature data to avoid an overestimation of the number of carrion-visiting Muscidae taxa. For example, Alves et al. [41] miscalculated carrion-visiting muscids in South America, erroneously listing Hydrotaea aenescens and its two junior synonyms H. argentina (Bigot) and Ophyra argentina as well as Synthesiomyia nudiseta and its junior synonym S. brasiliana Brauer & Bergenstamm as three and two valid species, respectively, rather than as just two species overall. For the majority of species reported to be visiting carrion as adults, carrion is not documented as a breeding medium. Hence, immature stages of only 25 muscid species and 8 taxa identified to genus level only have been found to develop in this habitat, either feeding on the putrefying tissues or preying on other necrophagous larvae. Some Muscidae that are predatory in the larval stage reside in the soil beneath or close to cadavers (e.g. Phaonia Robineau-Desvoidy, Helina Robineau-Desvoidy) and may erroneously be considered as a component of the carrion fauna, when they are actually preying on larvae dispersing from the cadaver. The most regular and frequent muscid components of the carrion-breeding community are species of the genera Atherigona Rondani, Hydrotaea Robineau-Desvoidy, Musca Linnaeus, Muscina Robineau-Desvoidy and Synthesiomyia Brauer & Bergenstamm. Some of these species have a wide geographic distribution and have been reported as elements of carrion communities in different regions of the world, e.g. A. orientalis, H. aenescens, H. capensis, H. ignava, H. dentipes, M. domestica, M. stabulans.

In the family Muscidae, some species have recently been shown to be common components of the carrion fauna [16], while many others are just casual visitors as adults [12, 16], attracted less frequently/predictably to decomposing tissues for feeding purposes. Furthermore, Matuszewski et al. [16] revealed that even regular carrion-visiting species may be of little or no forensic value, and the medico-legal usefulness of each taxon requires a detailed study before a firm assessment can be made of its potential forensic significance. However, researchers should be aware of the possible occurrence among the typical carrion-visiting and carrion-breeding species of numerous more rarely attracted taxa. This may be the case for some of the Muscidae listed here, since for about 80 taxa, we found only a single reference reporting the presence of adults on carrion (Electronic supplementary material 1). For this reason, application of identification keys for adult muscids with a broad taxon coverage for the geographic region of interest is recommended in forensic entomology surveys instead of those exclusively oriented towards identification of ‘forensically important’ species [42].

We included all references to carrion-visiting muscids that we are aware of, but may have missed some less obvious reports; nevertheless, we consider the set of western Palaearctic taxa presented here, particularly the narrow set of carrion breeders, to be complete. However, we expect that further species will be named that are either rare visitors as adults or breed in carrion and cadavers, in particular in the latter group from the genera Azelia Robineau-Desvoidy and Morellia.

The larvae of muscids that are commonly considered as forensically important are either truly necrophagous or display a predacious behaviour as they mature. In the latter case (e.g. A. orientalis, Hydrotaea spp., Muscina spp., S. nudiseta), they can considerably lower the abundance of other necrophagous species by preying on their larvae, similar to some predatory blow flies and flesh flies [1]. Muscidae are considered to arrive at cadavers and carrion just after the blow flies and flesh flies [5]. However, in some cases, muscids were the only insects reported to colonize decomposing bodies, especially if access to a corpse was denied to the primary carrion colonizers [1]. Muscids are generally considered to breed in carrion in the later stages of decomposition, and they tend to occur at the moist advanced decay stage [5]. Under certain circumstances, muscids can occur on cadavers as pioneer colonizers. Smith [1] stated that Musca autumnalis usually occurs in the early stages of cadaver decomposition and reported a similar occurrence for representatives of the genus Muscina, but according to Thomson [43], species of the latter genus prefer cadavers already colonized by other flies. Certain synanthropic species, e.g. Musca domestica and Muscina stabulans, are likely to be associated with cadavers in domestic conditions and under certain circumstances may be the sole colonizers of a body [1]. The majority of species are not synanthropic and probably do not inhabit human dwellings, being associated instead with rural and forest habitats [12, 16]. Muscidae are not frequently referred to in forensic studies, despite the fact that many Muscidae are regularly attracted to carrion (Electronic supplementary material 1). However, recently, some authors have revealed a high diversity of Muscidae among arthropods attracted to decomposing carrion in rural and forest habitats of Central Europe [12, 16, 44]. In these habitats, muscid species significantly outnumbered Sarcophagidae, commonly considered as one of the most forensically important groups of insects [16, 45]. Matuszewski et al. [16] found a significant association of adults of H. aenescens, H. armipes, H. cyrtoneurina (Zetterstedt), H. dentipes, H. ignava, H. pilipes and H. similis and larvae of H. ignava and H. dentipes with the bloated stage of carrion decomposition.

According to Smith [1], Musca domestica and Muscina spp. are more readily attracted to bodies contaminated with faeces rather than to those not so contaminated. Indeed, Benecke and Lessig [46] reported child neglect preceding death due to the presence of M. stabulans larvae attracted to faeces. The occurrence of Muscidae on a cadaver prior to death is of importance and could happen, not only because the flies were attracted by faeces present on the body but also from infected wounds, because some muscids are known to be involved in cases of secondary myiasis in humans and animals [47], e.g. M. domestica, Muscina levida, M. prolapsa, M. stabulans and S. nudiseta.

Restricted access of arthropods to a dead body has been recognized as one of the most important factors affecting the breakdown of cadaver. Concealed remains, e.g. buried bodies, can still be colonized by insects, but even a relatively thin layer of soil, just 5–10 cm, may either disturb or inhibit colonization by some typical necrophagous species [48]. Although some authors have reported flies (Calliphora vicina Robineau-Desvoidy) ovipositing on the soil covering a body buried at a depth of 30 cm [14], such observations are not consistent with the ability of larvae to reach a buried corpse [48]. Some Muscidae, particularly of the genera Muscina and Hydrotaea, together with some Phoridae and Sarcophagidae, are among the few dipterans known for their ability to exploit buried remains [20, 37, 48, 49], and in some cases, Muscidae have even been described as predominant on buried remains [49, 50]. Nuorteva [51] observed females of H. dentipes ovipositing on a human corpse partly covered with snow, and Anderson [52] reported a Hydrotaea sp. colonising a body placed in a car trunk and Shin et al. [53] reported H. obscurifrons also from a body in a car trunk. According to Mariani et al. [54], H. aenescens and M. stabulans are able to develop through several generations on a buried cadaver. A similar phenomenon has been observed for H. capensis colonising bodies in buried coffins [55]. Skidmore [8] reported that H. dentipes and H. ignava overwinter in the larval or pupal stage, and recently, Mądra et al. [44] revealed those two species and H. pilipes overwintering on pig carcasses in their immature stages. Two species of Muscidae (H. capensis, M. prolapsa) have been reported to develop in pig heads concealed in zipped suitcases [56].

Precise species identification of larvae inhabiting dead bodies is a crucial first step in the analysis of insect evidence in any forensic case [2]. The literature concerning larval morphology of Muscidae is extensive, but has a strong bias towards species of sanitary, medical, veterinary and agricultural importance. This bias causes difficulties in the identification of third instar larvae of a broader range of Muscidae, ultimately severely restricting the analysis of such entomological material in forensic cases and carrion succession experiments. Until now, the only reliable alternative method for identification of larval material has been rearing to adults in the laboratory. Although rearing can be simple, it requires carefully sampled live specimens. Also, it takes from 2 to 5 weeks and may be unsuccessful if, for example, the larvae have been contaminated by insecticides or injured through desiccation [51]. Species identification of preserved larvae is therefore of considerable practical importance. Some authors claim that because identification based on morphological methods requires specialized taxonomic knowledge, only some specialists are able to identify larvae of forensically relevant insects to species level [2]. For this reason, other methods of species identification have been developed, such as molecular approaches. However, molecular libraries for identification of Muscidae have not yet been sufficiently developed and currently do not allow identification of the full set of taxa here recognized as breeding in carrion and human cadavers [57‐61]. Hence, identification keys based on morphological characters of larvae remain an important tool in forensic entomology. In particular, well-illustrated keys should be developed that are readily available (open access) to the non-entomologist, relying as much as possible on easily recognizable characters.

Species of Muscidae occurring in the western Palaearctic region breeding in carrion and cadavers have been shown here to differ sufficiently in their third instar larval morphology to allow for their discrimination. Musca sorbens, known as the bazaar fly, is a species closely associated with humans in all areas of its occurrence. There is no confirmed report of its larvae breeding either in animal carrion or human cadavers. However, the species is closely associated with animal dung and human faeces, and therefore, the presence of its larvae on cadavers contaminated with faeces is possible and the species has been included in the present key. Similar to M. sorbens is the case of Stomoxys calcitrans. Although this species has not been reported breeding in carrion and cadavers, we found a report of unidentified representative of the genus Stomoxys from a human cadaver in France [62]. Because S. calcitrans is the only Palaearctic species from this genus and, similarly to M. sorbens, is closely associated with human dwellings, we included it in the key. As the genus Morellia has only been recorded once from decomposing carrion [14], and then only from North America and without a specific identification, this taxon has been excluded from detailed study. However, because Morellia species occurring in the western Palaearctic region share a similar immature biology with the North American fauna, and furthermore one North American representative, M. podagrica (Loew), is known from the western Palaearctic, the genus has been included in the identification key provided here. Although larvae of some species of Helina and Phaonia have been reported from decomposing carrion, they have here been considered as rare visitors without forensic importance. Representatives of Helina and Phaonia are obligatory predators [8], living e.g. in humus-rich soil or under tree bark. In these habitats, they are active predators that prey on other arthropod larvae. However, due to possible accidental occurrence in and around decomposing cadavers, they have also been included in the identification key.

Some serious discrepancies with the present study and misinterpretations have been revealed in the medical and veterinary entomology literature, including textbooks concerning the identification of third instar larvae of forensically important Muscidae. All these cases are discussed in detail in the attached appendix (Electronic supplementary material 2).

We incorporated in the identification key larval morphology characters widely used hitherto and also others not previously recognized as valuable for taxonomic purposes. Since the presence or absence of spines covering thoracic and abdominal segments in Muscidae may be difficult to observe because of their lack of colour, previous authors did not describe details of the spinulation pattern nor did they include it in identification keys, with few exceptions [63, 64]. Although often difficult to observe and with some intraspecific variability, the spinulation pattern has been recognized as useful for taxonomic purposes, particularly for discrimination of representatives of Muscina [28]. Some students of Muscidae third instar morphology identified the importance of the distance separating posterior spiracles for taxonomic purposes [64, 65]. However, this has been revealed insufficient for taxonomic purposes in third instar larvae [28]. On the other hand, valuable characters for identification of third instars of muscids are found in details of posterior spiracles, since the spiracular scar and respiratory slits exhibit great variation in their shape and arrangement (Fig. 3a–h). However, the pigmentation of the posterior spiracles and, in some species, of the adjoining area increases during larval growth, and so it is not a useful character (for details see Grzywacz et al. [28]). The cephaloskeleton, a structure reflecting the larval feeding strategy, differs between saprophagous, and both facultative and obligatory carnivores and is of primary importance for taxonomic purposes (Fig. 2a–d). In species with asymmetric mouthhooks, the apical parts appose closely, appearing as one structure, whereas in those with symmetric mouthhooks, the apical parts adjoin but are clearly separated. The basal parts of mouthhooks in the former group are joined through the broadened unpaired sclerite, while in the latter group, the basal parts are distinctly separated.