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

Abstract

The Muscidae is one of the main dipteran families recognized as important for medico-legal purposes. Although an association of adult flies with decomposing human and animal bodies is documented for about 200 taxa worldwide, cadavers and carrion represents a breeding habitat for considerably fewer species. Species that do colonize dead human bodies can do so under diverse environmental conditions and, under certain circumstances, Muscidae may be the only colonizers of a body. Because of difficulties in identification, many studies have identified immature and/or adult muscids only to the genus or family level. This lack of detailed species-level identifications hinders detailed investigation of their medico-legal usefulness in carrion succession-oriented experiments. Identification to species level of third instars of Muscidae of forensic importance and the utility of larval morphological characters for taxonomic purposes were subjected to an in-depth revision. A combination of characters allowing for the discrimination of third instar muscids from other forensically important dipterans is proposed. An identification key for third instar larvae, which covers the full set of cadaver-colonising species of Muscidae from the western Palaearctic (Europe, North Africa, Middle East), is provided. This key will facilitate more detailed and species-specific knowledge of the occurrence of Muscidae in forensic entomology experiments and real cases. The carrion-visiting Muscidae worldwide are catalogued, and those species breeding in animal carrion and dead human bodies are briefly discussed with regard to their forensic importance.

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ORIGINAL ARTICLE
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
Andrzej Grzywacz
1
&Martin J. R. Hall
2
&Thomas Pape
3
&Krzysztof Szpila
1
Received: 31 May 2016 /Accepted: 8 November 2016 /Published online: 7 December 2016
#The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract The Muscidae is one of the main dipteran families
recognized as important for medico-legal purposes. Although
an association of adult flies with decomposing human and
animal bodies is documented for about 200 taxa worldwide,
cadavers and carrion represents a breeding habitat for consid-
erably fewer species. Species that do colonize dead human
bodies can do so under diverse environmental conditions
and, under certain circumstances, Muscidae may be the only
colonizers of a body. Because of difficulties in identification,
many studies have identified immature and/or adult muscids
only to the genus or family level. This lack of detailed species-
level identifications hinders detailed investigation of their
medico-legal usefulness in carrion succession-oriented exper-
iments. Identification to species level of third instars of
Muscidae of forensic importance and the utility of larval mor-
phological characters for taxonomic purposes were subjected
to an in-depth revision. A combination of characters allowing
for the discrimination of third instar muscids from other fo-
rensically important dipterans is proposed. An identification
key for third instar larvae, which covers the full set of cadaver-
colonising species of Muscidae from the western Palaearctic
(Europe, North Africa, Middle East), is provided. This key
will facilitate more detailed and species-specific knowledge
of the occurrence of Muscidae in forensic entomology exper-
iments and real cases. The carrion-visiting Muscidae world-
wide are catalogued, and those species breeding in animal
carrion and dead human bodies are briefly discussed with
regard to their forensic importance.
Keywords Forensic entomology .Muscidae .Immature
stages .Identification .Post-mortem interval
Introduction
Insects often play a major role in the decomposition of organic
matter. Generally, the most common arthropod inhabitants of
decomposing human cadavers and animal carrion are the lar-
vae of flies (Diptera), particularly those of the families
Calliphoridae, Sarcophagidae, Muscidae and Piophilidae [1].
Valuable conclusions for forensic investigations can be drawn
from the analysis of entomological material, either by means
of age estimation of the oldest immature insects inhabiting the
cadaver or by an analysis of arthropod species composition on
the body [2].
The Muscidae, commonly known as the house flies and
their relatives, is one of the dipteran families of recognized
forensic importance. Some textbooks still consider the
Fanniidae or lesser house flies as a subfamily within the
Muscidae [3–5], but substantial evidence has shown that they
warrant family status [6,7]. Muscids are small- to medium-
sized dipterans that can be found in a variety of terrestrial and
aquatic habitats, except for the most arid environments [8].
The association between man and Muscidae, for example
Musca domestica Linnaeus and M. sorbens Wiedemann, is
traceable to the earliest times of recorded history [9,10].
Due to their worldwide distribution and broad association
Electronic supplementary material The online version of this article
(doi:10.1007/s00414-016-1495-0) contains supplementary material,
which is available to authorized users.
*Andrzej Grzywacz
hydrotaea@gmail.com
1
Faculty of Biology and Environmental Protection, Nicolaus
Copernicus University, Lwowska 1, 87-100 Toruń, Poland
2
Department of Life Sciences, Natural History Museum, London, UK
3
Natural History Museum of Denmark, University of Copenhagen,
Copenhagen, Denmark
Int J Legal Med (2017) 131:855–866
DOI 10.1007/s00414-016-1495-0
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with human settlements (many are synanthropic species),
muscid flies are renowned for their agricultural, medical and
veterinary significance [5,8]. Most Muscidae hatch from the
egg into a first instar larva, which, after feeding for some time,
moults into a second and subsequently a third instar before
pupariating (so-called trimorphic condition). However, in spe-
cies of some genera, the incubation period of the egg is
prolonged, and the larva hatches from the egg as a second or
third instar (dimorphic and monomorphic conditions, respec-
tively). The reduction of free-living larval instars applies only
to species with obligatory carnivorous larvae, whereas facul-
tative carnivores are always trimorphic and can reach maturity
as non-predators [8].
There are a number of papers in which Muscidae have been
discussed in relation to forensic entomology experiments as
well as to real cases. Since the pioneer work of Mégnin [11],
who reported an association of certain muscids with
decomposing bodies, many further muscid taxa have been
documented to be attracted to dead human and animal bodies
(see Electronic supplementary material 1). Although fewer
muscids are known to colonize animal carrion and human
cadavers than species of other families, particularly
Calliphoridae [12], those that do colonize them can do so
under diverse environmental conditions. For example, corpses
may be colonized out- or indoors, in sunny or shaded sites, in
wet or dry ones, in exposed or concealed situations, and
muscids can be found associated with carcasses in both the
early and late stages of decomposition.
Muscids have received limited attention in forensic ento-
mology experiments mainly because of taxonomic issues. In
some studies, adults and/or immatures were identified to the
genus or family level only [13–15]. However, where studies
succeeded in identifying species and determining their abun-
dance, both adults and larvae of Muscidae were shown to be
very numerous [12,16,17]. Species-level identification of
entomological material is a prerequisite for a meaningful ap-
plication of entomological methods for PMI estimation pur-
poses. Thus, when taxonomic complexities or lack of identi-
fication tools prevent relatively easy and precise species iden-
tification, a broad application of the group for medico-legal
purposes is severely limited. The growing sophistication in
forensic entomology methodology has raised interest in the
larval morphology of dipterans colonising cadavers [18–21].
Although some of these studies concern muscid species as
well, they provide no new information and focus solely on
the identification of a very few species [19,21].
The aim of the present study is to provide a key allowing
the identification of the third instar larvae of Muscidae breed-
ing in carrion and dead human bodies in the western
Palaearctic region (Europe, North Africa and the Middle
East). Thus, we catalogue all carrion-visiting Muscidae world-
wide and recognize those species or taxa that additionally
breed on/in carrion. Subsequently, species breeding in animal
carrion and dead human bodies, and therefore of potential
forensic importance, were investigated for their geographical
distribution, and a detailed morphological study is presented
for species of the western Palearctic. Larval morphological
characters used by previous authors for taxonomic purposes
are subjected to an in-depth revision with the application of
the combined methods of light and scanning electron micros-
copy. We provide a set of characters allowing for the discrim-
ination of larvae of Muscidae from those of other forensically
relevant families, and a key is provided for the identification
of all studied species. Finally, the role of Muscidae in the
faunal succession of cadavers and their application for
medico-legal purposes is briefly discussed.
Material and methods
The selection of species for the present study involved two
criteria. First, species visiting carrion and cadavers were iden-
tified on the basis of the available literature and communica-
tions with practicing forensic entomologists. A taxon was rec-
ognized as of potential forensic importance if there was at least
one report of immature stages breeding in a human cadaver or
in animal carrion. Second, the geographic distribution of each
species was studied in the literature data [22–26], and only
species confirmed as occurring in the western Palaearctic were
included in the study.
Female muscids were collected from the field by hand-
netting and the use of carrion-baited traps, and larvae were
obtained by keeping those flies in the laboratory until ovipo-
sition. Specimens were reared, killed and preserved as de-
scribed by Grzywacz et al. [27,28], Grzywacz and Pape
[29] and Velásquez et al. [26]. A laboratory colony of
Hydrotaea aenescens (Wiedemann) was established from
adults emerged from c. 25 pupae obtained from the Institut
de Recherche Criminelle de la Gendarmerie, Fort de Rosny,
France. Third instar larvae of Synthesiomyia nudiseta (van der
Wulp) were obtained from a laboratory colony maintained at
the Department of Environmental Sciences and Natural
Resources, University of Alicante, Spain, and from the
Mexican-American Commission for the Eradication of
Screwworm (COMEXA), Chiapa de Corzo, Chiapas,
Mexico. Since attempts to obtain immature stages of Musca
autumnalis De Geer and Morellia Robineau-Desvoidy failed,
i.e. the collected females either did not oviposit or did not fully
develop their eggs, third instar larvae were collected directly
from cow manure and identified according to Stoffolano [30]
and Skidmore [8]. Larvae of Musca sorbens were obtained
from the collection of the Natural History Museum, London,
UK. The number of examined specimens is as follows:
Atherigona orientalis Schiner n=16, Helina sp. n=36,
Hydrotaea aenescens n = 476, H. armipes (Fallén) n=20,
H. capensis (Wiedemann) n=45, H. dentipes (Fabricius)
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n=602, H. ignava (Harris) n=512, H. pilipes Stein n=32,
H. similis Meade n=20, Morellia sp. n=3, Musca
autumnalis n = 27, M. domestica n = 61, M. sorbens n = 5,
Muscina levida (Harris) n=154, M. prolapsa (Harris) n=
38, M. stabulans (Fallén) n=165, Phaonia sp. n=41,
Stomoxys calcitrans (Linnaeus) n=27, and Synthesiomyia
nudiseta n = 158.
Material for SEM examination was prepared by dehydra-
tion through 80.0, 90.0 and 99.5% ethanol, with subsequent
critical point drying in CO
2
. Subsequently, larvae were
mounted on aluminium stubs and sputter-coated with plati-
num or gold. SEM images were taken with a JEOL scanning
electron microscope (JSM-6335F; JEOL Ltd, Tokyo, Japan)
oravariablepressureSEMLEO1455(CarlZeiss
Microscopy, Germany).
Light microscopy was performed with a Stemi 2000 ste-
reomicroscope (Carl Zeiss Light Microscopy, Germany).
Larvae were mounted on microscope slides in Hoyer’smedi-
um and examined with a Nikon Eclipse E200 microscope
(Nikon Corp., Tokyo, Japan). For additional observation of
details of the cephaloskeleton, larvae were dehydrated through
a 80.0, 96.0 and 99.5% ethanol series and studied with a
stereomicroscope 3 h after transfer to methyl salicylate [31].
After examination, the material was transferred back to a 70%
ethanol solution.
Photographs for light microscopy illustrations were taken
with a Nikon 8400 digital camera mounted either on a Nikon
Eclipse E200 microscope or Nikon SMZ 1500 stereomicro-
scope (Nikon Corp., Tokyo, Japan). Line drawings were pre-
pared by hand and subsequently digitized.
Results
Searching the available literature revealed 168 muscidspecies
and 31 taxa identified to genus level only that visit dead hu-
man bodies or animal carrion worldwide (Electronic
supplementary material 1). We expect that the many taxa iden-
tified to genus level only are represented among those identi-
fied to species level. Decomposing animal carrion and human
bodies have been documented as breeding habitats for consid-
erably fewer species, since our search revealed only 25 muscid
species and 8 taxa identified to genus level documented from
immature stages in forensic case reports and carrion succes-
sion experiments. Among these, 14 species and 3 taxa identi-
fied to genus level occur in the western Palaearctic region. The
present key covers all muscids reported from immature stages
from animal carrion and human bodies in the western
Palaearctic. Except for Hydrotaea chalcogaster
(Wiedemann), H. obscurifrons (Sabrosky) and H. spinigera
(Stein), for which we had no material available, the key will
work for the entire Holarctic carrion-breeding muscid fauna.
Identification key to third instar larvae of western
Palaearctic Muscidae of forensic importance
1. Fleshy projections covering the larval body absent and
body not flattened dorso-ventrally (Fig. 1a), parastomal
bars as well as distinct windows in both dorsal and ven-
tral cornua not developed (Fig. 2a). Unpaired sclerite,
either reduced or well developed, present between basal
parts of mouthhooks (Fig. 2b–d)→Muscidae 2
Fleshy projections covering the larval body present
(Fanniidae) (Fig. 1c), if absent then parastomal bars
and/or distinct windows in both dorsal and ventral cor-
nua present; mouthhooks always symmetric, accessory
stomal sclerites absent, respiratory slits in posterior spi-
racles never serpentine to tortuous →other forensically
important Diptera
2. Posterior spiracles distinctly raised on darkened stalks
(Fig. 1f), slits in posterior spiracles peripheral, bow-
shaped, never straight, mouthhooks always symmetric
(cf. Fig. 2d)→Atherigona orientalis
Posterior spiracles at most slightly raised, if markedly
then stalks not darkened and slits straight (Fig. 3a–d),
mouthhooks symmetric or asymmetric (Fig. 2b–d)→3
3. Anal division abruptly truncated, abdominal segments
1–7 covered by complete anterior and posterior bands
of dark spines (Fig. 1b)→Morellia sp.
Abdominal segments 1–7 without complete posterior
bands of spines →4
4. Slits in posterior spiracles serpentine (Fig. 3f–h)totor-
tuous (Fig. 3e); peritreme surrounding respiratory slits
often darkly pigmented (Fig. 1e)→5
Slits of posterior spiracles straight, never serpentine
(Fig. 3a–d); peritreme often clear and poorly pigmented
(Fig. 1d)→12
5. Posterior spiracles subtriangular (i.e. almost triangular in
shape, but with rounded corners), respiratory slits S-
shaped encircling spiracular scar and the scar shifted
towards central position (Fig. 3f)→Stomoxys calcitrans
Posterior spiracles not subtriangular, kidney-shaped
(Fig. 3e) or rounded (Fig. 3e, g, h), respiratory slits not
encircling spiracular scar and the scar placed in median
position (Fig. 3e, g, h)→6
6. Posterior spiracles kidney-shaped (Fig. 3e); extra-anal
and postanal papillae on anal division absent and
subanal papillae devoid of spines (for papillae position
see Fig. 1d); anterior rods and oral bars absent,
suprabuccal teeth devoid of distinct pigmentation, optic
lobe present (Fig. 2a), dental sclerites separated ventral-
ly, mouthhooks asymmetric (Fig. 2c)→Musca sp. 7
Posterior spiracles rounded (Fig. 3g, h); extra-anal
and postanal papillae on anal division well developed
and subanal papillae covered by spines (Fig. 1e);
mouthhooks symmetric (Fig. 2d), anterior rods and oral
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bars well developed, suprabuccal teeth distinctly dark
pigmented, optic lobe absent (Fig. 2a), dental sclerites
joined ventrally →9
7. Anal plate small, barely visible in lateral view (Fig. 2f);
dorsal surface of the anal division devoid of spines and
papillae surrounding posterior spiracles indistinguish-
able (Fig. 1a)→Musca domestica
Anal plate well visible in lateral view (Fig. 2e, g)and
papillae surrounding posterior spiracles well visible,
bulge or cone-shaped →8
8. Anal plate large, well visible in lateral view
(Fig. 2e); dorsal surface of the anal division cov-
ered by spines →Musca autumnalis
Anal plate narrow, not angular (Fig. 2g); anal
division covered entirely with minute spicules →
Musca sorbens
Fig. 1 Third instar larvae: aMusca domestica with artificial colours; b
Morellia sp. with artificial grey lines indicating segment borders and
arrows pointing to the spines on anterior and posterior margins; c
Fannia canicularis;dHydrotaea dentipes, anal division in posterior
view; eSynthesiomyia nudiseta, anal division with papillae surrounding
posterior spiracles (arrows) in posterior view; fAtherigona orientalis,
anal division, lateral view. Abbreviations: a1–7abdominal segments,
ad anal division, ex extra-anal papilla, pa postanal papilla, paa para-
anal papilla, pc pseudocephalon, ps posterior spiracle, sa subanal papilla,
t1–3, thoracic segments
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9. Slits in posterior spiracles S-shaped (Fig. 3h); anal plate
small, triangular in ventral view; para-anal papillae well
developed; papillae surrounding posterior spiracles
cone-shaped (Fig. 1e)→Synthesiomyia nudiseta
Slits crescent shaped (Fig. 3g); anal plate extended
laterally in ventral view; para-anal papillae indistin-
guishable; papillae surrounding posterior spiracles at
most in the form of small bulges (cf. Fig. 1d)→
Muscina spp. (for details see Grzywacz et al. [28]) 10
10. Postero-ventral surface of abdominal segments 5–6at
most with a single up to two rows of about five
spines each (Figs. 1f,7BinGrzywaczetal.
[28]) →Muscina prolapsa
Postero-ventral surface of abdominal segments 5–6
with about ten rows of about five small, light spines
each (Figs. 1e, g and 4d; 9B in Grzywacz et al.
[28]) →11
11. Spines on the lateral surface of the third abdominal seg-
ment reaches well above the upper margin of the lateral
creeping welt (Fig. 1e, 4a in Grzywacz et al. [28]) and
anterior spinose band on the fourth abdominal segment
(a4) reaches at least the middle of lateral creeping
Fig. 2 ThirdinstarlarvaeofMuscidae:astructures present in Muscidae
cephaloskeleton; bcephaloskeleton of predatory species in dorsal view
with asymmetric mouthhooks (arrow) and well-developed unpaired
sclerite (us, grey); ccephaloskeleton of saprophagous species in dorsal
view with asymmetric mouthhooks (arrow) and well-developed unpaired
sclerite (grey); dcephaloskeleton of predatory species in dorsal view with
symmetric mouthhooks (arrow) and reduced unpaired sclerite (grey); e
Musca autumnalis, posterior body end with enlargedand broad anal plate
(ap, grey); fMusca domestica, posterior body end with small anal plate
(ap, grey); gMusca sorbens, posterior body end with enlarged but narrow
anal plate (grey); Abbreviations: a6–7sixth and seventh abdominal seg-
ments, acc accessory stomal sclerite, accs supplementary accessory sto-
mal sclerite, ad anal division, ap anal plate, aro anterior rod, cut cutane-
ous teeth, db dorsal bridge, dc dorsal cornu, ds dental sclerite, es
epistomal plate, is intermediate sclerite, ls labial sclerite, mh mouthhook,
ob oral bar, ol optic lobe, sub suprabuccal teeth, us unpaired sclerite, vc
ventral cornu, vb ventral bridge, vp vertical plate, xsensory organ X
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Fig. 3 Posterior spiracles of Muscidae third instar larvae of forensic
interest: aHydrotaea dentipes;bH. pilipes;cH. capensis;dH. ignava;
eMusca domestica;fStomoxys calcitrans;gMuscina stabulans;h
Synthesiomyia nudiseta. Scale bar 0.1 mm. Abbreviations: pperitreme,
rs respiratory slit, ss spiracular scar, st spiracular tuft
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Fig. 4 Scanning electron microscopy images of the third instar larval morphology of Muscidae of forensic interest: aHydrotaea dentipes, posterior
body end in ventral view with posterior surface, behind anal papillae, devoid of spines (arrow); bH. similis, posterior body end in ventral view with
posterior surface, behind anal papillae, covered with spines (arrow); cH. armipes, body surface of the first abdominal segment; dH. pilipes,body
surface of the first abdominal segment; eH. aenescens, anterior body end, lateral view with spinose band on the first thoracic segment not uniformly
broad (arrow); fHydrotaea capensis, anterior body end, lateral view with the spinose band on the first thoracic segment uniformly broad (arrow)
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welt →Muscina levida
Spines on the lateral surface of the third abdominal seg-
ment reaches at most slightly above the lateral cree- ping
welt (Fig. 1g, 9A in Grzywacz et al. [28]) and anterior
spinose band on the fourth abdominal segment hardly pres-
ent (Fig. 1g in Grzywacz et al. [28]) →Muscina stabulans
12. Mouthhooks symmetric (Fig. 2d)→Helina spp. &
Phaonia spp.
Mouthhooks asymmetric (Fig. 2b)→Hydrotaea sp.
13
13. Posterior spiracles ovoid, spiracular scar in inferior po-
sition (Fig. 3a)→14
Posterior spiracles rounded, scar in middle position
(Fig. 3b–d)→15
14. Posterior surface of anal division, behind anal papillae,
devoid of spines (Fig. 4a,arrow)→Hydrotaea dentipes
Posterior surface of anal division, behind anal
papillae, covered by spines (Fig. 4b,arrow)→
Hydrotaea similis
15. Surface of the anal division surrounding anal papillae
smooth, without distinct spines; complete spinose band
present only on the first thoracic segment; virtual projec-
tions of respiratory slits in posterior spiracles distinctly
convergent (Fig. 3b)→16
Surface of the anal division surrounding anal papillae
covered with distinct spines; complete spinose bands not
restricted to the first thoracic segment; slits of posterior
spiracles parallel (Fig. 3c) or slightly convergent
(Fig. 3d)→17
16. Larval body covered with distinct longitudinal ridges
(Fig. 4c)→Hydrotaea armipes
Larval body smooth, devoid of distinct longitudinal
ridges (Fig. 4d)→Hydrotaea pilipes
17. Spinose band on the first thoracic segment not uniformly
broad, broadened ventrally by an additional patch of
spines (Fig. 4e,arrow)→Hydrotaea aenescens
Spinose band on the first thoracic segment uniformly
broad (Fig. 4f,arrow)→18
18.Upper and lower respiratory slits in posterior spiracles
parallel (Fig. 3c), at most slightly convergent towards the
median scar →Hydrotaea capensis
Upper and lower respiratory slits in posterior spiracle
distinctly convergent towards the median scar
(Fig. 3d)→Hydrotaea ignava
Discussion
Forensically important species
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 identi-
fication, 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 overesti-
mation of the number of carrion-visiting Muscidae taxa. For ex-
ample, 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 spe-
cies, 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 commu-
nity 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 re-
gion of interest is recommended in forensic entomology
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surveys instead of those exclusively oriented towards identi-
fication 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 re-
ports; 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 spe-
cies 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.
Muscidae in forensic context
The larvae of muscids that are commonly considered as forensi-
cally important are either truly necrophagous or display a preda-
cious 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 rep-
resentatives 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 occur-
rence 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 rec-
ognized as one of the most important factors affecting the break-
down 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 typ-
ical necrophagous species [48]. Although some authors have re-
ported 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 abil-
itytoexploitburiedremains[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].
Identification of third instar larvae
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 alter-
native 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
Int J Legal Med (2017) 131:855–866 863
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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 identifica-
tion 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-en-
tomologist, relying as much as possible on easily recognizable
characters.
Species of Muscidae occurring in the western Palaearctic re-
gion 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 isthecaseofStomoxys 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 mis-
interpretations have been revealed in the medical and veteri-
nary entomology literature, including textbooks concerning
the identification of third instar larvae of forensically impor-
tant 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 pres-
ence or absence of spines covering thoracic and abdominal seg-
ments 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 taxo-
nomic 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 ar-
rangement (Fig. 3a–h). However, the pigmentation of the poste-
rior 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 sapropha-
gous, and both facultative and obligatory carnivores and is of
primary importance for taxonomic purposes (Fig. 2a–d). In spe-
cies 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.
Conclusions
A high species diversity has been revealed for the community
of carrion-visiting Muscidae. Recent studies have significant-
ly added to this set of muscids known to be attracted to
decomposing carrion/cadavers. Some species have been
shown to regularly either visit carrion as adults or to breed
in/on decomposing bodies and carrion. Although the value
of some muscid species as forensic indicators has been docu-
mented, particularly in Central European habitats, the poten-
tial value of many other species still needs to be studied in
detail. The key for the identification of third instar larvae pro-
vided here will allow for precise identification of all muscid
species known to breed in human cadavers in the western
Palaearctic, facilitating a better understanding of the role
played by Muscidae as forensic entomology indicators. Data
to estimate the age of the oldest cadaver colonizers, thereby
864 Int J Legal Med (2017) 131:855–866
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

the minimum PMI, includes developmental models of a par-
ticular species under specific conditions gathered by laborato-
ry studies. Though such data have been provided for some
muscids [26,66–69], the majority of species still need to be
studied.
Acknowledgments The present work was supported financially by the
Polish National Science Centre (grant no. N N303 470838), the Ministry
of Science and Higher Education grant IUVENTUS PLUS (grant no.
0146/IP1/2015/73) and the SYNTHESYS Projects http://www.
synthesys.info/, which are financed by the European Community
Research Infrastructure Action under the FP7 Integrating Activities
Programme (grants nos. DK-TAF-5412 and GB-TAF-924) to the first
author. We thank Ms. Ana Farinha, Ms. Nina Feddern, Dr. Heike
Fremdt, Mr. Mateusz Jarmusz and Dr. Szymon Matuszewski granting
us their unpublished records. We would like to express our appreciation
to Dr. Yelitza Velásquez (University of Alicante, Alicante, Spain), Ms.
Socorro Gomez (Chiapa de Corzo, Chiapas, Mexico) and Mr. Thierry
Pasquerault (Institut de Recherche Criminelle de la Gendarmerie, Fort
de Rosny, France) for the aid in obtaining larval material of some species.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interests.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
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