Forensic Science International 242 (2014) e22–e30

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Case Report

The first report of Telomerina flavipes (Meigen, 1830) (Diptera,

Sphaeroceridae) in a forensic case, with redescription of its pupa

Marıa-Isabel Arnaldos a,b, Nicolas Ubero-Pascal a,b, Rafael Garcıa c, Miguel Carles-Tolra d,Juan-Jose Presa a,b, Marıa-Dolores Garcıa a,b,*a Area of Zoology, Department of Zoology and Physical Anthropology, University of Murcia, 30100 Murcia, Spainb Unit of Service of Forensic Entomology and Evidence Microscopic Analysis, External Service of Forensic Sciences and Techniques (SECYTEF),

University of Murcia, 30100 Murcia, Spainc Murcia Institute of Legal Medicine, Luis Fontes Pagan, 2, 30003 Murcia, Spaind Avda. Prıncipe de Asturias 30, atico 1, 08012 Barcelona, Spain

A R T I C L E I N F O

Article history:

Received 21 December 2013

Received in revised form 5 July 2014

Accepted 22 July 2014

Available online 30 July 2014

Keywords:

Telomerina flavipes

Sphaeroceridae

Forensic case

PMI

Iberian Peninsula

A B S T R A C T

This paper presents a forensic investigation that took place in the city of Murcia (SE Spain) and shows

how the entomological specimens collected at the scene were extremely helpful for estimating the

minimum post-mortem interval (PMImin). The occurrence of Telomerina flavipes (Meigen, 1830) (Diptera:

Sphaeroceridae) is reported here for the first time in a forensic case. Additionally, the importance of other

entomological evidence in this case is discussed. The first known images of the puparium are provided, as

well as its redescription and that of the cephalopharyngeal skeleton recovered from the puparium.

� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The contribution of forensic entomology to forensic practice hasbeen widely documented and in some cases has providedinteresting results on the fauna related to corpses (e.g. [1–8]among many others). The most interesting application to forensicpractice deals with the estimation of a minimum post-morteminterval (PMImin) on the basis of entomological evidence recoveredfrom the corpse or the forensic scene. Such estimation can be madetaking into account the larval development of the species feedingon the corpse, as well as the succession model of the sarcosapro-phagous fauna. Following Carvalho et al. [9], insects of mostforensic importance are those that develop in the corpse becausethey afford an estimation of the minimum time of death if thedevelopmental time of their preimaginal stages is known. Thus,one of the primary goals of forensic entomology studies is toidentify those species associated with corpses and, for this goal,actual forensic cases provide excellent substrates for these studies.Diptera are the group most often recovered in actual forensic cases,

* Corresponding author at: Area de Zoologıa, Facultad de Biologıa, Universidad de

Murcia, 30100 Murcia, Spain. Tel.: +34 868 884207; fax: +34 868883963.

E-mail address: mdgarcia@um.es (M.-D. Garcıa).

http://dx.doi.org/10.1016/j.forsciint.2014.07.023

0379-0738/� 2014 Elsevier Ireland Ltd. All rights reserved.

both in their adult and preimaginal stages. Species of somedipteran families (e.g. Calliphoridae, Muscidae, Sarcophagidae) areusually considered for PMImin estimation since the life-cycle ofsome of the more common species and its duration is well known(e.g. [10–13] among others). While such species compose most ofthe evidence collected in forensic cases, families of smaller sizedspecies, although present on a corpse, are less noticeable and,therefore, are usually not taken into consideration for PMIestimation. This is the case for Sphaeroceridae, for which veryfew references related to human corpses exist and only in some ofthem are the specimens identified at species level. Although thefamily is quite often referred to in relation with animal carrion (e.g.[14–21]), this is not the case with human corpses. To ourknowledge, Motter [22] cited genus Limosina Macquart, 1835from a corpse disinterred after more than three years; Smeetonet al. [23] cited mature larvae of Leptocera Olivier 1813 on a5-month-old corpse; Smith [24] cited Kimosina (Alimosina)

empirica (Hutton, 1901) and commented about the few dataavailable on the biology and developmental rates of Sphaerocer-idae; Lord [25] mentioned two cases in which Sphaerocerids(probably Copromyza Fallen, 1810) were involved; Bourel et al. [26]reported Leptocera caenosa (Rondani, 1880) as pupae and adultsassociated with buried human corpses and Lefebvre and Gaudry[27] reported larval Sphaerocerids at family level in seven cases.

M.-I. Arnaldos et al. / Forensic Science International 242 (2014) e22–e30 e23

The aim of the present study is to describe a forensic case fromthe South-east of the Iberian Peninsula in which the entomologicalspecimens recovered were used to estimate the minimum time ofdeath. These included numerous fly puparia that were identified asTelomerina flavipes (Meigen, 1830) (Diptera, Sphaeroceridae). Thisis the first time this species has been identified from a humancorpse, and this is the first record from a forensic case. In addition,its puparia had not previously been well characterized. Thus, thepupa is newly described and illustrated here for the first time. Aredescription of the cephalopharyngeal skeleton is also presented,providing images and details that are useful for identificationpurposes.

2. Case report

In February 2013, the body of a middle-aged man was located ina recess under a staircase attached to the outside of house situatedin the outskirts of the city of Murcia (Fig. 1A). The corpse was foundin a right lateral decubitus position, having been hidden by foliageand cardboard, plastic and other refuse. The corpse was dressed,with its head wrapped in a blood-stained cloth (Fig. 1B). The bodywas partially mummified and colonized by abundant cadavericfauna.

The identification of the victim was carried out by members ofthe fingerprinting section of the National Police after hydration ofthe distal phalanx of the right index finger and corresponded to a44 years old Caucasian male. The autopsy revealed death bystrangulation with a rope and an incised-contused wound in theleft zygomatic region. The site inspection revealed that thedeceased suffered the attack in a room of the same house.

Samples of cadaveric fauna were taken before performing theautopsy. The fauna was primarily located at the right hemi thorax,oral cavity and upper limbs.

The man in question was last seen alive almost 2 months (56days) prior to the completion of the autopsy.

3. Results and discussion

The entomological evidence provided on 12 February by themedical examiner consisted of four samples: two contained livingspecimens and two consisted of specimens preserved in 70%ethanol. One live sample contained puparia found in the body-bagcontaining the corpse and associated clothing. The other livingsample, provided by the pathologist, came from the autopsyprocedure and consisted of specimens in larval stage fed withcorpse lung tissue. The two samples with preserved specimens

[(Fig._1)TD$FIG]

Fig. 1. (A) General view of the place where the corpse

were also collected both from the body-bag containing thecorpse and clothing and on the corpse during the autopsyprocedure. The living samples were kept in a growth chamber(Climas Grow 470/HR), at 25 8C and 60% RH using commercial pigliver as feeding substrate for the larvae.

The taxa identified in the preserved samples were: Calliphor-idae: Calliphora vicina Robineau-Desvoidy, 1830: larvae II, larvaeIII, hatched and unhatched puparia, adult female; Chrysomya

albiceps (Wiedemann, 1818): larvae II, larvae III; Muscidae:Hydrotaea capensis (Wiedemann, 1818): larvae III, unhatchedpuparia; Synthesiomya nudiseta (van der Wulp, 1883): larvae II,larvae III, hatched and unhatched puparia; Fanniidae: larva III,unhatched puparia; Drosophilidae: unhatched puparia; Phoridae:larva III, hatched and unhatched puparia; Sphaeroceridae:T. flavipes: unhatched puparia, adult; Staphylinidae: larvae andadults; Nitidulidae: adults; Acarida.

Specimens obtained from the living samples belonged to:C. vicina (adults began emerging on 13 February); C. albiceps (beganemerging on 18 February); H. capensis (began emerging on 15February); S. nudiseta (began emerging on 14 February); Fanniidae(began emerging on 22 February); Phoridae (began emerging on 15February); and T. flavipes (began emerging on 15 February).

Specimens are kept at the Zoology Area, Department of Zoologyand Physical Anthropology of the University of Murcia.

The fauna recovered and its developmental stage was indicativeof the advanced stage of decomposition of the corpse [24,28–32].The estimation of PMImin was made on the basis of theCalliphoridae and Muscidae, which are usually the best indicators,taking into account the meteorological data recorded throughoutthe period after the disappearance of the subject (dailymean � 14 8C). C. vicina is a winter early colonizer of corpses inthe geographical area considered. Its life-cycle duration has beenstudied by different authors (see e.g. [33,34] for a summary ofreferences to developmental publications). Since empty pupariawere recovered from the corpse, the estimation of the PMImin wasequivalent to at least the whole life cycle duration, from egg toadult. Anderson [10] studied the life cycle duration under differentthermal regimes, the lowest being 15.8 8C, at which temperaturethe time elapsing between oviposition and adult emergence wasabout 37 days, clearly less than the period elapsing since lastknown sighting of the deceased. Nevertheless, this result should beconsidered with caution, since developmental variations havebeen detected in different geographical populations of flies as wellas variations observed in ADD/ADH at temperatures below about15 8C [35]. As regards S. nudiseta, recently recorded in forensiccases in the Iberian Peninsula [5], its life cycle duration was studied

was found; (B) original position of the dead body.

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by Velasquez et al. [36]. The most applicable data were thoseobtained at 15 8C, which pointed to a period of about 47 days fromegg laying to adult emergence. This species is usually considered asecondary fly and Lord et al. [37] mentioned a case in which larvaeof this species were collected together with pupae of C. vicina.Because of all the above, it would seem to be reasonable to add acertain time to the PMI considering a delay in accessing to thecorpse and the oviposition. The biology of H. capensis is poorlyknown, although the species has been frequently recovered inforensic cases (e.g. [5,38–40]). Lefebvre and Pasquerault [38]studied its development at three different temperatures, thelowest being 17 8C, when adults emerged after about 62 days. Inour case at 14 8C, these data provide an indication of the veryminimum life cycle duration. Since we recovered pupae, fromwhich adults emerged two days later (albeit at 25 8C), it seemedreasonable to estimate the time of colonization of H. capensis asbeing close to the date when the victim was last seen alive.

The presence of T. flavipes (1 adult, 145 unhatched puparia, 34 ofwhich hatched between 15 and 19 February) is interesting forseveral reasons. To start with, this is the first forensic casewhere this species has been recorded. The discovery of puparia onthe body during the autopsy shows it had developed on the corpseas a necrophagous species and could thereafter be taken intoconsideration for estimating the minimum PMI.

3.1. Notes on T. flavipes, including redescription of the puparium

This species (Fig. 2A–E) is predominantly necrophagous and isalmost cosmopolitan because of its partial synanthropy [41],surviving outside its natural range because of the highertemperatures around human dwellings. It has frequently beencited in archaeological studies [42–48], where its presence helps

[(Fig._2)TD$FIG]

Fig. 2. Adults of T. flavipes. (A) Habitus of male. (B) Habitus of female. (C) Wing

confirm that material has originated from human settlements. Itspresence in this case suggests a similar origin in a confined space,as indicated by Bourel et al. [26], both for other sphaerocerids andfor species such as H. capensis, suggesting the corpse had not beenmoved between the time of death and when it was found.

In the Iberian Peninsula it has been recorded in animal carrion[49–51], in the north, northeast and west areas, and has recentlybeen collected in the southeast [21]. Though mainly thought tobreed in vertebrate carrion, this species also breeds in otherdecaying animal matter, fungi and dung, and it also occurs in cavesand mammal burrows [52–54]. However, until now, it had notbeen recorded from human corpses, although, being a smallspecies, it may have been overlooked.

Regarding the preimaginal stages of this species, Marshall andRohacek [54] have provided descriptions of the third instar larvaand puparium, but only the larva has been illustrated, so somepuparia collected from this case were studied using both light andscanning electron microscopy (SEM).

For light microscopy, five puparia were dehydrated, dissectedand mounted on slides using Canada balsam as mounting medium.Cephalopharyngeal skeletons were extracted from puparia andmounted following the same methodology. In both cases, noclearing technique was necessary. Samples were analyzed using aNikon Eclipse i80 microscope with Nomarski interference contrast.Pictures were taken using a Nikon DS-Fi1 digital camera of5-megapixel CCD and Nis-elements software.

For SEM, 10 puparia fixed in 70% ethanol were dehydrated,desiccated and sputter coated using the techniques and devicesused by Panos et al. [55], and observed in a Jeol 6100 SEM. Pictureswere directly obtained and digitized from the SEM. For correctdiagnosis of the puparia features, five specimens were cut intothree parts: thoracic segments, abdominal segments and anal

venation. (D) Detail of mesothoracic tibia. (E) Detail of the right surstylus.

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division. These were arranged in different positions in order toeasily observe various parts of the body, especially the posteriorspiracles. The other five specimens were preserved entire forviewing from ventral, lateral, and dorsal positions.

The body length of 2.35 mm and brownish colouration of thepuparia fits the description given for this species by Marshall andRohacek [54]. The general body shape conforms to the generalfeatures of the puparia of Sphaeroceridae given by Rohacek [56]and Smith [57], with a barrel-like habitus, slightly flatteneddorsoventrally, extremely wrinkled, with a blunt anterior tip andthe posterior tip prolonged by the posterior spiracles, like twoshort horns (Fig. 3A and B). The surface of puparia are often soiled,obscuring morphological characters, and thus making themdifficult to study using SEM (as in Figs. 3D, E and H and 4F).However, SEM was successfully used for puparia with little or nosoiling, allowing comparison with the third-instar larval char-acters described by Marshall and Rohacek [54]. Light microscopywas used as a complementary technique to SEM in the presentstudy as suggested by Ubero-Pascal et al. [58], and proved criticalfor the examination of features such as the ventral creeping welts(Fig. 3G and I).

The anterior spiracles are positioned at the anterior corners ofthe puparium (Fig. 3B and D), as the larval pseudocephalon andanterior part of the first thoracic segment collapse internallyduring pupariation. The presence of debris may make the anteriorspiracles appear bent or damaged when studied using lightmicroscopy (Fig. 3C and D). However, SEM indicated that whenthey are free of debris they preserve the morphology given byMarshall and Rohacek [54] for the third instar larvae: a long stemwith seven to eight branches or finger-like papillae (Fig. 3D–F).According to these authors, the base of the anterior spiracles,adjacent to the puparium, is strongly pigmented (Fig. 3A and C). Inmarked contrast to that found by Marshall and Rohacek [54],creeping welts of abdominal segments are easily distinguished byboth light and scanning electron microscopy (Fig. 3G–J), and theirfeatures are consistent with those observed in the third instarlarvae. The creeping welts consist of several rows of spines andprotuberances (Fig. 3G–J), so that an anterior row of wide andmainly rounded spaced protuberances can be distinguished in themiddle area of each segment, followed by three rows of minutespines in a comb-like arrangement, a different row of wide androunded protuberances close to one another and uniformlyaligned, and three more rows of tiny spines arranged in a comb-like pattern. The spines are triangular-shaped and pointed,sometimes bifid, although some are blunt in the central area ofthe anterior rows (Fig. 3H and J). However, when observed by lightmicroscopy, they may appear blunt due to the more sclerotizedbase (Fig. 3I). Some protuberances may also be pointed and evenbifid, in agreement with the description given by Marshall andRohacek [54] for the third instar larva. The second and third rows ofthe posterior set of spines cannot be clearly observed by SEM dueto debris or because they are hidden between folds on the surfaceof the puparium (Fig. 3J). The anal pad in the pupa appearsdifferentiated by both microscopy techniques (Fig. 4A and B), butthe apparently bulbous form of the perianal pad described in thirdinstar larvae by Marshal and Rohacek [54] is collapsed (Fig. 4B).Notwithstanding, the clusters of large hook-shaped spines on theposterior margin of the anal pad are clearly observable by SEM (Fig.4B). The spiracular processes of the puparium appear somewhatcollapsed compared to those of the third instar larva, being darkpigmented distally with the spiracular plates facing slightlyinwards (Fig. 4A), each plate having three slit-like openings withfour long and multibranched peristigmatic tufts between them(Fig. 4D). The darkly pigmented area of the distal tip and itsspherical appearance are present in the pupae (Fig. 4A and C). Theslits are about one and a half times longer than the width of the

base and are constricted half way along their length. The spiracularslits are disposed perimetrally, showing a trabecular structure bylight microscopy (Fig. 4C and E). By SEM, however, the spiracularslits appear as a simple line displaying small protuberances at theends (Fig. 4G). Unfortunately, it has not been possible to determinethe number of spiracular openings of the pupa given by Marshalland Rohacek [54]. The peristigmatic tufts cover the entire surfaceof the spiracular process, except its inner area (Fig. 4F).

The third-instar larval cephalopharyngeal skeleton can easilybe dissected from the puparium (Fig. 5A). According to Marshalland Rohacek [54], the features and arrangement of larval scleritesremain the same in the structure obtained from the puparium(Fig. 5A). In addition, we identified sclerites that were clearlyoverlooked by Marshall and Rohacek [54], such as a pair of dentalsclerites and a complex labial sclerite (Fig. 5A and B). Themouthhooks are sclerotized, with a sharpened tooth (Fig. 5B) abasal part with a rounded window that is clearly visible whenmouthhooks are not overlapping (Fig. 5B and C). The dorsal edge ofthe basal part is concave and has a posterior prominence (Fig. 5B),while, ventrally, it shows a fossette in which the dental sclerite isarticulated, which is not observable in the lateral view (Fig. 5B).The dental sclerite is triangular inshape with rounded angles(Fig. 5C). The intermediate or hypopharyngeal sclerite is approxi-mately 1.3 times the length of the mouthhook; it is H-shaped whenviewed dorsally, strongly sclerotized, and has a ventral protrusionin the middle (Fig. 5B and D). The anterior arms of the intermediatesclerite are shorter than the posterior ones. They have roundedtips, while the tips of the posterior arms are pointed (Fig. 5D–F).The bridge of the intermediate sclerite is concave and slightlypigmented (Fig. 5F). The labial sclerite is situated between theanterior arms of the intermediate sclerite, with the posterior partraised and extending over the bridge of the intermediate sclerite(Fig. 5B, D and E). With the exception of the lateral edges, theanterior part of the labial sclerite is slightly pigmented especially inthe ventral area (Fig. 5F) which articulates with the intermediatesclerite. The labial sclerite has two small holes in the centre(Fig. 5E), the posterior branches articulating with the basal ortentoropharyngeal sclerite (Fig. 5D). The parastomal bars are thinand elongate, situated above the intermediate sclerite andapparently fused with the basal sclerite (Fig. 5D–F). The basalsclerite has a well pigmented vertical plate, dorsal cornua anddorsal bridge; however, the ventral cornua are partly pigmented,while the ventral and posterior areas are slightly pigmented. Thedorsal bridge is separated from the dorsal cornua and fused to thevertical plate by a thin and curved bar, the distal end of which iswider than the proximal one. Dorsally, the bridge forms a broadperforated plate, with four hollows in each side. The dorsal cornu isa thin and straight prolongation of the vertical plate and has a smalland elongated window at the posterior tip. The ventral cornu is aslong as the dorsal cornu but the posterior two thirds are weaklypigmented. They present a protuberance like a shark-fin on theirdorsal edge (Fig. 5).

Distinguishing puparia of Sphaeroceridae from those of othersmall Diptera which sometimes occur on human corpses, such asPiophilidae and Drosophilidae, is reasonably straightforward[27,57,59]. Puparia of T. flavipes can easily be distinguished fromthose of Piophila casei, P. megastigmata, or Prochyliza nigrimana,due to the presence of long and branched anterior spiracles, thecharacteristic arrangement of spines and protuberances in theventral creeping welts, and the posterior spiracles in the form oflong body processes. Piophilids, on the other hand, lack longanterior spiracles; their creeping welts have just two/three rows ofspines, and show a pair of posterior papillae forming ventralprocesses [55,60]. Concerning Drosophilidae, the presence of rowsof spines in the dorsal area of the abdominal segments and thearrangement on the anal division of both posterior papillae and

[(Fig._3)TD$FIG]

Fig. 3. Puparium, photographed using light and scanning electron microscopy (LM and SEM). (A) Ventral view (LM) of mature specimen with developing adult visible inside;

(B) habitus, ventral view (SEM); (C) anterior spiracle adhering to the surface of the puparium by debris (LM), with associated tracheal tube visible; (D) head end showing

anterior spiracles (SEM), one in normal position and the other adhering to the surface of the puparium by debris; (E) detail of the anterior spiracle (SEM) covered by debris but

in the natural position and showing its characteristic shape with finger-like papillae; (F) detail of a finger-like papilla (SEM); (G and H) general view of the ventral creeping

welts by LM and SEM, respectively, in which their characteristic arrangement is distinguished; (I and J) detail of the different structural elements of the creeping welts by LM

and SEM, respectively; the spines of anterior and posterior rows and the rounded protuberances. Abbreviation: af, developing adult; an, anal pad; as, anterior spiracles; asl,

anterior spiracle bent; asn, anterior spiracle in natural position; cs, cephalopharyngeal skeleton; cw, creeping welt; db, debris; fp, finger-like papilla; ps, posterior spiracles;

rp, rounded protuberance; rs, row of spines; st, stem of anterior spiracle; tt, tracheal tube.

M.-I. Arnaldos et al. / Forensic Science International 242 (2014) e22–e30e26

[(Fig._4)TD$FIG]

Fig. 4. Posterior spiracles and anal region. (A) Ventral view of anal region and posterior spiracles (LM); (B) detail of the anal pad by SEM; (C) spiracular plate, showing

arrangement of slits (LM); (D) same, photographed using SEM, showing peristigmatic tufts; (E) lateral view of spiracular process (LM); (F) same, photographed using SEM; (G)

detail of a spiracular slit by SEM. Abbreviation: an, anal pad; ao, anal opening; db, debris; hs, hook-shaped spines; pb, perianal bulbous collapsed; ps, posterior spiracles; psp,

posterior spiracle process; p, protuberance; pt, peristigmatic tuft; sl, spiracular slit; sp, spiracular plate.

M.-I. Arnaldos et al. / Forensic Science International 242 (2014) e22–e30 e27

spiracles [61] allow their pupae to be easily distinguished fromthose of Sphaeroceridae.

This is the first forensic case on record involving T. flavipes.Further studies on the life cycle development and the generaldelay between death and arrival of this species are needed to

increase its potential value to aid in minimum PMI estimation.Our findings enlarge the global list of Diptera of forensicimportance. Moreover, we provide new data on the morpholog-ical features of preimaginal stages. In addition, and as pointedout by Magni et al. [62], our work emphasizes the need to

[(Fig._5)TD$FIG]

Fig. 5. Cephalopharyngeal skeleton (all LM). (A) Whole skeleton, lateral view; (B) lateral view of mouth-hooks and intermediate sclerite; (C) Detail of the mouthhooks in the

dorsal view; (D–F) detail of the area of the intermediate sclerite, ventral view; (G) detail of the dorsal bridge in the lateral view; (H) detail of the dorsal plate of dorsal bridge;

(I) tips of dorsal and ventral cornua. Abbreviation: db, dorsal bridge; dbb, dorsal bridge bar; dbh, dorsal bridge hole; dc, dorsal cornu; ds, dental sclerite; fav, forearm of vertical

plate; is, intermediate sclerite; isb, intermediate sclerite bridge; ls, labial sclerite; lsh, labial sclerite hole; mh, mouthhook; pb, parastomal bar; sfp, shark-fin protuberance; vc,

ventral cornu; vp, vertical plate; w, window.

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identify all entomological evidence to the deepest possible levelin forensic practice, since the recognition of new speciesinvolved in this process could provide additional informationrelated to the time of death.

Acknowledgments

The authors are grateful to the Murcia Institute of LegalMedicine to provide evidence of actual forensic cases and byrelying on the SECYTEF for their expert report and to theanonymous referees and the editor for their comments andsuggestion to improve the manuscript.

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