Morphological characterization of puparia and molecular analysis of Heleomyza serrata (Linnaeus, 1758) (Diptera: Heleomyzidae): a species of potential forensic interest

Abstract

In medicolegal forensic entomology, the estimation of the minimum postmortem interval and any other evaluation depend on the accuracy of the species identification. Although the most common species received a lot of attention in the last years, other species in less common families still remain poorly investigated and sometimes the immature stages, especially puparia, are not described. The finding of several specimens of Heleomyza serrata (Linnaeus, 1758) (Diptera: Heleomyzidae) during an experiment with rabbit cadavers provided the opportunity to describe the puparium of this species. Molecular analysis based on the COI region enabled the evaluation of the monophyly of the genus and on the other side highlighted the usefulness of this sequence for the identification of this taxon. In general, this work offers a morphological and molecular overview about a species that has been underestimated in forensic investigations being potentially present on human and other animals’ cadavers during the cold seasons.

Keywords:

• Forensic entomology
• winter
• Diptera
• identification
• body colonization

Introduction

Diptera and Coleoptera are considered the most important taxa colonizing bodies after death, with Diptera being the first colonizer of a body. Among Diptera, species in the families Calliphoridae, Sarcophagidae, Muscidae, Fanniidae, Piophilidae and Phoridae are constantly reported from human and animal cadavers during the decomposition process all over the world (Tomberlin & Adler 1998; Schroeder et al. 2003; Vanin et al. 2008; Grisales et al. 2010), whereas other families are reported only sporadically from bodies discovered in specific habitat and/or seasons, e.g.: Trichoceridae, Milichidae, Sphaeroceridae (Smith 1986; Giordani et al. 2019b; Giordani & Vanin 2020). Among the species that are only occasionally found on humans, animals and in traps deployed for forensic purposes or for studying the entomofauna associated with decomposing bodies, the family Heleomyzidae must be mentioned (Vanin 2012; Farinha et al. 2014; Sessa et al. 2019). It is also worth mentioning that their occurrence on cadavers seems to be particularly relevant in the cold season. (Hwang & Turner 2005; Martin‐Vega & Baz 2013). Members of this family are often underestimated because of the lack of tools for the identification of their immature stages and for this reason reported only at the family level, as already highlighted for several families of potential forensic interest. Moreover, because of the presence of puparia of this family in funerary archaeoentomological context, the possibility of their correct identification based on morphological characters (DNA may be too degraded in these contexts) may provide useful information for archaeological reconstruction.

The family Heleomyzidae consists of flies that range from minute to large in size (1.2–12 mm) and from yellow to dark grey or brown in colour. Their wings often show distinctly longer bristles mixed with shorter bristles along the leading edge of the costal vein.

The family includes approximately 600 species that occur in all the biogeographical regions of the world with the exception of Antarctica; 153 European species are known (Woźnica 2006; Lo Giudice & Woźnica 2013; SoszyńskaMaj & Woźnica 2016). According to Gorodkov (1984), the Palearctic Heleomyzidae are divided into three subfamilies: Suilliinae, Heteromyzinae and Heleomyzinae (Gorodkov 1984; Papp 1998; SoszyńskaMaj & Woźnica 2016). The description of puparia of species in this family is very limited despite the very interesting study by Rotheray (2012). Among Heteromyzinae, Heleomyza serrata (Linnaeus, 1758) [Syn: Musca serrata Linnaeus, 1758; Helomyza geniculata Zetterstedt, 1838] was reported from excrements, bird nests, fungi, dung, chicken manure, decomposing plant remains and cadavers (Gill 1962; Skidmore 1962; Garnett & Foote 1966; Krivosheina 2008; Carles-Tolrá 2011). Heleomyza serrata, as many other heleomyzids, is a cold-adapted species, active in winter and often collected on the surface of snow in Poland (Soszyńska 2004; Soszyńska-Maj & Woźnica 2012) and Scandinavia (Hågvar & Greve 2003) and it has often been found in caves in Norway, Austria and Poland (Kjærandsen 1993; Christian & Spötl 2010; Østbye & Lauritzen 2013). Adults are often confused with Heleomyza captiosa (Gorodkov, 1962) as highlighted by Chandler (1998), from which it can be separated by the analysis of the male genitalia or by the shape of spermathecae (for more details see Gorodkov 1962; Soszyńska-Maj & Woźnica 2016).

In order to improve knowledge about this species and to help in its identification, morphological descriptions of adults and puparia are provided and discussed, together with molecular analyses useful for identification.

Materials and methods

Puparia were collected in 2017 from completely skeletonized rabbits exposed in 2014 on a layer of sand a few centimetres deep used in a decomposition experiment as previously reported (Tuccia et al. 2019).

The dead rabbits (2.75–3.50 kg) were placed in plastic boxes on the roof of the Science Building at the University of Huddersfield (West Yorkshire, UK, 53°38′36.5″ N 1°46′40.1″ W).

Puparia were stored at room temperature in jars covered with laboratory paper until adult emergence.

A Keyence VHX-S90BE digital microscope, equipped with Keyence VH-Z250R and VH-Z20R lens and VHX-2000 Ver. 2.2.3.2 software (Keyence, Japan) was used for the observation of both adults and puparia. To improve the pictorial archive available for H. serrata, air-dried cleaned puparia were observed with a FEI QUANTA 650 FEG scanning electron microscope (SEM) (Thermo Scientific, USA). Images were directly digitized from the SEM.

Adult identification was provided by Dr Andrzej J. Woźnica (Instytut Biologii, Uniwersytet Przyrodniczy we Wrocławiuand – Poland).

Three adult flies were used for molecular analyses. Manufacturer’s instructions of QIAamp® DNA Mini kit were followed. The amount of proteinase K was increased from 20 to 40 μl, previous extractions showed better results with this amount. Each specimen was submerged and incubated overnight at 56 °C into the extraction buffer preventing morphological destruction. The commercial kit “Quant-iT™ Qubit™ dsDNA High-Sensitivity Assay Kit” (Invitrogen, United States) was used on a Qubit® 3.0 Fluorometer (Invitrogen, United States) for DNA quantification. The universal primers LCO-1490 and HCO-2198 designed by Folmer et al. (1994) were used for the amplification of the mitochondrial COI Barcoding region. A total reaction volume of 20 μl was prepared using Promega GoTaq® Flexi Polymerase protocol: 4 μl of Colourless GoTaq Flexi Buffer (5×), 2 μl of MgCl₂ (25 mM), 0.5 μl of each primer (10 pmol/μl), 0.5 μl of dNTPs Mix (10 mM), 0.25 μl GoTaq DNA Polymerase (5 u/μl) and 2 μl of DNA template. The reactions, including positive and negative controls, were assembled under a Purair PCR-36 laminar flow cabinet (AirScience®, Florida, USA) in order to prevent cross contaminations between samples.

The thermal cycler BioRad C1000 (Bio-Rad Laboratories, Inc.) was used to perform the amplification setting the following program: initial heat activation step at 95 °C for 10 min, 35 cycles of 95 °C for 1 min, 49.8 °C for 1 min, 72 °C for 1 min and a final extension step at 72 °C for 10 min. Positive and negative DNA templates were used as controls for the reaction. The quality of the reactions was qualitatively assessed by 1.5% (w/v) agarose gel electrophoresis stained with Midori Green Advanced DNA Stain (Geneflow Ltd., Lichfield, UK.) Amplicons were purified using QIAquick PCR Purification Kit® (Qiagen, Hilden, Germany) following the manufacturer’s instructions, eluted in 40 μl of EB and sequenced by Eurofins Genomics (Ebersberg, Germany).

The newly produced sequences have been deposited in GenBank (Table I). Sequences of COI gene from Heleomyzidae species were downloaded from BOLD online system and included in the phylogenetic analyses (Table II). Sequences were aligned with Clustal Omega (Sievers et al. 2011). The phylogenetic trees were built using the Neighbour Joining method on MEGA 7.0 (Kumar et al. 2016). Fifty sequences of 563 bp were used for the phylogenetic analysis. Drosophila melanogaster Meigen, 1830 (Diptera, Drosophilidae) was used as an outgroup.

Table I. GenBank codes of COI sequences produced in this work.

Family	Species	GenBank code
Heleomyzidae	Heleomyza serrata (Linnaeus, 1758)	MH825674 MH825675 MH825676

Table II. COI sequences of Heleomyzidae downloaded from BOLD and included in the analyses.

Family	Species	BOLD code
Heleomyzidae	Tephrochlamys flavipes (Zetterstedt, 1838)	GMGMF066-14 GMGMG599-14 GMGMF169-14 GMGMF070-14 GMGMF067-14	GMGMF048-14 GMGMF118-14 GMGME1399-14 GMGME593-14
	Tephrochlamys rufiventris (Meigen, 1830)	NSMTP094-15 NSMTP095-15 MAMTA1456-12 CDFD034-12	GMGMH242-14 GMGMG513-14 GMGMG530-14
	Tephrochlamys tarsalis (Zetterstedt, 1847)	SSBAA5552-12 SSBAC1964-12 CNTNK076-14	JWDCE454-10 SMTPM2063-15
	Borboropsis puberula (Zetterstedt, 1838)	SSDWA6374-15	SSECC323-15
	Heleomyza bisetata (Garrett, 1922)	SMTPB143-13 SSKOA1062-15 NGNAA2145-13 SMTPM1735-15	CNFDL185-14 SMTPM1173-15 JSDIP575-10
	Heleomyza brachypterna (Loew, 1873)	PHDIP677-11
	Heleomyza serrata (Linnaeus, 1758)	MOBIL1324-16 ARTYT100-12 ARTYT111-12 SMTPB133-13 SMTPM6945-15	SMTPM6375-15 GMGMI1145-14 GMGMG448-14 HEAPR1900-12
	Suillia apicalis (Loew, 1862)	ARTYT128-12
	Suillia quinquepunctata (Say, 1823)	RRMFC2068-15
	Suillia barberi (Darlington, 1908)	SSECC023-15
	Suillia nemorum (Meigen, 1830)	ARTYT129-12	ARTYT131-12
	Trixoscelis fumipennis Melander, 1913	SSWLB7408-13	SSWLB7503-13
Drosophilidae (Outgroup)	Drosophila melanogaster Meigen, 1830	CYTC5404-12	GBDP2877-06

Results

Morphological analysis

Adults

In the analysed samples, adults body length was 4.3 ± 0.3 mm (N = 7) while the length of the wings was 4.9 ± 0.3 mm (Figure 1A). Males show long L shape gonostyli (Figure 1B). Adults are grey flies with an orange frons and the eye height is more than double the gena height. The head shows a grey ocellar triangle with two divergent ocellar setae and two converging postvertical bristles (Figure 1C,D). Two outer vertical bristles, three pairs of fronto-orbital bristles and numerous minute scattered bristles in the frons are present. Lunula is bare. The hind part of the head is grey (Figure 1D). The lower part of the head and the gena are yellowish-light brown with one strong black vibrissa and two additional smaller bristles (Figure 1C). The thorax has four dorsocentral bristles, one short prescutellar acrostichal bristle, one humeral bristle, one posthumeral bristle, two notopleural bristles and two post-alar bristles (Figure 1E). Prosternum is shiny grey with four to six lateral bristles (Figure 1F). The katepisternum has one strong posterodorsal black bristle and six small anterior setulae (Figure 1G,H). The scutellum has four long and strong black marginal setae (Figure 1E). This species can be distinguished from Heleomyza captiosa (Gorodkov, 1962), a species also present in the UK, using the male genitalia or by the shape of the spermathecae.

Figure 1. Heleomyza serrata adult (male). Lateral view (a), magnification of male genitalia (red box 1a)(b), head chaetotaxy in frontal view (c), head chaetotaxy in dorsal view (d), thorax chaetotaxy in dorsal view (e), prosternum (f), thorax chaetotaxy in lateral view (g) and magnification of the katepisternum (red box 1g)(h). Observation performed using a Keyence VHX-S90BE digital microscope (scale bar 500 µm).

Puparia

The average length of the puparia, in the analysed sample, was 5.24 ± 1.14 mm (N = 20). Puparia are yellowish-brown in colour, wrinkled and covered by spines with anterior and posterior spiracles clearly detectable (Figures 2A–C, 3 A–E, 4A–D). The anal plate is light brown and has short expansions upward directed (wings). The post and subanal papillae are present (Figure 3A, Figure 4A). The posterior spiracles are allocated on two parallel tubercules. The slits are radiate, and the scar is in upper position (Figure 3B,C, Figure 4C). Dorsal muscle scars are well defined and visible in SEM observation (Figure 4C). Anterior spiracles in the analysed samples have a branch shape with 11–13 prospiracular lobes (Figure 3D, Figure 4D). The ventral welt of abdominal segment seven is made up of small flame-shaped spines, some of which bifid, placed in disorganized rows (Figure 3E, Figure 4B). Some rows are directed toward the anal plate, some other toward the anterior part.

Figure 2. Heleomyza serrata puparium. Ventral (a), dorsal (b) and lateral (c) views. Observation performed using a Keyence VHX-S90BE digital microscope (scale bar 500 μm).

Figure 3. Heleomyza serrata puparium details. Anal plate (a), posterior spiracles (b,c), anterior spiracle (d) and intersegmental spicules (e). Observation performed using a Keyence VHX-S90BE digital microscope (scale bar 500 μm).

Figure 4. Heleomyza serrata puparium details. Anal plate (a), intersegmental spicules (b), posterior spiracle (c) and anterior spiracle (d). Observation performed using a SEM (scale bar is reported in each frame).

Molecular analysis

The total amount of DNA extracted from the adults ranged from 460 to 1290 ng. A phylogenetic reconstruction was performed to confirm the morphological identification.

The analysis of the sequences obtained from the analysed specimens confirmed the morphological identification as Heleomyza serrata. All the sequences obtained an extremely high value of identity with the sequences of the same species present in the databases (100–97.58%, 0.0 E). The NJ analysis of 50 sequences of 563 bp belonging to different genera (Borboropsis Czerny, 1902, Heleomyza Fallen, 1810, Suillia Robineau-Desvoidy, 1830, Tephrochlamys Loew, 1862 and Trixoscelis Rondani, 1856) in the Heleomyzidae family brought about a phylogenetic tree with well-supported clades at both genus and species levels (Figure 5). However, the low level of bootstrap at the basal nodes of the tree does not allow for any statement about the relationship between and among genera.

Figure 5. Phylogenetic tree of Heleomyzidae family. Neighbour joining method analysis of 563 bp sequence of the COI gene. The green spots and the number at each node indicate the bootstrap support. Sequences from this study are reported in red.

All the sequences of H. serrata cluster together, and the sequences obtained from this study (MH825676, MH825675 and MH825674) branch together with two sequences of H. serrata coded in BOLD as MOBIL1324-16 and ARTYT100-12 from Alaska and Canada.

Discussion and conclusion

Time since death and cadaver displacement from the primary scene are among the information that insects can provide from a crime scene (Amendt et al. 2007, 2011; Gennard 2007; Tomberlin & Benbow 2015) as long as the correct species identification is provided. Extensive literature is available about the morphology of adults and larvae of insects of sanitary, medical, veterinary, agricultural and forensic interest (Barros de Carvalho & Antunes de Mello-Patiu 2008; Rochefort et al. 2015; Szpila et al. 2015) while puparia are still an under examined subject except for few taxa of forensic interest (Giordani et al. 2018, 2019a, 2019b, 2023; Giordani & Vanin 2020; Tuccia et al. 2021).

Although puparia's general shape and size allow family-level identification, these features are not sufficient to reach the species level. Some forensic scientists do not feel confident in the use of morphological characters for identification of immatures (Amendt et al. 2011; Grzywacz et al. 2017) preferring to rear the immatures until fully develop of the adults or relying on the molecular method. The high probability of encountering empty puparia or dead pupae, and the consequent impossibility to rear the specimens to adults, is a limitation of this strategy, in addition to the time and space required. On the other hand, molecular databases may lack information or contain misidentified species sequences, thereby failing in the identification of numerous taxa of forensic interest (Vanin 2008). This is particularly evident for “secondary families” not largely investigated. The synergy between the two approaches – morphological and molecular – is therefore the only way to reach a reliable identification result for many species. To do that, open-access, well-illustrated keys need to be available for non-specialists to rely on easily recognizable characters (Grzywacz et al. 2017) and it is also vital the use of DNA extraction methods that prevent the morphological destruction of the specimens. A method to perform DNA extraction preserving the morphology on 3^rd instar larvae of Calliphoridae, Muscidae and Phoridae has been reported (Tuccia et al. 2016), but nothing has been published yet about empty puparia.

From a morphological point of view, the images reported in the paper show peculiar shapes and structures of the spiracles, of the spinose bands and also of the anal region of the puparium that can be considered and used, as in other taxa, for example in the genus Hydrotaea (family Muscidae) and in the family Piophilidae, as robust diagnostic characters for the puparia identification (Giordani et al. 2019a, 2023).

Some important and inclusive molecular studies have been performed for numerous families of insects of forensic interest (Kits et al. 2013; Marshall et al. 2015; Zajac et al. 2016) but, to the knowledge of the authors, none for Heleomyzidae. A high number of sequences are available in GenBank for the Heleomyzidae family but unfortunately referring to a few species, reflecting the absence of phylogenetic work. The lack of sequences of H. captiosa in BOLD system and NCBI GenBank prevents the possibility to differentiate the two species and it underlines the actual weakness of the molecular approach in poorly investigated families.

It is worth mentioning that the phylogenetic reconstruction shows the monophyly of the analysed genera (Tephrochlamys, Borboropsis, Heleomyza and Suilla) and a well-supported differentiation of co-generic species. Additional sequences are needed to discuss the phylogenetic relationships among and between the taxa of this family.

This work offers a morphological and molecular overview of H. serrata that has been under-evaluated or non-identified in forensic contexts, especially in cold seasons. Further studies of habitat preference and the rate of development at different temperatures are fundamental to make this species useful in medico-legal and veterinary forensic entomology.

Acknowledgements

The authors are grateful to Dr Andrzej J. Woźnica (Instytut Biologii, Uniwersytet Przyrodniczy we Wrocławiu – Poland) for the species identification.

The work of G. Giordani was funded by the Leverhulme Trust Doctoral Scholarship program, and the results here presented are part of her PhD Thesis at the University of Huddersfield http://eprints.hud.ac.uk/id/eprint/34893/; https://core.ac.uk/download/pdf/200757534.pdf.

Disclosure statement

No potential conflict of interest was reported by the author(s).