The dorsoventral muscle attachment sites (MAS) patterns are described for six species of the tribe Piophilini (Diptera: Piophilidae): Centrophlebomyia furcata (Fabricius), Liopiophila varipes (Meigen), Piophila casei (Linnaeus), Piophila megastigmata McAlpine, Prochyliza nigrimana (Meigen) and Stearibia nigriceps (Meigen). Comparison between the MAS patterns of Piophilini and previous descriptions for Calliphoridae (Diptera) revealed differences in the muscle equipment between the larvae of both taxa. Among the Piophilini, the MAS patterns were highly conserved and only a genus-specific pattern for Piophila species and a species-specific pattern for C. furcata were found. Nevertheless, these differences in MAS patterns were subtle and some intraspecific variability was observed; hence, the MAS patterns do not appear to be suitable as diagnostic characters allowing for species determination of Piophilini larvae.

Larval morphology, Forensic entomology, Phylogeny, Identification, Centrophlebomyia furcata , Liopiophila varipes , Piophila casei , Piophila megastigmata , Prochyliza nigrimana , Stearibia nigriceps

Lacking legs or prolegs, the larvae of DipteraCyclorrhapha move by the contraction of longitudinal and dorsoventral muscles, increasing the haemolymph hydrostatic pressure (Roberts 1971). However, in spite of its importance, the anatomy of these muscles has only been described for some cyclorrhaphous species; see for example the works of Hooper (1986), Bate (1990) and Wipfler et al. (2013) on Drosophila melanogaster Meigen, Hewitt (1908) on Musca domestica Linnaeus, and Crossley (1965) and Hanslik et al. (2010) on Calliphora vicina Robineau-Desvoidy. All of those studies described a great number of longitudinal muscles usually extending between two segmental borders, and a small number of dorsoventral muscles in the lateral, ventrolateral and dorsolateral regions of each segment (Wipfler et al. 2013).

Recently, Niederegger and Spieβ (2012), and Niederegger et al. (2013, 2015) studied the larval dorsoventral muscles and their cuticular attachments in forensically important blow fly species (Diptera: Calliphoridae). These muscular attachment sites (MAS) are easily visualized as clusters of dots in the larval cuticle and form distinct and both genus- and species-specific patterns in blow fly larvae, allowing for species identification (Niederegger and Spieβ 2012; Niederegger et al. 2013, 2015). Furthermore, the MAS patterns are constant throughout larval development and the length of the MAS rows is linearly correlated with the larval body length (Niederegger et al. 2013). Reliable identification of the material collected is particularly crucial in forensic investigations as the immature stages of necrophagous insects are usually the only entomological evidence collected at autopsies and crime scenes (Amendt et al. 2007). However, the identification of immature stages remains a difficult task for some forensically important insect groups like the commonly named ‘skipper flies’ (Diptera: Piophilidae). This common name is due to the skipping behaviour showed by the third-instar larvae, which is mediated by contraction of the musculature. According to the phylogeny proposed by McAlpine (1977), the family Piophilidae includes two subfamilies: Neottiophilinae, which includes ectoparasite species of nestling birds, and Piophilinae, which includes two tribes: the Mycetaulini, whose larvae develop mainly on rotten fungi, and the Piophilini, whose larvae develop mainly on decaying organic matter and which is divided in two subtribes: Piophilina and Thyreophorina. Both subtribes Piophilina and Thyreophorina are typically associated with cadavers in advanced stages of decay and can be potentially useful as forensic indicators; moreover, some Piophilina species can also be major pests for the food industry and agents of human myiasis (Martín-Vega 2011). Given this forensic, economic and medical importance, methods for the identification of piophilid immature stages are strongly needed. However, barcode sequences for molecular identification are still only available for a few species (e.g. Boehme et al. 2012) and, although several recent studies have yielded new insights into the morphology of the immature stages of the Piophilidae (e.g. Martín-Vega et al. 2012, 2014; Paños et al. 2013; Martín-Vega and Baz 2014), the larval morphology of most piophilid genera and species remains undescribed. As a consequence, some diagnostic characters within existing identification keys must be used with caution (Martín-Vega et al. 2014). It is therefore desirable to explore additional morphological characters which may increase not only the accuracy and reliability of the species identification (which is particularly essential for a correct use of insects as forensic indicators), but also may support the reconstruction of phylogenetic relationships.

The aims of the current study were (i) to describe the dorsoventral muscle equipment in a representative set of piophilid species from the tribe Piophilini, comparing them with previous data on the anatomy of Cyclorrhapha larval muscles; and (ii) to determine if those patterns are genus- or species-specific and can thus be used as an additional tool for Piophilidae species determination.

Six target species were selected for the current study: five species belonging to subtribe Piophilina—Liopiophila varipes (Meigen), Piophila casei (Linnaeus), Piophila megastigmata McAlpine, Prochyliza nigrimana (Meigen) and Stearibia nigriceps (Meigen); and one species belonging to subtribe Thyreophorina—Centrophlebomyia furcata (Fabricius). These six species are among the most common piophilid species occurring on carrion and show wide geographical distributions (Martín-Vega 2011).

Adult males and females of C. furcata, L. varipes, P. casei, P. megastigmata and P. nigrimana were collected on animal carcasses and carrion baits in different habitats of central Spain, identified using published taxonomical keys (McAlpine 1977, 1978) and subsequently used to start laboratory cultures. Details on collection sites and on the conditions and maintenance of the laboratory colonies for each species can be found in Martín-Vega et al. (2012, 2014) and Martín-Vega and Baz (2014). For each species, larvae were reared to the third-instar, killed in near-boiling water and then preserved in 70% ethanol for more than 24 hours to allow the muscles to detach from the cuticle (see Niederegger and Spieβ 2012). This fixation and storage method is also recommended to achieve best preservation of larval samples (Amendt et al. 2007). Furthermore, larvae of S. nigriceps were obtained from a human corpse being object of a current investigation in the Institute of Legal Medicine of the Friedrich-Schiller-University of Jena (Germany). Additional larvae of S. nigriceps were kindly supplied by Dr Krzysztof Szpila (Nicolaus Copernicus University, Poland). Several third-instar larvae were killed and preserved following the aforementioned method, while the remaining larvae were placed in a plastic container containing minced meat and reared to adulthood. Ten third-instar larvae of each target species were randomly collected and dissected for the study. Before dissection, the length and diameter of the larvae were measured (accuracy ± 0.1 mm) using a calibrated ocular micrometre.

Preparation of larvae and evaluation of the MAS patterns followed the methodology described in Niederegger and Spieβ (2012) and Niederegger et al. (2013, 2015). The dorsally dissected cuticle was cleaned and stained with Coomassie brilliant blue solution (Sigma, 1% in tap water), cutting off the first segment (Fig. 1). The stained cuticles were flattened and covered with a glass slide cover and mounted onto the dissecting microscope. After study, the cuticles were stored in 70% ethanol at the Institute of Legal Medicine of the Friedrich-Schiller-University of Jena.

Each MAS is visible on the cuticle as a ‘dot’. The dots are grouped in distinct clusters which are arranged symmetrically along the ventral midline (Figs 1, 2). Following Niederegger and Spieβ (2012), a cluster of dots is called ‘row’ and the term ‘pattern’ refers to the shape of a row. All rows in segments 2–11 were documented and labelled following Niederegger et al. (2015): rows were numbered according to the segment and the position within the segment, starting at the centre (Fig. 2). The individual rows were photographed for each segment using a digital camera (BeyTec, Moticam 1000) attached to the dissecting microscope, using identical magnification in every preparation. Then, on the computer, each MAS dot was covered with a semitransparent coloured circle using graphic software (Adobe Photoshop CS). The resulting rows of circles were stacked, using the most anterior circle (in longitudinal rows) or the left circle (in transverse rows) as reference point. The resulting areas with a high degree of overlap were marked as ‘condensed pattern’ to allow the direct comparison between species. Moreover, the number of MAS per row was documented for each specimen, and the average number of MAS per row and the standard deviation were computed for each species (see Suppl. material 1).

Figures 1–2. 

Liopiophila varipes (Meigen), third-instar larva. 1. Dorsal view of a pinned larva before dissection (left) and stained larval cuticle (right), showing the symmetrical muscular attachment sites on segments S2 to S11; 2. Detail of the abdominal segment S5 showing the label for each muscular attachment site pattern.

We are grateful to Dr Roland Spieβ (Jena, Germany) for his assistance with the preparation of larval specimens and his helpful comments on the present manuscript. We also thank Dr Krzysztof Szpila (Nicolaus Copernicus University, Toruń, Poland) for kindly providing larval specimens of Stearibia nigriceps, and James Alexander Stone for his corrections on the English language. Dominique Zimmermann and two reviewers provided constructive input on an earlier version of the manuscript. This work was supported by the DAAD (Deutscher Akademischer Austausch Dienst; scholarship no. A/13/71361 awarded to the first author). Open access is available thanks to the support of the Museum für Naturkunde, Berlin.