First record of Phormia regina (Meigen, 1826) (Diptera: Calliphoridae) from mummies at the Sant’Antonio Abate Cathedral of Castelsardo, Sardinia, Italy

Geographical and Historical context

The small town of Castelsardo is located in the Northern coast of Sardinia (Italy), 114 m asl, as part of Sassari province (40°54′52″N  8°42′46″E) overlooking the Asinara gulf (Fig. 1). The climate is typically Mediterranean and has been classified as Csa type (Köppen & Geiger, 1930; Peel, Finlayson & McMahon, 2007). The average annual temperature is 16.1 °C, with a maximum of 28.9 °C and a minimum of 6−7 °C during the summer and winter seasons respectively (Metereological station of Alghero Fertilia, Sassari, Sardinia; Pinna, 1945). The average annual rainfall is 588 mm (https://it.climate-data.org).

The Sant’Antonio Abate Cathedral, an ancient Romanic Church which became the seat of the bishop of Ampurias in 1503, is located in the old town center. The modern building is the result of the reconstruction which was carried on between 1597 and 1606 due to the transformation of the original Church into Cathedral. Architectonic elements from the Renaissance renovation and original Romanic ones define the style of the Cathedral. The interior structure is based on the Latin cross plan, with a single nave with barrel vaults, side chapels and transept. The old basements (hypógheios) of the Church were left unburied and used as sepulchral crypts of the new cathedral.

Modern renovation works began in 2011 to expand the area of the Diocesan museum located in the underground crypts. During the excavations, human remains were found in the so called “Room 3”. Heaps of bones and mummified bodies have been found in different positions and places of the crypt, allowing to reconstruct the relative chronology of the depositions.

Two individuals, conventionally called “Bob and Mary”, are believed to be the most recent inhumations since they have been found at the top of the excavation in close proximity to the floor of the Cathedral. Considering that from 1804 intra muros burials were no longer permitted (Napoleon’s Saint Cloud Edict), “Bob and Mary” represent not only the most recent inhumations but probably even the last. At the same time, despite the still uncertainty about the time of the other previous burials, the practice of underground burials in the Cathedral can be dated between 1597 and 1806, when the Saint Cloud Edict was applied in Italy.

The archaeological material was studied by a multidisciplinary international scientific team including archaeologists, anthropologists, biologists, and immunologists in order to investigate about the sanitary condition of the population and to better understand the burial practices carried out in this region.

Diptera

Of the total number of entomological fragments, 95% were Diptera puparia, all of them sampled in the area where the bodies of “Bob & Mary” were located. The number of puparia fragments clearly indicate that this was not a sporadic finding but it is consistent with the colonization by flies in the early phase of decomposition. It was not possible to associate the puparia from one body or the other because of the archaeological context, and because post-feeding maggots tend to move away from the body to find a protected place where the pupariation and later the pupation take place (Lewis & Benbow, 2011; Martín-Vega, Hall & Simonsen, 2016).

Puparia of four different morphotypes were isolated among the studied material: the largest three morphotypes were recognized as members of the families Calliphoridae (Calliphorinae and Chrysomyinae) and Sarcophagidae, the smallest belonging to Muscidae. From the morphology of the posterior region, the shape and position of the posterior spiracles, the structure of the anal plate and the shape and disposition of the intersegmental spines, Calliphoridae puparia were identified as P. regina (Fig. 2) and Calliphora vicina (Robineau-Desvoidy, 1830) (Fig. 3). Both species are typical of the first body colonization wave and are reported mainly from exposed bodies (Smith, 1986).

Phormia regina puparia show a rough appearance and a characteristic posterior region with the posterior spiracle, big and rounded, located in a superficial invagination (Fig. 2), and minute, sharp intersegmental spines arranged in short rows. The lack of big and robust anal papillae with a crow shape is useful for distinguishing this species from Protophormia terraenovae (Robineau-Desvoidy, 1830) which has been often reported in association with P. regina (e.g., Vanin et al., 2009).

Classified within the subfamily Chrysomyiinae, P. regina is a forensically important fly. This widespread, hemisynanthropic¹ species is associated with decomposing material (dung and carrion) (Byrd & Allen, 2001; Greenberg, 1985; Nabity, Higley & Heng-Moss, 2006). In temperate regions, this species is abundant in spring and summer whereas its presence decreases in cooler areas (Goddard & Lago, 1985; Greenberg, 1985; Hall, 1948). Relatively tolerant to cold weather, the minimum and maximum thresholds for this species range from 10–12.7 °C to 35 °C (Deonier, 1942; Haskell, 1993). Rare nocturnal oviposition has been recorded in natural environments (Berg & Benbow, 2013). This species is reported from almost the whole continental Europe, with the exception of the Iberian Peninsula, the Denmark, the Hellenic Peninsula and the Italian main islands (Sardinia and Sicily) (http://www.fauna-eu.org). If this lack of reporting the species from Sardinia is due to a real absence or a misidentification with P. terraenovae remains to be evaluated. The finding of this species is particularly important because no modern records of the species are available in the specific literature from Sardinia neither modern specimens from this region are stored in museum collections in Italy. In contrast the species is widespread in Italy, mainland, where it was reported from archaeological and forensic works (Vanin et al., 2008; Vanin et al., 2009; Vanin et al., 2011; Benelli et al., 2014). It is worth mentioning that an important part of the past Sardinian economy was based on the sheep farming and that P. regina was reported as cause of myiasis on livestock in North America; however, this species was never been reported from goat and sheep in Europe (Hall & Wall, 1995; Sotiraki & Hall, 2012).

Phormia regina was reported from different forensic context in North America and Central Europe (Italy, Germany, Poland, etc.) both on animal carcasses (pigs and fishes) and human bodies (Anton, Niederegger & Beutel, 2011; Cianci & Sheldon, 1990; Goddard & Lago, 1985; Greenberg, 1985; Matuszewski et al., 2008; Matuszewski et al., 2010a; Matuszewski et al., 2010b; Matuszewski et al., 2011; Schroeder, Klotzbach & Puschel, 2003; Sharanowski, Walker & Anderson, 2008; Vanin et al., 2008; Vanin et al., 2011). From archaeological contexts it was reported only from the remains of a WWI soldier in the Italian front (North-Eastern Italy) (Vanin et al., 2009) and on pre-historic graves in Canada by Teskey & Turnbull (1979). In both cases, the authors suggested a considerable lapse of time between death and interment (Teskey & Turnbull, 1979; Vanin et al., 2009).

Puparia of C. vicina appear smoother and of a lighter brown colour if compared to the previous species (Fig. 3). The ratio between the major dimension of the spiracle and the interspiracle distance allow to distinguish this species from the cogeneric Calliphora vomitoria (Linnaeus, 1758). In fact this ratio is <1 (big spiracles, small interspiracle distance) in C.vomitoria and >1 (smaller spiracle, bigger interspiracle distance) in C. vicina (Emden, 1965).

Calliphora vicina is a common forensically important blow fly, belonging to the subfamily Calliphorinae. This species has an almost cosmopolite, very common in urban habitat, closely associated with humans (Wallman, 2001) but also present in hedgerow or woodland habitats (Smith & Wall, 1997a). Calliphora vicina was reported over the whole year (Schroeder, Klotzbach & Puschel, 2003), although in regions with hot summer, its seasonal population curve is typically bimodal with spring and fall peaks where the temperatures are around 13–24 °C (Greenberg, 1985; Salvetti et al., 2012; Zumpt, 1965). Because of its promptness in reaching and colonizing the body after death, C. vicina is currently considered as one of the most important fly species in forensic entomology (Davies, 1990; Isiche, Hillerton & Nowell, 1992; Smith & Wall, 1997a; Smith & Wall, 1997b). Its presence on dead bodies or animal carcasses has been deeply studied from all around the world (North America, Europe, Asia and Africa) (Bonacci et al., 2009; Bugelli et al., 2015; Dupont et al., 2011; Greenberg, 1985; Keshavarzi et al., 2016; Moemenbellah-Fard et al., 2015). From archaeological contexts, this species was reported from Central Italy, on partially mummified bodies found in a crypt under a Church (Vanin, 2012) in a circumstance similar to the one described in this work. However, in that context some openings (small windows) allowed the insect arrival and body colonization from the external environment. The ability to colonize bodies in ipogeic context is confirmed by the finding of Faucherre and colleagues (1999). These authors reported the presence of C. vicina eggs on a human cadaver found in a 10 m deep cave in the Swiss Jura mountains at an altitude of 1,260 m asl. However, in colonization experiments this species showed a limited ability to colonise buried remains, not deeper than 10 cm (Gunn & Bird, 2011) and it is not reported from buried human remains. In general, the presence of this species indicates a colonization of exposed bodies: before their burial or their transfer into a crypt.

Three puparia fragments (Table 1, Fig. 4) were identified as belonging to a species in the genus Sarcophaga Meigen, 1826, in the family Sarcophagidae. Puparia of Sarcophagidae are bigger than the Calliphoridae ones and are characterized by having the posterior spiracles hidden in a crateriform-shaped cavity located in the upper part of the last abdominal segment. The distribution and shape of the spiracle slides and the open peritreme are also diagnostic characters for the family identification (Fig. 4).

Sarcophagidae family includes about 2,600 species worldwide distributed (Pape et al., 2009). Sarcophaga females are larviparous and directly deposit first instar larvae on carrions and cadavers. They usually arrive at corpses slightly later than the Calliphoridae and are able to colonize exposed and buried remains (Pastula & Merritt, 2013; Lo Pinto et al., 2017; Bugelli et al., 2015). In Italy the species of this genus are reported being active during the warmer season of the year (Bugelli et al., 2015; Lo Pinto et al., 2017; Vanin et al., 2008). Only few Sarcophagidae records, mainly undetermined species, refer to archaeological contexts (Huchet, 2010; Huchet et al., 2013b).

The majority of the analysed puparia were smaller than the Calliphoridae ones and showed respiratory horns, a characteristic anal plate and spiracles weakly protruding from the posterior region with three subparallel slits (Fig. 5). All these characters allow the identification of the puparia as belonging to the Muscidae fly, Hydrotaea capensis (Wiedemann, 1818). Puparia of H. capensis share several features with the very similar puparia of Hydrotaea aenescens (Wiedemann, 1830), species introduced in Europe from South America after the XV century and widely distributed in the Mediterranean region. An accurate analysis of the posterior spiracle slits and of the anal plate allows the distinction of the two species (Skidmore, 1985).

Hydrotaea capensis is a synantropic species known from a wide variety of habitats, except for the arid ones (Grzywacz et al., 2017). The species was reported from different archaeological and forensic contexts all over the Europe (France, Portugal, Italy, Germany, Spain, etc.). In exposed conditions, Hydrotaea capensis is present during the advanced stages of decomposition whereas with buried or concealed bodies it tends to be one of the first to colonize (Greenberg, 1991; Greenberg & Kunich, 2002; Huchet et al., 2013a; Smith, 1986).

In archaeological context the species was reported from mummified bodies in religious burials (Couri et al., 2009; Morrow et al., 2015; Vanin, 2012) and from WWI battlefields (Huchet et al., 2013a).

Lepidoptera

Few moth cocoons were also isolated from the entomological samples (Fig. 6, Table 1). Because of their shape, structure and composition they were identified as belonging to the family Tineidae. The most common species of this family associated with carrion but as well with biodegradation of textiles, with some implications as well in cultural heritage conservation, are Tinea pellionella (Linnaeus, 1758) and Tineola bisseliella (Hummel, 1823). These species are typical of the last phases of the human decomposition when they can feed on dry tissues and hair (Smith, 1986; Mazzarelli et al., 2015).

Coleoptera

A single specimen of beetle in the family Histeridae and a single elytron, potentially of a species in the family Tenebrionidae, were also present (Table 1). The hister beetle (Fig. 7) was identified as Saprinus (Saprinus) semistriatus (Scriba, 1790). This species with a Palearctic distribution was reported also from India and Taiwan and is commonly found under animal carcasses and decomposing fishes, manure heaps and rubbish dumps (Vienna, 1980). The species was reported from small and big animal carrion in Europe where it represents the most abundant taxon among Histeridae species during the spring and summer seasons (Bajerlein, Matuszewski & Konwerski, 2011; Kočárek, 2003). In Northern Italy, it was reported from burned and unburned pig carrions during the summer period but not in the winter season (Vanin et al., 2013). Matuszewski & Szafalowicz (2013) found that S. semistriatus in addition to Thanatophilus sinuatus Linnaeus, 1758, Necrodes littoralis (Linnaeus, 1758), Necrobia rufipes (De Geer, 1775), Necrobia violacea (Linnaeus, 1758), Creophilus maxillosus (Linnaeus, 1758), Philonthus politus (Linnaeus, 1758), Saprinus planiusculus Motschulsky, 1849 and Margarinotus brunneus (Fabricius, 1775) can be used to estimate the pre-appearance interval (PAI)—the time before the appearance of a species on the cadaver/carrion—from temperature. This approach can be used because S. semistriatus shows a close relationship between PAI and the environmental temperature.