The Sarcophagidae (Diptera) of Kermanshah, Western Iran, with the first record of Sarcophaga (Helicophagella) bellae Lehrer from Iran

Introduction

The flesh fly family, Sarcophagidae, is one of the most diverse families in the order Diptera. Systematically, Sarcophagidae belongs to the Calyptrata group within the Schizophora, infraorder Cyclorrhapha (Moscomorpha), and suborder Brachycera (Pape et al., 2011; Buenaventura & Pape, 2013; Khosravi et al., 2024). Members of this family are relatively large compared to the housefly, Musca domestica Linnaeus, 1758, with a body coloration that is predominantly gray, especially in the genus Sarcophaga Meigen, 1826. Like many families within Cyclorrhapha, Sarcophagidae exhibits diverse physiological and developmental strategies. Notably, their feeding behaviors underscore their medical, veterinary, and forensic importance (Byrd, 2002). Sarcophagidae flies deposit either first-instar larvae or eggs, depending on the availability of resources (Hediyeloo et al., 2024). They typically follow the family Calliphoridae as the second wave of colonizers on carcasses (Haskell, 1997). The maggots primarily feed on cadavers or exposed meat, but some also consume excrement in varied ecological niches. Feeding behaviors can be species-specific, with certain species exhibiting indoor or outdoor activity preferences while others are active in decomposing buried bodies. In a previous study from Tehran, Sarcophaga argyrostoma Robineau-Desvoidy, 1830 maggots were collected exclusively from human cadavers, indicating an indoor association for this species (Haskell, 1997; Talebzadeh et al., 2017; Jafari et al., 2019, Mirzakhanlou et al., 2025).

Alongside postmortem interval estimation, determining the place of death can be a valuable contribution of entomological data to forensic investigations, sometimes providing unique clues for cadaver tracking. Forensic entomologists can identify region-specific species as biomarkers, which may indicate geographical origins and support the repositioning of cadavers (Matuszewski et al., 2013). Some fly species are restricted to specific regions or environmental conditions; for example, Chrysomya marginalis Wiedemann, 1830 (Diptera: Calliphoridae) and Sarcophaga ruficornis Fabricius, 1794 (Diptera: Sarcophagidae) have been recorded only in southern Iran (Sanei-Dehkordi et al., 2020). A comprehensive database documenting fly diversity by locality is essential for accurate cadaver relocation analysis. The Sarcophagidae family comprises around 2,828 species across 118 genera and 221 subgenera (Pape et al., 2025). These flies are medically significant due to their potential to cause myiasis in humans and animals (Najjari et al., 2020; Ren et al., 2013). In Iran, human myiasis is considered an occupational disease (Akbarzadeh et al., 2012; Alizadeh et al., 2014), with 68 species of the genus Sarcophaga reported. The diversity of this family remains unexplored in many parts of Iran, suggesting the possibility of new species records for the Iranian fly fauna (Akbarzadeh et al., 2018).

Various methods are available for identifying Sarcophagidae flies, with male genitalia being the most critical morphological characteristic (Szpila et al., 2015; Brown, 2009). Molecular markers, due to their reliability, cost-effectiveness, and ease of use, have become popular tools for species identification, genetic analysis, phylogenetic studies, and forensic investigations. These molecular techniques are now used to support the morphological identification of Sarcophagidae species (Jafari et al., 2019; Talebzadeh et al., 2020). While some studies on necrophagous flies in Iran exist (Jafari et al., 2019), the fauna in the western parts of the country remains undocumented. This study investigates the diversity of the Sarcophagidae family in Kermanshah city, Kermanshah province, Western Iran. As part of this effort, we document the species Sarcophaga bellae as a new record for the Iranian Sarcophagidae diversity database.

Materials and methods

Study site: Kermanshah Province, Kermanshah City

Kermanshah (34.31° N, 47.07° E, avg. elev. 1,391 m) is the largest city in western Iran and the capital of Kermanshah province (Fig. 1). Located along the Qarahsoo River, it lies between Farokhshad and Paraw Mountains (north), Teq Bostan (northwest), and Sefidkuh (south). The region borders Kurdistan (north), Ilam and Lorestan (south), Hamedan (east), and Iraq (west). Kermanshah experiences hot, arid summers and cold, partly cloudy winters, with temperatures ranging from -3°C to 38°C, rarely falling below -8°C or exceeding 40°C.

Fig. 1. Map of districts in Kermanshah, western Iran, surveyed for Sarcophagidae fly collection in 2018.

Sample Collection 

Members of the family Sarcophagidae were collected using a standard fly net from three regions of Kermanshah city during the summer of 2018 (Table 1, Fig. 1). Samples were collected bi-weekly over a three-month period. To maintain consistency, all collections were conducted by the same researcher. Each specimen was placed in a 1.5 mL microtube with a small hole in the lid to allow air circulation, preserving the flies’ morphological integrity until arrival at the laboratory. Specimens were transported to the lab at the end of each collection session. Male flies were killed by freezing, after which their external genitalia were dissected. Species identification was performed by comparing the morphology of genital structures with previously validated keys (Pape, 1996; Povolny, 1997; Pape, 2025). Some specimens were mounted on pins and stored in collection boxes, while others were preserved for molecular identification. Fresh samples were stored in 70% ethanol at -20°C prior to DNA extraction (Jafari et al., 2019).

DNA Extraction and PCR 

Among the 35 S. bellae specimens, three males were randomly selected for molecular analysis, focusing on the COI gene. Genomic DNA was extracted from the thoracic muscles of identified adult male sarcophagid specimens using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The DNA was resuspended in 30 µl of TE buffer (1 mM Tris–HCl, pH 8; 0.1 mM EDTA) and stored at 4°C. The COI gene was amplified with the primer pair LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al., 1994) following the thermal cycling conditions described by Hashemi-Aghdam et al. (2017), yielding a product of approximately 711 bp. PCR products were visualized on a 1.2% agarose gel stained with ethidium bromide under a UV transilluminator. All PCR reactions were conducted in a total volume of 25μL, using the Taq DNA Polymerase 2x Master Mix RED, Ampliqon (Denmark), with the following reagents: 1–2μL of DNA extract (10–25 ng), 12.5μL of Master mix, 1μL of each primer (10mM), and 8.5–9.5μL of sterile water. For COI regions, the PCR thermal profile was 2 min at 94°C, followed by 5 cycles of 94°C (30s), 45°C (40s), and 72°C (60s), and then 35 cycles of 94°C (30s), 51°C (40s), and 72°C (60s), with a final extension at 72°C for 10 min.

PCR amplicons from the three male specimens, which were initially identified morphologically, were purified using the MRN-10 GenElute PCR Purification Kit to remove unincorporated dNTPs and primers. Briefly, the protocol involved binding DNA to a spin column, washing with a supplied buffer, and eluting the purified DNA in nuclease-free water. The purified products were sequenced on an automated sequencer (Seqlab, Goettingen, Germany). Bidirectional sequences were edited with ChromasPro (Version, 1.33; www.technelysium.com.au/ChromasPro.html) and aligned using ClustalW2 (Larkin, 2007). Homology was assessed using the FASTA search tool (http://www.ebi.ac.uk/fasta33/). The consensus COI sequences were submitted to GenBank, with unambiguous sequences trimmed to a length of 635 to 673 bp.

Phylogenetic Analysis

Due to the identical sequences of the three specimens obtained in this study, a single sequence was used as a representative for Iranian S. bellae. This representative sequence was combined with 44 previously reported sarcophagid sequences from GenBank (Table 2). Chrysomya albiceps (GenBank ID: MW200912) was designated as the outgroup. Phylogenetic analysis was conducted using MEGA X software (version 11; Kumar et al., 2018). Trees were reconstructed using Maximum Likelihood (ML) method. The evolutionary distances were computed using the Kimura 2-parameter (K2P) model (Kimura, 1980).

Results

Morphological Species Identification

In this study, 142 male flesh fly specimens from various parts of the city were examined, comprising eight Sarcophagidae species identified using taxonomic keys specific to sarcophagid males (Table 3). Sarcophaga variegata Scopoli, 1763 was the most commonly observed species in the area. Identifications were based on sarcophagid morphological characteristics, focusing on male genitalia, which were the only reliable feature for species-level identification. Among the specimens, 35 were identified as Sarcophaga bellae Lehrer, 2000. The theme of the adults is gray with obvious checker pattern on abdomen and three conspicuous dark strips on the thorax. The adult can be categorized as big Sarcophagidae with a length about 8 – 12 mm. All parts the external genitalia such as epandrium, cercus, surstylus and phallic tube are blackish brown and have condensed hairs. The aedeagous is curved with a circular end (Z).

Sequence Analysis

 We successfully amplified and sequenced a 711 bp fragment of the COI gene of the three selected male specimens, obtaining reliable sequences of approximately 635, 668, and 673 bp, which were deposited in GenBank (accession numbers OL589153–OL589155). The average A+T content for the COI region was 68.5%. Blast search confirmed a 100% identity between the Iranian S. bellae specimens and S. bellae from Türkiye (GenBank ID: KU746540).

Phylogenetic Relationships

With a few exceptions, phylogenetic analysis using either Neighbor-Joining or Maximum Likelihood produced similar topology and grouped the Sarcophagidae species into distinct clades corresponding to their taxonomic series or subgenera (Fig. 3). The Iranian S. bellae formed a clade with S. bellae from Türkiye, and this combined group was sister to a clade containing S. noverca, S. rosellei, and S. agnata, all within the Helicophagella subgenus.

Discussion

This study documented eight Sarcophaga species in Kermanshah city; all newly recorded for this area in western Iran. These findings emphasize the diversity of necrophagous flies in the region and highlight the influence of varied biogeographical zones on Iran’s fauna. Although Iran is part of the Eastern Mediterranean Region, it is classified within the southern Palearctic zone according to international biogeographical categorizations.

Fig. 2. General appearance (A) and male genitalia (B) of Sarcophaga (Helicophagella) bellae (Sarcophagidae: Diptera), collected in Kermashah, Iran, 2018.

Fig. 3: Maximum Likelihood phylogenetic tree of Sarcophaga species based on mtDNA-COI sequences (644 bp). The tree was reconstructed in MEGA X (Kumar et al., 2018) using the Kimura 2-parameter model (Kimura, 1980) and includes a representative Iranian Sarcophaga bellae sequence (highlighted with a red circle) along with 44 other Sarcophaga sequences from the GenBank. Chrysomya albiceps was used as the outgroup. Bootstrap values equal or greater than 25% are shown at the nodes. Taxonomic series (subgenera) are indicated on the right. The final dataset comprised 46 nucleotide sequences.

The country’s geographic diversity incorporates influences from both the Oriental zone (in the southeast) and the Afrotropical zone (in the south), contributing to a wide range of species, including those within Arthropoda. Such regional data on necrophagous fly diversity are essential for forensic entomological surveys, as they help establish local species baselines. Akbari et al. (2023) reported on the diversity of necrophagous flies in Ilam province, also in western Iran, using pooled data from various families such as Calliphoridae, Sarcophagidae, Muscidae, and Oestridae to calculate species diversity indices. The current study provides preliminary data specifically on the Sarcophaga genus (Diptera: Sarcophagidae) in Kermanshah, a province with high religious and historical tourism. This baseline data contributes to a broader understanding of regional fly fauna in Iran. Sarcophaga (Helicophagella) bellae (Lehrer, 2000) (Diptera: Sarcophagidae) is reported here for the first time among Iranian flesh fly fauna. This report expands the known distribution of S. bellae in the Palearctic region, extending it from Greece, the Middle East, and Türkiye (Verves & Khrokalo, 2018) to Iran. While Kermanshah does not border Türkiye, its close proximity suggests that S. bellae may have migrated from Türkiye into Iran and potentially into Iraq, which shares borders with both Türkiye and Iran. Given the lack of data on Iraqi Sarcophagidae, this finding of S. bellae in western Iran (east of Iraq) suggests that it may also be present in Iraq. Our findings significantly expand the known dipteran fauna of Kermanshah province. While S. bellae represents a new country record, the seven other species collected, though previously documented in other parts of Iran (Akbarzadeh et al., 2012, 2018; Sanei-Dehkordi et al., 2020; Motevalli Haghi et al., 2021), are all newly recorded for this province. This underscores the substantial gap in regional biodiversity data that this study begins to address.

The species assemblage we observed provides insight into the sarcophagid community in western Iran. The prevalence of widespread species like S. argyrostoma, S. africa, S. aegyptica, and the dominant S. variegata—all reported from diverse biogeographical zones across the country—suggests a degree of ecological generality. Their confirmed presence in Kermanshah aligns with their status as common members of Iran's sarcophagid fauna. Of particular forensic significance is S. argyrostoma, which to date remains the only species in Iran with confirmed activity on human corpses in indoor settings (Talebzadeh et al., 2017). The ubiquity of these species marks them as key forensically relevant taxa whose life-history patterns warrant further detailed study. In contrast, we also recorded species with more restricted distributions, such as S. flagellifera and S. crassipalpis. Their presence, previously noted primarily in southern provinces like Fars and Sistan and Baluchestan (Rafinejad et al., 2014; Nateghpour & Akbarzadeh, 2017), indicates a more specialized biogeographical pattern. Such species could become valuable forensic indicators for cases of cadaver relocation once a more comprehensive, nation-wide distribution database is established. Furthermore, our study adds Kermanshah to the known range of W. nuba, a species confirmed as a myiasis agent in other parts of the country (Akbarzadeh, 2012; Maleki Ravasan et al., 2012). This new record is crucial for understanding the potential public and animal health risks associated with this fly in western Iran. Finally, the discovery of S. bellae is not merely a new provincial record but elevates the total number of documented Sarcophaga species in Iran from 20 to 21 (Akbarzadeh et al., 2018). This highlights that even among a relatively well-studied family, significant taxonomic and distributional discoveries remain to be made, particularly in under-surveyed regions like Kermanshah.

The main problem for the low number of reported species of the genus Sarcophaga is the limited morphological characters for their identification. The only reliable character is the shape of male genital organ. In almost all studies on the fauna of Sarcophagidae flies, a noticeable number of samples were left unknown because of their gender (Szpila et al., 2015). Identification of female members of the family Sarcophagidae based on external morphology is almost impossible. Due to this issue, most precious samples especially in investigations in the field of forensic entomology, remain unusable (Babapour et al., 2016). In this study, about 17% of the collected flies of the genus Sarcophaga were female and unidentified. With sampling in various geographical conditions and approving new methods for identifying such as molecular methods, finding new records could be expected.  Cadaver displacement is one of the most important types of information that can be derived from the fauna of flies on the cadavers (Catts, 1992). Knowing the fauna of each locality is needed to locate the origin of flies found on cadavers (Fakoorziba et al., 2017). The results of this study revealed that S. bellae can be mentioned as representative of western regions of Iran.

Using molecular studies based on COI has been confirmed to be effective for many groups of insects (Oshaghi et al., 2007; Naddaf et al., 2012; Karimian et al., 2014; Sharma et al., 2015; Hashemi-Agdam et al., 2017; Koosha et al., 2017; Jafari et al., 2019; Khanzadeh et al., 2020) as well as members of family Sarcophagidae (Sharma et al., 2015; Talebzadeh et al., 2020). The 623 bp COI region sequence data used for phylogenetic analysis of the S. bellae species in this study showed this marker is a powerful molecular tool for better understanding of the species identification and its geographical distribution. Results of this study suggest that spatial distribution of S. bellae has been extended from Türkiye toward western parts of Iran. As a conclusion, this study represents the first documentation of S. (Helicophagella) bellae in Iran, expanding its known distribution in the Palearctic region to include western Iran. Given the geographical proximity of Kermanshah province to Iraq and Türkiye, this finding highlights the need for further studies in Iraq to clarify the species' full range. In addition to S. bellae, the study identified seven others Sarcophaga species, all newly recorded in Kermanshah province, underscoring the regional diversity of necrophagous flies and the influence of varied biogeographical zones on Iran’s fauna. The results further emphasize the utility of COI molecular markers for accurate species identification, particularly for forensic applications where species differentiation is crucial. Enhanced sampling and molecular techniques are likely to reveal additional species within this genus, aiding both biodiversity assessments and forensic investigations in the region.