Key for the identification of third instar larvae of African blowflies (Diptera: Calliphoridae) of forensic importance in death investigations.

Blowfly larvae are the insects primarily responsible for the active stage of decomposition of exposed vertebrate remains and are the most frequently collected entomological evidence during forensic investigations of death. The necrophagous calliphorids in continental Africa that consistently develop on large vertebrate carrion include 11 species belonging to four genera: Calliphora, Chrysomya, Hemipyrellia and Lucilia. Most of these species are widespread in Africa and frequently reported on large animal carcasses and carrion and human corpses. A few keys have been compiled for identification of their third instar larvae, but none of them covers the complete set of taxa. Therefore, we provide a new comprehensive key with original illustrations of all taxonomically significant characters. The key is based on characters that should be easily observable even in poorly equipped local laboratories and is a reliable taxonomic tool for material collected in either urban or rural areas where synanthropic species predominate. However, it should be used with some caution in areas with relatively pristine natural habitats, where additional carrion-breeding species may occur. The publication of the key will significantly facilitate both medical and forensic entomological research and practice in Africa.

Larval morphology of the avian parasitic genus Passeromyia: playing hide and seek with a parastomal bar.

The enigmatic larvae of the Old World genus Passeromyia Rodhain & Villeneuve, 1915 (Diptera: Muscidae) inhabit the nests of birds as saprophages or as haematophagous agents of myiasis among nestlings. Using light microscopy, confocal laser scanning microscopy and scanning electron microscopy, we provide the first morphological descriptions of the first, second and third instar of P. longicornis (Macquart, 1851) (Diptera: Muscidae), the first and third instar of P. indecora (Walker, 1858) (Diptera: Muscidae), and we revise the larval morphology of P. heterochaeta (Villenueve, 1915) (Diptera: Muscidae) and P. steini Pont, 1970 (Diptera: Muscidae). We provide a key to the third instar of examined species (excluding P. steini and P. veitchi Bezzi, 1928 (Diptera: Muscidae)). Examination of the cephaloskeleton revealed paired rod-like sclerites, named 'rami', between the lateral arms of the intermediate sclerite in the second and third instar larva. We reveal parastomal bars fused apically with the intermediate sclerite, the absence of which has so far been considered as apomorphic for second and third instar muscid larvae. Examination of additional material suggests that modified parastomal bars are not exclusive features of Passeromyia but occur widespread in the Muscidae, and rami may occur widespread in the Cyclorrhapha.

Wing measurements are a possible tool for the identification of European forensically important Sarcophagidae.

The flesh flies are a group of insects well known for their forensic importance. Reliable identification of these flies relies on the use of either molecular markers or the morphology of the male genital apparatus. Identification of female flesh flies is more time consuming and less reliable than their male counterparts. This is particularly problematic for forensic entomology practitioners, because female flesh flies are more abundant than males in carrion arthropod assemblages. As such, it is critical that alternative methods for flesh fly identification are established that are equally effective for both sexes. One promising technique is the use of wing measurements, which have been shown to be reliable for the identification of some groups of necrophagous Diptera from several geographical regions. We applied this method to the European Sarcophagidae for the first time, using a dataset of 881 specimens representing 29 species and 5 genera. Species identifications were based on 15 landmarks located at wing vein junctions. We also combined our results with data from previous studies of Calliphoridae and Muscidae which utilised the same methodology, enabling the testing of family level identification using wing morphometrics. Species identifications using wing measurements had varied success. While some species were successfully identified without error, others, particularly from the genus Sarcophaga, were often misclassified. Notably, in several species wing measurements successfully identified both males and females. The results presented here suggest that wing measurements are a promising complementary method to other methods for the identification of necrophagous Sarcophagidae especially in material unsorted at the family level. It can also be used to double check identification performed by a taxonomist using traditional methods.

Linking bacteria, volatiles and insects on carrion: the role of temporal and spatial factors regulating inter-kingdom communication via volatiles.

Multi-kingdom community complexity and the chemically mediated dynamics between bacteria and insects have recently received increased attention in carrion research. However, the strength of these inter-kingdom interactions and the factors that regulate them are poorly studied. We used 75 piglet cadavers across three forest regions to survey the relationship between three actors (epinecrotic bacteria, volatile organic compounds (VOCs) and flies) during the first 4 days of decomposition and the factors that regulate this interdependence. The results showed a dynamic bacterial change during decomposition (temperature-time index) and across the forest management gradient, but not between regions. Similarly, VOC emission was dynamic across a temperature-time index and the forest management gradient but did not differ between regions. However, fly occurrence was dynamic across both space and time. The strong interdependence between the three actors was mainly regulated by the temperature-time index and the study regions, thereby revealing regulation at temporal and spatial scales. Additionally, the actor interdependence was stable across a gradient of forest management intensity. By combining different actors of decomposition, we have expanded our knowledge of the holistic mechanisms regulating carrion community dynamics and inter-kingdom interactions, an important precondition for better describing food web dynamics and entire ecosystem functions.

Host-trailing satellite flight behaviour is associated with greater investment in peripheral visual sensory system in miltogrammine flies.

Insect sensory systems are the subjects of different selective pressures that shape their morphology. In many species of the flesh fly subfamily Miltogramminae (Diptera: Sarcophagidae) that are kleptoparasitic on bees and wasps, females perch on objects close to the host nests and, once a returning host is detected, they follow it in flight at a fixed distance behind until reaching the nest. We hypothesized that such satellite (SAT) flight behaviour, which implies a finely coordinated trailing flight, is associated with an improved visual system, compared to species adopting other, non-satellite (NON-SAT) strategies. After looking at body size and common ancestry, we found that SAT species have a greater number of ommatidia and a greater eye surface area when compared to NON-SAT species. Ommatidium area is only affected by body size, suggesting that selection changes disproportionately (relative to body size variation) the number of ommatidia and as a consequence the eye area, instead of ommatidium size. SAT species also tend to have larger ocelli, but their role in host-finding was less clear. This suggests that SAT species may have a higher visual acuity by increasing ommatidia number, as well as better stability during flight and motion perception through larger ocelli. Interestingly, antennal length was significantly reduced in SAT species, and ommatidia number negatively correlated with antennal length. While this finding does not imply a selection pressure of improved antennal sensory system in species adopting NON-SAT strategies, it suggests an inverse resource (i.e. a single imaginal disc) allocation between eyes and antennae in this fly subfamily.

Evolution of sexual conflict in scorpionflies.

Sexual conflict - opposite reproductive/genetic interests between sexes - can be a significant driver of insect evolution. Scorpionflies (Insecta: Mecoptera) are models in sexual conflict research due to their large variety of mating practices, including coercive behaviour and nuptial gift provisioning. However, the role of palaeontology in sexual conflict studies remains negligible, namely due to the paucity of well-preserved fossils. Here, we describe three male scorpionflies from Cretaceous and Eocene ambers. The structure of notal and postnotal organs is analysed in extant and extinct forms; a depression below the base of the notal organ in different panorpid species spatially matches the anterior fold of the female's wing. Based on disparate abdominal configurations and correlations in extant relatives, we posit that each new fossil taxon had a different mating approach along a nuptial gifting-coercive spectrum. The Eocene specimen possesses extreme female clamping abdominal armature, suggesting a degree of sexual coercion greater than in any other known scorpionfly, extinct or extant. The fossil record of abdominal modifications in male scorpionflies documents a relatively late evolution (Eocene) of long notal organs indicating oppressive behaviour toward a female during mating. Our findings reveal a wider array of mating-related morphological specialisations among extinct Panorpoidea, likely reflecting more diversified past mating strategies and behaviours in this group, and represent first steps towards gaining a deep-time perspective on the evolution of sexual conflict over mating among insects.

Integrative taxonomy reveals remarkable diversity in Australian Protomiltogramma (Diptera: Sarcophagidae).

Protomiltogramma Townsend is the largest and most diverse genus of miltogrammine flesh flies in Australia. However, no comprehensive taxonomic work had been completed on the Australian members of this genus in almost a century. This study presents the first taxonomic revision of all Australian species of Protomiltogramma (Sarcophagidae: Miltogramminae), completed using an integrative approach combining molecular and morphological data. Eight new species endemic to Australia are described: P. dalbiensis sp. n., P. grandis sp. n., P. incana sp. n., P. kapnos sp. n., P. nigrisensa sp. n., P. popularis sp. n., P. rubra sp. n. and P. subtilis sp. n. In addition, P. australis Malloch, 1930 is synonymised with P. cincta Townsend, 1916, syn. n. and P. mallochi Verves, 1987 is synonymised with P. laticeps Malloch, 1930, syn. n. Molecular phylogenetics is used to place the Australian Protomiltogramma among the miltogrammine genera of the world.

DNA Barcoding Identifies Unknown Females and Larvae of Fannia R.-D. (Diptera: Fanniidae) from Carrion Succession Experiment and Case Report.

Application of available keys to European Fanniidae did not facilitate unequivocal species identification for third instar larvae and females of Fannia Robineau-Desvoidy, 1830 collected during a study of arthropod succession on pig carrion. To link these samples to known species, we took the advantage of molecular identification methods and compared newly obtained cytochrome oxidase subunit I (COI) barcode sequences against sequences deposited in reference databases. As an outcome of the results obtained, we describe for the first time a third instar larva of Fannia nigra Malloch, 1910 and Fannia pallitibia (Rondani, 1866) and a female of Fannia collini d'Assis-Fonseca, 1966. We provide combinations of characters allowing for discrimination of described insects from other Fanniidae. We provide an update for the key by Rozkosný et al. 1997, which allows differentiation between females of F. collini and other species of Fanniidae. Additionally, we provide a case of a human cadaver discovered in Southern Poland and insect fauna associated with it as the first report of F. nigra larvae developing on a human body.

New Species of Soldier Fly-Sargus bipunctatus (Scopoli, 1763) (Diptera: Stratiomyidae), Recorded from a Human Corpse in Europe-A Case Report.

The only European Stratiomyidae species known for feeding on human corpses was the black soldier fly Hermetia illucens (Linnaeus, 1758). Analysis of fauna found on a human corpse, discovered in central Poland, revealed the presence of feeding larvae of another species from this family: the twin-spot centurion fly Sargus bipunctatus (Scopoli, 1763). The investigated corpse was in a stage of advanced decomposition. The larvae were mainly observed in the adipocere formed on the back and lower limbs of the corpse, and in the mixture of litter and lumps of adipocere located under the corpse. Adult specimens and larvae were identified based on morphological characters, and final identification was confirmed using DNA barcoding. Implementing a combination of morphological and molecular methods provided a reliable way for distinguishing the larvae of S. bipunctatus and H. illucens. The potential of S. bipunctatus for practical applications in forensic entomology is currently difficult to assess. Wide and reliable use of S. bipunctatus in the practice of forensic entomology requires further studies of the bionomy of this fly.

First instar larvae of endemic Australian Miltogramminae (Diptera: Sarcophagidae).

The first instar larva of a species of the Australian endemic genus Aenigmetopia Malloch is described for the first time, along with the first instar larvae of three other Australian species representing the genera Amobia Robineau-Desvoidy and Protomiltogramma Townsend. Larval morphology was analysed using a combination of light microscopy, confocal laser scanning microscopy and scanning electron microscopy. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, modifications of thoracic and abdominal segments, anal region, spiracular field, posterior spiracles and details of the cephaloskeleton. Substantial morphological differences are observed between the three genera, most notably in the labrum and mouthhooks of the cephaloskeleton, sensory organs of the pseudocephalon, spinulation, sculpture of the integument and form of the spiracular field. The first instar larval morphology of Aenigmetopia amissa Johnston, Wallman, Szpila & Pape corroborates the close phylogenetic affinity of Aenigmetopia Malloch with Metopia Meigen, inferred from recent molecular analysis. The larval morphology of Amobia auriceps (Baranov), Protomiltogramma cincta Townsend and Protomiltogramma plebeia Malloch is mostly congruent with the morphology of Palaearctic representatives of both genera.

Life History of Oxybelus variegatus Wesmael, 1852 (Hymenoptera: Crabronidae) with a Description of the Mature Larva.

The first comprehensive information on the bionomics of the digger wasp Oxybelus variegatus Wesmael, 1852 is presented. Females nested in small aggregations in crevices between paving stones of a frequently used pedestrian pathway in lowland agricultural wasteland. Nests were dug in the ground using mandibles, legs and abdomen. The nest consists of a main burrow with one or, rarely, two cells. The mature larva is described for the first time. The egg stage lasts for about two days before the larva hatches. The female provisioned each cell with an average of 11 paralysed male flies of Delia platura (Meigen, 1826) (Diptera: Anthomyiidae). Numerous females of dipteran kleptoparasites were observed in the nesting area of O. variegatus. However, only a few nests were infested by larvae of Senotainia conica (Fallen, 1810).

Massive Orbital Myiasis Caused by Sarcophaga argyrostoma Complicating Eyelid Malignancy.

Purpose. To report a case of massive orbital myiasis caused by the larvae of Sarcophaga argyrostoma, complicating eyelid malignancy. Observations. A 98-year-old man first presented to our clinic noted to have a fast-growing lesion on his right upper and lower eyelids. Squamous cell carcinoma of the eyelids was highly suspected, and surgical excision was advised, but the patient refused any surgical or nonsurgical intervention. For the next eight months, the patient's family members continued to observe a high rate of tumor growth accompanied by deterioration of the general condition. During this whole period, the patient rejected admission to the hospital and was observed by nursing home staff. He was admitted to the emergency room in cachexic, unresponsive condition with fetid discharge and multiple live maggots crawling out from a large necrotic mass over the right orbit. On examination, no eyelids, eyeball, or other ocular tissue could be seen, while an extension of necrotic mass to forehead and midcheek was noted. Manual removal of larvae was performed. The patient passed away eight hours after his admission and larval removal. The maggots were identified as the third-instar larvae of Sarcophaga argyrostoma. Conclusions and Importance. This is the first reported case of home-acquired, massive orbital myiasis by S. argyrostoma. This case illustrates the crucial role of fly control as part of medical and home care in immobile patients. Moreover, it shows the importance of awareness by nursing home staff, paramedical, and medical personnel of possible myiasis, especially in bed-bound patients with skin malignancies and open wounds.

Redescription of Sphecapatoclea excisa Villeneuve, 1909 (Diptera: Sarcophagidae).

Sphecapatoclea excisa Villeneuve, 1909, the type species of the Palaearctic genus Sphecapatoclea Villeneuve, 1909, is redescribed based on a female syntype and on material from Makhtesh Ramon National Park, Israel, and its first instar larva is described for the first time. The species is sexually dimorphic, with much darker adult males. The male genital apparatus is unique by its compressed and sclerotised epiphallus. The morphology of the first instar larva is in accordance with the recently suggested position of the genus Sphecapatoclea in the Old World clade of the "lower" Miltogramminae. Two COI mini-barcodes are provided for S. excisa, and molecular data are in agreement with sequences for Sphecapatoclea spp. available in GenBank. Morphology supports a broad concept of the genus, as S. excisa presents a mixture of character states traditionally used to diagnose either Sphecapatoclea (s. str.) or Parthomyia Rohdendorf, 1925. Available morphological keys for genera of Palaearctic Miltogramminae are compared for functionality, and possible autapomorphies from both adult and larval morphology are discussed.

Muscle attachment site patterns for species determination in West Palaearctic Wohlfahrtia (Diptera: Sarcophagidae) of medical and veterinary importance.

The flesh fly genus Wohlfahrtia Brauer & Bergenstamm contains at least six species of medical and veterinary importance. Traditional methods of species identification in specimens of Wohlfahrtia, however, are restricted mostly to adult forms. Muscle attachment site (MAS) patterns allow for species determination in larval forms. MAS patterns in third instar larvae of six common West Palearctic species of Wohlfahrtia have been analyzed for this study. As in previously investigated Calliphoridae and Sarcophagidae, MAS patterns were found to be species specific. A genus pattern was established to be used as base for comparison in further species determination. For the first time a tool is provided for species identification of such broad range in larvae of Wohlfahrtia species. Wohlfahrtia patterns are composed of a significantly higher number of MAS than patterns found in Sarcophaga. Specifics of the six species analyzed are explained in detail. The larvae of the well-known species W. magnifica, an obligate traumatic myiasis agent, had to be excluded from the analysis as a great number of spines on the outside obscure muscle attachment sites on the inside of the cuticle.

Correction to: A formulation of neem and hypericum oily extract for the treatment of the wound myiasis by Wohlfahrtia magnifica in domestic animals.

The given names and family names of the authors of the originally published version of this article was inadvertently interchanged. Correct presentation of the author names are presented here.

A formulation of neem and hypericum oily extract for the treatment of the wound myiasis by Wohlfahrtia magnifica in domestic animals.

Untreated, traumatic, or wound myiases can cause severe consequences to animal health and welfare as well as economic losses to livestock productions. For healing myiasis-caused wounds, disinfectant such as creolin is wrongly but currently used in association with insecticides. Though effective, creolin is highly toxic to the patients, is inadequate with respect to the repellent effect, and may delay the healing of treated wounds. In this uncontrolled study, the efficacy of the patented plant-derived formulation 1 Primary Wound Dressing© (1-PWD), composed of neem oil (Azadirachta indica A. Juss.) and the oily extract of Hypericum perforatum (L.) flowers, has been investigated. Forty-four domestic animals of different species suffering from wound myiasis lasting for up to 25 days, at different parts of the body, were enrolled in the study. No systemic or local antibiotic or disinfectants' treatment was administered. Larvae recovered on open wounds and adults reared from mature larvae were identified as Wohlfahrtia magnifica. All the treated wounds healed in a range of 10 to 32 days without further infestation. None of the recruited animals presented bacterial complications. Data herein presented indicate that the tested natural plant-derived formulation is able to manage the infestation caused by W. magnifica larvae and the healing process of traumatic infested wounds in several domestic animal species, without any side effect on the living tissue and without the need to use local or systemic chemical or other products.

Wing measurement can be used to identify European blow flies (Diptera: Calliphoridae) of forensic importance.

Correct species identification is a crucial step in research and the practice of forensic entomology. At the moment the most common and widely used tool for this purpose are taxonomic keys based on morphological characters. During recent decades molecular methods have been used more and more often for species identification. Automated species identification can also be based on wing measurements. This method proved to be reliable in the case of some groups of necrophagous Diptera from various geographical regions. Up to now, this method has not been applied to a wide range of species of European Calliphoridae. We have used 968 specimens representing 15 species and 6 genera. Identification was based on 15 landmarks located at wing vein junctions. Wing shape differed markedly between the studied species. The overall correct classification rate at the level of species was 97.1%. Seven species, including Calliphora vomitoria, Chrysomya albiceps, Phormia regina, Protophormia terraenovae and Lucilia sericata were identified without error. Lucilia caesar and Lucilia illustris, which are considered relatively difficult to distinguish, were correctly classified in 93.0% and 94.1% cases, respectively. The correct classification rate at the level of genera was 99.2%. The results presented here suggest that wing measurements are a promising alternative to other methods of identification of necrophagous Calliphoridae. This method can be particularly useful for identification of large samples of females collected during carrion succession studies. It can also be used to double check identification performed by a taxonomist using traditional methods.

Species identification of adult African blowflies (Diptera: Calliphoridae) of forensic importance.

Necrophagous blowflies can provide an excellent source of evidence for forensic entomologists and are also relevant to problems in public health, medicine, and animal health. However, access to useful information about these blowflies is constrained by the need to correctly identify the flies, and the poor availability of reliable, accessible identification tools is a serious obstacle to the development of forensic entomology in the majority of African countries. In response to this need, a high-quality key to the adults of all species of forensically relevant blowflies of Africa has been prepared, drawing on high-quality entomological materials and modern focus-stacking photomicroscopy. This new key can be easily applied by investigators inexperienced in the taxonomy of blowflies and is made available through a highly accessible online platform. Problematic diagnostic characters used in previous keys are discussed.

Molecular phylogeny of Miltogramminae (Diptera: Sarcophagidae): Implications for classification, systematics and evolution of larval feeding strategies.

Miltogramminae is one of the phylogenetically most poorly studied taxa of the species-rich family Sarcophagidae (Diptera). Most species are kleptoparasites in nests of solitary aculeate wasps and bees, although parasitoids and saprophagous species are also known, and the ancestral miltogrammine life habit remains unsettled. Here, we present for the first time a comprehensive phylogenetic tree consisting of 58 representatives of Miltogramminae, reconstructed using sequence data from three mitochondrial (COI, cytB, ND4) and one nuclear (Ef-1α) genes. Our phylogenetic hypothesis suggests that: (1) Miltogramminae are sister to Paramacronychiinae, (2) Miltogramminae can be divided into the "lower miltogrammines" containing two clades of mainly saprophages and a clade of "higher miltogrammines" with mainly kleptoparasitic species, (3) only three genera turn out to be non-monophyletic: Miltogramma, Senotainia and Pterella and (4) the genus Sarcotachina, which traditionally has been considered as belonging to the Paramacronychiinae, is placed in one of the clades of "lower miltogrammines". Ancestral state reconstruction of larval feeding strategy and five larval characters reveals that the ancestor of Miltogramminae was likely a saprophage retaining plesiomorphic oral ridges and a cephaloskeleton with sclerotized dorsal bridge. Synapomorphies like large pseudocephalic sensory organs and well-developed cuticular sculpture suggest that the ancestral first instar larva actively searched for a buried food supply.

Wound Myiasis Caused by Sarcophaga (Liopygia) Argyrostoma (Robineau-Desvoidy) (Diptera: Sarcophagidae): Additional Evidences of the Morphological Identification Dilemma and Molecular Investigation.

In Mediterranean countries, Sarcophaga (Liopygia) crassipalpis, Sarcophaga (L.) argyrostoma, and Sarcophaga (L.) cultellata share the same ecological niche and can be responsible of myiasis. In this study, the main morphological characters of a larva found in a hospitalized woman were described and illustrated by light and SEM microscopy and the features discussed. Then, a fragment within the mitochondrial encoded cytochrome c oxidase subunit I (coxI) gene of ~735 bp was amplified and sequenced. The molecular investigation was necessary to confirm the species Sarcophaga (Liopygia) argyrostoma (99% of identity). Our findings showed that morphological descriptions of larvae of three Mediterranean species of Liopygia available in several papers might not be clear enough to allow for comparison and correct identification. Until results of reliable comparative studies of larvae of all three species will be available, the use of molecular tools is crucial, to avoid misleading or incomplete identification, and in particular when a myiasis becomes a legal issue.