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.

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.

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.

Muscidae (Diptera) of forensic importance-an identification key to third instar larvae of the western Palaearctic region and a catalogue of the muscid carrion community.

The Muscidae is one of the main dipteran families recognized as important for medico-legal purposes. Although an association of adult flies with decomposing human and animal bodies is documented for about 200 taxa worldwide, cadavers and carrion represents a breeding habitat for considerably fewer species. Species that do colonize dead human bodies can do so under diverse environmental conditions and, under certain circumstances, Muscidae may be the only colonizers of a body. Because of difficulties in identification, many studies have identified immature and/or adult muscids only to the genus or family level. This lack of detailed species-level identifications hinders detailed investigation of their medico-legal usefulness in carrion succession-oriented experiments. Identification to species level of third instars of Muscidae of forensic importance and the utility of larval morphological characters for taxonomic purposes were subjected to an in-depth revision. A combination of characters allowing for the discrimination of third instar muscids from other forensically important dipterans is proposed. An identification key for third instar larvae, which covers the full set of cadaver-colonising species of Muscidae from the western Palaearctic (Europe, North Africa, Middle East), is provided. This key will facilitate more detailed and species-specific knowledge of the occurrence of Muscidae in forensic entomology experiments and real cases. The carrion-visiting Muscidae worldwide are catalogued, and those species breeding in animal carrion and dead human bodies are briefly discussed with regard to their forensic importance.

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.

Effect of body mass and clothing on carrion entomofauna.

Carcass mass largely affects pattern and rate of carrion decomposition. Supposedly, it is similarly important for carrion entomofauna; however, most of its likely effects have not been tested experimentally. Here, simultaneous effects of carcass mass and clothing are analyzed. A factorial block experiment with four levels of carcass mass (small carcasses 5-15 kg, medium carcasses 15.1-30 kg, medium/large carcasses 35-50 kg, large carcasses 55-70 kg) and two levels of carcass clothing (clothed and unclothed) was made in a grassland habitat of Western Poland. Pig carcasses (N = 24) were grouped into spring, early summer, and late summer blocks. Insects were sampled manually and with pitfall traps. Results demonstrate that insect assemblages are more complex, abundant, and long-lasting on larger carcasses, whereas clothing is of minor importance in this respect. Only large or medium/large carcasses were colonized by all guilds of carrion insects, while small or medium carcasses revealed high underrepresentation of late-colonizing insects (e.g., Cleridae or Nitidulidae). This finding indicates that carcasses weighing about 23 kg-a standard in forensic decomposition studies-give an incomplete picture of carrion entomofauna. Residencies of all forensically relevant insects were distinctly prolonged on larger carcasses, indicating that cadaver mass is a factor of great importance in this respect. The pre-appearance interval of most taxa was found to be unrelated to mass or clothing of a carcass. Moreover, current results suggest that rate of larval development is higher on smaller carcasses. In conclusion, this study demonstrates that carcass mass is a factor of crucial importance for carrion entomofauna, whereas the importance of clothing is small.

Muscle attachment site (MAS) patterns for species determination in five species of Sarcophaga (Diptera: Sarcophagidae).

To further establish species determination using the muscle attachment site (MAS) pattern method, third instar larvae of five forensically important species of Sarcophaga Meigen were investigated: Sarcophaga argyrostoma (Robineau-Desvoidy), Sarcophaga caerulescens Zetterstedt, Sarcophaga melanura Meigen, Sarcophaga albiceps Meigen and Sarcophaga similis Meade. As in the previously investigated Calliphoridae, patterns were found to be species specific. The main feature of the Sarcophaga patterns is the divided central horizontal row of segment four. A genus pattern was established to be used as base for comparison in further species determination.

Muscle attachment site (MAS) patterns for species determination in European species of Lucilia (Diptera: Calliphoridae).

Species identification is generally assessed to be more difficult in larval stages than in adult forms. Especially closely related species such as Lucilia caesar and Lucilia illustris are difficult to identify. The aim of this study was to simplify species determination in Lucilia larvae for entomological and forensic purposes. Muscle attachment site (MAS) patterns were previously found to be a good tool for species determination in blowfly larvae. Here, distinctive MAS patterns are presented for European Lucilia ampullacea, L. caesar, L. illustris, L. richardsi, L. sericata, and L. silvarum. A joint pattern for the genus Lucilia is provided for a quick classification of a larva to the genus.

Third instar larvae of flesh flies (Diptera: Sarcophagidae) of forensic importance--critical review of characters and key for European species.

Necrophagous Sarcophagidae are among the insects most frequently reported from human corpses. The broad forensic application of flesh flies is restricted by the lack of reliable tools for species identification of larval stages and mass breeding of collected flesh fly larvae to the adult stage, and more recently DNA-based methods are usually recommended for precise species identification. To overcome this situation, the following study was implemented: (1) original larval material was obtained of the European flesh flies of confirmed or potential forensic importance; (2) larval morphology was studied and documented using a combination of standard light microscopy, image-stacking stereomicroscopy and SEM; and (3) larval characters used in previously published keys were critically revised. The taxonomic value of the following characters was considered insignificant: (1) differences in level of sclerotisation of particular parts of the cephaloskeleton, (2) level of sclerotisation of the posterior spiracular peritreme and (3) the shape of posterior spiracular slits. A high taxonomic value was noticed for the general shape of anterior spiracles, pattern of arrangement of their lobes, and distribution and shape of spines/warts on the inter-band area of segments. Two character states-long window in the dorsal cornu of cephaloskeleton and deep spiracular cavity on anal division-are not found in the Miltogramminae and therefore cannot be considered as family-specific for the entire Sarcophagidae. As a comprehensive result of our studies, an identification key is presented for the third instar larvae of European flesh flies of forensic importance. The key is user-friendly and requires no dissections of larvae, as soaking the material in methyl salicylate will allow observation of all diagnostic details of the cephaloskeleton. A simple stereomicroscope (magnification about ×50) is sufficient for the observation of all characters presented in the key. This key may be systematically extended by the addition of species present in adjacent geographical regions.

Flesh flies (Diptera: Sarcophagidae) colonising large carcasses in Central Europe.

Sarcophagidae are an important element of carrion insect community. Unfortunately, results on larval and adult Sarcophagidae from forensic carrion studies are virtually absent mostly due to the taxonomic problems with species identification of females and larvae. The impact of this taxon on decomposition of large carrion has not been reliably evaluated. During several pig carcass studies in Poland, large body of data on adult and larval Sarcophagidae was collected. We determined (1) assemblages of adult flesh flies visiting pig carrion in various habitats, (2) species of flesh flies which breed in pig carcasses, and (3) temporal distribution of flesh fly larvae during decomposition. Due to species identification of complete material, including larvae, females, and males, it was possible for the first time to reliably answer several questions related to the role of Sarcophagidae in decomposition of large carrion and hence define their forensic importance. Fifteen species of flesh flies were found to visit pig carcasses, with higher diversity and abundance in grasslands as compared to forests. Sex ratio biased towards females was observed only for Sarcophaga argyrostoma, S. caerulescens, S. similis and S. carnaria species group. Gravid females and larvae were collected only in the case of S. argyrostoma, S. caerulescens, S. melanura and S. similis. Sarcophaga caerulescens and S. similis bred regularly in carcasses, while S. argyrostoma was recorded only occasionally. First instar larvae of flesh flies were recorded on carrion earlier or concurrently with first instar larvae of blowflies. Third instar larvae of S. caerulescens were usually observed before the appearance of the third instar blowfly larvae. These results contest the view that flesh flies colonise carcasses later than blowflies. Sarcophaga caerulescens is designated as a good candidate for a broad forensic use in Central European cases.

Species identification of Middle Eastern blowflies (Diptera: Calliphoridae) of forensic importance.

The lack of reliable tools for species identification of necrophagous blowflies of the Middle East is a serious obstacle to the development of forensic entomology in the majority of countries of this region. Adding to the complexity of diagnosing the regional fauna is that species representing three different zoogeographical elements exist in sympatry. In response to this situation, a high-quality key to the adults of all species of forensically relevant blowflies of the Middle East has been prepared. Thanks to the modern technique of image-stack stereomicroscopy and high-quality entomological materials, this new key can be easily applied by investigators inexperienced in the taxonomy of blowflies. The major technical problems relating to the species identification of necrophagous blowflies of the Middle East are also discussed.

Confocal laser scanning microscopy as a valuable tool in Diptera larval morphology studies.

Larval morphology of flies is traditionally studied using light microscopy, yet in the case of fine structures compound light microscopy is limited due to problems of resolution, illumination and depth of field, not allowing for precise recognition of sclerites' edges and interactions. Using larval instars of cyclorrhaphan Diptera, we show the usefulness of confocal laser scanning microscopy (CLSM) for studying the morphological characters of immature stages by taking advantage of the autofluorescent properties of cephaloskeleton structures. We compare data obtained from killed but unprepared larvae with those from larvae prepared by clearing according to two commonly used methods, either with potassium hydroxide or with Hoyer's medium. We also evaluated the CLSM application for examining already slide-mounted larvae stored in museum collections and those freshly prepared. Our results indicate that CLSM and 3D reconstruction are excellent for visualizing small, compound structures of cylrorrhaphan larvae cephaloskeleton, if appropriate clearing techniques, i.e. the application of KOH, are used. Maximum intensity projection of confocal data sets obtained from material freshly prepared and that stored in museum collection does not differ. Because of this and the fact that KOH is commonly used as a clearing method to examine the cephaloskeleton of Diptera larvae, it is possible, and highly recommended, to use slides already prepared with this method for re-examination by CLSM. We conclude that CLSM application can be an invaluable source of data for studies of larval morphology of Cyclorrhapha by way of taxonomic diagnoses, character identification and improvement in characters homologization.

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.

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.

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.

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.

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.

Morphology and identification of first instars of African blow flies (Diptera: Calliphoridae) commonly of forensic importance.

Scanning electron microscopy images of the first instars of Calliphora croceipalpis Jaennicke, 1876; Chrysomya chloropyga (Wiedemann, 1818); Chrysomya marginalis (Wiedemann, 1830); and Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) are presented for the first time, and the following morphological structures are documented: pseudocephalon, antenna, maxillary palpus, facial mask, labial lobe, thoracic and abdominal spinulation, spiracular field, posterior spiracles, and anal pads. Light microscopy photographs and line illustrations are provided for the cephaloskeleton in lateral and ventral views, and the "ectostomal sclerite" and "chitinized teeth" of the cephaloskeleton are recognized as integral parts of the mouthhooks. New diagnostic features of the cephaloskeleton and the spinulation of the abdominal segments are described. These results allow refinement, clarification, and correction of earlier descriptions, which are reviewed. The relative taxonomic importance of various morphological characters of the first instars of necrophagous blow flies is discussed, and details of the cephaloskeleton and the spinulation of the abdominal segments are highlighted as the characters most useful for species identification. Finally, a key for identifying first instars of common African carrion blow flies is provided.