Andrzej Grzywacz

Morphology and identification of first instars of the European and Mediterranean blowflies of forensic importance. Part II. Luciliinae.

First instars of Lucilia ampullacea Villeneuve, Lucilia caesar Linnaeus, Lucilia cuprina Weidemann, Lucilia richardsi Collin, Lucilia sericata Meigen and Lucilia silvarum Meigen (Diptera: Calliphoridae) are thoroughly documented with scanning electron microscopy images, light microscopy photographs and line drawings. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, cephaloskeleton, thoracic and abdominal spinulation, spiracular field, and posterior spiracles. New diagnostic features of the cephaloskeleton are presented and the spinulation of the abdominal segments is described. Earlier descriptions are summarized and major discrepancies with the current study are discussed. The present results allow for the clarification, correction and, especially, complementing existing information provided by numerous authors. The first instar larva of L. richardsi is described for the first time and an identification key to the first instars of European species of Lucilia Robineau‐Desvoidy of forensic importance is presented.

Long-term study of pig carrion entomofauna

Long-term changes of carrion entomofauna are poorly understood. No single carrion study lasted longer than one year. We studied entomofauna of large pig carcasses in the second and the third year postmortem. Ten carcasses were exposed in xerothermic grasslands of Western Poland in spring, early and late summer of 2012. Entomofauna was monitored until September of 2014. 72 species were found in the second year, and six in the third year. In the second or the third year carcasses from the late summer block revealed larger number of taxa compared to carcasses from the spring or the early summer block. Taxa differed in pattern of multiple larval colonizations. Only necrophagous taxa breeding in long-lasting carrion parts as well as predators feeding on arthropods present in these parts were found to recolonize. Recolonizations were always recorded in the second or the third year after death. Patterns of multiple colonization depended on the time of carcass exposure in the first year. Residencies of larvae were unexpectedly long in the second year. Several forensically important flies overwintered on carcasses in the larval stage. Current results support the view that insect fauna of cadavers with long post-mortem interval (PMI) may be used for PMI estimation.

A large-scale molecular phylogeny of flesh flies (Diptera: Sarcophagidae)

The available data for Sarcophagidae in GenBank were analysed in order to reconstruct the most comprehensive phylogeny to date. GenBank was explored for nine markers that are commonly used in various molecular and phylogenetic studies of flesh flies. We obtained data for 187 species and constructed an aligned dataset with 9241 characters. However, the matrix suffered from 74% missing data due to a low number of sequences for some markers and in most of the cases only short fragments of the analysed genes were available. The reconstructed tree was taxonomically biased towards the subfamilies Paramacronychiinae (12% of the described species) and Sarcophaginae (8.6% of the described species) and specifically the genus Sarcophaga. The third subfamily Miltogramminae was represented by only 0.7% of described species. Moreover, about half of the included species were of forensic importance, while the percentage of such species in the entire family was estimated at 7%. Many nodes had very low support, so in order to increase the support and thereby identify a ‘core topology’, we pruned ‘rogue’ taxa and applied different substitution models. Both strategies improved support considerably, although some nodes still were left unresolved. An analysis of the distribution of bootstrap values across chronograms showed that the weakest phylogenetic signal is restricted to that part of the tree which coincides with the onset of rapid radiations mainly within the genus Sarcophaga. Our study is concordant with phylogenies obtained by other authors, with the most noteworthy exception being the subfamily Paramacronychiinae emerging as paraphyletic with regard to the Miltogramminae, which is in strong conflict with morphological evidence. We discuss the new findings in the light of traditional taxonomical classifications of Sarcophagidae and recent molecular studies.

Temperature-dependent appearance of forensically useful flies on carcasses.

Flies are frequently used for postmortem interval (PMI) estimations. These estimates are usually based on the age of larval or pupal specimens. However, the age defines only the minimum PMI. In order to move forensic entomology further, a method useful for the estimation of an interval preceding insect appearance on a corpse called the pre-appearance interval (PAI) is needed. Recently, it was demonstrated that the PAI of several carrion beetles is closely related to the temperature prevailing throughout this interval. Hence, it was postulated to estimate PAI from temperature. In order to check premises for using this approach with flies, a test of the relationship between adult or oviposition PAI and temperature was made for nine species of European flies. Data on PAI originated from pig carcasses decomposing under various temperatures. Adult PAI of Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis, Phormia regina, and Stearibia nigriceps and oviposition PAI of S. nigriceps were exponentially related to temperature. Only S. nigriceps revealed a close relationship, demonstrating solid premises for PAI estimation from temperature alone. Adult and oviposition PAI of Calliphora vomitoria and adult PAI of Hydrotaea pilipes were not related to temperature. Adult and oviposition PAI of Lucilia sericata and Lucilia caesar responded similarly, with an abrupt and large increase in a narrow range of low temperatures and no response in a broad range of high temperatures. Probably, different mechanisms form the basis for the response of PAI to temperature in flies colonizing carcasses shortly after death and flies colonizing carcasses later in the decomposition process.

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.

Larval morphology of the lesser housefly, Fannia canicularis

The morphology of all larval instars of Fannia canicularis (Linnaeus) (Diptera: Fanniidae) is documented using a combination of light and scanning electron microscopy. The following structures are documented for all instars: antennal complex; maxillary palpus; facial mask; cephaloskeleton; ventral organ; anterior spiracle; Keilins organ; posterior spiracle; fleshy processes, and anal pad. Structures reported for the first time for all instars include: two pairs of lateral prominences on the prothoracic segment; additional ventrolateral prominences on the second thoracic segment, and a papilla at the base of the posterior spiracle. Other structures reported for the first time are anterior spiracles in the first instar and a serrated tip on the mouthhook in the second instar. A trichoid sensillum on the posterior spiracular plate, representing a sensory organ otherwise unknown in the Calyptratae, is described in the second and third instars. Results are discussed and compared with existing knowledge on dipteran larval morphology.

Morphology of immature stages of Atherigona reversura (Diptera: Muscidae), with notes on the recent invasion of North America

The muscid shoot-fly Atherigona reversura Villeneuve (Diptera: Muscidae), recently introduced to North America, is reported for the first time from the Neotropical Region: Mexico, Chiapas, Chiapa de Corzo. Information about distribution throughout the continent is summarized. Morphology of the second and third instar larva and puparium is described using light microscopy techniques, including photographs of posterior spiracles and cephaloskeleton as well as line drawings of the latter. Detailed scanning electron microscopy documentation is provided for the following morphological structures of the third instar larva: pseudocephalon; antennal complex; maxillary palpus; facial mask; ventral organ; thoracic and abdominal spinulation; bubble membrane; anal division; and anal plate. Previous descriptions of immature stages of Atherigona Rondani, particularly details of the cephaloskeleton, are revised.

Egg morphology of nine species of pollenia robineau-desvoidy, 1830 (Diptera: Calliphoridae)

Egg morphology of nine species of the cluster fly genus Pollenia Robineau‐Desvoidy was studied with scanning electron microscopy. Funnel‐shaped micropylar area, chorion covered with centrally depressed hexagons, well developed hatching pleats on the dorsal surface encompassing the median area were found in eggs of all species: P. amentaria (Scopoli), P. angustigena Wainwright, P. atramentaria (Meigen), P. labialis Robineau‐Desvoidy, P. mayeri Jacentkovsky, P. pediculata Macquart, P. rudis (Fabricius), P. similis (Jacentkovsky), and P. vagabunda (Meigen). Untypical for Calliphoridae, a dual morphological structure of plastron‐bearing area is described here for the first time. On the basis of our observations, all species studied share the presence of a plastron respiratory function on the entire surface between the hatching lines. Differences between species were found in the shape of the longitudinal hatching pleats and the proportion between ‘island pattern’ and ‘hexagonal pattern’ of their chorionic surface, and in the intensity of perforation of hexagons of the median area. Comparisons of Pollenia egg morphology with that of other representatives of Calliphoridae revealed its unique structure, allowing easy differentiation from other representatives of the family. Microsc. Res. Tech. 2012.

Morphology successfully separates third instar larvae of Muscina

Three representatives of the muscid genus Muscina Robineau‐Desvoidy (Diptera: Musicidae), Muscina levida (Harris), Muscina prolapsa (Harris) and Muscina stabulans (Fallén), are well known for their medical, veterinary and forensic importance. However, data in the literature provide contradictory information for the identification of third instar larvae of these species. This hinders easy species differentiation because it requires the rearing of material to adult stages in order to facilitate reliable identification. Third instar larvae of these three Muscina species were studied in detail using light and scanning electron microscopy to provide thorough redescriptions of their morphology. Existing information concerning third instar morphology, with particular reference to its value for taxonomy and identification, is revised and discussed. Emdens spiracular distance factor (SDF) is considered here as inappropriate for identification purposes in third instar larvae of Muscina spp. because its values are not constant ratios but increase during larval maturation and overlap in the examined species. These species were, instead, discriminated here by differences in the spinulation patterns of their abdominal segments.

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.