Review of Synthesiomyia nudiseta (Diptera: Muscidae) as a useful tool in forensic entomology.

Synthesiomyia nudiseta (van der Wulp, 1883) is a synanthropic muscid found in tropical and subtropical zones around the world. The larvae of this species are a secondary agent of myiasis with necrophagous habits and play an important role in forensic entomology, as they are used as an indicator of post-mortem interval. Adults can be considered vectors of etiological agents such as Escherichia coli and Shigella dysenteriae. Due to its ability to adapt to different environmental conditions, its high dispersal capacity (shown by its introduction to Europe), its predatory habits in the last larval stage and the difficulty of identifying it, a very important goal is to update our knowledge about this species. Therefore, the main objective of this paper is to review the identification, geographical distribution and biology of this species in order to provide better support to investigations involving this fly.

Methods for Surveying Stable Fly Populations.

Stable flies are among the most important pests of livestock throughout much of the world. Their painful bites induce costly behavioral and physiological stress responses and reduce productivity. Stable flies are anthropogenic and their population dynamics vary depending on agricultural and animal husbandry practices. Standardized sampling methods are needed to better identify the factors controlling stable fly populations, test novel control technologies, and determine optimal management strategies. The current study reviewed methods used for a long-term study of stable fly population dynamics in the central Great Plains. An additional study compared the relative size of flies sampled from the general population with that of flies sampled emerging from substrates associated with livestock production. Flies developing in livestock associated substrates are significantly larger than those in the general population indicating that other types of developmental sites are contributing significant numbers of flies to the general population. Because efforts to identify those sites have yet to be successful, we speculate that they may be sites with low densities of developing stable flies, but covering large areas such as croplands and grasslands. The stable fly surveillance methods discussed can be used and further improved for monitoring stable fly populations for research and management programs.

Laboratory Methods for Rearing Horn Flies (Diptera: Muscidae).

The horn fly, Haematobia irritans (L.), is an obligate hematophagous ectoparasite of cattle, and one of the most important pests of cattle causing unrealized gains or losses in meat and milk production. The present study describes the difficulties that arise when research programs have attempted to maintain this pest, both on-host and off-host, in a laboratory environment. Suggestions aimed at assisting future researchers in successfully colonizing horn flies in the laboratory are provided.

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.

Effects of four commercial fungal formulations on mortality and sporulation in house flies (Musca domestica) and stable flies (Stomoxys calcitrans).

The house fly Musca domestica L. (Diptera: Muscidae) and stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae) are major pests of livestock. Biological control is an important tool in an integrated control framework. Increased mortality in filth flies has been documented with entomopathogenic fungi, several strains of which are commercially available. Three strains of Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and one strain of Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) were tested in commercial formulations for pathogenicity against house flies and stable flies. There was a significant increase in mortality of house flies with three of the formulations, BotaniGard® ES, Mycotrol® O, and Met52® EC, during days 4-9 in comparison with balEnce™ and the control. In stable flies, mortality rates were highest with Met52® EC, followed by Mycotrol® O, BotaniGard® ES and, finally, balEnce™. There was a significant fungal effect on sporulation in both house flies and stable flies. Product formulation, species differences and fungal strains may be responsible for some of the differences observed. Future testing in field situations is necessary. These commercial biopesticides may represent important tools in integrated fly management programmes.

The larval development of Habronema muscae (Nematoda: Habronematidae) affects its intermediate host, Musca domestica (Diptera: Muscidae).

Although the life cycle of the equid stomach parasite Habronema muscae was disclosed more than 100 years ago, little is known about the effect of the developing nematode larvae in its intermediate host, Musca domestica. In a series of experiments, freshly hatched M. domestica larvae were exposed to H. muscae eggs contained in a faecal sample of a naturally infected horse. In daily intervals, 50 fly larvae were removed and transferred on a parasite-free larval rearing medium where they completed their development. Hatched flies were examined for the presence of Habronema third-stage larvae. In two subsequent control groups, flies spend their entire larval life in contaminated horse faeces and in a parasite-free larval rearing medium, respectively. Out of the 700 fly larvae used in the infection experiments, 304 developed into adult flies of which 281 were infected. The average nematode larval burden rose from 3.6 in the group with the shortest exposure to more than 25 in the groups with the longest exposure. The proportion of larvae that developed into the adult insect fell from 82 % in the uninfected control group to 27 % in the positive control group. The pupae of the positive control group were smaller and lighter than those of the uninfected control group. Lower pupal size and weight in the positive control group as well as a lower insect developing rate might be attributed to the destruction of adipose cells in the maggots by Habronema larvae.

Augmenting Laboratory Rearing of Stable Fly (Diptera: Muscidae) Larvae With Ammoniacal Salts.

Stable flies are blood feeding parasites and serious pests of livestock. The immature stages develop in decaying materials which frequently have high ammonium content. We added various ammonium salts to our laboratory stable fly rearing medium and measured their effect on size and survival as well as the physical properties of the used media. The addition of ammonium hydroxide, ammonium phosphate and ammonium sulfate reduced larval survival. These compounds decreased pH and increased ammonium content of the used media. Ammonium bicarbonate had no effect on pH and marginally increased ammonium while increasing survival twofold. The optimal level of ammonium bicarbonate was 50 g (0.63 mol) per pan. Larval survival decreased when pH was outside the range of 8.5 to 9.0.

Dividing the pie: differential dung pat size utilization by sympatric Haematobia irritans and Musca autumnalis.

Horn flies [Haematobia irritans (Diptera: Muscidae) (L.)] and face flies [Musca autumnalis (Diptera: Muscidae) De Geer] use the same larval resource, but their interactions are poorly studied. Dung pats (n = 350) were core sampled in the summers of 2012 and 2013 from irrigated pastures in Pomona, California, U.S.A. (34°03'N, 117°48'W) and held for face fly and horn fly emergence. Surface areas and estimated weights were recorded for each whole pat. Almost half (42.0%) of the pat cores yielded neither fly, 29.7% yielded horn flies only, 12.9% yielded face flies only and 15.4% yielded both flies. Of the fly-positive pats, surface area and mass were larger for face fly-occupied pats, whereas horn fly-occupied pats were smaller. Pats shared by the two species were intermediate. Horn flies per positive core were unaffected by the absence/presence of face flies, but half as many face flies emerged when pats were co-inhabited by horn flies. Face flies inhabited larger pats, which might better resist heating and drying, to which they are susceptible; horn flies inhabited a broad pat size range. Horn fly tolerance of lower dung moisture probably allows horn flies to colonize and survive in a wide range of pats in dry areas like southern California.

Rearing Larvae of the Avian Nest Parasite, Philornis downsi (Diptera: Muscidae), on Chicken Blood-Based Diets.

Captive rearing of insect pests is necessary to understand their biology and to develop control methods. The avian nest fly, Philornis downsi Dodge and Aitken, is a blood-sucking parasite during its larval stage and a serious threat to endemic birds in the Galapagos Islands where it is considered invasive. In order to procure large numbers of flies for biological studies, rearing media and diets were trialed for rearing the larval stage of P. downsi under controlled conditions in the absence of its avian host. P. downsi eggs were obtained from field-caught female flies, and once eggs hatched they were reared on chicken blood for the first 3 d. Following this, three diets were tested on second- and third-instar larvae: 1) chicken blood only; 2) chicken blood, hydrolyzed protein and dried milk powder; and 3) chicken blood, hydrolyzed protein and brewer's yeast. Out of 385 P. downsi larvae tested, we were able to rear 50 larvae to the adult stage. The highest level of mortality was found in the first-instar larvae. Survivorship of second- and third-instar larvae was similar irrespective of diet and diet did not significantly influence larval or pupal development times; though larvae fed the diet with brewer's yeast developed marginally faster. Pupal weights were similar to those of larvae that had developed on bird hosts in the field. To our knowledge, this is the first effective protocol for rearing a hematophagous parasitic avian fly from egg to adult in the absence of a living host.

Oviposition Deterrence and Immature Survival of Filth Flies (Diptera: Muscidae) When Exposed to Commercial Fungal Products.

Filth flies are pests of livestock, and can transmit pathogens that cause disease to animals and their caretakers. Studies have shown successful infection of adult filth flies following exposure to different strains and formulations of entomopathogenic fungi. This study aimed to examine the effects of commercial formulations of Beauveria bassiana (Balsamo) (Moniliales: Moniliaceae) (i.e., BotaniGard ES, Mycotrol O, balEnce), and Metarhizium brunneum (Metsch.) (Ascomycota: Hypocreales) (i.e., Met52 EC), on filth fly oviposition and immature fly survival after exposure. House flies, Musca domestica L., laid significantly fewer eggs on Met52 EC-treated surfaces than on surfaces treated with all other products and the control. Similar numbers of eggs were laid on surfaces treated with all B. bassiana products, but egg production was half of the control. Stable flies, Stomoxys calcitrans (L.), laid the fewest eggs on Met52 EC- and Mycotrol O-treated surfaces. This species did not distinguish between the remaining products and the control. In a second experiment, house fly eggs were placed on treated cloths so that hatched larvae contacted the treatment prior to development. Met52 EC had the greatest effect on immature survival with a significant reduction in recovered pupae at the medium and high doses of fungi. Overall, Met52 EC, containing M. brunneum, had the greatest effect on house fly and stable fly oviposition deterrence and immature development of house flies. Management implications are discussed.

Complete tribal sampling reveals basal split in Muscidae (Diptera), confirms saprophagy as ancestral feeding mode, and reveals an evolutionary correlation between instar numbers and carnivory.

With about 5000 species in ca. 180 genera, the Muscidae is the most species-rich family in the muscoid grade of Calyptratae (Diptera: Cyclorrhapha), the others being the Fanniidae, Scathophagidae and Anthomyiidae. Muscidae is remarkable for its young age, high species diversity in all biogeographic regions, and an unusually diverse range of feeding habits at the larval stage (e.g., saprophagy, phytophagy, carnivory, endoparasitism, haematophagy). We here review muscid classification and biology and present a molecular phylogeny based on four mitochondrial genes (12S, 16S, COI, CYTB) and three nuclear genes (28S, Ef1a, and CAD) for 84 species from 40 genera. Our analysis is the first to include species from all biogeographic regions and all currently recognised muscid subfamilies and tribes. We provide strong support for the monophyly of the Muscidae, and for the first time also for the first split within this family. The ancestral larval feeding habit is reconstructed to be saprophagy with more specialised coprophagous saprophagy, phytophagy, and carnivory evolving multiple times from saprophagous ancestors. The origins of carnivory in larvae are significantly correlated with a reduction of the number of larval instars from three (ancestral) to two and one. The genus Achanthiptera which was previously in its own subfamily is shown to be closely related to Azeliini. However, it appears that Azeliinae is paraphyletic because Muscinae is sister-group to the Azeliini while the azeliine Reinwardtiini are polyphyletic. Coenosiinae and Muscinae are monophyletic, but Muscini is paraphyletic with regard to Stomoxyini. Because many subfamilies are apparently para- or even polyphyletic, we review the history of muscid classification in order to reveal how the currently used classification originated.

Multiple colonization of a cadaver by insects in an indoor environment: first record of Fannia trimaculata (Diptera: Fanniidae) and Peckia (Peckia) chrysostoma (Sarcophagidae) as colonizers of a human corpse.

We describe here a case of multiple colonization of a male cadaver found indoors in the municipality of Jaboatao dos Guararapes, Brazil. The body was colonized by six species of Diptera: Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria (Calliphoridae), Megaselia scalaris (Phoridae), Fannia trimaculata (Fanniidae), and Peckia (Peckia) chrysostoma (Sarcophagidae). The most abundant species were C. albiceps (65.0 % of all emerged adults) and C. megacephala (18.6 %). The case illustrates the ability of six insect species to simultaneously colonize a corpse in an indoor environment and represents the first collaboration between the forensic police and entomologists in Northeastern Brazil. We provide here the first record of two species, F. trimaculata and Peckia (P.) chrysostoma colonizing a human cadaver. We also report the first case of cadaver colonization by C. putoria and M. scalaris in Northeastern Brazil. Information on the development time of two species, C. albiceps and C. megacephala, were used to discuss the estimation of the post-mortem interval. Considering that the region harbors the highest rates of homicide in Brazil, implications of these findings for the consolidation of forensic entomology in the region are discussed.

GC-MS analysis of cuticular lipids in recent and older scavenger insect puparia. An approach to estimate the postmortem interval (PMI).

An analytical method was developed to characterize puparia cuticular lipids (hydrocarbons, waxes) and to compare the molecular distribution patterns in the extracts from either recent or older puparia. Acid-catalyzed transesterification and solvent extraction and purification, followed by combined gas chromatography coupled to mass spectrometry, were optimized for the determination of hydrocarbons and fatty acid ethyl esters from transesterified waxes, extracted from a single species of a fly scavenger (Hydrotaea aenescens Wiedemann, 1830). Comparison between recent (2012) or older (1997) puparia contents has highlighted significant composition differences, in particular, a general decrease of the chain length in the n-alkane distribution pattern and, on the contrary, an increase of the ester chain length. Both extracts contain traces of three hopane hydrocarbon congeners. Preliminary results evidence the change in puparia lipid composition over time, thus potentially providing new indices for estimating postmortem interval.

Efficacy and longevity of newly developed catnip oil microcapsules against stable fly oviposition and larval growth.

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), is one of the most important pests of cattle and costs U.S. cattle producers billions of dollars in losses annually. In this study, the efficacy of catnip oil encapsulated in gelatin in oviposition deterrence and larval growth inhibition in stable flies was examined under laboratory conditions. More than 98% inhibition of stable fly larval growth and female oviposition was observed in larval and oviposition media treated with encapsulated catnip oil (0.5 g). Further, dose-response tests showed that as little as 0.1 g of encapsulated catnip oil provided > 85% oviposition deterrence. The release of nepetalactones from the capsules was more rapid when the capsules were placed on a moist substrate rather than a dry substrate. Encapsulated catnip oil also exhibited antibacterial activity, supporting the hypothesis that its inhibition of larval growth may be based on its killing of the bacteria on which larvae feed. The use of encapsulated catnip oil can provide an alternative control strategy for stable fly management.

Droppings from captive Coturnix coturnix (Galliformes: Phasianidae) as a fly breeding resource.

The aim of this study was to describe the fauna of flies associated with captive Coturnix coturnix (L.) (Galliformes: Phasianidae) droppings. Samples of 150 g of quail droppings were exposed in the quail house for 48 h in plastic containers to promote eventual access of flies, and then placed in emergence traps. The number of adults and species emerging was recorded daily. This procedure was carried out in spring 2008 and spring and autumn 2009. In total, 2,138 adults belonging to Muscidae, Calliphoridae, Piophilidae, Phoridae, Fanniidae, and Milichiidae families were collected. The most numerous family was Muscidae (representing >82% of the total specimens), with Musca domestica L. being the most abundant species followed by Ophyra aenescens (Wiedemann) (both Diptera: Muscidae). Quail breeding should include adequate droppings management policies to avoid potential sanitary issues related to fly production.

An illustrated identification key to early instar larvae of forensically important Muscidae (Diptera) of the western Palaearctic region.

There is a significant gap in the availability of comprehensive identification keys for the early larval stages of forensically important fly species. While well-documented identification keys exist for the third instar larvae, particularly for the Calliphoridae, Muscidae and Sarcophagidae families, there is a notable scarcity of keys for the first, except Calliphoridae, and the second instar larvae, with no such resources available for muscid species. The second instar larvae suffer the most from the lack of morphological descriptions and available identification keys. The Muscidae is one of the most frequently reported dipteran families of forensic importance colonising animal cadavers and human corpses. Nevertheless, descriptions of the morphology of their early instars remain scarce and limited to only a few species, thus their larval identification is challenging or impossible. Considering the numerous challenges associated with studying small-sized entomological material, we tested whether it is feasible to identify muscid flies to the species or at least genus level based predominantly on the details of the cephaloskeleton. To overcome the obstacle of observing details of small sclerites, especially their shapes and interconnections, we effectively employed confocal laser scanning microscopy (CLSM) as a supplementary method for light microscopy (LM). This study provides an identification key for first and second instar larvae of forensically important muscid species from the western Palaearctic (Europe, North Africa, Middle East). The proposed key primarily utilises details of the cephaloskeleton with only addition of external morphology.

Larval morphology, development and forensic importance of Synthesiomyia nudiseta (Diptera: Muscidae) in Europe: a rare species or just overlooked?

The muscid Synthesiomyia nudiseta (van der Wulp, 1883) is a species with forensic importance in tropical and subtropical regions of the world. This fly has recently been introduced into southern Europe and, until now, had not been recorded in forensic cases in this region. Here, morphology of all larval instars of S. nudiseta is documented in detail by using a combination of light and scanning electron microscopy. Literature data concerning larval morphology are revised and characters allowing identification from other forensically important Muscidae are listed. The life cycle of this species was studied at four constant temperatures: 15, 20, 25 and 30 °C. Total development varied between 46.50 ± 0.97 days at 15 °C and 15.39 ± 0.32 days at 30 °C. Moreover, we report this species breeding in human corpses, for the first time in Europe, in forensic cases from autopsies at the Anatomical Forensic Institute of Madrid and the Institute of Legal Medicine of Alicante, Spain.

Field measurements of stable fly (Diptera: Muscidae) demography, fecundity, and survival based on daily trap catches at a beef farm in southern Ontario over a 5-yr period.

We sampled stable fly, Stomoxys calcitrans (L.), populations using a CO2 baited cloth trap (Nzi trap) each day throughout the summer and autumn at a beef farm near Peterborough, Ontario, Canada, from 1997 to 2001. Females (2,512) were dissected for ovarian age-grading, to produce a demographic profile of farm populations. The number of follicles were counted to produce fecundity estimates. The developmental periods of adult female stages, measured as accumulated degree-days above 10 degrees C, were determined for a lab colony of stable flies. These measurements were used to calculate survival in terms of degree-days of the farm populations each year. Of the 2,512 females caught, 42.4% were nulliparous on average each year. The median follicle size at insemination was 305 microm in the field populations, and 495 microm in the colony. Farm caught females had an average of 49.15 follicles per ovary overall, with the body size (leg length) and fecundity increasing slightly with age. On average, 44.5% (SE 3.2%) of nulliparous females survived to become parous, and of these, 45.7% (SE 2.1%) survived the uniparous state to become multiparous. Years of higher rainfall had increased fecundity; rainfall did not appear to affect survival.

Effects of pyriproxyfen and buprofezin on immature development and reproduction in the stable fly.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most significant biting flies that affect cattle. The use of traditional insecticides for stable fly control has only a limited success owing to the insect's unique feeding behaviours and immature development sites. A laboratory study was conducted to evaluate the effects of two insect growth regulator (IGR) products, pyriproxyfen and buprofezin, on the development of the immature stages of the stable fly and the effects of pyriproxyfen on oviposition and egg hatch. Both pyriproxyfen and buprofezin had significant inhibitory effects on immature development. The LC(50) s of pyriproxyfen and buprofezin were 0.002 and 18.92 p.p.m., respectively. Topical treatment of adult females with different doses of pyriproxyfen had significant negative effects on both female oviposition and egg hatching when 1- and 3-day-old females were treated, and the effects were dose dependent. A significant reduction in the mean number of eggs laid was observed only at the highest pyriproxyfen dose (8 µg/fly) and egg hatch was unaffected by pyriproxyfen treatment when 5-day-old females were treated. Results from the present study indicate that pyriproxyfen has the potential to be used as part of an integrated stable fly management programme.

Mortality of adult Stomoxys calcitrans fed isolates of Bacillus thuringiensis.

We examined the ability of five isolates of Bacillus thuringiensis Berliner to cause mortality in adult stable flies, Stomoxys calcitrans (L.). Isolates Bacillus thuringiensis tolworthi 4L3 (serotype 9), Bacillus thuringiensis darmstadiensis 4M1 (serotype 10a10b), Bacillus thuringiensis thompsoni 401 (serotype 12), Bacillus thuringiensis thuringiensis HD2 (serotype 1), and Bacillus thuringiensis kurstaki HD945 (serotype 3a3b3c) were administered to adult flies in diets containing blood only, sugar only, and both sugar and blood combined. B. t. tolworthi 4L3 had no effect on adult mortality regardless of the feeding substrate. The remaining isolates tended to cause the greatest mortality when administered in blood alone. B. t. thompsoni 401 was the only isolate that consistently caused adult mortality when fed in blood at concentrations ranging from 0.21 to 50.0 microg of protein per ml of blood. This isolate also caused mortality when applied topically. The time to 50% mortality declined with dose and reached a lower asymptote at approximately equal to 1.3 d at an oral dose of 8.75 microg/ml and at a topical dose of 0.14 microg per fly.