Intrapuparial development of Sarcophaga (Liopygia) ruficornis (Diptera: Sarcophagidae), a species of medical-veterinary and forensic importance, under laboratory conditions.

Sarcophaga (=Parasarcophaga) (Liopygia) ruficornis (Fabricius, 1794) is a species of medical-veterinary and forensic importance, as its immatures cause myiasis in humans and animals and colonize carcasses and cadavers. Therefore, investigations into the biology and morphology of this species, with a particular focus on pupae that constitute ≥50% of the developmental period for the immatures, are pertinent. Although there are biological and morphological studies of pupae, the intrapuparial development at different temperatures has not yet been analysed. Therefore, the present study aimed to describe how temperature (22, 27 and 32 ± 1°C) affects the development and morphology of S. (L.) ruficornis pupae at 60 ± 10% relative humidity and a 12-h photoperiod. Ten pupae were collected, euthanized and fixed every 4 h from pupariation until 24 h and every 8 h until the emergence of the first adult. Emergence occurred at 440, 272 and 232 h at 22, 27 and 32°C, with 590, 380 and 330 pupae, respectively. The highest mortality occurred at 32°C. Eight periods were defined, and sex was determined in pharate adult stage; in addition, 40 key morphological characteristics to estimate pupal age were presented. These findings can serve as a basis for studies on the biology and morphology of the pupa of S. (L.) ruficornis, particularly in bionomics, control and forensics, helping researchers and experts.

Sarcophaga (=Parasarcophaga) (Liopygia) ruficornis (Fabricius, 1794) é uma espécie de importância médico­veterinária e forense, pois seus imaturos causam miíase em humanos e animais e colonizam carcaças e cadáveres. Portanto, são pertinentes as investigações sobre a biologia e morfologia dessa espécie, com foco especial nas pupas que constituem ≥50% do período de desenvolvimento dos imaturos. Embora existam estudos biológicos e morfológicos das pupas, o desenvolvimento intrapuparial em diferentes temperaturas ainda não foi analisado. Logo, o presente estudo teve como objetivo descrever como a temperatura (22, 27 e 32 ± 1°C) afeta o desenvolvimento e a morfologia das pupas de S. (L.) ruficornis a 60 ± 10% de umidade relativa e fotoperíodo de 12 horas. Dez pupas foram coletadas, eutanasiadas e fixadas a cada quatro horas desde a pupariação até 24 horas e a cada oito horas até a emergência do primeiro adulto. A emergência ocorreu em 440, 272 e 232 horas a 22, 27 e 32°C, com 590, 380 e 330 pupas, respectivamente. A maior mortalidade ocorreu a 32°C. Foram definidos oito períodos e o sexo foi determinado na fase de adulto farato; além disso, foram apresentadas 40 características morfológicas importantes para estimar a idade das pupas. Essas descobertas podem servir de base para estudos sobre a biologia e a morfologia da pupa de S. (L.) ruficornis, especialmente em bionomia, controle e ciência forense, ajudando pesquisadores e especialistas.

Evaluation of environmental and local factors on urban sarcosaprophagous Diptera: a case study with Sarcophagidae.

The urbanization process affects the structure of insect communities, favouring biotic homogenization processes by promoting adequate conditions for the establishment of synanthropic species. Considering the scarce knowledge about the ecology of Neotropical sarcophagids, we compared the flesh fly community from two different urban zones in two contrasting seasons and examined the response of the Sarcophagidae assemblage to landscape and microhabitat variables. Adult flies were collected in 13 sites with two traps (total number of traps 26) baited with 200 g of hydrated bone meal. A total of 2312 adult sarcophagids were collected belonging to 27 species; Oxysarcodexia paulistanensis (Mattos, 1919) represented >48% of the total sample. The local factor of insolation explained both the abundance and species richness of sarcophagids, collections being higher and richer in traps exposed to sunlight compared with traps placed in the shade regardless of the season. Richness was also affected by the environment, being higher in traps exposed in suburban rather than urban sites. The combination of these factors (sub-urban environment and availability of open areas) could explain the Sarcophagidae assemblage. The average number of sarcophagid species collected was related to the season, providing a good opportunity to assess the temporal variability of this family in the region.

Establishment of correctly focused eyes may not require visual input in arthropods.

For proper function, vertebrate and invertebrate visual systems must be able to achieve and maintain emmetropia, a state where distant objects are in focus on the retina. In vertebrates, this is accomplished through a combination of genetic control during early development and homeostatic visual input that fine-tunes the optics of the eye. While emmetropization has long been researched in vertebrates, it is largely unknown how emmetropia is established in arthropods. We used a micro-ophthalmoscope to directly measure how the lens projects images onto the retina in the eyes of small, live arthropods, allowing us to compare the refractive states of light-reared and dark-reared arthropods. First, we measured the image-forming larval eyes of diving beetles (Thermonectus marmoratus), which are known to grow rapidly and dramatically between larval instars. Then, we measured the image-forming principal anterior-median eyes of jumping spiders (Phidippus audax) after emergence from their egg cases. Finally, we measured individual ommatidia in the compound eyes of flesh flies (Sarcophaga bullata) that had developed and emerged under either light or dark conditions. Surprisingly, and in sharp contrast to vertebrates, our data for this diverse set of arthropods suggest that visual input is inconsequential in regard to achieving well-focused eyes. Although it remains unclear whether visual input that is received after the initial development further improves focusing, these results suggest that at least the initial coordination between the lens refractive power and eye size in arthropods may be more strongly predetermined by developmental factors than is typically the case in vertebrates.

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.

Immunosuppressive effects of the limonoid azadirachtin, insights on a nongenotoxic stress botanical, in flesh flies.

The tetranortriterpenoid azadirachtin (Aza) is a well-known insect growth disruptor of plant origin. Although its actions on insects have been extensively studied; fragmentary reports are available from the immunological point of view. Therefore, in the present study, total (THC) and differential hemocyte counts (DHC), nodulation, phenoloxidase (PO) activity, immune-reactive lysozymes and inducible nitric oxide (NO) were assessed, as measures of immune responses, in Sarcophaga argyrostoma 3rd instars challenged individually with M. luteus or Aza, or in combination with both compared to the control larvae. THC was significantly declined after 12 h and 24 h of treatment with Aza. DHC varied considerably; in particular, plasmatocytes were significantly decreased after 36 h and 48 h of treatment with Aza; whereas granulocytes were significantly increased. Nodulation was significantly increased with the increase of time after all treatments. Challenging with M. luteus significantly increased the activity of PO in hemocytes and plasma; whereas such activity was significantly decreased after treatment with Aza or combined Aza and M. luteus. Treatment with Aza or M. luteus alone or in couple significantly increased lysozyme activity of fat body, hemocytes and plasma. However, challenging with M. luteus significantly increased NO concentration in the same tissues. A hypothetical model of Aza as a potential mutagen is presented. However, no genotoxic effect was observed through tracking apoptosis-associated changes in Aza-treated hemocytes via flow cytometry-based apoptosis detection. Our study suggests that the integration of Aza, as an eco-friendly pesticide, with bacterial biopesticides may be a successful approach for controlling insect pests.

Molecular identification and detection of Wohlfahrtia magnifica in ovine vulvar myiasis in Gansu, China.

Traumatic myiasis causes substantial economic losses to farmers worldwide. In the present study, six flocks of sheep (2261 sheep) were investigated in Gansu, China, and 207 of 552 larvae were genetically characterized based on three genes, including cyt b, EF-1α, and white gene, by polymerase chain reaction and sequence analysis. A survey of sheep in China revealed that the prevalence of vulvar myiasis of six sheep flocks was 5.00% (11/220, Flock1), 4.85% (10/206, Flock2), 4.50% (9/200, Flock3), 5.00% (15/300, Flock4), 4.68% (15/320, Flock5), 0% (0/1015, Flock6), respectively. The sequence and phylogenetic analysis showed that only Wohlfahrtia magnifica was detected in the field samples. This is the first report of ovine vulvar myiasis caused by W. magnifica in Gansu, China. Some prophylactic measures are strongly recommended to reduce the risk of sheep acquiring traumatic myiasis in Gansu, China.

Phonotactic flight of the parasitoid fly Emblemasoma auditrix (Diptera: Sarcophagidae).

The parasitoid fly Emblemasoma auditrix locates its hosts using acoustic cues from sound producing males of the cicada Okanagana rimosa. Here, we experimentally analysed the flight path of the phonotaxis from a landmark to the target, a hidden loudspeaker in the field. During flight, the fly showed only small lateral deviations. The vertical flight direction angles were initially negative (directed downwards relative to starting position), grew positive (directed upwards) in the second half of the flight, and finally flattened (directed horizontally or slightly upwards), typically resulting in a landing above the loudspeaker. This phonotactic flight pattern was largely independent from sound pressure level or target distance, but depended on the elevation of the sound source. The flight velocity was partially influenced by sound pressure level and distance, but also by elevation. The more elevated the target, the lower was the speed. The accuracy of flight increased with elevation of the target as well as the landing precision. The minimal vertical angle difference eliciting differences in behaviour was 10°. By changing the elevation of the acoustic target after take-off, we showed that the fly is able to orientate acoustically while flying.

Clock and Hormonal Controls of an Eclosion Gate in the Flesh Fly Sarcophaga crassipalpis.

The eclosion gate in insect development is controlled by the circadian clock and hormonal cascade. To study mechanisms underlying the eclosion gate, we examined eclosion-timing signals from the circadian clock, and the role of 20-hydroxyecdysone in the eclosion gate of the flesh fly, Sarcophaga crassipalpis. Phase responses of the eclosion rhythm were examined by applying a low-temperature pulse in the day prior to the first eclosion peak. A low-temperature pulse applied about 5.4 h before eclosion advanced an eclosion peak by 0.9 h. This indicates that an interval from the Zeitgeber (external environmental cues) input to the behavioral output by the circadian clock is 4.5 h. Signals released by the circadian clock in the last 4.5 h before eclosion could change eclosion time. In the prothoracic gland, daily changes in immunoreactivity against a circadian clock protein PERIOD were observed in the last two days before eclosion. Hemolymph titers of 20-hydroxyecdysone were very low in the last two days of the pupal period. 20-hydroxyecdysone injections caused a delay, not an advancement, in eclosion time in a time dependent manner: pharate adults were sensitive to 20-hydroxyecdysone about 20 and 16 h before eclosion, whereas no significant effects were observed about 12 and 8 h before eclosion. These results suggest that 20-hydroxyecdysone is not a timing signal submitted by the circadian clock but an indicator to suppress premature eclosion. The circadian clock in the prothoracic gland presumably sends a signal distinct from ecdysteroids from several hours before eclosion to time the onset of eclosion.

Facultative myiasis of domestic cats by Sarcophaga argyrostoma (Diptera: Sarcophagidae), Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) in northern Italy.

We describe five cases of myiasis of domestic cats, Felis silvestris catus L. (Carnivora: Felidae), reported in 2016 in northern Italy and caused by three Diptera species: Sarcophaga argyrostoma (Robineau-Desvoidy) (Sarcophagidae), Calliphora vicina Robineau-Desvoidy and Lucilia sericata (Meigen) (Calliphoridae). Three were cases of traumatic myiasis, one by S. argyrostoma and two by L. sericata, one was a case of auricular myiasis by C. vicina and one was a case of ophthalmomyiasis caused by an association of L. sericata and C. vicina. The myiasis by S. argyrostoma is the first reported case of this species in a cat, whereas the two myiases by C. vicina are the first reported cases in cats in Italy.

PHARMACOLOGICAL REGULATION OF DIGESTION IN THE ANAUTOGENOUS FLESH FLY, Sarcophaga crassipalpis, BY SIMPLE INJECTION OF 6-HYDROXYDOPAMINE.

Female anautogenous Sarcophaga flesh flies need a protein meal to start large-scale yolk polypeptides (YPs) production and oocyte maturation. Protein meal rapidly elicits a brain-dependent increase in midgut proteolytic activity. Trypsin and chymotrypsin together represent over 80% of protease activity in liver-fed flies. Abdominal injection of 6-hydroxydopamine (6-OHDA) dose-dependently prohibits this increase in proteolytic activity at translational level in a similar way as post liver feeding decapitation. Delayed injection of 6-OHDA later than 6 h post liver meal has no effect. In flesh flies, chemical decapitation by 6-OHDA, by interrupting the brain-gut dopaminergic signaling, can be used as tool for the controlled inhibition of midgut proteolytic activity and subsequent ovarial development. Inhibition of ovarial development is probably indirect due to a deficit in circulating amino acids needed for YPs synthesis.

Calcium signaling mediates cold sensing in insect tissues.

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

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.

Effect of bait decomposition on the attractiveness to species of Diptera of veterinary and forensic importance in a rainforest fragment in Brazil.

Insects associated with carrion can have parasitological importance as vectors of several pathogens and causal agents of myiasis to men and to domestic and wild animals. We tested the attractiveness of animal baits (chicken liver) at different stages of decomposition to necrophagous species of Diptera (Calliphoridae, Fanniidae, Muscidae, Phoridae and Sarcophagidae) in a rainforest fragment in Brazil. Five types of bait were used: fresh and decomposed at room temperature (26 °C) for 24, 48, 72 and 96 h. A positive correlation was detected between the time of decomposition and the abundance of Calliphoridae and Muscidae, whilst the abundance of adults of Phoridae decreased with the time of decomposition. Ten species of calliphorids were registered, of which Chrysomya albiceps, Chrysomya megacephala and Chloroprocta idioidea showed a positive significant correlation between abundance and decomposition. Specimens of Sarcophagidae and Fanniidae did not discriminate between fresh and highly decomposed baits. A strong female bias was registered for all species of Calliphoridae irrespective of the type of bait. The results reinforce the feasibility of using animal tissues as attractants to a wide diversity of dipterans of medical, parasitological and forensic importance in short-term surveys, especially using baits at intermediate stages of decomposition.

Eavesdropping to Find Mates: The Function of Male Hearing for a Cicada-Hunting Parasitoid Fly, Emblemasoma erro (Diptera: Sarcophagidae).

Females of several species of dipteran parasitoids use long-range hearing to locate hosts for their offspring by eavesdropping on the acoustic mating calls of other insects. Males of these acoustic eavesdropping parasitoids also have physiologically functional ears, but so far, no adaptive function for male hearing has been discovered. I investigated the function of male hearing for the sarcophagid fly Emblemasoma erro Aldrich, an acoustic parasitoid of cicadas, by testing the hypothesis that both male and female E. erro use hearing to locate potential mates. I found that both male and nongravid female E. erro perform phonotaxis to the sounds of calling cicadas, that male flies engage in short-range, mate-finding behavior once they arrive at a sound source, and that encounters between females and males at a sound source can lead to copulation. Thus, cicada calling songs appear to serve as a mate-finding cue for both sexes of E. erro Emblemasoma erro's mate-finding behavior is compared to that of other sarcophagid flies, other acoustic parasitoids, and nonacoustic eavesdropping parasitoids.

The Auditory System of the Dipteran Parasitoid Emblemasoma auditrix (Sarcophagidae).

Several taxa of insects evolved a tympanate ear at different body positions, whereby the ear is composed of common parts: a scolopidial sense organ, a tracheal air space, and a tympanal membrane. Here, we analyzed the anatomy and physiology of the ear at the ventral prothorax of the sarcophagid fly, Emblemasoma auditrix (Soper). We used micro-computed tomography to analyze the ear and its tracheal air space in relation to the body morphology. Both tympana are separated by a small cuticular bridge, face in the same frontal direction, and are backed by a single tracheal enlargement. This enlargement is connected to the anterior spiracles at the dorsofrontal thorax and is continuous with the tracheal network in the thorax and in the abdomen. Analyses of responses of auditory afferents and interneurons show that the ear is broadly tuned, with a sensitivity peak at 5 kHz. Single-cell recordings of auditory interneurons indicate a frequency- and intensity-dependent tuning, whereby some neurons react best to 9 kHz, the peak frequency of the host's calling song. The results are compared to the convergently evolved ear in Tachinidae (Diptera).

Sex-biased Patterns of Saprophagous Calyptratae (Diptera) Collected With Different Baits of Animal Origin.

The aim of the present study was to explore the response behavior of males and females of different families of Calyptratae (Diptera) to two different baits (rotten liver and feces) as separate attractants. We describe the sex bias toward these baits for species of Calliphoridae, Muscidae, and Sarcophagidae and compare the general patterns of this sex bias according to the family or trophic guild. In total, 15 species of Sarcophagidae, 10 species of Muscidae, and 9 species of Calliphoridae were analyzed. A female-biased pattern was observed for most calliphorids and for all muscids, whereas a male-biased pattern was more frequent among sarcophagids. The female captures on each kind of bait were assessed as a potential indicator of potential breeding substrates of the species. Three different trophic guilds and their pattern of sex bias were compared. The results obtained allow hypothesizing on the biological traits of saprophagous species, especially on their potential breeding substrates, and assessing proper sampling methods.

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.

Effects of juvenoid Pyriproxyfen on reproduction and F1 progeny in myiasis causing flesh fly Sarcophaga ruficornis L. (Sarcophagidae: Diptera).

Freshly emerged virgin female Sarcophaga ruficornis were topically treated with different doses of pyriproxyfen to test the efficacy on reproduction and subsequent F1 progeny. The results included mortality of the treated adults, significant reduction in fecundity, more than 90% inhibition in larvae production, mortality in F1 generation during larval instars, reduction in pupariation, and adult emergence and production of deformed adults. There was a dose-dependent response showing high degree of correlation in the doses administered and deformities observed. The effects in F1 generation show that an intraovarial transfer of pyriproxyfen was responsible for the various deformations observed, showing the potency of juvenoid pyriproxyfen for longer durations across generations. This is the first study that clearly demonstrates the efficacy of pyriproxyfen against reproduction in S. ruficornis and its potential for the management of this notorious pest of medico-veterinary importance.

Traumatic Myiasis Caused by an Association of Sarcophaga tibialis (Diptera: Sarcophagidae) and Lucilia sericata (Diptera: Calliphoridae) in a Domestic Cat in Italy.

We describe here a rare case of traumatic myiasis occurred in August 2014, caused by an association of 2 Diptera species, Sarcophaga tibialis Macquart (Diptera: Sarcophagidae) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), in a domestic cat in northern Italy. Species identification was based on adult male morphology. The present case is the first report of S. tibialis as an agent of myiasis in Italy, and also the first ever report of myiasis caused by an association of S. tibialis and L. sericata. The cat developed an extensive traumatic myiasis in a large wound on the rump, which was treated pharmacologically and surgically. The biology, ecology, and distribution of S. tibialis and L. sericata are also discussed. A literature review is provided on cases of myiasis caused by S. tibialis, and cases of myiasis by L. sericata involving cats worldwide and humans and animals in Italy.

Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis.

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within 4h post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies 5h post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16% up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction.