Fly Pupae Research Articles

Sodalis praecaptivus subsp. spalangiae subsp. nov., a nascent bacterial endosymbiont isolated from the parasitoid wasp, Spalangia cameroni.

An endosymbiotic bacterium of the genus Sodalis, designated as strain HZT, was cultured from the parasitoid wasp Spalangia cameroni, which develops on the pupae of various host flies. The bacterium was detected in S. cameroni developed on houseflies, Musca domestica, in a poultry facility in Hazon, northern Israel. After culturing, this bacterium displayed no surface motility on Luria-Bertani agar and was rod-shaped and irregular in size, ~10-30 nm in diameter and 5-20 µm in length. Phylogenetic analyses revealed that strain HZT is closely related to Sodalis praecaptivus strain HST, a free-living species of the genus Sodalis that includes many insect endosymbionts. Although these bacteria maintain >98% sequence identity in shared genes, genomic characterization revealed that strain HZT has undergone substantial reductive evolution, such that it lacks many gene functions that are maintained in S. praecaptivus strain HST. Based on the results of phylogenetic, genomic and chemotaxonomic analyses, we propose that this endosymbiont should be classified in a new subspecies as S. praecaptivus subsp. spalangiae subsp. nov. The type strain for this new subspecies is HZT (=ATCC TSD-398T=NCIMB 15482T). The subspecies Sodalis praecaptivus subsp. praecaptivus strain HST is created automatically with the type strain ATCC BAA-2554T (=DSMZ 27494T).

Age estimation of Phormia regina pupae based on ATR-FTIR and chemometrics

Accurate postmortem interval estimation is vital in the investigation of homicides, suicides, and accidental deaths. It is key in narrowing suspect lists, improving crime-solving efficiency, and offering solace to bereaved families. The intra-puparial period, comprising about half of a fly’s developmental cycle, presents challenges for morphological age estimation. External changes are limited to color shifts and the appearance of respiratory horns on the puparium only within several hours after pupariation, while detailed internal development analysis often requires invasive methods like removing the puparium, which can be damaging. Additionally, these techniques usually depend on a forensic entomologist’s expertise, which lead to subjective biases. This study employed attenuated total reflection-fourier transform infrared spectroscopy, a rapid, non-destructive method for analyzing proteins, chitosan, and chitin in puparia. Data showed a consistent reduction in the concentration of the amide I band within the puparium during the intra-puparial development at five constant temperatures (19 °C, 22 °C, 25 °C, 28 °C and 31 °C). This trend in the spectral data effectively distinguishes pupae at various stages of intra-puparial development, facilitating precise age estimation, which is critical for the estimation of the minimum postmortem interval (PMImin). Finally, this work combined the total reflection-fourier transform infrared spectroscopy with chemometric analysis and successfully developed a partial least squares discriminant analysis model and a random forest model, with accuracies of 88 % and 81 %, respectively. These models enable the non-invasive age estimation of P. regina in its intra-puparial period, a stage traditionally difficult to assess morphologically, thus laying the groundwork for PMImin estimation using fly pupae.

Evaluating impacts of radiation-induced sterilization on the performance and gut microbiome of mass-reared Mediterranean fruit fly (Ceratitis capitata) in Hawai'i.

Sterile insect technique (SIT) is a useful strategy for preventing and mitigative establishment of invasive insect species. SIT of the pest tephritid Mediterranean fruit fly, Ceratitis capitata (Wiedemann, 1824)WiedemannWiedemann, has been effective in preventing population establishment in vulnerable agricultural areas of the United States. However, irradiation-induced sterilization can have detrimental impacts resulting in reduced performance metrics. Mediterranean fruit fly males reared for SIT have been shown to have differences in their microbiomes relative to other population sources, which has been postulated to be a factor in how well flies compete with wild conspecifics. To identify baseline performance metrics on the effects of irradiation on the gut microbiome of mass-reared flies in Hawai'i, a study was performed to assess performance metrics and microbiome (bacterial 16S rRNA) variation across multiple timepoints. Mediterranean fruit fly pupae were selected from mass-reared trays intended for release, and paired samples were either irradiated or remained as controls and transported to the laboratory for evaluation. Irradiated flies exhibited fewer successful fliers, more rapid mortality rates, and were less active relative to control nonirradiated flies. Contrary to initial expectations, irradiation did not exert substantial impacts on the composition or diversity of bacterial reads. Samples were primarily comprised of sequences classified as Klebsiella and there were low levels of both read and taxonomic diversity relative to other 16S surveys of medfly. Although this study does not demonstrate a strong effect of irradiation alone on the Mediterranean fruit fly microbiome, there are several explanations for this discrepancy.

Development and Competition of Three Parasitoid Wasps, Brachymeria podagrica, Dirhinus himalayanus, and Nasonia vitripennis, in Their Host, Sarcophaga dux, in Single and Mixed Infections.

Laboratory trials were carried out to investigate the development of three entomophagous parasitoid wasps in preimaginal stages of Sarcophaga dux in monoinfections and mixed infections. Laboratory-raised postfeeding S. dux third-stage larvae were exposed to Brachymeria podagrica. After pupation, 50 of these fly puparia were brought in contact with pupal parasitoid Dirhinus himalayanus and 50 with Nasonia vitripennis, and the remaining 50 puparia were left as Brachymeria monoinfection. In three further trials, each set of 50 freshly pupated host puparia from the same source was exposed to N. vitripennis and D. himalayanus, as monoinfections and mixed infections, respectively. The uninfected control group consisted of 50 S. dux larvae that were kept separately under the same conditions. The percentages of successfully developed B. podagrica and D. himalayanus in monoinfections were 56 and 86%, respectively, and progeny of N. vitripennis hatched from 88% of the exposed host puparia. In mixed infections, N. vitripennis dominated over B. podagrica and D. himalayanus with rates of successfully infected hosts of 50 and 94%, respectively. The number of Nasonia progeny in these groups ranged from 4 to 49 and 5 to 43, respectively. Dirhinus himalayanus did not develop in the simultaneous infection with N. vitripennis. Not a single S. dux eclosed in the six experimental groups, while in the uninfected control group, 46 (92%) adult flies eclosed 11 to 14 days after the start of pupation. Since the three parasitoids emerge from flesh fly pupae, these insects can become important in criminal forensic investigations when corpses are in an advanced stage of decay. More data on their preimaginal development at different temperatures are necessary.

Study of Population Dynamics of Syrphid Fly (Order- Diptera, Family- Syrphidae) in ITM University Gwalior, India

An intriguing ecological interaction can exist between aphids and Syrphid flies, particularly in the setting of millet crops. Aphids (Order Hemiptera: Family Aphididae) are a problem variety of crops, including all types of millets. Syrphid flies (Order Diptera: Family Syrphidae) naturally prey on aphids when they are in their larval stage. Syrphid fly larvae are referred to as "aphid lions" or "hoverfly larvae." Aphids are the main source of food for these larvae, and they manage the aphid population in millet crops. Therefore, Syrphids are used as natural enemies and biological control agents and syrphid flies are advantageous insects in agriculture. The present study was conducted in the month of August 2021 to March 2022 (summer and winter season) to find out the population dynamic of syrphid flies in agricultural farms and the campus of ITM University. The target crops like maize, sorghum, pearl millets, okra, mustard crop, cabbage cauliflower, marigold and brinjal were tagged and marked and Syrphid fly larvae and pupa were counted four times in a month. The larvae and pupa were also collected and kept for adult emergence. The maximum number of larvae were collected from the Turari campus and were as a minimum found in CRC 2. The other predator like lady bird beetles were also observed but their population was very minor. The population of syrphid flies and larvae depends on the climatic conditions and the availability of foods and their prey.

SOME BIOLOGICAL AND ECOLOGICAL FEATURES OF HYPODERMA LATREILLE, 1818 IN TAVUSH REGION OF ARMENIA

Background. Cattle hypodermosis is a chronic parasitic disease (entomosis) that occurs as a result of parasitizing the larvae of warble flies in the body of animals. Disease is common in more than 55 countries of the world, causing great economic damage. It is considered as a parasitic disease with a certain zoonotic potential. Purpose. To conduct research on biological and ecological features of the warble flies that determines certain epidemiological traits of the caused pathology in Tavush Region of Armenia. Materials and methods. The observations were carried out in Tavush Region, and the experiments were carried out at the Research Center for Veterinary Medicine and Veterinary Sanitary Expertise at the Armenian National Agrarian University. The warble flies marked with stain, were released at a distance of 0.5 to 1.5 km from livestock farms with subsequent search and trapping of insects, located on pastures and on the skin of animals. Generally, 275 warble flies were released. Warble fly activity was evaluated visually counting the comparative number of flying insects at the fixed point per minute at different times of the day, as well as animal anxiety state caused by warble flies was identified. Statistical processing of experimental data was performed by means of “Vassart.net” online calculator. Results. Female warble flies Hypoderma bovis flew up to 12.1 km, while female warble flies H. lineatum did it up to 8.6 km. Females of H. bovis and H. lineatum demonstrated various spatial and circadian flying activity that depends on season, and altitude above the sea level. Spring generation of warble flies is more resistant to low air temperatures compared to the summer generation. Mild and humid summers, as well as an increase in the number of animals, contribute to an increase in the population of warble flies, while high temperature and high soil moisture contribute to the mass death of warble fly pupae developing in the ground. Conclusion. The research data cited in the article, detect the presence of a wide range of biocenotic factors that determine the epidemiology of cattle hypodermosis in the Tavush region of the Republic of Armenia. Cattle hypodermosis control plan was designed based on the results of the research, as well as natural and climatic conditions of Tavush region. Pasture prevention activities and periodic treatment of the cattle with insecticides were emphasized.

A multispectral imaging system developed based on the spectral feature selection method for identification of housefly pupae

Hyperspectral imaging systems can simultaneously obtain spectral and spatial information of fly pupae, especially achieving a large area of spectral information collection in a short period. However, hyperspectral imaging systems are expensive and require much computation. This study attempts to develop a four-channel spectral imaging system composed of a camera and four filters to achieve spectral discrimination of housefly pupae over a large area. It also aims to verify the feasibility of using spectral feature selection methods to guide the development of low-cost specialized spectral systems. Meanwhile, a strategy combining spectral degradation with the traversal method was proposed for determining the parameter indicators of optical components. Firstly, use a microspectrometer to acquire the spectra of fly pupae. Secondly, the successive projections algorithm (SPA) extracts four feature wavelengths related to fly pupa recognition from the full spectrum variables. Then, based on the strategy of spectral degradation combined with the traversal method, the parameters of the four filters are determined. Finally, a 4-channel spectral imaging system was constructed, and a prediction model was built with random forest (RF), while the spectral index (SI) method was used to improve model performance. The results indicate that this low-cost multispectral imaging system can achieve high-performance discrimination of housefly pupae (accuracy = 97.44 %). At the same time, SPA, spectral degradation, and traversal methods can provide effective central wavelength and resolution indicators for constructing low-cost multispectral imaging systems, offering a practical and feasible example to guide the development of specialized low-cost spectral devices using spectral feature selection methods.

Evaluation of the Entomopathogenic Fungus Beauveria bassiana on Different Stages of Phlebotomus papatasi (Diptera: Psychodidae), Vector of Zoonotic Cutaneous Leishmaniasis in Iran.

Zoonotic cutaneous leishmaniasis is a major public health problem in Iran with the main vector of Phlebotomus papatasi. The use of entomopathogenic fungi for biological control of the vector is a potential substitute for the current methods which are being used. The purpose of the current study was to assess the virulence of two local isolates of Beauveria bassiana (OZ2 and TV) against Ph. papatasi. To perform the bioassay test, fungal suspensions were applied for every stage of the sand fly life cycle. The mortality rate, longevity, and number of eggs laid were determined. Also, the probability of fungal survival on the surface of rodent's body was assessed. The longevity of infected adult sand flies with both isolates of B. bassiana was significantly lower (P< 0.05) in comparison to the negative control. The estimated Lethal concentration 50 (LC50) values for adult female and male sand flies treated with OZ2 isolate were 1.4×106 and 2.2×107 conidia/ml, respectively, while they were 6.8×106 and 2.3×108 conidia/ml for TV isolate, respectively. Both isolates of B. bassiana exhibited nonsignificant mortality rates in sand fly larvae and pupae and fecundity rate (P> 0.05). According to our findings for both isolates, the fungus continued to spread throughout the surface of the rodent's body for 144 hours after spraying. The current study demonstrated that both isolates of B. bassiana have considerable biological control capacity against adult sand flies.

Exploring the impact of culture media on life history characteristics of the house fly, Musca domestica L. (Diptera: Muscidae)

Insects, particularly house fly pupae (M. domestica L.), are increasingly promoted as a rich and sustainable protein source, presenting a practical solution for organic waste management. These insects possess the capability to transform animal waste into valuable biomass, underscoring the necessity to evaluate housefly larval growth on diverse waste mixtures. The primary objective was to comprehend how different culture media influence the life history traits of this species. To address this, the research aimed to assess the growth and development performance of housefly larvae reared on various waste mixtures. Adult house flies (M. domestica) were sourced from meat and fish markets in Rajshahi city, Bangladesh, employing systematic sampling from different market sections for a representative selection. Experimental diets were formulated with combinations of cattle blood, cattle manure, rice bran, wheat bran, and kitchen waste, categorized into Diet-A (kitchen waste:cattle manure:wheat bran), Diet-B (cattle blood:rice bran), Diet-C (cattle manure:wheat bran), and Diet-D (cattle blood:kitchen waste:rice bran). Additionally, a control group comprised a mixture of waste materials, including yeast, egg white, milk powder, and wheat bran. The findings revealed that larvae, pupae, and adults reared on Diet-B, incorporating cattle blood and rice bran, demonstrated increased length compared to other diets. This varied diet also produced the heaviest larvae, pupae, and adults, exhibiting the highest morphometric parameters. The moisture content of pupae varied across the different diets, ranging from 53.23±0.17 % to 65.47±1.32 %. The pupae reared on Diet-B had the highest levels of crude protein (53.44±0.05 %, p < 0.05), while those reared on Diet-C had the highest levels of fat (18.56±0.05 %, p < 0.05 except for diet A). The findings demonstrate that housefly larvae can be reared on diets containing rice bran and cattle blood, which have different nutritional profiles and biological capacities. However, the ideal rearing strategy should depend on how M. domestica will be utilized in the future. The study highlights insects, especially housefly pupae, as a sustainable protein source and an organic waste management solution. Therefore, the results of this study contribute to the broader understanding of Musca domestica and offer practical implications for both scientific research and pest management practices.

Functional response and mutual interference in the parasitoid Coptera haywardi (Oglobin) (Hymenoptera: Diapriidae) attacking Anastrepha ludens (Loew) (Diptera: Tephritidae) pupae.

Functional response and mutual interference are important attributes of natural enemies that should be analysed in species with the potential to be used as biological control agents in order to increase the predictive power of the possible benefits and/or consequences of their release in the field. Our main objective was to determine the functional response and mutual interference of Coptera haywardi (Oglobin), a pupal parasitoid of economically important fruit flies (Diptera: Tephritidae). The functional response of C. haywardi on A. ludens pupae corresponded to a type II model, with an attack rate of 0.0134 host pupa/h and a handling time of 1.843 h, which reveals a meticulous selection process of pupal hosts. The effect of mutual interference among foraging females was negatively correlated with increased parasitoid density in the experimental arena, showing a gradual decline in attack rate per individual female. The increase in the number of foraging females also had an impact on the number of oviposition scars per pupa and the number of immature parasitoids per dissected pupa, but not on the percentage of adult emergence or the sex ratio. Our results suggest that C. haywardi could act as a complementary parasitoid in the control of fruit fly pupae, since the random distribution of these pupae in the soil would decrease the possibility of aggregation and mutual interference between foraging females.

The relationship between overwintering peach fruit fly pupae Bactrocera zonata (Saunders) (Diptera: Tephritidae) and some pathogenies in citrus orchard soils in Diyala province

A field and laboratory study was conducted for soil samples in citrus orchards in the Muqdadiya region - Diyala province in year 2021, for three depths (3, 5, and 7) cm, to detect overwintering peach fruit fly pupae Bactrocera zonata (Saunders) its infecting by Beauveria bassiana (Bals) Vuill. . The results showed that inverse (negative) correlation between the rates of the numbers of fungal colonies and the different soil depths, as the correlation value was (-0.906).

Green Production of Chitin from Black Soldier Fly Pupae Using a Natural Deep Eutectic Solvents

Natural deep eutectic solvents (NADES) are eco-sustainable, non-toxic, non-volatile, renewable, reusable, and biodegradable, and are composed of natural compounds. NADES were developed as a new-generation solvent for extracting chitin from black soldier fly (BSF) pupae, and its effectiveness for demineralization and deproteinization was determined. Here, two promising NADES, consisting of mixtures of choline chloride-betaine-xylitol (NADES A) and choline chloride-malic acid-water (NADES B), were tested. Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were used to investigate the changes in the chemical composition of the extracted chitin. α-chitin revealed at wavenumbers 1660-1500 cm-1, in the amide group and decomposed at 330-350°C. NADES A and NADES B have a crystalline index of 91.65% and 90.65%, respectively. The chitin-NADES A and chitin-NADES B surfaces reveal the repetition of square, pentagonal, and hexagonal units (250× magnification) and fibrils (25,000× magnification). This study provides a green approach for chitin production from BSF and reveals the potential of NADES for extracting bio-polymers from natural sources.

Effect of X-ray Irradiation on Pupal Development of Bactrocera zonata and Bactrocera dorsalis under Laboratory Conditions

The studies were performed to check the effect of X-ray doses such as 20, 40, 60, 80 and 100 Gy on the Bactrocera zonata (Saunders) and Bactrocera dorsalis (Hendel) pupae reared on different diets. The laboratory strains of the Peach fruit fly and Oriental fruit fly reared on Guava, Mango and artificial diets. Results showed a significantly (P&lt;0.05) greater number of deformed adults (Males and Females) of B. zonata at 100 Gy dose of x-ray irradiation followed by B. dorsalis obtained from artificial diets. Less deformity in adults of both fruit fly species was found at the same dosage of irradiation when maggots tended on mango. Similarly, a reduced number of normal adults of both fruit flies were recorded at 100 Gy dose of X-rays reared on an artificial diet. Reduced pupal emergence was recorded on an artificial diet. From the above results, it is determined that B. zonata is more tolerant to different irradiation doses. Results of the present investigation would be useful to control or target fruit fly pupae in fruits of trade commodities as these pests are universally regulated as quarantine pests.

Automatic classification of parasitized fruit fly pupae from X-ray images by convolutional neural networks

The parasitoid wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae) has been used for the biological control of fruit fly larvae in many countries. Due to the large scale of production, quality control systems must be utilized to assess insect quality to optimize prerelease activities. However, the parasitism rate, and the unparasitized puparia, which become waste pupae, can only be known after waiting for the emergence of the adults from sampled pupae (ca. 16 days at 25 °C). This waiting time can result in resources being wasted. A way to speed up the quality control of pupal batches and increase the accuracy of the production process would be to combine digital radiography of the parasitized pupae and deep learning methods for the automatic classification of images. Therefore, the purpose of this work was to discriminate parasitized from waste pupae through X-ray images and to test the suitability of 7 Convolutional Neural Networks to classify the pupal images. Radiographic images from 11-day-old pupae allowed the in vivo identification of immature stages of the parasitoid, preserving the viability of the wasp. A positive correlation was found between the number of emerged parasitoids and the ones identified in the X-ray images. In terms of performance metrics, the accuracies achieved by all CNN-based neural architectures were higher than 97%, indicating a high predictive classification power. At least three of the CNNs tested showed great potential as classifiers of X-ray images of parasitized pupae and can be indicated for practical applications in quality control programs.

Chemical and population genetic analysis show no evidence of ecotype formation in a European population of the parasitoid wasp Nasonia vitripennis

Ecotypes, subpopulations or strains of a single species locally adapted to divergent ecological conditions within the same habitat are often considered to be the first steps in sympatric speciation. It has been suggested that two ecotypes are distinguishable in Nasonia vitripennis, a prominent model organism for parasitic Hymenoptera, with one ecotype parasitizing fly pupae in bird nests, and the other one parasitizing fly pupae on carrion. This differentiation into two ecotypes has been hypothesized to indicate incipient sympatric speciation in populations of this globally distributed species. In the present study, we investigated the differentiation into these two distinct ecotypes focusing on chemical profiles and the population genetic divergence in a wild N. vitripennis population from the Netherlands. Isofemale lines were obtained from bird nest boxes and from deer carrion, respectively, representing both microhabitats. To test for phenotypic differentiation, we determined the surface cuticular hydrocarbon (CHC) profiles from wasps of both host patches. Using a panel of 14 microsatellites, we concordantly determined the population genetic structure and tested for genetic differentiation between foundresses obtained from both microhabitats. Both the phenotypic as well as the genetic datasets show no evidence for any kind of separation based on the postulated two ecotypes, but rather suggest free interbreeding with no gene flow interruption between the two distinct host patches. Our findings challenge previous assumptions on clearly distinguishable ecotypes in N. vitripennis, and demonstrate how a chemical ecological assessment coupled with population genetics can be instrumental in re-evaluating the potential of ecological differentiation and incipient speciation mechanisms in parasitoid wasps.

Identification of fruit fly (Tephritidae) pupae parasitized by hymenoptera parasitoids

AbstractOne of the major problems in orchards is the presence of Tephritidae fly maggots, especially in Mirtaceae species. Various biological control tools include the use of Hymenoptera parasitoids, which naturally control fly populations. However, identification of the host species of parasitoids is challenging, because only the puparium remains after the parasitoid hatches. Therefore, the aim of this work was to determine the host species of Hymenoptera parasitoids, through the puparium. Host species were identified by amplification and sequencing of cytochrome C oxidase subunit I (COI) of mitochondrial DNA. The results of the present study show that COI DNA sequencing was able to reliably identify host pupae to the genus level, allowing the identification of the Anastrepha genus in this study. This suggests that this method can be used to identify agriculturally relevant hosts through its puparium remains, since this genus has already been described as a pest of Campomanesia adamantium. Knowledge of the species that cause damage to crop is extremely important to study the biological and ecological aspects of these pests and their control. In this way, our results suggest that this method can be used to identify economically relevant host pupal species since the genus identified in this study has already been described as a pest of C. adamantium.

Upper thermal limits of Rhagoletis indifferens (Diptera: Tephritidae) pupae and pteromalid parasitoids (Hymenoptera: Pteromalidae) inside fly puparia.

Determining upper thermal limits of tephritid fly pupae can have practical implications for disinfesting soils and for predicting differential impacts of global warming on flies and their parasites. Here, upper thermal limits of Rhagoletis indifferens Curran (Diptera: Tephritidae) pupae and pteromalid wasps (Hymenoptera: Pteromalidae) inside puparia were determined. Puparia receiving sufficient chill to terminate pupal diapause were exposed to temperatures ramped linearly over 6 h from 21 °C to 47.8, 49.4, 51.1, 55.0, or 60.0 °C for a 0-h hold time. Flies eclosed when pupae were exposed to 47.8 °C but not to 49.4, 51.1, 55.0, or 60.0 °C nor in a separate test to 47.8 °C for 1-3 h hold times. All fly pupae in treatments where no eclosion occurred were dead based on puparial dissections. In contrast, adult wasps eclosed when puparia were exposed to 49.4 and 51.1 °C for 0 h and to 47.8 °C for 1- and 2-h hold times. Despite the greater upper thermal limits of wasps, heat delayed eclosion times of both adult flies and wasps, in 47.8 and 51.1 °C treatments, respectively. In separate tests, longevity of flies exposed as pupae to 47.3-48.6 °C was greater than of control flies, while longevity of control wasps and wasps exposed as immatures to 47.8-51.1 °C did not differ. Flies exposed as pupae to 47.2-48.6 °C produced as many eggs and puparia as control flies. Results suggest heat could be used to disinfest soils of puparia while sparing parasitoids. In addition, extreme heat waves due to global warming might be more detrimental to fly pupae than immature wasps.

The son-killer microbe Arsenophonus nasoniae is a widespread associate of the parasitic wasp Nasonia vitripennis in Europe

Heritable microbes that exhibit reproductive parasitism are common in insects. One class of these are the male-killing bacteria, which are found in a broad range of insect hosts. Commonly, our knowledge of the incidence of these microbes is based on one or a few sampling sites, and the degree and causes of spatial variation are unclear. In this paper, we examine the incidence of the son-killer microbe Arsenophonus nasoniae across European populations of its wasp host, Nasonia vitripennis. In preliminary work, we noticed two female N. vitripennis producing highly female biased sex ratios in a field study from the Netherlands and Germany. When tested, the brood from Germany was revealed to be infected with A. nasoniae. We then completed a broad survey in 2012, in which fly pupal hosts of N. vitripennis were collected from vacated birds’ nests from four European populations, N. vitripennis wasps allowed to emerge and then tested for A. nasoniae presence through PCR assay. We then developed a new screening methodology based on direct PCR assays of fly pupae and applied this to ethanol-preserved material collected from great tit (Parus major) nests in Portugal. These data show A. nasoniae is found widely in European N. vitripennis, being present in Germany, the UK, Finland, Switzerland and Portugal. Samples varied in the frequency with which they carry A. nasoniae, from being rare to being present in 50% of the pupae parasitised by N. vitripennis. Direct screening of ethanol-preserved fly pupae was an effective method for revealing both wasp and A. nasoniae infection, and will facilitate sample transport across national boundaries. Future research should examine the causes of variation in frequency, in particular testing the hypothesis that N. vitripennis superparasitism rates drive the variation in A. nasoniae frequency through providing opportunities for infectious transmission.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

Descriptive analyses of differentially expressed proteins during intrapuparial stage based on the label-free proteomics technique between Chrysomya megacephala and Synthesiomyia nudiseta

The lack of rapid and accurate species identification methods on pupae restricts the practical application of forensic entomology. It is a new idea to construct portable and rapid identification kits based on the principle of antigen/antibody interaction. Screening differentially expressed proteins (DEPs) of fly pupae is a basis of solving the problem. Here, we used the label-free proteomics technique to discover the DEPs and further validate using the parallel reaction monitoring technique (PRM) in the common flies. In this study, we reared the Chrysomya megacephala and Synthesiomyia nudiseta at constanttemperature, and then we sampled at least four pupae at 24 h intervals until the end of the intrapuparial stage. We found 132 DEPs between Ch. megacephala, and S. nudiseta groups, with 68 and 64 proteins being up-regulated and down-regulated between the two groups. Among the 132 DEPs, we selected five proteins having potential for further development and utilization, such as C1-tetrahydrofolate synthase, Malate dehydrogenase, Transferrin, Protein disulfide-isomerase, and Fructose-bisphosphate aldolase, for further validation using PRM-targeted proteomics, with the trends of PRM results being consistent with the label-free data for corresponding proteins. The present study investigated DEPs via the label-free technique during the pupal development in the Ch. megacephala, and S. nudiseta and provided reference data for development of rapid and accurate identification kits.