Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

Seasonal Blowfly Distribution and Abundance in Fragmented Landscapes. Is It Useful in Forensic Inference about Where a Corpse Has Been Decaying?

Blowflies are insects of forensic interest as they may indicate characteristics of the environment where a body has been laying prior to the discovery. In order to estimate changes in community related to landscape and to assess if blowfly species can be used as indicators of the landscape where a corpse has been decaying, we studied the blowfly community and how it is affected by landscape in a 7,000 km(2) region during a whole year. Using baited traps deployed monthly we collected 28,507 individuals of 10 calliphorid species, 7 of them well represented and distributed in the study area. Multiple Analysis of Variance found changes in abundance between seasons in the 7 analyzed species, and changes related to land use in 4 of them (Calliphora vomitoria, Lucilia ampullacea, L. caesar and L. illustris). Generalised Linear Model analyses of abundance of these species compared with landscape descriptors at different scales found only a clear significant relationship between summer abundance of C. vomitoria and distance to urban areas and degree of urbanisation. This relationship explained more deviance when considering the landscape composition at larger geographical scales (up to 2,500 m around sampling site). For the other species, no clear relationship between land uses and abundance was found, and therefore observed changes in their abundance patterns could be the result of other variables, probably small changes in temperature. Our results suggest that blowfly community composition cannot be used to infer in what kind of landscape a corpse has decayed, at least in highly fragmented habitats, the only exception being the summer abundance of C. vomitoria.

Disruption of Chrysomya megacephala Growth Caused by Lignan Grandisin

The toxicity of tetrahydrofuran lignan grandisin was evaluated against larvae of Chrysomya megacephala F. (Diptera: Calliphoridae). The bioassay involved topical treatment on larvae, topical treatment oil egg masses, and incorporation in the larval diet. Grandisin showed inhibition of postembryonic development by ovicidal (30%) and larvicidal (38%) effects and reduced larval weight (4 mg), when topically applied oil egg masses and starving larvae (L1) at a concentration of 100 mu g/mu l. These findings elucidated the effect of grandisin on the C. megacephala life cycle and its potential to control C. megacephala populations.

The determination of morphine in the larvae of calliphora stygia using flow injection analysis and HPLC with chemiluminescence detection

Selective determination of morphine in the larvae of Calliphora stygia (Fabricius) (Diptera: Calliphoridae) using acidic potassium permanganate chemiluminescence detection coupled with flow injection analysis and high-performance liquid chromatography (HPLC) is described. Larvae of C. stygia were reared on minced meat substrates that had been spiked with varying concentrations of morphine. Morphine concentrations were chosen to reflect typical levels in human tissues from opiate overdose victims. After maturing on substrates, larvae were analyzed for the presence of morphine using chemiluminescence detection coupled to flow injection analysis and a rapid HPLC method. Analysis of the larval matrix by flow injection analysis with chemiluminescence detection indicated the presence of interferants capable of generating chemiluminescence. A rapid chromatographic separation with a monolithic column allowed selective determination of morphine in larvae using postcolumn chemiluminescence detection. Larvae of C. stygia reared on substrates containing morphine at concentrations of 500 and 1000 ng/g did not sequester morphine at detectable concentrations. Larvae reared on substrates containing morphine concentrations of 2500, 5000, and 10,000 ng/g tested positive for the drug at concentrations of 765, 2720, and 3010 ng/g, respectively.

Effects of bait age, larval chemical cues and nutrient depletion on colonization by forensically important calliphorid and sarcophagid flies

Species colonization patterns on corpses and the frequency of carrion fly oviposition and larviposition are affected by decomposition stage and previous maggot colonization. This study investigated these effects on meat bait colonization by Victorian Diptera of forensic importance. Bait treatments were: 'aged' (aged for 4 days at 22 °C, allowing some decomposition); 'nutrient-depleted' [aged for 4 days at 22 °C with feeding Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) larvae]; 'extract' (fresh bait mixed with liquid formed by feeding C. vicina larvae), and 'fresh' (untreated control bait). Statistical analysis (α = 0.05) revealed that colonization frequency differed significantly among treatments (Welch's F 3,18.83 = 4.66, P < 0.05). Post hoc tests showed that fresh and extract baits were colonized extensively throughout the experiment with no significant difference, whereas the colonization of nutrient-depleted baits was significantly lower. This suggests that larval digestive enzymes, larval excreta and cuticular hydrocarbons have less effect on colonizing Diptera than the nutritional content of meat. The colonization of aged baits did not differ significantly from that of fresh, extract or nutrient-depleted baits. A further experiment testing 'very aged' (aged for 8 days at 28 °C), 'larvae-added' (fresh bait with C. vicina larvae added before placement) and 'fresh' (untreated control) baits revealed that very aged baits were colonized significantly less frequently than either fresh or larvae-added baits (Welch's F 2, 6.17 = 17.40, P < 0.05).

Optical coherence tomography : age estimation of Calliphora vicina pupae in vivo?

Necrophagous blowfly pupae are valuable contributors to the estimation of post-mortem interval, should an accurate age estimate be obtained. At present, this is reliant on a combination of rearing and destructive methods conducted on preserved samples, including morphological observation and gene expression analyses. This study demonstrates the use of optical coherence tomography (OCT) as a tool for in vivo morphological observation and pupal age estimation. Using a Michelson OCT microscope, alive and preserved four and ten-day old Calliphora vicina pupae were scanned in different orientations. Two and three-dimensional images were created. Morphological characteristics such as the brain, mouthparts and legs were identifiable in both living and preserved samples, with distinct differences noted between the two ages. Absorption of light by the puparium results in a vertical resolution of 1-2 mm, preventing observation of deeper tissues. The use of contrast agents or a longer wavelength laser would improve the images obtainable. At present, the data suggests OCT provides a primary view of external and internal morphology, which can be used to distinguish younger and older pupae for further analysis of age and PMI estimation.

Larval predation on different instars in blowfly populations

Chrysomya albiceps (Diptera: Calliphoridae) é uma predadora facultativa sobre outras moscas-varejeiras, durante o terceiro instar larval. Nesse estudo, nos investigamos a taxa de predação de C. albiceps sobre larvas de primeiro, segundo e terceiro instar de C. megacephala e C. macellaria comparando a vulnerabilidade dos instares larvais frente à predadora. Para as presas de primeiro e segundo instar, C. albiceps apresentou maior taxa de predação sobre C. megacephala. Já sobre larvas de terceiro instar a predadora consumiu mais C. macellaria. O comportamento de C. albiceps sobre as duas espécies de presas sugere uma mudança na estratégia de forrageio da predadora e essa mudança pode ter influencia sobre a comunidade de dípteros necrófagos.

Insects on decomposing carcasses of small rodents in a secondary forest in Southeastern Brazil

The decomposition of small carcasses in the open is frequently neglected although it may provide information of forensic importance. This paper describes an experimental study of arthropod species associated with carcasses of mouse, Mus musculus (Linnaeus, 1758) and rat, Rattus norvegicus (Berkenhout, 1769) (Rodentia: Muridae). Four carcasses were left inside iron cages in sunlit and shady areas in a secondary forest in Southeastern Brazil twice a season for four seasons (n = 16 carcasses of each rodent). The carcasses were removed when arthropods ceased to visit them. The visiting and colonizing invertebrates were collected daily and identified. Immatures were also collected and reared in a laboratory for identification. We collected 6,514 arthropods (820 adults and 5,694 juvenile forms) belonging to 53 species from the families Sarcophagidae, Calliphoridae, Muscidae, Fanniidae, Syrphidae, Richardiidae, Sepsidae, Micropezidae, Otitidae, Drosophilidae, Phoridae, Dolichopodidae, Anthomyiidae, Asilidae and Lauxaniidae (Diptera), Formicidae, Ichneumonidae, Encyrtidae and Apidae (Hymenoptera), Staphylinidae (Coleoptera) and Gonyleptidae (Opiliones). Lucilia eximia (Wiedemann, 1819) (Diptera: Calliphoridae) and Peckia (Pattonella) intermutans (Walker, 1861) (Diptera: Sarcophagidae) deserve special attention because both adult and immature forms were collected in all seasons and in both areas. Our results indicate that the frequency of occurrence of these arthropods was positively associated with carcass size (mouse or rat); no marked insect succession on the carcasses occurred; and the diversity of Calliphoridae and Sarcophagidae was high, irrespective of season.

Behavior of Ectatomma brunneum (Formicidae: Ectatomminae) Preying on Dipterans in Field Conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of temperature on the behavior of burrowing in larvae of the blowflies, Chrysomya albiceps and Lucilia cuprina, under controlled conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Artificial neural networks: A novel approach to analysing the nutritional ecology of a blowfly species, Chrysomya megacephala

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Foraging by Polybia (Trichothorax) ignobilis (Hymenoptera, Vespidae) on flies at animal carcasses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A preliminary study of insect fauna on pig carcasses located in sugarcane in winter in southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct and indirect top-down effects of previous contact with an enemy on the feeding behavior of blowfly larvae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)