Beyond barcoding : a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

Composition of chemical attractants affects trap catches of the Australian sheep blowfly, lucilia cuprina, and other blowflies

Numbers of Lucilia cuprina (Australian sheep blowfly), Chrysomya spp., and Calliphora spp. blowflies caught on sticky traps baited with various synthetic attractants or a standard liver/sodium sulfide attractant in western Queensland were recorded. Numbers of each genus collected were influenced by the composition of the chemical attractants. Attractant mixtures based on 2-mercaptoethanol, indole, butanoic/pentanoic acid, and a sodium sulfide solution gave 5- to 20-fold higher L. cuprina catches than the liver standard. These blends attracted similar numbers of Chrysomya spp. (0.85–2.7× ) and fewer Calliphora spp. (0.02–0.2× ) compared to the liver standard. These synthetic attractants were more effective and selective for L. cuprina than the standard liver/sodium sulfide attractant, and they can be packaged in controlled-release dispensers to generate constant, prolonged release of the attractant.

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin).

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.

Neighborhood Interactions and Larval Dispersal Behavior in Blowflies

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

Stochastic dynamics in exotic and native blowflies: an analysis combining laboratory experiments and a two-patch metapopulation model

In this study we explored the stochastic population dynamics of three exotic blowfly species, Chrysomya albiceps, Chrysomya megacephala and Chrysomya putoria, and two native species, Cochliomyia macellaria and Lucilia eximia, by combining a density-dependent growth model with a two-patch metapopulation model. Stochastic fecundity, survival and migration were investigated by permitting random variations between predetermined demographic boundary values based on experimental data. Lucilia eximia and Chrysomya albiceps were the species most susceptible to the risk of local extinction. Cochliomyia macellaria, C. megacephala and C. putoria exhibited lower risks of extinction when compared to the other species. The simultaneous analysis of stochastic fecundity and survival revealed an increase in the extinction risk for all species. When stochastic fecundity, survival and migration were simulated together, the coupled populations were synchronized in the five species. These results are discussed, emphasizing biological invasion and interspecific interaction dynamics.

Fuzzy subset approach in coupled population dynamics of blowflies

This paper is a study on the population dynamics of blowflies employing a density-dependent. non-linear mathematical model and a coupled population formalism. In this Study, we investigated the coupled population dynamics applying fuzzy subsets to model the Population trajectory. analyzing demographic parameters such as fecundity, Survival, and migration. The main results suggest different possibilities in terms of dynamic behavior produced by migration in coupled Populations between distinct environments and the rescue effect generated by the connection between populations. It was possible to conclude that environmental heterogeneity can play an important role in blowfly metapopulation systems. The implications of these results for population dynamics of blowflies are discussed.

Ovipositional behavior in predator and prey blowflies

In this study we analyzed the ovipositional behavior of C. albiceps, C. megacephala and L. eximia in response to previous presence of larvae of different species, both predator and prey. The preference for substrates that previously had had no larvae was predominant for all species. However, the experiments showed that C. megacephala and L. eximia avoid laying eggs principally in patches with previous presence of C. albiceps larvae. The implications of these results for the necrophagous Diptera community dynamics are discussed.

Dynamics of experimental populations of native and introduced blowflies (Diptera: Calliphoridae): mathematical modelling and the transition from asymptotic equilibrium to bounded oscillations

The equilibrium dynamics of native and introduced blowflies is modelled using a density-dependent model of population growth that takes into account important features of the life-history in these flies. A theoretical analysis indicates that the product of maximum fecundity and survival is the primary determinant of the dynamics. Cochliomyia macellaria, a blowfly native to the Americas and the introduced Chrysomya megacephala and Chrysomya putoria, differ in their dynamics in that the first species shows a damping oscillatory behavior leading to a one-point equilibrium, whereas in the last two species population numbers show a two-point limit cycle. Simulations showed that variation in fecundity has a marked effect on the dynamics and indicates the possibility of transitions from one-point equilibrium to bounded oscillations and aperiodic behavior. Variation in survival has much less influence on the dynamics.

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)

A review of postfeeding larval dispersal in blowflies: implications for forensic entomology

Immature and adult stages of blowflies are one of the primary invertebrate consumers of decomposing animal organic matter. When the food supply is consumed or when the larvae complete their development and migrate prior to the total removal of the larval substrate, they disperse to find adequate places for pupation, a process known as postfeeding larval dispersal. Several important ecological and physiological aspects of this process were studied since the work by Green (Ann Appl Biol 38:475, 1951) 50 years ago. An understanding of postfeeding larval dispersal can be useful for determining the postmortem interval (PMI) of human cadavers in legal medicine, particularly because this interval may be underestimated if older dispersing larvae or those that disperse longer, faster, and deeper are not taken into account. In this article, we review the process of postfeeding larval dispersal and its implications for legal medicine, in particular showing that aspects such as burial behavior and competition among species of blowflies can influence this process and consequently, the estimation of PMI.

Nutritional ecology of blowflies (Diptera, Calliphoridae): estimates of critical larval weight for pupation on two different diets

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Study of the combined radial post-feeding dispersion of the blowflies Chrysomya megacephala (Fabricius) and C. albiceps (Wiedemann) (Diptera, Calliphoridae)

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

Behavior of the combined radial post-feeding larval dispersal of the blowflies Chrysomya megacephala and Chrysomya albiceps (Diptera, Calliphoridae) and implications for forensic entomology

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

Theoretical Dynamics of Experimental Populations of Introduced and Native Blowflies (Diptera: Calliphoridae)

Equilibrium dynamics in experimental populations of Chrysomya megacephala (F.) and C. putoria (Wiedemann), which have recently invaded the Americas, and the native species Cochliomyia macellaria (F.), were investigated using nonlinear difference equations. A theoretical analysis of the mathematical model using bifurcation theory established the combination of demographic parameters responsible for producing shifts in blowfly population dynamics from stable equilibria to bounded cycles and aperiodic behavior. Mathematical modeling shows that the populations of the 2 introduced Chrysomya species will form stable oscillations with numbers fluctuating 3-4 times in successive generations. However, in the native species C. macellaria, the dynamics is characterized by damping oscillations in population size, leading to a stable population level.

Diffusion Model Applied to Postfeeding Larval Dispersal in Blowflies (Diptera: Calliphoridae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)