Comprehensive evaluation of DNA barcoding for the molecular species identification of forensically important Australian Sarcophagidae (Diptera)

Carrion-breeding Sarcophagidae (Diptera) can be used to estimate the post-mortem interval (PMI) in forensic cases. Difficulties with accurate morphological identifications at any life stage and a lack of documented thermobiological profiles have limited their current usefulness of these flies. The molecular-based approach of DNA barcoding, which utilises a 648-bp fragment of the mitochondrial cytochrome oxidase subunit I gene, was previously evaluated in a pilot study for the discrimination between 16 Australian sarcophagids. The current study comprehensively evaluated DNA barcoding on a larger taxon set of 588 adult Australian sarcophagids. A total of 39 of the 84 known Australian species were represented by 580 specimens, which includes 92% of potentially forensically important species. A further eight specimens could not be reliably identified, but included as six unidentifable taxa. A neighbour-joining phylogenetic tree was generated and nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model. All species except Sarcophaga (Fergusonimyia) bancroftorum, known for high morphological variability, were resolved as reciprocally monophyletic (99.2% of cases), with most having bootstrap support of 100. Excluding S. bancroftorum, the mean intraspecific and interspecific variation ranged from 0.00-1.12% and 2.81-11.23%, respectively, allowing for species discrimination. DNA barcoding was therefore validated as a suitable method for the molecular identification of the Australian Sarcophagidae, which will aid in the implementation of this fauna in forensic entomology.

The complete mitochondrial genome of the flesh fly, Sarcophaga impatiens Walker (Diptera: Sarcophagidae)

Approximately 2500 fly species comprise the Sarcophagidae family worldwide. The complete mitochondrial genome of the carrion-breeding, forensically important Sarcophaga impatiens Walker (Diptera: Sarcophagidae) from Australia was sequenced. The 15,169 bp circular genome contains the 37 genes found in a typical Metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. It also contains one non-coding A+T-rich region. The arrangement of the genes was the same as that found in the ancestral insect. All the protein initiation codons are ATN, except for cox1 that begins with TCG (encoding S). The 22 tRNA anticodons of S. impatiens are consistent with those observed in Drosophila yakuba, and all form the typical cloverleaf structure, except for tRNA-Ser(AGN) that lacks the DHU arm. The mitochondrial genome of Sarcophaga presented will be valuable for resolving phylogenetic relationships within the family Sarcophagidae and the order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.

Utility of COI, CAD and morphological data for resolving relationships within the genus Sarcophaga (sensu lato) (Diptera: Sarcophagidae) : a preliminary study

Currently there are ~3000 known species of Sarcophagidae (Diptera), which are classified into 173 genera in three subfamilies. Almost 25% of sarcophagids belong to the genus Sarcophaga (sensu lato) however little is known about the validity of, and relationships between the ~150 (or more) subgenera of Sarcophaga s.l. In this preliminary study, we evaluated the usefulness of three sources of data for resolving relationships between 35 species from 14 Sarcophaga s.l. subgenera: the mitochondrial COI barcode region, ~800. bp of the nuclear gene CAD, and 110 morphological characters. Bayesian, maximum likelihood (ML) and maximum parsimony (MP) analyses were performed on the combined dataset. Much of the tree was only supported by the Bayesian and ML analyses, with the MP tree poorly resolved. The genus Sarcophaga s.l. was resolved as monophyletic in both the Bayesian and ML analyses and strong support was obtained at the species-level. Notably, the only subgenus consistently resolved as monophyletic was Liopygia. The monophyly of and relationships between the remaining Sarcophaga s.l. subgenera sampled remain questionable. We suggest that future phylogenetic studies on the genus Sarcophaga s.l. use combined datasets for analyses. We also advocate the use of additional data and a range of inference strategies to assist with resolving relationships within Sarcophaga s.l.

Occurrences of flesh flies (Diptera: Sarcophagidae) on human cadavers in Switzerland, and their importance as forensic indicators.

From 1993 to 2008, criminal investigations were conducted in the western part of Switzerland with special attention to blowfly and flesh fly species in order to estimate the post-mortem interval when requested by the police authorities. Flesh flies were found in only 33 cases out of 160. Five species of the genus Sarcophaga were identified (S. africa, S. argyrostoma, S. caerulescens, S. similis and S. sp.). The main species found on corpses (larval stage) was S. argyrostoma. The thermal constant (K) calculated for this species in Switzerland is 380.6 ± 16.3 (mean ± S.D.) degree-days. With the exception of S. caerulescens, found three times in the larval stage on corpses, the three other species are of minor forensic importance. S. argyrostoma is found during summer and indoors. This species colonises dead bodies, usually the same day as blowfly species, and it could be used to estimate the post-mortem interval. Other species are discussed in the light of current knowledge on their biology and ecology. It is recommended that voucher material be deposited in a museum, allowing further studies by relevant specialists, thereby helping investigators and avoiding misidentifications.

Occurrence of Microcerella halli (Engel) (Diptera, Sarcophagidae) in snake carrion in southeastern Brazil

Occurrence of Microcerella halli (Engel) (Diptera, Sarcophagidae) in snake carrion in southeastern Brazil. The occurrence of 27 second-instar larvae of the flesh fly Microcerella halli (Engel, 1931) (Diptera, Sarcophagidae) in a carcass of a snake usually called as Urutu, Bothrops alternatus (Dumeril, Bibron & Dumeril, 1854) (Serpentes, Viperidae, Crotalinae) is reported. The snake was kept in captivity in a snake farm in Morungaba, São Paulo state, Brazil. Descriptions of reptile carcass colonization by insects and general biological data of this flesh fly are scarce and this necrophagic behavior is described for the first time in literature.

Revisão taxonômica e filogenia das espécies do gênero Nephochaetopteryx Townsend, 1934 (Diptera: Sarcophagidae)

Nephochaetopteryx Townsend, 1934 é um gênero de moscas da família Sarcophagidae, da tribo Sarothromyiini, predominantemente Neotropical, com somente uma espécie Neártica. Este gênero compreende espécies pequenas (4 a 7,3 mm), caracterizadas principalmente por possuírem veia R₁ completamente coberta por sétulas na superfície dorsal, presença de cerda orbital proclinada nos machos e fêmur mediano com ctenídio. Além disso, algumas espécies apresentam asa com mácula escura entre a parte apical das veias R₂₊₃ e C. A revisão taxonômica do gênero resultou em 34 espécies válidas, sendo que sete destas são novas para a ciência. Nephochaetopteryx calida não pertence ao gênero, N. shannoni foi considerada sinônimo júnior de N. flavipalpis, e N. linharensis sinônimo júnior de N. pallidifacies. Uma chave para os espécimes machos de todas as espécies válidas (com exceção de N. juquiana) foi apresentada, bem como ilustrações detalhadas da terminália e esternito 5 dos machos. Além disso, é proposta também uma hipótese de relacionamento filogenético para o gênero, baseada em 24 caracteres da morfologia externa dos machos adultos, principalmente da terminália. Foram utilizados 32 táxons terminais, sendo 29 de Nephochaetopteryx. A monofilia do gênero foi corroborada, e uma nova sinapomorfia foi proposta dentro do contexto de Sarothromyiini: presença de lóbulo mediano no esternito 5 dos machos. Baseado na análise filogenética, o gênero foi dividido em quatro grupos de espécies: angustifrons, biculcita, cyaneiventris e pallidiventris. O grupo angustifrons é o grupo irmão do clado formado pelas demais espécies.

Revisão taxonômica das espécies brasileiras do gênero Argoravinia (Diptera : Sarcophagidae)

A família Sarcophagidae apresenta distribuição cosmopolita e compreende cerca de 2.600 espécies descritas, com aproximadamente 800 espécies registradas para a região Neotropical. Argoravinia Townsend, 1917 é um gênero de Sarcophagidae (Diptera, Oestroidea) pertencente à subfamília Sarcophaginae com espécies Neotropicais e Neárticas, com exceção de duas que foram introduzidas nas Ilhas Marshal (região Australasiana/Oceânica). O gênero compreende espécies de tamanho pequeno a médio (4 - 9,5 mm), com R₁ cerdosa na metade basal e tronco da veia R₂₊₃, na face ventral, com uma ou mais cerdas, apódema do ducto ejaculatório muito desenvolvido, falo com processo mediano longo e sinuoso e base do estilo lateral livre. O gênero Argoravinia era composto por somente duas espécies até que o gênero Raviniopsis Townsend, 1918, com quatro espécies, foi considerado sinônimo de Argoravinia no catálogo da família Sarcophagidae. Sendo assim, o gênero compreende atualmente seis espécies formalmente descritas: A. alvarengai Lopes, 1976; A. aurea (Townsend, 1918); A. brasiliana Lopes, 1976; A. candida (Curran, 1928); A. rufiventris (Wiedemann, 1830) e A. timbarensis (Lopes, 1988); no entanto, somente quatro destas são registradas para o Brasil. No presente estudo, as espécies brasileiras do gênero Argoravinia são revisadas e redescritas: A. alvarengai, A. aurea, A. brasiliana e A. rufiventris. Duas espécies novas são descritas para o estado do Pará: A. catiae sp. nov. e A. paraensis sp. nov. A. alvarengai é registrada pela primeira vez para o estado do Pará e A. rufiventris é um novo registro para os estados da Bahia e Maranhão. São apresentadas chaves de identificações para os espécimes machos e fêmeas, ilustrações da morfologia da terminália de machos e fêmeas, fotos de microscopia eletrônica de varredura , além de mapas de distribuição geográfica.

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.

Suppression of populations of Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), with a novel blowfly trap

The Australian sheep blowfly, Lucilia cuprina initiates more than 85% of fly strikes on sheep in Australia with an estimated average annual cost of A$280 million to the Australian wool industry. LuciTrap® is a commercially available, selective trap for L. cuprina consisting of a plastic bucket with multiple fly entry cones and a synthetic attractant. The impact of LuciTrap on populations of L. cuprina on sheep properties in five Australian states was evaluated by comparing L. cuprina populations on paired properties with and without LuciTraps over seasons when significant fly populations could be expected. Twenty-four comparisons (trials) were conducted over four years. During times of ‘higher fly density’ (when the 48 h geometric mean of trap catches on the control property was greater than five L. cuprina), the overall geometric mean trap catches for control and trapped properties differed significantly (P<0.001) with mean trap catches of 19.4 and 7.74 L. cuprina respectively. The selectivity of the LuciTrap was confirmed with 59% of all trapped flies being L. cuprina. Chrysomya spp. and Calliphora spp. constituted 9.3% and 1.1% of the catches with a variety of other flies (mainly Sarcophagidae and Muscidae) providing the remainder (31%). L. sericata was only trapped in Tasmania and made up 7.7% of the Lucilia spp. catch in this State. Seventy-two percent of the trapped L. cuprina were female. The deployment of LuciTrap on sheep properties at one trap per 100 sheep from the beginning of the anticipated fly season suppressed the populations of L. cuprina by 60% compared to matched control properties. The LuciTrap is a selective and easy to use fly trap and constitutes an effective, non-insecticidal tool for use in integrated management programs for L. cuprina.

The phylogeny and evolutionary timescale of muscoide (Diptera: Brachycera: Calyptratae) inferred from mitochondrial genomes

Muscoidea is a significant dipteran clade that includes house flies (Family Muscidae), latrine flies (F. Fannidae), dung flies (F. Scathophagidae) and root maggot flies (F. Anthomyiidae). It is comprised of approximately 7000 described species. The monophyly of the Muscoidea and the precise relationships of muscoids to the closest superfamily the Oestroidea (blow flies, flesh flies etc) are both unresolved. Until now mitochondrial (mt) genomes were available for only two of the four muscoid families precluding a thorough test of phylogenetic relationships using this data source. Here we present the first two mt genomes for the families Fanniidae (Euryomma sp.) (family Fanniidae) and Anthomyiidae (Delia platura (Meigen, 1826)). We also conducted phylogenetic analyses containing of these newly sequenced mt genomes plus 15 other species representative of dipteran diversity to address the internal relationship of Muscoidea and its systematic position. Both maximum-likelihood and Bayesian analyses suggested that Muscoidea was not a monophyletic group with the relationship: (Fanniidae + Muscidae) + ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)), supported by the majority of analysed datasets. This also infers that Oestroidea was paraphyletic in the majority of analyses. Divergence time estimation suggested that the earliest split within the Calyptratae, separating (Tachinidae + Oestridae) from the remaining families, occurred in the Early Eocene. The main divergence within the paraphyletic muscoidea grade was between Fanniidae + Muscidae and the lineage ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)) which occurred in the Late Eocene

Identificación y descripción de los principales insectos rastreros asociados al cultivo de marañon (Anacardium occidentale L.), orgánico y convencional en León, Nicaragua

El marañón (Anacardium occidentale L).) Es originario del noroeste de Brasil, este árbol pertenece a la familia de las Anacardiáceas y al género Anacardium. Actualmente en la zona de occidente de Nicaragua, se han incrementado las plantaciones de marañón manejado bajo dos modalidades orgánicas y convencionales, estas plantaciones se encuentran en manos de pequeños productores los cuales presentan una serie de problemas fitosanitarios. El presente estudio se realizó con el objetivo de identificar y describir el rol de los principales insectos rastreros asociados al cultivo del marañón orgánico y convencional. Este trabajo se realizó durante el período entre Agosto 2009 y Marzo del 2010, en la comarca de Chacraseca, del departamento de León, para esto se seleccionaron tres fincas orgánicas y tres fincas convencionales de 0.7 ha cada una, se realizaron muestreos semanales de insectos en cada finca, se seleccionaron cinco sitios por finca, en estos se realizaron capturas manuales de especímenes a través de trampas de caída libre (Pitfall traps). Todos los insectos colectados en el campo se llevaron al laboratorio para su respectiva identificación. Los resultados obtenidos en este estudio fueron los siguientes: encontrándose de manera general, un mayor número total de insectos en las parcelas de marañón convencional que en la orgánica , por el contrario, se encontró un mayor número de familias de insectos en las parcelas orgánicas que en las parcelas convencionales , se identificaron las principales familias de insectos asociados al cultivo del marañón entre ellos: Acrydidae, Coreidae, Cydnidae, Largidae, Carabidae, Curculionidae, Scarabaeidae, Tenebrionidae, Noctuidae, Sarcophagidae, Apidae, Formicidae, Tiphiidae, Vespidae y Arañas. Se encontró que los órdenes Orthóptera, Hemíptera, Coleóptera, Lepidóptera, Díptera, Hymenóptera y Aránea fueron los organismos más comunes encontrados en el marañón orgánico y convencional, aunque los órdenes Hemíptera, Coleóptera e Hymenóptera son los que en mayor número fueron encontrados en ambos tipos de parcelas. Este es el primer estudio en Nicaragua donde se compara la diversidad de insectos en parcelas de marañón orgánico y convencional, además, esta es la primera vez que se identifican las principales familias, géneros y especies de insectos asociados al cultivo de marañón orgánico y convencional.

Satellite flies (Leucophora personata, Diptera : Anthomyiidae) and other dipteran parasites of the communal bee Andrena agilissima (Hymenoptera : Andrenidae) on the island of Elba, Italy

Solitary and presocial aculueate Hymenoptera are parasitized by a range of dipteran species in the families Axithomyiidae, Bombyliidae, Conopidae, Phoridae, and Sarcophagidae that are likely to impact on their hosts. We undertook a study over several years of a univoltine and communal bee, Andrena agilissima, and its main dipteran parasites, in particular the satellite fly Leucophora personata (Diptera: Anthomyiidae). Behavioural and ecological data were collected from one nesting aggregation of the host bee on the island of Elba, Italy, from 1993 to 2003, and from a foraging site of the bee, ca 5 km from the nesting aggregation. Other Diptera associated with A. agilissmia at the field site were the bee fly Bombylius fimbriatus (Bombyliidae), the conopid fly Zodion cinereum (Conopidae), and the scuttle fly Megaselia andrenae (Phoridae). The phenology of the Diptera broadly overlapped with that of their host across the season of activity (end of April and all of May). Diurnal activity patterns differed slightly; L. personata in particular was active at the host's nesting site before A. agilissima. Female satellite flies also showed a range of behaviours in gaining entry to a host nest. We summarize published data on this and other Leucophora species that parasitize Andrena host bees. Host bees returning to their nests occasionally undertook zig-zag flight manoeuvres if followed by a satellite fly that were generally successful in evading the fly. Satellite flies that entered a nest, presumably to oviposit, were less likely to remain therein if another host bee entered the same nest, suggesting that one advantage to communal nesting for this host is a reduction in brood cell parasitism by L. personata. We provide the first clear evidence for parasitism by a Zodion of any Andrena host. Both L. personata and M. andrenae concentrated their parasitic activities in the zone of the host nesting aggregation with highest nest densities. Three of the Diptera, L. personata, B. fimbriatus, and Z. cinereum, seemed to have extremely low rates of parasitism whilst that of M. andrenae appeared low. Though they have refined parasitic behaviour that allows them to gain entry into host nests (L. personata, B. fimbriatus, and M. andrenae) or to parasitize adults (Z. cinercum), these parasites seem not to impact upon the dynamics of the host A. agilissima at the nesting aggregation, and the host possesses traits to reduce parasitism.

First evidence of insect attraction by a Southern Hemisphere Splachnaceae: The case of Tayloria dubyi Broth. in the Reserve Biosphere Cape Horn, Chile

The moss Tayloria dubyi (Splachnaceae) is endemic to the subantarctic Magallanes ecoregion where it grows exclusively on bird dung and perhaps only on feces of the goose Chloephaga picta, a unique habitat among Splachnaceae. Some species of Splachnaceae from the Northern Hemisphere are known to recruit coprophilous flies as a vector to disperse their spores by releasing intense odors mimicking fresh clung or decaying corpses. The flies land on the capsule, and may get in contact with the protruding mass of spores that stick to the insect body. The dispersal strategy relies on the spores falling off when the insect reaches fresh droppings or carrion. Germination is thought to be rapid and a new population is quickly established over the entire substrate. The objectives of this investigation were to determine whether the coprophilous T. dubyi attracts flies and to assess the taxonomic diversity of the flies visiting this moss. For this, fly traps were set up above mature sporophyte bearing populations in two peatlands on Navarino Island. We captured 64 flies belonging to the Muscidae (Palpibracus chilensis), Tachinidae (Dasyuromyia sp) and Sarcophagidae (not identified to species) above sporophytes of T. dubyi, whereas no flies were captured in control traps set up above Sphagnum mats nearby.

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.

Comparative cytogenetic study in Muscidae flies

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