James F. Wallman

Using COI barcodes to identify forensically and medically important blowflies

Abstract The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658‐bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura‐two‐parameter distance model and a neighbour‐joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0–0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458–9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.

The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera : Calliphoridae) in southeastern Australia

The applicability of mitochondrial DNA (mtDNA) sequencing was investigated for the identification of the following forensically important species of blowflies from southeastern Australia: Calliphora albifrontalis, C. augur, C. dubia, C. hilli hilli, C. maritima, C. stygia, C. vicina, Chrysomya rufifacies, Ch. varipes and Onesia tibialis. All breed in carrion except O. tibialis, which is an earthworm parasitoid. Emphasis was placed on Calliphora species because they predominate among the carrion-breeding blowfly fauna of southern Australia and their immatures are difficult to identify morphologically. A partial sequence of the mitochondrial COII gene was determined for all species and for COI for C. albifrontalis, C. augur, C. dubia and C. stygia only. Five other species of blowflies, Chrysomya albiceps, Ch. rufifacies, Protophormia terraenovae, Lucilia illustris and L. sericata, for which sequence data were already available, were also included. Analysis of the COI and COII sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, i.e. the species within the C. augur and C. stygia groups. The molecular data support the existing species group separation of the taxa within Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach, with the analysis of additional taxa and genes, is likely to enable the reliable identification of carrion-breeding blowflies in Australia.

Molecular systematics of Australian carrion-breeding blowflies (Diptera : Calliphoridae) based on mitochondrial DNA

Carrion-breeding blowflies have substantial ecological and forensic importance. Because morphological recognition of their immatures is difficult, sequencing of the mtDNA of these flies may assist with their identification. Molecular phylogenetic analysis based on DNA sequences can also clarify evolutionary relationships. In this study, the mitochondrial genes CO1, CO2, ND4 and ND4L were sequenced for 34 species of blowflies, among which are almost all species known or suspected to breed in carrion in Australia. The resulting sequences were analysed using parsimony and maximum-likelihood Bayesian techniques. The results showed that the combination of these four genes should identify most species reliably, although some very closely related taxa could still be misdiagnosed. The data also helped clarify the life histories of Calliphora centralis Malloch, 1927, C. fuscofemorata Malloch, 1927 and C. gilesi Norris, 1994, which have hitherto only been suspected carrion breeders, and revealed that the current subgeneric assignment of taxa within Calliphora Robineau-Desvoidy, based on morphology, requires revision. Unexpectedly, both Chrysomya rufifacies (Macquart, 1843) and Lucilia cuprina (Wiedemann, 1830) were paraphyletic; each probably comprises two distinct species. The application of a molecular-clock approach to the study of the evolutionary divergence of the carrion-breeding blowflies suggests that the speciation of at least the endemic Australian taxa may have been the result of increasing aridification in Australia during the last five million years.

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.

DNA-based identification of forensically important Australian Sarcophagidae (Diptera).

The utility of the forensically important Sarcophagidae (Diptera) for time since death estimates has been severely limited, as morphological identification is difficult and thermobiological histories are inadequately documented. A molecular identification method involving the sequencing of a 658-bp ‘barcode’ fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 85 specimens, representing 16 Australian species from varying populations, was evaluated. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining phylogenetic tree generated. All species were resolved as reciprocally monophyletic, except Sarcophaga dux. Intraspecific and interspecific variation ranged from 0.000% to 1.499% (SE = 0.044%) and 6.658% to 8.983% (SE = 0.653%), respectively. The COI ‘barcode’ sequence was found to be suitable for the molecular identification of the studied Australian Sarcophagidae: 96.5% of the examined specimens were assigned to the correct species. Given that the sarcophagid fauna is poorly described, it is feasible that the few incorrectly assigned specimens represent cryptic species. The results of this research will be instrumental for implementation of the Australian Sarcophagidae in forensic entomology.

Third-instar larvae of common carrion-breeding blowflies of the genus Calliphora (Diptera : Calliphoridae) in South Australia

Detailed morphological study was conducted on the third-instar larvae of seven common carrion-breeding species of blowflies of the genus Calliphora Robineau-Desvoidy (Diptera : Calliphoridae) found in South Australia. The larval morphology of five species is revised: C. stygia (Fabricius),C. dubia (Macquart), C. augur (Fabricius), C. hilli hilli Patton and C. vicina Robineau-Desvoidy, while that of C. albifrontalis Malloch and C. maritima Norris is described for the first time. Examination of features neglected in descriptions by previous workers, particularly cuticular spinulation, has provided characters that make it possible to identify all species. An illustrated key is provided. Overall, substantial morphological distinctness exists only at the species-group level. Morphological differences at this level largely support species-group and subgeneric arrangements previously proposed for these taxa, as well as the separate status of C. vicina. However, the sister species within the C. stygia- and augur-groups are very difficult to separate, confirming the need for molecular identification in certain cases.

Identification of forensically important Chrysomya (Diptera: Calliphoridae) species using the second ribosomal internal transcribed spacer (ITS2)

The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. In contrast to most previous studies, the utility of a nuclear rather than mitochondrial genetic marker was investigated to solve this problem. The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) was amplified and sequenced from all nine Chrysomya species known from Australia. Difficulties encountered with direct sequencing of ITS2 for Chrysomya flavifrons necessitated cloning prior to sequencing for this species, which revealed a low level (0-0.23%) of intraindividual variation. Five restriction enzymes (DraI, BsaXI, BciVI, AseI and HinfI) were identified that were able to differentiate most members of the genus by polymerase chain reaction (PCR) restriction fragment length polymorphism (PCR-RFLP). The PCR-RFLP analysis revealed characteristic restriction profiles for all species except the closely related species pairs Chrysomya latifrons+Chrysomya semimetallica and Chrysomya incisuralis+Chrysomya rufifacies. Ch. incisuralis and Ch. rufifacies were able to be separated using the size differences resulting from amplification of the entire ITS region. The lack of intraspecific ITS2 sequence variation among eight Ch. incisuralis specimens was verified by the identical restriction profiles generated from these specimens. A DNA-based approach, such as PCR-RFLP, has the capacity to be useful for the identification of forensic entomological evidence in cases where morphological characters are unreliable.

Influence of substrate tissue type on larval growth in Calliphora augur and Lucilia cuprina (Diptera: Calliphoridae)

ABSTRACT: The size of fly larvae is an important variable in the use of these insects to estimate postmortem interval. Furthermore, the nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect of type of food substrate on larval growth in two species of forensically important Australian blowflies. After collection on sheeps liver in the laboratory, different groups of larvae of Lucilia cuprina (Wiedemann) and Calliphora augur (Fabricius) were grown on sheeps liver, meat, and brains, and their body lengths compared. Results indicated that the development of larvae fed sheeps liver was adversely affected compared with larvae fed meat and brain; they moulted later, reached maximum length more slowly and sometimes produced significantly smaller pupae. These findings, similar to those of another recent study, have obvious implications for postmortem interval determinations. Estimates may be considerably skewed if the site of collection of larvae at a death scene contains tissue types different to those used in reference experiments. We therefore recommend caution in forensic analyses that interpret crime scene data using developmental studies performed with a single type of larval food substrate.

A key to the adults of species of blowflies in southern Australia known or suspected to breed in carrion

Abstract. The reliable morphological identification of carrion‐breeding blowflies is important ecologically, as well as for medical, veterinary and forensic reasons. To date, no comprehensive key has been available to make this possible for workers in southern Australia. An illustrated key is presented to the adults of all species of blowflies (Diptera: Calliphoridae) south of 30° S known or suspected to breed in carrion (species exclusive to Queensland and/or the Northern Territory are excluded).

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)†

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.