Pigs vs people: the use of pigs as analogues for humans in forensic entomology and taphonomy research.

Most studies of decomposition in forensic entomology and taphonomy have used non-human cadavers. Following the recommendation of using domestic pig cadavers as analogues for humans in forensic entomology in the 1980s, pigs became the most frequently used model cadavers in forensic sciences. They have shaped our understanding of how large vertebrate cadavers decompose in, for example, various environments, seasons and after various ante- or postmortem cadaver modifications. They have also been used to demonstrate the feasibility of several new or well-established forensic techniques. The advent of outdoor human taphonomy facilities enabled experimental comparisons of decomposition between pig and human cadavers. Recent comparisons challenged the pig-as-analogue claim in entomology and taphonomy research. In this review, we discuss in a broad methodological context the advantages and disadvantages of pig and human cadavers for forensic research and rebut the critique of pigs as analogues for humans. We conclude that experiments using human cadaver analogues (i.e. pig carcasses) are easier to replicate and more practical for controlling confounding factors than studies based solely on humans and, therefore, are likely to remain our primary epistemic source of forensic knowledge for the immediate future. We supplement these considerations with new guidelines for model cadaver choice in forensic science research.

Molecular phylogeny of Chondrocyclus (Gastropoda: Cyclophoridae), a widespread genus of sedentary, restricted-range snails.

The genus Chondrocyclus Ancey, 1898 contains the majority of southern African members of the Cyclophoridae, a large family of operculate land snails. We present the first molecular phylogeny of the genus based on two mitochondrial genes (16S and CO1) and complement this with an appraisal of morphological characters relating to the shell and soft parts. Worn shells on which some descriptions and records of different species were based appear to be indistinguishable morphologically, creating taxonomic confusion. We show that Chondrocyclus s.l. underwent two major radiations, one Afromontane and the other largely coastal. Accordingly, we recommend a revision recognising two genera. Chondrocyclus s.s. contains four monophyletic lineages, each characterized by a combination of morphological features. The Afromontane group is shown to be a species complex; relationships within this complex could not be resolved due to insufficient DNA sequence data. The molecular data confirms the monophyly of seven currently recognised species and provides evidence for at least twelve undescribed species; the morphological data are broadly consistent with this finding. The morphological data suggest that the two species from countries to the north of South Africa should be removed from the genus, and that Chondrocyclus sensu lato is endemic to South Africa. The historical biogeography of this group of microhabitat specialists with poor dispersal abilities contributes an additional, phylogenetically independent taxon to our understanding of the processes generating biodiversity in southern Africa, a natural laboratory for palaeobiogeography. All taxa are narrow-range endemics, underlining the importance of conserving South Africa's threatened forest habitats.

Species identification of adult African blowflies (Diptera: Calliphoridae) of forensic importance.

Necrophagous blowflies can provide an excellent source of evidence for forensic entomologists and are also relevant to problems in public health, medicine, and animal health. However, access to useful information about these blowflies is constrained by the need to correctly identify the flies, and the poor availability of reliable, accessible identification tools is a serious obstacle to the development of forensic entomology in the majority of African countries. In response to this need, a high-quality key to the adults of all species of forensically relevant blowflies of Africa has been prepared, drawing on high-quality entomological materials and modern focus-stacking photomicroscopy. This new key can be easily applied by investigators inexperienced in the taxonomy of blowflies and is made available through a highly accessible online platform. Problematic diagnostic characters used in previous keys are discussed.

Forensic entomotoxicology revisited-towards professional standardisation of study designs.

Forensic entomotoxicology is the use of insects as evidence of whether a toxicant is present in an environment such as a corpse, river or landscape. The earliest overtly forensic study was published in 1977, and since then, at least 63 papers have been published, most of them focused on the detection of toxicants in insects or on effects of toxicants on diverse insect indicator taxa. A comprehensive review of the published literature revealed various inconsistencies between studies that could be addressed by introducing standard protocols for such studies. These protocols could include selecting widespread and common model organisms (such as Lucilia sericata, Calliphora vicina, Chrysomya megacephala and Dermestes maculatus) and model toxicants (e.g. morphine and amitriptyline) to build up comparative databases; developing a standard matrix for use as a feeding substrate; setting guidelines for statistically adequate sample sizes; and deploying more sophisticated analytical methods from the general field of toxicology. Future studies should then be aimed at refining standardised protocols to improve experimental results, and make these results more comparable between studies.

Nocturnal oviposition behavior of blowflies (Diptera: Calliphoridae) in the southern hemisphere (South Africa and Australia) and its forensic implications.

Published research has offered contradictory evidence of the occurrence of nocturnal oviposition by carrion-breeding blowflies, a behavior that can affect the interpretation of forensic estimates of a minimum post mortem interval (minPMI) by up to 12 hours, depending on latitude and season. The majority of published studies are from the northern hemisphere. Field experiments were conducted in South Africa and Australia that extend observations to species of the southern hemisphere. Various vertebrate carrion was exposed at night in summer under different lunar phases and/or artificial lighting, and in woodland and pasture areas. Three laboratory experiments were also conducted. No nocturnal oviposition occurred outdoors in Berry, Australia, but Lucilia cuprina, Lucilia sericata and Chrysomya megacephala laid eggs outdoors at night in Grahamstown and Durban, South Africa. In laboratory experiments L. sericata, L. cuprina, Chrysomya chloropyga and Chrysomya putoria laid eggs and Calliphora augur deposited larvae under nocturnal conditions. Chrysomya albiceps and C. chloropyga laid eggs in darkness with increasing likelihood as ambient temperature increased. This study shows that nocturnal ovi/larviposition by carrion-breeding blowflies is possible in both South Africa and Australia. The forensic issue is therefore not whether nocturnal oviposition occurs, but rather whether the conditions of a particular case are more or less conducive to it. Circadian rhythms and physiological thresholds (particularly temperature and humidity) appear to act individually and in conjunction to stimulate or inhibit nocturnal laying. The significance of carcass size, freezing and handling of carcasses and comprehensive quantification for experimental design is discussed, and recommendations are made for future laboratory and case scene experiments.

Effect of temperature on development of the blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae).

The blowfly Lucilia cuprina is a primary colonizer of decaying vertebrate carrion, and its development provides a temperature-dependent clock that may be used to estimate the post-mortem interval of corpses and carcasses in medicolegal forensic investigations. This study uses the development of L. cuprina raised on a substrate of chicken liver at six constant temperatures from 18 to 33 °C to calibrate a thermal accumulation model of development for forensic applications. Development was optimal near 24 °C; above this temperature, survival of post-feeding life stages was increasingly compromised, while below it, development was increasingly retarded. The lower developmental threshold (~12 °C) and thermal summation constants of L. cuprina are distinct from those reported for Lucilia sericata, verifying that it is essential to identify African Lucilia specimens accurately when using them to estimate post-mortem intervals.

South African nose flies (Diptera, Calliphoridae, Rhiniinae): taxonomy, diversity, distribution and biology.

Background: Rhiniinae (Diptera, Calliphoridae) is a taxon of nearly 400 known species, many of them termitophilous. Approximatelly 160 valid species in 16 genera are Afrotropical, with over 60 of them occurring in South Africa. The taxonomy of this group is outdated, as most studies of the South African taxa were conducted 40 to 70 years ago (mostly by Salvador Peris and Fritz Zumpt). Published information on their biology and ecology is also scarce. New information: An annotated checklist of 73 species of Rhiniinae for South Africa was developed, based on the holdings of sixteen entomological collections in Africa, Europe and North America. Over 3,700 specimens were examined, revealing nine new species records for South Africa (Cosminaundulata Malloch, 1926, Isomyiacuthbertsoni (Curran, 1938), Rhyncomyabotswana Zumpt, 1974, R.tristis Séguy, 1933, Stomorhinaapta Curran, 1931, S.malobana (Lehrer, 2007), Thoraciteskirkspriggsi Kurahashi, 2001, Th.sarcophagoides Kurahashi, 2001 and Trichoberialanata (Villeneuve, 1920)). We propose one new combination Eurhyncomyiametzi (Zumpt, 1981) comb. nov. (= Rhyncomyametzi Zumpt, 1981)). Additionally, evidence is presented to remove Rhyncomyaviduella Villeneuve, 1927 stat. rev. from synonymy with Rhyncomyacassotis (Walker, 1849). Relevant novel biological and seasonality information, historical occurrence maps and high-definition photographs for each species are compiled.

Cryptic variation in an ecological indicator organism: mitochondrial and nuclear DNA sequence data confirm distinct lineages of Baetis harrisoni Barnard (Ephemeroptera: Baetidae) in southern Africa.

BACKGROUND: Baetis harrisoni Barnard is a mayfly frequently encountered in river studies across Africa, but the external morphological features used for identifying nymphs have been observed to vary subtly between different geographic locations. It has been associated with a wide range of ecological conditions, including pH extremes of pH 2.9-10.0 in polluted waters. We present a molecular study of the genetic variation within B. harrisoni across 21 rivers in its distribution range in southern Africa. RESULTS: Four gene regions were examined, two mitochondrial (cytochrome c oxidase subunit I [COI] and small subunit ribosomal 16S rDNA [16S]) and two nuclear (elongation factor 1 alpha [EF1α] and phosphoenolpyruvate carboxykinase [PEPCK]). Bayesian and parsimony approaches to phylogeny reconstruction resulted in five well-supported major lineages, which were confirmed using a general mixed Yule-coalescent (GMYC) model. Results from the EF1α gene were significantly incongruent with both mitochondrial and nuclear (PEPCK) results, possibly due to incomplete lineage sorting of the EF1α gene. Mean between-clade distance estimated using the COI and PEPCK data was found to be an order of magnitude greater than the within-clade distance and comparable to that previously reported for other recognised Baetis species. Analysis of the Isolation by Distance (IBD) between all samples showed a small but significant effect of IBD. Within each lineage the contribution of IBD was minimal. Tentative dating analyses using an uncorrelated log-normal relaxed clock and two published estimates of COI mutation rates suggest that diversification within the group occurred throughout the Pliocene and mid-Miocene (~2.4-11.5 mya). CONCLUSIONS: The distinct lineages of B. harrisoni correspond to categorical environmental variation, with two lineages comprising samples from streams that flow through acidic Table Mountain Sandstone and three lineages with samples from neutral-to-alkaline streams found within eastern South Africa, Malawi and Zambia. The results of this study suggest that B. harrisoni as it is currently recognised is not a single species with a wide geographic range and pH-tolerance, but may comprise up to five species under the phylogenetic species concept, each with limited pH-tolerances, and that the B. harrisoni species group is thus in need of taxonomic review.

Molecular systematics of the African snake family Lamprophiidae Fitzinger, 1843 (Serpentes: Elapoidea), with particular focus on the genera Lamprophis Fitzinger 1843 and Mehelya Csiki 1903.

The snake family Lamprophiidae Fitzinger (Serpentes: Elapoidea) is a putatively Late Eocene radiation of nocturnal snakes endemic to the African continent. It incorporates many of the most characteristic and prolific of Africa's non-venomous snake species, including the widespread type genus Lamprophis Fitzinger, 1843 (house snakes). We used approximately 2500 bases of mitochondrial and nuclear DNA sequence data from 28 (41%) of the approximately 68 recognised lamprophiid species in nine of the eleven genera to investigate phylogenetic structure in the family and to inform taxonomy at the generic level. Cytochrome b, ND4 and tRNA gene sequences (mitochondrial) and c-mos sequences (nuclear) were analysed using Maximum Likelihood, Bayesian Inference and Maximum Parsimony methods. The genus Mehelya Csiki, 1903 was paraphyletic with respect to Gonionotophis Boulenger, 1893. To address this, the concept of Gonionotophis is expanded to include all current Mehelya species. The genus Lamprophis emerged polyphyletic: the enigmatic Lamprophis swazicus was sister to Hormonotus modestus from West Africa, and not closely related to its nominal congeners. It is moved to a new monotypic genus (Inyoka gen. nov.). The remaining Lamprophis species occur in three early-diverging lineages. (1) Lamprophis virgatus and the widely distributed Lamprophis fuliginosus species complex (which also includes Lamprophis lineatus and Lamprophis olivaceus) formed a clade for which the generic name Boaedon Duméril, Bibron & Duméril, 1854 is resurrected. (2) The water snakes (Lycodonomorphus) were nested within Lamprophis (sensu lato), sister to Lamprophis inornatus. We transfer this species to the genus Lycodonomorphus Fitzinger, 1843. (3) We restrict Lamprophis (sensu strictissimo) to a small clade of four species endemic to southern Africa: the type species of Lamprophis Fitzinger, 1843 (Lamprophis aurora) plus Lamprophis fiskii, Lamprophis fuscus and Lamprophis guttatus.

Morphology and identification of first instars of African blow flies (Diptera: Calliphoridae) commonly of forensic importance.

Scanning electron microscopy images of the first instars of Calliphora croceipalpis Jaennicke, 1876; Chrysomya chloropyga (Wiedemann, 1818); Chrysomya marginalis (Wiedemann, 1830); and Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) are presented for the first time, and the following morphological structures are documented: pseudocephalon, antenna, maxillary palpus, facial mask, labial lobe, thoracic and abdominal spinulation, spiracular field, posterior spiracles, and anal pads. Light microscopy photographs and line illustrations are provided for the cephaloskeleton in lateral and ventral views, and the "ectostomal sclerite" and "chitinized teeth" of the cephaloskeleton are recognized as integral parts of the mouthhooks. New diagnostic features of the cephaloskeleton and the spinulation of the abdominal segments are described. These results allow refinement, clarification, and correction of earlier descriptions, which are reviewed. The relative taxonomic importance of various morphological characters of the first instars of necrophagous blow flies is discussed, and details of the cephaloskeleton and the spinulation of the abdominal segments are highlighted as the characters most useful for species identification. Finally, a key for identifying first instars of common African carrion blow flies is provided.

Revision of the Afrotropical genus Fainia Zumpt, 1958, with notes on the morphology of Rhiniidae subfamilies (Diptera, Oestroidea).

The taxonomy and diversity of Fainia Zumpt, 1958, an exclusive Afrotropical genus, had not been reviewed recently. The genus included six nominal species, but the status of several of them was debated. Identification of most Fainia species depends on characters of the male terminalia; females are poorly known and, in several cases, are not adequately diagnosed. We conducted a taxonomic revision of the genus and generated identification tools. Based on the study of type material and specimens available in entomological collections in Africa and Europe, we recognise here three of the six species as valid (F. albitarsis (Macquart, 1846), F. elongata (Bezzi, 1908) and F. inexpectata Zumpt, 1973). We also provide an identification key to both sexes, redescriptions of the species, updated distribution records and high resolution photographs of males' and females' habitus and male terminalia. The description of Fainia kagerana Lehrer, 2007a nom. nud. is an invalid nomenclatural act in terms of ICZN Article 13.1.1. Based on examinations of their holotypes, F. sambura Lehrer, 2008 syn. nov. is proposed as a junior synonym of F. albitarsis; F. kirinyaga Lehrer, 2007b syn. nov. is proposed as a junior synonym of F. inexpectata; and Fainia giriama Lehrer, 2007b is moved from the genus Fainia to the genus Rhinia Robineau-Desvoidy, as Rhinia giriama (Lehrer, 2007b) comb. nov.. We propose two apomorphies that support the status of the subfamily Rhiniinae.

An accidental but safe and effective use of Lucilia cuprina (Diptera: Calliphoridae) in maggot debridement therapy in Alexandria, Egypt.

The calliphorid fly, Lucilia cuprina (Wiedemann), is known to cause serious malign myiasis in animals, whereas its sibling species Lucilia sericata (Meigen) is commonly a carrion breeder and is used in maggot debridement therapy (MDT). The current study reports an accidental involvement of L. cuprina in MDT in Alexandria, Egypt, that has proved to be safe and effective. In November 2008, the laboratory colonies of L. sericata (the species regularly used in MDT) at the Faculty of Science, Alexandria University were renewed by Lucilia flies collected as third instar larvae on exposed rabbit carcasses. Flies from the new colonies were successfully used to heal the diabetic foot wounds of two patients at Alexandria Main University Hospital. Analysis of DNA sequences and adult and larval morphology then revealed that these flies were and still are L. cuprina. Breeding of this species in carrion in Alexandria is a new record. Despite the safety of this strain of L. cuprina in MDT, entomologists rearing blow flies for the purpose of wound debridement should regularly maintain high quality assurance of their species' identity to avoid possible clinical complications that may result from the introduction of an unexpected and invasive species to their laboratory colonies.

A new genus and three new species of South African Cicadettini (Hemiptera: Cicadidae: Cicadettinae).

Ingcainyenzane irhiniensis n. gen., n. sp. and Ingcainyenzane nolukhanyoensis n. gen., n. sp. are described from Eastern Cape and Ingcainyenzane umgeniensis n. gen., n. sp. is described from KwaZulu-Natal, South Africa. Notes on its biology of the species and a key to species of the genus are also provided.

Afromelampsalta, a new genus, a new species, and five new combinations of African cicadettine cicadas (Hemiptera: Cicadidae: Cicadettinae).

Afromelampsalta n. gen. is described for African species currently assigned to the genera Melampsalta Kolenati, 1857. Cicadetta Kolenati, 1857 and Pauropsalta Goding Froggatt, 1904, and the new species Afromelampsalta luteofasciata n. gen., n. sp. is described. Afromelampsalta aethiopica (Distant, 1905) n. comb., A. cadisia (Walker, 1850) n. comb. and A. leucoptera (Germar, 1830) n. comb. are reassigned from Melampsalta to Afromelampsalta n. gen., A. limitata (Walker, 1852) n. comb. is transferred from Cicadetta Kolenati, 1857 and A. mimica (Distant ,1907) n. comb. is transferred from Pauropsalta Goding Froggatt, 1904 to Afromelampsalta n. gen. Notes on the biology of the new species, a description of the exuvia of A. mimica n. comb., and a key to the species of African Cicadettini are provided.

Habitat requirements affect genetic variation in three species of mayfly (Ephemeroptera, Baetidae) from South Africa.

This study investigates genetic diversity in three species of Ephemeroptera, one eurytopic and therefore widespread (Afroptilum sudafricanum) and two stenotopic and thus endemic (Demoreptus natalensis and Demoreptus capensis) species, all of which co-occur in the southern Great Escarpment, South Africa. Mitochondrial DNA was analysed to compare the genetic diversity between the habitat generalist and the two habitat specialists. Afroptilum sudafricanum showed no indication of population genetic structure due to geographic location, while both Demoreptus species revealed clear genetic differentiation between geographic localities and catchments, evident from phylogenetic analyses and high FST values from AMOVA. In addition, the phylogenetic analyses indicate some deeper haplotype divergences within A. sudafricanum and Demoreptus that merit taxonomic attention. These results give important insight into evolutionary processes occurring through habitat specialisation and population isolation. Further research and sampling across a wider geographic setting that includes both major mountain blocks of the Escarpment and lowland non-Escarpment sites will allow for refined understanding of biodiversity and associated habitat preferences, and illuminate comparative inferences into gene flow and cryptic speciation.

Phylogeny, biogeography and classification of the snake superfamily Elapoidea: a rapid radiation in the late Eocene.

The snake superfamily Elapoidea presents one of the most intransigent problems in systematics of the Caenophidia. Its monophyly is undisputed and several cohesive constituent lineages have been identified (including the diverse and clinically important family Elapidae), but its basal phylogenetic structure is obscure. We investigate phylogenetic relationships and spatial and temporal history of the Elapoidea using 94 caenophidian species and approximately 2300-4300 bases of DNA sequence from one nuclear and four mitochondrial genes. Phylogenetic reconstruction was conducted in a parametric framework using complex models of sequence evolution. We employed Bayesian relaxed clocks and Penalized Likelihood with rate smoothing to date the phylogeny, in conjunction with seven fossil calibration constraints. Elapoid biogeography was investigated using maximum likelihood and maximum parsimony methods. Resolution was poor for early relationships in the Elapoidea and in Elapidae and our results imply rapid basal diversification in both clades, in the late Eocene of Africa (Elapoidea) and the mid-Oligocene of the Oriental region (Elapidae). We identify the major elapoid and elapid lineages, present a phylogenetic classification system for the superfamily (excluding Elapidae), and combine our phylogenetic, temporal and biogeographic results to provide an account of elapoid evolution in light of current palaeontological data and palaeogeographic models. © The Willi Hennig Society 2009.

Effect of the killing method on post-mortem change in length of larvae of Thanatophilus micans (Fabricius 1794) (Coleoptera: Silphidae) stored in 70% ethanol.

It is recommended that insect larvae collected for forensic purposes should be killed using the same method as was used to create existing models for rate of development. Certain killing methods have been shown to be preferable because they cause less distortion of the specimens, but these are not always practicable in a particular case, and so a method of correcting for effect of killing method is required. Larvae of all instars of Thanatophilus micans (Fabricius 1794) (Coleoptera: Silphidae) were measured and then killed by immersion in ethanol, immersion in hot water or freezing. Samples were re-measured immediately after death, then stored in excess 70% ethanol and re-measured after 1 week and again after 4 weeks. The change in length was significantly different from zero in all samples (t = -9.07022, p < 0.001). An analysis of covariance showed that instar, killing method and storage time all had a significant effect on the change in length. The results showed that T. micans larvae have a great potential for change in length during storage but that the change is not predictable, as the magnitude and sign of the change are variable.

Development of Thanatophilus micans (Fabricius 1794) (Coleoptera: Silphidae) at constant temperatures.

Thanatophilus micans is capable of finding corpses at least as quickly as most fly species and, as the most widespread species of the Silphidae in Africa, offers a useful model for estimating post-mortem interval. Larvae were reared at ten constant temperatures from 15 degrees C to 35 degrees C and their length measured at 4, 8, or 12-h intervals depending on their instar. Length generally increased with increased rearing temperature, but decreased at extremely high temperatures. Note was made of the age at which individuals progressed past developmental milestones. Development took longer at lower temperatures. These results are presented as a combined isomegalen and isomorphen diagram. Developmental constants were generated for each milestone using major axis regression. Developmental threshold values did not differ significantly between milestones. Development took longer than in blow flies, but was faster than in Dermestidae. The three models presented here, therefore, cover an important time frame in estimating minimum PMI once fly larvae have matured to the point of leaving a corpse, and, therefore, provide a tool that was not previously available to forensic entomologists.

The uses of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) in forensic entomology.

Chrysomya megacephala (Fabricius, 1794) occurs on every continent and is closely associated with carrion and decaying material in human environments. Its abilities to find dead bodies and carry pathogens give it a prominence in human affairs that may involve prosecution or litigation, and therefore forensic entomologists. The identification, geographical distribution and biology of the species are reviewed to provide a background for approaches that four branches of forensic entomology (urban, stored-product, medico-criminal and environmental) might take to investigations involving this fly.

A molecular phylogeny of the cicadas (Hemiptera: Cicadidae) with a review of tribe and subfamily classification.

A molecular phylogeny and a review of family-group classification are presented for 137 species (ca. 125 genera) of the insect family Cicadidae, the true cicadas, plus two species of hairy cicadas (Tettigarctidae) and two outgroup species from Cercopidae. Five genes, two of them mitochondrial, comprise the 4992 base-pair molecular dataset. Maximum-likelihood and Bayesian phylogenetic results are shown, including analyses to address potential base composition bias. Tettigarcta is confirmed as the sister-clade of the Cicadidae and support is found for three subfamilies identified in an earlier morphological cladistic analysis. A set of paraphyletic deep-level clades formed by African genera are together named as Tettigomyiinae n. stat. Taxonomic reassignments of genera and tribes are made where morphological examination confirms incorrect placements suggested by the molecular tree, and 11 new tribes are defined (Arenopsaltriini n. tribe, Durangonini n. tribe, Katoini n. tribe, Lacetasini n. tribe, Macrotristriini n. tribe, Malagasiini n. tribe, Nelcyndanini n. tribe, Pagiphorini n. tribe, Pictilini n. tribe, Psaltodini n. tribe, and Selymbriini n. tribe). Tribe Tacuini n. syn. is synonymized with Cryptotympanini, and Tryellina n. syn. is synonymized with an expanded Tribe Lamotialnini. Tribe Hyantiini n. syn. is synonymized with Fidicinini. Tribe Sinosenini is transferred to Cicadinae from Cicadettinae, Cicadatrini is moved to Cicadettinae from Cicadinae, and Ydiellini and Tettigomyiini are transferred to Tettigomyiinae n. stat from Cicadettinae. While the subfamily Cicadinae, historically defined by the presence of timbal covers, is weakly supported in the molecular tree, high taxonomic rank is not supported for several earlier clades based on unique morphology associated with sound production.