Key for the identification of third instar larvae of African blowflies (Diptera: Calliphoridae) of forensic importance in death investigations.

Blowfly larvae are the insects primarily responsible for the active stage of decomposition of exposed vertebrate remains and are the most frequently collected entomological evidence during forensic investigations of death. The necrophagous calliphorids in continental Africa that consistently develop on large vertebrate carrion include 11 species belonging to four genera: Calliphora, Chrysomya, Hemipyrellia and Lucilia. Most of these species are widespread in Africa and frequently reported on large animal carcasses and carrion and human corpses. A few keys have been compiled for identification of their third instar larvae, but none of them covers the complete set of taxa. Therefore, we provide a new comprehensive key with original illustrations of all taxonomically significant characters. The key is based on characters that should be easily observable even in poorly equipped local laboratories and is a reliable taxonomic tool for material collected in either urban or rural areas where synanthropic species predominate. However, it should be used with some caution in areas with relatively pristine natural habitats, where additional carrion-breeding species may occur. The publication of the key will significantly facilitate both medical and forensic entomological research and practice in Africa.

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 The taxonomy of this group is outdated, as most studies of the South African taxa were conducted 40 to 70 years ago (mostly by Salvador Peris and Fritz Zumpt).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.

A confirmed feeding attempt by the haematophagous horse fly Philoliche (Philoliche) rondani (Bertoloni, 1861) (Diptera: Tabanidae) on fresh carrion.

Background: Many flies have specially evolved feeding mechanisms to imbibe liquids of specific viscosities. Observations of feeding on atypical liquids are notable because of their rarity. New information: We report the first record of intrusive fluid feeding on vertebrate carrion by Philolicherondani.

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.

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.

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.

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.

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.

Metrological framework for selecting morphological characters to identify species and estimate developmental maturity of forensically significant insect specimens.

Accurate age estimates of immature necrophagous insects associated with a human or animal body can provide evidence of how long the body has been dead. These estimates are based on species-specific details of the insects' aging processes, and therefore require accurate species identification and developmental stage estimation. Many professionals who produce or use identified organisms as forensic evidence have little training in taxonomy or metrology, and appreciate the availability of formalized principles and standards for biological identification. Taxonomic identifications are usually most readily and economically made using categorical and qualitative morphological characters, but it may be necessary to use less convenient and potentially more ambiguous characters that are continuous and quantitative if two candidate species are closely related, or if identifying developmental stages within a species. Characters should be selected by criteria such as taxonomic specificity and metrological repeatability and relative error. We propose such a hierarchical framework, critique various measurements of immature insects, and suggest some standard approaches to determine the reliability of organismal identifications and measurements in estimating postmortem intervals. Relevant criteria for good characters include high repeatability (including low scope for ambiguity or parallax effects), pronounced discreteness, and small relative error in measurements. These same principles apply to individuation of unique objects in general.Key pointsMetrological rigour can increase in forensic entomology by selecting measurements based on their metrological qualities.Selection of high-quality features for morphological identification of organisms should consider these criteria: (1) pronounced discreteness of features (minimising group overlap or maximizing interval); (2) high repeatability of assessment (such as symmetrical width rather than asymmetrical length); (3) small relative error in measurement (selecting the physically largest continuous rigid feature for measurement).These metrological principles also apply to individuation of unique objects in general.

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.

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.

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.

Dung beetle (Coleoptera: Scarabaeoidea) assemblages in the western Italian Alps: benchmark data for land use monitoring.

BACKGROUND: Traditional agro-pastoral practices are in decline over much of the Alps (MacDonald et al. 2000), leading to shrub and tree encroachment, and this represents one of the main threats for the conservation of alpine biodiversity, as many plant and animal species are dependent on the presence of semi-natural open habitats. However, quantifying this environmental change and assessing its impact on biodiversity may be difficult, especially in the context of sparse historical survey data. The accessibility of contemporary data about local biodiversity surveys in general, and indicator taxa in particular, is an essential consideration for planning future evaluations of conservation status in the Alps and for conservation plans that use ecological indicators to monitor temporal changes in biodiversity. Dung beetles are important ecosystem service providers (Nichols et al. 2008) that have been assessed as a good ecological indicator taxon in several studies (reviewed by Nichols and Gardner 2011), and although the Alps is perhaps one of the best-studied regions in respect of dung beetles, there are still only eight readily-accessible publications. We have augmented and comprehensively reviewed the data from these publications. NEW INFORMATION: We first provide data about changes on a temporal scale of seasons in a dung beetle community in the western Italian Alps, an issue that has to be addressed in the local assemblages because it would affect regional biomonitoring and conservation research. This survey of 12 099 individuals belonging to 22 species illustrates a distinct seasonal pattern at a single site. Second, we collate the results of 13 published surveys of the presence of 46 species of dung beetles in 11 valleys in the western Italian Alps in the period from 2005 to 2012, a period of accelerated change in land use that started around 1945 (MacDonald et al. 2000). Because ten of the surveys used baited pitfall traps and four more used manual collection of specimens, the abundance data were not strictly comparable and they were therefore transformed to binary data (presence-or-absence records) with measures of sampling effort. The results illustrate both spatial variation and temporal variation at the scale of years.Because of the importance of dung beetles in agro-pastoral ecosystems and the high sensitivity of montane ecosystems to climate change, these spatially and temporally explicit data sets provide important baseline information about western Italian Alpine dung beetles for investigations of the effects of land use change under ongoing climate change scenarios.

Heat accumulation and development rate of massed maggots of the sheep blowfly, Lucilia cuprina (Diptera: Calliphoridae).

Blowfly larvae aggregate on exposed carcasses and corpses and pass through three instars before wandering from the carcass and pupating. The developmental landmarks in this process can be used by forensic entomologists to estimate the time since the insects colonised the carcass, which sets a minimum post mortem interval. Large aggregations of feeding larvae generate a microclimate with temperatures up to 15°C above ambient conditions, which may accelerate larval development and affect forensic estimates of post-mortem intervals. This study investigated the effects of heat accumulated by maggot masses of Lucilia cuprina at aggregations of 20, 50 and 100 larvae, each at incubation temperatures of 18°C, 24°C and 30°C, using body length and life stage as developmental indicators. Aggregation temperatures reached up to 18.7°C above ambient temperature, with significant effects of both size and temperature of the aggregation on the development time of its larvae. Survivorship was highest for all life stages at 24°C, which is near the developmental optimum of L. cuprina. The results of this study provide a broadly applicable method of quantifying heat accumulation by aggregations of a wide range of species of forensic importance, and the results obtained from such studies will demonstrate that ambient temperature cannot be considered the only source of heat that blowfly larvae experience when they develop on a carcass. Neglect of temperatures within larval aggregations will result in an overestimation of post-mortem intervals and thus have far-reaching medicolegal consequences.

Two new species of Xosopsaltria Kirkaldy, 1904, with a key to the species of South African pygmy bladder cicadas (Hemiptera: Cicadidae: Cicadettinae: Tettigomyiini).

Two species, Xosopsaltria brachyptera n. sp. and Xosopsaltria vitripennis n. sp., are described as new. Notes on their biology and a key to the South African pygmy bladder cicadas are also provided.

Phylogenetic radiation of the greenbottle flies (Diptera, Calliphoridae, Luciliinae).

The subfamily Luciliinae is diverse and geographically widespread. Its four currently recognised genera (Dyscritomyia Grimshaw, 1901, Hemipyrellia Townsend, 1918, Hypopygiopsis Townsend 1916 and Lucilia Robineau-Desvoidy, 1830) contain species that range from saprophages to obligate parasites, but their pattern of phylogenetic diversification is unclear. The 28S rRNA, COI and Period genes of 14 species of Lucilia and Hemipyrellia were partially sequenced and analysed together with sequences of 11 further species from public databases. The molecular data confirmed molecular paraphyly in three species-pairs in Lucilia that hamper barcode identifications of those six species. Lucilia sericata and Lucilia cuprina were confirmed as mutual sister species. The placements of Dyscritomyia and Hypopygiopsis were ambiguous, since both made Lucilia paraphyletic in some analyses. Recognising Hemipyrellia as a genus consistently left Lucilia s.l. paraphyletic, and the occasionally-recognised (sub)genus Phaenicia was consistently paraphyletic, so these taxa should be synonymised with Lucilia to maintain monophyly. Analysis of a matrix of 14 morphological characters scored for adults of all genera and for most of the species included in the molecular analysis confirmed several of these findings. The different degrees of parasitism were phylogenetically clustered within this genus but did not form a graded series of evolutionary stages, and there was no particular relationship between feeding habits and biogeography. Because of the ubiquity of hybridization, introgression and incomplete lineage sorting in blow flies, we recommend that using a combination of mitochondrial and nuclear markers should be a procedural standard for medico-criminal forensic identifications of insects.

Phylogeographic, morphometric and taxonomic re-evaluation of the river sardine, Mesobola brevianalis (Boulenger, 1908) (Teleostei, Cyprinidae, Chedrini).

The river sardine, Mesobola brevianalis (Boulenger, 1908), is the type species of Mesobola Howes, 1984. Standard phylogenetic analyses of partial sequences of the cytochrome oxidase I gene of individuals from populations across southern Africa that are currently identified as Mesobola brevianalis showed that these populations represent four genetically distinct allopatric lineages. Furthermore, Engraulicypris sardella (Günther, 1868), the type species of Engraulicypris Günther, 1894, was convincingly nested amongst these clades. These findings support synonymisation of Engraulicypris and Mesobolasyn. n.; restoration of Engraulicypris gariepinus (Barnard, 1943), stat. rev. for the lower Orange River population; description of two new species, Engraulicypris ngalalasp. n. and Engraulicypris howesisp. n. from the Rovuma and Kunene river systems, respectively; affirmation of the synonymy of Engraulicypris brevianalis (Boulenger, 1908), comb. n. sensu stricto and Engraulicypris whitei van der Horst, 1934; and restoration of Engraulicypris bredoi Poll, 1945, stat. rev. and Engraulicypris spinifer Bailey & Matthes, 1971, stat. rev. from Mesobola. Discriminant function analysis of a truss network of five traditional morphometric measurements and 21 morphometric measurements that characterised the shape of the fishes was used to seek morphological markers for the genetically distinct populations. Only Engraulicypris gariepinus was morphometrically distinctive, but live colouration differed between the lineages. Detailed taxonomic descriptions and an identification key for the species are provided.