Forensic parasitology: a new frontier in criminalistics.

Parasites are ubiquitous, diverse, and have close interactions with humans and other animals. Despite this, they have not garnered significant interest from forensic scientists, and their utility as indicators in criminal investigations has been largely overlooked. To foster the development of forensic parasitology we explore the utility of parasites as forensic indicators in five broad areas: (i) wildlife trafficking and exploitation, (ii) biological attacks, (iii) sex crimes, (iv) criminal neglect of humans and other animals, and (v) indicators of movement and travel. To encourage the development and growth of forensic parasitology as a field, we lay out a four-step roadmap to increase the use and utility of parasites in criminal investigations.

The blowfly Chrysomya latifrons inhabits fragmented rainforests, but shows no population structure.

Climate change and deforestation are causing rainforests to become increasingly fragmented, placing them at heightened risk of biodiversity loss. Invertebrates constitute the greatest proportion of this biodiversity, yet we lack basic knowledge of their population structure and ecology. There is a compelling need to develop our understanding of the population dynamics of a wide range of rainforest invertebrates so that we can begin to understand how rainforest fragments are connected, and how they will cope with future habitat fragmentation and climate change. Blowflies are an ideal candidate for such research because they are widespread, abundant, and can be easily collected within rainforests. We genotyped 188 blowflies (Chrysomya latifrons) from 15 isolated rainforests and found high levels of gene flow, a lack of genetic structure between rainforests, and low genetic diversity - suggesting the presence of a single large genetically depauperate population. This highlights that: (1) the blowfly Ch. latifrons inhabits a ~ 1000 km stretch of Australian rainforests, where it plays an important role as a nutrient recycler; (2) strongly dispersing flies can migrate between and connect isolated rainforests, likely carrying pollen, parasites, phoronts, and pathogens along with them; and (3) widely dispersing and abundant insects can nevertheless be genetically depauperate. There is an urgent need to better understand the relationships between habitat fragmentation, genetic diversity, and adaptive potential-especially for poorly dispersing rainforest-restricted insects, as many of these may be particularly fragmented and at highest risk of local extinction.

How does mass loss compare with total body score when assessing decomposition of human and pig cadavers?

Providing accurate and reliable measures of decomposition is paramount for forensic research where decomposition progress is used to estimate time of death. Mass loss is routinely used as a direct measure of biomass decomposition in ecological studies, yet few studies have analysed mass loss in a forensic context on human cadavers to determine its usefulness for modelling the decomposition process. Mass loss was examined in decomposing human and pig cadavers, and compared with other common decomposition metrics, such as total body score (TBS). One summer and one winter field decomposition experiment was conducted using human and pig cadavers, as pigs are often used as proxies for human cadavers in forensic research. The two measures of decomposition revealed two contrasting patterns of decomposition on pigs and humans, particularly in winter where TBS stabilised at similar values, but mass loss differed greatly. Mass loss was found to be faster in pigs than humans during early decomposition. Pigs lost 75% of their mass in winter, while humans lost less than 50%; however, in summer, both lost around 80% of their mass. TBS displayed similar patterns in both experiments, with TBS increasing more rapidly in pigs compared with humans but both eventually reaching similar TBS values in late decomposition. Measuring mass loss can provide additional information about decomposition progress that is missed if using TBS only. Key differences in decomposition progress between cadaver types were also observed, suggesting caution when extrapolating data from pigs to humans for forensic research and decomposition modelling.

Priority effects and density promote coexistence between the facultative predator Chrysomya rufifacies and its competitor Calliphora stygia.

Highly competitive ephemeral resources like carrion tend to support much greater diversity relative to longer-lived resources. The coexistence of diverse communities on short-lived carrion is a delicate balance, maintained by several processes including competition. Despite this balance, few studies have investigated the effect of competition on carrion, limiting our understanding of how competition drives coexistence. We investigated how priority effects and larval density influence coexistence between two blowfly species, the facultative predator Chrysomya rufifacies and its competitor Calliphora stygia, which occupy broadly similar niches but differ in their ecological strategies for exploiting carrion. We examined how adult oviposition, larval survival, developmental duration, and adult fitness were affected by the presence of differently aged heterospecific larval masses, and how these measures varied under three larval densities. We found C. rufifacies larval survival was lowest in conspecific masses with low larval densities. In heterospecific masses, survival increased, particularly at high larval density, with priority effects having minimal effect, suggesting a dependency on collective exodigestion. For C. stygia, we found survival to be constant across larval densities in a conspecific mass. In heterospecific masses, survival decreased drastically when C. rufifacies arrived first, regardless of larval density, suggesting C. stygia is temporally constrained to avoid competition with C. rufifacies. Neither species appeared to completely outcompete the other, as they were either constrained by density requirements (C. rufifacies) or priority effects (C. stygia). Our results provide new mechanistic insights into the ecological processes allowing for coexistence on a competitively intense, ephemeral resource such as carrion.

Insect abundance patterns on vertebrate remains reveal carrion resource quality variation.

Resource quality is a key driver of species abundance and community structure. Carrion is unique among resources due to its high nutritional quality, rapidly changing nature, and the diverse community of organisms it supports. Yet the role resource quality plays in driving variation in abundance patterns of carrion-associated species remains poorly studied. Here we investigate how species abundances change with a measure of resource change, and interpret these findings to determine how species differ in their association with carrion that changes in quality over time. We conducted field succession experiments using pigs and humans over two winters and one summer. We quantified the effect of total body score, an objective measure of resource change, on adult insect abundance using generalised additive models. For each species, phases of increasing abundance likely indicated attraction to a high-quality resource, and length of abundance maxima indicated optimal oviposition and feeding time. Some species such as the beetle Necrobia rufipes had a rapid spike in abundance, suggesting a narrow window of opportunity for carrion resource exploitation, while species like the wasp Nasonia vitripennis had a gradual change in abundance, indicating a wide window of resource exploitation. Different abundance patterns were also observed between species occurring on pigs and humans, suggesting cadaver type is an important aspect of resource quality. Our findings show that species abundances, unlike species occurrences, can reveal additional detail about species exploitation of carrion and provide information about how resource quality may drive competition and variation in insect community succession.

Integrative taxonomy reveals remarkable diversity in Australian Protomiltogramma (Diptera: Sarcophagidae).

Protomiltogramma Townsend is the largest and most diverse genus of miltogrammine flesh flies in Australia. However, no comprehensive taxonomic work had been completed on the Australian members of this genus in almost a century. This study presents the first taxonomic revision of all Australian species of Protomiltogramma (Sarcophagidae: Miltogramminae), completed using an integrative approach combining molecular and morphological data. Eight new species endemic to Australia are described: P. dalbiensis sp. n., P. grandis sp. n., P. incana sp. n., P. kapnos sp. n., P. nigrisensa sp. n., P. popularis sp. n., P. rubra sp. n. and P. subtilis sp. n. In addition, P. australis Malloch, 1930 is synonymised with P. cincta Townsend, 1916, syn. n. and P. mallochi Verves, 1987 is synonymised with P. laticeps Malloch, 1930, syn. n. Molecular phylogenetics is used to place the Australian Protomiltogramma among the miltogrammine genera of the world.

Field succession studies and casework can help to identify forensically useful Diptera.

Fly development rates, and to a lesser extent succession data, can be used to provide an estimate of a minimum postmortem interval (mPMI). Yet, these data are most useful when a full account of species' ecology, seasonality, and distribution is known. We conducted succession experiments on human cadavers over different seasons near Sydney, Australia, to document forensically useful information, including the pre-appearance interval for carrion flies. We also compiled a detailed record of flies identified in casework collected in 156 cases distributed across New South Wales, Australia. We then compared the occurrence of fly species from both field and casework datasets to identify any consistencies or gaps to determine how useful species might be for forensic investigations. In the field experiments, we found differences in species diversity and abundance between seasons, as well as yearly variation between two winter seasons. Most fly species we recorded ovipositing showed a 2- or 3-day delay between adult arrival and oviposition in summer, with a longer delay in winter. Species that were previously encountered in casework, such as Calliphora augur (Fabricius, 1775) and Calliphora ochracea Schiner, 1868, were confirmed as forensically useful, with their colonization behavior and seasonal preferences documented here. Although not encountered in casework, we confirmed Hemipyrellia fergusoni Patton, 1925 as a primary colonizer of human cadavers. Our study emphasizes the need to link field and casework data for a complete understanding of all aspects of a carrion fly's ecology to assist forensic investigators in mPMI estimations.

Temperature dynamics in different body regions of decomposing vertebrate remains.

The decomposition of vertebrates is controlled largely by external temperature, yet internal temperatures can also play an important role but are generally poorly documented. In this study, we compared continuous hourly temperature recordings from the mouth, under the head, right chest and right abdomen, and in the rectum of one refrigerated human and one fresh pig cadaver during 29 days of decomposition. Each cadaver differed in its internal starting temperature, thus providing two contrasting case studies for examining temperature dynamics among body regions. We used time-series analysis methods common to hydrology to reveal key differences in internal temperature dynamics. Within both cadavers, the chest region experienced the highest average temperatures, and the mouth experienced the highest maximum hourly temperature. Temperatures exceeded 30 °C inside the pig for between 40% (rectum) and 75% (chest) of the duration of the study, but for only 20% (rectum) and 35% (chest) of the time in the human. Our study provides evidence of the different thermal trajectories occurring in different body regions, and some similarities between two cadavers despite their different starting thermal conditions. These results improve our understanding of why decomposition occurs at different rates within the same cadaver, and that the location of blowfly larvae collections should be noted to improve estimates of the post-mortem interval.

Is Resource Change a Useful Predictor of Carrion Insect Succession on Pigs and Humans?

Carrion is a dynamic and nutrient-rich resource that attracts numerous insect species that undergo succession due to the rapid change in the carrion resource. Despite this process being well-understood, few studies have examined resource change as a driver of carrion insect succession, and instead have focused on the effects of time per se, or on coarse, qualitative measures such as decay stage. Here we report on three field succession experiments using pig carcasses and human cadavers encompassing two winters and one summer. We quantified the effects of resource change (measured as total body score, TBS), carrion type, initial carrion mass, ambient temperature, and season on insect species richness and community composition. We found that all variables had an effect on different taxonomic or trophic components of the insect community composition, with the exception of initial carrion mass which had no effect. We found significant positive effects of TBS on beetle species richness and composition, while fly species richness was not significantly affected by TBS, but was by ambient temperature. TBS had a significant positive effect on all trophic groups, while ambient temperature also had a significant positive effect on the necrophages and predator/parasitoids. Our study indicates that resource change, as indicated by TBS, is an important driver of carrion insect species turnover and succession on carrion, and that TBS can provide information about insect ecological patterns on carrion that other temporal measures of change cannot.

Love at first flight: wing interference patterns are species-specific and sexually dimorphic in blowflies (Diptera: Calliphoridae).

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genus Chrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females and demonstrate sexual dimorphisms in several species. These data suggest that WIPs have diversified substantially in blowflies as a result of either sexual or ecological selection.

First instar larvae of endemic Australian Miltogramminae (Diptera: Sarcophagidae).

The first instar larva of a species of the Australian endemic genus Aenigmetopia Malloch is described for the first time, along with the first instar larvae of three other Australian species representing the genera Amobia Robineau-Desvoidy and Protomiltogramma Townsend. Larval morphology was analysed using a combination of light microscopy, confocal laser scanning microscopy and scanning electron microscopy. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, modifications of thoracic and abdominal segments, anal region, spiracular field, posterior spiracles and details of the cephaloskeleton. Substantial morphological differences are observed between the three genera, most notably in the labrum and mouthhooks of the cephaloskeleton, sensory organs of the pseudocephalon, spinulation, sculpture of the integument and form of the spiracular field. The first instar larval morphology of Aenigmetopia amissa Johnston, Wallman, Szpila & Pape corroborates the close phylogenetic affinity of Aenigmetopia Malloch with Metopia Meigen, inferred from recent molecular analysis. The larval morphology of Amobia auriceps (Baranov), Protomiltogramma cincta Townsend and Protomiltogramma plebeia Malloch is mostly congruent with the morphology of Palaearctic representatives of both genera.

Contrasting insect activity and decomposition of pigs and humans in an Australian environment: A preliminary study.

Non-human vertebrate animals, primarily domestic pigs, have been widely used in forensic science research as analogues for humans due to ethical and logistical constraints. Yet the suitability of pigs to mimic human decomposition and entomological patterns remains largely untested, and explicit comparative research in this area is lacking. We compared the decomposition rates and insect communities found at pig and human remains during summer and winter at the Australian Facility for Taphonomic Experimental Research (AFTER). Pigs decomposed faster than humans, with pigs entering active decay earlier in both summer and winter, and humans undergoing desiccation rather than skeletonisation. There was also a delay in the colonisation of humans by both flies and beetles. Species richness of these necrophagous taxa was between two and five times higher during the first two weeks of decomposition on pigs compared to humans during both summer and winter. Insect species composition was also significantly different between pigs and humans in each season. We interpret our findings to mean that the difference between humans and pigs, such as their mass, diet, medical history, or their microbiomes, might be causing different decomposition processes and altered timing or production of chemical cues for insect colonisation. Although preliminary, our results suggest that pigs might not be accurate substitutes for humans in particular fields of taphonomy and forensic entomology. Our findings also have broader implications for the reliability of forensic studies using pigs as models for humans, and highlight the need to recognise intrinsic differences between animal models and humans.

Soil chemical markers distinguishing human and pig decomposition islands: a preliminary study.

The decomposition of vertebrate cadavers on the soil surface produces nutrient-rich fluids that enter the soil profile, leaving clear evidence of the presence of a cadaver decomposition island. Few studies, however, have described soil physicochemistry under human cadavers, or compared the soil between human and non-human animal models. In this study, we sampled soil to 5 cm depth at distances of 0 cm and 30 cm from cadavers, as well as from control sites 90 cm distant, from five human and three pig cadavers at the Australian Facility for Taphonomic Experimental Research (AFTER). We found that soil moisture, electrical conductivity, nitrate, ammonium, and total phosphorus were higher in soil directly under cadavers (0 cm), with very limited lateral spread beyond 30 cm. These patterns lasted up to 700 days, indicating that key soil nutrients might be useful markers of the location of the decomposition island for up to 2 years. Soil phosphorus was always higher under pigs than humans, suggesting a possible difference in the decomposition and soil processes under these two cadaver types. Our preliminary study highlights the need for further experimental and replicated research to quantify variability in soil properties, and to identify when non-human animals are suitable analogues.

Major Transitions in Cuticular Hydrocarbon Expression Coincide with Sexual Maturity in a Blowfly (Diptera: Calliphoridae).

In many animals, there is a prolonged pre-reproductive period prior to sexual maturity. To avoid premature mating attempts, it is common for phenotypic changes to occur during this period that signal the onset of reproductive viability. Among the insects, pre-reproductive phases can last for up to 50% of the adult lifespan, but little is known about the accompanying phenotypic changes that signal sexual maturity. Contact pheromones such as cuticular hydrocarbons (CHCs) may fulfil this role, as they are known to change rapidly with age in many insects. Despite this, few studies have investigated CHC development in the context of sexual maturity or considered differences in CHC development between sexes. The blowflies (Diptera: Calliphoridae) provide an ideal system for such studies because CHCs are known to change rapidly with age and likely play an important role in sexual behaviour. As such, using the small hairy maggot blowfly Chrysomya varipes, we investigate whether there are age- and sex-specific changes in CHCs over the course of adult blowfly maturation. We show that: (1) major qualitative transitions in CHC expression coincide with the onset of sexual maturity and (2) these changes occur more slowly in females - in line with their extended pre-reproductive phase. We suggest that CHCs may play an important role in signalling sexual maturity in the small hairy maggot blowfly and that this species will likely serve as a useful model for understanding the complex ontogeny of cuticular hydrocarbons in insects.

The evolution of sexually dimorphic cuticular hydrocarbons in blowflies (Diptera: Calliphoridae).

Cuticular hydrocarbons (CHCs) are organic compounds found on the cuticles of all insects which can act as close-contact pheromones, while also providing a hydrophobic barrier to water loss. Given their widespread importance in sexual behaviour and survival, CHCs have likely contributed heavily to the adaptation and speciation of insects. Despite this, the patterns and mechanisms of their diversification have been studied in very few taxa. Here, we perform the first study of CHC diversification in blowflies, focussing on wild populations of the ecologically diverse genus Chrysomya. We convert CHC profiles into qualitative and quantitative traits and assess their inter- and intra-specific variation across 10 species. We also construct a global phylogeny of Chrysomya, onto which CHCs were mapped to explore the patterns of their diversification. For the first time, we demonstrate that blowflies express an exceptional diversity of CHCs, which have diversified in a nonphylogenetic and punctuated manner, are species-specific and sexually dimorphic. It is likely that both ecological and sexual selection have shaped these patterns of CHC diversification, and our study now provides a comprehensive framework for testing such hypotheses.

First record of Miltogramminae from New Caledonia: a new species of Protomiltogramma (Diptera: Sarcophagidae).

New Caledonia is described as one of Earth's key biodiversity hotspots, with high species endemism, particularly for plants and vertebrate animals (Myers et al. 2000; Kier et al. 2009). The distinct biodiversity of this country has been attributed to the Gondwanan origins of the New Caledonian archipelago, its relative isolation from neighbouring countries (such as Australia) and the stability of its climate. These factors are suggested to have led to the accumulation of many Gondwanan species, with few instances of extinction, which has allowed for marked species diversification (Espeland Murienne 2011).

Delayed reception of live blowfly (Calliphora vicina and Chrysomya rufifacies) larval samples: implications for minimum postmortem interval estimates.

Forensic entomology evidence collected by police and mortuary staff may be delayed in getting to the entomologist. Live samples may continue developing and alter minimum postmortem interval (PMImin) estimates. This study investigated development of simulated evidential samples of Calliphora vicina Robineau-Desvoidy and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) maggots. Maggots of each species were studied in three developmental classes: "small" (late second/early third instar), "mid" (mid third instar) and "large" (late third instar). Seven replicates of 11 maggots in each class were assigned without food to four treatments: (1) 24 h at 20 °C; (2) 24 h at 4 °C; (3) 48 h at 20 °C and (4) 48 h at 4 °C. There was a significant difference in absolute length change across treatments, reflecting size for C. vicina, and interaction between size, time and temperature for Ch. rufifacies. Calliphora vicina maggots showed minimal mortality, and most second instar larvae moulted by the experiment's end. Chrysomya rufifacies showed heavy mortality and minimal moulting from the second to third instar. Only "large" Ch. rufifacies maggots kept at room temperature for 48 h pupariated. Since these results confirm that development can continue in live unfed maggot samples after their collection, it is not advisable to delay their preservation.

Body Odor and Sex: Do Cuticular Hydrocarbons Facilitate Sexual Attraction in the Small Hairy Maggot Blowfly?

Cuticular hydrocarbons (CHCs) play an important role as contact pheromones in insects, particularly in flies. However, for many fly taxa our understanding of the importance of CHCs in sexual communication is limited. Within the family Calliphoridae (blowflies), sex-specific differences in CHCs have been reported for several species, but there is no evidence that CHCs facilitate sexual behavior. In order to elucidate the function of CHCs in Calliphoridae, studies combining behavioral and chemical analyses are required. The present study used gas chromatography/mass spectrometry, along with behavioral assays, to assess whether CHCs facilitate sexual attraction in the small hairy maggot blowfly, Chrysomya varipes. The specific aims were to: 1) determine if CHCs differ between the sexes and 2) assess whether flies exhibit positive chemotaxis to CHCs of the opposite sex. Fifty-two hydrocarbons common to both sexes were identified, and quantitative differences for numerous CHCs were observed between the sexes. However, behavioral assays provided no evidence that flies were attracted to CHCs of the opposite sex, challenging the hypothesis that CHCs facilitate sexual attraction in Ch. varipes. In contrast to other blowflies, Ch. varipes males invest heavily in courtship displays and ornamentation, so we speculate that visual communication in this species may have relaxed sexual selection for chemical communication. More broadly, our findings support suggestions that CHCs may not always facilitate insect sexual communication.

Monitoring the extent of vertical and lateral movement of human decomposition products through sediment using cholesterol as a biomarker.

Due to the lack of human decomposition research facilities available in different geographical regions, the extent of movement of human decomposition products from a cadaver into various sedimentary environments, in different climates, has not been able to be studied in detail. In our study, a human cadaver was placed on the surface of a designated plot at the Australian Facility for Taphonomic Experimental Research (AFTER), the only human decomposition facility in Australia, where the natural process of decomposition was allowed to progress over 14days in the Australian summer. Sediment columns (approximately 1m deep) were collected at lateral distances of 0.25m, 0.5m, 1.0m and 2.5m in each of four directions from the centre of the torso. Plot elevation and weather data were also collected. Each sediment column was subdivided, dried and homogenised. A sample was isolated from each sediment subdivision, extracted with hexane, and the hexane extract cleaned with citrate buffer (pH 3), filtered and spiked with cholesterol-D7 internal standard. After derivatisation with BSTFA+1% TMCS, cholesterol was monitored in the samples using targeted gas chromatography tandem mass spectrometry analysis. A positive result for decomposition products was given if the cholesterol abundance in the test sample was higher than that detected in the 'control' samples of a similar substrate type collected prior to cadaver placement. Within the confines of the experimental design and the measured parameters, lateral leaching was observed over distances of up to 2.5m from the centre of the torso, which was the maximum distance tested in the study. Vertical leaching was detected to depths of up to 49cm below the ground surface. Such data can aid the development of policies related to plot sizing and sediment renewal and regeneration at other human decomposition facilities and at cemeteries. The density and distribution of cholesterol surrounding the cadaver in this study can also help forensic investigators interpret cases involving remains that have been moved or scavenged.