Necrophagous Blowflies Research Articles

Can Monitoring Data of Forensically Important Blow Flies Be Transferred Between Cities?

ABSTRACTThe use of monitoring data to build prediction models for the abundance and activity of necrophagous blow flies is common practice in forensic entomology, but its advantages and disadvantages are still debated. A frequently asked question is the transferability of such species prediction models. So far no study has examined whether the assumption of low transferability of these data between cities and seasons holds true. In the present study, we evaluate whether models calibrated with a specific training data set from a specific place and time can be transferred to other data sets for different time periods and locations. We developed models using five different algorithms to predict the activity and abundance of four forensically relevant blow fly species (Calliphora vicina Robineau‐Desvoidy, Lucilia ampullacea Villeneuve, Lucilia caesar (Linnaeus), Lucilia sericata (Meigen)). The training data set was obtained from a single city, and the transferability of the models was evaluated using monitoring data from this and three other cities. The geographic transferability of the models was confirmed for all algorithms, but only for two species, C. vicina and L. sericata, and for two of the four cities. Lucilia caesar and L. ampullacea were rare in the test data set, and their species‐specific adaptation to environmental parameters was not captured by the models. Cities that did not work differed from the training data set in terms of climate and habitat features. To build generalised predictive models of blow fly abundance and activity, we need training data sets based on monitoring data from different regions, seasons and years to cover a wide range of environmental conditions. This is essential for describing and predicting natural variability.

On the influence of environmental factors on the oviposition activity of necrophagous flies

Abstract1. Locating an optimal oviposition site can be a challenging task for female insects, especially when dealing with a patchy, unpredictable and ephemeral food source such as carrion. Understanding the biotic and abiotic parameters that influence the oviposition behaviour of necrophagous flies is not just of great biological importance but also essential for their application in legal investigations.2. In this study, we monitored the oviposition activity of necrophagous flies (Calliphoridae, Sarcophagidae) using mouse carcasses in an urban (city) and a rural (mixed forest) habitat in Frankfurt/Germany over a 2‐year period.3. Over 240 sampling days, 220,963 larvae of 4 blow fly species and 1 flesh fly were sampled. The most abundant species was the blow fly Lucilia ampullacea, followed by its family members Calliphora vicina and Lucilia caesar, the flesh fly Sarcophaga caerulescens and Lucilia sericata. Up to seven environmental parameters were statistically significant predictors for a colonisation of the carcasses, leading to unique patterns of seasonal and daily oviposition activity for all five species.4. Overall, the analysis showed that the seasonal adaption (the phenology of each species), the habitat (rural vs. urban) as well as temperature are the most important factors influencing the oviposition behaviour and activity of necrophagous blow flies and flesh flies.

Spatio-temporal distribution and habitat preference of necrophagous Calliphoridae based on 160 real cases from Switzerland

Necrophagous blowflies (Diptera: Calliphoridae) are of great importance particularly during investigations of suspicious deaths. Many studies have analyzed the distribution of blowflies based on pig experiments and baited trapping; however, data from real case scenarios are rarely used. In this article, the distribution of blowflies found during investigations of 160 real cases during 1993–2007 in Switzerland is evaluated based on habitat, altitude, and season. Ten species of blowflies were present in 145 out of the 160 cases. The most common species was Calliphora vicina, which occurs throughout the year and was present in 69 % of all cases. Lucilia sericata, Calliphora vomitoria, and L. caesar were identified among the rest of the flies as species of great forensic importance mainly due to their distributional patterns. After a comparison with a similar dataset from Frankfurt, Germany, some surprising differences were determined and discussed. The biggest discrepancies between our dataset and the German dataset were in the occurrences of L. sericata (30 % vs. 86 %, respectively), Phormia regina (5 % vs. 43 %), and L. ampullacea (1 % vs. 45 %). The life-history strategies and intraspecific behavioral variability of blowflies remain understudied, although they can be essential for an unbiased approach during a death investigation. Further research and comparison of occurrence patterns across the area of distribution of blowflies are therefore needed and recommended.

Intrapuparial Development and Age Estimation of Calliphora grahami (Diptera: Calliphoridae) for Postmortem Interval Estimation.

Calliphora grahami (Aldrich, 1930) (Diptera: calliphoridae) is a forensically important blow fly that is widely distributed across Asia, North America, Russia, and Mexico. Calliphora grahami is frequently found on corpses during the spring, autumn, and winter seasons. It is among the early colonizers of cadavers during the cold season, and sometimes, the only necrophagous blow fly on cadavers. Therefore, this species is of forensic significance, although very few studies have explored the application of its intrapuparial age for PMI estimation. This study aimed to examine the intrapuparial development of C. grahami and establish a method for estimating its intrapuparial age. Herein, the C. grahami puparia were studied under six different temperatures (13, 16, 19, 22, 25, and 28°C) for the intrapuparial age estimation, and a total of 5776 puparia were sampled. The morphological changes were divided into 11 stages based on the 1) development of legs and wings, 2) differentiation of the head, thorax, and abdomen, 3) growth and color of the bristles, and 4) color changes of the compound eyes. The corresponding time of each stage was determined. Moreover, the observation and classification of individual morphological features, including compound eyes, antennae, mouthparts, thorax, abdomen, legs, and wings was used to improve the precision of intrapuparial age estimation. The findings of this study provide important information on the use of C. grahami intrapuparia to estimate the minimum postmortem interval (PMImin).

First Life Table of Synthesiomyia nudiseta (Diptera: Muscidae) From Palearctic Region for Analyzing the Effect on Its Dispersal Ability.

To better understand the population dynamics and dispersal ability of insect species, it is often helpful to derive a life table containing fundamental demographic data. The aim of this study was to determine a life table for the predatory necrophagous species Synthesiomyia nudiseta (van der Wulp, 1883) on a pig liver diet and under controlled laboratory conditions (29.5 ± 2. 5°C, RH 50 ± 15%, and a photoperiod of 12:12). This species has medical and veterinary importance and its distribution extends in tropical and subtropical areas and now it has been established in the southwestern of Europe. The mean adult longevity was 36. 18 ± 2. 06 d and the net reproduction rate, R, was 27.65 offspring/female, the mean generation time, T, was 22. 09 d, the finite rate of increase, λ, was 1. 16 d-1, and the intrinsic rate of increase, r, was 0. 15 d-1. These results indicate that S. nudiseta cannot be considered an r-strategist as the most common synanthropic necrophagous blowflies due to its predatory behavior; however, its invasive and colonist abilities are discussed. This is the first life table study of this species from Palearctic region to analyze the effect of its dispersal ability.

Blow flies, synanthropy and sex ratio: Are the deviations in the sex proportion linked to human transformation of landscapes?

In most species, several factors like time of emergence, age at maturation, reproductive life span, survival of males and females, mating behavior, differential resource use, and migration patterns may affect the adult sex ratio. Anthropogenic landscape transformation is known to change diversity, favoring colonization by exotic species but other populational parameters, such as the sex ratio, have not been assessed. The aim of the present study was to describe the sex-ratio patterns of adult necrophagous blow flies captured using carrion-baited traps along habitats representing different levels of human impact. We describe the sex bias for four species: the exotic Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria, and the native Cochliomyia macellaria. Three types of habitats were selected: highly anthropized (urbanized), moderately anthropized (rural), and native forest (natural) within a humid subtropical ecoregion in Northeastern Argentina. We found an overall trend to female bias among the four species when considering the total number of each sex. However, our study showed a changing sex-ratio pattern along the gradient of human influence. Our results indicate that a higher likelihood of trapping more males in unsuitable habitats seems to be widespread among blow flies. Urban exploiters, such as Ch. megacephala and Ch. putoria, locally shifted their trends, becoming male-biased in natural habitats. The opposite trend was detected in the urban avoider Co. macellaria (which shifted to male-biased in urban habitats). The exception was Ch. albiceps, whose sex proportion did not shift to a male-biased sex ratio. The results of this study highlight the changing sex-ratio patterns displayed by Calliphoridae in response to different conditions along a human-influence gradient. The biological traits and underlying mechanisms promoting the intraspecific changes of the sex ratio are discussed.

Assemblages of Necrophagous Blow Flies (Diptera: Calliphoridae) in Four Contrasting Ecoregions of Los Angeles County, California, United States.

An ecological study to evaluate necrophagous species richness, abundance, and diversity was conducted in four contrasting ecoregions of Los Angeles County, California, United States: a highly anthropized area, two moderately populated areas, and a sparsely populated area. Our hypothesis states that there will be higher abundance of exotic species in greatly anthropized ecoregions and higher diversity of native calliphorid flies in sparsely populated sites. A total of 2,883 blow flies belonging to 12 species were captured from the four ecoregions. The exotic Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) was the most abundant species, representing 72.84% of all Calliphoridae collected. The highest abundance was in the highly anthropized ecoregion. Differences of the blow fly assemblages across environmentally and anthropically different ecoregions were found, and that the relative abundance of the exotic species increased with anthropization. Our results suggest that both anthropization and elevation alter the structure of blow fly assemblages, modifying the abundance and species richness of Calliphoridae. The results of this study support the hypothesis of biotic homogenization, since we found a higher proportional abundance of exotic species and lower diversity of native blow flies in ecoregions heavily modified by human action. Additionally, the hypothesis of intermediate disturbance was supported as the highest diversity was found in the ecoregion with moderate anthropization mixed with natural elements. This is the first study to conduct an intensive survey of the Calliphoridae assemblages in Los Angeles County, which can be potentially used as a tool in human and veterinary health and forensic sciences.

Benefits of heterospecific aggregation on necromass: influence of temperature, group density, and composition on fitness-related traits.

Necrophagous blowflies (Diptera: Calliphoridae) such as Calliphora vicina, a cold-tolerant species, and Lucilia sericata, a warm-adapted species, are pioneer carrion-breeder. Although these two species have different temperature preferences, larvae aggregate actively and often feed simultaneously on carrion. The hypothesis to be tested was that L. sericata benefits from the association with C. vicina at lower temperatures (i.e., 15 °C) and that C. vicina derives greater benefits from this association at higher temperatures (i.e., 28 °C). Therefore, both species were raised at these two constant temperatures from first instars to adults under three different conditions: monospecific low-density, monospecific high-density, and heterospecific high-density. The time until larval migration, surface area of puparia, and survival rates were determined for each condition. Differences between these fitness-related traits were found between species, temperatures, group densities, and species compositions. At 28 °C, C. vicina larvae bred in heterospecific groups migrated significantly earlier and in higher numbers than that under same density conspecific conditions, with a lower mortality rate. At 15 °C, both species benefited from high-density heterospecific associations, expressed by faster development and larger puparia. In conclusion, necrophagous larvae benefited from heterospecific aggregations at suboptimal temperatures by adapting their migration time to that of the faster species. Since temperature changes throughout the day and over the year, the beneficiary of such a collective association also changes. The costs involved and deviations to the temperature-size rule highlight the complexity of the carrion ecosystem.

New approach to application of mid-infrared photoacoustic spectroscopy in forensic analysis: Study with the necrophagous blow fly Chrysomya megacephala (Diptera: Calliphoridae)

Insects can provide clues in a variety of ways to assist in criminal investigations. The FTIR-PAS technique has been successfully used to assess the cuticular chemical profiles of insect samples from different groups and for several goals. However, until now, it has never been used to evaluate samples of forensic interest, despite providing faster results, compared to the methods currently used. In this study, mid-infrared photoacoustic spectroscopy was employed to assess the cuticular chemical profiles of different stages of development of the blow fly Chrysomya megacephala sampled from two distinct populations. The results showed that this technique enabled detection of significant differences between the main vibrational modes of the chemical bonds present in the cuticles of the two populations and the different stages of development of the fly. The method enables identification of the age of individuals collected at the crime scene, as well as the distinction of different populations. Therefore, this methodology could assist in forensic investigations, in both estimating the Postmortem Interval and determining the location where the crime occurred, or whether the body had suffered some type of translocation. The technique provides high reproducibility and fast analysis, so its application for analysis of C. megacephala is a viable option in forensic crime investigations.

Development of Necrobia rufipes (De Geer, 1775) (Coleoptera: Cleridae) under constant temperatures and its implication in forensic entomology

After the death of humans or animals, the odors released at different stages of decay attract various insects, and other arthropods, to the corpses. Therefore, the development of insects, and other arthropods present on corpses, can be assessed to estimate the minimum postmortem interval since death. In general, necrophagous blow flies are the insects that first colonize corpses. With progressing decay, other necrophagous and predatory insects arrive at the corpses, which will develop on or around these either by feeding directly on the corpses or by prey on other immature insects. Beetles (Coleoptera) mainly arrive at the corpses during the later stages of decay, and play important roles in cases with longer postmortem intervals. Necrobia rufipes (De Geer, 1755) (Coleoptera: Cleridae) is an important stored-product species with world-wide distribution. Moreover, it is also a forensically important insect species. At temperatures of 22, 25, 28, 31, 34, and 36°C (±0.5°C), the developmental periods from egg to adult were 113.20±2.96, 66.16±3.22, 50.61±1.95, 38.26±2.48, 37.97±2.40, and 31.20±2.11 days, respectively. In vivo measurements obtained the morphological indexes of larvae. The growth curve and the equation of the relationship between development time, body lengths, and mesonotum widths were simulated. The isomorphen diagram model, the isomegalen diagram model, and the thermal summation model were established. In addition, the widths of head capsules and pronota of larvae at different instars were determined by cluster analysis. Classifiers were created and validated by linear discriminant analysis.

Collective exodigestion favours blow fly colonization and development on fresh carcasses

Necrophagous flies breeding on carcasses face high selection pressures and therefore provide interesting opportunities to study social adaptations. We postulated that gregariousness in necrophagous blow fly larvae is an adaptive response to the environmental constraints of fresh carcasses. Cooperation is indeed believed to be key to the global success of social species. To test this idea, the development of Lucilia sericata (Diptera: Calliphoridae) larvae growing on low- or high-digestibility food substrate (control or trypsin-added ground beef muscle, respectively) at different larval densities was monitored. Results showed that larvae developed faster and had decreased mortality at high than low larval density. Furthermore, aggregation had no deleterious effect on the morphological characteristics (e.g. size) of postfeeding larvae and adult flies. We concluded that increased density positively affected population fitness, which is a conclusion consistent with the predictions of the Allee effect. Compared with those fed on regular food, larvae fed on high-digestibility food had reduced mortality and faster development on average. From these results, we postulated that collective exodigestion might be an adaptive response allowing blow flies to colonize fresh carcasses before the arrival of other insects and the multiplication of microbes. This hypothesis is consistent with the idea that cooperation may enable species to expand their niches.

A preliminary study about the spatiotemporal distribution of forensically important blow flies (Diptera: Calliphoridae) in the area of Bern, Switzerland

To assess the species composition of necrophagous blow flies (Diptera: Calliphoridae) in the area around the federal city of Switzerland, Bern, sampling with baited bottle traps was performed over the course of 34 weeks in 2014. Six locations ranging from urban to forest habitats were sampled weekly or rather biweekly in the winter period. 5580 individuals belonging to 16 species were identified with Lucilia sericata as the most dominant and frequent species, followed by Calliphora vicina. While most individuals were found in the urban habitats, species richness was highest in the forest. Species richness and Chao–Shen entropy estimator peaked in most locations in the summer. In winter only two species were documented (C. vicina, Calliphora vomitoria). The species Lucilia illustris was found to be an indicator species for summer and autumn, while C. vomitoria was found to be an indicator species for the forest location. Entomological cases of the same time period conducted in the Institute of Forensic Medicine Bern were included to compare the species composition. Six blow fly species were found on human bodies which are in line with the monitoring. Of these, L. sericata, C. vicina and Protophormia terraenovae were the most frequent.

Description of Larval Instars To Fill a Gap in Forensic Entomology: The Larvae of Paralucilia pseudolyrcea (Diptera: Calliphoridae).

A fundamental assumption of forensic entomology for estimating the postmortem interval is that insect species are accurately identified, which depends on diagnostic morphological characters. Larvae of the blow fly Paralucilia pseudolyrcea (Mello, 1969) (Diptera: Calliphoridae) were sampled from four corpses in the state of Paraná, Brazil, but despite the forensic importance of this species, morphological data for the identification of its larval instars are lacking, limiting its usefulness in such cases. Thus, the main goal of this study was to describe the larval instars of P. pseudolyrcea. The material was obtained from a colony established by larvae collected from a corpse of a murder case. Overall, the distribution of spines is a key character for identifying this species in the first, second and third instars. Other characteristics, such as the presence of an accessory oral sclerite, the small cirri, the number of lobes of the anterior spiracle and the morphology of posterior spiracles, separates P. pseudolyrcea from other necrophagous blow flies. The detailed morphological description provided here facilitates the identification of larval instars of P. pseudolyrcea and their differentiation from those of other calliphorid species.

The effect of antifreeze (ethylene glycol) on the survival and the life cycle of two species of necrophagous blowflies (Diptera: Calliphoridae)

Entomotoxicology involves the analysis of the presence and the effects of toxicological substances in necrophagous insects. Results obtained by entomotoxicological studies may assist in the investigation of both the causes and the time of death of humans and animals. Ethylene glycol (EG) is easy to purchase, sweet and extremely toxic. It may be consumed accidentally or purposefully, in an attempt to cause death for suicidal or homicidal intent. Several cases report fatalities of humans and animals. The present study is the first to examine the effects of EG on the survival, developmental rate and morphology of two blowfly species, (Diptera: Calliphoridae) typically found on corpses and carcasses: Lucilia sericata (Meigen) and L. cuprina (Wiedemann). Both species were reared on substrates (beef liver) spiked with three different concentrations of EG that could cause death in either a human or cat: 1/2LD50 (T1), LD50 (T2), 2LD50 (T3), in addition to a control treatment (C) with no EG.Results of this research show that: a) both species are unable to survive if reared on a food substrate spiked with the highest concentration of EG (T3), while lower and medium concentrations (T1, T2) affect, but not prevent, the survival and the completion of the life cycle of such species; b) adults of L. sericata eclose only in C and T1, while adults of L. cuprina in both C, T1, T2; however, c) the developmental time of both species reared in T1 and T2 is statistically slower than the control; d) the body length of the immatures of both of the species reared in T1 and T2 is statistically smaller than the control.

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.

First documentation of early preimaginal stages of the blowfly Bengalia (Diptera: Calliphoridae)

Adult blowflies of the large tropical genus Bengalia present a peculiar predacious behaviour, while breeding habits and morphology of their preimaginal instars have remained almost entirely unknown. Light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were successfully adopted for the first documentation of morphology of eggs and first instar larvae of two species of Bengalia obtained in West Bengal, India. General egg morphology resembles the ground plan known from necrophagous blowflies, with similar form and structure of median area, hatching pleats and micropyle. The structure of the chorion, with subducted central parts of hexagons, is most similar to eggs of parasitic Pollenia and Rhinophoridae. Morphology of the first instar larvae of Bengalia is entirely different from known larvae of necrophagous blowflies. The complex anterior part of the cephaloskeleton in first instar Bengalia is unique within the Calyptratae, although the pseudocephalic sensory organs resemble those found in parasitic species of Calliphoridae and Sarcophagidae with a free-living first instar larva penetrating the soil environment. However, larval biology in Bengalia and in particular the function of their unique mouthhooks need further studies with an integrated field and laboratory approach.

Evaluating Sarconesiopsis magellanica blowfly-derived larval therapy and comparing it to Lucilia sericata-derived therapy in an animal model

Larval therapy is used as alternative treatment for hard-to-heal chronic and infected wounds. Lucilia sericata is the most used blowfly species. However, it has been shown recently that Sarconesiopsis magellanica larval excretions and secretions have potent antibacterial activity; this blowfly belongs to the Calliphoridae family.The present work has dealt with evaluating larval therapy using S. magellanica on wounds induced in diabetic rabbits and its action was compared to the effect induced by L. sericata. Twelve New Zealand White rabbits (Oryctolagus cuniculus) were used; they were divided into 4 groups, the first two being treated with larval therapy derived from both aforementioned necrophagous blowflies, an antibiotic was used in the third and the fourth was used as control. All the animals were wounded on the back and infected with Pseudomonas aeruginosa and Staphylococcus aureus.Samples of the secretion from each animal’s infected wound were taken and sown on blood agar. The colony forming units were then counted. The PUSH scale was used for the macroscopic evaluation of the wounds.Bacterial control was encountered 48h post-treatment in the treatments involving larval therapy and to a lesser extent with the antibiotic. Likewise, wound debridement was quicker and more efficient with larval therapy compared to the antibiotic group; however, wound closing time was 23 days in all treatments. The group treated with S. magellanica larvae had relatively quicker evolution until the proliferation phase and the start of maturation, even though there were no significant differences between both blowfly species evaluated here regarding treatments by the end of the treatment period.The present study has validated the diabetic rabbit model for inducing chronic wounds regarding larval therapy and has likewise confirmed the effectiveness of S. magellanica-derived larval therapy as an alternative for curing and healing wounds.

Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds?: Experimental data and implications for forensic entomology

This study was designed to examine the common belief that necrophagous blowflies lay their eggs in wounds.The egg-laying behaviour of Lucilia sericata was observed under controlled conditions on wet, artificially wounded or short-haired areas of rat cadavers. Flies laid significantly more eggs on the wet area and the area with short hair than on the dry area or area with long hair. No eggs were observed inside the wounds in any of the replicates.The effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae was also investigated. In low water condition, an average of 72.7±7.9% of the larvae survived and they reached a mean length of 7.5±0.6mm. In contrast, submerging eggs under a high volume of water strongly affected their survival rate (25±3.7%) and development. Similar results were observed using unfrozen pig blood instead of water.These data question the information found in the literature regarding the preferential egg-laying behaviour of Calliphorids flies in wounds.

Species identification of Middle Eastern blowflies (Diptera: Calliphoridae) of forensic importance.

The lack of reliable tools for species identification of necrophagous blowflies of the Middle East is a serious obstacle to the development of forensic entomology in the majority of countries of this region. Adding to the complexity of diagnosing the regional fauna is that species representing three different zoogeographical elements exist in sympatry. In response to this situation, a high-quality key to the adults of all species of forensically relevant blowflies of the Middle East has been prepared. Thanks to the modern technique of image-stack stereomicroscopy and high-quality entomological materials, this new key can be easily applied by investigators inexperienced in the taxonomy of blowflies. The major technical problems relating to the species identification of necrophagous blowflies of the Middle East are also discussed.

An experimental investigation into the colonization of concealed cadavers by necrophagous blowflies.

We used seven baited boxes with different combinations of access holes and odor diffusion surfaces to study the arrival of necrophagous flies. During laboratory experiments, 30 gravid Lucilia sericata females were kept in a chamber with one of the boxes. The box with the largest odor diffusion surface (99 cm2) combined with the lowest accessibility (one 1 cm2 entrance hole) was entered least (5 ± 3.7 flies per run). In contrast, the most frequently entered box (one 9 cm2 entrance hole with no additional odor diffusion surface) caught a mean of 24.6 ± 3.4 flies per run. These results indicate that 1) L. sericata entered nearly inaccessible places and 2) both odor diffusion and accessibility impacted the number of flies caught. During field experiments, the seven boxes were placed together outdoors. The box with the most entrances (ten 9-cm2 holes) caught the most flies (55.6–99.4% of the total). Only a few flies entered the other boxes. Access to the less accessible boxes (poor odor diffusion and small entrances) was also delayed. The major conclusions of the field experiments are that 1) boxes with low accessibility took longer to be accessed; 2) larger odor diffusion surfaces were more attractive to flies; and 3) flies accessed boxes more readily through larger holes than through an equivalent surface area made up of smaller holes. With these conclusions in mind, attempts to quantify the preappearance interval or to interpret the number of flies observed in indoor forensic entomology cases should be approached with caution.