A methodological approach to age estimation of the intra-puparial period of the forensically relevant blow fly Calliphora vicina via Fourier transform infrared spectroscopy.

Estimating the age of immature blow flies is of great importance for forensic entomology. However, no gold-standard technique for an accurate determination of the intra-puparial age has yet been established. Fourier transform infrared (FTIR) spectroscopy is a method to (bio-)chemically characterise material based on the absorbance of electromagnetic energy by functional groups of molecules. In recent years, it also has become a powerful tool in forensic and life sciences, as it is a fast and cost-effective way to characterise all kinds of material and biological traces. This study is the first to collect developmental reference data on the changes in absorption spectra during the intra-puparial period of the forensically important blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Calliphora vicina was reared at constant 20°C and 25°C and specimens were killed every other day throughout their intra-puparial development. In order to investigate which part yields the highest detectable differences in absorption spectra throughout the intra-puparial development, each specimen was divided into two different subsamples: the pupal body and the former cuticle of the third instar, that is, the puparium. Absorption spectra were collected with a FTIR spectrometer coupled to an attenuated total reflection (ATR) unit. Classification accuracies of different wavenumber regions with two machine learning models, i.e., random forests (RF) and support vector machines (SVMs), were tested. The best age predictions for both temperature settings and machine learning models were obtained by using the full spectral range from 3700 to 600 cm-1. While SVMs resulted in better accuracies for C. vicina reared at 20°C, RFs performed almost as good as SVMs for data obtained from 25°C. In terms of sample type, the pupal body gave smoother spectra and usually better classification accuracies than the puparia. This study shows that FTIR spectroscopy is a promising technique in forensic entomology to support the estimation of the minimum post-mortem interval (PMImin), by estimating the age of a given insect specimen.

The Entomopathogenic Fungus Conidiobolus coronatus Has Similar Effects on the Cuticular Free Fatty Acid Profile of Sensitive and Resistant Insects.

The mechanisms underlying the recognition of a susceptible host by a fungus and the role of cuticular compounds (CCs) in this process remain unclear; however, accumulated data suggest that this is influenced to a great degree by cuticular lipids. Two insect species differing in their sensitivity to fungal infection, viz. the highly sensitive Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) and the resistant Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), exhibited significant qualitative and quantitative changes in cuticular free fatty acid (FFA) profiles after exposure to Conidiobolus coronatus (Constantin) Batko (Entomopthorales). Despite being systematically distant, leading different lifestyles in different habitats, both insect species demonstrated similar changes in the same FFAs following exposure to the fungus (C12:0, C13:0, C14:0, C15:0, C16:1, C16:0, C18:1, C18:0), suggesting that these are involved in a contact-induced defense response. As it was not possible to distinguish the share of FFAs present in the conidia that were attached to the cuticle from the FFAs of the cuticle itself in the total number of extracted FFAs, further research is necessary.

Establishing and characterising a new cell line from Calliphora vicina (diptera: calliphoridae) fly embryonic tissues.

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

Characterization of the complete mitochondrial genome of the New Zealand parasitic blowfly Calliphora vicina (Insecta: Diptera: Calliphoridae).

In this study, the complete mitochondrial (mt) genome of the New Zealand parasitic blowfly Calliphora vicina (blue bottle blowfly) field strain NZ_CalVic_NP was generated using next-generation sequencing technology and annotated. The 16,518 bp mt genome consists of 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a 1689 bp non-coding region, similar to the two other available C. vicina and most metazoan mt genomes. Phylogenetic analysis showed that C. vicina NZ_CalVic_NP forms a monophyletic cluster with the remaining three Calliphorinae species. The complete mt genome sequence of C. vicina NZ_CalVic_NP is a resource to facilitate future species- and strain-level identification research and investigations into the evolutionary provenance within the Calliphoridae.

Cutaneous myiasis in cats and dogs: Cases, predisposing conditions and risk factors.

Two cases of cutaneous myiasis diagnosed in 2018 in Emilia-Romagna region (northern Italy) were reported. The first one, described in a domestic cat Felis silvestris catus L. (Carnivora: Felidae) and caused by Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), was the first one of this type ever reported in Italy in cats. The second one was described in a domestic dog Canis lupus familiaris L. (Carnivora: Canidae) and caused by Lucilia sericata (Meigen) (Diptera: Calliphoridae) and was unusual because it occurred in absence of lesions. An extensive literature search on cutaneous myiasis in these two domestic animal species was performed in order to draw attention to predisposing conditions and risk factors.

An Analysis of Forensically Important Insects Associated with Human Remains in Indiana, U.S.A.

Research documenting insect colonization of human remains is limited in North America, and currently nonexistent for the American Midwest. Such research is essential for forensic entomologists to identify species of research interest in a region. In this study, we collected insects from human remains in 24 cases across Indiana from June 2016 through September 2018. We analyzed species composition across scene type and season. Eight species of blow flies were collected as larvae from human remains, with Phormia regina and Lucilia sericata as the two predominant colonizers. Phormia regina was the most numerous species collected from outdoor scenes (73.6% of total collections) while L. sericata was the most numerous from the indoor scenes (60.4% of total collections). With scene types pooled, Calliphora vicina and Cochliomya macellaria were the predominant species in the fall (55.6% and 42.2%, respectively); P. regina was the dominant colonizer in the spring (68.6%); and P. regina and L. sericata were the predominant colonizers (46.5% and 44.4%, respectively) in the summer. In addition to these findings, we confirmed the first record of Lucilia cuprina colonizing human remains in Indiana having collected this species from three cases. A single adult Chrysomya megacephala was collected from an indoor scene in southern Indiana, which represents its second collection in the state. Beetles belonging to the families Staphylinidae, Silphidae, Histeridae, Cleridae, Trogidae, Dermestdae, and Nitidulidae were also collected from two outdoor scenes. This study provides important baseline data for forensic entomologists in Indiana, as well as surrounding states with similar environments.

How should living entomological samples be stored?

Sampling and storing insect evidence alive are important tasks in forensic entomology as it can impact survival and growth rates. To investigate the effect of cooling and storing of insect evidence before its arrival in the laboratory, samples of all three larval stages of the blow fly species Lucilia sericata and Calliphora vicina were analyzed. A first group was stored at room temperature and a second one in a refrigerator (~ 5 °C) for 16 h, all without air, supply of food, and sawdust. Afterwards, they were kept at 6-8 °C in a Styrofoam box for 8 h, simulating a transport situation. Mortality rate (MR) was calculated and 25% of the surviving larvae were killed and measured to check for interim growth. The remaining alive specimens were reared at 25 °C until adult's eclosion for estimating a possible storage impact on survival during later development. The results were then compared with a control which was not temporarily stored and chilled but left feeding in boxes with an air-permeable lid on food substrate at 25 °C.A 24-h temporary storage stopped the larval growth in comparison with the control especially in early larval stages in both species. A high MR of up to 100% for third instar (L3) larvae stored both at room temperature and in a cold environment without air supply was found. Oxygen supply can reduce significantly the MR at least for L3 larvae of L. sericata. Findings provide scientific evidence for the recommendation to store larval samples at cold temperatures with both oxygen and food supply. The high MR for samples of the last larval stage clearly shows the need for a fast delivery after sampling and a more sophisticated storage procedure like, e.g., providing air supply. Storing live samples at room temperature without air access should be avoided.

Predatory behavior of Synthesiomyia nudiseta larvae (Diptera: Muscidae) on several necrophagous blowfly species (Diptera: Calliphoridae).

Intraguild interactions play a prominent role in sarcosaprophagous communities, so intraspecific and interspecific competition phenomena between fly larvae take place. Synthesiomyia nudiseta is a species that has recently been recorded on human corpses in Europe, and it has been cited in forensic cases in Spain and Italy. The mature larvae are known to be facultative predators on necrophagous fly larvae, but their role as predators is unknown. The aim of this paper is to investigate the predatory behavior of S. nudiseta on the most abundant blowfly larvae in sarcosaprophagous communities in southwestern Europe (i.e., Chrysomya albiceps, Lucilia sericata, and Calliphora vicina). Intraspecific and interspecific competition experiments were carried out to study the effect of this species in the necrophagous diptera community. First-instar larvae were placed in plastic cups with the same amount of pig liver (15 g) at different densities (50, 100, 150, and 300 individuals/cup). Mortality in blowflies increased when S. nudiseta larvae were present: up to 98.40% in Ch. albiceps, 89.60% in L. sericata, and 84.93% in C. vicina. Pre-imaginal developmental time and adult wing size were analyzed and compared under each of the intraspecific and interspecific conditions; both variables were affected by competition in all cases. The implications of the presence of S. nudiseta for sarcosaprophagous community dynamics and its effect on the estimation of postmortem interval (PMI) are discussed.

Histological age estimation of the eggs of Calliphora vicina Robineau Desvoidy (Diptera: Calliphoridae).

Aging blow fly eggs can be critical to a forensic investigation, but there are currently no forensically useful timelines describing internal anatomical changes in embryological development. This is partly due to the lack of an economical, rapid and technically simple histological technique to allow mass production of slides for research and casework. We present a histological method that uses a slightly modified standard laboratory processing run with 1 h fixation in 10% formalin, 2 h softening in Molliflex and Haemotoxylin and Eosin (H&E) staining. We also present a summary of the internal anatomical changes that can be visualized using our technique in the developing eggs of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). We examined eggs from at least three different females grown at 15 °C and sampled at 6 h intervals, and eggs grown at 20 °C and sampled at 3 h intervals. Blind aging trials demonstrated that it is possible to accurately age material grown at 20 °C to within 6 h (but attempts to further narrow this interval resulted in errors in one-third of cases). It was also possible to see sufficient anatomical detail to age eggs preserved for forensic casework 5, 8, 9, 10 and 11 years previously. Additionally, we determined that section quality was improved by 5 s fixation in hot water prior to preservation in ethanol. However, hot water fixation for longer than this increased the level of section artefact.

Sexual size dimorphism in three species of forensically important blowflies (Diptera: Calliphoridae) and its implications for postmortem interval estimation.

Forensic entomologists rely on insects present in human remains to establish a minimum postmortem interval (PMImin). Blowflies have been widely used in these estimates because they lay eggs on the victim's body shortly after death and, within hours, larvae hatch and grow at predictable rates. Hence, isomegalen diagrams based on larval size and local temperature are considered good models to estimate PMImin. Still, most professional do not account for size sexual dimorphism in blowflies, which add uncertainty to predictive models by two mechanisms: (1) males and females might grow up to different sizes, and; (2) males and females might grow at different rates. In this study, we investigate sexual dimorphism and biases on the prediction of adult size in three species of blowflies (Lucilia sericata, Calliphora vicina and Calliphora vomitoria) reared under different larval densities. Estimated size range, stablished with and without sex discrimination, showed that females were larger than males in the three species. The ultimate size of adult stage, however, was more difficult to predict. C. vicina and C. vomitoria decrease as density raises but at different rates, and even males and females of the same species react differently to density increase. Adult size of L. sericata, in contrast, shows a slight increase with density. Except for C. vomitoria females, estimated size ranges are lower when species are divided by sex. Our results show that sex is an important factor to consider in PMImin estimates. Scenarios for all three species shorten their estimated size ranges when compared to databases with no sex identification. Therefore, computing data by sex raises accuracy in size based predictive models.

Biofilm infections between Scylla and Charybdis: interplay of host antimicrobial peptides and antibiotics.

PURPOSE: The aim of this study is to improve the anti-biofilm activity of antibiotics. We hypothesized that the antimicrobial peptide (AMP) complex of the host's immune system can be used for this purpose and examined the assumption on model biofilms. METHODS: FLIP7, the AMP complex of the blowfly Calliphora vicina containing a combination of defensins, cecropins, diptericins and proline-rich peptides was isolated from the hemolymph of bacteria-challenged maggots. The complex interaction with antibiotics of various classes was studied in biofilm and planktonic cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii by the checkerboard method using trimethyl tetrazolium chloride cell viability and crystal violet biofilm eradication assays supplemented with microscopic analysis. RESULTS: We found that FLIP7 demonstrated: high synergy (fractional inhibitory concentration index <0.25) with meropenem, amikacin, kanamycin, ampicillin, vancomycin and cefotaxime; synergy with clindamycin, erythromycin and chloramphenicol; additive interaction with oxacillin, tetracycline, ciprofloxacin and gentamicin; and no interaction with polymyxin B. The interaction in planktonic cell models was significantly weaker than in biofilms of the same strains. The analysis of the dose-effect curves pointed to persister cells as a likely target of FLIP7 synergistic effect. The biofilm eradication assay showed that the effect also caused total destruction of S. aureus and E. coli biofilm materials. The effect allowed reducing the effective anti-biofilm concentration of the antibiotic to a level well below the one clinically achievable (2-3 orders of magnitude in the case of meropenem, ampicillin, cefotaxime and oxacillin). CONCLUSION: FLIP7 is a highly efficient host antimicrobial system helping antibiotics to overcome biofilm barriers through persisters' sensitization and biofilm material destruction. It is promising for the treatment of biofilm infections as an adjuvant of various small-molecule antibiotics.

Effects of phase correlations in naturalistic stimuli on quantitative information coding by fly photoreceptors.

Natural visual scenes are rarely random. Instead, intensity and wavelength change slowly in time and space over many regions of the scene, so that neighboring temporal and spatial visual inputs are more correlated and contain less information than truly random signals. It has been suggested that sensory optimization to match these higher order correlations (HOC) occurs at the earliest visual stages, and that photoreceptors can process temporal natural signals more efficiently than random signals. We tested this early-stage hypothesis by comparing the information content of Calliphora vicina photoreceptor responses to naturalistic inputs before and after removing HOC by randomizing phase. Forty different, 60-s long, naturalistic sequences (NS) were used, together with randomized-phase versions of the same sequences to give pink noise (PN) so that each input pair had identical means, variances, mean contrasts, and power spectra. We measured the information content of inputs and membrane potential responses by three different methods: coherence, mutual information, and compression entropy. We also used entropy and phase statistics of each pair as measures of HOC. Responses to randomized signals generally had higher gain, signal-to-noise ratio, and information rates than responses to NS. Information rate increased with a strong, positive, linear correlation to phase randomization within sequence pairs. This was confirmed by varying the degree of phase randomization. Our data indicate that individual photoreceptors encode input information by Weber's law, with HOC within natural sequences reducing information transfer by decreasing the number of local contrast events that exceed the noise-imposed threshold. NEW & NOTEWORTHY Natural visual scenes feature statistical regularities, or higher order correlations (HOC), both in time and space, to encode surfaces, textures, and object boundaries. Visual systems rely on this information; however, it remains controversial whether individual photoreceptors can discriminate and enhance information encoded in HOC. Here we show that the more HOC the stimulus contains, the lower the information transfer rate of photoreceptors. We explain our findings by applying the Weber's paradigm of differential signal perception.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

Evaluation of blowfly larvae (Diptera: Calliphoridae) as possible reservoirs and mechanical vectors of African swine fever virus.

In 2014, highly virulent African swine fever virus (ASFV) was introduced into the Baltic States and Poland, with new cases being reported almost every week from wild boar and also from domestic pigs. Contrary to initial predictions that the disease would either die out due to the high virulence of the virus strain or spread rapidly in westerly direction, the infection became endemic and spread slowly. The unexpected disease epidemiology led to the hypothesis that hitherto unconsidered factors might contribute to virus persistence and dispersal. To check whether arthropod species feeding and developing on infected carcasses might be involved, larvae of two commonly found blowfly species, Lucilia sericata and Calliphora vicina, were experimentally bred on ASFV-infected spleen tissue. After different time intervals, developing larvae and pupae were tested for infectious virus and viral DNA. By qPCR, contamination of the blowfly larvae and pupae with ASFV-DNA could be demonstrated even after several washing steps, proving the uptake of virus during feeding in the larval stage. However, infectious virus could never be isolated. By contrast, the larvae appeared to have inactivated ASFV in the offered tissue, which might be explained by the known anti-biotic effect of salivary secretions. It is concluded that immature blowfly stages do not play a relevant role as reservoirs or mechanical vectors of ASFV.

Facultative myiasis of domestic cats by Sarcophaga argyrostoma (Diptera: Sarcophagidae), Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) in northern Italy.

We describe five cases of myiasis of domestic cats, Felis silvestris catus L. (Carnivora: Felidae), reported in 2016 in northern Italy and caused by three Diptera species: Sarcophaga argyrostoma (Robineau-Desvoidy) (Sarcophagidae), Calliphora vicina Robineau-Desvoidy and Lucilia sericata (Meigen) (Calliphoridae). Three were cases of traumatic myiasis, one by S. argyrostoma and two by L. sericata, one was a case of auricular myiasis by C. vicina and one was a case of ophthalmomyiasis caused by an association of L. sericata and C. vicina. The myiasis by S. argyrostoma is the first reported case of this species in a cat, whereas the two myiases by C. vicina are the first reported cases in cats in Italy.

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus.

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

Anti Leishmania activity of Lucilia sericata and Calliphora vicina maggots in laboratory models.

Use of sterile fly larvae (maggots) of blow flies for the treatment of many different types of skin and soft tissue wounds is called Maggot debridement therapy (MDT). The larvae of blow flies secrete a broad spectrum of compounds with diverse mechanisms of action in the gut and salivary glands called excretion/secretion (ES) products which showed to have antimicrobial activities against Gram negative and positive bacteria. Cutaneous leishmaniasis (CL) which is the common form of leishmaniasis is difficult to treat. In this study, the effect of ES from 2nd and 3rd stages of L. sericata and C. vicina larvae on in vitro Leishmania major amastigote growth in macrophage was evaluated. The effect of ES on Leishmania growth was estimated by assessing the rate of macrophage infection and the number of amastigotes per infected macrophages. In addition, the anti Leishmania activities of larval and ES of L. sericata and C. vicina on the skin lesion induced by L. major infection was evaluated in susceptible BALB/c mice. The results showed that ES of both flies reduced the number of infected macrophages; 2.6 and 1.5-fold using L. sericata ES and C. vicina ES, respectively, and inhibited amastigotes growth in macrophages; 2.03 and 1.36-fold by L. sericata ES and C. vicina ES, respectively as compared to the control group. The results showed that L. sericata ES was significantly more effective than C. vicina ES to inhibit in vitro L. major amastigotes growth, The size of lesion was significantly smaller in BALB/c mice treated with L. sericata ES than treated with C. vicina ES. The results of in vivo experiments suggested that pre-treatment with ES derived from L. sericata may have some protective effects on the development of L. major lesion. Therefore, it seems that maggot ES might be considered as a possible candidate for the treatment of cutaneous leishmaniasis.

Evaluating the Biological Cycle and Reproductive and Population Parameters of Calliphora vicina (Diptera: Calliphoridae) Reared on Three Different Diets.

This work was aimed at evaluating Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) life cycle, life tables, and reproductive and population parameters. This species is a necrophagous, synanthropic, cosmopolitan fly with broad distribution in the Neotropical region. Six successive generations from adult specimens collected in Bogotá, Colombia, were analyzed in laboratory conditions. The fly's life cycle was evaluated, using a natural diet of pig's liver (L) and two artificial diets-milk powder and egg (ME) and powdered liver (PL). The species' longest life cycle duration (in days), from egg to pupa, was obtained with the ME diet (24.6 ± 6.7) whereas the lowest value was recorded with the L diet (18.8 ± 3.6). Female adult forms achieved high longevity values, this being highest in ME (102.8 ± 3.6). The most efficient diets (L and ME) led to the following average reproductive and population values: 226.2 and 465.4 net reproductive rate (Ro), 22.2 and 47.1 cohort generation time (Tc), 0.24 and 0.13 intrinsic population growth rate (rm), and 1.27 and 1.13 finite rate of increase (λ). The set of results (in terms of relatively high average values concerning the biological characteristics analyzed) showed the species' adaptability to the environmental and nutritional conditions established in a laboratory setting, thereby enabling their colonization.

Calculating time since death in a mock crime case comparing a new computational method (ExLAC) with the ADH method.

We compared the results of calculating a minimum post-mortem interval (PMImin) in a mock crime case using two different methods: accumulated degree hours (ADH method) and a newly developed computational model called ExLAC. For the ADH method we further applied five reference datasets for the development time of Calliphora vicina (Diptera: Calliphoridae) from 5 different countries and our results confirmed the following: (1) Reference data for blowfly development that has not been sampled using a local blowfly colony should not, in most circumstances, be used in estimating a PMI in real cases; and (2) The new method ExLAC might be a potential alternative to the ADH method.

Establishment of developmental charts for the larvae of the blow fly Calliphora vicina using quantile regression.

Developmental data of necrophagous blow fly species can be used to estimate a minimum postmortem interval (PMImin) in death investigations by estimating the age of larvae sampled from the cadaver. The most important parameter used in this age estimation is the increase in larval length during growth. Larval length can be compared to species-specific reference data to get an age estimate for the larval specimen. The exploration of this type of data and the use of an appropriate statistical method are the major challenges in evaluating forensic entomological data sets. In Europe, Calliphora vicina is one of the most dominant species with forensic entomological relevance. Despite its frequency and importance, there are currently no published developmental studies for German populations of this blow fly that can be referenced for larval age estimations. This is regrettable because the geographical origin of different populations may lead to phenotypic plasticity in the same species and population-specific growth patterns that differ from published data sets. To address this shortcoming, the objective of the present research was to generate growth data for C. vicina which can be used for age estimation in German casework. We present, for the first time, local developmental data for the larval stages of German C. vicina, reared at three constant temperatures (15, 20 and 25°C) and compare the results with published studies on C. vicina development from elsewhere. To analyse the development we chose a quantile mixed effects model because of its robustness and insensitivity towards outliers. Quantile regression was developed as an extension of the linear model to estimate rates of change in all parts of the distribution of a response variable and to discover more useful predictive relationships between variables. By applying a linear quantile mixed effect model, we estimated the 0.1 and 0.9 quantile functions of the larval age for each temperature. Graphically, these quantile functions act like reference bands and therefore, plots for larval stages 1-3 can be used to estimate the age of a larva. Nevertheless, the post-feeding stage still remains difficult to describe with linear models and need to be further investigated. A comparison of the results from this present study with the currently applied methods for age estimation of C. vicina still demonstrates the importance of collecting growth rates for local blow fly populations and of standardising experimental designs and statistical analyses.