The nocturnal oviposition behaviour of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications

Numerous factors may cause delayed colonisation of a corpse by blowflies, leading to a discrepancy between the entomologically determined post-mortem interval (PMI) and the time of death. Blowflies, for example, are considered to be inactive at night, however, published observations are contradictory. In the present study, several field experiments and one type of indoor experiment were conducted in summer of 2004 and 2005 in order to investigate the nocturnal ovipositional behaviour of blowflies. In the field, two types of bait, dead hedgehogs and fresh beef liver, were placed at night in different urban and rural locations in Frankfurt and in Munich, Germany. For the indoor-experiments beef liver was placed in small plastic boxes containing caged Lucilia sericata females in the evening and left overnight. At night, no ovipositon was observed in the field (n=51, T=10-24 degrees C). Nocturnal oviposition in complete darkness occurred in the plastic boxes in two of six cases (T=25 degrees C). Considering the behavioural and physiological characteristics of flies we suggest that nocturnal oviposition of blowflies appears to be unlikely under natural conditions in Central Europe but may occur under certain circumstances, such as unusual high nightly temperatures and the presence of gravid flies with an appropriate arousal threshold.     

Larval-mass effect: Characterisation of heat emission by necrophageous blowflies (Diptera: Calliphoridae) larval aggregates

Numerous Calliphoridae species larvae are necrophageous and develop on animal cadavers. During the feeding stages, a strong gregarious behaviour leads to the formation of large larval masses, allowing larvae to share digestive fluids. Furthermore, a mass of larvae emits heat, resulting in a local increase of temperature. Differences greater than 20°C between ambient and larval mass temperatures have already been observed, and the temperature of the mass can reach 50°C. Thus, larvae could benefit from this increased local temperature to speed up their development. This study focuses on the dynamic and characterisation of heat emission by Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) larval masses. Experiments were performed under controlled conditions using several ambient temperatures and different numbers of larvae. Results indicate that heat emission depends on the instar and is strongly affected by available amount of food. Furthermore, according to the experimental data, the heat emission is also relative to the weight of the larval mass, the larvae number and the local temperature. These results also demonstrate that optimum ambient-temperature values ranging between 22°C and 25°C produce maximal heat-emission per larva. Furthermore, a feedback loop, involving heat exchanges and physiological and behavioural thermoregulation processes, appears inside aggregates. In the context of forensic cases and post-mortem interval (PMI) estimation, these results indicate that heat emission can occur even with "small" masses if they are composed of high number of second stage larvae. Furthermore, particular attention should be paid on cases involving L. sericata larvae at ambient temperature ranging from 22°C to 25°C, which appears to maximise the heat-emission process.     

The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia.

The applicability of mitochondrial DNA (mtDNA) sequencing was investigated for the identification of the following forensically important species of blowflies from southeastern Australia: Calliphora albifrontalis, C. augur, C. dubia, C. hilli hilli, C. maritima, C. stygia, C. vicina, Chrysomya rufifacies, Ch. varipes and Onesia tibialis. All breed in carrion except O. tibialis, which is an earthworm parasitoid. Emphasis was placed on Calliphora species because they predominate among the carrion-breeding blowfly fauna of southern Australia and their immatures are difficult to identify morphologically. A partial sequence of the mitochondrial COII gene was determined for all species and for COI for C. albifrontalis, C. augur, C. dubia and C. stygia only. Five other species of blowflies, Chrysomya albiceps, Ch. rufifacies, Protophormia terraenovae, Lucilia illustris and L. sericata, for which sequence data were already available, were also included. Analysis of the COI and COII sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, i.e. the species within the C. augur and C. stygia groups. The molecular data support the existing species group separation of the taxa within Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach, with the analysis of additional taxa and genes, is likely to enable the reliable identification of carrion-breeding blowflies in Australia.     

Morphology of second and third instars of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a fly species of forensic importance

The morphology of the second and third instars of Chrysomya villeneuvi Patton, a fly species of forensic importance, was presented by use of light microscopy. Both instars were of hairy appearance, bearing elongated tubercles along the abdominal and caudal segments. The anterior spiracle had 13-15 papillae. Minute dark spots were observed to thoroughly cover the tubercle's surface, with 4-6 strong dark tips. Regarding the third instar, the intersegmental spines between the prothorax and mesothorax were heavily pigmented. The posterior spiracle had a thick and heavily pigmented incomplete peritreme. The surface and tip of the tubercles was covered with heavily pigmented sharp spines. The integument of the body was covered with numerous distinct net-like patches. A comparison with another well-known hairy maggot, Chrysomya rufifacies (Macquart), was discussed.     

Spread and habitat selection of Chrysomya albiceps (Wiedemann) (Diptera Calliphoridae) in Northern Italy: forensic implications

Abstract: Habitat selection exploited by Chrysomya albiceps during its initial spread in Northern Italy was analyzed in relation to landscape structure. The results of two short studies and a case report are here discussed. C. albiceps was not found on experimental pig carcasses in the urban area of Pavia. It was missing in the woody mountains surrounding Lecco, but it was found in the same area, at a lower altitude, within the typical “urban sprawl” landscape. It was then recorded in a natural reserve, among a rich carrion‐fly population. Indications coming from habitat selection suggest that C. albiceps has not yet saturated its potential ecological niche in newly colonized areas of Northern Italy. Factors like temperature, altitude, and interspecific competition can act as limiting factors, affecting habitat selection and distribution in newly colonized areas; the influence of those factors has to be taken into account for forensic purposes.     

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.     

Skipping clues: forensic importance of the family Piophilidae (Diptera)

Among the insects which are typically considered of forensic interest, the family Piophilidae (Diptera) is frequently cited because of its common occurrence on carcasses in different stages of decay. Piophilids are mainly known from the cosmopolitan species Piophila casei, which can be also a major pest for the food industry and an agent of myiasis. However, many other species of Piophilidae occur frequently on carrion, including human corpses; hence, it is essential to ensure a careful identification of specimens. Reviews of relevant published information about the Piophilidae species of potential forensic use, including recent interesting records, are presented.     

Influence of rain and malathion on the oviposition and development of blowflies (Diptera: Calliphoridae) infesting rabbit carcasses in Kelantan, Malaysia

The influence of rain and malathion on the initial oviposition as well as development of blowfly species infesting rabbit carcasses decomposing in sunlit and shaded habitats were studied over a period of 1 year in Kelantan, Malaysia. Chrysomya megacephala (Fabricius) was the most dominant species that infested the carcasses, followed by Chrysomya rufifacies (Macquart). In general, rain, depending on its intensity, delayed initial oviposition by 1–2 days and prolonged the pupation period by 1–3 days. The presence of malathion in the carcasses delayed initial oviposition by 1–3 days and prolonged the pupation period by 2–3 days. These findings deserve consideration while estimating postmortem interval since rain is a commonplace occurrence in Malaysia and malathion is one of the common poisons as an agent for choice to commit suicide.     

DNA typing for identification of some species of Calliphoridae. An interest in forensic entomology.

To determine precisely post mortem interval, larvae and puparium species found on a corpse have to be identified. Among more than 200 cases examined at the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale, two-thirds concerned corpses less than one month old. Therefore, insects from first and second screwworms are the most frequently found [1]. Some species commonly found in France, such as different Lucilia and Calliphora vicina Robineau-Desvoidy, are easily identifiable at an adult stage, but are almost impossible to differentiate at immature stages when only fragments of puparium or necrosed first instar larvae are available. For this reason, an easy and objective method of identification was thus searched by genetic analysis of these insects. Sequencing of partial gene of sub unit I of cytochrome oxydase has been used to predict restriction sites. Restriction enzyme cleavage of PCR products with Dde I allowed us to differentiate these species.     

Central pontine myelosis. Morphology and forensic importance

Central pontine myelinolysis (CPM) evidently occurs more frequently than had been assumed up to now owing to the cases that have been substantiated solely on the basis of pathological anatomy. Its genesis is still unclarified. Computed tomography and magnetic resonance methods allow detection of the foci while the affected person is still alive. They are clearly capable of regression and are not automatically accompanied by a poor prognosis. Since an iatrogenic factor (forced compensation of hyponatremia) is increasingly under discussion as the cause of CPM, the condition also has substantial significance from a forensic point of view. On the basis of a prospective study on CPM confirmed in 100 brains of alcoholics, as well as 4 further cases from the forensic autopsy material, it is shown that hyponatremia is not likely to be the sole triggering factor. The course of the condition in the cases investigated shows that the capacity for action may be preserved up to death (which has occurred for other reasons) in not very pronounced CPM. In questionable violent and other unclear deaths, CPM must also be considered a possible cause of death. The various hypotheses on its etiology in the extensive literature are compared with the findings in our own cases and discussed.     

The forensic application of allozyme electrophoresis to the identification of blowfly larvae (Diptera: Calliphoridae) in southern Australia

Most known carrion-breeding species of blowflies in southern Australia are of the genus Calliphora. The morphological similarity of the immatures of these species means that correctly identifying them poses a challenge for forensic entomologists. This study investigates the potential of allozyme analysis to assist with this task. Molecular profiles of third-instar larvae and adults representing four of these carrion-breeding species, Calliphora stygia, C. dubia, C. hilli hilli, and C. vicina, were compared at 42 allozyme loci. The two life stages were found to display almost identical allozyme profiles in each species (93% of loci were expressed in both life history stages), enabling the reliable identification of larvae in these four species. Integration of these results with data from a previous study indicates that allozyme analysis would also be suitable for rapid, species-level identification of the larvae of six other carrion-breeding Calliphora species occurring in southern Australia. This is the first report of the application of allozyme data to the identification of forensically important blowflies.     

Lucilia silvarum Meigen, 1826 (Diptera: Calliphoridae)-A new species of interest for forensic entomology in Europe

In Europe, the blowfly genus Lucilia is represented in Forensic Entomology mainly by the species L. ampullacea, L. caesar, L. illustris and L. sericata. In the US, Lucilia silvarum is rarely recorded as a carrion breeding species but usually as a more or less exclusive parasite of frogs and toads. We present three forensic cases from different European countries reporting, for the first time, L. silvarum on human bodies that were found close to lakes, wetlands, or riversides. To use this species for post-mortem interval estimations, thermal development data is needed. The first step is accurate identification by morphological and molecular means. Therefore, we analysed a 611bp part of the mitochondrial COI region for 23 specimens of L. silvarum from 9 different geographical regions, all of which give the same haplotype. Differences within the haplotype varied by up to 0.2%. Comparison between the haplotype found and those published on GenBank showed up to 1.2% variance. Moreover, we present an updated key for the morphological identification of the third larval instars of European Lucilia spp. of forensic importance, adding not only L. silvarum, but also L. cuprina which was recorded in Europe for the first time about 20 years ago.     

Genetic profiling of a central Venezuelan population using 15 STR markers that may be of forensic importance

The AmpFlSTR Identifiler kit has recently been accepted for use in DNA databasing of forensic samples in the FBI's National DNA Index System. In the present study, we used this kit to analyze the allele distribution of 15 short tandem repeat markers (STR) in individuals living in Caracas city, Venezuela. The allele frequencies of two of these STR, D2S1338 and D19S433, have not previously been reported for this or any other Latin American population. The results indicate that for the population here studied, the 15 STR tested are useful markers for paternity testing and forensic casework.     

Effects of refrigeration on the biometry and development of Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations

The aim of this study was to simulate the low temperatures that insects could experience between the time being sampled from cadavers and their arrival in the laboratory. This was in order to investigate the effect of low temperature on development of maggots. At different stages of development, individuals of Protophormia terraenovae (Robineau-Desvoidy) reared at 24 degrees C were submitted to a temperature of 4.0+/-0.5 degrees C for a period varying from 1 to 10 days. Independent of the stage of development at which the insects were refrigerated, the treatment induced significant changes on the duration of development. The effect of low temperature on the developmental time between the return to 24 degrees C to adult emergence depended on the larval stage that was refrigerated. When first instar larvae and prepupae were refrigerated, the time to emergence at 24 degrees C decreased with an increase of duration of the refrigeration period. Time to emergence increased under the same conditions when second instar larvae and pupae were refrigerated. These results indicate that keeping larvae of P. terraenovae at 4 degrees C does not just simply lead to a cessation of metabolism but disturbs the regular development. Ten days of cooling induced an error in estimating post-mortem interval (PMI) of more than 6h.     

Multivariate analysis ('forensiometrics')--a new tool in forensic medicine. Differentiation between firearm-related homicides and suicides.

A forensiometric model to discriminate between homicidal and suicidal firearm-fatalities is presented. It is based on a survey of all 45 homicidal and 251 suicidal firearm-fatalities that were examined at the Department of Forensic Medicine in Stockholm, Sweden during the period 1983-92. The model used 15 variables to describe 19.8% of the variation in the data and its goodness of prediction, Q2, was 0.742. The variables ranked in falling order of covariation with homicide (i.e. correlation with or importance in the prediction of homicide) were: presence of other injuries than firearm-wounds, entrance wound in the front part of the chest except precordium, bullet path through clothes, entrance wound in upper extremity, female victim, entrance wound in abdomen, entrance wound in head except temples, central forehead and mouth, and entrance wound in back. The variables most correlated to suicide were: used firearm found close to victim's body, reported suicidal ideation, victim's age, presence of contact wound(s), male victim, presence of farewell letter and entrance wound in mouth. The model was thereafter validated on a test-set of 18 homicides and 84 suicides during 1993-95. All suicides and 16 homicides were classified by the model in agreement with the previous police and forensic medical examinations. Thus, the model's sensitivity to classify homicides is estimated to be 89% and its specificity 100%.     

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.     

The importance of forensic evidence for decisions on criminal guilt

Recent studies have found that the general public perceives forensic evidence to be relatively inaccurate and to involve high levels of human judgement. This study examines how important the general public finds forensic evidence by comparing decisions on guilt and punishment in criminal cases that involve forensic versus eyewitness testimony evidence and examining whether a CSI effect exists. Specifically, this experimental survey study utilized a 2 (crime type: murder or rape) × 4 (evidence type: DNA, fingerprint, victim eyewitness testimony, or bystander eyewitness testimony) ? 1 (no victim testimony for murder scenario) design, yielding seven vignettes scenarios to which participants were randomly assigned. Results indicate that forensic evidence was associated with more guilty verdicts and higher confidence in a guilty verdict. Forensic evidence did not change the expected sentence length and did not generally affect the ideal sentence length. However, for rape, respondents believed that the defendant should receive a longer sentence when forensic evidence was presented but forensic evidence did not alter likely sentence that respondents expected the defendant to receive. The results of this study did not support a CSI effect. Overall, this study suggests that forensic evidence – particularly DNA – has a stronger influence during the verdict stage than the sentencing stage.