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.     

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.     

Genetic relationships between blowflies (Calliphoridae) of forensic importance.

Phylogenetic relationships among blowfly (Calliphoridae) species of forensic importance are explored using DNA sequence data from the large sub-unit (lsu, 28S) ribosomal RNA (rRNA) gene, the study includes representatives of a range of calliphorid species commonly encountered in forensic analysis in Britain and Europe. The data presented provide a basis to define molecular markers, including the identification of highly informative intra-sequence regions, which may be of use in the identification of larvae for forensic entomology. Phylogenetic analysis of the sequences also provides new insights into the different evolutionary patterns apparent within the family Calliphoridae which, additionally, can provide a measure of the degree of genetic variation likely to be encountered within taxonomic groups of differing forensic utility.     

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.     

Influence of substrate tissue type on larval growth in Calliphora augur and Lucilia cuprina (Diptera: Calliphoridae)

The size of fly larvae is an important variable in the use of these insects to estimate postmortem interval. Furthermore, the nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect of type of food substrate on larval growth in two species of forensically important Australian blowflies. After collection on sheep's liver in the laboratory, different groups of larvae of Lucilia cuprina (Wiedemann) and Calliphora augur (Fabricius) were grown on sheep's liver, meat, and brains, and their body lengths compared. Results indicated that the development of larvae fed sheep's liver was adversely affected compared with larvae fed meat and brain; they moulted later, reached maximum length more slowly and sometimes produced significantly smaller pupae. These findings, similar to those of another recent study, have obvious implications for postmortem interval determinations. Estimates may be considerably skewed if the site of collection of larvae at a death scene contains tissue types different to those used in reference experiments. We therefore recommend caution in forensic analyses that interpret crime scene data using developmental studies performed with a single type of larval food substrate.     

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.     

Carrion fly (Diptera: Calliphoridae) larval colonization of sunlit and shaded pig carcasses in West Virginia, USA

Two pig (Sus scrofta L.) carcasses were placed in sunlit and shaded plots in September 2003, and again in May 2004. Mean ambient temperatures between sunlit and shaded plots were not significantly different in either September or May, but mean ambient temperatures at sunlit and shaded plots in 2004 were significantly higher than corresponding means for sunlit and shaded plots in 2003. Mean maggot mass temperatures were significantly higher than ambient plot temperatures for all four experimental plots (i.e., sunlit and shaded carcasses in both 2003 and 2004). In addition, maggot mass temperatures on sunlit carcasses were positively, and significantly, correlated with ambient temperatures, whereas there was no significant correlation between maggot mass and ambient temperatures at shaded plots. Carcass decomposition proceeded more rapidly in 2004 in the presence of higher ambient temperatures, and sunlit carcasses decomposed faster than shaded ones in both 2003 and 2004 experiments. Phaenecia coeruleiviridis (Macquart) and Phormia regina (Meigen) third instars dominated collections on all four carcasses, but there was little temporal overlap between these species with third instars of the former dominating collections in the early portion (approximately 40%) of each experimental period (with the exception of the shaded carcass in 2004 where both species were co-dominant), and the latter assuming dominance in the latter portion (approximately 60%). Lower accumulated degree hour values were calculated for instar development on 2004 carcasses subjected to higher ambient temperatures.     

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.     

DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae).

Identifying an insect specimen is an important first step in a forensic-entomological analysis. However, diagnostic morphological criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic analysis to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely separated geographic locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.     

Responses of Lucilia sericata Meigen (Diptera: Calliphoridae) to cadaveric volatile organic compounds

Flies of the Calliphoridae Family are the most forensically important insects because of their abundance on the decedent during the first minutes following death. Necrophagous insects are attracted at a distance by a decomposing body, through the use of volatile chemical cues. We tested the possible attractive role of some volatile organic chemicals (VOCs) released by decaying cadavers, on male and female of Lucilia sericata Meigen (Diptera: Calliphoridae). Two complementary approaches were used. Electroantennography (EAG) allowed identifying the semiochemicals that are detected by the olfactory system of L. sericata. Dose-response tests with EAG showed that dimethyl disulfide (DMDS) and butan-1-ol elicited the highest responses. Behavioral assays showed that, among the VOCs tested, DMDS and butan-1-ol are attractive for L. sericata, while the other VOCs are repulsive or do not cause any behavior. Our results may have potential implications in a better understanding of attractiveness of blowflies toward a corpse.     

Methadone determination in puparia and its effect on the development of Lucilia sericata (Diptera, Calliphoridae)

This paper describes a sensitive UPLC-MS/MS method for quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in single empty puparial case of Lucilia sericata. Larvae were reared on substrates spiked with different concentrations of methadone (0-4 μg/g). Methadone was quantified in puparia reared on high concentrated substrates (0.8-4 μg/g). The major metabolite of methadone (EDDP) was not detected, confirming rapid elimination of metabolites by the larvae before pupation. The effects of methadone on the development of L. sericata were also investigated. No effect on sex ratio was detected. A significant difference was calculated for emerged adults but no trends could be observed. Concerning the developmental curve, a significant difference was observed between control and high methadone concentrations using the Kolmogorov-Smirnov test.     

Effectiveness of wound cleansing treatments on maggot (Diptera, Calliphoridae) mortality

Myiasis is defined as an infestation of the organs and/or tissues of human and other animals by fly maggots. Fly species that normally breed in meat or carrion (Diptera: Calliphoridae, Sarcophagidae) may become involved in cutaneous myiasis by colonizing preexisting wounds. Reports of human wound myiasis contracted in hospitals and nursing homes, especially when patients are chronically ill or bed-ridden, are not uncommon across North America and often result in cases of neglect and civil litigation. Based on a case history dealing with this latter situation and circumstances surrounding the treatment of maggot infestation, we designed an experiment to assess the effectiveness of wound cleansing solutions on maggot mortality. Treatments, consisting of four commonly used cleaning solutions (isopropyl alcohol, Dakin's solution, iodine, and hydrogen peroxide) and a control (deionized water), were applied to experimental units (n=5), with each unit consisting of groups of actively feeding Lucilia sericata maggots (Diptera: Calliphoridae). Every 24h, treatments were applied and mortality was assessed for the duration of the study (14 days). Total mean mortality increased over the duration of the experiment, with an initial large increase (10-25%) after the first treatment application, followed by a gradual increase over the remainder of the study. General differences among treatments indicated greatest mean total mortality for Dakin's solution (sodium hypochlorite) (46%), followed by isopropyl alcohol (42%), Betadine (37%), hydrogen peroxide (33%) and lowest mortality for the control (25%); however, no statistically significant differences were observed among treatments and no treatment resulted in 100% maggot mortality. Traditional wound cleansing solutions may not be sufficient for maggot infestations of pre-existing wounds and supplemental treatments may be necessary to effectively treat cases of wound myiasis.     

Minimum and maximum development rates of some forensically important Calliphoridae (Diptera)

Blow fly development rates are frequently used to estimate elapsed time since death in homicide investigations in the first few weeks after death. However, in order to make more precise estimates of time since death, accurate developmental data must be generated for all carrion species, and at temperatures that are comparable with those found at crime scenes. This paper presents developmental rates determined for five forensically important species in British Columbia, Phormia regina (Meigen), Phaenicia sericata (Meigen), Eucalliphora latifrons (Hough), Lucilia illustris (Meigen), and Calliphora vicina Robineau-Desvoidy, at several temperatures.     

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.     

Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) established in the vicinity of Knoxville, Tennessee, USA

The hairy maggot blow fly, Chrsomya rufifacies (Macquart) (Diptera: Calliphoridae) was collected in large numbers as both adults and immatures in the Knoxville, Tennessee, area during 1998 and is likely established there. The distribution of this species in the Old World, isothermal data, and its collection from mid-Michigan during 1998 suggest that it will eventually occupy most of the U.S. The forensic importance of C. rufifacies makes it probable that it will factor into an increasing number of medicolegal cases, but the expanding distribution of this species decreases its utility as a geographic indicator when postmortem movement of decedents is suspected.     

Effects of morphine in decomposing bodies on the development of Lucilia sericata (Diptera: Calliphoridae)

This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Student's T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.     

Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.     

Calliphora vicina (Diptera: Calliphoridae) collected from a human corpse above 3400 m in elevation

In the summer of 2005 an adult hiker was killed in a fall in Rocky Mountain National Park, Colorado (U.S.A.). His body was subsequently colonized by the blowfly Calliphora vicina (Diptera: Calliphoridae). The body was discovered 9 days later at an elevation of 3475 m (11,400 feet). This is the first report of C. vicina on a human corpse at this elevation in Colorado. This paper provides additional confirmation of this fly colonizing a human corpse at high elevations in Colorado.     

PCR-RFLP identification of Diptera (Calliphoridae,Muscidae and Sarcophagidae)--a generally applicable method

A simple, rapid method using restriction fragment length polymorphisms (RFLPs) within the internal transcribed spacer (ITS) regions of the ribosomal DNA gene repeat allows identification of insects and other organisms. We used the method to identify the morphologically similar Diptera larvae that are important in forensic entomology for estimating the time and location of death. Polymerase chain reaction (PCR) was used to amplify a region from the 18S to the 28S rRNA genes. The ITS1 and ITS2 regions provided variation between species and homogeneity within species, with the exception of Cochliomya macellaria. Combinations of the restriction enzymes DdeI, HinfI and Sau3AI provided diagnostic bands for identification of the ten species from three families of Diptera (Calliphoridae, Muscidae and Sarcophagidae).     

Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Immature development times of the blow fly Protophormia terraenovae (Robineau-Desvoidy, 1830) were studied in the laboratory at five different constant temperatures (15, 20, 25, 30, 35 degrees C). The minimal duration of development from oviposition to adult emergence was inversely related to temperature, ranging from 9.19+/-0.3 days at 35 degrees C to 37.78+/-2.96 days at 15 degrees C. From linear regression of development rates at the five studied constant temperature regimes, it followed that the minimum development threshold (t(L)) for total immature development is 8.95 degrees C ( approximately 9 degrees C) and the overall thermal constant (K) for P. terraenovae is 240.2+/-9.3 day-degrees (DD) above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 degrees C. However, it is possible that developmental time from oviposition to adult eclosion might be different in various regions of the world, and that the thermal constant of a holarctic species like P. terraenovae is not same everywhere. Additionally, as the present paper shows, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework.