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.     

Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae)

By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the post-mortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25°C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48h. Only the larvae with a dispersal time of 24h (total time of development 325.2h; median) and 48h (total time of development 347.7h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately.     

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.     

Determination of drug levels in larvae of Lucilia sericata (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

This study concerns the determination of morphine concentrations in fly larvae reared on rabbits administered different concentrations of morphine and a correlation between concentrations of the drug in larvae and tissues. Three rabbits (R1, R2 and R3) were given dosages of 12.5, 25.0 and 50.0 mg/h of morphine over a 3 h period via continuous ear artery perfusion. These dosages and time of perfusion were calculated to create tissue concentrations of morphine similar to those encountered in human death due to overdose. Morphine blood level plateau was attained after 1 h of perfusion. A fourth rabbit was used as a control. To evaluate drug concentrations, tissues were sampled using a coelioscopic technique. Approximately 400 eggs of Lucilia sericata, all of the same age category, were placed in eyes, nostrils and mouth of each rabbit carcass. Larvae and puparia were regularly collected from each rabbit for toxicological analysis. The concentrations of the drug in the tissues sampled were determined to be similar to those normally encountered in human overdoses and were correlated with the dosage of morphine that had been administered. Morphine was detected in all larvae and pupae fed on tissues from carcasses administered morphine, except for puparia from the colony fed on the R1 animal which received 12.5 mg/h dosage of morphine. All samples from the control rabbit were negative for morphine. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were 30 to 100 times lower than the concentrations found in the tissues. A correlation between the tissue concentrations and larval concentrations was found in only 3rd instar larvae (80 to 140 h following hatching). No correlations were found between administered dosages, tissue concentrations and younger larvae, prepuparial larvae or puparia.     

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.     

Alteration of expirated bloodstain patterns by Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) through ingestion and deposition of artifacts

Abstract: Bloodstain pattern analysis can provide insight into a sequence of events associated with a violent crime. However, bloodstain pattern analysis can be confounded by the feeding activity of blow flies. We conducted two laboratory experiments to investigate the relationships between Lucilia sericata (green bottle fly) and Calliphora vicina (blue bottle fly), expirated bloodstains, and pooled bloodstains on a range of surfaces (linoleum, wallpaper, textured paint). C. vicina and L. sericata changed bloodstain pattern morphology through feeding and defecation. They also deposited artifacts in rooms where blood was not present originally. Chemical presumptive tests (Hemastix^®, phenolphthalein, leucocrystal violet, fluorescein) were not able to differentiate between insect artifacts and bloodstains. Thus, C. vicina and L. sericata can confound bloodstain pattern analysis, crime scene investigation, and reconstruction. Crime scene investigators should be aware of these fundamental behaviors, and the effects that blow flies can have on expirated and pooled bloodstain patterns.     

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.     

Stability of prostate specific antigen (PSA), and subsequent Y-STR typing, of Lucilia (Phaenicia) sericata (Meigen) (Diptera: Calliphoridae) maggots reared from a simulated postmortem sexual assault.

Rape-homicide represents one of the most heinous crimes, but which are also the hardest to solve due to the high occurrence of stranger-to-stranger interaction. This is the first case of obtaining P30 and Y-STR typing from a simulated postmortem sexual assault. 2, 3.5 and 6 microl of liquid semen was added to a liver substrate and Lucilia (Phaenicia) sericata (Meigen) eggs added. The larvae fed upon the semen coated substrate and were removed for testing after 48 and 145h after initial liquid semen deposition. P30 was recorded from whole postfeeding larvae after 145h, with correct Y-STR profiles obtained from the crop of actively feeding second instar larvae after 48h of initial semen deposition. The ability to obtain P30 and Y-STR profiles from larvae infesting a cadaver, with the suspicion of sexual assault having occurred prior to death, provides a new avenue to aid in the solving of such crimes.     

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.     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

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.     

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.     

Effect of temperature on development of Liopygia (= Sarcophaga) argyrostoma (Robineau-Desvoidy) (Diptera: Sarcophagidae) and its forensic implications

The temperature-dependent development of the forensically important flesh fly Liopygia (Thomsonea) argyrostoma (Robineau-Desvoidy) (=Sarcophaga argyrostoma) was studied at six constant temperature regimes in the laboratory. Total developmental time of L. argyrostoma from larviposition to adult emergence was 54.9+/-1.45, 31.3+/-1.1, 22.2+/-0.67, 16.3+/-0.54, and 14.9+/-0.4 days (+/-S.D.) when reared at 15, 20, 25, 30, and 35 degrees C, respectively. At 8 degrees C, larval development was not completed. From linear regression of development rates of five of the six studied constant temperature regimes, it was determined that the minimum development threshold (tL) for total immature development is 7.4 degrees C, and the overall thermal constant (K) for L. argyrostoma is 396.4+/-19.18 (mean +/- S.D.) day-degrees (DD) above the threshold.     

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.     

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.     

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.     

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.     

丝光绿蝇的发育速度和有效积温的实验观察及其法医学应用

根据丝光绿蝇在自然条件下的发育速度及其发育过程中的有效积温推断尸体的死亡时间。对在尸体腐败过程中最常见、国内分布极广的丝光绿蝇在自然条件下的发育速度及其发育过程中的有效积温进行观察、记录结果和统计学处理 ,分别确立了K1=2 0 880 + 0 80 14X1、K2 =5 4 0 917-2 8814X2 、K3=133 2 180 -2 6 312X3 等 3个有效积温与长度 (蛆 )和卵重的线性回归方程。应用丝光绿蝇发育速度与有效积温的回归方程 ,能比较准确地进行死亡时间的推断