Benoit Bourel

Entomofauna of buried bodies in northern France

Autopsies of exhumed cadavers can reveal important evidence for clarification of medical insurance and social issues. This study concerns insects sampled on 22 exhumed cadavers in the Lille area. For each corpse, the species and the stages of development were noted, as well as the time elapsed after burial, the location of the cemetery, the stage of decay and possible preservation treatment. A total of eight Diptera and two Coleoptera species were sampled on the corpses. The relationships between entomofauna and conditions of burial are discussed. Three species were regularly found because of their preference for underground environments or closed environments: Conicera tibialis, typically associated with buried bodies, Leptocera caenosa which is known to be associated with human faeces, water closets, caves and cracked soil pipes, and Ophyra capensis, sometimes found on human bodies kept indoors for several months, where blowflies have not had access. Triphleba hyalinata, which is associated with human bodies in wooden coffins, was found only twice.

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 Students 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.

Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure. This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results. Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists.

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.

Flies eggs: a new method for the estimation of short-term post-mortem interval?

Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).

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.

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.

Repellent effect of some household products on fly attraction to cadavers

The most common task of a forensic entomologist is to determine an accurate minimum post-mortem interval (PMI) using necrophagous fly larvae found on carrion. More often, blowflies (Diptera: Calliphoridae) are the first insects to detect the cadaver and, if the circumstances are favourable, to leave eggs on the body. However, several studies reveal that products such as gas or paint found on the cadaver induce a delay in the colonisation of the body, leading to an under-estimate of the PMI. Six common household products (gas, mosquito citronella repellent, perfume, bleach, hydrochloric acid and soda) were added to dead rats (Rattus norvegicus) in a field (Lille Forensic Institute, France). The presence of necrophagous flies was checked at regular intervals during 1 month. This experiment was repeated at the same period for four consecutive years. Results clearly showed the repellent effect of three of the six tested substances: gas (petroleum spirit), perfume and mosquito citronella repellent, which resulted in a mean delay of several days in the appearance of the first Dipteran species. Experiments were then carried out in controlled conditions in order to confirm previous observations. An olfactometer was specially designed to observe the behaviour of female Calliphora vicina (Diptera: Calliphoridae) in response to mice (Mus musculus) cadaver odour stimuli combined with household products. Dead mouse odour was a strong attractive stimulus for most of the tested individuals. Furthermore, it was noticed that the presence of mosquito citronella repellent, perfume, hydrochloric acid and paradichlorobenzene produced a significant repellent effect on female flies. All these results together confirm the repellent effect of some household products on flies and the necessity for forensic entomologists to consider this hypothesis when estimating the PMI.

Effect of body length and temperature on the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae)

This work investigates variation in the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae) as a function of body length and ambient temperature. A video-tracking system was used to follow the movement of larvae in an experimental arena, and to estimate their average crawling speed. Recordings were carried out at various temperatures using larvae of different developmental stages. As temperature increased, the larvae crawled at a faster speed. Furthermore, speed increased as a function of body length. We present a general allometric relationship to express the average crawling speed of larvae as a combined function of temperature and body length, expressed in the following equation: speed (cm/min)=5.45 x log[length (mm)]+0.66 x temperature (degrees C)-12.8. These results are particularly useful in forensic entomology for estimating the time that elapsed following the larvas departure from the corpse. This information can provide more accuracy for post-mortem interval estimations and a better comprehension of larvae behaviour and displacement.

Cordylobia anthropophaga (Diptera: Calliphoridae) outside Africa: a case of furuncular myiasis in a child returning from Congo.

Abstract International travel to tropical countries accounts for an increasing incidence of imported diseases. An unusual case of furuncular myiasis due to Cordylobia anthropophaga (Blanchard) is reported in northern France in a 9-mo-old infant, after a 4-mo stay in Congo. A review has been made of the major cases of imported furuncular myiasis due to Cordylobia, as well as identification of second larval instars and management of the myiasis. RESUMÉ Les trajets internationaux ont pour résultat une augmentation des pathologies importées des zones tropicales. Les auteurs présentent un cas peu courant de myiase furonculeuse causée par des larves de Cordylobia anthropophaga (Blanchard) sur un jeune enfant de neuf mois qui revenait du congo. Une revue de la littérature concernant les cas d’importation de myiases furonculeuses à Cordylobia est faite. L’identification a été possible à partir de larves au second stade. Le traitement de la myiase est également présenté.