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.     

Determination of drug levels in larvae of Protophormia terraenovae and Calliphora vicina (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

Two species of blow flies (Diptera: Calliphoridae) were reared on tissues from rabbits administered different dosages of morphine. These species, Protophormia terraenovae and Calliphora vicina are among the first wave of insects colonizing a dead body. Two series of 3 rabbits were given dosages of 10, 20, and 40 mg/h of morphine over a 3 h period via ear artery perfusion. A morphine blood level plateau was attained after 1 h of perfusion. Two other rabbits were used as controls. Samples of tissues collected from rabbits using a coelioscopic technique were determined to have morphine concentrations similar to those encountered in human overdoses and were correlated with dosages of morphine administered. All samples from control rabbits were negative for morphine. Larvae and puparia of both species were regularly collected from each rabbit for toxicological analysis. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were significantly lower than concentrations found in the tissues. There was a decrease in concentration in morphine observed in transition from feeding 3rd instar larva to puparium. A correlation between larval concentration and tissue concentration was found only in feeding 3rd instar larvae.     

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.     

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.     

Bionomics of two forensically important blowfly species Chrysomya megacephala and Chrysomya putoria (Diptera: Calliphoridae) reared on four types of diet

The use of heterogeneous animal tissues for the rearing of necrophagous insect species can produce uneven biological data, which can compromise the determination of larval age and, consequently, estimates for post-mortem intervals. We investigated the development of two species, Chrysomya megacephala and Chrysomya putoria (Diptera: Calliphoridae), reared on four substrates: minced beef (control) and semi-synthetic diets with the addition of sardine, rumen or chicken eggs. No differences in total developmental times were detected among larvae reared on different diets. Length and width of larvae were partially affected by the type of food. Third instar larvae and pupae of both species were heavier on beef treatment when compared with other substrates. Overall mortality was lower when beef was used as food. Longevity of adults and sex ratio were not negatively affected by the use of diets. Egg-based diet was the least effective for both species. Given the fact that several bionomical parameters of larvae reared on diets were close to those obtained when minced beef was offered, and considering the putrid odour, frequency of contamination and lack of homogeneity of animal tissue, semi-synthetic diets can be used for rearing C. megacephala and C. putoria.     

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.     

Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China

The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion. Correlations were found between administered dosage, tissue concentrations and younger or prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius) were also reported. C. megacephala is the most common fly species found on corpses in South China during the early stages of decomposition. Significant differences in larval growth rate were both observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on tissues from rabbits treated with Malathion were smaller and attained maximum length later than those from the control colony. Duration of the larval and pupal stages was both significantly prolonged for larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of the duration of the larval and pupal stages together from the muscle colonies would alter the postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.     

Effect of heroin in decomposing tissues on the development rate of Boettcherisca peregrina (Diptera, Sarcophagidae) and implications of this effect on estimation of postmortem intervals using arthropod development patterns

Larvae of the flesh fly Boettcherisca peregrina (Robineau-Desvoidy) were reared on the tissues of rabbits to study the effects of heroin on the development rates of this species. The rabbits were given 6, 12, 18, and 24 mg of heroin by cardiac puncture. From Hours 18 to 96, larvae feeding on tissues containing heroin (as morphine) developed more rapidly than those feeding on tissues from the control. The time required for pupation was significantly greater for colonies fed on tissues from heroin-dosed rabbits than for the control colony. The differences observed in the rates of development were sufficient to alter postmortem interval estimates based on larval development by up to 29 h and estimates based on pupal development by 18 to 38 h.     

Preliminary observations of the effect of methamphetamine in decomposing tissues on the development rate of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect on the estimations of postmortem intervals.

Larvae of Parasarcophaga ruficornis (Fabricius) (Diptera: Sarcophagidae) were reared on tissues from rabbits administered different dosages of methamphetamine to study the effects of this drug on development of this species. The rabbits were given 37.5, 71.4, and 142.9 mg of methamphetamine via ear vein infusion. From Hours 30 to 60, larvae feeding on tissues from rabbits receiving 71.4 and 142.9 mg of methamphetamine developed more rapidly than larvae from the control colony and those feeding on tissues from the rabbit receiving 37.5 mg of methamphetamine. The time required for pupariation was significantly greater for colonies fed on tissues from methamphetamine-dosed rabbits than for the control. These differences were sufficient to alter postmortem interval estimates based on larval development by up to 18 h and estimates based on puparial development by up to 48 h. The presence of methamphetamine or amphetamine could not be detected in Diptera larvae in this experiment using radioimmunoassay techniques, as there was a nonspecific reaction, resulting in a false positive.     

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.     

Mitochondrial DNA sequencing of beetle larvae (Nitidulidae: Omosita) recovered from human bone

The isolation, amplification, and characterization of human DNA from hematophagous (blood feeding) and necrophagous (carrion feeding) arthropods have been advanced significantly by the development of polymerase chain reaction (PCR) DNA sequencing methodologies. Historically, DNA technology has been successfully utilized to identify individual hosts upon which species of hematophagous arthropods have fed. The analysis of hematophagous insects' gut content blood meals has led to major advances in medical entomology and vector-borne disease epidemiology. In the forensic arena, the ability to apply similar techniques to insects recovered from badly decomposed remains has been greatly enhanced through the advent of mitochondrial DNA (mtDNA) techniques. Mitochondrial DNA analyses have been utilized to identify both the human remains upon which fly larvae (maggots) have fed and the species of the larvae themselves. The preliminary work detailed here demonstrates, for the first time, the successful application of mtDNA sequencing techniques to the analysis of necrophagous beetle larvae. A small sample of sap beetle larvae, Omosita spp. (Coleoptera: Nitidulidae), was collected from human skeletal remains during anthropological examination and analyzed for human DNA using mtDNA sequencing. The beetle larvae yielded mtDNA matching that of the host human bone. The results detailed here further demonstrate the robust nature of human mtDNA and the ability to recover valuable mtDNA evidence from forensically important, late decompositional stage insect species.     

Determination of drug levels and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil.

Larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) were reared on tissues from rabbits administered twice the lethal dosage of diazepam in order to study the effects of this drug on the development of these two species. The rabbits were given 50mg of diazepam via ear vein infusion. From 18 to 54h, larvae feeding on tissues containing the drug developed more rapidly than larvae from the control colony for both fly species. The time required for pupariation and adult emergence was significantly greater for colony fed on tissues from diazepam dosed rabbits than for the control ones. These differences are significant for they are large enough to alter the estimate of postmortem interval based on fly development. The presence of diazepam could be detected through gas chromatography-mass spectrometer (GC-MS) in all rabbit samples and in almost all diptera samples in this experiment.     

石河子春季常见尸食性昆虫种类及丝光绿蝇发育观察

目的观察新疆石河子市区尸食性昆虫的种类及温度对优势种丝光绿蝇生长发育的影响。方法 2007年和2008年的4月15日至6月15日在石河子市（E44.18°、N86.00°）3个不同地点诱集尸食性昆虫,并鉴定分类。将优势种丝光绿蝇在不同温度下饲养,观察其体长、体重、羽化率、雌雄比、历期等生长发育特点。结果石河子地区春季3个不同地点共诱集到尸食性昆虫6目14科16种,其中以膜翅目、双翅目和鞘翅目为主;丝光绿蝇幼虫体长、体重随着温度升高而增长、增加,化蛹时又有所缩短、减轻;不同的温度对幼虫的羽化率和雌雄比影响不明显。结论本文所得数据可为新疆石河子地区的法医昆虫学及相关研究和实践提供一定参考。     

Morphine perfused rabbits: a tool for experiments in forensic entomotoxicology

In order to establish an animal model for entomotoxicological studies, the kinetics of morphine elimination from blood after a single intravenous injection of morphine and the concentration of morphine in tissues following a continuous perfusion were studied. The aim of these experiments was to obtain controlled morphine tissue concentrations similar to those encountered in fatal human heroin overdoses. These tissues can be used as a food source for developing fly larvae in entomotoxicological studies. In the single injection experiment, seven rabbits were administered 1 or 2 mg/kg body weight of morphine chlorhydrate via the main ear artery. Blood samples of 200 microL were removed regularly via a catheter. Morphine concentration was determined using RIA techniques. Morphine was found to be first rapidly distributed and then slowly eliminated, following a two-exponential equation. Elimination of morphine from blood can be described as a two-compartment model. Constants of the equation were determined using the Kaleidagraph program. Using those constants, the main pharmacokinetics parameters were calculated. Results of these parameters showed the following: clearance from 13.3 to 16.2 L.h.1, half-life of the distribution phase from 0.6 to 0.9 min, and half-life of the elimination phase from 21 to 26 min. These results were used to calculate the rate of perfusion of morphine for rabbits to obtain desired, controlled, and constant concentrations of morphine in tissues. In the second experiment, three rabbits received a perfusion of morphine intravascularly at a rate of 2 mg/kg/h for a period of 3 h. These rabbits were sacrificed and analyses performed on several abdominal and thoracic organs. Results showed that the concentrations of morphine differed according to the organ analyzed, but were reproducible for organs between animals. These concentrations were similar to those normally encountered in cases of human death due to heroin overdoses.     

不同温度下铜绿蝇蛹期发育形态观察

目的观察不同温度下铜绿蝇蛹形态变化特征规律并进行发育阶段划分。方法在16℃、20℃、24℃、28℃、32℃下饲养铜绿蝇，于化蛹起，每隔12h取样1次，直至半数羽化。解剖样本，在体视显微镜下观察其体表形态变化。结果铜绿蝇蛹形态发育过程包含Ⅰ蛹体分头、胸、腹；Ⅱ头咽骨贴壁；Ⅲ原头出现；Ⅳ呼吸角破壳；Ⅴ翅与足出现及褐化；Ⅵ鬃毛出现及褐化；Ⅶ复眼轮廓形成及色素化等7个时间标志性特征，并据此将蛹期划分为9个阶段。结论在已知现场环境温度的前提下，蝇蛹形态变化具有阶段性特征，以此划分蛹期阶段，可望作为推断死后间隔时间（PMI）的参考。     

Growth rates of the blowfly, Lucilia sericata, on different body tissues

Estimates of post-mortem interval based on forensic analysis of the age of blowfly larvae on a corpse, are generated from standardised larval development curves. These are derived from studies of the growth of larvae reared, usually, on liver and at a range of temperatures. However, it is possible that the species or organ on which the larvae feed, might significantly alter the rate of growth. The present study therefore compared the development of the blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae) fed on lung, liver and heart, from both cows and pigs. Half of the tissue samples used were liquidised while half were not, to assess whether structural differences in the food source were important. Larvae grew significantly faster and gave rise to larger adults when reared on pig compared to cow tissue and when reared on lung and heart compared to liver. Larvae completed feeding and wandered from the food source 31h earlier and grew 2mm longer when reared on lung compared to liver. Tissue structure had no or little effect on development. For the forensic entomologist, these results highlight the importance of recording the position of larvae removed from a body and the care that should be used when extrapolating development rates from standard curves based on larvae fed on only a single medium, particularly where that is liver.     

A preliminary study of forensic entomology in Medellín, Colombia.

This is the first report of an ongoing study of insect succession on carrion carried out in Medellín, Colombia, using pigs (Sus scrofa) as a model to determine the insect sequence over 207 days. During this period, 2314 insects belonging to the following orders and families were collected: Diptera: Calliphoridae, Muscidae, Piophilidae, Sarcophagidae, Syrphidae, Otitidae; Hymenoptera: Apidae, Formicidae, Halictidae, Mutilidae, Vespidae; Coleoptera: Staphylinidae, Histeridae, Carabidae, Scarabaeidae, Silphidae, Dermestidae, Cleridae, Nitidulidae; Dermaptera: Forficulidae; Hemyptera: Gelastocoridae, Coreidae; Lepidoptera: Hesperiidae.Five decomposition stages were observed (fresh, bloated, active decay, advanced decay, and dry remains) and four insect ecological categories (necrophagous, predators, omnivorous, and incidental). During the fresh stage, the first insects that appeared were flies of the families Sarcophagidae and Muscidae and specimens of Formicidae (Hymenoptera). During the bloated period, species of Calliphoridae (Diptera) were predominant and the first to oviposit. During the third and fourth stages (active decay and advanced decay), the most abundant families were Calliphoridae and Muscidae, although Staphilinidae (Coleoptera) also stood out. During the last stage (dry remains), the dominant family was Formicidae (Hymenoptera) followed by Dermestidae (Coleoptera) with a large number of immature insects.     

Seasonal structure and dynamics of sarcosaprophagous fauna on pig carrion in a rural area of Cordoba (Argentina): their importance in forensic science

Four experiments, one in each season, were carried out during 2004 in a rural area of Córdoba, central Argentina. Two pigs (Sus scrofa L.), weighing approximately 8 kg each, were used in each of the four experiments. The animals were killed by a sharp blow to the head and immediately placed in an appropriate arthropod trap. One pig was placed in the shade and the other under direct sunlight. This research was conducted to determine the seasonal structure and dynamics of arthropods that constitute the sarcosaprophagous community in a given area of the central region of Argentina. The decomposition process was divided into five stages: fresh, bloated, decay, advanced decay and dry. The duration of each stage varied in different seasons, showing that the temperature and humidity are the most important variables that influence this process. A total of 51,500 adults and 36,909 immature were collected. More than 80% of the specimens collected belong to Insecta, and within this, Diptera were the most abundant order. The flies were mainly represented by adults and immature of the following species: Chrysomya albiceps (Wiedemann) (Calliphoridae), Musca domestica Linnaeus (Muscidae) and Fannia femoralis (Stein) (Fanniidae). Coleoptera were mainly represented by adults and immature of the species Creophilus maxillosus (Linnaeus) (Staphylinidae) and Dermestes maculatus (Geer) (Dermestidae). More than 50% of hymenopteran fauna collected belong to Formicidae. The trophic levels associated with the decomposition of the remains included five categories: necrophagous, parasites and predators of the necrophagous species, omnivores, opportunists and adventives. During faunal succession a strong dominance of the necrophagous species of the family Calliphoridae and Muscidae was observed, mainly during the early stages of decay process in all seasons.     

北京地区尸食性蝇类调查

目的了解北京地区目前尸食性蝇类数目及常见尸食性蝇类的季节分布。方法采用动物尸体或组织诱捕和饲养的方法采集尸食性蝇类标本,对其进行分类保存后进行观察和统计。结果北京地区尸食性蝇类为4科9亚科21属46种,其中有12种是北京新记录种。尸食性蝇类具有区域性分布和季节性分布的特性。结论北京地区尸食性蝇类及常见的尸食性蝇类季节分布数据,可为相关研究提供参考依据。     

Initial study of arthropods succession and pig carrion decomposition in two freshwater ecosystems in the Colombian Andes

Entomological succession and trophic roles of arthropods associated with different stages of carcass decomposition were studied to estimate the post-mortem submersion interval in two freshwater ecosystems in the Colombian Andes, at an altitude of 2614 m. Pig carcasses were employed as models placed 68 m apart, one in a stream (lotic) and another in an artificial lake (lentic). Decomposition time to skeletal remains was 74 days in the lake and 80 days in the stream. Six phases of decomposition were established: submerged fresh, early floating, floating decay, bloated deterioration, floating remains and sunken remains. A total of 18,832 organisms associated with the carcasses were collected: 11,487 in the lake (four orders, 19 families and 33 species) and 7345 in the stream (eight orders, 15 families and 25 species). Organisms were classified in the following ecological categories: shredders, collectors, predators, necrophagous, sarcosaprophagous and opportunists. Physical and chemical properties of the habitats, such as water temperature, CO(2) and conductivity, varied according to rainfall. In the lake, shredders (Coleoptera: Tropisternus sp. and Berosus sp.) and collectors (Diptera: Chironomus sp.) were found to be associated with submerged phases. Predators (Odonata) were only present during the first phases. Coleoptera (Dytiscidae) were found during floating decay and bloated deterioration stages. In the stream, shredders (Hyalella sp.) and collectors (Simulium sp.) were found during all stages, whereas the predator Oxelytrum discicolle was found exclusively during the floating stages, during which body temperature increased in a fashion similar to active decay in terrestrial environments.