The Effect of Buscopan® on the Development of the Blow Fly Chrysomya megacephala (F.) (Diptera: Calliphoridae)

Abstract:  This work investigated the effects of butylscopolamine bromide, a drug present in the pharmaceutical formulation Buscopan^®, on the development of Chrysomya megacephala , a blow fly species of considerable forensic and medical importance in Brazil. Larvae exposed to the drug showed a decreased rate of development, with higher drug concentrations further retarding the development. Besides, larvae reared on the presence of the drug showed smaller body weight and body length when compared with larvae reared on the absence of Buscopan^®. The drug also affected the mortality of the species.     

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.     

Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval.

Larvae of Sarcophaga (Curranea) tibialis (S. tibialis) were reared at constant temperature on chicken liver treated with a steroid or a barbiturate at concentrations that would be lethal, half-lethal and twice-lethal doses for humans. Trends to greater mortality at higher drug concentrations were not statistically significant. Larvae exposed to either drug took significantly longer to reach pupation compared to those in the control, while larvae exposed to sodium methohexital passed through pupation significantly faster than those in the control. No systematic relationship was found between drug concentration and development time of larvae or pupae. The total developmental period from hatching to eclosion did not differ between treatments, implying that estimates of post mortem intervals- (PMI) based on the emergence of adult flies will not be affected by the involvement of these drugs in a case. On the other hand, anomalous pupation spans may indicate the presence of barbiturates. These findings are compared with patterns found in another fly fed other contaminants.     

Blowfly Larval Tissues as a Secondary Detector for Determining Paraquat-Related Death in Rabbit Carcass

Paraquat poisoning is commonly associated with suicide or homicide in Malaysia. In a case involving advanced body decomposition, pathological analysis regarding the cause of death may become difficult or almost impossible. Insects serve as common alternative matrix for poison detection in forensic analysis. Paraquat detection via secondary bioaccumulation in fly larvae tissue has never been reported. In this study, tissues from blowfly larvae collected from a rabbit carcass with paraquat poisoning were analyzed for secondary bioaccumulation. Larvae samples were collected and analyzed using liquid–liquid extraction. The detection was performed via reduction of quaternary ammonium presence in paraquat and analyzed using gas chromatography–mass spectrometry (GC-MS) with selected ion monitoring mode (SIM mode). GC-MS showed the elution of reduced paraquat was at retention time 12.8 min. Blowfly larvae tissue has proven useful as a secondary detector in paraquat-related deaths.     

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.     

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.     

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 evidential value of intra-alveolar haemosiderin-macrophages in cases of sudden infant death syndrome (SIDS)

This study aimed to determine the effect of cocaine on the development and growth of immature and adult blowflies, in an attempt to better understand the impacts of such effects on postmortem interval (PMI) estimation. Twice the lethal dose of cocaine was injected into rabbits. The control animals were injected only with saline solution. Experimental and control rabbits were autopsied, and portions of their livers were exposed to newly eclosed larvae of Chrysomya putoria and Chrysomya albiceps. Larvae were weighed individually every 6 h, up 54 h of exposure. The larvae were then placed on an artificial diet to continue their development. Pupariation time, adult emergence and adult longevity were also analysed. The larvae of both species that fed on the cocaine-containing livers developed faster than those that fed on the livers of the control animals, leading to the conclusion that cocaine influences and stimulates larval growth. The difference in growth between the control and treated flies was best observed from 12 h of exposure onward. This finding has important implications for forensic investigations.     

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.     

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.     

The effects of larval crowding and food type on the size and development of the blowfly, Calliphora vomitoria

The use of entomological evidence in the estimation of the post mortem interval (PMI) often depends on the size and developmental stage of blowfly larvae collected from a corpse. Therefore, factors which can have an effect on the larval size and growth rate can have implications for reliable PMI determinations. This study explores the competitive effects of larval overcrowding on Calliphora vomitoria reared on three different pig tissues--liver, brain and muscle. The competitive feeding environment within the more crowded larval cultures resulted in increased development rates and the production of undersized larvae and adults. Variation in the extent of these effects was observed on each of the three body tissues, highlighting the importance of documenting the positions from which entomological evidence is recovered from a corpse.     

A model for the aging of fly larvae in forensic entomology

A method is described for determining the time of hatching of blowfly larvae on a corpse with respect to temperature. Temperature is known to have a profound effect on the rate of growth of fly larvae, and it is suggested that past assumptions that the growth of larvae found in a corpse has taken place at one approximated ambient temperature may have led to large overestimates of the development time of the larvae, and thus overestimates of time of death. Larvae of Lucilia cuprina, Calliphora vicina (= erythrocephala), Calliphora stygia and Calliphora hilli were cultured under constant temperatures, and their growth rates modeled with a logistic growth curve. Two programmes have been developed, one to compute the parameters for the logistic equations, the other is used to estimate the time elapsed from the time at which a sample is removed from a body to the estimated hatching time of the larvae comprising the sample, with temperatures and species as variables.     

Human Cutaneous Myiasis by the Australian Sheep Blowfly,Lucilia cuprina (Diptera: Calliphoridae), in Oklahoma

This case report describes an incident of myiasis in Oklahoma City, Oklahoma. Paramedics treated a 53‐year‐old man in the field when he suffered cardiac arrest. He was intubated by the paramedics and transported to an emergency room, where he received two stents and a balloon pump. He was found to have a GCS of three and remained in the hospital for 7 days before passing away. After his death, the breathing tube was removed, revealing the presence of several maggots. On closer inspection, the dipteran larvae were found in both the oral and nasal cavities. Four of these larvae were reared to adulthood and identified as Lucilia cuprina (Weidemann) (Diptera: Calliphoridae), commonly known as the Australian sheep blowfly, by the Biology Department of the University of Oklahoma. Based on the fly's life cycle and rate of development, the infestation is suspected to be hospital acquired.     

法医器官组织特异性circRNAs的筛选及确证

目的 筛选并确证器官组织特异性circRNAs分子标记物,应用于法医器官组织溯源.方法 从TSCD数据库及文献中,筛选出器官组织特异性circRNAs,设计并验证引物.收集心脏、大脑、肝脏、皮肤、骨骼肌、肺脏等人体组织样本,经总RNA提取、RT-qPCR、琼脂糖凝胶电泳等步骤对候选circRNAs进行组织表达水平分析,...     

In Vitro Effects of Household Products on Calliphoridae Larvae Development: Implication for Forensic Entomology

Several parameters can delay the first arrival of flies on a corpse and the subsequent development of the larvae. This study focuses on the development of Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) on household chemical‐contaminated substrates. bleach, perfume, hydrochloric acid, caustic soda, insecticide, mosquito repellent, and gasoline in quantities consistent with an amount that could possibly be spilled on a corpse were mixed with beef liver to simulate contaminated fleshes. Larvae were bred at 25°C on these media until emergence. Four developmental parameters were followed: survival rates, development times, sex ratios, and adult sizes. Hydrochloric acid, insecticide, and gasoline killed all larvae. In low quantities, caustic soda and mosquito repellent increased the development time and decreased the adult size. However, high quantities of these chemicals killed all larvae. Lastly, bleach and perfume did not affect the survival rate and barely impacted the development time or adult size. These results demonstrate common household products spilled on a corpse can strongly affect the development of Calliphoridae larvae. The effects of such products should be considered in forensic entomology cases.     

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.     

Post-feeding larval behaviour in the blowfly, Calliphora vicina: effects on post-mortem interval estimates

Using the rate of development of blowflies colonising a corpse, accumulated degree hours (ADH), or days (ADD), is an established method used by forensic entomologists to estimate the post-mortem interval (PMI). Derived from laboratory experiments, their application to field situations needs care. This study examines the effect of the post-feeding larval dispersal time on the ADH and therefore the PMI estimate. Post-feeding dispersal in blowfly larvae is typically very short in the laboratory but may extend for hours or days in the field, whilst the larvae try to find a suitable pupariation site. Increases in total ADH (to adult eclosion), due to time spent dispersing, are not simply equal to the dispersal time. The pupal period is increased by approximately 2 times the length of the dispersal period. In practice, this can introduce over-estimation errors in the PMI estimate of between 1 and 2 days if the total ADH calculations do not consider the possibility of an extended larval dispersal period.     

HE染色切片组织的STR检测及法医学应用

目的建立并优化HE染色切片组织STR分型方法,评估HE染色切片组织的STR分型有效性。方法用QIAgen法提取2例尸检人体的心、肝、肺、肠等8种组织的HE染色切片DNA,用TaqMan荧光探针法通过荧光域值循环数(Ct值)测定获得各提取液中的DNA质量浓度,以阳性内对照(internal posi-tive control,IPC)监测PCR过程中的抑制水平。再用Identifiler试剂盒扩增,在3100-Avant上进行STR片段分析。结果在本研究建立优化的STR分型技术条件下,8种人体组织的HE染色切片DNA提取液质量浓度均可达到1ng/μL以上,其IPC的Ct值提示无PCR抑制剂。HE染色切片组织的STR分型有效性与石蜡包埋组织相当,其DNA随时间延续而缓慢降解。结论在一定保存时限内,HE染色切片的DNA质和量可以满足STR分型检测需要,但受残余甲醛固定剂的影响,HE染色切片的分型成功率随保存时间延长而降低。     

Retrograde blood aspiration: a vital reaction

Blood aspiration, which is based on an intact breathing activity, is one of the forensically most important vital reactions. On the example of 11 forensic cases it is shown that the aspiration of blood into the lung tissue is possible not only when the bleeding source is situated above the trachea or main bronchi, but also in a retrograde way, taking its origin from an injury of the peripheral lung tissue itself. Equally to 'classical' blood aspiration, the findings of retrograde blood aspiration are to be diagnosed by means of macroscopical and histological examination of the lung tissue. The lung tissue presents with roundish, reddish spots, which are situated close to, but separate from a lung contusion or other lung tissue injury, and microscopical examination reveals blood in the alveoli and the bronchioli. Retrograde blood aspiration may be a helpful vital sign in the forensic assessment of thorax trauma cases where no other vital reactions are present.