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.     

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.     

Effects of cocaine and heroin,and their combination,on the development rate of Calliphora vomitoria (Diptera: Calliphoridae)

Insects present on or near decomposing bodies are collected by forensic entomologists and used to estimate the post-mortem interval. Drugs metabolized by a person before death may affect the rate of development of insects feeding on the corpse. This study aimed to determine the effects of cocaine and heroin main metabolites on the development rate of the Calliphora vomitoria (Diptera: Calliphoridae) and their implications on minimum post-mortem interval determination. Groups of 250 eggs each were placed into four separate pots of 150 g of minced pork meat being either un-spiked, or spiked with benzoylecgonine, morphine, or a combination of both. Larval length (mm) and weight (mg) measurements were taken twice daily and the rate of development of the insects’ life cycle was monitored until eclosion. Results show that cocaine-fed larvae developed less in length and weight than the control group. Heroin-fed larvae showed a more fluctuating pattern, being smaller and lighter than the control group for most of their larval cycle, but overtaking them in both parameters towards pupation. Combination-fed larvae seemed to favour the effects of cocaine. The three conditions also had a significant impact on the length of the insects’ life cycle. Cocaine and drug combination treatments increased the length of the second and third instar stages, but led to the shortening of pupation and accelerated eclosion. Conversely, heroin treatment led to lengthier pupation. Interestingly, the effects of the drug combination seemed to mirror more precisely those of cocaine.These findings indicate that both cocaine and heroin, singularly and in combination, have sizable effects on blowflies’ development rates, potentially biasing post-mortem interval estimations.     

Morphological observation of puparia of Chrysomya nigripes (Diptera: Calliphoridae) from human corpse

This article presents Chrysomya nigripes Aubertin as a blow fly species of forensic importance in Thailand, and morphological observation of fly puparia using scanning electron microscopy (SEM). Morphologically, we focused on the characteristics of puparia used to accurately identify fly species. Numerous puparia of C. nigripes were found aggregated, adhering side by side, on the tibia of a skeletonized corpse, which was recovered from a forested area of Chiang Mai, northern Thailand. In the triangular shape of the anterior end of the puparia, three thoracic segments and broad hairy patches beginning dorsolaterally at the sixth segments were distinguishing characteristics. This study, showing pupariation of the flies along the bone of a corpse as well as morphological features, provides important guidance in identifying C. nigripes puparia. A key to differentiate puparia of C. nigripes from the other flies of forensic importance in Thailand is given.     

Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length

A record of the length of the largest larvae collected from a corpse can be used to estimate the age of the oldest larvae present and, therefore, give an estimate of minimum time since death. Consequently, factors that affect post-mortem larval length will impact on any estimate of PMI based on it. Methods used to kill and preserve larvae are known to affect post-mortem length. This study looks at the effects of different preservatives, and variations in the protocol used for killing larvae by immersion in a hot water bath = [hot water killed; HWK], on the length of dead larvae of two common blowfly species. Post-feeding third instar Calliphora vomitoria and Lucilia sericata larvae were either HWK in boiling water and then placed in 80% ethanol or 10% formaldehyde solution, or placed live into the preservatives. For both species, choice of preservative and method of killing significantly affected post-mortem length. There were significant interspecific differences in their response to identical methods of killing and preservation. Additional experiments were carried out where C. vomitoria larvae were HWK in water at 80 and 100 degrees C for 1, 30, 60 and 90 s duration. Both temperature and duration significantly affected post-mortem length. Maximum length was attained after at least 60 s immersion. The amount of post-mortem decomposition that occurred after the larvae were placed in preservative could be greatly reduced by increasing the duration of immersion and/or increasing the water temperature. For the HWK larvae, it was possible to record their length immediately after death and before they had been placed in preservative. This data revealed that where 80% ethanol was used as a preservative the larvae expanded in the preservative. The timing of this expansion was investigated with a sample of C. vomitoria, HWK at 100 degrees C for 30 s and recording post-mortem length immediately after death and again after 3, 6, 9, 12, 24, 27, 30 and 33 h storage in 80% ethanol. Maximum length was recorded after 12 h storage and the rate of expansion was highest during the first 3 h in this preservative. After long-term storage (290 days), larvae killed and preserved in the same way were on average 0.7% longer than immediately after death and 0.6% (0.11 mm) smaller than when last measured (after 28 days storage).     

根据绿蝇属昆虫推断死亡时间的法医学研究进展

尸源性昆虫在某些刑事案件的侦破中占有重要地位,对于推断死者的死亡时间和推断尸体是否有转移有重要的作用。双翅目中的丽蝇科绿蝇属昆虫是尸体上常见的昆虫之一,通常最先到达尸体,而且最喜阳光,该特点以及在尸体上发现的绿蝇属昆虫幼虫的生长发育阶段,可以为刑事案件的侦破提供十分有力的证据。本文主要从生物形态学和分子生物学对绿蝇的种属鉴定以及绿蝇日龄的推断等方面,综述了根据绿蝇属昆虫推断死亡时间的法医学应用研究进展。     

Flight activity of the blowflies, Calliphora vomitoria and Lucilia sericata, in the dark

Many species of insects are able to fly at night or in very low light intensities. The question of whether calliphorid blowflies are also able to do this to locate a corpse and oviposit nocturnally is of considerable forensic importance. However, to date studies of this behaviour have been contradictory. Here, the activity and number of Calliphora vomitoria L. and Lucilia sericata (Meigen) (Diptera: Calliphoridae) caught on sticky-traps were examined in a slow-speed wind tunnel, at different intensities of artificial light. The traps were either unbaited or baited-with liver. The number of both species caught, decreased incrementally as light intensity was reduced. While the responses of the two species were broadly similar, L. sericata were significantly more active than C. vomitoria, especially at higher light intensities. The number of flies of both species that were caught was higher in the presence of liver bait, but the presence of the liver bait did not change the shape of the relationship between catch and light intensity. Hence, light intensity acts as an independent exogenous stimulus for activity and although liver volatiles increase activity levels, they are not necessary as an activation stimulus. Comparison of the numbers caught in small or large enclosures suggests that any flies caught in darkness probably alighted on the trap by chance and that in darkness, while flies may be activated by carrion odours, they do not appear to be able to navigate effectively to the source of that odour. The results presented here suggest that in darkness, the probability of oriented flight leading to oviposition on a corpse by either species, is relatively low.     

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.     

The succession and development of insects on pig carcasses and their significances in estimating PMI in south China

The succession of insect communities on carrion varies at local and global spatial scales. As such, ecological succession data obtained from corpses at one geographic location cannot necessarily be applied to other locations. Our study describes this succession in the far southern part of China to provide such data for forensic cases in this region. A total of 18 pig carcasses were placed in the field in four seasons, and the timing of the following events were recorded: appearance of larvae, onset of larval wandering, when most larvae had wandered, onset of pupariation, when most larvae had pupariated, onset of eclosion and end of eclosion. Our results indicated that all of the evaluated events could be used as accurate indicators of postmortem interval (PMI). The carcasses decayed fairly quickly in spring, summer and autumn, taking 225+/-75 h, 183+/-44 h, and 247+/-70 h, respectively, to decay from the fresh stage to skeletonisation. In winter, carcasses needed longer (1180+/-291) to decay as much. Carcasses attracted 47 species of insect, with flies predominating. The larvae were mainly Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart) and Hydrotaea (Ophyra) spinigera (Stein). Most necrophagous insects were found all year around, and there were no marked differences in species among the four seasons, except that Dermestes maculatus (De Geer) was absent in winter. Blowflies produced only one generation on a carcass before it became skeletonised, which simplified the estimation of PMIs.     

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.     

温度对4种常见尸食性蝇类生长发育的影响及其法医学意义

为查明温度对尸食性蝇类生长发育的影响，便于推断死亡时间，在实验室恒温条件下，以常见的4种尸食性蝇类，即巨尾阿丽蝇Aldrichinagrahami、丝光鲜蝇Luciliasericata、大头金蝇Chrysomyamegacephala和肥须亚麻蝇Parasarcophagacrassipalpis为材料进行研究。结果表明，在实验温度范围内，除巨尾阿丽蝇二、三龄幼虫和丝光绿蝇三龄幼虫之外，各种类各发育阶段的历期均随温度增高而缩短，巨尾阿丽蝇蛹在12℃和30℃时停止发育。根据试验结果，利用线性回归方程对发育速率与温度间的关系进行了模拟，结果均有良好的相关性。研究结果具有推断死亡时间的法医学意义。     

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.     

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.     

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).     

Use of Lucilia species for forensic investigations in Southern Europe

The aim of this study was to highlight the importance of evaluating entomological evidence in forensic investigations on a regional scale. To evaluate climatic, geographical and environmental influences on the selection of carrion-breeding fauna in Northern Italy and consequently on inferred forensic data (post-mortem intervals and post-mortem transfer), we present details of six indoor-outdoor cases. Results show that the most abundant species was Lucilia sericata, together with other fly species of entomo-forensic interest, belonging to the Calliphoridae and Sarcophagidae families. In particular, for the first time in Italy, we report finding Phormia regina, Lucilia ampullacea, Lucilia caesar and Sarcophaga (Pandelleana) protuberans on fresh cadavers. The active period of L. sericata in Northern Italy, according to previous findings in Southern Europe, revealing clearcut differences with phenologies in Northern Europe, has important consequences in estimating the period (season, months) of death in cases of long post-mortem intervals (several months or years) if empty puparia of this fly are found. According to our results, the distribution of L. sericata in areas with urban sprawl, like Northern Italian regions, cannot be used to evaluate post-mortem transfer from an urban area to a rural one.     

Forensic entomo-toxicology.

Drugs present in a decomposing corpse may be identified through analysis of maggots feeding off it. Case reports in forensic entomo-toxicology are sparse and the data base is unstructured. Drug concentrations should be measured in residual skeletal muscle, the principal food source for fly larvae, as well as in washed maggots, and the fly species should be determined. An untested possibility is the analysis of puparia or puparial cases which could extend the time frame for analysis into years or even into palaeopathology. In deaths indoors, the analysis of flies known to have emerged from the corpse is a theoretical possibility. To what extent drugs are retained in successive levels of the food chain is entirely unknown; drugs might be detectable in beetles feeding off fly larvae.     

Larval-mass effect: Characterisation of heat emission by necrophageous blowflies (Diptera: Calliphoridae) larval aggregates

Numerous Calliphoridae species larvae are necrophageous and develop on animal cadavers. During the feeding stages, a strong gregarious behaviour leads to the formation of large larval masses, allowing larvae to share digestive fluids. Furthermore, a mass of larvae emits heat, resulting in a local increase of temperature. Differences greater than 20°C between ambient and larval mass temperatures have already been observed, and the temperature of the mass can reach 50°C. Thus, larvae could benefit from this increased local temperature to speed up their development. This study focuses on the dynamic and characterisation of heat emission by Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) larval masses. Experiments were performed under controlled conditions using several ambient temperatures and different numbers of larvae. Results indicate that heat emission depends on the instar and is strongly affected by available amount of food. Furthermore, according to the experimental data, the heat emission is also relative to the weight of the larval mass, the larvae number and the local temperature. These results also demonstrate that optimum ambient-temperature values ranging between 22°C and 25°C produce maximal heat-emission per larva. Furthermore, a feedback loop, involving heat exchanges and physiological and behavioural thermoregulation processes, appears inside aggregates. In the context of forensic cases and post-mortem interval (PMI) estimation, these results indicate that heat emission can occur even with "small" masses if they are composed of high number of second stage larvae. Furthermore, particular attention should be paid on cases involving L. sericata larvae at ambient temperature ranging from 22°C to 25°C, which appears to maximise the heat-emission process.     

北京地区尸生性蝇类研究及其在法医鉴定中的应用

为提供法医学尸体死亡时间鉴定的准确性，对北京地区人尸上蝇类区系进行了系统调查（1994～1996）。发现本地区共有双翅目尸生性蝇类3科、12属、14种。其中常见种有红头丽蝇、丝光绿蝇、大头金蝇、肥须亚麻蝇、急钓亚麻蝇、棕尾别麻蝇和家蝇7种。对其常见种的幼虫进行不同温度和湿度下培养，观察常见环境因素，如日历期、风雨天气等因素对其生长发育速率的影响。结果表明，蝇类幼虫在其适宜生长发育的温度范围内，随着温度的升高发育速率加快，历期缩短；在同一温度和同一饲养条件下，发育速率因种而异；平均温度在28℃左右时，蝇类幼虫发育最快，尤其是1～2龄幼虫，时间为1天左右，且较稳定。这些数据对法医学推断死亡时间具有参考价值。     

Gene expression during blow fly development: improving the precision of age estimates in forensic entomology

Forensic entomologists use size and developmental stage to estimate blow fly age, and from those, a postmortem interval. Since such estimates are generally accurate but often lack precision, particularly in the older developmental stages, alternative aging methods would be advantageous. Presented here is a means of incorporating developmentally regulated gene expression levels into traditional stage and size data, with a goal of more precisely estimating developmental age of immature Lucilia sericata. Generalized additive models of development showed improved statistical support compared to models that did not include gene expression data, resulting in an increase in estimate precision, especially for postfeeding third instars and pupae. The models were then used to make blind estimates of development for 86 immature L. sericata raised on rat carcasses. Overall, inclusion of gene expression data resulted in increased precision in aging blow flies.     

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.