Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

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.     

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.     

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。     

The Effect of Flunitrazepam (Rohypnol®) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology

This study investigated the potential effects of flunitrazepam (known as “date rape drug”) on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal–Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.     

Effect of Temperature on Six Different Developmental Landmarks within the Pupal Stage of the Forensically Important Blowfly Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (t_L) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework.     

Postmortem interval (PMI) determined by study sarcophagous biocenoses: three cases from the province of Venice (Italy).

This paper presents and discusses three cases of cadavers found, unburied, in the province of Venice, Northern Italy. In each case, all insect species playing some role in carrion decay processes, both sarcophagous and their predators, found on or in the bodies, were collected and determined. In one case, many larvae of the black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae) were found and are reported, for the first time in Italy, as necrophagous. Experimental breeding of some fly species was also carried out in controlled temperature and humidity conditions. In particular, it was of interest to know how the Neotropical black soldier fly modified its life-cycle to adapt to a temperate climate. It was thus possible to establish the rate of larval growth and timing of pupation, emergence, oviposition and hatching in various external conditions. The postmortem interval (PMI) established by identifying the life-cycle stages of dipterofauna were later confirmed in all cases through police investigations and the results of forensic analyses.     

大鼠死后组织细胞DNA含量变化与死亡时间的相关性研究

目的应用流式细胞术研究大鼠死后组织细胞DNA含量变化与死亡时间的相关性。方法SD大鼠断颈处死后于不同时间段取心、肝、脾、肾器官组织,经胰蛋白酶消化等处理后制成单细胞悬液,应用流式细胞术,在620nm波长处检测各组不同器官组织细胞DNA含量,观察其变化规律。结果大鼠各器官组织细胞DNA含量随死亡时间延长均呈下降趋势。其中以脾组织细胞DNA含量变化趋势与死亡时间最具相关性,肝、肾次之,而心最差;死后48h,各器官组织细胞仅存微量完整的细胞核DNA。结论机体死亡后,各器官组织细胞核DNA含量随死亡时间的延长逐渐减少,具有一定的变化规律,可应用组织细胞DNA含量变化来推断死亡时间。     

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.     

Maggot development during morgue storage and its effect on estimating the post-mortem interval

When insect evidence is obtained during autopsy, forensic entomologists make decisions regarding the effects of low-temperature (-1 degrees C to 4 degrees C) storage of the body and associated insects when estimating the post-mortem interval (PMI). To determine the effects of storage in a morgue cooler on the temperature of maggot masses, temperatures inside and outside of body bags containing a human cadaver and porcine cadavers (seven replicates) were measured during storage. Temperatures remained significantly higher (p<0.05) inside of the body bags relative to the cooler, and remained at levels sufficient for maggot feeding and development. If the assumption that no insect development takes place during preautopsy refrigeration is made, potential error rates in PMI estimation of 8.6-12.8% occur. The potential for blow fly larvae to undergo significant development while being stored in the morgue is a possibility that forensic entomologists should consider during an investigation involving samples collected from autopsy. Case and experimental evidence also demonstrate that substantial tissue loss can occur from maggot feeding during morgue storage.     

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.     

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.     

利用COI序列序列鉴定常见嗜尸性蝇种

目的 检测嗜尸性蝇类线粒体DNA(mtDNA)细胞色素氧化酶辅酶Ⅰ(COI)中278bp序列,鉴定蝇科3属5种嗜尸性蝇,解决其形态学种类鉴定的难题,为死亡时间推断提供技术支持.方法 从15省、市(地区)室外草地家兔尸体上采集蝇科3属5种共计18个蝇类成蝇样本,提取mtDNA进行PCR扩增,序列测定且上传GENBANK,...     

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.     

家兔眼玻璃体液21例元素含量PMI关系的研究

目的寻找一种精确推定PMI的方法。方法应用电感耦合等离子体质谱(ICP-MS),系统研究了家兔死后96h内眼玻璃体液21种元素含量与PMI的相关性。结果眼玻璃体液Co、Mo、Cd、Sb、I、Pb、Bi、Li共8种元素与PMI无关。K、Mg、Fe、P、Na、Al、Ca、Ti、Ni、Cu、Zn、Sr、Ba共13种元素含量与PMI有关。结论眼玻璃体液K、Mg、Fe、P、Na、Al、Ca、Ti、Ni、Cu、Zn、Sr、Ba共13种元素与PMI有关。为研究人尸体眼玻璃体液元素与PMI的关系奠定了选择指标的基础。     

DNA定量技术在死亡时间推断中的研究进展

死亡时间推断是法医病理学研究的热点及难点之一.随着分子生物学技术的发展,DNA的定量已广泛应用于死亡时间推断的研究.本文主要对机体死后不同组织和器官中DNA含量随时间的降解情况,以及单细胞凝胶电泳、Feulgen染色图像分析技术、流式细胞仪等近年来新的DNA定量技术应用于死亡时间推断研究中的进展进行了综述.     

Yearly activity patterns in southern Victoria (Australia) of seasonally active carrion insects

Some seasonally active carrion invertebrates leave remnants of their presence that remain long after the completion of flesh decay. Remnants may include fly puparia (pupal cases) or insect exoskeletal elements. The presence of these remnants among old, decomposed remains can therefore indicate possible months in which death occurred. A large-scale study of the patterns of neonatal piglet decomposition and carrion insect succession in a forest was carried out in 1999 and 2000. Five fresh piglet carcasses were exposed once per season in both years and visited frequently throughout decay. Ten seasonally active taxa, likely to leave durable remnants of their presence were selected, and profiles were constructed of their activity times over the 2-year study.     

Survival of submerged blowfly species and their parasitoids: implications for postmortem submersion interval

Pupal survival of three blowfly species, Chrysomya albiceps, Chrysomya megacephala, and Chrysomya putoria (Diptera: Calliphoridae) and the parasitoid species Nasonia vitripennis (Hymenoptera: Pteromalidae) was studied after the pupae were experimentally submerged in water. Non-parasitized pupae at different developmental stages, 0, 24, 48, and 72 h, and parasitized pupae after 3, 8, 10, and 12 days of development were submerged for 6, 24, 48, or 72 h. Control groups for each pupal developmental stage (parasitized or not), which were not submerged, were also observed in order to compare the adult emergence rates. The survival of white pupae (0 h/age) decreased with time of submergence for all three blowfly species, showing the lowest rates compared with other experimental pupa groups. For the three blowfly species, non-parasitized pupae at 24 and 48 h of age showed survival rates above 60%. However, for pupae at 72 h of age, the survival rates decreased with increased underwater time, with less than 30% survival after 72 h in C. putoria and C. albiceps. The survival of parasitoids inside blowfly pupae that were submerged during their larval stage (3 days/age) decreased with the increase of submergence time. After the parasitoids reached the pre-pupal life stage, the survival was higher for all underwater periods. These observations can be useful in investigations of the decomposition of partially submerged bodies, or in cases of pupae found adhering to decaying flesh, hair, or clothes of corpses that were submerged after the larvae had developed and pupated.     

Growth of Chrysomya megacephala (Fabricius) Maggots in a Morgue Cooler*

Abstract: In estimating the postmortem interval (PMI) using maggots obtained during autopsy, the forensic entomologist makes decisions regarding the effects of low‐temperature storage of the body on the insects. In this case report, a corpse was found in an abandoned house in the residential area of Bukit Mertajam, Penang, Malaysia. The maggots were found to be alive inside the mouth of the deceased although the corpse had been in the morgue cooler for 12 days. The maggots were reared and identified as Chrysomya megacephala (Fabricius). The emerged adult flies were kept as a stock colony, and the duration of development under the indoor fluctuating temperature regime was studied. The total duration of developmental process of this species was 9.5 ± 0.5 days, and the PMI estimated was 3.2 ± 0.6 days. This case report demonstrates the survival of Ch. megacephala maggots for 12 days and their growth inside the morgue cooler.