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.     

Wildlife forensic entomology: determining time of death in two illegally killed black bear cubs.

Forensic entomology is now commonly used to determine time of death in human death investigations. However, it can be equally applicable to wildlife crimes. This paper describes the use of entomology to determine time of death in the illegal killing of two young bear cubs in Manitoba, Canada. Two cubs were found shot, disemboweled, with their gall bladders removed. Natural Resource officers (Conservation Officers) and a Royal Canadian Mounted Police (R.C.M.P.) officer examined the remains, and the R.C.M.P. officer collected insect evidence. The only insects on the remains were adult blow flies coming to lay eggs and the blow fly eggs themselves (Diptera: Calliphoridae). The time of hatch was recorded and the insects were reared to adulthood. Time of hatch, together with species identification, macro and micro climate and lab developmental data were used to determine the time of death. The time was consistent with the time that the defendants were seen at the scene and was used in their conviction. This case illustrates that insect evidence can be equally as valuable in poaching cases as in homicide cases. However, in most cases Conservation Officers are unaware of this science. It is therefore, extremely important for more Conservation Officers to be educated about this field.     

Distinction of bloodstain patterns from fly artifacts

Forensic scientists may encounter blood spatter at a scene which may be pure or a mixture of fly artifacts and human bloodstains. It is important to be able to make an informed identification, or at least advanced documentation of such stains since the mechanics of production of fly artifacts are not determinable to the crime scene reconstructionist from regular police forces. We describe three cases in which experiments and crime scene reconstruction led to additional information. Case 1: Above the position of a victim, numerous blood stains of the low-high-velocity type were found. Exclusion of these stains being caused by force (but instead caused by the activity of adult blow flies) by use of the following observations that were confirmed in experiments: (a) sperm-/tadpole-like structure with length > width, (b) random directionality, and (c) mixture of round symmetrical and teardrop shaped stains. Case 2: A reddish spatter field was found on a fan chain two rooms away from the place where a dead woman was found. Localization of the spatter on the bottom end of the surface hinted strongly towards fly activity. Case 3: Double homicide; submillimeter stains were found on a lamp between the two corpses. Activity of flies was less likely compared to alternative scenario of moving lampshade and violent stabbing.     

Minimum and maximum development rates of some forensically important Calliphoridae (Diptera)

Blow fly development rates are frequently used to estimate elapsed time since death in homicide investigations in the first few weeks after death. However, in order to make more precise estimates of time since death, accurate developmental data must be generated for all carrion species, and at temperatures that are comparable with those found at crime scenes. This paper presents developmental rates determined for five forensically important species in British Columbia, Phormia regina (Meigen), Phaenicia sericata (Meigen), Eucalliphora latifrons (Hough), Lucilia illustris (Meigen), and Calliphora vicina Robineau-Desvoidy, at several temperatures.     

Child neglect and forensic entomology.

Close co-operation between forensic scientists, medico-legal doctors, and police forces made it possible to estimate not only the post-mortem interval but also the time since a child was neglected. On the skin surface under the diaper (anal-genital area), third instar larvae of the false stable fly Muscina stabulans FALLEN, and the lesser house fly Fannia canicularis L. were found. F. canicularis adults are attracted to both feces and urine. From the face, larvae of the bluebottle fly Calliphora vomitoria L. were collected. C. vomitoria maggots are typical early inhabitants of corpses. From the developmental times of the flies, it was estimated that the anal-genital area of the child had not been cleaned for about 14 days (7-21 day range), and that death occurred only 6-8 days prior to discovery of the body. This is the first report where an examination of the maggot fauna on a person illustrated neglect that had occurred prior to death.     

利用积温和昆虫发育历期推测死亡时间的研究

目的积累尸食性蝇类虫态发育历期和积温的资料。方法采用动物尸体作诱饵,分别对自然飞到诱饵上繁殖生长的6种尸食性蝇类的虫态发育历期和积温进行观察研究。结果经过观察,取得了棕尾别麻蝇、丝光绿蝇、紫绿蝇、宽丽蝇、巨尾阿丽蝇、大头金蝇等6种尸食性蝇类的虫态发育历期和积温的基础数据。并采用倒计积温的方法推测实际案例中死者死亡时间,破案后证实推测时间与实际死亡时间一致。结论本文数据可为利用尸食性蝇类虫态育历期的积温推测死亡时间提供依据。     

Caddisflies assist with homicide case: determining a postmortem submersion interval using aquatic insects

Although few indicators of time since death for corpses found in aquatic ecosystems are comparable in precision to the insect indicators used in terrestrial cases, there are observations that can be useful in suggesting or ruling out an approximate PMSI (postmortem submersion interval). For example, the time intervals required for certain growth phases of aquatic insects, such as caddisflies, that may attach themselves to the submerged remains can be used to estimate a minimum PMSI. Approximately 8 of the 13 orders of insects containing species with aquatic or semi-aquatic stages are likely to be associated with carrion or corpses in aquatic habitats. We present a case study in which portions of a body from an adult male were discovered in a south central Michigan stream. The body was dismembered and portions were recovered from two bags floating and submerged in the stream. Insect specimens collected from mesh and plastic bags consisted of one fly larva belonging to the family Muscidae, and caddisfly larvae belonging to two families: the Limnephilidae. (case-makers) and the Hydropsychidae, (net spinners). We used unique case-building behaviors of the limnephilid caddisflies found on the remains to elucidate a PMSI range consistent with the disappearance of the victim. It is important for forensic investigators to understand that although some precision is lost in estimating a PMSI with aquatic insects, these organisms should not be ignored in gathering evidence from aquatic crime scenes, and in fact, they can provide valuable details in estimating a PMSI.     

The development of the black blow fly, Phormia regina (Meigen).

The black blow fly, Phormia regina (Meigen) is a primary species commonly utilized to indicate a postmortem interval, or more appropriately a "time since colonization". Due to the importance of this species as a secondary myiasis producer in livestock operations, and more recently as a time since death indicator in the field of forensic entomology, a considerable amount of data on its growth and development has been generated. However, the developmental time as reported by these studies varies greatly, and current more detailed data is needed for use in medicocriminal entomology. Hourly developmental data is presented under constant temperatures of 10, 15, 20, 25, 30, 35 and 40 degrees C, and cyclic temperatures of 10-15, 15-25, 25-35 and 35-45 degrees C. This study is in agreement with the results reported by Kamal [Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera). I. Bionomics, Ann. Entomol. Soc. Am. 51 (1958) 261] and Melvin [Incubation period of eggs of certain musciod flies at different constant temperatures, Ann. Entomol. Soc. Am. 27 (1934) 406] only at temperatures of 25 degrees C and below. Bishopp [Flies which cause myiasis in man and animals: some aspects of the problem, J. Econ. Entomol. 8 (1915) 317] reported a shorter developmental duration for larval stages than what was produced with our laboratory rearings.     

Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal-adult development of synchronised animals using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: "fresh" pupae, kept frozen until analysis and "experimentally dried" pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in "experimentally dried" pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic entomology are discussed.     

Influence of electricity on post-mortal body temperature

In a case of suicidal application of electricity differences between the rectal temperature of the body and the suspected time of death were observed. In order to answer the question whether an electric current from hand to hand over >30 min led to a rise in body temperature FEM-based computer simulations and animal experiments were carried out. Both resulted in a warming of the soft parts in the arm without warming the body core. Thus a temperature-based estimation of the time since death can also be used in cases with electricity as the cause of death. Besides, in the animal experiment we found a spontaneous rise in the body core temperature even without application of electricity which may be a reason for the typical temperature plateau after death.     

Insects found on a human cadaver in central Italy including the blowfly Calliphora loewi (Diptera, Calliphoridae), a new species of forensic interest

In the case of unidentified bodies the estimation of the period since death or of the season of death plays an important role to focus the attention on a reduced number of people among the ones reported missing. Forensic entomology can be one of the most important methods for these estimations, as occurred in this case. Flies are typically the first insects to colonize a dead body. The case reported here concerns the colonisation by insects of a male body in advanced decay found during the winter in Central Italy. This case is of particular interest as few data are available on the entomological evidence in the cold season. In particular, in this case we recovered Calliphora loewi (Calliphoridae), a species never collected before on dead bodies in Southern Europe. Larvae of the black soldier fly Hermetia illucens (Stratiomyidae), pupae and larvae belonging to genus Hydrothea (Muscidae), and Necrobia rufipes (Cleridae) specimens were also collected. The estimated PMI enabled identification of the cadaver, confirmed by DNA analysis.     

Influence of electricity on post-mortal body temperature

In a case of suicidal application of electricity differences between the rectal temperature of the body and the suspected time of death were observed.In order to answer the question whether an electric current from hand to hand over >30 min led to a rise in body temperature FEM-based computer simulations and animal experiments were carried out. Both resulted in a warming of the soft parts in the arm without warming the body core. Thus a temperature-based estimation of the time since death can also be used in cases with electricity as the cause of death. Besides, in the animal experiment we found a spontaneous rise in the body core temperature even without application of electricity which may be a reason for the typical temperature plateau after death.     

Generalized additive models and Lucilia sericata growth: assessing confidence intervals and error rates in forensic entomology

Forensic entomologists use blow fly development to estimate a postmortem interval. Although accurate, fly age estimates can be imprecise for older developmental stages and no standard means of assigning confidence intervals exists. Presented here is a method for modeling growth of the forensically important blow fly Lucilia sericata, using generalized additive models (GAMs). Eighteen GAMs were created to predict the extent of juvenile fly development, encompassing developmental stage, length, weight, strain, and temperature data, collected from 2559 individuals. All measures were informative, explaining up to 92.6% of the deviance in the data, though strain and temperature exerted negligible influences. Predictions made with an independent data set allowed for a subsequent examination of error. Estimates using length and developmental stage were within 5% of true development percent during the feeding portion of the larval life cycle, while predictions for postfeeding third instars were less precise, but within expected error.     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.     

Width as an alternative measurement to length for post-mortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae

The length of fly larvae collected from corpses is often used to help provide an entomological estimate of time since death. However, 'head-curling' by larvae can affect the accuracy of length measurements. To investigate a possible resolution to this problem, larvae of Calliphora augur were grown on sheep's liver at two constant temperatures (20 and 25 degrees C). Replicate samples were collected at set time intervals until pupation. Body length and width were measured for individual larvae and examined as predictors of age. It was found that body width, as measured at the junction of the fifth and sixth abdominal segments, is comparable with body length for age prediction of maggots of C. augur grown at these temperatures. Furthermore, conversion of width to length can be done with 95% accuracy from a simple linear model.     

Maggot-induced postmortem changes simulating gunshot wounds

In the warm season, human cadavers often are the breeding ground of fly larvae. In special conditions, the damage by maggots can take on the appearance of bullet holes. Preexisting injuries allow the maggots to penetrate the skin also in parts other than the body apertures commonly infested. Round tissue damage similar to gunshot-wounds are favored by the fact that the larvae of some types of flies bundle together to form feeding communities. The enzymes from the salivary glands of the fly larvae may in some rare instances even lead to perforation of bone lamellae (e.g. in the region of the orbital roofs). The case reports deal with 2 cases in which changes due to damage by maggots primarily gave the impression of gunshot-wounds.     

Changes in cause of death determination due to the new Baden-Wurttemberg burial regulation

In the Burial Ordinance of the State of Baden-Württemberg taking effect on 1 November 2000 some aspects of the post-mortem examination have been revised. One of these revisions concerns the determination of the manner of death: In addition to the alternatives available so far ("natural death" and "clues to unnatural death") there is now the option to classify the death as "unclear"; in this case the physician is obliged to inform the local police without delay. The emergency doctors working in the ambulance service may restrict themselves to the determination of death; if there are grounds suggesting an unnatural death, the emergency doctor has to inform the ambulance control station immediately, which will then inform the police. Another new regulation is that the post-mortem examination has to be performed at the place of death or the place where the body was found. Moreover the Ordinance standardizes the obligation to perform a "thorough" examination on the undressed body under adequate light conditions. If there are clues pointing to an unnatural death or if the body is unidentified no changes must be made, in particular the body must not be undressed. The new legal situation is described and discussed with regard to its practical implications related to the determination of death.     

Three species of insects collected from an adult human corpse above 3300 m in elevation: a review of a case from Colorado

We report on the colonization of an adult human corpse by three insect species at 3350 m (11,000 ft) in elevation. The adult silphid Thanatophilus coloradensis (Wickham), adults of the blow fly Calliphora coloradensis (Hough), and larvae and adults of Lucilia silvarum (Meigen) were all collected from the victim's body which had been wrapped in plastic. The victim was found in late June in the Rocky Mountains of Colorado. This paper provides additional confirmation of the taxa utilizing a human corpse at high elevations in Colorado.     

DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae).

Identifying an insect specimen is an important first step in a forensic-entomological analysis. However, diagnostic morphological criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic analysis to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely separated geographic locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.