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.     

Factors affecting decomposition and Diptera colonization.

Understanding the process of corpse decomposition is basic to establishing the postmortem interval (PMI) in any death investigation even using insect evidence. The sequence of postmortem changes in soft tissues usually gives an idea of how long an individual has been dead. However, modification of the decomposition process can considerably alter the estimate of the time of death. A body after death is sometimes subject to depredation by various types of animals among which insects can have a predominant role in the breakdown of the corpse thus, accelerating the decomposition rate. The interference of the insect community in the decomposition process has been investigated by several experimental studies using animal models and very few contributions directly on cadavers. Several of the most frequent factors affecting PMI estimates such as temperature, burial depth and access of the body to insects are fully reviewed. On account of their activity and world wide distribution, Diptera are the insects of greatest forensic interest. The knowledge of factors inhibiting or favouring colonization and Diptera development is a necessary pre-requisite for estimating the PMI using entomological data.     

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).     

Use of PCR-RFLP for differentiation of calliphorid larvae (Diptera,Calliphoridae) on human corpses

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.     

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.     

The Influence of Preburial Insect Access on the Decomposition Rate

Abstract: This study compared total body score (TBS) in buried remains (35 cm depth) with and without insect access prior to burial. Sixty rabbit carcasses were exhumed at 50 accumulated degree day (ADD) intervals. Weight loss, TBS, intra‐abdominal decomposition, carcass/soil interface temperature, and below‐carcass soil pH were recorded and analyzed. Results showed significant differences (p << 0.001) in decomposition rates between carcasses with and without insect access prior to burial. An approximately 30% enhanced decomposition rate with insects was observed. TBS was the most valid tool in postmortem interval (PMI) estimation. All other variables showed only weak relationships to decomposition stages, adding little value to PMI estimation. Although progress in estimating the PMI for surface remains has been made, no previous studies have accomplished this for buried remains. This study builds a framework to which further comparable studies can contribute, to produce predictive models for PMI estimation in buried human remains.     

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.     

A Forensic Entomological Analysis Can Yield an Estimate of Postmortem Interval,and Not Just a Minimum Postmortem Interval: An Explanation and Illustration Using a Case

Several authors claimed that a forensic entomological analysis can never be interpreted as the postmortem interval (PMI), but that it can be interpreted as a minimum postmortem interval (PMI_min), or dead for not less than X amount of time. Because in most instances, a PMI estimate should be a range, that is, the set of values from maximum postmortem interval (PMI_max) to PMI_min, objections to PMI estimation are objections to PMI_max estimation. The arguments for this position did not address the substantial body of literature describing estimation of both PMI_max and PMI_min using insect succession analysis. This report reviews the scientific issues and presents a recent homicide investigation in which several forensic entomology experts used the absence of carrion insects on the corpse, a kind of succession analysis, to estimate PMI or PMI_max.     

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.     

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。     

An investigation into the persistence of textile fibres on buried carcasses

A significant amount of research has been carried out on fibres to aid the forensic scientist in determining the significance of these when found on a victim or suspect. This work has focused on open-air environments, and as such no research has been undertaken to examine the persistence of fibres on bodies in the burial environment.Wool and cotton fibres, known to fluoresce under ultraviolet (UV) light, were transferred onto the skin of four porcine (Sus scrofa) carcasses (two carcasses per fibre type). The number of fibres transferred was recorded from images taken under UV light. The remains were subsequently placed in four burial sites and left interred for 14 days. After this period the carcasses were excavated and lightly brushed down to remove the soil layer that had adhered to the skin. Once again photography under UV light was used to record the number of fibres which persisted on the skin.Results showed that after 14 days, wool and cotton fibres remain on the surface of the buried carcasses. In no circumstance was there a total loss of fibres suggesting that in such scenarios, the likelihood of finding fibres is high but the initial number of fibres transferred would be strongly diminished. This has important implications for both the excavation protocol for buried remains and the subsequent analysis for physical evidence.     

深圳地区夏季室内外猪尸体上嗜尸性昆虫的群落演替

目的：根据深圳地区夏季室内外猪尸体上主要嗜尸性昆虫的生长发育和群落演替规律来推断死亡时间（PMI）。方法从2013年5月初到8月初,在深圳市公安局法医检验中心模拟的室内、外场地分别放置成年猪尸体两头,观察尸体腐败的过程和猪尸体上出现主要昆虫的种类,记录猪尸体腐败各阶段的历期,以及主要昆虫在尸体上的群落演替。结果室内、外的猪尸体分别经历了412.5 h和325 h进入白骨化阶段。主要的嗜尸性昆虫,蝇类以大头金蝇、绯颜裸金蝇、星岛金蝇,甲虫类以大隐翅甲、赤颈郭公甲、丽腐阎甲、白腹皮蠹为优势代表性种类。室外猪尸体在较多雨水的影响下,优势蝇群明显产生二代,室内的猪尸体上未见较大规模二代产生。结论深圳地区夏季室内外猪尸体上主要昆虫的群落演替有规律性,7种代表性昆虫及其幼虫整体的活动规律对推断PMI有较大的意义。     

Insect fauna visiting carrion in Southwest Virginia

Successional patterns of insect fauna on pig carcasses were studied in southwest Virginia. The objective was to identify and qualitatively assess the major taxa of forensic importance in this region. Studies were conducted in spring and summer 2001 and 2002, and fall 2002. Over 50 taxa were collected and identified. Phormia regina was the dominant fly species in the spring (>90%) and co-dominant with Phaenicia coeruleiviridis in the summer. Phaenicia sericata, Lucilia illustris, and Calliphora spp. were collected in spring and summer, but less frequently. Eleven species of Sarcophagidae also were collected with Sarcophaga utilis and Helicobia rapax the most common. In the fall, the dominant fly species were Calliphora vomitoria, L. illustris, and P. coeruleiviridis. The primary beetle species collected in spring and summer included three Staphylinidae (Creophilis maxillosus, Platydracus maculosus, and Aleochara lata) and three Silphidae (Oiceoptoma noveboracense, Necrodes surinamensis, and Necrophila americana). No beetles were collected in the fall.     

Forensic entomology exemplified by a homicide. A combined stain and postmortem time analysis]

The combined analysis of both ant and blow fly evidence recovered from a corpse, and from the boot of a suspect, suggested that an assumed scenario in a high profile murder case was likely to be true. The ants (Lasius fuliginous) were used as classical crime scene stains that linked the suspect to the scene. Blow fly maggots (Calliphora spec.) helped to determine the post mortem interval (PMI) with the calculated PMI overlapping with the assumed time of the killing. In the trial, the results of the medico-legal analysis of the insects was understood to be crucial scientific evidence, and the suspect was sentenced to 8 years in prison.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.     

Retrospective analysis of factors affecting rate of skeletonization within a tropical climate

Estimating the post mortem interval (PMI) in skeletonized cases is an extremely challenging exercise. Sri Lanka lacks adequate taphonomic research which is a serious limitation when assessing PMI in forensic death investigations. Methods that have been proposed to estimate PMI using the total body score (TBS) and accumulated degree days (ADD) are mainly based on data from continental and temperate climates and have shown less reliability in tropical climates. With the intention of developing a region-specific, evidence-based guide which would be applicable to tropical climates like Sri Lanka, we selected thirteen skeletonized remains with known PMIs from forensic case records and analysed their taphonomy in relation to selected weather data. We also compared the ADD values within our dataset with reference ranges calculated using published formula.All except one were found from outdoor locations. The TBS ranged from 24 to 32 and had a weak positive correlation with the PMI. The earliest appearance of skeletonization was 15 days in a body found indoors. The highest rate of skeletonization was seen in a body with a TBS of 32 and a PMI of 23 days. The average daily temperature and relative humidity were similar across all the cases however, the amount of rainfall varied. Bodies exposed to monsoon rains (n = 6) had a lower mean rate of skeletonization compared to those that were not exposed (n = 4) suggesting lower rates of decomposition during periods of heavy rainfall.No correlation was found between ADD and TBS. In 9 (69.2%) cases, the actual ADD was much lower than reference ADD ranges for TBS values, indicating poor applicability of TBS and ADD based formulae in estimating PMI within the Sri Lankan climate. Our study shows a strong need for taphonomic and entomological research in tropical climates to further explore the impact of monsoons on biotic and abiotic factors affecting skeletonization.     

Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae) in Human Skull: A New Fly Species of Forensic Importance

A body of an unknown adult female was found within a shallow burial ground in Malaysia whereas the skull was exposed and visible on the ground. During autopsy examination, nine insect larvae were recovered from the interior of the human skull and subsequently preserved in 70% ethanol. The larvae were greyish in appearance, each with a posterior elongated breathing tube. A week after the autopsy, more larvae were collected at the burial site, and some of them were reared into adults. Adult specimens and larvae from the skull and from the burial site were sequenced to obtain DNA barcodes. Results showed all adult flies reared from the burial site, as well as the larvae collected from the skull were identified as Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae). Here, we report the colonization of E. arvorum larvae on a human corpse for the first time.     

An initial study of insect succession and carrion decomposition in various forest habitats of Central Europe

At the end of summer and beginning of fall 2005 insect succession and pig carrion decomposition were studied in pine-oak forest, hornbeam-oak forest, and alder forest in Western Poland (Central Europe). Five stages of decomposition were recognized: fresh stage, bloated stage, active decay stage, advanced decay stage, and remains stage. The highest rate of decomposition was observed in the alder forest and the lowest in the pine-oak forest. A similar composition of carrion entomofauna was found in each forest. Numerous adults of Lucilia caesar, Phormia regina and Calliphora vomitoria (Calliphoridae), Hydrotaea spp. (Muscidae), Stearibia nigriceps and Parapiophila vulgaris (Piophilidae), Necrodes littoralis and Thanatophilus rugosus (Silphidae), Creophilus maxillosus, Omalium rivulare, Oxypoda acuminata and Philonthus spp. (Staphylinidae) as well as larvae of L. caesar, P. regina, C. vomitoria, Fannidae (Diptera) and N. littoralis, C. maxillosus, Philonthus (Coleoptera) were collected. There were no differences between forests in the sequence of insect occurrence on carrion. However, differences between forests in occurrence time and activity period of some taxa were found. Implications of these results for forensic entomology are discussed.     

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.     

An investigation of model forensic bone in soil environments studied using infrared spectroscopy

Infrared spectroscopy has been used to examine changes to bone chemistry as a result of soil burial. Pig carcasses were buried as part of a controlled field study, and pig bone was used in soil environments established in the laboratory. The variables of species type, bone pretreatment, soil type and pH, moisture content, temperature, and burial time were investigated. The crystallinity index (CI) and the organic and carbonate contents of the bones were monitored. The data revealed decreasing trends in the organic and carbonate contents and an increase in the CI of the bone with burial time. An acidic soil environment and soil type are the factors that have the most influence on bone chemistry as a result of burial. The study demonstrates the potential of infrared spectroscopy as a straightforward method of monitoring the changes associated with aging of bones in a variety of soil environments.