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.     

The use of mitochondrial cytochrome oxidase I gene (COI) to differentiate two UK blowfly species -- Calliphora vicina and Calliphora vomitoria

Traditionally identification of forensically important insects has been carried out based upon morphological differences between species. However insect evidence found at a crime scene may on occasion be difficult to distinguish by morphological techniques and under these circumstances another method of accurate identification is required. This work utilises a cytochrome oxidase I partial mitochondrial gene region (COI) to distinguish the two of the main UK blowfly species -- Calliphora vicina (Robineau Desvoidy) and Calliphora vomitoria (Linnaeus) (Diptera:Calliphoridae). Seventeen interspecific differences in COI sequence were located. Use of the restriction enzyme SfcI on this gene region provides a simple method for distinguishing between C. vicina and C. vomitoria.     

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): a comparative study

A comparative morphological study of preimaginal stages (larvae and pupae) of Calliphora vicina Robineau-Desvoidy, 1830 is presented. The entomological samples came from laboratory colonies bred under controlled environmental conditions (25°C and 60% relative humidity). In this study, a recently published technique to clear Diptera larvae for light microscopy and a standard protocol for scanning electron microscopy were used. For the morphological comparison of larval instars I, II and III, and pupae of C. vicina, different larval regions (cephalic, thoracic and abdominal, including anal division), as well the internal chitinised cephalopharyngeal skeleton, were considered separately. Our results focus on showing the changes observed throughout development for the most important structures in the cephalic region (sensilla of maxillary palpus, antennae and oral ridges), the thoracic region (the first segment and its anterior spinose band) and in the anal division of the abdominal region (posterior spiracles and shape of the papillae). In addition, some morphological structures are described or pictured for the first time, such as the ventral organ and the anterior spiracle of larva I and the antenna sensilla, Keilin's organ and wrinkled area of the anal division of all instars. The cephalopharyngeal skeleton is an important structure for the taxonomy of Diptera larvae in all instars, including Calliphoridae. Our observations in C. vicina indicate that an in-depth review of the sclerite composition is needed. Pupae and larvae stages can only be compared by following the segmentary spinose bands and the anal segment, where the morphology of the posterior spiracles and papillae can be observed, in some cases despite the reduced condition of the latter.     

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).     

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.     

Calliphora vicina (Diptera: Calliphoridae) collected from a human corpse above 3400 m in elevation

In the summer of 2005 an adult hiker was killed in a fall in Rocky Mountain National Park, Colorado (U.S.A.). His body was subsequently colonized by the blowfly Calliphora vicina (Diptera: Calliphoridae). The body was discovered 9 days later at an elevation of 3475 m (11,400 feet). This is the first report of C. vicina on a human corpse at this elevation in Colorado. This paper provides additional confirmation of this fly colonizing a human corpse at high elevations in Colorado.     

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.     

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.     

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.     

Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram.

Developmental behavior of eggs, larva and pupa of the blowfly species Lucilia sericata (Meigen) were studied under 10 different temperature regimes. Data from these studies were used to construct the isomegalen-diagram. In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. If the temperature is roughly constant, as is the case with corpses found indoors, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable, an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. Equally, the isomorphen-diagram representing all morphological stages from oviposition to eclosion should be used, if maggots in the migratory phase or pupae or puparia are recovered from the scene. The isomegalen- and the isomorphen-diagrams could facilitate a quick and more precise estimate of the postmortem interval even for the inexperienced investigator. In addition, our results vary from those of other investigators, suggesting a different thermal behavior of the holarctic blowfly L. sericata in various zoogeographic regions.     

The forensic application of allozyme electrophoresis to the identification of blowfly larvae (Diptera: Calliphoridae) in southern Australia

Most known carrion-breeding species of blowflies in southern Australia are of the genus Calliphora. The morphological similarity of the immatures of these species means that correctly identifying them poses a challenge for forensic entomologists. This study investigates the potential of allozyme analysis to assist with this task. Molecular profiles of third-instar larvae and adults representing four of these carrion-breeding species, Calliphora stygia, C. dubia, C. hilli hilli, and C. vicina, were compared at 42 allozyme loci. The two life stages were found to display almost identical allozyme profiles in each species (93% of loci were expressed in both life history stages), enabling the reliable identification of larvae in these four species. Integration of these results with data from a previous study indicates that allozyme analysis would also be suitable for rapid, species-level identification of the larvae of six other carrion-breeding Calliphora species occurring in southern Australia. This is the first report of the application of allozyme data to the identification of forensically important blowflies.     

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.     

Tracking Movement and Temperature Selection of Larvae of Two Forensically Important Blow Fly Species Within a “Maggot Mass”

The current study responds to the lack of understanding about the temperatures experienced by individual blow fly larvae within “maggot masses.” The temperature selection of both aggregating (in a mass) and nonaggregating larvae was compared and their pattern of movement assessed. Infrared imaging determined the temperatures within a mass and in the vicinity of the constituent individual larvae, whose movements were tracked by dyeing their tissues red. Individual Chrysomya rufifacies larvae selected temperatures above 27°C, significantly higher than the temperature selected by Calliphora vicina larvae (24.5°C). However, this same difference was not seen within a mass, with both species selecting temperatures around 28°C. Larval movement in a mass was nonrandom, indicating that larvae actively select their position in a mass. Furthermore, larvae have a strong tendency to select the hottest part of a mass; therefore, maximum mass temperatures might provide a reliable proxy for the actual temperatures experienced by larvae.     

Effects of Ketamine on the Development of forensically important Blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its Forensic Relevance

This study investigated effects of ketamine on the development of Chrysomya Megacephala (Diptera: Calliphoridae) at three different temperatures. Larvae of the C. Megacephala were exposed to different concentrations of drugs and temperatures. The larval lengths, weights, and developmental durations of each stage were observed. This study demonstrated that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala (p < 0.001). The time that the larvae in all the treatments achieved the maximum length/weight was significantly delayed (p < 0.05), and that resulted in prolonged duration of larval and prepupal stages especially at low temperature. However, no linear correlations were discovered between ketamine concentration and growth rate of larval length/weight.     

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。     

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16–34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D₀ and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D₀ = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.     

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.     

The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia.

The applicability of mitochondrial DNA (mtDNA) sequencing was investigated for the identification of the following forensically important species of blowflies from southeastern Australia: Calliphora albifrontalis, C. augur, C. dubia, C. hilli hilli, C. maritima, C. stygia, C. vicina, Chrysomya rufifacies, Ch. varipes and Onesia tibialis. All breed in carrion except O. tibialis, which is an earthworm parasitoid. Emphasis was placed on Calliphora species because they predominate among the carrion-breeding blowfly fauna of southern Australia and their immatures are difficult to identify morphologically. A partial sequence of the mitochondrial COII gene was determined for all species and for COI for C. albifrontalis, C. augur, C. dubia and C. stygia only. Five other species of blowflies, Chrysomya albiceps, Ch. rufifacies, Protophormia terraenovae, Lucilia illustris and L. sericata, for which sequence data were already available, were also included. Analysis of the COI and COII sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, i.e. the species within the C. augur and C. stygia groups. The molecular data support the existing species group separation of the taxa within Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach, with the analysis of additional taxa and genes, is likely to enable the reliable identification of carrion-breeding blowflies in Australia.     

Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation

The identification of insects found on a dead body can lead to the estimation of the time of death (postmortem interval). We report an updated version of an established method based on sequence analysis of PCR products from a region of the cytochrome b oxidase subunit I mitochondrial gene of different members of the family Calliphoridae, by sequencing six European species: Lucilia sericata (Meigen), Lucilia caesar (Linné), Lucilia illustris (Meigen), Calliphora vicina (Robineau-Desvody), Calliphora vomitoria (Linné), Protophormia terraenovae (Robineau-Desvody) and one Guianese species: Cochliomyia macellaria (Fabricius). This technique provided clear results when applied to the larvae and we also report the identification of empty puparia.     

Opiate analysis in cadaveric blowfly larvae as an indicator of narcotic intoxication

Specimens of liver were collected from 40 cases in which the cause of death had been determined to be opiate intoxication. Rearings of Calliphora vicina larvae were then promoted on the decomposing liver. A control group of 10 decomposed liver specimens from non-opiate deaths was treated similarly. Analysis of larvae and liver for opiates (morphine) was conducted by radioimmunoassay. Good qualitative and quantitative correlation was observed in both the positive and negative groups. Regression analysis comparing the concentrations of opiates found in the larvae with those found in the liver in the positive group resulted in a correlation of r = 0.790.