Quantifying the Temperature of Maggot Masses and its Relationship to Decomposition

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single‐species aggregations (Lucilia sericata) of various sizes (50–2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p‐value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.     

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.     

Preliminary study of the succession pattern of necrobiont insects, colonising species and larvae on pig carcasses in Temuco (Chile) for forensic applications

Knowledge regarding the succession patterns of insects that visit carcasses as well as the other arthropod that colonise them and analysis of the parameters that are associated with larvae allow calculation of the minimum postmortem interval (PMI). This information is obtained from experiments carried out under specific geoclimatic conditions, which determine their application in forensic environments under similar conditions. The field study presented here is the first in Chile to analyse the decomposition process of pig carcasses and the associated succession of insects, colonising species and parameters related to larval masses. All of the larvae obtained from daily samples were measured (in mm), and their mean, range, standard deviation and stage of development (instars) were determined. The carcasses reached the dry remains stage in only 11 days. Seven species of Diptera visited the carcass during the process, but only two species colonised it by means of egg deposition followed by development of larvae: Cochliomyia macellaria (Fabricius) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), which exhibited a duration of the development cycle from egg to adult of 21 days. The collected Coleoptera correspond to five predator species (Staphilinidae and Histeridae). From the results of this study, it can be concluded that only some of the insect species present in this region can provide information that can be used in forensic entomology and that analysis of larval masses of colonising species can be a valuable tool for determining the PMI(min) in this region of Chile.     

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.     

Decomposition and insect succession of clothed and unclothed carcasses in Western Australia

The effect of clothing on carcass decomposition and patterns of insect succession onto remains were investigated in two separate years during autumn in Western Australia. The progression of decomposition differed between clothed and unclothed carcasses in both years of the study. The presence of clothing markedly prolonged the wet decay stage in both years with larval feeding occurring across the moist skin surface underneath clothing, as well as within and under the carcasses. Ambient temperatures were higher in the second year of the study and corresponded to marginally faster rates of decay throughout decomposition. Within years, insect arrival and oviposition were largely consistent between clothed and unclothed carcasses with a few notable exceptions. The green blow fly, Lucilia sericata Meigen (Diptera: Calliphoridae) oviposited one day earlier on clothed than unclothed carcasses in both years of the study. The black carrion fly, Australophyra rostrata Robineau-Desvoidy, (Diptera: Muscidae) colonised clothed carcasses in two distinct waves of succession but only one wave of ovipoistion was observed on unclothed carcasses in either year. Correspondingly, clothed carcasses supported larval feeding by A. rostrata for a longer duration than unclothed carcasses. Finally, dipteran larval masses were more widely distributed across the carcass surface and were present for a longer period of time on clothed carcasses than on unclothed carcasses in both years. Forensically relevant data detailing the seasonal pattern of insect succession onto clothed and unclothed decomposing remains in Western Australia are reported.     

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.     

Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae)

By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the post-mortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25°C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48h. Only the larvae with a dispersal time of 24h (total time of development 325.2h; median) and 48h (total time of development 347.7h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately.     

Temperature variations in a parked vehicle

There were two reasons why this work was conducted. The first was to help determine the time of death of suicide and homicide victims inside vehicles. The second was to investigate the serious threat to life of children or pets left in stationary vehicles on a hot summers day. This paper demonstrates that when a vehicle is parked in the sun, temperature levels in the cabin of the vehicle can be more than 20°C above the ambient temperature. A simple 'greenhouse' model for predicting the daily internal vehicle temperatures, using readily available local meteorological data, was developed. This statistical model was calibrated using meteorological data and temperature data collected on parked vehicles over several summer seasons. The model uses environmental temperature and radiation data as input, and is shown to predict cabin temperatures to within about 1°C. Both the data collected and the model developed show that the temperature inside the cabin of a black vehicle is typically 5°C higher than that inside a white vehicle on a hot summer day. Also lowering the driver's window of the vehicle by 2.5 cm typically reduces cabin temperatures by about 3°C, which is not sufficient to reduce significantly the safety concerns for children or pets left in parked vehicles.     

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.     

石河子春季常见尸食性昆虫种类及丝光绿蝇发育观察

目的观察新疆石河子市区尸食性昆虫的种类及温度对优势种丝光绿蝇生长发育的影响。方法 2007年和2008年的4月15日至6月15日在石河子市（E44.18°、N86.00°）3个不同地点诱集尸食性昆虫,并鉴定分类。将优势种丝光绿蝇在不同温度下饲养,观察其体长、体重、羽化率、雌雄比、历期等生长发育特点。结果石河子地区春季3个不同地点共诱集到尸食性昆虫6目14科16种,其中以膜翅目、双翅目和鞘翅目为主;丝光绿蝇幼虫体长、体重随着温度升高而增长、增加,化蛹时又有所缩短、减轻;不同的温度对幼虫的羽化率和雌雄比影响不明显。结论本文所得数据可为新疆石河子地区的法医昆虫学及相关研究和实践提供一定参考。     

Growth behavior of the blue blowfly Calliphora vicina maggots

Fifty-three clusters of blowfly eggs of the genus Calliphora vicina were observed in the laboratory up to the hatching stage under reproducible and virtually field-like conditions. Rearing the larvae was then continued up to pupation, the larval growth in length being recorded several times a day. As the object was to study the dependence of the larvae increase in length on the temperature conditions in vitro, the substratal humidity and food supply were kept unchanged during the entire study. The temperature ranged from 6.5 degrees C to 35 degrees C, with the temperature for the individual cluster kept constant during the entire developmental process. Data on about 5500 measured larvae were statistically evaluated. The basic result established was that in the case of the blowfly of the genus Calliphora vicina in vivo, all developmental stages relevant to the entomologic determination of the time of death depend on the temperature conditions: (1) the duration of the egg stage increases with decreasing temperature; (2) the speed of larval growth is slower at lower temperatures; (3) the maximal larval length is reached earlier at higher temperatures; (4) the mean value of maximal length decreases with increasing temperature; (5) larvae under all temperature conditions decrease in size after having reached their maximal length, the decrease in length being more rapid at higher temperatures; (6) constant temperatures over 30 degrees C lead to "stunted forms" which do not pupate and die; (7) constant temperatures under approximately 16 degrees C after the peak of growth has been reached inhibit the readiness to pupate, which causes the larvae to fall into a stationary state of rest, which will be interrupted only when the temperature is raised and resumption of the metamorphosis is thus induced. To allow rapid reconstruction of the larval age in general practice, the established growth data were set out in the form of a diagram designated isomegalendiagram, which permits temperature-fluctuation-related entomologic determination of the time of death with a maximum degree of accuracy.     

Responses of Lucilia sericata Meigen (Diptera: Calliphoridae) to cadaveric volatile organic compounds

Flies of the Calliphoridae Family are the most forensically important insects because of their abundance on the decedent during the first minutes following death. Necrophagous insects are attracted at a distance by a decomposing body, through the use of volatile chemical cues. We tested the possible attractive role of some volatile organic chemicals (VOCs) released by decaying cadavers, on male and female of Lucilia sericata Meigen (Diptera: Calliphoridae). Two complementary approaches were used. Electroantennography (EAG) allowed identifying the semiochemicals that are detected by the olfactory system of L. sericata. Dose-response tests with EAG showed that dimethyl disulfide (DMDS) and butan-1-ol elicited the highest responses. Behavioral assays showed that, among the VOCs tested, DMDS and butan-1-ol are attractive for L. sericata, while the other VOCs are repulsive or do not cause any behavior. Our results may have potential implications in a better understanding of attractiveness of blowflies toward a corpse.     

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。     

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (D_z) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17–37 ⁰C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (D_z ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.     

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.     

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.     

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 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.     

Potential Use of Hydrocarbons for Aging Lucilia sericata Blowfly Larvae to Establish the Postmortem Interval

Previous studies on Diptera have shown the potential for the use of cuticular hydrocarbons' analysis in the determination of larval age and hence the postmortem interval (PMI) for an associated cadaver. In this work, hydrocarbon compounds, extracted daily until pupation from the cuticle of the blowfly Lucilia sericata (Diptera: Calliphoridae), have been analyzed using gas chromatography–mass spectrometry (GC–MS). The results show distinguishing features within the hydrocarbon profile over the period of the larvae life cycle, with significant chemical changes occurring from the younger larvae to the postfeeding larvae. Further interpretation of the chromatograms using principal component analysis revealed a strong correlation between the magnitudes of particular principal components and time. This outcome suggests that, under the conditions of this study, the cuticular hydrocarbons evolve in a systematic fashion with time, thus supporting the potential for GC–MS analysis as a tool for establishing PMI where such a species is present.     

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.