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.     

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.     

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.     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

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.     

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.     

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.     

Influence of storage on larval length and age determination of the forensically important blow fly Lucilia sericata (Diptera: Calliphoridae)

One of the main tasks in forensic entomology is the determination of the minimum post-mortem interval (PMI_min) based on the age of the juvenile insects feeding and developing on the dead body. An important task is to store the evidence appropriately so that the evaluation and expert report can be used in court. However, existing recommendations can be contradictory or lacking scientific validation, e.g. by proposing various preservation liquids without knowing whether and to what extent the period of storage in such a liquid has an effect on the length of the preserved larvae. Storage time can be an issue since, due to technical and procedural circumstances, killed larvae may be stored for hours, days, weeks or even longer prior length measurement. A changed body length would have consequences for the entomological report, as the age of the larvae is usually derived from their length.This study investigates the effect of four differently concentrated ethanol solutions (70%, 80%, 90% and 96%) during a storage period of up to 196 days on the body length of stored larvae of the forensically important blow fly species L. sericata (Diptera: Calliphoridae). Larvae of different ages (24 h, 48 h and 72 h after hatching) were killed by immersion in hot, non-boiling water (≥80 °C) for at least 30 s. Their lengths were measured immediately. Subsequently samples were stored in ethanol of appropriate concentration at room temperature (approx. 22 °C). Further length measurements were made at 16 different storage intervals between 1 and 196 days.Many specimens showed a length decrease for most storage conditions and all larval ages. However, there was a tendency for 48 h- and 72 h-old larvae to increase in length after the first days of storage of up to 1.1 mm which may lead to an erroneous overestimation of the PMI_min using this kind of specimens. All changes in length within each cohort over total time were in the range of +7% to −9.1%. Significant differences in length changes within the first days of storage were found mainly in larvae stored in 70%- and 80%-ethanol, but larvae stored in 90%- and 96%-ethanol showed first significant differences on day 56 at the earliest.Our results lead to the recommendation that the measurements of fly larvae samples should be taken immediately after killing and before storage to avoid any effects. Ethanol ≥90% should be used for storage.     

Effects of Different Temperatures on the Development of Dermestes Frischii and Dermestes Undulatus (Coleoptera,Dermestidae): Comparison Between Species

Dermestidae could be useful in forensic investigations to assess the PMI as adults and larvae colonize dried remains. We reared two species of Dermestidae (Dermestes frischii and Dermestes undulatus) to understand the effects of different temperatures on the length of their whole life cycle and on their immature stages. Both species were reared at 23°C ± 0.5, RH 75% and at 26°C ± 0.5, 75% RH. Our result shows that the temperature is the main factor that influences the development of those species; in fact, increasing temperature leads to a shorter development cycle (59.8 ± 0.5 and 38.1 ± 0.2 for D. frischii; 50.6 ± 0.6 and 36.2 ± 0.2 for D. undulatus). Furthermore, we found that the number of the molts before the pupa decreases from 5–7 to 5–6 for D. frischii and from 4–6 to 4–5 for D. undulatus, respectively, at 23°C and 26°C.     

Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real-Time Polymerase Chain Reaction High-Resolution Melt Duplex Assay,

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used “legal high” opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR^® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant^™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen^® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/μL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen^® PLUS.     

The Effect of Temperature and Exposure Time on Stability of Cholesterol and Squalene in Latent Fingermarks Deposited on PVDF Membrane

Cholesterol and squalene are fatty materials of latent fingermarks that can be utilized for dating methodologies and visualization techniques. Previous studies have suggested these compounds undergo degradation in fingermarks as a function of time (days) and light at ambient temperature. However, studies assessing how their composition changes at low and high temperatures over short periods of time (hours) have not been published previously. Here, we performed quantitative analysis of cholesterol and squalene in natural fingermark residue using PVDF membrane, after exposure to a range of temperatures (−20 to 100°C) for 4 and 8 h. We found that levels of both fatty materials remained constant at −20 to 60°C, but both showed significant reduction at 100°C, over short exposure times. These results indicate that cholesterol and squalene are detectable at −20 to 60°C, whereas at 100°C or higher, both are lost due to rapid thermal degradation.     

An Experiment to Characterize the Decomposer Community Associated with Carcasses (Sus scrofa domesticus) on Oahu,Hawaii

Pig (Sus scrofa domesticus) carcasses were decomposed on the soil surface of a terrestrial habitat on the island of Oahu, Hawaii to begin characterizing the decomposer community. Results showed that carcasses can decompose rapidly on Oahu, primarily due to the activity of fly larvae, with ~80% of mass lost by 8 days (~220 ADD) postmortem. Scavenging was conducted exclusively by the small Indian mongoose (Herpestes javanicus), first feeding on larvae then feeding on the remains. Carcasses were habitats of warm temperature, little to no oxygen, slightly acidic/neutral pH, and high sodium concentration. Larval masses selected for a microbial community comprised of multiple bacterial taxa from phyla Firmicutes and Proteobacteria, particularly genera Clostridium, Proteus, and Providencia. These larval masses were well established from 3 to 8 days (~90 to ~220 ADD) postmortem. These data provide helpful, novel insight into the structure and activity of carcass decomposer communities on Oahu.     

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.     

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.     

Validation of a new method of providing case-specific time-of-death estimates using cadaver temperatures

The near ubiquitous presence of numerical simulation has made case-specific calculations of body temperatures following death possible so that accurate calculations of body temperatures can provide valuable information for estimating the time of death and can aid in forensic investigations. Here, a computational approach is described that has been validated against multiple, independent, and experimental investigations. The approach only requires one subjective input parameter (the heat transfer coefficient). A simple algorithm serves as a guidepost to the selection of this parameter. The algorithm incorporates clothing and the space in which the body is housed. Heat transfer coefficients that range from h = 2 W/m²/°C for bodies that are heavily clothed to h = 9 W/m²/°C for bodies that are nude (in air). The method also requires setting of ambient temperature conditions (ambient temperature)—however, that input is often available. The paucity of inputs makes this technique remarkably easy to employ. The new method is also able to calculate cadaver cooling rates for situations where the cadaver is in a timewise or spatially changing thermal environment (diurnal temperature variations, bodies partially submerged in water, changes to weather, insolation, etc.). Results from the present calculations are compared with a large body of measurements from the literature; it was found that the predictions and measurements were in excellent agreement, regardless of the ambient temperature conditions and the nature of the clothing of the body. This new calculation approach can be used with reasonable accuracy for determining cadaver cooling and time since death.     

Time- and Temperature-Dependent Changes in Cytochrome c Oxidase Activity and Cyanide Concentration in Excised Mice Organs and Mice Cadavers

Postmortem stability of cyanide biomarkers is often disputed. We assessed the time and temperature-dependent changes in cytochrome c oxidase (CCO) activity and cyanide concentration in various organs of mice succumbing to cyanide. Immediately after death, excised mice organs and mice cadavers were stored at room temperature (35°C ± 5°C) or in frozen storage (−20°C ± 2°C). At various times after death, CCO activity and cyanide concentrations were measured in excised mice organs or organs removed from mice cadavers. The study revealed that (i) measuring both the biomarkers in mice cadavers was more reliable compared to excised mice organs, (ii) measuring temporal CCO activity and cyanide concentration in vital organs from mice cadavers (room temperature) was reliable up to 24 h, and (iii) CCO activity in the brain and lungs and cyanide concentration in organs from mice cadavers (frozen) were measurable beyond 21 days. This study will be helpful in postmortem determination of cyanide poisoning.     

Viability of Human Articular Chondrocytes Harvested Postmortem: Changes with Time and Temperature of In Vitro Culture Conditions

Different studies of long‐term chondrocytes viability have shown a gradual reduction as a function of time and ambient temperature. The aim of our in vitro study was to establish chondrocyte postmortem viability curves for 4°C, 11°C, 23°C, 35°C during 63 days after the donors' death. Osteochondral cylinders were procured from the knees of 16 male donors (20–47 years), stored in preservation media that was not changed, and analyzed in 3‐day intervals using a confocal laser scanning microscope. A significant influence of time on viability was found from Day 9 (p = 0.0029) and onwards (p < 0.0001). The lowest overall chondrocyte viability was at 35°C, followed by 4°C (p < 0.0001). The conditions used in this in vitro analysis suggest that similar viabilities may occur while in situ in the decedent. Further studies of chondrocyte viability from individuals with known postmortem intervals may show premise to help evaluate time since death in the late postmortem interval.     

Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China

The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion. Correlations were found between administered dosage, tissue concentrations and younger or prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius) were also reported. C. megacephala is the most common fly species found on corpses in South China during the early stages of decomposition. Significant differences in larval growth rate were both observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on tissues from rabbits treated with Malathion were smaller and attained maximum length later than those from the control colony. Duration of the larval and pupal stages was both significantly prolonged for larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of the duration of the larval and pupal stages together from the muscle colonies would alter the postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.     

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.