Taphonomic Marks on Pig Tissue Due to Cadaveric Coleoptera Activity Under Controlled Conditions

The aim of this work was to study taphonomic marks that cadaveric coleopteran can produce under controlled conditions. To evaluate this, pig trotters were initially exposed to adults of Dermestes maculatus De Geer at 21 ± 5°C and a 12:12‐h day/night cycle. Observations were made and photographs taken every 4–5 days for 9 months. When feeding and reproducing, D. maculatus produced, in both adult and larvae stages, different types of marks such as holes, striations, scratches, and pits in several kinds of tissue such as integumental, connective, and muscular, in both their fresh and dried stages. Bite marks were also evident. The results in this study provide not only taphonomic but also biological and forensic information. This is the first time that this kind of experiment has been performed.     

The Effect of Temperature and Laboratory Rearing Conditions on the Development of Dermestes maculatus (Coleoptera: Dermestidae)

Experiments were conducted to study the life cycle of Dermestes maculatus and to establish the total developmental time and the developmental time of immature stages, in relation with six different temperatures. We also analyzed the variations in size, morphology, and other indicators of temporal variation during life cycle of D. maculatus, in relation with temperature. One hundred larvae were selected per experiment, reared individually. The remaining larvae were reared to evaluate and establish temporal variations among the instars (length, cephalic width, and dry weight). In all trials, survivorship was greater than 50% and seven larval instars were found. Data of the average developmental time of immature stages and of the total cycle, at different temperatures, are provided. This is of relevance when estimating particularly, a minimum PMI. No relation between morphometric parameters and temperature was found, suggesting that other random factors may have been involved. Thus, this indicates that the method of isomegalen diagrams could not be used for calculating PMI.     

Recommendations for Maintaining a Dermestid Beetle Colony (Dermestes maculatus) for Processing Human Remains

Dermestid beetles (Dermestes maculatus De Geer 1774) are small carrion insects characterized by a rounded or oval-shaped body and white abdomen with black markings. Given their natural propensity to consume soft tissue throughout various stages of decomposition, biological anthropologists have sought to use dermestids as a forensic processing method in addition to traditional chemical tissue removal techniques. Although useful, most of the existing academic literature regarding the upkeep of dermestid colonies for skeletal remains processing either lack specificity or are outdated. Additionally, nonacademic sources that contain information regarding dermestid maintenance are often disjointed, resulting in a difficulty to replicate habitat construction and ideal environmental conditions. Therefore, this technical note presents recommendations for anthropologists interested in establishing and maintaining a D. maculatus population. These recommendations are based on our experiences using several dermestid colonies to process five unembalmed human heads, procured from an anatomical gift company for a larger study on gunshot trauma. Aspects of the dermestids’ environment that are crucial for the management of a healthy colony include the type of bedding, food, water, and containment method used, in addition to maintaining appropriate temperature ranges (24–27°C) and humidity levels (35–73%). Although habitat construction and dermestid maintenance involve materials that are relatively inexpensive and readily available, setting up and maintaining a D. maculatus colony can be laborious and time consuming and should only be undertaken when the volume of casework is such that this investment would be offset.     

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.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

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.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.     

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.     

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.     

A Preliminary Study of Insect Succession in Al‐Ahsaa Oasis,in the Eastern Region of the Kingdom of Saudi Arabia

Rabbit carcass decomposition was examined in four seasons in Al‐Ahsaa Oasis. Decomposition rate was significantly faster in summer compared with other seasons. Fourteen insect species from four orders and seven families were recorded: Calliphoridae, Muscidae, Sarcophagidae, Formicidae, Histeridae, Dermestidae, and Tenebrionidae. Most of the fly maggots collected were Calliphoridae with only one species from both Muscidae and Sarcophagidae. Maggots of Calliphora vicina (R.‐D.) and Chrysomya albiceps (Wiedemann) colonized the carcasses in all seasons while both Ch. bezziana (Villeneuve) and Lucilia sericata (Meigen) were found in all seasons except for winter. Maggots of Chrysomya megacephala (F.) and Phormia regina (Meigen) were only collected in the summer and autumn, respectively. Four coleopteran species were collected: Hymenorus sp., Saprinus chacites (Illiger), Dermestes maculatus DeGeer and Blaps sp. One formicidian species, Pheidole megacephala (F.), was observed in all seasons. Insect richness was greater, and decomposition rate was faster in summer compared with other seasons.     

The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy

This study investigated the effect of soft tissue and different exposure times on the prediction of burning temperatures of bone when using Fourier transform infrared spectroscopy (FTIR). Ovis aries rib bones were burnt at different temperatures and for varying time intervals. Results of a linear regression analysis indicated that burn temperatures can be predicted with a standard error of ±70°C from defleshed bone spectra. Exposure time does not have a significant impact on prediction accuracy. The presence of soft tissue has a significant impact on heat‐induced changes of the bone matrix in low (<300°C) as well as high temperatures (>800°C), slowing down combustion in the former and accelerating it in the latter (p < 0.05). At medium temperatures, no significant difference was noted. These results provide forensic investigators a new perspective with which to interpret the results of crystallinity measures derived from burnt bone.     

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.     

Dermestes maculatus (Coleoptera: Dermestidae) development under fluoxetine effect using two drug administration models

Fluoxetine is a selective serotonin reuptake inhibitor, commonly used for the treatment of a variety of psychopathological conditions. As such, fluoxetine may be expected to appear in clinical and forensic cases. Dermestes maculatus De Geer (Coleoptera: Dermestidae) has been recognized as a relevant component of the insect fauna associated with decomposing human and animal remains. Experiments were conducted to study the effect of fluoxetine on developing D. maculatus using two‐drug administration models: a non‐living animal model (pork muscle) and a living one (Sus scrofa L. pigs). We assessed the duration of immature stages and total life cycle, as well as morphological parameters (body length, cephalic width, and weight). The effect of fluoxetine was studied at an overdose concentration: In the non‐living animal model the drug was mixed with macerated pork muscle (2000 mg/kg) and in the living animal model, pigs were given the drug orally (833 mg/kg). A control was used for each model. Daily observations were performed from the beginning to the end of the experiments. GC‐MS was used for drug detection and quantification. There were no statistically significant differences in the duration of immature stages, life cycle, larval mortality, morphological parameters, or sex ratio, between treatment and control, regardless of the drug administration model. Given that fluoxetine had no detectable effect on the development of D. maculatus, detection of this drug in forensic situations would not compromise the accuracy of PMI estimations.     

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 Influence of Clothing and Wrapping on Carcass Decomposition and Arthropod Succession During the Warmer Seasons in Central South Africa*

Abstract: The influence of clothing and wrapping on carcass decomposition and arthropod succession was investigated to provide data to enable estimated postmortem interval in homicide investigations. Six pig carcasses, Sus scrofa, were divided into three sample groups, each with a clothed carcass wrapped and a carcass wrapped with no clothes. Two more carcasses, one with no clothes or wrapping, the other with clothes and no wrapping were used as controls. The clothed or wrapped carcasses had larger visible maggot masses, which moved more freely and these carcasses took longer to dry out. The blow fly maggot masses were dominated by Chrysomya marginalis and Chrysomya albiceps. Oviposition occurred simultaneously on all carcasses. High temperatures in one case caused significant maggot mortality. The Coleoptera community was dominated by Silphidae, Thanathopilus micans larvae, Dermestidae, Dermestes maculatus adults and larvae, and Cleridae, Necrobia rufipes.     

Diurnal and Nocturnal Flight Activity of Blow Flies (Diptera: Calliphoridae) in a Rainforest Fragment in Brazil: Implications for the Colonization of Homicide Victims

Nocturnal flight of blow flies (Diptera: Calliphoridae) is a controversial issue in forensic entomology. We performed two field experiments to investigate the diurnal and nocturnal activity of six blow fly species in a rainforest fragment in Brazil. Initially, nocturnal (17:30–05:30) versus diurnal (05:30–17:30) flight activity was investigated. Only 3.9% of adults were collected at night, mostly the native species Mesembrinella bicolor, and nocturnal oviposition did not occur. In the second experiment, collection of adults took place at the following intervals: 05:30–08:30, 08:30–11:30, 11:30–14:30, and 14:30–17:30. The proportions of adults did not differ significantly among the four diurnal intervals, except for Hemilucilia segmentaria, which was captured more frequently in the early morning. Calliphoridae has predominantly diurnal behavior, not laying eggs in darkness. The association of the native species M. bicolor, Hemilucilia semidiaphana, and H. segmentaria to forested areas reinforces the forensic relevance of data on their flight pattern.     

Repetitive TASER X26 Discharge Resulted in Adverse Physiologic Events with a Dose–Response Relationship Related to the Duration of Discharge in Anesthetized Swine Model

The objectives of our study were to investigate the dose–response relationship of the TASER X26 discharge duration in an anesthetized swine model. Fourteen swines were anesthetized and then exposed to TASER X26 discharge for 5 sec (n = 5) or for 10 sec (n = 6). The sham control group (n = 3) was anesthetized and studied using the same protocol except TASER X26 discharges during the experiments. Hemodynamic parameters were obtained. Blood pressure and total peripheral resistance decreased significantly after TASER discharge and returned to baseline value at 15 min after 5 sec of TASER discharge but did not return to baseline values during the 30‐min observation period after 10 sec of TASER discharge. Repetitive TASER X26 discharge resulted in adverse physiologic events with a dose–response relationship related to the duration of TASER X26 discharge in an anesthetized swine model.     

Growth of Chrysomya megacephala (Fabricius) Maggots in a Morgue Cooler*

Abstract: In estimating the postmortem interval (PMI) using maggots obtained during autopsy, the forensic entomologist makes decisions regarding the effects of low‐temperature storage of the body on the insects. In this case report, a corpse was found in an abandoned house in the residential area of Bukit Mertajam, Penang, Malaysia. The maggots were found to be alive inside the mouth of the deceased although the corpse had been in the morgue cooler for 12 days. The maggots were reared and identified as Chrysomya megacephala (Fabricius). The emerged adult flies were kept as a stock colony, and the duration of development under the indoor fluctuating temperature regime was studied. The total duration of developmental process of this species was 9.5 ± 0.5 days, and the PMI estimated was 3.2 ± 0.6 days. This case report demonstrates the survival of Ch. megacephala maggots for 12 days and their growth inside the morgue cooler.     

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.     

Determination of drug levels in two species of necrophagous Coleoptera reared on substrates containing morphine

Two species of necrophagous Coleoptera: Dermestes frischi (Dermestidae) and Thanatophilus sinuatus (Silphidae), were reared on substrates containing different amounts of morphine. Colonies of D. frischi were reared on rabbit carcasses which had been given 10, 20, and 40 mg/h of morphine hydrochloride via ear artery perfusion over a 3 h period prior to death. A fourth rabbit served as a control. T. sinuatus was reared on minced beef spiked with morphine hydrochloride to give concentrations of 1,000, 2,500, 5,000, and 10,000 ng/g and one control colony. These dosages were calculated to create tissue concentrations of morphine similar to those encountered in human deaths due to morphine overdose. Larvae. pupae, and adults (except for T. sinuatus) were analyzed for morphine content. All developmental stages of D. frischi were positive for morphine and concentrations correlated with cadaveric tissue concentrations during larval stages and to a lesser extent in the adult stage. For T. sinuatus, the best correlations were found in 2nd and 3rd instar larvae. This study demonstrates the potential for use of necrophagous Coleoptera, as well as Diptera larvae, as alternate specimens for toxicological analyses.