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.     

Using Frons Width to Differentiate Blow Fly Species (Diptera: Calliphoridae) Phormia regina (Meigen) and Protophormia terraenovae (Robineau‐Desvoidy)

Protophormia terraenovae (Robineau‐Desvoidy) (Diptera: Calliphoridae) and Phormia regina (Meigen) (Diptera: Calliphoridae) are morphologically similar blow fly species commonly used for estimating postmortem intervals. Field collection and storage of adults can result in color changes, in particular on calypters and palps; often collected specimens show damage such as wing fray or fungal growth. We measured the frons width: total head width ratio using photographs (ImageJ version 1.49) to differentiate these two species. Both sexes were distinguishable to species, with the greatest difference between males: 12.34% P. terraenovae versus 1.62% P. regina, less so for females: 40.25% P. terraenovae, versus 33.65% P. regina. Incorporating this feature into future blow fly keys would help with distinguishing field‐caught specimens when other features are obstructed.     

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.     

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.     

A Test of Age Estimation Methods on Impacted Third Molars in Males

This study tests the relationship between third molar impaction and its concomitant effect on age estimation methods. Data were collected on radiographs of males analyzed in the Defense POW/MIA Accounting Agency Laboratory (n = 114). Radiographs of skeletonized individuals were scored for dental development, and age was assigned based on the appropriate ancestry‐based method. Differences between identified age and estimated dental age were assessed to determine whether molar impaction affected root development and age estimations. Results indicate that impacted teeth tend to be underdeveloped and result in age estimates that are too low. While these results are of note to anthropologists and odontologists performing dental age estimates, more work is needed to explore the effect of impaction on development among a more diverse sample.     

Gene expression during blow fly development: improving the precision of age estimates in forensic entomology

Forensic entomologists use size and developmental stage to estimate blow fly age, and from those, a postmortem interval. Since such estimates are generally accurate but often lack precision, particularly in the older developmental stages, alternative aging methods would be advantageous. Presented here is a means of incorporating developmentally regulated gene expression levels into traditional stage and size data, with a goal of more precisely estimating developmental age of immature Lucilia sericata. Generalized additive models of development showed improved statistical support compared to models that did not include gene expression data, resulting in an increase in estimate precision, especially for postfeeding third instars and pupae. The models were then used to make blind estimates of development for 86 immature L. sericata raised on rat carcasses. Overall, inclusion of gene expression data resulted in increased precision in aging blow flies.     

DEET (N,N‐Diethyl‐meta‐toluamide) Induced Delay of Blowfly Landing and Oviposition Rates on Treated Pig Carrion (Sus scrofa L.)

Abstract: The question of whether the insect repellent N,N‐Diethyl‐meta‐toluamide (DEET) affected fly attraction, oviposition, and larval development was investigated; in part, to determine whether the common habit of wearing DEET as a repellent could affect the rate of human decomposition. Experiments using pig surrogates of human decedents were carried out in a rural environment. Dead piglets were sprayed with DEET, and fly behavior, colonization levels, and maggot development were compared with those in nonsprayed controls. Piglets treated with DEET experienced significant delays in fly visitation and oviposition and delayed appearance of each larval instar, as well as reduced total larval numbers (p < 0.01 for all variables), with subsequently reduced decomposition (p < 0.05). Such changes in fly behavior and larval population development would significantly impact the estimation of the period following the death from entomological evidence in decedents wearing DEET at the time of their death.     

Investigating the Potential of Fluorescent Fingerprint Powders as a Marker for Blow Fly Larvae (Diptera: Calliphoridae)

Four fluorescent fingerprint powders (RedWop^?, GreenWop^?, Basic Yellow^?, and Yellow Powder^?) were evaluated as a marker for blow fly larvae. Administration methods included ingestion (high vs. low concentration) or topical. Ingestion of high concentrations of Basic Yellow^? and RedWop^? caused higher larval mortality. Basic Yellow^? delayed development and adult emergence while RedWop^? and Yellow Powder^? had a significant effect on particular stages of development, however, emergence time was not altered. Optimal administration is through ingestion at low concentration levels (<10%) or topically, with GreenWop^? demonstrating minimal adverse effects. Optimum wavelength for discrimination between powders was 450 nm. This research can aid in investigative training to increase visibility of larval and pupal blow flies. It can also be used in entomological studies to differentiate between larval blow flies (or other dipteran) species or individuals to further understand complex interactions and behavior during larval development.     

The Food Preferences of the Blow Fly Lucilia cuprina Offered Human Blood,Semen and Saliva,and Various Nonhuman Foods Sources

As human DNA profiles can be obtained from blow fly artifacts, this study aimed to establish the feeding preferences of Lucilia cuprina (Wiedemann) blow flies when offered human biological fluids and nonhuman food sources. One‐day‐old and 3‐day‐old blow flies of both sexes were simultaneously offered human blood, semen and saliva, pet food, canned tuna and honey, and the number and length of visits documented over 6 h. One‐day‐old flies visited pet food and honey most often, but stayed longest on honey and semen. Three‐day‐old flies visited semen and pet food most often, and stayed longest on these food sources. Blood and saliva were the least preferred options for all flies. Overall, flies preferred dry blood and semen to the wet forms. These findings demonstrate that even when other food sources are available, flies at a crime scene may feed on human biological fluids if present, potentially transferring human DNA.     

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.     

Age Estimation from the Teeth Using a Modified Demirjian System*

Abstract: The estimation of age at time of death is often an important step in the identification of human remains. The purpose of this study was to test the applicability of the Demirjian system on a sample of the Sydney child population and to develop and test age‐prediction models using a large sample of Sydney children (1624 girls, 1637 boys). The use of the Demirjian standards resulted in consistent overestimates of chronological age in children under the age of 14 by as much as a mean of 0.99 years. Of the alternative predictive models derived from the Sydney sample, those that provided the most accurate age estimates are applicable for the age ranges 2–14 years, with R‐square = 0.94 and a 95% confidence interval of ±1.8 years. The Sydney‐based standards provided significantly different and more accurate estimates of age for that sample when compared to the published standards of Demirjian.     

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.     

Stature and Body Weight Estimation from Various Footprint Measurements Among Egyptian Population

Abstract: Analysis of footprints can reveal very important clues which can be used as a forensic evidence and help in the estimation of stature and body weight of an individual. In this work, bilateral footprints were obtained from 50 male Egyptian medical students ranging in age between 18 and 25. Nine measurements were taken on each footprint. The result revealed significant bilateral asymmetry (p < 0.001) except foot breadth at ball. The significant and positive highest correlation coefficients with stature were shown by toe‐5 length on right side (R = 0.58) and with body weight by foot breadth at ball on left side (R = ?0.52). Regression equations presented smaller standard errors of estimate (3.52–4.69) in determination of stature than those in estimation of body weight (4.05–5.28). In conclusion, this study has provided equations that help to estimate stature and body weight from footprint measurements among Egyptians.     

Rib Variation at Multiple Locations and Implications for Histological Age Estimation*

Existing histological age estimation methods using the rib were developed mainly from the midshaft; however, in forensic practice, uncertainty of sampling location often arises due to fragmented or previously sampled ribs. The potential for error increases when sampling location is uncertain and utilizing a section beyond the midshaft (either anterior or posterior) may result in erroneous age estimates. Additionally, there is debate within the field regarding the minimum number of sections needed for accurate age estimation. The aim of this research is to determine the importance of the midshaft distinction for age-at-death assessment and the necessity of analyzing serial sections by evaluating histological variables at sampling locations along the length of the rib. Three seriated histological sections at three sampling locations (anterior, midshaft, and posterior) were obtained from sixth ribs of ten postmortem human subjects. Cortical area (Ct.Ar) and osteon population density (OPD) were collected from each section (n = 90). Significant differences were determined in Ct.Ar between sampling locations, demonstrating the variation present along the length of the rib. A comparison of OPD at sampling locations revealed significant differences, suggesting that sampling site is critical to accurate age estimates. When sampling location is uncertain, a more anterior section should be taken. Analysis of serial sections within locations revealed no significant differences in OPD or Ct.Ar, supporting the practice of collecting data from one section for age estimation. While an age estimate can be achieved through the analysis of one section, best practice suggests reading two sections to capture intraindividual variation.     

Validity Comparison of Three Dental Methods for Age Estimation Based on Tooth Root Translucency

Age estimation of nonliving subjects plays a very important role in identifying human remains. When adult remains are in question, this process becomes even more complex. Among the numerous dental methods that exist for adults aging, the evaluation of root translucency is one of the most useful. This study studies the correlation between age and root translucency by investigating monoradicular teeth affected by pathological processes using the equations proposed by Lamendin, Prince & Ubelaker and from Singhal et al. The average age values calculated according to the three methods were compared with the values of known age. Age estimation using the Student's t‐test resulted as more accurate in the methods by Singhal et al. and by Prince & Ubelaker, with respect to that of Lamendin. Age estimation based solely on the percentage of radicular dentinal translucency has been found to be more accurate when diseased teeth are involved.     

The Reliability of the Greulich and Pyle Atlas When Applied to a Modern Scottish Population

This study examines the reliability of age estimation utilizing the Greulich and Pyle atlas in relation to a modern Scottish population. A total of 406 left‐hand/wrist radiographs (157 females and 249 males) were age‐assessed using the Greulich and Pyle atlas. Analysis showed that there was a strong correlation between chronological age and estimated age (females R² = 0.939, males R² = 0.940). When age groups were broken down into year cohorts, the atlas over‐aged females from birth until 13 years of age. The pattern for males showed that the atlas under‐estimated age until 13 years of age after which point it consistently over‐aged boys between 13 and 17 years of age. This study showed that the Greulich and Pyle atlas can be applied to a modern population but would recommend that any analysis takes into account the potential for over‐ and under‐aging shown in this study.     

Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMI_min) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post‐feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9–33 carbons, alkenes (18%), and methyl‐branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post‐feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMI_min estimations. However, future research is needed to validate these results under natural conditions in the field.     

Validation of the Acetabulum As a Skeletal Indicator of Age at Death in Modern European‐Americans,

Progressive changes in the acetabulum have been used in modern skeletal age estimation, but they have not been completely understood. If their age correlations are weakened by the influence of factors like physical activity and obesity, acetabular changes should not be used for age estimation. To investigate their utility for aging, the acetabular variables of Rissech et al. (2006) were analyzed in 409 modern European‐Americans (Bass Collection, Tennessee). Correlation tests assessed potential associations between acetabular data, osteoarthritis scores (collected per Jurmain, 1990), and documented demographic information (age, body mass index [BMI], metabolic intensity of physical activities). Acetabular changes had statistically significant, positive correlations with osteoarthritis (p < 0.001 in most joints/regions) and age (p < 0.001), indicating their degenerative nature and relevance for age estimation. Acetabular changes showed no associations with BMI or metabolic values, suggesting resistance to obesity and activity effects. These results suggest that acetabular degeneration is a valid skeletal age‐at‐death indicator.     

Introducing Computed Tomography Standards for Age Estimation of Modern Australian Subadults Using Postnatal Ossification Timings of Select Cranial and Cervical Sites,

Contemporary, population‐specific ossification timings of the cranium are lacking in current literature due to challenges in obtaining large repositories of documented subadult material, forcing Australian practitioners to rely on North American, arguably antiquated reference standards for age estimation. This study assessed the temporal pattern of ossification of the cranium and provides recalibrated probabilistic information for age estimation of modern Australian children. Fusion status of the occipital and frontal bones, atlas, and axis was scored using a modified two‐ to four‐tier system from cranial/cervical DICOM datasets of 585 children aged birth to 10 years. Transition analysis was applied to elucidate maximum‐likelihood estimates between consecutive fusion stages, in conjunction with Bayesian statistics to calculate credible intervals for age estimation. Results demonstrate significant sex differences in skeletal maturation (p < 0.05) and earlier timings in comparison with major literary sources, underscoring the requisite of updated standards for age estimation of modern individuals.