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.     

Generalized additive models and Lucilia sericata growth: assessing confidence intervals and error rates in forensic entomology

Forensic entomologists use blow fly development to estimate a postmortem interval. Although accurate, fly age estimates can be imprecise for older developmental stages and no standard means of assigning confidence intervals exists. Presented here is a method for modeling growth of the forensically important blow fly Lucilia sericata, using generalized additive models (GAMs). Eighteen GAMs were created to predict the extent of juvenile fly development, encompassing developmental stage, length, weight, strain, and temperature data, collected from 2559 individuals. All measures were informative, explaining up to 92.6% of the deviance in the data, though strain and temperature exerted negligible influences. Predictions made with an independent data set allowed for a subsequent examination of error. Estimates using length and developmental stage were within 5% of true development percent during the feeding portion of the larval life cycle, while predictions for postfeeding third instars were less precise, but within expected error.     

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.     

Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

Time since death. An entomological study on corpses

After death, during disintegration, various environmental conditions and fauna play an important role, indicating "time since death." The natural openings of the host body provide the most important breeding place for an agent which could consume the body very quickly, e.g., flies. Wounds are preferred over the natural openings by this agent. In such cases, antemortem wounds become unidentifiable. This paper presents the study of 25 cases in which inferences relating to the maturation of blow fly life stages and the total developmental time of an adult blow fly in breeding experiments in the prevailing environment are drawn. These can be used to help estimate the "time since death" of infested bodies.     

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.     

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae),

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high‐content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti‐ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High‐content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.     

The Effect of Buscopan® on the Development of the Blow Fly Chrysomya megacephala (F.) (Diptera: Calliphoridae)

Abstract:  This work investigated the effects of butylscopolamine bromide, a drug present in the pharmaceutical formulation Buscopan^®, on the development of Chrysomya megacephala , a blow fly species of considerable forensic and medical importance in Brazil. Larvae exposed to the drug showed a decreased rate of development, with higher drug concentrations further retarding the development. Besides, larvae reared on the presence of the drug showed smaller body weight and body length when compared with larvae reared on the absence of Buscopan^®. The drug also affected the mortality of the species.     

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.     

Maggot development during morgue storage and its effect on estimating the post-mortem interval

When insect evidence is obtained during autopsy, forensic entomologists make decisions regarding the effects of low-temperature (-1 degrees C to 4 degrees C) storage of the body and associated insects when estimating the post-mortem interval (PMI). To determine the effects of storage in a morgue cooler on the temperature of maggot masses, temperatures inside and outside of body bags containing a human cadaver and porcine cadavers (seven replicates) were measured during storage. Temperatures remained significantly higher (p<0.05) inside of the body bags relative to the cooler, and remained at levels sufficient for maggot feeding and development. If the assumption that no insect development takes place during preautopsy refrigeration is made, potential error rates in PMI estimation of 8.6-12.8% occur. The potential for blow fly larvae to undergo significant development while being stored in the morgue is a possibility that forensic entomologists should consider during an investigation involving samples collected from autopsy. Case and experimental evidence also demonstrate that substantial tissue loss can occur from maggot feeding during morgue storage.     

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.     

A Four‐Stage Method of Age at Death Estimation for Use in the Subadult Rib Cortex*,†

Abstract: Age estimation in the subadult skeleton can be rather precise when the epiphyses and dentition are present, but incomplete or commingled remains still present a challenge. Histomorphometric age‐at‐death estimation methods developed for use on adults are based on the age‐associated accumulation of osteons. In the growing skeleton, there is a poor correlation between osteon numbers and age until the latter half of the second decade. As a result, there has been no histological aging method for use in subadults. The analysis of the rib cortex of 72 subadults ranging in age from 2 to 21 years has identified a series of developmental changes in the bone microstructure that can be used to estimate age. This qualitative method utilizes the systematic changes in rib cortical morphology to classify ribs into one of four age phases. This method can be applied to immature skeletons in forensic, archaeological, and paleontological contexts.     

Blind validation of postmortem interval estimates using developmental rates of blow flies

This study evaluated the use of different degree day (DD) models, developmental thresholds and developmental data sources for estimating postmortem interval (PMI) based on developmental rates of blow flies (Diptera: Calliphoridae). Blow fly evidence was collected from three mock crime scenes and sent for blind analysis of PMI. PMI estimates were calculated using averaging, single sine, double sine, single triangle, and double triangle models of DD calculation with minimum developmental thresholds of 0, 6 and 10 °C and Anderson's, Byrd and Allen's, Greenberg's, Nishida's and Kamal's developmental data sets. These estimates were compared with the actual PMI to determine which combination of DD model, developmental threshold and developmental data provided the most accurate estimate. For all three cases, at least one method gave a PMI estimation that was within 1 day of the actual date of death. There was no variability between PMI estimated using the five models of DD calculation. Overall, as lower developmental threshold increased, the PMI interval estimates increased. Depending on the developmental data set used, increasing the lower developmental threshold caused some overestimations of the date of oviposition, with estimates given that were prior to the actual death. Future PMI estimates should include error ranges, so that overestimation of PMI is avoided.     

Blow Flies from Forest Fragments Embedded in Different Land Uses: Implications for Selecting Indicators in Forensic Entomology

Given the general expectation that forest loss can alter biodiversity patterns, we hypothesize that blow fly species abundances differ in a gradient of native vegetation cover. This study was conducted in 17 fragments across different landscapes in central Brazil. Different land cover type proportions were used to represent landscape structure. In total, 2334 specimens of nine species of Calliphoridae were collected. We used principal component analysis (PCA) to reduce dimensionality and multicollinearity of the landscape data. The first component explained 70%, and it represented a gradient of forest‐pasture land uses. Alien species showed a wide distribution in different fragments with no clear relationship between the abundance values and the scores of PCA axes, whereas native species occurred only in areas with a predominance of forest cover. Our study revealed that certain native species may be sensitive to forest loss at the landscape scale, and they represent a bioindicator in forensic entomology.     

Alteration of expirated bloodstain patterns by Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) through ingestion and deposition of artifacts

Abstract: Bloodstain pattern analysis can provide insight into a sequence of events associated with a violent crime. However, bloodstain pattern analysis can be confounded by the feeding activity of blow flies. We conducted two laboratory experiments to investigate the relationships between Lucilia sericata (green bottle fly) and Calliphora vicina (blue bottle fly), expirated bloodstains, and pooled bloodstains on a range of surfaces (linoleum, wallpaper, textured paint). C. vicina and L. sericata changed bloodstain pattern morphology through feeding and defecation. They also deposited artifacts in rooms where blood was not present originally. Chemical presumptive tests (Hemastix^®, phenolphthalein, leucocrystal violet, fluorescein) were not able to differentiate between insect artifacts and bloodstains. Thus, C. vicina and L. sericata can confound bloodstain pattern analysis, crime scene investigation, and reconstruction. Crime scene investigators should be aware of these fundamental behaviors, and the effects that blow flies can have on expirated and pooled bloodstain patterns.     

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.     

Influence of substrate tissue type on larval growth in Calliphora augur and Lucilia cuprina (Diptera: Calliphoridae)

The size of fly larvae is an important variable in the use of these insects to estimate postmortem interval. Furthermore, the nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect of type of food substrate on larval growth in two species of forensically important Australian blowflies. After collection on sheep's liver in the laboratory, different groups of larvae of Lucilia cuprina (Wiedemann) and Calliphora augur (Fabricius) were grown on sheep's liver, meat, and brains, and their body lengths compared. Results indicated that the development of larvae fed sheep's liver was adversely affected compared with larvae fed meat and brain; they moulted later, reached maximum length more slowly and sometimes produced significantly smaller pupae. These findings, similar to those of another recent study, have obvious implications for postmortem interval determinations. Estimates may be considerably skewed if the site of collection of larvae at a death scene contains tissue types different to those used in reference experiments. We therefore recommend caution in forensic analyses that interpret crime scene data using developmental studies performed with a single type of larval food substrate.     

Correlation of Molecular Expression with Diel Rhythm of Oviposition in Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) and Implications for Forensic Entomology

This study explored the molecular mechanisms potentially underlying blow fly nocturnal oviposition. A behavioral study revealed that Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) possesses a diel rhythm of oviposition in light under 12:12 light/dark conditions. Reversal to 12:12 dark/light resulted in oviposition behavior changing to align with the adjusted regime in most females, but four of 59 experimental females lacked a diel rhythm of oviposition (were arrhythmic). Real-time PCR was used to monitor the molecular expression levels of known circadian genes per and tim in C. vicina to determine whether gene expression and behavior correlated. As with behavior, reversing light/dark conditions changed rhythmic gene expression to align with an adjusted light regime. This suggests that although it is unlikely that C. vicina will colonize dead bodies at night, arrhythmic females and oviposition in the dark was demonstrated.     

Mandibular morphology as an indicator of human subadult age: interlandmark approaches

The dentition is widely recognized as the set of developmental markers that appear to show the least variability against chronological age; these markers are thus widely used in forensic anthropological investigations. As a possible alternative, we investigate here the potential of mandibular morphology as a developmental marker for estimating age at death in subadults. The sample analyzed comprises 79 known age and sex subadult individuals of South African Bantu and African American origin. Linear measurements of ramus height were obtained from the mathematical conversion of three-dimensional landmark data. A series of regression analyses were then performed to predict age by using the measurement of ramus height; results were cross-validated using a jackknife procedure. Our results show that ramus height can be used to predict age in the subadult skeleton with accuracy, closely approaching that of standards based on the dentition (standard error rates are between +/-1.1 years and +/-2.4 years).     

Current practices by forensic anthropologists in adult skeletal age estimation

When determining an age estimate from adult skeletal remains, forensic anthropologists face a series of methodological choices. These decisions, such as which skeletal region to evaluate, which methods to apply, what statistical information to use, and how to combine information from multiple methods, ultimately impacts the final reported age estimate. In this study, a questionnaire was administered to 145 forensic anthropologists, documenting current trends in adult age at death estimation procedures used throughout the field. Results indicate that the Suchey-Brooks pubic symphysis method (1990) remains the most highly favored aging technique, with cranial sutures and dental wear being the least preferred, regardless of experience. The majority of respondents stated that they vary their skeletal age estimate process case-by-case and ultimately present to officials both a narrow and broad possible age range. Overall, respondents displayed a very high degree of variation in how they generate their age estimates, and indicated that experience and expertise play a large role in skeletal age estimates.