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.     

The Effect of Flunitrazepam (Rohypnol®) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology

This study investigated the potential effects of flunitrazepam (known as “date rape drug”) on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal–Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.     

Influence of Paraquat on Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) Infesting Minced‐beef Substrates in Kelantan,Malaysia

This study investigated the influence of paraquat, a prevalent poison used by suicides, on initial oviposition and development of Chrysomya megacephala (Fabricius) using minced‐beef substrates. Paraquat in lethal dose for human (40 mg/kg), two times the lethal dose (80 mg/kg) and five times the lethal dose (200 mg/kg) were mixed thoroughly with respective minced‐beef substrates (1 kg each) that were decomposed in a shaded habitat fully protected from rain. Results of four replications of the above experiment revealed that the presence of paraquat neither delayed initial oviposition nor prolonged the developmental stages of C. megacephala. Therefore, estimation of postmortem interval (PMI) based on empirical baseline data obtained using animal models devoid of any poisons would still be appropriate for estimating PMI in paraquat‐related deaths.     

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.     

Postfeeding Larval Dispersal Behavior of Late Season Blow Flies (Calliphoridae) in Southern Ontario,Canada

Postfeeding dispersal involves migration of larvae away from their food source in order to pupate. Puparia are difficult to find, yet are important for estimating PMI, and missing puparia during collection can result in inaccurate estimations. This study investigates the late season maggot dispersal patterns for blow flies at coyote carcasses in two habitats with an aim to improving puparia collection procedures. Puparia samples collected from various dispersal distances and directions tested the spatial distribution patterns of the various species using the variance/mean ratio (VMR). Lucilia illustris was the most common species to emerge, with a preferred minimum dispersal distance of more than 50.8 cm and an overall VMR value of 14.91, indicating this species had a clumped distribution pattern. These findings highlight that current collection procedures that use random sampling from under carcasses do not adequately account for the spatial distribution of larvae.     

Human Cutaneous Myiasis by the Australian Sheep Blowfly,Lucilia cuprina (Diptera: Calliphoridae), in Oklahoma

This case report describes an incident of myiasis in Oklahoma City, Oklahoma. Paramedics treated a 53‐year‐old man in the field when he suffered cardiac arrest. He was intubated by the paramedics and transported to an emergency room, where he received two stents and a balloon pump. He was found to have a GCS of three and remained in the hospital for 7 days before passing away. After his death, the breathing tube was removed, revealing the presence of several maggots. On closer inspection, the dipteran larvae were found in both the oral and nasal cavities. Four of these larvae were reared to adulthood and identified as Lucilia cuprina (Weidemann) (Diptera: Calliphoridae), commonly known as the Australian sheep blowfly, by the Biology Department of the University of Oklahoma. Based on the fly's life cycle and rate of development, the infestation is suspected to be hospital acquired.     

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.     

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.     

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 Use of the Developmental Rate of the Aquatic Midge Chironomus riparius (Diptera,Chironomidae) in the Assessment of the Postsubmersion Interval

Nonbiting midges (Diptera, Chironomidae) are the most abundant members of the fauna associated with submerged carcasses, but their use in the medicolegal context is very restricted because of their complex ontogeny. In this case, the corpse of a woman was recovered in late spring from a river in Granada (Iberian Peninsula). It showed obvious signs of long permanence in the aquatic environment and, along with pulmonary and microscopical analyses, led to the conclusion that the cause of death was drowning. Several larvae‐like specimens were sampled from the scalp and later identified by morphological external features as IV instar larvae of Chironomus riparius Meigen, 1804 (Diptera, Chironomidae). Sequencing of cytochrome oxidase subunit I was performed to confirm the identification. The knowledge of the biology of C. riparius at low temperatures was critical to assess a postsubmersion interval of 16–17 days.     

Sequences of the Cytochrome C Oxidase Subunit I (COI) Gene are Suitable for Species Identification of Korean Calliphorinae Flies of Forensic Importance (Diptera: Calliphoridae)

Abstract:  Calliphorinae fly species are important indicators of the postmortem interval especially during early spring and late fall in Korea. Although nucleotide sequences of various Calliphorinae fly species are available, there has been no research on the cytochrome c oxidase subunit I (COI) nucleotide sequences of Korean Calliphorinae flies. Here, we report the full-length sequences of the COI gene of four Calliphorinae fly species collected in Korea (five individuals of Calliphora vicina , five Calliphora lata , four Triceratopyga calliphoroides and three Aldrichina grahami ). Each COI gene was amplified by polymerase chain reaction and directly sequenced and the resulting nucleotide sequences were aligned and analyzed by MEGA4 software. The results indicate that COI nucleotide sequences can be used to distinguish between these four species. Our phylogenetic result coincides with recent taxonomic views on the subfamily Calliphorinae in that the genera Aldrichina and Triceratopyga are nested within the genus Calliphora .     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

Analysis of the Effect of Cyclophosphamide and Methotrexate on Chrysomya megacephala (Diptera: Calliphoridae),

Forensic entomotoxicology investigates the effects of chemical substances in the development of scavenger insects and the reflection on estimating the minimum postmortem interval (PMI). To evaluate the impact of the chemotherapeutic drugs cyclophosphamide (CF) and methotrexate (MTX) on the postembryonic development of Chrysomya megacephala, controlled experiments were performed by rearing the larvae on spiked minced beef with different concentrations of these drugs. The results indicated that CF significantly decreased their developmental rate up to 28 h but had no significant effect on larval and adult sizes, survival rate, and sex ratio, whereas MTX decreased larval and adult sizes, survival rate, and there was a deviation in the expected sex ratio toward females in MTX‐exposed larvae but had no significant impact on developmental rate. These negative interference factors should be considered in cases of suspected death of people that have undergone chemotherapy.     

Determination of Calliphoridae (Diptera) Fauna and Seasonal Distribution on Carrion in Ankara Province*

Abstract: This study was conducted from March 2006 to 2007. The aim of the study was to determine the forensically significant Calliphoridae (Diptera) species and their seasonal distribution in Ankara province at Beytepe Campus. Pig carcasses were killed by a veterinary using pentobarbital sodium with intervals from 2 to 3 weeks to a month. Samples were collected from the carcass everyday in the morning, in the afternoon, and at sunset. Temperature and parameters of weather, such as cloudy, sunny, rainy, and snowy, were recorded at each visit. Phaenicia sericata (Meigen), Chrysomya albiceps (Wiedemann), Calliphora vomitoria (Linnaeus), and Calliphora vicina (Robineau‐Desvoidy) species, which belong to Calliphoridae family, were collected. It was shown that the seasonal distribution of the collected species was different from each other. The species were determined on carcass between the following times; P. sericata, from April to November; C. albiceps, from May to November; Ca. vomitoria, from February to June and from September to December; and Ca. vicina, from February to May and in June and September. Calliphoridae fauna and its annual seasonal distribution in Turkey on 12 pig carcasses were reported for the first time in this study.     

Nocturnal Oviposition Behavior of Forensically Important Diptera in Central England

Timing of oviposition on a corpse is a key factor in entomologically based minimum postmortem interval (mPMI) calculations. However, there is considerable variation in nocturnal oviposition behavior of blow flies reported in the research literature. This study investigated nocturnal oviposition in central England for the first time, over 25 trials from 2011 to 2013. Liver-baited traps were placed in an urban location during control (diurnal), and nocturnal periods and environmental conditions were recorded during each 5-h trial. No nocturnal activity or oviposition was observed during the course of the study indicating that nocturnal oviposition is highly unlikely in central England.     

A Checklist of Calliphoridae Blowflies (Insecta,Diptera) Associated with a Pig Carrion in Central Brazil*

Abstract: Although the Cerrado is the second major Brazilian biome, few studies have been undertaken about its entomofauna. Blowflies have an important role in forensic entomology, helping in the determination of postmortem intervals. The main goal of this exploratory study was to identify and to catalog the blowfly species associated with a pig carcass. The study was conducted in a pasture in Brasília, Distrito Federal. A pig (Sus scrofa) was killed with a .22 caliber shot in the frontal region of the head. Adult blowflies were surveyed daily from June 1 through 30, 2004. A total of 14,910 adult calliphorids were collected, representing eight species: Chrysomya albiceps, C. megacephala, Cochliomyia macellaria, Chloroprocta idioidea, Hemilucilia semidiaphana, H. segmentaria, Lucilia cuprina, and L. eximia. C. albiceps was the most frequent species, amounting to 94.76% of the catch. Five decomposition stages were observed, and for calliphorids, the most attractive stage was the bloated one.     

In Vitro Effects of Household Products on Calliphoridae Larvae Development: Implication for Forensic Entomology

Several parameters can delay the first arrival of flies on a corpse and the subsequent development of the larvae. This study focuses on the development of Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) on household chemical‐contaminated substrates. bleach, perfume, hydrochloric acid, caustic soda, insecticide, mosquito repellent, and gasoline in quantities consistent with an amount that could possibly be spilled on a corpse were mixed with beef liver to simulate contaminated fleshes. Larvae were bred at 25°C on these media until emergence. Four developmental parameters were followed: survival rates, development times, sex ratios, and adult sizes. Hydrochloric acid, insecticide, and gasoline killed all larvae. In low quantities, caustic soda and mosquito repellent increased the development time and decreased the adult size. However, high quantities of these chemicals killed all larvae. Lastly, bleach and perfume did not affect the survival rate and barely impacted the development time or adult size. These results demonstrate common household products spilled on a corpse can strongly affect the development of Calliphoridae larvae. The effects of such products should be considered in forensic entomology cases.     

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.     

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.     

Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.