Black soldier fly (Diptera: Stratiomyidae) colonization of pig carrion in south Georgia

The black soldier fly, Hermetia illucens (L.), is thought to colonize corpses 20-30 days postmortem. However, recent observations indicate this might not be true for all cases. Therefore, we conducted a study examining colonization by the black soldier fly and other Diptera on pig carrion in a plowed field in southern Georgia from 20 September through 21 February. Our data indicate black soldier flies could colonize a corpse within the first week after death. Knowing this information could prevent a serious mistake in estimating the time at which a corpse is colonized by this species. This study also represents the first record of Chrysomya rufifacies in Georgia.     

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.     

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.     

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

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

Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) established in the vicinity of Knoxville, Tennessee, USA

The hairy maggot blow fly, Chrsomya rufifacies (Macquart) (Diptera: Calliphoridae) was collected in large numbers as both adults and immatures in the Knoxville, Tennessee, area during 1998 and is likely established there. The distribution of this species in the Old World, isothermal data, and its collection from mid-Michigan during 1998 suggest that it will eventually occupy most of the U.S. The forensic importance of C. rufifacies makes it probable that it will factor into an increasing number of medicolegal cases, but the expanding distribution of this species decreases its utility as a geographic indicator when postmortem movement of decedents is suspected.     

Larval-mass effect: Characterisation of heat emission by necrophageous blowflies (Diptera: Calliphoridae) larval aggregates

Numerous Calliphoridae species larvae are necrophageous and develop on animal cadavers. During the feeding stages, a strong gregarious behaviour leads to the formation of large larval masses, allowing larvae to share digestive fluids. Furthermore, a mass of larvae emits heat, resulting in a local increase of temperature. Differences greater than 20°C between ambient and larval mass temperatures have already been observed, and the temperature of the mass can reach 50°C. Thus, larvae could benefit from this increased local temperature to speed up their development. This study focuses on the dynamic and characterisation of heat emission by Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) larval masses. Experiments were performed under controlled conditions using several ambient temperatures and different numbers of larvae. Results indicate that heat emission depends on the instar and is strongly affected by available amount of food. Furthermore, according to the experimental data, the heat emission is also relative to the weight of the larval mass, the larvae number and the local temperature. These results also demonstrate that optimum ambient-temperature values ranging between 22°C and 25°C produce maximal heat-emission per larva. Furthermore, a feedback loop, involving heat exchanges and physiological and behavioural thermoregulation processes, appears inside aggregates. In the context of forensic cases and post-mortem interval (PMI) estimation, these results indicate that heat emission can occur even with "small" masses if they are composed of high number of second stage larvae. Furthermore, particular attention should be paid on cases involving L. sericata larvae at ambient temperature ranging from 22°C to 25°C, which appears to maximise the heat-emission process.     

Determination of drug levels in larvae of Lucilia sericata (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

This study concerns the determination of morphine concentrations in fly larvae reared on rabbits administered different concentrations of morphine and a correlation between concentrations of the drug in larvae and tissues. Three rabbits (R1, R2 and R3) were given dosages of 12.5, 25.0 and 50.0 mg/h of morphine over a 3 h period via continuous ear artery perfusion. These dosages and time of perfusion were calculated to create tissue concentrations of morphine similar to those encountered in human death due to overdose. Morphine blood level plateau was attained after 1 h of perfusion. A fourth rabbit was used as a control. To evaluate drug concentrations, tissues were sampled using a coelioscopic technique. Approximately 400 eggs of Lucilia sericata, all of the same age category, were placed in eyes, nostrils and mouth of each rabbit carcass. Larvae and puparia were regularly collected from each rabbit for toxicological analysis. The concentrations of the drug in the tissues sampled were determined to be similar to those normally encountered in human overdoses and were correlated with the dosage of morphine that had been administered. Morphine was detected in all larvae and pupae fed on tissues from carcasses administered morphine, except for puparia from the colony fed on the R1 animal which received 12.5 mg/h dosage of morphine. All samples from the control rabbit were negative for morphine. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were 30 to 100 times lower than the concentrations found in the tissues. A correlation between the tissue concentrations and larval concentrations was found in only 3rd instar larvae (80 to 140 h following hatching). No correlations were found between administered dosages, tissue concentrations and younger larvae, prepuparial larvae or puparia.     

Influence of rain and malathion on the oviposition and development of blowflies (Diptera: Calliphoridae) infesting rabbit carcasses in Kelantan, Malaysia

The influence of rain and malathion on the initial oviposition as well as development of blowfly species infesting rabbit carcasses decomposing in sunlit and shaded habitats were studied over a period of 1 year in Kelantan, Malaysia. Chrysomya megacephala (Fabricius) was the most dominant species that infested the carcasses, followed by Chrysomya rufifacies (Macquart). In general, rain, depending on its intensity, delayed initial oviposition by 1–2 days and prolonged the pupation period by 1–3 days. The presence of malathion in the carcasses delayed initial oviposition by 1–3 days and prolonged the pupation period by 2–3 days. These findings deserve consideration while estimating postmortem interval since rain is a commonplace occurrence in Malaysia and malathion is one of the common poisons as an agent for choice to commit suicide.     

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.     

Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal-adult development of synchronised animals using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: "fresh" pupae, kept frozen until analysis and "experimentally dried" pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in "experimentally dried" pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic entomology are discussed.     

Determination of drug levels in larvae of Protophormia terraenovae and Calliphora vicina (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

Two species of blow flies (Diptera: Calliphoridae) were reared on tissues from rabbits administered different dosages of morphine. These species, Protophormia terraenovae and Calliphora vicina are among the first wave of insects colonizing a dead body. Two series of 3 rabbits were given dosages of 10, 20, and 40 mg/h of morphine over a 3 h period via ear artery perfusion. A morphine blood level plateau was attained after 1 h of perfusion. Two other rabbits were used as controls. Samples of tissues collected from rabbits using a coelioscopic technique were determined to have morphine concentrations similar to those encountered in human overdoses and were correlated with dosages of morphine administered. All samples from control rabbits were negative for morphine. Larvae and puparia of both species were regularly collected from each rabbit for toxicological analysis. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were significantly lower than concentrations found in the tissues. There was a decrease in concentration in morphine observed in transition from feeding 3rd instar larva to puparium. A correlation between larval concentration and tissue concentration was found only in feeding 3rd instar larvae.     

Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Immature development times of the blow fly Protophormia terraenovae (Robineau-Desvoidy, 1830) were studied in the laboratory at five different constant temperatures (15, 20, 25, 30, 35 degrees C). The minimal duration of development from oviposition to adult emergence was inversely related to temperature, ranging from 9.19+/-0.3 days at 35 degrees C to 37.78+/-2.96 days at 15 degrees C. From linear regression of development rates at the five studied constant temperature regimes, it followed that the minimum development threshold (t(L)) for total immature development is 8.95 degrees C ( approximately 9 degrees C) and the overall thermal constant (K) for P. terraenovae is 240.2+/-9.3 day-degrees (DD) above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 degrees C. However, it is possible that developmental time from oviposition to adult eclosion might be different in various regions of the world, and that the thermal constant of a holarctic species like P. terraenovae is not same everywhere. Additionally, as the present paper shows, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework.     

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (D_z) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17–37 ⁰C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (D_z ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.     

Effectiveness of wound cleansing treatments on maggot (Diptera, Calliphoridae) mortality

Myiasis is defined as an infestation of the organs and/or tissues of human and other animals by fly maggots. Fly species that normally breed in meat or carrion (Diptera: Calliphoridae, Sarcophagidae) may become involved in cutaneous myiasis by colonizing preexisting wounds. Reports of human wound myiasis contracted in hospitals and nursing homes, especially when patients are chronically ill or bed-ridden, are not uncommon across North America and often result in cases of neglect and civil litigation. Based on a case history dealing with this latter situation and circumstances surrounding the treatment of maggot infestation, we designed an experiment to assess the effectiveness of wound cleansing solutions on maggot mortality. Treatments, consisting of four commonly used cleaning solutions (isopropyl alcohol, Dakin's solution, iodine, and hydrogen peroxide) and a control (deionized water), were applied to experimental units (n=5), with each unit consisting of groups of actively feeding Lucilia sericata maggots (Diptera: Calliphoridae). Every 24h, treatments were applied and mortality was assessed for the duration of the study (14 days). Total mean mortality increased over the duration of the experiment, with an initial large increase (10-25%) after the first treatment application, followed by a gradual increase over the remainder of the study. General differences among treatments indicated greatest mean total mortality for Dakin's solution (sodium hypochlorite) (46%), followed by isopropyl alcohol (42%), Betadine (37%), hydrogen peroxide (33%) and lowest mortality for the control (25%); however, no statistically significant differences were observed among treatments and no treatment resulted in 100% maggot mortality. Traditional wound cleansing solutions may not be sufficient for maggot infestations of pre-existing wounds and supplemental treatments may be necessary to effectively treat cases of wound myiasis.     

PCR-RFLP identification of Diptera (Calliphoridae,Muscidae and Sarcophagidae)--a generally applicable method

A simple, rapid method using restriction fragment length polymorphisms (RFLPs) within the internal transcribed spacer (ITS) regions of the ribosomal DNA gene repeat allows identification of insects and other organisms. We used the method to identify the morphologically similar Diptera larvae that are important in forensic entomology for estimating the time and location of death. Polymerase chain reaction (PCR) was used to amplify a region from the 18S to the 28S rRNA genes. The ITS1 and ITS2 regions provided variation between species and homogeneity within species, with the exception of Cochliomya macellaria. Combinations of the restriction enzymes DdeI, HinfI and Sau3AI provided diagnostic bands for identification of the ten species from three families of Diptera (Calliphoridae, Muscidae and Sarcophagidae).     

Morphology of second and third instars of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a fly species of forensic importance

The morphology of the second and third instars of Chrysomya villeneuvi Patton, a fly species of forensic importance, was presented by use of light microscopy. Both instars were of hairy appearance, bearing elongated tubercles along the abdominal and caudal segments. The anterior spiracle had 13-15 papillae. Minute dark spots were observed to thoroughly cover the tubercle's surface, with 4-6 strong dark tips. Regarding the third instar, the intersegmental spines between the prothorax and mesothorax were heavily pigmented. The posterior spiracle had a thick and heavily pigmented incomplete peritreme. The surface and tip of the tubercles was covered with heavily pigmented sharp spines. The integument of the body was covered with numerous distinct net-like patches. A comparison with another well-known hairy maggot, Chrysomya rufifacies (Macquart), was discussed.     

Minimum and maximum development rates of some forensically important Calliphoridae (Diptera)

Blow fly development rates are frequently used to estimate elapsed time since death in homicide investigations in the first few weeks after death. However, in order to make more precise estimates of time since death, accurate developmental data must be generated for all carrion species, and at temperatures that are comparable with those found at crime scenes. This paper presents developmental rates determined for five forensically important species in British Columbia, Phormia regina (Meigen), Phaenicia sericata (Meigen), Eucalliphora latifrons (Hough), Lucilia illustris (Meigen), and Calliphora vicina Robineau-Desvoidy, at several temperatures.     

Spread and habitat selection of Chrysomya albiceps (Wiedemann) (Diptera Calliphoridae) in Northern Italy: forensic implications

Abstract: Habitat selection exploited by Chrysomya albiceps during its initial spread in Northern Italy was analyzed in relation to landscape structure. The results of two short studies and a case report are here discussed. C. albiceps was not found on experimental pig carcasses in the urban area of Pavia. It was missing in the woody mountains surrounding Lecco, but it was found in the same area, at a lower altitude, within the typical “urban sprawl” landscape. It was then recorded in a natural reserve, among a rich carrion‐fly population. Indications coming from habitat selection suggest that C. albiceps has not yet saturated its potential ecological niche in newly colonized areas of Northern Italy. Factors like temperature, altitude, and interspecific competition can act as limiting factors, affecting habitat selection and distribution in newly colonized areas; the influence of those factors has to be taken into account for forensic purposes.     

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): a comparative study

A comparative morphological study of preimaginal stages (larvae and pupae) of Calliphora vicina Robineau-Desvoidy, 1830 is presented. The entomological samples came from laboratory colonies bred under controlled environmental conditions (25°C and 60% relative humidity). In this study, a recently published technique to clear Diptera larvae for light microscopy and a standard protocol for scanning electron microscopy were used. For the morphological comparison of larval instars I, II and III, and pupae of C. vicina, different larval regions (cephalic, thoracic and abdominal, including anal division), as well the internal chitinised cephalopharyngeal skeleton, were considered separately. Our results focus on showing the changes observed throughout development for the most important structures in the cephalic region (sensilla of maxillary palpus, antennae and oral ridges), the thoracic region (the first segment and its anterior spinose band) and in the anal division of the abdominal region (posterior spiracles and shape of the papillae). In addition, some morphological structures are described or pictured for the first time, such as the ventral organ and the anterior spiracle of larva I and the antenna sensilla, Keilin's organ and wrinkled area of the anal division of all instars. The cephalopharyngeal skeleton is an important structure for the taxonomy of Diptera larvae in all instars, including Calliphoridae. Our observations in C. vicina indicate that an in-depth review of the sclerite composition is needed. Pupae and larvae stages can only be compared by following the segmentary spinose bands and the anal segment, where the morphology of the posterior spiracles and papillae can be observed, in some cases despite the reduced condition of the latter.     

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.