Electrophysiological and Behavioral Responses of Thanatophilus sinuatus Fabricius (Coleoptera: Silphidae) to Selected Cadaveric Volatile Organic Compounds

Soon after death, carcasses release volatile chemicals that attract carrion insects including Silphidae. Nevertheless, it is not known which chemical cues are involved in the attractiveness of the carcass. So far, little information is available on the chemical ecology of carrion beetles, particularly concerning the subfamily of Silphinae. The biological role of selected cadaveric volatile organic compounds including dimethyldisulfide (DMDS), butan‐1‐ol, n‐butanoic acid, indole, phenol, p‐cresol, putrescine, and cadaverine on the silphine species, Thanatophilus sinuatus Fabricius, was investigated using both electrophysiological and behavioral techniques. Among the tested cadaveric compounds, butan‐1‐ol and DMDS elicited the strongest electroantennography (EAG) from both T. sinuatus male and female antennae. In a two‐arm olfactometer, males and females were significantly attracted to DMDS for both tested doses, whereas only males were attracted to p‐cresol at 100 ng. Putrescine was repellent to males at the dose of 1 μg.     

Host‐habitat Location by the Parasitoid,Nasonia vitripennis Walker (Hymenoptera: Pteromalidae)

This study investigated the role of odorant cues used during host‐habitat location by the generalist parasitoid, Nasonia vitripennis Walker. Nasonia vitripennis is a common parasitoid of Dipteran pupae found in association with decaying carrion. Behavioral assays were used to investigate the host‐habitat searching behavior under different scenarios. First, we demonstrated N. vitripennis to be significantly attracted toward odorant cues associated with decaying meat. The biological activity of nine of the volatile molecules constituting the odor of decaying meat was tested on the searching behavior of parasitoid females through two complementary chemoecological approaches: electroantennography (EAG) and olfactometry bioassays. Butanoic acid and butan‐1‐ol elicited high olfactory responses, but no attraction was induced by these two chemicals. Behavioral assays showed that, among the VOCs tested, methyldisulfanylmethane (DMDS) was the only volatile chemical to induce attraction in N. vitripennis.     

Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication.

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure.This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results.Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.     

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.     

Associative Learning of Nasonia vitripennis Walker (Hymenoptera:Pteromalidae) to Methyldisulfanylmethane

Traditional methods of volatile detection used by police typically consist of reliance on canine olfaction. However, dogs have some limitations such as cost of training and time of conditioning. The possibility of using parasitic wasps for detecting explosives and narcotics has been developed. Moreover, wasps are cheap to produce and can be conditioned with impressive speed for a specific chemical detection task. We examined the ability of Nasonia vitripennis Walker to learn and respond to methyldisulfanylmethane (DMDS), a volatile discriminator of cadaver. The training aimed to form an association between an unconditioned stimulus (pupae) and the conditioned stimulus (odor source). After the training, the time spent by conditioned wasps in the DMDS chamber was measured. Statistical analysis showed that the increasing concentrations involved an increase in the time spent in the chamber containing DMDS. This study indicates that N. vitripennis can respond to DMDS, which provide further support for its development as a biological sensor.     

Flight activity of the blowflies, Calliphora vomitoria and Lucilia sericata, in the dark

Many species of insects are able to fly at night or in very low light intensities. The question of whether calliphorid blowflies are also able to do this to locate a corpse and oviposit nocturnally is of considerable forensic importance. However, to date studies of this behaviour have been contradictory. Here, the activity and number of Calliphora vomitoria L. and Lucilia sericata (Meigen) (Diptera: Calliphoridae) caught on sticky-traps were examined in a slow-speed wind tunnel, at different intensities of artificial light. The traps were either unbaited or baited-with liver. The number of both species caught, decreased incrementally as light intensity was reduced. While the responses of the two species were broadly similar, L. sericata were significantly more active than C. vomitoria, especially at higher light intensities. The number of flies of both species that were caught was higher in the presence of liver bait, but the presence of the liver bait did not change the shape of the relationship between catch and light intensity. Hence, light intensity acts as an independent exogenous stimulus for activity and although liver volatiles increase activity levels, they are not necessary as an activation stimulus. Comparison of the numbers caught in small or large enclosures suggests that any flies caught in darkness probably alighted on the trap by chance and that in darkness, while flies may be activated by carrion odours, they do not appear to be able to navigate effectively to the source of that odour. The results presented here suggest that in darkness, the probability of oriented flight leading to oviposition on a corpse by either species, is relatively low.     

常见嗜尸性甲虫及蝇类虫体不同部位mtDNA的提取效果

目的探讨改良后十六烷基三甲基溴化铵(cetyltriethylammnonium bromide,CTAB)法对常见嗜尸性昆虫虫体不同部位mtDNA的提取效果。方法随机采集放置在呼和浩特地区室外草地家兔尸体上丽蝇科的丝光绿蝇[Lucilia sericata(Meigen)]、埋葬甲科的Nicrophorus fossor(Erichson)各13例,采用改良CTAB法分别提取每只昆虫头部、胸肌、腿部、翅膀4个部位的mtDNA,核酸蛋白测定仪检测DNA纯度及质量浓度,琼脂糖凝胶电泳检测其PCR产物,测定PCR产物序列,上传GenBank。结果 13例丝光绿蝇[Lu-cilia sericata(Meigen)]的胸肌均提取到了DNA,10例头部样本、6例腿部样本、4例翅膀样本得到DNA;13例Nicrophorus fossor(Erichson)胸肌DNA均提取成功,5例头部样本、8例腿部样本及3例翅膀样本提取出DNA。结论改良CTAB法可用于对嗜尸性昆虫胸肌及其他部位进行DNA的提取。     

Use of Lucilia species for forensic investigations in Southern Europe

The aim of this study was to highlight the importance of evaluating entomological evidence in forensic investigations on a regional scale. To evaluate climatic, geographical and environmental influences on the selection of carrion-breeding fauna in Northern Italy and consequently on inferred forensic data (post-mortem intervals and post-mortem transfer), we present details of six indoor-outdoor cases. Results show that the most abundant species was Lucilia sericata, together with other fly species of entomo-forensic interest, belonging to the Calliphoridae and Sarcophagidae families. In particular, for the first time in Italy, we report finding Phormia regina, Lucilia ampullacea, Lucilia caesar and Sarcophaga (Pandelleana) protuberans on fresh cadavers. The active period of L. sericata in Northern Italy, according to previous findings in Southern Europe, revealing clearcut differences with phenologies in Northern Europe, has important consequences in estimating the period (season, months) of death in cases of long post-mortem intervals (several months or years) if empty puparia of this fly are found. According to our results, the distribution of L. sericata in areas with urban sprawl, like Northern Italian regions, cannot be used to evaluate post-mortem transfer from an urban area to a rural one.     

Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation

The identification of insects found on a dead body can lead to the estimation of the time of death (postmortem interval). We report an updated version of an established method based on sequence analysis of PCR products from a region of the cytochrome b oxidase subunit I mitochondrial gene of different members of the family Calliphoridae, by sequencing six European species: Lucilia sericata (Meigen), Lucilia caesar (Linné), Lucilia illustris (Meigen), Calliphora vicina (Robineau-Desvody), Calliphora vomitoria (Linné), Protophormia terraenovae (Robineau-Desvody) and one Guianese species: Cochliomyia macellaria (Fabricius). This technique provided clear results when applied to the larvae and we also report the identification of empty puparia.     

青岛地区常见嗜尸性蝇种及其季节性分布研究

目的观察青岛地区嗜尸性蝇种季节性演替规律,为死亡时间推断提供参考。方法在室外设立田间观察场所,2012年4月1日开始至2013年4月1日,每月第1天6时放置猪尸,20min后开始观察,至18时,期间每小时观察1次;第3天以后,分别于7时、12时、18时各观察1次。观察时记录尸体上出现的昆虫种类、虫态、数量并采集相应标本,进行分类鉴定。结果可以观察到有嗜尸性蝇类侵袭尸体的月份为4月至11月,共发现3科23种蝇类,其中丝光绿蝇、大头金蝇和亮绿蝇数量位列前三,分别占全部观察数量的40.71%、31.95%和7.12%。丝光绿蝇、大头金蝇中午时段出现的数量显著多于早上和傍晚。一年中各种蝇类在中午出现的数量均多于早上及晚上。4月上旬,蝇类早、晚与中午出现的数量相差显著;而在7、8月份差别较小。大部分嗜尸性蝇种集中在新鲜期和肿胀期。结论本文观察数据可为利用嗜尸性蝇类演替规律进行法医学死亡时间推断,以及建立地区嗜尸性蝇种类数据提供参考。     

Decomposing Human Blood: Canine Detection Odor Signature and Volatile Organic Compounds

The admissibility of human “odor mortis” discrimination in courts depends on the lack of comprehension of volatile organic compounds (VOCs) during the human decay process and of the lack in standardized procedures in training cadaver dogs. Blood was collected from four young people who died from traffic accidents and analyzed using HS‐SPME/GC‐MS at different decompositional stages. Two dogs, professionally trained, were tested to exactly locate blood samples, for each time point of the experiment. We found a long list of VOCs which varied from fresh to decomposed blood samples, showing differences in specific compounds. Dog performance showed a positive predictive value between 98.96% and 100% for DOG A, and between 99.47% and 100% for DOG B. Our findings demonstrated that decomposing human blood is a good source of VOCs and a good target for canine training.     

新乡地区夏季常见嗜尸性昆虫群落演替规律初步研究

目的研究新乡地区夏季嗜尸性昆虫的种类及其群落演替规律。方法观察法研究新乡医学院动物房后偏僻草地上6只大白鼠和5只家兔尸体腐败过程中嗜尸性蝇类的主要物种及其群落演替规律。结果该地区主要出现嗜尸性蝇类为双翅目3科7种,包括家蝇、大头金蝇、丝光绿蝇、棕尾别麻蝇、黑尾黑麻蝇、厩腐蝇、元厕蝇,且呈现出演替规律性。结论研究结果可作为新乡地区对尸体死亡时间的推断依据。     

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.     

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.     

Decomposition and Arthropod Succession in Whitehorse,Yukon Territory,Canada

Forensic arthropod succession patterns are known to vary between regions. However, the northern habitats of the globe have been largely left unstudied. Three pig carcasses were studied outdoors in Whitehorse, Yukon Territory. Adult and immature insects were collected for identification and comparison. The dominant Diptera and Coleoptera species at all carcasses were Protophormia terraneovae (R‐D) (Fam: Calliphoridae) and Thanatophilus lapponicus (Herbst) (Fam: Silphidae), respectively. Rate of decomposition, patterns of Diptera and Coleoptera succession, and species dominance were shown to differ from previous studies in temperate regions, particularly as P. terraenovae showed complete dominance among blowfly species. Rate of decomposition through the first four stages was generally slow, and the last stage of decomposition was not observed at any carcass due to time constraints. It is concluded that biogeoclimatic range has a significant effect on insect presence and rate of decomposition, making it an important factor to consider when calculating a postmortem interval.     

Flies eggs: a new method for the estimation of short-term post-mortem interval?

Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).     

Forensic Entomology and the Estimation of the Minimum Time Since Death in Indoor Cases

Eight cases that occurred indoors in which the insects played an important role in the mPMI estimation are presented. The bodies of socially isolated people and old people living alone were discovered in central Italy between June and November. mPMI ranged from a few days to several weeks. Insects were collected during the body recovery and the postmortem. Climatic data were obtained from the closest meteorological stations and from measurements performed on the site. Sarcophagidae and Calliphoridae species were present in 75% of the cases with Lucilia sericata and Chrysomya albiceps collected in 50% of the cases. Chrysomya albiceps was always found in association with Lucilia species. Scuttle flies (Phoridae) were found in 37.5% of the cases, confirming the ability of these species in indoor body colonization. We show that if sealed environment may delay, the insect arrival dirty houses may create the environment where sarcosaprophagous insects are already present.     

Preliminary study of the succession pattern of necrobiont insects, colonising species and larvae on pig carcasses in Temuco (Chile) for forensic applications

Knowledge regarding the succession patterns of insects that visit carcasses as well as the other arthropod that colonise them and analysis of the parameters that are associated with larvae allow calculation of the minimum postmortem interval (PMI). This information is obtained from experiments carried out under specific geoclimatic conditions, which determine their application in forensic environments under similar conditions. The field study presented here is the first in Chile to analyse the decomposition process of pig carcasses and the associated succession of insects, colonising species and parameters related to larval masses. All of the larvae obtained from daily samples were measured (in mm), and their mean, range, standard deviation and stage of development (instars) were determined. The carcasses reached the dry remains stage in only 11 days. Seven species of Diptera visited the carcass during the process, but only two species colonised it by means of egg deposition followed by development of larvae: Cochliomyia macellaria (Fabricius) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), which exhibited a duration of the development cycle from egg to adult of 21 days. The collected Coleoptera correspond to five predator species (Staphilinidae and Histeridae). From the results of this study, it can be concluded that only some of the insect species present in this region can provide information that can be used in forensic entomology and that analysis of larval masses of colonising species can be a valuable tool for determining the PMI(min) in this region of Chile.     

Volatile Emission of Decomposing Pig Carcasses (Sus scrofa domesticus L.) as an Indicator for the Postmortem Interval

This study aimed at correlating selected carcass borne volatile organic compounds (VOCs) with the postmortem interval (PMI). Selected volatiles should 1st be reliably emitted during vertebrate decay, 2nd be emitted at high concentrations, and 3rd show a reproducible quantitative dynamic during the decaying process. Four pigs (Sus scrofa domesticus L.) were placed in a deciduous forest in different seasons and volatiles emitted during the decaying process were sampled. Seventeen compounds were identified and quantified by GC-MS. Electrophysiological experiments on the antenna of female Calliphora vicina and additional data of Dermestes maculans were used as an evolutionary tuned information filter to evaluate the 1st criterion. The relative quantitative emission of hexanal, nonanal, dimethyl disulfide, dimethyl trisulfide, 1-butanol, and phenol were correlated with the PMI, and the observed stages of decay and the limitations of this model were discussed.     

呼和浩特地区夏秋季常见嗜尸性蝇类的研究

目的研究呼和浩特地区常见嗜尸性苍蝇种类及其出现规律。方法观察法研究7～10月份兔尸上嗜尸性苍蝇种类变化。结果该地区兔尸上出现的双翅目嗜尸性苍蝇3科8属10种,主要有蝇科的家蝇、舍蝇、开普黑蝇、刺足齿股蝇、厩腐蝇;丽蝇科的丝光绿蝇、大头金蝇、铜绿蝇;麻蝇科的棕尾别麻蝇、肥须亚麻蝇。上述蝇类在尸体上出现时间及部位表现出很强的规律性。结论研究结果有望成为法医在呼和浩特地区对尸体进行死亡时间推断的依据。