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Forensic entomological evidence is employed to estimate minimum postmortem interval (PMImin), location, and identification of fly samples or human remains. Traditional forensic DNA analysis (i.e., STR, mitochondrial DNA) has been used for human identification from the larval gut contents. Forensic DNA phenotyping (FDP), predicting human appearance from DNA-based crime scene evidence, has become an established approach in forensic genetics in the past years. In this study, we aimed to recover human DNA from Lucilia sericata (Meigen 1826) (Diptera: Calliphoridae) gut contents and predict the eye and hair color of individuals using the HIrisPlex system. Lucilia sericata larvae and reference blood samples were collected from 30 human volunteers who were under maggot debridement therapy. The human DNA was extracted from the crop contents and quantified. HIrisPlex multiplex analysis was performed using the SNaPshot minisequencing procedure. The HIrisPlex online tool was used to assess the prediction of the eye and hair color of the larval and reference samples. We successfully genotyped 25 out of 30 larval samples, and the most SNP genotypes (87.13%) matched those of reference samples, though some alleles were dropped out, producing partial profiles. The prediction of the eye colors was accurate in 17 out of 25 larval samples, and only one sample was misclassified. Fourteen out of 25 larval samples were correctly predicted for hair color, and eight were misclassified. This study shows that SNP analysis of L. sericata gut contents can be used to predict eye and hair color of a corpse., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Forensic entomology requires knowledge of the developmental rates of the species that colonize a body after death to estimate the postmortem interval (PMI). These developmental rates may vary depending not only on the species but also on the geographic location due to population differences. Therefore, the objectives of this work were to determine the developmental duration of the forensically important fly Chrysomya megacephala under constant controlled and field condition temperatures and to compare these results, through a meta-analysis, with data reported by other authors on populations from different localities. For this, C. megacephala colonies were established in the laboratory, and the duration of the life cycle was studied at two controlled temperatures (25 °C and 27 °C) and field conditions (27.5 ± 3.2 °C). Analysis of variance was performed to determine differences in developmental time and larval length between constant laboratory temperatures and field conditions. A generalized linear model was performed with predictor variables extracted from the literature (diet, relative humidity, latitude, longitude) to evaluate the effect of population variation on developmental times. The results showed significant differences in developmental times between 25 and 27 °C. As expected, the complete life cycle of C. megacephala was shorter at 27 °C. Finally, the meta-analysis suggested differences between the developmental times of different populations, based on temperature and geographic location. The results of this study provide fundamental developmental data to use C. megacephala in PMI estimations. Finally, we suggest that, when making expert reports, information from local populations should be used to determine a more accurate and reliable PMI., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Many authors produced carrion insect development data for predicting the age of an insect from a corpse. Under some circumstances, this age value is a minimum postmortem interval. There are no standard protocols for such experiments, and the literature includes a variety of sampling methods. To our knowledge, there has been no investigation of how the choice of sampling method can be expected to influence the performance of the resulting predictive model. We calculated 95 % inverse prediction confidence limits for growth curves of the forensically important carrion flies Chrysomya megacephala and Sarconesia chlorogaster (Calliphoridae) at a constant temperature. Confidence limits constructed on data for entire age cohorts were considered to be the most realistic and were used to judge the effect of various subsampling schemes from the literature. Random subsamples yielded predictive models very similar to those of the complete data. Because taking genuinely random subsamples would require a great deal of effort, we imagine that it would be worthwhile only if the larval measurement technique were especially slow and/or expensive. However, although some authors claimed to use random samples, their published methods suggest otherwise. Subsampling the largest larvae produced a predictive model that performed poorly, with confidence intervals about an estimate of age being unjustifiably narrow and unlikely to contain the true age. We believe these results indicate that most forensic insect development studies should involve the measurement of entire age cohorts rather than subsamples of one or more cohorts.

Forensic entomology case reports are the product of rapid development in the field, the widespread acceptance of the science and the application of forensic entomological knowledge. In this study, we retrospectively summarized information derived from 307 forensic entomology case reports from 1935 to 2022 from a global perspective. Our checklist of relevant information included insect species, specific indoor or outdoor preferences, preferred temperatures, and stages of body decomposition. Finally, a concept and calculation method for postmortem interval (PMI) estimation accuracy was proposed. There were 232 cases using insect developmental data and 28 cases using succession patterns to estimate PMI. A total of 146 species of insects were involved in the cases, of which 62.3% were Diptera and 37.7% were Coleoptera. Postmortem intervals were estimated from eggs in 4 cases, larvae in 180 cases, pupae in 45 cases, and puparia in 38 cases. The majority of cases were from June to October, and the average number of species mentioned in the cases was more at 15-30 °C. Considering the standardization of application, in the majority of cases, insect evidence was collected by other personnel and sent to forensic entomologists, there was a delay in the sampling, and the scene or meteorological data were directly used without correcting. Our data shows that there are still many shortcomings in the universality and standardization of forensic entomology in its practical application., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Forensic scientists are increasingly using DNA to identify the species of a tissue sample. However, little attention has been paid to basic experimental design issues such as replication and the selection of taxa when designing a species diagnostic test. We present an example using the forensically important fly genus Lucilia in which an increasingly larger sample size revealed that species diagnosis based on the commonly used cytochrome oxidase I gene (COI) was less straightforward than we initially thought. This locus may still be useful for diagnosing Lucilia specimens, but additional knowledge other than the genotype will be required to reduce the list of candidate species to include only forms that can be distinguished by COI. We believe that these results illustrate the importance of study design and biological knowledge of the study species when proposing a DNA-based identification test for any taxonomic group.

Many authors have proposed DNA-based methods for identifying an insect specimen associated with human remains. However, almost no attempt has been made to validate these methods using additional observations. We tested a protocol for identifying insects in the blow fly subfamily Chrysomyinae (Diptera: Calliphoridae) often found to be associated with a human corpse in Canada or the USA. This method uses phylogenetic analysis of DNA sequence from a short segment of the mitochondrial gene for cytochrome oxidase one (COI). Test chrysomyine COI sequences were obtained from 245 newly sequenced specimens and 51 specimens from the published literature. Published sequences from representatives of nonchrysomyine genera were also included to check for the possibility of a false positive identification. All of the chrysomyine test haplotypes were correctly identified with strong statistical support, and there were no false positives. This method appears to be an accurate and robust technique for identifying chrysomyine species from a death investigation in this geographic region. The far northern species Protophormia atriceps was not evaluated; therefore, caution is required in applying this method at very high latitudes in North America.

Megaselia scalaris (Schmitz, 1938) (Diptera: Phoridae) is a common insect in forensic science that is frequently found in indoor cases, particularly on corpses in closed environments. Although this species is useful for estimating the minimum postmortem interval (PMImin) in the absence of Calliphoridae, there is a lack of data on its development in China. Herein, we studied the development of M. scalaris exposed to seven constant temperatures ranging from 16 to 34 °C. The mean (± SD) developmental durations of M. scalaris from egg to adult stage at 16, 19, 22, 25, 28, 31, and 34 °C were 1486.9 ± 75.3, 823.7 ± 42.8, 448.2 ± 59.8, 417.7 ± 19.7, 297.2 ± 27.3, 272.9 ± 10.4, and 253.0 ± 5.0 h, respectively. The mean (± SE) lower developmental threshold temperature (TL) and the thermal summation constant (K) were determined by a linear model as 12.69 ± 0.3 °C and 4965.8 ± 227.9-degree hours, respectively. A nonlinear model estimated the lower developmental threshold temperature, intrinsic optimum temperature, and upper lethal developmental threshold temperature as 14.58, 21.00, and 34.15 °C, respectively. We established three development models to estimate the age of the immature insect, namely the isomegalen diagram, isomorphen diagram, and thermal summation model. In addition, a regression analysis of the relationship between body length and total development time from hatching to pupariation was performed. Our findings provide a basis for applications of M. scalaris in PMImin estimations. [ABSTRACT FROM AUTHOR]

Chrysomya megacephala and C. rufifacies are globally predominant species of blowflies that are amongst the primary colonisers of decomposing carcasses. The development of blowflies has been studied extensively to estimate the minimum postmortem interval (PMImin). This article evaluates the effect of aluminium phosphide (AlP) on the larval morphometry and life cycles of C. megacephala and C. rufifacies. The rabbits were given different dosages of AlP and their livers were excised to rear C. megacephala and C. rufifacies. Through multiple-regression analysis, we assessed the overall impact of varying dosages of AlP on larvae of both species. AlP accelerated development until pupation, whilst the time until emergence remained the same. At higher concentrations, AlP had a positive effect on the development of these species. Since both species are abundant, forensic entomology studies examining the effect of AlP on morphometry and developmental stages would provide valuable information to investigators assessing PMImin. [ABSTRACT FROM AUTHOR]

Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae) is a colonizer of cadavers in the Neotropical Region. Nevertheless, data on development for the P. (P.) chrysostoma (e.g., instar duration) and behavioral strategies used by the species for locating and colonizing a corpse are scant. We aimed to explore bionomic and reproductive aspects of the flesh fly P. (P.) chrysostoma, and in this article we: (a) provide quantitative data on the life cycle of P. (P.) chrysostoma; (b) present bionomic measurements (length and weight) of larvae and pupae; (c) describe intrauterine egg and larvae development; and (d) analyze the ovo/larviposition behavior by gravid females. Females showed ovaries with discernible eggs and larvae between 8 and 10 days (x̅ = 23.3 eggs/female). This study reports the first observation of egg deposition, an atypical behavior for the species. The average development time for immature stages was 22.24 h and 21.36 h for 1st and 2nd respectively, and 3rd showed an average development time of 80.47 h. Pupa had the longest duration (x̅ = 295.69 h). A direct increase was observed in weight (P < 0.05) and length (P < 0.05) throughout time. The average survival time of males and females is approximately 30 days. This study expands the knowledge on P. (P.) chrysostoma, such as facultative ovoviviparity under laboratory conditions and the life cycle, which may benefit future studies for accuracy in entomology-based estimation of minimum post-mortem interval (min PMI)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Age determination of insects collected from vertebrate remains is an essential step in estimating time since colonization as related to the post-mortem interval. Long-established methods for making such estimates rely on determining age related to stage of development at the time of collection in relation to conditions experienced. However, such estimates are based on the completion of a stage of development. Methods allowing for more precise estimates of age (i.e., within a stage of development) are sorely needed. This study examined the potential of volatile organic compounds emitted by blow fly, Ch. rufifacies (Macquart), immatures to determine stage of development, which could potentially be used to estimate the age. Volatile organic compounds (VOCs) from the larval and pupal stages of Ch. rufifacies were collected by headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry (GC-MS). Analyses indicated 37 compounds shift quantitatively, as well as qualitatively, as the larvae and pupae age. Furthermore, compounds, such as 2-ethyl-1-hexanol, phenol, butanoic acid, hexadecanoic acid, octadecanoic acid, 2-methyl propanamide, and 2-methyl butanoic acid, serve as indicator compounds of specific stages within Ch. rufifacies development. This information could be important to determine if these compounds can be used in the field to predict the presence of certain developmental stages, in order to determine the potential of using volatile markers to estimate time of colonization.

Stearibia (=Piophila) nigriceps (Meigen) occurs in the Palearctic Region from Europe and Far East Russia, and in the Nearctic, the Neotropical, and the Oriental regions. In this study, we report the first record of Stearibia nigriceps from Africa collected in Algeria. In addition, our results provide DNA sequences of Piophila casei (Linnaeus) and S. nigriceps that may serve as reference data for future identification. A region of the cytochrome c oxidase I (COI) gene marker of 473 bp was amplified and DNA was sequenced. Intra- and interspecific genetic distances were calculated. A phylogenetic tree was generated by the maximum likelihood (ML) method using 1000 bootstrap replicates based on the Tamura-Nei model. A total number of 9 adults of S. nigriceps were collected together with 72 adults of P. casei. The intraspecific variation of the species barcodes analysed in our study was < 3% and the interspecific nucleotide divergence of the same species was > 3%. The 473 bp COI region was sufficient to identify and distinguish unambiguously between the two species. P. casei and P. nigriceps individuals from Algeria clustered separately from each other. They were also separated from the individuals of the same species originating from other countries with a 100% high bootstrap value. There was no correlation between the clades and the geographic origins of the haplotypes. Our results contribute to update the knowledge of the distribution of Piophilidae family. They also contribute to the build-up of online DNA sequence databases.

Estimating postmortem interval (PMI) is one of the most challenging tasks in forensic practice due to the effects of many factors. Here, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with chemometrics was utilized to evaluate the effects of causes of death when estimating PMI and to establish a partial least square (PLS) regression model, which can precisely predict PMI under different causes of death. First, the sensitivities to causes of death (brainstem injury, mechanical asphyxia, and hemorrhage shock) of seven kinds of organs were evaluated based on their degrees of cohesion and separation. Then, the liver was selected as the most sensitive organ to establish a PMI estimation model to compare the predicted deviations from different causes of death. It turns out that the cause of death has no significant effect on estimating PMI. Next, a PLS regression model was built with kidney tissues, which have the lowest sensitivity, and this model showed a satisfactory predictive ability and wide applicability. This study demonstrates the feasibility of using ATR-FTIR spectroscopy in conjunction with chemometrics as a powerful alternative for detecting changes in biochemistry and estimating PMI. A new perspective was also provided for evaluating the effect of causes of death when predicting PMI.

Abstract  Forensic scientists are increasingly using DNA to identify the species of a tissue sample. However, little attention has been paid to basic experimental design issues such as replication and the selection of taxa when designing a species diagnostic test. We present an example using the forensically important fly genus Lucilia in which an increasingly larger sample size revealed that species diagnosis based on the commonly used cytochrome oxidase I gene (COI) was less straightforward than we initially thought. This locus may still be useful for diagnosing Lucilia specimens, but additional knowledge other than the genotype will be required to reduce the list of candidate species to include only forms that can be distinguished by COI. We believe that these results illustrate the importance of study design and biological knowledge of the study species when proposing a DNA-based identification test for any taxonomic group. [ABSTRACT FROM AUTHOR]

This study highlights the importance of insect evidence by evaluating 949 insect-associated cases, including 139 entomological reports, from 2001 to 2019 at the Institute of Legal Medicine Frankfurt/Germany. With a high number of cases in the summer months and a low number in the colder season, 78.5% of the bodies were found indoors, regardless of year or month. In more than 80% of the cases, where PMI information was available (n = 704), the presumed PMI ranged from 1 to 21 days, a period during which entomological evidence can provide a day-specific estimate of PMImin. In cases where insects have been identified to species level (n = 279), most bodies were infested by one or two species with a maximum of 10 different species. Overall, a total of 55 insect species were found. Information on biology, activity and distribution of the most abundant taxa is given and applied for 5 case histories estimating different PMImins of up to over 6 months. Despite proved importance and scientific development of forensic entomology, insects are still rarely considered as a tool in forensic case work. The main reasons are a lack of awareness and (too) late involvement of a forensic entomologist. Our work shows that forensic entomology is an independent discipline that requires specialist expertise. [ABSTRACT FROM AUTHOR]

Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5–15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data. [ABSTRACT FROM AUTHOR]

Fly artifacts resulting from insect activity could act as confounding factors on a crime scene and interfere with bloodstain pattern analysis interpretation. Several techniques have been proposed to distinguish fly artifacts from human bloodstains based on morphological approach and immunological assay, but a DNA-based method has not been developed so far. Even if in forensic genetic investigations the detection of human DNA is generally the primary goal, fly artifacts can provide useful information on the dynamics of crime events. The present study provides a molecular method to detect fly DNA from artifacts deposited by Calliphora vomitoria after feeding on human blood through the analysis of the mitochondrial cytochrome oxidase gene subunit I (COI). Fly artifacts originated from digestive process and of different morphology spanning from red and brownish/light brown, circular and elliptical stains to artifacts with sperm-like tail or a tear-shaped body were collected. The COI amplification was successfully obtained in 94% of fly artifact samples. The method showed high sensitivity and reproducibility, and no human DNA contamination was observed, offering specificity for use in confirmatory test. This molecular approach permits the distinction of fly artifacts from genuine bloodstains and the identification of fly's species through the COI region sequencing by protocols usually applied in forensic genetic laboratories. [ABSTRACT FROM AUTHOR]

Synthesiomyia nudiseta (van der Wulp, 1883) is a synanthropic muscid found in tropical and subtropical zones around the world. The larvae of this species are a secondary agent of myiasis with necrophagous habits and play an important role in forensic entomology, as they are used as an indicator of post-mortem interval. Adults can be considered vectors of etiological agents such as Escherichia coli and Shigella dysenteriae. Due to its ability to adapt to different environmental conditions, its high dispersal capacity (shown by its introduction to Europe), its predatory habits in the last larval stage and the difficulty of identifying it, a very important goal is to update our knowledge about this species. Therefore, the main objective of this paper is to review the identification, geographical distribution and biology of this species in order to provide better support to investigations involving this fly. [ABSTRACT FROM AUTHOR]

Postmortem interval (PMI) determination is an important part of criminal investigations, but it is still subject to uncertainty. Degradation of mRNA in PMI determination has been studied in decays; however, some studies have reported no correlation between PMI and RNA degradation. Thus, we aimed to determine whether RNA quantity was correlated with PMI. Heart and brain tissues were separated from a mouse model of a 0–48 h PMI with 29 time points. We then coextracted the DNA and RNA in one tube with Bioteke coextraction kits and selected some mRNA markers associated with cell oxygen deprivation and apoptosis as target genes, such as hypoxia-associated factor (HAF), apoptosis-inducing factor (AIF), hypoxia-inducible factor 2 alpha (HIF2a), and factor inhibiting HIF (FIH). We measured the quantity of these markers using real-time quantitative PCR (qPCR), and Caspase-3 DNA and 18S were each used for normalization. The results showed that in the heart tissue, the degradation of HIF2a, AIF, and FIH was correlated with PMI, as was the degradation of HIF2a, FIH, and AIF in brain tissue when normalized with Caspase-3 DNA. However, when normalized with 18S, only the degradation of HIF2a in brain tissue was correlated with PMI. Interestingly, the quantity of HAF in brain tissue was found to increase after death with either 18S or Caspase-3 DNA normalization, and it was significantly correlated with 0–48 h PMI. These results indicated that mRNA quantity can be used to determine PMI and that Caspase-3 DNA is feasible for PMI estimation. In summary, we established mathematical models for PMI determination using multiple mRNA markers and multiple tissues and further studies are needed to validate and investigate these markers and mathematical models in human tissues. Duo Peng and Meili Lv contributed equally to this work. [ABSTRACT FROM AUTHOR]

Post-mortem interval (PMI) determination is one of the most important issues in forensic sciences. In the past, forensic scientists provided different approaches (physical, chemical, and entomological) for the estimation of PMI without success. However, advances in molecular biology over the last two decades have allowed us to assess the time-dependent degradation of biological markers (e.g., proteins, DNA, and RNA). Thus, the aim of the manuscript is to provide a review of the recent progress in the estimation of PMI using molecular biology methods, mainly focusing on the potential usefulness of RNA markers. To this end, 29 studies have been systematically reviewed, each one chosen according to specific inclusion/exclusion criteria. The selected studies evaluated the contribution of endogenous reference genes in different biological samples in order to determine the PMI based on post-mortem RNA degradation as a function of other influencing factors such as time, cause of death, and environmental conditions. [ABSTRACT FROM AUTHOR]

Baseline data on the insect successional patterns on carcasses can be a valuable estimation tool in the investigations of suspicious deaths, particularly when the post-mortem interval is longer than months or years. However, although carrion insect succession is a recurrent topic in forensic science research, the duration of the published studies is typically shorter than 1 year, with only one published study from central Europe documenting successional patterns beyond the first year of decomposition. We provide here the first data on the long-term insect successional patterns in southern Europe, using pig carcasses exposed in the four seasons of the year and documenting the carrion entomofauna during the second and third year of decomposition. Our results confirmed previous observations from central Europe that several Coleoptera species are able to recolonize cadavers during the second and third year after death, with their larvae residing on the remains for long periods. The season of cadaver exposure appears to be a main factor determining the composition of the insect fauna that exploits the remains during subsequent years. Our results suggest that it might be possible to estimate the year and season of death analyzing the composition of the insect fauna occurring on a cadaver. The present data highlight the need for further studies on the long-term insect succession on cadavers in different seasons and geographical areas, in order to fully understand its patterns and ensure its proper application in PMI estimations. [ABSTRACT FROM AUTHOR]

In forensic entomology, evaluation of a possible delay between a person's death and insect colonization is crucial. We monitored the seasonal flight activities of the most abundant blow flies in an urban habitat in Frankfurt/Germany based on 152 sampling days between April and October 2017. Thirty-six thousand female specimens of 12 necrophagous taxa were sampled as a possible groundwork for establishing a prediction tool for the activity of certain forensically relevant taxa. The most abundant taxon was Lucilia sericata (n = 19,544), followed by Lucilia caesar (n = 8025), Calliphora vicina (n = 5224), and Lucilia ampullacea (n = 1834). Up to six environmental parameters were statistically significant predictors of fly presence, leading to unique patterns of seasonal and daily activity for all four species. In detail, our analysis proved that L. sericata is a sun-loving, high-summer species that dominates the warmer months and is mostly influenced by mean day temperature. In contrast, L. caesar seems to be a shade-loving species that dominates in autumn resp. late-season and is mainly influenced by mean day temperature and wind speed. The activity of L. ampullacea was highly related to mean day temperature and relative humidity. In contrast to all other species, C. vicina behaved differently, particularly due to its occurrence throughout the entire sampling interval and the higher tolerance limits for the measured abiotic parameters, especially temperature. The present study is groundwork for establishing a prediction tool for the flight and oviposition activity of forensically relevant taxa. [ABSTRACT FROM AUTHOR]

Intraguild interactions play a prominent role in sarcosaprophagous communities, so intraspecific and interspecific competition phenomena between fly larvae take place. Synthesiomyia nudiseta is a species that has recently been recorded on human corpses in Europe, and it has been cited in forensic cases in Spain and Italy. The mature larvae are known to be facultative predators on necrophagous fly larvae, but their role as predators is unknown. The aim of this paper is to investigate the predatory behavior of S. nudiseta on the most abundant blowfly larvae in sarcosaprophagous communities in southwestern Europe (i.e., Chrysomya albiceps, Lucilia sericata, and Calliphora vicina). Intraspecific and interspecific competition experiments were carried out to study the effect of this species in the necrophagous diptera community. First-instar larvae were placed in plastic cups with the same amount of pig liver (15 g) at different densities (50, 100, 150, and 300 individuals/cup). Mortality in blowflies increased when S. nudiseta larvae were present: up to 98.40% in Ch. albiceps, 89.60% in L. sericata, and 84.93% in C. vicina. Pre-imaginal developmental time and adult wing size were analyzed and compared under each of the intraspecific and interspecific conditions; both variables were affected by competition in all cases. The implications of the presence of S. nudiseta for sarcosaprophagous community dynamics and its effect on the estimation of postmortem interval (PMI) are discussed. [ABSTRACT FROM AUTHOR]

Determination of insect species and their instars, occurring on human remains, is important information that allows us to use insects for estimation of postmortem interval and detect possible manipulation with the body. However, larvae of many common species can be identified only by molecular methods, which is not always possible. The instar determination is even more challenging, and qualitative characters that would allow a more precise identification are mostly unknown. Thanatophilus rugosus (Linnaeus, 1758) is a common necrophagous beetle in the whole Palaearctic region from Europe to Japan. The species is often encountered on corpses of large vertebrates including humans, and its potential to become a useful bioindicator for forensic entomology is therefore high. Adults can be easily distinguished from other species; however, larvae were never thoroughly described to allow species and instar identification. The aim of this study was to provide reliable morphological characters that would allow species and instar identification of T. rugosus larvae. The material for morphological study was obtained from rearing under controlled conditions (20 °C and 12:12 h of light/dark period), and specimens that were not studied morphologically were allowed to complete their development. Quantitative and qualitative morphological characters for instar and species identification are described and illustrated. Additionally, we report observations of biology and developmental length for all stages of the species. [ABSTRACT FROM AUTHOR]

Estimation of the post-mortem interval (PMI) of unknown skeletal remains is a common forensic task. Boaks and colleagues demonstrated a new method for PMI estimation in showing a reduction of the collagen to non-collagen content (Co/NCo ratio) in porcine bones after a PMI of 12 months using the Sirius Red/Fast Green Collagen Staining Kit from Chondrex in 2014 (Boaks et al. Forensic Sci Int 240: 104-110, 2014). The aim of our study was to reproduce this method and to investigate if the method could be used for forensic issues. Sixteen fresh porcine bones were placed in prepared boxes where they were treated regularly with distilled water or with water from hay infusions. For determining the Co/NCo ratio, we used the Sirius Red/Fast Green Collagen Staining Kit from Chondrex, which stains collagenous (Co) proteins red and non-collagenous (NCo) proteins green Chondrex Inc. (2008). After a PMI of 1-3 months, an analysis of porcine bone thin sections was performed on the one hand with spectrophotometry, on the other hand with stereomicroscopy. Using spectrophotometry, we go low and partially negative Co/NCo ratios which were up to 100-fold lower than the results we expected to get. The data we got by stereomicroscopy and calculating the Co/NCo ratio from extracting the red and green content with the software MATLAB and so calculating the Co/NCo ratio showed a correlation between PMI and the Co/NCo ratio in the porcine bone samples. Regular addition of distilled water or water from a hay infusion did not produce any significant differences so that an increased presence of microorganisms had obviously no influence on collagen degradation. [ABSTRACT FROM AUTHOR]

Differences in size between males and females, called the sexual size dimorphism, are common in insects. These differences may be followed by differences in the duration of development. Accordingly, it is believed that insect sex may be used to increase the accuracy of insect age estimates in forensic entomology. Here, the sex-specific differences in the development of Creophilus maxillosus were studied at seven constant temperatures. We have also created separate developmental models for males and females of C. maxillosus and tested them in a validation study to answer a question whether sex-specific developmental models improve the accuracy of insect age estimates. Results demonstrate that males of C. maxillosus developed significantly longer than females. The sex-specific and general models for the total immature development had the same optimal temperature range and similar developmental threshold but different thermal constant K, which was the largest in the case of the male-specific model and the smallest in the case of the female-specific model. Despite these differences, validation study revealed just minimal and statistically insignificant differences in the accuracy of age estimates using sex-specific and general thermal summation models. This finding indicates that in spite of statistically significant differences in the duration of immature development between females and males of C. maxillosus, there is no increase in the accuracy of insect age estimates while using the sex-specific thermal summation models compared to the general model. Accordingly, this study does not support the use of sex-specific developmental data for the estimation of insect age in forensic entomology. [ABSTRACT FROM AUTHOR]

Necrophagous blowflies can provide an excellent source of evidence for forensic entomologists and are also relevant to problems in public health, medicine, and animal health. However, access to useful information about these blowflies is constrained by the need to correctly identify the flies, and the poor availability of reliable, accessible identification tools is a serious obstacle to the development of forensic entomology in the majority of African countries. In response to this need, a high-quality key to the adults of all species of forensically relevant blowflies of Africa has been prepared, drawing on high-quality entomological materials and modern focus-stacking photomicroscopy. This new key can be easily applied by investigators inexperienced in the taxonomy of blowflies and is made available through a highly accessible online platform. Problematic diagnostic characters used in previous keys are discussed. [ABSTRACT FROM AUTHOR]

The rate of decomposition and insect succession onto decomposing pig carcasses were investigated following burning of carcasses. Ten pig carcasses (40-45 kg) were exposed to insect activity during autumn (March-April) in Western Australia. Five replicates were burnt to a degree described by the Crow-Glassman Scale (CGS) level #2, while five carcasses were left unburnt as controls. Burning carcasses greatly accelerated decomposition in contrast to unburnt carcasses. Physical modifications following burning such as skin discolouration, splitting of abdominal tissue and leathery consolidation of skin eliminated evidence of bloat and altered microambient temperatures associated with carcasses throughout decomposition. Insect species identified on carcasses were consistent between treatment groups; however, a statistically significant difference in insect succession onto remains was evident between treatments (PERMANOVA F = 14.23, p < 0.01) during an 8-day period that corresponds with the wet stage of decomposition. Differences were noted in the arrival time of late colonisers (Coleoptera) and the development of colonising insects between treatment groups. Differences in the duration of decomposition stages and insect assemblages indicate that burning has an effect on both rate of decomposition and insect succession. The findings presented here provide baseline data for entomological casework involving burnt remains criminal investigations. [ABSTRACT FROM AUTHOR]

Over the last decades, studies on juvenile development of forensically important Diptera were performed by using non-human tissues (e.g., beef liver) as a nutrition medium. Such developmental data are used as a reference in determining the age of juvenile necrophagous insects sampled from a human body and thereby to estimate the minimum postmortem interval (PMI). Despite the acceptance of these studies in the forensic community, some might ask whether such data appropriately reflect the growth of blow flies on human tissue. We, therefore, studied larval growth rates and development times of Calliphora vicina (Diptera: Calliphoridae) on human muscle tissue as well as on pork loin, pork liver, and minced pork at 25 °C. Larval growth rates were significantly ( p < 0.001) slower on pork loin and pork liver compared to human muscle tissue. Nonetheless, the time at which the examined developmental landmarks 'post-feeding larvae,' 'pupae,' and 'adult fly' were reached was similar for all tissues, with significant delays in first fly eclosion only for specimens reared on pork loin ( p = 0.027) and pork liver ( p = 0.036). Our results highlight the fact that not all porcine tissues are similarly suitable for producing sound growth data for necrophagous Diptera. At present, we recommend the use of minced pork as a non-human nutrition medium, since our results show no developmental differences on this diet compared to human tissue. [ABSTRACT FROM AUTHOR]

There are many qualitative indicators for postmortem interval (PMI) of human or animal cadavers. When such indicators are uniformly spaced over PMI, the resultant distribution may be very useful for the estimation of PMI. Existing methods of estimation rely on indicator persistence time that is, however, difficult to estimate because of its dependence on many interacting factors, of which forensic scientists are usually unaware in casework. In this article, an approach is developed for the estimation of PMI from qualitative markers in which indicator persistence time is not used. The method involves the estimation of an interval preceding appearance of a marker on cadaver called the pre-appearance interval (PAI). PMI is delineated by PAI for two consecutive markers: the one being recorded on the cadaver (defining lower PMI) and the other that is next along the PMI timeline but yet absent on the cadaver (defining upper PMI). The approach was calibrated for use with subsequent life stages of carrion insects and tested using results of pig cadaver experiments. Results demonstrate that the presence and absence of the subsequent developmental stages of carrion insects, coupled with the estimation of their PAI, gives a reliable and easily accessible knowledge of PMI in a forensic context. [ABSTRACT FROM AUTHOR]

Awareness of postmortem degradation processes in a human body is fundamental to develop methods for forensic time since death estimation (TDE). Currently, applied approaches are all more or less limited to certain postmortem phases, or have restrictions on behalf of circumstances of death. Novel techniques, however, rarely exceed basic research phases due to various reasons. We report the first application of a novel method, based on decay of muscle proteins, in a recent case of murder-suicide, where other TDE methods failed to obtain data. We detected considerably different protein degradation profiles in both individuals involved and compared the data to our presently available database. We obtained statistical evidence for un-simultaneous death and therefore received valuable information to trace the progression of events based on protein degradation. Although we could not sensibly convert the data to respective times of death, this case highlights the potential for future application and elucidates the necessary further steps to develop a viable TDE method. [ABSTRACT FROM AUTHOR]

Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans. [ABSTRACT FROM AUTHOR]

In forensic entomology, the developmental duration and larval body length of sarcosaprophagous flies are presently the two major approaches to estimate minimal postmortem interval (PMImin). A full characterization of the developmental stages of Calliphoridae, a predominating species on corpses, is especially important for PMI estimation. Chrysomya nigripes Aubertin, which appears on the corpse mainly during the decay and the post-decay stages, is of significant value for PMImin estimation. However, there are limited detailed reports on the development of such species. Hence, this study was conducted to address the development pattern of C. nigripes, in order to provide the basic data related to PMI estimation. In this study, the larvae of C. nigripes were collected from pig carcasses that were placed in the field. The colonies were cultured at constant temperatures of 16, 20, 24, 28, and 32 (±1) °C. The biological features of C. nigripes, the developmental pattern, and the body length with time at different temperatures were studied. The results showed that the average developmental duration of the C. nigripes larvae at 20, 24, 28, and 32 (±1) °C were 608.0 ± 68.0 h, 327.0 ± 53.8 h, 254.0 ± 36.5 h, and 217.0 ± 28.0 h, respectively. There were two phases of body length increment with developmental time: growing phase and plateau phase. The maximal body length was 11-12 mm, and the relationship between body length and developmental time can be simulated using the following equation: L = a + bT + cT + dT. The data for developmental duration and larval body length at 16 °C were not obtained. In summary, this study comprehensively studied the developmental biology of C. nigripes, which has a significant value for estimating PMI from highly decayed carcasses. [ABSTRACT FROM AUTHOR]

Post-mortem interval (PMI) is the amount of time elapsed since the time of death. Over the years, many methods were developed to assess PMI, but their precision and time frame of applicability are often limited. Our present pilot study aimed to prove if RNA degradation of human dental pulp can be used for PMI estimation. RNA was isolated from the pulps of healthy wisdom teeth and premolars. RNA degradation was determined as RNA integrity number (RIN) with Agilent Bioanalyzer and subsequently by amplification of different length products by PCR after reverse transcription. The RNA integrity analysis allowed us to determine the time of post-mortem interval with high confidence level in the first 21 days. With the PCR-based method, we were able to perform a crude estimation of incubation time of teeth between 20 and 42 days post extraction. These results show that this method might be a promising new tool for PMI estimation despite the limitations. [ABSTRACT FROM AUTHOR]

Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies. [ABSTRACT FROM AUTHOR]

Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), also known as the 'shore sexton beetle,' is a common silphid beetle that visits and breeds on large vertebrate cadavers. This study describes, for the first time, the involvement of N. littoralis on human corpses based on a large dataset of 154 French forensic cases. Various parameters regarding corpse location, decomposition stages, and entomofauna were extracted from each file. Compared to all of the forensic entomology cases analyzed between 1990 and 2013 (1028), N. littoralis was observed, on average, in one case out of eight; most of these cases occurred during spring and summer (73.5 %). More than 90 % of the cases were located outdoors, especially in woodlands, bushes, and fields. The decomposition stage of the corpse varied among cases, with more than 50 % in the advanced decomposition stage, 36 % in the early decomposition stage, and less than 10 % in the fresh, mummified, or skeletonized stages. Regarding other necrophagous species sampled with N. littoralis, Calliphorid flies were found in 94 % of the cases and Fanniidae/Muscidae in 65 % of the cases. Chrysomya albiceps, a heliophilic species mostly located in the Mediterranean area, was present in 34 % of the cases (only 20 % in the whole dataset). The most common coleopteran species were Necrobia spp. (Coleoptera: Cleridae) and Creophilus maxillosus (Coleoptera: Staphylinidae); these beetles were observed in 27 % of the cases. The over-representation of these species is likely due to similar requirements regarding the climate and decomposition stage. As N. littoralis is frequently observed and tends to become more common, we conclude that the developmental data for this species would be a precious tool for forensic entomologists in Europe. [ABSTRACT FROM AUTHOR]

The length or stage of development of blow fly (Diptera: Calliphoridae) larvae may be used to estimate a minimum postmortem interval, often by targeting the largest individuals of a species in the belief that they will be the oldest. However, natural variation in rate of development, and therefore length, implies that the size of the largest larva, as well as the number of larvae longer than any stated length, will be greater for larger cohorts. Length data from the blow flies Protophormia terraenovae and Lucilia sericata were collected from one field-based and two laboratory-based experiments. The field cohorts contained considerably more individuals than have been used for reference data collection in the literature. Cohorts were shown to have an approximately normal distribution. Summary statistics were derived from the collected data allowing the quantification of errors in development time which arise when different sized cohorts are compared through their largest larvae. These errors may be considerable and can lead to overestimation of postmortem intervals when making comparisons with reference data collected from smaller cohorts. This source of error has hitherto been overlooked in forensic entomology. [ABSTRACT FROM AUTHOR]

Carcass mass largely affects pattern and rate of carrion decomposition. Supposedly, it is similarly important for carrion entomofauna; however, most of its likely effects have not been tested experimentally. Here, simultaneous effects of carcass mass and clothing are analyzed. A factorial block experiment with four levels of carcass mass (small carcasses 5-15 kg, medium carcasses 15.1-30 kg, medium/large carcasses 35-50 kg, large carcasses 55-70 kg) and two levels of carcass clothing (clothed and unclothed) was made in a grassland habitat of Western Poland. Pig carcasses ( N = 24) were grouped into spring, early summer, and late summer blocks. Insects were sampled manually and with pitfall traps. Results demonstrate that insect assemblages are more complex, abundant, and long-lasting on larger carcasses, whereas clothing is of minor importance in this respect. Only large or medium/large carcasses were colonized by all guilds of carrion insects, while small or medium carcasses revealed high underrepresentation of late-colonizing insects (e.g., Cleridae or Nitidulidae). This finding indicates that carcasses weighing about 23 kg-a standard in forensic decomposition studies-give an incomplete picture of carrion entomofauna. Residencies of all forensically relevant insects were distinctly prolonged on larger carcasses, indicating that cadaver mass is a factor of great importance in this respect. The pre-appearance interval of most taxa was found to be unrelated to mass or clothing of a carcass. Moreover, current results suggest that rate of larval development is higher on smaller carcasses. In conclusion, this study demonstrates that carcass mass is a factor of crucial importance for carrion entomofauna, whereas the importance of clothing is small. [ABSTRACT FROM AUTHOR]

Most studies of decomposition in forensic entomology and taphonomy have used non-human cadavers. Following the recommendation of using domestic pig cadavers as analogues for humans in forensic entomology in the 1980s, pigs became the most frequently used model cadavers in forensic sciences. They have shaped our understanding of how large vertebrate cadavers decompose in, for example, various environments, seasons and after various ante- or postmortem cadaver modifications. They have also been used to demonstrate the feasibility of several new or well-established forensic techniques. The advent of outdoor human taphonomy facilities enabled experimental comparisons of decomposition between pig and human cadavers. Recent comparisons challenged the pig-as-analogue claim in entomology and taphonomy research. In this review, we discuss in a broad methodological context the advantages and disadvantages of pig and human cadavers for forensic research and rebut the critique of pigs as analogues for humans. We conclude that experiments using human cadaver analogues (i.e. pig carcasses) are easier to replicate and more practical for controlling confounding factors than studies based solely on humans and, therefore, are likely to remain our primary epistemic source of forensic knowledge for the immediate future. We supplement these considerations with new guidelines for model cadaver choice in forensic science research.

The minimum postmortem interval (PMI) is commonly estimated using calliphorid larvae, for which there are established age estimation methods based on morphological and development data. Despite the increased duration and sedentary nature of the pupal stage of the blowfly, morphological age estimation methods are poorly documented and infrequently used for PMI determination. The aim of this study was to develop a timeline of metamorphosis, focusing on the development of external morphology (within the puparium), to provide a means of age and PMI estimation for Calliphora vicina (Rob-Desvoidy) pupae. Under controlled conditions, 1,494 pupae were reared and sampled at regular time intervals. After puparium removal, observations of 23 external metamorphic developments were correlated to age in accumulated degree hours (ADH). Two age estimation methods were developed based on (1) the combination of possible age ranges observed for each characteristic and (2) regression analyses to generate age estimation equations employing all 23 characteristics observed and a subset of ten characteristics most significantly correlated with age. Blind sample analysis indicated that, using the combination of both methods, pupal age could be estimated to within ±500 ADH with 95 % reliability. [ABSTRACT FROM AUTHOR]

Understanding the onset and duration of adult blow fly activity is critical to accurately estimating the period of insect activity or minimum postmortem interval (minPMI). Few, if any, reliable techniques have been developed and consequently validated for using adult fly activity to determine a minPMI. In this study, adult blow flies (Diptera: Calliphoridae) of Cochliomyia macellaria and Chrysomya rufifacies were collected from swine carcasses in rural central Texas, USA, during summer 2008 and Phormia regina and Calliphora vicina in the winter during 2009 and 2010. Carcass attendance patterns of blow flies were related to species, sex, and oocyte development. Summer-active flies were found to arrive 4-12 h after initial carcass exposure, with both C. macellaria and C. rufifacies arriving within 2 h of one another. Winter-active flies arrived within 48 h of one another. There was significant difference in degree of oocyte development on each of the first 3 days postmortem. These frequency differences allowed a minPMI to be calculated using a binomial analysis. When validated with seven tests using domestic and feral swine and human remains, the technique correctly estimated time of placement in six trials. [ABSTRACT FROM AUTHOR]

Flies are frequently used for postmortem interval (PMI) estimations. These estimates are usually based on the age of larval or pupal specimens. However, the age defines only the minimum PMI. In order to move forensic entomology further, a method useful for the estimation of an interval preceding insect appearance on a corpse called the pre-appearance interval (PAI) is needed. Recently, it was demonstrated that the PAI of several carrion beetles is closely related to the temperature prevailing throughout this interval. Hence, it was postulated to estimate PAI from temperature. In order to check premises for using this approach with flies, a test of the relationship between adult or oviposition PAI and temperature was made for nine species of European flies. Data on PAI originated from pig carcasses decomposing under various temperatures. Adult PAI of Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis, Phormia regina, and Stearibia nigriceps and oviposition PAI of S. nigriceps were exponentially related to temperature. Only S. nigriceps revealed a close relationship, demonstrating solid premises for PAI estimation from temperature alone. Adult and oviposition PAI of Calliphora vomitoria and adult PAI of Hydrotaea pilipes were not related to temperature. Adult and oviposition PAI of Lucilia sericata and Lucilia caesar responded similarly, with an abrupt and large increase in a narrow range of low temperatures and no response in a broad range of high temperatures. Probably, different mechanisms form the basis for the response of PAI to temperature in flies colonizing carcasses shortly after death and flies colonizing carcasses later in the decomposition process. [ABSTRACT FROM AUTHOR]

Forensic entomologists rely on published developmental datasets to estimate the age of insects developing on human remains. Currently, these datasets only represent populations of targeted insects from specific locations. However, recent data indicate that populations can exhibit genetic variation in their development, including signatures of local adaptation demonstrated by regionally distinct plastic responses to their environments. In this study, three geographically distinct populations of the secondary screwworm, Cochliomyia macellaria Fabricius (Diptera: Calliphoridae; College Station, Longview, and San Marcos, TX, USA), a common blow fly collected from human remains in the southern USA, were reared in two distinct environments (cool 21 °C, 65 % relative humidity (RH); and warm 31 °C, 70 % RH) over 2 years (2011 and 2012) in order to determine differences in development time and mass. Significant differences in immature and pupal development time, as well as pupal mass, were shown to exist among strains derived from different populations and years. For immature development times, there was evidence of only an environmental effect on phenotype, while genotype by environment interactions was observed in pupal development times and pupal mass. College Station and San Marcos populations exhibited faster pupal development and smaller pupal sizes in the cooler environment relative to the Longview population, but showed an opposite trend in the warm environment. Rank order for College Station and Longview populations was reversed across years. Failure to take genetic variation into consideration when making such estimates can lead to unanticipated error and bias. These results indicate that genetics will have little impact on error when working with Texas genotypes of C. macellaria at ~30 °C and 70 % RH, but will have a more meaningful impact on error in postmortem interval estimates with this species in cooler, drier environments. [ABSTRACT FROM AUTHOR]

The estimation of the minimum postmortem interval can be implemented by age estimation of corpse-associated primary colonizers such as the blow flies (Diptera: Calliphoridae). In cases where pupae represent the oldest stages found on a body, their age must be added to the duration of prepupal development to estimate the minimum postmortem interval. Although methods to age blow fly larvae have been well established using morphology, length or weight and age estimation of pupae has proved challenging. In a previous work, we quantified the changes in mRNA levels of four differentially expressed genes during the metamorphosis of Calliphora vicina pupae, hence representing molecular markers for pupal age (i.e., time elapsed since pupariation). Here, we demonstrate how these data can be used to estimate pupal age with inverse prediction. We present three blind studies conducted under various conditions and show that age of C. vicina pupae can be well estimated based on gene expression data. As these data are quantitative and can be processed automatically, gene expression has the potential to outperform morphological analysis in age estimation of forensically relevant blow fly pupae. [ABSTRACT FROM AUTHOR]

We describe here a case of multiple colonization of a male cadaver found indoors in the municipality of Jaboatao dos Guararapes, Brazil. The body was colonized by six species of Diptera: Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria (Calliphoridae), Megaselia scalaris (Phoridae), Fannia trimaculata (Fanniidae), and Peckia (Peckia) chrysostoma (Sarcophagidae). The most abundant species were C. albiceps (65.0 % of all emerged adults) and C. megacephala (18.6 %). The case illustrates the ability of six insect species to simultaneously colonize a corpse in an indoor environment and represents the first collaboration between the forensic police and entomologists in Northeastern Brazil. We provide here the first record of two species, F. trimaculata and Peckia (P.) chrysostoma colonizing a human cadaver. We also report the first case of cadaver colonization by C. putoria and M. scalaris in Northeastern Brazil. Information on the development time of two species, C. albiceps and C. megacephala, were used to discuss the estimation of the post-mortem interval. Considering that the region harbors the highest rates of homicide in Brazil, implications of these findings for the consolidation of forensic entomology in the region are discussed. [ABSTRACT FROM AUTHOR]

Unequivocal identification of insect specimens is an essential requirement in forensic entomology. With the development of molecular identification, spate of discussions about the feature of the DNA fragments have been raised. Relying solely on single DNA fragment for delimiting closely related species is supposed to be dangerous. Aiming at obtaining more reliable markers that might be universally used, we explore the utility of 700-bp COI fragment and 678-bp period gene fragment in the identification of Sarcophagidae (Diptera). Thirty-six sarcophagid fly specimens were collected from 19 locations in 11 Chinese provinces. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into nine species with relatively strong supporting values, which indicated the possibility of separation congeneric species with COI and period gene fragments. The difference between intraspecific threshold and interspecific divergence confirmed that the combination of nuclear and mitochondrial genes for species identification is much more accurate. The results of this research will be instrumental for implementation of the Chinese Sarcophagidae database. [ABSTRACT FROM AUTHOR]

The identification of species of the forensically important genus Sarcophaga is very difficult and requires strong taxonomic expertise. In this study, we sequenced the mitochondrial cytochrome c oxidase subunit I ( COI) gene of 126 specimens of 56 W European Sarcophaga species and added GenBank data to our database to yield a total dataset of 270 COI sequences from 99 Sarcophaga species to evaluate the COI gene as a molecular diagnostic tool for species identification in this genus. Using two simple criteria (Best Match, BM and Best Close Match, BCM), we showed that the identification success using a mini-barcode region of 127 bp was very low (80.7-82.5 %) and the use of this region is not recommended as a species identifier. In contrast, identification success was very high using the standard barcode region (658 bp) or using the entire COI region (1,535 bp) (98.2-99.3 %). Yet, there was a low interspecific sequence divergence (<2 %) in six species groups so that for 16 out of the 99 species (nine of which are of forensic importance), the use of COI barcodes as species identifier should be done with care. For these species, additional markers will be necessary to achieve a 100 % identification success. We further illustrate how such reference databases can improve local reference databases for forensic entomologists. [ABSTRACT FROM AUTHOR]

Developmental data of juvenile blow flies (Diptera: Calliphoridae) are typically used to calculate the age of immature stages found on or around a corpse and thus to estimate a minimum post-mortem interval (PMI). However, many of those data sets don't take into account that immature blow flies grow in a non-linear fashion. Linear models do not supply a sufficient reliability on age estimates and may even lead to an erroneous determination of the PMI. According to the Daubert standard and the need for improvements in forensic science, new statistic tools like smoothing methods and mixed models allow the modelling of non-linear relationships and expand the field of statistical analyses. The present study introduces into the background and application of these statistical techniques by analysing a model which describes the development of the forensically important blow fly Calliphora vicina at different temperatures. The comparison of three statistical methods (linear regression, generalised additive modelling and generalised additive mixed modelling) clearly demonstrates that only the latter provided regression parameters that reflect the data adequately. We focus explicitly on both the exploration of the data-to assure their quality and to show the importance of checking it carefully prior to conducting the statistical tests-and the validation of the resulting models. Hence, we present a common method for evaluating and testing forensic entomological data sets by using for the first time generalised additive mixed models. [ABSTRACT FROM AUTHOR]

Necrophagous blow fly larvae can provide accurate estimates of the minimum postmortem interval in death investigations. During larval development, predictable morphological changes occur and measurements of weight, length, and width are compared to species-specific growth curves for reliable age estimates. However, aging blow fly pupae is more challenging because morphological and anatomical changes are not visible with the naked eye. Thus, delicate preparation of the pupae or rearing to the adult stage seems unavoidable. Conversely, metamorphosis evokes a remodelling of the larval shape to adult structures, and gene expression analysis potentially serves as a molecular tool to mirror the ageing process of a pupa. The present study focuses on the differential expression of two newly described, arbitrarily named genes ( 15_2, 2014192) and two previously identified genes ( actin, arylphorin receptor) during Calliphora vicina (Diptera: Calliphoridae) metamorphosis. Quantification through real-time PCR revealed significant up- and downregulation of these transcripts found to be temperature dependent and age specific, hence, a new possibility to age forensically important blow fly pupae. [ABSTRACT FROM AUTHOR]

The utility of the forensically important Sarcophagidae (Diptera) for time since death estimates has been severely limited, as morphological identification is difficult and thermobiological histories are inadequately documented. A molecular identification method involving the sequencing of a 658-bp 'barcode' fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 85 specimens, representing 16 Australian species from varying populations, was evaluated. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining phylogenetic tree generated. All species were resolved as reciprocally monophyletic, except Sarcophaga dux. Intraspecific and interspecific variation ranged from 0.000% to 1.499% (SE = 0.044%) and 6.658% to 8.983% (SE = 0.653%), respectively. The COI 'barcode' sequence was found to be suitable for the molecular identification of the studied Australian Sarcophagidae: 96.5% of the examined specimens were assigned to the correct species. Given that the sarcophagid fauna is poorly described, it is feasible that the few incorrectly assigned specimens represent cryptic species. The results of this research will be instrumental for implementation of the Australian Sarcophagidae in forensic entomology. [ABSTRACT FROM AUTHOR]

Entomological evidence is often used in forensic cases for post-mortem interval (PMI) calculation. The most dominant species present on a corpse are typically blowflies. However, several cases have been reported where access to a corpse has been restricted for blowflies (e.g., on a buried or wrapped cadavers) but species of the family Phoridae were abundant. It has also been reported that some phorid species that exploit human corpses may also feature in cases of myiasis acquired ante-mortem. In all these cases, they may provide decisive evidence. As for blowflies, the precise identification of a phorid species collected from a corpse is necessary when estimating the PMI. Since morphological determination is often hampered due to similar characteristics especially in the larval and pupal stage, we used DNA-based methods to identify six phorid species ( Megaselia scalaris, Megaselia giraudii, Megaselia abdita, Megaselia rufipes, Conicera tibialis, and Puliciphora borinquenensis) on the molecular level. We focused on a 658-bp-long region of the cytochrome oxidase I gene ( COI), the most common molecular marker in forensic entomology. The amplified fragment is also used in DNA barcode approaches and was found to be suitable for identification of a wide range of insect taxa. The present study demonstrates that this region is also sufficient to distinguish between several species of scuttle flies. [ABSTRACT FROM AUTHOR]