Accurate Postmortem Interval Research Articles

Multimodal Approaches Based on Microbial Data for Accurate Postmortem Interval Estimation

Accurate postmortem interval (PMI) estimation is critical for forensic investigations, aiding case classification and providing vital trial evidence. Early postmortem signs, such as body temperature and rigor mortis, are reliable for estimating PMI shortly after death. However, these indicators become less useful as decomposition progresses, making late-stage PMI estimation a significant challenge. Decomposition involves predictable microbial activity, which may serve as an objective criterion for PMI estimation. During decomposition, anaerobic microbes metabolize body tissues, producing gases and organic acids, leading to significant changes in skin and soil microbial communities. These shifts, especially the transition from anaerobic to aerobic microbiomes, can objectively segment decomposition into pre- and post-rupture stages according to rupture point. Microbial communities change markedly after death, with anaerobic bacteria dominating early stages and aerobic bacteria prevalent post-rupture. Different organs exhibit distinct microbial successions, providing valuable PMI insights. Alongside microbial changes, metabolic and volatile organic compound (VOC) profiles also shift, reflecting the body’s biochemical environment. Due to insufficient information, unimodal models could not comprehensively reflect the PMI, so a muti-modal model should be used to estimate the PMI. Machine learning (ML) offers promising methods for integrating these multimodal data sources, enabling more accurate PMI predictions. Despite challenges such as data quality and ethical considerations, developing human-specific multimodal databases and exploring microbial–insect interactions can significantly enhance PMI estimation accuracy, advancing forensic science.

Age estimation of Phormia regina pupae based on ATR-FTIR and chemometrics

Accurate postmortem interval estimation is vital in the investigation of homicides, suicides, and accidental deaths. It is key in narrowing suspect lists, improving crime-solving efficiency, and offering solace to bereaved families. The intra-puparial period, comprising about half of a fly’s developmental cycle, presents challenges for morphological age estimation. External changes are limited to color shifts and the appearance of respiratory horns on the puparium only within several hours after pupariation, while detailed internal development analysis often requires invasive methods like removing the puparium, which can be damaging. Additionally, these techniques usually depend on a forensic entomologist’s expertise, which lead to subjective biases. This study employed attenuated total reflection-fourier transform infrared spectroscopy, a rapid, non-destructive method for analyzing proteins, chitosan, and chitin in puparia. Data showed a consistent reduction in the concentration of the amide I band within the puparium during the intra-puparial development at five constant temperatures (19 °C, 22 °C, 25 °C, 28 °C and 31 °C). This trend in the spectral data effectively distinguishes pupae at various stages of intra-puparial development, facilitating precise age estimation, which is critical for the estimation of the minimum postmortem interval (PMImin). Finally, this work combined the total reflection-fourier transform infrared spectroscopy with chemometric analysis and successfully developed a partial least squares discriminant analysis model and a random forest model, with accuracies of 88 % and 81 %, respectively. These models enable the non-invasive age estimation of P. regina in its intra-puparial period, a stage traditionally difficult to assess morphologically, thus laying the groundwork for PMImin estimation using fly pupae.

Evaluation of the effect of clonazepam and its metabolites on the life cycle of Megaselia scalaris (Loew) (Diptera: Phoridae)

Fly colonization patterns and development are crucial in estimating the post-mortem interval (PMI) of decomposing corpses. Understanding the potential effects of xenobiotics on species development in cadaveric entomofauna is essential for accurate PMI estimation, given their presence in decomposing bodies. Benzodiazepines, commonly prescribed for their anxiolytic, hypnotic, and muscle relaxant effects, are of forensic interest due to their potential for abuse, dependence, intoxication, and overdose-related deaths. This study aimed to explore the effect of clonazepam and its metabolites on Megaselia scalaris, a species commonly used to estimate PMI, the alteration of which could impact the accuracy of said estimation. The S9 biotransformation fraction, an in vitro model consisting of an array of metabolic enzymes, was used to generate phase I and II metabolites for evaluating their effect on M. scalaris development, representing an innovative approach to this type of study. Megaselia scalaris larvae were reared in synthetic growth media under controlled conditions. The study compared different groups: control, clonazepam, and clonazepam with S9 fraction. Larvae were measured daily to determine growth rate, and clonazepam concentrations were analyzed using HPLC-DAD. Results showed that larvae grown in media containing clonazepam or clonazepam with S9 fraction developed faster than control larvae, reaching their pupal stage earlier. Growth rates were also altered in treated groups. In conclusion, the presence of clonazepam and its metabolites accelerated the life cycle of M. scalaris, potentially impacting the accuracy of PMI estimation. These findings underscore the importance of considering xenobiotics in forensic entomological studies for precise post-mortem interval determination.

The effects of insect repellent on initial Dipteran colonization of decomposing pig remains.

Numerous factors influence insect colonization and development on decomposing remains and can influence postmortem interval (PMI) estimations when insects are used in investigations of suspicious deaths. This study aimed to determine whether insect repellent influences insect colonization of decomposing remains. Pig feet were divided into 4 groups: a control not sprayed with repellent, 40% DEET repellent, 98% DEET repellent, and lemon eucalyptus repellent. The feet were placed in containers, and insect visitation was observed over the course of 3 days, then insects were collected at the end of the third day. This was repeated twice a month from April through November 2022. The analysis focused on the order Diptera. Pairwise comparison determined that there was a significant delay in visitation time for remains with 98% DEET compared to remains with no repellent and 40% DEET. There were significantly fewer adult Diptera collected and more remains with no larvae or eggs present among remains treated with 98% DEET. It was concluded that low concentrations of DEET and lemon eucalyptus repellent did not delay colonization, but that high concentrations of DEET delayed visitation, reduced the number of adult flies collected from the remains, and reduced the likelihood of collecting eggs or larvae from the remains. This contributes to the understanding of factors that can influence insect colonization and development that allow for more accurate PMI estimations by suggesting that insect repellent does not need to be accounted for in PMI estimation, but a high concentration of DEET can deter insects in the short term.

Recognition of the Presence of Bone Fractures Through Physicochemical Changes in Diagenetic Bone.

Much research has focused on attempting to understand the drivers of bone diagenesis. However, this sensitive process is easily influenced by various factors, particularly the condition of the remains (i.e., whether they have been subjected to trauma). Previous research demonstrates that trauma can influence soft tissue decomposition, yet to date, no studies have looked at how bone fractures could affect bone diagenesis. To address this gap, two short timescale studies were conducted to investigate the influence of bone fractures on the physicochemical composition of disarticulated, partially fleshed animal remains. Disarticulated porcine bones were either fractured using blunt force or sharp force whilst fresh (producing perimortem damage), at 60 days producing postmortem damage (postmortem interval (PMI)), or left intact and left outside for up to 180 days post-fracture/240 days PMI. Retrieved bone sections were then analyzed for physicochemical differences using non-destructive methods, i.e., scanning electron microscopy energy dispersive spectroscopy and Fourier transform infrared spectroscopy with attenuated total reflectance. It was hypothesized that differences would be found in the physicochemical composition between the bones with fractures and those without after undergoing diagenetic change. The bone fractures significantly affected the elemental composition of bone over time, but structural composition initially remained stable. It was also possible to distinguish between perimortem and postmortem fractures using these two analytical techniques due to physicochemical differences. This research shows bone fractures can significantly alter the physicochemical composition of the bone during the postmortem period and have the potential to facilitate more accurate PMI estimations in forensic contexts.

Evaluating the Time of Death (Post Mortem Interval, PMI) through Forensic Entomo-toxicological Investigations of Maggots and Flies

The estimation of the post mortem interval (PMI) is a critical component of forensic science, aiding investigations and resolving missing persons cases. This abstract explores the innovative approach of forensic entomotoxicological investigations, focusing on the analysis of maggots and flies in PMI estimation. Maggots and flies are key players in the decomposition process, and their lifecycles are closely linked to the stages of decay. Challenges and limitations, such as environmental factors and variable drug metabolism, are considered. Two compelling case studies exemplify the practical applications of forensic entomotoxicology in solving criminal cases and missing persons investigations.
 The primary goal of this paper is to provide guidance to knowledgeable researchers and investigators in the realm of forensic entomotoxicology. It underscores the need for a heightened focus on forensic Entomotoxicology, particularly in investigations involving the effects of drugs on the growth stages of insect development, for more accurate postmortem interval estimations, especially in cases involving decomposed remains. In conclusion, forensic entomotoxicological investigations of maggots and flies present a dynamic and invaluable approach to PMI estimation, promising to enhance the accuracy and reliability of forensic science in determining the timing of death.

Nomogram based Postmortem-interval estimation compared with the Actual death-interval in Tropical environmental conditions

BackgroundEstimating an accurate postmortem interval (PMI) is essential for death investigators to confirm the offence timing and suspects' alibis. Compared to traditional methods, the Nomogram-based method is claimed to estimate nearly accurate PMI, but most of such studies are based on the Western population. Adequate data for bodies exposed to tropical climate of India and the Indian subcontinent are not available. AimTo assess the accuracy and goodness-of-fit of Nomogram based PMI estimation in bodies exposed to Indian climatic conditions after death. Material and methodsThis is a 3-year-long study on 200 bodies with known death times. The exact PMI was recorded from direct sources: family members, police and hospital records. Before autopsy, the ambient temperature, body weight, length, and rectal temperature were measured, and the details of clothing, sex, and age, were used on a nomogram to calculate the PMI (tN). One-way ANOVA correlation and Mann-Whitney U test were used to compare the variables. Linear regression analysis was used to establish the relationship between the rectal temperature (Tr) and nomogram estimated PMI (tN) and the known PMI(t). The consistency and agreement between the tN and t were measured using Intra-class correlation coefficient (ICC). Bland-Altman (BA) plot was used to compare the level of agreement between direct(t) and nomograms method PMIs. ResultsNomogram-based PMI estimation showed a statistically significant strong relationship with rectal temperature (R 0.77, R2 0.74, adjusted R2 0.76, t-stat 25.83 p < 0.000) and exhibited a relatively consistent agreement with the known death interval (ICC 0.935). Regressing the tN over t showed that the nomogram method can predict PMI with 95 % accuracy. The BA plot between Direct and Nomogram methods exhibited a consistent agreement in PMI estimation though the limits of agreement (LoA) were wide: range 39.09–121.18 min. ConclusionThe accuracy and reliability of the Nomogram method in PMI estimation is high and recommended for the South Indian population. However, the presence of systematic differences between tN and t can't be ruled out due to wider LoA in BA plot. Hence, these findings highlight the need for further investigation and potential refinement of the PMI estimation methods to enhance accuracy and reduce discrepancies.

Expression of human cardiac-specific genes: a novel method for post-mortem interval estimation.

In legal medicine, the determination of post-mortem interval (PMI) is not only an important but also one of the most difficult aspects. Several methods are used to estimate PMI such as physicochemical, entomological, biochemical, metabolic, autolytic, and physical methods. These methods provide a wide range of PMI as they are affected by different factors. The approach behind the present study is to calculate an accurate PMI by using mRNA degradation and fold change expression (FCE) of cardiac-specific genes viz. N-terminal pro-B-type natriuretic peptide (NPPB) and cardiac troponin I (TNNI3). Seventeen cadaver heart tissues were analysed within a time frame of up to 12 hours from the time since death, at different time intervals at room temperature. Gene expression was determined and the data were analysed using the value of average delta Ct (ΔCt) value of the assessed gene and housekeeping gene. Delta delta Ct (ΔΔCt) method was used to calculate the FCE at the different 7-time groups. The FCE of TNNI3 was almost stable till 15 hours of PMI and then after 15 hours, expression shows a decrease up to 24 hours after death; whereas, NPPB shows that FCE was stable till 12 hours of PMI and then after 12 hours, expression shows a decrease up to 24 hours after death. The FCE of NPPB and TNNI3 was almost stable till 12 hours. Thus, the estimation of PMI by analysis of the FCE of cardiac-specific genes can be a new promising method in forensic medicine.

Proteome alterations in human autopsy tissues in relation to time after death

Protein expression is a primary area of interest for routine histological diagnostics and tissue-based research projects, but the limitations of its post-mortem applicability remain largely unclear. On the other hand, tissue specimens obtained during autopsies can provide unique insight into advanced disease states, especially in cancer research. Therefore, we aimed to identify the maximum post-mortem interval (PMI) which is still suitable for characterizing protein expression patterns, to explore organ-specific differences in protein degradation, and to investigate whether certain proteins follow specific degradation kinetics. Therefore, the proteome of human tissue samples obtained during routine autopsies of deceased patients with accurate PMI (6, 12, 18, 24, 48, 72, 96 h) and without specific diseases that significantly affect tissue preservation, from lungs, kidneys and livers, was analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the kidney and liver, significant protein degradation became apparent at 48 h. For the lung, the proteome composition was rather static for up to 48 h and substantial protein degradation was detected only at 72 h suggesting that degradation kinetics appear to be organ specific. More detailed analyses suggested that proteins with similar post-mortem kinetics are not primarily shared in their biological functions. The overrepresentation of protein families with analogous structural motifs in the kidney indicates that structural features may be a common factor in determining similar postmortem stability. Our study demonstrates that a longer post-mortem period may have a significant impact on proteome composition, but sampling within 24 h may be appropriate, as degradation is within acceptable limits even in organs with faster autolysis.

New Trends in Immunohistochemical Methods to Estimate the Time since Death: A Review.

The identification of a reliable and accurate post-mortem interval (PMI) is a major challenge in the field of forensic sciences and criminal investigation. Several laboratory techniques have recently been developed that offer a better contribution to the estimation of PMI, in addition to the traditional physical or physico-chemical (body cooling, lividity, radiocarbon dating, rigor mortis), chemical (autolysis), microbiological (putrefaction), entomological, as well as botanical parameters. Molecular biology (degradation pattern of macromolecules such as proteins, DNA, RNA), biochemical analysis of biological fluids (such as blood, cerebrospinal fluid, and vitreous humor), and immunohistochemistry are some of the most recent technological innovations. A systematic review of the literature was performed with the aim of presenting an up-to-date overview on the correlation between the immunohistochemical (IHC) expression of specific antigenic markers at different PMIs. The systematic review was performed according to PRISMA guidelines. Scopus and PubMed were used as search engines from January 1, 1998 to March 1, 2022 to evaluate the effectiveness of immunohistochemistry in estimating PMI. The following keywords were used: (immunohistochemical) OR (immunohistochemistry) AND (time since death) OR (post-mortem interval) OR (PMI). A total of 6571 articles were collected. Ultimately, 16 studies were included in this review. The results of this systematic review highlighted that IHC techniques, in association with traditional methods, add, in Bayesian terms, additional information to define a more accurate time of death and PMI. However, current IHC results are numerically limited and more data and studies are desirable in the near future.

Prevalence of mouthpart sensilla and protease producing symbiotic gut bacteria in the forensic fly Chrysomya megacephala (Fabricius, 1794): Insight from foraging to digestion

The blow fly, Chrysomya megacephala (Fabricius, 1794) is a globally prevalent forensically important species that helps to estimate accurate postmortem interval since the death. This fly occasionally causes cutaneous myiasis and transmits several pathogenic bacteria. To understand their ability of corpse detection and digestion of protein-rich meal, the present study describes the mouthpart sensilla and assessment of protease producing symbiotic gut bacteria. Scanning electron microscopy (SEM) showed the prevalence of trichoid sensilla (Tr), basiconic sensilla (Ba) and microtrichia (Mr) on labellar lobes, haustellum and maxillary palps of mouthparts. Bacterial particles of both rod (small and large) and spherical shaped were detected in the gut of C. megacephala using SEM. The bacterial density was higher on the foregut and midgut in comparison to the hindgut. From 72 bacterial isolates, 10 isolates from the foregut region showed considerable protease-producing efficacy ranging between 3.98 – 6.83 GHR and 9.73 – 34.68 U/ml protease. Among these, the most promising protease-producing bacterial isolate showed 16S rDNA sequence similarity (99.85%) with Chryseobacterium artocarpi DNA. This bacterium was the first report from flies. The findings of the study might help in better understanding of the role of sensilla in host perception and foregut symbiotic bacterial association in protein digestion in C. megacephala.

Decomposition variability between the scene and autopsy examination and implications for post-mortem interval estimations

Forensic experts rely on scene and/or autopsy photographs to estimate the post-mortem interval (PMI) when an in-situ assessment of decomposition is unfeasible. The degree of decomposition may vary between the scene and autopsy, which importantly could affect estimations of the unknown PMI in forensic casework. This study aimed to investigate decomposition variability between the scene and autopsy and assess the subsequent effect on the accuracy of PMI estimations. Scene and autopsy photographs from 94 cases with known PMI were used from the Allegheny County Office of the Medical Examiner in Pittsburgh, United States. The total decomposition scoring (TDS) method measured the overall decomposition level, and 28 markers of decomposition were recorded as a percentage of the total body surface area (TBSA). In 60% of cases the TDS had increased at autopsy causing significant overestimations of the autopsy PMI and 86% of decomposition markers varied between the scene and autopsy. Decomposition progressed during mortuary time lags (MTL) of 3–44 h, where bodies were stored in a pre-autopsy refrigerator at 4 °C, suggesting that refrigeration may not always delay decomposition. This research also assisted in validating photographs as a proxy for real-time decomposition assessments. While the autopsy photographs conferred higher quality than the scene photographs, the scene photographs produced more accurate PMI estimations. Forensic experts should exhibit caution when estimating the PMI from autopsy photographs alone, as they may not accurately reflect scene decomposition. To prevent misinterpretation of the PMI estimation, both scene and autopsy photographs should always be requested.

Calliphorids as forensic indicator to facilitate PMI estimation: A case study from Chhattisgarh, India

ObjectivesCalliphorids especially blowflies are act as important forensic indicators as they are foremost visitors of the corpse and act as silent witness after the accomplishment of crime. MethodsIn present case study immature stages of blowflies utilized for PMI estimation. Postmortem Interval (PMI) of a highly putrefied male corpse in advanced stage of decomposition was estimated. Corpse was found under the culvert of Bijapur – Bhopalpatnam road, Village Modakpal, District Bijapur, State Chhattisgarh, India. PMI was determined with the help of Accumulated Degree Hours (ADH) method based on developmental period of third instar larvae of Blowfly Chrysomya megacephala (Fabricius). Tentative PMI of corpse is 5.5 days. Apart from Chrysomya megacephala immature stages as well as adults of Chrysomya rufifaces (Macquart) were also collected from deceased body. Entomological samples were sent for identification to the department of Zoology and Environmental Sciences, Punjabi University, Patiala, India. Results and conclusionsLarval stages were identified based on anterior and posterior spiracles and cephalophrangeal apparatus. Adults were identified based on their morphological characters. For accurate PMI estimation correct insect identification and meteorological data of crime scene are key parameters.

Research Progress on Developmental Biology of Sarcosaprophagous Insects.

Forensic entomology provides a feasible way to estimate postmortem interval (PMI), of which the growth and development of sarcosaprophagous insects is the most widely used indicator in forensic practice. Over the years, forensic entomologists have carried out a large number of studies on the development biology of sarcosaprophagous insects. This paper illustrates the main factors that affect the development of sarcosaprophagous insects, including temperature, humidity, light, food types and poisons. The development indicators of sarcosaprophagous insects were reviewed from the perspectives of morphology, differential gene expression and biochemical characteristics. It is emphasized that future research of development biology on sarcosaprophagous insects should fully absorb and integrate the methods of artificial intelligence and omics, and the research object also needs further expansion in order to establish a more objective and more accurate PMI estimation method.

Molecular Aspect of Post-mortem interval (PMI)

Post-mortem interval (PMI) is a major component of investigation in forensic science. It is one of the fundamental tasks for forensic pathologist when a body is found. According to criminal law point of view, a correct estimation of the PMI allows authentication of witness statements, thus restrict the number of suspects. Accurate PMI estimation is difficult because of intrinsic and extrinsic factors. Analysis of the biochemical changes in glucose and electrolytes for PMI estimation has made significant progress. Molecular ways in which measurable or quantifiable technique for degradation of nucleic acid molecule such as DNA and RNA, may be the good indicator for PMI. Several studies have tried to determine changes in molecular markers to provide more useful information for PMI. The studies demonstrated mixed results that showed the influence of ante and post-mortem factors on nucleic acid degradation. More studies would be required in order to standardize on human cases under standard parameters. Thus, molecular aspects of forensic involves this application of omicsin medical sciences to investigate cause of death and its process at the genetic basis and biological molecular level. Keywords: Post-mortem interval (PMI); Molecular biology; DNA and RNA quantification; Forensic Genetic identification.

Human Bone Proteomes before and after Decomposition: Investigating the Effects of Biological Variation and Taphonomic Alteration on Bone Protein Profiles and the Implications for Forensic Proteomics.

Bone proteomic studies using animal proxies and skeletonized human remains have delivered encouraging results in the search for potential biomarkers for precise and accurate post-mortem interval (PMI) and the age-at-death (AAD) estimation in medico-legal investigations. The development of forensic proteomics for PMI and AAD estimation is in critical need of research on human remains throughout decomposition, as currently the effects of both inter-individual biological differences and taphonomic alteration on the survival of human bone protein profiles are unclear. This study investigated the human bone proteome in four human body donors studied throughout decomposition outdoors. The effects of ageing phenomena (in vivo and post-mortem) and intrinsic and extrinsic variables on the variety and abundancy of the bone proteome were assessed. Results indicate that taphonomic and biological variables play a significant role in the survival of proteins in bone. Our findings suggest that inter-individual and inter-skeletal differences in bone mineral density (BMD) are important variables affecting the survival of proteins. Specific proteins survive better within the mineral matrix due to their mineral-binding properties. The mineral matrix likely also protects these proteins by restricting the movement of decomposer microbes. New potential biomarkers for PMI estimation and AAD estimation were identified. Future development of forensic bone proteomics should include standard measurement of BMD and target a combination of different biomarkers.

Predicting the postmortem interval of burial cadavers based on microbial community succession

Previous studies have demonstrated that microbial community succession during the decomposition of cadavers could be used to estimate the postmortem interval (PMI). However, the vast majority of the existing studies focused on exposed cadavers. In fact, burial cadavers are common scenarios for forensic investigations. In this study, the microbial communities from gravesoil, rectum and skin of burial SD rat cadavers during decomposition were characterized using 16S rRNA gene high-throughput sequencing. We predicted PMI based on the microbial community succession. Obvious differences in microbial community structures were observed between different stages of decomposition. Later decay stages had a lower alpha diversity compared to earlier decay stages. Significant linear relationships between similarities of the microbial communities and postmortem intervals were observed, manifesting regular succession over the course of decomposition. Furthermore, we combined random forest models with postmortem microbial features to predict PMI. The model explained 86.83%, 84.55% and 81.67% of the variation in the microbial community, with a mean absolute error of 1.82, 2.06 and 2.13 days within 60 days of decomposition for gravesoil, rectum and skin of burial cadavers, respectively. Overall, our results suggested that postmortem microbial community data could serve as a potential forensic tool to estimate accurate PMI of burial cadavers.

An electrochemical biosensor for hypoxanthine detection in vitreous humor: A potential tool for estimating the post-mortem interval in forensic cases

In this study, a simple electrochemical method was developed for fast and convenient detection of hypoxanthine (Hx) and subsequent estimation of the post-mortem interval (PMI) in forensic cases. A polydopamine-modified carbon nanofibers (CNFs) electrode was utilized with xanthine oxidase (XOD) for Hx detection in vitreous humor (VH) samples for the first time. Fourier transform infrared (FT-IR) spectra and electrochemical characterization revealed the PDA-coated CNFs exhibited good biocompatibility for XOD immobilization and fast electron transfer. Under the optimal conditions, acceptable stability and reproducibility of the assay were achieved. Additionally, this enzymatic electrochemical biosensor showed a good linear relationship between the current response and Hx concentrations in the range of 5 − 60 µM and a detection limit of 2 μM. Consequently, the possible interferences in VH samples were studied to verify the selectivity of this device, and the findings confirmed the excellent detection specificity of this assay for the analysis of real samples. Moreover, the recycling experiments conducted in VH samples showed the practical application of this microdevice. In addition, the analysis of Hx concentration allowed for accurate PMI prediction, compared with known PMI values. These results indicated that the Hx biosensor may be a promising tool for the estimation of PMI in forensic investigations.

A comparison of endogenous and exogenous RNA reference marker as relevant for accurate Post-Mortem Interval estimation

The Post Mortem-Interval (PMI), also known as time since death, is of relevance for forensic routine casework and serves primarily as the basis for the determination of the time that has passed since the time of death. The existing, “classical” approaches are characterised by certain limitations because they are predominantly based on morphological examinations.The present study deals with the assessment of two molecular messengerRNA-markers in different kinds post-mortem human specimens concerning their ability to use them for the estimation of the time of death. Moreover, a comparison between carefully selected endogenous and one exogenous reference marker for the normalisation of the gene expression data as relevant for accurate PMI estimation is drawn.The evaluation showed that the used approach is methodologically useful but on consideration of the achieved results it was only possible to determine slightly meaningful tendencies. The tested exogenous reference marker was not suitable for sufficient data normalisation whereas the endogenous reference markers showed a stable expression. However, it was not possible to recognise a specific expression level of the genes within a PMI group when endogenous reference markers were used.

Description of immature stages of Thanatophilus sinuatus (Coleoptera: Silphidae).

Necrophagous beetles of genus Thanatophilus are well recognized as a group of beetles with a high potential utility in forensic entomology. They can be used to estimate postmortem interval (PMI) or validate the value for other groups of insects commonly encountered on human remains, like blowflies (Calliphoridae). However, reliable tools for instar and species identification of their larvae are needed as such information is crucial for allowing accurate PMI estimate. One of the most common species of the genus Thanatophilus in Europe is Thanatophilus sinuatus. This species occurs frequently on human remains and its larvae feed on decaying tissues throughout their development. Therefore, the larvae could become useful bioindicators for forensic entomology, although their current description does not allow reliable instar or species identification. Our goal was to provide morphological characters for species and instar identification of all larval stages of T. sinuatus. The larvae were obtained from laboratory rearing under controlled conditions (20°C and 16:8h of light/dark period). Qualitative and quantitative morphological instar and species-specific characters are described and illustrated. Additionally, we report observations of biological and developmental lengths for all stages of the species. We also compared these morphological characters with recent description of T. rugosus and provided an identification key of these two similar and often co-occurring species. We noticed that some characters for instar identification were shared between T. sinuatus and T. rugosus and were confirmed by comparison with larvae of T. dentigerus that they can be applied to other species of the genus.