Estimation Of Postmortem Interval Research Articles

Novel use of a servosphere to study apodous insects: Investigation of blow fly post-feeding larval dispersal.

Blow flies (Diptera: Calliphoridae) are arguably the most important providers of an estimate of minimum post-mortem interval in forensic investigations. They usually undergo a post-feeding dispersal from the body. While previous studies have looked at dispersal of groups of larvae, recording the dispersal activity of individual larvae has not previously been demonstrated. A servosphere was used here to record the speed, directionality and phototaxis of individual post-feeding larvae of two species of blow fly on a smooth plastic surface over time. The servosphere rotates to compensate for the movement of an insect placed at its apex, thereby enabling its unimpeded locomotion in any direction to be studied and behavioural changes to external stimuli recorded. To our knowledge, the servosphere has not previously been used to study apodous insects. The objective of our study was to compare dispersal behaviour of Calliphora vicina Robineau-Desvoidy and Protophormia terraenovae (Robineau-Desvoidy), both common primary colonisers of human and animal cadavers, but showing different post-feeding dispersal strategies. Larvae of C. vicina generally disperse from the body while those of P. terraenovae remain on or close to the body. Our aims were to study (1) changes in dispersal speed over a 1-h period; (2) changes in dispersal speed once a day for 4 days, between the end of feeding and onset of pupariation; and (3) response of dispersing larvae to light. We demonstrated that (1) the movement of three C. vicina larvae tracked for 1 continuous hour on 1 day slowed from an average of 3 to <1.7 mms-1; (2) the average speed of 20 larvae of C. vicina (4.08 mms-1) recorded for 5 min once per day over a 4-day period between onset of dispersal and pupariation was significantly greater than that of P. terraenovae (2.36 mms-1; p < 0.0001), but that speed of both species increased slightly over the 4 days; (3) the responses of larvae of C. vicina to changes in light direction from the four cardinal directions of the compass, showed that they exhibited a strong negative phototactic response within 5 s, turning to move at approximately 180° away from the new light position. While conducted to observe larval calliphorid post-feeding behaviour, the results of this proof of concept study show that apodous insects can be studied on a servosphere to produce both qualitative and quantitative data.

Using Stable Nitrogen Isotope Ratios from Human Skeletal Muscle Tissue for Postmortem Interval (PMI) Estimation (Part 1)

Human skeletal muscle tissue was analyzed for stable nitrogen isotope analysis (δ15N) to test the applicability of changes in values (Δ15N) through the putrefaction process for postmortem interval (PMI) estimation. Seasonal differences in Δ15N values of decom-posing skeletal muscle tissue were analyzed from 20 human donors at the University of Tennessee Anthropology Research Facility. This study revealed that change in the δ15N ratios of decomposing muscle tissue has potential as a novel method for PMI estimation. In winter months, when identifiable muscle tissue can be retained on a body for months, the data indicate that there is a systematic increase in Δ15N values with increasing temperatures when putrefaction progresses faster with values shifting as much as 3.0‰. This initial study indicates there is value in further method development of this quantitative approach to PMI estimation for recently deceased individuals, but further work is needed. While the analysis only requires a small amount of tissue, has a user- friendly collection and analysis protocol, and is less subjective than visual methods, there is still significant research to be done prior to widespread applications as a PMI estimation method. Understanding variability in δ15N across decomposing muscle tissue groups, individual variation (i.e., muscle mass, disease, body chem-istry), and an accurate approximation for a perimortem δ15N value necessary to calculate Δ15N all need to be further researched prior to generation of a reliable regression formula and error rates.

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.

Influence of photoperiod on the developmental times of the forensically relevant blow fly species Calliphora vicina (Diptera: Calliphoridae)

Blow flies (Diptera: Calliphoridae) are frequently used in forensic investigations due to their rapid colonization of cadavers. As with other insects, environmental temperature strongly influences their developmental rates. While published research has typically explored not only the impact of the environmental temperature, but also of other factors like tissue type and drug presence on developmental rates, the influence of photoperiod on the developmental rates of forensically relevant blow fly species has remained largely underexplored. Understanding the relationship between photoperiod and developmental times is crucial, as neglecting this aspect could compromise the accuracy of minimum post-mortem interval (minPMI) estimations. The present study investigates the impact of three photoperiod conditions (0:24, 8:16, and 12:12 light:darkness) on the developmental rates of Calliphora vicina, focusing on the duration of the different immature stages and on the total developmental time. Our results revealed significant variation in the intra-puparial stage and total development time across different photoperiods. Notably, a 12:12 photoperiod led to a significantly prolonged intra-puparial stage and total development time compared to the 0:24 photoperiod, suggesting that Calliphora vicina develops faster in total darkness. These findings highlight the importance of considering photoperiod in both laboratory rearing protocols and forensic casework to improve the accuracy and reliability of minPMI estimations. In this regard, preliminary guidelines and recommendations are provided.

The role of miRNA-21 and hypoxia inducible factor-1 in predicting post mortem interval in cardiac muscles of aluminum phosphide deaths

BackgroundThe assessment of the postmortem interval (PMI) represents one of the major challenges in forensic pathology. Because of their stability, microRNAs, or miRNAs, are anticipated to be helpful in forensic research. ObjectiveTo see if estimation of PMI is possible using miRNA-21 and Hypoxia-inducible factor-1α (HIF-1α) expression levels in the heart samples from aluminum phosphide toxicity (Alpt). MethodsThis was a cross sectional study on 60 post-mortem samples (heart tissues) collected at different intervals during forensic autopsies. The two groups were allocated equally according to the cause of death into Group I (non-toxicated deaths, n = 30): Deaths caused by other than toxicity, and Group II (toxicated deaths, n = 30): Deaths due to Alpt. MDA (Malondialdehyde) and GSH (Glutathione), were measured in heart tissues using ELIZA. MiRNA- 21and HIF-1α expression levels were measured in heart tissues at different PMI using RT-Q PCR. ROC curve for detection of toxicated deaths using miRNA-21 and HIF was carried out. ResultsmiRNA-21 and HIF-1α expression levels in Alp deaths were up regulated while GSH was downregulated with statistically significant difference. There was positive correlation between miRNA-21, HIF-1α and MDA with PMI while there was negative correlation between GSH and PMI in Alp deaths. In prediction of post mortem interval in Alp deaths miRNA-21 sensitivity and specificity were (75.9 %, 51.7 %, respectively) while HIF-1α sensitivity and specificity were 100 %. ConclusionPMI can be calculated using the degree to which particular miRNA-21 and HIF-1α are expressed in the heart tissue. The combination of miRNA-21 with HIF-1α in post mortem estimation is precious indicators.

Intrapuparial stage aging and PMI estimation based on the developmental transcriptomes of forensically important Aldrichina grahami (Diptera: Calliphoridae) gene expression

BackgroundThe expression profiles of differentially expressed genes (DEGs) during pupal development have been demonstrated to be vital in age estimation of forensic entomological study. Here, using forensically important Aldrichina grahami (Diptera: Calliphoridae), we aimed to explore the potential of intrapuparial stage aging and postmortem interval (PMI) estimation based on characterization of successive developmental transcriptomes and gene expression patterns. MethodsWe collected A. grahami pupae at 11 successive intrapuparial stages at 20 °C and used the RNA-seq technique to build the transcriptome profiles of their intrapuparial stages. The DEGs were identified during the different intrapuparial stages using comparative transcriptome analysis. The selected marker DEGs were classified and clustered for intrapuparial stage aging and PMI estimation and then further verified for transcriptome data validation. Ultimately, we categorized the overall gene expression levels as the dependent variable and the age of intrapuparial A. grahami as the independent variable to conduct nonlinear regression analysis. ResultsWe redefined the intrapuparial stages of A. grahami into five key successive substages (I, II, III, IV, and V), based on the overall gene expression patterns during pupal development. We screened 99 specific time-dependent expressed genes (stage-specific DEGs) to determine the different intrapuparial stages based on comparison of the gene expression levels during the 11 developmental intrapuparial stages of A. grahami. We observed that 55 DEGs showed persistent upregulation during the development of intrapuparial A. grahami. We then selected four DEGs (act79b, act88f, up and ninac) which presented consistent upregulation using RT-qPCR (quantitative real-time PCR) analysis, along with consideration of the maximum fold changes during the pupal development. We conducted nonlinear regression analysis to simulate the calculations of the relationships between the expression levels of the four selected DEGs and the developmental time of intrapuparial A. grahami and constructed fitting curves. The curves demonstrated that act79b and ninac showed continuous relatively increasing levels. ConclusionsThis study redefined the intrapuparial stages of A. grahami based on expression profiles of developmental transcriptomes for the first time. The stage-specific DEGs and those with consistent tendencies of expression were found to have potential in age estimation of intrapuparial A. grahami and could be supplementary to a more accurate prediction of PMI.

Post-mortem intervals estimation by determination of insect diversity

ABSTRACT Dipteran flies have been employed to measure the post-mortem period examination with the help of decomposition of the carcasses. The current study attempted to update the baseline data of the insects to determine the post-mortem interval. Five decomposition stages were observed but the complete decomposition of (Oryctolagus cuniculus) rabbits in winter took place in 21 days and in spring in 12 to 15 days. Decomposition period in both trials varied due to temperature fluctuations. Odours were released at different stages of decay and blow flies were the first insect to come on carcasses. The species that identified were the blowfly (Calliphoridae) (34.4% | 49.12%), blue fly (Calliphora) (17.24% | 41.22%), flesh fly Sarcophagidae (20.68% | 5.26%), and house fly (Muscidae) (27.58% | 4.38%) in the first trial and second trial respectively. The timing of the various arthropod species’ arrivals and their correlation with various decompositional phases throughout both trials suggested that they could be useful for determining the PMI (post-mortem interval) in forensic investigations. This study showed that insect fauna on the corpse is a tool for estimating post-mortem interval and plays an important role in forensic entomology to analyse the suspicious suspect.

A fundamental study on postmortem submersion interval estimation by metabolomics analyzing of gastrocnemius muscle from submersed rat models in freshwater.

In forensic practice, determining the postmortem submersion interval (PMSI) and cause-of-death of cadavers in aquatic ecosystems has always been challenging task. Traditional approaches are not yet able to address these issues effectively and adequately. Our previous study proposed novel models to predict the PMSI and cause-of-death based on metabolites of blood from rats immersed in freshwater. However, with the advance of putrefaction, it is hardly to obtain blood samples beyond 3 days postmortem. To further assess the feasibility of PMSI estimation and drowning diagnosis in the later postmortem phase, gastrocnemius, the more degradation-resistant tissue, was collected from drowned rats and postmortem submersion model in freshwater immediately after death, and at 1day, 3 days, 5 days, 7 days, and 10 days postmortem respectively. Then the samples were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the dynamic changes of the metabolites. A total of 924 metabolites were identified. Similar chronological changes of gastrocnemius metabolites were observed in the drowning and postmortem submersion groups. The difference in metabolic profiles between drowning and postmortem submersion groups was only evident in the initial 1day postmortem, which was faded as the PMSI extension. Nineteen metabolites representing temporally-dynamic patterns were selected as biomarkers for PMSI estimation. A regression model was built based on these biomarkers with random forest algorithm, which yielded a mean absolute error (± SE) of 5.856 (± 1.296) h on validation samples from an independent experiment. These findings added to our knowledge of chronological changes in muscle metabolites from submerged vertebrate remains during decomposition, which provided a new perspective for PMSI estimation.

Estimating postmortem interval based on oral microbial community succession in rat cadavers

The accurate estimation of the postmortem interval has been one of the crucial issues to be solved in forensic research, and it is influenced by various factors in the process of decay. With the development of high-throughput sequencing technology, forensic microbiology has become the major hot topic in forensic science, which provides new research options for postmortem interval estimation. The oral microbial community is one of the most diverse of microbiomes, ranking as the second most abundant microbiota following the gastrointestinal tract. It is remarkable that oral microorganisms have a significant function in the decay process of cadavers. Therefore, we collected outdoor soil to simulate the death environment and focused on the relationship between oral microbial community succession and PMI in rats above the soil. In addition, linear regression models and random forest regression models were developed for the relationship between the relative abundance of oral microbes and PMI. We also identified a number of microorganisms that may be important to estimate PMI, including: Ignatzschineria, Morganella, Proteus, Lysinibacillus, Pseudomonas, Globicatella, Corynebacterium, Streptococcus, Rothia, Aerococcus, Staphylococcus, and so on.

Decomposition in an extreme cold environment and associated microbiome-prediction model implications for the postmortem interval estimation.

The accurate estimation of postmortem interval (PMI), the time between death and discovery of the body, is crucial in forensic science investigations as it impacts legal outcomes. PMI estimation in extremely cold environments becomes susceptible to errors and misinterpretations, especially with prolonged PMIs. This study addresses the lack of data on decomposition in extreme cold by providing the first overview of decomposition in such settings. Moreover, it proposes the first postmortem microbiome prediction model for PMI estimation in cold environments, applicable even when the visual decomposition is halted. The experiment was conducted on animal models in the second-coldest region in the United States, Grand Forks, North Dakota, and covered 23 weeks, including the winter months with temperatures as low as -39°C. Random Forest analysis models were developed to estimate the PMI based either uniquely on 16s rRNA gene microbial data derived from nasal swabs or based on both microbial data and measurable environmental parameters such as snow depth and outdoor temperatures, on a total of 393 samples. Among the six developed models, the best performing one was the complex model based on both internal and external swabs. It achieved a Mean Absolute Error (MAE) of 1.36 weeks and an R2 value of 0.91. On the other hand, the worst performing model was the minimal one that relied solely on external swabs. It had an MAE of 2.89 weeks and an R2 of 0.73. Furthermore, among the six developed models, the commonly identified predictors across at least five out of six models included the following genera: Psychrobacter (ASV1925 and ASV1929), Carnobacterium (ASV2872) and Pseudomonas (ASV1863). The outcome of this research provides the first microbial model able to predict PMI with an accuracy of 9.52 days over a six-month period of extreme winter conditions.

The influence of rehydration on decomposition in the Highveld region of South Africa-Using a pig model.

Researchers have observed that rainfall may re-initiate decomposition in desiccated tissue; however, no conclusive research-based evidence exists on the specific effects of rehydration on decomposition. Therefore, this study aimed to assess the effects of artificial rehydration on the progression of decomposition following the advanced stage of decomposition. Twelve adult pig cadavers (8 experimental; 4 controls) were placed in the central Highveld of South Africa during cooler (April-July 2021) and warmer (August-November 2021) months. Decomposition was scored approximately biweekly to obtain the total body score, and accumulated degree days (ADD) were calculated for each pig. All pig cadavers were covered by chicken wire cages with transparent tarps to control for natural rehydration and scavenging. Once the experimental pig cadavers reached a three-visit stasis in the advanced phase of decomposition, they were artificially rehydrated, and changes in the progression of decomposition between the control and experimental groups were plotted (ADD against TBS) for observation. The rehydrated experimental pig cadavers showed re-initiation of decay and insect re-colonization, while the control cadavers mainly remained in a state of stasis with insect activity ceased altogether. Greater cadaver decomposition islands and a color change post-rehydration were also noted in some experimental cadavers. This supports the need for future research on the impact of rehydration, including associated soil moisture on decomposition rates, progression, and invertebrate colonization, which will enhance our understanding of the effects these environmental factors have on the accuracy of post-mortem interval estimation.

Bionomics, reproductive traits and assessment of forensic relevance of Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae).

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 22.24h and 21.36h for 1st and 2nd respectively, and 3rd showed an average development time of 80.47h. Pupa had the longest duration (x̅ = 295.69h). 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).

Estimation of postmortem interval based on scene environment and plant evidence： A case report

Estimation of postmortem interval based on scene environment and plant evidence： A case report

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Succession of medico-legal important flesh flies (Diptera: Sarcophagidae) in the temporal gradient of pig decomposition in the Brazilian Cerrado

Flies belonging to the Sarcophagidae family play a significant role in forensic investigations by aiding in the estimation of post-mortem interval through the assessment of the developmental time of their immature stages and/or the succession patterns of species on carcasses and cadavers. In this regard, this study aimed to investigate the ecological succession of these flies on pig carcasses within Cerrado of Brazil. The progression of fly succession was examined over a 10-day period using the Threshold Indicator Taxa Analysis (TITAN) approach. Six Z + species (Oxysarcodexia thornax (Walker), Peckia (Sarcodexia) lambens (Wiedemann), Peckia (Euboettcheria) collusor (Curran & Walley), Ravinia belforti (Prado & Fonseca), Tricharaea (Sarcophagula) canuta (Wulp) and Tricharaea (Sarcophagula) occidua (Fabricius)) were identified, with change points ranging from 2.5 to 3 days during the dry season and 2.5 to 5.5 days during the rainy season. Two Z− species (Dexosarcophaga carvalhoi (Lopes) and Peckia (Sarcodexia) tridentata (Hall)) were present, with a change point of 6.5 days during the rainy season. This study provides a continuous analysis of the temporal succession of flies, enabling an investigation into species progression based on their change points and directions (Z + and Z− ), thereby offering methodological advancements to avoid the arbitrary categorization of inherently continuous data.

Estimation of Post-mortem Interval Based on Livor Mortis using a Colorimeter in Thai Populations

OBJECTIVE Livor mortis is a helpful and widely used method of estimating postmortem interval (PMI) in Thailand. This study aimed to investigate the value of a colorimeter as a tool for estimating the PMI. METHODS The color of livor mortis and control skin in 80 cadavers whose PMI was within 12 hours was measured by a colorimeter. The L* (brightness), a*b* (chroma and hue), and ∆E* values were compared to the control skin values. Statistical analysis was performed to determine the relationship between PMI and skin color before and after application of a specific pressure. RESULTS The results showed that colorimetric parameters were only weakly correlated with the PMI. An univariable analysis of ∆E* values was performed and showed good discriminatory power, with an area under the ROC curve of 0.82. The recommended cut-off value of ∆E* was 14 for the discrimination between early PMI (less than 6 hours) and late PMI (6-12 hours), in which the sensitivity and specificity were 72.5% and 80%. CONCLUSIONS The findings in this study reinforce the utility of colorimetric measurements in PMI estimation. With additional study and a larger sample size, the estimation of PMI could be established for general use in forensic practice.

Visualization Analysis of Artificial Intelligence Literature in Forensic Research.

To analyze the literature on artificial intelligence in forensic research from 2012 to 2022 in the Web of Science Core Collection Database, to explore research hotspots and developmental trends. A total of 736 articles on artificial intelligence in forensic medicine in the Web of Science Core Collection Database from 2012 to 2022 were visualized and analyzed through the literature measuring tool CiteSpace. The authors, institution, country (region), title, journal, keywords, cited references and other information of relevant literatures were analyzed. A total of 736 articles published in 220 journals by 355 authors from 289 institutions in 69 countries (regions) were identified, with the number of articles published showing an increasing trend year by year. Among them, the United States had the highest number of publications and China ranked the second. Academy of Forensic Science had the highest number of publications among the institutions. Forensic Science International, Journal of Forensic Sciences, International Journal of Legal Medicine ranked high in publication and citation frequency. Through the analysis of keywords, it was found that the research hotspots of artificial intelligence in the forensic field mainly focused on the use of artificial intelligence technology for sex and age estimation, cause of death analysis, postmortem interval estimation, individual identification and so on. It is necessary to pay attention to international and institutional cooperation and to strengthen the cross-disciplinary research. Exploring the combination of advanced artificial intelligence technologies with forensic research will be a hotspot and direction for future research.

Reply to the comment on “Assessing the extent of bone bioerosion in short timescales – A novel approach for quantifying microstructural loss [Quat. Int., in press, https://doi.org/10.1016/j.quaint.2023.01.011

In our original article we evaluated diagenetic changes to rat cortical bone osteocyte lacunae over increasing post-mortem intervals. We suggested that with further, more intensive research, it may be possible to exploit these changes for the purposes of post-mortem interval estimation of skeletal remains. In their comment to our paper, Miszkiewicz et al. (in press) raise concerns about the project design and interpretation of the results. Here, we reply to their comments and offer answers to their concerns.

Attraction of common Australian blowflies to algal affected remains, and the specific effect of algae present in the food substrate on growth and survival of Calliphora stygia

ABSTRACT Entomology is a well-established approach for estimating the post-mortem interval (PMI) in terrestrial decomposition, however it is of limited value in aquatic environments due to little research into insect attraction and colonization in aquatic environments. This study aimed to investigate the effect algae in the feeding substrate has on the growth and survival of Calliphora stygia, and on the attraction of a range of common Australian blowfly species. Growth and survival studies were conducted under controlled laboratory conditions, with growth measured through larval length and developmental stage, and survival estimated by percentage of adult eclosion. A walk olfactometer was used to assess attraction to an algal affected substrate, observing that algae was a strong attractant for females of multiple blowfly species. Species of the genus Calliphora had the highest rate of attraction at 95–100%, with Chrysomya rufifacies the lowest at 75%. Both developmental stage and survival of C. stygia were not significantly impacted by the presence of algae. However, larvae fed algae-soaked liver were significantly longer when compared with the controls. This study highlights the potential for skewed PMI estimations in forensic casework where entomologists may rely on larval length for PMI estimations, and the attractive effect of algae on blowflies.

Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.