In the medico-legal application of forensic entomology, estimating the time of death is critical and traditionally relies on changes in observable traits of carrion feeding insect larvae. Traits such as size, weight, and morphology can be used to predict the insect specimen age and help define the minimum time since death. The blowfly Phormia regina Meigen (Diptera: Calliphoridae) is a key forensic insect, yet age estimation for older maggots in this and other carrion-feeding species is particularly challenging due to the limited morphological changes in the late-stage larvae. To enhance age-estimation precision, we employed transcriptomic profiling on blowfly maggots, aiming to identify genes as markers for time of death estimation. Our study characterized maggot development, reinforcing that weight and behavior cannot precisely determine age between 100 and 130 hours at 27.5 °C. We built a chromosomal scale annotated genome, establishing a reliable database for uncovering transcriptomic signatures during larval development. Applying differential gene expression analyses, weighted gene co-expression network analysis, and the generalized linear model, we identified nine candidate genes (y5078, y5076, agt2, ech1, dhb4, asm, gabd, acohc, ivd) that delineate the age of otherwise indeterminate maggots. This research introduces a molecular approach to address a longstanding problem in forensic entomology and promises to increase precision in determining the time of death at a crime scene.

Deep learning method based on image recognition for intra-puparial age and postmortem interval estimation in the forensically important Sarcophaga peregrina (Diptera: Sarcophagidae).

Abstract Forensic entomology utilizes the age of the larvae to estimate the minimum post-mortem interval (mPMI), a critical aspect of death where improving the accuracy and reliability of developmental age estimated is essential. Traditional method often rely on larval length, which can be influenced by preservation method, environmental and genetic factors. This study investigates the changes in juvenile hormone III bisepoxide (JHB III) concentrations during the larval development of Chrysomya megacephala , Chrysomya rufifacies , and Musca domestica– three fly species of forensic importance in Southeast Asia and other tropical regions. JHB III was quantified using High-Performance Liquid Chromatography (HPLC). Pearson’s r correlation analysis examined relationships among larval weight, raw and normalized JHB III concentrations, and larval developmental age. Linear regression models were generated forsignificant correlations( p < 0.05) between normalized JHB III and larval developmental age. A fluctuating pattern of normalized JHB III concentrations throughout development was observed, with moderate negative correlations to larval age ( r = − 0.55 to − 0.67). These suggest that quantifying JHB III concentrations could supplement traditional morphological measurements like larval length, potentially increasing the accuracy and reliability of larval age estimates for mPMI calculation within the geographical range where these species are the forensic indicators.

In forensic entomology, accurate species identification is essential for calculating the exact minimum postmortem interval (minPMI), as insect developmental rates are highly species-specific. Sarcophaga (Liopygia) ruficornis (Fabricius, 1794) is a species of medical and forensic significance. Recently, it was recorded from Egypt for the first time. The current study aimed to conclusively identify S. ruficornis in Egypt through DNA barcoding and morphological examination of both adult males and females, for the first time, to our knowledge. A segment of the mitochondrial cytochrome oxidase subunit one gene (COI) of S. ruficornis was amplified, and a sequence length of 817 bp was obtained and submitted to GenBank. The genitalia of both sexes and the diagnostic morphological characters of the species were examined and illustrated. In addition, the phylogenetic relationship of the Egyptian population of S. ruficornis was investigated. This study demonstrates the utility of DNA barcoding for investigating the genetic composition and variation of S. ruficornis populations and provides essential data for the identification of S. ruficornis in Egypt, which makes it possible to identify a specimen correctly even when only limited morphological evidence is available.

Accurate estimation of the time of colonization (TOC) is a cornerstone of forensic entomology, yet direct validation of laboratory-derived development datasets in field contexts remains rare. Within validation studies, when the TOC is unknown, the time of placement (TOP)-the point at which remains become accessible to insects (e.g,. Diptera)-can serve as a proxy. This study evaluated the accuracy of Cochliomyia macellaria (Diptera: Calliphoridae) development estimates under varying ecological conditions by calculating the estimated-to-actual TOP ratio (%). Factors examined included carcass type (small, medium, and large mammals; large birds), thermal summation method (accumulated degree days [ADD] vs. accumulated degree hours [ADH]), emergence timing (first vs. last emergence), development dataset tissue source (porcine vs. equine), and species coexistence with Chrysomya rufifacies (Diptera: Calliphoridae). A full-factorial, aligned rank-transformed ANOVA was conducted using three replicates per carcass type across two Texas field sites in 2023 and 2024. Using ADD, first-emergence data, and porcine-derived tissue consistently yielded the highest TOP ratios (93.9%, 96.3%, and 93.4%, respectively). Precision was greatest in small mammals and lowest in large mammals. Although ADD-based estimates generally outperformed ADH, ADH occasionally demonstrated greater consistency under certain contexts. Species coexistence and trial year showed context-dependent influences on TOP ratio outcomes. These findings underscore the importance of incorporating ecological and biological variability when applying development studies to forensic casework and highlight the robustness of ADD calculations and early-emergence sampling for estimating TOC in the field.

Blow flies (Diptera: Calliphoridae) play an important role in forensic entomology (using insects and other arthropods to criminal investigations). An early step for this application is accurate species identification. The 2 main approaches are morphological and molecular, but each has its limitations, making it necessary to use alternative or supplementary tools. Since outline-based geometric morphometrics (GM) is widely applied with insect identification, this study examined this method for species identification of 800 third-instar larvae of 8 blow fly species Chrysomya chani Kurahashi, Chrysomya megacephala (Fabricius), Chrysomya (Ceylonomyia) nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya (Achoetandrus) rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia cuprina (Wiedemann), and Lucilia porphyrina (Walker). Successful classification based on the cephaloskeleton demonstrated high reclassification scores ranging from 89% to 100%. This indicates that outline-based GM of the larval cephaloskeleton contour offers a significant advantage in identifying fly specimens. It can complement traditional methods, especially when encountering certain limitations-such as incomplete or damaged larvae-and can also help lower costs associated with molecular analyses.

Simple SummaryThis study examines blow fly (Diptera: Calliphoridae) communities across the Georgia Fall Line (GFL), a geologic boundary with a distinct elevation gradient, across winter/spring and summer/fall transitions, highlighting implications for forensic entomology. By deploying baited traps at two locations north and south of the GFL, we show that blow fly community composition differed by regional biogeography more than immediate weather conditions. These findings challenge simplistic summer- vs. winter-active blow fly categorizations and underscores the need for more nuanced, geographically informed models in forensic casework.Forensic entomologists use insect development, especially in blow flies (Diptera: Calliphoridae), to estimate the minimum postmortem interval (mPMI). Since insect activity is driven mainly by temperature, understanding geographic and seasonal variation in community composition is critical. In the southeastern United States, approximately 10 blow fly species dominate, generally classified as “summer-active” or “winter-active” flies. We studied their presence and abundance during winter/spring and summer/fall transitions across the Georgia Fall Line (GFL), a major geophysical boundary separating the Piedmont and Coastal Plain. Here we show that community structure was shaped more by regional biogeography and seasonal transitions, than by current temperature. Three species; Calliphora livida, Lucilia coeruleiviridis, and Cochliomyia macellaria accounted for over 70% of seasonal variation. Fly communities differed sharply across the GFL and shifted between seasonal transitions. Recognizing these geographic and temporal patterns can help forensic entomologists produce more accurate mPMI estimates in death investigations.

Estimating the pupal age of Dermestes ater (Coleoptera: Dermestidae) using Fourier transform infrared spectroscopy combined with Chemometrics.

Medicolegal forensic entomology relies largely on blow fly (Diptera: Calliphoridae) activity to estimate a minimum postmortem interval (mPMI). The prevailing assumption, that blow flies are inactive at night, leads to the exclusion of nocturnal colonization from mPMI estimates. Artificial light at night (ALAN), however, especially from the increasing use of light emitting diode (LED) streetlights, may alter this innate behavior. Baited traps were deployed at two sites: one natural site that was dark at night (unlit) and one undeveloped but with infrastructure site that was LED-illuminated at night (lit). Samples were collected during daytime and nighttime periods. A total of 1,544 blow flies representing seven species were collected. Only six flies (0.4% of the total) were captured at night, four in unlit and two in lit environments, indicating minimal nighttime activity. These results suggest that, under current conditions, ALAN is unlikely to induce nocturnal blow fly behavior and impact mPMI estimations. Further research across diverse taxa and environments is needed to better understand the effects of ALAN on insects of forensic importance.

The developmental patterns of the oldest developing flies are commonly used by forensic entomologists to estimate the minimum postmortem interval (PMImin). Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) are widely distributed in the Australian and Palearctic regions and are closely associated with human corpses. Here, the developmental pattern of H. ligurriens was studied under laboratory conditions at 7 different constant temperatures between 16 and 34 °C. Five models were established for the study of H. ligurriens, including an isomorphen diagram, an isomegalen diagram, a linear thermal summation model, a nonlinear SSI model, and a logistic model. The lower developmental threshold and thermal summation constant of H. ligurriens evaluated by the linear thermal summation models were 8.99 °C and 4,967.35 degree hours, respectively. The lower developmental threshold, intrinsic optimum temperature, and upper lethal developmental threshold obtained by Optim SSI models were 12.56, 19.91, and 33.21 °C, respectively. The expression levels of 5 genes (Hsp83, Hsp60, USP, wg, and amKI) were analyzed at sequential developmental time points to estimate the pupal formation time of H. ligurriens under constant temperatures of 16 and 25 °C, and to verify the temporal regularity of this process. This study provides a foundation for forensic entomologists to accurately estimate the PMImin using H. ligurriens in future death investigations.

Blowfly species, which play a crucial role in forensic investigations as primary colonizers of cadavers, are influenced by environmental factors. However, most research conducted on blowfly species in South Korea remains limited to a single province. We investigated the spatiotemporal distribution of forensically relevant blowflies (Diptera: Calliphoridae) in the southern provinces of South Korea to enhance forensic entomology databases and improve postmortem interval (PMI) estimation. Overall, 3,934 adult blowflies representing 14 species across 5 genera were collected from 4 regions (Changnyeong, Pohang, Yeosu, and Jeju) over a 1-yr period using baited traps. The dominant species included Chrysomya megacephala (Fabricius), Lucilia illustris (Meigen), Lucilia caesar (Linnaeus), and Lucilia sericata (Meigen); Ch. megacephala exhibited a significantly higher abundance in Jeju than in the other regions, particularly during the warmer seasons. A self-organizing map (SOM) and principal component analysis (PCA) were employed to visualize and validate the spatiotemporal clustering of blowfly populations, confirming that seasonal factors strongly influence distribution patterns. The combination of SOM and PCA effectively distinguished seasonal and regional clustering patterns, demonstrating the influence of environmental factors on species-specific distributions. These findings emphasize the importance of considering regional and seasonal variations in forensic casework and the need to expand entomological databases to reflect geographic differences. Furthermore, the observed regional differences in species dominance underscore the need to incorporate environmental variability into forensic models to improve the accuracy of PMI estimates. This study provides fundamental data for improving forensic applications based on insect evidence, particularly PMI estimation and crime scene reconstruction.

Determining the time since death (postmortem interval, PMI) is one of the key challenges in forensic practice, as the accuracy of this estimation directly affects the reliability of expert conclusions and the efficiency of investigative procedures. Traditional methods based on morphological signs and thermometric measurements have limited accuracy, particularly at extended intervals. Recent research focuses on the development of innovative approaches, including molecular technologies, microbiome analysis, multi-omics strategies, and the integration of artificial intelligence for big data processing. This article provides an overview of the latest methods for estimating PMI, with particular attention to nucleic acids (DNA, RNA), proteomics, metabolomics, and microbiome profiling. The potential of immunohistochemical markers, mass spectrometry, and nuclear magnetic resonance for quantitative assessment of biochemical changes in tissues and biological fluids is discussed. Advances in forensic entomology are also highlighted, including the application of molecular and chemical techniques for validating insect developmental stages. A separate section addresses the implementation of machine learning and deep learning algorithms to build predictive models based on multifactorial datasets, including microbiome profiles, imaging data, and environmental parameters. Examples of combined approaches integrating biomolecular markers with computational technologies are presented, demonstrating improved accuracy in PMI estimation during both early and advanced decomposition stages. The integration of traditional and innovative methods, the development of standardized protocols, and interdisciplinary collaboration open new opportunities for forensic medicine, forming a basis for creating reliable, reproducible, and universal algorithms for PMI assessment.

Simple SummaryAccurate identification of the insect species is the initial step in forensic entomology case analysis. Phorid flies are significant forensic insects found on indoor and buried corpses. Due to their minute size, species identification of their immature stages presents certain challenges. This study utilized Fourier transform infrared (FTIR) combined with multivariate analytical methods to identify three common species of necrophagous phorid fly immatures. The research demonstrated that using spectral data from the fingerprint region (1800–900 cm−1) coupled with partial least squares-discriminant analysis (PLS-DA) effectively discriminates the species. This study provides an effective tool for the rapid identification of immature species in forensic entomology.Phorid flies serve as the main colonizers of human remains in both indoor and burial environments. Their developmental patterns can be utilized to estimate the minimum postmortem interval (minPMI). Accurate species identification, particularly for immature stages, is essential before utilizing their developmental data to estimate minPMI. This study employed Fourier transform infrared spectroscopy (FTIR) coupled with principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to investigate species identification of eggs (0 h, 8 h, 16 h), larvae (12 h, 60 h, 84 h), and pupae (1 d, 5 d, 10 d) of three necrophagous Phoridae species, Dohrniphora cornuta, Diplonevra funebris, and Megaselia scalaris at 24 °C. The results showed that the FTIR spectra within the fingerprint region (1800–900 cm−1) differed among the three immature phorid flies. These differences were primarily manifested in absorption peak intensities. The PLS-DA analysis successfully distinguished the three species at the same developmental stage. This study demonstrated the feasibility of utilizing FTIR spectroscopy coupled with chemometric methods to both rapidly identify the species of immature small flies and simultaneously estimate their age.

Objective: This study aimed to evaluate the synanthropy of blow flies (Diptera: Calliphoridae) in northwestern Paraná, Brazil, through simultaneous collections in three different environments (urban, rural, and forest) using baited traps. Theoretical Framework: Blow flies are found in a wide range of environments and are strongly associated with human-modified areas. This characteristic, along with their feeding habits, gives them medical, veterinary, and forensic importance. Method: Three baited traps were installed in each environment and remained active for 72 hours, with biweekly collections from November 2022 to November 2023. The traps used protein-based attractants to capture adult flies. Results and Discussion: A total of 577 individuals belonging to six species were collected, with no significant difference in species abundance among environments. Chrysomya albiceps was the most abundant and highly synanthropic species (+97.3). Lucilia eximia was considered hemisynanthropic (-28.4), while Chrysomya putoria (+2.6) and Chrysomya megacephala (+30.8) showed preference for populated areas. Hemilucilia semidiaphana showed strong aversion to anthropized areas (-53.7), and only one Cochliomyia macellaria specimen was recorded. Abiotic variables had no influence on species abundance or seasonality. Research Implications: The results provide valuable data on the distribution, abundance, and synanthropic behavior of blowflies, contributing to a better understanding of their ecological roles and impacts on human and animal health. Originality/Value: This study presents novel data on the blow fly fauna of northwestern Paraná, highlighting the synanthropic tendencies of native and invasive species, with potential applications in forensic entomology, public health, and environmental monitoring.

The examination of insect species inhabiting decaying cadavers often yields critical forensic insights, particularlyregarding the determination of the postmortem interval (PMI), or the time of death. The life cycle of an insectoperates akin to a precise chronometer; it commences its relentless ticking mere minutes or hours followingdemise. The intricacies of insect life cycles profoundly influence the calculation of postmortem intervals, evenin instances where alternative methodologies are recognized for providing pertinent information. The presentfindings illuminate the distinctive characteristics of the local insect fauna associated with human corpses invarious regions of India, as well as their ecological dynamics. Specimens of entomofauna were meticulouslycollected during autopsy procedures from the deceased. This paper discusses ten case studies spanning fromhistorical to contemporary contexts within India. Among the subjects, whose ages ranged from a few monthsto 52 years, six were male and four were female. Both suicide and homicide constituted the causes of death. Theprimary objectives of this study were to gather data concerning the potential application of necrophagous insectsin criminal investigations and to identify the specific insect species colonizing human remains.

Estimating time of death based on entomological evidence commonly relies on the "law of total effective temperature", which requires developmental parameters of specific insect taxa. These are often calculated using the method of Ikemoto and Takai. However, this approach has key limitations. Most importantly, the lack of interval estimates may give the false impression of population homogeneity, which contradicts the substantial variation typically observed in insect populations. In this study, we propose an alternative method. It estimates interval values for developmental parameters while simultaneously identifying component populations within a dataset. The method involves fitting a finite mixture of Weibull distributions to development time data using the Expectation-Maximization (EM) algorithm. This allows for the inclusion of individual-level variability in the estimation process. We tested the method using previously published developmental data on two beetle species, Creophilus maxillosus and Necrodes littoralis (Staphylinidae). Our approach yielded 95% intervals with coverage close to the nominal level, in contrast to Ikemoto and Takai's method, which captured only 59% and 75% of actual cases, respectively. These findings suggest that our method improves the accuracy of insect-based postmortem interval estimates in forensic entomology and, more broadly, provides a general framework for interval estimation of developmental parameters applicable in thermal ecology and applied entomology.

Forensic entomology is a branch of science that uses insects as evidence in medicolegal investigations as they could provide valuable information regarding the cause and time since death specially in the case of buried bodies. The present study was the first attempt in India to compare the decomposition and associated insects on pig carcasses above the ground and those buried at two different depths (40 and 80 cm) during spring season over two consecutive years. Two pigs were buried at each depth and one pig carcass was placed above the ground. One of the buried carcasses was exhumed frequently and other one was kept undisturbed to investigate the decomposition rate and insect access to the carcass. The rate of decomposition was found to be faster above the ground as compared to the buried environment. Also, the carcass buried at 80 cm depth exhibited slower decomposition rates and had limited insect activity as compared to the one buried at 40 cm. Species within the Calliphoridae and Sarcophagidae families were dominant on the surface-exposed carcass, whereas Phoridae were more prevalent on the buried carcasses even at a depth of 80 cm. The findings of the study highlights the significant influence of burial depth and exhumation on decomposition rate and insect arrival and colonization.

Background: Forensic entomology has introduced new methods that are recommended to determine the cause, manner, and time since death, particularly for deteriorated, putrid bodies discovered at unexpected places. This scientific field provides new techniques that are applicableto molecular studies, such as DNA analysis based on entomological data. Numerous techniquesare used to estimate the minimum postmortem interval, depending on the location and state ofthe corpse as well as the circumstances surrounding its discovery.Objectives: Since forensic pathologists always face the difficult task of excluding misleadingevidence when investigating cases of unnatural deaths or when the corpse has been movedfrom the original crime scene, this review aimed to provide a broad overview of the potentialapplications and advantages of forensic entomology data in criminal investigations in courts. Inaddition to highlighting the factors that can negatively influence the use of these entomologicalevidences.Conclusion: Various factors could affect the entomological data collected from a crime scene butcareful analysis accompanied by thorough observations and evaluations could overcome suchobstacles in an investigation.

The chemical profiles of the cuticle of adult flies are highly influenced by environmental factors and generational variation, although the extent and mechanisms of these influences are still poorly understood. This research rigorously investigates the influence of rearing environment and generational changes on the cuticular hydrocarbon (CHC) profiles of adult flies collected from the natural environment in Swindon (UK), in June 2019. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the hydrocarbon profiles. Then, chemometric analysis was applied to determine the chemical variation patterns, allowing the samples to be classified according to their chemical fingerprints. Significant differences in hydrocarbon composition were found between laboratory-maintained and field-collected specimens, underscoring the impact of environmental conditions on CHC expression. Additionally, gradual modifications in hydrocarbon content were detected across generations raised in the controlled environment, suggesting the involvement of adaptive physiological or epigenetic mechanisms. These findings contribute valuable insights into cuticle plasticity, highlighting its relevance in forensic entomology, chemical ecology, and insect evolutionary biology. The implications also extend to forensic investigations, where cuticular hydrocarbon profiles (CHCs) demonstrate potential for enhancing postmortem interval (PMI) estimation accuracy and species identification in criminal cases. By demonstrating quantifiable differences in CHC composition across rearing conditions and generations (AUC values ≥0.92 for all comparisons), this study provides a foundation for the broader application of chemical markers in forensic investigations.

The time elapsed after death is known as the postmortem interval (PMI). After death, the body undergoes changes such as algor mortis, rigor mortis, livor mortis, and putrefaction. In forensic medicine, PMI is traditionally determined using standard methods and forensic entomology, both of which can be affected by environmental conditions. Recently, genetic materials, which are less influenced by the environment, have gained attention in PMI studies. Cell-free DNA (cfDNA) comprises short DNA fragments released into the bloodstream due to cell necrosis and apoptosis. This study quantitatively analyzed cfDNA and specific microRNAs (miRNAs) - miR-122, miR-133, miR-1, miR-16, and let-7e - in serum samples from rats at various time intervals to assess their correlation with PMI. The results were analyzed using Ct, ΔCt, ΔΔCt, and fold change methods. Statistically, miR-16 and miR-122 had lower ΔCt values at all time points. miR-16 showed a lower ΔΔCt average at 24 and 48h and the highest fold change at 24h. Reference genes GAPDH and ACTB declined over time, with higher averages at 0, 6, and 12h compared to 24 and 48h. This research is the first to quantitatively explore cfDNA's potential role in PMI estimation.