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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.

Phormia regina (Meigen, 1826; Diptera: Calliphoridae) is a Holarctic species that rapidly colonizes carcasses and has been used as an indicator for determining the minimum postmortem interval. However, studies using morphological methods to estimate the intrapuparial age of P. regina are lacking. In this study, morphological changes within the puparium were observed under a stereomicroscope at 7 constant temperatures ranging from 16 °C to 34 °C.The intrapuparial period was categorized into 12 substages. Morphological indicators, including compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were recorded in detail. The observed morphological changes were divided into 6–10 substages, and the duration of each substage was also recorded in detail.The results of this study provide primary data for using the intrapuparial morphology of P. regina when pupae are collected at a crime scene and estimating the minimum postmortem interval.

Benzodiazepines are commonly identified in drug overdose deaths worldwide. However, research on their effects on the most common necrophagous insect species is limited. In this context, the current study investigated the effects of clonazepam and flunitrazepam on the development cycle of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera: Calliphoridae). Three blow fly colonies were reared under controlled laboratory conditions (24℃, 50% humidity, 12:12 light-dark cycle), and the experiment was carried out in triplicate. A solution of 4 mg of clonazepam and 2 mg of flunitrazepam, each dissolved in 50 mL of ultrapure water, was added to minced beef liver. The development cycle and growth rate were monitored daily, with a total of 2700 specimens weighed, covering each developmental stage except for the egg clusters. Statistical analyses using aligned rank transform (ART) ANOVA revealed significant interactions between the drug and developmental stages. Larvae exposed to benzodiazepines had higher median weights compared to controls, with more pronounced effects observed during the transition from the third instar larvae to the pupae stage. However, no differences were observed regarding the development cycle length between the three colonies. The findings suggest that clonazepam and flunitrazepam influence C. vicina morphology, particularly weight, which, when size is considered, has potential implications for forensic entomology in estimating the minimum postmortem interval (minPMI).

Necrophagous blow flies are a commonly used forensic tool to estimate the minimum postmortem interval (PMImin), where researchers collect development data under constant temperature regimes and construct models to estimate PMImin. However, the ambient temperatures of real death scenes are often fluctuant, which limits the reliability of data obtained under constant temperature regimes. Here we investigate the possible differences in the development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), an important species in forensic entomology. Chrysomya megacephala was exposed to nine temperature regimes with both large and small fluctuations, including a 19℃ constant temperature (19CT), 19℃ average with fluctuating temperatures 1 (19FT1) (16–22℃), a 19℃ average with fluctuating temperatures 2 (19FT2) (12–30℃), as well as 25CT (25℃), 25FT1 (22–28℃), 25FT2 (16–34℃), 28CT (28℃), 28FT1 (25–31℃) and 28FT2 (18–38℃). The total developmental durations of C. megacephala were 463.94 h (19CT), 534.50 h (19FT1), 507.78 h (19FT2), 273.70 h (25CT), 293.80 h (25FT1), 302.89 h (25FT2), 230.00 h (28CT), 245.84 h (28FT1) and 265.17 h (28FT2), respectively. The results showed that the developmental time of C. megacephala under fluctuating temperatures was longer than at constant temperatures. Except for 19℃, large amplitude fluctuating temperatures resulted in more developmental delays. Although there were numerical differences in the developmental time of almost all the stages of C. megacephala at fluctuating temperatures compared with constant temperatures, the Kruskal-Wallis test only found statistically significant differences during a few developmental stages (p < 0.05). We also used the larval body length equations constructed with constant temperature data to verify larval growth at fluctuating temperatures and found that there was a deviation between the estimated development time and the actual development time. These results are reference for the application and correction of C. megacephala data, aiding in the accurate estimation of PMImin. •Development of Chrysomya megacephala under fluctuating temperatures was studied.•Both small and large fluctuating amplitude were included.•The accuracy of the constant temperature body length equations was tested.•Differences were found under fluctuating temperatures that may affect PMI estimation.

Addressing global food waste requires innovative and sustainable solutions. This study investigates the potential of Chrysomya megacephala (Diptera: Calliphoridae) larvae to convert food waste into valuable byproducts, while evaluating the antimicrobial properties of larval extracts. Under controlled laboratory conditions, the larvae reduced the weight of food waste by 21.87%, demonstrating their effectiveness in waste degradation. The optimal food waste-to-sawdust ratio was identified as 10:1. The bioconversion process resulted in 111.60-fold increase in larval biomass when reared on food waste and a 153.20-fold increase on fresh pork liver, highlighting their efficiency in converting protein-rich substrates. Larval extracts demonstrated significant antimicrobial activity against Bacillus subtilis and Pseudomonas aeruginosa , with minimum inhibitory concentrations (MICs) of 100 µg/ml. Proteomic analysis revealed proteins with potential antimicrobial and antioxidative properties. Furthermore, the extracts promoted cell growth in vitro without showing cytotoxic effects on HaCaT cell lines, suggesting potential applications in wound healing and infection control. These findings highlight the capacity of C. megacephala larvae to reduce food waste while generating antimicrobial agents, offering a sustainable approach to waste management with promising implications in medical applications.

Many disciplines are utilized within the field of veterinary forensic sciences, including forensic entomology. Understanding the initial colonization period by flies of forensic importance can contribute to estimating the minimum postmortem interval. There is limited data regarding the time of colonization of animals with fur, and the interpretation of this data is difficult due to the variation in animal models used. The purpose of this study was to examine the initial insect colonization of cats (Felis catus), with light and dark fur. Twelve domestic short-haired cats were placed in cages 15.2 m apart in a grassy field in West Lafayette, IN, United States. Weather data (temperature, precipitation, sun/cloud exposure, humidity), insect activity, time to oviposition, and decomposition changes were documented. Eggs from initial oviposition events were collected and reared to identify the primary colonizing species. Although the time of first oviposition event was not different between the treatments, fur color did affect fly colonization, and cats with dark fur had more oviposition events than cats with light fur (t = 2.639, df = 4, P = 0.029). Three species of Lucilia (Diptera: Calliphoridae) colonized the cats on the initial day of placement. Further studies in cats should include the decompositional studies to understand the unique characteristics that occur during each stage of decomposition, which could aid in developing a scoring system for animals with fur. Additional studies could include analyzing how fur length would affect colonization.

The genetic structure of forensically important blow fly (Brauer & Bergenstamm) (Diptera: Calliphoridae) populations has remained elusive despite high relatedness within wild-caught samples. This research aimed to determine if the implementation of a high-resolution spatiotemporal sampling design would reveal latent genetic structure among blow fly populations and to elucidate any environmental impacts on observed patterns of genetic structure. Adult females of the black blow fly, Phormia regina (Meigen) (Diptera: Calliphoridae), were collected from 9 urban parks in Indiana, USA over 3 yr and genotyped at 6 polymorphic microsatellite loci. The data analysis involved 3 clustering methods: principal coordinate analysis (PCoA), discriminant analysis of principal components (DAPC), and STRUCTURE. While the PCoA did not uncover any discernible clustering patterns, the DAPC and STRUCTURE analyses yielded significant results, with 9 and 4 genetic clusters, respectively. Visualization of the STRUCTURE bar plot revealed N = 11 temporal demarcations indicating barriers to gene flow. An analysis of molecular variance of these STRUCTURE-inferred populations supported strong temporally driven genetic differentiation (FST = 0.048, F'ST = 0.664) relative to geographic differentiation (FST = 0.009, F'ST = 0.241). Integrated Nested Laplace Approximation and Boosted Regression Tree analyses revealed that collection timepoint and 4 main abiotic factors (temperature, humidity, precipitation, and wind speed) were associated with the genetic subdivisions observed for P. regina. A complex interplay between environmental conditions, the unique reproductive strategies of the blow fly, and the extensive dispersal abilities of these organisms likely drives the strong genetic structure of P. regina in the Midwestern US.

Estimation of the time of colonization (TOC) is often based on laboratory studies that document arthropod development. Precise data for forensically important species, such as blow flies (Diptera: Calliphoridae), are essential for accuracy in the estimate of TOC. Calliphorid development is a quantitative trait and thus depends on a host of variables. In calliphorids, studies showed photoperiod can play a role in development. However, there has been little research to date on the effects of photoperiod, and available data indicate the impact is species-specific. In this study, the effects of photoperiod on the development of Chrysomya rufifacies (Macquart), were examined. Chrysomya rufifacies is a fly of great medical and legal importance and is often encountered on vertebrate remains in temperate and tropic regions throughout the world, including North and Central America, Asia, and Australia. Larvae were reared under light regimes of 12, 16, and 24 h of light at 28.5 ± 0.0°C, 86.2 ± 0.3 RH. Minimum development time for each stage did not differ significantly for the applied photoperiods, nor were there significant differences in total minimum postembryonic development time. Photoperiod did not significantly affect larval size or growth rate. The data suggest that light durations investigated in this study do not influence the development of C. rufifacies. This indicates that photoperiod may not be a concern for forensic entomologists in Texas, United States, or other areas with similar conditions when estimating the TOC for this species. Validation efforts are encouraged to verify this conclusion.

Forensic entomologists use the maturity of necrophagous larvae to estimate the minimum post-mortem interval (PMImin), ideally taking account of effects that xenobiotics in the corpse may have on insect maturation. Forensic toxicologists may employ larvae to detect drugs in drug-related deaths when human samples are unavailable. Yet current pre-analytical practices of these two professions differ significantly, impeding the successful use of the same samples. Potential benefits of shared pre-analytical practices and opportunities for enhanced collaboration have yet to be fully explored. We employed Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae, raised in the presence of diazepam, to investigate the effects of two standard investigative practices on larvae for drug detection and for quantifying mass and length as proxies of age. Specimens were killed by either blanching or freezing and stored at −20℃ for either intermediate or long periods. Blanched larvae showed smaller changes in size and body integrity during storage, thereby producing the most reproducible estimates of PMImin. Consequently, data obtained from blanched larvae were used to evaluate the impact of diazepam on larval development. Diazepam exerted no significant effect on larval mass, and a weak effect on length. Diazepam recovery was significantly higher from blanched larvae, suggesting that freeze-killing causes drug loss. This model system demonstrates the value to forensic entomologists of the standard technique of blanching larvae, followed by storage at −20℃ for toxicological analysis. We recommend that forensic toxicologists consider blanching to kill larvae before storage at low temperatures, at least for certain drugs. This approach offers the dual benefit of high-quality specimens for both PMI estimation and drug detection. •Model systems can inform the development of standard entomotoxicological methods.•Killing samples of larvae by freezing causes artefacts in estimates of their maturity.•Blanched larvae produce better diazepam recoveries than larvae killed by freezing.•Shared, unified methodological guidelines are proposed for forensic entomotoxicology.•Diazepam has no significant effect on L. sericata larval mass and a weak effect on their length.

Studies under constant temperatures are the most common to estimate the Postmortem Interval (PMI). It is imperative that forensic sciences have data from studies carried out in the field. Therefore, this work aims to: (1) evaluate the parameters (weight, length, development time) associated with the life cycles of Lucilia ochricornis (Wiedemann) (Diptera: Calliphoridae) and Lucilia purpurascens (Walker) under experimental conditions in the field considering fluctuating temperatures, and (2) compare these results with those known and published by the same authors for cultures realized in the laboratory under constant temperatures; which will permit us to contrast the most widely used existing methodologies for forensic application in estimating the minimum postmortem interval (PMImin). For each season of the year, cultures of both species were made in the field, collecting information on temperature, humidity, and photoperiod to perform laboratory cultures, later comparing: development time, length, weight, and Accumulated Degree-Hours (ADH) in both types of cultures. Methods for estimating the PMI were obtained and validated with the information of the cultures grown in the field. The two types of cultures showed differences between each other for both species. The forensic use methods to estimate PMI were enhanced and their precision increased when maximum larval length data were used, and it was also concluded that feeding larval stages are the most accurate to be used in making estimates because the larva is growing. The estimation of the PMI through the use of necrophagous flies development remains reliable for obtaining the PMImin.