Development of Megaselia scalaris at constant temperatures and its significance in estimating the time of death.

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

A novel mathematical model and application software for estimating the age of necrophagous fly larvae.

The change in larval body length of necrophagous flies during their development is a key indicator for estimating larval age. However, existing forensic entomological models have limitations in this regard. In this study, a logistic algorithm was used to establish a general model for estimating the minimum postmortem interval (PMImin) using larval body length. The new model was used to simulate the relationship between larval body length and developmental time of eight species of necrophagous flies. The model parameters of body length variation with developmental time of the different species were calculated. Computer software was developed based on the established logistic model. The advantage of the new model is that each parameter has a biological meaning and can be used to estimate the age of larvae at any temperature and any larval body length. Cross-validation of the model showed that the overall mean accuracy of the fitted growth curves for the eight necrophagous fly larvae was 82.7%, the mean accuracy of age extrapolations for seven necrophagous fly species ranged from 76.8% to 92.9%, while the accuracy of age extrapolations for only one species was lower (i.e., 63.3%). This study provides a new method to estimate the PMImin based on larval body length, and the developed computer software will facilitate its application in forensic entomology.

A global perspective of forensic entomology case reports from 1935 to 2022.

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

Temperature-Dependent Development of Nitidula rufipes (Linnaeus, 1767) (Coleoptera: Nitidulidae) and Its Significance in Estimating Minimum Postmortem Interval.

Coleoptera, including the family Nitidulidae, are valuable for estimating long-term postmortem intervals in the late stage of body decomposition. This study showed that, under seven constant temperatures of 16, 19, 22, 25, 28, 31, and 34 °C, the developmental durations of Nitidula rufipes (Linnaeus, 1767) from oviposition to eclosion were 71.0 ± 4.4, 52.9 ± 4.1, 40.1 ± 3.4, 30.1 ± 2.1, 24.2 ± 2.0, 21.0 ±2.3, and 20.8 ± 2.4 days, respectively. The morphological indexes of body length, the widths of the head capsules, and the distance between the urogomphi of the larvae were measured in vivo. The regression model between larval body length and developmental durations was simulated for larval aging, and the head capsule width and the distance between the urogomphi at different instars were cluster-analyzed for instar discrimination. Based on the developmental durations, larval body length and thermal summation data were obtained, and the isomorphen diagram, isomegalen diagram, linear thermal summation models, and curvilinear Optim SSI models were established. The lower developmental threshold and thermal summation constant of N. rufipes evaluated by the linear thermal summation models were 9.65 ± 0.62 °C and 471.40 ± 25.46 degree days, respectively. The lower developmental thresholds, intrinsic optimum temperature, and upper lethal developmental threshold obtained by Optim SSI models were 10.12, 24.15, and 36.00 °C, respectively. The study of the immature stages of N. rufipes can provide preliminary basic developmental data for the estimation of minimum postmortem interval (PMImin). However, more extensive studies are needed on the effects of constant and fluctuating temperatures on the development of N. rufipes.

Estimating the intra-puparial period of Hydrotaea spinigera (Stein,1910) (Diptera: Muscidae) with morphological and gene expression changes.

Hydrotaea spinigera (Stein, 1910) (Diptera: Muscidae) is a forensically important sarcosaprophagous species widely distributed throughout the Oriental and Australasian regions. At the advanced decomposition stage or the skeletonize stage, the immature stages of H. spinigera, especially the pupae, can still be found in large quantities and could be used as important indicators to estimate the minimum postmortem interval (PMImin). However, there have been no studies on the intra-puparial period of this species. Herein, we studied morphological and differential gene expression changes during the intra-puparial development of H. spinigera, aiming to accurately estimate the intra-puparial age of H. spinigera. The intra-puparial morphological changes of H. spinigera were observed at seven constant temperatures ranging from 16 °C to 34 °C and divided into 12 sub-stages. Structures that could be used to estimate the intra-puparial age, such as compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were observed in detail, and the developmental process of each structure was divided into 5 to 10 stages. The time range of each sub-stage, or when a structure appeared, was recorded. For the gene expression section, the most suitable reference genes were screened by geNorm, NormFinder, BestKeeper, and ΔCt methods. Based on the selected reference genes, real-time quantitative PCR (RT qPCR) was used to detect the expression changes of the white and hsp90 genes with developmental time at 19 °C, 25 °C, and 31 °C. Results showed that the trend of hsp90 gene expression under different temperatures was not consistent, while white genes exhibited regular changes during development, and could thus be used for age estimation of H. spinigera. This study provides an important basis for forensic entomology to use morphological and differential gene expression for estimating the age of H. spinigera during the intra-puparial period. Moreover, the combination of the two methods can produce a more accurate minimum postmortem interval (PMImin) estimate compared to when each method is used separately.

A Preliminary Study of Body Decomposition and Arthropod Succession in an Arid Area in Northwest China During Summer.

The study of insect fauna and their development and succession patterns on decaying cadavers is crucial to promoting insect evidence as a useful tool in forensic science, particularly for estimating the postmortem interval (PMI). Body decomposition and arthropod succession are affected by many factors and exhibit substantial regional variations; therefore, detailed succession studies in different biogeographic regions are required for understanding the successional patterns of insects in various environments. This study was conducted in the summer of 2021 using three domestic pig carcasses (Sus scrofa domestica L., 1758) in the field of Shizuishan City, Ningxia, Northwest China. A total of 40 species of necrophagous insects belonging to three orders and 16 families were collected. Among Diptera, Lucilia sericata (Meigen,1826), Chrysomya megacephala (Fabricius,1794), and Phormia regina (Meigen,1826) (Diptera: Calliphoridae) were the dominant species. As for Coleoptera, the dominant species changed throughout the process of carcass decomposition from Saprinus semipunctatus (Fabricius,1792) (Coleoptera: Histeridae) to Dermestes maculatus DeGeer,1774 and Dermestes frischii Kugelann,1792 (Coleoptera: Dermestidae). The carcasses desiccated rapidly and reached the remains stage under extreme conditions of high temperatures and low humidity, after which a large amount of dried tissue of the carcasses attracted populations of Coleoptera, particularly Dermstidae, which were abundant and remained until the end of the experiment on day 50. The current study is the first forensic entomological investigation of succession in Northwest China and provides basic data for the estimation of PMI during summer in this region.

Succession patterns of sarcosaprophagous insects on pig carcasses in different months in Yangtze River Delta, China.

The Yangtze River Delta is one of the most economically developed regions in China, and many cases in the area require an estimation of the postmortem interval each year. This article reports forensic entomological research conducted in this region in different months. A total of 18 domestic pigs were used to study carcass decomposition, species composition, development events, and succession patterns of insects in different months from 2015 to 2021. A total of 53 arthropod species were identified, of which 38, 40, 41, and 25 were observed in April, June, September, and November, respectively. We demonstrated that insects vary in number and species from month to month, and the same species vary in number among the different months. Larvae of Calliphoridae mainly drove the decomposition of carcasses, and the decomposition rate of carcasses varied over the months. Particularly, the decomposition rate was highest in June and September, slower in April, and slowest in November. The predominant species on the carcasses also varied from month to month; Calliphora grahami Aldrich, Chrysomya pinguis (Walker), Lucilia sericata (Meigen) (Diptera: Calliphoridae), and Hydrotaea spinigera Stein (Diptera: Muscidae) were most predominant in April; Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were most predominant in June and September; and Ca. grahami was most predominant in November. In addition, the seven dominant insect species displayed four developmental events, which could be used to determine minimum postmortem interval, including adult arrival (T1), larvae wandering (T2), pupation (T3), and eclosion (T4) time on the carcasses. Insect succession patterns on carcasses also varied across months. For example, Ch. megacephala appeared later in the composition stages and was not numerically dominant in April but appeared earlier and was dominant in June and September. The results of this study guide the use of insect evidence in different months in the Yangtze River Delta region of China for estimating the postmortem interval of carcasses.

Assembly and phylogenetic analysis of the mitogenome of Piophila megastigmata (Diptera: Piophilidae), a forensically important species.

Piophila megastigmata (McAlpine, 1978) (Diptera: Piophilidae) is a forensically important species, but it was not found in China until recently. In this study, the first complete mitogenome of P. megastigmata was sequenced using the next-generation sequencing, which was also the first mitogenome of Piophilidae. Our mitochondrial assembly has a length of 15,410 bp, which consists of 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes, and a non-coding control region. This study provides a molecular basis to infer the minimum postmortem interval with P. megastigmata and a better understanding of the phylogeny of the order Diptera.

Development of Necrobia ruficollis (Fabricius) (Coleoptera: Cleridae) under Different Constant Temperatures.

Necrobia ruficollis (Fabricius, 1775) (Coleoptera: Cleridae) is an important cosmopolitan storage pest, and also frequently appears on highly decomposed and skeletonized corpses. It is a forensically important species expected to indicate a longer postmortem interval (PMI). Therefore, we investigated the development of N. ruficollis at five constant temperatures between 22 °C and 34 °C. Under temperatures of 22, 25, 28, 31, and 34 °C, the mean (±SD) developmental durations from eggs to adults were 93.00 ± 1.63, 70.67 ± 0.94, 65.33 ± 3.40, 47.33 ± 0.94, and 56.66 ± 8.73 days, respectively. According to the developmental time and accumulated degree hours results, an isomorphen diagram and thermal summation model were generated. The calculated values of developmental threshold temperature and accumulated temperature constant were estimated by a linear model to be 14.51 ± 0.52 °C and 684.12 ± 33.85 degree days, respectively. Lower developmental thresholds, intrinsic optimum temperature, and upper lethal developmental threshold temperature were estimated by a nonlinear model to be 14.61, 25.90, and 34.94 °C. Morphological indexes of larvae were obtained by in vivo measurements. A growth curve and an equation of the relationship between development time and body length were simulated. In addition, the widths of the head capsules and the distance between the urogomphi of larvae at different instars were determined by cluster analysis. Classifiers were created and validated by linear discriminant analysis. These results provide important basic developmental data for using N. ruficollis to estimate the minimum postmortem interval (minimum PMI). However, this study was only conducted under constant temperature, and the applicability of these data to variable temperature conditions needs to be further confirmed.

The Postmortem Interval of Two Decedents and Two Dog Carcasses at the Same Scene Based on Forensic Entomology.

In this paper, we report the estimation of the minimum PMIs of two human corpses and two dog carcasses using entomological evidence. Corpses of an elderly couple and carcasses of four dogs were found scattered on different floors in a house. The scene was very dirty. In addition, there were 12 emaciated live dogs at the scene. The corpses had been eaten by the dogs to different degrees, but the damage was greater on the man's corpse. After forensic examination, it was concluded that both individuals died of natural causes. The minimum PMIs of the two individuals and the two dogs were estimated using entomological evidence. The minimum PMIs of the other two dogs were not estimated because of the risk of contamination with the human corpses. Different insect species were found on each of the corpses and carcasses. The minimum PMIs were estimated as about 8.75 days for the woman, 4.17 days for the man, 3.13 days for the dog found in the stairwell and about 28.80 days for the dog found in the toilet. These estimations coincided with the time the woman stopped communicating with her daughter and when the electricity consumption at the house decreased significantly.