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Forensic entomology is an important field of forensic science that utilizes insect evidence in criminal investigations. Blow flies (Diptera: Calliphoridae) are among the first colonizers of remains and are frequently used in determining the minimum postmortem interval (mPMI). Blow fly development is influenced by a variety of factors including temperature and feeding substrate. Unfortunately, dietary fat content remains an understudied factor in the development process, which is problematic given the high rates of obesity in the United States. To study the effects of fat content on blow flies we investigated survivorship, adult weight, and development time (overall and by sex) of Lucilia sericata (Meigen) and Phormia regina (Meigen) on ground beef with 10%, 20%, or 27% fat. As fat content increased, survivorship decreased across both species with significant impact to P. regina. While P. regina adults were generally larger than L. sericata across all fat levels, only L. sericata demonstrated a significant (P < 0.05) difference in weight by sex. Overall development times did not vary by fat content, excluding 27% for P. regina. Additionally, development times did not vary by sex for P. regina but did differ (P < 0.05) for L. sericata with females taking longer to develop. These findings provide insight into the effect of fat content on blow fly development, a factor that should be considered when estimating an mPMI. By understanding how fat levels affect the survivorship and development of the species studied here, we can begin improving the practice of insect evidence analysis in casework.

Lhasa is located on the Qinghai–Tibet Plateau, with an altitude of 3,650 m, and a unique geography. Its climate is dry and cold all year round. Forensic entomological studies of the region are scarce. In this study, the diversity and seasonality of necrophagous flies in eight counties among Lhasa region were determined, and succession of necrophagous flies colonizing on rabbits in the Chengguan area of Lhasa was studied, so as to provide reference data for estimating postmortem interval (PMI) and location of death. In total, 22 species of necrophagous flies, belonging to six families were identified in Lhasa. Protophormia terraenovae (Robineau-Desvoidy, 1830) (Diptera: Calliphoridae) was the dominant species throughout the year, the diversity index (4.5834) indicated that the study on necrophagous flies in the Lhasa region is representative.

The most widely used entomological method of determining the time since death (minimum postmortem interval, mPMI) has been calculating the developmental time of blow flies (Diptera: Calliphoridae) on the deceased body. However, because blow flies are known to be diurnal, nocturnal oviposition has been excluded from standard mPMI calculations. This has been challenged by recent studies demonstrating nocturnal oviposition due to an unknown reason. Therefore, this study investigated the role of chronobiology. We recorded the locomotion amount and pattern of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) under different chronobiological conditions and examined whether Ch. megacephala can oviposit under nighttime conditions in field and laboratory settings. Subjects were found to have a daily activity pattern under normal darkness conditions (12:12 L:D) and under continuous darkness (DD), but they exhibited no pattern under continuous light (LL). Free-running period was approximately 1,341 min/d (22.35 h/d). In the field, no flies were observed during nighttime. Oviposition occurred in the laboratory setting during daytime with no lights and during nighttime with artificial lights. Free-running subjects oviposited in both active and resting periods, with more eggs laid during active than resting periods. The result of this study indicates it is possible to induce oviposition behavior during evening hours on Ch. megacephala. However, this was only observed in the laboratory setting and could only happen during the flies’ subjective day.

The geographic ranges of forensically informative taxa on decomposing remains vary across regions. To determine which calliphorid flies would be expected to occur in Minnesota and the upper Midwest, individual freshly killed pig carcasses (Sus scrofa L.) were placed in the field in St. Paul, MN, at monthly intervals from May to October 2017 and May to September 2018. Aerial nets, forceps, and pitfall traps were used to collect and preserve associated adult and immature insects. Sixty-four forensically informative insect taxa were recorded, representing three insect orders and 14 families. Ten informative calliphorid species were recorded on carcasses, adding four new Minnesota records. Comparison of species lists from 26 human forensic cases indicated agreement between the two lists, except for Lucilia coeruleiviridis Macquart, Calliphora vomitoria (L.), and Cynomya cadaverina Robineau-Desvoidy, which occurred on pig carcasses but not human remains, and Calliphora livida Hall, which occurred on human remains, but not carcasses. The composite fauna list from cadavers agreed largely with the 2-yr list from pig carcasses.

An ecological study to evaluate necrophagous species richness, abundance, and diversity was conducted in four contrasting ecoregions of Los Angeles County, California, United States: a highly anthropized area, two moderately populated areas, and a sparsely populated area. Our hypothesis states that there will be higher abundance of exotic species in greatly anthropized ecoregions and higher diversity of native calliphorid flies in sparsely populated sites. A total of 2,883 blow flies belonging to 12 species were captured from the four ecoregions. The exotic Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) was the most abundant species, representing 72.84% of all Calliphoridae collected. The highest abundance was in the highly anthropized ecoregion. Differences of the blow fly assemblages across environmentally and anthropically different ecoregions were found, and that the relative abundance of the exotic species increased with anthropization. Our results suggest that both anthropization and elevation alter the structure of blow fly assemblages, modifying the abundance and species richness of Calliphoridae. The results of this study support the hypothesis of biotic homogenization, since we found a higher proportional abundance of exotic species and lower diversity of native blow flies in ecoregions heavily modified by human action. Additionally, the hypothesis of intermediate disturbance was supported as the highest diversity was found in the ecoregion with moderate anthropization mixed with natural elements. This is the first study to conduct an intensive survey of the Calliphoridae assemblages in Los Angeles County, which can be potentially used as a tool in human and veterinary health and forensic sciences.

The present study aimed to investigate the impact of gunshot residue (GSR) components (Pb, Sb, and Ba) on the life history parameters of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae). This experiment was carried out at the Zoology Department, the Ondokuz Mayıs University in 2020. About 50 larvae, respectively were exposed to the chicken liver with four different concentrations of GSR particles and life history parameters recorded. A two-way analysis of variance (Two way ANOVA) was used on the larval weight, and length, development time, percentage of pupal and larval survival. Pearson’s correlation coefficient was used to assess the association between life-history parameters and the concentration of GSR. The total development times decreased with increasing Sb, Ba, and Pb concentrations in the diets 1–3 d when compared to the control The results showed that Ba and Sb significantly decreased larval and pupal survival and GSR decreased the pupa and adult weight as compared to the control. We concluded that life-history parameters of L. sericata are sensitive to GSR residue and heavy metal changes in the environment. The presence of gunshot residue in the corpse should be kept in mind in a criminal investigation.

Carrion is a dynamic and nutrient-rich resource that attracts numerous insect species that undergo succession due to the rapid change in the carrion resource. Despite this process being well-understood, few studies have examined resource change as a driver of carrion insect succession, and instead have focused on the effects of time per se, or on coarse, qualitative measures such as decay stage. Here we report on three field succession experiments using pig carcasses and human cadavers encompassing two winters and one summer. We quantified the effects of resource change (measured as total body score, TBS), carrion type, initial carrion mass, ambient temperature, and season on insect species richness and community composition. We found that all variables had an effect on different taxonomic or trophic components of the insect community composition, with the exception of initial carrion mass which had no effect. We found significant positive effects of TBS on beetle species richness and composition, while fly species richness was not significantly affected by TBS, but was by ambient temperature. TBS had a significant positive effect on all trophic groups, while ambient temperature also had a significant positive effect on the necrophages and predator/parasitoids. Our study indicates that resource change, as indicated by TBS, is an important driver of carrion insect species turnover and succession on carrion, and that TBS can provide information about insect ecological patterns on carrion that other temporal measures of change cannot.

Entomotoxicology is an emerging science in the forensic investigation of death; it now influences judicial activities. Briefly, carrion flies ingest various chemicals as they feed on a cadaver. In entomotoxicology, such toxicological compounds are qualitatively and quantitatively analyzed in insects recovered from carrion. Some of these chemicals can disrupt flies’ cellular physiology, leading to acceleration or delay in their developmental rate. In this study, the effect of zolpidem-contaminated tissue on the dipteran flies Chrysomya megacephala (Fabricius 1794) and Chrysomya saffranea (Bigot 1877) was assessed. Zolpidem tartrate is a sedative-hypnotic compound commonly used to treat anxiety and sleeping disorders, but it is associated with death from suicide. The effects were tested by providing the flies with liver tissue treated with various concentrations of zolpidem tartrate. The morphological parameters and development rate for all life cycle stages of treated insects were determined and used to estimate postmortem intervals. Results showed that the morphological parameters, i.e., weight, width, and length and rate of development of C. megacephala and C. saffranea was negatively associated with zolpidem tartrate concentration of C. saffranea, were negatively associated with the concentration of zolpidem tartrate, indicating that arthropod evidence evaluation can help establish the cause and time of death.

Holometabolous insects undergo complete metamorphosis, and hence, they have different phases of development (egg, larva, pupa, and adult), which occupy distinct ecological niches. The pupae of several fly species are surrounded by the puparium, which is a rigid structure, usually formed by the integument of the last larval instar. The puparium presents unique characteristics distinct from those of the larval and adult phases. During intrapuparial development, it is possible to distinguish at least four fundamental and continuous steps, namely: 1) larval–pupal apolysis, 2) cryptocephalic pupa, 3) phanerocephalic pupa, and 4) pharate adult. The objective of this work was to describe the external morphology of the distinct phase of development for five species that were collected, identified, and raised in the laboratory; intrapuparial development was studied by fixing immature specimens at regular intervals; the morphological analyses were performed with the aid of both light and scanning electron microscopy. Under the conditions established (27 ± 1.0 or 23 ± 1.0°C, 60 ± 10% relative humidity, 12 h of photoperiod), the minimum time for intrapuparial development was: 252 h for Megaselia scalaris (Loew 1966) (Phoridae), 192 h for Piophila casei (Linnaeus 1758) (Piophilidae), Fannia pusio (Wiedemann 1830) (Fanniidae), and Musca domestica (Linnaeus 1758) (Muscidae), and 96 h for Chrysomya megacephala (Fabricius 1794) (Calliphoridae). Intrapuparial development has defined steps, and distinct species responded differently to the same environmental conditions. In addition, it is possible to establish a sequential rule without ignoring the specific characteristics of each taxon.

Fire ants (Solenopsis spp.) have increasingly been reported from carrion in the southeastern United States and are now a part of the normal succession community. There have been previous observations of these ants altering carrion and preying on other carrion-attendant fauna; however, the overall effects of these activities on carrion decomposition rates, community composition, and blow fly larval development are poorly understood. Alteration of these ecological processes by fire ants could affect the forensic interpretation of entomological data. We conducted a study in Mississippi and Florida whereby portions of the succession fauna were excluded from access to pig carrion to study the relative effects of fire ants and blow flies on carrion decomposition and succession: a control with all fauna having access, a second treatment where fire ants and other geophilic taxa were excluded, and a third treatment in which blow flies and other large organisms were excluded. Fire ants inflicted lesions in the carrion, buried portions that touched the ground, and preyed on some members of the succession fauna. Their exclusion did not affect carrion decomposition rates that were measured but slightly affected the overall carrion community, and strongly affected the oviposition and development of blow flies. Despite the presence of fire ants early in the control, blow flies were eventually able to overcome predation of eggs and larvae, continue colonization, and complete development; however, the delay in the colonization of blow flies on carrion could affect the determination of postmortem intervals when development rates of blow flies are considered in the calculation.

Cluster flies are represented by the genus Pollenia Robineau-Desvoidy, 1830 of the family Polleniidae Brauer and Bergenstamm, 1889. Their larvae are known to be internal parasites or predators of earthworms. Herein, we report for the first time the occurrence of the cluster flies Pollenia rudis Fabricius, 1794 and Pollenia vagabunda (Meigen, 1826) (Diptera: Polleniidae) on carcasses in Algeria and identify them through DNA barcoding. A region of the mitochondrial cytochrome c oxidase I gene (COI) was amplified and sequenced. Genetic distances were determined. A phylogenetic tree was constructed with the maximum parsimony method using 10 000 bootstrap replicates. A total number of 157 adults of P. rudis were collected together with 325 adults of Pollenia vagabunda. The occurrence of Pollenia on animal carcasses does not seem to be correlated with a particular stage of decomposition. All the sequences were correctly identified using the BLASTn tool from the GenBank database and the BOLD identification engine. Intra- and interspecific sequence divergence values were less than 1% and greater than 3%, respectively. COI barcodes obtained from this study were robust enough to identify and distinguish unambiguously between P. rudis and P. vagabunda. In the tree-based analysis, the cluster flies were all assigned to their respective species separately from each other confirming the morphological identification. These results provide DNA barcodes that contribute to the growth of reference databases and allow fast and accurate identification.

Chrysomya megacephala (Fab. 1794) (Diptera: Calliphoridae) is a very important species for forensic entomology, mainly contributing estimations of the postmortem interval (PMI) in judicial investigations. There are some doubts about the nocturnal oviposition of these flies, which could lead to errors in the PMI calculation. This study aimed to monitor the nocturnal oviposition behavior of this species through four experimental conditions carried out in laboratory. Ten cages, each containing five males and females (n = 100), were kept in a fume hood and subjected to total darkness or to artificial light for 11 consecutive hours. Two verifications were performed to determine whether the females deposited eggs on the substrate of ~20 g of chicken gizzards per cage. The first verification occurred at 9:00 pm in nocturnal experiments and at 09:00 am in diurnal experiments. The second verification occurred at 05:00 am in nocturnal experiments and at 05:00 pm in diurnal experiments. Each experiment lasted 5 d. Chrysomya megacephala deposited eggs at night under artificial light and in total darkness, but the amount of eggs was significantly lower when compared with the daytime experiments in dark conditions and under natural light. Oviposition occurred when the average temperature was around 25°C (± 2°C) and relative humidity around 73% (± 6%). Night oviposition is a possibility which should not be ruled out. Thus, future experiments are recommended.

Chrysomya albiceps (Wiedemann 1819) is one of the most important insects in forensic entomology. Its larval developmental and survival rates are influenced by nutritional resources, temperature, humidity, and geographical regions. The present study investigated the possibility of relying on body size and cuticular hydrocarbon composition as indicators for age estimation of the different larval instars of C. albiceps. Larvae were maintained in standardized laboratory conditions at different experimental temperatures. All larval instars (first, second, and third) were randomly collected for measuring their body sizes and for estimating their cuticular hydrocarbons at different rearing temperatures (30, 35, 40, and 45°C) using gas chromatography–mass spectrometry (GC–MS). Results indicated that the duration of larval stage was temperature dependent as it gradually decreased on increasing the rearing temperature (30, 35, and 40°C) except 45°C at which larval development was ceased. In contrary, larval body size, in terms of length, width, and weight, was temperature dependent as it gradually increased with larval development on increasing rearing temperature except at 45°C at which larval development was ceased. The GC–MS showed a significant difference in the extracted components of cuticular hydrocarbons between different larval instars reared in the same temperature and between the same larval instar that reared at different temperatures. Furthermore, the highest and lowest amounts of cuticular hydrocarbons were detected at 35 and 40°C, respectively. Overall, larval body size and cuticular hydrocarbon components were temperature dependent within the range 30–40°C, which may suggest them as possible reliable age indicators for estimating the postmortem interval in the field of medicolegal entomology.

Detecting and locating a carrion resource is critical for the reproduction of necrophagous insects and initiating forensically important timelines. Blow flies (Diptera: Calliphoridae) primarily use olfactory cues in the form of volatile organic compounds to locate a suitable resource. Factors governing detecting and locating a resource have been studied using various behavior assays with modifications to suit the experiment design, such as the dual-choice cube olfactometer, which was examined in the current study. Systems optimization ensures biologically relevant and consistent results across replicates. In this study, two responses were measured: 1) leaving cube for either control or treatment and 2) choice between control and treatment. Phenotype (e.g., male, non-gravid, gravid) and total blow fly, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), number to respond were measured. Four parameters were assessed for their impact on response: 1) adjustment time in cube before trial, 2) trial length, 3) sugar/water presence, and 4) screening type in arms. Approximately, 70% of all phenotypes responded to liver with the 30-min adjustment period where only 50% responded with other adjustment periods. Trial length had a significant impact on response (35% increase in the 8 h trial compared to shorter durations); however, significant response to treatment was lost by increasing trial length. The presence of sugar/water decreased gravid and non-gravid response by 35% but did not impact males. Screening had no influence on overall or treatment response. Data indicate experiment design impacts fly response. Future studies should optimize parameters for their given fly population prior to initiating experiments.

This study investigates the succession of insects and the length of the decomposition of rabbit carcasses injected with heroin. In total, 24 rabbits (2–2.5 kg, Oryctolagus cuniculus domesticus L.) were used in the study. Twelve carcasses were exposed to insect activity in winter (January and February) and 12 in summer (July and August) in Riyadh, Saudi Arabia. Three replicate experiments were conducted in each season. For each experiment, three rabbits were injected with varying doses of heroin, and the fourth rabbit was used as a control, being injected only with saline solution. Insects belonging to the order Diptera, Coleoptera, and Hymenoptera were attracted to the carcasses. In both of the investigated seasons, heroin appeared to delay the decomposition process but did not have a significant impact on the number of insects. In general, the number of flies in both seasons was substantially higher than that of beetles and ants. During the summer, flies were more attracted to treated carcasses with a higher dose of heroin. Some flies such as Musca domestica L. (Diptera: Muscidae) and Chrysomya albiceps Wiedemann (Diptera: Calliphoridae) and some beetles such as Pimelia boyeri Solier (Coleoptera: Tenebrionidae) were represented on the carcasses by different developmental instars (larvae, pupae, and adults). Overall, heroin has not potential effects on the insect succession patterns of insect, however, influences the decomposition of rabbit carcasses. These findings are important while using insects in medicolegal investigation, since heroin is one of the common poisons used as an addictive agent.

The early arrival and colonization of species belonging to the family Calliphoridae (Insecta: Diptera) on a corpse represent one of the most reliable means of estimating minimum postmortem interval (PMImin). However, information on the development and life cycles of some Argentine species in this family is not complete. The objective of this work was to contribute new information regarding the larval body size of neotropical species that allow, through the construction of forensic methods, the estimation of a more precise and specific PMImin. This work was conducted on laboratory cultures of larvae of Lucilia ochricornis (Wiedemann) and Lucilia purpurascens (Walker) using as average temperatures: 13.4, 15.1, 22.6, and 23.3°C, which represent the four seasons of the year for the province of Salta. With this information, we constructed isomegalen diagrams and growth models for the obtained variables of larval length and body weight. The mean values of length and body weight differ between both species, indicating that L. purpurascens exceeded L. ochricornis in both variables. In contrast, within each species the mean length and weight remained unchanged between culture temperatures for the three larval instars. Isomegalen diagrams can be used for the entire range of temperatures worked in the laboratory, but the body size entered is approximate. The growth models allow the use of point data but are specific for each culture temperature used.

Avoiding competition is thought to explain insect successional patterns on carrion, but few studies have looked at competition directly. We use replacement series experiments with three species of blow flies: Phormia regina (Meigen) (Diptera: Calliphoridae), Lucilia sericata (Meigen) (Diptera:Calliphoridae), and Chrysomya rufifacies (Macquart) (Diptera:Calliphoridae) to characterize competitive relationships. From experimental results, P. regina showed a significant competitive advantage over L. sericata. Infestation of carrion differs between L. sericata and P. regina; specifically, L. sericata oviposits on carrion without any delay, while P. regina typically delays oviposition. Our findings are consistent with the notion that differences in oviposition times represent a mechanism for L. sericata to avoid potential competition. Competition by C. rufifacies differs since C. rufifacies, in the event of a limited food supply, will prey on other maggot species. In replacement series experiments, C. rufifacies killed all P. regina in mixed treatments, representing an ultimate competitive advantage. In the United States, these two species do not often overlap because of differences in seasonal distribution. However, with climate change, phenological separation may grow less distinct. Surprisingly, in replacement series experiments with C. rufifacies and L. sericata, no competitive interactions were observed. In other studies, L. sericata has been shown to form clusters away from predaceous maggots, allowing improved survival, which may account for the absence of predation by C. rufifacies. Finally, this study shows that replacement series models are useful in measuring competition, supporting the notion that interspecific competition between necrophagous insect species may have driven life history traits of those species.

Accurate age estimates of immature necrophagous insects associated with a human or animal body can provide evidence of how long the body has been dead. These estimates are based on species-specific details of the insects’ aging processes, and therefore require accurate species identification and developmental stage estimation. Many professionals who produce or use identified organisms as forensic evidence have little training in taxonomy or metrology, and appreciate the availability of formalized principles and standards for biological identification. Taxonomic identifications are usually most readily and economically made using categorical and qualitative morphological characters, but it may be necessary to use less convenient and potentially more ambiguous characters that are continuous and quantitative if two candidate species are closely related, or if identifying developmental stages within a species. Characters should be selected by criteria such as taxonomic specificity and metrological repeatability and relative error. We propose such a hierarchical framework, critique various measurements of immature insects, and suggest some standard approaches to determine the reliability of organismal identifications and measurements in estimating postmortem intervals. Relevant criteria for good characters include high repeatability (including low scope for ambiguity or parallax effects), pronounced discreteness, and small relative error in measurements. These same principles apply to individuation of unique objects in general.Key pointsMetrological rigour can increase in forensic entomology by selecting measurements based on their metrological qualities.Selection of high-quality features for morphological identification of organisms should consider these criteria: (1) pronounced discreteness of features (minimising group overlap or maximizing interval); (2) high repeatability of assessment (such as symmetrical width rather than asymmetrical length); (3) small relative error in measurement (selecting the physically largest continuous rigid feature for measurement).These metrological principles also apply to individuation of unique objects in general.

This study determined the spatial and temporal dynamics of two native neotropical species flies of forensic interest, belonging to the Lucilia (Robineau-Desvoidy) genus. The study focused on their abundance and reproductive behaviors associated with different habitats and phenological parameters. In the Province of Salta, Argentina, monthly samplings were performed over 1 yr in urban, rural, and native habitats, at morning, mid-day, and afternoon periods, controlling the oviposition of captured specimens. Environmental variables were also assessed: cloudiness, precipitation, relative humidity, temperature, and tree cover. Lucilia purpurascens (Walker) appeared to be associated with native habitats, whereas Lucilia ochricornis (Wiedemann) was mainly associated with rural habitats, exhibiting distinct habitat preferences. Two ecotones were also identified: rural–urban and rural–native, suggesting rural habitats promoted habitable conditions at its margins. Both species were recorded at the end of winter to the middle of autumn, with an initial peak in early spring, and a second peak at late summer. These peaks were associated with the highest numbers of laid eggs. Lucilia purpurascens preferred high tree coverings, whereas L. ochricornis resisted areas with intermediate sun, suggesting limited sun exposure was important. The latter was also associated with daily flight activities; during the warm season, the densest catches occurred at morning and afternoon periods, whereas during the cold season, they occurred at mid-day. Climatic variables explained 77% of variability in terms of abundance and oviposition. Synergistic effects were observed between these variables, suggesting that these variables conditioned insect distribution and reproduction, and not just temperature per se.

Diptera Mesembrinellidae present great ecological importance as potential bioindicators and Calliphoridae stand out for their medical-sanitary importance as potential vectors of pathogens and causes of human and animal diseases such as myiasis. They beneficially aid in elucidating crimes in forensic entomology, contributing to determine the postmortem interval (PMI) and assisting in the healing process of necrotic wounds in larval therapy. This study aimed to inventory the Calliphoridae and Mesembrinellidae fauna in order to evaluate structural parameters and to apply faunal indexes in catching species in the Itaipu–Piratininga lagoon complex, in Niterói (Rio de Janeiro), Brazil. The collections were carried out monthly from September 2015 to August 2016 with fish bait (sardines) exposed for 48 h, totaling six traps installed in three physiognomies (mangrove, ombrophilous forest, and restinga). In total, 9,773 individuals were captured, divided into two families, five genera, and 11 species. Chrysomya megacephala (Fabricius, 1794) was the predominant species, while Mesembrinella bellardiana (Aldrich, 1922) was the least representative species, indicating the preservation level of this ecosystem. Ombrophilous forest was the physiognomy with greater diversity and restinga was the physiognomy with greater species dominance. The similarity index indicated greater similarity among the mangrove and restinga populations.

One of the problems faced by entomotoxicological studies is the large variability of experimental set-ups and the absence of harmonized protocols to compare the data and results obtained by different research groups. Among the wide range of influencing factors on the development and growth of insects, food substrates are remarkably relevant. This article proposes a standardized growth medium to be employed in future entomotoxicological studies on the scuttle fly Megaselia scalaris (Loew, 1866), (Diptera: Phoridae). This species plays an important role in forensic cases related to the decomposition of human remains found indoors and/or in concealed environments, because of their small size and ability to enter these locations at an earlier time than many other insects. Because of these traits, it can be used for the estimation of the minimum postmortem interval (mPMI). We achieved the formulation of a medium that overcomes two traditional disadvantages of culture media used to raise necrophagous Diptera, the unknown media composition and different growth rates from those reared in tissue. The proposed medium is a known composition formulation, free of xenobiotics, in which M. scalaris shows growth rates and development times similar to those obtained when it is reared in tissue. This new diet might be used in future studies to test the effect of substances of forensic interest (prescription or illicit drugs, poisons, etc.) on the larval development time, helping to adjust the estimation of mPMI based on the presence of such substances. Additionally, the formulation revealed some interesting data about nutritional requirements of this species.

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 usually estimate the minimum postmortem interval (PMImin) using the time required for the oldest immature insects found on the corpse to grow to its development stage and age at the time of discovery. The sheep blow fly Lucilia sericata (Meigen, 1826) is a carrion fly found nearly worldwide, and important in forensics. We studied the development time of L. sericata from egg to adult at constant temperatures of 16, 19, 22, 25, 28, 31, and 34°C, and found that the times required are 913.2 ± 19.4, 588.8 ± 35.8, 459.8 ± 15.2, 373.2 ± 15.3, 308.0 ± 9.7, 272.5 ± 9.2, and 267.5 ± 10.5 h, respectively. We established three development models to infer the age of the immature insect: isomegalen diagram, isomorphen diagram, and thermal summation model. In addition, a regression analysis was performed on the relationship between body length and total development time from hatching to dispersing. The thermal summation constant during the development of L. sericata is 6023.2 degree hours and development threshold temperature is 9.19°C. The results of this experiment provide a basis for the use of L. sericata in the estimation of PMImin.

Despite the fact that necrophagous flies are known to alter bloodstains and create unique artifacts, no research has occurred to date that has examined the characteristics of insect stains on textiles or fabrics. This study represents the first effort to characterize artifacts produced by adult Calliphora vicina Robineau-Desvoidy deposited on a range of shirt fabrics that varied in type, color, orientation, and yarn tension. In general, artifact morphology on any type of fabric was distorted in comparison to those observed on smooth and/or nonporous surfaces in previous studies. Consequently, distinction of artifact type could only be made broadly as digestive artifacts and transfer patterns, in which the latter was predominantly detected as tarsal tracks. None of the artifacts displayed satellite stains typical of human bloodstains found on textiles. Wicking was evident on all fabrics but was most pronounced with dri wick and jersey knit polyester in comparison to cotton knit. Digestive artifacts on any colored fabric, but especially with green and yellow shirt samples, resembled the reported color, size, and morphology of bloodstains generated in laboratory studies on a range of fabrics. Unique digestive artifacts were also detected as small, black, and nearly spherical. These defecatory stains did not appear to wet or wick into any of the fabrics. Digestive artifacts and tarsal tracks differentially interacted with front face stitch loops of clothing fabrics to yield distinct stain patterns. The implications of these observations in reference to bloodstain pattern analysis at crime scenes are discussed.

On rabbit carcasses (Oryctolagus cuniculus L.) exposed in open and shaded habitats, the relative abundance of insects and species richness were observed. The decomposition process was classified into four stages: fresh, bloated, decayed, and dry. Except for the decayed stage, the elapsed time for each stage of decomposition was similar between open and shaded habitats, with all carcasses reaching dryness in 13 and 14 d, respectively. In total, 2009 insects were collected during the decomposition process with 1,863 flies belonging to seven families and 15 species, and 146 beetles belonging to six species and three families. Insect abundances rose between the fresh and decay stages. The dominant families of insects included Calliphoridae and Muscidae (80.9% of the collected insects) and accounting for 38.07% of the species richness, whereas Histeridae accounted for 4.3% of the collected insects and 14.29% of the species richness. The open habitat attracted 1,233 insects belonging to 18 families, including 1,142 flies and 91 beetles, whereas the shaded habitat attracted 776 insects belonging to 18 families, including 721 flies and 55 beetles. Diversity level and ratios of exclusive species are also reported for each habitat (open 61.4%; shaded 38.6%). Between habitats, there were substantially separate insect communities, however. In addition, there was a substantial difference in the insect number and species between decomposition stages. This study demonstrates that the exposure status needs to be evaluated and examined when estimating the time since death.

Age determination of necrophagous flies serves as an important tool for postmortem interval (PMI) estimation in forensic investigations. Drugs or toxins in cadavers may alter the developmental time of larvae, and lead to deviation in PMI estimation. Methamphetamine (MA), as one of the most abused psychostimulant drugs in Asia and North America, is often involved in forensic entomotoxicological cases. This study investigated the effects of MA (0, 45, 90, and 180 ng/mg) on the developmental rate, morphology, and survival of Aldrichina grahami (Aldrich, 1930). The results showed that 1) the developmental time to reach the pupal instar was statistically slower for the larvae reared on rabbit mince containing MA than for the control; 2) the mean length of the larvae exposed to MA concentrations was longer than those of the control; 3) the mean weight of the pupae exposed to the highest concentration of MA was significantly lighter than those of the control; 4) the GC–MS method can detect the content of MA in A. grahami immatures and empty puparia (EP).

The age of a blow fly larval specimen found on a body can be used to estimate a minimum postmortem interval by comparing a physical feature of a larva (e.g., length) to a reference growth curve created under similar conditions. A better understanding of factors known to influence growth rates would lead to more precise estimates of larval age. A factor known to influence insect development, but almost always lacking in blow fly larval growth curves, is sex. We wanted to understand how sex might affect larval growth rates and therefore lead to more precise age estimates, in the forensically important blow fly Chrysomya megacephala (F.). We examined sex for an entire age cohort at 70 h and found on average, males were longer than females. To assess whether this difference would cause an investigator to interpret the results differently if comparing a larval specimen of one sex based on reference data from the other sex, we made sex-specific 95% prediction intervals. We did not find a difference when comparing a male specimen with the female prediction interval, but did find more females outside the interval than expected using the male interval. All females outside the male interval were shorter. Investigators commonly remove the largest individuals from a body to estimate age, so these females are less likely to be chosen. These results do not support the generation of sex-specific growth curves for this species, though this study’s narrow scope means more information is required before making a final conclusion.

Ecological succession of necrophilous insects follows a predictable sequence, related to their differential attraction to changing odor profiles associated with carrion and colonizing insects. However, the dependency of insect arrival on the duration of the carrion’s residency at a location has not been investigated. To assess the fidelity of necrophilous insects to carrion of specific decomposition ages, independent of its location, we monitored the decomposition of neonate pigs in one field and then simultaneously relocated carcasses of different decomposition ages to an ecologically similar but remote field. We examined the effects of decomposition age and relocation on the assembly of the necrophilous insect community, using a novel vented-chamber trap, which excluded all sensory cues except odors. Community composition differed over a 4-d decomposition period, showing that insects were differentially attracted to pigs of different decomposition ages. There was overall concordance between respective decomposition ages in the two fields, with similar relative abundances of taxa before and after transfer. Although different decomposition ages continued to attract different insects, differentiation of the necrophilous insect communities relative to the age of decomposition was less pronounced after transfer. The results of this study demonstrate that translocating a decomposing body to a new, but geographically and ecologically similar location continues the predicted insect succession, albeit with greater variance, based on olfactory cues alone. Several rare taxa were sampled only prior to relocation, including the first documentation of the invasive hairy maggot blow fly, Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae), in central North Carolina.

In forensic entomology, the minimum postmortem interval (PMImin) estimative is usually based on the oldest immature recovered from a local of death. The time spent by fly immatures in the intrapuparial period comprises more than 50% of their complete life cycles. An accurate estimate of the duration of this period will improve PMImin estimates. The blow fly Hemilucilia semidiaphana (Rondani 1850) was found in six criminal cases in the city of Curitiba. Even though there is data on the morphology of the larval instars and developmental rate of H. semidiaphana, the intrapuparial period has not been investigated. Here, we provide a detailed description of the intrapuparial morphological changes of H. semidiaphana, which might be useful to estimate minimum PMI. Samples of H. semidiaphana in the intrapuparial period were obtained from immatures reared on an artificial diet in incubators adjusted to 25°C or 20°C temperature regimes. Blow fly puparia of H. semidiaphana were fixed at intervals of 3 and 6 h until emergence of the adult. The external morphological traits of sampled immatures were analyzed using light microscopy. Our analysis of the intrapuparial period of H. semidiaphana provided 21 traits from which nine were age informative. These nine characteristics divide the developmental time (144 h at 25°C and 192 h at 20°C) into smaller sections. The developmental data provided, together with the time line allows a practical way to make interspecific comparisons as well as to estimate the age of H. semidiaphana based on the intrapuparial development.

Techniques for the survey of necrophagous dipterans are frequently designed for adult flies and often neglect forensically important information, such as the postfeeding larval dispersal following carcass colonization. In this study, we propose and test a novel trap design for the collection of dipteran larvae at the postfeeding stage. The Trap for Dispersing Larvae (TDL) consists of two semiindependent parts: 1) a 2-liter PET bottle, containing the attractant (carcass); and 2) a plastic box, which selectively captures postfeeding dispersing larvae. Based on a field test in a dry forest fragment in Brazil, the TDL trap captured ca. 1,500 larvae of 13 species of Calliphoridae, Muscidae, Fanniidae, and Sarcophagidae. The species richness of dispersing larvae recorded in our study was ca. 60% of that previously recorded in this area. As the TDL trap can assess larval dispersal without interfering with carcass decomposition, we demonstrate that it can be used to assess temporal variation in the dynamics of colonization. In the present study, most larval dispersal occurred at 5-d postexposure, but the traps remained efficient until 7 d, by which time rat carcasses had skeletonized. We conclude that the TDL trap captures the dispersing dipteran larvae with little interference in carcass decomposition processes. The trap also provides a representative sample of species and is simple to use and of low cost.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

The seasonal activity of adults of eight forensically important blow fly species [Calliphora croceipalpis (Diptera: Calliphoridae), Jaennicke, Lucilia sericata (Meigen) (Diptera: Calliphoridae), L. cuprina (Wiedemann) (Diptera: Calliphoridae), Chrysomya chloropyga (Wiedemann) (Diptera: Calliphoridae), Ch. albiceps (Wiedemann) (Diptera: Calliphoridae), Ch. marginalis (Wiedemann) (Diptera: Calliphoridae), Ch. putoria (Wiedemann) (Diptera: Calliphoridae), Ch. megacephala (Fabricius) (Diptera: Calliphoridae)] was quantified from collections in Makhanda, South Africa. Flies were collected in traps baited with chicken liver and placed in the field at eight locations for four consecutive days each fortnight for 14 mo. The seasonal distribution of each species compared well to published seasonal distributions of these blow flies elsewhere in South Africa, with evidence of year-to-year variation within seasons that might be explained by weather. This information is important for determining when and where certain species are likely to occur and will be of use in forensic investigations and myiasis management plans.

The age of insects found on corpses is often used to estimate the minimum postmortem interval by forensic entomologists. Insect development is affected by biotic and abiotic factors, and temperature is the most important environmental factor that determines the length of insect development. Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) is a globally distributed fly that is commonly found on corpses, and this study investigated the development of C. rufifacies from China at various constant temperatures. At 16, 19, 22, 25, 28, 31, and 34°C, the developmental time from egg to adult was 870.17 ± 11.50, 566.20 ± 8.79, 406.38 ± 10.98, 291.14 ± 4.71, 232.59 ± 5.96, 192.47 ± 3.45, and 160.48 ± 7.15 h, respectively. We established three developmental models for C. rufifacies to estimate the age of the developing insect: the isomorphen diagram model, the isomegalen diagram model and the thermal summation model. Regression analysis was conducted to obtain the relationship between body length and development time of the larvae from hatching to wandering. The developmental threshold temperatures of the egg, 1st instar, 2nd instar, 3rd instar, and wandering larvae, and intra-puparial stages were 12.28 ± 0.30, 11.74 ± 0.95, 12.70 ± 0.55, 11.68 ± 0.96, 10.53 ± 1.53, and 12.51 ± 0.41°C, respectively. The developmental threshold temperature, and the thermal summation constant during the entire developmental period were 3759.95 ± 170.80 degree hours and 11.96 ± 0.38°C, respectively. This study provides an improved dataset to estimate the postmortem interval of corpses using C. rufifacies.

The decomposition rate and insects attraction to carcasses are affected by a number of factors such as temperature, body mass, the presence of poisons, humidity, and insect activity. The present study was planned to investigate the influence of clothing on both the decomposition process and the presence of insects on rabbit carcasses. The study was performed in May 2016 and involved the use of 18 live mature rabbits (Oryctolagus cuniculus L.), which were euthanized by chloroform. Rabbit carcasses were divided into three replicates with six rabbits in each replicate. Rabbits in each replicate were further subdivided, so that three rabbits were wrapped with cloth, whereas the remaining three were left unclothed. Both clothed and unclothed carcasses reached the dry stage within 6 d. The results of this study showed that clothes neither affect the decomposition process nor the presence of insects (flies and beetles). The pattern of flies and beetles, however, did vary significantly in the different decomposition stages. In total, 2,849 insects, including 2,459 flies and 390 beetles, were collected and sorted during the decomposition process. Among the dominant species, flies were assigned to the families Calliphoridae and Muscidae, whereas beetles were assigned to the families Dermestidae and Histeridae. Lucilia sericata Meigen, Musca domestica L., Dermestes maculatus DeGeer, and Saprinus moyses Marseul were the most abundant species occurring on both the clothed and unclothed carcasses. In conclusion, this study emphasized that, the clothes have no effect when dealing with decay process and insect attraction to the carrions.

Determining the age of an insect collected in a corpse is a key element to estimate the minimum postmortem interval in forensic entomology. Along with models of accumulated degree-hours, the estimation of the age based on larval weight and length is among the most common methods for this purpose. Sarconesia chlorogaster (Wiedemann, 1830), along with other Calliphoridae, is an important species in forensics in South America. However, studies analyzing the weight and length of S. chlorogaster larvae during development have not yet been done. In this work, we develop growth models based on the weight and length of larvae in different temperatures and provide formulas that can be used to estimate the age of the larvae based on the weight and length for each temperature. Larvae were reared at six temperatures (10, 15, 20, 25, 30, 35°C), and the measurements of larval length and weight were taken during all development until they stopped feeding. For the measurements, we attempted to use methodologies that can be easily replicated and do not require equipment that could be of difficult access. The results of this work come to complement and improve the use of S. chlorogaster in forensic applications.

Coleoptera is one of the largest taxon among animals and exhibits diverse eating habits. When associated with decaying corpses, beetles can be of great value in estimating the postmortem interval. In order to consolidate a useful database for the forensic field, it is necessary to study the entomological fauna associated with the carcasses in different geographical regions since the diversity of insects varies according to the biogeoclimate zone. Thus, this study aimed to assess the influence of environmental and ecological factors on the composition and succession of beetles associated with pig carcasses exposed in southern Brazil. Collections were carried out during the hot/dry and cold/wet seasons. A total of 415 specimens belonging to 18 Coleoptera families were sampled. The highest total abundance (n = 329) and diversity (n = 44 taxa) were recorded in the cold/wet season, corresponding to approximately 80% of the total sampled from the two seasons. Dermestidae (26.7%) was the family most abundantly sampled. Regarding eating habits, in an increasing order of importance were necrophagous (43.3%), predator (31.6%), and omnivorous (0.05%). In the hot/dry season, there was no faunal succession. In the cold/wet season, the succession was more associated with differences in abundance than to the presence or absence of a specific taxon by decomposition stage. Considering all the factors analyzed in the current study, three species of beetles, Dermestes maculatus DeGeer (Dermestidae), Euspilotus azureus (Sahlberg, 1823) (Histeridae), and Oxelytrum discicolle Brullé, 1840 (Silphidae), could be identified as being of the greatest forensic relevance in this biogeoclimatic zone.

Hydrotaea spinigera Stein is a necrophagous species, widely distributed in Oriental and Australasian regions. Considering that the postfeeding larvae or puparia of this species can still be found in abundance at the advanced decomposition stage or even the skeleton stage of remains, it can serve as a good supplementary indicator for estimating the minimum postmortem interval (PMImin). This could also extend the range of PMImin when the primary colonizers are no longer associated with the corpse or have emerged as adults. This study investigated the development duration, accumulated degree hours, and larval body length changes of H. spinigera at seven constant temperatures ranging from 16 to 34°C, and established three development models for estimating PMImin, including isomorphen diagram, isomegalen diagram, and thermal summation model. At 16, 19, 22, 25, 28, 31, and 34°C, the development durations of H. spinigera from egg to adult stage were 1,412.6 ± 62.9, 867.4 ± 14.9, 657.1 ± 22.9, 532.3 ± 10.1, 418.8 ± 21.3, 379.8 ± 16.6, and 340.0 ± 20.3 h, respectively. The lower developmental threshold L0 was estimated as 10.50 ± 0.20°C, and the thermal summation constant K was 7,648.83 ± 146.74 degree hours. Using regression analysis, equations were obtained modeling the change of larval body length with time after hatching at different temperatures. This study provided basic data based on the growth and development of H. spinigera for the estimation of PMImin in forensic science.

The postmortem interval (PMI) estimation, in cases where the body was discovered in an advanced stage of decomposition, is predominantly based on entomological evidence. However, very few forensically important species are sufficiently known in detail to allow a practical application. One of them is the carrion beetle, Necrophila (Calosilpha) brunnicollis (Kraatz, 1877). Its development from egg to adulthood was studied under a range of ecologically relevant constant temperatures to find parameters of thermal summation models. Developmental sexual dimorphism and the presence of developmental rate isomorphy were investigated. Herein we present the lower developmental thresholds and sum of effective temperatures for all developmental stages of N. brunnicollis (egg, first–third larval instar, postfeeding stage, and pupae). We did not find any evidence of developmental sexual dimorphism nor was the presence of developmental rate isomorphy confirmed. Our results present the first thermal summation model of the East Asian carrion beetle that can be used for the PMI estimation.

Precise data regarding feeding habits of necrobiont species are a key element of food web and evolutionary ecology. They can also be used to assess the utility and value of those species for forensic entomology, where obligatory necrophagous species in particular are considered good bioindicators of postmortem or preappearance interval. However, the feeding habits of many species are known only from anecdotal field observations, often reduced to vaguely defined categories—predatory, necrophagous, or omnivorous. To address this issue, we designed a simple, in vitro behavioral experiment allowing the quantification of food preferences. Next, we applied it on Necrophila (Calosilpha) brunnicollis (Kraatz, 1877), which is a common carrion beetle of East Asia with unresolved food preferences. The results suggest that this species is preferentially necrophagous, thus valuable for forensic research. Importantly, however, our experimental design allowed us to reveal that it also readily feeds on larvae of Diptera, although they compose a minor proportion of its diet. This methodology can be applied to other species, and it could provide evidence for future decision making in forensic research.

Forensic entomology focuses on the analysis of insect larvae present at crime scenes to help identify unknown cadavers. Carrion-feeding maggots store food in a crop located at the anterior end of the gut. DNA recovered from the crop can be amplified, sequenced, and identified to determine the origin of the food. This information could help investigators to identify a missing victim if maggots are discovered at a crime scene in the absence of a corpse. Chrysomya albiceps (Wiedemann) and Musca domestica (Linnaeus) are primary forensic species. Little or no information on the identification of unknown cadavers using C. albiceps and M. domestica larvae is available, and we aimed to compare the effectiveness of using the crop contents of instars of C. albiceps and M. domestica larvae to identify corpses at different time intervals. Two hundred and forty larvae of both species were reared on rat and/or mouse liver, and DNA from crop contents was extracted after different time intervals from different instar larvae. DNA was amplified using specific primers that match the mitochondrial cytochrome oxidase subunit I (mt COI) gene of the food source. Results showed that food provided to larvae affected life span and the ability to be used to identify unknown cadavers. Chrysomya albiceps larvae proved more useful than M. domestica larvae. Moreover, crop contents of third instar larvae of both species fed for 24 h are more useful than contents from other time intervals in identifying unknown tissues. Results are promising and may help investigators to identify unknown/missing victims.

Background Blowflies (Diptera: Calliphoridae) are the most commonly found entomological evidence in forensic investigations. Distinguished from other blowflies, Aldrichina grahami has some unique biological characteristics and is a species of forensic importance. Its development rate, pattern, and life cycle can provide valuable information for the estimation of the minimum postmortem interval. Findings Herein we provide a chromosome-level genome assembly of A. grahami that was generated by Pacific BioSciences sequencing platform and chromosome conformation capture (Hi-C) technology. A total of 50.15 Gb clean reads of the A. grahami genome were generated. FALCON and Wtdbg were used to construct the genome of A. grahami, resulting in an assembly of 600 Mb and 1,604 contigs with an N50 size of 1.93 Mb. We predicted 12,823 protein-coding genes, 99.8% of which was functionally annotated on the basis of the de novo genome (SRA: PRJNA513084) and transcriptome (SRA: SRX5207346) of A. grahami. According to the co-analysis with 11 other insect species, clustering and phylogenetic reconstruction of gene families were performed. Using Hi-C sequencing, a chromosome-level assembly of 6 chromosomes was generated with scaffold N50 of 104.7 Mb. Of these scaffolds, 96.4% were anchored to the total A. grahami genome contig bases. Conclusions The present study provides a robust genome reference for A. grahami that supplements vital genetic information for nonhuman forensic genomics and facilitates the future research of A. grahami and other necrophagous blowfly species used in forensic medicine.

Geographical regions have a major effect on the arrival times of different insect species on carrion. This means that data generated in one region should not be used to determine time of death in a different region. In the present study, we demonstrate the effect of geographical location on the diversity of carrion beetles in Saudi Arabia, whereas the mitochondrial cytochrome c oxidase subunit I (mtCOI) barcodes were used as a marker for molecular identification of the carrion beetles at a dry stage on sheep carrion. We analyzed 819 adult beetles belonging to nine species originating from Riyadh (609 beetles), Jazan (157 beetles), and Arar (53 beetles). In Riyadh, results showed the presence of six species belonging to three different families. On the other hand, in Jazan five species belonging to four families were collected. From Arar, five species belonging to three families were collected. By comparing between individuals of the same species from different regions, individuals of Necrobia rufipes DeGeer (Coleoptera: Cleridae) showed the highest intraspecific variations 0-20%, while individuals of Saprinus splendens Paykull and Saprinus semistriatus Scriba (Coleoptera: Histeridae) showed the lowest intraspecific variations 0-1%. Interspecific variability was also measured between collected and identified species, with differences revealed to be in the range of 3.8-29.8%. The results are important from an ecological point of view and for Medico-Legal Forensic Entomology.

Forensic entomology is a complementary tool for penal procedures, mainly on estimating postmortem interval. Study of cadaveric fauna in various environments is primary as source of information to support this science. This study collected information about the fauna of Calliphoridae associated to carcasses of Rattus rattus in the Tijuca National Park, RJ. Four collections were conducted, one for each season of 2015, exposing six carcasses at georeferenced points in each collection. The carcasses were placed 550 m from the boarder and equidistant by 100 m. Five decomposition stages were identified, and 10,559 individuals of Calliphoridae belonging to 10 species were collected. The most abundant species were Hemilucilia semidiaphana (Rondani) (Diptera: Calliphoridae) and Lucilia eximia (Wiedemann) (Diptera: Calliphoridae). L. eximia was the most abundant species during the Swelling and Black Putrefaction stages, succeeded in the next stages by two species of the genera Hemilucilia. H. semidiaphana was the dominant species in the last two stages, followed by Hemilucilia segmentaria (Fabricius) (Diptera: Calliphoridae). The genus Mesembrinella (Diptera: Calliphoridae) occurred mainly during the Black putrefaction stage. Mesembrinella bellardiana (Aldrich) was more abundant, with higher occurrence during the Black putrefaction and Dry decay stages. Mesembrinella peregrina (Aldrich) occurred in the two last stages with low abundance. Huascaromusca aeneiventris (Wiedemann) (Diptera: Calliphoridae) occurred during all the observed stages, mainly during the Butyric fermentation stage. Huascaromusca purpurata (Aldrich) (Diptera: Calliphoridae) occurred only during the Dry decay stage and in low abundance. A succession pattern in the carcasses colonization was observed, providing relevant information for the resolution of criminal investigations in this environment.

A fundamental assumption of forensic entomology for estimating the postmortem interval is that insect species are accurately identified, which depends on diagnostic morphological characters. Larvae of the blow fly Paralucilia pseudolyrcea (Mello, 1969) (Diptera: Calliphoridae) were sampled from four corpses in the state of Paraná, Brazil, but despite the forensic importance of this species, morphological data for the identification of its larval instars are lacking, limiting its usefulness in such cases. Thus, the main goal of this study was to describe the larval instars of P. pseudolyrcea. The material was obtained from a colony established by larvae collected from a corpse of a murder case. Overall, the distribution of spines is a key character for identifying this species in the first, second and third instars. Other characteristics, such as the presence of an accessory oral sclerite, the small cirri, the number of lobes of the anterior spiracle and the morphology of posterior spiracles, separates P. pseudolyrcea from other necrophagous blow flies. The detailed morphological description provided here facilitates the identification of larval instars of P. pseudolyrcea and their differentiation from those of other calliphorid species.

Studies of the pre-colonization interval and mechanisms driving necrophilous insect ecological succession depend on effective sampling of adult insects and knowledge of their diel and successional activity patterns. The number of insects trapped, their diversity, and diel periodicity were compared with four sampling methods on neonate pigs. Sampling method, time of day and decomposition age of the pigs significantly affected the number of insects sampled from pigs. We also found significant interactions of sampling method and decomposition day, time of sampling and decomposition day. No single method was superior to the other methods during all three decomposition days. Sampling times after noon yielded the largest samples during the first 2 d of decomposition. On day 3 of decomposition however, all sampling times were equally effective. Therefore, to maximize insect collections from neonate pigs, the method used to sample must vary by decomposition day. The suction trap collected the most species-rich samples, but sticky trap samples were the most diverse, when both species richness and evenness were factored into a Shannon diversity index. Repeated sampling during the noon to 18:00 hours period was most effective to obtain the maximum diversity of trapped insects. The integration of multiple sampling techniques would most effectively sample the necrophilous insect community. However, because all four tested methods were deficient at sampling beetle species, future work should focus on optimizing the most promising methods, alone or in combinations, and incorporate hand-collections of beetles.

Forensic entomology could provide valuable data for the minimum postmortem interval (PMImin) estimation and other relevant information, such as causes and circumstances of death. Some representatives of flesh flies are one of the dominant necrophagous insects during early stages of decomposition, demonstrating unique biological characteristics compared with other necrophagous flies. Moreover, they lead to global health concerns as carriers of various pathogenic micro-organisms, and dominantly result in the traumatic myiasis. Thus, sarcophagid flies are considered important in decomposition processes for PMImin estimation. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. The aim of this paper is to provide a brief review on the species, distribution and biological habit of forensically important sarcophagid flies. In addition, the relation between traumatic myiasis and flesh flies, molecular identification methods and developmental pattern of flesh flies are summarized.

Aging blow fly eggs can be critical to a forensic investigation, but there are currently no forensically useful timelines describing internal anatomical changes in embryological development. This is partly due to the lack of an economical, rapid and technically simple histological technique to allow mass production of slides for research and casework. We present a histological method that uses a slightly modified standard laboratory processing run with 1 h fixation in 10% formalin, 2 h softening in Molliflex and Haemotoxylin and Eosin (H&E) staining. We also present a summary of the internal anatomical changes that can be visualized using our technique in the developing eggs of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). We examined eggs from at least three different females grown at 15 °C and sampled at 6 h intervals, and eggs grown at 20 °C and sampled at 3 h intervals. Blind aging trials demonstrated that it is possible to accurately age material grown at 20 °C to within 6 h (but attempts to further narrow this interval resulted in errors in one-third of cases). It was also possible to see sufficient anatomical detail to age eggs preserved for forensic casework 5, 8, 9, 10 and 11 years previously. Additionally, we determined that section quality was improved by 5 s fixation in hot water prior to preservation in ethanol. However, hot water fixation for longer than this increased the level of section artefact.

Forensic entomology evidence collected by police and mortuary staff may be delayed in getting to the entomologist. Live samples may continue developing and alter minimum postmortem interval (PMImin) estimates. This study investigated development of simulated evidential samples of Calliphora vicina Robineau-Desvoidy and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) maggots. Maggots of each species were studied in three developmental classes: “small” (late second/early third instar), “mid” (mid third instar) and “large” (late third instar). Seven replicates of 11 maggots in each class were assigned without food to four treatments: (1) 24 h at 20 °C; (2) 24 h at 4 °C; (3) 48 h at 20 °C and (4) 48 h at 4 °C. There was a significant difference in absolute length change across treatments, reflecting size for C. vicina, and interaction between size, time and temperature for Ch. rufifacies. Calliphora vicina maggots showed minimal mortality, and most second instar larvae moulted by the experiment's end. Chrysomya rufifacies showed heavy mortality and minimal moulting from the second to third instar. Only “large” Ch. rufifacies maggots kept at room temperature for 48 h pupariated. Since these results confirm that development can continue in live unfed maggot samples after their collection, it is not advisable to delay their preservation.

The value of the Formicidae (Insecta, Hymenoptera) community in forensic investigations is poorly studied in Portugal. In order to better understand the structure and dynamics of this group of insects in cadavers and their putative value in forensic investigations, studies were carried out in Lisbon area during one year. Piglet carcasses were used as a model of human decomposition. The entomofauna attracted to the carcasses was collected over a period of about 2 months in each season of the year. The collection of ants was performed at regular intervals, daily in the first 23 days and then with intervals of 2, 3 or 5 days until the end of the experiment. Five stages of cadaveric decomposition were recognized with the ants being present in all of them. Three hundred and nine specimens were captured: 7 in autumn, 6 in winter, 90 in spring and 206 in summer. These specimens belong to three subfamilies and seven different species. Four of them had never been mentioned before as being associated to cadavers in this geographical area. Spring and summer had the greater diversity of species and a larger number of individuals. Temnothorax luteus was the dominant species in spring and Tapinoma nigerrimum in summer. Corroborating the information of other similar studies carried out in the Iberian Peninsula, the sarcosaprophagous Formicidae community found in Lisbon is unique and different from other studied locations, which supports the need to perform regional studies. Our results shown that ants do not present a definite pattern of succession, but some species have the potential to be seasonal or geographic indicators.

Each biome has its own fauna and intrinsic local conditions that determine the succession patterns of insects on carcasses. For this reason, regional studies are very important to forensics. This study deals with the flies that visit carcasses of Rattus rattus (L., 1758) in the Atlantic Forest remnant of Floresta da Tijuca, Rio de Janeiro, Brazil. We compare the diversity and relative abundance of fly families and species throughout the four seasons of the year and test for correlations between certain environmental variables and fly richness. In each of the four seasons, we exposed the carcasses of three rats. The carcasses were put in traps that were installed on the ground, separated by 100 m. The flies were collected from the carcasses on a daily basis, and were taken to the laboratory and kept in a climate chamber (28 °C day and 26 °C night). A total of 5,537 flies were captured, identified into 12 families: Calliphoridae (4,884 specimens), followed by Sarcophagidae, Micropezidae, Fannidae, Muscidae, Phoridae, and Neridae. Canonical correspondence and redundancy analyses of the environmental variables indicated no relationship between the seasons and fly abundance. Carcass decomposition lasted longer in the winter (12 d) than in the summer (8 d). The Margalef richness index indicated higher richness in the spring and winter, and lower richness in the summer. The Shannon-Wiener index and Simpson dominance indicated greater diversity in the spring. Diptera colonized all stages of decomposition. The families Calliphoridae and Sarcophagidae were more prevalent during the Fermentation stage. Our data have important implications for forensic entomology.