Effects of ivermectin on development of Calliphora vicina, Robineau-Desvoidy 1830 (Diptera, Calliphoridae).

Ivermectin is one of the most widely used drugs for parasite control. Previous studies have shown a reduction in the abundance and diversity of "non-target" coprophilous organisms due to the presence of ivermectin (IVM) in bovine faecal matter (FM). Due to its breadth of behavioural habits, Calliphora vicina is a suitable dipteran species to evaluate the effects of IVM in FM. The aim of this work was to evaluate the effect of five concentrations of IVM in FM (3000, 300, 100, 30, and 3 ng/g) on the development of C. vicina. The following endpoints were evaluated: survival (between the first larval stage and emergence of new adults), larval development times to pupation and pupation times to adult, and adult emergence (% sex) and LC50. Sampling was performed from larval hatching at 60 and 120 min and at 3, 4, 5, and 12 h, and every 24 h specimens were weighed until pupae were observed. Data were analysed by ANOVA using a non-parametric Kruskal-Wallis test and as a function of elapsed development time and accumulated degree hours (ADH). Mortality at 3000 and 300 ng/g was 100% and 97%, respectively. There were statistically significant delays in adult emergence time (p = 0.0216) and in the ADH (p = 0.0431) between the control group (C) and 100 ng/g. The LC50 was determined at 5.6 ng/g. These results demonstrate the lethal and sub-lethal effects of IVM on C. vicina, while highlighting the usefulness of this species as a bioindicator for ecotoxicological studies.

The influence of altitude on the abundance and occurrence of species of the family Calliphoridae (Diptera) in corpses in a Mediterranean area.

A carcass is defined as a temporary resource that can support high levels of diversity compared to other resources. The level of diversity often depends on the environmental conditions in which the corpse is found. Calliphoridae (Diptera) are the most important necrophagous insects used in forensic investigations because this family is common, widespread, abundant, and usually the first to colonize a corpse. However, understanding the processes responsible for variation in calliphorid species abundance and richness along gradients in ecology remains a key challenge. In this study, we analyzed the influence of altitude on the abundance and diversity of Calliphoridae species in corpses at three different altitudes (400, 900, and 1500 m a.s.l.) in a mountainous area of southeastern Spain. The results revealed a gradient of decreasing abundance with increasing altitude. An altitudinal substitution of species was observed, with Chrysomya albiceps being found at the lowest altitude and Calliphora vicina at the highest altitude. Seasonal variation was also noted, with Ch. albiceps being the dominant species in the warmer months and C. vicina being the dominant species in winter. Our results confirm the importance of Calliphorids as seasonal, altitudinal, and environmental indicators, given the wide distribution and abundance of this family. This information is of great interest on the interpretation of data in forensic practice.

Morphological changes of larvae and pupae of Lucilia sericata (Diptera: Calliphoridae) reared at two temperatures and on three food types.

Determining the minimum postmortem interval (minPMI) from an entomological perspective relies mainly on development data recorded for various species of flies collected from a crime scene or suspicious death. This study focused on the larval and pupal development of Lucilia sericata (Meigen), with an emphasis on the changes of the external morphology of the puparium and its pupal content throughout the duration of metamorphosis. Colonies of L. sericata were reared on 3 types of swine tissue (skeletal muscle, liver tissue, and heart tissue) at 2 different temperature regimes; 24 ±â€…1 °C and 30 ±â€…1 °C. The overall developmental time, larval width and length, and inner and outer pupal morphology changes were observed and recorded. The results show that: (i) temperature significantly influenced overall development time, as well as changes in larval width and length, but this effect was not dependent on tissue type; (ii) larval development duration was longest on heart tissue, and shortest on skeletal muscle for both temperatures; and (iii) pupation was longest for larvae reared on skeletal muscle at 24 ±â€…1 °C, and on liver tissue at 30 ±â€…1 °C, while those larvae reared on liver tissue at 24 ±â€…1 °C and heart tissue at 30 ±â€…1 °C had the shortest pupation period. A seven-character checklist plus 4 landmark stages were developed comprising the external morphology of the puparium and pupal content changes of L. sericata. In conclusion, the study provides larval and pupal development timetables, as well as checklists and photo guides for pupal character development that may be useful for future postmortem determinations.

Initial blow fly (Diptera: Calliphoridae) colonization of cats (Felis catus) in Indiana.

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

Distinguishing Four Calliphoridae Species (Diptera) from Jamaica Using the Cephalopharyngeal Skeleton: Application to Forensic Investigations.

Flies of the family Calliphoridae, commonly called blow flies, are important in the decomposition process. Knowledge on their succession pattern on corpses, species identification and the duration of their life cycle stages can be useful in forensic investigations especially when estimating the post-mortem interval. We performed linear-based morphometrics on the cephalopharyngeal skeleton of four blow fly species found in Jamaica to distinguish species and determine larval development stage. We collected eggs from pigs' heads used as bait in the field and conducted rearing exercises in the laboratory. We used the internal skeletonized structure, the cephalopharyngeal skeleton, to develop a practical and efficient method for species identification. For the first instar, we found species can be differentiated using all the measurements analysed in the study. We found that the mouth hook length may be useful in distinguishing larvae in the second instar. For the larvae in the third instar, the whole length of the skeleton, from mouth hook to length of the dorsal cornue, may be useful for separating species. We provide information on the cephalopharyngeal skeleton of Lucilia lucigerens (James), a blow fly species endemic to Jamaica, for the first time. Our work provides relevant information that could be utilized for species identification and life stage determination if fly evidence is to be incorporated in forensic investigations in Jamaica.

The microbiome of wild and mass-reared new world screwworm, Cochliomyia hominivorax.

Insect population control through continual releases of large numbers of sterile insects, called sterile insect technique (SIT), is only possible if one can mass-rear large quantities of healthy insects. Adaptation of insect stocks to rearing conditions and artificial feeding systems can have a multitude of negative effects such as inbreeding depression, reduced compatibility with wild strains, unintentional selection for traits that lower fitness after release, and an altered microbiome. Changes to insect microbiomes can have many effects on insects ranging from a reduction in sex pheromones or reduced fitness. Thus understanding these systems is important for mass rearing and the performance of the sterile insect control programs. In this study we explored the microbiome of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) an economically important parasite of warm-blooded animals. Samples from myiases in cows and wild adults were compared to and mass-reared flies used by the SIT program. Significant differences were observed between these treatments, with wild captured flies having a significantly more diverse microbial composition. Bacteria known to stimulate oviposition were found in both wild and mass-reared flies. Two bacteria of veterinary importance were abundant in wild flies, suggesting screwworm is a potential vector of these diseases. Overall, this study provides the screwworm eradication program a platform to continue exploring the effects associated bacteria have on screwworm fitness.

Postmortem Interval Estimation and Validation Through a Comparative Study of South American Flies Reared in the Field Versus Laboratory Conditions.

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

Initial Work on Sex-Specific Growth Curve Guidelines for Forensically Important Blow Flies Using Chrysomya megacephala (Diptera: Calliphoridae) Entire Age Cohorts.

Sex is a factor influencing development in many insect species, but is not widely studied in forensically important blow flies. If sex influences blow fly development, knowing the sex of a larva from a corpse can increase precision in estimates of that larva's age. The improved prediction of larval age will make estimates of time since death using entomological evidence better. Larvae lack sexually dimorphic morphological characteristics, so sex is not immediately known visually. To generate sexually dimorphic reference growth curves, a subsample must be large enough to ensure enough males and females are present for comparison. Using two entire age Chrysomya megacephala (Fabricius) cohorts, we evaluated the minimum sample number needed to have enough individuals of both sexes for comparison using 95% prediction intervals. Through a simulation of three trials of 1000 random replicates, we determined that a sample size of 19 would prevent any instance of a comparison not occurring because of insufficient sampling from one sex. As the current method for molecular sex determination can be expensive, we also compared how the results of various subsampling percentages compare those of the entire age cohorts. We found that subsampling at least 50% of an entire cohort leads to almost identical results in comparison to the entire age cohort. Together, these findings will help establish guidelines for generating sex-specific reference growth curves. A uniform approach to generating these sex-specific growth curves will lead to more consistency in age estimates made from them.

Calliphoridae (Diptera) on Decomposing Pig Carcasses and Human Cadavers in the Upper Midwest of North America.

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.

Oral Toxicity of Pseudomonas protegens against Muscoid Flies.

The bioinsecticidal action of Pseudomonas protegens has so far been reported against some target insects, and the mode of action remains unclear. In this study, the pathogenicity potential of a recently isolated strain of this bacterial species against fly larvae of medical and veterinary interest was determined. Preliminary experiments were conducted to determine the biocidal action by ingestion against Musca domestica and Lucilia caesar larvae, which highlighted a concentration-dependent effect, with LC50 values of 3.6 and 2.5 × 108 CFU/mL, respectively. Bacterial septicaemia was observed in the body of insects assuming bacterial cells by ingestion. Such rapid bacterial reproduction in the hemolymph supports a toxin-mediated mechanism of action involving the intestinal barrier overcoming. In order to gain more information on the interaction with the host, the relative time-course expression of selected P. protegens genes associated with virulence and pathogenicity, was determined by qPCR at the gut level during the first infection stage. Among target genes, chitinase D was the most expressed, followed by pesticin and the fluorescent insecticidal toxin fitD. According to our observations and to the diversity of metabolites P. protegens produces, the pathogenic interaction this bacterium can establish with different targets appears to be complex and multifactorial.

Age-dependent gene expression of Calliphora vicina pupae (Diptera: Calliphoridae) at constant and fluctuating temperatures.

Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5-15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data.

Balance between larval and pupal development time in carrion blowflies.

Several studies have highlighted the impact of environmental factors such as food type or larval density on the development of blowfly larvae. We investigated how changes in development speed (due to larval density and group composition) are divided among feeding and post-feeding stages. Even if these parameters impinge only on feeding larvae, they may ultimately also affect their subsequent development, and especially metamorphosis duration. Therefore, this study analysed the effect of larval density and group composition on the rhythm of necrophagous blowfly development. Based on laboratory studies, we highlighted that Calliphora vicina individuals with a fast development during their feeding phase developed slower in the later post-feeding phase (i.e., they had a compensatory effect). Lucilia sericata, a calliphorid species also frequently found on carrion at the same time as C. vicina, showed a different developmental strategy by not making its post-larval development speed dependent on the larval development speed. Finally, while a compensatory effect may exist, variations in the development rate more often accumulate through life-stages and resulted in a larger variability for later development instars. In this respect, the inclusion of detailed development duration covering all life stages, including post-feeding, is recommended in future studies, especially those dedicated to forensic entomology.

Life Cycle of Chrysomya rufifacies (Diptera: Calliphoridae) Under Semi-Controlled Laboratory Conditions.

Adult Calliphoridae flies, as well as their immature stages collected from carcasses, have been used as evidence in forensic investigations to estimate the postmortem interval (PMI), particularly those of the genus Chrysomya as it is one of the first genera to colonize a corpse. Chrysomya rufifacies (Macquart 1842), due to its appearance in cadaveric remains, plays a fundamental role in the study of forensic entomology. For this reason, we determined the biological cycle of C. rufifacies under semicontrolled laboratory conditions: uncontrolled average fluctuating temperature of 29. 76 ± 3. 22°C, uncontrolled average fluctuating humidity of 48. 91 ± 11.13%, and a controlled photoperiod of 12/12 (L/O). We established that the total development time from oviposition to adult emergence of C. rufifacies was 6. 5 d. The eggs took 12 h to hatch after oviposition. The complete larval stage took 60 h (instar 1 = 12 h, instar 2 = 12 h, instar 3 = 24 h, instar 3 post-feeding = 12 h). The pupa had a duration of 84 h. The species needed a total of 4642.8(±4.59) accumulated degree-hours (ADH) to complete its biological cycle.

Myiasis in humans: Case reports in Northeastern Brazil including multispecies co-infestation by Sarcophagidae.

Myiasis is a parasitosis characterized by an infestation of living vertebrates (humans and other animals) by Diptera larvae, whose occurrence and etiological identification are still neglected by health professionals. Here we analyzed the human myiasis cases registered from 2010 to 2018 in health care units in the municipality of Natal, Northeast region of Brazil. Specifically, we aimed to: I) analyze the medical records of cases documented from 2010 to 2017; II) list the patients predisposing factors; and III) monitor the recent cases diagnosed in health units between August 2017 and March 2018 and report the taxonomic identity of the infesting species. Our data revealed that myiases mainly affect the elderly and individuals with predisposing medical conditions (e.g. senility, filariasis). Regarding the new cases, larvae of Calliphoridae (C. hominivorax (Coquerel, 1858)) and Sarcophagidae [Sarcophaga (Liopygia) ruficornis (Fabricius, 1794), Peckia (Sarcodexia) lambens (Wiedemann, 1830), and Helicobia morionella (Aldrich, 1930)] were identified. Furthermore, this was the first report of a co-infestation with three Sarcophagidae species and also the presence of H. morionella in a human wound. These results reinforce that myiasis is an underreported parasitosis, which may underestimate the ability of previously unreported fly species to feed on living human tissues in the Neotropical Region.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Calliphora vomitoria L. (Diptera: Calliphoridae) and effects on the determination of the post-mortem interval.

The determination of the post-mortem interval (PMI) is one of the main tasks of forensic entomology, where growth and stages of development of arthropods are used for PMI determination. It is well acknowledged that maggot development is significantly influenced by temperature. Attention has also been paid to the microbial populations of the cadaver, because toxic substances contained in the substrate can influence the microorganisms and affect arthropods growth and development. However, little is known about the influence of antibiotics taken during lifetime of a person on insect development after that persons death. The aim of this study was to test the hypothesis that the antibiotics ceftriaxone and levofloxacin cause inhibition of growth and delay of pupation of the blow fly Calliphora vomitoria, which would then lead to an incorrect determination of the post-mortem interval in forensic cases. It was found that maggot development was delayed by levofloxacin mixed in minced pork, where a mixture of both antibiotics increased this effect. The maggot growth in the samples with ceftriaxone was not delayed. Pupation was delayed in treatments with a mixture of both antibiotics. The mortality was reduced by separate or combined application of ceftriaxone and levofloxacin, which we attribute to a bactericidal effect of the antibiotics on maggot pathogens. Depending on the concentration of the antibiotics, an underestimation of the post-mortem interval between 24 and 48 h could be suspected. We conclude that antibiotics need to be considered if instar stages are to be used to determine the PMI and that some antibiotics may improve the breeding conditions of maggots.

Assemblages of Necrophagous Blow Flies (Diptera: Calliphoridae) in Four Contrasting Ecoregions of Los Angeles County, California, United States.

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 Effects of Gunshot Residue Components (Pb, Ba, and Sb) on the Life History Traits of Lucilia sericata (Diptera: Calliphoridae).

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 Mayis 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.

Effect of a novel juvenoid fenoxycarb on the pupal-adult transformation in the blowfly, Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

Chrysomya megacephala (Fabricius, 1794) (Diptera, Calliphoridae) acts as a vector of many disease-causing pathogens. It causes myiasis in human beings and other living vertebrates. In the present study, the effect of a juvenile hormone analog (JHA), fenoxycarb, was evaluated on pupal-adult transformation by exposing pupae (0 and 1-day-old) of blowfly. Pupae were topically treated with different concentrations of the compound, viz., 20, 40, and 80 µg/µl applied on the posterior dorsum with the help of a micropipette. The effects comprised various developmental aberrations, such as delayed pupal-adult ecdysis duration, pupal mortality, formation of pupal-adult intermediates, eclosion failure, reduced adult emergence, and formation of abnormal adults. The freshly molted pupae (0 day) were more susceptible as compared to 1-day-old pupae. Pupal mortality was highest at 80 µg. Normal adult emergence was completely inhibited at day 0 pupae treated with 80 µg of the fenoxycarb. These results demonstrate that fenoxycarb was capable of successfully inhibiting the pupal-adult transformation, and thus, it can be used to control this myiasis-causing agent.

Effect of Type of Tissue on the Development of Chrysomya rufifacies (Diptera: Calliphoridae) in Sri Lanka.

Chrysomya rufifacies (Macquart), the hairy maggot blow fly, is of great importance for the field of forensic entomology due to its habit as an early colonizer of decomposing vertebrate remains and myiasis producer. Development studies on this species have been conducted in scattered regions of the world, using types of tissue from several species of animals as a rearing medium. Despite the commonality of C. rufifacies in Sri Lanka, developmental studies have never been performed in this region. As well, the effects of diet on development have not been tested. In the current study, C. rufifacies immatures were reared on skeletal muscle, liver, and heart from domestic swine, with flies from colonies maintained at 25 and 28°C. The minimum time needed to complete each stage at 25°C on liver (224.14 h) was fastest followed by skeletal muscle (249.33 h) and heart (251.64 h) respectively, whereas at 28°C, fly development was quickest on heart muscle (178.27 h) followed by liver (178.50 h) and skeletal muscle (186.17 h) respectively. A significant difference in total development time was determined for temperature, while the rearing medium was not significant. Temperature also showed a significant effect on the length and the width of the larvae, while the type of tissue statistically impacted only the width.

Effect of Zolpidem Tartrate on the Developmental Rate of Forensically Important Flies Chrysomya megacephala (Diptera: Calliphoridae) and Chrysomya saffranea.

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.