Development of mixed linear models to analyze and describe the impact of malathion on the larval growth of Megaselia scalaris (Diptera: Phoridae) under various feeding media and environmental conditions.

Forensic entomology plays a crucial role in estimating the minimum postmortem interval through the study of insect larvae found at crime scenes. The precision of this estimation relies on various biotic and abiotic elements that simultaneously influence insect growth and development, encompassing factors such as temperature, humidity, photoperiod, diet, and the existence of xenobiotics in decomposing tissues. Despite numerous studies on the influence of these factors, including the impact of xenobiotics, there are currently no robust tools available for making corrections to this estimation considering concurrently all variables. In an attempt to propose an exploratory and descriptive statistical model to analyze the simultaneous effect and interaction of different variables on larval growth, this study aimed to compare the effect of malathion on the growth of Megaselia scalaris (Loew, 1866) (Diptera: Phoridae) raised in malathion-spiked porcine muscle, under controlled and uncontrolled temperature and humidity conditions (environmental conditions). Larvae were also reared using various growth media. A split-plot design that combined crossed and nested factors was employed; 2 linear mixed models were developed to assess the relationships between the variables. The model provides valuable insights into the complex interactions among xenobiotics, growth media, and environmental conditions in the size and development of M. scalaris.

Evaluation of the effect of clonazepam and its metabolites on the life cycle of Megaselia scalaris (Loew) (Diptera: Phoridae).

Fly colonization patterns and development are crucial in estimating the post-mortem interval (PMI) of decomposing corpses. Understanding the potential effects of xenobiotics on species development in cadaveric entomofauna is essential for accurate PMI estimation, given their presence in decomposing bodies. Benzodiazepines, commonly prescribed for their anxiolytic, hypnotic, and muscle relaxant effects, are of forensic interest due to their potential for abuse, dependence, intoxication, and overdose-related deaths. This study aimed to explore the effect of clonazepam and its metabolites on Megaselia scalaris, a species commonly used to estimate PMI, the alteration of which could impact the accuracy of said estimation. The S9 biotransformation fraction, an in vitro model consisting of an array of metabolic enzymes, was used to generate phase I and II metabolites for evaluating their effect on M. scalaris development, representing an innovative approach to this type of study. Megaselia scalaris larvae were reared in synthetic growth media under controlled conditions. The study compared different groups: control, clonazepam, and clonazepam with S9 fraction. Larvae were measured daily to determine growth rate, and clonazepam concentrations were analyzed using HPLC-DAD. Results showed that larvae grown in media containing clonazepam or clonazepam with S9 fraction developed faster than control larvae, reaching their pupal stage earlier. Growth rates were also altered in treated groups. In conclusion, the presence of clonazepam and its metabolites accelerated the life cycle of M. scalaris, potentially impacting the accuracy of PMI estimation. These findings underscore the importance of considering xenobiotics in forensic entomological studies for precise post-mortem interval determination.

Forensic significance of Roundup Full® II effect on the development of Dermestes maculatus (Coleoptera: Dermestidae) and Lucilia sericata (Diptera: Calliphoridae).

The most widely used herbicides in the world are glyphosate formulations. These have been used in self-poisoning and accidental poisoning. Dermestes maculatus (Coleoptera: Dermestidae) and Lucilia sericata (Diptera: Calliphoridae) are associated with decomposing human and animal remains. The aim of the study was to evaluate Roundup Full® II effects on saprophagous insects´ development. For this, green bottle flies and hide beetles were exposed to different doses of the herbicide. The duration of life cycles was recorded by observations made every 8 and 24 h. To register possible variations in size, external morphology, and female proportion, specimens of all developmental stages were collected. The duration of developmental stages in L. sericata did not change due to the herbicide, but this reduced all size parameters of puparium with pupae inside, the weight of L1, and of adults. The larval stage and total duration in D. maculatus were decreased by the highest dose, while size parameters of hide beetles remained unchanged at all development stages. In both flies and beetles, there was no evidence of abnormal external morphology and the female proportion did not change in response to the herbicide. Finally, this study exhibited an impact on L. sericata and D. maculatus, so the possibility that the herbicide may affect other forensically important species should be considered, but more work is needed. Taxonomic determination could be done as usual; however, caution should be exercised when estimating the postmortem interval. This study represents the first to analyze a glyphosate-based herbicide associated with Forensic Entomology.

Effect of diazinon (organophosphate) on the composition and succession of Calliphoridae assemblages in rabbit carcasses in the Eastern Amazon.

Fatal poisonings with diazinon may occur both accidentally or intentionally in suicide cases. Forensic entomotoxicology can aid in understanding these deaths by detecting and analyzing the interference of toxic substances in the biology of necrophagous insects. Hence, this study sought to evaluate diazinon's effect on the composition and succession of calliphorid species in the tropical savannas of the Amazon. Nine rabbit carcasses were divided into three groups: one control and two diazinon treatments (100 mg/kg and 300 mg/kg); each group had three replicates. Three fragments of the Amazon tropical savanna were selected for the experiments. Daily collections of adult and immature calliphorids were performed. Five decomposition stages were observed: fresh, bloated, active decay co-occurring, advanced decay, and dry. Eight species of Calliphoridae were identified among the collected adults: Chloroprocta idioidea (0.1%), Chrysomya albiceps (58.3%), Chrysomya megacephala (14.2%), Chrysomya putoria (2.6%), Cochliomyia hominivorax (1.3%), Cochliomyia macellaria (0.5%), Lucilia eximia (19.8%), and Paralucilia paraensis (3.3%). The adult specimens in the control group with the highest abundance were observed only from the advanced decay stage onward. In the dry stage, abundance was higher in control than in treated carcasses. From the sampled 941 Calliphorid immatures, three species were identified: C. albiceps (76,3%), C. putoria (1%), and L. eximia (22,7). The number of immatures was higher in control than in treated carcasses. Therefore, diazinon interferes with the putrefaction timeframe in carcasses, slowing the decomposition stages and affecting their colonization by immature forms of Calliphoridae.

Detection of terbufos in cases of intoxication by means of entomotoxicological analysis using ATR-FTIR spectroscopy combined with chemometrics.

The detection of toxic substances in larvae from carcasses in an advanced stage of decomposition may help criminal expertise in elucidating the cause of death in suspected cases of poisoning. Terbufos (Counter®) or O,O-diethyl-S-[(tert-butylsulfanyl)methyl] phosphorodithioate is an insecticide and systemic nematicide, which has very high toxicity from an acute point of view (oral LD50 in rodents ranging from 1.4 to 9.2 mg/kg) that has been marketed irregularly and indiscriminately in Brazil as a rodenticide, often being used to practice homicides. The present study aims to evaluate the use of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to detect traces of terbufos pesticide in fly larvae (Sarcophagidae). ATR-FTIR spectra of scavenger fly larvae from control (n = 31) and intoxicated (n = 80) groups were collected and submitted to chemometric analysis by means of multivariate classification using principal component analysis with quadratic discriminant analysis (PCA-QDA), successive projections algorithm with quadratic discriminant analysis (SPA-QDA) and genetic algorithm with quadratic discriminant analysis (GA-QDA) in order to distinguish between control and intoxicated groups. All discriminant models showed sensitivity and specificity above 90%, with the GA-QDA model showing the best performance with 98.9% sensitivity and specificity. The proposed methodology proved to be sensitive and promising for the detection of terbufos in scavenger fly larvae from intoxicated rat carcasses. In addition, the non-destructive nature of the ATR-FTIR technique may be useful in preserving the forensic evidence, meeting the precepts of the chain of custody and allowing for counter-proof.

Effect of thiamethoxam (organophosphate) on the flies and beetle visitation and cadaveric decomposition process

ABSTRACT Entomofauna associated with decaying cadavers may be useful in criminal investigation, either through the development of immature insects of interest or through entomological succession in corpses. These factors may vary if the insects are exposed to toxic substances that can modify the insect's developmental cycle, as well as its own occurrence, which would imply significant changes in the results of any investigation. However, there are few studies on how contamination by toxic compounds can affect the action of insects on carcasses and their consequence for forensic expertise. Therefore, this study aimed to test the hypothesis that the normal visitation of flies and beetles is altered in insecticide-contaminated carcasses. The experiment was carried out in a sugarcane plantation, using pig carcasses contaminated with insecticide and the same number of carcasses without any type of contamination as a control. In all experiments, the contaminated carcasses reached the final phase of decomposition in a longer time than the uncontaminated carcasses of the control group. A total of 2.767 specimens were collected and identified, 2.103 individuals from the order Diptera and 664 from the order Coleoptera. There was a significant geometric regression adjustment during the decomposition phases only for the control group, different from the contaminated carcasses in which this pattern did not occur. Results show that contamination by insecticide alters decomposition time and phase, altering the action of flies and beetles, affecting the abundance, composition of species as well as their activities, which can alter the data used by experts in criminal experts.

Effect of Terbufos (Organophosphate) on the Cadaveric Colonization Process: Implications for Postmortem Interval Calculation.

The determination of necrophagous fly specie's development time is considered an accurate method for estimating postmortem interval (PMI). However, pesticides and other chemicals can alter the flies' life cycle, inducing errors in PMI estimation. Thus, this work aimed to evaluate the effect of different doses of Terbufos (Organophosphates) on the temporal dispersion pattern and development of immature dipterans associated with decaying rat carcasses. For this, 150 g female Wistar rats received, via gavage, 200 µl of Terbufos (5 or 10 mg/kg) or distilled water (control) and, after 30 min of administration, the animals were euthanized and distributed in suspended traps to decompose under environmental conditions. The dispersing immatures were collected daily, and their development time was monitored until the emergence of adult flies. After data analysis, it was observed that Terbufos altered 1) the temporal pattern of larval dispersion; 2) the composition and structure of the colonizing assemblage (emerged adults); 3) species' development time, accelerating or delaying their cycle, depending on the dose used; and 4) the califorids and sarcophagids emergence rate, increasing the mortality of pupae from intoxicated carcasses. Thus, this work demonstrates experimentally that Terbufos directly influences the development of flies with forensic potential and discusses the implications for PMI estimation, which can assist in future investigative processes with suspected poisoning by this organophosphate.

Dermestes maculatus (Coleoptera: Dermestidae) development under fluoxetine effect using two drug administration models.

Fluoxetine is a selective serotonin reuptake inhibitor, commonly used for the treatment of a variety of psychopathological conditions. As such, fluoxetine may be expected to appear in clinical and forensic cases. Dermestes maculatus De Geer (Coleoptera: Dermestidae) has been recognized as a relevant component of the insect fauna associated with decomposing human and animal remains. Experiments were conducted to study the effect of fluoxetine on developing D. maculatus using two-drug administration models: a non-living animal model (pork muscle) and a living one (Sus scrofa L. pigs). We assessed the duration of immature stages and total life cycle, as well as morphological parameters (body length, cephalic width, and weight). The effect of fluoxetine was studied at an overdose concentration: In the non-living animal model the drug was mixed with macerated pork muscle (2000 mg/kg) and in the living animal model, pigs were given the drug orally (833 mg/kg). A control was used for each model. Daily observations were performed from the beginning to the end of the experiments. GC-MS was used for drug detection and quantification. There were no statistically significant differences in the duration of immature stages, life cycle, larval mortality, morphological parameters, or sex ratio, between treatment and control, regardless of the drug administration model. Given that fluoxetine had no detectable effect on the development of D. maculatus, detection of this drug in forensic situations would not compromise the accuracy of PMI estimations.

Analysis of the Effect of Cyclophosphamide and Methotrexate on Chrysomya megacephala (Diptera: Calliphoridae).

Forensic entomotoxicology investigates the effects of chemical substances in the development of scavenger insects and the reflection on estimating the minimum postmortem interval (PMI). To evaluate the impact of the chemotherapeutic drugs cyclophosphamide (CF) and methotrexate (MTX) on the postembryonic development of Chrysomya megacephala, controlled experiments were performed by rearing the larvae on spiked minced beef with different concentrations of these drugs. The results indicated that CF significantly decreased their developmental rate up to 28 h but had no significant effect on larval and adult sizes, survival rate, and sex ratio, whereas MTX decreased larval and adult sizes, survival rate, and there was a deviation in the expected sex ratio toward females in MTX-exposed larvae but had no significant impact on developmental rate. These negative interference factors should be considered in cases of suspected death of people that have undergone chemotherapy.

The Effect of Flunitrazepam (Rohypnol(®) ) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology.

This study investigated the potential effects of flunitrazepam (known as "date rape drug") on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal-Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.

Standardization of histological procedures for the detection of toxic substances by immunohistochemistry in Dipteran larvae of forensic importance.

Immunohistochemistry (IHC) technique is an alternative toxicological analysis to detect drugs in insects of forensic importance, but it requires thorough histological procedures. In this study, we tested different fixatives--phosphate-buffered paraformaldehyde 4% (PP), Carnoy's fluid (CF), Kahle's solution (KS), ethanol in different concentrations, and ethanol associated to PP and CF, time of fixation and histological processes for dipteran larvae's tissue, aiming to develop a sample preparation protocol for IHC application. A suitable fixation was achieved using PP for 12 and 24 h, CF for 3 h, 70% ethanol for 19 days, and 70% ethanol/CF for 2 h/3 h. Postfixation using negative pressure, two immersions in xylene for 30 min each, and one in xylene plus paraffin for 45 min increased tissue preservation. An immunohistochemical test for cocaine detection was performed using monoclonal benzoylecgonine antibody from mouse, peroxidase-conjugated anti-mouse IgG and visualized by 3,3'-diaminobenzidine method showed these histological procedures didn't compromise antigenicity.

Influence of insecticide on insect fauna of forensic importance in rat carcasses (Rattus norvegicus) exposed in a suburban area near Atlantic rainforest fragments of Southeastern Brazil / Influência de inseticidas sobre a entomofauna de importância forense em carcaças de ratos (Rattus norvegicus) expostos em área suburbana no domínio da Mata Atlântica, no Sudeste do Brasil

Forensic entomology is the science that applies the study of insects, in combination with other forensic disciplines, in investigations. Chemical compounds, common in deaths linked to accidental or deliberate use of poisons or other toxic substances may alter the succession pattern and development of insects presents in corpses, leading to errors in estimating the postmortem interval. Carcasses of rats (Rattus norvegicus) were sprayed with insecticides of pyrethrins and pyrethroids group and exposed in a suburban area in the Atlantic Rainforest domain. The results indicated that pesticides alter the entomological succession, affecting the activity and development of insects found in dead bodies, modifying the duration of stages of decomposition and may lead to an error in estimating the postmortem interval.

A entomologia forense é a ciência que aplica o estudo dos insetos, em associação a outros procedimentos criminalísticos, em investigações de âmbito cível e criminal.Compostos químicos, frequentes em mortes ligadas ao uso acidental ou proposital de venenos, ou outras substâncias tóxicas, podem alterar o padrão de sucessão e o desenvolvimento da entomofauna presente nos cadáveres, levando a erros na estimativa do intervalo pós-morte. Carcaças de ratos (Rattus norvegicus) foram pulverizadas com inseticidas do grupo das piretrinas e piretróides e expostas em área suburbana no Domínio da Mata Atlântica. Os resultados indicaram que inseticidas alteram a sucessão entomológica, prejudicando a atividade e o desenvolvimento dos insetos presentes nos cadáveres, modificando aduração dos estágios da decomposição e podendo levar a um erro na estimativa do intervalo pós-morte.

Influence of insecticide on insect fauna of forensic importance in rat carcasses (Rattus norvegicus) exposed in a suburban area near Atlantic rainforest fragments of Southeastern Brazil / Influência de inseticidas sobre a entomofauna de importância forense em carcaças de ratos (Rattus norvegicus) expostos em área suburbana no domínio da Mata Atlântica, no Sudeste do Brasil

Forensic entomology is the science that applies the study of insects, in combination with other forensic disciplines, in investigations. Chemical compounds, common in deaths linked to accidental or deliberate use of poisons or other toxic substances may alter the succession pattern and development of insects presents in corpses, leading to errors in estimating the postmortem interval. Carcasses of rats (Rattus norvegicus) were sprayed with insecticides of pyrethrins and pyrethroids group and exposed in a suburban area in the Atlantic Rainforest domain. The results indicated that pesticides alter the entomological succession, affecting the activity and development of insects found in dead bodies, modifying the duration of stages of decomposition and may lead to an error in estimating the postmortem interval.(AU)

A entomologia forense é a ciência que aplica o estudo dos insetos, em associação a outros procedimentos criminalísticos, em investigações de âmbito cível e criminal.Compostos químicos, frequentes em mortes ligadas ao uso acidental ou proposital de venenos, ou outras substâncias tóxicas, podem alterar o padrão de sucessão e o desenvolvimento da entomofauna presente nos cadáveres, levando a erros na estimativa do intervalo pós-morte. Carcaças de ratos (Rattus norvegicus) foram pulverizadas com inseticidas do grupo das piretrinas e piretróides e expostas em área suburbana no Domínio da Mata Atlântica. Os resultados indicaram que inseticidas alteram a sucessão entomológica, prejudicando a atividade e o desenvolvimento dos insetos presentes nos cadáveres, modificando aduração dos estágios da decomposição e podendo levar a um erro na estimativa do intervalo pós-morte.(AU)