Effect of Lorazepam on the Development of the Hairy Maggot Blow Fly, Chrysomya rufifacies (Macquart): Implication for Forensic Entomology

Entomotoxicology is based on using insect evidence recovered from a dead body to find out the cause and time of the death. Drugs can accumulate in fly larvae when they ingest the flesh of deceased persons and alter the normal development of the fly causing implications in calculating postmortem intervals. Lorazepam is an antidepressant generally used to treat anxiety. Larvae of Chrysomya rufifacies were fed on the beef liver mixed with lorazepam to study the effect of lorazepam on the developmental rate of larvae and to count delay in postmortem interval. Larvae grown on the beef liver with different doses of lorazepam showed delayed development as compared to normal larvae. The life cycle durations in experimental cultures with different concentrations of lorazepam completed in 1 ppm (272.56 hrs), 2 ppm (289.23 hrs), 3 ppm (324.10 hrs), and 4 ppm (350.72 hrs), while in the control culture life cycle completed in 257.26 hrs. The length, weight, and width of the larvae treated with lorazepam were smaller than the untreated culture. Length, weight, and width decreased with increased concentration of lorazepam. This delay in development ultimately affects the postmortem interval. That is why prior knowledge of the life cycle of flies with respect to various drugs needs to be studied, and these baseline data can be used to calculate postmortem interval and cause of death.


Introduction
Insect's life cycle acts as a precise clock that begins soon after death. Tat is why insects are called the frst entity to witness death and are used to calculate postmortem intervals. Forensic entomology makes use of insects and insect evidence to help law enforcement to determine the accurate time and cause of the death [1]. When using entomological evidence, two considerations are most important. First, insects often lay eggs within a few minutes or hours of death. Tat is why the cycle of development of the oldest maggots feeding on the corpse shows the most approximate time since death [2]. Second, the pattern of insect succession is also a good indicator of PMI because insects arrive at predictable and successive waves based on stages of decomposition [3,4].
Generally, Calliphoridae fies colonize frst on a cadaver because they have the ability to travel over a 15 km distance after getting attracted by the odor produced during decomposition. Females start to oviposit within the frst few hours after death [5]. When traditional specimens such as blood, urine, or muscle tissues are unavailable, insects and insect remnants can also be used for toxicological examinations [6][7][8]. Entomotoxicology is another important and interesting area to investigate the efects of drugs and toxins on arthropod development [9]. Te pattern of cadaveric changes in soft tissue structures tends to indicate how long a person has been dead. However, changing the breakdown of organic matter can signifcantly alter the estimated time of death [10]. When dipteran larvae feed on intoxicated tissue they in turn metabolize the substance and incorporate it into their own tissue. Such larvae can also be useful in the toxicological analysis as secondary bioaccumulation occurs in the larval body; it can tell us which toxin was present in the body. During diferent experimental studies, a large number of poisonous chemicals were recovered from maggots that feed upon animals, which died due to the intake of certain chemicals. Because of the rise in drugrelated deaths, it is essential to know how all these drugs infuence the development of fies that feed on cadavers in order to prevent mistakes in PMI estimates [11].
In the present study, the efect of lorazepam on the development of forensic fy C. ruffacies is studied. Tese baseline data are valuable as it can contribute to the forensic entomologists to calculate accurate PMI. Knowledge of the drug-specifc life cycle of a fy saves time and takes an entomologist a direct conclusion to calculate PMI. Many times, it is observed that the suicidal or murderer victims have been given sedative drugs and under such conditions, it is found that these drugs accumulate in larval body tissue and these drugs can afect the duration of the life cycle stages. In such conditions, it becomes hard to fnd correct postmortem intervals (PMI). Tat is why it is necessary to have the standard data related to such drugs and their efects on the duration of the life cycle stages and the impact on their morph metric measurement.

Lorazepam.
Lorazepam is an antianxiety agent belonging to the class benzodiazepine. Te FDA approves it for short-term relief of anxiety symptoms such as anxiety disorders, anxiety-associated insomnia, anesthesia premedication in adults to relieve anxiety or to produce amnesia, and treatment of status epilepticus [12]. Overdose of lorazepam may cause CNS and respiratory depression. It also leads to hypotension, ataxia, confusion, and coma and can be fatal. Each tablet contains 0.5 mg, 1 mg, or 2 mg of lorazepam [13].

Collection of Sample.
Sample collection was carried out in the Osmanabad district of Maharashtra state, India. Te fresh meat was purchased from the local slaughterhouse and allowed to be partially putrefed. Partially putrefed meat was exposed to the air and the fies were attracted to it within a few minutes. Flies were collected with the help of an insect net. Similarly, maggots and adult fies were also collected from diferent road cadavers of diferent animals. Flies were collected with the help of an insect net and larvae were collected with the help of forceps and kept in the 500 ml beaker. Collected fies and larvae were brought to the laboratory and reared in laboratory conditions.

Laboratory Rearing.
Adults were fed daily on fresh liver and honey mixed in water. Te fresh liver was used as an oviposition site for females. Fresh liver and honey water were provided in a separate Petri dish. Hygiene and cleanliness were maintained in the rearing boxes to avoid any infection of the culture. Daily Petri dishes were cleaned and dried to avoid infection of the culture [14].
Cultures of the fies were kept under continuous observation and after each hour it was checked to ensure egg laying. In the beginning, all fies were kept in a cage but to maintain species-specifc culture proper identifcation was important. As soon as the fy laid eggs, eggs were separated and kept in the separate cage to maintain the pure culture of each isofemale. Temperature and humidity data were recorded throughout the experiment. Te frst, second, and third instar were dissected to ensure correct species identifcation based on morphological characters. Adult fies are also examined to ensure correct identifcation using previously published standard identifcation keys [15,16]. After morphology-based identifcation, DNA barcoding of mtDNA using cytochrome oxidase subunit I was performed to confrm species identifcation. Te sequence was uploaded to the BOLD to confrm the identifcation of molecular analysis of the data. Te GenBank accession number of C. ruffacies is (MG816778) [17]. To measure morphological parameters larvae were immersed in hot water to aid the straightening which helped in proper measurement.

Experimental Setup.
Larvae of C. ruffacies were collected from the pure culture maintained at the laboratory. 500 gm of the fresh beef liver was purchased from the slaughterhouse and it was fnely chopped. Lorazepam 1 mg was used for the present study. Te concentrations of lorazepam were set as 1 ppm, 2 ppm, 3 ppm, and 4 ppm, respectively. One set was maintained as a control free from lorazepam doses. 20 larvae were introduced to each set and development was studied till the emergence of an adult. Te duration required for the development of each stage in diferent concentrations was recorded with respect to temperature and humidity. A total of four replicates were taken of the present research. Temperature and humidity records are presented in the form of graphs.

Statistical Analysis.
A statistical analysis is performed using SPSS. One-way ANOVA with post hoc test was performed for comparison.  Figure 1). Multiple comparisons done by using a post hoc test showed that there were a highly signifcant diferences between each developmental stage in the lorazepam-treated culture and the control culture. Developmental time varied signifcantly in all treated cultures. Details of data of the post hoc test and results can be found in supplementary fles (https://osf.io/zd269/?view_only= d3081187a13040d5b04c26048901f1c4). Figure 1 shows the graphical representation of the developmental time of different stages of C. ruffacies in treated and untreated cultures. Whereas Table 2 shows the developmental time parameters of diferent stages of C. ruffacies in treated and untreated cultures. Te time required to complete the development is higher in treated cultures than in untreated cultures and it is delayed with the increased concentration of lorazepam. Te life cycle of the fy was disturbed due to the increase in the concentration of lorazepam. With the increases in doses of lorazepam, the larval development was slowed and the pupal development was also delayed. In the control set development was normal and fies emerged frst from it. In treated cultures, the life cycle duration increased with the increase in concentrations of lorazepam.

Results
Morphometric parameters such as weight, length, and width of diferent stages in control were slightly bigger than in the treated cultures, even in treated cultures sizes varied depending on the concentration of the drug in the cultures. Te sizes of diferent stages decreased with an increase in the concentration of lorazepam. Larvae in high concentration were smaller in length, weight, and width than the larvae in low concentration.
Results of ANOVA showed that there was a signifcant diference in each developmental stage of larvae when the control group compared with the treated group (1 ppm, 2 ppm, 3 ppm, and 4 ppm). In the control set, 1 ppm, and 2 ppm no mortality was observed and the emergence of the adult fies was 100% but in treated cultures of 3 ppm and 4 ppm emergence was 50% only and adults were very small in size as compared to the control set. Detailed data regarding the variation in the developmental time and morphological parameters in the treated and untreated cultures can be found here. Figures 2-4 show the graphical representation of means of the length, width, and weight of diferent development stages of C. ruffacies in treated and untreated cultures. Observed length, width, and weight in untreated cultures are higher than in treated cultures and it decreases with the increased concentration of lorazepam. Te least length, width, and weight were recorded in culture with a high dose of lorazepam. Colours below the bars indicate the results of the Tukey comparison, where the same colours did not difer statistically to α � 0.05.

Journal of Toxicology
Larvae in all treated cultures showed very fast and random movement. Most of the time they were trying to move away from the food and gather at the edges of the beaker. Tis movement was faster in the cultures with high concentrations of the drug as compared to the least concentration and controlled culture. Larvae in the untreated cultures were voracious feeders in feeding stages, whereas the rate of feeding was decreased in the cultures treated with lorazepam. Larvae treated in 3 ppm and 4 ppm having high doses of lorazepam showed decreased feeding than that to control, 1 ppm, and 2 ppm treated cultures.

Discussion
In the present research, the efect of lorazepam on the developmental duration and morphological parameters is performed. Beyer et al. [6] frst proposed the use of insects in a toxicological analysis and since then many researchers have been using insects as evidence to determine the cause of death. Carrion fies, especially dipteran fies Calliphoridae and Sarcophagidae are extensively used in toxicological analysis. Insects have been demonstrated to be an efective alternative technique for toxicological analysis, especially when the dead body is only a skeleton. Drugs and toxins can be detected in larvae whenever their metabolic accumulation rate exceeds the excretion rate and alter the development of the fy life cycle. Alterations in the life cycle of the fy should be considered to calculate PMI accurately (de Carvalho) [18]. Gof and Lord [9] and Kintz et al. [19,20] have demonstrated the detection of the prescription of drugs through the analysis of fy larvae feeding upon human remains. Toxicological tests were performed on the remains of a male decedent having a known postmortem interval of 67 days. Liquid chromatography was employed in the analysis of heart, liver, lungs, spleen, and kidney tissues as well as Calliphoridae fy larvae collected from the victim. Results of this analysis revealed the presence of fve drugs namely triazolam, oxazepam, phenobarbital, alimemazine, and clomipramine in both the tissues and fy larvae examined. All drugs were present in aforementioned tissues except trizolam. Trizolam is not detected in either the spleen or the kidney samples. All fve drugs were isolated from the developing fy larvae. It was not possible here to establish any quantitative correlations between the concentrations of the drugs detected in the fy larvae and the drugs present in human tissues. Dayananda [21] have studied the efect of morphine and heroin on the development of fies. Morphine and heroin both slow down the rate of fy development. As per the examination, heroin has an efect on the development of fies and it actually speeds up larval growth and development. Ten, it decreases the developmental rate of the pupal stage. Tis leads to an increase in the overall timing of development from an egg to an adult. Some efects of toxins on arthropods depend on the concentration of the toxin while others simply depend on the presence of the toxin. For example, the lethal dose of cocaine causes larvae to "develop more rapidly in between 36 and 76 hours after hatching." Te amount of growth and development depends on the concentration of cocaine in the area being fed. Te amount of methamphetamine, on the other hand, afects the rate of pupal development. Similar results are obtained by Carvalho et al. [22], who studied the efect of diazepam on larvae of C. albiceps and C. putoria of the Calliphoridae family. Te time required for pupariation and adult emergence was signifcantly greater than the control set of larvae. Similarly, morphological parameters (length, weight, and width) were also afected in treated cultures.
Kharbouche et.al. [23] studied the presence of codeine accumulation and elimination in larvae, pupae, and imago of the blowfy Lucilia sericata and its efect on its development. Results showed a 29 hrs interval bias on the evaluation of the larval stage duration calculated from the larvae weight that has to be considered if codeine was present in the larvae substrate. Similarly, a 21 hrs interval bias on the total duration of development, from egg to imago, has to be considered if codeine was present in the larvae substrate. Kanesarajah and Turner [10] have also shown the larval growth of the blowfy, Calliphora vicina was signifcantly faster by as much as 2 days on lung, kidney, heart, or brain tissues as compared to liver tissues which signifcantly causes the postmortem interval in a forensic cases.
A similar study performed by Andrade-Herrera et al. [24], they have also studied the efect of lorazepam on the development of larvae of Calliphora vicina and Calliphora loewi. Teir fndings showed that larvae feeding on drug-containing tissues in lower concentrations developed more rapidly and the emergence of adults was greater but with the increase in concentration developmental rate was delayed. Pupation was normal for the  93 h). Morphological parameters such as weight, length, and width of C. megacephala decreased with the increased concentration of zolpidem tartrate. Morphological parameters of C. safranea showed a negative association with increased concentration of zolpidem tartrate. Somehow larval development did not show much variation in 1 ppm and 2 ppm due to the least concentration but as concentration increased the growth pattern was disturbed. While calculating the PMI concentration of drugs also needs to be considered for the correct estimation of time since death. A similar study done by Gof [26] has proved efects of cocaine and benzoylecognine on growth performance of larvae of Boettcherisca peregrina (Robineau-Desvoidy). Trough intravenous injection, the rabbits were given 35, 69, and 137 mg of cocaine. Larvae developed faster on tissue containing cocaine, benzoylecognine, or both from rabbits injected with 69 and 137 mg of cocaine than on tissue from rabbits injected with 35 mg of cocaine or no cocaine from hours 30 to 70. Total development times for pupation and adult eclosion were reduced accordingly. Tese diferences observed in the rate of development alter postmortem interval and creates inconvenience to the forensic entomologist.
Studies done by Bansode et al. [27,28] showed that temperature plays a vital role in insect growth and development. Lower temperature and high humidity increase the life cycle duration whereas high temperature and low

Conclusion
Insects can prove to be a valuable tool in the investigation of homicides, suicides, and other unattended death. Drugs and toxic chemicals present in a dead body accumulating in the larval body can afect its development and morphological parameters. Toxicological analysis of carrion insects can reveal the cause of death and also helps in calculating postmortem intervals. Carrion fies may give diferent responses to diferent drugs and chemicals. Lorazepam has shown a signifcant efect on the development and

Data Availability
Te data and the results used to support the fndings of this study are uploaded to OSF and can be assessed using the given link (https://osf.io/zd269/?view_only=d30811 87a13040d5b04c26048901f1c4).

Conflicts of Interest
Te authors declare that they have no conficts of interest.