Effect of the sedative drug zolpidem tartrate on the immature and mature stages of carrion flies Chrysomya rufifacies and Chrysomya indiana

1 Introduction

Forensic medical investigation of death involves the analysis of samples such as urine, blood, and gastric contents. Collecting these samples from decaying bodies can be challenging. Arthropod evidence can be critical in such cases, especially when investigating suspected poisoning (Goff et al., 1988). Entomotoxicology entails the qualitative and quantitative evaluation of toxicological substances in arthropod witnesses of death, and has become widely accepted in medico-legal death investigations (Chophi et al., 2019). Insects provide an alternative source of toxicological samples that can aid the identification of the cause and time of death. The larvae of carrion flies feed on dead, decaying, or living animal tissues. Carrion flies colonize cadavers in succession (El-Kady et al., 1994). Flies of the family Calliphoridae, that feed on fresh flesh, were reported as the first visitors to a scene immediately after a death (Early and Goff, 1986). In poisoning cases, the chemicals that caused death in the victim can be transferred into the calliphorid’s tissues as they feed on the intoxicated body.

Various growth and developmental stages in arthropods are affected by the chemicals ingested with their cadaveric meals. Recent studies (Goff et al., 1988) have identified chemicals in some cadaveric flies’ feeding stages. Other researchers have established an association between these chemicals, variations in the arthropods’ life cycle, and the estimation of MPI. Several studies have confirmed the effects of different chemicals on the development of insects and the estimation of PMI. (Boon et al., 2008) found that malathion decreases the growth rate of carrion flies. (Bourel et al., 2001) confirmed the effects of insects’ consumption of opiates and morphine. (Leclercq and Vaillant, 1992) investigated the effects of exposing carrion flies to lead arsenate contaminated cadavers. This research established that cadaveric flies recovered at the scene are worth investigation, in order to detect, identify, and quantify chemicals associated with the individual’s death (Chophi et al., 2019). This broad applicability explains why entomotoxicology is highly valued in medico-legal death investigations.

In the current study we evaluated the effects of zolpidem tartrate (Zolfresh) on growth, as reflected by changes in morphological parameters, and developmental rate of the Calliphoridae C. rififacies and C. indiana. Zolfresh is a sedative-hypnotic medicinal substance used for treating insomnia and anxiety. Among other studies, (Darke et al., 2012; Ben-Hamou et al., 2011) linked Zolfresh to cases of suicide.

2 Materials and methods

2.2

2.2 Experimental design

We used the sedative drug Zolfresh, produced by Indian Acme Formulation Pvt. Ltd. A 1 g/mL Zolfresh solution was prepared by dissolving a 5 mg tablet in 5.0 mL distilled water to prepare 1 mg/ml solution. From the prepared solution, different volumes (0.05 mL, 0.1 mL, 0.15 mL, and 0.20 mL) were taken and mixed with 50 gm of minced fresh liver tissue to prepare food containing 1 ppm, 2 ppm 3 ppm, and 4 ppm of zolpidem. These treated foods were kept in four cleaned beakers, and the fifth baker was kept as a control, containing 50 gms of untreated minced liver. Simultaneously, about 80–90 first instar maggots were released in the treated and untreated media to minimize the overcrowding of the larvae within the containers. Each developmental stages were monitored and recorded, to estimate the developmental duration of each stage. The experiments were carried out at the same room temperature and relative humidity (Fig. 1), with a 12 h dark/12 h light cycle.

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Fig. 1

Summary of temperature and humidity data obtained during the study.

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The maggots were dipped in boiling water (Adams and Hall, 2003) to perform the morphological analysis at full relaxation. The length and width, while the weight was measured (Richards et al., 2013; Rosilawati et al., 2014; Tantawi and Greenberg, 1993).

3 Results

4 Discussion

In this study two hairy maggot species were used to evaluate the effects of different concentrations of the sedative drug Zolfresh (zolpidem tartrate) on the estimation of PMI and the morphological parameters of the carrion insects. In the treated cultures of C. rufifacies the PMI (the total life cycle) were delayed compared to the control culture, this delay also shown among the treated cultures with different concentration. In the 1 ppm treated culture the delay was 5 h, and in the 4 ppm culture the delay was about 68 h, while in the cultures treated with 2 ppm and 3 ppm delays were about 36 h and 52 h, respectively.

Many various studies agreed with the results obtained in the current study. (Rashid et al., 2013) investigated the effect of ketum extract on the developmental rate of C. rufifacies and C. megacephala and reported that the duration of development through the post-feeding and feeding stages was prolonged for about 62 and 42 h in C. rufifacies and C. megacephala, respectively. Also C. ruffacies completed development more slowly when treated with Alprazolam and Diazepam than in the control culture, and the time of development increased as the Diazepam concentration increased (Mali, 2011). Similarly the C. megacephala, L. cuprina, and L. sericata developmental duration were increases when the Diazepam concentration were increases (Pawar, 2011). (Bourel et al., 2001) found that the Lucilia sericata life cycle was affected when the flies were reared on rabbit carcass injected with different doses of morphine. They reported that the presence of morphine could lead to a underestimation of PMI determination by up to 24 h. (Tabor et al., 2005) also reported significant variations in PMI duration and the length among maggots of Phormia regina treated with varying concentrations of ethanol. Such findings are in concurrence with those of the current study.

Each species of Calliphoridae flies may respond differently to various chemical compounds, leading to acceleration or retardation of the development of calliphorids. (Patil, 2010) investigated the effect of Alprazolam and Diazepam on C. bezziana. The Alprazolam results were contrary to those of the current study. Patil found that control cultures without Alprazolam of C. bezziana could complete their life cycle in 12 days, maggots in 0.4 ppm Alprazolam spent 11 days, and both 1.2 and 1.6 ppm concentrations of Alprazolam led to a life cycle duration of 10 days. The study concluded that Alprazolam accelerates the development of C. bezziana. (Goff and Lord, 2001) studied the development of larvae of the flesh fly Boettcherisca peregrine reared on tissue obtained from rabbits that received varying doses of heroin before death. PMI estimates based on larval and pupal development were altered by up to 29 h and 18–38 h, respectively. They concluded that the arthropod-based estimation of PMI may be compromised by drug use prior to death. Similarly, the current study found that the estimated PMI varied in the treated culture of C. rufifacies, and the duration was prolonged as the concentration of the zolpidem increased. Decreases in the size of morphological features of different developmental stages as the zolpidem concentration increased. The greatest sizes were observed in 1 ppm culture among all the treated cultures, but it was smaller than the sizes of different stages in the control culture.

C. indiana shown similar results also. The minimum estimated PMI in the control culture was 217 h while in cultures treated with 1 ppm, 2 ppm, 3 ppm or 4 ppm the estimated PMIs were 244 h, 260 h, 270 h, and 284 h, so the duration was prolonged by about 27 h, 43 h, 53 h, and 67 h, respectively. When the concentration increased the duration of the life cycle also increased. These findings are in agreement with those of (Rashid et al., 2013) on the effect of extraction of ketum on the development of Chrysomya rufifacies at different doses, they reported that the alkaloid Mitragynine delayed the developmental rate by up to 62 h.

Similarly, the treated cultures of C. indiana showed variations in the morphological parameters compared to the control culture.

The most extensive developmental stage for each calliphorid species, including C. rufifacies and C. indiana, aided in estimating the minimum PMI value. These results are in agreement with a previous study on the influence of methylphenidate hydrochloride on the development of the forensically significant blowfly Chrysomya chloropyga in South Africa (Visser, 2016).

These chemicals may significantly disrupt the cellular physiology of carrion flies (Introna et al., 2001). Such disruptions may accelerate or retard developmental rates through various life cycle stages. They may extend or reduce the duration spent in certain developmental stages (de Carvalho, 2010).

Toxicological analysis of calliphorid arthropods collected from death scenes can lead to forensic determination of cause and time of death (Anderson et al., 2001; Babu et al., 2013). The rate of development through the various stages of an insect’s life cycle, and the measurements of morphological parameters facilitates insect-based PMI estimation, as these insects colonize cadavers (Gennard, 2012). Different developmental stages of C. rufifacies and C. indiana were delayed by exposure to Zolfresh and the growth retardation associated with a declined in the morphological parameters (weight, width, and length).

Factors such as the presence of different drugs in the cadaver should be considered in any insect-based forensic investigation.

5 Conclusions

Calliphorid flies usually feed on cadavers. The sedative drug zolpidem produced adverse effects on the developmental rate and morphological parameters of treated Calliphorid species. The duration of development through the feeding and post-feeding stages of C. rufifacies and C. indiana was significantly prolonged depending on the concentration of Zolfresh. While the morphological parameters were significantly reduced in the treated cultures. The measurements facilitate the estimation of PMI and can consequently aid in determining the time of death. This research supports the extensive use of arthropod evidence in forensic death investigations.