1. Observations on the Effect of Benzylpiperazine on the Larval Development Rate of Calliphora vomitoria
and its Implications on the Estimation of Post Mortem Intervals.
J Zorba,
Faculty of Science and Technology, Bournemouth University, Poole Dorset, BH12 5BB
The identification of insect species present on a cadaver can help in estimating the Post Mortem Interval (PMI). Several studies have however
demonstrated that insect development rates may be altered, when insects consume drugs or toxins present in their food source. Subsequently, PMI
estimations based up on insect evidence in cases of drug related deaths may be unreliable. Benzylpiperazine was found to significantly increase the
length of C. vomitoria larvae throughout each day of sampling. The time to pupation was also reduced, as was the time to the third instar stage. The
differences observed were sufficient to influence PMI estimations by up to 24 hours.
The post mortem interval (PMI) is the time interval from the infestation of insects on a corpse to the
time of discovery. Using insect evidence to determine the PMI contributes a valuable aspect of
forensic entomology. Blowflies are typically the first group of insects to colonize a cadaver and
therefore the focus of PMI estimates is upon them. The blow fly Calliphora vomitoria (figure 1) is of
significant forensic importance throughout the UK, as oviposition frequently begins within minutes
after death. Larvae develop at a predictable, species-specific, temperature mediated growth rate, by
which, within limits, their age can be estimated from their length. Studies have, however, shown that
some drugs may alter insect development rates. For example, cocaine and methamfetamine have
been found to increase larval development, whereas ethanol has been found to retard it. These
effects can subsequently result in either an over or underestimation of the PMI, which must be taken
into consideration to accurately determine the true time of death. Benzylpiperazine (BZP) has been
used for non-medical recreational purposes, as a replacement for amfetamine derived designer
drugs. There is currently no literature available regarding the effects of BZP on larval development
rates from which an estimation of the PMI in cases of BZP related deaths cannot be made.
The experimental design consisted of four BZP treatments; 0 (control), 1, 3 and 6 µg/ml, which were divided into pre-labeled plastic sample pots (⌀
5cm, height 11cm). The experiment consisted of 12 sample pots for each treatment (total 48), labeled according to the treatment and the day of
sacrifice. The experiment lasted for a total of 288 hours (12 days), of which one sample pot from each treatment was sacrificed every 24 hours. The
food substrate for each treatment was prepared by homogenizing 250g of organic pork liver with the desired BZP spiking solution. Solutions were
prepared to a final volume of 25ml in water (0.1ml of water to every gram of liver); the control contained 25ml of water only. 20g of liver was then
transferred into each sample pot and 20 newly hatched C. vomitoria larvae were added. Sample pots were sealed with a gauze net and placed into an
incubator at 25°C with a photoperiod of 16 hours. Larvae were sacrificed every 24 hours and killed by immersion into hot water at 60°C for 1 minute.
Larval length was immediately measured using digital calipers under a microscope.
Results
All treatments of BZP were found to significantly increase median
larval length (recorded at 24 hour time intervals) in comparison to
the control (Figure 2). No significant differences were, however,
found when comparing the median length of larvae between each
treatment of BZP (1, 3 and 6µg/ml). Larvae reared on all
treatments of BZP were also found to pupate before the control
with an earliest pupation time of 192 hours. The time taken for
larvae reared on each treatment of BZP to reach the third instar
stage, was also reduced in comparison to the control. Larvae
reared on BZP reached the third instar stage 24 hours earlier
than the control, equating to 528 accumulated degree hours
(ADH) in comparison to 1056 ADH for the control.
Discussion
The results (figure 2) show that BZP accelerated the larval development rate of C. vomitoria. Larvae reared on all treatments of BZP were longer than
the control throughout each day of sampling. In addition, although the difference was not statistically significant, larvae reared on 6µg/ml of BZP had
noticeably larger median lengths than those reared on 1 and 3µg/ml. A dose dependent effect therefore appears to be present, yet as the differences
were not significant, these effects may have also occurred by chance. The time to pupation was also reduced for larvae reared on all treatments of BZP.
The presence of BZP could have resulted in larvae accumulating sufficient nutrients required for pupation quicker than those of the control, thus
pupating at a quicker rate (Nijhout, 1984; Davidowitz et al., 2003; Caldwell et al., 2005; Colombani et al., 2005; Mirth et al., 2005). This is supported by
the fact that larvae reared on BZP reached the third instar life stage 24 hours faster than those of the control. This could result in a potential
overestimation of the PMI by up to 24 hours. These results coincide with other studies in the literature which also show drugs such as diazepam
cocaine, methamfetamine, heroin, ketamine and paracetamol to increase larval development rates. It is possible that lipid soluble compounds such as
BZP (Antia et al. 2009a,b), are readily absorbed by fly species with high fat contents, as they able to cross the lipid bi-layer of larval cells and increase
the metabolic rate. An accelerated metabolic rate could manifest as an increase in the rate of development, as well as enlarged larvae, pupae and
adults (Mullany et al. 2014).
Conclusion
The presence of 1, 3 and 6µg/ml of BZP in the food source of C.
vomitoria larvae, not only caused larval length to be increased,
but also reduce the duration of the larval stage. The differences
observed between larvae reared on liver spiked with BZP
compared to the control, are sufficient to alter PMI estimations by
up to 24 hours. The PMI in BZP related deaths could
subsequently be theoretically overestimated by up to 24 hours.
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• Caldwell, P., Walkiewicz, M. and Stern, M., 2005. Ras Activity in the Drosophila Prothoracic Gland Regulates Body Size and Developmental
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629-633.
Introduction
Method
Abstract
Figure 1. The life cycle stages of C. vomitoria.
Figure 2. Median length (mm) of larvae recorded at 24 hour time intervals
when reared on liver spiked with 0 (control), 1, 3 and 6µg/ml of BZP. Error
bars represent the 95% confidence interval around the median.
References