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Forensic Science International 175 (2008) 6164 www.elsevier.

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The nocturnal oviposition behaviour of blowies (Diptera: Calliphoridae) in Central Europe and its forensic implications
J. Amendt a,*, R. Zehner a, F. Reckel b
b a Institute of Legal Medicine, Goethe-University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany Bavarian State Bureau of Investigation, Department of Microtraces and Biology, Maillingerstrasse 15, D-80636 Munich, Germany

Received 13 June 2006; received in revised form 10 May 2007; accepted 10 May 2007 Available online 22 June 2007

Abstract Numerous factors may cause delayed colonisation of a corpse by blowies, leading to a discrepancy between the entomologically determined post-mortem interval (PMI) and the time of death. Blowies, for example, are considered to be inactive at night, however, published observations are contradictory. In the present study, several eld experiments and one type of indoor experiment were conducted in summer of 2004 and 2005 in order to investigate the nocturnal ovipositional behaviour of blowies. In the eld, two types of bait, dead hedgehogs and fresh beef liver, were placed at night in different urban and rural locations in Frankfurt and in Munich, Germany. For the indoor-experiments beef liver was placed in small plastic boxes containing caged Lucilia sericata females in the evening and left overnight. At night, no ovipositon was observed in the eld (n = 51, T = 1024 8C). Nocturnal oviposition in complete darkness occurred in the plastic boxes in two of six cases (T = 25 8C). Considering the behavioural and physiological characteristics of ies we suggest that nocturnal oviposition of blowies appears to be unlikely under natural conditions in Central Europe but may occur under certain circumstances, such as unusual high nightly temperatures and the presence of gravid ies with an appropriate arousal threshold. # 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic entomology; Blowies; Lucilia sericata; Nocturnal ovipositon; Post-mortem interval; Central Europe

1. Introduction The main application of forensic entomology is the estimation of the post-mortem interval (PMI) by identifying the necrophagous species present and calculating the age of the oldest immature insect stages feeding on a corpse [13]. An essential question is whether the dead body has been colonised by ies shortly after death or whether oviposition was delayed. Several factors may cause a delay in colonisation of a body by insects such as rain, low temperatures or hampered accessibility. The knowledge of these factors may improve the interpretation of entomological ndings [4,5]. The diurnal activity pattern and the oviposition behaviour of blowies at night are two other important aspects that are presently under discussion. Generally, calliphorid ies are

* Corresponding author. Tel.: +49 69 6301 7571; fax: +49 69 6301 5882. E-mail address: amendt@em.uni-frankfurt.de (J. Amendt). 0379-0738/$ see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.05.010

considered to be inactive at night [6,7]. As a result, a victim killed during the night would not be colonised before the following morning. This may lead to a considerable discrepancy between the calculated PMI and the actual time of death of up to 12 h. Few studies exist on the nocturnal activity and oviposition in blowies, however, these report contradictory results. While Nuorteva [6], Haskell et al. [8], Tessmer et al. [9] and Spencer [10] did not observe any oviposition at night, Greenberg [11] as well as Singh and Bharti [12] found clear evidence for a nocturnal egg-deposition. Baldridge et al. [13] report limited nocturnal oviposition on a bloated pig at a lighted rural site. However, this was the case in only one out of numerous experiments. Recently, Wooldridge et al. [14] demonstrated a decrease in ight activity of two blowy species in the laboratory, linked with reduced light intensity. In the present study, experiments were conducted to investigate the nocturnal oviposition behaviour of blowies in Central Europe.

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J. Amendt et al. / Forensic Science International 175 (2008) 6164 For indoor experiments in the laboratory, a strain of Lucilia sericata was collected from a corpse at the Institute of Legal Medicine in Frankfurt in 2004 and was reared in a stock culture for 1 year. The colony was subject to an articial diurnal rhythm of 14h light and 10h darkness at a constant temperature of 25 1 8C. Bait was offered to 1015 females (810 days old), which were caged in a clear plastic box (size: 19 cm 11 cm 14.5 cm), from 09:00 p.m. to 07:00 a.m. The box was ventilated by air holes. A wet sponge provided moisture and some sugar cubes provided food. During the placement of the bait the light was switched on for a period of about 1 min. The next morning, the liver was inspected for eggs. The gravid status of the ies used was conrmed at the end of the day by inspecting the bait for eggs again (Table 2). This experiment was performed six times.

2. Materials and methods


Field experiments were carried out in Frankfurt am Main (508070 18N, 88520 50E) and in Munich (488070 23N, 118380 26E), Germany. All times given in this study are Central European Time (CET). In August 2004 (Table 1), ve frozen hedgehogs (obtained from a station, where they died during hibernation) were thawed and placed at different urban and rural locations in Frankfurt at 10:30 p.m. (sunset between 08:25 and 08:56 p.m.). Before sunrise (at 04:30 a.m.) the baits were inspected for nocturnal oviposition. In the evening of the following day, shortly before sunset, the bait was inspected again to check for ovipositon during daytime. In addition in 2005 (May till September), samples of raw fresh beef liver (approximately 250 g) were placed out of each of 28 and 23 single nights, respectively (Table 1) at two urban locations in Frankfurt and/or Munich. The bait was placed out at 10:30 p.m. (sunset between 08:07 and 08:46 p.m.) on the ground of a site located in a garden near a hornbeam hedge (Munich), protected by a rodent cage (size: 46 cm 29 cm 23 cm) and on a window sill 2.5 m above ground, belonging to a terraced house with a small garden and mixed vegetation (Frankfurt), respectively. The next morning at 04:30 a.m., the liver was inspected for eggs. Each bait sample was left at the same place and inspected again before sunset.

3. Results and discussion In 57 eld and indoor experiments, oviposition was recorded on only two occasions at night in darkness, both times when caged females of L. sericata oviposited in the small plastic boxes in the laboratory at 25 8C, i.e. under unnatural conditions.

Table 1 Nocturnal oviposition (eld experiments) on two types of carrion bait (in 2004: hedgehogs, $ 500 g; in 2005: beef liver $ 250 g) in Munich (Mu) and Frankfurt (Fr) +: oviposition; : no oviposition; n.d.: not determined; = 22:30; = 04:30; night = 22:3004:30; day = 04:30 following daysunset; lighting conditions: source www.wunderground.com Date Oviposition Night Mu 09/10 16/17 19/20 23/24 24/25 02/03 13/14 20/21 03/04 08/09 11/12 13/14 16/17 17/18 20/21 21/22 August 2004 August 2004 August 2004 August 2004 August 2004 May 2005 May 2005 May 2005 June 2005 June 2005 June 2005 June 2005 June 2005 June 2005 June 2005 June 2005 n.d n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Fr n.d. n.d. Day Mu n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. + + + + + + + + + + + + + n.d. n.d. n.d. + + + + + Fr + + + + n.d. n.d. + n.d. n.d. n.d. n.d. n.d. + + + n.d. n.d. + + n.d. + + + n.d. + + n.d. n.d. n.d. n.d. n.d. n.d. Temperature (8C) / Mu n.d. n.d. n.d. n.d. n.d. $18/$15 >10/<10 15/10 21/17 n.d. n.d. 15/13 17/17 19/18 18/15 20/18 19/15 22/22 20/19 22/21 21/19 20/14 18/15 22/17 21/16 n.d. n.d. n.d. 18/15 21/17 24/20 21/16 20/18 / Fr 24/18 19/18 21/19 18/15 18/15 20/15 15/10 20/14 18/16 13/10 13/10 17/12 21/18 22/20 24/18 24/20 23/16 24/22 22/16 24/20 24/21 23/17 23/18 23/18 22/16 24/20 19/17 19/12 20/18 24/21 24/20 n.d. n.d. Clear; waning moon Cloudy (rain on 17.08); waxing moon, Clear; waxing moon Slightly cloudy; waxing moon Clear; rst quarter of the moon Slightly cloudy; last quarter of the moon illuminated Clear; waxing moon Cloudy; in the morning rain; waxing moon Cloudy; beginning rain at the morning; waning moon Clear; new moon Clear; waxing moon Clear; waxing moon Cloudy (Mu)clear (Fr); waxing moon Cloudy; waxing moon Clear; waxing moon Slightly cloudy; waxing moon; egg deposal at 6.00 a.m. (L. sericata) Slightly cloudy; full moon Clear; partly windy; waning moon; temperature. at 24:00:24 8C Clear; waning moon Clear; waning moon Slightly cloudy; waning moon Slightly cloudy; waning moon Clear; waxing moon Slightly cloudy; rst quarter of the moon Slightly cloudy; waxing moon Cloudy; waxing moon clear; waxing moon clear; waning moon Clear (Mu)cloudy (Fr); waning moon Clear (Mu)stormy (Fr); waning moon, Clear; waning moon Clear; waning moon Cloudy; waning moon Lighting conditions and comments

22/23 June 2005 24/25 June 2005 26/27 June 2005 27/28 June 2005 28/29 June 2005 03/04 July 2005 14/15 July 2005 15/16 July 2007 17/18 July 2005 18/19 July 2005 19/20 July 2005 23/24 July 2005 26/27 July 2005 27/28 July 2005 28/29 July 2005 31 August 2005/01 September 2005 01/02 September 2005

J. Amendt et al. / Forensic Science International 175 (2008) 6164 Table 2 Nocturnal oviposition (indoor experiments: plastic box in the laboratory) in 2005; night = 20:0007:00; day = 07:00 following daysunset, +: oviposition, : no oviposition Date Oviposition Night 05/06 10/11 11/12 16/17 19/20 23/24 May May May May May May 2005 2005 2005 2005 2005 2005 + + Day + + + + + + Temperature (8C) 25 25 25 25 25 25 Lighting conditions Darkness Darkness Darkness Darkness Darkness Darkness

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In the eld experiments, oviposition by blowies took place only during the day (in almost every case) indicating the presence of gravid blowies in the locality and their ability to nd and colonise the offered bait. The blowy species reared from eggs oviposited during the day were identied as: L. sericata, L. ampullacea, L. caesar, Calliphora vicina in Munich and L. sericata, L. caesar, Calliphora vicina and Protophormia terraenovae in Frankfurt. In Munich, one female blowy was observed laying eggs at 6 a.m., shortly after sunrise (T = 18.7 8C). The adults which were bred from these eggs were identied as L. sericata. In Frankfurt, several blowies were observed interrupting oviposition and departing one by one at nightfall. No obvious external disturbance could be noted. After dispersing, no blowy returned to the bait. Both observations support the ndings of the present studies, i.e. that calliphorid ies do not visit baits to oviposit during nighttime. It is generally accepted that Calliphoridae primarily use olfaction to detect carcasses, especially over long distances. But for short-range-detection, their well-developed visual system plays an important role as well [7,15,16]. Whereas olfaction should not be limited at night, vision denitely is, due to the low light conditions. The compound eye of the blowy is far less sensitive to light than the true optical superposition eye design, which is present in nocturnal moths or beetles [17]. In contrast to some nocturnal lower Diptera, e.g. mosquitoes [18,19], for blowies neither optical nor spectral adaptation for night-vision has been described. Nevertheless, the entering of dark rooms like caves or trunks of cars during daytime, followed by oviposition, has been demonstrated [7,11,20]. But entering a dark room during an active period (day) of the y is a different matter to oviposition during a presumably inactive period (night). In ies, locomotor activity shows a robust circadian rhythm, which has been studied in many species, including forensically important species such as C. vicina and Musca domestica [21,22]. Circadian rhythms can also regulate the sensitivity of the insect eye [23] and sensitivity variations seem to correlate with the activity of the ies [21,24,25]. In Drosophila a circadian rest-activity cycle has been shown, together with a sleep-like rest behaviour that can last up to several hours. During the sleep-like period, the ies become immobile and are relatively unresponsive to sensory stimuli [2629]. Warman et al. [30] showed the existence of a

Drosophila-like circadian system in the calliphorid L. cuprina, conrming the relevance of this system as a general model for circadian rhythms in ies. This behaviour could explain our observations that without any obvious disturbance at sunset several blowies, which were laying eggs, stopped oviposition and ew away one by one. Perhaps they started searching for a resting place for the night. But even if these observations are valid for forensically important species, they might only reect the status of the sensory system of ies kept under unnatural conditions (e.g. immobilised in a recording set-up in the laboratory). It has yet to be shown whether these data can be applied to populations in the eld. Moreover, light, mechanical or even acoustical stimulation can awake sleeping Drosophila [31], and this might be valid for calliphorids as well. The change from day to night is associated with the decrease of ambient temperature. Temperature is considered to be one of the most important constraints on blowy activity and oviposition [7,32]. Species-specic thresholds are not well-known, but it is accepted that below 12 8C, oviposition is rather unlikely ([7,33,34], but see [20]) which may explain especially in the cold season that there is no nocturnal oviposition at night. On the other hand, exceptional high temperatures at a bloody scene of crime or light and mechanical stimulations might attract some gravid females with a high appetence for oviposition and could thus lead to the colonisation of a corpse at night. Such desynchronisation may explain some of the contradictory results in literature. While Haskell et al. [8], Tessmer et al. [9] and Spencer [10] could not observe nocturnal oviposition in Indiana/USA, the southwest of Britain and southern Louisiana/USA, respectively, Greenberg [11] and Singh and Barthi [12] reported egg-deposition at night in Chicago/USA and Patiala/India, respectively. Nightly temperatures during the experiments in India and Chicago, though insufciently documented, were much higher compared to those in Central Europe. But in southern Louisiana no oviposition on lighted and unlighted baits occurred under comparably high temperatures. Baldridge et al. [13] observed nocturnal oviposition in central Texas, however, limited to just one case on a light-exposed pig in the bloated stage of decomposition. Unfortunately no temperatures were reported for that night. The light-exposition of the cadaver is one factor that might be responsible for the nocturnal colonisation in this case. But see [9], where even light exposition did not result in nocturnal oviposition. In conclusion, nocturnal oviposition by blowies appears to be unlikely in Central Europe under natural conditions. However, considering the results of the laboratory experiments, egg-deposition at night may occur under certain circumstances. The bait used in the present experiments and in other experiments was small. A human corpse may be much more attractive for a gravid blowy. In conned rooms, providing a close spatial association between gravid blowies and the corpse, nocturnal oviposition cannot denitely be excluded. Further indoor studies and the use of larger carcasses are necessary to improve the present knowledge of the nocturnal activity of blowies.

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Acknowledgements We thank Dr. Grunwald, Bavarian State Bureau of Investigation, Munich, Dr. Hall, The Natural History Museum, London, and Prof. Mebs, Institute of Legal Medicine, Frankfurt, and one anonymous reviewer for reading the manuscript and their helpful comments. References
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