Flight Activity of the Blowflies, Calliphora Vomitoria and Lucilia Sericata, in the Dark

Forensic Science International 172 (2007) 94–97 www.elsevier.com/locate/forsciint

Flight activity of the blowﬂies, Calliphora vomitoria and Lucilia sericata, in the dark J. Wooldridge, L. Scrase, R. Wall * School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK Received 14 April 2006; received in revised form 7 December 2006; accepted 20 December 2006 Available online 30 January 2007

Abstract Many species of insects are able to fly at night or in very low light intensities. The question of whether calliphorid blowflies are also able to do this to locate a corpse and oviposit nocturnally is of considerable forensic importance. However, to date studies of this behaviour have been contradictory. Here, the activity and number of Calliphora vomitoria L. and Lucilia sericata (Meigen) (Diptera: Calliphoridae) caught on sticky- traps were examined in a slow-speed wind tunnel, at different intensities of artificial light. The traps were either unbaited or baited-with liver. The number of both species caught, decreased incrementally as light intensity was reduced. While the responses of the two species were broadly similar, L. sericata were significantly more active than C. vomitoria, especially at higher light intensities. The number of flies of both species that were caught was higher in the presence of liver bait, but the presence of the liver bait did not change the shape of the relationship between catch and light intensity. Hence, light intensity acts as an independent exogenous stimulus for activity and although liver volatiles increase activity levels, they are not necessary as an activation stimulus. Comparison of the numbers caught in small or large enclosures suggests that any flies caught in darkness probably alighted on the trap by chance and that in darkness, while flies may be activated by carrion odours, they do not appear to be able to navigate effectively to the source of that odour. The results presented here suggest that in darkness, the probability of oriented flight leading to oviposition on a corpse by either species, is relatively low. # 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Activity; Dark; Diptera; Forensic entomology; Post-mortem interval

1. Introduction oviposition was observed by both C. vicina and Lucilia sericata (Meigen) on carrion both placed directly on the ground [6] and on In forensic entomology, when the age of insect larvae is used carrion raised off the ground to ensure that only flying insects to estimate the minimum post-mortem interval (PMI), knowl- were likely to reach it [7]. However, studies in the dark and edge of the factors that are likely to delay the time between death artificially illuminated areas during the night found no evidence and oviposition will clearly be of critical importance. One of the of nocturnal oviposition [8], neither did 2-year study of carrion key questions relates to the effects of periods of darkness. Most colonisation in Indiana, USA [9]. Oviposition was found to be blowflies are usually considered to be largely diurnal and synchronised with the onset of the photophase following a period relatively inactive at night [1]. Hence a body left exposed at night of darkness [3]. would not attract flies until the next morning [2,3].However,a Clearly whether or not flies are active at night is an important number of anecdotal observations suggest that nocturnal factor which may significantly affect the estimate of PMI. The oviposition may occur, particularly with species of Calliphora. aim of the present work therefore was to undertake a For example, Calliphora vicina R.-D. were observed laying eggs comparative investigation of the question of whether blowflies in a slaughterhouse at night [4] and on a cadaver in a dark cave are active in darkness, using two species of forensic [5]. However, experimental studies which have attempted to test importance, Calliphora vomitoria L. and L. sericata. these observations have given contradictory results. Nocturnal 2. Methods

* Corresponding author. Tel.: +44 117 928 9182. Fly behaviour was investigated in the laboratory using a slow-speed wind E-mail address: [email protected] (R. Wall). tunnel, constructed of aluminium with clear reinforced-glass sides and ceiling

[10]. The wind-tunnel had a working section length of 2 m, width of 1.3 m and height of 1 m and was illuminated by one 1.5 m, 32 kHz, diffused fluorescent light. Air was drawn into the wind tunnel through a vertical bed of activated charcoal by a centrifugal fan and blown upwards into a secondary horizontal bed of activated charcoal filter. The airflow was then rotated through 908 by a series of aluminium vanes and straightened by passing through two sheets of steel mesh before entering the working section of the wind tunnel. Air exited the wind tunnel via a sheet of 2 mm diameter nylon mesh covering the downwind end of the working section. The wind tunnel was maintained in a small room at 25 8C (range Æ1 8C). Larval C. vomitoria and L. sericata to be used in experimental trials were obtained from a local fishing-bait shop and kept in the laboratory to pupate. The adults that emerged were maintained in a cage (30 cm Â 30 cm Â 30 cm) supplied with granulated sucrose and water ad libitum, and at 24–25 8C and under a 16 h light:8 h dark cycle. Female adults only were used in experiments, when 6 days-of-age. Prior to use they were briefly cooled, to allow then to be counted and sexed. Sticky-traps were constructed from a square of cardboard (10 cm Â 10 cm) covered by a white plastic sheet coated with polybutene-based, high-tack, non- setting adhesive (AgriSense BCS Ltd., Pontypridd). The cardboard base and sticky plastic had a hole in its centre, under which was placed a plastic beaker (4 cm Â 6 cm) covered with mesh. The beakers were either empty or contained Fig. 1. The mean number (ÆS.E.) of Lucilia sericata females caught on a white 50 g of liver. The liver used was bought from a local retailer, cut into pieces the sticky-trap under a range of different light intensities (W m2) in the presence same day and left for 3 days at ambient laboratory conditions prior to use. (*) or absence of (&) liver bait.

2.1. Small cage trials P < 0.001; Fig. 1). There was no significant interaction between light intensity and the presence or absence of liver bait (F = 2.70, Batches of 30 adult female C. vomitoria or L. sericata were transferred to P = 0.05), indicating that the responses of flies to light intensity the wind tunnel in a plastic cup and placed into each six mesh cages (35 cm Â 35 cm Â 35 cm) positioned in the centre of the wind tunnel in two followed the same pattern in both liver and control treatment rows of three, perpendicular to the air flow. One sticky-trap had been placed in groups. Tukey multiple range tests showed a significant the centre of each mesh cage prior to the release of the flies, which were then left difference between the 100% (345 W m2) and 32% for 3 h before the number of flies caught on each sticky-target was counted. (110 W m2) treatments and between the two higher and the Light intensities within the wind tunnel were changed using neutral density two lower light intensities, but no significant difference between filters wrapped around the fluorescent light source, to give light intensities of approximately 100%, 32% and 12%. The experiment was also run with lights the number caught at the lowest light intensity or in darkness. off. The precise irradiances were confirmed using a light intensity meter and found to be: 345 W m2 (full illumination), 110, 40 and 0 W m2 (darkness). Four 3.2. C. vomitoria replicates of this experiment were performed for the four light intensities for each species, two with liver, and two without. A greater number of flies was caught when traps were baited 2.2. Large scale trial with liver than when unbaited (F = 22.66, P < 0.001; Fig. 2) and more flies were caught at higher light intensities In the dark in the small cages, it was possible that if flies were spontaneously active they might have alighted on the sticky-traps at random rather than having demonstrated any specific orientation towards the liver bait. To allow this possibility to be considered further, in a second trial 200 female C. vomitoria or L. sericata were released directly into the entire wind tunnel with a larger sticky- trap (30 cm Â 30 cm) and 100 g liver as bait. Flies were again left for 3 h before the numbers caught were counted.

2.3. Data analysis

All data were tested for normality and, where required, log10 transformation was used to normalise the residuals. The log10 (+1) of the number of ﬂies caught was then subjected to analysis of variance with the light intensity and bait presence or absence as ﬁxed factors. Tukey pairwise comparisons were used to compare the means of within treatment groups.

3. Results

3.1. L. sericata

A greater number of ﬂies was caught when traps were baited Fig. 2. The mean number (ÆS.E.) of Calliphora vomitoria females caught on a with liver than when unbaited (F = 20.19, P < 0.001; Fig. 1)and white sticky-trap under a range of different light intensities (W m2) in the more ﬂies were caught at higher light intensities (F = 43.08, presence (*) or absence of (&) liver bait. 96 J. Wooldridge et al. / Forensic Science International 172 (2007) 94–97

was reduced. It is notable however, that in the small cages used, captures were not reduced to zero even in, what was assumed to be total darkness. The room in which the wind tunnel was placed had no windows and no other form of illumination such as infra-red lighting; however, the possibility of some low level illumination, for example, leaking from under the laboratory door, can not be completely ruled out. If this was the case it was certainly at a level that was to low to be measured by the light intensity meter used here. Nevertheless, the graded response to light intensity, particularly when the liver bait was present, is surprising. From an adaptive perspective, it might have been anticipated that flies would remain active down to a threshold below which their eyes could not function effectively, and that an all-or-nothing relationship between activity and light intensity would have been observed. The ommatidia of compound eyes are capable of complex photoreceptor dark adaptation, although resolu- Fig. 3. The mean number (ÆS.E.) of female Lucilia sericata (^) and Calli- tion may decrease as the signal to noise ratio increases at lower phora vomitoria (^) landing on a white sticky-trap in the presence or absence light intensities [13,14]. of a carrion bait (3-day-old liver), with all light intensity treatments pooled. The responses of the two species were broadly similar, but L. sericata were significantly more active than C. vomitoria, (F = 18.63, P < 0.001; Fig. 2). Again, there was no significant especially at the higher light intensities and particularly at interaction between light intensity and the presence or absence 110 W m2. As a result, the decline in activity of L. sericata of liver bait (F = 1.26, P = 0.29). Tukey multiple range tests appeared to show something of a step response, with a showed that there was no significant difference in the number substantive decline in activity between 110 and 40 W m2 caught at the two highest light intensities (3.45 and 1.1 W m2. whereas the decline in activity seen in C. vomitoria appeared to However, there was a significant difference between the be more gradual. This difference may be related to their numbers caught at two higher and the two lower light intensity different behaviours in the field; C. vomitoria occur more treatment groups and between at the lowest light intensity and commonly in shaded and edge-habitats found in woodland and darkness. hedgerows. These environments have a lower level of illumination than the open, field habitats where L. sericata 3.3. Comparison between species are more commonly caught [15–17]. The numbers of flies of both species that were caught was Fewer C. vomitoria were caught than L. sericata overall higher in the presence of liver, but the presence of the liver did (P < 0.05) and in both control and liver-bait treatment groups not change the shape of the relationship between activity and (Fig. 3). light intensity. This shows that light intensity acts as an exogenous independent stimulus for activity and that although 3.4. Large scale trial liver odours increase the level of activity of the flies, they are not necessary as an activating stimulus. Hence fly activity can When flies were released into the entire wind tunnel in the be modulated directly by light intensity, without the need for dark, rather than the small mesh cages, a significantly lower volatile odours to trigger activation. The fact that L. sericata mean percentage of flies was caught than when they were were more active than C. vomitoria both in the presence and released in the dark into the smaller cages (x2 = 30.01; absence of the liver bait also suggests that the difference in P < 0.005). Means of 1.04% and 24.22% of the L. sericata activity level is not causally related to any difference in the way were caught and 3.0% and 14.72% of the C. vomitoria were these two species respond to odour cues. caught, in the wind tunnel and cages, respectively. In the small cages, the fact that flies continued to be caught in the dark, particularly on the liver-baited sticky-trap, 4. Discussion suggested that either that there was undirected flight and that flies, moving at random, eventually alighted by chance on the Many insects, such as moths, flies, beetles and bees, are sticky surface, or that the flies were capable of oriented flight well known to be able to fly at night in very low light towards the odour source in darkness. To attempt to distinguish intensities [11,12]. Nocturnal flight activity is often strongly between these alternative possibilities, trials were carried out in related to ambient temperature. However, to date, the debate the much larger volume of the entire wind tunnel. In these trials, over the ability of calliphorids to locate a corpse and oviposit captures were reduced to almost zero, suggesting that the nocturnally has been inconclusive. 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