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Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae)
Forensic Science International 221 (2012) 65–69
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae) Madeleine Mai, Jens Amendt * Institute of Forensic Medicine, Kennedyallee 104, 60596 Frankfurt am Main, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 May 2011 Received in revised form 3 April 2012 Accepted 4 April 2012 Available online 1 May 2012
By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the postmortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25 8C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48 h. Only the larvae with a dispersal time of 24 h (total time of development 325.2 h; median) and 48 h (total time of development 347.7 h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4 h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48 h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic entomology Blowflies Lucilia sericata Post-feeding period Developmental time
1. Introduction One of the main tasks of forensic entomology is the estimation of the minimum time since death or post-mortem interval (PMI) [1]. The most important methods are the examination of the insect succession on corpses and the estimation of age of the immature stages of the necrophagous fauna developing on the cadaver [2]. Identifying the species, the stage of its development and knowing the environmental conditions at the scene of death may enable the expert to calculate the age of the oldest specimen and, thereby, estimate the minimum PMI [3]. Blowflies (Diptera: Calliphoridae) colonize a cadaver soon after death and therefore are the most important insects in forensic entomology [4]. For several species there exist published data dealing with the growth of the species
* Corresponding author. Tel.: +49 69 6301 7571; fax: +49 69 6301 5882. E-mail addresses: [email protected] (M. Mai), [email protected] (J. Amendt). 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.04.001
and the time they need for the different stages of development at a range of temperatures [3,5]. Those estimations are based upon the assumption that the immature insects (eggs, larvae, pupae) are of a certain species and accumulate a stage specific thermal input calculated as the product of time and temperature and given in accumulated degree hours (ADH) or days (ADD) [3,5,6]. One of the most interesting stages in the blowfly lifecycle is the so called post-feeding stage between the late larval period and the pupal stage. The larva stops feeding and starts to search for a suitable site for pupariation. As the majority of fly species leave the corpse on which they had been feeding, this stage of development is also called the dispersal or wandering stage. Arnott and Turner [7] pointed out that precise details about this stage are lacking and, when present, are primarily descriptive. This is regrettable because the post-feeding stage is potentially the most variable regarding its duration. While searching for a place for pupariation the larvae may have to crawl several meters if the ground is hard and without any cover or cracks. This not only applies to dry soil environments but also to indoor scenarios, where the larvae might have to move
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M. Mai, J. Amendt / Forensic Science International 221 (2012) 65–69
up to 30 m or more (Green [18]; Amendt, unpublished data) before starting the process of pupariation. Reference data for the time of development are normally established by laboratory experiments where under controlled conditions the larvae usually do not need to search for a suitable pupariation medium since one is offered right after leaving the food substrate (e.g. sawdust or vermiculite). Subsequently, larvae in laboratory settings get the opportunity to pupate just when leaving the food source and the time spent in dispersal might be very short in laboratory settings according to Arnott and Turner [7] and is usually ignored in the overall calculation of ADH. Natural variations in environmental conditions may lead to a prolonged dispersal time including an energetic cost. Gomes and Von Zuben [8] showed that longer dispersal time in the blowfly Chrysomya albiceps leads to a reduction of its pupal weight. According to Arnott and Turner [7] a longer post-feeding period simply causes further accumulation of degree hours and, as more energy is used, leads to a reduction in resources available for the pupa and the adult stages. This process may also influence the time of development for the blowfly Calliphora vicina. In this paper the effect of different dispersal periods for postfeeding larvae on the development of the forensically important blowfly Lucilia sericata (Diptera: Calliphoridae), a widespread and common species of urban areas in temperate regions, was analyzed and the consequences for PMI estimations are discussed. 2. Materials and methods
Fig. 1. Dispersal arena for the post-feeding-larvae (four different tubes enabled the simultaneous start of different post-feeding groups).
The experiments were repeated four times (4 32 = 128 larvae per experiment, n = 512). 2.3. Mortality rate and body size The mortality rate was recorded for the post feeding larvae and the pupae: dead = larvae which died in the tube or pupae which did not hatch. Adult flies were killed afterwards and the cross vein dmcu of the left wing was measured (Mikroskop Zeiss HbO 50/HC, Camera Olympus SC, Cell Imaging Software for Life Sciences Microscopy, Olympus GmbH 2007) as an indicator for body size [16].
2.1. Rearing The colony of L. sericata used in this study was descended from 200 larvae from a specialty medical supplier (BioMonde GmbH) and had been maintained in the laboratory for three generations at the start of this study. The adult flies were kept in a temperature-controlled room (25 8C, SD = 0.8 8C) with constant light and air humidity (33%, SD = 5%). Adult flies were provided with sugar and water ad libitum in a cage covered with fly nets and eventually stimulated to oviposit by adding 150 g of pork liver. Oviposition was documented for a timeframe of about 3 h. The liver samples with eggs were transferred into plastic cups (Ø 9 cm; h = 13 cm) with about 200 g of minced pork meat and pork liver. The plastic cups were put in larger plastic boxes (l = 20 cm; b = 10 cm; h = 15 cm), filled with sawdust, and covered with gauze. There the hatched larvae were fed until they reached the post-feeding stage. 2.2. Postfeeding and pupariation The actual definition of the onset of the post-feeding phase, namely the leaving of the food source [10,11], was supplemented by three more preconditions to assure that the specimens had definitely reached this stage when used in the experiments. Firstly, as an initial reference point, an average minimum age of the larvae had to be fulfilled; according to Grassberger and Reiter [9], this is reached after 85 h at 25 8C by L. sericata. Secondly, the experimental setting was modified by offering additional food sources as a back-up for larvae that left the food source before completing feeding: Schu¨tte [13] showed that larvae of C. vicina sometimes left the food source but then started to feed again later, illustrating that leaving the food source cannot be the only criterion for the post-feeding stage. Hence, just the larvae leaving the second food source were accepted as post-feeding larvae. Finally, a third reference point was set by observing the crop of the larva [12,14] and using only larvae with a visibly empty crop. Although it is known [1] that larvae with empty crops are not the sole larvae found in the post-feeding phase and that larvae might be erroneously precluded because of their crop content, this was a supporting indicator for making sure that just post-feeding larvae were used in the experiments. 32 of the selected post-feeding larvae were transferred in transparent plastic boxes with sawdust for pupariation (control group), three other groups (each with 32 larvae) were placed into an environment unfavorable for pupariation: four transparent tubes (l = 150 cm; Ø 2.5 cm inside; wall 0.3 cm) under permanent light with ends looped around and joined to form a circle simulating an infinite circular path and provoking an extended search for a place suitable for pupariation (Fig. 1). There they stayed for 12 h, 24 h or 48 h until being transferred into the transparent boxes for pupariation. Under permanent light the eclosion of adult flies in the boxes was recorded using a camera (Sony HDR-SR12 Camcorder, 120 GB HDD hard disc), enabling recording of this event to an accuracy of 1 min. The total time of development was estimated in ADH based on a lower developmental threshold of 9 8C [15]. The time of pupariation was not determined in order to avoid disturbances of the process and possible errors in the measurement of the total developmental time.
2.4. Statistics All tests (Pearson correlation coefficient, Hahn prediction interval, Kruskal– Wallis, Jonckheere–Terpstra test, chi-square) were performed with the statistical software BiAS 9.12 [17].
3. Results 3.1. Total developmental time With increasing time in the arena the total time of development increased (Fig. 2). However, this was significant only for the 24 h (Kruskal–Wallis: p < 0.03) and 48 h dispersal groups (Kruskal– Wallis: p < 0.01). A positive trend of the increasing total developmental time was confirmed by a Pearson-regression (Fig. 3) and Hahn prediction interval (p < 0.01) and Jonckheere– Terpstra test (p < 0.01). There were differences of 6.9 h (or 109.6 ADH) between the control group (median 318.4 h) and 24 h post feeding phase (median 325.2 h) and 29.4 h (or 469.6 ADH) between the control group and 48 h post feeding phase (median 347.7 h). No significant differences were detected between control and the shorter dispersal time of 12 h. 3.2. Body size The size, based on wing measurements (cross vein dmcu), was analyzed for 444 adults. Since the wings of 68 flies were not fully extended, these specimens were excluded from the analysis. The length of the vein and thus the body size decreases with increasing time in the arena (Fig. 4). Jonckheere–Terpstra test (p < 0.01), Pearson-regression (Fig. 5) and Hahn prediction interval (p < 0.01) confirmed this observation as significant. 3.3. Mortality rate Mortality rate shows values up to 18% (control: 14.1%; 12 h: 10.9%; 24 h: 13.3%; 48 h: 17.2%), but differences between dispersal times were not statistically significant (x2-contingency, p < 0.6).
M. Mai, J. Amendt / Forensic Science International 221 (2012) 65–69
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Fig. 2. Influence of the dispersal time on the total developmental time (x-axis, groups 1–4 = dispersal time in the arena (0–48 h); y-axis, total developmental time in ADH based on a lower developmental threshold of 9 8C [15]).
Fig. 3. Pearson-regression for the influence of the dispersal time on the total developmental time (x-axis, dispersal time in the arena (0–48 h); y-axis, total developmental time in ADH based on a lower developmental threshold of 9 8C [15]).
Fig. 4. Influence of the dispersal time on the adult body size (x-axis, groups 1–4 = dispersal time in the arena (0–48 h); y-axis, length of vein dmcu (in mm)).
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Fig. 5. Pearson-regression for the influence of the dispersal time on the adult body size (x-axis, dispersal time in the arena (0–48 h); y-axis, length of vein dmcu (in mm)).
4. Discussion The major entomological method to determine the minimum time since death is the estimation of the age of the oldest immature insect stages developing on the dead body. This is done by comparing certain indicators, such as size and accumulated degree hours/days, of the collected evidence with reference values based on data established in laboratory studies. Those studies are usually performed under controlled, ideal conditions, e.g. constant temperatures and food ad libitum. One could argue that such conditions do not appropriately mirror reality and lead to incorrect estimations when calculating the age of a specimen in natural scenarios using those lab-based data sets for reference. As such, there is the need to consider possible influencing factors. One so far neglected factor is the period of post-feeding dispersal in nature. Under laboratory conditions, pupariation substrate is made available immediately and cannot be the determining parameter for the duration of post-feeding larval dispersal. However, previous studies have shown that larvae appear to have different postfeeding periods under differing circumstances [18,19]. For instance, high larval densities or waterlogged soil can delay the start of pupariation and the question arises whether such delays lead to an increase in the overall development time. The only study of this aspect so far published is that by Arnott and Turner [7]. They investigated the post-feeding behavior of the blowfly C. vicina and found that greater dispersal times significantly increased total developmental time with a maximal overestimation of PMI of 2.1 days following a 24 h dispersal period. However, this extreme was not confirmed in the present study although significant differences in total developmental time were found for a dispersal period of 24 h or more, with a maximum extension of development time by approximately 29 h after a dispersal period of 48 h. Apart from our use of another species of blowfly (L. sericata) and, thereby, possible species-related differences, the present study was based on a greater number of specimens (128 per time unit, in total 512) and a more exact method of measuring eclosion than was found in [7]. These two factors were responsible for the more robust data set presented here. As no significant difference between the control group and the 12 h post feeding phase was found, this indicates that even the larvae in the control group, having right from the beginning been provided with suitable substrate, did not immediately pupariate and also spent up to at least approximately 12 h wandering in the box. After that period of time there was no 1:1 relationship
between the extension of the period of dispersal and the resultant prolongation of development time, i.e. a 24 h increase in dispersal did not result in a 24 h increase in total development time. Nevertheless there was a significant delay of about 29 h following 48 h of post-feeding dispersal, which could lead to an inaccurate estimation of the PMI. For evaluating the consequences of the present results and those by Arnott and Turner [7] further studies are required dealing with the post-feeding behavior of larvae in both laboratory and field settings as well as a clearer definition for the onset and termination of the post-feeding phase. Surprisingly, the definition of the time when a larva reaches its post-feeding stage in the laboratory is still problematic and is insufficiently discussed in the literature. Boldrini et al. [19], who studied the interactions of post-feeding larvae, defined the onset of postfeeding as when the first larva began to leave the food source. In other studies [e.g. 7,10,11] it is unclear whether the starting point of post-feeding is defined from when all larvae or only the first one started leaving the food source. It is also unclear whether other characteristics, e.g. an empty gut, were used as markers in those studies. To further complicate matters, it was found [13] that not all larvae of the blowfly C. vicina that left the food source had reached the post-feeding phase (i.e. when provided with a food source some larvae started to feed again). This phenomenon (a premature departure from the food source) was either not taken into consideration [20] or was not realized as a potential problem [e.g. 21,22] when choosing presumed post-feeding larvae. In the experiments reported here the only larvae used were those which were fully fed, had left the food source and showed no gut contents. Using this procedure we achieved a large number of post-feeding specimens which was essential for this study as it allowed us to eliminate the error associated with an insufficient feeding time. It remains unclear whether other definitions of post-feeding would lead to different results. A general guideline and definition of the start of the post-feeding phase in laboratory studies is important both to compare the results of different studies and for the routine interpretation of entomological findings at a crime scene. The same is true for describing the termination of the post-feeding dispersal, which is not adequately defined in some studies. Some studies also include in their methodology the regular physical disturbance of prepupae, for assessment of their stage of development, which possibly disrupts the onset of pupariation. The present study shows that a prolongation of dispersal time (for whatever reason) of a post-feeding larva can lead to a prolongation of the total development time. However, what stimulates larvae to disperse
M. Mai, J. Amendt / Forensic Science International 221 (2012) 65–69
for longer than expected in nature? In our experiments we created an extreme situation with permanent light and no opportunity for larvae to hide for pupariation. However, even under such unfavorable conditions for pupariation, some specimens left in the experimental chamber beyond the 48 h when most specimens were removed started pupariation (data not shown), possibly because the termination of this dispersal process is not only related to parameters of the surroundings at the scene but also strongly connected with a group of toxins produced by the larva itself called paralysins, which appear to be universal in this class of insect [23]. The concentration of these toxins is low in young larvae and increases during development peaking just after pupariation. A possible role of these molecules, with a paralytic effect in the late immature stages, could be the immobilization of the larvae just before pupariation [24]. So it seems that it is not just a behavioral response of the larva to its environment to either continue with dispersal or to start pupariation, but also a chemical process. It would be helpful to estimate the maximum time of dispersal for species of forensic importance because it might reveal a window of time within which pupariation must take place. The introduction of dispersal arenas for post-feeding larvae in laboratory studies could contribute to a better understanding of the post-feeding behavior and more accurate calculations of minimum PMI. Last but not least, field studies that analyze the post-feeding behavior of larvae at different sites and under different climatic conditions could reveal further parameters which are relevant for different dispersal times and, therefore, different development times. Acknowledgements We thank Hanns Ackermann for his statistical support, Dietrich Mebs and Chris Freeman for improving the language of the article, three anonymous reviewers for helpful comments and Stefan Leuchtenberg for drawing Fig. 1. References [1] B. Greenberg, J.C. Kunich, Entomology and the Law: Flies as Forensic Indicators, Cambridge, 2002. [2] J. Amendt, R. Zehner, C.P. Campobasso, C.S. Richards, M.J.R. Hall, Forensic entomology: applications and limitations, Forensic Sci. Med. Pathol. 7 (2011) 379–392. [3] J. Amendt, C.P. Campobasso, E. Gaudry, C. Reiter, H.N. LeBlanc, M.J.R. Hall, Best practice in forensic entomology – standards and guidelines, Int. J. Legal Med. 121 (2007) 90–104. [4] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 (1991) 565–577.
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[5] L.G. Higley, N.H. Haskell, Insect development and forensic entomology, in: J.H. Byrd, J.L. Castner (Eds.), Forensic Entomology. The utility of Arthropods in Legal Investigations, CRC Press, Boca Raton, 2010, pp. 287–302. [6] M.H. Villet, C.S. Richards, J.M. Midgley, Contemporary precision bias and accuracy of minimum post-mortem intervals estimated using development of carrion-feeding insects, in: J. Amendt, C.P. Campobasso, M.L. Goff, M. Grassberger (Eds.), Current Concepts in Forensic Entomology, Springer, Dordrecht, 2010, pp. 109–137. [7] S. Arnott, B. Turner, Post-feeding larval behaviour in the blowfly Calliphora vicina: effects on post-mortem interval estimates, Forensic Sci. Int. 177 (2008) 162–167. [8] L. Gomes, C.J. Von Zuben, Postfeeding radial dispersal in larvae of Chrysomya albiceps (Diptera: Calliphoridae): implications for forensic entomology, Forensic Sci. Int. 155 (2005) 61–64. [9] M. Grassberger, C. Reiter, Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram, Forensic Sci. Int. 120 (2001) 32–36. [10] M. Strehler, H. Klotzbach, B. Madea, S. Reibe, Entwicklung von Calliphora vicina unter Nahrungszugabe von Methadon, Rechtsmedizin 18 (2008) 425–430. [11] D.M. Day, J.M. Wallman, Width as an alternative measurement to length for postmortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae, Forensic Sci. Int. 159 (2006) 158–167. [12] C. Reiter, Zum Wachstumsverhalten der Maden der blauen Schmeißfliege Calliphora vicina, Z. Rechtsmed. 91 (1984) 295–308. [13] A. Schu¨tte, Molekularbiologische Altersbestimmung der nekrophagen Fliegenart Calliphora vicina im Post-Feeding-Stadium. Unpublished thesis, University of Bonn, 2008. [14] C. Reiter, P. Hajek, Zum altersabha¨ngigen Wandel der Darmtraktfu¨llung bei Schmeißfliegenmaden. Eine Untersuchung im Rahmen der forensischen Todeszeitpunktbestimmung, Z. Rechtsmed. 92 (1984) 39–45. [15] M.I. Marchenko, Medicolegal relevance of cadaver entomofauna for the determination of the time of death, Forensic Sci. Int. 120 (2001) 89–109. [16] S. Ireland, B.D. Turner, The effects of larval crowding and food type on the size and development of the blowfly, Calliphora vomitoria, Forensic Sci. Int. 159 (2006) 175–181. [17] H. Ackermann, BiAS fu¨r Windows, Epsilon-Verlag Hochheim, Darmstadt, 1989– 2011. [18] A.A. Green, The control of blowflies infesting slaughterhouses. I. Field observations on the habits of blowflies, Ann. Appl. Biol. 38 (1951) 475–494. [19] J.L. Boldrini, R.C. Bassanezi, A.C. Moretti, C.J. Von Zuben, W.A.C. Godoy, F.J. Von Zuben, S.F. Reis, Non-local interactions and the dynamics of dispersal in immature insects, J. Theor. Biol. 185 (1997) 523–531. [20] M.B. Gallagher, S. Sandhu, R. Kimsey, Variation in developmental time for geographically distinct populations of the common green bottle fly, Lucilia sericata (Meigen), J. Forensic Sci. (2010). [21] C.S. Richards, I.D. Paterson, M.H. Villet, Estimating the age of immature Chrysomya albiceps (Diptera: Calliphoridae), correcting for temperature and geographical latitude, Int. J. Legal Med. 122 (2007) 271–279. [22] S.E. Donovan, M.J.R. Hall, B.D. Turner, C.B. Moncrieff, Larval growth rates of the blowfly, Calliphora vicina, over a range of temperatures, Med. Vet. Entomol. 20 (2006) 106–114. [23] S.-J. Chiou, A. Cerstiaens, S.P. Kotanen, A. De Loof, L. Schoofs, Insect larvae contain substances toxic to adults: the discovery of paralysins, J. Insect. Physiol. 44 (1998) 405–411. [24] S. Kotanen, J. Huybrechts, A. Cerstiaens, K. Zoltan, D. Daloze, G. Baggerman, P. Forgo, A. De Loof, L. Schoofs, Identification of tryptophan and b-carboline as paralysins in larvae of the yellow mealworm, Tenebrio molitor, Biochem. Biophys. Res. Commun. 310 (2003) 64–71.
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae) Madeleine Mai, Jens Amendt * Institute of Forensic Medicine, Kennedyallee 104, 60596 Frankfurt am Main, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 May 2011 Received in revised form 3 April 2012 Accepted 4 April 2012 Available online 1 May 2012
By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the postmortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25 8C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48 h. Only the larvae with a dispersal time of 24 h (total time of development 325.2 h; median) and 48 h (total time of development 347.7 h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4 h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48 h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic entomology Blowflies Lucilia sericata Post-feeding period Developmental time
1. Introduction One of the main tasks of forensic entomology is the estimation of the minimum time since death or post-mortem interval (PMI) [1]. The most important methods are the examination of the insect succession on corpses and the estimation of age of the immature stages of the necrophagous fauna developing on the cadaver [2]. Identifying the species, the stage of its development and knowing the environmental conditions at the scene of death may enable the expert to calculate the age of the oldest specimen and, thereby, estimate the minimum PMI [3]. Blowflies (Diptera: Calliphoridae) colonize a cadaver soon after death and therefore are the most important insects in forensic entomology [4]. For several species there exist published data dealing with the growth of the species
* Corresponding author. Tel.: +49 69 6301 7571; fax: +49 69 6301 5882. E-mail addresses: [email protected] (M. Mai), [email protected] (J. Amendt). 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.04.001
and the time they need for the different stages of development at a range of temperatures [3,5]. Those estimations are based upon the assumption that the immature insects (eggs, larvae, pupae) are of a certain species and accumulate a stage specific thermal input calculated as the product of time and temperature and given in accumulated degree hours (ADH) or days (ADD) [3,5,6]. One of the most interesting stages in the blowfly lifecycle is the so called post-feeding stage between the late larval period and the pupal stage. The larva stops feeding and starts to search for a suitable site for pupariation. As the majority of fly species leave the corpse on which they had been feeding, this stage of development is also called the dispersal or wandering stage. Arnott and Turner [7] pointed out that precise details about this stage are lacking and, when present, are primarily descriptive. This is regrettable because the post-feeding stage is potentially the most variable regarding its duration. While searching for a place for pupariation the larvae may have to crawl several meters if the ground is hard and without any cover or cracks. This not only applies to dry soil environments but also to indoor scenarios, where the larvae might have to move
66
M. Mai, J. Amendt / Forensic Science International 221 (2012) 65–69
up to 30 m or more (Green [18]; Amendt, unpublished data) before starting the process of pupariation. Reference data for the time of development are normally established by laboratory experiments where under controlled conditions the larvae usually do not need to search for a suitable pupariation medium since one is offered right after leaving the food substrate (e.g. sawdust or vermiculite). Subsequently, larvae in laboratory settings get the opportunity to pupate just when leaving the food source and the time spent in dispersal might be very short in laboratory settings according to Arnott and Turner [7] and is usually ignored in the overall calculation of ADH. Natural variations in environmental conditions may lead to a prolonged dispersal time including an energetic cost. Gomes and Von Zuben [8] showed that longer dispersal time in the blowfly Chrysomya albiceps leads to a reduction of its pupal weight. According to Arnott and Turner [7] a longer post-feeding period simply causes further accumulation of degree hours and, as more energy is used, leads to a reduction in resources available for the pupa and the adult stages. This process may also influence the time of development for the blowfly Calliphora vicina. In this paper the effect of different dispersal periods for postfeeding larvae on the development of the forensically important blowfly Lucilia sericata (Diptera: Calliphoridae), a widespread and common species of urban areas in temperate regions, was analyzed and the consequences for PMI estimations are discussed. 2. Materials and methods
Fig. 1. Dispersal arena for the post-feeding-larvae (four different tubes enabled the simultaneous start of different post-feeding groups).
The experiments were repeated four times (4 32 = 128 larvae per experiment, n = 512). 2.3. Mortality rate and body size The mortality rate was recorded for the post feeding larvae and the pupae: dead = larvae which died in the tube or pupae which did not hatch. Adult flies were killed afterwards and the cross vein dmcu of the left wing was measured (Mikroskop Zeiss HbO 50/HC, Camera Olympus SC, Cell Imaging Software for Life Sciences Microscopy, Olympus GmbH 2007) as an indicator for body size [16].
2.1. Rearing The colony of L. sericata used in this study was descended from 200 larvae from a specialty medical supplier (BioMonde GmbH) and had been maintained in the laboratory for three generations at the start of this study. The adult flies were kept in a temperature-controlled room (25 8C, SD = 0.8 8C) with constant light and air humidity (33%, SD = 5%). Adult flies were provided with sugar and water ad libitum in a cage covered with fly nets and eventually stimulated to oviposit by adding 150 g of pork liver. Oviposition was documented for a timeframe of about 3 h. The liver samples with eggs were transferred into plastic cups (Ø 9 cm; h = 13 cm) with about 200 g of minced pork meat and pork liver. The plastic cups were put in larger plastic boxes (l = 20 cm; b = 10 cm; h = 15 cm), filled with sawdust, and covered with gauze. There the hatched larvae were fed until they reached the post-feeding stage. 2.2. Postfeeding and pupariation The actual definition of the onset of the post-feeding phase, namely the leaving of the food source [10,11], was supplemented by three more preconditions to assure that the specimens had definitely reached this stage when used in the experiments. Firstly, as an initial reference point, an average minimum age of the larvae had to be fulfilled; according to Grassberger and Reiter [9], this is reached after 85 h at 25 8C by L. sericata. Secondly, the experimental setting was modified by offering additional food sources as a back-up for larvae that left the food source before completing feeding: Schu¨tte [13] showed that larvae of C. vicina sometimes left the food source but then started to feed again later, illustrating that leaving the food source cannot be the only criterion for the post-feeding stage. Hence, just the larvae leaving the second food source were accepted as post-feeding larvae. Finally, a third reference point was set by observing the crop of the larva [12,14] and using only larvae with a visibly empty crop. Although it is known [1] that larvae with empty crops are not the sole larvae found in the post-feeding phase and that larvae might be erroneously precluded because of their crop content, this was a supporting indicator for making sure that just post-feeding larvae were used in the experiments. 32 of the selected post-feeding larvae were transferred in transparent plastic boxes with sawdust for pupariation (control group), three other groups (each with 32 larvae) were placed into an environment unfavorable for pupariation: four transparent tubes (l = 150 cm; Ø 2.5 cm inside; wall 0.3 cm) under permanent light with ends looped around and joined to form a circle simulating an infinite circular path and provoking an extended search for a place suitable for pupariation (Fig. 1). There they stayed for 12 h, 24 h or 48 h until being transferred into the transparent boxes for pupariation. Under permanent light the eclosion of adult flies in the boxes was recorded using a camera (Sony HDR-SR12 Camcorder, 120 GB HDD hard disc), enabling recording of this event to an accuracy of 1 min. The total time of development was estimated in ADH based on a lower developmental threshold of 9 8C [15]. The time of pupariation was not determined in order to avoid disturbances of the process and possible errors in the measurement of the total developmental time.
2.4. Statistics All tests (Pearson correlation coefficient, Hahn prediction interval, Kruskal– Wallis, Jonckheere–Terpstra test, chi-square) were performed with the statistical software BiAS 9.12 [17].
3. Results 3.1. Total developmental time With increasing time in the arena the total time of development increased (Fig. 2). However, this was significant only for the 24 h (Kruskal–Wallis: p < 0.03) and 48 h dispersal groups (Kruskal– Wallis: p < 0.01). A positive trend of the increasing total developmental time was confirmed by a Pearson-regression (Fig. 3) and Hahn prediction interval (p < 0.01) and Jonckheere– Terpstra test (p < 0.01). There were differences of 6.9 h (or 109.6 ADH) between the control group (median 318.4 h) and 24 h post feeding phase (median 325.2 h) and 29.4 h (or 469.6 ADH) between the control group and 48 h post feeding phase (median 347.7 h). No significant differences were detected between control and the shorter dispersal time of 12 h. 3.2. Body size The size, based on wing measurements (cross vein dmcu), was analyzed for 444 adults. Since the wings of 68 flies were not fully extended, these specimens were excluded from the analysis. The length of the vein and thus the body size decreases with increasing time in the arena (Fig. 4). Jonckheere–Terpstra test (p < 0.01), Pearson-regression (Fig. 5) and Hahn prediction interval (p < 0.01) confirmed this observation as significant. 3.3. Mortality rate Mortality rate shows values up to 18% (control: 14.1%; 12 h: 10.9%; 24 h: 13.3%; 48 h: 17.2%), but differences between dispersal times were not statistically significant (x2-contingency, p < 0.6).
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Fig. 2. Influence of the dispersal time on the total developmental time (x-axis, groups 1–4 = dispersal time in the arena (0–48 h); y-axis, total developmental time in ADH based on a lower developmental threshold of 9 8C [15]).
Fig. 3. Pearson-regression for the influence of the dispersal time on the total developmental time (x-axis, dispersal time in the arena (0–48 h); y-axis, total developmental time in ADH based on a lower developmental threshold of 9 8C [15]).
Fig. 4. Influence of the dispersal time on the adult body size (x-axis, groups 1–4 = dispersal time in the arena (0–48 h); y-axis, length of vein dmcu (in mm)).
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Fig. 5. Pearson-regression for the influence of the dispersal time on the adult body size (x-axis, dispersal time in the arena (0–48 h); y-axis, length of vein dmcu (in mm)).
4. Discussion The major entomological method to determine the minimum time since death is the estimation of the age of the oldest immature insect stages developing on the dead body. This is done by comparing certain indicators, such as size and accumulated degree hours/days, of the collected evidence with reference values based on data established in laboratory studies. Those studies are usually performed under controlled, ideal conditions, e.g. constant temperatures and food ad libitum. One could argue that such conditions do not appropriately mirror reality and lead to incorrect estimations when calculating the age of a specimen in natural scenarios using those lab-based data sets for reference. As such, there is the need to consider possible influencing factors. One so far neglected factor is the period of post-feeding dispersal in nature. Under laboratory conditions, pupariation substrate is made available immediately and cannot be the determining parameter for the duration of post-feeding larval dispersal. However, previous studies have shown that larvae appear to have different postfeeding periods under differing circumstances [18,19]. For instance, high larval densities or waterlogged soil can delay the start of pupariation and the question arises whether such delays lead to an increase in the overall development time. The only study of this aspect so far published is that by Arnott and Turner [7]. They investigated the post-feeding behavior of the blowfly C. vicina and found that greater dispersal times significantly increased total developmental time with a maximal overestimation of PMI of 2.1 days following a 24 h dispersal period. However, this extreme was not confirmed in the present study although significant differences in total developmental time were found for a dispersal period of 24 h or more, with a maximum extension of development time by approximately 29 h after a dispersal period of 48 h. Apart from our use of another species of blowfly (L. sericata) and, thereby, possible species-related differences, the present study was based on a greater number of specimens (128 per time unit, in total 512) and a more exact method of measuring eclosion than was found in [7]. These two factors were responsible for the more robust data set presented here. As no significant difference between the control group and the 12 h post feeding phase was found, this indicates that even the larvae in the control group, having right from the beginning been provided with suitable substrate, did not immediately pupariate and also spent up to at least approximately 12 h wandering in the box. After that period of time there was no 1:1 relationship
between the extension of the period of dispersal and the resultant prolongation of development time, i.e. a 24 h increase in dispersal did not result in a 24 h increase in total development time. Nevertheless there was a significant delay of about 29 h following 48 h of post-feeding dispersal, which could lead to an inaccurate estimation of the PMI. For evaluating the consequences of the present results and those by Arnott and Turner [7] further studies are required dealing with the post-feeding behavior of larvae in both laboratory and field settings as well as a clearer definition for the onset and termination of the post-feeding phase. Surprisingly, the definition of the time when a larva reaches its post-feeding stage in the laboratory is still problematic and is insufficiently discussed in the literature. Boldrini et al. [19], who studied the interactions of post-feeding larvae, defined the onset of postfeeding as when the first larva began to leave the food source. In other studies [e.g. 7,10,11] it is unclear whether the starting point of post-feeding is defined from when all larvae or only the first one started leaving the food source. It is also unclear whether other characteristics, e.g. an empty gut, were used as markers in those studies. To further complicate matters, it was found [13] that not all larvae of the blowfly C. vicina that left the food source had reached the post-feeding phase (i.e. when provided with a food source some larvae started to feed again). This phenomenon (a premature departure from the food source) was either not taken into consideration [20] or was not realized as a potential problem [e.g. 21,22] when choosing presumed post-feeding larvae. In the experiments reported here the only larvae used were those which were fully fed, had left the food source and showed no gut contents. Using this procedure we achieved a large number of post-feeding specimens which was essential for this study as it allowed us to eliminate the error associated with an insufficient feeding time. It remains unclear whether other definitions of post-feeding would lead to different results. A general guideline and definition of the start of the post-feeding phase in laboratory studies is important both to compare the results of different studies and for the routine interpretation of entomological findings at a crime scene. The same is true for describing the termination of the post-feeding dispersal, which is not adequately defined in some studies. Some studies also include in their methodology the regular physical disturbance of prepupae, for assessment of their stage of development, which possibly disrupts the onset of pupariation. The present study shows that a prolongation of dispersal time (for whatever reason) of a post-feeding larva can lead to a prolongation of the total development time. However, what stimulates larvae to disperse
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for longer than expected in nature? In our experiments we created an extreme situation with permanent light and no opportunity for larvae to hide for pupariation. However, even under such unfavorable conditions for pupariation, some specimens left in the experimental chamber beyond the 48 h when most specimens were removed started pupariation (data not shown), possibly because the termination of this dispersal process is not only related to parameters of the surroundings at the scene but also strongly connected with a group of toxins produced by the larva itself called paralysins, which appear to be universal in this class of insect [23]. The concentration of these toxins is low in young larvae and increases during development peaking just after pupariation. A possible role of these molecules, with a paralytic effect in the late immature stages, could be the immobilization of the larvae just before pupariation [24]. So it seems that it is not just a behavioral response of the larva to its environment to either continue with dispersal or to start pupariation, but also a chemical process. It would be helpful to estimate the maximum time of dispersal for species of forensic importance because it might reveal a window of time within which pupariation must take place. The introduction of dispersal arenas for post-feeding larvae in laboratory studies could contribute to a better understanding of the post-feeding behavior and more accurate calculations of minimum PMI. Last but not least, field studies that analyze the post-feeding behavior of larvae at different sites and under different climatic conditions could reveal further parameters which are relevant for different dispersal times and, therefore, different development times. Acknowledgements We thank Hanns Ackermann for his statistical support, Dietrich Mebs and Chris Freeman for improving the language of the article, three anonymous reviewers for helpful comments and Stefan Leuchtenberg for drawing Fig. 1. References [1] B. Greenberg, J.C. Kunich, Entomology and the Law: Flies as Forensic Indicators, Cambridge, 2002. [2] J. Amendt, R. Zehner, C.P. Campobasso, C.S. Richards, M.J.R. Hall, Forensic entomology: applications and limitations, Forensic Sci. Med. Pathol. 7 (2011) 379–392. [3] J. Amendt, C.P. Campobasso, E. Gaudry, C. Reiter, H.N. LeBlanc, M.J.R. Hall, Best practice in forensic entomology – standards and guidelines, Int. J. Legal Med. 121 (2007) 90–104. [4] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 (1991) 565–577.
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