Forensic Science International 120 2001) 32±36 Effect of temperature on Lucilia sericata Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram Martin Grassberger *, Christian Reiter Institute for Gerichtliche Medizin der Universitaet, Sensengasse 2, Wien 1090, Austria Abstract Developmental behavior of eggs, larva and pupa of the blow y species Lucilia sericata Meigen) were studied under 10 different temperature regimes. Data from these studies were used to construct the isomegalen-diagram. In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. If the temperature is roughly constant, as is the case with corpses found indoors, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable, an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. Equally, the isomorphen-diagram representing all morphological stages from oviposition to eclosion should be used, if maggots in the migratory phase or pupae or puparia are recovered from the scene. The isomegalen- and the isomorphen-diagrams could facilitate a quick and more precise estimate of the postmortem interval even for the inexperienced investigator. In addition, our results vary from those of other investigators, suggesting a different thermal behavior of the holarctic blow y L. sericata in various zoogeographic regions. # 2001 Published by Elsevier Science Ireland Ltd. Keywords: Lucilia sericata; Calliphoridae; Blow ies; Forensic entomology; Postmortem interval 1. Introduction Lucilia sericata Meigen) Phaenicia of American authors), originally described in 1826, is a synanthropic y very common around human habitations in the Holarctic Region but occurs throughout the world. Since MeÂgnin [1], synanthropic ies, particularly calliphorids, are recognized as the rst wave of the faunal succession on human cadavers [2,3]. They are therefore the primary and most accurate forensic indicators of time of death. Due to the recent increase in the number of cases in Europe and the United States involving forensic entomology, detailed development data are needed to allow more precise postmortem interval PMI) estimates. Previous studies on growth behavior and PMI estimates concerning L. sericata have been carried out by Kamal [4], Nuorteva [2], Introna et al. [5] and Greenberg [6]. However, data obtained * Corresponding author. through these studies were not always consistent. Our study puts special emphasis on the isomegalen-diagram published earlier for Calliphora vicina [7] and the new isomorphendiagram, which should facilitate a quicker and more precise estimate of the postmortem interval even for the inexperienced investigator. 2. Material and methods Eggs, larvae and adults of L. sericata were collected from human cadavers and from liver baits in and around the city of Vienna during the y-active period of the years 1992±1999. Adults and larvae were identi ed, using the morphological characters described by Aubertin [8], Smith [3] and Holloway [9] including the comparison of male genitalia. The ies were held in an insectary at 22±258C with approximately 60% RH and a photoperiod h) of 12:12 L:D). New ies were added from time-to-time. About 300 adult ies were kept in screen cages 40 cm 30 cm 30 cm) and fed dry 0379-0738/01/$ ± see front matter # 2001 Published by Elsevier Science Ireland Ltd. PII: S 0379-0738 01)00413-3
M. Grassberger, C. Reiter / Forensic Science International 120 2001) 32±36 33 granular sugar and a mixture of powdered milk 70%) and brewer's yeast 20%) in gelatine 10%). Water was supplied by inversion of a beaker on a Petri dish covered with a lter paper. 2.1. Egg period under different constant temperature regimes To study the time range of the egg period i.e. time from oviposition to emergence of rst instar larvae) under different constant temperatures, eggs were collected within 30 min of oviposition, using black 35 mm lmcups baited with decaying beef liver. This provided a dark and moist environment preferred by the female adults for oviposition. The eggs were separated from each other by soaking them in sodium sulphite solution 1%). After shaking vigorously, the egg-clusters are usually broken apart within 5 min. Eggs were spread on Columbia agar plates containing 5% sheep blood BioMeÂrieux) using a Pasteur pipette. The resulting egg-monolayer facilitated recognition of larval emergence and the moisture of the agar prevented the eggs from drying out, an important detail at higher temperatures. The agar plates were put in the incubator at one of the six desired temperatures 15, 20, 25, 30, 35 and 408C) and incubated plates were checked at half an hour intervals. For each temperature regime, ve plates at different times of day were prepared to ensure early recognition. 2.2. Growth under different constant temperature regimes Eggs were collected within 30 min of oviposition, as mentioned above. Samples of about 100 eggs were spread on 250 g raw beef liver, cut in approximately 1 cm thick slices, and subsequently transferred into plastic jars 25 cm 25 cm 7 cm) covered with a gauze-net. Using this procedure, we achieved a more two-dimensional and disseminated feeding behavior, which is essential to prevent maggot mass formation. The bottom of the jars was covered with sawdust, to provide a dry place for pupation. This is important, because it is considered that larvae could delay pupation under suboptimal conditions [10]. The jars were then placed into a precision incubator KB 115, WTB Binder, Germany) at one of the 10 desired temperature regimes 15, 17, 19, 20, 21, 22, 25, 28, 30 and 348C, respectively). This procedure was repeated 10 times for each temperature regime. Twice a day we recorded the mean temperature within the center of actively feeding maggots using a digital thermometer 0.18C). Four of the largest looking maggots were removed from the plastic jars every 4 h. When the rst maggots stopped feeding, we removed those in the migratory phase for measurement purposes, until 10% of the maggots underwent pupation. After peakfeeding samples were removed for every 6 h. Measuring the largest individuals i.e. the oldest, before peakfeeding) is regarded as common practice in forensic entomology [11]. Specimens were killed in boiling water to prevent shrinkage, as might be the case with other killing and preservative solutions [12]. Measurement was followed immediately under the microscope in 0.2 mm units. 3. Results 3.1. Growth curves from constant temperature regimes The means of the maximal measured lengths of all rearings were plotted against time for each of the constant temperature regimes beginning with the emergence of the larvae) Fig. 1). After peakfeeding, variation in maximal length occurred until the onset of pupation, resulting in a undulating curve. The duration of each developmental stage under all temperature regimes is presented in Table 1. At 158C, no emergence of adult ies was observed. In the center of actively feeding 3rd instars, the recorded temperature was sporadically 0.5±18C above the desired temperature regime. First and second molting always occurred at least under optimal trophic conditions) within a certain range of larval length about 3.8 and 8 mm). Around molting, the maggot stops growing, which results in a cascade-like shape of the growth curve arrows in Fig. 1). Microscopical examination Table 1 L. sericata: average minimum duration of developmental stages n ˆ 10 for each temperature regime) a Duration h) Stage 158C 178C 198C 208C 218C 228C 258C 288C 308C 348C Eggs 31 28 24 22 19 17 14 11 10 8.5 1st Instar 56 39 27 24 23 19 16 11 10 9.5 2nd Instar 70 54 42 35 29 26 19 16 15 12 3rd Instar 115 79 60 53 47 46 36 30 27 27 Postfeeding 340 200 118 108 103 94 87 87 87 82 Pupa a) 442 293 209 158 137 125 120 119 120 Total ± 842 564 451 379 339 297 275 268 259 a No emergence of adults.
34 M. Grassberger, C. Reiter / Forensic Science International 120 2001) 32±36 Fig. 1. Development of L. sericata from hatching to pupation at 10 different temperature regimes. Arrows indicate 1st and 2nd molting. Fig. 2. Isomegalen-diagram for L. sericata larvae from hatching to peakfeeding. Time is plotted against temperature, each line representing identical larval length mm). The small graph shows the analogous egg-periods between 15 and 408C.
M. Grassberger, C. Reiter / Forensic Science International 120 2001) 32±36 35 Fig. 3. Isomorphen-diagram for L. sericata, showing all stages from oviposition to eclosion 15±348C). Areas between lines represent identical morphological stages at various temperatures. Where a ˆ egg; b ˆ 1st instar; c ˆ 2nd instar; d ˆ 3rd instar; e ˆ postfeeding larva i.e. prepupa); f ˆ pupa; g ˆ imago. Each line represents identical morphological changes of this holometabolous insect. shortly before and during molting revealed the new posterior spiracular slits underneath the old spiracles. 3.2. Isomegalen-diagram Data from the growth curves were used to construct the isomegalen-diagram Fig. 2). In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. 3.3. Isomorphen-diagram Similar to the isomegalen-diagram, all developmental data from oviposition to eclosion are represented in the isomorphen-diagram. Areas between lines represent identical morphological stages of the blow y L. sericata Fig. 3). This diagram is especially useful when postfeeding larvae or pupae are recovered from the corpse, a condition under which length is no longer a useful criterion of age. 4. Discussion 4.1. Use of the isomegalen- and isomorphen-diagrams Entomological evidence found on and around the corpse should be collected and preserved according to medico-legal standard procedures [14]. On site microclimatic temperatures prevailing in the maggots' immediate environment should be established and correlated retrospectively with the air temperature records. Assuming an average constant temperature, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. When postfeeding larvae or pupae are recovered from the scene, live specimens should be stored at constant temperature, until they pupate or the rst adults emerge. Their age can then be determined retrospectively, using the isomorphen-diagram. 4.2. Varying developmental data Greenberg [6] points out that the developmental times from oviposition to eclosion might possibly differ in various regions of the world. He raises the question whether it is valid to assume that the thermal constant of a holarctic species is the same everywhere. For comparison of developmental data at 228C from Greenberg [6] and the present study see Table 2. Greenberg also reports a personal communication with Marchenko from Leningrad, whose laboratory data time from egg to adult) differs from his own to a maximum of 1.3 days at 228C. Whether this inhomogenity in developmental data is an artifact or due to different regional
36 M. Grassberger, C. Reiter / Forensic Science International 120 2001) 32±36 Table 2 Developmental data 228C) from Greenberg [6] compared to data of the present study average minimum duration of each stage) Duration h) at 228C Egg 1st Instar 2nd Instar 3rd Instar Postfeeding Pupa Total immature Greenberg [6] 23 27 22 22 106 143 343 Present study 17 19 26 46 94 137 339 bionomics i.e. different biological strains) is unknown. In the genus Lucilia considerable variation in myiasis behavior exists both between and within individual species [15]. Additionally, the genus Lucilia is a small, relatively homogenous group of at least 27 species, all of which bear a very close resemblance to each other [9,13]. The species L. richardsi Collin for example is almost identical to L. sericata). This could be the cause for misidenti cation of specimens. Efforts are under way to solve this problem of identi cation, using DNA typing techniques [16,17]. 5. Conclusion If the temperature is roughly constant, as is the case with corpses found indoors the use of the isomegalen- and isomorphen-diagrams could provide a quick and precise estimate for the PMI. Since biological systems under eld conditions are rarely predictable with the precision attainable in the laboratory, the greatest care must be taken in interpretation of the results. Moreover, it must be borne in mind that a Holarctic blow y species might not necessarily exhibit the same growth pattern in different zoogeographic regions. Acknowledgements We are indebted to Franz Huemer University of Vienna) for assistance in our laboratory. References [1] J.P. MeÂgnin, La faune des cadavres: application de l'entomologie aá la meâdicine leâgale, EncyclopeÂdie scienti que des Aide-meÂmoires, Masson et Gauthier-Villars, Paris, 1894. [2] P. Nuorteva, Sarcosaprophagous insects as forensic indicators, in: C.G. Tedeschi Ed.), Forensic Medicine: A Study in Trauma and Environmental Hazards, Vol. II, Saunders, Philadelphia, 1977, pp. 1072±1095. [3] K.G.V. Smith, A manual of forensic entomology, British Museum, Natural History, London, and Cornell University Press, Ithaca, NY, 1986. [4] A.S. Kamal, Comparative study of 13 species of Sarcosaprophagous Calliphoridae and Sarcophagidae Diptera). 1. Bionomics, Ann. Entomol. Soc. Am. 51 1958) 261±271. [5] F.J. Introna, B.M. Altamura, A. Dell'Erba, V. Datoli, Time since death de nition by experimental reproduction of Lucilia sericata cycles in growth cabinet, J. Forensic Sci. 34 1989) 478±480. [6] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 1991) 565±577. [7] C. Reiter, Zum Wachstumsverhalten der Maden der blauen Schmeiû iege Calliphora vicina, Z. Rechtsmed. 91 1984) 295±308. [8] D. Aubertin, Revision of the genus Lucilia R.-D. Diptera, Calliphoridae), Linnaean Soc. J. Zool. 38 1933) 389±463. [9] B.A. Holloway, Morphological characters to identify adult Lucilia sericata Meigen, 1826) and L. cuprina Wiedmann, 1830) Diptera: Calliphoridae), New Zealand J. Zool. 18 1991) 415±420. [10] J.D. Wells, H. Kurahashi, Chrysomya megacephala Fabricius) Diptera: Calliphoridae) development: rate, variation and the implications for forensic entomology, Jpn. J. Sanit. Zool. 45 1994) 303±309. [11] J.H. Byrd, J.F. Butler, Effects of temperature on Sarcophaga haemorrhoidalis Diptera: Sarcophagidae) development, J. Med. Entomol. 35 1998) 694±698. [12] T.I. Tantawi, B. Greenberg, The effect of killing and preservative solutions on estimates of maggot age in forensic cases, J. Forensic Sci. 38 1993) 702±707. [13] J. Stevens, R. Wall, Classi cation of the genus Lucilia Diptera: Calliphoridae): a preliminary parsimony analysis, J. Natural History 30 1996) 1087±1094. [14] N.H. Haskell, R.D. Hall, V.J. Cervenka, M.A. Clark, On the body: insects' life stage presence and their postmortem artifacts, in: W.D. Haglund, M.H. Sorg Eds.), Forensic Taphonomy, The Postmortem Fate of Human Remains, CRC Press, LLC, Boca Raton, 1997, pp. 436±441. [15] J. Stevens, R. Wall, The evolution of Ectoparasitism in the genus Lucilia Diptera: Calliphoridae), Int. J. Parasitol. 27 1997) 51±59. [16] F.A. Sperling, G.S. Anderson, D.A. Hickey, A DNA-based approach to the identi cation of insect species used for postmortem interval estimation, J. Forensic Sci. 39 1994) 418±427. [17] M. Benecke, Random ampli ed polymorphic DNA RAPD) typing of necrophagous insects Diptera, Coleoptera) in criminal forensic studies: validation and use in practice, For. Sci. Int. 98 1998) 157±168.