Effect of temperature on development of the ...

Forensic Science International 128 (2002) 177–182

Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) Martin Grassbergera,b,*, Christian Reitera a

Department of Medical and Forensic Entomology, Institute of Forensic Medicine, University of Vienna, Sensengasse 2, A-1090 Vienna, Austria b Department of Anthropology, University of Vienna, Vienna, Austria

Received 13 February 2002; received in revised form 15 May 2002; accepted 29 May 2002

Abstract Immature development times of the blow fly Protophormia terraenovae (Robineau-Desvoidy, 1830) were studied in the laboratory at five different constant temperatures (15, 20, 25, 30, 35 8C). The minimal duration of development from oviposition to adult emergence was inversely related to temperature, ranging from 9:19 0:3 days at 35 8C to 37:78 2:96 days at 15 8C. From linear regression of development rates at the five studied constant temperature regimes, it followed that the minimum development threshold (tL) for total immature development is 8.95 8C (9 8C) and the overall thermal constant (K) for P. terraenovae is 240:2 9:3 day-degrees (DD) above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 8C. However, it is possible that developmental time from oviposition to adult eclosion might be different in various regions of the world, and that the thermal constant of a holarctic species like P. terraenovae is not same everywhere. Additionally, as the present paper shows, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Protophormia terraenovae; Forensic entomology; Post-mortem interval; Development time

1. Introduction Since Me´gnin [1], synanthropic flies, particularly calliphorids, are recognised as the first wave of the faunal succession on human cadavers [2,3]. They are therefore the primary and most accurate forensic indicators of time of death. Protophormia terraenovae (Robineau-Desvoidy) has a Holarctic distribution and is very common in the cooler regions. Abundant in the Arctic, it has been found within 550 miles of the North Pole [3]. Cool weather favours development and this species is the most cold tolerant of all calliphorid species [4]. Benecke [5] found a single adult

* Corresponding author. Tel.: þ43-699-113-26-708; fax: þ43-1-4277-9657. E-mail address: [email protected] (M. Grassberger).

and larvae inside the skull of a 29-day-old corpse in Cologne during May. Introna et al. [6] collected second instar larvae from the charred remains of a body in the month of August in a rural area of the city of Brindisi (southern Italy), showing the broad ecological potential of this species. In Austria, it is most abundant during spring and autumn, but can also be collected from corpses during summer in higher, elevations (Grassberger, unpublished data). Since P. terraenovae (Robineau-Desvoidy) is one of the early invaders of carrion and especially attracted to human cadavers [7], the temperature dependent development of this fly species can be used to pinpoint time since death postmortem interval (PMI). The temperature dependent development of this species has been studied by Green [8], Kamal [9], Greenberg and Tantawi [10] and Marchenko [11]. Since the developmental time of P. terraenovae reported in these studies varies to a certain extent, or is studied only under a single constant temperature, there is a continuing need to

0379-0738/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 2 ) 0 0 1 9 9 - 8

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M. Grassberger, C. Reiter / Forensic Science International 128 (2002) 177–182

refine and improve these values. Due to its forensic importance in many regions [3–6,11–13], a detailed study of its developmental duration under different constant temperature regimes was undertaken. Detailed Information on the developmental rate, minimum development threshold and thermal constant of P. terraenovae will provide background data for future forensic-entomological casework.

2. Material and methods Eggs, larvae and adults of P. terraenovaewere collected from human cadavers and from pig carcasses in and around the city of Vienna during the fly-active period of the years 1999–2001. Adults and larvae were identified, using the morphological characters described by Smith [3] and Crosskey and Lane [14]. The flies were held in an insectary at 22–25 8C with approx. 60% RH and a photoperiod of 14:10 (L:D) hours. New flies were added from time to time. About 300 adult flies were kept in screen cages (40 cm 30 cm 30 cm) and fed a mixture of dry granular sugar, powdered milk and brewer’s yeast. Water was supplied by inversion of a beaker on a Petri dish, which was covered with a filter paper. 2.1. Egg period under different constant temperature regimes

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 [15]. However, if the substrate is relatively dry and not exposed to bright light, larvae of P. terraenovae pupate on the surface. The jars were then placed into a precision environmental chamber (KBK/LS 4330, Ehret, Germany) at one of the five desired temperature regimes (15, 20, 25, 30 and 35 8C, respectively), with a relative humidity set to 65–70%. This procedure was repeated 10 times for each temperature regime. Twice a day, we recorded the mean temperature within the centre of actively feeding maggots using a digital thermometer. Four of the largest maggots were removed from the plastic jars every 4 h. When the first maggots stopped feeding, we removed those in the migratory phase for measurement purposes, until 10% of the maggots underwent pupation. After peak feeding samples were removed every 6 h. Measuring the largest individuals (i.e. the oldest, before peak feeding) is regarded as common practice in forensic entomology [16]. Specimens were killed in hot water to prevent shrinkage, as might be the case with other killing and preservative solutions [17]. Measurement was followed immediately under the binocular in 0.1 mm units using a Vernier caliper. 2.3. Developmental threshold and thermal constant

To study the time range of the egg period (i.e. time from oviposition to emergence of first instar larvae) under different constant temperatures, eggs were collected within 30 min of oviposition, using black 35 mm film cups filled 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 (BioMe´ rieux) using a Pasteur pipette. The resulting monolayer of eggs 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 five temperatures (15, 20, 25, 30 and 35 8C) and incubated plates were checked at half hour intervals. For each temperature regime, five plates were prepared at different times of the day to ensure early recognition. 2.2. Growth under different constant temperature regimes Eggs were collected within 30 min of oviposition. 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 behaviour, which

Lower thresholds (tL) for development were estimated from the linear regression (Microcal Origin1) of the developmental rates (y ¼ 1/developmental time) on constant temperature (x) [18,19]. The thermal constant K was calculated from the equation K ¼ yðt tL Þ, where y is the developmental time (days), t is the rearing temperature (8C), and tL is the lower developmental threshold temperature (8C). The thermal constant was calculated for each of the five constant temperatures (for total immature development) to obtain the overall K (mean S:D:). Values of K represent the number of degree-days (DD) above the threshold (tL) needed for total immature development.

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 (Fig. 1). The mean minimum duration of development (S.D.) from oviposition to egg-hatching, from oviposition to pupariation and from oviposition to eclosion (total immature development) at each of the five studied temperature regimes is given in Table 1. Development time from oviposition to adult eclosion was shortest at 35 8C (mean 9:19 0:3 days) and longest at 158C (mean


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Fig. 1. Development of P. terraenovae from oviposition to pupariation at five different temperature regimes (means of the maximal measured lengths).

Table 1 Minimal developmental times (mean days S.D.) of P. terraenovae life stages at five constant temperature regimes Temperature (8C)

Minimal developmental time (days) from oviposition to Egg hatch

15 20 25 30 35

2.87 1.3 0.79 0.46 0.38

0.11 0.05 0.03 0.03 0.04

Pupariation 22.3 13.1 9.58 6.1 5.17

1.91 1.1 0.53 0.13 0.29

Eclosion 37.78 21.96 15.8 11.48 9.19

2.96 1.45 1.19 0.66 0.3

37.78 2.96 days). In the centre of actively feeding second and third instars the recorded temperature was 1–1.3 8C above the desired temperature regime. First and second molting always occurred within a certain range of larval length (about 3.8 and 8 mm). 3.2. Isomorphen-diagram Similar to the previously published isomegalen-diagram [20,21], all developmental data from oviposition to eclosion are represented in the isomorphen-diagram. In this diagram, time from oviposition to eclosion is plotted against temperature, each line representing morphological changes. Areas between lines represent identical morphological stages of the blow fly P. terraenovae (Fig. 2). 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.

3.3. Developmental threshold and thermal constant The rate of total immature development increased with temperature, with development rates of 0.0265, 0.0455, 0.0633, 0.0871 and 0.1088 at 15, 20, 25, 30 and 35 8C, respectively, and r ¼ 0:99, P < 0:0001; y ¼ 0:00412x 0:03686. From the regression line plotted in Fig. 3, it was calculated that the minimum development threshold (tL) for total immature development was 8.9 8C (9 8C) and the overall thermal constant (K) for P. terraenovae was 240:2 9:3 DD above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 8C (10 8C).

4. Discussion 4.1. Use of the isomorphen-diagram Entomological evidence found on and around the corpse should be collected and preserved according to medico-legal standard procedures [22]. 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, as is the case with corpses found indoors, larvae or pupae recovered from the scene should be stored at a constant temperature, until they pupate or the first adults emerge. Their age can then be determined retrospectively, using the isomorphen-diagram. Where temperature is variable, an age range can be estimated between the points where

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Fig. 2. Isomorphen-diagram for P. terraenovae, showing the egg, larval and pupal stage between 15 and 35 8C. Each line represents identical morphological changes of this holometabolous (i.e. the immature stage is substantially different from the adult in structure and biology) insect.

Fig. 3. Linear regression of rearing temperature and rate of development (from oviposition to pupariation and from oviposition to adult eclosion) of P. terraenovaefrom the Viennese area.

the observed morphological change (pupation or eclosion) cuts the graph at the maximum and minimum temperatures recorded. If the temperature is roughly constant, as is the case with corpses found indoors the use of the growthcurve and the isomorphen-diagram could provide a quick and precise minimal estimate for the PMI. Since biological systems under field conditions are rarely predictable with

the precision attainable in the laboratory, the greatest care must be taken in interpretation of the results. 4.2. Temperature summation model The development of poikilotherms (i.e. animals that are unable to regulate their body temperature metabolically and


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Table 2 Developmental data from [11] compared to data of the present study (average minimum duration) Temperature (8C)

15 20 25 30 a b

Minimum total immature deviation (days)

Calculated DD

Present study

[11]

Present studya

[11]b

37.8 21.9 15.8 11.5

34.9 20.6 14.6 11.3

226.7 241.6 252.8 241.1

251.3 251.3 251.2 250.9

Calculated development threshold: 8.95 8C. Calculated development threshold: 7.82 8C.

maintain their body temperature by absorbing heat from the surrounding environment) as P. terraenovae is widely described using the temperature summation model which is valid for the linear proportion of the sigmoidal development curve [18]. In our experiments, development of P. terraenovae was linearly related to temperature (r ¼ 0:99, P < 0:0001) between 15 and 35 8C, and expressed by the overall thermal constant K ¼ 240:2 9:3 DD above the threshold of 8.95 8C. A comparable threshold temperature (108) was roughly calculated by Higley and Haskell [23] using the data from Kamal [9]. However, using the same data [9] they calculated an overall thermal constant of 209 DD for total development of P. terraenovae. Greenberg and Tantawi [10] calculated 1133.7, 319.7, 314.6 and 322 DD at temperatures of 12.5, 23, 29 and 35 8C, respectively, but for unknown reasons used the rearing temperature t instead of the effective temperature (t tL ) for their calculations. However, at 35 8C they found an average minimum duration of development of 9.2 days, which corresponds to the duration at the same temperature in the present study (9:19 0:3 days). Marchenko [11] successfully reared P. terraenovae from the Leningrad area and from the Kaunas region of Lithuania at constant temperatures from 11 to 30 8C. Using his data, we calculated a minimum development threshold (tL) for total immature development of 7.82 8C and an mean thermal constant (K) of 251 0:3 DD. Between 15 and 30 8C, development times of P. terraenovae from the Viennese area (present study) were in general similar to those of Marchenko [11] (Table 2).

5. Conclusion We conclude that developmental times from oviposition to adult eclosion might possibly differ in various regions of the world. This raises the question whether it is valid to assume that the thermal constant of a holarctic species is the same everywhere. However, larger differences do not necessarily have to be attributed to variation in experimental method (extrinsic factors). Geographic adaptation (intrinsic factors) could explain a difference in temperature dependent development. Nevertheless, there is a continuing need to

refine and improve these values. Precise values for developmental minima and degree-day estimates by stage (egg, total larva and pupa) are important areas of improvement [23]. Additionally, as our experiments show, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework.

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