Effects of Refrigeration on the Biometry and Development of Protophormia Terraenovae (Robineau–Desvoidy) (Diptera: Calliphorid

Home , Calliphoridae, Protophormia terraenovae

Forensic Science International 125 (2002) 254–261

Effects of refrigeration on the biometry and development of Protophormia terraenovae (Robineau–Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations Jean-Bernard Myskowiaka,*, Claudie Doumsb aInstitut de Recherche Criminelle de la Gendarmerie Nationale, 1 Bd The´ophile Sueur, 93111 Rosny-sous-Bois Cedex, France bLaboratoire d’e´cologie de l’Universite´ Pierre-et-Marie-Curie, UMR 7625, BaˆtA7e`me e´tage, 7 quai St Bernard, 75252 Paris Cedex, France Received 17 July 2001; received in revised form 31 October 2001; accepted 19 December 2001

Abstract

The aim of this study was to simulate the low temperatures that insects could experience between the time being sampled from cadavers and their arrival in the laboratory. This was in order to investigate the effect of low temperature on development of maggots. At different stages of development, individuals of Protophormia terraenovae (Robineau–Desvoidy) reared at 24 8C were submitted to a temperature of 4:0 0:5 8C for a period varying from 1 to 10 days. Independent of the stage of development at which the insects were refrigerated, the treatment induced signiﬁcant changes on the duration of development. The effect of low temperature on the developmental time between the return to 24 8C to adult emergence depended on the larval stage that was refrigerated. When ﬁrst instar larvae and prepupae were refrigerated, the time to emergence at 24 8C decreased with an increase of duration of the refrigeration period. Time to emergence increased under the same conditions when second instar larvae and pupae were refrigerated. These results indicate that keeping larvae of P. terraenovae at 4 8C does not just simply lead to a cessation of metabolism but disturbs the regular development. Ten days of cooling induced an error in estimating post-mortem interval (PMI) of more than 6 h. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Forensic entomology; Temperature; Development rate; Weight; Length; Protophormia terraenovae

1. Introduction when corpses are discovered, and during initial investigations, insects are collected directly on the remains by scene-of- At the end of the 19th century, the French entomologist crime ofﬁcers before being conveyed as soon as possible to a Megnin and Benecke used sarcosaprophagous insects that entomology laboratory. When a rapid transport is impossi- had developed on dead horses lying in a ﬁeld to gather ble, the insects are placed in a refrigerator for a varying forensic information [1,2]. Today, forensic entomology, period depending on legal procedures. Low temperatures including the study of the ecology and development of are known to stop insect development [19], but this treat- necrophagous insects colonizing a corpse, is commonly ment could still induce a physiological disturbance involv- used in investigations of death [3–17]. In practice, the ing post-mortem interval (PMI) errors. For instance, if the accuracy of this method is about 1 day for a 1-month-old insect development could be fastened after a refrigeration corpse and 1 week for a 6-month-old corpse [18]. In France period, this could lead to an underestimation of the time of death. Effects of low temperature on insect development are now * Corresponding author. Tel.: þ33-1-4935-5862; well known reviewed in [20–23]. Diapause and quiescence are fax: þ33-1-4935-5027. the two main physiological responses to low temperatures.

0379-0738/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0379-0738(02)00003-8 J.-B. Myskowiak, C. Doums / Forensic Science International 125 (2002) 254–261 255

Diapause is a delay in development evolved in response to (Sm ¼ 7:8 8C) [34]. Its complete development requires regularly recurring periods of adverse environmental condi- 10–13 days at 27 8C [35]. tions. In temperate regions, it facilitates winter survival. So, P. terraenovae was established in the laboratory from diapause is a characteristic feature of the life cycle of insects specimens bought in a fishing tackle shop. Adults were synchronizing development with cyclic and seasonal con- reared at ambient temperature and allowed to oviposit on ditions [21], whereas quiescence mainly stops development the surface of fresh beef muscle of uniform quality. The meat for a short time and acts like an anaesthetic. Quiescence is was observed regularly and was removed when enough eggs induced by the immediate effect of temperatures ranging were obtained. Meat with eggs was placed on sand in a from 0 to 10 8C and involves a deceleration of insect plastic box. This box was held at a mean temperature of metabolic activity [22]. Experiments on cold temperature 24 1 8C and a relative humidity of 75% in a FIRLABO show that cooled insects recover and develop normally if 140 H incubator. returned to a high temperature after a short interval at low Four times per day, approximately 20 immature stages temperatures [20]. Some studies have been specifically were randomly selected and examined briefly under a designed to address the potential effect of low temperature LEICA dissecting microscope to determine their develop- in insects used for PMI estimates in forensic science ment stages from the larval structures (anterior and posterior [24–28]. These few studies were mainly concerned with spiracles and crop size). Basically, the first two larval stages the effect of cyclic or alternating temperatures (never lower differ by the appearance of anterior spiracles. The posterior than 10 8C) on the growth curves of different insects used in spiracles of the third larval stage have three slits, whereas forensic science. Johl and Anderson simulated the chilling only two slits are visible at the second larval stage. In encountered by insects collected from cadavers in a mor- addition to these morphological criteria, the prepupal stage gue. They showed that keeping individuals of Calliphora is characterized by migration out of the feeder substrate to vicina (Robineau–Desvoidy) at 3 8C for 24 h induced a prepare for pupation in the sand. When the developmental 24 h delay in adult emergence without mortality no matter in stage (egg, first, second and third instar larvae, prepupa and which stage the refrigeration took place. Here, low tempera- pupa) needed for each different experiment was reached, 30 ture induced quiescence without affecting the insect physiol- individuals were randomly removed and placed on a piece of ogy when the individuals were returned to 24 8C. This is what beef in a small polystyrene box. The procedure was ran- is assumed by investigators in France when they advise domly repeated to obtain 33 random samples. These samples keeping the insect in a refrigerator while waiting for legal were submitted to 11 experimental treatments (T) (three decisions or during transport from the field to the laboratory. samples/treatment). The first treatment T0 was a control with However, in France, the time spent by insects in a refrigerator a rearing temperature of 24 8C in an incubator with 75% RH. can often be longer than 24 h. For treatments T1–T10, the samples were subjected to a The aim of this study was to analyze the consequences on constant cooling in a pharmaceutical refrigerator (SANYO the PMI estimation of the storage of insects in a refrigerator MPR 161 D) at 4 0:5 8C and 75% RH for a period varying for different durations. We simulated a period of refrigeration from 1 to 10 days, respectively. After the cooling periods, the at 4 8C varying from 24 to 240 h on the different immature samples were returned to the incubator at 24 8C. This stages of Protophormia terraenovae (Robineau–Desvoidy) experiment was performed on each stage of the insect (Diptera: Calliphoridae) in order to (a) analyze the effect of developmental cycle (egg, L1, L2, L3, prepupa and pupa). refrigeration on weight and length of pupae and (b) to test Every day, the samples were sprayed with as much tap whether the period of refrigeration affected the development water as necessary to keep the meat moist. The number of rate once the insect was returned to a normal rearing tem- pupae and eclosions was recorded to determine two aspects perature of 24 8C. P. terraenovae is a blowflyregularly of the development rate: (a) the larval development rate found in forensic entomological cases, usually observed in between the return to 24 8C and pupation; and (b) the European cool regions but also recorded as far as in southern nymphal development rate between pupation and eclosed Italy [7,11,21,29,30]. This fly is a good indicator of the time adults. Weight and length of all 2-day-old pupae was mea- of death, because its larvae are primary consumers of the sured. carcass during the first month post-mortem [31]. In order to investigate the effect of refrigeration on the development rate and biometry (weight and length of pupae), the analyses were performed separately for each stage. The 2. Materials and methods effect of refrigeration on development and biometry was analyzed by a two-way nested analysis of variance (ANOVA) P. terraenovae is a holometabolous insect (total meta- with replicates (three samples per treatment) nested within morphosis between the last larval stage and the adult eclo- treatment (control and 10 treatments). A weighted linear sion) [32] with an induction of its reproductive diapause regression was used to test for a linear effect of increasing by the photoperiod [33]. This insect accumulates a sum of the period of refrigeration on the different development daily temperatures (accumulated degree-days ¼ 251 8C) and biometry measurements [36] (treatment means were [34] associated with a minimal development threshold weighted by the number of individuals per treatment). 256 .B ykwa,C om oescSineItrainl15(02 254 (2002) 125 International Science Forensic / Doums C. Myskowiak, J.-B.

Table 1 Summary of the statistical results of cooling on the different measurements for each developmental stages

Developmental stage

L1 L2 L3 Prepupa Pupa

ANOVA Regression ANOVA Regression ANOVA Regression ANOVA Regression ANOVA Regression a a b b (FA [9,20]) (FR [1,8]) (FA [10,22]) (FR [1,9]) (FA [10,22]) (FR [1,9]) (FA [10,22]) (FR [1,9]) (FA [6,14]) (FR [1,5]) Weight 8.02*** NS NS NS 7.69*** NS 14.03*** 10.00*** & 55.30*** 53.89*** & Length 3.44** NS 4.59*** NS 19.07*** NS 7.33*** NS 4.84** NS Total cycle 5.80*** 74.45*** & 2.45* 17.09**% 2.57* NS 13.50*** 21.02*** & 14.23*** 14.47*% Larval development 9.00*** 60.66*** & 2.00* 24.09*** % 3.80** NS 6.91*** 8.35*& 0c 0 Metamorphosis NS NS NS NS 2.80* NS NS NS 14.23*** 14.47*%

The F-value of the treatment effect in the hierarchical ANOVA (FA) and the F-value of the weighted regression (FR) are given for each measure (see Section 2 for more details on the analyses). When the weighted regression was signiﬁcant, the direction of slope is indicated by the arrow. a The number of degrees of freedom is different in L1 and pupae experiments because data of 10 days cooling treatment of L1 were not used in statistical analysis because of a very high mortality. b The number of degrees of freedom is different in L1 and pupae experiments because data of pupae after 6 days cooling were not used for the same reason. c No data. *** P < 0:001. **P < 0:01. *P < 0:05. – 261 J.-B. Myskowiak, C. Doums / Forensic Science International 125 (2002) 254–261 257

These analyses were performed using the procedure general The results of the other developmental stages are sum- linear model (GLM) of the software SAS, v.6.12 (SAS marized in Table 1. The detailed output of statistical analyses Institute Inc.) The ‘‘type III’’ sum of squares was used to is available from the authors upon request. A significant calculate the F-statistics. effect of refrigeration was observed on biometry (nine significant effects recorded out of ten measurements of weight and length) and on development rate (five out of 3. Results five), for all developmental stages. For most of the ANOVA performed, there was a significant differences between A very high mortality rate (70% for the control and 95% replicates (the three boxes) within treatments for both the for the other treatments) was observed after 24 h of refrig- development rate and the biometry (data not shown). This eration on the experiment performed with blowfly eggs. This heterogeneity did not affect our results as it was taken into high mortality was probably induced during handling as it account in the nested ANOVA when the effect of treatment was also observed for the control treatment. Therefore, was tested. data from the experiments on the egg stage were not used. The refrigeration significantly affected the biometry of Another lethal effect of refrigeration was observed after 2-day-old pupae, except for weight when L2 were refrigera- 10 days of cooling in the L1 experiment (100%). A very ted. However, the variation in biometry within treatments high mortality (81%) was also recorded when pupae were was not a function of the time spent at 4 8C, as shown by refrigerated after only 6 days of refrigeration. This strong the non-significant weighted regression (except for weight, mortality rate also prevented the use of this data. which decreased significantly with refrigeration time when

Fig. 1. Variation of the mean weight of 2-day-old pupae per treatment as a function of the time spent at 4 8C for the cooling of prepupal (a) and pupal (b) stages of P. terraenovae. The mean weight, expressed as 104 g, is calculated from back-transformed data of mean weight depicted with lower and upper 95% CL. The equation of the weighted regression is given for each graph. 258 J.-B. Myskowiak, C. Doums / Forensic Science International 125 (2002) 254–261 prepupae and pupae were refrigerated) (Table 1). This It also varied significantly linearly with the time spent at decrease in weight was particularly strong for refrigeration 4 8C, as shown by the significant weighted regression, except of the pupal stage (Fig. 1b). when the L3 stage was refrigerated. The direction of slopes The total developmental time, from end of cooling to depended on the stage refrigerated. The developmental time eclosion, was significantly affected by treatment for all decreased when L1 and prepupae (Fig. 2a and c) were developmental stages that had been refrigerated (Table 1). refrigerated but increased when L2 and pupae were refri-

Fig. 2. Variation of the total development rate as a function of time spent at 4 8C for the cooling of L1 (a); L2 (b); prepupa (c); and pupa (d) stages of P. terraenovae (between end of cooling to emergence). The mean total development rate, expressed in hours, is calculated from back-transformed data of mean rate depicted with lower and upper 95% CL. The equation of the weighted regression is given for each graph. J.-B. Myskowiak, C. Doums / Forensic Science International 125 (2002) 254–261 259

Fig. 2. (Continued ). gerated (Fig. 2b and d). The average values of developmental end of refrigeration and pupation at 24 8C), rather than on time are given for the control, 1-day refrigeration and the metamorphosis (time between pupation and eclosion at maximum cooling period of 10 days, for all the experiments 24 8C). Indeed, the statistical results on the larval develop- in Table 2. After a day at 4 8C, the developmental time is mental time were similar to the results on total develop- already significantly different from the control for all the mental time with an identical direction of regression slopes experiments except for prepupae (Table 2). (Table 1). In contrast, treatment had no significant effect Rearing an unspecified immature stage at low tempera- on metamorphosis except for L3 and pupae, although the tures could therefore induce a significant error in PMI regression was not significant (Table 1). estimate. For instance, 10 days of refrigeration induced a decrease of the total developmental time of 56 and 18 h for L1 and prepupae and an increase of 15 h for L2. For 4. Discussion refrigerated pupae, after 6 days of refrigeration, the developmental time increased by 9 h approximately. The effect of The aim of the experiments was to simulate the refrig- cooling on the total developmental time was primarily due eration of insects used in forensic investigation before arrival to its effect on larval developmental time (time between at the laboratory. Even though low temperatures are sup- posed to induce quiescence [22,37], our results show that refrigeration of the necrophagous fly, P. terraenovae can also affect the development after the refrigeration period after Table 2 only 1 day of refrigeration. Also, adult biometry is affected, Comparison of development rates after (a) 1 day and (b) 10 days of depending on the developmental stage refrigerated. There- refrigeration with the control fore, if the forensic investigator assumes an absence of Meancontrol Mean1 day ts (ddl) P-value development during storage and a complete return to normal development after storage, this may result in errors in PMI (a) 7 7 estimation. Our results differ from those of Johl and Ander- L1 5.56 10 5.19 10 4.85(92) <0.0001 7 7 son who observed that keeping C. vicina at 3 8C during 24 h L2 3.17 10 2.88 10 3.14(90) <0.01 7 7 L3 2.75 10 2.97 10 4.86(110) <0.0001 resulted in a proportional delay in the time to eclosion 6 6 Prepupa 9.04 10 8.82 10 0.96(158) >0.05 whatever the stage refrigerated. The effect of refrigeration 6 6 Pupa 2.38 10 2.55 10 5(155) <0.0001 on developmental time is already significant after 1 day of (b) refrigeration on the different parameters measured except in 7 7 prepupa experiments (Table 2). Therefore, a storage period L1 5.56 10 3.49 10 33.66(81) <0.0001 7 7 L2 3.17 10 3.62 10 3.18(57) <0.01 at 4 8C is not advised for P. terraenovae (Table 2). 7 7 L3 2.75 10 2.35 10 6.43(128) <0.0001 The relationships between the time spent at 4 8C and the 6 6 Prepupa 9.04 10 6.92 10 10.5(132) <0.0001 development rate after refrigeration differed according to the 6 6 Pupa 2.38 10 2.80 10 2.8(102) <0.01 stage refrigerated. (i) It is often assumed that the time spent For each developmental stage, the mean development rate, at low temperatures has no influence on development rate expressed in hours, is given for the control 1 and 10 days of after cooling; during refrigeration, the insect becomes refrigeration in (a) and (b), respectively. The value of a Student test quiescent and returns to a standard development rate

(ts) and its probability are given. when returned to 24 8C. Therefore, forensic entomologists, 260 J.-B. Myskowiak, C. Doums / Forensic Science International 125 (2002) 254–261 in France, advise keeping the sample at low temperatures 5. Conclusion for storage. However, in our experiments, this was observed only for L3 larvae. (ii) The developmental rate after refrig- One to ten days of refrigeration can modify the weight and eration decreases with the time spent at 4 8C. This was length of pupae and the development rate whatever the observed when first instars (L1) and prepupae were refri- immature stages of P. terraenovae refrigerated. These gerated. Although the rearing temperature of 4 8C is lower experiments should be useful for forensic investigations than the minimal development threshold (7.8 8C) of P. as they show that keeping the specimen collected on a terraenovae, L1 and prepupa do not stop their development corpse at 4 8C do not simply result in the cessation of insect completely during the refrigeration period. The decrease of development as generally expected. Refrigerating and hand- the developmental rate after refrigeration compared to the ling induced a modification of development rate after the control was 56 and 18 h in L1 and prepupae from the return refrigeration period depending on the refrigerated immature to 24 8C to adult emergence. (iii) The developmental rate stage. Generally, at 24 8C, P. terraenovae completes its after refrigeration increases with the time spent at 4 8C. This development in 15.5 days, 10 days of refrigeration led to was observed for L2 larvae and pupae. In this case, flies a variation of 9–56 h. This result must be taken into account appear not to recover properly from the refrigeration treat- in criminal investigations. Necrophagous: Calliphoridae ment. After 10 days of cooling at 4 8C, an increase of generally lay theirs eggs, on the corpse, nearby the death, development rate of 15 and 9 h was found for L2 and pupae, so an error more than 6 h prevents the precise estimation of respectively. the crime day. A precise knowledge of development rate The weight and length of 2-day-old pupae were also after refrigeration, such as the data collected in this studied, strongly affected after the maximal period of refrigeration. should therefore improve the estimate of the PMI. However, this effect was not a linear function of the time Even though weight and length measurements of pupae spent at 4 8C except when prepupae and pupae were refri- are not systematically used in criminal investigation, they gerated. Only then the weight decreased with the time spent can be considered when anomalies of the development are at 4 8C especially in pupae. Leopold et al. [38], when suspected such as after a refrigeration period. In such case, examining cold resistance of house flies, showed that the biometry measurement may allow the discrimination most sensitive developmental stages to low temperature between problems during development due to the refrigera- were those closest to the end of pupation and the onset of tion or to nutritional deficiency caused, for example, by a adult emergence. This sensitivity was explained by the post-mortem handling of the corpse by the murderer (dis- intense activity of tissue remodeling. For instance, during memberment of the victim). pupation, the insect undergoes extensive histolysis and Finally, our result showed that the stress induced by histogenesis to ultimately form the adult structures. In our refrigeration was especially large in pupae. To allow rearing experiment, the loss of weight, caused by prolonged refrig- at the laboratory, refrigeration is therefore not recommended eration (more than 3 days), was associated with a heavy during metamorphosis as it leads to a significant loss of mortality (50% after 3 days). The insect therefore could not weight, always associated with a heavy mortality, even after enter a phase of quiescence during the refrigeration period. 3 days of refrigeration. Further experiments should examine the causes of this loss of weight during metamorphosis of P. terraenovae treated under the same conditions. Acknowledgements Our experiment also showed a large uncontrolled variability among the three samples within treatments for all We thank Michel Veuille and Serge Caillet for their help measured parameters. An experiment with only one sample and advice. 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