Forensic Science International 160 (2006) 27–34 www.elsevier.com/locate/forsciint

Study of steroidogenesis in pupae of the forensically important blow fly terraenovae (Robineau-Desvoidy) (Diptera: ) Emmanuel Gaudry a,*, Catherine Blais b,1, Annick Maria b, Chantal Dauphin-Villemant b a Institut de Recherche Criminelle de la Gendarmerie Nationale, 1 Boulevard The´ophile Sueur, F-93111 Rosny-Sous-Bois Cedex, France b Biogene`se des ste´roı¨des, FRE 2852, CNRS, Universite´ Pierre et Marie Curie, Case 29, 7 quai St. Bernard, F-75252 Paris Cedex 05, France

Received 23 August 2004; accepted 20 June 2005 Available online 23 September 2005

Abstract

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal–adult development of synchronised using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: ‘‘fresh’’ pupae, kept frozen until analysis and ‘‘experimentally dried’’ pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in ‘‘experimentally dried’’ pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic are discussed. # 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: ; Ecdysteroids; ; Pupal–adult development

1. Introduction

Forensic entomology is described as the field where study of (and more generally ) interacts with legal matters [1]. It can help to estimate the time elapsed * Corresponding author. Tel.: +33 1 58 66 50 92; fax: +33 1 58 66 50 27. since death, the so-called ‘‘post-mortem interval’’ (PMI) [2], E-mail addresses: [email protected] by determining the age of immature stages of necrophagous (E. Gaudry), [email protected] (C. Blais). insects collected on a human cadaver and around, from the 1 Tel.: +33 1 44 27 22 63; fax: +33 1 44 27 23 61. scene of crime and autopsy.

0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.06.014 28 E. Gaudry et al. / Forensic Science International 160 (2006) 27–34

Necrophagous insects (mainly flies) are attracted by the Thanks to the advent of immunoassays and high-perfor- odours produced by a decaying corpse. Females lay eggs or mance chromatographic methods (see ref. [10]), quantitative larvae directly into natural openings and wound sites. Me´g- and qualitative analyses of ecdysteroids have been done nin [3] described a succession pattern of insects and other during post-embryonic development of various spe- arthropods on a cadaver, eight ‘‘waves’’ from death until the cies, but only in a small number of calliphorid flies, mainly skeleton stage, due to modifications induced by the decom- Calliphora sp. [11]. The presence of both ecdysone and 20- position. (Diptera) are the first insects to invade a hydroxyecdysone in pupae of two Calliphora has cadaver and are predominant. This is why determination of been demonstrated [12] and ecdysteroid metabolism has when necrophagous flies started to colonise a corpse (ovi- been extensively studied in the blue blow fly Calliphora position or larviposition) gives an estimate of the PMI. vicina (review in refs. [11,13]). In P. terraenovae, only Dipterans undergo a post-embryonic development com- ecdysteroids present in adult females have so far been prising several stages separated by moults. Larvae live on or studied [14,15]. Identification and quantification of ecdys- in the corpse, which constitutes their nutritional substrate. At teroids in pupae could possibly help to determine their age the end of the third larval instar, migrate away from and thus give an estimate of PMI. In order to examine this the corpse in order to find an adequate site to pupate, either in question, we studied the level and the nature of ecdysteroids the soil when outdoors or under furniture, carpets and so throughout the pupal stage of P. terraenovae. forth when indoors. Metamorphosis is achieved during the pupal stage and at its conclusion the adult fly (imago) emerges from the puparium. The duration of this develop- 2. Materials and methods ment cycle varies according to species and environmental conditions, especially temperature. 2.1. Insects Marchenko [4] reported that the embryonic and post- embryonic development rate of necrophagous flies can be P. terraenovae were bought in a fishing tackle shop near estimated using temperature summation, the accumulated Paris, and came from the south of France. Specimens were degree days (ADD) model. Above a specific threshold, each reared until adult emergence in plastic boxes (260 species needs a specific amount of degree days to develop. mm  130 mm  77 mm, with a 20–30 mm thick layer of The blow fly Protophormia terraenovae (Robineau-Des- sand) placed inside an incubator (Snijders Economic Delux) voidy) (Diptera: Calliphoridae) has a holarctic distribution at 24 8C with 75–95% relative humidity and a photoperiod of and is often associated with the necrophagous fauna, in the 12-h light:12-h dark hours. The rearing substrate was fresh first waves of insect succession [3]. This species is known to beef muscle of uniform quality. Development time from egg oviposit shortly after death and its temperature dependent to adult lasted about 330 h. About 30 adults were transferred development [5,6] can be used to estimate the PMI. Pupation into plastic cages (300 mm  500 mm  500 mm) at ambi- takes place on the surface of the nutritive medium or very ent temperature (20 Æ 2 8C) for mating and were provided close to it [7]. The duration of the pupal stage is relatively with sugar and water. After 1 week, beef blood was supplied long, representing 43% of the total developmental time, in for 2 h in the cages to stimulate maturation of eggs of adult comparison to the third larval instar: 13% for the feeding females that fed on it. One day later, sugar was replaced by a stage and 22% for the post-feeding phase [5]. piece of fresh beef muscle of uniform quality. Within 30 min Rearing pre-imaginal stages of necrophagous insects, of oviposition, eggs and substrate were transferred into species identification, study of their developmental time plastic boxes. First instar larvae were distributed to different and analysis of the temperature data, together provide an plastic boxes containing 40 g of beef muscle, which was estimation of the PMI [2]. But sometimes, forensic ento- replaced regularly, to avoid food shortage competition. mologists [8] receive dead pupae or animals which died during rearing. Dissection and examination of pupae can 2.2. Synchronisation of immature instars give some information on their age ([9]; Pasquerault, unpub- lished data). However, it would be beneficial to find an The larval instars were identified by observation of the alternative and/or complementary method to estimate the posterior spiracular slits under a binocular microscope. At age of pupae. the beginning of the third instar, larvae were transferred into Ecdysteroids are polyhydroxylated steroid hormones that new boxes (50 individuals with 100 g of beef muscle, are essential for insect development and reproduction. They supplied regularly). When they stopped feeding, they were trigger transitions between developmental stages, control- transferred into another set of plastic boxes, containing sand ling moulting and metamorphosis processes, via their per- only. iodical fluctuations. 20-Hydroxyecdysone (20E) is regarded From the first appearance of the immobile white puparia as the principal active ecdysteroid in most species. It comes until adult emergence, pupae were collected every 6 h from the transformation by peripheral tissues, of the ‘pro- (Æ1 h) and transferred into a freezer at À22 8C. Pupae were hormone’ ecdysone (E), synthesised and secreted by ecdy- not sexed, as it was not possible to remove them undamaged sial glands, the endocrine glands producing ecdysteroids. from the puparia. Experimentally dried specimens were E. Gaudry et al. / Forensic Science International 160 (2006) 27–34 29 also studied. They were obtained by leaving thawed pupae with a flow rate of 1 mL/min. Some analyses were per- from all ages, at ambient temperature (20 Æ 2 8C) for 5 formed by reverse-phase HPLC (RP-HPLC) on a Spherisorb weeks. ODS2 column (AIT), with a flow rate of 1 mL/min, using a linear gradient of solvent B (acetonitrile/propan-2-ol, 5:2, v/ 2.3. Extraction of ecdysteroids v) in solvent A (0.1% trifluoroacetic acid in water). Fractions of 0.5 mL were collected, evaporated until dry and resus- Each pupa was cleaned, weighed, cut with scissors into pended in EIA buffer for ecdysteroid quantification. two pieces and homogenised in 70% methanol (Merck) The retention times of immunoreactive fractions were (1 mL/10 mg) in Eppendorf tubes with a plastic pestle. compared to the following reference ecdysteroids: 20E, The homogenate was centrifuged and the pellet resus- ecdysone (E), 3-dehydro-ecdysone (3DE) and 3-dehydro- pended in 70% methanol, for a second extraction (see 20-hydroxyecdysone (3D20E). Section 2.4). For further high-pressure liquid chromatogra- phy (HPLC) analyses, large samples (20 pupae) were ground in a glass-Teflon homogeniser. After evaporation 3. Results of the methanol phase, a partition with chloroform/water (1:1, v/v) was performed twice. The aqueous phase was 3.1. Ecdysteroid levels during the pupal stage purified on a C18 Sep–Pak cartridge as previously described [16]. Ecdysteroids were eluted successively by 5 mL of In ‘‘fresh’’ pupae, the ecdysteroid titre increased from 30% methanol (polar fraction), 5 mL of 70% methanol (free 36 h after pupariation and peaked at 48–54 h, reaching ecdysteroid fraction) and 5 mL of absolute methanol (apo- 582 fmol 20E equivalents per mg fresh weight (i.e. 28 pmol lar fraction). per ) (Fig. 1A). It then decreased until 96 h after

2.4. Ecdysteroid quantification

Ecdysteroids were quantified by an enzyme immunoas- say (EIA) adapted from the method described by Porcheron et al. [17]. The enzymatic tracer was 2-succi- nyl-20-hydroxyecdysone coupled to peroxidase [18].We used a polyclonal anti-20-hydroxyecdysone antiserum, AS4919, provided by Dr. P. Porcheron [17], which presents approximately an equal affinity for 20E and E. In routine experiments, calibration curves were generated with 20- hydroxyecdysone (Simes, Milan, Italy) (range 32– 4000 fmol). Dried samples were resuspended in EIA buffer solution and each determination was made in dupli- cate. Results were expressed as 20-hydroxyecdysone equivalents.

2.5. Enzymatic hydrolyses

Samples were incubated with b-glucuronidase (400 enzyme units per 100 mL of type H-1 from Helix sp., Sigma) for 24 h at 30 8C, in a sodium acetate buffer (50 mM, pH 5.3). Other samples were incubated in the presence of esterase from porcine liver (Sigma, 15 units per 100 mL) for 20 h at 37 8C in a sodium borate buffer (100 mM, pH 8). After incubation, ecdysteroids were adsorbed on a C18 Sep– Pak cartridge and eluted with 5 mL of absolute methanol. Samples were evaporated until dry for further analysis.

Fig. 1. Ecdysteroid titres during pupal development. (A) Fresh 2.6. HPLC pupae, reared at 24 8C and (B) experimentally dried pupae, frozen at different ages then left for 5 weeks at 20 8C. Each time point Ecdysteroids were analysed by normal-phase HPLC represents the mean (ÆS.D.) of 3–10 individual measurements, (NP-HPLC) using a silica column (250 mm length, internal expressed in 20E equivalents. Solid line: fmol per mg weight diameter 4.6 mm, Zorbax SIL, Hypersil) and dichloro- (left-hand y-axis); dashed line: pmol per animal (right-hand y-axis). methane/propan-2-ol/water (100:40:2.5, v/v/v) as solvent, WP, white puparia; AE, adult eclosion. 30 E. Gaudry et al. / Forensic Science International 160 (2006) 27–34 pupariation, to reach a level of approximately 200 fmol 20E equivalents per mg fresh weight, which was maintained until the time of adult eclosion. In experimentally dried pupae, the developmental profile of ecdysteroids was different (Fig. 1B). A first surge of ecdysteroids was detected at 48 h after pupariation, as in ‘‘fresh’’ pupae, but it was followed by alternating low and high values, with large interindividual variations. Levels remained high in experimentally dried pupae at the end of the stage. The highest values were in the range of 600 fmol 20E equivalents per mg dry weight, i.e. 11 pmol per animal. This concentration per animal is less than half the value found in a ‘‘fresh’’ pupa. The mean weight of ‘‘fresh’’ pupae was about 2.5 times greater than the mean weight of ‘‘dried’’ pupae: 53.0 Æ 6.8 mg compared to 21.2 Æ 3.8 mg.

3.2. Nature of ecdysteroids

The nature of ecdysteroids was investigated in large samples of fresh pupae (20–40 animals) from three different ages, i.e. in early pupae (24 Æ 6 h post-pupariation) at the time of the ecdysteroid peak (60 Æ 12 h) and in late pupae (140 Æ 12 h). Ecdysteroids were analysed by HPLC, both in normal and reverse phase (data not shown), by comparing retention times of immunoreactive compounds and refer- ences run separately. Free ecdysteroids were directly separated by HPLC followed by EIA quantification. At all pupal ages, the major ecdysteroid corresponded to 20E retention time, represent- ing about 36.4, 41.2 and 46.8% of total immunoreactive compounds in 24, 60 and 140-h-old pupae, respectively (Fig. 2A–C). Another immunoreactive peak corresponded to the retention time of reference E, in a lesser proportion (16, 19 and 8.3%, respectively, for the three ages). Some immunoreactivity was also detected at the retention times of 3DE and 3D20E. Enzymatic hydrolysis of polar and apolar fractions eluted from Sep–Pak cartridges allowed a check for the eventual presence of conjugated ecdysteroids in pupae. After b-glu- curonidase hydrolysis of polar fractions, there was no sig- Fig. 2. NP-HPLC/EIA analysis of free ecdysteroids from early nificant modification of the immunoreactivity (Table 1). On pupae (24 h after pupariation) (A); 60-h-old pupae (B) and 140- the contrary, it appears that apolar conjugates were present in h-old pupae (C). HPLC solvent system: dichloromethane/propan-2- pupal extracts, because after treatment of apolar fractions by ol/water (120:40:2.5, v/v/v) at a flow rate of 1 mL/min. Arrows esterase, immunoreactivity was increased by 4.5, 5.8 and 11 indicate the retention times of known ecdysteroid references. 3DE, times in the case of 24, 60 and 140-h-old pupae, respectively 3-dehydro-ecdysone; 3D20E, 3-dehydro-20-hydroxyecdysone; E, (Table 1). HPLC/EIA analyses showed that 20E and 3D20E ecdysone; 20E, 20-hydroxyecdysone. Ecdysteroids were quantified were liberated after hydrolysis, both in 24 and 60-h-old pupae in 0.5-min fractions as 20E equivalents, they are expressed as percentage of total immunoreactivity. (Fig. 3A). We did not get enough late pupae to analyse their ecdysteroids after hydrolysis. In order to be sure that any apolar conjugate would not have been discarded in the chloro- form phase during sample purification (see Section 2.3), the different from the previous one (Fig. 3A), because in addition partition step was omitted for a sample of 60-h-old pupae. The to 3D20E and 20E, two other immunoreactive compounds partially dried methanol extract was diluted in water (final 5% appeared, corresponding to 3DE and E retention times. 3DE methanol) and purified on a Sep–Pak cartridge as described in was the major one (45% of total immunoreactivity). This Section 2.3. The apolar fraction was hydrolysed by esterase, suggests that apolar conjugates of 3DE and, to a lesser extent then analysed by HPLC/EIA. The profile (Fig. 3B) was of E were also present in pupae. E. Gaudry et al. / Forensic Science International 160 (2006) 27–34 31

Table 1 Ecdysteroid levels in pupae from different ages before and after enzymatic hydrolysis Age (hours Æ 12 Ecdysteroids (pmola) Ecdysteroids (pmola) post-pupariation) before hydrolysis after hydrolysisb Total extract 24 9 – 60 58 – 140 11.5 – Polar fractionsb 24 7 7.2 60 31 29 140 8.4 9.6 Apolar fractionsb 24 1.5 6.7 60 0.24 1.4 140 0.25 2.7 a pmol 20E equivalents per pupa. b Fractions were obtained from Sep–Pak purification; polar fractions were hydrolysed by b-glucuronidase, apolar fractions by esterase.

RP-HPLC/EIA analyses of free ecdysteroids present in pupae, respectively. Other analyses after separation by NP- experimentally dried pupae are shown in Fig. 4. In both 24 HPLC (not shown) confirmed that ecdysone was the major and 60-h-old pupae, two immunoreactive peaks were immunoreactive ecdysteroid present in dried pupae. No 3- detected, corresponding to the retention times of E and dehydroecdysteroids could be detected. 20E references. There was a higher proportion of ecdysone, 79 and 42% of total immunoreactivity for 24 and 60-h-old 4. Discussion

4.1. Titres and nature of ecdysteroids in fresh pupae

At the beginning of pupal development (white puparia) in P. terraenovae, ecdysteroid levels are relatively high when expressed per animal (Fig. 1) and reach values similar to those measured in larvae just before pupariation (Gaudry and Blais, unpublished results). Titres then decrease during the first day of pupal development (Fig. 1). This pattern could correspond to the decrease of the prepupal surge of ecdysteroids which has been described in all insect species investigated so far and is responsible for pupariation (see refs. [11,19–21] for Diptera).

Fig. 4. RP-HPLC/EIA analysis of free ecdysteroids from experimen- Fig. 3. NP-HPLC/EIA analysis of ecdysteroids after hydrolysis of tally dried pupae, killed by freezing at 24 h (white bars) and 60 h apolar fractions by esterase. (A) 24 and 60-h-old pupae; purification (black bars). HPLC solvent system: linear gradient from 10 to 90% of of samples included a chloroform partitioning and (B) 60-h-old acetonitrile/propan-2-ol (5:2, v/v) in 0.1% trifluoroacetic acid in water pupae, without chloroform partitioning. Same HPLC conditions and for 30 min, at a flow rate of 1 mL/min. Same ecdysteroid references as ecdysteroid references as in Fig. 2. Extracts were treated overnight in Fig. 2. Ecdysteroids were quantified in 0.5-min fractions as 20E eq- by esterase before HPLC analysis. uivalents, they are expressed as percentage of total immunoreactivity. 32 E. Gaudry et al. / Forensic Science International 160 (2006) 27–34

From 36 h after pupariation, ecdysteroid levels rise again and and might represent a storage form of ecdysteroids in eggs. In peak at 48–54 h, then gradually decrease to reach basal values P.terraenovae, presence of non-polar conjugates has not been at about 96 h after pupariation. A similar large peak of investigated in female adults by Wilps and Zo¨ller [14].In ecdysteroid activity has been reported in other dipteran spe- another study, E metabolism was analysed in Phormia sp. cies during the first part of the pupal stage, for example, in the flies, using tritiated ecdysteroid [26]. It was reported that, after blow fly C. vicina [11], in the flesh fly Sarcophaga bullata [19] ingestion of tritiated E and a 24-h incubation, 50–60% of the and in Drosophila melanogaster (review in ref. [21]). This radioactivity present in animals and faeces corresponded to pupal ecdysteroid pulse is considered to be responsible for apolar compounds, considered as ester-type conjugates [26]. development of adult structures. Another later sustained peak However, these ‘‘putative esters’’ could not be hydrolysed was reported in Sarcophaga only, concomitant with the enzymatically and were not identified. synthesis and secretion of adult cuticle [19].InDrosophila, After enzymatic hydrolysis by Helix pomatia juice, con- a first smaller pulse of ecdysteroids has been described in a few jugated ecdysteroids (E and 20E) were revealed in ovaries of studies, at the time of head eversion, marking the completion P. terraenovae females [14]. In the present study, we did not of the pupa [21]. Such a precocious small peak was not detect any increase of immunoreactivity after treatment by detected in P. terraenovae. glucuronidase of pupal extracts (Table 1). We did not detect The most prominent ecdysteroids were characterised by any immunoreactive compo- und more polar than 20E in RP- the use of HPLC, in two chromatographic systems and EIA. HPLC/EIA analyses of polar fractions from Sep–Pak purifi- At any pupal age studied, ‘‘putative’’ 20E appears to be the cation (data not shown). Formation of conjugates is often con- major free ecdysteroid; other minor immunoreactive com- sidered as a mean for inactivation/storage of ecdysteroids [10]. pounds correspond to reference E and 3-dehydroecdyster- In young P.terraenovae females, polar conjugates appeared to oids (Fig. 2). In most insect species, 20E is the major be the only form present in ovaries [14]; in contrast, we were circulating ecdysteroid. The sterol precursor of ecdysteroids unable to detect such conjugates in pupae, even in the is supplied by the food, as insects are unable to synthesize oldest ones. sterols de novo (see refs. [10,22]). Zoosterols being essen- tially cholesterol, necrophagous insects get cholesterol 4.2. Ecdysteroids in ‘‘experimentally dried’’ pupae directly, which is subsequently converted into the C27 hor- mone 20E. Both 20E and E can be converted into 3-dehy- In ‘‘experimentally dried’’ pupae, the developmental pro- droecdysteroids [10]. This metabolic pathway has been file of ecdysteroids was different from the profile in ‘‘fresh’’ shown a major one in several Dipterans, such as pupae of pupae. The surge in ecdysteroid titre in the first part of the C. vicina and D. melanogaster by in vitro experiments [23] instar was not observed to the same degree. In early ‘‘dried’’ and third instar Drosophila larvae by in vivo conversion of pupae, as in ‘‘fresh’’ pupae, ecdysteroid levels decreased to labelled cholesterol [24]. In adult females of P. terraenovae, reach low values, around 150–200 fmol/mg. Then, titres the nature of ecdysteroids has been previously investigated increased with the age of pupae, oscillating between 600 in hemolymph and ovaries by TLC/radioimmunoassay [14] and 300 fmol/mg. In contrast with ‘‘fresh’’ pupae, there and E and 20E were the major free forms when ovaries were was no decrease to basal levels at the end of the stage. The mature. Some minor products were either less polar than E or nature and the relative proportions of free ecdysteroids dif- more polar than 20E: they accumulated with increasing fered significantly from those of fresh pupae. At all ages tested ovariandevelopment[14],buttheyhavenotbeencharacterised. (24 and 60-h-old pupae in Fig. 4), the only immunoreactive In pupae of P. terraenovae, the four ecdysteroids detected compounds detected were putative E and 20E, E being the as free forms were also present as non-polar conjugates, major one. This is based on retention times similar to refer- which could be hydrolysed by esterase. After this enzymatic ences in two different HPLC systems (data not shown for treatment, immunoreactivity increased by a factor 4.5–11 normal-phase HPLC). The presence of E in these pupae is (Table 1) depending on the increasing age of pupae, and nevertheless puzzling, because de novo production is impos- ecdysteroids could be detected by HPLC/EIA (Fig. 3). Their sible and dehydroxylation of 20E to E could only be per- nature and relative proportions varied according to the formed by bacteria. Such dehydroxylations are already extraction protocol. When apolar compounds from the documented for bile acids in mammals, concerning essentially methanol extract of pupae were preserved, 3DE and E 7a-dehydroxylation [27–29], and in the case of ecdysteroids, conjugates were revealed in addition to 20E and 3D20E it was described only for the 14-dehydroxylation, caused by conjugates (compare Fig. 3A and B). 3DE conjugates bacteria from the gut lumen [30]. Thus, it remains possible that appeared as a major form, at least in pupae at the peak time the major immunoreactive compound is some unknown (Fig. 3B). Their precise chemical nature was not investigated metabolite of 20E. To our knowledge, no study of ecdysteroid in our study. In other Diptera, apolar conjugates have been degradation in dead insects by biotransformation has been reported and identified as sulphate and phosphate esters in made. The kinetics and even the nature of ecdysteroid degra- larvae and prepupae of C. vicina (see review in ref. [11]). 22- dation could vary between individuals, and could be a possible fatty acid acyl esters were characterised in D. melanogaster explanation for the high interindividual variations noticed in adults [25]. They appeared to be only produced by adult flies ecdysteroid levels of ‘‘dried pupae’’. E. Gaudry et al. / Forensic Science International 160 (2006) 27–34 33

4.3. Measurement of ecdysteroids as a tool in mology, The Utility of Arthropods in Legal Investigation, CRC forensic entomology Press, London, New York, 2001, pp. 1–15. [2] K.G.V. Smith, A Manual of Forensic Entomology, Trustees of EIA determinations of ecdysteroids are relatively easy the British Museum of Natural History, London, 1986. [3] P. Me´gnin, La Faune Des Cadavres, Masson, Paris, 1894. and rapid to perform. The high sensitivity of the method [4] M.I. Marchenko, Medicolegal relevance of cadaver entomo- allows the quantification of ecdysteroids in only one pupa. fauna for the determination of the time of death, Forensic Sci. However, assays must be performed with standardised con- Int. 120 (2001) 89–109. ditions (tracer, antiserum and so forth) in order to make [5] B. Greenberg, T. Tantawi, Different developmental strategies comparisons. As tools for estimation of PMI from specimens in two boreal blow flies (Diptera: Calliphoridae), J. Med. of P. terraenovae collected at a scene of death, ecdysteroid Entomol. 30 (1993) 481–484. concentrations >400 fmol per mg fresh weight would indi- [6] M. Grassberger, C. Reiter, Effect of temperature on develop- cate that a post-pupariation period of between 2 and 3 days ment of the forensically important holarctic blow fly Proto- had elapsed for fresh pupae, in which the whole pupal period phormia terraenovae (Robineau-Desvoidy) (Diptera: lasted 6 days at 24 8C. If pupae have died a short time ago Calliphoridae), Forensic Sci. Int. 128 (2002) 177–182. [7] P. Nuorteva, Empty puparia of Phormia terraenovae R.-D. (for instance, during transport between the collection site (Diptera Calliphoridae) as forensic indicators, Ann. Entomol. and the laboratory), the same conclusion could be drawn. In Fenn. 53 (1987) 53–56. order to perform ecdysteroids quantification in pupae, one [8] E. Gaudry, J.-B. Myskowiak, B. Chauvet, T. Pasquerault, F. can advise death scene technicians to freeze insects imme- Lefebvre, Y. Malgorn, Activity of the forensic entomology diately after collection. If pupae are dead and dried, high department of the French Gendarmerie, Forensic Sci. Int. 120 values (>5 pmol of ecdysteroids per animal) would only (2001) 68–71. indicate that pupariation has taken place at least 36 h before [9] B. Greenberg, J.C. Kunich, Entomology and the Law: Flies as animal death, and low values (<5 pmol per animal) would Forensic Indicators, Cambridge University Press, Cambridge, correspond to early pupae (36 h). However, the time 2002. elapsed since the death of pupae would be unknown. To [10] R. Lafont, C. Dauphin-Villemant, J.T. Warren, H. Rees, Ecdys- teroid chemistry and biochemistry, in: L.I. Gilbert, K. Iatrou, perform an HPLC/EIA analysis of ecdysteroids, 15–20 S.S. Gill (Eds.), Comprehensive Molecular Insect Science, animals were necessary, which can be found in field con- Elsevier, Oxford, 2005, pp. 125–195. ditions in many cases. However, our results showed that no [11] J. Koolman, Ecdysteroids in the blowfly Calliphora vicina, in: significant differences in the nature and the relative propor- J.A. Hoffmann (Ed.), Progress in Ecdysone Research, Elsevier/ tions of ecdysteroids exist in relation with the age of pupae. North-Holland, Amsterdam, 1980, pp. 187–209. In conclusion, quantification of ecdysteroids could be [12] M.N. Galbraith, D.H.S. Horn, J.A. Thomson, G.J. Neufeld, used within limits, to estimate the age of fresh pupae of P. R.J. Hackney, Insect moulting hormones: crustecdysone in terraenovae collected on a cadaver, and therefore could give Calliphora, J. Insect Physiol. 15 (1969) 1225–1233. hints on the PMI. If pupae had just died—at the time of [13] M.F. Meister, H.M. Brandtner, J. Koolman, J.A. Hoffmann, Con- collection or during transfer—and rearing was therefore not version of a radiolabelled putative ecdysone precursor 2,22,25- trideoxyecdysone (5b-ketodiol) on larvae and pupae of Calli- possible, determination of ecdysteroid content could give phora vicina, Int. J. Invert. Reprod. Dev. 12 (1987) 13–28. some temporal information, to complement observations of [14] H. Wilps, T. Zo¨ller, Meaning and titre of ecdysteroids during the pupa’s morphology. life span of Phormia terraenovae females, J. Insect Physiol. 35 (1989) 175–181. [15] T. Zo¨ller, H. Wilps, Ecdysteroids in the blowfly Phormia Acknowledgements terraenovae, the ovaries as a storage site, Invert. Reprod. Dev. 18 (1990) 134–135. The authors thank Dr. R. Lafont for his generous gift of [16] R. Lafont, J.-L. Pennetier, M. Andrianjafintrimo, J. Claret, J.-F. ecdysteroid references and his helpful advises, Dr. P. Porch- Modde, C. Blais, Sample processing for high-performance eron for providing the antiserum AS4919. liquid chromatography of ecdysteroids, J. Chromatogr. 236 Emmanuel Gaudry wishes to thank Colonel S. Caillet, F. (1982) 137–149. Cottin, B. Fre`re and his colleagues from the Department of [17] P.Porcheron, M. Morinie`re, J. Grassi, P.Pradelles, Development Forensic Entomology (especially T. Pasquerault and J.-B. of an enzyme immunoassay for ecdysteroids using acetylcho- Myskowiak) for their support. linesterase as label, Insect Biochem. 19 (1989) 117–122. [18] H.G. Marco, C. Blais, D. Soyez, G. Ga¨de, Characterisation of The authors lastly wish to acknowledge Dr. M. Hall for moult-inhibiting activities of sinus glands of the spiny lobster his precious advices. Jasus lalandii, Invert. Reprod. Dev. 39 (2001) 99–107. [19] S.L. Wentworth, B. Roberts, J.D. O’Connor, Ecdysteroid titres during post-embryonic development of Sarcophaga bullata References (Sarcophagidae: Diptera), J. Insect Physiol. 27 (1981) 435– 440. [1] R.D. Hall, Introduction: perception and status of forensic [20] B. Roberts, L.I. Gilbert, W.E. Bollenbacher, In vitro activity entomology, in: J.H. Byrd, J.L. Castner (Eds.), Forensic Ento- of dipteran ring glands and activation by the prothoracico- 34 E. Gaudry et al. / Forensic Science International 160 (2006) 27–34

tropic hormone, Gen. Comp. Endocrinol. 54 (1984) 469– [26] J. Sutter, Etude compare´edume´tabolisme de l’ecdysone chez 477. trois Dipte`res adultes (Phormia species, Lucilia [21] L.M. Riddiford, Hormones and Drosophila development, in: cuprina et L. sericata) apre`s injection ou ingestion de [3H]- M. Bate, A. Martinez-Arias (Eds.), The Development of ecdysone, Dissertation, Neuchaˆtel University, 1986. Drosophila melanogaster, Cold Spring Harbor Press, New [27] O. Bortolini, A. Medici, S. Poli, Biotransformations on steroid York, 1993, pp. 899–939. nucleus of bile acids, Steroids 62 (1997) 564–577. [22] R. Lafont, C. Dauphin-Villemant, C. Blais, Ecdysteroids, [28] P.B. Hylemon, J. Harder, Biotransformation of monoterpenes, overview, in: H.L. Henry, A.W. Norman (Eds.), Encyclopedia bile acids, and other isoprenoids in anaerobic ecosystems, of Hormones, Elsevier, 2003, pp. 471–476. FEMS Microbiol. Rev. 22 (1999) 475–488. [23] J. Koolman, Ecdysone oxidase in insects, Hoppe-Seyler’s Z. [29] J.E. Wells, K.B. Williams, T.R. Whitehead, D.M. Heuman, Physiol. Chem. 359 (1978) 1315–1321. P.B. Hylemon, Development and application of a polymerase [24] G. Somme´-Martin, J. Colardeau, R. Lafont, Conversion of chain reaction assay for the detection and enumeration of bile ecdysone and 20-hydroxyecdysone into 3-dehydroecdyster- acid 7a-dehydroxylating bacteria in human feces, Clin. Chim. oids is a major pathway in third instar Drosophila melanoga- Acta 331 (2003) 127–134. ster larvae, Insect Biochem. 18 (1988) 729–734. [30] K.H. Hoffmann, E. Thiry, R. Lafont, 14-Deoxyecdysteroids in [25] V. Grau, R. Lafont, Metabolism of ecdysone and 20-hydro- an insect (Gryllus bimaculatus), Z. Naturforsch. 45c (1990) xyecdysone in adult Drosophila melanogaster, Insect Bio- 703–708. chem. Mol. Biol. 24 (1994) 49–58.