Int J Legal Med DOI 10.1007/s00414-014-0968-2
ORIGINAL ARTICLE
The analysis of pupal development period in Lucilia sericata (Diptera: Calliphoridae) forensically important insect Tuçe Karabey & Osman Sert
Received: 18 April 2013 / Accepted: 16 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Forensic entomology is a study that insect evidence is used for the determination of postmortem interval (PMImin). While determining PMImin, the pupal period is given as the duration between the beginning of the pupal period and the stage of adult emergence. The pupal period constitutes approximately 50 % of the immature development, and with detailed examination it would be possible to present a much more accurate PMImin. In this study, the pupal period of Lucilia sericata (Meigen, 1826), which is one of the species used in PMImin estimation in forensic entomology, was investigated. Studies about the pupal period of development are considerably limited in the forensic entomology world. Stages and duration of the pupal period of Lucilia sericata were studied at temperatures of 20 °C, 25 °C, and 30 °C. For all temperatures, pupae specimens were dissected and photographed hourly. According to findings based on all three temperatures, it was determined that as the temperature increases, the duration of development shortens. Also, in addition to 11 pre-determined development stages, nine new development stages were identified.
Keywords Lucilia sericata . Pupae . Development . PMImin . Temperature
This article is the main part of Tuçe Karabey’s master thesis, which was supervised by Assoc. Prof. Dr. Osman Sert and accepted on 11 April 2012. T. Karabey : O. Sert (*) Faculty of Science Department of Biology, Hacettepe University, Beytepe Campus, 06532 Ankara, Turkey e-mail: [email protected]
Introduction Insects arrive on carrion less than 5–10 min after death [1, 2]. Therefore, the method of estimating the postmortem interval (PMImin) by using the life cycles of some holometabolous insects plays a very important role in the legal system. In particular, the larval stage of the insect is used to estimate the time of death in cases where this is unknown [3]. In the larval stage, morphological structures can be identified from the outside and this data makes the PMImin estimation possible. In the forensic field, many researchers have gathered data on the larval periods and adult stages of forensic insects [2, 4–9]. There are many studies regarding the time when pupation begins and when the adult emerges, but few studies have dealt with the stages of the pupal period and their sequences. However, the pupal period constitutes approximately 50 % of the immature development and therefore, it is thought that the age of the pupae may serve as an important tool for PMImin estimation [10]. Determining the age of the pupae is much more difficult compared to determining the age of larvae, since it is not possible to observe morphological differences such as length and weight from the outside [4]. Changes in the color of the puparium from white to dark brown provide useful information concerning the age of pupae for the first 10 h [6]. However, after this first 10 h, there is no way of detecting the age of pupae because the color remains unchanged [4]. Regarding the development of the insect, because of the fact that morphological differentiations in the pupal stage cannot be observed from the outside and because the use of the gene expression approach for age determination is expensive and time consuming, the remains of pupae collected from crime scenes are like a black box for forensic entomologists [6]. Three fundamental studies were conducted on the progress of the pupal period. Agrell and Lundquist [11] generally divided the pupal period in the three stages of histolytic change, histogenetic change, and differentiation, without
Int J Legal Med
Fig. 1 a Dorsal view of the prepupae. b Shattered prepupae cannot be removed as a whole when dissected
specifying species or genus. Finell and Jarvilehto [12] distinguished the pupal stages of Calliphora erythrocephala as histolysis, juvenile stage, histogenetic stage, immature stage, mature stage, and the last differentiation stage. Greenberg and Kunich [6] distinguished the pupal period into11 stages. In their study, which is the first published paper dealing with this issue at the species level, they determined the pupal development stages and durations of the stages of Phormia regina (Meigen, 1826) at 22 °C and 29 °C. Until 2012, among forensically important species besides Phormia regina, no other species were studied for pupal development stages and durations of these stages. However, in the same time period along with our study, two other papers were published. The pupal development stages of Calliphora vicinia [13, 14] and Lucilia sericata [14] were described using morphological and histological methods. Brown [13] conducted her study at 22 °C, whereas Zajac and Amendt [14] conducted their study at 25 °C. An MSc. thesis conducted on the species of Calliphora vomitoria and Chrysomya albiceps [15] should be mentioned as an additional study on this topic. In our study, pupal development stages and the durations of these stages of Lucilia sericata were determined at three different temperatures: 20 °C, 25 °C, and 30 °C. With respect to this species, nine development stages and the durations of these stages were established different from Greenberg and Kunich [6]. Our findings on Lucilia sericata are novel for this research area. Fig. 2 a Lateral view of the pupae. b Ventral view of the pupae. Leg formation was observed (leg buds in circles)
Fig. 3 Ventral view of the pupae. Wing formation was observed at the lateral part of the pupae (wing formation area in circle)
Materials and method Specimens were collected by placing beef liver at woodlands at Hacettepe University, Beytepe Campus. Lucilia sericata specimens were identified using a Leica MZ16 A stereoscopic microscope system according to the identification keys by Zumpt [16], Greenberg and Kunich [6] at the Biocriminal Entomology Laboratory in the Hacettepe University biology department. Lucilia sericata specimens were reared in a square, net cage measuring 21×21×21 cm in a Sanyo MIR-253 cooled incubator. Each cage was supplied with cotton soaked with sugary water for feeding (sugary water was prepared as approximately 20 g of sugar mixed in 0.5 l water, according to Catts and Haskell [17]). In addition, fresh beef liver was placed for oviposition. For the pupation process of the postfeeding larvae, 26×16×15-cm and 17×11×7-cm rectangular rearing containers supplied with sawdust were used. After the postfeeding larvae pupated, they were sacrificed using hot water. From the prepupae stage, they were removed
Int J Legal Med Fig. 4 a Lateral view of the pupae. Respiratory horns were brown (in circle). b Ventral view of the pupae. Cephalopharyngeal skeleton was embedded in the anterior part of the pupae
from the sawdust using forceps and put into a 3-cm-diameter screw cap with labels. On average, one or two individuals were dissected and photographed hourly. When the features of the pupae couldn’t be seen, the dissected specimen number was increased. In this study, the prepupae stage was taken as point zero for time calculations for each of three temperatures; when the puparium was white and when the pseudocephalon, which is located at the anterior part of the larvae, was completely withdrawn [6]. Pupae were killed, dissected, and photographed according to the hour calculations based on the elapsed time between point zero and adult emergence, and then stored in 80 % alcohol [3]. Lucilia sericata is a warm-adapted species, therefore, it is common between April–November in Ankara [8]. Considering the summer season monthly temperature averages during the summer season for Ankara, 20°, 25°, and 30° were preferred for our study, with 25° believed to be the optimal development temperature.
Fig. 5 a Dorsal view of the pupae. The head was pushed outwards, separation of head– thorax–abdomen. b Ventral view of the pupae. Leg and wing development continued
Results Pupal stages and development times at 20 °C Prepupae: Hour 0: Larvae that are going to pupate after postfeeding take the form of pupae by shortening and thickening. The cephalopharyngeal skeleton appears to move under the puparium when the larvae are first pupated. They are considered to be in the prepupae stage when this structure remains constant (Fig. 1a). Because the prepupae are very tightly bound to the puparium and cephalopharyngeal skeleton, the pupae could not be removed as a whole during dissection (Fig. 1b). 12th–14th hours: Distinctly articulated between anterior segments, and the beginning of the leg bud formation was observed (Fig. 2a, b). Pupae shaped as larvae. The cephalopharyngeal skeleton was cut out during dissection.
Int J Legal Med
Fig. 6 Dorsal view of the pupae. With the head pushed outwards, the respiratory horns were pushed anterolaterally to the back of the compound eyes (compound eyes in circle, respiratory horn indicated with arrow) Fig. 8 Dorsal view of the pupae. Abdomen has adopted its adult form
19th–21st hours: Wing buds were observed laterally (Fig. 3). Because the cephalopharyngeal skeleton was not bound to puparium, the pupae could easily be removed from the puparium, and this was considered to be the beginning of the Cryptocephalic stage. 26th–29th hours: The cephalopharyngeal skeleton started to embed in the apical area and in accordance with this, the apical part could easily be removed at dissection. The color of the respiratory horns turned light brown. 29th–33rd hours: The color of the respiratory horns turned brown and the cephalopharyngeal skeleton was completely embedded in the apical area (Fig. 4). The growth of the legs and wings continued. 41st–43rd hours: The head was pushed outwards and separation of head–thorax–abdomen was observed (Fig. 5a). Wings and legs grew on thorax (Fig. 5b). Respiratory horns were pushed anterolaterally to the back of the compound eyes. Mouth parts and compound eyes formed on the head (Fig. 6). Abdomen was observed as U-shaped (Fig. 7a) and looked like larvae. This stage in which the head was pushed forward is the beginning of the Phanerocephalic stage.
Fig. 7 a Dorsal view of the pupae. Apical constriction of the abdomen. b Lateral view of the pupae (stigma openings in circle)
45th–48th hours: The formation of stigma openings was observed on the abdomen (Fig. 7b). 46th–48th hours: Abdomen contraction occurred at the posterior part, the abdomen appeared to adopt its adult form (Fig. 8). 77th–80th hours: The formation of the maxillar palpus was observed (Fig. 9a, b). 132nd–136th hours: The antenna was seen to adopt its adult form. Development of the maxillar palpus continued. The beginning of the abdomen segmentation was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 145th–147th hours: Maxillar palpus on the head had adopted its adult form (Fig. 11a, b). 163rd–164th hours: Color of compound eyes was observed as yellow and their contour became significant (Fig. 12). 187th–190th hours: When examined, the pigmented part of the eye could not be seen dorsally (Fig. 13a), but could be seen ventrally (Fig. 13b). Compound eyes had begun to turning pink from the posterior part (Fig. 13c).
Int J Legal Med Fig. 9 a Ventral view of the pupae. b Formation of the maxillar palpus (maxillar palpus in circle)
This stage is accepted as the beginning of the pigmentation stage of the eye’s posterior. 190th–192nd hours: The color of the compound eyes was observed as light pink (Fig. 14a). The ommatidia of the eye were significant (Fig. 14b). 203th–205th hours: The color of the compound eyes was observed as maroon red (Fig. 15). 216th–218th hours: The formation of bristles was observed on the thorax (Fig. 16). 218th–221st hours: The formation of bristles was observed on the head. This stage is the beginning of the head and thorax bristles tanning stage (Fig. 17). 223rd–225th hours: The formation of bristles was observed on the abdomen. This stage is the beginning of the abdominal bristles tanning stage. The pigmentation of bristles was not observed (Fig. 18).
240th–244th hours: Bristles prolonged and darkened on the head, thorax and legs (Fig. 19a, d, e). The color of antenna was dark grey. Mouth parts thickened, bristles observed on palpus and were pigmented (Fig. 19b). The costal vein of the wing became bristled and thickened, and pigmentation was observed through the wing (Fig. 19f). All of the bristles were black on the abdomen (Fig. 19c). 252nd–255th hours: Bristles on the head completely darkened. Mouthparts chitinized and maxillar palpus darkened with bristles. Thorax and legs covered with black bristles. Wing grey colored, with the costal wing thickened and covered with darkened bristles. All of the abdomen bristles were black colored. 256th–258th hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 281st–284th hours: Pupal cuticle on the pupae could easily be removed and mostly remained in the puparium during dissection (Fig. 21). 314th–318th hours: Adult emergence was observed.
Pupal stages and development times at 25 °C
Fig. 10 Dorsal view of the pupae. Segmentation of the abdomen (segment lines indicated with arrows)
Prepupae: hour 0: Larvae that were going to pupate after postfeeding took the form of pupae by shortening and thickening. Cephalopharyngeal skeleton appeared to move under the puparium when larvae first pupated. It is considered to be a prepupae when this structure remains constant (Fig. 1a). Because the prepupae is very tightly bound to the puparium and cephalopharyngeal skeleton, the pupae could not be removed as a whole during dissection (Fig. 1b).
Int J Legal Med Fig. 11 a Ventral view of the pupae. b Mouthparts and maxillar palpus
8th–12th hours: The formation of aspiratory tubes on the apical area and the beginning of the formation of the leg buds (Fig. 2a) on the ventral part of the anterior area were observed. The cephalopharyngeal skeleton was cut out during dissection. 11th–12th hours: While development and growth of the legs continued (Fig. 2b), the formation of wing buds was observed (Fig. 3). Respiratory horns were white. Because of the withdrawal of the cephalopharyngeal skeleton, it was not tightly bound to the puparium and thus, the pupae could easily be removed from the puparium. This stage was the beginning of the Cryptocephalic stage.
Fig. 12 Lateral view of the pupae. Compound eye contour significant (upper and lower borders indicated with arrows)
19th–21st hours: Respiratory horns were brown and the cephalopharyngeal skeleton was completely embedded in the apical area (Fig. 4a, b). 23rd–25th hours: The head was pushed outwards and the separation of the head–thorax–abdomen was observed (Fig. 5a). The formation of wings and legs continued (Fig. 5b). As the head was pushed outwards, the respiratory horns were pushed to anterolateral side of the pupae. Compound eyes and mouthparts formed on the head (Fig. 6). The abdomen was observed to be U-shaped (Fig. 7a). This stage was the beginning of the Phanerocephalic stage. 26th–28th hours: The formation of stigma openings was observed on the lateral side of the abdomen (Fig. 7b). 27th–30th hours: Abdomen contraction occurred at the anterior, and the abdomen adopted its adult form (Fig. 8). 44th–47th hours: The beginning of the formation of the maxillar palpus was observed on the head (Fig. 9a, b). 67th–69th hours: Abdomen segmentation was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 73rd–75th hours: The maxillar palpus completely developed (Fig. 11). 76th–79th hours: The color of the compound eyes became yellow (Fig. 12) 100th–103rd hours: The compound eyes had begun turning pink, starting from the posterior part. This stage is accepted as the beginning of the pigmentation stage of the eye’s posterior (Fig. 13b, c). 120th–123rd hours: The color of the compound eyes darken and turned to maroon red (Fig. 15).
Int J Legal Med
Fig. 13 a Dorsal view of the pupae. Pigmented part of the eye could not be seen. b Ventral view of the pupae. Pigmented part could be seen. c Lateral view of the pupae. The pigmentation of the eyes started from the ventral part (pigmented parts indicated with arrows in b and c)
123rd–127th hours: The formation of bristles was observed on the thorax (Fig. 16). 126th–129th hours: Bristles were observed on the head and thorax. This stage was the beginning of the head and thorax bristles tanning stage (Fig. 17). 127th–130th hours: The formation of bristles was observed on the abdomen. This stage was the beginning of the abdomen bristles tanning stage (Fig. 18). 138th–141st hours: Arista of antennae was black colored. Mouthparts were chitinized, the palpus developed and was covered with black bristles (Fig. 19b). The wing was grey colored, the costal vein thickened and was covered with black bristles (Fig. 19f). The legs thickened and became pigmented (Fig. 19a). All of the abdomen bristles were black colored (Fig. 19c). 141st–143rd hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 165th–167th hours: Pupal cuticle could easily be removed from the pupae. During dissection it remained primarily in the puparium (Fig. 21). 171st–175th hours: Adult emergence was observed. Fig. 14 a Lateral view of the pupae. b Significant ommatidia on compound eyes
Pupal stages and development times at 30 °C Prepupae: Hour 0. 7th–9th hours: Leg formation was observed at the anterior area of the pupae (Fig. 2). The cephalopharyngeal skeleton was bound to the puparium. White respiratory horns were observed on the apical part of the pupae. 10th–11th hours: All legs were observed at the anterior area. The formation of the wing was observed on the lateral side (Fig. 3). Respiratory horns were white and the cephalopharyngeal skeleton was withdrawn (Fig. 4). This stage was the beginning of the Cryptocephalic stage. 14th–16th hours: The cephalopharyngeal skeleton was completely embedded in the anterior area. Respiratory horns were brown. The growth of the wings and legs continued (Fig. 4). 18th–21st hours: With the head pushed outwards, separation of the head–thorax–abdomen was observed (Fig. 5a). The formation of wings on the lateral side and legs on the ventral side was observed (Fig. 5b). The formation of compound eyes and mouthparts was also observed (Fig. 6). The
Int J Legal Med
Fig. 15 Lateral view of the pupae. Maroon red eye
wing was seen as a membrane, and the abdomen was U-shaped. This marked the beginning of the Phanerocephalic stage. 19th–21st hours: The abdomen here was U-shaped and the stigma openings began to be visible in the form of projections (Fig. 7). 34th–37th hours: The formation of the maxillar palpus was observed (Fig. 9). 40th–42nd hours: The beginning of the segmentation of the abdomen was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 48th–49th hours: The maxillar palpus developed and adopted its adult form (Fig. 11a, b). 69th–71st hours: The color of the compound eyes was yellow (Fig. 12).
Fig. 16 Dorsal view of the pupae. Formation of the bristles on the thorax (in circle)
79th–83rd hours: The compound eyes started to darken significantly on the ventral area (Fig. 13c). This stage marked the beginning of the pigmentation stage of the eye’s posterior. 89th–92nd hours: The color of the compound eyes was maroon red (Fig. 15). 93rd–94th hours: The formation of bristles was observed on the thorax (Fig. 16). 96th–98th hours: The formation of bristles on the head was observed. This stage is the beginning of the head and thorax bristles tanning stage (Fig. 17). 100th–102nd hours: The formation of bristles at the wing base and darkening of the wing were observed. The formation of bristles on the abdomen was observed (Fig. 18). This stage was the beginning of the abdomen bristles tanning stage. 103rd–105th hours: Mouthparts thickened and became apparent. Numerous bristles were observed on the leg (Fig. 19a). The color of the antenna was grey (Fig. 19b). All the bristles on the body darkened. All the bristles on the head and thorax were black, while the abdomen bristles near to the end were brown, and the bristles close to the thorax were black (Fig. 19d, e). The base of the wings was bristled and the costal vein was thickened and bristled (Fig. 19f). 106th–109th hours: All bristles on the abdomen were darkened. All limbs were also darkened and chitinized. 109th–111th hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 114th–115th hours: Pupal cuticle could easily be removed from the pupae during dissection (Fig. 21). 121st–123rd hours: Adult emergence was observed.
Int J Legal Med Fig. 17 Dorsal view of the pupae. Darkened bristles on the head and thorax
Discussion On the basis of this research, we aimed to determine how long the 11 pre-determined development stages that were given in Greenberg and Kunich [6] would take at 20 °C, 25 °C, and 30 °C. Nine out of the 11 pre-determined development stages were observed and nine new development stages were also identified. The newly identified stages for Lucilia sericata are: the formation of the first leg buds, the formation of wing buds, the embedding of the cephalopharyngeal skeleton in the apical area, stigma
Fig. 18 Dorsal view of the pupae. Bristle formation on the abdomen
openings becoming clear on the abdomen, the formation of the maxillar palpus, the color of the compound eyes becoming yellow, a darkening and thickening of the limbs, the pupal cuticle becoming thinner on pupae, and the beginning of removal of the pupal cuticle. In this study, pupal developments of Lucilia sericata were examined at 20 °C, 25 °C, and 30 °C (Table 1). According to findings based on all three temperatures, we determined that as the temperature increases, the duration of development shortens. L. sericata was development completed in 13 days, 2 h at 20 °C, in 7 days, 3 h at 25 °C, and in 5 days, 1 h at 30 °C.
Int J Legal Med
Fig. 19 a Thickened and pigmented legs. b Pigmented mouthparts and antenna. c Pigmented bristles on the abdomen. d Darkened bristles on the thorax. e Darkened bristles on the head. f Thickened costal vein (a–b ventral view; c–d–e dorsal view; costal vein (indicated with arrow))
In Zajac and Amendt’s study [14], the authors examined the pupal morphology of Lucillia sericata at 25 °C, and their results are consistent with our findings on the changes in pupal morphology. Based on our findings, there are 18 different morphological stages in the pupal development of Lucilia sericata, whereas Zajac and Amendt [14] described ten stages. Furthermore, Zajac and Amendt [14] carried out their study only on one temperature (25 °C), while in our study the differences Fig. 20 a Darkened bristles on the abdomen. b Darkened bristles on the thorax. c Darkened bristles on the head. d Thickened and darkened legs, darkened wings. a–d The pupal cuticle on the pupae became thinner (a–b–c dorsal view; d ventral view)
between pupal development periods and progression were documented at three different temperatures: 20 °C, 25 °C, and 30 °C. However, because their examinations were performed once every 24 h, we feel that our morphological evaluations are thus more detailed. For example, the observation of respiratory horns pushed laterally and forming the anterior stigma is not evaluated as a single stage in an hourly-examined study, but in a 24-h study, it appears as the formation of the anterior stigma stage.
Int J Legal Med
It is generally assumed that the pupal stage duration across temperatures has a linear relationship [6]. In this study, for all three temperatures conducted, a slight non-linear change was observed in the observation of the leg bud, Cryptocephalic, and wing formation stages. These stages correspond to the early pupal development period, in which the equilibrium between destruction and production did not occur, and thereby, we believe that these early stages are mostly temperaturedependent stages. In contrast, in looking at the advanced stages of the pupal development period, a more linear change could be seen.
Fig. 21 Dorsal view of the pupae (removed pupal cuticle indicated with arrow)
When our results are compared with Richards et al.’s study [18], in which the authors examined the pupal morphology of Calliphora vicina at 23 °C, it could be seen that they examined the pupal process in four quarters: 24 h, 96 h, 168 h and 240 h. They determined eight different stages at these four quarters. Most of the stages are in reference to the characteristics related to color, and correlate with our findings except for the fully-melanized labrum stage. Staging was done in a more detailed manner in our study, in which pupae were dissected hourly and the times when changes occurred were more apparent.
Conclusion From our observations of the pupae development stages, all stages were related to the coloration of body parts, except for the structural changes, such as the formation of legs and wings, stigma openings on the abdomen and mouthparts, and the thinning and removal of pupal cuticles. Therefore, it is thought that our results are important for the determination of the age of pupae, because of the detailed evaluations concerning color changes. With respect to the pupal period that is used to determine the postmortem interval, the development stages of L. sericata at three different temperatures were described for the first time. We believe that the data presented in this study will
Table 1 The development stages of Lucilia sericata in puparium (newly identified stages indicated with asterisk (*)) Stage
20 °C
20 °C ADH
25 °C
25 °C ADH
30 °C
30 °C ADH
Prepupae *Observation of the leg buds Cryptocephalic stage *Wing formation *Cephalopharyngeal skeleton embedded in the apical area Phanerocephalic stage *Stigma formation on the abdomen *Formation of maxillar palpus Segmentation of the abdomen *Yellow-colored compound eyes Coloration of the posterior portion of the eye Maroon red-colored compound eyes Bristles darkened on head and thorax Bristles darkened on abdomen *Limbs darkened and thickened *Thinning of pupal cuticle *Removal of pupal cuticle Adult emergence
0 12th–14th hours 19th–21st hours 19th–21st hours 26th–29th hours
0 240–280 380–420 380–420 520–580
0 8th–12th hours 11th–12th hours 11th–12th hours 19th–21st hours
0 200–300 275–300 275–300 475–525
0 7th–9th hours 10th–11th hours 10th–11th hours 14th–16th hours
0 210–270 300–330 300–330 420–480
41st–43rd hours 45th–48th hours 77th–80th hours 132nd–136th hours 163rd–164th hours 187th–190th hours
820–860 900–960 1,540–1,600 2,640–2,720 3,260–3,280 3,740–3,800
23rd–25th hours 26th–28th hours 44th–47th hours 67th–69th hours 76th–79th hours 100th–103rd hours
575–625 650–700 1,100–1,175 1,675–1,725 1,900–1,975 2,500–2,575
18th–21st hours 19th–21st hours 34th–37th hours 40th–42nd hours 69th–71st hours 79th–83rd hours
540–630 570–630 1,020–1,110 1,200–1,260 2,070–2,130 2,370–2,490
203rd–205th hours 218th–221st hours 223rd–225th hours 252nd–255th hours 256th–258th hours 281st–284th hours 314th–318th hours
4,060–4,100 4,360–4,420 4,460–4,500 5,040–5,100 5,120–5,160 5,620–5,680 6,280–6,360
120th–123rd hours 126th–129th hours 127th–130th hours 138th–141st hours 141st–143rd hours 165th–167th hours 171st–175th hours
3,000–3,075 3,150–3,225 3,175–3,250 3,450–3,525 3,525–3,575 4,125–4,175 4,275–4,375
89th–92nd hours 96th–98th hours 100th–102nd hours 106th–109th hours 109th–111th hours 114th–115th hours 121st–123rd hours
2,670–2,760 2,880–2,940 3,000–3,060 3,180–3,270 3,270–3,330 3,420–3,450 3,630–3,690
Int J Legal Med
provide significant contributions to the field of forensic entomology. Acknowledgments We would like to thank Assoc. Prof. Dr. Hilal Özdağ from the Ankara University Institute of Biotechnology for proofreading and corrections. We also would like to thank Res. Assist. Dr. Burcu Şabanoğlu and Res. Assist. Dr. Senem Firat, and appreciate their help. Conflict of interest The authors declare that they have no conflicts of interest.
References 1. Goff ML (2001) A fly for the prosecution: how insect evidence helps solve crimes. Harvard University Press, Cambridge 2. Şabanoğlu B (2007) Determination of Calliphoridae (Diptera) Fauna on Carrion and Morphological Studies in the Point of Systematic Aspect in Ankara Province. Dissertation, Hacettepe University, Institute of Graduate Studies in Science 3. Adams ZJO, Hall MJR (2003) Methods used for killing and preservation of blowfly larvae and their effect on post mortem length. Forensic Sci Int 138(1–3):50–61 4. Amendt J, Kretteck R, Zehner R (2004) Forensic entomology. Naturwissenshaften 91:51–65 5. Grassberger M, Reiter C (2001) Effect of temperature on Lucilia sericata (Diptera:Calliphoridae) development with special reference to the isomegalen and isomorphen-diagram. Forensic Sci Int 120:32–36 6. Greenberg B, Kunich JC (2002) Entomology and the law: flies as forensic indicators. Cambridge University Press, Cambridge, p 306 7. Mandeville DJ (1988) Rearing Phaenicia sericata (Diptera: Calliphoridae) on dry cat food with CSMA. J Med Entomol 25(3): 197–198
8. Şabanoğlu B, Sert O (2010) Determination of Calliphoridae (Diptera) fauna and seasonal distribution on carrion in Ankara province. J Forensic Sci 55:1003–1007 9. Tantawi TI, Greenberg B (1993) The effects of killing and preserving solutions on estimates of larval age in forensic cases. J Forensic Sci 38:303–309 10. Zehner R, Amendt J, Boehme P (2009) Gene expression analysis as a tool for age estimation of blowfly pupae. For Sci Int Genet Suppl 2: 292–293 11. Agrel IPS, Lundquist AM (1973) Physiological and biochemical changes during insect development. In: Rockstein M (ed) The physiology of insecta. Academic, New York 12. Finell N, Jarvilehto M (1983) Development of compound eyes of the blowfly Calliphora erythrocephala: changes in morphology and function during metamorphosis. Ann Zool Fenn 20:223–234 13. Brown KE (2012) Utility of the Calliphora vicina (Diptera: Calliphoridae) Pupal Stage for Providing Temporal Information for Death Investigations. Dissertation, University of Portsmouth 14. Zajac BK, Amendt J (2012) Bestimmung des Alters Forensisch Relevanter Fliegenpuppen. Rechtsmedizin 22:456–465. doi:10. 1007/s00194-012-0854-5 15. Ergil C (2012) The Analysis of Pupal Developments in Calliphora vomitoria (Linnaeus, 1758) and Chrysomya albiceps (Wieddemann, 1819) Species which Belong to Calliphoridae Family that is Among the Forensically Important Insect Groups. Dissertation, Hacettepe University, Institute of Graduate Studies in Science 16. Zumpt F (1965) Myiasis in man and animals in the old world. Butterworths, London 17. Catts EP, Haskell NH (1990) Entomology and death: a procedural guide, 1st edn. Joyce’s Print Shop, South Carolina, pp 1–182 18. Richards CS, Simonsen TJ, Abel RL, Hall MJR, Schwyn DA, Wicklein M (2012) Virtual forensic entomology: improving estimates of minimum post-mortem interval with 3D micro-computed tomography. For Sci Int 220(1–3):251–264. doi:10.1016/j.forsciint. 2012.03.012
ORIGINAL ARTICLE
The analysis of pupal development period in Lucilia sericata (Diptera: Calliphoridae) forensically important insect Tuçe Karabey & Osman Sert
Received: 18 April 2013 / Accepted: 16 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Forensic entomology is a study that insect evidence is used for the determination of postmortem interval (PMImin). While determining PMImin, the pupal period is given as the duration between the beginning of the pupal period and the stage of adult emergence. The pupal period constitutes approximately 50 % of the immature development, and with detailed examination it would be possible to present a much more accurate PMImin. In this study, the pupal period of Lucilia sericata (Meigen, 1826), which is one of the species used in PMImin estimation in forensic entomology, was investigated. Studies about the pupal period of development are considerably limited in the forensic entomology world. Stages and duration of the pupal period of Lucilia sericata were studied at temperatures of 20 °C, 25 °C, and 30 °C. For all temperatures, pupae specimens were dissected and photographed hourly. According to findings based on all three temperatures, it was determined that as the temperature increases, the duration of development shortens. Also, in addition to 11 pre-determined development stages, nine new development stages were identified.
Keywords Lucilia sericata . Pupae . Development . PMImin . Temperature
This article is the main part of Tuçe Karabey’s master thesis, which was supervised by Assoc. Prof. Dr. Osman Sert and accepted on 11 April 2012. T. Karabey : O. Sert (*) Faculty of Science Department of Biology, Hacettepe University, Beytepe Campus, 06532 Ankara, Turkey e-mail: [email protected]
Introduction Insects arrive on carrion less than 5–10 min after death [1, 2]. Therefore, the method of estimating the postmortem interval (PMImin) by using the life cycles of some holometabolous insects plays a very important role in the legal system. In particular, the larval stage of the insect is used to estimate the time of death in cases where this is unknown [3]. In the larval stage, morphological structures can be identified from the outside and this data makes the PMImin estimation possible. In the forensic field, many researchers have gathered data on the larval periods and adult stages of forensic insects [2, 4–9]. There are many studies regarding the time when pupation begins and when the adult emerges, but few studies have dealt with the stages of the pupal period and their sequences. However, the pupal period constitutes approximately 50 % of the immature development and therefore, it is thought that the age of the pupae may serve as an important tool for PMImin estimation [10]. Determining the age of the pupae is much more difficult compared to determining the age of larvae, since it is not possible to observe morphological differences such as length and weight from the outside [4]. Changes in the color of the puparium from white to dark brown provide useful information concerning the age of pupae for the first 10 h [6]. However, after this first 10 h, there is no way of detecting the age of pupae because the color remains unchanged [4]. Regarding the development of the insect, because of the fact that morphological differentiations in the pupal stage cannot be observed from the outside and because the use of the gene expression approach for age determination is expensive and time consuming, the remains of pupae collected from crime scenes are like a black box for forensic entomologists [6]. Three fundamental studies were conducted on the progress of the pupal period. Agrell and Lundquist [11] generally divided the pupal period in the three stages of histolytic change, histogenetic change, and differentiation, without
Int J Legal Med
Fig. 1 a Dorsal view of the prepupae. b Shattered prepupae cannot be removed as a whole when dissected
specifying species or genus. Finell and Jarvilehto [12] distinguished the pupal stages of Calliphora erythrocephala as histolysis, juvenile stage, histogenetic stage, immature stage, mature stage, and the last differentiation stage. Greenberg and Kunich [6] distinguished the pupal period into11 stages. In their study, which is the first published paper dealing with this issue at the species level, they determined the pupal development stages and durations of the stages of Phormia regina (Meigen, 1826) at 22 °C and 29 °C. Until 2012, among forensically important species besides Phormia regina, no other species were studied for pupal development stages and durations of these stages. However, in the same time period along with our study, two other papers were published. The pupal development stages of Calliphora vicinia [13, 14] and Lucilia sericata [14] were described using morphological and histological methods. Brown [13] conducted her study at 22 °C, whereas Zajac and Amendt [14] conducted their study at 25 °C. An MSc. thesis conducted on the species of Calliphora vomitoria and Chrysomya albiceps [15] should be mentioned as an additional study on this topic. In our study, pupal development stages and the durations of these stages of Lucilia sericata were determined at three different temperatures: 20 °C, 25 °C, and 30 °C. With respect to this species, nine development stages and the durations of these stages were established different from Greenberg and Kunich [6]. Our findings on Lucilia sericata are novel for this research area. Fig. 2 a Lateral view of the pupae. b Ventral view of the pupae. Leg formation was observed (leg buds in circles)
Fig. 3 Ventral view of the pupae. Wing formation was observed at the lateral part of the pupae (wing formation area in circle)
Materials and method Specimens were collected by placing beef liver at woodlands at Hacettepe University, Beytepe Campus. Lucilia sericata specimens were identified using a Leica MZ16 A stereoscopic microscope system according to the identification keys by Zumpt [16], Greenberg and Kunich [6] at the Biocriminal Entomology Laboratory in the Hacettepe University biology department. Lucilia sericata specimens were reared in a square, net cage measuring 21×21×21 cm in a Sanyo MIR-253 cooled incubator. Each cage was supplied with cotton soaked with sugary water for feeding (sugary water was prepared as approximately 20 g of sugar mixed in 0.5 l water, according to Catts and Haskell [17]). In addition, fresh beef liver was placed for oviposition. For the pupation process of the postfeeding larvae, 26×16×15-cm and 17×11×7-cm rectangular rearing containers supplied with sawdust were used. After the postfeeding larvae pupated, they were sacrificed using hot water. From the prepupae stage, they were removed
Int J Legal Med Fig. 4 a Lateral view of the pupae. Respiratory horns were brown (in circle). b Ventral view of the pupae. Cephalopharyngeal skeleton was embedded in the anterior part of the pupae
from the sawdust using forceps and put into a 3-cm-diameter screw cap with labels. On average, one or two individuals were dissected and photographed hourly. When the features of the pupae couldn’t be seen, the dissected specimen number was increased. In this study, the prepupae stage was taken as point zero for time calculations for each of three temperatures; when the puparium was white and when the pseudocephalon, which is located at the anterior part of the larvae, was completely withdrawn [6]. Pupae were killed, dissected, and photographed according to the hour calculations based on the elapsed time between point zero and adult emergence, and then stored in 80 % alcohol [3]. Lucilia sericata is a warm-adapted species, therefore, it is common between April–November in Ankara [8]. Considering the summer season monthly temperature averages during the summer season for Ankara, 20°, 25°, and 30° were preferred for our study, with 25° believed to be the optimal development temperature.
Fig. 5 a Dorsal view of the pupae. The head was pushed outwards, separation of head– thorax–abdomen. b Ventral view of the pupae. Leg and wing development continued
Results Pupal stages and development times at 20 °C Prepupae: Hour 0: Larvae that are going to pupate after postfeeding take the form of pupae by shortening and thickening. The cephalopharyngeal skeleton appears to move under the puparium when the larvae are first pupated. They are considered to be in the prepupae stage when this structure remains constant (Fig. 1a). Because the prepupae are very tightly bound to the puparium and cephalopharyngeal skeleton, the pupae could not be removed as a whole during dissection (Fig. 1b). 12th–14th hours: Distinctly articulated between anterior segments, and the beginning of the leg bud formation was observed (Fig. 2a, b). Pupae shaped as larvae. The cephalopharyngeal skeleton was cut out during dissection.
Int J Legal Med
Fig. 6 Dorsal view of the pupae. With the head pushed outwards, the respiratory horns were pushed anterolaterally to the back of the compound eyes (compound eyes in circle, respiratory horn indicated with arrow) Fig. 8 Dorsal view of the pupae. Abdomen has adopted its adult form
19th–21st hours: Wing buds were observed laterally (Fig. 3). Because the cephalopharyngeal skeleton was not bound to puparium, the pupae could easily be removed from the puparium, and this was considered to be the beginning of the Cryptocephalic stage. 26th–29th hours: The cephalopharyngeal skeleton started to embed in the apical area and in accordance with this, the apical part could easily be removed at dissection. The color of the respiratory horns turned light brown. 29th–33rd hours: The color of the respiratory horns turned brown and the cephalopharyngeal skeleton was completely embedded in the apical area (Fig. 4). The growth of the legs and wings continued. 41st–43rd hours: The head was pushed outwards and separation of head–thorax–abdomen was observed (Fig. 5a). Wings and legs grew on thorax (Fig. 5b). Respiratory horns were pushed anterolaterally to the back of the compound eyes. Mouth parts and compound eyes formed on the head (Fig. 6). Abdomen was observed as U-shaped (Fig. 7a) and looked like larvae. This stage in which the head was pushed forward is the beginning of the Phanerocephalic stage.
Fig. 7 a Dorsal view of the pupae. Apical constriction of the abdomen. b Lateral view of the pupae (stigma openings in circle)
45th–48th hours: The formation of stigma openings was observed on the abdomen (Fig. 7b). 46th–48th hours: Abdomen contraction occurred at the posterior part, the abdomen appeared to adopt its adult form (Fig. 8). 77th–80th hours: The formation of the maxillar palpus was observed (Fig. 9a, b). 132nd–136th hours: The antenna was seen to adopt its adult form. Development of the maxillar palpus continued. The beginning of the abdomen segmentation was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 145th–147th hours: Maxillar palpus on the head had adopted its adult form (Fig. 11a, b). 163rd–164th hours: Color of compound eyes was observed as yellow and their contour became significant (Fig. 12). 187th–190th hours: When examined, the pigmented part of the eye could not be seen dorsally (Fig. 13a), but could be seen ventrally (Fig. 13b). Compound eyes had begun to turning pink from the posterior part (Fig. 13c).
Int J Legal Med Fig. 9 a Ventral view of the pupae. b Formation of the maxillar palpus (maxillar palpus in circle)
This stage is accepted as the beginning of the pigmentation stage of the eye’s posterior. 190th–192nd hours: The color of the compound eyes was observed as light pink (Fig. 14a). The ommatidia of the eye were significant (Fig. 14b). 203th–205th hours: The color of the compound eyes was observed as maroon red (Fig. 15). 216th–218th hours: The formation of bristles was observed on the thorax (Fig. 16). 218th–221st hours: The formation of bristles was observed on the head. This stage is the beginning of the head and thorax bristles tanning stage (Fig. 17). 223rd–225th hours: The formation of bristles was observed on the abdomen. This stage is the beginning of the abdominal bristles tanning stage. The pigmentation of bristles was not observed (Fig. 18).
240th–244th hours: Bristles prolonged and darkened on the head, thorax and legs (Fig. 19a, d, e). The color of antenna was dark grey. Mouth parts thickened, bristles observed on palpus and were pigmented (Fig. 19b). The costal vein of the wing became bristled and thickened, and pigmentation was observed through the wing (Fig. 19f). All of the bristles were black on the abdomen (Fig. 19c). 252nd–255th hours: Bristles on the head completely darkened. Mouthparts chitinized and maxillar palpus darkened with bristles. Thorax and legs covered with black bristles. Wing grey colored, with the costal wing thickened and covered with darkened bristles. All of the abdomen bristles were black colored. 256th–258th hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 281st–284th hours: Pupal cuticle on the pupae could easily be removed and mostly remained in the puparium during dissection (Fig. 21). 314th–318th hours: Adult emergence was observed.
Pupal stages and development times at 25 °C
Fig. 10 Dorsal view of the pupae. Segmentation of the abdomen (segment lines indicated with arrows)
Prepupae: hour 0: Larvae that were going to pupate after postfeeding took the form of pupae by shortening and thickening. Cephalopharyngeal skeleton appeared to move under the puparium when larvae first pupated. It is considered to be a prepupae when this structure remains constant (Fig. 1a). Because the prepupae is very tightly bound to the puparium and cephalopharyngeal skeleton, the pupae could not be removed as a whole during dissection (Fig. 1b).
Int J Legal Med Fig. 11 a Ventral view of the pupae. b Mouthparts and maxillar palpus
8th–12th hours: The formation of aspiratory tubes on the apical area and the beginning of the formation of the leg buds (Fig. 2a) on the ventral part of the anterior area were observed. The cephalopharyngeal skeleton was cut out during dissection. 11th–12th hours: While development and growth of the legs continued (Fig. 2b), the formation of wing buds was observed (Fig. 3). Respiratory horns were white. Because of the withdrawal of the cephalopharyngeal skeleton, it was not tightly bound to the puparium and thus, the pupae could easily be removed from the puparium. This stage was the beginning of the Cryptocephalic stage.
Fig. 12 Lateral view of the pupae. Compound eye contour significant (upper and lower borders indicated with arrows)
19th–21st hours: Respiratory horns were brown and the cephalopharyngeal skeleton was completely embedded in the apical area (Fig. 4a, b). 23rd–25th hours: The head was pushed outwards and the separation of the head–thorax–abdomen was observed (Fig. 5a). The formation of wings and legs continued (Fig. 5b). As the head was pushed outwards, the respiratory horns were pushed to anterolateral side of the pupae. Compound eyes and mouthparts formed on the head (Fig. 6). The abdomen was observed to be U-shaped (Fig. 7a). This stage was the beginning of the Phanerocephalic stage. 26th–28th hours: The formation of stigma openings was observed on the lateral side of the abdomen (Fig. 7b). 27th–30th hours: Abdomen contraction occurred at the anterior, and the abdomen adopted its adult form (Fig. 8). 44th–47th hours: The beginning of the formation of the maxillar palpus was observed on the head (Fig. 9a, b). 67th–69th hours: Abdomen segmentation was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 73rd–75th hours: The maxillar palpus completely developed (Fig. 11). 76th–79th hours: The color of the compound eyes became yellow (Fig. 12) 100th–103rd hours: The compound eyes had begun turning pink, starting from the posterior part. This stage is accepted as the beginning of the pigmentation stage of the eye’s posterior (Fig. 13b, c). 120th–123rd hours: The color of the compound eyes darken and turned to maroon red (Fig. 15).
Int J Legal Med
Fig. 13 a Dorsal view of the pupae. Pigmented part of the eye could not be seen. b Ventral view of the pupae. Pigmented part could be seen. c Lateral view of the pupae. The pigmentation of the eyes started from the ventral part (pigmented parts indicated with arrows in b and c)
123rd–127th hours: The formation of bristles was observed on the thorax (Fig. 16). 126th–129th hours: Bristles were observed on the head and thorax. This stage was the beginning of the head and thorax bristles tanning stage (Fig. 17). 127th–130th hours: The formation of bristles was observed on the abdomen. This stage was the beginning of the abdomen bristles tanning stage (Fig. 18). 138th–141st hours: Arista of antennae was black colored. Mouthparts were chitinized, the palpus developed and was covered with black bristles (Fig. 19b). The wing was grey colored, the costal vein thickened and was covered with black bristles (Fig. 19f). The legs thickened and became pigmented (Fig. 19a). All of the abdomen bristles were black colored (Fig. 19c). 141st–143rd hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 165th–167th hours: Pupal cuticle could easily be removed from the pupae. During dissection it remained primarily in the puparium (Fig. 21). 171st–175th hours: Adult emergence was observed. Fig. 14 a Lateral view of the pupae. b Significant ommatidia on compound eyes
Pupal stages and development times at 30 °C Prepupae: Hour 0. 7th–9th hours: Leg formation was observed at the anterior area of the pupae (Fig. 2). The cephalopharyngeal skeleton was bound to the puparium. White respiratory horns were observed on the apical part of the pupae. 10th–11th hours: All legs were observed at the anterior area. The formation of the wing was observed on the lateral side (Fig. 3). Respiratory horns were white and the cephalopharyngeal skeleton was withdrawn (Fig. 4). This stage was the beginning of the Cryptocephalic stage. 14th–16th hours: The cephalopharyngeal skeleton was completely embedded in the anterior area. Respiratory horns were brown. The growth of the wings and legs continued (Fig. 4). 18th–21st hours: With the head pushed outwards, separation of the head–thorax–abdomen was observed (Fig. 5a). The formation of wings on the lateral side and legs on the ventral side was observed (Fig. 5b). The formation of compound eyes and mouthparts was also observed (Fig. 6). The
Int J Legal Med
Fig. 15 Lateral view of the pupae. Maroon red eye
wing was seen as a membrane, and the abdomen was U-shaped. This marked the beginning of the Phanerocephalic stage. 19th–21st hours: The abdomen here was U-shaped and the stigma openings began to be visible in the form of projections (Fig. 7). 34th–37th hours: The formation of the maxillar palpus was observed (Fig. 9). 40th–42nd hours: The beginning of the segmentation of the abdomen was observed. This stage was the beginning of the segmented abdomen stage (Fig. 10). 48th–49th hours: The maxillar palpus developed and adopted its adult form (Fig. 11a, b). 69th–71st hours: The color of the compound eyes was yellow (Fig. 12).
Fig. 16 Dorsal view of the pupae. Formation of the bristles on the thorax (in circle)
79th–83rd hours: The compound eyes started to darken significantly on the ventral area (Fig. 13c). This stage marked the beginning of the pigmentation stage of the eye’s posterior. 89th–92nd hours: The color of the compound eyes was maroon red (Fig. 15). 93rd–94th hours: The formation of bristles was observed on the thorax (Fig. 16). 96th–98th hours: The formation of bristles on the head was observed. This stage is the beginning of the head and thorax bristles tanning stage (Fig. 17). 100th–102nd hours: The formation of bristles at the wing base and darkening of the wing were observed. The formation of bristles on the abdomen was observed (Fig. 18). This stage was the beginning of the abdomen bristles tanning stage. 103rd–105th hours: Mouthparts thickened and became apparent. Numerous bristles were observed on the leg (Fig. 19a). The color of the antenna was grey (Fig. 19b). All the bristles on the body darkened. All the bristles on the head and thorax were black, while the abdomen bristles near to the end were brown, and the bristles close to the thorax were black (Fig. 19d, e). The base of the wings was bristled and the costal vein was thickened and bristled (Fig. 19f). 106th–109th hours: All bristles on the abdomen were darkened. All limbs were also darkened and chitinized. 109th–111th hours: Pupal cuticle on the pupae became thinner (Fig. 20a–d). 114th–115th hours: Pupal cuticle could easily be removed from the pupae during dissection (Fig. 21). 121st–123rd hours: Adult emergence was observed.
Int J Legal Med Fig. 17 Dorsal view of the pupae. Darkened bristles on the head and thorax
Discussion On the basis of this research, we aimed to determine how long the 11 pre-determined development stages that were given in Greenberg and Kunich [6] would take at 20 °C, 25 °C, and 30 °C. Nine out of the 11 pre-determined development stages were observed and nine new development stages were also identified. The newly identified stages for Lucilia sericata are: the formation of the first leg buds, the formation of wing buds, the embedding of the cephalopharyngeal skeleton in the apical area, stigma
Fig. 18 Dorsal view of the pupae. Bristle formation on the abdomen
openings becoming clear on the abdomen, the formation of the maxillar palpus, the color of the compound eyes becoming yellow, a darkening and thickening of the limbs, the pupal cuticle becoming thinner on pupae, and the beginning of removal of the pupal cuticle. In this study, pupal developments of Lucilia sericata were examined at 20 °C, 25 °C, and 30 °C (Table 1). According to findings based on all three temperatures, we determined that as the temperature increases, the duration of development shortens. L. sericata was development completed in 13 days, 2 h at 20 °C, in 7 days, 3 h at 25 °C, and in 5 days, 1 h at 30 °C.
Int J Legal Med
Fig. 19 a Thickened and pigmented legs. b Pigmented mouthparts and antenna. c Pigmented bristles on the abdomen. d Darkened bristles on the thorax. e Darkened bristles on the head. f Thickened costal vein (a–b ventral view; c–d–e dorsal view; costal vein (indicated with arrow))
In Zajac and Amendt’s study [14], the authors examined the pupal morphology of Lucillia sericata at 25 °C, and their results are consistent with our findings on the changes in pupal morphology. Based on our findings, there are 18 different morphological stages in the pupal development of Lucilia sericata, whereas Zajac and Amendt [14] described ten stages. Furthermore, Zajac and Amendt [14] carried out their study only on one temperature (25 °C), while in our study the differences Fig. 20 a Darkened bristles on the abdomen. b Darkened bristles on the thorax. c Darkened bristles on the head. d Thickened and darkened legs, darkened wings. a–d The pupal cuticle on the pupae became thinner (a–b–c dorsal view; d ventral view)
between pupal development periods and progression were documented at three different temperatures: 20 °C, 25 °C, and 30 °C. However, because their examinations were performed once every 24 h, we feel that our morphological evaluations are thus more detailed. For example, the observation of respiratory horns pushed laterally and forming the anterior stigma is not evaluated as a single stage in an hourly-examined study, but in a 24-h study, it appears as the formation of the anterior stigma stage.
Int J Legal Med
It is generally assumed that the pupal stage duration across temperatures has a linear relationship [6]. In this study, for all three temperatures conducted, a slight non-linear change was observed in the observation of the leg bud, Cryptocephalic, and wing formation stages. These stages correspond to the early pupal development period, in which the equilibrium between destruction and production did not occur, and thereby, we believe that these early stages are mostly temperaturedependent stages. In contrast, in looking at the advanced stages of the pupal development period, a more linear change could be seen.
Fig. 21 Dorsal view of the pupae (removed pupal cuticle indicated with arrow)
When our results are compared with Richards et al.’s study [18], in which the authors examined the pupal morphology of Calliphora vicina at 23 °C, it could be seen that they examined the pupal process in four quarters: 24 h, 96 h, 168 h and 240 h. They determined eight different stages at these four quarters. Most of the stages are in reference to the characteristics related to color, and correlate with our findings except for the fully-melanized labrum stage. Staging was done in a more detailed manner in our study, in which pupae were dissected hourly and the times when changes occurred were more apparent.
Conclusion From our observations of the pupae development stages, all stages were related to the coloration of body parts, except for the structural changes, such as the formation of legs and wings, stigma openings on the abdomen and mouthparts, and the thinning and removal of pupal cuticles. Therefore, it is thought that our results are important for the determination of the age of pupae, because of the detailed evaluations concerning color changes. With respect to the pupal period that is used to determine the postmortem interval, the development stages of L. sericata at three different temperatures were described for the first time. We believe that the data presented in this study will
Table 1 The development stages of Lucilia sericata in puparium (newly identified stages indicated with asterisk (*)) Stage
20 °C
20 °C ADH
25 °C
25 °C ADH
30 °C
30 °C ADH
Prepupae *Observation of the leg buds Cryptocephalic stage *Wing formation *Cephalopharyngeal skeleton embedded in the apical area Phanerocephalic stage *Stigma formation on the abdomen *Formation of maxillar palpus Segmentation of the abdomen *Yellow-colored compound eyes Coloration of the posterior portion of the eye Maroon red-colored compound eyes Bristles darkened on head and thorax Bristles darkened on abdomen *Limbs darkened and thickened *Thinning of pupal cuticle *Removal of pupal cuticle Adult emergence
0 12th–14th hours 19th–21st hours 19th–21st hours 26th–29th hours
0 240–280 380–420 380–420 520–580
0 8th–12th hours 11th–12th hours 11th–12th hours 19th–21st hours
0 200–300 275–300 275–300 475–525
0 7th–9th hours 10th–11th hours 10th–11th hours 14th–16th hours
0 210–270 300–330 300–330 420–480
41st–43rd hours 45th–48th hours 77th–80th hours 132nd–136th hours 163rd–164th hours 187th–190th hours
820–860 900–960 1,540–1,600 2,640–2,720 3,260–3,280 3,740–3,800
23rd–25th hours 26th–28th hours 44th–47th hours 67th–69th hours 76th–79th hours 100th–103rd hours
575–625 650–700 1,100–1,175 1,675–1,725 1,900–1,975 2,500–2,575
18th–21st hours 19th–21st hours 34th–37th hours 40th–42nd hours 69th–71st hours 79th–83rd hours
540–630 570–630 1,020–1,110 1,200–1,260 2,070–2,130 2,370–2,490
203rd–205th hours 218th–221st hours 223rd–225th hours 252nd–255th hours 256th–258th hours 281st–284th hours 314th–318th hours
4,060–4,100 4,360–4,420 4,460–4,500 5,040–5,100 5,120–5,160 5,620–5,680 6,280–6,360
120th–123rd hours 126th–129th hours 127th–130th hours 138th–141st hours 141st–143rd hours 165th–167th hours 171st–175th hours
3,000–3,075 3,150–3,225 3,175–3,250 3,450–3,525 3,525–3,575 4,125–4,175 4,275–4,375
89th–92nd hours 96th–98th hours 100th–102nd hours 106th–109th hours 109th–111th hours 114th–115th hours 121st–123rd hours
2,670–2,760 2,880–2,940 3,000–3,060 3,180–3,270 3,270–3,330 3,420–3,450 3,630–3,690
Int J Legal Med
provide significant contributions to the field of forensic entomology. Acknowledgments We would like to thank Assoc. Prof. Dr. Hilal Özdağ from the Ankara University Institute of Biotechnology for proofreading and corrections. We also would like to thank Res. Assist. Dr. Burcu Şabanoğlu and Res. Assist. Dr. Senem Firat, and appreciate their help. Conflict of interest The authors declare that they have no conflicts of interest.
References 1. Goff ML (2001) A fly for the prosecution: how insect evidence helps solve crimes. Harvard University Press, Cambridge 2. Şabanoğlu B (2007) Determination of Calliphoridae (Diptera) Fauna on Carrion and Morphological Studies in the Point of Systematic Aspect in Ankara Province. Dissertation, Hacettepe University, Institute of Graduate Studies in Science 3. Adams ZJO, Hall MJR (2003) Methods used for killing and preservation of blowfly larvae and their effect on post mortem length. Forensic Sci Int 138(1–3):50–61 4. Amendt J, Kretteck R, Zehner R (2004) Forensic entomology. Naturwissenshaften 91:51–65 5. Grassberger M, Reiter C (2001) Effect of temperature on Lucilia sericata (Diptera:Calliphoridae) development with special reference to the isomegalen and isomorphen-diagram. Forensic Sci Int 120:32–36 6. Greenberg B, Kunich JC (2002) Entomology and the law: flies as forensic indicators. Cambridge University Press, Cambridge, p 306 7. Mandeville DJ (1988) Rearing Phaenicia sericata (Diptera: Calliphoridae) on dry cat food with CSMA. J Med Entomol 25(3): 197–198
8. Şabanoğlu B, Sert O (2010) Determination of Calliphoridae (Diptera) fauna and seasonal distribution on carrion in Ankara province. J Forensic Sci 55:1003–1007 9. Tantawi TI, Greenberg B (1993) The effects of killing and preserving solutions on estimates of larval age in forensic cases. J Forensic Sci 38:303–309 10. Zehner R, Amendt J, Boehme P (2009) Gene expression analysis as a tool for age estimation of blowfly pupae. For Sci Int Genet Suppl 2: 292–293 11. Agrel IPS, Lundquist AM (1973) Physiological and biochemical changes during insect development. In: Rockstein M (ed) The physiology of insecta. Academic, New York 12. Finell N, Jarvilehto M (1983) Development of compound eyes of the blowfly Calliphora erythrocephala: changes in morphology and function during metamorphosis. Ann Zool Fenn 20:223–234 13. Brown KE (2012) Utility of the Calliphora vicina (Diptera: Calliphoridae) Pupal Stage for Providing Temporal Information for Death Investigations. Dissertation, University of Portsmouth 14. Zajac BK, Amendt J (2012) Bestimmung des Alters Forensisch Relevanter Fliegenpuppen. Rechtsmedizin 22:456–465. doi:10. 1007/s00194-012-0854-5 15. Ergil C (2012) The Analysis of Pupal Developments in Calliphora vomitoria (Linnaeus, 1758) and Chrysomya albiceps (Wieddemann, 1819) Species which Belong to Calliphoridae Family that is Among the Forensically Important Insect Groups. Dissertation, Hacettepe University, Institute of Graduate Studies in Science 16. Zumpt F (1965) Myiasis in man and animals in the old world. Butterworths, London 17. Catts EP, Haskell NH (1990) Entomology and death: a procedural guide, 1st edn. Joyce’s Print Shop, South Carolina, pp 1–182 18. Richards CS, Simonsen TJ, Abel RL, Hall MJR, Schwyn DA, Wicklein M (2012) Virtual forensic entomology: improving estimates of minimum post-mortem interval with 3D micro-computed tomography. For Sci Int 220(1–3):251–264. doi:10.1016/j.forsciint. 2012.03.012
