Forensic Sci Med Pathol (2017) 13:328–335 DOI 10.1007/s12024-017-9892-3 ORIGINAL ARTICLE Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions Anna Mądra-Bielewicz 1 & Katarzyna Frątczak-Łagiewska1,2 & Szymon Matuszewski1 Accepted: 26 May 2017 /Published online: 1 July 2017 # The Author(s) 2017. This article is an open access publication Abstract Despite widely accepted standards for sampling Introduction and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently The importance of insect evidence in criminal investigations in forensic investigations. Here, we report the results of labo- has increased substantially over recent decades [1]. However, ratory studies on the survival of Lucilia sericata and in practice it is extremely rare that a qualified forensic ento- Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial mologist is present at the crime scene. Usually, insects are forms in hermetic containers, which were stimulated by a collected by crime scene technicians or medical examiners. recent case. It is demonstrated that the survival of blowfly Although there are widely accepted protocols for sampling intra-puparial forms inside airtight containers is dependent and preservation of insect evidence [1–5], unrepresentative on container volume, number of puparia inside, and their samples or improperly preserved evidence are encountered age. The survival in both species was found to increase with frequently in forensic investigations [1, 6]. an increase in the volume of air per 1 mg of puparium per day The blowfly puparium is an opaque, barrel-like structure; a of development in a hermetic container. Below 0.05 ml of air, prepupa, a pupa or a pharate adult (i.e. intra-puparial forms) no insect survived, and above 0.2 ml of air per 1 mg of pupar- develop inside it [7, 8]. When puparia contain developing ium per day, survival reached its maximum. These results insects, it is recommended to preserve some of them for lab- suggest that blowflies reveal a single, general pattern of sur- oratory rearing and some as dead specimens [1, 5]. “Living” vival under decreasing oxygen conditions and that this pattern puparia should be kept on a damp soil (or a similar substrate) is a product of number of developing insects, their age and the with constant air access and should be transferred within 24 h initial amount of available air. Implications for forensic ento- for rearing in laboratory conditions [1, 4, 5]. The other portion mology are discussed. should be killed with boiling water and kept in 70%–95% ethanol in the fridge if possible, after piercing the puparium to allow the preservative to enter inside [9, 10]. Despite these Keywords Forensic entomology . Post-mortem interval . standards, puparia are sometimes improperly preserved, for Insect evidence . Preservation techniques . Hypoxia example, in a hermetic container with no preservative inside or in a leaking container resulting in evaporation of preserva- tive and dehydration of specimens [6]. Incorrect preservation may affect the scope of laboratory procedures to be performed using intra-puparial forms. Moreover, in some instances it may elicit extra questions, which are irrelevant in cases with * Szymon Matuszewski [email protected] properly preserved insect samples. Here we report results of experiments provoked by a recent 1 Laboratory of Criminalistics, Adam Mickiewicz University, Św. case in which fly puparia were improperly preserved. The Marcin 90, 61-809 Poznań,Poland senior author received the insect evidence preserved in an 2 Department of Animal Taxonomy and Ecology, Adam Mickiewicz airtight glass jar with no preservative inside. Almost four University, Umultowska 89, 61-614 Poznań,Poland months passed between insect sampling at the crime scene Forensic Sci Med Pathol (2017) 13:328–335 329 and the arrival of specimens at the laboratory. Inspection re- Materials and methods vealed that the jar contained 14 adult insects (11 true flies, among them a single specimen of the flesh fly Sarcophaga General protocol sp., and three specimens of parasitoid wasp Nasonia vitripennis (Walker, 1836)), 18 closed puparia with decaying Lucilia sericata and Calliphora vomitoria (Linnaeus, 1758) pupae inside, two puparia with small holes and decaying pu- were chosen for the experiments as they are frequently report- pae inside, and a single empty puparium. All puparia belonged ed from forensic cases [20, 21] and pig carcass experiments in to Sarcophaga argyrostoma (Robineau-Desvoidy, 1830). The Central Europe [22–24]. Larvae were purchased from a fish- report from the crime scene indicated that crime technicians ing shop before each trial. Experiments were performed in sampled 21 puparia and 10 adult flies, the latter only from the insect incubators at 22.5 °C for a photoperiod (h) of 12:12 families Calliphoridae, Muscidae and Fannidae. Accordingly, (L:D). Glass jars (twist type with a metal lid) were used as asinglespecimenofadultSarcophaga and three specimens of containers. Only fresh puparia (sampled shortly after puparial adult N. vitripennis must have emerged inside the jar. Because darkening had started, within 5 h from pupariation) were se- the oldest specimen was the puparium of S. argyrostoma from lected for the experiments. Upon termination of each trial, which the adult emerged in the jar, minimum post-mortem adult flies that emerged were counted. Tenerals, which died interval was inferred from the total immature development during emergence, were also counted. Closed puparia were of S. argyrostoma minus the period of development in the left in open containers for an additional seven days to jar. No scientific data on the survival of intra-puparial forms double-check the mortality. inside hermetic containers were available at the time of case analysis. Accordingly, the senior author estimated (based on jar volume and number of puparia inside) that insects might Experiment 1: factors affecting the survival have been developing in the jar for no more than 5 days. of intra-puparial forms in hermetic containers However, the estimate was subjective and therefore prone to error. Consequently, it was decided to make a basic study to In order to choose factors of interest and test the protocol, answer the following questions provoked by the circum- several preliminary studies were conducted. The main exper- stances of the case. How long may intra-puparial forms sur- iment was performed according to a factorial block design. vive inside a hermetic container? Does the number of puparia Three blocks (trials) were separated in time. Based on results in the container affect their survival? Does the age of insect of preliminary studies, it was decided to test effects of the inside the puparium affect its ability to survive inside the con- following factors: blow fly species, number of puparia, con- tainer under conditions of decreasing oxygen? Are forensical- tainer volume, damp paper substrate, and container condi- ly important species equally sensitive to hypoxia? tions, with two levels each (Table 1). Puparia of both species Living organisms can experience hypoxia at high altitudes were placed in each container (ratio 1:1) and containers [11], inside mammalian stomachs [12], in dung [13]orcarrion were either left open or hermetically sealed (hermetic). [14], in hermetic containers [15], and in temporarily immersed Single fold paper towels (25 × 23 cm) were used as a substrates [11, 16]. In the case of holometabolic insects, the paper substrate, hydrated with 3 ml of water at the onset ability to survive in hypoxic conditions is often stage-specific of the study in sealed containers. In the case of open [11]. Metabolically highly active stages, for example, pupae, containers the same size of paper towels were used and are most sensitive to hypoxia. The survival rate of larvae and they were hydrated with 3 ml of water once a day during intra-puparial forms after submergence in water was studied the study. Each trial included 16 containers and was ter- for several forensically important blowflies: Phormia regina minated after 14 days. (Meigen, 1826) [17, 18], Protophormia terraenovae (Robineau-Desvoidy, 1830), Calliphora vicina Robineau- Desvoidy, 1830, Cochliomyia macellaria (Fabricius, 1775), Experiment 2: survival of intra-puparial forms of various Lucilia sericata (Meigen, 1826) [18], Chrysomya albiceps ages in hermetic containers (Wiedemann, 1819), C. megacephala (Fabricius, 1794), and C. putoria (Wiedemann, 1830) [19]. These studies indicated This experiment included two factors: age of intra-puparial that the survival is affected by the amount of time a life stage is forms (10 levels) and number of puparia (three levels) submerged and its age at submergence [17, 18]. The survival (Table 1). A block design was used with three blocks (trials), of intra-puparial forms of blowflies was, however, not studied separated in time. Containers of 315 ml were used with no under decreasing oxygen conditions as experienced in airtight paper inside. Puparia of both species (ratio 1:1) were put twice containers. Different conditions in hermetic containers, com- daily into the containers, which were then sealed, starting from pared to underwater environments, may result in different sur- 3- to 7.5 day-old insects. Each trial included 30 containers and vival rates of forensically important blowflies. was terminated after 14 days. 330 Forensic Sci Med Pathol (2017) 13:328–335 Table 1 Factors included in the experiments Factor Levels E1 E2 Species I. Lucilia sericata ++ II. Calliphora vomitoria ++ Number of puparia I. 10 + + II. 20 − + III. 30 + + Container volume I. 315 ml + + II. 900 ml + − Damp paper substrate I. Present + − II. Absent + + Container conditions I. Open + − II. Hermetic + + Age of intra-puparial forms 10 levels (from 3 days old until 7.5 days old) − + E1, E2 - experiment 1 and 2 Data analyzes of intra-puparial forms inside containers (Table 2). The sur- vival rate was distinctly lower in hermetic containers, and this Results of experiment 1 were tested using factorial analysis of was the largest observed effect.