Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions

The importance of insect evidence in criminal investigations has increased substantially over recent decades [1]. However, in practice it is extremely rare that a qualified forensic entomologist is present at the crime scene. Usually, insects are collected by crime scene technicians or medical examiners. Although there are widely accepted protocols for sampling and preservation of insect evidence [1‐5], unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations [1, 6].

The blowfly puparium is an opaque, barrel-like structure; a prepupa, a pupa or a pharate adult (i.e. intra-puparial forms) develop inside it [7, 8]. When puparia contain developing insects, it is recommended to preserve some of them for laboratory rearing and some as dead specimens [1, 5]. “Living” puparia should be kept on a damp soil (or a similar substrate) with constant air access and should be transferred within 24 h for rearing in laboratory conditions [1, 4, 5]. The other portion 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 standards, puparia are sometimes improperly preserved, for example, in a hermetic container with no preservative inside or in a leaking container resulting in evaporation of preservative 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 properly preserved insect samples.

Here we report results of experiments provoked by a recent case in which fly puparia were improperly preserved. The senior author received the insect evidence preserved in an airtight glass jar with no preservative inside. Almost four months passed between insect sampling at the crime scene and the arrival of specimens at the laboratory. Inspection revealed that the jar contained 14 adult insects (11 true flies, among them a single specimen of the flesh fly Sarcophaga sp., and three specimens of parasitoid wasp Nasonia vitripennis (Walker, 1836)), 18 closed puparia with decaying pupae inside, two puparia with small holes and decaying pupae inside, and a single empty puparium. All puparia belonged to Sarcophaga argyrostoma (Robineau-Desvoidy, 1830). The report from the crime scene indicated that crime technicians sampled 21 puparia and 10 adult flies, the latter only from the families Calliphoridae, Muscidae and Fannidae. Accordingly, a single specimen of adult Sarcophaga and three specimens of adult N. vitripennis must have emerged inside the jar. Because the oldest specimen was the puparium of S. argyrostoma from which the adult emerged in the jar, minimum post-mortem interval was inferred from the total immature development of S. argyrostoma minus the period of development in the jar. No scientific data on the survival of intra-puparial forms 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 have been developing in the jar for no more than 5 days. However, the estimate was subjective and therefore prone to error. Consequently, it was decided to make a basic study to answer the following questions provoked by the circumstances of the case. How long may intra-puparial forms survive inside a hermetic container? Does the number of puparia in the container affect their survival? Does the age of insect inside the puparium affect its ability to survive inside the container under conditions of decreasing oxygen? Are forensically important species equally sensitive to hypoxia?

Living organisms can experience hypoxia at high altitudes [11], inside mammalian stomachs [12], in dung [13] or carrion [14], in hermetic containers [15], and in temporarily immersed substrates [11, 16]. In the case of holometabolic insects, the ability to survive in hypoxic conditions is often stage-specific [11]. Metabolically highly active stages, for example, pupae, are most sensitive to hypoxia. The survival rate of larvae and intra-puparial forms after submergence in water was studied for several forensically important blowflies: Phormia regina (Meigen, 1826) [17, 18], Protophormia terraenovae (Robineau-Desvoidy, 1830), Calliphora vicina Robineau-Desvoidy, 1830, Cochliomyia macellaria (Fabricius, 1775), Lucilia sericata (Meigen, 1826) [18], Chrysomya albiceps (Wiedemann, 1819), C. megacephala (Fabricius, 1794), and C. putoria (Wiedemann, 1830) [19]. These studies indicated that the survival is affected by the amount of time a life stage is submerged and its age at submergence [17, 18]. The survival of intra-puparial forms of blowflies was, however, not studied under decreasing oxygen conditions as experienced in airtight containers. Different conditions in hermetic containers, compared to underwater environments, may result in different survival rates of forensically important blowflies.

This study has revealed that container volume, number of puparia inside, and their age are the factors of highest importance for the survival of blowfly intra-puparial forms inside hermetic containers. Experiments with submerged puparia indicated that survival is determined by the time of submergence and the age of the insect [18, 19]. Moreover, in submersion conditions, the pattern of survival was found to inversely track oxygen consumption during metamorphosis, with decrease of survival in the white puparial stage and pharate adult stage [18]. This study demonstrated that in hermetic containers survival increases with the increase of insect’s age at the moment of placement inside the container. These differences may be explained by different stress agents to which insects are exposed in hermetic containers and after submersion under water. In underwater conditions, oxygen deprivation seems to be the most important and the only permanent stress agent (water temperature or water contamination with toxic substances are not permanent stressors). However, in airtight containers, the set of permanent stress agents is more complex. Apart from gradual oxygen deprivation, gradual increase of carbon dioxide and other by-products of insect metabolism are permanent stressors [15]. Moreover, in such conditions, some insects will inevitably die, resulting in putrefaction of their bodies and resultant accumulation of volatile by-products of decay. All these accumulating substances are toxic for insects. Accordingly, their gradual increase in hermetic containers may be similarly or even more important for survival than gradual decrease of oxygen level. From this point of view, large effects of container volume and number of puparia inside, as revealed in this study, may simply result from the higher rate of toxins accumulation in small containers and at higher densities of puparia inside.

Intra-puparial C. vomitoria revealed lower survival rate in hermetic containers, compared to L. sericata, which is in line with the results of previous studies [18]. The intra-puparial development lasts longer in C. vomitoria than in L. sericata [25‐28]. Therefore, in airtight containers, it can be assumed that puparia of C. vomitoria had not completed development by the time the atmosphere had become lethal, whereas L. sericata, due to its shorter intra-puparial development time, usually completed its development before the conditions in the container became lethal.

We believe that the synthetic measure developed during this study (i.e. the air volume per 1 mg of puparium per day of intra-puparial development) may be used to describe the survival of all forensically relevant intra-puparial forms inside hermetic containers. The survival patterns of L. sericata and C. vomitoria were surprisingly similar when plotted against this measure. This finding suggests that the survival of intra-puparial forms of blowflies in airtight containers may be described by a single general model. We think that the model given in this article (Figs. 3 and 4) reasonably approximates this general one. However, the question of whether the general “window for survival” lies between 0.05 and 0.2 ml of air (per mg of puparium per day) needs to be studied further with more species.

The measure discussed in the previous section has clear practical advantages. It may easily be calculated for any container and any number of puparia inside. Therefore, in cases as the one being an inspiration for this research, that is, when intra-puparial forms had died or adult flies had emerged inside the container before arrival to the laboratory, it may be used to roughly estimate the age of intra-puparial forms when they were placed into the container. Based on the number of adult insects, which has emerged in the container, the amount of air per puparial weight per day may be estimated using the models given in this article or by simply inferring the values from the graphs (Figs 3 and 4). Then, having the knowledge of the container volume and the number of puparia inside, one may infer the amount of time insects have been developing in the container. For example, if all puparia of C. vomitoria in the container gave adult flies (i.e. the survival was 100%), we may deduce from Figs. 3 and 4 that the amount of air per 1 mg of puparium per day must have been above 0.09 ml and most probably above 0.2 ml. If the container is 300 ml in volume and there are 10 puparia inside (in total 852 mg), the amount of air per 1 mg of puparium will be 0.352 ml. Keeping in mind the daily amounts of air, which provides 100% survival, we may calculate that at 0.09 ml of air per day, insects might have been developing in the container for no more than about 3.9 days, and at 0.2 ml of air per day, the development would occur for no more than about 1.8 days. The survival patterns presented in this article may also be useful to calculate the maximum possible development in the given container and at a given number of puparia inside. As no adult emerged below 0.05 ml of air per day, this value may be the lower limit for survival and it may be used to calculate the maximum possible development time in the container. If for example, 21 puparia of S. argyrostoma (about 3150 mg) were put into a 300 ml container, about 0.095 ml of air would be available per 1 mg of puparium inside the container. Using the lower limit for survival, we get 1.9 days of maximum possible development in the container. Accordingly, if the current results would have been available when the case had been analyzed and assuming that temperature conditions had been similar to the experimental temperature from this study (i.e. about 22.5 °C), the PMI estimate would be more accurate by about 3 days (the estimate changed from 5 days in the jar to 2 days in the jar).

Future studies should address the effect of temperature on the survival rate of blowflies under the decreasing oxygen conditions. Due to the importance of temperature for all aspects of insect life [29], the survival of intra-puparial forms inside hermetic containers will be under some influence of temperature conditions inside the container.