Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions
Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions
Anna Mądra-Bielewicz 0 1
Katarzyna Frątczak-Łagiewska 0 1
Szymon Matuszewski 0 1
0 Department of Animal Taxonomy and Ecology, Adam Mickiewicz University , Umultowska 89, 61-614 Poznań , Poland
1 Laboratory of Criminalistics, Adam Mickiewicz University , Św. Marcin 90, 61-809 Poznań , Poland
2 Szymon Matuszewski
Despite widely accepted standards for sampling and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations. Here, we report the results of laboratory studies on the survival of Lucilia sericata and Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial forms in hermetic containers, which were stimulated by a recent case. It is demonstrated that the survival of blowfly intra-puparial forms inside airtight containers is dependent on container volume, number of puparia inside, and their age. The survival in both species was found to increase with an increase in the volume of air per 1 mg of puparium per day of development in a hermetic container. Below 0.05 ml of air, no insect survived, and above 0.2 ml of air per 1 mg of puparium per day, survival reached its maximum. These results suggest that blowflies reveal a single, general pattern of survival under decreasing oxygen conditions and that this pattern is a product of number of developing insects, their age and the initial amount of available air. Implications for forensic entomology are discussed.
Forensic entomology; Post-mortem interval; Insect evidence; Preservation techniques; Hypoxia
The importance of insect evidence in criminal investigations
has increased substantially over recent decades [
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 [
samples or improperly preserved evidence are encountered
frequently in forensic investigations [
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 [
]. When puparia contain developing
insects, it is recommended to preserve some of them for
laboratory rearing and some as dead specimens [
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 [
]. 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 . 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
], inside mammalian stomachs [
], in dung [
] or carrion
], in hermetic containers [
], and in temporarily immersed
]. In the case of holometabolic insects, the
ability to survive in hypoxic conditions is often stage-specific
. 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) [
], Protophormia terraenovae
(Robineau-Desvoidy, 1830), Calliphora vicina
RobineauDesvoidy, 1830, Cochliomyia macellaria (Fabricius, 1775),
Lucilia sericata (Meigen, 1826) , Chrysomya albiceps
(Wiedemann, 1819), C. megacephala (Fabricius, 1794), and
C. putoria (Wiedemann, 1830) [
]. These studies indicated
that the survival is affected by the amount of time a life stage is
submerged and its age at submergence [
]. 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.
Materials and methods
Lucilia sericata and Calliphora vomitoria (Linnaeus, 1758)
were chosen for the experiments as they are frequently
reported from forensic cases [
] and pig carcass experiments in
Central Europe [
]. Larvae were purchased from a
fishing shop before each trial. Experiments were performed in
insect incubators at 22.5 °C for a photoperiod (h) of 12:12
(L:D). Glass jars (twist type with a metal lid) were used as
containers. Only fresh puparia (sampled shortly after puparial
darkening had started, within 5 h from pupariation) were
selected for the experiments. Upon termination of each trial,
adult flies that emerged were counted. Tenerals, which died
during emergence, were also counted. Closed puparia were
left in open containers for an additional seven days to
double-check the mortality.
Experiment 1: factors affecting the survival of intra-puparial forms in hermetic containers
In order to choose factors of interest and test the protocol,
several preliminary studies were conducted. The main
experiment was performed according to a factorial block design.
Three blocks (trials) were separated in time. Based on results
of preliminary studies, it was decided to test effects of the
following factors: blow fly species, number of puparia,
container volume, damp paper substrate, and container
conditions, with two levels each (Table 1). Puparia of both species
were placed in each container (ratio 1:1) and containers
were either left open or hermetically sealed (hermetic).
Single fold paper towels (25 × 23 cm) were used as a
paper substrate, hydrated with 3 ml of water at the onset
of the study in sealed containers. In the case of open
containers the same size of paper towels were used and
they were hydrated with 3 ml of water once a day during
the study. Each trial included 16 containers and was
terminated after 14 days.
Experiment 2: survival of intra-puparial forms of various ages in hermetic containers
This experiment included two factors: age of intra-puparial
forms (10 levels) and number of puparia (three levels)
(Table 1). A block design was used with three blocks (trials),
separated in time. Containers of 315 ml were used with no
paper inside. Puparia of both species (ratio 1:1) were put twice
daily into the containers, which were then sealed, starting from
3- to 7.5 day-old insects. Each trial included 30 containers and
was terminated after 14 days.
Results of experiment 1 were tested using factorial analysis of
variance. After analyzing individual factors, the number of
puparia and the container volume were transformed into air
volume per puparial weight. For this purpose, the volume of
air inside the container at the onset of the trial was divided by
the total mass of puparia put into the container. Consequently,
air volume per 1 mg of puparium was obtained. Average mass
of a fresh puparium was used in these calculations (28.9 mg
for L. sericata and 85.2 mg for C. vomitoria).
In experiment 2, the survival of intra-puparial forms was
used as a response variable and air volume per 1 mg of
puparium per day as a predictor variable. To obtain the predictor
variable, air volume per 1 mg of puparium (see the previous
section) was divided by a predicted period of development in
the container. The predicted development in the container is
the total intra-puparial development (10.3 days at 23 °C for
C. vomitoria [
] and 6.2 days at 22 °C for L. sericata [
minus the age of intra-puparial forms at the time of placement
in the container. The relationship was analyzed using
nonlinear regression. The polynomial model (y = b0 + b1x + b2x2)
was fitted with the Levenberg–Marquardt procedure for
In the analysis 5% level of significance was accepted.
Calculations were made using Statistica 10 (StatSoft, Inc.,
Factors affecting the survival of intra-puparial forms in hermetic containers
Container conditions, container volume, species, number of
puparia, and paper substrate significantly affected the survival
of intra-puparial forms inside containers (Table 2). The
survival rate was distinctly lower in hermetic containers, and this
was the largest observed effect. Moreover, container
conditions significantly interacted with all the other factors
(Table 2). In all cases, the differences between treatments in
hermetic containers were clearly larger than the differences in
open containers (Fig. 1). Lower container volume and higher
density of puparia resulted in a substantial decrease of survival
(Fig. 1, Table 2). Lucilia sericata revealed higher survival
rates than C. vomitoria (Fig. 1). Damp paper reduced the
survival rate in airtight containers (Fig. 1). Transforming the
container volume and the number of puparia into air volume per
puparial weight revealed that the survival of intra-puparial
forms inside hermetic containers is positively related to the
volume of air per 1 mg of puparium at the time of placement
in the container (Fig. 2).
Survival of intra-puparial forms of various ages in hermetic containers
Survival was found to increase with the initial age of an insect
inside the puparium. It increased with increase in the volume
of air per 1 mg of puparium per day of the intra-puparial
development inside the container (Fig. 3). The pattern of
changes was similar for both species, with polynomial increase
of survival between 0.05 and 0.2 ml of air per 1 mg of
puparium per day (L. sericata, y = (−70.41) + (2172.28) × x +
(−7086.6) × x2, P < 0.001, r2 = 0.85; C. vomitoria,
y = (−73.804) + (1580.01) × x + (−3772.7) × x2, P < 0.01 for
b0 and b1, P = 0.09 for b2, r2 = 0.58; where y is the survival
of intra-puparial forms and x is air volume per 1 mg of
puparium per day; Fig. 4). Below 0.05 ml of air per 1 mg
of puparium per day, no insect survived, and above 0.2 ml
of air per 1 mg of puparium per day, the survival reached
its maximum (Fig. 3).
Fig. 1 Effect of container volume (a), number of puparia inside the
container (b), species (c) and presence of damp paper substrate inside
the container (d) on mean survival of intra-puparial blowflies inside
open (■) and hermetic containers (●). Vertical bars represent 95%
confidence intervals. Data were pooled to show main effects
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 [
]. 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
. 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 [
]. Moreover, in such conditions, some
Fig. 4 The polynomial models
for the relationship between
survival of intra-puparial forms
(Lucilia sericata or Calliphora
vomitoria) in hermetic containers
and air volume per 1 mg of
puparial weight per day inside
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 [
]. The intra-puparial
development lasts longer in C. vomitoria than in L. sericata
]. 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
intrapuparial 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 [
], the survival of intra-puparial forms
inside hermetic containers will be under some influence of
temperature conditions inside the container.
No control over initial stages of development We used
larvae bought in a fishing shop and therefore had no control over
the conditions of rearing at the initial stages of insect
development. However, the survival recorded in this study in open
containers was high, similar to the laboratory rearing in
optimal conditions. Accordingly, specimens used in this study
were a realistic representation of wild populations.
Developmental rate in the containers The developmental
rate in hermetic containers may be higher than that in optimal
]. Therefore, the time of intra-puparial
development used to calculate the predicted development in the
container might have inaccurately represented the times of
development in airtight containers. Accordingly, air volumes per
puparial weight per day, which were obtained during the
experiments, may also be inaccurate (too low). However, at
present, better models are out of our reach, as there are no
scientific data on developmental rate of blowflies in hermetic
1. The survival of intra-puparial forms in hermetic
containers was studied for Lucilia sericata and Calliphora
2. For both species survival in such conditions was largely
affected by the container volume, the number of puparia
inside, and their age.
3. Below 0.05 ml of air per 1 mg of puparium per day of the
development inside the container no insect survived. At or
above 0.2 ml of air per 1 mg of puparium per day 100% of
The study has indicated that blowflies reveal a single,
general pattern of survival in hermetic conditions.
Acknowledgments We thank the reviewers for their helpful comments
on earlier versions of the manuscript.
Compliance with ethical standards
Funding This study was conducted without any external funding.
Conflict of interest Authors declare that they have no conflict of
Ethical approval All applicable international, national, and/or
institutional guidelines for the care and use of animals were followed. This
article does not contain any studies with human participants performed
by any of the authors.
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