Available online at www.sciencedirect.com
Forensic Science International 177 (2008) 162 167
www.elsevier.com/locate/forsciint
Post-feeding larval behaviour in the blowfly, Calliphora vicina:
Effects on post-mortem interval estimates
*
Sophie Arnott, Bryan Turner
Department of Forensic Science and Drug Monitoring, King s College London, Franklin-Wilkins Building, 150 Stamford Street,
London SE1 9NH, United Kingdom
Received 9 July 2007; received in revised form 26 September 2007; accepted 5 December 2007
Available online 19 February 2008
Abstract
Using the rate of development of blowflies colonising a corpse, accumulated degree hours (ADH), or days (ADD), is an established method used
by forensic entomologists to estimate the post-mortem interval (PMI). Derived from laboratory experiments, their application to field situations
needs care. This study examines the effect of the post-feeding larval dispersal time on the ADH and therefore the PMI estimate. Post-feeding
dispersal in blowfly larvae is typically very short in the laboratory but may extend for hours or days in the field, whilst the larvae try to find a suitable
pupariation site.
Increases in total ADH (to adult eclosion), due to time spent dispersing, are not simply equal to the dispersal time. The pupal period is increased
by approximately 2 times the length of the dispersal period. In practice, this can introduce over-estimation errors in the PMI estimate of between 1
and 2 days if the total ADH calculations do not consider the possibility of an extended larval dispersal period.
# 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Dispersal; Accumulated degree hours; ADH; Accumulated degree days; ADD; Forensic entomology; PMI; Blowflies; Calliphora
1. Introduction have a longer dispersal phase. Nuorteva [1] provides an
interesting link between distance and time by noting that
In the blowfly lifecycle (from egg, through three feeding Lucilia sericata in Finland travels at the slow rate of about 1 m
larval stages, a pupal and finally the adult stage) there is an per day over a moss covered forest floor, but in most other cases
important late larval period when feeding ceases and the so there is no link available between distance and time, or
called post-feeding larvae move away from the corpse on which importantly with temperature.
they have been feeding to find a suitable site for pupariation. A Some studies, specifically on this post-feeding larval
number of previously published forensic entomology studies dispersal phase [2 5], have emphasised the patterns and spatial
have recorded details of the length of time or the distance distribution of larval movement. Whilst this is of considerable
travelled during the post-feeding phase of larval blowflies. use in forensic entomology, particularly in identifying the most
These are summarised in Table 1. Often precise details are probable areas to look for puparia in relation to a corpse, only
lacking, as to, for instance, which species the measurements Gomes and Von Zuben [3] touch on the relevance of the
relate to, and in others the account is primarily descriptive. dispersal stage to the estimation of the post-mortem interval
What is clear is that some blowfly species (e.g. Protophormia (PMI). They show that dispersal puts an energetic cost on the
terraenovae, Fannia sp. Chrysomya rufifacies and Chrysomya post-feeding larvae which causes a reduction in pupal weight
albiceps, listed towards the top of the table) appear to spend less with increasing distance dispersed.
time dispersing since they remain close to or on the larval food One common, but by no means foolproof method (see for
source, whilst others (eg. Lucilia and Calliphora spp.) appear to example [6]) used by forensic entomologists to estimate the
PMI, is the calculation of the accumulated degree hours (ADH)
necessary to reach a specific point in the blowfly s development
(see Higley and Haskell in [7]). Such baseline data are normally
* Corresponding author.
E-mail address: [e-mail] (B. Turner). established in the laboratory where, in controlled conditions, it
0379-0738/$  see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2007.12.002
S. Arnott, B. Turner / Forensic Science International 177 (2008) 162 167 163
Table 1
Published information on the length of time or distance travelled in the post-feeding dispersal phase in larval blowflies
Taxon Time Location/distance Ref.
Protophormia terraenovae On corpse [14]
Fannia sp. On corpse [1]
Chrysomya rufifacies In clothing on corpse [7]
Calliphorinae   Under or near [15]
Chrysomya rufifacies  Under or near [16]
Blowfly larvae  In nearby soil [17]
Chrysomya albiceps  Under and around [18]
Chrysomya albiceps <20 cm (experimental arena) [3]
Cochliomyia hominivorax 0.6 2 m [19]
 Prepuparial Diptera 3 m [16]
Cochliomyia macellaria, Lucilia sericata >4.6 m [4]
and Phormia regina
Calliphorinae >6m(>20 ft) [20]
Lucilia sericata 6.4 m over soil [21]
Calliphoridae and Sarcophagidae   Normally pupation occurs in soil but in domestic situations suitable sites may be difficult to locate [22]
and fully grown maggots may be found wandering in quite unlikely situations some distance from
their larval food 
Lucilia sericata and Calliphora vicina 3 8.1 m [23]
Chrysomya rufifacies <3.3 m [23]
Cochliomya macellaria <5.1 m [23]
Chrysomya megacephala 7 m at one site and approx. 25 m at another on lava [16]
 Blowflies >30 m over hard ground [8]
Lucilia illustris and L. caesar 3 days 3 m [1]
Lucilia sericata 3 4 days (depending on temperature) [24]
 Blowflies 24 72 h in culture [7]
Calliphora vicina 5 14 days [11]
Calliphorids Up to 4 days Some climbed 1 2 m to litter in tree forks [25]
2. Methods
is usual that a suitable pupariation medium (sawdust, soiless
compost or similar) is readily available and adjacent to the
2.1. General
feeding larvae. The time spent in dispersal is therefore normally
very short in laboratory settings and, in the overall calculation
Wild C. vicina adults were trapped at several sites in London using bottle
of ADH, usually ignored.
traps (see [9], for the design) baited with pig s liver and 30% sodium sulphide
This contrasts with situations in the field at least for some
solution. These blowflies were cultured in cages and provided with sugar and
species. Post-feeding larvae may stay on the corpse or find water ad libitum. Egg-laying was induced by providing liquid liver exudate for
several days followed by solid pig s liver as an egg laying stimulant. Larvae
suitable pupariation substrates very close to the corpse (for
were then grown on pig s liver in small chambers at 20 8C with an excess of food
example if the corpse is in an area of well drained friable soil) or
to avoid competition effects [10], to provide a source of post-feeding larvae for
they may have to travel many metres if the ground is hard and
the experiments.
unyielding, as has been observed in the post-feeding disperal of
The experiments made use of the lid of a large plastic box as the arena
larvae from a corpse at the Anthropological Facility in (Fig. 1).
The lid has a large peripheral indented groove, which normally fits the rolled
Tennessee (Amoret Whitaker, personal communication) and
top of the plastic box on which it fits. When the lid is placed upside down the
noted by Green [8] Thus, in situations where pupariation is
groove forms a continuous track 25 mm wide by 37 mm deep. One circuit round
delayed until a suitable site has been found, there can be marked
the lid is exactly 2 m although the number of circuits made by larvae was not
differences in ADH estimates based on pupal and adult stages
recorded. Larvae introduced into the track continued to travel around it and did
between field and laboratory material. During long post- not attempt to scale the track walls. The arena was placed on the laboratory
bench. Laboratory temperature was 21 1.0 8C. Humidity was not controlled
feeding dispersal periods time passes (and therefore degree-
for, but was held by the building s air conditioning system at approximately
hours accumulate) and energy is used up, leading to a reduction
50%rh. Following the specified time in the arena the larvae were returned to a
in resources available for the pupa and adult stages.
constant 20 8C and given moist peat to pupate in.
This paper focuses on the impact of differing dispersal
periods for larvae (both at the normal post-feeding time and
2.1.1. Experiment 1
also at an earlier stage of growth) on the further development
This experiment explored the effects of differing lengths of time spent in
with particular reference to the levels of error that such post-feeding dispersal on the time to eclosion and the size of the adults were
examined. Larvae were collected from the cultures as they were leaving the food
situations might create in PMI estimates using ADH methods.
source. These post-feeding larvae were  run in the trackway in small groups for
The experimental blowfly species used in this study was the
1, 2, 4, 6, 8, or 24 h before being placed in plastic boxes containing damp soil-
blue bottle Calliphora vicina Robinseau-Desvoidy, a forensi-
less compost as a pupariation medium and returned to the incubators to
cally important and widespread species of urban areas in
complete development. Control individuals (0 h disperal time) were placed
temperate regions. immediately on the compost. The numbers for each experiment, were governed
164 S. Arnott, B. Turner / Forensic Science International 177 (2008) 162 167
Fig. 1. The plastic box lid arena with a close up of the channel used as the dispersal route. Dimensions are given in the text.
by the numbers of fresh post-feeding larvae available from the cultures Between
which at 20 8C is 460ADH. Whereas the difference in total
60 and 87 individual post-feeding larvae were  run for each time period.
ADH to eclosion, seen in Fig. 2 (9777ADH for a dispersal
The ADH to eclosion was recorded for all flies in the experiment.
period of 1 h and 10800ADH for 24 h dispersal) between 1 and
24 h is 1023ADH, more than twice what would be expected by
2.1.2. Experiment 2
simple addition.
The second experiment was similar to the first but used 2nd instar feeding
Time spent dispersing as larvae clearly uses resources that
larvae. This was considered as a simulation of the effects of being withdrawn
from the food source through competition, rising water levels or some other
would otherwise be available for pupal development and so it is
occurrence which breaks the feeding cycle. Larvae for this experiment were
not surprising that adult size, as measured by the d-cu cross vein
collected from the cultures as 2nd instars. They were  run in the trackway for 1,
in the wing (see [10], for details of method), is reduced with
2, 4, 6, 16, and 48 h. They were then returned to their food supply and followed
longer post-feeding larval dispersal time (Fig. 3) (F = 14.62,
through to adult emergence. In a further experiment 100 2nd instar larvae were
p<0.001).
removed from their food and left to run in the track for 96 h to see whether in the
absence of further food sources they could or would pupate.
In experiment 2, 2nd larvae were forced to move away from
their food for periods of up to 48 h and then returned to their
2.1.3. Experiment 3
food to develop as normal with no further dispersal delays. In
The behaviour of larvae when dispersing, in particular whether they follow
addition, 100 2nd instar larvae were left to circulate in the
trails left by previous individuals, was examined using the flat central area of the
arena, simulating total and final removal from the food source.
plastic lid which was marked out into 4 quadrants by two lines drawn at right
Only 7 survived to 96 h and these seven all pupated, having
angles between the mid points of opposite sides. Post-feeding larvae were
individually placed in the centre of the lid and their direction of travel traced on
moulted into 3rd instars just prior to pupariation. These 7
a paper template of the arena. Where they dropped into the peripheral groove
individuals have been added to the data for the 2nd instar larvae
was recorded. Between each larva the plastic lid was cleaned with an alcohol
starved for up to 48 h, and plotted in Fig. 4. Overall there is a
wipe and left to dry. It was also randomly rotated to obviate any directional
significant reduction in ADH with time spent off the food
effects that might have been induced by slightly non-uniform overhead lighting.
(F = 37.02, p<0.001) but, using Fishers PLSD post hoc test
Ninety larvae were tested in this way.
This was then repeated but without the alcohol washing between each larva.
indicates that there was no significant effect on the ADH to
eclosion in the larvae with up to a 48 h delay in their
2.1.4. Experiment 4
In a second behavioural experiment two temporary aluminium foil walls
were used to define a track along the long axis of the arena through the centre
point. Several hundred larvae where confined to this track to create a distinct
larval trail. Larvae were then individually placed in the centre of the arena and
their path recorded as being on or off the trail.
3. Results
The results from experiment 1 show that time spent in post-
feeding dispersal does influence the overall ADH to eclosion
(Fig. 2). There is a significant tendency (F = 369.4, p<0.001)
for the total ADH estimate to be increased with longer dispersal
times, although this is only apparent when dispersal times
exceed 5 h (Fisher s PLSD post hoc test gives non-significant p-
values in pair-wise comparisons between the 0, 1, 2, and 4 h
dispersal times).
The increase in total ADH is not a simple addition of the
Fig. 2. Effect of the post-feeding dispersal period on the accumulated degree
hours spent dispersing. For example to take the extremes in
hours to eclosion in Calliphora vicina. The error bars are 1.96 S.D. from the
mean.
Fig. 2, between 1 and 24 h dispersal time, the difference is 23 h,
S. Arnott, B. Turner / Forensic Science International 177 (2008) 162 167 165
Fig. 3. Effect of time spent in larval dispersal on the final adult size, as
measured by the d-cu crossvein, in Calliphora vicina. The error bars are 1.96
Fig. 5. Effect of disruption to development by enforced dispersal in the 2nd
S.D. from the mean.
instar stage on Calliphora vicina adult size. The error bars are 1.96 S.D. from
the mean.
development programme. It is only the inclusion of the seven
larvae that pupated after 96 h that gives an indication that the 2.8, n.s.). Without cleaning the arena between larvae there was a
ADH to adult eclosion is reduced by this interruption in the 2nd distinct attraction to the quadrant to which the first larva
instar stage. The impact of this disruption is more clearly travelled (Chi-square = 17.1, p<0.001).
indicated in Fig. 5, which shows a significant decrease in adult Where a previously generated trail was present, made by
size with increased time away from the food source in the 2nd confining a number of larvae between two temporary walls of
instar stage (F = 3.97, p<0.003). Again the ANOVA is aluminium foil across the arena, then the experimental larvae
strongly influenced by the small size of the 96 h cohort of placed in the centre of the arena showed a significant attraction
adults, with Fisher s PLSD post hoc test indicating no to the pre-laid trail compared with being  off trail (Chi-
significant size difference between pair-wise comparisons of square = 11.1, p<0.001).
the adults from other groups.
The simple behavioural observations (experiments 3 and 4) 4. Discussion
were designed to see whether an individual larva s dispersal is
influenced by other larvae, by reacting to any chemical trails The estimation of the post mortem interval is frequently
that might be present. Such behaviours on the one hand might calculated using ADH or ADD methods. In passing it might be
reduce the time spent in locating suitable pupariation areas but observed that the use of the former is to be preferred over the
conversely would lead to very high densities of pupae in latter because of its greater sensitivity to diurnal temperature
particular areas with possible predation/parasitisation con- fluctuations. ADH calculations are invariably calculated for a
sequences. specific species from laboratory studies and these constrained
Where the larvae were individually started in the centre of and simplified conditions often do not reflect features that are
the arena and the surface was cleaned between each larval run common in the real world. Ames and Turner [6] showed the
the larvae took off in random directions and there was no non-intuitive effect of short cold episodes (which would also
significant attraction to one quadrant or another (Chi-square = include the cold storage of larvae recovered from a crime scene
prior to being sent or examined by an entomologist) on ADH
calculations and this present study considers another area where
there is a potentially frequent difference in the laboratory and
field conditions, namely the ready availability of a suitable
pupariation site very close to the corpse.
Calculations of adult emergence ADH, based on laboratory
studies, do not normally consider the post-feeding larval
dispersal time separately. It is usually included in the total
larval ADH (as for example in Table 1 in [6]), and in any case
the dispersal time is usually short as the larvae leave the food
and burrow into the adjacently provided pupariation medium.
This present study suggests several areas that need to be
considered in ADH calculations. The length of the post-feeding
dispersal stage is important not only for the time it takes, but
also for its impact on the following pupal development stage.
No single value can be applied as the dispersal time will depend
Fig. 4. Impact on the total ADH to eclosion in Calliphora vicina of the removal
on the dispersal behaviour of the specific blowfly species and
of 2nd instar larvae from their food for differing periods of time. The error bars
are 1.96 S.D. from the mean.
the terrain where the corpse was positioned. This study has
166 S. Arnott, B. Turner / Forensic Science International 177 (2008) 162 167
highlighted an area of the blowfly lifecycle that can introduce Acknowledgements
errors into the use of insect development beyond the larval stage
to estimate PMI. Further studies are needed for each of the main We thank Carole Ames for her input to this study. We would
species to relate post-feeding dispersal to both temperature and also like to thank the two anonymous reviewers of the initial
terrain so as to provide a more accurate estimate of the actual manuscript for their valued comments. This study was carried
ADH and therefore PMI. out by S.A. in partial fulfilment of her M.Sc. in Forensic
The experimental arena was of smooth plastic and whilst this Science at King s College London.
might be similar to vinyl or polished wooden floors it is quite
different to the rougher textures of carpeting or out of doors
References
surfaces. In these latter cases it might be expected that greater
energy expenditure would be needed by larvae to cover a
[1] P. Nuorteva, Sarcosaprophagous insects as forensic indicators, in: C.G.
particular distance and that progress generally would be Tedeschi (Ed.), Forensic Medicine a Study in Trauma and Environmental
Hazards, vol. 3, Saunder, Philadelphia, 1977, pp. 1072 1095.
slowed.
[2] R.C. Bassanezi, M.B.F. Leite, W.A.C. Godoy, C.J. Von Zuben, F.J. Von
Pupal duration, in individuals that had a long post-feeding
Zuben, S.F. Dosreis, Diffusion model applied to post-feeding larval
dispersal is lengthened relative to those with a short post-
dispersal in blowflies (Diptera Calliphoridae), Memorias Do Instituto
feeding dispersal period; and the resultant adults are reduced in
Oswaldo Cruz 92 (1997) 281 286.
size. This becomes a significant feature if the post-feeding [3] A. Gomes, C.J. Von Zuben, Postfeeding radial dispersal in larvae of
Chrysomya albiceps (Diptera; Calliphoridae): implications for forensic
larval dispersal is longer than 5 h and is likely to be the result of
entomology, Forensic Sci. Int. 155 (2005) 61 64.
the loss of resources during the dispersal period. This finding
[4] J.W. Tessmer, C.L. Meek, Dispersal and distribution of Calliphoridae
might not be of any relevance to scenes of crime in moist
(Diptera) immatures from animal carcasses in Southern Louisiana, J. Med.
woodlands but if the soils are hard and dried out, or
Entomol. 33 (1996) 665 669.
alternatively the scene of crime is indoors, then the larvae [5] C.J. von Zuben, R.C. Bassanezi, S.F. dosReis, W.A.C. Godoy, F.J. von
Zuben, Theoretical approaches to forensic entomology.1. Mathematical
may well spend considerable periods of time, counted in days,
model of postfeeding larval dispersal, J. Appl. Entomol.-Zeitschrift Fur
finding somewhere to pupate. Robinson [11] gives figures of 5
Angewandte Entomologie 120 (1996) 379 382.
14 days for the post-feeding dispersal period for C. vicina
[6] C. Ames, B. Turner, Low temperature episodes in the development of
which would add very considerable error to the PMI
blowflies: implications for postmortem interval estimation, Med. Vet.
calculations. Long post-feeding dispersal uses resources that Entomol. 17 (2003) 178 186.
[7] L.G. Higley, N.H. Haskell, Insect development and forensic entomology,
would otherwise have produced normal sized adults. Wild
in: J.H. Byrd, J.L. Castner (Eds.), Forensic Entomology: the utility of
caught C. vicina do show a considerable range in adult size,
arthropods in legal investigations, CRC Press, Boca Raton, 2001.
compared with the much more constant C. vomitoria. Further
[8] A.A. Green, The control of blowflies infesting slaughterhouses I. Field
experimentation is needed before adult size in C. vicina can be
observations on the habits of blowflies, Ann. Appl. Biol. 38 (1951) 475 494.
related to dispersal times or competition or partitioned between [9] C.-C. Hwang, B. Turner, Spatial and temporal variability of necrophagous
Diptera from urban to rural areas, Med. Vet. Entomol. 19 (2005) 379 391.
them.
[10] S. Ireland, B. Turner, The effects of crowding and food type on the size and
Previous studies have suggested that larvae dispersing from
development of Calliphora vomitoria, Forensic Sci. Int. 159 (2006) 175
a food source is random [3] and the simple experiments
181.
described here support this for individual C. vicina larvae.
[11] W.H. Robinson, Urban Insects and Arachnids A Handbook of Urban
However, this does not hold for sequentially leaving larvae; Entomology, CUP, Cambridge, 2005.
[12] M.P. Hassell, The Dynamics of Predator Prey Systems, Princeton Uni-
later ones are strongly influenced by the trails of those that have
versity Press, New Jersey, 1977.
gone before them. The evolutionary value in following a trail is
[13] R.J Taylor, Predation, Chapman & Hall, London, 1984.
not clear since the trail of one larva may not lead to a good
[14] Y.Z. Erzinclioglu, Blowflies, Richmond Publishing Co. Ltd., Slough,
pupariation site. Such behaviour is seen in larvae dispersing in
1996.
huge numbers from human corpses at the Anthropological [15] C.N. Coyler, C.O. Hammond, Flies of the British Isles, Fredericke Warne
& Co. Ltd., London, 1968.
facility, Tennessee (Amoret Whitaker, personal communica-
[16] E.N. Richards, M.L. Goff, Arthropod succession on exposed carrion in
tion) and leads to areas of soil with very high densities of
three contrasting tropical habitats on Hawaii island, Hawaii, J. Med.
puparia. Such high densities may increase the chance of
Entomol. 34 (1997) 328 339.
survival although it is well known that high prey density attracts
[17] W.C. Rodriguez, W.M. Bass, Insect activity and its relationship to decay
high numbers of predators and parasitoids [12,13]. rates of human cadavers in East Tennessee, J. Forensic Sci. 28 (1983) 423
432.
Differences between the observed and expected ADH values
[18] T.I. Tantawi, E.M. Elkady, B. Greenberg, H.A. ElGhaffar, Arthropod
(from Fig. 2) indicate that considerable errors may be
succession on exposed rabbit carrion in Alexandria, Egypt, J. Med.
introduced by a variable post-feeding dispersal period when
Entomol. 33 (1996) 566 580.
using this method to estimate PMI. Assuming the constant
[19] B.V. Travis, E.F. Knipling, A.L. Brody, Lateral migration and depth of
20 8C conditions of the growth of these experimental pupation of the larvae of the primary screw-worm Cochliomyia amer-
icana, J. Econ. Entomol. 33 (1940) 847 857.
calliphorids, a migration lasting 6 h will give a total ADH
[20] K.G.V. Smith, An introduction to the immature stages of British Flies,
that is would over-state the PMI estimate by 1.25 days, an 8 h
Handbooks for the Identification of British Insects, vol. 10, Royal
dispersal time overstates by 1.5 days, and a 24 h dispersal by 2.1
Entomological Society of London, 1989, pp. 1 280.
days. These experiments were not run for the extended period
[21] J.B. Cragg, The natural history of sheep blowflies in Britain, Ann. Appl.
of 5 14 days quoted by Robinson [11] for C. vicina dispersal. Biol. 42 (1955) 197 207.
S. Arnott, B. Turner / Forensic Science International 177 (2008) 162 167 167
[22] K.G.V. Smith, Insects and Other Arthropods of Medical Importance, [24] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 (1991)
British Museum of Natural History, London, 1973. 565 577.
[23] B. Greenberg, Behavior of postfeeding larvae of some Calliphoridae [25] K. Tullis, M.L. Goff, Arthropod succession in exposed carrion in a
and a muscid (Diptera), Ann. Entomol. Soc. Am. 83 (1990) 1210 tropical rainforest on O ahu Island, Hawaii, J. Med. Entomol. 24
1214. (1987) 332 339.