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

In the blowfly lifecycle (from egg, through three feeding

larval stages, a pupal and finally the adult stage) there is an
important late larval period when feeding ceases and the so
called post-feeding larvae move away from the corpse on which
they have been feeding to find a suitable site for pupariation. A
number of previously published forensic entomology studies
have recorded details of the length of time or the distance
travelled during the post-feeding phase of larval blowflies.
These are summarised in

Table 1

. Often precise details are

lacking, as to, for instance, which species the measurements
relate to, and in others the account is primarily descriptive.
What is clear is that some blowfly species (e.g. Protophormia
terraenovae, Fannia sp. Chrysomya rufifacies and Chrysomya
albiceps, listed towards the top of the table) appear to spend less
time dispersing since they remain close to or on the larval food
source, whilst others (eg. Lucilia and Calliphora spp.) appear to

have a longer dispersal phase. Nuorteva

[1]

provides an

interesting link between distance and time by noting that
Lucilia sericata in Finland travels at the slow rate of about 1 m
per day over a moss covered forest floor, but in most other cases
there is no link available between distance and time, or
importantly with temperature.

Some studies, specifically on this post-feeding larval

dispersal phase

[2–5]

, have emphasised the patterns and spatial

distribution of larval movement. Whilst this is of considerable
use in forensic entomology, particularly in identifying the most
probable areas to look for puparia in relation to a corpse, only
Gomes and Von Zuben

[3]

touch on the relevance of the

dispersal stage to the estimation of the post-mortem interval
(PMI). They show that dispersal puts an energetic cost on the
post-feeding larvae which causes a reduction in pupal weight
with increasing distance dispersed.

One common, but by no means foolproof method (see for

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

established in the laboratory where, in controlled conditions, it

www.elsevier.com/locate/forsciint

Available online at www.sciencedirect.com

Forensic Science International 177 (2008) 162–167

* Corresponding author.

E-mail address:

[e-mail]

(B. Turner).

0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:

10.1016/j.forsciint.2007.12.002

is usual that a suitable pupariation medium (sawdust, soiless
compost or similar) is readily available and adjacent to the
feeding larvae. The time spent in dispersal is therefore normally
very short in laboratory settings and, in the overall calculation
of ADH, usually ignored.

This contrasts with situations in the field at least for some

species. Post-feeding larvae may stay on the corpse or find
suitable pupariation substrates very close to the corpse (for
example if the corpse is in an area of well drained friable soil) or
they may have to travel many metres if the ground is hard and
unyielding, as has been observed in the post-feeding disperal of
larvae from a corpse at the Anthropological Facility in
Tennessee (Amoret Whitaker, personal communication) and
noted by Green

[8]

Thus, in situations where pupariation is

delayed until a suitable site has been found, there can be marked
differences in ADH estimates based on pupal and adult stages
between field and laboratory material. During long post-
feeding dispersal periods time passes (and therefore degree-
hours accumulate) and energy is used up, leading to a reduction
in resources available for the pupa and adult stages.

This paper focuses on the impact of differing dispersal

periods for larvae (both at the normal post-feeding time and
also at an earlier stage of growth) on the further development
with particular reference to the levels of error that such
situations might create in PMI estimates using ADH methods.
The experimental blowfly species used in this study was the
blue bottle Calliphora vicina Robinseau-Desvoidy, a forensi-
cally important and widespread species of urban areas in
temperate regions.

2. Methods

2.1. General

Wild C. vicina adults were trapped at several sites in London using bottle

traps (see

[9]

, for the design) baited with pig’s liver and 30% sodium sulphide

solution. These blowflies were cultured in cages and provided with sugar and
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
were then grown on pig’s liver in small chambers at 20 8C with an excess of food
to avoid competition effects

[10]

, to provide a source of post-feeding larvae for

the experiments.

The experiments made use of the lid of a large plastic box as the arena

(

Fig. 1

).

The lid has a large peripheral indented groove, which normally fits the rolled

top of the plastic box on which it fits. When the lid is placed upside down the
groove forms a continuous track 25 mm wide by 37 mm deep. One circuit round
the lid is exactly 2 m although the number of circuits made by larvae was not
recorded. Larvae introduced into the track continued to travel around it and did
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

for, but was held by the building’s air conditioning system at approximately
50%rh. Following the specified time in the arena the larvae were returned to a
constant 20 8C and given moist peat to pupate in.

2.1.1. Experiment 1

This experiment explored the effects of differing lengths of time spent in

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
source. These post-feeding larvae were ‘run’ in the trackway in small groups for
1, 2, 4, 6, 8, or 24 h before being placed in plastic boxes containing damp soil-
less compost as a pupariation medium and returned to the incubators to
complete development. Control individuals (0 h disperal time) were placed
immediately on the compost. The numbers for each experiment, were governed

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

and Phormia regina

>4.6 m

[4]

Calliphorinae

>6 m (>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
and fully grown maggots may be found wandering in quite unlikely situations some distance from
their larval food’’

[22]

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]

S. Arnott, B. Turner / Forensic Science International 177 (2008) 162–167

163

by the numbers of fresh post-feeding larvae available from the cultures Between
60 and 87 individual post-feeding larvae were ‘run’ for each time period.

The ADH to eclosion was recorded for all flies in the experiment.

2.1.2. Experiment 2

The second experiment was similar to the first but used 2nd instar feeding

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
occurrence which breaks the feeding cycle. Larvae for this experiment were
collected from the cultures as 2nd instars. They were ‘run’ in the trackway for 1,
2, 4, 6, 16, and 48 h. They were then returned to their food supply and followed
through to adult emergence. In a further experiment 100 2nd instar larvae were
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.

2.1.3. Experiment 3

The behaviour of larvae when dispersing, in particular whether they follow

trails left by previous individuals, was examined using the flat central area of the
plastic lid which was marked out into 4 quadrants by two lines drawn at right
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
a paper template of the arena. Where they dropped into the peripheral groove
was recorded. Between each larva the plastic lid was cleaned with an alcohol
wipe and left to dry. It was also randomly rotated to obviate any directional
effects that might have been induced by slightly non-uniform overhead lighting.
Ninety larvae were tested in this way.

This was then repeated but without the alcohol washing between each larva.

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

hours spent dispersing. For example to take the extremes in

Fig. 2

, between 1 and 24 h dispersal time, the difference is 23 h,

which at 20 8C is 460ADH. Whereas the difference in total
ADH to eclosion, seen in

Fig. 2

(9777ADH for a dispersal

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
simple addition.

Time spent dispersing as larvae clearly uses resources that

would otherwise be available for pupal development and so it is
not surprising that adult size, as measured by the d-cu cross vein
in the wing (see

[10]

, for details of method), is reduced with

longer post-feeding larval dispersal time (

Fig. 3

) (F = 14.62,

p

< 0.001).

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
food to develop as normal with no further dispersal delays. In
addition, 100 2nd instar larvae were left to circulate in the
arena, simulating total and final removal from the food source.
Only 7 survived to 96 h and these seven all pupated, having
moulted into 3rd instars just prior to pupariation. These 7
individuals have been added to the data for the 2nd instar larvae
starved for up to 48 h, and plotted in

Fig. 4

. Overall there is a

significant reduction in ADH with time spent off the food
(F = 37.02, p < 0.001) but, using Fishers PLSD post hoc test
indicates that there was no significant effect on the ADH to
eclosion in the larvae with up to a 48 h delay in their

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.

Fig. 2. Effect of the post-feeding dispersal period on the accumulated degree
hours to eclosion in Calliphora vicina. The error bars are

1.96 S.D. from the

mean.

S. Arnott, B. Turner / Forensic Science International 177 (2008) 162–167

164

development programme. It is only the inclusion of the seven
larvae that pupated after 96 h that gives an indication that the
ADH to adult eclosion is reduced by this interruption in the 2nd
instar stage. The impact of this disruption is more clearly
indicated in

Fig. 5

, which shows a significant decrease in adult

size with increased time away from the food source in the 2nd
instar stage (F = 3.97, p < 0.003). Again the ANOVA is
strongly influenced by the small size of the 96 h cohort of
adults, with Fisher’s PLSD post hoc test indicating no
significant size difference between pair-wise comparisons of
the adults from other groups.

The simple behavioural observations (experiments 3 and 4)

were designed to see whether an individual larva’s dispersal is
influenced by other larvae, by reacting to any chemical trails
that might be present. Such behaviours on the one hand might
reduce the time spent in locating suitable pupariation areas but
conversely would lead to very high densities of pupae in
particular areas with possible predation/parasitisation con-
sequences.

Where the larvae were individually started in the centre of

the arena and the surface was cleaned between each larval run
the larvae took off in random directions and there was no
significant attraction to one quadrant or another (Chi-square =

2.8, n.s.). Without cleaning the arena between larvae there was a
distinct attraction to the quadrant to which the first larva
travelled (Chi-square = 17.1, p < 0.001).

Where a previously generated trail was present, made by

confining a number of larvae between two temporary walls of
aluminium foil across the arena, then the experimental larvae
placed in the centre of the arena showed a significant attraction
to the pre-laid trail compared with being ‘off’ trail (Chi-
square = 11.1, p < 0.001).

4. Discussion

The estimation of the post mortem interval is frequently

calculated using ADH or ADD methods. In passing it might be
observed that the use of the former is to be preferred over the
latter because of its greater sensitivity to diurnal temperature
fluctuations. ADH calculations are invariably calculated for a
specific species from laboratory studies and these constrained
and simplified conditions often do not reflect features that are
common in the real world. Ames and Turner

[6]

showed the

non-intuitive effect of short cold episodes (which would also
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
on the dispersal behaviour of the specific blowfly species and
the terrain where the corpse was positioned. This study has

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

S.D. from the mean.

Fig. 4. Impact on the total ADH to eclosion in Calliphora vicina of the removal
of 2nd instar larvae from their food for differing periods of time. The error bars
are

1.96 S.D. from the mean.

Fig. 5. Effect of disruption to development by enforced dispersal in the 2nd
instar stage on Calliphora vicina adult size. The error bars are

1.96 S.D. from

the mean.

S. Arnott, B. Turner / Forensic Science International 177 (2008) 162–167

165

highlighted an area of the blowfly lifecycle that can introduce
errors into the use of insect development beyond the larval stage
to estimate PMI. Further studies are needed for each of the main
species to relate post-feeding dispersal to both temperature and
terrain so as to provide a more accurate estimate of the actual
ADH and therefore PMI.

The experimental arena was of smooth plastic and whilst this

might be similar to vinyl or polished wooden floors it is quite
different to the rougher textures of carpeting or out of doors
surfaces. In these latter cases it might be expected that greater
energy expenditure would be needed by larvae to cover a
particular distance and that progress generally would be
slowed.

Pupal duration, in individuals that had a long post-feeding

dispersal is lengthened relative to those with a short post-
feeding dispersal period; and the resultant adults are reduced in
size. This becomes a significant feature if the post-feeding
larval dispersal is longer than 5 h and is likely to be the result of
the loss of resources during the dispersal period. This finding
might not be of any relevance to scenes of crime in moist
woodlands but if the soils are hard and dried out, or
alternatively the scene of crime is indoors, then the larvae
may well spend considerable periods of time, counted in days,
finding somewhere to pupate. Robinson

[11]

gives figures of 5–

14 days for the post-feeding dispersal period for C. vicina
which would add very considerable error to the PMI
calculations. Long post-feeding dispersal uses resources that
would otherwise have produced normal sized adults. Wild
caught C. vicina do show a considerable range in adult size,
compared with the much more constant C. vomitoria. Further
experimentation is needed before adult size in C. vicina can be
related to dispersal times or competition or partitioned between
them.

Previous studies have suggested that larvae dispersing from

a food source is random

[3]

and the simple experiments

described here support this for individual C. vicina larvae.
However, this does not hold for sequentially leaving larvae;
later ones are strongly influenced by the trails of those that have
gone before them. The evolutionary value in following a trail is
not clear since the trail of one larva may not lead to a good
pupariation site. Such behaviour is seen in larvae dispersing in
huge numbers from human corpses at the Anthropological
facility, Tennessee (Amoret Whitaker, personal communica-
tion) and leads to areas of soil with very high densities of
puparia. Such high densities may increase the chance of
survival although it is well known that high prey density attracts
high numbers of predators and parasitoids

[12,13]

.

Differences between the observed and expected ADH values

(from

Fig. 2

) indicate that considerable errors may be

introduced by a variable post-feeding dispersal period when
using this method to estimate PMI. Assuming the constant
20 8C conditions of the growth of these experimental
calliphorids, a migration lasting 6 h will give a total ADH
that is would over-state the PMI estimate by 1.25 days, an 8 h
dispersal time overstates by 1.5 days, and a 24 h dispersal by 2.1
days. These experiments were not run for the extended period
of 5–14 days quoted by Robinson

[11]

for C. vicina dispersal.

Acknowledgements

We thank Carole Ames for her input to this study. We would

also like to thank the two anonymous reviewers of the initial
manuscript for their valued comments. This study was carried
out by S.A. in partial fulfilment of her M.Sc. in Forensic
Science at King’s College London.

References

[1] P. Nuorteva, Sarcosaprophagous insects as forensic indicators, in: C.G.

Tedeschi (Ed.), Forensic Medicine a Study in Trauma and Environmental
Hazards, vol. 3, Saunder, Philadelphia, 1977, pp. 1072–1095.

[2] R.C. Bassanezi, M.B.F. Leite, W.A.C. Godoy, C.J. Von Zuben, F.J. Von

Zuben, S.F. Dosreis, Diffusion model applied to post-feeding larval
dispersal in blowflies (Diptera Calliphoridae), Memorias Do Instituto
Oswaldo Cruz 92 (1997) 281–286.

[3] A. Gomes, C.J. Von Zuben, Postfeeding radial dispersal in larvae of

Chrysomya albiceps (Diptera; Calliphoridae): implications for forensic
entomology, Forensic Sci. Int. 155 (2005) 61–64.

[4] J.W. Tessmer, C.L. Meek, Dispersal and distribution of Calliphoridae

(Diptera) immatures from animal carcasses in Southern Louisiana, J. Med.
Entomol. 33 (1996) 665–669.

[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
model of postfeeding larval dispersal, J. Appl. Entomol.-Zeitschrift Fur
Angewandte Entomologie 120 (1996) 379–382.

[6] C. Ames, B. Turner, Low temperature episodes in the development of

blowflies: implications for postmortem interval estimation, Med. Vet.
Entomol. 17 (2003) 178–186.

[7] L.G. Higley, N.H. Haskell, Insect development and forensic entomology,

in: J.H. Byrd, J.L. Castner (Eds.), Forensic Entomology: the utility of
arthropods in legal investigations, CRC Press, Boca Raton, 2001.

[8] A.A. Green, The control of blowflies infesting slaughterhouses I. Field

observations on the habits of blowflies, Ann. Appl. Biol. 38 (1951) 475–494.

[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.

[10] S. Ireland, B. Turner, The effects of crowding and food type on the size and

development of Calliphora vomitoria, Forensic Sci. Int. 159 (2006) 175–
181.

[11] W.H. Robinson, Urban Insects and Arachnids—A Handbook of Urban

Entomology, CUP, Cambridge, 2005.

[12] M.P. Hassell, The Dynamics of Predator–Prey Systems, Princeton Uni-

versity Press, New Jersey, 1977.

[13] R.J Taylor, Predation, Chapman & Hall, London, 1984.
[14] Y.Z. Erzinclioglu, Blowflies, Richmond Publishing Co. Ltd., Slough,

1996.

[15] C.N. Coyler, C.O. Hammond, Flies of the British Isles, Fredericke Warne

& Co. Ltd., London, 1968.

[16] E.N. Richards, M.L. Goff, Arthropod succession on exposed carrion in

three contrasting tropical habitats on Hawaii island, Hawaii, J. Med.
Entomol. 34 (1997) 328–339.

[17] W.C. Rodriguez, W.M. Bass, Insect activity and its relationship to decay

rates of human cadavers in East Tennessee, J. Forensic Sci. 28 (1983) 423–
432.

[18] T.I. Tantawi, E.M. Elkady, B. Greenberg, H.A. ElGhaffar, Arthropod

succession on exposed rabbit carrion in Alexandria, Egypt, J. Med.
Entomol. 33 (1996) 566–580.

[19] B.V. Travis, E.F. Knipling, A.L. Brody, Lateral migration and depth of

pupation of the larvae of the primary screw-worm Cochliomyia amer-
icana, J. Econ. Entomol. 33 (1940) 847–857.

[20] K.G.V. Smith, An introduction to the immature stages of British Flies,

Handbooks for the Identification of British Insects, vol. 10, Royal
Entomological Society of London, 1989, pp. 1–280.

[21] J.B. Cragg, The natural history of sheep blowflies in Britain, Ann. Appl.

Biol. 42 (1955) 197–207.

S. Arnott, B. Turner / Forensic Science International 177 (2008) 162–167

166

[22] K.G.V. Smith, Insects and Other Arthropods of Medical Importance,

British Museum of Natural History, London, 1973.

[23] B. Greenberg, Behavior of postfeeding larvae of some Calliphoridae

and a muscid (Diptera), Ann. Entomol. Soc. Am. 83 (1990) 1210–
1214.

[24] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 (1991)

565–577.

[25] K. Tullis, M.L. Goff, Arthropod succession in exposed carrion in a

tropical rainforest on O’ahu Island, Hawaii, J. Med. Entomol. 24
(1987) 332–339.

S. Arnott, B. Turner / Forensic Science International 177 (2008) 162–167

167