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Medical and Veterinary Entomology (2003) 17, 178–186

Low temperature episodes in development of blowflies:


implications for postmortem interval estimation
C . A M E S and B . T U R N E R
Department of Life Sciences, King’s College London, U.K.

Abstract. Traditionally the calculation of accumulated degree days or hours


(ADD or ADH) involves the concept of a minimum threshold temperature
below which development ceases. Hence in fluctuating conditions, where tempera-
tures drop below this threshold, there may be periods of time when development is
taken to be zero. This has important implications when the calculation of post-
mortem interval (PMI) is based on the ADD or ADH of larval dipterans. Normal
development of larvae of the blowflies Calliphora vicina Robineau-Desvoidy and
C. vomitoria L. (Diptera: Calliphoridae) at 20 C was interrupted by cold episodes.
The expectation was that total development time would increase by the period at
low (therefore no development) temperature but the total ADD or ADH should
be the same as non-cold treated cohorts. The results, however, showed that total
ADH for both species decreased linearly with increasing temperature with no
evidence of any minimum threshold temperature effect. The increased ADH at
low temperatures may be due to either continued but reduced development or a
delay in development restarting after the cold episode. Use of ADH in PMI
estimations has shortcomings particularly during the winter period where low
temperatures are involved or where there are sudden summer cold spells during
the development period. As blowfly development progresses from egg to pupa
such errors will be compounded.
Key words. Calliphora vicina, Calliphora vomitoria, development threshold tem-
perature, forensic entomology, postmortem interval.

Introduction Northern Europe. In favourable conditions these species lay


their eggs around natural orifices or wounds on the fresh
In forensic science, insect evidence is most commonly used corpse within a short time after death. These hatch to
to estimate the time of death of a corpse (the postmortem larvae, which grow through three stages. During the
interval, PMI). Some of the first insects to invade a corpse latter part of the third stage, larvae stop feeding, migrate
in the U.K. are Calliphoridae (Diptera), including species of from the corpse and burrow down into the soil to pupate.
Calliphora, Protophormia and Lucilia (Lane, 1975). In many The pupal stage has the largest duration before adults
temperate regions Calliphora species are considered emerge.
the most important, with Calliphora vicina (Robineau- Being poikilothermic, the rate of development in insects is
Desvoidy) (¼ C. erythrocephala Meigen) and Calliphora governed by ambient temperature; the higher the tempera-
vomitoria (Linnaeus) being widely distributed throughout ture the faster development occurs. Thus by knowing the
ambient temperature and the progress of blowfly develop-
ment, an estimation of time since the eggs were laid can be
made.
Correspondence: Dr B. D. Turner, Department of Life Sciences, Two methods to estimate PMI using blowfly develop-
King’s College London, Franklin Wilkins Building, 150 Stamford St., ment are available. Larval size, usually length, can be
London SE1 9NN, U.K. E-mail: bryan.turner@kcl.ac.uk compared with data on larval size related to temperature

178 # 2003 The Royal Entomological Society


Low temperature and blowfly development 179

and time in an isomegalendiagram (Grassberger & Reiter, room temperature (20–22 C). Flies were supplied with
2001). There are a number of difficulties with this approach, granulated sugar and water ad libitum. New flies were
including the actual size, the limited number of published added to these populations as the experiment progressed.
isomegalendiagrams and the restriction of this method to Each cage only contained approximately 100 flies to avoid
the larval stages only. The other approach is to measure the overcrowding (Saunders, 1997).
accumulated degree days or hours (ADD or ADH) needed A few days before eggs were required, liquid liver exudate
to reach a particular stage of development. Accumulated was provided as a protein source for the flies. Fresh pigs’
degree days or hours are the product of temperature above liver was then introduced as an oviposition and larval food
a species’ (and possibly geographical population) minimum source. Females massed upon the liver and oviposition
developmental threshold and the time spent at that would normally begin 30 min to 1 h later. The time of
temperature. Prediction of insect development focuses on oviposition was noted and the liver and eggs were removed
the range where the relationship between development rate from the adult cage. Eggs were then separated onto new
and temperature is constant and is based on the work of liver in a disposable weighing boat. To prevent any larval
Abrami (1972), Allen (1976), Arnold (1960), and Baskerville & mass effects which might cause a localized temperature
Emin (1969) (see also Wagner et al., 1984). One difficulty elevation (Vinogradova & Marchenko, 1984 cited in Catts &
with this linear day/hour degree model is that it only truly Goff, 1992; Turner & Howard, 1992), eggs were separated
applies to the thermal range where temperature is directly into small clumps of about two to five and spread over the
proportional to development. The calculated ADD/ADH liver. The liver, which had been kept refrigerated, was
required for development will be too low at temperatures allowed to warm up to room temperature before the eggs
near the lower threshold and too high near the optimum were placed on it. Approximately 30–40 eggs were used in
temperature (Wagner et al., 1984). Additionally, although it each experimental replicate.
is accepted that development ceases at low temperatures The weighing boats, containing liver and eggs, were
(Laudien, 1973 cited in Lamb et al., 1984), the lower placed on a 2-cm layer of soil-less compost in individual
threshold temperature is often very difficult to determine plastic containers (7  8  11 cm) with a plastic lid. The
accurately because insects survive for long periods with near compost provided a medium in which the post-feeding
zero development. The Development Threshold Tempera- larvae could pupate. The liver was changed regularly so
ture (DTT) is most commonly estimated by measuring that food for the larvae was always in excess, to avoid
developmental rates at a range of temperatures and fitting competition. The containers were then kept at 20 C (Cooled
a regression line to the results. This can then be extrapolated Incubator, LMS, Sevenoaks, Kent, U.K.) and a second
to the x-axis where development is zero. incubator (Cooled Incubator, Sanyo-Gallenkamp, U.K.)
According to Higley & Haskell (2001) who reworked was used when needed for the alternative temperature.
Kamal’s data (Kamal, 1958), the DTT for C. vicina and The laboratory was air-conditioned at 20 C  1 C so
C. vomitoria is 6 C, yet recent studies suggest this varies that the removal of larvae from the 20 C incubator for
with locality (Saunders & Hayward, 1998). The majority of measurement did not vary their temperature regime. The
past research on the effects of temperature on development second incubator (monitored using a Tiny Tag, Gemini data
on species in the Calliphoridae has been done at tempera- loggers, Chichester, U.K.) returned to the set temperature
tures close to the optimum, mainly over 20 C (Dallwitz, within 10 min of opening and closing the door to put in the
1984; Greenberg & Tantawi, 1993; Anderson, 2000). experimental larvae and was then left unopened until the
Vinogradova & Marchenko (1984), however, did include end of the exposure period. Larvae were kept in continuous
lower temperatures. In Britain and Northern Europe, dark. Statistical analyses were carried out using the SAS
temperatures for the greater part of the year are relatively package STATVIEW v. 5.01.
low. Calliphora vicina and C. vomitoria are able to develop at The effects of low temperature episodes were explored in
these low temperatures and C. vicina is active through the year. two series of experiments on both blowfly species. Specific
This study reports on a series of laboratory experiments criteria for each series were influenced by previous experi-
that explore the effects of short periods of low temperature mental experience. Development was followed from the egg
on the development of two locally common blowfly species stage through to adult eclosion.
with particular reference to ADD/ADH estimation. A recent
study (Myskowiak & Doums, 2002), considering the impact
of the practice of refrigeration of insect evidential material Series 1 – Low temperature period at different development
(using Protophormia terraenovae Robineau-Desvoidy) to stages
temporarily halt development until passed to a forensic
entomologist, is complementary to this present study. Egg development took place at 20 C. Individual cohorts
of larvae were then subjected to cold episodes, of 5 C for
5 days, at one of the following developmental stages – larval
Materials and methods stages one, two or three (feeding) or the pupal stage.
Following the cold episode the blowflies were returned to
Wild populations of C. vicina and C. vomitoria were raised 20 C for the remainder of their development. Controls
and maintained in gauze covered cages (22  38  27 cm) at remained at 20 C for the entire developmental duration.

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
180 C. Ames and B. Turner

Series 2 – Differing low temperatures for a constant time degree hours to move from stage to stage. Therefore,
period regardless of where the cold episode occurred, whilst the
developmental time will be longer (to make up the deficit of
All experiments were kept at 20 C for 3 days following 1800 ADH), the ADH value to reach a specific stage should
oviposition. By this time the larvae were late second stage. not be significantly different from the corresponding con-
Cohorts of these larvae were placed at 1 , 3 , 5 or 10 C for trol ADH value.
5 days, after which they were returned to 20 C for the This was not the case for the ‘post cold episode’ stages of
remainder of development. Again, controls remained at either species (Fig. 2). Bartlett’s test for homogeneity of
20 C for the entire developmental duration. variances among treatments indicated that the group vari-
Three replicate cohorts, each starting with approximately ances were not equal for either blowfly species (C. vicina
30 eggs, were individually followed in each treatment within F ¼ 8.3, P < 0.001; C. vomitoria F ¼ 10.1, P < 0.001). As this
an experimental series. Following oviposition, each cohort renders the standard one-way ANOVA inappropriate, Welch’s
of eggs was checked half hourly, until all were hatched. one-way ANOVA test for data containing groups with
Subsequently they were observed every 2 h between unequal variances was used instead. This showed that
10.00 hours and 18.00 hours each day. Different starting there are significant differences between the treatments for
times were used to co-ordinate hatching and metamorphosis both species (C. vicina W ¼ 102.5, P < 0.001; C. vomitoria
events with the daytime observational periods. Immature W ¼ 46.5, P < 0.001). This is seen in Fig. 2 by comparing the
stages were examined under dissecting microscope to deter- Control ADH values with the treatment ADH values where
mine developmental stage. On pupariation, the puparia there is a difference of approximately 600 ADH, suggesting
were removed from within the compost and placed upon that no development took place in either species during the
the surface. As the adults emerged, they were counted and 5 C period: that is the DTT is around 5 C.
removed from the containers. During all experiments, the The data were reanalysed using 5 C as the DTT. Welch’s
time in hours since oviposition when the insects progressed test, applied to the recalculated ADH values in each treat-
from one stage to another was recorded. ment, showed that for both species there were significant
To calculate the ADH from the developmental time the differences between ADH values (C. vicina W ¼ 3.2,
following formulae was used: P ¼ 0.016; C. vomitoria W ¼ 2.7, P ¼ 0.033). For C. vicina,
the developmental stage at which the cold episode occurs
ADH ¼ {[development time (h)  time in cold episode appears important. Scheffé’s post hoc test (Table 2) shows a
(h)]  20} þ [time in cold episode  temperature significant difference only between the 1st stage larva and
of cold episode ( C)]. pupal treatments, but low P-values between the 1st stage
larval treatment and all the others for C. vicina.
As a result of the uncertainty of the concept of a DTT, This effect is not seen in C. vomitoria. Scheffé’s test shows
initially no correction for this was applied to any of the data no significant differences between the treatments in this
for either species. species. The ADH values to reach adult emergence are all
similar to each other (P-values all > 0.1) regardless of when
the 5-day cold episode occurs in the developmental period.
Results

Development at 20 C – the controls Experimental series 2

Each of the experimental series included a set of controls As the temperature of the 5-day cold episode increased,
at 20 C. The duration and cumulated values for the control the development time (in hours) to reach the adult stage for
data have been combined in Table 1. At 20 C, the timings both species decreased linearly (Fig. 3). Both slopes are
for the egg stage and 1st and 2nd stage larvae are quite significantly different from zero (Fig. 3). There was an
similar for C. vicina and C. vomitoria. The 3rd stage larvae increase of approximately 8 h on the development time for
period for C. vomitoria takes about 50% more time than for each  C decrease in the 5-day cold episode.
C. vicina, whilst the pupal stage is some 12% shorter If the DTT is 5 C (based on Experimental series 1), we
(Fig. 1). Thus overall the total development time for would expect there to be no significant difference in the
C. vomitoria is 34 h longer than C. vicina, 683 more ADH, developmental times for the 1, 3 and 5 C data. However,
assuming both have a DTT of 0 C. a significant difference between the development times for
each temperature was seen using Welch’s test on the data
sets of each species (C. vicina W ¼ 2027.2, P < 0.001;
Experimental series 1 C. vomitoria W ¼ 1529.4, P < 0.001). Using Scheffé’s multiple
comparison test on these data indicated significant differ-
Excluding the controls, each treatment had a 5-day period ences (P < 0.001) between all temperature combinations in
at 20 C (¼ 2400 ADH) replaced by a 5-day cold episode of both species. This appears to be at variance with the find-
5 C (¼ 600 ADH). The linear hour-degree model suggests ings of the first experimental series (although this was
that insects need to have accumulated a specific number of limited to only one temperature for the cold period) and

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
#
Table 1. Mean duration, standard deviation, range and accumulated degree hours of the egg, larval and pupal stages of Calliphora vicina and Calliphora vomitoria at 20 C.

C. vicina C. vomitoria

Mean Mean Mean Mean


duration cumulative cumulative cumulative duration cumulative Cumulative Cumulative ANOVA of
of stage duration duration duration of stage duration duration duration ADH C. vicina vs.
Stage n (h) (h) SD min–max ADH n (h) (h) SD min–max ADH C. vomitoria
Egg 160 24.7 24.7 1.3 21.8–22.8 493 113 23.6 23.6 1.2 20.5–26.3 472 **
Larval 1 97 17.9 42.5 4.9 36.3–50.5 851 150 15.5 39.2 4.2 35.0–47.5 783 **
Larval 2 104 44.1 86.6 5.7 74.0–119.0 1733 103 47.2 86.4 5.8 70.0–96.5 1728 P ¼ 0.74
Larval 3 140 123.4 210.0 12.9 179.0–252.8 4200 70 195.5 281.9 13.1 269.0–335.0 5638 **
Pupae 106 328.5 538.4 12.5 501.0–563.0 10769 111 290.7 572.6 18.1 540.0–636.0 11452 **

**Significant at 1% significance level.

2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
Low temperature and blowfly development
181
182 C. Ames and B. Turner

C. vicina a)

24.65 17.89
44.1 12000

ADH
11000
123.36
328.45
10000

Pupal
Control

Larval 1

Larval 2

Larval 3
Stage cold episode received

C. vomitoria b)

23.62 15.53
47.23 13000

ADH
195.5 12000

290.73 egg stage


lar va 1
11000
lar va 2
lar va 3

Pupal
Control

Larval 1

Larval 2

Larval 3
pupa

Fig. 1. Comparison of relative proportions of the egg, larval and Stage cold episode received
pupal stages to the total developmental time in Calliphora. vicina
and Calliphora. vomitoria life cycles. Figures in the pie charts are Fig. 2. Boxplots of total ADH against the stage at which cold
the mean duration of each developmental stage in hours at 20 C. episode (5 C/5 days) occurred. (a) Calliphora vicina, (b) C. vomitoria.
*Indicates outliers. The line across the box is the median. The
suggests there is no clear cut minimum development thresh- bottom of the box is at the first quartile (Q1), and the top is at the
old for either blowfly species within the temperature range third quartile (Q3) value. The lines that extend from the top and
bottom of the box show the range of the distribution.
covered in these experiments (1–20 C).
Rather than the expected consistent ADH values, across
the range of temperatures (that is an expectation of gradient (C. vicina W ¼ 130.0, P < 0.001; C. vomitoria W ¼ 325.0,
b ¼ 0), adult ADH values for both species follow a decreas- P < 0.001). Applying Scheffé’s post hoc test to these data
ing linear relationship with increasing temperature for the shows that the ADH values for the 1 C and 3 C treatments
5-day cold episode, which are significantly different from are each significantly different to those of all the other
zero (Fig. 4). temperatures (Table 3).
Using Welch’s test on each species’ ADH and tempera- Whilst statistically the ADH values for 5, 10 and 20 C are not
ture data set (and taking the DTT point to be 5 C) reveals significantly different (mean value for C. vicina ¼ 8057 ADH
that, for both species, there is a significant difference and for C. vomitoria ¼ 8549 ADH), the ADH for the 1 and
between 5-day cold temperature episodes and ADH values 3 C treatments were higher (mean for 1 C cold period,

Table 2. Scheffé’s multiple comparison significance table between total ADHs for differing treatments of cold episodes (5 C/5 days) during
different life-cycle stages for Calliphora vicina (top right) and Calliphora vomitoria (bottom left). Bold indicates significant at the 5% level.

Larva 1 Larva 2 Larva 3 Pupa Control


Larva1 0.050 0.134 0.009 0.088
Larva 2 0.973 0.960 >0.999 0.905
Larva 3 0.789 0.977 0.886 >0.999
Pupa 0.907 0.999 0.996 0.723
Control 0.943 0.417 0.105 0.175

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
Low temperature and blowfly development 183

a) a)
13000
800
12500
600
12000

ADH
y = –8.1251x + 699.17
ADH

11500
400 R2 = 0.8765
11000

200 10500 y = –42.501x + 11583


2
R = 0.3269
10000
0 0 2 4 6 8 10 12 14 16 18 20
0 2 4 6 8 10 12 14 16 18 20
Temperature of 5-day cold episode (°C)
Temperature of 5-day cold episode (°C)
b)
b) 13000
800
12500

600 12000

ADH
y = –8.1224x + 732.35
ADH

400 R2 = 0.9482 11500


y = –42.447x + 12247
2
11000 R = 0.5558

200
10500
0 2 4 6 8 10 12 14 16 18 20
0 Temperature of 5-day cold episode (°C)
0 2 4 6 8 10 12 14 16 18 20
Temperature of 5-day cold episode (°C) Fig. 4. The effect of temperature of a 5-day cold episode on total
ADH. Calliphora vicina: n ¼ 265, t ¼ 11.3, P < 0.001. (b) Calliphora
Fig. 3. The effect of temperature of a 5-day cold episode on duration vomitoria: n ¼ 214, t ¼ 16.3, P < 0.001.
of development to adult. (a) Calliphora vicina: n ¼ 365, t ¼ 43.2,
P < 0.001. (b) Calliphora vomitoria: n ¼ 214, t ¼ 62.3, P < 0.001. DTT value affects the controls at 20 C because the hourly
multiplier is the difference between the DTT and control
temperature. Thus lowering the DTT from 5 to 0 C
C. vicina ¼ 8619 ADH and C. vomitoria ¼ 9084 ADH; mean for increases the hourly multiplier from 15 to 20. Lowering
3 C cold period ¼ 8341 ADH and C. vomitoria ¼ 8823 ADH). the minimum threshold also changes the shape of the rela-
As the DTT value was set at 5 C, the impact of the 5-day tionship and removes the obvious elbow point (interpretable
period at 1 or 3 C should have had no numerical input to as some sort of threshold) seen when the DTT is set at 5 C.
the ADH calculation. However, the data clearly shows that
the development rate outside the cold episodes was affected
by the low temperature experience. The cold episodes below
the DTT had a negative influence, having some unknown Discussion
effect, delaying development, resulting in an increased total
ADH. Davies & Ratcliffe (1994) suggest that ADH values would
The impact of the level set for the DTT on the ADH be expected to be approximately similar for two congeneric
estimate can be seen in Fig. 5. Two things are evident. As a species of similar size, such as C. vicina and C. vomitoria.
result of the mathematics of calculating ADH, changing the The current study found that only the ADH to reach the 3rd

Table 3. Scheffé’s multiple comparison significance table between total ADH for differing treatments of 5-day cold episodes at various
temperatures during the late 2nd larval stage for Calliphora vicina (top right) and Calliphora vomitoria (bottom left). Bold indicates significant
at the 5% level.

1 C 3 C 5 C 10 C 20 C
1 C <0.001 <0.001 <0.001 <0.001
3 C <0.001 <0.001 <0.001 0.004
5 C <0.001 <0.001 0.829 0.903
10 C <0.001 <0.001 0.996 0.440
20 C <0.001 <0.001 0.999 0.983

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
184 C. Ames and B. Turner

a) below 3.5 C as hatching of C. vicina eggs occurred at this


12000 temperature. They also found that adults emerged at 5 C in
11500 their experiment. The authors then went on to suggest that
11000 if the minimum threshold for C. vicina is taken as 2 C, after
10500 Vinogradova & Marchenko (1984; cited in Davies &
10000 DTT 0 °C Ratcliffe, 1994), assuming that C. vicina and C. vomitoria
ADH

9500 DTT 1 °C have equal ADH above the thresholds, that the lower
DTT 3 °C
9000
DTT 5 °C
threshold for C. vomitoria can be estimated as 6 C. These
8500 points are not supported by this study, based on mater-
8000 ial caught in SE England. In their recent paper on the
7500
effects of refrigeration on the development of Protophormia
7000
0 2 4 6 8 10 12 14 16 18 20 terraenovae, Myskowiak & Doums (2002) also showed that
Temperature of 5-day cold episode (°C) development during the 4 C cold period is not totally
b) arrested as is normally assumed by forensic investigators.
13000 The concept of ADH assumes that there is a fixed quan-
12500
tity of metabolic activity, controlled by time and tempera-
12000
11500 ture, that is necessary to complete development. The linear
11000 model assumes that the time/temperature relationship is
10500 DTT 0 °C measured in terms of ADH, where one ADH unit is equal
ADH

10000 DTT 1 °C
to 1 (1  1 h) across all temperatures. Figure 5 shows that,
9500 DTT 3 °C
9000 DTT 5 °C irrespective of what temperature is taken as the DTT, total
8500 ADH increases as a result of 5-day low temperature cold
8000 episodes. This may be interpreted in either of two mechan-
7500
istic ways. Either the value of the ADH unit decreases with
7000
0 2 4 6 8 10 12 14 16 18 20 lower temperatures or the unit remains a constant but
Temperature of 5-day cold episode (°C) development is slowed so that there is an overall increase
in the total ADH required. Either way, if the standard ADH
Fig. 5. The effect of using different development temperature method is used to estimate PMI where there have been
thresholds (DTT) on the total ADH calculations (mean  1.96 SE)
periods of low temperature then it is likely that the PMI
for (a) Calliphora vicina and (b) Calliphora vomitoria for 5-day cold
episodes of various temperatures.
will be underestimated.
The estimation of the developmental threshold tempera-
ture DTT is normally calculated by extrapolating back the
larval stage was statistically similar (Table 1) between the straight line plot of daily developmental rate against
two blowfly species. Davies & Ratcliffe (1994) postulated temperature to the point where it reaches zero (Williams &
that the difference between the development times for Richardson, 1984; Wall et al., 1992; Grassberger & Reiter,
C. vicina and C. vomitoria is due to the development thresh- 2002). This method can be traced back at least to
old temperature for C. vomitoria being higher than that for Wigglesworth (1965), who points out (p. 617) that at low
C. vicina. No difference was seen in the effects of tempera- temperatures the development velocity curve becomes non-
ture on the developmental rates of these two blowfly species linear and curves less steeply ‘so that the ‘‘development
in these experiments. At 20 C, Table 1 and Fig. 1 show that zero’’ is not in fact the true threshold of development; the
whilst overall C. vomitoria had a higher ADH value to reach temperature at which development ceases lies appreciably
the adult stage, this was due to an extended time in the 3rd lower’. This is exactly as seen in Fig. 5 where development
larval stage. Time in the pupal stage for C. vomitoria is either continues during the cold period but at a lower rate,
slightly shorter than in C. vicina but not sufficient to com- or stops completely during the cold period and has some
pensate for the longer third stage larval period. negative effect after the larvae are returned to 20 C: either
Published ADH values for a number of blowfly species way there is an increase in the total ADH for individuals
have been collated by Higley & Haskell (2001). Their values subjected to low temperature episodes.
for C. vicina and C. vomitoria (derived from data from It could be postulated that the increase in development
Kamal, 1958 and Greenberg, 1991) show considerable after the cold interval might be due to a period of diapause.
variability both within their table (9.1) and with the data However, once in diapause, specific stimuli are needed for
presented here. This suggests that the use of generic ADH development to resume. Vinogradova & Zinovjeva (1972)
values is suspect and may well vary on a climatic, locality, studied the factors leading to induction of diapause in
population or even possibly egg batch basis. C. vicina. It appears that diapause is only fully expressed
Kamal (1958) and Greenberg (1991) took 6 C as the in conditions below 15 C. Calliphora vicina larvae then
minimum development threshold temperature for both enter diapause in the pre-pupal state and are characterized
C. vicina and C. vomitoria. Using species obtained from by greatly delayed pupariation. Vinogradova & Zinovjeva
NE England, Davies & Ratcliffe (1994), however, suggested (1972) also showed that the main contributing factor to
that C. vicina’s minimum development threshold must be C. vicina diapause is maternal exposure to short days. In

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186
Low temperature and blowfly development 185

this current study, which took place during the summer species. The whole concept of DTT and its input to the
months, the adult flies experienced long days and therefore ADH calculation needs an urgent review in the light of the
diapause is probably not the cause of increased developmen- findings presented here and those of Myskowiak & Doums
tal times for those insects experiencing 5 C and below in (2002).
larval stage 1. This is supported by the work of Vaz Nunes & The use of ADH is extensively used in PMI estimations
Saunders (1989) who discovered that for C. vicina it was in the and frequently provides results that are of value in forensic
post-feeding stage that temperature was critical for averting investigations. Its use, however, can become misleading
diapause. In this project a cold spell experienced in larval 3 when there are prolonged periods of cold weather. In one
is not significantly different to larval 2 or pupal stages and murder investigation, BDT used the ADH approach to find
therefore diapause is unlikely to be the cause of lengthened a minimum time of death using blowfly pupae from soil
development after a cold episode in larval 1. adjacent to a skeletonized corpse discovered in February in
Johl & Anderson (1996) conducted a series of experi- southern England. The calculation, which involved quite
ments where Canadian C. vicina larvae were chilled at long periods in November and January–February where
different lifestages for 24 h at 3 C. They discovered that the temperature was below 5 C, proved to be in error by
for those larvae chilled in larval stages 1, 2 and 3, develop- about a month. In this case the ADH method underesti-
ment to adult emergence was delayed by about 1 day com- mated the amount of development that had occurred and so
pared to the control. Myskowiak & Doums (2002) showed lengthened the PMI. Based on the ADH calculations, death
highly variable effects of refrigeration on the development was minimally estimated to be in mid September of the
of P. terraenovae, depending on the stage when cooling previous year whereas the murdered woman was in fact
occurred. Paradoxically, they indicated that development still alive a month later in mid October.
time decreased if refrigeration occurred in the first or third
larval stage but it increased if the cold spell was in the
second larval or pupal stages. Acknowledgements
Previous comparisons of temperature effects of different
species have noted differences in development at various We are grateful to Martin Hall for his valued comments on
temperatures and related this to species distribution. Davies an earlier draft of this paper. This work was carried out by
& Ratcliffe (1994) compared C. vicina with C. alpina Ring- CA in partial fulfilment of her MSc degree in Forensic
dahl (a Northern Holarctic species that coexists with Science at King’s College London.
C. vicina in the uplands of Britain) and suggested that
C. vicina was cold-adapted. On this basis, it is possible that
the same species from different places may also have different
References
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(Robineau Desvoidy) (Diptera: Calliphoridae) and its conse- Accepted 21 February 2003

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186

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