low temp blowflies

Low temperature episodes in development of blowflies:implications for postmortem interval estimation

C. AMES and B . TURNERDepartment 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 temperaturebelow which development ceases. Hence in fluctuating conditions, where tempera-tures drop below this threshold, there may be periods of time when development istaken 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. Normaldevelopment of larvae of the blowflies Calliphora vicina Robineau-Desvoidy andC. vomitoria L. (Diptera: Calliphoridae) at 20�C was interrupted by cold episodes.The expectation was that total development time would increase by the period atlow (therefore no development) temperature but the total ADD or ADH shouldbe the same as non-cold treated cohorts. The results, however, showed that totalADH for both species decreased linearly with increasing temperature with noevidence of any minimum threshold temperature effect. The increased ADH atlow temperatures may be due to either continued but reduced development or adelay in development restarting after the cold episode. Use of ADH in PMIestimations has shortcomings particularly during the winter period where lowtemperatures are involved or where there are sudden summer cold spells duringthe development period. As blowfly development progresses from egg to pupasuch errors will be compounded.

Key words. Calliphora vicina, Calliphora vomitoria, development threshold tem-perature, forensic entomology, postmortem interval.

Introduction

In forensic science, insect evidence is most commonly used

to estimate the time of death of a corpse (the postmortem

interval, PMI). Some of the first insects to invade a corpse

in the U.K. are Calliphoridae (Diptera), including species of

Calliphora, Protophormia and Lucilia (Lane, 1975). In many

temperate regions Calliphora species are considered

the most important, with Calliphora vicina (Robineau-

Desvoidy) (¼C. erythrocephala Meigen) and Calliphora

vomitoria (Linnaeus) being widely distributed throughout

Northern Europe. In favourable conditions these species lay

their eggs around natural orifices or wounds on the fresh

corpse within a short time after death. These hatch to

larvae, which grow through three stages. During the

latter part of the third stage, larvae stop feeding, migrate

from the corpse and burrow down into the soil to pupate.

The pupal stage has the largest duration before adults

emerge.

Being poikilothermic, the rate of development in insects is

governed by ambient temperature; the higher the tempera-

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.

Two methods to estimate PMI using blowfly develop-

ment are available. Larval size, usually length, can be

compared with data on larval size related to temperature

Correspondence: Dr B. D. Turner, Department of Life Sciences,

King’s College London, Franklin Wilkins Building, 150 Stamford St.,

London SE1 9NN, U.K. E-mail: [email protected]

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

178 # 2003 The Royal Entomological Society

and time in an isomegalendiagram (Grassberger & Reiter,

2001). There are a number of difficulties with this approach,

including the actual size, the limited number of published

isomegalendiagrams and the restriction of this method to

the larval stages only. The other approach is to measure the

accumulated degree days or hours (ADD or ADH) needed

to reach a particular stage of development. Accumulated

degree days or hours are the product of temperature above

a species’ (and possibly geographical population) minimum

developmental threshold and the time spent at that

temperature. Prediction of insect development focuses on

the range where the relationship between development rate

and temperature is constant and is based on the work of

Abrami (1972), Allen (1976), Arnold (1960), and Baskerville &

Emin (1969) (see also Wagner et al., 1984). One difficulty

with this linear day/hour degree model is that it only truly

applies to the thermal range where temperature is directly

proportional to development. The calculated ADD/ADH

required for development will be too low at temperatures

near the lower threshold and too high near the optimum

temperature (Wagner et al., 1984). Additionally, although it

is accepted that development ceases at low temperatures

(Laudien, 1973 cited in Lamb et al., 1984), the lower

threshold temperature is often very difficult to determine

accurately because insects survive for long periods with near

zero development. The Development Threshold Tempera-

ture (DTT) is most commonly estimated by measuring

developmental rates at a range of temperatures and fitting

a regression line to the results. This can then be extrapolated

to the x-axis where development is zero.

According to Higley & Haskell (2001) who reworked

Kamal’s data (Kamal, 1958), the DTT for C. vicina and

C. vomitoria is 6�C, yet recent studies suggest this varies

with locality (Saunders & Hayward, 1998). The majority of

past research on the effects of temperature on development

on species in the Calliphoridae has been done at tempera-

tures close to the optimum, mainly over 20�C (Dallwitz,

1984; Greenberg & Tantawi, 1993; Anderson, 2000).

Vinogradova & Marchenko (1984), however, did include

lower temperatures. In Britain and Northern Europe,

temperatures for the greater part of the year are relatively

low. Calliphora vicina and C. vomitoria are able to develop at

these low temperatures andC. vicina is active through the year.

This study reports on a series of laboratory experiments

that explore the effects of short periods of low temperature

on the development of two locally common blowfly species

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

(using Protophormia terraenovae Robineau-Desvoidy) to

temporarily halt development until passed to a forensic

entomologist, is complementary to this present study.

Materials and methods

Wild populations of C. vicina and C. vomitoria were raised

and maintained in gauze covered cages (22� 38� 27 cm) at

room temperature (20–22�C). Flies were supplied with

granulated sugar and water ad libitum. New flies were

added to these populations as the experiment progressed.

Each cage only contained approximately 100 flies to avoid

overcrowding (Saunders, 1997).

A few days before eggs were required, liquid liver exudate

was provided as a protein source for the flies. Fresh pigs’

liver was then introduced as an oviposition and larval food

source. Females massed upon the liver and oviposition

would normally begin 30min to 1 h later. The time of

oviposition was noted and the liver and eggs were removed

from the adult cage. Eggs were then separated onto new

liver in a disposable weighing boat. To prevent any larval

mass effects which might cause a localized temperature

elevation (Vinogradova & Marchenko, 1984 cited in Catts &

Goff, 1992; Turner & Howard, 1992), eggs were separated

into small clumps of about two to five and spread over the

liver. The liver, which had been kept refrigerated, was

allowed to warm up to room temperature before the eggs

were placed on it. Approximately 30–40 eggs were used in

each experimental replicate.

The weighing boats, containing liver and eggs, were

placed on a 2-cm layer of soil-less compost in individual

plastic containers (7� 8� 11 cm) with a plastic lid. The

compost provided a medium in which the post-feeding

larvae could pupate. The liver was changed regularly so

that food for the larvae was always in excess, to avoid

competition. The containers were then kept at 20�C (Cooled

Incubator, LMS, Sevenoaks, Kent, U.K.) and a second

incubator (Cooled Incubator, Sanyo-Gallenkamp, U.K.)

was used when needed for the alternative temperature.

The laboratory was air-conditioned at 20�C� 1�C so

that the removal of larvae from the 20�C incubator for

measurement did not vary their temperature regime. The

second incubator (monitored using a Tiny Tag, Gemini data

loggers, Chichester, U.K.) returned to the set temperature

within 10min of opening and closing the door to put in the

experimental larvae and was then left unopened until the

end of the exposure period. Larvae were kept in continuous

dark. Statistical analyses were carried out using the SAS

package STATVIEW v. 5.01.

The effects of low temperature episodes were explored in

two series of experiments on both blowfly species. Specific

criteria for each series were influenced by previous experi-

mental experience. Development was followed from the egg

stage through to adult eclosion.

Series 1 – Low temperature period at different development

stages

Egg development took place at 20�C. Individual cohortsof larvae were then subjected to cold episodes, of 5�C for

5 days, at one of the following developmental stages – larval

stages one, two or three (feeding) or the pupal stage.

Following the cold episode the blowflies were returned to

20�C for the remainder of their development. Controls

remained at 20�C for the entire developmental duration.

Low temperature and blowfly development 179

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

Series 2 – Differing low temperatures for a constant time

period

All experiments were kept at 20�C for 3 days following

oviposition. By this time the larvae were late second stage.

Cohorts of these larvae were placed at 1�, 3�, 5� or 10�C for

5 days, after which they were returned to 20�C for the

remainder of development. Again, controls remained at

20�C for the entire developmental duration.

Three replicate cohorts, each starting with approximately

30 eggs, were individually followed in each treatment within

an experimental series. Following oviposition, each cohort

of eggs was checked half hourly, until all were hatched.

Subsequently they were observed every 2 h between

10.00 hours and 18.00 hours each day. Different starting

times were used to co-ordinate hatching and metamorphosis

events with the daytime observational periods. Immature

stages were examined under dissecting microscope to deter-

mine developmental stage. On pupariation, the puparia

were removed from within the compost and placed upon

the surface. As the adults emerged, they were counted and

removed from the containers. During all experiments, the

time in hours since oviposition when the insects progressed

from one stage to another was recorded.

To calculate the ADH from the developmental time the

following formulae was used:

ADH ¼ {[development time (h) � time in cold episode

(h)] � 20} þ [time in cold episode � temperature

of cold episode (�C)].

As a result of the uncertainty of the concept of a DTT,

initially no correction for this was applied to any of the data

for either species.

Results

Development at 20�C – the controls

Each of the experimental series included a set of controls

at 20�C. The duration and cumulated values for the control

data have been combined in Table 1. At 20�C, the timings

for the egg stage and 1st and 2nd stage larvae are quite

similar for C. vicina and C. vomitoria. The 3rd stage larvae

period for C. vomitoria takes about 50% more time than for

C. vicina, whilst the pupal stage is some 12% shorter

(Fig. 1). Thus overall the total development time for

C. vomitoria is 34 h longer than C. vicina, 683 more ADH,

assuming both have a DTT of 0�C.

Experimental series 1

Excluding the controls, each treatment had a 5-day period

at 20�C (¼ 2400ADH) replaced by a 5-day cold episode of

5�C (¼ 600ADH). The linear hour-degree model suggests

that insects need to have accumulated a specific number of

degree hours to move from stage to stage. Therefore,

regardless of where the cold episode occurred, whilst the

developmental time will be longer (to make up the deficit of

1800ADH), the ADH value to reach a specific stage should

not be significantly different from the corresponding con-

trol ADH value.

This was not the case for the ‘post cold episode’ stages of

either species (Fig. 2). Bartlett’s test for homogeneity of

variances among treatments indicated that the group vari-

ances were not equal for either blowfly species (C. vicina

F¼ 8.3, P< 0.001; C. vomitoria F¼ 10.1, P< 0.001). As this

renders the standard one-way ANOVA inappropriate, Welch’s

one-way ANOVA test for data containing groups with

unequal variances was used instead. This showed that

there are significant differences between the treatments for

both species (C. vicina W¼ 102.5, P< 0.001; C. vomitoria

W¼ 46.5, P< 0.001). This is seen in Fig. 2 by comparing the

Control ADH values with the treatment ADH values where

there is a difference of approximately 600ADH, suggesting

that no development took place in either species during the

5�C period: that is the DTT is around 5�C.The data were reanalysed using 5�C as the DTT. Welch’s

test, applied to the recalculated ADH values in each treat-

ment, showed that for both species there were significant

differences between ADH values (C. vicina W¼ 3.2,

P¼ 0.016; C. vomitoria W¼ 2.7, P¼ 0.033). For C. vicina,

the developmental stage at which the cold episode occurs

appears important. Scheffe’s post hoc test (Table 2) shows a

significant difference only between the 1st stage larva and

pupal treatments, but low P-values between the 1st stage

larval treatment and all the others for C. vicina.

This effect is not seen in C. vomitoria. Scheffe’s test shows

no significant differences between the treatments in this

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.

Experimental series 2

As the temperature of the 5-day cold episode increased,

the development time (in hours) to reach the adult stage for

both species decreased linearly (Fig. 3). Both slopes are

significantly different from zero (Fig. 3). There was an

increase of approximately 8 h on the development time for

each �C decrease in the 5-day cold episode.

If the DTT is 5�C (based on Experimental series 1), we

would expect there to be no significant difference in the

developmental times for the 1, 3 and 5�C data. However,

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 W¼ 2027.2, P< 0.001;

C. vomitoria W¼ 1529.4, P< 0.001). Using Scheffe’s multiple

comparison test on these data indicated significant differ-

ences (P< 0.001) between all temperature combinations in

both species. This appears to be at variance with the find-

ings of the first experimental series (although this was

limited to only one temperature for the cold period) and

180 C. Ames and B. Turner


Tab

le1.Meanduration,standard

deviation,rangeandaccumulateddegreehours

oftheegg,larvalandpupalstages

ofC

allip

hora

vici

naand

Callip

hora

vom

itoriaat20� C

.

C.vi

cina

C.vo

mitoria

Mean

duration

ofstage

Mean

cumulative

duration

cumulative

duration

cumulative

duration

Mean

duration

ofstage

Mean

cumulative

duration

Cumulative

duration

Cumulative

duration

ANOVAof

ADH

C.vi

cinavs.

Stage

n(h)

(h)

SD

min–max

ADH

n(h)

(h)

SD

min–max

ADH

C.vo

mitoria

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

**

Larval1

97

17.9

42.5

4.9

36.3–50.5

851

150

15.5

39.2

4.2

35.0–47.5

783

**

Larval2

104

44.1

86.6

5.7

74.0–119.0

1733

103

47.2

86.4

5.8

70.0–96.5

1728

P¼0.74

Larval3

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

**

**Significantat1%

significance

level.



suggests there is no clear cut minimum development thresh-

old for either blowfly species within the temperature range

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

b¼ 0), adult ADH values for both species follow a decreas-

ing linear relationship with increasing temperature for the

5-day cold episode, which are significantly different from

zero (Fig. 4).

Using Welch’s test on each species’ ADH and tempera-

ture data set (and taking the DTT point to be 5�C) revealsthat, for both species, there is a significant difference

between 5-day cold temperature episodes and ADH values

(C. vicina W¼ 130.0, P< 0.001; C. vomitoria W¼ 325.0,

P< 0.001). Applying Scheffe’s post hoc test to these data

shows that the ADH values for the 1�C and 3�C treatments

are each significantly different to those of all the other

temperatures (Table 3).

Whilst statistically theADHvalues for 5, 10and20�Carenot

significantly different (mean value for C. vicina¼ 8057ADH

and for C. vomitoria¼ 8549ADH), the ADH for the 1 and

3�C treatments were higher (mean for 1�C cold period,

C. vicina

24.65 17.89

44.1

123.36

328.45

C. vomitoria

23.62 15.5347.23

195.5

290.73 egg stagelar va 1lar va 2lar va 3pupa

Fig. 1. Comparison of relative proportions of the egg, larval and

pupal stages to the total developmental time in Calliphora. vicina

and Calliphora. vomitoria life cycles. Figures in the pie charts are

the mean duration of each developmental stage in hours at 20�C.

a)

Pup

al

Larv

al 3

Larv

al 2

Larv

al 1

Con

trol

12000

11000

10000

AD

H

Stage cold episode received

b)

11000

12000

13000

AD

H

Pup

al

Larv

al 3

Larv

al 2

Larv

al 1

Con

trol

Stage cold episode received

Fig. 2. Boxplots of total ADH against the stage at which cold

episode (5�C/5days) occurred. (a) Calliphora vicina, (b) C. vomitoria.

*Indicates outliers. The line across the box is the median. The

bottom of the box is at the first quartile (Q1), and the top is at the

third quartile (Q3) value. The lines that extend from the top and

bottom of the box show the range of the distribution.

Table 2. Scheffe’s multiple comparison significance table between total ADHs for differing treatments of cold episodes (5�C/5 days) duringdifferent 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



C. vicina¼ 8619ADHandC. vomitoria¼ 9084ADH;mean for

3�C cold period¼ 8341ADH and C. vomitoria¼ 8823ADH).

As the DTT value was set at 5�C, the impact of the 5-day

period at 1 or 3�C should have had no numerical input to

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

effect, delaying development, resulting in an increased total

ADH.

The impact of the level set for the DTT on the ADH

estimate can be seen in Fig. 5. Two things are evident. As a

result of the mathematics of calculating ADH, changing the

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�Cincreases the hourly multiplier from 15 to 20. Lowering

the minimum threshold also changes the shape of the rela-

tionship and removes the obvious elbow point (interpretable

as some sort of threshold) seen when the DTT is set at 5�C.

Discussion

Davies & Ratcliffe (1994) suggest that ADH values would

be expected to be approximately similar for two congeneric

species of similar size, such as C. vicina and C. vomitoria.

The current study found that only the ADH to reach the 3rd

a)

y = –8.1251x + 699.17R2 = 0.8765

0

200

400

600

800

20

b)

0

200

400

600

800

0 2 4 6 8 10 12 14 16 18

Temperature of 5-day cold episode (°C)

200 2 4 6 8 10 12 14 16 18


y = –8.1224x + 732.35R2 = 0.9482

AD

HA

DH

Fig. 3. The effect of temperature of a 5-day cold episode on duration

of development to adult. (a) Calliphora vicina: n¼ 365, t¼ 43.2,

P< 0.001. (b) Calliphora vomitoria: n¼ 214, t¼ 62.3, P< 0.001.

13000

12500

12000

11500

11000

10500

100000 2 4 6 8 10 12 18 2014 16

Temperature of 5-day cold episode ( C)°

y = –42.501x + 11583

R = 0.32692

AD

H

13000

12500

12000

11500

11000

10500

y = –42.447x + 12247

R = 0.55582

AD

H

0 2 4 6 8 10 12 18 2014 16

Temperature of 5-day cold episode ( C)°

a)

b)

Fig. 4. The effect of temperature of a 5-day cold episode on total

ADH. Calliphora vicina: n¼ 265, t¼ 11.3, P< 0.001. (b) Calliphora

vomitoria: n¼ 214, t¼ 16.3, P< 0.001.

Table 3. Scheffe’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



larval stage was statistically similar (Table 1) between the

two blowfly species. Davies & Ratcliffe (1994) postulated

that the difference between the development times for

C. vicina and C. vomitoria is due to the development thresh-

old temperature for C. vomitoria being higher than that for

C. vicina. No difference was seen in the effects of tempera-

ture on the developmental rates of these two blowfly species

in these experiments. At 20�C, Table 1 and Fig. 1 show that

whilst overall C. vomitoria had a higher ADH value to reach

the adult stage, this was due to an extended time in the 3rd

larval stage. Time in the pupal stage for C. vomitoria is

slightly shorter than in C. vicina but not sufficient to com-

pensate for the longer third stage larval period.

Published ADH values for a number of blowfly species

have been collated by Higley & Haskell (2001). Their values

for C. vicina and C. vomitoria (derived from data from

Kamal, 1958 and Greenberg, 1991) show considerable

variability both within their table (9.1) and with the data

presented here. This suggests that the use of generic ADH

values is suspect and may well vary on a climatic, locality,

population or even possibly egg batch basis.

Kamal (1958) and Greenberg (1991) took 6�C as the

minimum development threshold temperature for both

C. vicina and C. vomitoria. Using species obtained from

NE England, Davies & Ratcliffe (1994), however, suggested

that C. vicina’s minimum development threshold must be

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

temperature. They also found that adults emerged at 5�C in

their experiment. The authors then went on to suggest that

if the minimum threshold for C. vicina is taken as 2�C, afterVinogradova & Marchenko (1984; cited in Davies &

Ratcliffe, 1994), assuming that C. vicina and C. vomitoria

have equal ADH above the thresholds, that the lower

threshold for C. vomitoria can be estimated as 6�C. Thesepoints are not supported by this study, based on mater-

ial caught in SE England. In their recent paper on the

effects of refrigeration on the development of Protophormia

terraenovae, Myskowiak & Doums (2002) also showed that

development during the 4�C cold period is not totally

arrested as is normally assumed by forensic investigators.

The concept of ADH assumes that there is a fixed quan-

tity of metabolic activity, controlled by time and tempera-

ture, that is necessary to complete development. The linear

model assumes that the time/temperature relationship is

measured in terms of ADH, where one ADH unit is equal

to 1 (1� � 1 h) across all temperatures. Figure 5 shows that,

irrespective of what temperature is taken as the DTT, total

ADH increases as a result of 5-day low temperature cold

episodes. This may be interpreted in either of two mechan-

istic ways. Either the value of the ADH unit decreases with

lower temperatures or the unit remains a constant but

development is slowed so that there is an overall increase

in the total ADH required. Either way, if the standard ADH

method is used to estimate PMI where there have been

periods of low temperature then it is likely that the PMI

will be underestimated.

The estimation of the developmental threshold tempera-

ture DTT is normally calculated by extrapolating back the

straight line plot of daily developmental rate against

temperature to the point where it reaches zero (Williams &

Richardson, 1984; Wall et al., 1992; Grassberger & Reiter,

2002). This method can be traced back at least to

Wigglesworth (1965), who points out (p. 617) that at low

temperatures the development velocity curve becomes non-

linear and curves less steeply ‘so that the ‘‘development

zero’’ is not in fact the true threshold of development; the

temperature at which development ceases lies appreciably

lower’. This is exactly as seen in Fig. 5 where development

either continues during the cold period but at a lower rate,

or stops completely during the cold period and has some

negative effect after the larvae are returned to 20�C: eitherway there is an increase in the total ADH for individuals

subjected to low temperature episodes.

It could be postulated that the increase in development

after the cold interval might be due to a period of diapause.

However, once in diapause, specific stimuli are needed for

development to resume. Vinogradova & Zinovjeva (1972)

studied the factors leading to induction of diapause in

C. vicina. It appears that diapause is only fully expressed

in conditions below 15�C. Calliphora vicina larvae then

enter diapause in the pre-pupal state and are characterized

by greatly delayed pupariation. Vinogradova & Zinovjeva

(1972) also showed that the main contributing factor to

C. vicina diapause is maternal exposure to short days. In

a)

7000

7500

8000

8500

9000

9500

10000

10500

11000

11500

12000

10 12 14 16 18 20

AD

H

b)

700075008000850090009500

10000105001100011500120001250013000

0 4 8 10 16 18 20

AD

H

DTT 0 °CDTT 1 °CDTT 3 °CDTT 5 °C

0 2 4 6 8


2 6 12 14


DTT 0 °CDTT 1 °CDTT 3 °CDTT 5 °C

Fig. 5. The effect of using different development temperature

thresholds (DTT) on the total ADH calculations (mean� 1.96 SE)

for (a) Calliphora vicina and (b) Calliphora vomitoria for 5-day cold

episodes of various temperatures.



this current study, which took place during the summer

months, the adult flies experienced long days and therefore

diapause is probably not the cause of increased developmen-

tal times for those insects experiencing 5�C and below in

larval stage 1. This is supported by the work of Vaz Nunes &

Saunders (1989) who discovered that for C. vicina it was in the

post-feeding stage that temperature was critical for averting

diapause. In this project a cold spell experienced in larval 3

is not significantly different to larval 2 or pupal stages and

therefore diapause is unlikely to be the cause of lengthened

development after a cold episode in larval 1.

Johl & Anderson (1996) conducted a series of experi-

ments where Canadian C. vicina larvae were chilled at

different lifestages for 24 h at 3�C. They discovered that

for those larvae chilled in larval stages 1, 2 and 3, develop-

ment to adult emergence was delayed by about 1 day com-

pared to the control. Myskowiak & Doums (2002) showed

highly variable effects of refrigeration on the development

of P. terraenovae, depending on the stage when cooling

occurred. Paradoxically, they indicated that development

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.

Previous comparisons of temperature effects of different

species have noted differences in development at various

temperatures and related this to species distribution. Davies

& Ratcliffe (1994) compared C. vicina with C. alpina Ring-

dahl (a Northern Holarctic species that coexists with

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

thermal constant ADH values. Greenberg (1991) compared

adult ADH values for Lucilia (Phaenicia) sericata (Meigen)

from the American Midwest to Marchenko’s (1980) data for

the same species in Russia. The flies from the Midwest

reached adult in 7684ADH compared with 8395ADH for

the Russian strain.

Our study assumed that the control temperature (20�C)was itself a favourable temperature for both blowfly species

and that 20�C falls on the linear region of the developmen-

tal rate/temperature relationship, where the ADH unit has a

value of 1. This is a reasonable assumption for these two

species as 20�C falls into the climatic conditions to which

they would be adapted since both are temperate species.

Campbell et al. (1974) consider insects to be so well adapted

to their particular environment that ‘exposure to inescap-

able temperature extremes is rare’. They too assume that

field temperatures will lie within the linear part of the

temperature/development rate curve.

These findings add to the concerns expressed by

Myskowiak & Doums (2002) that care should be exercised

when applying ADH estimates to the calculation of PMI.

Where cold periods, either natural or artificial (refrigeration),

are included in the experience of the developing fly larvae or

pupae, it should not be assumed that no growth has occurred

during these cold periods.

Higley & Haskell (2001) have pointed to the paucity of

information on DTT values for forensically important fly

species. The whole concept of DTT and its input to the

ADH calculation needs an urgent review in the light of the

findings presented here and those of Myskowiak & Doums

(2002).

The use of ADH is extensively used in PMI estimations

and frequently provides results that are of value in forensic

investigations. Its use, however, can become misleading

when there are prolonged periods of cold weather. In one

murder investigation, BDT used the ADH approach to find

a minimum time of death using blowfly pupae from soil

adjacent to a skeletonized corpse discovered in February in

southern England. The calculation, which involved quite

long periods in November and January–February where

the temperature was below 5�C, proved to be in error by

about a month. In this case the ADH method underesti-

mated the amount of development that had occurred and so

lengthened the PMI. Based on the ADH calculations, death

was minimally estimated to be in mid September of the

previous year whereas the murdered woman was in fact

still alive a month later in mid October.

Acknowledgements

We are grateful to Martin Hall for his valued comments on

an earlier draft of this paper. This work was carried out by

CA in partial fulfilment of her MSc degree in Forensic

Science at King’s College London.

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Accepted 21 February 2003



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