Medical and Veterinary (2003) 17, 178–186

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

C. AMES andB. TURNER Department of Life Sciences, King’s College London, U.K.

Abstract. Traditionallythe calculation of accumulated degree daysor 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 maybe 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 vicina Robineau-Desvoidyand C. vomitoria L. (Diptera: ) at 20C was interrupted bycold episodes. The expectation was that total development time would increase bythe 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 decreased linearlywith increasing temperature with no evidence of anyminimum threshold temperature effect. The increased ADH at low temperatures maybe due to either continued but reduced development or a delayin development restarting after the cold episode. Use of ADH in PMI estimations has shortcomings particularlyduring the winter period where low temperatures are involved or where there are sudden summer cold spells during the development period. As blowflydevelopment progresses from egg to such errors will be compounded. Key words. , , development threshold tem- perature, , postmortem interval.

Introduction Northern Europe. In favourable conditions these species lay their eggs around natural orifices or wounds on the fresh In forensic science, evidence is most commonlyused 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 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, 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 byambient temperature; the higher the tempera- vomitoria (Linnaeus) being widelydistributed throughout ture the faster development occurs. Thus byknowing the ambient temperature and the progress of blowflydevelop- 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 blowflydevelop- King’s College London, Franklin Wilkins Building, 150 Stamford St., ment are available. Larval size, usuallylength, can be London SE1 9NN, U.K. E-mail: [email protected] 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 (20–22C). 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 onlycontained approximately100 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 possiblygeographical population) minimum source. Females massed upon the liver and oviposition developmental threshold and the time spent at that would normallybegin 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 anylarval 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. Approximately30–40 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 verydifficult to determine plastic containers (7 Â 8 Â 11 cm) with a plastic lid. The accuratelybecause 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 regularlyso ture (DTT) is most commonlyestimated bymeasuring 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 20C (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 laboratorywas air-conditioned at 20 C Æ 1Cso C. vomitoria is 6C, yet recent studies suggest this varies that the removal of larvae from the 20C incubator for with locality(Saunders & Hayward,1998). The majorityof measurement did not varytheir temperature regime. The past research on the effects of temperature on development second incubator (monitored using a TinyTag, 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, mainlyover 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 blowflyspecies. Specific This studyreports on a series of laboratoryexperiments criteria for each series were influenced byprevious experi- that explore the effects of short periods of low temperature mental experience. Development was followed from the egg on the development of two locallycommon blowflyspecies 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 Robineau-Desvoidy) to stages temporarilyhalt development until passed to a forensic entomologist, is complementaryto this present study. Egg development took place at 20C. Individual cohorts of larvae were then subjected to cold episodes, of 5C 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 20C for the remainder of their development. Controls and maintained in gauze covered cages (22 Â 38 Â 27 cm) at remained at 20C 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 20C for 3 days following 1800 ADH), the ADH value to reach a specific stage should oviposition. Bythis time the larvae were late second stage. not be significantlydifferent from the corresponding con- Cohorts of these larvae were placed at 1,3,5 or 10C for trol ADH value. 5 days, after which they were returned to 20C 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 homogeneityof 20C 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 blowflyspecies ( C. vicina 30 eggs, were individuallyfollowed 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 Subsequentlytheywere observed every2 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 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 bycomparing 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 approximately600 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, theywere counted and 5C period: that is the DTT is around 5C. removed from the containers. During all experiments, the The data were reanalysed using 5C 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) À time in cold episode appears important. Scheffe´ ’s post hoc test (Table 2) shows a (h)]  20} þ [time in cold episode  temperature significant difference onlybetween the 1st stage 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 uncertaintyof the concept of a DTT, This effect is not seen in C. vomitoria. Scheffe´ ’s test shows initiallyno correction for this was applied to anyof 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-daycold episode occurs in the developmental period. Results

Development at 20C – the controls Experimental series 2

Each of the experimental series included a set of controls As the temperature of the 5-daycold episode increased, at 20C. 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 20C, the timings both species decreased linearly(Fig. 3). Both slopes are for the egg stage and 1st and 2nd stage larvae are quite significantlydifferent from zero (Fig. 3). There was an similar for C. vicina and C. vomitoria. The 3rd stage larvae increase of approximately8 h on the development time for period for C. vomitoria takes about 50% more time than for each C decrease in the 5-daycold episode. C. vicina, whilst the pupal stage is some 12% shorter If the DTT is 5C (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 5C data. However, assuming both have a DTT of 0C. 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 Scheffe´ ’s multiple comparison test on these data indicated significant differ- Excluding the controls, each treatment had a 5-dayperiod ences (P < 0.001) between all temperature combinations in at 20C(¼ 2400 ADH) replaced bya 5-daycold episode of both species. This appears to be at variance with the find- 5C(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 onlyone temperature for the cold period) and

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 178–186 # 03TeRylEtmlgclSociety, Entomological Royal The 2003 eia n eeiayEntomology Veterinary and Medical

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 20C.

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 17

178–186 , 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 ** o eprtr n lwl development blowfly and temperature Low

**Significant at 1% significance level. 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

195.5 12000 ADH 290.73 egg stage

lar va 1 11000 lar va 2

lar va 3 pupa Pupal Control Larval 1 Larval 2 Larval 3 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 (5C/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 blowflyspecies within the temperature range third quartile (Q3) value. The lines that extend from the top and covered in these experiments (1–20C). bottom of the box show the range of the distribution. 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 Scheffe´ ’s post hoc test to these data ing linear relationship with increasing temperature for the shows that the ADH values for the 1C and 3C treatments 5-daycold episode, which are significantlydifferent from are each significantlydifferent to those of all the other zero (Fig. 4). temperatures (Table 3). Using Welch’s test on each species’ ADH and tempera- Whilst statisticallythe ADH values for 5, 10 and 20 C are not ture data set (and taking the DTT point to be 5C) reveals significantlydifferent (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-daycold temperature episodes and ADH values 3Ctreatmentswerehigher(meanfor1C cold period,

Table 2. Scheffe´ ’s multiple comparison significance table between total ADHs for differing treatments of cold episodes (5C/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) 800 13000 12500 600 12000

y = –8.1251x + 699.17 2 11500 400 R = 0.8765 ADH ADH 11000

200 10500 y=–42.501x + 11583 R2 = 0.3269 10000 0 0 24681012 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

y = –8.1224x + 732.35 ADH 2 R = 0.9482 11500 400 y=–42.447x + 12247 ADH 2 11000 R = 0.5558 200 10500 0 24681012 14 16 18 20 0 Temperature of 5-day cold episode (°C) 0 2 4 6 8 101214161820 Temperature of 5-day cold episode (°C) Fig. 4. The effect of temperature of a 5-daycold 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-daycold 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 20C because the hourly multiplier is the difference between the DTT and control temperature. Thus lowering the DTT from 5 to 0C C. vicina ¼ 8619 ADH and C. vomitoria ¼ 9084 ADH; mean for increases the hourlymultiplier from 15 to 20. Lowering 3 Ccoldperiod¼ 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 5C. the ADH calculation. However, the data clearlyshows that the development rate outside the cold episodes was affected bythe 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 approximatelysimilar 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 studyfound that onlythe ADH to reach the 3rd

Table 3. Scheffe´ ’s multiple comparison significance table between total ADH for differing treatments of 5-daycold 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.

1C3C5C10C20C 1C <0.001 <0.001 <0.001 <0.001 3C <0.001 <0.001 <0.001 0.004 5C <0.001 <0.001 0.829 0.903 10C <0.001 <0.001 0.996 0.440 20C <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. Theyalso found that adults emerged at 5 Cin 11500 their experiment. The authors then went on to suggest that 11000 if the minimum threshold for C. vicina is taken as 2C, after 10500 Vinogradova & Marchenko (1984; cited in Davies & 10000 DTT 0 °C Ratcliffe, 1994), assuming that C. vicina and C. vomitoria 9500 DTT 1 °C have equal ADH above the thresholds, that the lower ADH ° 9000 DTT 3 C threshold for C. vomitoria can be estimated as 6C. These DTT 5 °C 8500 points are not supported bythis 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 0246810 12 14 16 18 20 terraenovae, Myskowiak & Doums (2002) also showed that Temperature of 5-day cold episode (°C) development during the 4C cold period is not totally b) arrested as is normallyassumed byforensic investigators. 13000 The concept of ADH assumes that there is a fixed quan- 12500 tityof metabolic activity,controlled bytime and tempera- 12000 11500 ture, that is necessaryto 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 ° 10000 DTT 1 C to 1 (1 1 h) across all temperatures. Figure 5 shows that, ADH Â 9500 DTT 3 °C irrespective of what temperature is taken as the DTT, total 9000 DTT 5 °C 8500 ADH increases as a result of 5-daylow temperature cold 8000 episodes. This maybe interpreted in either of two mechan- 7500 istic ways. Either the value of the ADH unit decreases with 7000 02 4 6 8 10 12 14 161820 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 Æ 1.96 SE) periods of low temperature then it is likelythat the PMI for (a) Calliphora vicina and (b) Calliphora vomitoria for 5-daycold will be underestimated. episodes of various temperatures. The estimation of the developmental threshold tempera- ture DTT is normallycalculated byextrapolating back the larval stage was statisticallysimilar (Table 1) between the straight line plot of dailydevelopmental rate against two blowflyspecies. 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 velocitycurve becomes non- ture on the developmental rates of these two blowflyspecies linear and curves less steeply‘so that the ‘‘development in these experiments. At 20C, 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 exactlyas 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, slightlyshorter than in C. vicina but not sufficient to com- or stops completelyduring the cold period and has some pensate for the longer third stage larval period. negative effect after the larvae are returned to 20C: either Published ADH values for a number of blowflyspecies waythere is an increase in the total ADH for individuals have been collated byHigley& 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 . variabilityboth 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 maywell varyon a climatic, locality, studied the factors leading to induction of diapause in population or even possiblyegg batch basis. C. vicina. It appears that diapause is onlyfullyexpressed Kamal (1958) and Greenberg (1991) took 6C as the in conditions below 15C. 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 bygreatlydelayedpupariation. 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 probablynot the cause of increased developmen- findings presented here and those of Myskowiak & Doums tal times for those insects experiencing 5C and below in (2002). larval stage 1. This is supported bythe work of Vaz Nunes & The use of ADH is extensivelyused in PMI estimations Saunders (1989) who discovered that for C. vicina it was in the and frequentlyprovides 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 significantlydifferent to larval 2 or pupal stages and murder investigation, BDT used the ADH approach to find therefore diapause is unlikelyto be the cause of lengthened a minimum time of death using blowflypupae from soil development after a cold episode in larval 1. adjacent to a skeletonized corpse discovered in Februaryin 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 3C. Theydiscovered that the temperature was below 5C, 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 highlyvariable effects of refrigeration on the development was minimallyestimated 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 mayalso have different References thermal constant ADH values. Greenberg (1991) compared adult ADH values for Lucilia (Phaenicia) sericata (Meigen) Abrami, G. (1972) Optimum mean temperature for plant growth from the American Midwest to Marchenko’s (1980) data for calculated bya new method of summation . Ecology, 53, the same species in Russia. The flies from the Midwest 893–900. reached adult in 7684 ADH compared with 8395 ADH for Allen, J.C. (1976) A modified sine wave method for calculating day the Russian strain. degrees. Environmental Entomology, 5, 388–396. Our studyassumed that the control temperature (20 C) Anderson, G.S. (2000) Minimum and maximum development rates was itself a favourable temperature for both blowflyspecies of some forensicallyimportant Calliphoridae (Diptera) . Journal and that 20C falls on the linear region of the developmen- ofForensic Sciences , 45, 824–832. tal rate/temperature relationship, where the ADH unit has a Arnold, C.Y. (1960) Maximum–minimum temperatures as a basic value of 1. This is a reasonable assumption for these two for computing heat units. Proceedings for the American Society ofHorticultural Science , 76, 682–692. species as 20C falls into the climatic conditions to which Baskerville, G.L. & Emin, P. (1969) Rapid estimation of heat theywould be adapted since both are temperate species. accumulation from maximum and minimum temperatures. Campbell et al. (1974) consider insects to be so well adapted Ecology, 50, 514–517. to their particular environment that ‘exposure to inescap- Campbell, A., Frazer, B.D., Gilbert, N., Gutierrez, A.P. & able temperature extremes is rare’. Theytoo assume that MacKauer, M. (1974) Temperature requirements of some aphids field temperatures will lie within the linear part of the and their parasites. Journal ofApplied Ecology , 11, 431–438. temperature/development rate curve. Catts, E.P. & Goff, M.L. (1992) Forensic entomologyin criminal These findings add to the concerns expressed by investigations. Annual Review ofEntomology , 37, 253–272. Myskowiak & Doums (2002) that care should be exercised Dallwitz, R. (1984) The influence of constant and fluctuating when applying ADH estimates to the calculation of PMI. temperatures on developmental rate and survival of pupae of the Australian sheep blowflyLucilia cuprina . Entomologica Experi- Where cold periods, either natural or artificial (refrigeration), mentalis et Applicata, 36, 89–95. are included in the experience of the developing flylarvae or Davies, L. & Ratcliffe, G.G. (1994) Development rates of some pupae, it should not be assumed that no growth has occurred pre-adult stages in blowflies with reference to low temperatures. during these cold periods. Medical and Veterinary Entomology, 8, 245–254. Higley& Haskell (2001) have pointed to the paucityof Grassberger, M. & Reiter, C. (2001) Effect of temperature on information on DTT values for forensicallyimportant Lucilia sericata (Diptera: Calliphoridae) development with

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