The development rate of immature Calliphora vicina reared on decomposed liver was significantly slower, by as much as 30 h (55.4 % of total development time) for mid-sized larvae, and 71 h (35.0 %) and 58 h (14.6 %) if using times to the onset of pupariation and eclosion, respectively, than those of immatures that developed on fresh whole pig's liver. Development rates of larvae reared on decomposed liver were also slower than those of larvae reared on minced pig's liver and frozen/thawed pig's liver. These results suggest that any estimate of minimum post-mortem interval may result in an over estimate if the blowflies used were developing on an already decomposed body.Open Access This article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Cuttiford, Lue

Grimsley, R

Hall, MJR

Richards, CS

Rowlinson, CC

International Journal of Legal Medicine

English

The development rate of immature Calliphora vicina reared on decomposed liver was significantly slower, by as much as 30 h (55.4 % of total development time) for mid-sized larvae, and 71 h (35.0 %) and 58 h (14.6 %) if using times to the onset of pupariation and eclosion, respectively, than those of immatures that developed on fresh whole pig's liver. Development rates of larvae reared on decomposed liver were also slower than those of larvae reared on minced pig's liver and frozen/thawed pig's liver. These results suggest that any estimate of minimum post-mortem interval may result in an over estimate if the blowflies used were developing on an already decomposed body.Open Access This article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.NHM Repositor

Natural History Museum Repository

SHORT COMMUNICATIONDecomposed liver has a significantly adverse affecton the development rate of the blowfly Calliphora vicinaCameron S. Richards & Catherine C. Rowlinson &Lue Cuttiford & Rebecca Grimsley & Martin J. R. HallReceived: 16 January 2012 /Accepted: 2 April 2012 /Published online: 26 April 2012# The Author(s) 2012. This article is published with open access at Springerlink.comAbstract The development rate of immature Calliphoravicina reared on decomposed liver was significantly slower,by as much as 30 h (55.4 % of total development time) formid-sized larvae, and 71 h (35.0 %) and 58 h (14.6 %) ifusing times to the onset of pupariation and eclosion, respec-tively, than those of immatures that developed on freshwhole pig's liver. Development rates of larvae reared ondecomposed liver were also slower than those of larvaereared on minced pig's liver and frozen/thawed pig's liver.These results suggest that any estimate of minimum post-mortem interval may result in an over estimate if the blow-flies used were developing on an already decomposed body.Keywords Calliphora vicina . Development rates . Diet .Forensic entomologyIntroductionModels describing the development rates of blowfly larvaeare the primary tools used by forensic entomologists toestimate a minimum time of death or minimum post-mortem interval (PMImin) in cases of suspicious death [4].There are numerous biotic factors that may affect the devel-opment rates of blowfly larvae, and an understanding ofthese effects is necessary in order to make accurate correc-tions to a resulting PMImin estimate [2, 8, 12]. One suchfactor is the effect of diet on the development of immatureblowflies and this has been the subject of several recentstudies [5–7, 9, 10]. The study by Kaneshrajah and Turner[10] was the first significant contribution to this area andreported differences in the development of Calliphoravicina larvae of up to 2 days, after the first 5 days ofdevelopment at 20 °C, when larvae reared on pig's liverwere compared to those reared on pig's brain, heart, kidneyor lung tissue. Several notable studies in 2006 looked at arange of diets including the effects of: different organs ofone species of animal [6]; the same organ of differentanimals [5]; different treatments of one organ type, i.e.frozen/thawed vs fresh [7]; and larval densities on a rangeof different food sources [9]. All these studies have addedsignificant insight into the effects of blowfly larval diet ondevelopment, but more work is needed.It is well documented that blowflies colonise a body soonafter death and it is for this reason that they are a good toolto use to estimate PMImin [1, 4, 11]. However, these insectsmay not always feed on fresh tissue, e.g. in cases where accessto a body is delayed, or second and subsequent waves of insectcolonisers are used to establish a timeline of events in a case. Inthese scenarios, the insects will be feeding on decomposedtissue which may have a reduced nutritional value. If this isthe case, then it is likely that the development will be retarded,and failure to account for this may have a significant effect onresultant estimates.This paper investigates the differences in developmentrates of C. vicina larvae reared on fresh minced pig's liverand whole pig's liver that was either fresh, thawed fromfrozen or decomposed.C. S. Richards (*) :C. C. Rowlinson : L. Cuttiford :M. J. R. HallDepartment of Entomology,Natural History Museum,Cromwell Rd,London SW75BD, UKe-mail: c.richards@nhm.ac.ukC. S. Richardse-mail: cam.richards@gmail.comR. GrimsleyDepartment of Life Sciences, Imperial College London,Silwood Park,Ascot SL5 7TG, UKInt J Legal Med (2013) 127:259–262DOI 10.1007/s00414-012-0697-3Materials and methodsAdult C. vicina larvae were trapped in the Wildlife Garden ofthe Natural History Museum and then maintained on sugarand water ad libitum at 21 °C (±2 °C). They were fed 2–4 mlof blood each day for 10 days, after which, they were providedwith a ±250-g pork chop as an oviposition medium. Within1 h, numerous eggs were laid, which hatched within 24 h.Fresh pig's liver (20 g) was placed into each of ten plasticcups (250 ml). Twenty newly hatched larvae were then addedinto each cup. These cups were then placed into a LMS (seriesA1 80 L) incubator set at 23 °C (mean023.2 °C; n0673;SD00.2). Five larvae, one from each of five cups, wererandomly sampled every 6 h for the first 84 h, after which,five larvae were sampled once a day until pupariation. Larvaewere killed by immersing them in boiling water for at least30 s, after which, the length and instar were recorded at eachsampling event using a Dinolight Pro2 digital microscope anda Wild M5 light microscope, respectively.Approximately 5 cm depth of dampened soil was addedinto each experimental cup, as a pupariation medium, oncelarvae started showing signs of entering the wandering phase,e.g. emptying of the crop and/or regularly crawling up thesides of the experimental cups. The food and larvae of eachcup were placed in a Petri dish, in the experimental cup, on topof the soil. Half of the experimental cups were used to con-tinue sampling larval development, in the manner describedabove, while the other half of the experimental cups were usedto assess pupariation and eclosion. This was achieved bycounting the number of larvae, pupae and eclosed adults inone experimental cup at each sampling event. This samplingprotocol limits the summary statistic for developmental eventsdata to median data rather than average data.The average time taken to reach maximum larval size, themedian times taken to reach first ecdysis and second ecdy-sis, the onset of pupariation and the onset of eclosion werecalculated. These data and those detailing the developmentrates of larvae (calculated from length data) were analysedin a Krusal–Wallis (one-way) ANOVA using the statisticalsoftware package Statistica 9.The procedure described above was replicated three times,and the whole experiment was repeated using frozen/thawedpig's liver, fresh minced pig's liver and pig's liver decomposedfor 7 days (at 21±2 °C). Liver in the latter treatment was leftinside vacuum packaging to prevent drying. Gases generatedby the decomposition processes caused the packaging toballoon to its maximum possible size by day 7.Results and discussionThere was no significant difference between the three replicateswithin treatments for all four diet treatments, i.e. fresh wholeliver (F01.32; degrees of freedom (DF)030; p>0.13), freshminced liver (F01.02; DF030; p>0.45), frozen/thawed liver(F01.77; DF030; p>0.12) and decomposed liver (F01.08;DF034; p>0.37). Therefore, all replicates within treatmentswere combined (n015 for each sample time per diet treatment)and used to test significant differences between treatments.Differences between length data for larvae rearedon different dietsThere was a significant difference in the duration of develop-ment for larvae reared on different diets (F038.1; DF045;p>0.00). ATukey post-hoc test indicated that larval develop-ment was significantly slower on decomposed liver than onthe other three diets. This significant difference amounted toas much as 30 h of development or 55.6 % of total develop-ment time for mid-sized larvae (approx. 7 mm long), but was aconsistent difference of 1 day for feeding larvae that were2 days or older. No significant differences in length data wereobserved between the other three diets, similar to the findingsof Day and Wallman [7] (Fig. 1).The development rates of larvae (derived from length data)up to second ecdysis were very similar for larvae reared onwhole fresh liver (slope00.19), freshminced liver (slope00.18)and frozen/thawed liver (slope00.17) (Fig. 1) (although thosereared on fresh liver were statistically significantly faster (F3,8066.61; p<0.02) than those reared on frozen/thawed liver, weconsider this difference biologically insignificant because theyall reached second ecdysis within 5 h of one another (Table 1)).However, larvae, up to second ecdysis, reared on decomposedliver (slope00.11) developed statistically and biologically sig-nificantly slower (F3,8066.61; p<0.00) than those reared on theother diets (Fig. 1). On the other hand, the development rates offeeding third instar larvae, larger than 8 mm, reared on all dietswere not significantly different (F3,802.02; p>0.18) from oneanother: whole fresh liver (slope00.28), fresh minced liver(slope00.29), frozen/thawed liver (slope00.28) and on decom-posed liver (slope00.28) (Fig. 1).These data suggest that first, second and early third instarlarvae are the larval feeding stages that are adversely affect-ed when reared on decomposed liver, while larvae largerthan 8 mm have the ability to develop equally well ondecomposed liver as on all other diets tested. A reason forthis might be because larger (i.e. oldest) larvae are the mostlikely to be naturally exposed to decomposed diets and areperhaps best adapted to tolerate them.Differences between the duration to developmental eventsfor larvae reared on different dietsLarvae reared on the four different diets reached first ecdysiswithin 3 h of one another. Larvae reared on fresh whole, frozen/thawed and minced liver reached second ecdysis within 5 h of260 Int J Legal Med (2013) 127:259–262one another, while those reared on decomposed liver tooksignificantly longer (another 7 h) (F3,807.88; p<0.00). Devel-opment times to reach the maximum larval length were thesame for larvae reared on frozen/thawed and minced liver anddiffered by only 3 h (2.1 % of total development time) and 8 h(1.9 %) for pupariation and eclosion. On the other hand, larvaedeveloping on whole fresh liver were significantly (14.3 and7.6 %) quicker than larvae reared on other food sources inreaching the maximum larval length (F3,80310.33; p<0.00)and eclosion (F3,8027.22; p<0.00), respectively. Larvae rearedon decomposed liver took significantly longer to reach thesedevelopmental landmarks, including pupariation (F3,80161.56;p<0.00), when compared to those reared on the other diets(Table 1). In particular, larvae reared on decomposed liver took71 h (35.0 %) and 58 h (14.6 %) longer, respectively, to reachpupariation and eclosion when compared to those reared onwhole fresh liver (Table 1).The duration of second instar (F3,807.41; p<0.01), feedingthird instar (F3,8053.53; p<0.00) and the wandering phase(F3,8050.58; p<0.00) were all significantly longer for larvaereared on decomposed liver than the other three diets tested.However, the duration of pupariation (F3,8024.76; p<0.00) forlarvae reared on decomposed liver was significantly shorterthan those larvae reared on fresh minced liver (p<0.00) andFig. 1 Larval development ofC. vicina on fresh whole (emptycircle), frozen/thawed (filledsquare), fresh minced (filleddiamond) and decomposed (for7 days) (empty triangle) pig'sliver up to 126 h. Error barsrepresent +1 standard deviation.The filled and empty arrows onthe graph represent the timingof the first and second ecdysisrespectively for larvae reared onthe (a) fresh whole liver, (b)frozen/thawed liver, (c) freshminced liver and (d)decomposed liverTable 1 Accumulative median times taken for development of C.vicina to five developmental eventsDevelopmentaleventDevelopment time (h) on different dietsFreshwholeFrozen/thawedFreshmincedDecomposedFirst ecdysis 13a 10a 10a 10aSecondecdysis33a 28a 31a 40bMaximumlength70a 84b 84b 112cPupariation 130a 143b 140ab 201cEclosion 397a 430b 422b 455cAccumulative median times are the time for 50 % of the experimentalpopulation to reach a developmental event; durations to all develop-mental events exclude egg incubation (n015; pooled data from threereplicates of five samples each). Significant difference within rows (p<0.05) was calculated using one-way ANOVA and denoted by super-script letters accordinglyTable 2 Median duration of each developmental stage for C. vicina(n015; pooled data from three replicates of five samples each)DevelopmentalstageDevelopment time (h) on different dietsFreshwholeFrozen/thawedFreshmincedDecomposedFirst instar 13a 10a 10a 10aSecond instar 20a 18a 21a 30bFeeding thirdinstar37a 56b 53b 72cWanderingphase60a 59a 56a 89bPupariation 267ab 287c 282ac 254bSignificant difference within rows (p<0.05) calculated by one-wayANOVA and denoted by superscript letters accordinglyInt J Legal Med (2013) 127:259–262 261frozen/thawed liver (p<0.00), but not significantly differentfrom those reared on whole fresh liver (p>0.05) (Table 2).The reason for a lack of effect of decomposed liver on theduration of pupariation may be due to the fact that meta-morphosis is not regulated by diet, as the larvae had alreadyfinished feeding, but rather by other developmental process.Why the post-feeding larvae were affected after a diet ofdecomposed liver is presently unknown.ConclusionsDecomposed liver had a significantly adverse effect on thedevelopment rate of C. vicina, when compared to the devel-opment on fresh whole liver, frozen/thawed liver andminced liver. It is important to note that, in most cases,these differences in rates of development will be unlikelyto affect estimates of PMImin, because larvae or other stages(i.e. the oldest specimens) used to estimate PMImin will bedeveloping on fresh bodies due to the promptness withwhich adult parent blowflies can locate a dead body [3].However, estimates in cases where there was a significantdelay in colonisation of the body or those used to establish atimeline of events, e.g. the date of relocation of a badlydecomposed body, could result in an over estimate of up to30 h (55.6 %) if using larvae and up to 71 h (35.0 %) and58 h (14.6 %) if using times to the onset of pupariation andeclosion, respectively.It is assumed that the decomposition of pig's liver inthis study was primarily due to anaerobic bacteria residingin the liver due to the sealed packaging in which the liverwas stored. It is possible that decomposition by aerobicbacteria might have a different effect on the developmentrates of blowflies, but this was outside the scope of thisstudy. This is something that could be included in futurestudies, as well as assessing the effects of different stagesof decomposition on the development of blowflies andother forensically important insects and determining whatfactors in decomposed liver are responsible for the observedeffects.Larvae reared on fresh liver required the least amount oftime to reach maximum larval length, pupariation and eclo-sion than those larvae reared on all other diets. Therefore,altering the food substance that blowfly larvae feed on, byeither bacterial (represented by the decomposed treatment)or mechanical means (represented by the minced and frozen/thawed treatment) appears to have a negative effect ofdevelopment.Acknowledgments Wewould like to thank the Public Sector ResearchExploitation Fund, from the Department of Business Innovation andSkills, for funding this project.Open Access This article is distributed under the terms of the Crea-tive Commons Attribution License which permits any use, distribution,and reproduction in any medium, provided the original author(s) andthe source are credited.References1. Amendt J, Campobasso CP, Goff ML, Grassberger M (2010)Current concepts in forensic entomology. Springer, London, p 3762. Amendt J, Richards CS, Campobasso CP, Zehner R, Hall MJR(2011) Forensic entomology: applications and limitations. ForensicSci Med Path. doi:10.1007/s12024-010-9209-23. Anderson GS, Cervenka VJ (2002) Insects associated with the body:their use and analyses. In: HaglundWD, Sorg MH (eds) Advances inforensic taphonomy:method, theory and archaeological perspectives.CRC Press, London, pp 174–2004. Catts EP (1992) Problems in estimating the postmortem interval indeath investigations. J Agric Entomol 9:245–2555. Clark K, Evans L, Wall R (2006) Growth rates of the blowfly,Lucilia sericata, on different body tissues. Forensic Sci Int156:145–1496. Day DM, Wallman JF (2006) Influence of substrate tissue type onlarval growth in Calliphora augur and Lucilia cuprina (Diptera:Calliphoridae). J Forensic Sci 51:657–6637. Day DM, Wallman JF (2006) A comparison of frozen/thawed andfresh food substrates in development of Calliphora augur (Diptera:Calliphoridae) larvae. Int J Legal Med 120:391–3948. Hall MJR, Richards CS, Whitaker AP (2010) Forensic entomologyadvances. Forensic Tech Review 1:140–1519. Ireland S, Turner B (2006) The effects of larval crowding and foodtype on the size and development of the blowfly, Calliphoravomitoria. Forensic Sci Int 159:175–18110. Kaneshrajah G, Turner B (2004) Calliphora vicina larvae grow atdifferent rates on different body tissues. Int J Legal Med 118:242–24411. Smith KGV (1986) A manual of forensic entomology. BritishMuseum (Natural History), University Printing House in Oxford,London, p 20512. Villet MH, Richards CS, Midgley JM (2010) Contemporary pre-cision, bias and accuracy of minimum post-mortem intervals esti-mated using development of carrion-feeding insects. In: Amendt J,Campobasso CP, Goff ML, Grassberger M (eds) Current conceptsin forensic entomology. Springer, London, pp 109–137262 Int J Legal Med (2013) 127:259–262

Decomposed liver has a significantly adverse affect on the development rate of the blowfly Calliphora vicina

Cameron S. Richards

Catherine C. Rowlinson

Lue Cuttiford

Rebecca Grimsley

Martin J. R. Hall

Springer - Publisher Connector

SHORT COMMUNICATION
Decomposed liver has a significantly adverse affect
on the development rate of the blowfly Calliphora vicina
Cameron S. Richards & Catherine C. Rowlinson &
Lue Cuttiford & Rebecca Grimsley & Martin J. R. Hall
Received: 16 January 2012 /Accepted: 2 April 2012 /Published online: 26 April 2012
# The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract The development rate of immature Calliphora
vicina reared on decomposed liver was significantly slower,
by as much as 30 h (55.4 % of total development time) for
mid-sized larvae, and 71 h (35.0 %) and 58 h (14.6 %) if
using times to the onset of pupariation and eclosion, respec-
tively, than those of immatures that developed on fresh
whole pig's liver. Development rates of larvae reared on
decomposed liver were also slower than those of larvae
reared on minced pig's liver and frozen/thawed pig's liver.
These results suggest that any estimate of minimum post-
mortem interval may result in an over estimate if the blow-
flies used were developing on an already decomposed body.
Keywords Calliphora vicina . Development rates . Diet .
Forensic entomology
Introduction
Models describing the development rates of blowfly larvae
are the primary tools used by forensic entomologists to
estimate a minimum time of death or minimum post-
mortem interval (PMImin) in cases of suspicious death [4].
There are numerous biotic factors that may affect the devel-
opment rates of blowfly larvae, and an understanding of
these effects is necessary in order to make accurate correc-
tions to a resulting PMImin estimate [2, 8, 12]. One such
factor is the effect of diet on the development of immature
blowflies and this has been the subject of several recent
studies [5–7, 9, 10]. The study by Kaneshrajah and Turner
[10] was the first significant contribution to this area and
reported differences in the development of Calliphora
vicina larvae of up to 2 days, after the first 5 days of
development at 20 °C, when larvae reared on pig's liver
were compared to those reared on pig's brain, heart, kidney
or lung tissue. Several notable studies in 2006 looked at a
range of diets including the effects of: different organs of
one species of animal [6]; the same organ of different
animals [5]; different treatments of one organ type, i.e.
frozen/thawed vs fresh [7]; and larval densities on a range
of different food sources [9]. All these studies have added
significant insight into the effects of blowfly larval diet on
development, but more work is needed.
It is well documented that blowflies colonise a body soon
after death and it is for this reason that they are a good tool
to use to estimate PMImin [1, 4, 11]. However, these insects
may not always feed on fresh tissue, e.g. in cases where access
to a body is delayed, or second and subsequent waves of insect
colonisers are used to establish a timeline of events in a case. In
these scenarios, the insects will be feeding on decomposed
tissue which may have a reduced nutritional value. If this is
the case, then it is likely that the development will be retarded,
and failure to account for this may have a significant effect on
resultant estimates.
This paper investigates the differences in development
rates of C. vicina larvae reared on fresh minced pig's liver
and whole pig's liver that was either fresh, thawed from
frozen or decomposed.
C. S. Richards (*) :C. C. Rowlinson : L. Cuttiford :M. J. R. Hall
Department of Entomology,
Natural History Museum,
Cromwell Rd,
London SW75BD, UK
e-mail: c.richards@nhm.ac.uk
C. S. Richards
e-mail: cam.richards@gmail.com
R. Grimsley
Department of Life Sciences, Imperial College London,
Silwood Park,
Ascot SL5 7TG, UK
Int J Legal Med (2013) 127:259–262
DOI 10.1007/s00414-012-0697-3
Materials and methods
Adult C. vicina larvae were trapped in the Wildlife Garden of
the Natural History Museum and then maintained on sugar
and water ad libitum at 21 °C (±2 °C). They were fed 2–4 ml
of blood each day for 10 days, after which, they were provided
with a ±250-g pork chop as an oviposition medium. Within
1 h, numerous eggs were laid, which hatched within 24 h.
Fresh pig's liver (20 g) was placed into each of ten plastic
cups (250 ml). Twenty newly hatched larvae were then added
into each cup. These cups were then placed into a LMS (series
A1 80 L) incubator set at 23 °C (mean023.2 °C; n0673;
SD00.2). Five larvae, one from each of five cups, were
randomly sampled every 6 h for the first 84 h, after which,
five larvae were sampled once a day until pupariation. Larvae
were killed by immersing them in boiling water for at least
30 s, after which, the length and instar were recorded at each
sampling event using a Dinolight Pro2 digital microscope and
a Wild M5 light microscope, respectively.
Approximately 5 cm depth of dampened soil was added
into each experimental cup, as a pupariation medium, once
larvae started showing signs of entering the wandering phase,
e.g. emptying of the crop and/or regularly crawling up the
sides of the experimental cups. The food and larvae of each
cup were placed in a Petri dish, in the experimental cup, on top
of the soil. Half of the experimental cups were used to con-
tinue sampling larval development, in the manner described
above, while the other half of the experimental cups were used
to assess pupariation and eclosion. This was achieved by
counting the number of larvae, pupae and eclosed adults in
one experimental cup at each sampling event. This sampling
protocol limits the summary statistic for developmental events
data to median data rather than average data.
The average time taken to reach maximum larval size, the
median times taken to reach first ecdysis and second ecdy-
sis, the onset of pupariation and the onset of eclosion were
calculated. These data and those detailing the development
rates of larvae (calculated from length data) were analysed
in a Krusal–Wallis (one-way) ANOVA using the statistical
software package Statistica 9.
The procedure described above was replicated three times,
and the whole experiment was repeated using frozen/thawed
pig's liver, fresh minced pig's liver and pig's liver decomposed
for 7 days (at 21±2 °C). Liver in the latter treatment was left
inside vacuum packaging to prevent drying. Gases generated
by the decomposition processes caused the packaging to
balloon to its maximum possible size by day 7.
Results and discussion
There was no significant difference between the three replicates
within treatments for all four diet treatments, i.e. fresh whole
liver (F01.32; degrees of freedom (DF)030; p>0.13), fresh
minced liver (F01.02; DF030; p>0.45), frozen/thawed liver
(F01.77; DF030; p>0.12) and decomposed liver (F01.08;
DF034; p>0.37). Therefore, all replicates within treatments
were combined (n015 for each sample time per diet treatment)
and used to test significant differences between treatments.
Differences between length data for larvae reared
on different diets
There was a significant difference in the duration of develop-
ment for larvae reared on different diets (F038.1; DF045;
p>0.00). ATukey post-hoc test indicated that larval develop-
ment was significantly slower on decomposed liver than on
the other three diets. This significant difference amounted to
as much as 30 h of development or 55.6 % of total develop-
ment time for mid-sized larvae (approx. 7 mm long), but was a
consistent difference of 1 day for feeding larvae that were
2 days or older. No significant differences in length data were
observed between the other three diets, similar to the findings
of Day and Wallman [7] (Fig. 1).
The development rates of larvae (derived from length data)
up to second ecdysis were very similar for larvae reared on
whole fresh liver (slope00.19), freshminced liver (slope00.18)
and frozen/thawed liver (slope00.17) (Fig. 1) (although those
reared on fresh liver were statistically significantly faster (F3,80
66.61; p<0.02) than those reared on frozen/thawed liver, we
consider this difference biologically insignificant because they
all reached second ecdysis within 5 h of one another (Table 1)).
However, larvae, up to second ecdysis, reared on decomposed
liver (slope00.11) developed statistically and biologically sig-
nificantly slower (F3,8066.61; p<0.00) than those reared on the
other diets (Fig. 1). On the other hand, the development rates of
feeding third instar larvae, larger than 8 mm, reared on all diets
were not significantly different (F3,802.02; p>0.18) from one
another: whole fresh liver (slope00.28), fresh minced liver
(slope00.29), frozen/thawed liver (slope00.28) and on decom-
posed liver (slope00.28) (Fig. 1).
These data suggest that first, second and early third instar
larvae are the larval feeding stages that are adversely affect-
ed when reared on decomposed liver, while larvae larger
than 8 mm have the ability to develop equally well on
decomposed liver as on all other diets tested. A reason for
this might be because larger (i.e. oldest) larvae are the most
likely to be naturally exposed to decomposed diets and are
perhaps best adapted to tolerate them.
Differences between the duration to developmental events
for larvae reared on different diets
Larvae reared on the four different diets reached first ecdysis
within 3 h of one another. Larvae reared on fresh whole, frozen/
thawed and minced liver reached second ecdysis within 5 h of
260 Int J Legal Med (2013) 127:259–262
one another, while those reared on decomposed liver took
significantly longer (another 7 h) (F3,807.88; p<0.00). Devel-
opment times to reach the maximum larval length were the
same for larvae reared on frozen/thawed and minced liver and
differed by only 3 h (2.1 % of total development time) and 8 h
(1.9 %) for pupariation and eclosion. On the other hand, larvae
developing on whole fresh liver were significantly (14.3 and
7.6 %) quicker than larvae reared on other food sources in
reaching the maximum larval length (F3,80310.33; p<0.00)
and eclosion (F3,8027.22; p<0.00), respectively. Larvae reared
on decomposed liver took significantly longer to reach these
developmental landmarks, including pupariation (F3,80161.56;
p<0.00), when compared to those reared on the other diets
(Table 1). In particular, larvae reared on decomposed liver took
71 h (35.0 %) and 58 h (14.6 %) longer, respectively, to reach
pupariation and eclosion when compared to those reared on
whole fresh liver (Table 1).
The duration of second instar (F3,807.41; p<0.01), feeding
third instar (F3,8053.53; p<0.00) and the wandering phase
(F3,8050.58; p<0.00) were all significantly longer for larvae
reared on decomposed liver than the other three diets tested.
However, the duration of pupariation (F3,8024.76; p<0.00) for
larvae reared on decomposed liver was significantly shorter
than those larvae reared on fresh minced liver (p<0.00) and
Fig. 1 Larval development of
C. vicina on fresh whole (empty
circle), frozen/thawed (filled
square), fresh minced (filled
diamond) and decomposed (for
7 days) (empty triangle) pig's
liver up to 126 h. Error bars
represent +1 standard deviation.
The filled and empty arrows on
the graph represent the timing
of the first and second ecdysis
respectively for larvae reared on
the (a) fresh whole liver, (b)
frozen/thawed liver, (c) fresh
minced liver and (d)
decomposed liver
Table 1 Accumulative median times taken for development of C.
vicina to five developmental events
Developmental
event
Development time (h) on different diets
Fresh
whole
Frozen/
thawed
Fresh
minced
Decomposed
First ecdysis 13a 10a 10a 10a
Second
ecdysis
33a 28a 31a 40b
Maximum
length
70a 84b 84b 112c
Pupariation 130a 143b 140ab 201c
Eclosion 397a 430b 422b 455c
Accumulative median times are the time for 50 % of the experimental
population to reach a developmental event; durations to all develop-
mental events exclude egg incubation (n015; pooled data from three
replicates of five samples each). Significant difference within rows (p<
0.05) was calculated using one-way ANOVA and denoted by super-
script letters accordingly
Table 2 Median duration of each developmental stage for C. vicina
(n015; pooled data from three replicates of five samples each)
Developmental
stage
Development time (h) on different diets
Fresh
whole
Frozen/
thawed
Fresh
minced
Decomposed
First instar 13a 10a 10a 10a
Second instar 20a 18a 21a 30b
Feeding third
instar
37a 56b 53b 72c
Wandering
phase
60a 59a 56a 89b
Pupariation 267ab 287c 282ac 254b
Significant difference within rows (p<0.05) calculated by one-way
ANOVA and denoted by superscript letters accordingly
Int J Legal Med (2013) 127:259–262 261
frozen/thawed liver (p<0.00), but not significantly different
from those reared on whole fresh liver (p>0.05) (Table 2).
The reason for a lack of effect of decomposed liver on the
duration of pupariation may be due to the fact that meta-
morphosis is not regulated by diet, as the larvae had already
finished feeding, but rather by other developmental process.
Why the post-feeding larvae were affected after a diet of
decomposed liver is presently unknown.
Conclusions
Decomposed liver had a significantly adverse effect on the
development rate of C. vicina, when compared to the devel-
opment on fresh whole liver, frozen/thawed liver and
minced liver. It is important to note that, in most cases,
these differences in rates of development will be unlikely
to affect estimates of PMImin, because larvae or other stages
(i.e. the oldest specimens) used to estimate PMImin will be
developing on fresh bodies due to the promptness with
which adult parent blowflies can locate a dead body [3].
However, estimates in cases where there was a significant
delay in colonisation of the body or those used to establish a
timeline of events, e.g. the date of relocation of a badly
decomposed body, could result in an over estimate of up to
30 h (55.6 %) if using larvae and up to 71 h (35.0 %) and
58 h (14.6 %) if using times to the onset of pupariation and
eclosion, respectively.
It is assumed that the decomposition of pig's liver in
this study was primarily due to anaerobic bacteria residing
in the liver due to the sealed packaging in which the liver
was stored. It is possible that decomposition by aerobic
bacteria might have a different effect on the development
rates of blowflies, but this was outside the scope of this
study. This is something that could be included in future
studies, as well as assessing the effects of different stages
of decomposition on the development of blowflies and
other forensically important insects and determining what
factors in decomposed liver are responsible for the observed
effects.
Larvae reared on fresh liver required the least amount of
time to reach maximum larval length, pupariation and eclo-
sion than those larvae reared on all other diets. Therefore,
altering the food substance that blowfly larvae feed on, by
either bacterial (represented by the decomposed treatment)
or mechanical means (represented by the minced and frozen/
thawed treatment) appears to have a negative effect of
development.
Acknowledgments Wewould like to thank the Public Sector Research
Exploitation Fund, from the Department of Business Innovation and
Skills, for funding this project.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribution,
and reproduction in any medium, provided the original author(s) and
the source are credited.
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Decomposed liver has a significantly adverse affect on the development rate of the blowfly Calliphora vicina

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