Effects of Temperature on Chrysomya rufifacies

(Diptera: Calliphoridae) Development

JASON H. BYRD AND JERRY F. BUTLER
Institute of Food and Agricultural Sciences, Department of Entomology and Nematology, University of Florida,
P.O. Box 110620, Building 970, Hull Road, Gainesville, FL 32611

J. Med. Entomol. 34(3): 353-358 (1997)

ABSTRACT Growth curves were studied for the egg, larva, and pupa of Chrysomya rufi-
fucics (Macquart) under mean cyclic temperatures of 15.6, 21.1, 26.7, and 35.0°C and a con-
stant temperature of 25.0°C. Development from egg to adult under all regimes ranged from
190 to 598 h. A constant temperature of 25°C produced a range of pupation times from 134
to 162 h, with adult emergence ranging from 237 to 289 h. The maximal preferential tem-
perature of 35.1°C was determined for maggots using a gradient system. Highly predictable

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developmental time, minimal larval length variation, and low cohort variation emphasize the
utility of this species in entomological-based postmortem interval determinations. Therefore,
C. rufifacies should be of primary forensic importance when recovered alone or in conjunction
with other species of Calliphoridae.

KEY WORDS Chrysomya rufifacies, temperature, insect development, forensic entomology,

forensic science, postmortem interval

ALTHOUGH THE FIELD of medicocriminal ento- Therefore, C. rufifacies is of profound forensic

mology has broad applicability, the use of ento-
mological evidence in legal investigations has its
most frequent use in the establishment of post-
mortem intervals (Schoenly et al. 1996). Because
entomologists are being used increasingly in crim-
inal investigations to assist in postmortem interval
determinations, the availability of accurate devel-
opmental and successional data on sarcophagous
insects is of primary importance. Such information
will enhance the use of arthropods in death inves-
tigations through an improved understanding of
development time and allow for accurate postmor-
tem interval estimations.
The hairy maggot blow fly, Chrysomya rufifacies
(Macquart), is indigenous to the Australian and
oriental regions of the old world tropics (Holdaway
1930, Greenberg 1971, Baumgartner and Green-
berg 1984, Baumgartner 1993) and has been rap-
idly expanding its distribution. This species was
found in the continental United States in 1981
(Gauge et al. 1982), and currently it is well estab-
lished in Louisiana, Texas, California, and Florida.
With rapid dispersal and preference for larger car-
casses (Anderson et al. 1988, Wells and Greenberg
1994), this species has become the dominate blow
fly on human cadavers in north and central Flori-
da, outnumbered only by C. megacephala (F.) in
southern Florida (J.H.B., unpublished data). As
these immigrant species of Chrysomya expand to
new ranges, the potential exists for displacement
of native species (Wells 1992; Wells and Greenberg
1992a, b).
0022-2585/97/0353-0358$02.00/0 © 1997 Entomological Society of America
importance and considered to be one of the "pri-
mary" flies (O'Flynn 1979) for use in postmortem
interval estimations because of its rapid coloniza-
tion of fresh corpses, oviposition activity through-
out the daylight hours, and its abundance in both
coastal and inland habitats. This study was con-
ducted to determine the developmental duration,
cohort variability, and temperature preference for
C. rufifacies.
Materials and Methods
Adult C. rufifacies were collected from human
cadavers and from inverted cone traps baited with
pork, fermenting sunflower and millet seed, and
chicken. These adults were held in an insectary at
25 ± 2°C, 75-80% RH, and continuous light. The
F 3 generation colonies used for this study were
held in screen cages (54 by 28 by 28 cm) with .250
adult flies fed a 50:50 mixture of table sugar and
powdered milk, and fresh water.
Before development studies were initiated, lar-
val preferences for beef, chicken, pork, and bovine
blood were determined. Larvae were placed in the
center of a circular arena (61 cm diameter) with
bait choices located in pitfall traps equidistant
form the periphery and left undisturbed. After 12
h, the number of larvae in each bait trap was
counted. This study revealed that 75-80% of the
larvae preferred center cut lean pork. Therefore,
the pork media were used exclusively for the es-
tablishment of larval growth curves.
354 JOURNAL OF MEDICAL ENTOMOLOGY
Cyclic and Constant Temperature Develop-
ment. Cyclic temperature regimes were used with
means of 15.6, 21.1, 26.7, and 32.2°C and an am-
plitude of 5.5°C. Temperature cycles were selected
to encompass the broadest range of temperatures
possible and remain within the physical limitations
of our test facility and labor requirements. All
treatments had a photoperiod of 12:12 (L:D) h and
75% RH. The photoperiod of 12:12 (L:D) h was
selected so that larval sampling would occur during
the period shift. Additional flies were allowed to
develop at constant 25°C and 24:0 (L:D) h.
Three groups of ^lOO eggs each <20 min old
were transferred into a S970 Dixie cup (8.5 cm
diameter, 11 cm high, James River, Norwalk, CT.)
containing 200 g of whole center cut lean pork,
and immediately were placed into a Percival en-
vironmental chamber (Boone, IA) with ± 1°C con-
trol. This procedure was repeated at 2-h intervals
until 6 replicates were prepared, with a 12-h in-
terval between the 1st and last of the 6 replicates.
A ratio of 1 larva per 2.0 g of pork was maintained
by adding meat twice daily so that the metabolic
heat generated by the feeding larvae would not be
higher than the growth chamber settings and
thereby accelerate growth rates. Although some
larval aggregations were noticed, no meaningful
temperature elevations of the rearing media were
recorded by a type-K thermocouple fluid probe in-
serted in each rearing cup.
Two of the largest maggots from each of the 3
subsamples within each of the 6 samples were re-
moved twice daily (at 12-h intervals) until 10% of
the cohort had pupated. Larval instar and stage
duration was based on the fastest developing 10%
of the sample population. Stage duration was re-
corded after 10% of the individuals had completed
development into that stage. Time of pupation was
characterized by failure of the larvae to elongate
and move in response to being disturbed (Byrd
1995).
For measurement purposes, maggots were killed
immediately in boiling water and transferred to
75% ethyl alcohol. Lengths were measured to the
nearest 0.1 mm using an electronic digital caliper.
Selection of this sampling method was justified be-
cause of the common practice of collecting a rep-
resentative sample of the insect fauna and then
selecting for largest maggots for measurement at a
death scene. In doing so, the investigating ento-
mologist assumes that the largest larvae of a par-
ticular species are the oldest individuals and prob-
ably developed from the 1st egg clutch.
Variation of Pupation and Emergence Times.
Range of pupation and emergence times were
studied to determine if this factor should be in-
corporated into the postmortem interval estima-
tion. Eight cohorts of 600 individuals each were
reared under a constant 25 ± 1°C with a photo-
period of 12:12 (L:D) h. Times of pupation and
adult emergence were recorded after 10% of the
Vol. 34, no. 3
population had completed the respective larval and
pupal stages.
Temperature Preference. This experiment de-
termined the temperature preferred by developing
larvae and established a baseline developmental
temperature for use in postmortem interval esti-
mations involving maggot masses in the event tem-
perature recordings were not made at the death
scene. Larvae can regulate body temperature by
positional effect within the mass so that a pre-
ferred developmental temperature is maintained.
To determine the temperature preferences of de-
veloping maggots, a temperature gradient was con-
structed along an aluminum plate. Eggs were
placed at 15-cm intervals in wooden troughs (76
cm long by 4.5 cm wide by 5 cm deep), which were
lined with plastic wrap and packed with ground
pork to a depth of 2.0 cm. These troughs then
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were placed on an aluminum plate (84 cm long by
46 cm wide) with a Brinkmann circulating cooler
(Atlanta, GA.) set at 20°C and a custom circulating
heater with a Cole Parmer (Niles, IL.) temperature
controller set at 45°C. The cooler and heater were
connected to opposite ends of the aluminum plate,
and thermal conduction produced a linear tem-
perature gradient of 0.29°C/cm along the length of
the plate. This test system was placed in a Hotpack
(Philadelphia, PA.) environmental chamber to reg-
ulate ambient temperature at 25 ± 1°C and 80%
RH.
The mean temperature of the maggot mass cen-
ter and the range of temperatures at which actively
feeding maggots were found on the mass periphery
were recorded at 24-h intervals. Temperatures
were recorded using a digital thermometer
(±0.1°C) with a type-K thermocouple fluid probe.
After maggots reached the start of the migrating
phase, they were transferred to an adjacent trough
on the plate that contained builders sand (1.0 cm
deep) for burrowing and pupation. The tempera-
ture ranges at which fully formed pupae were
found were recorded, and the pupae were held at
their mean temperature for adult emergence.
Results
Cyclic and Constant Temperature Develop-
ment. Maximal length of C. rujifacies larvae were
plotted against time for each of the cycling and
constant temperature regimes (Fig. 1). With the
exception of the 32.2°C regime, temperature had
little effect on peak larval length, although pupal
length progressively decreased with decreasing
temperatures. The mean pupal length in all ex-
perimental rearings was 8.1 ± 0.9 mm (mean ±
SD).
Incremental increases in the mean temperature
produced consistently shorter time requirements
for the first 10% of the population to complete
pupation and emergence (Table 1). Times for full
emergence and pupation under each of the 5 tem-
perature regimes are summarized in Table 2. An
May 1997 BYRD AND BUTLER: DEVELOPMENT OF C. rufifacies 355

18

16 -

14 -

12 -

10

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6 -

4 -

2 -

I I I I I I I I I I I 1 I I I I I I I I I I I I I I I 1 I I I I I I I I I [ I I I I I I I I
g 2
TIME (HOURS)
Fig. 1. Mean length of 6 largest C. rufifacies larvae measured at 2-h intervals and plotted as a function of cohort
age for each of 5 temperature regimes.

increase in the mean temperature decreased the
total duration of each life stage with the exception
of the 3rd instar between the 26.7°C and the
32.2°C regimes (Table 3). The 3rd instar required
4 additional h at 32.2°C than needed at 26.7°C.
Stage duration time was recorded once 10% of the
sample population completed development from
the previous stage.
The cycling temperature regime of 15.6°C pro-
duced the longest stage durations as well as the
smallest pupal size (mean ± SD) 6.8 ± 0.4 mm.
Maggot length was consistent throughout the feed-
ing stages, but varied increasingly as they entered
the migratory or postfeeding stage. After the onset
of migration, larval length gradually decreased
until pupation.
Increasing the rearing temperature to the
21.1°C mean cyclic temperature regime reduced
the duration of the egg stage by 10 h. This was the
largest hourly change in egg duration, because all
other temperatures produced hatch between 8 and
14 h, including the single constant temperature re-
gime. The constant temperature of 25°C produced
growth curves that were not unlike the cyclic tem-
peratures, although it noticeably prolonged the 3rd
instar.

Table 1. Time requirement for the first 10% of a C. Table 2. Mean ± SE time for C. rufifacies population
rufifacies population to complete pupation and emer- to complete pupation and emergence under 5 tempera-
gence under 5 temperature regimes ture regimes

Temp, °C Pupation, h Emergence, h Temp, °C Pupation, I) Emergence, li

15.6" 300 598 15.6" 350 ± 28 612: 48
21.1" 168 296 21.1" 174 ± 18 314 42
25.0*' 170 289 25.0'' 170 ± 11 289 26
26.7" 134 216 26.7" 140 ± 19 222 16
32.2" 114 190 32.2" 116 ± 16 180 19

" Cycling mean temperature (amplitude 5.5°C) and a photope- "Cycling mean temperature (amplitude 5.5°C) and a pliotope-
riod of 12:12 (L:D) h. riodof 12:12 (L:D) h.
'' Constant temperature and continuous light. " Constant temperature and continuous light.
356 JOURNAL OF MEDICAL ENTOMOLOGY
Table 3 . Stage duration of C. rufifacies based on first
10% of population to enter and complete the respective
life stage under 5 temperature regimes
Mean Stage duration, h
temp, 1st 2nd 3rd
°C Egg instar instar instar
15.6" 30 46 54 154
21.1" 20 26 30 92
25.0b 12 18 34 106
26.7" 14 18 24 78
32.2" 8 10 14 82
" Cycling mean temperature (amplitude 5.5°C) and a photope-
riod of 12:12 (L:D) h.
'' Constant temperature and continuous light.
The maximum mean temperature of 32.2°C de-
creased the egg stage by 4 h, and produced re-
markably short total developmental duration with
minimal length ranges. The most rapid larval
growth occurred before 64 h, with maximum
length attained at this time. Such rapid growth in-
dicates that early instars may find high tempera-
tures more favorable than expected.
Temperature Preference. Means of 4 replicate
experiments using larvae of C. rufifacies showed
that aggregations formed at a mean temperature
of 35.1 ± 0.4°C after 24 h (Fig. 2). During the
next 24 h, the larvae began to disperse and move
to cooler temperatures with a mean of 34 ± 1.5°C.
After 48 h, larvae continued to disperse but main-
tained a mean temperature of 31.0 ± 3.1°C. At 96
h, larvae were transferred to the adjacent sand
trough and left undisturbed for 24 h. This transfer
was necessary, because migrating larvae experi-
enced a 24-h delay from their expected pupation
times as they searched for a path to exit die pork.
After this 24-h period, the larvae were observed to
pupate normally either under, in, or on top of the
rearing media. At 120 h, pupation occurred in the
sand trough with larvae in a loose aggregation at a
mean temperature of 25 ± 1.1°C.
Variation of Pupation and Emergence Times.
Laboratory-reared C. rufifacies displayed minimal
developmental variation among pupal cohorts;
however, variation more than doubled among co-
horts at adult emergence when reared at a constant
25 ± 1°C. Time required for pupation ranged from
134 to 162 h (n = 8) with a mean of 146 ± 9 h
(±SD), 95% CI = 138-154 h, and 9% CV. Emer-
gence time ranged from 237 to 289 h (n = 8) with
a mean of 262 ± 20 h (±SD), 95% CI = 245-279
h, and 17% CV. This variation among cohorts was
minimal and illustrated the usefulness of this spe-
cies in postmortem interval estimations.
Discussion
Our reported times for egg eclosion agreed with
those reported by O'Flynn (1983); however,
O'Flynn reported that this species failed to pupate
Vol. 34, no. 3
U 33
Pupa
5
I 29
298 OS
128
119 w
82 H 27
76
20 30 40 90 80 TO 80 90 100 110 120
TIME (HOURS)
Fig. 2. Preferred mean development temperature of
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C. rufifacies larval mass plotted as a function of age. Er-
ror bars indicate range of temperature in which larvae
were observed to feed.
at 15°C. All larvae pupate normally in our 15.6°C
temperature regime. Total duration from egg to
adult for temperatures >20°C also agreed with the
findings of O'Flynn (1983). With the exception of
the 15.6°C temperature regime, our egg-to-adult
accumulated degree-hours were consistently short-
er than those of Greenberg (1990).
Data for egg eclosion, larval duration, and adult
emergence times will allow accurate postmortem
interval determination based on the temperature-
dependent development of this species. An impor-
tant and often overlooked stage for estimating lar-
val age is the migrating or wandering stage. The
onset of this stage is indicated by larval migration
away from the food substrate accompanied by a
slight reduction in body length. This is followed by
a rapid reduction in body length immediately be-
fore pupation. Although pupal lengths were pro-
gressively smaller under each cooler temperature
regime, the variation in larval length was minimal
throughout all feeding instars as well as the post-
feeding stage in all experimental rearings. Such
low variation will allow all life stages, including the
postfeeding stage, to be used effectively in estab-
lishing a postmortem interval.
Wells and Kurahashi (1994) reported that blow
fly larvae can delay pupation if conditions are sub-
optimal. Our study indicated that a 24-h delay in
pupation will occur if movement of C. rufifacies
larvae is restricted when they attempt to exit re-
mains during the migrating stage. Therefore, al-
tered developmental times must be considered in
death investigations where remains are tightly
wrapped, or if maggot migration is otherwise re-
stricted. Also, when constructing the postmortem
interval estimation, it is essential that laboratory
studies of insect development use temperatures
May 1097 BVHI) AND BUTLER: DEVELOPMENT OF C. rufifacies
with mean values comparable to those for the
death scene and the corpse.
Significantly smaller pupae than those reported
here should indicate starvation or suboptimal con-
ditions and those conditions should be factored
into the postmortem interval estimation. Although
we have observed larval cannibalism with this spe-
cies us noted by Fuller (1934), James (1947), Sub-
ramanian and Mohan (1980), and Goodbrod and
Goff (1990), none was observed during the course
of this study because of the constant availability of
sufficient rearing media.
The information obtained by comparing vari-
ability among replicate pupation and emergence
times under a constant temperature of 25°C will
aid in establishing confidence intervals for post-
mortem interval estimations. Larval growdi and to-
tal developmental duration under the constant
temperature regime was predictable and occurred
within the acceptable time ranges established by
tin4 cyclic temperature regimes. Therefore, devel-
opmental data obtained from constant tempera-
ture rcarings of this species could be applied to
cycling temperature conditions as long as mean
temperature values were comparable.
When dealing with C. rufifacies, confidence in-
tervals can be relatively narrow when compared
with the total postmortem interval. In addition, the
optimum temperature preferred by developing lar-
vae on the temperature gradient provided insight
into the temperature at which individual maggots
within a maggot mass regulate their temperature.
Therefore, known development rates at their pre-
ferred temperature can be used to calculate accel-
erated larval development and the subsequently al-
tered postmortem interval caused by maggot mass
formation. Although the larvae dispersed over a
relatively large range of preferred temperatures,
the mean temperature at which larval aggregations
were found was much cooler than expected. This
temperature adjustment lessened the effect of
maggot mass formation on temperature, which
may alter larval growth rates considerably.
Accurate documentation of ambient conditions
at the death scene and the microclimate of the
corpse is critical in postmortem interval estima-
tions. Controlled laboratory rearing may be re-
quired for accurate determination, and it is essen-
tial that such developmental studies be conducted
under temperatures with mean values comparable
to the conditions recorded on the body and at the
scene.
Acknowledgments
We thank Jon Allen, Dale Ilaheck (University of Flor-
ida), and Jeffrey D. Wells (University of California,
Berkeley) for careful reviews of the manuscript. We also
thank William Maples (C. A. Pound Human Identifica-
tion Laboratory, University of Florida) and William Ham-
ilton (District <S Medical Examiner's Office, Gainesville,
FL) for use of their facilities for specimen collection. This
357
article is published as Florida Agricultural Experiment
Station Journal Series No. R-05315.
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