Accepted Manuscript

Title: Insect succession on remains of human and animals in

Shenzhen, China

Author: <ce:author id="aut0005"

author-id="S0379073816305643-
fc3552a04dcca29272459958d5dbd33a"> Yu Wang<ce:author
id="aut0010" author-id="S0379073816305643-
46fc5552412aa089b0ba8f80e18a7a45"> Meng-yun
Ma<ce:author id="aut0015" author-id="S0379073816305643-
9f378deb197b17f2154a11a9ca233463"> Xin-yu
Jiang<ce:author id="aut0020"
author-id="S0379073816305643-
6a82ea75dc5cee0b1299cb2f5056adda"> Jiang-feng
Wang<ce:author id="aut0025"
author-id="S0379073816305643-
f41ef66083a87ee4c1f208249bbb1405"> Liang-liang
Li<ce:author id="aut0030" author-id="S0379073816305643-
493fa935ebdcadf5d5a656ddded1a221"> Xiao-jun
Yin<ce:author id="aut0035"
author-id="S0379073816305643-
d485975d616b1f0e0ab95ac1155daa3c"> Min
Wang<ce:author id="aut0040"
author-id="S0379073816305643-
d881f04d3b37e58d6aa8fac20190369d"> Yue Lai<ce:author
id="aut0045" author-id="S0379073816305643-
e53dab1b44860b71d1f42b2146c80eb7"> Lu-yang
Tao

PII: S0379-0738(16)30564-3
DOI: http://dx.doi.org/doi:10.1016/j.forsciint.2016.12.032
Reference: FSI 8710

To appear in: FSI

Received date: 11-11-2016

Revised date: 20-12-2016
Accepted date: 20-12-2016

Please cite this article as: Yu Wang, Meng-yun Ma, Xin-yu Jiang, Jiang-feng Wang,
Liang-liang Li, Xiao-jun Yin, Min Wang, Yue Lai, Lu-yang Tao, Insect succession
on remains of human and animals in Shenzhen, China, Forensic Science International
http://dx.doi.org/10.1016/j.forsciint.2016.12.032

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Title: Insect succession on remains of human and animals in Shenzhen, China
Yu Wanga,d,1, Meng-yun Mab,1, Xin-yu Jiangc, Jiang-feng Wanga,*, Liang-liang Lia, Xiao-jun Yina, Min
Wangd, Yue Laib , Lu-yang Taoa
a
Department of Forensic Medicine, Soochow University, Ganjiang East Road, Suzhou 215000, China.
b
Criminal Police Branch, Shenzhen Public Security Bureau, Jiefang Road, Shenzhen 518000, China.
c
Criminal Police Branch, Guangyuan Public Security Bureau, Chaoyang Road, Guangyuan 628000,
China.
d
Department of Entomology, College of Agriculture, South China Agricultural University, Wushan
Road, Guangzhou 510642, China.
*
Corresponding author at: Department of Forensic Medicine, Soochow University, Ganjiang East Road,
Suzhou 215000, China. Tel.: +86 181 5111 6801.
E-mail address: jfwang@suda.edu.cn (J. F. Wang).
1
The first two authors contributed equally to the study.

1
Highlights
Decomposition process of human, pig and rabbit were studied (118 days experiment).
Decomposition duration is in order of human > large pig > small pig > rabbit.
Insect assemblages (include life stages) were compared.
Development rates of the same fly species on all carcasses were consistent.
Basic entomological succession data of Shenzhen (China) was provided.

2
 Insect succession on remains of human and animals in Shenzhen, China

ABSTRACT Most forensic entomological succession studies have been carried out using pig or rabbit
carcasses; however, there have been few studies on the differences between insect succession patterns
on human cadavers and on animal carcasses. In order to clarify the differences between decomposition
and insect succession patterns of human cadavers and animal carcasses, one 49.5 kg human cadaver,
two large pig carcasses (45 and 48 kg), two small pig carcasses (23 and 25 kg) and two rabbit carcasses
(both 1.75 kg) were placed in the same field conditions in Shenzhen, China for a comparative study on
August, 2013. The results indicated that: (1) The duration from fresh to skeletonization is in order of
human cadaver > large pig carcasses > small pig carcasses > rabbit carcasses; (2) Insect assemblages
(including developmental stages) are more complex on larger carcasses, in order of human cadaver =
large pig carcasses > small pig carcasses > rabbit carcasses; (3) The developmental rates of the same
forensically important fly species on all carcasses are consistent; (4) All identified species of
Calliphoridae can complete development of one generation on human cadaver, and both large and small
pig carcasses, while on rabbit carcasses, only a subset of the Calliphoridae species can finish
development of one generation; (5) Beetles can generate offspring on human cadaver, and both large
and small pig carcasses, while they do not generate offspring on rabbit carcasses. This study provides
useful comparative data for decomposition and insect succession pattern of human cadaver with animal
carcasses.

Keywords Forensic entomology; Postmortem interval; Insect succession; Human cadaver; Carrion
decomposition; Comparative forensic medicine

3
Introduction
Investigating insect succession, along with developmental study of carrion insects, constitutes some of
the most fundamental and important work of forensic entomology. In the time since insect succession
theory on carcasses was first proposed by Mégnin [1], there have been a great number of studies on this
field. Through these studies, the succession patterns under different types of environment have been
observed, including land exposure [2,3], burying [4,5], indoor environments [6,7], vehicle
environments [8], ponds and rivers [9,10], oceans [11], dry environments [12,13], rainy seasons [14,15].
In further studies the effects of other factors have been examined, including the effects of burning
[16,17], drugs and toxins [18-20], hanging [21], clothing and covering [22-24], carcass size [24-28],
lime [29], penetrating injuries [30], scavengers [31], inter-species competition [32-34], altitude [35],
different seasons [36,37], and freezing [38,39].
Research methods in succession studies have been improved continuously, and standards for
setting replicates and controls that allow generation of more confident statistics have been proposed
[40,41]. There have also been some relevant studies of insect sampling methods [42-44], optimal
animal model [45-47], effects of daily sampling [48], the optimal inter-carcass distance [49], the effect
of repeated experiments at the same location [50], effects of freezing and thawing [39], and effects of
investigator disturbance [51].
Even though forensic entomology has achieved great accomplishments in the studies of succession,
unfortunately, most of these studies have drawn conclusions from animal carcasses [43,52,53]. There
exist differences between humans and other animals used in studies of succession, for example, humans
have longer living spans, walk upright, feed on a wide variety of food sources, wear clothing, have less
hair covering the body surface, take different kinds of medicines, take baths frequently and so on. Only
in a handful of areas around the world have human cadavers been used to conduct studies on forensic
insect succession [43,52,54,55], and in this aspect the „body farm‟ Anthropological Research Facility at
the University of Tennessee is widely renowned [56]. Early in 1983, Rodriguez and Bass [52] used four
human cadavers to study insect succession, and by comparing these results with the succession results
of 43 dogs by Reed [57] in the same area, they showed that the various insect species observed on
human cadavers were found to be the same as those on dog carcasses. A study by Schoenly et al. [43]
indicated that carrion insects had negligible preference for human cadavers versus pig carcasses, and
argued that pig carcasses weighing about 23-27 kg can substitute for human cadavers for experiments.
Since forensic entomology studies are mainly applied to estimating minimum postmortem interval
(PMImin) of humans, the use of human cadavers, if conditions allow, to conduct insect succession
studies is the best way to obtain accurate primary data for forensic entomology applications. The insect
succession patterns of human cadavers can vary dramatically, especially when we consider the variety
of different climatic zones on the earth, thus diverse studies using human cadavers are necessary.
Secondly, most forensic entomological succession studies around the world are carried out using pig or
rabbit carcasses, however, there is a dearth studies on the potential differences between insect
succession on pig and rabbit carcasses and that on human cadavers. Although the theory of 23-27 kg
pig carcasses as optimal models has been proposed for a long time and widely accepted, the proposal of
this theory also considered factors such as economy and operability; it is not necessarily true that pig

4
carcasses with such weights have exactly identical decomposing patterns and insect succession to
human cadavers. Therefore, it is necessary to carry out more comparative studies of decomposition and
succession between human cadavers and animal carcasses, and thus facilitate a comprehensive
reference for estimating PMImin. Moreover, comparative studies between human and animal remains
will aid in development of the discipline of comparative forensic medicine, leading to important
advances in understanding, and this will be of benefit to forensic disciplines of both human and animal
[58,59].

Materials and Methods

Experiment Site
This study was carried out in a forest of litchi Litchi chinensis Sonn. near the Forensic Autopsy
Centre of the Public Security Bureau of Shenzhen City. The litchi plants in the experiment site were
used as greening plants rather than fruit trees. The size of this experiment site is approximately 10 hm2
(500 m long and 200 m wide). There is an artificial lake about 150 m long and 50 m wide in the
experiment site, and the litchi forest and artificial grass were planted in a ring around the lake. There
are walls with height of about 5 m at the surrounding of the litchi forest. There are factories and
residential buildings around the experiment site, so generally the experiment site was in an urban area
(urban ecosystem) with frequent anthropogenic activities. The study area is located along the coast of
South China (22° 37′ 38″ N, 114° 09′ 30″ E), the altitude is 100 m, and it has a subtropical oceanic
monsoon climate with annual mean temperature being 23 °C. The summer in the area is very long and
lasts about 6 months, from May to December; the winter is warm, without snow, and with temperature
remaining above 0 °C.

Experimental Materials and Processing Methods

The cadaver of one human male of East Asian descent after postmortem examination in frozen
preservation (aged 19 years old, height of 170 cm, weighing 49.5 kg, Hm), two large pig carcasses with
weights close to that of the human cadaver: PgL1 and PgL2 weighing 48 and 45 kg, respectively, two
small pig carcasses whose weights were close to the recommended weights: PgS1 and PgS2 weighing
23 and 25 kg, respectively; two rabbit carcasses: Rt1 and Rt2 weighing both 1.75 kg were used in this
study. The human cadaver was donated by his family and the donation was strictly carried out and
registered according to the regulations of the Ethical Committee of Forensic center. Pigs Sus scrofa
domestica L. were bought from a commercial piggery and rabbits were purchased from the Laboratory
Animal Center of Southern Medical University. All animal studies were approved by the Institutional
Animal Care and Use Committee (IACUC) and carried out under the policies of Soochow University.
Animals were sacrificed on August 26th 2013 by blunt blow to the head at 8:00 to 9:00 am. The
human cadaver was thawed 12 h in advance in a closed room without insects. All the carcasses were
double-packaged with black plastic bags and transported to experiment field from 9:00 to 10:00 am.
The litchi trees are sparse, and the animal carcasses were placed in the open area within the woodland
where the sunlight could directly shine on the carcasses. The vegetation around the animal carcasses

5
consisted mainly of wild weeds. The experiment site was maintained so that the weeds in the litchi
forest were limited. The human cadaver was placed next to the Forensic Autopsy Centre where the
litchi trees meet the lawn, in order to facilitate our management. The vegetation around the human
cadaver was mainly artificial grass. The carcasses were placed with a north-south direction, and the
inter-carcass distance was ≥50 m. A metal cage (2 m×1.5 m×0.8 m) was placed on carcasses in order to
keep them from being eaten by scavengers. Iron fences with a height of 4.5 m were built around the
human cadaver to avoid disturbance.

Investigation Protocol
This study started on August 26th, 2013 and ended on December 21th, 2013. On the exact day of
placing carcasses, observation was conducted every two hours, and after that observation was
conducted twice every day at 9:00 am and 3:00 pm. After the carcasses became skeletonized,
observation was carried out once at 9:00 am every day. Insects were collected until no forensically
relevant taxa were present. When there was no forensically relevant taxon on carcasses, the
investigation was concluded. Field investigation tools were used as per the recommendation of Amendt
et al. [42].
The degree of carcass decay was observed, recorded, photographed and videoed. The degree of
decay of the three parts of carcass, i.e., head/neck, trunk, limbs were scored, according to the system
proposed by Megyesi et al. [60]. The scores of each part were added together to get the total body score
(TBS) (from a minimum of 3 to a maximum of 35 points), representing the decay degree of the total
carcass. Two Testo l75-H1 temperature and humidity recorders (Germany) were equipped 1.2 m above
the land surface in places sheltered from sun and rain, and temperature and humidity were recorded
automatically every hour.
Insect sampling was conducted along with observation. Samples were collected manually, and the
sampling objectives included eggs, larvae, pupae, adults and puparia. The observing and sampling areas
included above and beneath carcasses and above and below ground within the radius of 10 m
surrounding carcasses. During the sampling process, the impact on insects was minimized as far as
possible. For beetles that were easy to identify and were few in number, observation was conducted
without sampling. For insects above the ground, observation and sampling were conducted directly. For
sampling of insects distributed inside carcasses and those beneath carcasses, the carcasses were rolled
on to their side during sample collection. The sampling of insects in the soil was done by uncovering
soil carefully using long tweezers. Details such as appearance of pupation holes, eclosion holes and
exuvia, were recorded. The larval samples collected were placed into hot water with temperature ≥
90 °C for 30 s and then placed into 80% ethanol solution for preservation. Samples of pupae were
directly preserved in 80% ethanol solution. Adult insects were killed in a killing jar with ethyl acetate
and packaged with triangular paper bags. All collected samples were labeled and brought back to the
lab for identification.
Species identification was performed by visual examination in the field (only applying to insects
with very obvious characteristics, such as the Chrysomya rufifacies (Macquart), Necrobia ruficollis (F.)
and Creophilus maxillosus L.) and re-identification under a Zeiss Stemi 2000-C stereomicroscope

6
(Carlzeiss Germany) at the laboratory. Insects at different developmental stages were identified by
reference to Fan [61], Zhang et al. [62], Ji [63] and Wang [64].
We have previously carried out multiple studies of insect succession on pig carcasses in this region,
and identified insect species with forensic significance and the development process of each of these
species, and become familiar with their morphology. This meant that we were able to observe a high
level of detail in the current investigation, because, for example, we already knew the general time of
pupation and eclosion of C. megacephala, so in this study we could specifically focus on exactly when
these events would happen during the previously established time windows. In this study, we monitored
the key development time of important carrion insects on each carcass, including arrival of adults,
oviposition, hatching, start of wandering, peak of wandering, start of pupation, peak of pupation, start
of eclosion, peak of eclosion, etc. For insects of the same species, we only observed and recorded the
development time points of those insects that arrived first and grew fastest. We placed part of the eggs,
post-feeding larvae and pupae collected from carcasses and the surroundings into rearing boxes and
reared them beside bodies. The aim of the rearing is to remind us to ensure whether the development
events happened in the boxes were happened on the carcasses. For example, if a development event
such as pupation was happened in rearing box, and we would check the remains immediately to verify
if it was also happened on the remains. This method helps us to grasp the development events
accurately. The development data obtained on the remains were used in the data analysis. The rearing
boxes were well sheltered from rain.

Data Analysis
We adopted total body score (TBS) to evaluate the decomposition process of carcasses, and we
calculated accumulated degree days (ADD) by adding up average temperature every day. Data analysis
was conducted by using the Origin Pro 8.6. Linear regression analysis was performed by setting ADD
as the independent variable and TBS as the dependent variable.
Jaccard similarity index was used to evaluate the similarity in insect composition among different
types of carcasses, and the formula was as follows:
𝑁
Jaccard‟s index: q =
𝑁1 +𝑁2 −𝑁

N is the number of taxa shared between a pair of carcasses and N1 and N2 are the number of species
in each of the two carcasses.
A matrix was adopted and insect occurrence and succession on each carcass was displayed with a
day unit. Since carcasses were investigated twice a day before skeletonization, we combined the data of
the two investigations on the same day (e.g., if one species of insect occurred in both two investigations
or in either of investigations, the insect was considered as present, or else was considered absent). The
occurrences of adults, larvae, pupae and tenerals were all listed in the matrix. For the developmental
stage of some species that has never been observed was not listed in the matrix.
In the matrix, we used “▄”, “█” and blank to represent that a species was present on one carcass,
two carcasses and absent, and we used “-” to represent the circumstance that a species was expected to
be present on carrion but was not observed. For example, one species can be found on PgL carcass on
day 10, but cannot be found on day 11, and then be found on day 12. We believe that this species was

7
likely present on day 11 but was not found by us during that day‟s investigation.

Results
Environmental Conditions
The temperature was 7.6-34.1 °C (range 6.8 °C, mean 22.33 °C) and the humidity was 34.5-95.9%
RH (range 23.1% RH, mean 74.9% RH) during the study period. The temperature of the first 20 days
(during this period the bodies decomposed rapidly and the insects were most active) was 22.4-34.1 °C
(range 5.8 °C, mean 26.89 °C) and the average humidity was 76.9-93.7% RH (range 21.8% RH, mean
85.82% RH).

Comparison between Decaying Process of Human Cadaver and Animal Carcasses

The rabbit (Rt) and small pig (PgS) carcasses became completely skeletonized at the 5 d and 7 d
of postmortem and the decaying periods were very short, however, the human (Hm) cadaver and large
pig (PgL) carcasses still had relatively more mummified tissue at 20 d post-mortem. The mummified
tissue of carcass PgL completely decayed and disappeared until 80 d, while the mummified tissue on
the legs of carcass Hm remained until the end of investigation (118 d) (Fig. 2).

Total Body Scores of Different Remains in Different ADD

The changing relationship of TBS along with ADD is highly correlated with carcass size.
Carcasses with similar size have similar TBS changes patterns (Fig. 3). The decomposition rate of
rabbit carcasses was the fastest. The TBS of carcasses Rt1 and Rt2 reached the greatest value of 35
when their ADD values were 114.3 degree-days. The decomposition rate of small pig carcasses was
slightly slower than that of rabbit carcasses, and TBS values of carcasses PgS1 and PgS2 reached 35
when their ADD values were 161.3 and 187.1 degree-days. The decomposition rates of human cadaver
and large pig carcasses were the slowest and when their ADD values were within the range of
87.3-128.8 degree-days there existed a short plateau. TBS values of carcasses Hm, PgL1 and PgL2
reached 33 when their ADD values were 294.7, 267.8 and 294.7 degree-days, respectively. For a long
time after this point, the TBS values of the three carcasses did not change, therefore, the change of TBS
along with ADD change was not recorded in the figure thereafter.

Arthropod Composition on Different Carcasses

The carcasses yielded 42 species of arthropods, representing 5 orders and 18 families (Table 1),
among them Ptecticus aurifer (Walker) was a carrion insect reported on the carcasses in the insect
succession study for the first time. Carcasses Hm, PgL, PgS and Rt attracted 41, 40, 37 and 25 species
of arthropods, respectively.
As was shown in Table 2, the Jaccard similarity index between cadaver Hm and carcasses PgL was
the greatest with a value of 0.929, that between Hm and Rt was the lowest with a value of 0.610, while
that between Hm and PgS was modest at 0.881. These results indicated that the similarity in species
composition between human cadaver and large pig carcasses was the highest and that between human
cadaver and rabbit carcasses was the lowest.

8
 Combined with insect composition and the developmental stages that appeared on carcasses,
insect assemblages are more complex on larger carcasses in order of human cadaver = large pig
carcasses > small pig carcasses > rabbit carcasses (Table 3).

Comparison of Developmental Process of Important Carrion Insects on Different Carcasses

We used key developmental time points to evaluate the developmental process of insects and the
key time points adopted included: arrival of adults, oviposition, hatching, start of wandering, peak of
wandering, start of pupation, peak of pupation, start of eclosion, and peak of eclosion. Eleven key
development time points were recorded for Diptera (Fig. 4), and six key development time points were
recorded for Coleoptera (Fig. 5). We observed that five species of Dipteran: Chrysomya megacephala
(F.), Chrysomya rufifacies (Macquart), Chrysomya nigripes Aubertin, Hydrotaea spinigera Stein and
Hermetia illucens (L.) completed development on the Hm cadaver and PgL carcasses. Four insect
species of Diptera: C. megacephala, C. rufifacies, C. nigripes and H. spinigera completed development
on the PgS carcasses, while on rabbit carcasses only C. megacephala and C. rufifacies completed
development, and although the C. nigripes and H. spinigera arrived carcasses and colonized, they
didn‟t successfully finish development. Five Coleopteran insects all completed arrival and colonization
on the Hm cadaver and PgL carcasses. Although five species of insects arrived on PgS carcasses, only
Diamesus osculans Vigor completed colonization. The beetles selected did not generate offspring on
rabbit carcasses, therefore no key development time points were recorded in Fig. 5.
C. megacephala and C. rufifacies complete development on all the carcasses. We compared the
developmental process of the two fly species (Fig. 6). Results indicated that the time points of
wandering (T4), pupation (T6), eclosion (T8) of C. megacephala and C. rufifacies on Hm, PgL, PgS
and Rt carcasses were consistent: the two fly species entered into the next development phase on the
same day respectively, implying that the development rate of the same species of fly on different
carcasses were consistent. Our results also showed that the same species existed for a shorter period on
small carcasses than on large carcasses (T10 and T11).

Arthropod Succession Pattern on Different Carcasses

The arthropod succession on Hm cadaver was the most complicated and lasted for the longest time.
Arthropods with forensic significance could still be found at the end of investigation on 118 d (Table 4).
The arthropod succession on PgL carcasses was similar to that on Hm cadaver, but it lasted for a
relatively shorter time (82 d) (Table 5). Comparing the Hm cadaver and PgL carcasses, arthropod
succession on PgS carcasses was relatively simple and lasted for 33 d (Table 6). The arthropod
succession on Rt carcass was the simplest and lasted only for 12 d (Table 7).
The first insects arriving at and colonizing carcasses were C. megacephala and C. rufifacies. Their
larvae dominated on carcasses during first 3 d postmortem. Then a second wave of Diptera such as C.
nigripes and H. spinigera arrived at carcasses and began mass reproduction; and their larvae survived
until the early stages of skeletonization. He. illucens was the last wave Dipteran insect and its larvae
existed in the soil below carcasses, which was rich in humus, especially underneath the human cadaver.
At the end of the investigation there were still He. illucens larvae. Coleopteran adult insects arrived on

9
carcasses at 3 d postmortem and the occurrence time of their larvae was very late. Necrobia rufipes
(DeGeer), Necrobia ruficollis (F.), Dermestes maculates (DeGeer) were the dominant species during
the skeletonization period. There were also a large amount of mites and psocids found on skeletonized
carcasses.

Discussion
We adopted the system of TBS proposed by Megyesi et al. [60] instead of the system of
decomposition stage to evaluate the decomposition process of carcasses. This method enabled us to
compare the decomposition process of different carcasses in a more objective way, and the relationship
between TBS and ADD analyzed by linear regression can be used for estimating the PMI (Supp. Table
S1).
Several researchers have carried out studies on decomposition and insect succession of varying
sizes of pig carcasses. The unpublished data of Haskell indicates that the amount of carrion available
strongly influenced the decay rate (an infant corpse decomposed in 5 days, while the adult took 3-5
times longer) [53]. Komar and Beattie [27] found that the decomposition rate of small pig carcasses
(19-26 kg) was significantly faster than that of medium (36-80 kg) or large pig carcasses (156-162 kg).
Sutherland et al. [25] pointed out that the decomposition rate of small pig carcasses (<30 kg) was 2.82
times faster than that of large carcasses (60-90 kg). Similarly, Matuszewski et al. [24,28] compared four
types of pig carcasses (small carcasses 5-15 kg, medium carcasses 15.1-30 kg, medium/large carcasses
35-50 kg, large carcasses 55–70 kg), and concluded that the body size is a factor of key importance for
decomposition and carrion entomofauna. Compared with small carcasses, the insect assemblage was
more complex, abundant and lasted longer on large carcasses. In the present study, the decomposition
rates of carcasses with small weight were all faster than that of carcasses with large weight; the rabbit
carcasses decayed the fastest, small pig carcasses came second, and the large pig carcasses and human
cadaver decomposed the slowest, which is consistent with the previous studies mentioned above.
We found that the key time points such as pupation and eclosion of C. megacephala on all
carcasses were identical (evaluated by day, for example, the start of pupation and eclosion on all
carcasses happened on 5th and 11st d, respectively) based on our twice-daily investigation, implying that
the growth and development of the insects was not affected by the types of the carcass or its size.
However, take the hot weather of the study area and twice-daily investigation into consideration, there
may have differences if the investigations are conducted more frequently (for example 6 times daily) or
research is conduct under a cooler environment. As faster growth rates of larvae on smaller carcasses
were suggested by Matuszewski et al. [24], and larvae were found pupated earlier and emerged as
smaller adults when short of food by Greenberg [65]. Further research is needed.
Rabbits are the second most commonly used animals, behind pigs, for succession studies. This
study provided a comparison between succession on rabbit carcasses and that on a human cadaver. We
found that, compared with a human cadaver, the succession on rabbit carcasses was very simple and
lasted for a shorter time. We only found two species of beetles on rabbit carcasses, and both species of
beetle failed to generate offspring on carcasses. Few species of Coleoptera were reported using rabbits
as the model animal [66-68], which is consistent with our result.

10
 Different insects inhabit different habitats, therefore during crime investigation the insect
assemblage can be used to determine where death or decomposition happened [69]. In some
circumstances, however, even in the same habitat there may be differences in insect colonization and
body decomposition due to microenvironment differences. We found that the decomposition rates of
two pig carcasses (one pig was exposed under the sunlight, while the other was place under the shade
of a big tree) with similar weight were significantly different, even though they were placed in the same
forest in Zhongshan (southern China) (unpublished data). In the current study, we aimed to fully
control for the effect of microenvironment differences and kept the microenvironment of each carcass
as consistent as possible. This study was carried out in a small greening woodland in a highly urbanized
urban area. We therefore consider that the primary habitat of the experiment site was an urban
ecosystem. The insect activity was affected by frequent anthropogenic activities, and this may be
responsible for the relatively low diversity of insects found in this study. The insect assemblage was a
little different from the previous studies conducted in the same region, although the dominant species
were identical [70,71]. Further studies are needed concerning the effect of different habitats on insect
assemblage in this region.
In this study the human cadaver was observed closely for 118 days, and we conducted day-by-day
observation about the timing of cadaver decomposition phenomena and of skeletonization of each part
of the cadaver (Supp. Table S2 and Supp. Video S3), which provides precious data for forensic
pathology and forensic entomology. The human cadaver decomposed very fast due to the hot weather,
and the cadaver was largely skeletonized on day 11 (TBS reached 33; maximum 35). Similarly,
Campobasso and Introna [72] observed a case of per-skeletonization occurring as early as 10 days
postmortem in a young male found in September (mean temperature fluctuating between 20-26 °C) in
southern Italy. Galloway et al. [73] also described an exceptional case of per-skeletonization occurring
as early as 7 days after death. We identified a major difference in decomposition between the human
cadaver and pig carcasses, in that the soft tissue of the human lower limbs decomposed most slowly,
likely because fewer oviposition occurred in this region of the body, therefore the legs mummify as
they have fewer larvae feeding on them and speeding up decomposition.
In addition to the systematic comparison of decomposition and succession between human
cadaver and animal carcasses, this study also provides fundamental data for forensic entomological
succession in the coastal areas of southern China. This study has identified 42 carrion-associated
arthropods in total. We identified P. aurifer on the carcasses in our study, which is the first time that this
insect has been identified as a carrion insect in any insect succession study. This suggests that P. aurifer
may be able to be used as an indicator to estimate PMImin. The environmental characteristics of the
coastal areas in the southern China are hot weather, rain in all four seasons, no snowfall and frequent
anthropogenic activities. The succession characteristics in the area are: carcasses decay fast, insect
activities are not impacted by low temperatures, and due to human activities, diversity of carrion
insects is relatively low and dominant species are significant. The application of forensic entomology in
this area is different from that of other regions (frigid and temperate zones), for example, the estimated
PMImin based on insects is shorter than that in other temperate and frigid areas as the development of
insects is more rapid (e.g. it takes 5 days for C. megacephala to pupate and 11 days to eclosion), and in

11
this area we cannot estimate PMImin based on the seasonal change of insects [70].
As acquisition of human corpses is highly regulated by legal, ethical and religious challenges, and
involves complicated administrative approval processes, we were only able to obtain one human corpse
for this study, which is unfortunate as we intended to have two adult and two child human corpses in
order to have a biological replicate of the human corpse observation. Moreover, the cadaver has
undergone a postmortem examination and frozen, which may have affected the body decomposition
and insect succession. For further research, we aim to improve advocacy work and strive for more
human body donations to conduct further forensic investigations on human corpses, to take advantage
of further scientific investigation methods to conduct studies in other climatic zones of China, and to
provide a database for forensic entomology in China.

Acknowledgements
This study was supported by the grant from National Natural Science Foundation of China (No.
30870332, No. 81273352) and Priority Academic Program Development of Jiangsu Higher Education.
We thank the two anonymous reviewers for their comments and suggestions.

12
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17
 Table 1 The occurrence of arthropod species on different remains (August 2013, Shenzhen, China)

Order Family Species Hm PgL PgS Rt

n=1 n=2 n=2 n=2
Diptera Calliphoridae Chrysomya megacephala (F.) 1 2 2 2
Chrysomya rufifacies (Macquart) 1 2 2 2
Chrysomya nigripes Aubertin 1 2 2 2
Lucilia bazini Seguy 1 2 2 1
Lucilia sericata (Meigen) 1 2 2 2
Chrysomya villeneuvi Patton 1 2 2 2
Chrysomya chani Kurahashi 1 2 2 2
Hemipyrellia ligurriens (Wiedemann) 1 2 2 2
Sarcophagidae Boettcherisca peregrina (R.-D.) 1 2 2 2
Parasarcophaga crassipalpis (Macquart) 1 2 2 2
Muscidae Musca domestica L. 1 1 - 2
Hydrotaea spinigera Stein 1 2 2 2
Muscina stabulans (Fallen) 1 - 2 2
Stratiomyidae Hermetia illucens (L.) 1 2 1 -
Ptecticus aurifer (Walker) - 1 - -
Sepsidae Sepsis sp. 1 2 2 2
Phoridae Megaselia scalaris Loew 1 2 2 2
Coleoptera Histeridae Saprinus splendens (Paykull) 1 2 2 1
Merohister jekeli (Marseul) 1 2 2 -
Staphylinidae Creophilus maxillosus L. 1 2 2 -
Philonthus spinipes Sharp 1 2 2 -
Aleochara sp. 1 2 2 2
Scarabaeidae Onthophagus taurinus White 1 2 1 -
Onthophagus proletarius Harold 1 1 2 -
Cleridae Necrobia rufipes (DeGeer) 1 2 2 -
Necrobia ruficollis (F.) 1 2 2 -
Dermstidae Dermestes maculatus (DeGeer) 1 2 2 -
Dermesters tessellatocollis Motschulsky 1 1 2 -
Silphidae Diamesus osculans Vigor 1 2 2 -
Hydrophilidae Sphaeridium scarabaeoides (L.) 1 2 1 -
Hymenoptera Vespidae Vespa bicolor F. 1 2 2 2
Vespa velutina nigrithorax Buysson 1 2 2 2
Vespa affinis (L.) 1 2 2 1
Vespula flaviceps (Smith) 1 - 1 -
Formicidae Pheidologeton affinis (Jerdon) 1 2 1 1
Paratrechina longicornis (Latreille) 1 1 2 2
Camponotus variegatus (F. Smith) 1 2 2 2
Odontoponera transversa (Smith) 1 2 1 1
Pteromalidae Nasonia vitripennis (Walker) 1 2 2 2
Acarina Acaridae Acarus siro L. 1 2 - -
Tyroglyphus sp. 1 2 - -
Corrodentia Liposcelididae Liposcelis sp. 1 2 - -
1 represents that the species appeared on one carcass, 2 represents that the species appeared on two
carcasses, and - represents that the species did not appear on carcasses.

18
Table 2 Jaccard similarity index of arthropod community of different remains
Hm PgL PgS Rt
n=1 n=2 n=2 n=2
Hm 0.929 0.881 0.610

PgL 0.814 0.585

PgS 0.615

19
Table 3 The occurrence of developmental stages of key species on remains (August 2013, Shenzhen,
China)
Species Hm PgL PgS Rt
n=1 n=2 n=2 n=2
A L P A L P A L P A L P

C. megacephala √ √ √ √ √ √ √ √ √ √ √ √
C. rufifacies √ √ √ √ √ √ √ √ √ √ √ √
C. nigripes √ √ √ √ √ √ √ √ √ √ √
H. spinigera √ √ √ √ √ √ √ √ √ √ √
He. illucens √ √ √ √ √ √ √
D. maculatus √ √ √ √ √ √ √
N. rufipes √ √ √ √ √
N. ruficollis √ √ √ √ √
C. maxillosus √ √ √ √ √ √ √
S. splendens √ √ √ √ √
Di. osculans √ √ √ √ √ √ √ √
Sepsis sp. √ √ √ √
Philonthus sp. √ √ √ √
A. siro √ √
Tyroglyphus sp. √ √
Liposcelis sp. √ √
Total 16 11 8 16 11 8 16 5 4 6 4 2
√ represents that the taxon was found on the carcass, and blank represents that the taxon did not appear
on carcasses.

20
 Table 4 Arthropod succession on Hm cadaver in Shenzhen, China (August 2013, n=1)
PMI (d) Stage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 28 30 32 34 37 39 41 43 47 52 54 57 60 64 68 74 80 83 86 89 92 95 98 104 115 118
C.megacephala A/T ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

C. rufifacies A/T ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

C. nigripes A/T ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

H. spinigera A/T ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - ▄ ▄ ▄ ▄ - ▄ - - - ▄
L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - - - ▄
He. illucens A/T ▄ ▄ - - ▄ ▄ ▄ - - - ▄ ▄ - - ▄ ▄ - ▄ ▄ ▄ ▄ ▄ - - ▄ ▄ - ▄
L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ - ▄ - ▄ ▄
D. maculatus A/T ▄ - - ▄ ▄ ▄ ▄ ▄ ▄ ▄ - - ▄ - ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - - ▄ - ▄ ▄ ▄ ▄ ▄ ▄ ▄ - - ▄
L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄ - ▄ ▄ - - ▄ - ▄
Di. osculans A/T ▄ ▄ - ▄ ▄ - ▄ ▄ ▄ ▄

L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

P ▄ ▄ ▄ ▄

N. ruficollis A ▄ ▄ - - ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄
L ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - ▄ - ▄ ▄ ▄ ▄
Cr. maxillosus A/T ▄ - ▄ - ▄ ▄ ▄ - ▄ - ▄ ▄ ▄ ▄ ▄

L ▄ ▄ ▄ - ▄ ▄ - ▄ ▄
P ▄ ▄ - ▄ - - - - - ▄ - - ▄ ▄ - ▄
S. splendens A ▄ - - ▄ ▄ - - ▄ ▄ ▄ ▄ ▄ ▄ - ▄
L - ▄ ▄ - ▄ ▄ ▄ ▄ ▄ - ▄ ▄ ▄
Aleochara sp. A ▄ - - ▄ ▄ ▄ - - - ▄ - ▄ ▄ ▄ ▄ ▄ - ▄ ▄ - ▄
Sepsis sp. A ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - - - ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ - ▄ ▄ - ▄
A. siro A ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Liposcelis sp. A ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

: adults, : larvae, : pupae, : tenerals, A: adults, L: larvae, P: pupae, T: tenerals, ▄ represents the taxon appeared on human cadaver, - represents the taxon
was expected to appear but were not observed on cadaver during the investigation, and blank represents that the taxon didn‟t appear on human cadaver. N. rufipes and N.
ruficollis, A. siro and Tyroglyphus sp. appeared on the remains at the same time so data were merged during the statistical analysis.

21
 Table 5 Arthropod succession on PgL carcasses in Shenzhen, China (August 2013, n=2)
PMI (d) Stage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 28 30 32 34 37 39 41 43 47 52 57 64 71 74 79 82
C. megacephala A/T █ █ █ █ █ █ █ █ █ █ █ ▄

L █ █ █ █ █ █ █ █ █ █ ▄

P █ █ █ █ █ █ █ █ █ ▄

C. rufifacies A/T █ █ █ █ █ █ █ █ █ █ █ █

L █ █ █ █ █ █ █ █ █ █

P █ █ █ █ █ █ █ █ █ █ ▄

C. nigripes A/T █ █ - - █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ - █ - █ █

L █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █

P █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █

H. spinigera A/T █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ - █ █ █ █ █ █ █ █ █

L █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █

P █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █

He. illucens A/T ▄ - ▄ █ - - ▄ ▄ █ ▄ ▄

L █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ - █ █

P ▄ ▄ ▄ ▄

D. maculatus A/T ▄ █ █ █ █ █ █ - - █ █ █ █ █ █ █ - - █ - █ █ █ █ █ █

L █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ ▄ ▄

P ▄ █ █ ▄

D. osculans A/T ▄ - █ █ █ █ █

L █ █ █ █ █ █ █ █ █ ▄ ▄

P ▄ ▄ █ █ ▄ ▄

C. maxillosus A/T █ ▄ - █ █ ▄ █ █ █

L █ █ █ - █ █ █ - █ █ █

P ▄ █ ▄ - - - ▄ ▄ - - ▄ ▄ - █

N. rufipes A █ █ █ █ █ █ - █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ - █ - █ █ █ - - ▄ ▄ ▄

L █ █ █ █ █ █ █ █ █ █ █ - █ - █ ▄ ▄ ▄

S. splendens A █ ▄ █ █ █ █ - ▄ █ █ █ ▄ █ █ █

L █ █ - █ - █ █ - █ █ █ ▄ ▄ █

Aleochara sp. A █ █ █ █ █ █ █ █ ▄ █ █ █ - - █ █ █ █ ▄ █ - - █ - █ █ █ - █ █ - ▄ ▄

Sepsis sp. A █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ ▄ ▄ ▄

A. siro A █ █ █ █ █ █ █ █ █

Liposcelis sp. A █ █ █ █ █ █ █ █

: adults, : larvae, : pupae, : tenerals, A: adults, L: larvae, P: pupae, T: tenerals, ▄ represents that the taxon appeared on either of the two large pig carcasses,
█ represents that the taxon appeared on both of the two large pig carcasses, - represents the taxon was expected to appear but was not observed on carcasses during the
investigation, and blank represents that the taxon didn‟t appear on carcasses. N. rufipes and N. ruficollis, A. siro and Tyroglyphus sp. appeared on the remains at the same time
so data were merged during the statistical analysis.

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 Table 6 Arthropod succession on PgS carcasses in Shenzhen, China (August 2013, n=2)
PMI (d) Stage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 28 30 33
C. megacephala A/T █ █ █ █ █ █ █ █ █ █ ▄

L █ █ █ █ █

P █ █ █ █ █ █ █ █ █ █ █

C. rufifacies A/T █ █ █ █ █ █ █ █ █ █

L █ █ █ █ █ █ █ █ ▄

P █ █ █ █ █ █ █ █ █ ▄

C. nigripes A/T █ █ █ █ █ █ █ █

L █ █ █ █ █ █ █ █ █ █

P █ █ █ █ █ █ █ █ █ ▄

H. spinigera A/T █ █ █ █ █ █ █ █ █ █

L █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █ █

P █ █ █ █ █ █ █ █ █ █

He. illucens A █

C. maxillosus A █ █ - █

Di. osculans A █ █ █

L █ █ █ █ █

N. ruficollis A █ █ ▄ █ █ █ ▄ █ █ █ ▄

D. maculatus A █ █ █ █ █ █

S. splendens A █ █

Aleochara sp. A █ █ █ - █ █ █ - █ █ █ █ - - █ █ █

Sepsis sp. A █ █ █ █ █ █ - - █ █ - █ █ █ █ █ █ █ █ █ █ █

: adults, : larvae, : pupae, : tenerals, A: adults, L: larvae, P: pupae, T: tenerals, ▄

represents that the taxon appeared on either of the two small pig carcasses, █ represents that the

taxon appeared on both of the two small pig carcasses, - represents the taxon was expected to appear

but was not observed on carcasses during the investigation, and blank represents that the taxon didn‟t

appear on carcasses. N. rufipes and N. ruficollis appeared on the remains at the same time so data were

merged during the statistical analysis.

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 Table 7 Arthropod succession on Rt carcasses in Shenzhen, China (August, 2013, n=2)
PMI (d) Stage 1 2 3 4 5 6 7 8 9 10 11 12
C. megacephala A/T █ █ █ █ █

L █ █ █ ▄

P █ █ █ █ █ █ █

C. rufifacies A/T █ █ █ █

L █ █ █ █ ▄

P █ █ █ █ █

C. nigripes A █ █

L █ █ █ █

H. spinigera A █ █ █

L █ █ █ ▄

Aleochara sp. A █ - ▄

Sepsis sp. A █ ▄ █

: adults, : larvae, : pupae, : tenerals, A: adults, L: larvae, P: pupae, T: tenerals, ▄

represents that the taxon appeared on either of the two rabbit carcasses, █ represents that the taxon

appeared on both of the two rabbit carcasses, - represents the taxon was expected to appear but was not

observed on carcasses during the investigation, and blank represents that the taxon didn‟t appear on

carcasses.

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Figure Captions (Fig. 1-Fig. 6)

Fig. 1 Daily temperature and humidity at Shenzhen over the 118-d study period (26 August-21

December 2013)

Fig. 2 Comparison of decaying process of human cadaver and animal carcasses. (a) 1 d postmortem,

carcasses were fresh. (b) 3 d postmortem, human cadaver, large and small pig carcasses were bloated

while rabbit carcasses were highly decomposed. (c) 5 d postmortem, human cadaver and large pig

carcasses were bloated, small pig carcasses were highly decomposed, and rabbit carcasses were

skeletonized. (d) 7 d postmortem, human cadaver and large pig carcasses were highly decomposed, and

small pig carcasses and rabbit carcasses were skeletonized. (e) 10 d postmortem, human cadaver and

large pig carcasses were highly decomposed, small pig carcasses and rabbit carcasses were

skeletonized. (f) 15 d postmortem, human cadaver and large pig carcasses still remained mummified

tissue. (g) 20 d postmortem, remaining tissue on human cadaver and large pig carcasses decomposed

slowly. (h) 30 d postmortem. (i) 80 d postmortem, large pig carcasses were completely skeletonized

while there remained mummified tissue on the legs of the human cadaver. (j) 118 d postmortem.

Fig. 3 Scatter plot of TBS changing along with ADD for human cadaver and animal carcasses (August

2013, Shenzhen, China)

Fig. 4 Key developmental time points of Diptera on different remains (Mean±SD). (T1) Adults appear.

(T2) Adults laying eggs. (T3) Larvae appear. (T4) Start of wandering. (T5) Peak of wandering. (T6)

Start of pupation. (T7) Peak of pupation. (T8) Start of eclosion. (T9) Peak of eclosion. (T10) Larvae

disappear from remains. (T11) Adults disappear from remains. Each symbol represents the mean, and

error bar represents standard deviation.

Fig. 5 Key developmental time points of Coleoptera on different remains (Mean±SD). (T1) Adults

appear. (T2) Larvae appear. (T3) Start of pupation. (T4) Start of eclosion. (T5) Larvae disappear from

remains. (T6) Adults disappear from remains. N. rufipes and N. ruficollis appear on the remains at the

same time so data were merged during the statistical analysis. Each symbol represents the mean, and

error bar represents standard deviation.

Fig. 6 The comparison of the developmental time points of two fly species on different remains. (T1)

Adults appear. (T2) Adults laying eggs. (T3) Larvae appear. (T4) Start of wandering. (T5) Peak of

wandering. (T6) Start of pupation. (T7) Peak of pupation. (T8) Start of eclosion. (T9) Peak of eclosion.

(T10) Larvae disappear from remains. (T11) Adults disappear from remains. Each symbol represents

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the mean.

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