Accepted Manuscript Title: Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds? Experimental data and implications for forensic entomology Author: Damien Charabidze Aurore Depeme Cedric Devigne Valery Hedouin PII: DOI: Reference:
S0379-0738(15)00218-2 http://dx.doi.org/doi:10.1016/j.forsciint.2015.05.025 FSI 8017
To appear in:
FSI
Received date: Revised date: Accepted date:
12-11-2014 18-5-2015 19-5-2015
Please cite this article as: D. Charabidze, A. Depeme, C. Devigne, V. Hedouin, Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds? Experimental data and implications for forensic entomology, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.05.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Title Page (with authors and addresses)
Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds? Experimental data and implications for forensic entomology Damien Charabidze 1, 2, Aurore Depeme 1, 2, Cedric Devigne 1, 2, 3, Valery Hedouin 1, 2
2 Forensic Taphonomy Unit, UDSL, Lille, France
cr
3 Laboratoire Ecologie & Biodiversité, UCLILLE, FLST, Lille, France
ip t
1 Université Lille Nord de France, Lille, France
Ac
ce pt
ed
M
an
us
Contact: [email protected]
Page 1 of 22
*Highlights (for review)
1
Highlights
Egg-laying behaviour was studied under controlled conditions on rat cadavers.
3
No eggs were observed inside the wounds in any of the replicates.
4
Flies laid more eggs on the wet and short hair areas than on control areas.
5
Submerging eggs in liquid strongly affected their survival rate and development.
6
These data question the literature assertion that “blowflies lay their eggs in wounds”.
Ac ce p
te
d
M
an
us
cr
ip t
2
1 Page 2 of 22
*Manuscript (without author details)
Abstract
2
This study was designed to examine the common belief that necrophagous blowflies lay their
3
eggs in wounds.
4
The egg-laying behaviour of Lucilia sericata was observed under controlled conditions on wet,
5
artificially wounded or short-haired areas of rat cadavers. Flies laid significantly more eggs on
6
the wet area and the area with short hair than on the dry area or area with long hair. No eggs were
7
observed inside the wounds in any of the replicates.
8
The effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae
9
was also investigated. In low water condition, an average of 72.7 ± 7.9 % of the larvae survived
an
us
cr
ip t
1
and they reached a mean length of 7.5 ± 0.6 mm. In contrast, submerging eggs under a high
11
volume of water strongly affected their survival rate (25 ± 3.7 %) and development. Similar
12
results were observed using unfrozen pig blood instead of water.
13
These data question the information found in the literature regarding the preferential egg-laying
14
behaviour of Calliphorids flies in wounds.
d
te
Ac ce p
15
M
10
16
Keywords: oviposition; behaviour; colonization process; evidence interpretation; forensic
17
investigation
18 19
1 Page 3 of 22
This study investigate the factors that affect the oviposition sites of Lucilia sericata (Diptera:
21
Calliphoridae) (Meigen, 1826), a very common blow fly species of forensic interest. Blowflies
22
(Diptera: Calliphoridae) have an highly developed olfactory system that allows them to detect
23
corpses at a great distance (1–4). It is frequently reported in forensic entomology manuals
24
that once on the cadaver, blowflies lay their eggs in natural orifices and wounds (5–10). The
25
presence of other eggs, larvae or adult individuals can also act as an attractive signal to gravid
26
females and can increase the likelihood of oviposition in a given area (1,11–13).
27
Oviposition in natural openings, especially on the face, is often observed in field conditions. Due
28
to their weak mouth hooks, first instars are unable to attack hard tissues (e.g., muscle, skin, etc.)
29
and consequently feed on soft areas such as mucous membranes or brain (14–16). Nostrils offer
30
both protection and a suitable place for larvae to feed, and they are often heavily colonized. Byrd
31
& Tomberlin also note that when excrement-soiled clothing is present, flies often deposit their
32
eggs in these areas, a fact that highlights the importance of surface humidity in determining egg-
33
laying behaviour (in 8). On the other hand, larvae feeding on wounds are frequently observed in
34
the context of wound myiasis, i.e. infestation of living animals by fly larvae. In Europe, Lucilia
35
the main genus involved in human myiasis and is a common pest in sheep, responsible for costly
36
flies strike (17–19). It is also common in forensic pathology to observe deep alterations of wound
37
characteristics due to necrophagous larvae feeding at the injury site (20,21). But the observation
38
of larvae feeding in wounds does not require that eggs be laid in these areas. Actually, there
39
appears to be no experimental or quantified data in the literature to support the assertion that
40
blowflies lay their eggs in wounds.
Ac ce p
te
d
M
an
us
cr
ip t
20
41 42
To highlight the importance of this question in a forensic context, it is interesting to consider a
43
case that we analyzed some years ago. In 2009, the naked body of a young woman was 2
Page 4 of 22
discovered in a corn field a few hours after her disappearance. Several wounds caused by a sharp
45
object were present, and her hair was soaked with blood. Throttle traces were also observed on
46
the neck. An autopsy concluded that the victim was first throttled but did not die of asphyxiation
47
and was subsequently stabbed in the head following an attempted rape. The only forensic
48
entomology evidence was the unhatched fly eggs that were discovered in the eyes and nostrils of
49
the victim, which were identified as belonging to L. sericata. Because thousands were sampled
50
and even more were visible, it was clear that the colonization of these flies took place at a time
51
that the body was easily accessible (i.e., when it was in the corn field). Based on what has been
52
published in the literature, the complete lack of eggs on the bloody wounds would appear to
53
suggest that the victim had not already been stabbed when the Calliphorids flies oviposited on the
54
corpse. However, this scenario was inconsistent with subsequent investigations, and it was lastly
55
proven that the victim was stabbed before being deposited in the field where she was discovered.
56
Colonization by blowflies likely occurred at this time, but the flies did not lay any eggs in her
57
wounds.
cr
us
an
M
d
te
Ac ce p
58
ip t
44
59
On the basis of this case and to determine the reliability of the common belief that necrophagous
60
blowflies lay their eggs in wounds, the egg-laying behaviour of Lucilia sericata was observed
61
under controlled conditions on wet, artificially wounded or short-haired areas of rat cadavers. The
62
effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae was
63
also investigated.
64 65
Material & Methods
66
Experiments were performed on a laboratory L. sericata population raised and maintained in
67
50X50X50 cm gauze-covered cages. Inbreeding was reduced by adding monthly wild type 3
Page 5 of 22
(France) individuals. Adult flies (250±100) from a single emergence pool (hatching=D0) were
69
maintained at 20±2°C and 60±15% RH with a 12:12 photoperiod for a maximum of 20 days.
70
Minced beef liver was provided during the 7 first days to promote female ovarian development
71
and was then removed. After four more days, females were gravid and ready for experiment
72
(D11) (22).
ip t
68
cr
73 Oviposition on wet, wounded or short-haired cadavers
75
In this experiment, we investigated the egg-laying behaviour of L. sericata gravid females on
76
male laboratory rat cadavers (Ratus norvegicus). The setup was designed to test the likelihood
77
that females would lay eggs in wet, wounded or short-haired areas of the cadaver compared to
78
untreated control areas. Experiments were conducted from May until July between the hours of
79
10 a.m. and 3 p.m. (a 5 h period) in a laboratory room with artificial neon lighting. The room
80
temperature was kept at 24±2 °C and 60±15% RH.
81
For each tested condition, 20 gravid females and 10 males from the same pool were sorted and
82
placed in 30*30*30 cm gauze cages; caster sugar and water were provided ad libitum. A CO2-
83
killed, freshly unfrozen white rat cadaver (stored in the freezer during 3 to 5 month, 326±22 g)
84
was placed on a piece of cardboard in the cage on its ventral or dorsal side (the side was
85
alternated between replicates). Four experimental conditions were tested, each of which was
86
performed with 8 replicates (7 for Control):
87
Control: untreated rat cadavers.
88
Wet VS Dry: one half (longitudinally) of the rat cadaver was submerged in water for 15 s. The
89
wet side was alternated between experimental replicates.
90
Wounded VS Healthy: a 4 cm long transcutaneous wound was made with a surgical scalpel
91
horizontally on the flank of the rat. The entrails inside abdominal cavity were visible but not
Ac ce p
te
d
M
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us
74
4
Page 6 of 22
transected. The side of the wound (i.e., the right or left side of the rat) was alternated between
93
replicates.
94
Bare VS Haired: one half (longitudinally) of the rat cadaver was shaved using an electric shaver.
95
The hair was cut to a length of 1 mm, and the skin was carefully inspected to exclude any
96
possible skin lesions. The shaved side was alternated between replicates.
cr
97
ip t
92
After a 5 h period with the cadaver, all flies were removed and killed. Their size was measured
99
using the length of the posterior cross vein (dm-cu) (23). The eggs were removed using fine
100
forceps and a louse comb and placed in separate vials. These vials were kept at 3±1°C until
101
counting (24 h to 36 h after the start of the experiment).
102
The location of the eggs was noted as follows:
103
Control/tested side. For the wound condition, the location relative to the wound (in or outside)
104
was also reported.
105
Natural orifices (eggs located in the natural orifices were counted separately from those in the
106
corresponding body areas): face orifices (ears, nostrils, eyes, mouth) and anus.
107
Body area: (1) head, (2) fore legs, (3) abdomen, (4) hind legs and (5) tail and location
108
Beyond or underneath the cadaver.
an
M
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Ac ce p
109
us
98
110
Effects of egg immersion on the survival rate of blow fly larvae
111
This experiment was designed to assess the effect of body fluids, which can flow or accumulate
112
in wounds, on the survival rate off eggs and development of larvae. Fresh beef liver (25±5 g) was
113
introduced into fly-rearing cages and checked hourly until the first oviposition was observed (24).
114
Consequently, the oviposition time (T0) was known with an accuracy of ±30 min. The eggs were
115
immediately divided into 4 batches of approximately 50 eggs (determined gravimetrically). The 5
Page 7 of 22
eggs were then placed on a piece of filter paper in a sterile petri dish (5.5 cm in diameter) to
117
which either a low (1.5 mL, control) or a high (7.5 mL) volume of water or freshly thawed pig
118
blood (no additives) was added (Figure 1). At low volumes, the eggs simply sit on the wet paper,
119
but they were completely submerged under 1 cm of liquid in the high volume condition. The low
120
water condition was defined as the control.
121
Starting from this point (T=1 h 30 min), the petri dishes were incubated at 22±0.5°C (Binder ATP
122
climatic chamber) for 19 h, a duration that allowed the larvae to reach first instar (25). At T = 20
123
h 30 min, the petri dishes were opened, deposited vertically in 8.5x6 cm closed plastic boxes on
124
30±2 g freshly unfrozen beef liver and placed back in a climate-controlled chamber at 22±0.5 °C.
125
After 2 more days (T = 67 h 30 min), the larvae in each box were sorted, counted and killed in
126
hot water. The length of all larvae was also measured as an indicator of larval development.
127
Twelve replicates were performed for each treatment.
M
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116
te
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Fig. 1: experimental setup used to analyze the effects of egg immersion on the survival rate of
130
blow fly larvae.
131
Ac ce p
129
132
Results
133
Oviposition on wet, wounded or short-haired cadavers
134
Gravid females laid eggs on rat cadavers in all replicates of all conditions (N = 31). However, the
135
flies from two of the replicates laid significantly fewer eggs than those in the other replicates
136
(546 ± 296 eggs for these two replicates VS 3287 ± 1018 for the other 29 replicates, one-tailed
137
Mann-Whitney test, U = 225, p < 0.0001). As expected based on the literature, the females used
138
for these outlier replicates were significantly smaller than the others (105.8 ± 8 µm VS 131.5 ±
139
11 µm, one-tailed Mann-Whitney test, U = 746830, p < 0.0001). To reduce this effect of female 6
Page 8 of 22
size on the overall analysis of the results, these two replicates were excluded from the
141
calculations of the mean numbers of eggs, and comparisons between replicates were performed
142
using percent values (i.e., the number of eggs in a given area compared to the total number of
143
eggs laid on the rat during this replicate). In average, 17.7±30% of the eggs were laid in natural
144
orifices. However, because of their symmetry on the cadaver, eggs found in natural orifices were
145
removed from the analysis: only the numbers of eggs in the treated/untreated sides were
146
compared.
us
cr
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140
147
No significant effects of treatment on the total number of eggs laid on the cadaver were observed
149
(Kruskal-Wallis test with Dunn pairwise multiple comparison, K = 2.929, P = 0.417). Under
150
control conditions, females laid a mean of 3250 ± 911 eggs per replicate (N = 7). Under
151
experimental conditions, they laid an average of 3660 ± 798 eggs per replicate in the Dry VS Wet
152
experiment (N = 6), 2765 ± 941 eggs per replicate in the Wounded VS Healthy experiment (N =
153
6) and 3594 ± 1129 eggs per replicate in the Short VS Long hair experiment (N = 6). In control
154
condition, there was no difference in the number of eggs laid on the head (including the eyes,
155
nostrils, mouth and ears) and tail (including the anus) than on the fore legs, abdomen and hind
156
legs (two-tailed Wilcoxon paired test, α = 0.05, p = 0.82). The side of the rat cadaver (dorsal or
157
ventral, alternated between experiments) that was in contact with the cardboard did not affect the
158
number of eggs deposited (two-tailed Wilcoxon test, α = 0.05, p = 0.384).
Ac ce p
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an
148
159 160
No eggs were laid inside the wound in any of the 8 replicates. Eggs in the area surrounding the
161
wound (zone 3) were observed only three times and their number did not differ from controls
162
(Mann-Whitney test, p-value = 0.409). Significantly more eggs were oviposited on the portion of
163
the rat that was in contact with the cardboard than on the upper side: out of the total of 31 rats we 7
Page 9 of 22
investigated, we observed only four instances in which any eggs could be found on the upper side.
165
More eggs were laid on the wet side than on the dry side, with an average of 93±14% of the total
166
number of eggs per replicate (one-tailed Wilcoxon test, W = 36, p = 0.007) (Figure 2). Flies laid
167
significantly more eggs on the side with short hair side than on the side with long (natural) hair
168
(98.4 ± 3.2% of the eggs were observed on the side with short hair, N = 6 out of 8, Figure 3).
ip t
164
cr
169
Fig. 2: The observed distribution of eggs according to humidity. The grey bars represent the
171
percentage of eggs located on the treated side (i.e., the wet side), and the black bars denote the
172
percentage of eggs located on the control side (i.e., the dry side). The total number of eggs laid on
173
the rat for each replicate is reported on the left (note: the eggs located in natural orifices have
174
been excluded).
176
Fig. 3: The observed distribution of eggs according to hair size. The grey bars represent the
177
percentage of eggs located on the treated side (i.e., the short haired side), and the black bars
178
denote the percentage of eggs located on the control side (i.e., the long haired side). The total
179
number of eggs laid on the rat for each replicate is reported on the left (note: the eggs located in
180
natural orifices have been excluded).
te
Ac ce p
181
d
175
M
an
us
170
182
Effect of egg immersion on the survival rate of blow fly larvae
183
The eggs that were reared in control condition successfully hatched and developed. An average of
184
72.7 ± 7.9 % of the larvae survived until the end of the experiment, and they reached a mean
185
length of 7.5 ± 0.6 mm (3rd instar, N = 429) (Figure 4). Similar values were obtained for eggs
186
reared in the presence of a small volume of blood, which was characterized by an average
187
survival rate of 71.3 ± 7.8% and a mean larval length of 7.6 ± 0.7 mm (N = 424). Thus, placing 8
Page 10 of 22
eggs in a small volume of pig blood or water affects their development similarly (Kruskal-Wallis
189
with Dunn comparison; survival rate p = 0.92; length p = 0.25).
190
In contrast, submerging eggs under high volumes of water or pig blood strongly affected their
191
survival rate and the development of the larvae. The average survival rate observed in the high
192
volume of pig blood experiment was only 14.6 ± 1.9 %, and the survival rate in high water
193
condition was only marginally higher (25 ± 3.7 %). These two survival rates did not differ
194
significantly (Kruskal-Wallis with Dunn comparison; p = 0.24). The final length of the larvae
195
was also significantly reduced under high volume conditions, with a mean length of 6 ± 0.9 mm
196
for blood (3rd instar, N = 88) and 6.8 ± 0.7 mm for water (3rd instar, N = 149) (KW = 167; p
S0379-0738(15)00218-2 http://dx.doi.org/doi:10.1016/j.forsciint.2015.05.025 FSI 8017
To appear in:
FSI
Received date: Revised date: Accepted date:
12-11-2014 18-5-2015 19-5-2015
Please cite this article as: D. Charabidze, A. Depeme, C. Devigne, V. Hedouin, Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds? Experimental data and implications for forensic entomology, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.05.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Title Page (with authors and addresses)
Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds? Experimental data and implications for forensic entomology Damien Charabidze 1, 2, Aurore Depeme 1, 2, Cedric Devigne 1, 2, 3, Valery Hedouin 1, 2
2 Forensic Taphonomy Unit, UDSL, Lille, France
cr
3 Laboratoire Ecologie & Biodiversité, UCLILLE, FLST, Lille, France
ip t
1 Université Lille Nord de France, Lille, France
Ac
ce pt
ed
M
an
us
Contact: [email protected]
Page 1 of 22
*Highlights (for review)
1
Highlights
Egg-laying behaviour was studied under controlled conditions on rat cadavers.
3
No eggs were observed inside the wounds in any of the replicates.
4
Flies laid more eggs on the wet and short hair areas than on control areas.
5
Submerging eggs in liquid strongly affected their survival rate and development.
6
These data question the literature assertion that “blowflies lay their eggs in wounds”.
Ac ce p
te
d
M
an
us
cr
ip t
2
1 Page 2 of 22
*Manuscript (without author details)
Abstract
2
This study was designed to examine the common belief that necrophagous blowflies lay their
3
eggs in wounds.
4
The egg-laying behaviour of Lucilia sericata was observed under controlled conditions on wet,
5
artificially wounded or short-haired areas of rat cadavers. Flies laid significantly more eggs on
6
the wet area and the area with short hair than on the dry area or area with long hair. No eggs were
7
observed inside the wounds in any of the replicates.
8
The effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae
9
was also investigated. In low water condition, an average of 72.7 ± 7.9 % of the larvae survived
an
us
cr
ip t
1
and they reached a mean length of 7.5 ± 0.6 mm. In contrast, submerging eggs under a high
11
volume of water strongly affected their survival rate (25 ± 3.7 %) and development. Similar
12
results were observed using unfrozen pig blood instead of water.
13
These data question the information found in the literature regarding the preferential egg-laying
14
behaviour of Calliphorids flies in wounds.
d
te
Ac ce p
15
M
10
16
Keywords: oviposition; behaviour; colonization process; evidence interpretation; forensic
17
investigation
18 19
1 Page 3 of 22
This study investigate the factors that affect the oviposition sites of Lucilia sericata (Diptera:
21
Calliphoridae) (Meigen, 1826), a very common blow fly species of forensic interest. Blowflies
22
(Diptera: Calliphoridae) have an highly developed olfactory system that allows them to detect
23
corpses at a great distance (1–4). It is frequently reported in forensic entomology manuals
24
that once on the cadaver, blowflies lay their eggs in natural orifices and wounds (5–10). The
25
presence of other eggs, larvae or adult individuals can also act as an attractive signal to gravid
26
females and can increase the likelihood of oviposition in a given area (1,11–13).
27
Oviposition in natural openings, especially on the face, is often observed in field conditions. Due
28
to their weak mouth hooks, first instars are unable to attack hard tissues (e.g., muscle, skin, etc.)
29
and consequently feed on soft areas such as mucous membranes or brain (14–16). Nostrils offer
30
both protection and a suitable place for larvae to feed, and they are often heavily colonized. Byrd
31
& Tomberlin also note that when excrement-soiled clothing is present, flies often deposit their
32
eggs in these areas, a fact that highlights the importance of surface humidity in determining egg-
33
laying behaviour (in 8). On the other hand, larvae feeding on wounds are frequently observed in
34
the context of wound myiasis, i.e. infestation of living animals by fly larvae. In Europe, Lucilia
35
the main genus involved in human myiasis and is a common pest in sheep, responsible for costly
36
flies strike (17–19). It is also common in forensic pathology to observe deep alterations of wound
37
characteristics due to necrophagous larvae feeding at the injury site (20,21). But the observation
38
of larvae feeding in wounds does not require that eggs be laid in these areas. Actually, there
39
appears to be no experimental or quantified data in the literature to support the assertion that
40
blowflies lay their eggs in wounds.
Ac ce p
te
d
M
an
us
cr
ip t
20
41 42
To highlight the importance of this question in a forensic context, it is interesting to consider a
43
case that we analyzed some years ago. In 2009, the naked body of a young woman was 2
Page 4 of 22
discovered in a corn field a few hours after her disappearance. Several wounds caused by a sharp
45
object were present, and her hair was soaked with blood. Throttle traces were also observed on
46
the neck. An autopsy concluded that the victim was first throttled but did not die of asphyxiation
47
and was subsequently stabbed in the head following an attempted rape. The only forensic
48
entomology evidence was the unhatched fly eggs that were discovered in the eyes and nostrils of
49
the victim, which were identified as belonging to L. sericata. Because thousands were sampled
50
and even more were visible, it was clear that the colonization of these flies took place at a time
51
that the body was easily accessible (i.e., when it was in the corn field). Based on what has been
52
published in the literature, the complete lack of eggs on the bloody wounds would appear to
53
suggest that the victim had not already been stabbed when the Calliphorids flies oviposited on the
54
corpse. However, this scenario was inconsistent with subsequent investigations, and it was lastly
55
proven that the victim was stabbed before being deposited in the field where she was discovered.
56
Colonization by blowflies likely occurred at this time, but the flies did not lay any eggs in her
57
wounds.
cr
us
an
M
d
te
Ac ce p
58
ip t
44
59
On the basis of this case and to determine the reliability of the common belief that necrophagous
60
blowflies lay their eggs in wounds, the egg-laying behaviour of Lucilia sericata was observed
61
under controlled conditions on wet, artificially wounded or short-haired areas of rat cadavers. The
62
effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae was
63
also investigated.
64 65
Material & Methods
66
Experiments were performed on a laboratory L. sericata population raised and maintained in
67
50X50X50 cm gauze-covered cages. Inbreeding was reduced by adding monthly wild type 3
Page 5 of 22
(France) individuals. Adult flies (250±100) from a single emergence pool (hatching=D0) were
69
maintained at 20±2°C and 60±15% RH with a 12:12 photoperiod for a maximum of 20 days.
70
Minced beef liver was provided during the 7 first days to promote female ovarian development
71
and was then removed. After four more days, females were gravid and ready for experiment
72
(D11) (22).
ip t
68
cr
73 Oviposition on wet, wounded or short-haired cadavers
75
In this experiment, we investigated the egg-laying behaviour of L. sericata gravid females on
76
male laboratory rat cadavers (Ratus norvegicus). The setup was designed to test the likelihood
77
that females would lay eggs in wet, wounded or short-haired areas of the cadaver compared to
78
untreated control areas. Experiments were conducted from May until July between the hours of
79
10 a.m. and 3 p.m. (a 5 h period) in a laboratory room with artificial neon lighting. The room
80
temperature was kept at 24±2 °C and 60±15% RH.
81
For each tested condition, 20 gravid females and 10 males from the same pool were sorted and
82
placed in 30*30*30 cm gauze cages; caster sugar and water were provided ad libitum. A CO2-
83
killed, freshly unfrozen white rat cadaver (stored in the freezer during 3 to 5 month, 326±22 g)
84
was placed on a piece of cardboard in the cage on its ventral or dorsal side (the side was
85
alternated between replicates). Four experimental conditions were tested, each of which was
86
performed with 8 replicates (7 for Control):
87
Control: untreated rat cadavers.
88
Wet VS Dry: one half (longitudinally) of the rat cadaver was submerged in water for 15 s. The
89
wet side was alternated between experimental replicates.
90
Wounded VS Healthy: a 4 cm long transcutaneous wound was made with a surgical scalpel
91
horizontally on the flank of the rat. The entrails inside abdominal cavity were visible but not
Ac ce p
te
d
M
an
us
74
4
Page 6 of 22
transected. The side of the wound (i.e., the right or left side of the rat) was alternated between
93
replicates.
94
Bare VS Haired: one half (longitudinally) of the rat cadaver was shaved using an electric shaver.
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The hair was cut to a length of 1 mm, and the skin was carefully inspected to exclude any
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possible skin lesions. The shaved side was alternated between replicates.
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After a 5 h period with the cadaver, all flies were removed and killed. Their size was measured
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using the length of the posterior cross vein (dm-cu) (23). The eggs were removed using fine
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forceps and a louse comb and placed in separate vials. These vials were kept at 3±1°C until
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counting (24 h to 36 h after the start of the experiment).
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The location of the eggs was noted as follows:
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Control/tested side. For the wound condition, the location relative to the wound (in or outside)
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was also reported.
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Natural orifices (eggs located in the natural orifices were counted separately from those in the
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corresponding body areas): face orifices (ears, nostrils, eyes, mouth) and anus.
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Body area: (1) head, (2) fore legs, (3) abdomen, (4) hind legs and (5) tail and location
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Beyond or underneath the cadaver.
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Effects of egg immersion on the survival rate of blow fly larvae
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This experiment was designed to assess the effect of body fluids, which can flow or accumulate
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in wounds, on the survival rate off eggs and development of larvae. Fresh beef liver (25±5 g) was
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introduced into fly-rearing cages and checked hourly until the first oviposition was observed (24).
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Consequently, the oviposition time (T0) was known with an accuracy of ±30 min. The eggs were
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immediately divided into 4 batches of approximately 50 eggs (determined gravimetrically). The 5
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eggs were then placed on a piece of filter paper in a sterile petri dish (5.5 cm in diameter) to
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which either a low (1.5 mL, control) or a high (7.5 mL) volume of water or freshly thawed pig
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blood (no additives) was added (Figure 1). At low volumes, the eggs simply sit on the wet paper,
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but they were completely submerged under 1 cm of liquid in the high volume condition. The low
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water condition was defined as the control.
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Starting from this point (T=1 h 30 min), the petri dishes were incubated at 22±0.5°C (Binder ATP
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climatic chamber) for 19 h, a duration that allowed the larvae to reach first instar (25). At T = 20
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h 30 min, the petri dishes were opened, deposited vertically in 8.5x6 cm closed plastic boxes on
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30±2 g freshly unfrozen beef liver and placed back in a climate-controlled chamber at 22±0.5 °C.
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After 2 more days (T = 67 h 30 min), the larvae in each box were sorted, counted and killed in
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hot water. The length of all larvae was also measured as an indicator of larval development.
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Twelve replicates were performed for each treatment.
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Fig. 1: experimental setup used to analyze the effects of egg immersion on the survival rate of
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blow fly larvae.
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Results
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Oviposition on wet, wounded or short-haired cadavers
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Gravid females laid eggs on rat cadavers in all replicates of all conditions (N = 31). However, the
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flies from two of the replicates laid significantly fewer eggs than those in the other replicates
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(546 ± 296 eggs for these two replicates VS 3287 ± 1018 for the other 29 replicates, one-tailed
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Mann-Whitney test, U = 225, p < 0.0001). As expected based on the literature, the females used
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for these outlier replicates were significantly smaller than the others (105.8 ± 8 µm VS 131.5 ±
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11 µm, one-tailed Mann-Whitney test, U = 746830, p < 0.0001). To reduce this effect of female 6
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size on the overall analysis of the results, these two replicates were excluded from the
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calculations of the mean numbers of eggs, and comparisons between replicates were performed
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using percent values (i.e., the number of eggs in a given area compared to the total number of
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eggs laid on the rat during this replicate). In average, 17.7±30% of the eggs were laid in natural
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orifices. However, because of their symmetry on the cadaver, eggs found in natural orifices were
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removed from the analysis: only the numbers of eggs in the treated/untreated sides were
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compared.
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No significant effects of treatment on the total number of eggs laid on the cadaver were observed
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(Kruskal-Wallis test with Dunn pairwise multiple comparison, K = 2.929, P = 0.417). Under
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control conditions, females laid a mean of 3250 ± 911 eggs per replicate (N = 7). Under
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experimental conditions, they laid an average of 3660 ± 798 eggs per replicate in the Dry VS Wet
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experiment (N = 6), 2765 ± 941 eggs per replicate in the Wounded VS Healthy experiment (N =
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6) and 3594 ± 1129 eggs per replicate in the Short VS Long hair experiment (N = 6). In control
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condition, there was no difference in the number of eggs laid on the head (including the eyes,
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nostrils, mouth and ears) and tail (including the anus) than on the fore legs, abdomen and hind
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legs (two-tailed Wilcoxon paired test, α = 0.05, p = 0.82). The side of the rat cadaver (dorsal or
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ventral, alternated between experiments) that was in contact with the cardboard did not affect the
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number of eggs deposited (two-tailed Wilcoxon test, α = 0.05, p = 0.384).
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No eggs were laid inside the wound in any of the 8 replicates. Eggs in the area surrounding the
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wound (zone 3) were observed only three times and their number did not differ from controls
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(Mann-Whitney test, p-value = 0.409). Significantly more eggs were oviposited on the portion of
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the rat that was in contact with the cardboard than on the upper side: out of the total of 31 rats we 7
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investigated, we observed only four instances in which any eggs could be found on the upper side.
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More eggs were laid on the wet side than on the dry side, with an average of 93±14% of the total
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number of eggs per replicate (one-tailed Wilcoxon test, W = 36, p = 0.007) (Figure 2). Flies laid
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significantly more eggs on the side with short hair side than on the side with long (natural) hair
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(98.4 ± 3.2% of the eggs were observed on the side with short hair, N = 6 out of 8, Figure 3).
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Fig. 2: The observed distribution of eggs according to humidity. The grey bars represent the
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percentage of eggs located on the treated side (i.e., the wet side), and the black bars denote the
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percentage of eggs located on the control side (i.e., the dry side). The total number of eggs laid on
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the rat for each replicate is reported on the left (note: the eggs located in natural orifices have
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been excluded).
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Fig. 3: The observed distribution of eggs according to hair size. The grey bars represent the
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percentage of eggs located on the treated side (i.e., the short haired side), and the black bars
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denote the percentage of eggs located on the control side (i.e., the long haired side). The total
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number of eggs laid on the rat for each replicate is reported on the left (note: the eggs located in
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natural orifices have been excluded).
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Effect of egg immersion on the survival rate of blow fly larvae
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The eggs that were reared in control condition successfully hatched and developed. An average of
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72.7 ± 7.9 % of the larvae survived until the end of the experiment, and they reached a mean
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length of 7.5 ± 0.6 mm (3rd instar, N = 429) (Figure 4). Similar values were obtained for eggs
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reared in the presence of a small volume of blood, which was characterized by an average
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survival rate of 71.3 ± 7.8% and a mean larval length of 7.6 ± 0.7 mm (N = 424). Thus, placing 8
Page 10 of 22
eggs in a small volume of pig blood or water affects their development similarly (Kruskal-Wallis
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with Dunn comparison; survival rate p = 0.92; length p = 0.25).
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In contrast, submerging eggs under high volumes of water or pig blood strongly affected their
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survival rate and the development of the larvae. The average survival rate observed in the high
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volume of pig blood experiment was only 14.6 ± 1.9 %, and the survival rate in high water
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condition was only marginally higher (25 ± 3.7 %). These two survival rates did not differ
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significantly (Kruskal-Wallis with Dunn comparison; p = 0.24). The final length of the larvae
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was also significantly reduced under high volume conditions, with a mean length of 6 ± 0.9 mm
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for blood (3rd instar, N = 88) and 6.8 ± 0.7 mm for water (3rd instar, N = 149) (KW = 167; p
