In the field experiment, we applied different decay-aged pork liver in sequential days
to test the blow fly reaction. In this experiment, it may encounter raining or other weather
condition during the experiment. In addition, the experiments were lasted for 2 years, we didn’t consider the weather condition, so the seasonal changes and temperature
fluctuation will not be discussed in this study.
We found the oviposition performance of C. megacephala reached to the highest in
the day 3 with 2-day-old pork liver, which showed significant difference to fresh,
7-day-old and 8-day-7-day-old pork liver. Our results agree with the previous study, which indicated
the aged pork liver have greater attraction to blow flies than fresh pork liver (Bunchu et
al., 2008). However, in their study, only 1-day-old and 3-day-old decay treatments have
been tested, it could not provide the complete oviposition information for the continuous
process of decomposition. No eggs was laid on 8-day-old pork liver in our study, and the
similar results were also shown in George et al. (2012) which the very decayed
(8-day-old) treatments had lower oviposition and larviposition preference than fresh treatments,
however, they did find both oviposition and larviposition happened in 8-day-old
treatments in theirs experiment. Different results of 8-day-old treatment between our
study and previous study might cause by different species and the difference in geological
region (Amendt et al., 2004), which also indicate that different species and the reaction
of it while different decay-aged treatments were given should be tested.
In the oviposition preference test of different decay-aged pork liver for C.
megacephala, the results shows the 2-day-old and 4-day-old treatments have the highest
attraction to C. megacephala. In the post hoc analysis, the 4-day-old pork liver did not
show significantly different to the 2-day-old pork liver. In addition, the results show that
there is no eggs been laid on the 8-day-old pork liver. In our preference test, C.
megacephala did avoid to lay egg on the 8-day-old pork liver. However, we did not use
Y-tube to find out whether olfactory cues play an important role in this process. We simply
provided 5 different decay-aged treatments for oviposition selection. The factors that may
cause the results of the preference test is still unknown. How C. megacephala distinguish
the different decay-aged treatments need further investigation. In the previous studies,
researchers found the C. megacephala could use their visual, olfactory and tactile sensory
to locate the place to lay eggs, evaluate the resources from the information which acquired
from those sensory system (Tomberlin et al., 2011b). However, in our experiment, we did
not test any sensory organs of blow flies that involved in the preference of oviposition
process, so we can not reach any conclusion about which sensory organ plays the major
role in detecting conditions of different treatments.
However, Kasper et al. (2012) studied the different VOCs (volatile organic
compounds) emitted from the mouse carcasses under different storage time and weather
condition using GC-MS analysis. In their studies, researchers found that the concentration
and composition of VOCs emitted from mouse carcasses changed with different storage
times, from fresh, 10-day-old to 30-day-old treatment. The Sulphur compounds in the
VOCs have known to show great attraction to necrophagous insects including blow flies.
In addition, their results also showed the concentration of Sulphur compounds changed
when storage times or temperatures were different. That indicated the depletion of
methionine and cysteine during the decomposition process (Kasper et al., 2012). We can
conclude from their results that the proteolysis within decomposition cause the
degradation of Sulphur compounds. In another world, changes of the VOCs composition
in the decomposition process might be one of the factors that affect the oviposition
preference for blow flies on different decay-aged pork liver. In addition, during our pork
liver storage process (preparation of decay-aged treatment), some blow flies accidentally
invaded into the incubator and lay eggs on the fine mesh covered on the top of the
container of liver which were already stored for more than 8 days. Although the blow
flies which accidentally invaded into the incubator could only use the odor cues to detect
those decay-aged treatments. The oviposition behavior still could be observed. But in our
preference test, there is no oviposition behavior happened on that kind of the treatments
after 8-day-old. Thus, we believe not only the olfactory system plays in oviposition
decision process, but also other sensory system involved for helping blow flies to make
oviposition decision.
Gregarious oviposition might be a factor that caused the variation in egg numbers
on different treatments. Gregarious oviposition is defined as the female individuals which
have the tendency to produce collective egg mass aggregately (Aponte et al., 2003).
Chrysomya megacephala preferred to lay eggs on the media which already have eggs on
it (Esser, 1990), and the accumulation of the blow fly individuals also act as an attractant
to other blow flies (Norris, 1965). We did not use a single female but use 10 female adults
instead in our experiments, we have the following reasons for that: Chrysomya
megacephala seldom laid eggs when they are along (Yang and Shiao, 2014). Replacing
female individuals with male can trigger oviposition behavior as well (Yang and Shiao,
2014). However, we are afraid of the foraging preference of male blow flies will affect
aggregation behavior of females and influence their oviposition choices. And the average
fecundity of 10 females are not significantly different with that of 50 females (Yang and
Shiao, 2014). So we decided to use 10 female adults in our experiments. But the effects
from the female gregarious oviposition could not be controlled. However, in our raw data,
eggs clutches seldom appeared in a single medium, which indicated that the gregarious
oviposition is probably not a major factor that may influence the decision of C.
megacephala.
Comparison the field experiment and the preference test, no egg has been found on
the fresh pork liver in the field experiment. But were laid on the fresh pork liver in the
preference test. Why there is no egg laid on the fresh pork liver in the field experiment?
Bunchu et al. (2008) showed that fresh pork liver have attraction to C. megacephala than
the 3-day-old pork liver and 1-day-old pork liver, only about 60% of C. megacephala
could response to the fresh media. Although fresh pork liver also can attract blow flies,
some researchers believed that the amounts and concentration of Sulphur compounds
emitted from the protein resources which attract necrophagous insects are not high
enough in the very beginning of decomposition (Kasper et al., 2012; Tomberlin et al.,
2011b). In addition, when body was exposed in the nature environment, blow flies should
go through the exposure phase, detection phase and acceptance phase, than reach to the
oviposition stage (Tomberlin et al., 2011b). Wind speed, wind direction (Bunchu et al.,
2008) and the concentration of Sulphur compound may change the duration of exposure
phase and detection phase for blow flies(Tomberlin et al., 2011b). However, in our
laboratory environment, pork liver was directly provided to the blow flies inside the bug
cage. Therefore, in the laboratory environment, we can assume that we shorten the
duration of exposure phase and detection phase, but the acceptance phase remains
unchanged, which means blow flies have longer time to make their decisions to oviposit
or not. However, this argument need further studies to confirm.
Both field experiment and preference test showed no eggs on the 8-day-old pork
liver. What are the possible reasons? In our experiments, we removed the hardened
external layers of the media by homogenizing them in a blender. In addition, the fine mesh
covered on the medium provides the same contact surface and uniform texture. Therefore,
blow fly sensory system will not be affected by the different contact surface or texture.
Previous researches showed that the moisture of the oviposition media could affect the
larval development (Erzinclioglu, 1996). If the media was too moist, blow fly larvae may
drown to death, in contrast, if the media was too dry, it may damage the egg and cause
the low hatching rate. The dehydration of the media will cause the larvae dehydrate, and
the water loses will harden the surface of the media make larvae hard to penetrate the
surface (Erzinclioglu, 1996). It is possible that blow flies would avoid to lay eggs on the
dehydrated media (Erzinclioglu, 1996).
In the third experiments, we provided different oviposition media of different
decomposition level individually to test the reaction when C. megacephala has no other
media to choose, which is similar to the field situation. Since we believe there are not
many oviposition media in the field which can be chose by the blow flies in the same time.
In addition, what will happen if we simulate field selection situation in the laboratory
condition? The results showed most of the treatments do not have significance different
with each other, but the incidence of oviposition on 2-day-old pork liver was significantly
higher than that on the 11-day-old pork liver. Comparing the field and preference tests,
the peak of the incidence of oviposition also appeared on the 2-day-old pork liver
(40.74%), but significantly low incidence of oviposition was showed on 11-day-old pork
liver (0%). Those results of the 8-day-old, 9-day-old and 10-day-old pork livers do not
show significantly low incidence of oviposition. In another words, the effect of
decomposition media was delayed when single media was provided under laboratory
condition. We think this delay may refer to the natural reaction of C. megacephala. Under
the natural situation, insect with high fecundity such as C. megacephala, can continuously
reproduce during their life span, they do not really need to choose the very ideal
oviposition media to laid their eggs. In addition, scarce and limited resources like corpses
should be a highly competitive resource (Beaver, 1977), therefore, blow fly of C.
megacephala would always lay eggs when they encounter suitable oviposition media
except for some extremely unsuitable conditions. However, this argument needs further
investigation.
In the test of duration of acceptance phase, when oviposition media of different
decomposition levels were provided individually, the results showed no significant
difference was found among groups. That indicates under 95% confident interval, the
durations of acceptance phase have no significant difference in different decomposition
levels. However, the average mean duration is 9.83
1.92 hours, which is relativelylong if we compare with the assumption of “oviposit after arrive the corpse in a short
period”. Although most of the data we collected in each treatment are not enough for
statistic analysis, data collected in 1–day-old (9.43
2.31 hours) and 2-day-old (9.04
2.40 hours) pork livers should be reliable. In addition, the sex ratio is 1 : 9 (female :male) in this study, this ratio does not followed the natural condition of 1 : 1 (Das and
Dasgupta, 1982), whether the sex ratio could induce the early oviposition or not remains
unclear. However, the information of the delay oviposition in certain situation should be
concerned in further studies and crime investigation.
Different diet that larvae consumed will affect their development, including different
animal cadaver or different organs of the same cadaver to different physical conditions
(frozen / thawed) of oviposition media (Clark et al., 2006; Day and Wallman, 2006a; b;
Ireland and Turner, 2006; Kaneshrajah and Turner, 2004). Decomposed pork liver would
also retard the larval development (Richards et al., 2013). Prolonging of developmental
time may cause the larvae to increase the encounter risk of predators and parasites. The
decision made by a female blow fly to oviposit on a very decayed media would retard the
development rate of its offsprings and increase the risk of exposure to predators and
parasites might decrease the fitness of its offsprings. Therefore, it is reasonable to believe
that blow fly would avoid to lay eggs on the media when it has oviposition choice, which
might retard their larval development and lower their fitness.
On the other hand, microbe may also play an important role in attracting or inhibiting
the blow flies (Burkepile et al., 2006; Thompson et al., 2013; Tomberlin et al., 2012).
Usually, we thought the microbe on the corpse behaves as a decomposer, however, if the
microbe colonizes the corpse rapidly, it may also behave as a competitor to other
consumers (Burkepile et al., 2006). Necrophagous insects such as blow flies usually
represent the early visitor and colonizer of the carcass, in these cases, some microbes may
played as an attractant to the blow flies (Tomberlin et al., 2012). Nevertheless, we blocked
the invasion of insects during the decomposition process in our study, especially the
dipterans, therefore, the remaining media would be swarmed by the microbes. Previous
research indicated that releasing of the quorum sensing signal by specific bacteria to
interact with other conspecific bacteria, may attract some blow fly species that treated
this chemical signal as an attractant (Tomberlin et al., 2012). In that study, Tomberlin et
al. (2012) demonstrated the close connection of that blow fly Lucilia sericata detection
and the quorum sensing of the bacteria might be associated. In addition, along with the
decomposition process, nutrient quality and quantity change, indicate the
microenvironment related to the microbe change as will in the perspective of blow fly
(Burkepile et al., 2006). Composition of the microbe population was dynamic within this
dynamic process (Burkepile et al., 2006). Therefore, it is possible that different microbe
associated with the corpse would produce chemical compound which blow flies not prefer,
or the chemical cue emit from the medium which can attract C. megacephala is degrading.
Thus, it is reasonable to think that C. megacephala have different behavioral response to
different decay-aged treatment may cause by different microbe composition present on
the oviposition media.
Another study showed that chemical compound emitted during the decomposition
process of corpse itself could provide its information of physiological condition (Birkett
et al., 2004). Therefore, both the microbes on the media and the VOCs emitted by the
media might provide C. megacephala the information of inappropriate media, which
cause the reason the C. megacephala avoiding to lay eggs on the 8-day-old media in our
experiment. The relationships and interactions among bacteria, VOCs, humidity and blow
flies need further investigations.
Both ecological and evolution information of the target species are important in
forensic entomology (Tomberlin et al., 2011a). When we apply the experimental results
to the field, we must be aware of that our experimental design are not always matching
with the real situation of our target species in the field. Different ages of the adult blow
flies after eclosion may affect the oviposition strategies, and cause different decision
making when choosing oviposition media (Tomberlin et al., 2012). And not only the
nutritional acquisition of larvae affects adult fecundity, but also the quality and quantity
of adult protein intake play important roles on the fecundity (Tomberlin et al., 2012).
Interspecific interaction and the individual numbers of conspecific species might also
affect the fecundity and the decision making of oviposition (Norris, 1965; Yang and Shiao,
2014). To design a better experiment that can correspond to the field situation, we need
more investigation data on the biological and ecological information for those blow flies.
Smith (1986) have listed several critical issues that need further research one of them
are the “effect of postmortem treatment of the corpse”. Different postmortem treatments
usually make different insect succession patterns. Physical condition of the corpse such
as it has been burned or immersed in the water also showed different faunal composition.
In the case of burned corpse, some case studies showed that the charried corpse would
delay the blow fly invasion (Catts and Goff, 1992). However, in the immersed corpse,
only a few studies have been carried out. In the homicide cases that happened inside the
houses or automobiles which represent the closed environments, insects usually cannot
invade the body immediately and delay the colonization of blow flies. In addition, in the
situation that no insect invasion in the early decomposition also can be used to simulate
the hidden corpse (wrapped, bagged, etc.) and been re-exposed to the environment
Therefore, an investigator should not only consider the delay of invasion of insects caused
by closed environment, but also possible delayed in time when blow fly oviposit.
In this study, we applied pork liver as the oviposition media, rather than pig carcass,
however, the phenomenon of low incidence of oviposition happened on the decay-aged
media after 8-days is obvious. Such ecological and biological information might have the
application potentials in exploring repellent VOCs and the microbial utility. The possible
repellent VOCs and the microbe involved in the carcass decomposition need more studies
to reveal the mechanism behind these phenomena. In our research, we also found the fresh
treatments are not as attractive as we though before, and it need to be considered when
dealing with a freshly disposed corpse. The assumption that the blow fly will oviposit
right after they arrive the corpse should also be retested and reconsidered. Distribution,
population density, proportion of gravid females, age distribution in the field and adult
oviposition experience, many of these biological information even the evolutionary
information should be surveyed in different geographic regions, due to the different
species and environmental conditions may affect behavioral responses of blow flies. We
hope this research will be useful for further applications and help opening some new
aspects for forensic entomological researches.