These factors all play an important role in the insect life cycle as the can increase or .... was recorded and photographed as well, including pictures with a mm/cm.
All Bundled Up: Testing Oviposition Delay and Preference of Lucilia sericata Based on Common Restriction Methods Forrest Turner1 , Dr. Sherah Vanlaerhoven2, Patricia Okpara3
Abstract Blowflies are among the first carrion insects that will arrive upon a body while it decomposes, and these flies can help in forensic identification by giving a timeline of death, known as Post Mortem Interval, PMI. After arriving at the body the flies will feed and lay their eggs via ovipositor known as oviposition; and they will lay these eggs on mucus membranes upon the bodies such as the eyes, mouth, and nose. Restriction or any kinds of obstruction may block these mucus membranes or the body in general which may lead to a delay in the discovery and thus the oviposition by these flies. This delay might result in a delayed PMI than what would normally be expected, and a reduction in eggs laid by the flies because of the obstruction and difficulties to access the body. This experiment will investigate the response of obstruction methods presented by common household items to find the difference in oviposition by a common Southern Ontario blowfly species, Lucilia sericata. Restriction tests have been conducted in the past by M. Lee Goff in forensic investigations and this research is based on this concept (Goff, M.1992). These common household items; clothing, garbage bags, and a blanket are all items that may potentially be used in hiding a body which provides an outline on how L. Sericata will react and respond in the presence of an obstruction on a body, and if these different obstruction methods have a comparable difference in efficacy to keep L. Sericata away from the body, and create a significant delay, and reduction in oviposition.
Keywords: Entomology, Lucilia sericata, Oviposition, Post Mortem Interval, Restriction, Obstruction, Blowflies, Insects, Delay 45 | P a g e
Introduction Forensic entomology is the study of developmental stages, and successional patterns of arthropods in relation to the biology of criminal matters, mainly including human bodies. Arthropods, otherwise known as insects, can be useful to forensic matters by studying and recording their developmental stages, and successional patterns to discover a post mortem interval of the body, known as PMI, which is the time elapsed after death ( Goff, 1993). The main order of insects studied in these forensic matters as Diptera, the flies, mainly being Calliphoridae, which are the family of blowflies. These blowflies are significantly important as they are the first carrion insects to arrive upon a body and begin to colonize the body by feeding on the organic matter of the body and oviposit, which is there form of reproduction by laying eggs. Blowflies also have complete metamorphosis being a larval, pupation, and adult stage where we can observe significantly different life stages to help create a timeline of arrival of the insects, helping to discover PMI. There is a common equation used for this method it is insect stage of development + temperate records+ species = date eggs laid and minimum PMI, also in combination with the degree day/ hours equation to determine PMI which is ADH=T.(θ-θ°) (Sharma, R., Garg, R. K., & Gaur, J.;2015). There are many variables that can alter the life traits of insects, thus altering the PMI; these factors can include temperature, humidity, and availability of the body. These factors all play an important role in the insect life cycle as the can increase or decrease the activity, and life periods of insects which can be forensically important as these variables will be present upon a body. This research will focus on the availability of the body; if a body is restricted or covered in some way then it will be more difficult for the insects to find and colonize the body. and testing if common restriction methods will alter and delay the arrival of insects upon a body. Research has been done testing with this variable by Dr. Lee Goff in a forensic case involving the delay of PMI by a body warped in clothing, where the PMI was delayed by 2.5 days because of clothing present upon the body ( Goff, 1992). Other research has been conducted with this variable in Alberta by Anderson, G. S., Hobischak, N., Samborski, C., & Beattie, O., where the presence of clothing, and burial was tested to observe the delays of PMI, and showed there was significant delay because of this obstruction present ( Anderson, G. S., Hobischak, N., Samborski, C., & Beattie, O., 2002). Studies have also been done to test the burning of bodies to see what response arthropods will have to burnt carcasses, so there are many variables that can be tested for the research of arthropods and their relationship to decomposing bodies (Singh, R., Sharma, S., & Sharma, A., 2016). From these previous studies we can predict there is a significant delay of arrival and colonization of blowflies because of a restriction method such a clothing, resulting in a delay in PMI. This research explores the possibility of other restriction methods by testing common items that would be found around any normal household to possibly hide a body after a murder. These other items will give a wider variety of the knowledge of the response of blowflies to restriction methods other than clothing, and give an outline and understand the difficulties of how these items will delay and interfere with the estimation of PMI. Another variable that was also considered in this research is the preference of oviposition when presented with specific forms of restriction. Blowflies will commonly feed and oviposit on the mucus membranes of a body, these membranes include the ears, mouth, eyes, anus, and any 46 | P a g e
other openings of the body as blowflies wish to feed on the matter inside a body such as organs, and tissues ( Goff, 1992). Research has been done to test oviposition preference for different variables such as temperature, and the oviposition substrate but little research has been done to relate preference to restriction methods. These studies show blowflies do show a oviposition preference based on different variables present, which we can predict there should be a difference in oviposition location because of these restriction methods as well; studies have been done to test oviposition preference in different Diptera families using different substrates as their variable showing different species of flies prefer different areas and substrate to oviposit upon (Archer M. S. Elgar M. A, 2003). This oviposition preference could prove to be forensically significant as it provides the outline on the important areas forensic investigators, and entomologists to search and inspect for blowfly eggs/ larva as they would prefer this area because of the restriction methods. This research also provides useful information to general entomologists as well as it provides information on the behavioural response of blowflies to obstructions on their desired food and mating resource, and how this obstruction changes their overall behaviour of oviposition, and if they are able to overcome around the obstruction and access the body for the resources they require. This research will test the persistence of blowflies in the presence of restriction by common items on their preferred resource to observe if their behaviour, and time to access and oviposit will change; if so it provides significant information of these blowflies behaviour to overcome obstacles, and gives a forensic outline of which common items might alter the overall access and PMI of a body. Materials and Methods The location of this research was conducted in the Dr. Vanlearhoven Insect Behaviour and Ecology Laboratory, and this experiment took place in the greenhouse on top of Biology Building. Since the greenhouse is a dynamic environment in regards to temperature and humidity, which are important factors to insects, data loggers were placed in each of the three experimental cages to track the varying temperature, and humidity on a hour basis for every day that the experiment was being conducted. The experimental subjects of this research were fetal pigs as they are the closest test subjects to replicate human bodies because of their similar body structure and anatomy, and their lack of hair throughout their body (Catts and Goff; 1992). The blowflies used in the experiment were Lucilia sericata, a common blowfly found in Southwestern Ontario, and they were reared from eggs to adults in approximately 3 weeks by using liver as a food resource to develop. After reared to adulthood these flies were sorted by sex into three cages, each containing protein paste, water, sugar, and liver in order for them to thrive and sexually mature to prepare to reproduce. Each cage represents a different restriction method used; Cage #1 being child's sized clothing (Fig 1.0), Cage #2 being Kirkland brand garbage bags (Fig 1.1), and Cage #3 being a fleece throw blanket (Fig 1.2). 100 females, and 50 males were placed into cage #1 and #2, but only 50 females and 50 males were placed into cage #3 because of time restrictions and availability of L. Sericata flies. More females were placed in these cages to ensure reproductive success, cage #3 did have less females so we would expect less overall egg masses as a result. After the flies were sorted by sex they were released into their designated cages they fed off of liver for 3-4 days until egg masses were found to show they are ready to reproduce, then the experiment would begin. After the flies would become sexually mature the 47 | P a g e
fetal pigs were thawed from the freezer, weighed, and then placed in each cage with their restriction method wrapped around them, either being clothing, a garbage bag, or a blanket; the pig would be placed in the cage for 24 hours starting at 12:45pm, and then taken out the next day at the same time and placing a new pig in the cage to start another trial. The pigs were wrapped well to cover the entire pig in each method to simulate a realistic situation where a person would attempt to wrap a body with conviction in order to hide such body. After the pigs were taken out of the cage they were photographed, then unwrapped and photographed more to capture how many egg/ larval stages of L. Sericata were found on the pig, and where they were exactly found. Each maggot mass was recorded and photographed as well, including pictures with a mm/cm scale ruler to capture depth of the maggot masses. After all photographs and recordings of the pig were done the egg masses and larval stages were collected using fine point forceps, and a paintbrush into 70% ethanol in order to kill and preserve the flies for later recording and counting methods. It should be noted that egg/ larval stages found in different regions on the pig, either on the pig or on the restriction method itself were well photographed and collected in separate containers to count these specimens as they were found to find oviposition preference. This collection was done on each pig, and a total of 12 trials were, 4 for each restriction method to ensure validity of the experiment. After all trials were complete the collections of eggs and larval stages from each trial were placed under a microscope and counted individually based on their restriction method, trial number, and location found; either on the pig or the restriction method. After all trials were complete the pigs were placed back into a freezer, then discarded via incineration at a later time period. The data loggers that were placed in each cages were taken out after the experiment was done and all the information, temperature and humidity, were recorded and noted for later interpolation in comparison with the results as variable temperatures and humidities would cause significant changes in the behaviour of L. Sericata.
(Fig 1.0, Baby clothing used as Restriction method #1, in Cage #1)
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(Fig 1.1, Kirkland Garbage bags used as Restriction method #2, in Cage #2)
(Fig 1.2, Fleece Throw Blanket used a Restriction method #3, in Cage #3
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(Fig1.3, All restriction methods compared to each other wrapped around the fetal pig) Results All 12 trials were completed, each restriction method contained 4 trials each to ensure validity of the results, and the experiment. All the egg masses, and larval stages found on the pigs and the restriction methods were counted individually, and separately to find the preference of L. Sericata when presented with these restriction methods. The table below represents the number of eggs and larval stages found on each fetal pig at each of the trials (Fig 2.0) Restriction Method Clothing
Trial Number
Eggs/ Larval stages found 1
741
2
870
3
701
4
898 Avg 803
Garbage Bags
1
608
2
554
3
381
4
526 Avg 517
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Restriction Method
Trial Number
Eggs/ Larval stages found
Blanket
1
260
2
118
3
72
4
190 Avg 160
L. Sericata Eggs/ Larval stages Collected from Restricted Pigs of 4 Replicates Each 1,000
898
No. of Eggs/ Larva
870 750
741
701 608 554
500
526 381
250
260 190 118 72
0 C1 C2 C3 C4
G1 G2 G3 G4
B1 B2 B3 B4
Methods of Restriction ( Clothing, Garbage Bag, Blanket)
(Fig 2.1 Bar graph to demonstrate the differences in the egg/ larval stages found due to each of 1 2 the different restriction methods, Clothing SE= 48.097990256, Garbage Bag SE= 3 48.4997852229, Blanket SE= 41.2391399846)
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AVG Preference of L. Sericata of Oviposition on Clothing Method
35% 65%
Eggs/ Larval Stages collected from Pig Eggs/ Larval Stages collected from clothing
(Fig 2.1, The average oviposition location of L. Sericata due to the clothing restriction, showing overall preference on the fetal pig) AVG Preference of L. Sericata of Oviposition on Garbage Bag
49%
51%
Eggs/ Larval Stages collected from Pig Eggs/ Larval Stages collected from Garbage Bag
(Fig 2.2, The average oviposition location of L. Sericata due to the garbage bag restriction, showing a very close preference of the fetal pig to the garbage bag) AVG Preference of L. Sericata Oviposition on Blanket Method
100%
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Eggs/ Larval Stages collected from Pig Eggs/ Larval Stages collected from Blanket
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(Fig 2.3, The average oviposition location of L. Sericata due to the blanket restriction, showing no eggs or larval stages were found on the fetal pig but only the blanket) Clothing was found to have the most eggs, and larval stages on the pig, with the highest number found being 898 on trial #4, and an overall average of 803. Garbage bags had the second highest number of overall eggs, and larval stages with 608 on trial #1, and an overall average of 517. The blanket restriction method yielded the lowest number of eggs, and larval stages being 72 on trial #3, and an overall average of 160. All L. Sericata life stages were collected, and recorded but the locations of the stages were recorded as well to find the area where the flies would prefer to oviposit. This preference is displayed through these pie graphs below to show how many eggs/ larval stages were found on the pig itself, and found on the restriction method (Figs 2.2, 2.3, 2.4). Many photographs were also taken to capture the number of egg masses, and larva stages found on each of the pigs, and to capture the areas of oviposition or larval migration to observe the behavioural preference of L. Sericata when presented with obstacles upon their main resource.
( Fig 2.4, Trial #1 of Clothing, showing larva found in the mouth of the fetal pig)
(Fig 2.5, Trial #1 of Clothing, showing egg masses found on top of clothing , and around the collar of the shirt)
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(Fig 2.6, Trial #2 of Clothing, showing many egg masses on the front of the shirt and surrounding the collar)
(Fig 2.7, Trial #3 of Clothing, different shirt was used in this trial due to cleaning of original shirt, but shirts are same fabric and style; large egg masses found on neck of pig under collar of shirt)
(Fig 2.8, Trial #4 of Clothing, showing large egg masses in the mouth of fetal pig)
(Fig 2.9 Trial #4 of Clothing, showing egg masses on bottom of pants, this is the only trial to possess eggs found on the pants)
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(Fig 2. 10, Trial #1 of Garbage bags, showing many larval stages on the garbage, especially around pooling of blood and liquids)
(Fig 2.11, Trial #1 of Garbage bags, showing larva all throughout body especially on chest and under arms where body is moist from liquids)
(Fig 2.12, Trial #1 of Garbage bags, showing egg masses on the garbage bag within the creases)
(Fig 2.13 and 2.14, Trial #2 and Trial #4 of Garbage Bags, showing egg masses found within the garbage bag) 56 | P a g e
(Fig 2.14, Trial #2 of Garbage Bags, egg masses found under the mouth and on chin area where blood and fluids have pooled because of the garbage bag)
(Fig 2.15, Trial #2 of Garbage Bags, overall photograph of pig out of garbage bag where it can be observed the pig is very moist and covered in fluids from the bag, and the pig is very warm from the insulation of the bag)
(Fig 2.16, Trial #3 of Garbage bags, many larva found within garbage bag in pooling of fluids and creases of bag)
(Fig 2.17, Trial #1 of Blanket, showing blood spots upon blanket where egg masses are found)
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(Fig 2.18, Trial #1 of Blanket, no egg or larval masses found on the pig on the body or mucus membranes)
(Fig 2.19, Trial #4 of Blanket, small spots of egg masses found throughout blanket, but only on the outside)
(Fig 2.20, Trial #3 of Blanket, close up of pigs mouth where no eggs or larval stages were found)
Temperature and humidity are also factors that must be considered in this experiment, and to account for these data loggers were placed into each of the cages and recorded for the whole duration of the experiment until completed.
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(Fig 2.21 Graph of temperature, humidity, and intensity from 03-09/17- 03/24/17 of Cage #1, Clothing restriction)
(Fig 2.22, Graph of temperature, humidity, and intensity from 03/16/17- 03/24/17 of Cage #2, Garbage Bag restriction)
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(Fig 2. 23, Graph of temperature, humidity, and intensity from 03/16/17- 03/27/17 of Cage #3, Blanket restriction) Restriction Type, Trial No.
Avg Temperature (C°)
Avg RH, Humidity (%)
Clothing 1
27.85508
28.9
Clothing 2
30.12548
32.2
Clothing 3
28.44976
16.2
Clothing 4
31.75956
35.2
Garbage Bag 1
28.45371
24.1
Garbage Bag 2
27.12932
11.7
Garbage Bag 3
28.561
10.6
Garbage Bag 4
31.08024
18.7
Blanket 1
29.93415
30.0
Blanket 2
25.14736
63.5
Blanket 3
24.48316
81.9
Blanket 4
23.4015
68.4
(Fig 2.24, Table of average temperature and humidity on days of each trial was conducted)
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Discussion This research concluded with a clear pattern and result that demonstrates that a method of restriction upon a body does change the behaviour and accessibility of blowflies to aid in the field of forensic science. As seen in Fig 2.0, and 2.1 there is a clear linear pattern of number of eggs/ larval stages found based on each restriction method as clothing would always have the most activity of L. Sericata, and eggs/ larval stages, an average of 803, found on the fetal pig in each trial when compared to the other methods. Garbage was in middle in comparison to the other methods with an average of 517 eggs/ larval stages found, and blanket had the least number of eggs/ larval stages found with an average of 160 between the 4 trials conducted. This information shows that the blanket is the most effective restriction method out of the three tested as it resulted with least number of eggs/ larval stages found, and when the pig was unwrapped after each trial no eggs were found on the body, but only on the outside of the blanket. We would predict that the blowflies might be able to access the body even with the blanket restricting it, but this would take more than 24hours as these trials showed they could not access it; but more research can be done to show if this accessibility after 24 hours becomes more apparent. With the blanket showing significant restriction results we would expect this method would alter PMI by at least 24 hours as the flies are unable to access the body in a 24 hour time period. The other two methods, clothing and garbage bags did allow for L. Sericata to access the fetal pigs, but the garbage bags did yield a lower overall number of egg/ larval stages found which means that the garbage bag did create significant restriction in comparison to the clothing. Clothing did restrict the fetal pigs but not very effectively as this method yielded the highest number of egg/ larval stages, and the photographs from Fig 2.4, 2.7, and 2.8 show that L. Sericata did still oviposit in the major mucus membranes such as the mouth, and ears; but there was some oviposition in unpredicted areas such as the clothing which was very common around and under the collar in all trials. This oviposition of the collar of the clothing, and the pants in trial #4 was unexpected, and was very frequent, 35% between all trials, which is surprising as L. Sericata did have access to most of the mucus membranes of the fetal pig, but would choose to oviposit on the clothing in all of the trials conducted. This was also present in the garbage bag restriction as 49% of eggs/ larval stages were found inside the garbage bag, which is large percentage when considering the mucus membranes are available if the flies can overcome the restriction on the pig. It was noticed that the eggs and larva that were found in the garbage bag were usually located within large pools of fluid and blood that were created from the decomposing of the pig, and the heat created from the insulation of the garbage bag. With this knowledge it can be observed that L. Sericata does prefer these fluid pooling when they are available, and these areas did create preference of L. Sericata, which was not expected. This shows behavioural traits of L. Sericata to prefer areas of warm bodily fluid to the body itself in some instances; this gives us more information to the feeding and oviposition behaviour of the species L. Sericata when presented with certain variables such as restriction. It also gives us an outline on how to proceed with forensic entomological techniques to be careful when approaching a scene or body that has been restricted to check and investigate all areas of the restriction item as this research shows that the blowfly species L. Sericata does prefer to oviposit on shirt’s collar, pant cuffs, under the sleeves of shirts, and pooling of fluid and/ or blood in garbage bags. It must be noted that Cage #3, the 61 | P a g e
blanket restriction did have a lower overall colony of L. Sericata with 50 females:50 males because of time restrictions and the availability of these flies from the main colony in the
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laboratory, because of this lower ratio of females to males than the other cages we would expect the number of eggs/ larval stages found to be less than the other cages regardless of the restriction method used. The results still show significance as the blanket method had much less oviposition than the other methods predicting that even if the number of L. Sericata was even among all of the cages the blanket method would still have the overall lowest number of oviposition had between all methods. Temperature and humidity must also be noted in this experiment as well, even though temperature and humidity were not the key factors in this research but they play a major role as these two factors will always affect the oviposition and the life cycles of arthropods. The temperature amongst all the trials did remain relatively consistent showing a range of 21.7- 37.1C° ( +/- 15.4C°), and studies show that an ideal temperature for L. Sericata development is around 20-28C° (Shiravi, A., Mostafavi, R., Akbarzadeh, K., & Oshaghi, M., 2011), and this range was satisfied, but more research could be done testing varying controlled temperatures to observe if temperature will change the behaviour of L. Sericata when faced with restriction. Humidity must also be noted as humidity in this experiment did vary much more than temperature did as the range for humidity was 10.6-81.9% ( 71.3%+/-). The trials most affected by this increase in humidity would be the last blanket trials as this is when the humidity was the highest and this could have caused the lower oviposition results of these trials, but the restriction method of the blanket also cause this; but during the trials and this high humidity no mass deaths were noticed when checking on the cages daily so this large humidity increase could not have been a huge factor to the lower oviposition results. Another restriction of the research is the number of restriction methods tested as more items could be tested, and it was originally planned for a fourth method to be tested, plastic tarp, but because of time restraints it was removed from the experiment. This allows for future research to be done to test other items such as plastic tarp or other variables to restrict or obstruct a body to future explore the altering of PMI and oviposition preference by restriction methods. Conclusion In conclusion different common household items that would be used to restrict and hide a body show significant oviposition results as each restriction method yields a different number of eggs/ larval stages found on the body showing how accessibility the body was to the fly, and showed different locations of oviposition preference based on the different restriction methods presented to the blowfly species L. Sericata. References 1
Goff, M. (1992). Problems in Estimation of Postmortem Interval Resulting from Wrapping of the Corpse: A Case Study from Hawaii. J. Agric Entomology ,9(4), 237-243. 2
Singh, R., Sharma, S., & Sharma, A. (August 2016). Determinaton of post burial interval using entomology: A review. J Forensic Leg Med, 42, 37-40. 3
Anderson, G. S., Hobischak, N., Samborski, C., & Beattie, O. (2002). Insect Succession on Carrion in the Edmonton, Alberta Region of Canada . Technical Report Canadian Police Research Centre. 63 | P a g e
4
Goff, M., & Catts, E. (1992). Forensic Entomology in Criminal Investigation. Annual Review of Entomology, 37, 253-272. 5
Goff, M. (1993). Estimation of Postmortem Interval Using Arthropod Development and Successional Patterns. . Forensic Science Review , 5(2), 81-94. 6(Diptera):
a quantitative analysis. Australian Journal of Zoology 51, 165-174. Archer M. S. Elgar M. A. (2003) Female breeding-site preferences and larval feeding strategies of carrionbreeding Calliphoridae and Sarcophagidae 7Shiravi,
A., Mostafavi, R., Akbarzadeh, K., & Oshaghi, M. (2011). Temperature Requirements of Some Common Forensically Important Blow and Flesh Flies (Diptera) under Laboratory Conditions. Iranian Journal of Arthropod-Borne Diseases, 5(1), 54–62. 8Sharma,
R., Garg, R. K., & Gaur, J. (2015). Various methods for the estimation of the post mortem interval from Calliphoridae: A review. Egyptian Journal of Forensic Sciecnes, 5(1), 1-12. Acknowledgements Thank you to Dr. Vanlearhoven for allowing me to participate and conduct this research in her laboratory, and I am thankful of the help and guidance she provided to create and structure the experimental design and idea of this research. Thank you to Patricia Okpara for all the help she provided in the experimental design and labour for this research, without her I could not have completed the research.
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