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Examination of Decomposition and Insect Activity Variations of Sunlit and Shaded Carrion during Winter Forensic Research Project by Esther Breen Supervised by: Catherine Fitzgerald Bryan Lessard Jodie Ward

Abstract Little information exists in Canberra about the insect activity found on carrion in this region and about how the winter season and exposure to shaded or sunlit locations can affect the rate of insect development and decomposition of carrion. 8 pig ( Sus scrofa ) carrion in sunlit and shaded habitats were examined over approximately three months during the Canberra winter of 2009. No significant difference in mean daily temperatures and rate of decomposition between sunlit and shaded carrion. The insect population and species in sunlit and shaded habitats did show strong variations. Insect succession was different at this time of year. The Post Mortem Interval using Accumulated Degree Day calculations from the development threshold of Calliphora stygia was determined.

Background From decomposition, information can be gained about the mode of death, the season of year, the temperature and habitat of the area, the post mortem interval (PMI), the period of time that has passed since a body has been deposited and the approximate time of death, by observing the insect fauna activity of the body 1 . This is known as forensic entomology. The use of forensic entomology in forensic investigations has been recorded since 1235 A.D. in China and has continued to be a reliable method in providing valuable information for an investigation 2 . The stages of decomposition have been identified as 3 : Fresh Bloated Decay Post Decay and Skeletal 1 Geberth VJ, 2006 Estimating time of death. In: Practical homicide investigation – tactics, procedures and forensic techniques 4th Ed, p. 231-256, CRC Taylor and Francis Group, USA Merritt RW, 2009 The history of forensic entomology, American Board of Forensic Entomology, [viewed 19 April 2009] http://www.forensicentomologist.org/history.html Australian Museum, 2003 Decomposition: what happens to the body after death? [1 May 2009] http://www.deathonline.net/decomposition/index.htm

The colonisation of carrion by insects will occur successively over time during the stages of decomposition. the presence of a particular type of insect, coupled with the stage of decomposition can give an indication in estimating the Post Mortem Interval (PMI) 4 . The development of blowfly larvae can also be used to determine PMI. The Accumulated Degree Days/Hours (ADD/ADH) for the development of a blowfly at certain temperatures can be calculated using the following formula 5 : Time (hours) x (temperature – base temperature) = ADH Time (days) x (temperature – base temperature) = ADD This is the energy and time required for a maggot to go through a growth phase. The growth phase along with the base temperature threshold, the lowest temperature for larval development, can be linked to a time period 6 . 4 Wang J, Li Z, Chen Y, Chen Q, & Yin X, 2008 ‘The succession and development of insects on pig carcasses and their significances in estimating PMI in south china’, Forensic Science International, Vol. 179, no. 1, p. 11-18 Gennard DE, 2007 Forensic Entomology: An introduction, John Wiley and Sons Limited, England Haskell NH, Hall RD, Cervenka, VJ & Clark MA, 1997 On the body: insects’ life stage presence and their postmortem artefact. In: Forensic Taphonomy: the post-mortem fate of human remains , Haglund WD & Sorg MH(Eds.), p. 415-448, CRC Press LLC, USA

Blowfly Lifecycle 7 Myskowiak J, & Doums C, 2002 ‘ Effects of refrigeration on the biometry and development of Protophormia terranovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations’, Forensic Science International , Vol. 125, p. 254-261. Figure 1: The blowfly lifecycle 7 .

Previous Studies Eberhardt and Elliot 8 chose to examine the decomposition of pigs in three different habitats in Auckland, New Zealand. O’Flynn 9 examined the rate of development of several laboratory reared blowfly species at different temperatures. From his research the minimum development threshold or base temperature of the blowfly species was able to be determined. Norris 10 studied daily flight patterns of blowflies using fly traps in the Canberra region in which C. stygia was one of the most active blowfly species. Eberhardt TL, & Elliot DA, 2008 ,’ A preliminary investigation of insect colonisation and succession on remains in new zealand’ Forensic Science International, Vol. 176, no.2-3, p. 217-223, [viewed 20 April 2009] Elsevier, Science Direct O’Flynn MA, 1983 ‘The succession and rate of development of blowflies in carrion in southern Queensland and the application of these data to forensic entomology’ Journal of Australian Entomological Society , Vol. 22, p.137-148 10 Norris KR, 1966 ‘Daily patterns of flight activity of blowflies (Calliphoridae: Diptera) in the Canberra district as indicated by trap catches’, Australian Journal of Zoology , Vol. 14, p. 835-853

Methodology The experiment was conducted at the location of Spring Valley Farm, near Mt. Stromlo in Canberra. 8 pig carrion which had been euthanised, were examined. The pigs were male White Hybrids with a live-weight of between 20 and 30kg. The carrion were placed in a protective cage which were placed approximately 75 metres apart, with half in full shade (created by 70% shade cloth) and the other half in full sun and then allowed to decompose for 90 days. Visitations to the carrion for data recording and insect collection occurred regularly with observation of each pig location took 10-20 minutes to record data and collect insect samples.

HydrochronTM DS1923 temperature and humidity loggers were placed on each of the cages, in the middle of the site and were placed on top of and beneath each carrion to record the ambient temperature and humidity. A ThermochronTM DS1922L logger was also placed inside each carrion to monitor the internal temperature. Both devices recorded data at 2 hour intervals for the duration of the study. The collection methods used in the experiment involved field study collections with an entomology kit. The entomology kit contained: collection jars with labels, gloves, tweezers, 70% ethanol, butterfly net Data was recorded on a death scene form and photographs of the decomposition and insects were performed using a Nikon D70.

Figure 2: Death scene data form

Results Failures occurred in 6 out of the 24 data loggers which did not record temperature data from the internal and under body positions of each carrion. The recorded temperature used for data analysis was from the 10th of June to the 28th of August because this allowed more accurate daily data to be used. The mean temperatures for sunlit and shaded carrion over the experimental period showed similarities in temperature for all carrion locations.

Figure 3: Mean daily temperature for sunlit and shaded carrion. (a) represents mean daily ambient and body temperature of the sunlit carrion and (b) represents the mean daily ambient and body temperature shaded carrion. (a) showed similar daily mean temperatures in the sunlit carrion of P05, P07 and P09 with no strong differences. In contrast (b) had similar temperature in the shaded carrion in P06 and P08 with a spike of warmer daily temperature in P10. In comparison there are no strong differences in daily mean ambient and body temperature between the sunlit and shaded. b) a) Temperature

Decomposition Three distinct stages of decomposition were observed: described as Fresh, Bloat and Decay. The sunlit pigs became darker in colour and were more susceptible to mummification. The shaded carrion had duller skin from less exposure and appeared to have more fluids surrounding them with some adipocere on the underside of the body. The insect free control carrion P03 and P04 demonstrated a slower rate of decomposition compared to the other carrion.

Table 1: Sunlit and shaded comparison of P05 and P06 88 46 3 Shaded (Pig 6) Sunlit (Pig 5) Day

Table 2: Sunlit and shaded comparison of P07 and P08. 88 46 3 Shaded (Pig 8) Sunlit (Pig 7) Day

Table 3: Sunlit and shaded comparison of P09 and P10. 88 46 3 Shaded (Pig 10) Sunlit (Pig 9) Day

Figure A number of different insect orders were observed at each of the carrion locations with larger populations showing a strong preference for the sunlit locations of P09, P07 and P05. The Diptera showed the largest population with more abundance during the Bloat stage of decomposition. The Hymenoptera are the next most abundant order observed, followed by the Coleoptera and Acari in each stage. Diptera numbers were almost double that of shaded during Bloat. The total number of The Hymenoptera population was higher in abundance in the shaded carrion during Fresh and Bloat compared to that of the sunlit. Insect Population

Figure 4: Comparison of insect populations during Fresh stage of decomposition between sunlit and shaded carrion.

Figure 5: Comparison of insect populations during Bloat stage of decomposition between sunlit and shaded carrion.

Figure 6: Comparison of insect populations during Bloat stage of decomposition between sunlit and shaded carrion.

Table: Insect species found on carrion at each stage of decomposition.

Figure 7: Insect species observed on the carrion. Diptera: a) Calliphora stygia b) Muscidae sp. c) Sarcophaga aurifrons d) Piophila casei e) Chironomidae sp. Coleoptera: f) Scarabidae sp. g) Necrobia rufipes h) Cleridae sp. i) Curculionoidea sp. j) Paederus sp . Hymenoptera: k) Diapriidae sp. l) Rhytidoponera metallica m) Monomorium minimum . Acari: n) Tetranychidae sp. Gastrura: o) Gastrura sp . Photographs by Esther Breen. a) b) c) d) e) f) g) h) i) j) k) l) m) n) o) Insect Species Present

To determine PMI a scenario was developed using the meteorological station data. Using O’Flynn’s 9 data of C. stygia the base temperature threshold was determined to be 5.0°C and the development rate, at an average temperature of 9.5°C, would produce the following development times for each of the stages (in hours): Egg: 72.0 L1: 120.0 L2: 96.0 The total experimental time period to reach the third instar is 288 hours. Using the formula 5 : ADH = Time (hours) x (temperature – base temperature)  ADH = 288 x ( 9.5 – 5.0) = 1296.0 Post Mortem Interval 5 Gennard DE, 2007 Forensic Entomology: An introduction, John Wiley and Sons Limited, England 9 O’Flynn MA, 1983 ‘The succession and rate of development of blowflies in carrion in southern Queensland and the application of these data to forensic entomology’ Journal of Australian Entomological Society , Vol. 22, p.137-148

As a result it took 1296.0 accumulated degree hours for C. stygia to reach 3rd instar maggots. The ADD is then calculated: 1296.0 / 24 (the hours in a day) = 54.0 accumulated degree days. This, divided by the temperature data (9.5°C), results in 6.0 days for C. stygia to completely develop to the 3rd instar larvae stage 11 . Therefore, The estimated PMI lies between day 7 and 8 from when the carrion was placed at the location. From the knowledge of when the carrion was placed at the scene after death, this PMI is incorrect. When comparing the scenario PMI to the data from the current experiment, blowfly eggs were not observed on carrion until day 8. Therefore it is possible that the estimated PMI is correct in relation to the development rate of blowfly observed on the carrion. Anderson GS, 2001 Insect succession in carrion and its relationship to time since death In. Forensic Entomology: the utility of arthropods in legal investigations , Byrd JH & Castner JL (Eds.) CRC Press LLC, USA

Table 4:Calculation of the ADD for Calliphora stygia rate of development.

Discussion Temperature variations between sunlit and shaded carrion were not significantly different throughout the experiment. The temperature and humidity recorded by the data loggers from each carrion observed may have resulted in more significant figures if some had not failed. The data from the ThermochronTM logger, which was placed inside the carrion, and the underneath HydrochronTM logger would have resulted in maggot mass temperature variations which could have been compared. The rate of decomposition showed no significant differences between the sunlit and shaded carrion but decomposition was increased when warmer temperatures enhanced insect activity. This was obvious in P05 which had an abundance of insects present during most of the study. P06 showed substantial decomposition of the rear area, which received the most ambient warmth and sunlight through the shade cloth in the afternoons, whilst the upper portion of the body was in shade from a nearby tree. However the habitat also did appear to have an effect on the rate of decomposition. The soft tissue mummified in sunlit carrion, which would have made the skin tougher and harder for the insects and maggots to digest, slowing the rate of decomposition. Whereas, the soft tissue in the shaded carrion; developed adipocere around them which appeared to drown a number of insects trying to reach the carcass.

Shean et al. 12 determined that the ambient air temperature was the major factor that influenced variations in carrion decomposition between sunlit and shaded locations. This is supported by Joy et al. 13 who observed a greater rate of decomposition in higher ambient temperatures sunlit and shaded carrion decomposition. The sunlit carrion decomposed more rapidly compared to the shaded carrion, even when the ambient temperature was not significantly different. This may be related to variations in environmental conditions experienced by the carrion. The insect activity of the sunlit and shaded carrion presented very similar species with the sunlit carrion increasing in population for insects during all stages of decomposition, especially the Diptera species. The abundance of Diptera in sunlit locations was likely due to the warmer temperature increasing the activity of maggots and blowflies. Some species also showed a preference for the sunlit locations with no occurrence of these species on the shaded carrion. Hymenoptera were more abundant in the shaded location compared to the sunlit during the Bloat stage. But this may have been the result of increased larvae and eggs from the blowflies in the moist, shaded environment which would have provided more food and reproductive sources for certain species of parasitic wasp and ants. 12 Shean BS, Messinger L, & Papworth M, 1993 ‘Observtions of different decomposition on sun exposed v. shaded pig carrion in coastal washington state’, Journal of Forensic Science , Vol. 38, p. 938-949 13 Joy JE, Liette NL, & Harrah HL, 2006 ‘Carrion fly (Diptera: Calliphoridae) larval colonisation of sunlit and shaded pig carcasses in west virginia, USA’ Forensic Science International, Vol. 149, no.2-3, p. 57-65

The daily weather conditions were quite unpredictable with cold snaps, patchy rain and fog which then became warm and sunny or vice versa. The mean location temperature throughout the experimental period also showed no obvious linear increase in temperature as winter was heading into spring. These temperatures caused sudden active and dormant periods of insect activity with cooling and warming of the carrion as well as of the insects present. An examination on the effect of refrigeration on the development of blowflies and its impact for estimating PMI was performed by Myskowiak and Doums 14 . This research determined that development time of larvae was significantly reduced as well as larval weight when exposed to short periods of refrigeration at 4°C, before each growth phase which could cause significant errors in PMI. An alternative study by Ames and Turner 15 supporting this, found that cold temperatures caused variations in development rates between the same blowfly species, therefore reducing the reliability of ADH models that use a standard time frame for each development phase. They also suggest that larval growth still occurs during cold weather even if the species appears to be dormant. Myskowiak J, & Doums C, 2002 ‘ Effects of refrigeration on the biometry and development of Protophormia terranovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations’, Forensic Science International , Vol. 125, p. 254-261. Ames C, & Turner B, 2003 ‘Low temperature episodes in development of blowflies: implications for postmortem interval estimation’ Medical and Veterinary Entomology , Vol. 17, p.178-186.

The correct data set for the development of blowflies under different temperature conditions needs to be specific to the species, to reduce the chance of PMI errors 16 . The data used in the current experiment for the development rate of C. stygia were from laboratory reared samples collected in Queensland. Significant errors can arise from using an incorrect base temperature in which DD accumulations can be over-estimated. However, using an incorrect development minimum will only be problematic if daily temperatures are below the minimum threshold and the threshold is not set to zero 11 . The PMI using Gennard 6 ’s degree day calculations had consistent results with the development rate of C. stygia . Faults in the estimation to the time of carrion deposit could be contributed to by the cold temperatures causing late oviposition of blowfly eggs. Wallman 17 confirms the possibility of this occurring, where delays in oviposition can be from unsuitable climatic conditions; in which the extreme cold or heat will inhibit the thermal environment suitable for flies to seek out a body. The development rate of the blowfly larvae may also have been altered by maggot mass temperatures, which could have increased the temperature above ambient conditions 16 . Anderson GS 2000 ‘Minimum and maximum development rates of some forensically important Calliphoridae (Diptera)’, Journal of Forensic Science , Vol. 45, Is. 4, p. 824-832 Wallman JF, 2004 The application of entomology to criminal investigations. In: The practice of crime scene investigation , Horswell J(Ed.), p. 347-356, CRC Press LLC, USA

Limitations The temperature and humidity recorded by the data loggers from each carrion observed may have resulted in more significant figures if some had not failed. Sharing the carrion with other students also limited the data that I was able to collect as I was not able to disturb the carrion and collect liberally of insect samples. No maggots from the carrion were collected for laboratory rearing. The larvae of the blowfly species of the Canberra region could have been examined for variations to findings in the literature, such as the base development threshold of C. stygia and the effect of cold temperatures on the rate of development. The identification of insects was more difficult than expected with the likelihood of misidentifications without experienced entomologists.

Conclusion The current experiment demonstrated that the winter in the Canberra region reduced the rate of decomposition, which extended the stages of Fresh, Bloat and Decay over 90 days. The sunlit and shaded pig carrion showed no significant difference in rate of decomposition, however changes in soft tissue were observed showing mummification in the dry, sunlit habitats and the formation of adipocere in the moist, shaded habitats. Temperature variations between the two habitats showed no significant difference in ambient temperature or body temperature, but the control samples did demonstrated that the rate of decomposition was linked to the insect activity. When examining insect activity in sunlit and shaded locations, a significant difference between insect populations was observed. Diptera abundance was almost doubled at sunlit carrion sites throughout the stages of decomposition in comparison to shaded carrion, with slight variations in Hymenoptera and Coleoptera numbers. However, the Hymenoptera population was more abundant at shaded sites during Bloat. There also were observable preferences between some species for sunlit or shaded environments. when examining the development rate and ADD of blowflies at this time of year, errors in PMI due to cold weather which may delay the oviposition of eggs and reduce the growth rate of larvae can cause inaccuracies in PMI estimates. Therefore, temperature conditions which are below a species minimum temperature threshold and which stunt larvae development should be taken into consideration when estimating the PMI.

Future Directions and Research The current study had some limitations which could be improved in future sunlit and shaded carrion examination. Further consideration of location differences of sunlit and shaded carrion, and differences of seasonal and yearly data would contribute significantly to the research. Additional data from maggot mass temperatures, laboratory reared maggots and effective data loggers would also be beneficial. A comprehensive study of variations in Calliphora stygia larval weight, length, development rate and minimum temperature threshold for cold temperature conditions, which are collected from the Canberra region, could provide more information for estimating PMI during winter. Also the statistical error in delayed oviposition and the accuracy of PMI estimations could be examined to determine a standard error rate.

Acknowledgement I would like to thank my Supervisors for all there help and support: Catherine Fitzgerald Bryan Lessard Jodie Ward My appreciation also goes out to ANU and the manager of Spring Valley Farm, John Sullivan for allowing me access to the property. I would like to thank the Canberra Institute of Technology Forensic Studies staff for their help. A big thankyou goes out to all my family and friends for all their support with the project and especially those who took the time to help proof read the research project.

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