The blowflies

The blowflies Background The insects that have been most extensively studied in relation to their forensic uses are the blowflies, members of the Calliphoridae fly family in particular their larvae because they are the insects most commonly associated with corpses. Blowflies are usually the first to colonise a body after death, often within hours. The larval stage is the main period in which blowflies face limited food resources, when the fully grown third instar larvae stop feeding; they usually migrate in search of a place to pupate. Because blowfly pupae can provide useful forensic evidence it is important to know where the pupae are likely to be located. Methodology This study was carried out to investigate a variety of factors affecting the pupation behaviour of two forensically important species of blowfly larvae of Calliphora vomitoria and Lucilia sericata in soil. The burrowing behaviour of both species was studied in the laboratory under controlled conditions. Larvae of both Calliphora vomitoria and Lucilia sericata were used in six experiments for each condition. Principle findings The main findings were that most of the biological factors had an effect on the burial behaviour in Larvae of Calliphora vomitoria and Lucilia sericata. 1. Introduction Calliphora vomitoria and Lucilia sericata are two forensically important species of blowflies since they can arrive within few minutes (Payne 1965) or even few seconds (DeJong 1995) following corpse exposure. Because of this, the age of the oldest blowflies gives the most accurate evidence of the post mortem interval (PMI). Many other species of fly, beetle and wasp are also associated with corpses resulting in a succession of insects arriving at the body, but as they tend to arrive after the blowflies, they are less useful in establishing a PMI.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Blowfly infestations of human bodies are a natural outcome of the flies role in the environment as primary decomposers. The larval infestations are an essential component of the natural recycling of organic matter and, on human bodies; they can provide vital evidence to the timing and cause of death. Adult blowflies are well adapted to sensing and locating the sources of odours of decay, eggs are usually laid in dark and moist places such as the eyes, mouth and open sores. The eggs then quickly hatch into first instar larvae which feed rapidly, and shed their skin twice to pass through second and third instars until they finish feeding, or once the food resource has become unavailable.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  After the fully grown third instar larvae stop feeding and show no further response towards food, depending on the species the larvae leave in search of a suitable place to pupate. They may move many meters before burrowing into the soil. The larva then contracts and the cuticle hardens and darkens to form the puparium, within which the pupa transforms into an adult fly. When the fly emerges, the empty puparial case is left behind as evidence of the blowflies development. However, there are many biological factors that affect the pupation behaviour of larvae in soil. These factors include temperature, soil moisture content, soil compaction, as well as the effect of pre burial and high density. All the mentioned factors need to be considered when determining a PMI, however for many of them, little information is available. Furthermore, there are several studies on the influence of temperature on the behaviour of burrowing in larvae of blowflies such as the one done by Gomes (2009). The study of larvae burying behaviour is important to improve understanding of one of the process during larval dispersion, and to try and understand the influence of biological variables on this behaviour   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  The present study was conducted to investigate factors that influence the burial behaviour in post-feeding third instar blowfly larvae of Calliphora vomitoria and Lucilia sericata to evaluate if these two species have a different pupation pattern in the different treatments. 2. Materials and Methods C. vomitoria and L. sericata were collected; one thousand and sixty of each species in the final third instar stage were used for these experiments. The soil used was John Innes No 2 potting compost; all six experiments were carried out using the main materials mentioned. 2.1. Determination of normal burial depth and how this is affected by temperature Nine plastic containers were filled with soil to a depth of 24cm and were placed in an incubator so as to allow the soil to reach the temperatures required. Three of the containers had to reach 10 °C, the other three had to reach a temperature of 20 °C, and the remainder each at 28 °C. Fifteen larvae of Lucilia were then placed onto the soil surface of each of the containers; three at 10 °C, 20 °C and 28 °C. The same was done to the larvae of Calliphora, and the time of how long it took the larvae to burrow into the soil was observed, i.e., how long is it before the first and last larva burrows down. Similarly observations were made to see whether the larvae resurface and how if they do how soon. A total of eighteen containers were then covered with muslin cloth kept firmly in position by a rubber band and left for seven days. 2.2. Determination of the effect of moisture content Six plastic containers were filled with soil to a depth of 24cm, then 100ml of water was added to three of the containers and these were labelled as moist. 500ml of water was added to each of the remainder and these were labelled as wet. The containers were then left for 40 minutes in order for the water to be absorbed, after which fifteen larvae of Lucilia were added into each of the six containers, three wet and three moist. The same was done to the larvae of Calliphora, and then the time of how long it took for the larvae to burrow into the soil was counted and all twelve containers were placed into an incubator at 20 °C. 2.3. Determination of the effect of pre-burial 1 cm of soil was added to the bottom of a plastic container, and fifteen larvae of Lucilia were added and covered with 10cm layer of soil, and this was replicated twice. Also 10cm layer of soil was added to the bottom of another container, and fifteen larvae of Lucilia were added but this time they were covered with 20cm layer of soil and this was replicated twice. The exact same was done to the larvae of Calliphora. After the larvae were buried to a depth of 10cm or 20cm, observations were made to check how long it took for the first maggot to reach the surface, and the number of larvae on the surface was counted at 15, 30, 45 and 60 minutes. All 12 containers were then placed in an incubator at 20 °C. 2.4. Determination of the effect of soil compaction Soil was compacted into six containers to a depth of 24cm, and then fifteen larvae of Lucilia were added to each of the three containers. Also fifteen larvae of Calliphora were added to the other three containers, and observations were made to check how long it took for the larvae to burrow into the soil, i.e., how long was it before the first and last larva were burrowed. All six containers were incubated at 20 °C and then left for seven days. 2.5. Determination of the effect of larval density Three plastic containers were filled with highly dense soil to a depth of 24cm, and 150 larvae of Lucilia were added to each container. The same was done to the larvae of Calliphora, and observations were then made to see how long it took for the first and last larvae to burrow down. All six containers were covered with muslin cloth kept firmly in position by a rubber band and incubated at 20 °C. 2.6. Determination of the distance moved by the post-feeding stage of C. vomitoria and L. sericata from their feeding site 500 post-feeding larvae of the two species were released on a grassland area on the Byrom Street Campus, Liverpool John Moore University, UK. After 7 days soil core samples were taken from the surrounding soil and were searched in order to locate the pupae.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  After the larvae pupated in all of the experiments, they were located and removed from the soil as follows: a line was drawn every 2cm on the side of all the containers up until a soil depth of 24cm using a permanent marker pen, after which the number of pupae found on the surface was counted and removed. Moreover, each 2cm layer of soil was then carefully removed using a spatula and placed onto a plastic sheet where it was thoroughly searched, and the number of all the pupae of all the containers of the five experiments was calculated. All five experiments were undertaken at a lab temperature of 20 °C. However, there are many biological factors that affect the pupation behaviour of larvae in soil. These factors include temperature, soil moisture content, soil compaction, as well as the effect of pre burial and high density. All the mentioned factors need to be considered when determining a PMI, however for many of them, little information is available. Furthermore, there are several studies on the influence of temperature on the behaviour of burrowing in larvae of blowflies such as the one done by Gomes (2009). The study of larvae burying behaviour is important to improve understanding of one of the process during larval dispersion, and to try and understand the influence of biological variables on this behaviour   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  The present study was conducted to investigate factors that influence the burial behaviour in post-feeding third instar blowfly larvae of Calliphora vomitoria and Lucilia sericata to evaluate if these two species have a different pupation pattern in the different treatments. 2. Materials and Methods C. vomitoria and L. sericata were collected; one thousand and sixty of each species in the final third instar stage were used for these experiments. The soil used was John Innes No 2 potting compost; all six experiments were carried out using the main materials mentioned. 2.1. Determination of normal burial depth and how this is affected by temperature Nine plastic containers were filled with soil to a depth of 24cm and were placed in an incubator so as to allow the soil to reach the temperatures required. Three of the containers had to reach 10 °C, the other three had to reach a temperature of 20 °C, and the remainder each at 28 °C. Fifteen larvae of Lucilia were then placed onto the soil surface of each of the containers; three at 10 °C, 20 °C and 28 °C. The same was done to the larvae of Calliphora, and the time of how long it took the larvae to burrow into the soil was observed, i.e., how long is it before the first and last larva burrows down. Similarly observations were made to see whether the larvae resurface and how if they do how soon. A total of eighteen containers were then covered with muslin cloth kept firmly in position by a rubber band and left for seven days. 2.2. Determination of the effect of moisture content Six plastic containers were filled with soil to a depth of 24cm, then 100ml of water was added to three of the containers and these were labelled as moist. 500ml of water was added to each of the remainder and these were labelled as wet. The containers were then left for 40 minutes in order for the water to be absorbed, after which fifteen larvae of Lucilia were added into each of the six containers, three wet and three moist. The same was done to the larvae of Calliphora, and then the time of how long it took for the larvae to burrow into the soil was counted and all twelve containers were placed into an incubator at 20 °C. 2.3. Determination of the effect of pre-burial 1 cm of soil was added to the bottom of a plastic container, and fifteen larvae of Lucilia were added and covered with 10cm layer of soil, and this was replicated twice. Also 10cm layer of soil was added to the bottom of another container, and fifteen larvae of Lucilia were added but this time they were covered with 20cm layer of soil and this was replicated twice. The exact same was done to the larvae of Calliphora. After the larvae were buried to a depth of 10cm or 20cm, observations were made to check how long it took for the first maggot to reach the surface, and the number of larvae on the surface was counted at 15, 30, 45 and 60 minutes. All 12 containers were then placed in an incubator at 20 °C. 2.4. Determination of the effect of soil compaction Soil was compacted into six containers to a depth of 24cm, and then fifteen larvae of Lucilia were added to each of the three containers. Also fifteen larvae of Calliphora were added to the other three containers, and observations were made to check how long it took for the larvae to burrow into the soil, i.e., how long was it before the first and last larva were burrowed. All six containers were incubated at 20 °C and then left for seven days. 2.5. Determination of the effect of larval density Three plastic containers were filled with highly dense soil to a depth of 24cm, and 150 larvae of Lucilia were added to each container. The same was done to the larvae of Calliphora, and observations were then made to see how long it took for the first and last larvae to burrow down. All six containers were covered with muslin cloth kept firmly in position by a rubber band and incubated at 20 °C. 2.6. Determination of the distance moved by the post-feeding stage of C. vomitoria and L. sericata from their feeding site 500 post-feeding larvae of the two species were released on a grassland area on the Byrom Street Campus, Liverpool John Moore University, UK. After 7 days soil core samples were taken from the surrounding soil and were searched in order to locate the pupae.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  After the larvae pupated in all of the experiments, they were located and removed from the soil as follows: a line was drawn every 2cm on the side of all the containers up until a soil depth of 24cm using a permanent marker pen, after which the number of pupae found on the surface was counted and removed. Moreover, each 2cm layer of soil was then carefully removed using a spatula and placed onto a plastic sheet where it was thoroughly searched, and the number of all the pupae of all the containers of the five experiments was calculated. All five experiments were undertaken at a lab temperature of 20 °C. 3. Statistical Analysis The results were expressed as the mean and standard deviation (S.D). The Chi-Square test was performed to determine whether the observed frequency distribution differs significantly from the expected one. 4. Results discussion 4.1. Determination of normal burial depth and how this is affected by temperature The larvae of Calliphora vomitoria burrowed themselves deeper at a temperature of 10 °C to pupate, whereas the larvae of Lucilia sericata remained closer to the surface at the lower and higher temperatures used in this experiment. L.sericata shows normal distribution at 10 °C, however it stops burrowing at a depth of 14cm. In contrast, C.vomitoria continues to burrow to a depth of 24 but is not evenly distributed. The Chi square test was done for this experiment in order to see if there was a significant difference between the specific temperatures used as the graph didnt show clear differences. The results from the test showed that the distribution of C.vomitoria and L. sericata at a temp of 10 °C was significant ?2 (df 2) = 18.30 p>5.99, ?2 (df 2)= 17.85 p>5.99, also at a temperature of 20 °C for C.vomitoria it was found to be significant ?2 (df 2) = 6.49 p>5.99, and for L. Sericata ?2 (df 2) = 18.30 p>5.99 significant distribution. 4.2. Determination of the effect of moisture content The two species of larvae burrowed themselves up until a depth of 10cm; remained close to the surface to pupate in wet and moist soil conditions. However, the number of pupae of C.vomitoria was high in wet soil. In contrast, the number of pupae of L.sericata was high in moist soil. 4.3. Determination of the effect of pre-burial 4.4. Determination of the effect of soil compaction It is clear from the results that biological factors studied have a significant effect on the burying behaviour of the two species of larvae studied in this experiment. The rate of development of all insects is directly dependent on the ambient conditions, mainly temperature. Between upper and lower thresholds, which vary between species, the higher the temperature, the faster the insects will develop; the lower the temperature, the slower they will develop. If the ambient temperatures during the period of development are known, then the minimum PMI can be determined. Temperature affected the burrowing behaviour of larvae prior to pupation (Fig.1). At low temperatures, the metabolic rate may be markedly reduced and this could result in greater body weight and a tendency to burrow deeper in order to escape low temperatures (Grassberger and Reiter 2002) 5. Acknowledgement I would like to thank Dr Alan Gun for supporting the research reported by providing the data and equipment. I would also like to thank Dr Jeri Bird for his assistance in the data analysis. Thanks also to my lab partners and colleagues for their help and support. 6. References Clark, K., Evans, L. Wall, R. (2006) Growth rates of the blowfly Lucilia sericata on different body tissues. Forensic Science International 156, 145-149 DeJong GD. An Annotated Checklist of the Calliphoridae (Diptera) Of Colorado, With Notes on Carrion Associations and ForensicImportance. Journal of Kansas Entomological Society, 1995; 67(4): 378-385. Gomes,L., Gomes, G., Von Zuben, C.L. (2007) the influence of temperature on the behaviour of burrowing larvae of blowflies,Chrysomya albiceps and Lucilia cuprina, under controlled conditions. Journal of insect science.9, 1536-2442 Gomes, L., Sanches, M.R. Von Zuben, C.J. (2004) Dispersal and Burial Behaviour in Larvae of Chrysomya megacephala and Chrysomya albiceps (Diptera: Calliphoridae). Journal of insect behaviour 18, 282-292 Grassberger, M. Reiter, C. (2002) Effect of temperature of development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphordae). Forensic Science international 128, 177-182 Gunn, A. (2009) Essential Forensic Biology. 2nd edition, Wiley 214-251 Payne JA. A Summer Carrion Study of the Baby Pig Sus scrofa Linnaeus.Ecology, 1965; 46 (5): 592-602. Singh, D., Bala, M. (2009) the effect of starvation on the larval behaviour of two forensically important species of blow flies (Diptera: Calliphoridae). Forensic Science international 193, 118-121 Tullis K and Goff ML. Arthropod Succession in Exposed Carrion in tropical Rainforest on Ohau Island, Hawaii. Journal ofMedical Entomology, 1987; 24: 332-339. Wooldridge, J., Scrase, L., Wall, L. (2007) Flight activity of the blowflies, Calliphora vomitoria and Lucilia sericata, in the dark. Forensic Science International 172, 94-97 Cleveland Museum of Natural History, Blow fly life cycle. Available: http://www.nlm.nih.gov/visibleproofs//education/entomology/blow_fly_life_cycle.pdf Accessed 09/01/2010